Sleep research in space: expression of immediate early genes in forebrain structures of rats during the nasa neurolab mission (STS-90).

1. Electrophysiological and behavioural observations have shown that changes in the sleep-waking activity occur in astronauts during the space flight. Experiments performed in ground-based experiments have previously shown that the immediate early gene (IEG) c-fos, a marker of neuronal activation, can be used as a molecular correlate of sleep and waking. However, while Fos expression peaks within 2-4 hours after the stimulus and returns to baseline within 6-8 hours, other IEGs as the FRA proteins which are also synthetized soon after their induction, persist in the cell nuclei for longer periods of time, ranging from 1-2 days to weeks. 2. Both Fos and FRA expression were evaluated in several adult albino rats sacrificed at different time points of the space flight, i.e. either at FD2 and FD14, i.e. at launch and about two weeks after launch, respectively, or at R + 1 and R + 13, i.e. at the reentry and about two weeks after landing. The changes in Fos and FRA expression were then compared with those obtained in ground controls. These experiments demonstrate activation of several brain areas which varies during the different phases of the space flight. Due to their different time of persistence, Fos and FRA immunohistochemistry can provide only correlative observations. In particular, FRA expression has been quite helpful to identify the occurrence of short-lasting events such as those related either to stress or to REM-sleep, whose episodes last in the rat only a few min and could hardly be detected by using only Fos expression. 3. Evidence was presented indicating that at FD2 and FD14 Fos-labeled cells were observed in several brain areas in which Fos had been previously identified as being induced by spontaneous or forced waking in ground-based experiments. In contrast to these findings FLT rats sacrificed at R + 1 showed low levels of Fos immunostaining in the cerebral cortex (neocortex) and several forebrain structures such as the hypothalamus and thalamus. Some Fos staining was also present in limbic cortical areas, the septum, and the hippocampus. The main area of the forebrain of FLT rats sacrificed at R + 1, showing an increased expression of Fos, was the central nucleus of the amygdala (CeA) (cf. 127), as well as the noradrenergic locus coeruleus (LC) nucleus (cf. 122). At R + 13 Fos immunostaining was variable among FLT rats. However, none of these rats showed a significant number of Fos-positive cells in CeA. 4. Most of the rats studied for Fos expression were also tested for FRA expression. In particular, a scattered amount of FRA expression occurred at FD14 in different areas of the neocortex and in limbic forebrain regions (such as the cingulate, retrosplenial and entorhinal cortex). It included also the hippocampus, the lateral septum, the caudate/putamen, as well as some hypothalamic regions. At the reentry (R + 1) it was previously shown that a prominent increase in FRA expression occurred in the LC of FLT rats (cf. 122). This finding was associated with an increase in FRA expression which affected not only the nucleus paragigantocellularis lateralis of the medulla, which sends excitatory glutamatergic afferents to the LC (cf. 31 for ref.), but also structures which are known to produce corticotropin-releasing factor (CRF), a neuropeptide which activates the noradrenergic LC neurons during stress. 5. These findings which result from acceleration stress were followed by REMS episodes, which probably occurred after a long period of sleep deprivation following exposure to microgravity. It was previously shown that an increase in Fos and FRA expression occurred at the reentry in some pontine and medullary reticular structures (cf. 128), which are likely to be involved in both the descending (postural atonia) and the ascending manifestations of PS. These findings can be integrated by results of the present experiments showing that at the reentry high levels of FRA expression occurred in the hippocampus and the limbic system, i.e. in structures which are involved in the generalized pattern of EEG desynchronization and the theta activity, typical of REMS (cf. 83, 84). A prominent increase in FRA expression also affected at the reentry some components of the amygdaloid complex, particularly the CeA. as well as some related structures, such as the lateral parabrachial nucleus (cf. 122) and the nucleus of the tractus solitarius (cf. 127). These structures are known to contribute to the PGO waves, which drive the oculomotor system either directly or through the medial vestibular nuclei (128, cf. also 126). Unfortunately due to our brainstem transections we were unable to evaluate the changes in gene expression which could affect the dorsolateral pontine structures during the occurrence of REMS episodes. Further experiments are thus required to investigate the role that these pontine structures exert in determining adaptive changes following exposure to microgravity after launch as well as readaptation to the terrestrial environment after landing.

Gene expression in autonomic areas of the medulla and the central nucleus of the amygdala in rats during and after space flight.

During space flight astronauts show vestibular-related changes in balance, eye movements, and spontaneous and reflex control of cardiovascular, respiratory and gastrointestinal function, sometimes associated with space motion sickness. These symptoms undergo compensation over time. Here we used changes in the expression of two immediate-early gene (IEG) products to identify cellular and molecular changes occurring in autonomic brainstem regions of adult male albino rats killed at different times during the Neurolab Space Mission (STS-90). Both direct effects of gravitational changes, as well as indirect effects of gravitational changes on responses to light exposure were examined. Regions under the direct control of vestibular afferents such as the area postrema and the caudal part of the nucleus of the tractus solitarius (NTSC) were both directly and indirectly affected by gravity changes. These areas showed no changes in the expression of IEG products during exposure to microgravity with respect to ground controls, but did show a significant increase 24 h after return to 1 G (gravity). Exposure to microgravity significantly inhibited gene responses to light exposure seen after return to 1 G. A similar direct and indirect response pattern was also shown by the central nucleus of the amygdala, a basal forebrain structure anatomically and functionally related to the NTS. The rostral part of the NTS (NTSR) receives different afferent projections than the NTSC. This region did not show any direct gravity-related changes in IEG expression, but showed an indirect effect of gravity on IEG responses to light. A similar pattern was also obtained in the intermediate reticular nucleus and the parvocellular reticular nucleus. Two other medullary reticular structures, the dorsal and the ventral medullary reticular nuclei showed a less well defined pattern of responses that differed from those seen in the NTSC and NTSR. The short- and long-lasting molecular changes in medullary and basal forebrain gene expression described here are thought to play an important role in the integration of autonomic and vestibular signals that ultimately regulate neural adaptations to space flight.

Short-term (Fos) and long-term (FRA) protein expression in rat locus coeruleus neurons during the neurolab mission: contribution of altered gravitational fields, stress, and other factors.

Changes in immediate-early gene (IEG) expression during and after space flight were studied in the rat locus coeruleus (LC) during the NASA Neurolab mission. The LC sends widespread projections throughout the brain and releases the neuromodulator norepinephrine. LC neurons respond to natural vestibular stimulation; their firing rate also increases during waking and decreases or ceases during sleep. LC neurons express IEGs such as c-fos after activation. Adult male albino Fisher 344 rats were killed at four mission time points, and the number of Fos- and Fos-related antigen (FRA)-positive LC cells were counted in flight and ground-based control rats. Half of the subjects at each time point were exposed to light for 60 min prior to killing to standardize their sleep-waking state. FRA-expressing cells were more numerous than Fos-expressing cells in both flight- and ground-based subjects. The difference between FRA- and Fos-expressing cells within individuals was significantly larger 24 h after landing in subjects exposed to both space flight and light pulse than in all other subjects at any mission time point. Fos and FRA responses scaled in proportion to the maximum response observed in any single individual showed similar patterns of variation. Analysis of the scaled and combined responses showed that LC IEG levels responded to both gravity changes and light pulses. Subjects exposed to either single stimulus had equivalent responses, significantly greater than those of control subjects maintained in dim light. The combination of gravity change and light pulse gave significantly higher LC responses than either stimulus alone 24 h after takeoff, and to a lesser extent after 12 days in space; the highest responses were obtained 24 h after landing. By 14 days after landing, animals exposed to space flight and light pulse responded no differently than ground-based subjects. No difference in LC IEG expression was clearly attributable to changes in the sleep-waking state of subjects. Activity of noradrenergic LC neurons has been previously shown to modulate IEG expression in target structures. The increased IEG LC activity (seen most especially 24 h after landing) may reflect large-scale activation of noradrenergic neurons that may serve as a trigger for molecular changes in target structures, and be critical for adaptation to gravity changes.

Gene expression in rat vestibular and reticular structures during and after space flight.

Space flight produces profound changes of neuronal activity in the mammalian vestibular and reticular systems, affecting postural and motor functions. These changes are compensated over time by plastic alterations in the brain. Immediate early genes (IEGs) are useful indicators of both activity changes and neuronal plasticity. We studied the expression of two IEG protein products [Fos and Fos-related antigens (FRAs)] with different cell persistence times (hours and days, respectively) to identify brainstem vestibular and reticular structures involved in adaptation to microgravity and readaptation to 1 G (gravity) during the NASA Neurolab Mission (STS-90). IEG protein expression in flight animals was compared to that of ground controls using Fisher 344 rats killed 1 and 12 days after launch and 1 and 14 days after landing. An increase in the number of Fos-protein-positive cells in vestibular (especially medial and spinal) regions was observed 1 day after launch and 1 day after landing. Fos-positive cell numbers were no different from controls 12 days after launch or 14 days after landing. No G-related changes in IEG expression were observed in the lateral vestibular nucleus. The pattern of FRA protein expression was generally similar to that of Fos, except at 1 day after landing, when FRA-expressing cells were observed throughout the whole spinal vestibular nucleus, but only in the caudal part of the medial vestibular nucleus. Fos expression was found throughout the entire medial vestibular nucleus at this time. While both Fos and FRA expression patterns may reflect the increased G force experienced during take-off and landing, the Fos pattern may additionally reflect recent rebound episodes of rapid eye movement (REM) sleep following forced wakefulness, especially after landing. Pontine activity sources producing rhythmic discharges of vestibulo-oculomotor neurons during REM sleep could substitute for labyrinthine signals after exposure to microgravity, contributing to activity-related plastic changes leading to G readaptation. Reticular structures exhibited a contrasting pattern of changes in the numbers of Fos- and FRA-positive cells suggestive of a major influence from proprioceptive inputs, and plastic re-weighting of inputs after landing. Asymmetric induction of Fos and FRAs observed in some vestibular nuclei 1 day after landing suggests that activity asymmetries between bilateral otolith organs, their primary labyrinthine afferents, and vestibular nuclei may become unmasked during flight.

Immediate early gene expression in the vestibular nuclei and related vegetative areas in rats during space flight.

Changes in neuronal activity resulting in somatic and vegetative deficits occur during different space flight conditions. Immediate early genes (IEGs: c-fos and Fos-related antigen [FRA]) are useful indicators of changes in neuronal activity and plasticity. They are induced within minutes of several extracellular stimulations, while the corresponding proteins persist for hours (Fos) or days (FRAs). Changes in IEG expression are likely to contribute to adaptation to microgravity and readaptation to the terrestrial environment. During the NASA Neurolab Mission (STS-90), changes in IEG expression were studied in adult male albino rats (Fisher 344) sacrificed at flight day (FD) 2 (24 h after launch), FD14 and at similar time points after re-entry (R + 1, 24 h after re-entry, and R + 13). These time points were chosen to maximize the probability of detecting changes in IEG expression related to changes in gravitational fields occurring during the mission, e.g. (i) increase in gravitational force from 1 to 3 g during the launch, before reaching about 0 g at FD2; (ii) adaptation to 0 g at FD14; (iii) increase in gravity from 0 to approximately 1.5-1.8 g before reaching 1 g at R + 1; and (iv) readaptation to 1 g at R + 13. Fos- and FRA-positive cells were identified in the brainstem of flight rats and ground-based controls using immunocytochemistry. With respect to control rats, the number of labeled cells increased in flight animals in the medial and spinal vestibular nuclei (but not in the lateral vestibular nucleus) at FD2, decreased at FD14, greatly increased at R + 1 and returned to baseline levels at R + 13. Similar changes in IEG expression were also observed in the nucleus of the solitary tract, the area postrema and the central nucleus of the amygdala. In particular, in these vegetative areas the number of Fos-positive cells decreased in flight rats with respect to controls at FD14, i.e. after exposure to 0 g, but significantly increased at R + 1, i.e. after return to 1 g. Thus, altered gravitational fields produced molecular changes in vestibular nuclei controlling somatic functions, as well as in related medullary and basal forebrain structures regulating vegetative functions.

Fos-related antigens are involved in the transcriptional responses of locus coeruleus neurons to altered gravitational fields in rats.

Locus coeruleus (LC) neurons, which have widespread projections to the whole brain, respond to natural stimulation of macular receptors. Using immunocytochemistry we investigated whether rats exposed to altered gravitational fields showed changes in Fos and Fos-related antigen (FRA) protein levels in the LC. Fos protein is induced very rapidly and returns to basal levels within hours after stimulation, while FRAs persist for days or weeks after induction. Adult male albino rats (Fisher 344) were sacrificed at different time points during a space flight (NASA Neurolab Mission, STS-90) and the numbers of Fos- and FRA-positive cells in the LC were counted and compared to those in ground-based control rats. No significant changes in Fos protein expression were detected in the LC under different space flight conditions. In contrast, the number of FRA-positive cells increased on average to 167% of that of the controls at FD2, i.e. when gravity increased from 1 to 3 g during the launch before reaching about 0 g. FRA-labeled neurons then decreased to 46% of control values at FD14, i.e. after adaptation to 0 g, but increased again to 317% of control values at R + 1, when the animals were exposed to an increase in gravitational force from 0 to 1.5-1.8 g before reaching 1 g during landing. The number of labeled cells was 193% of the control values at R + 13, i.e. after readaptation to 1 g. Thus gravitational force appears to be very effective in inducing a long-term increase in FRA protein expression in the LC. Because activity in the noradrenergic LC neurons may increase Fos expression in several target structures, we postulate that the long-lasting induction of FRAs in the LC at FD2, and more prominently at R + 1, may contribute to the long-term molecular changes which probably occur in the brain during adaptation to 0 g and readaptation to 1 g.