1 Instituto Cajal (CSIC), Avenida Dr Arce, 37, 28002 Madrid, Spain, , 2 Universidad Europea CEES, Villaviciosa de Odón, 28670 Madrid, Spain and , 3 Department of Physiology and Neuroscience, New York University School of Medicine, New York, NY 10016, USA
Javier DeFelipe, Instituto Cajal (CSIC), Avenida Dr Arce, 37, 28002 Madrid, Spain. Email: defelipe{at}cajal.csic.es.
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Abstract |
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Introduction |
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The results from several studies suggest that a terrestrial gravitational field is necessary for the normal, early postnatal development of the motor system. For example, by simulating weightlessness in rats using a tail-suspension model (a system that allows the animals to move about the cage, extend and flex their hindlimbs), a critical period of development from postnatal day 13 (P13) to P31 has been identified (Walton et al., 1992). During this neonatal period, suspended animals are sensitive to hindlimb unloading and develop marked abnormalities in locomotion that persist into adulthood, particularly in the hindlimbs (Walton et al., 1992
). Studies on neonatal hindlimb muscle have shown that the soleus, a prototypical antigravity hindlimb muscle, becomes atrophied after tail suspension from P8 to P24 (Huckstorf et al., 2000
), and recent studies have reported changes in the expression of myosin isoforms in neonatal rats after spaceflight (Adams et al., 2000
; Ikemoto et al., 2001
). At the cortical level, spaceflight studies have revealed increases in the overall density of dendritic spines in the sensorimotor cortex of adult rats after 7 and 14 days in microgravity (Belichenko, 1988
; Belichenko and Krasnov, 1991
). However, these studies did not examine the tissue at ultrastructural level to determine quantitative changes in synaptic size or connectivity. In addition, the transitory or permanent nature of these changes has not been assessed. Electrophysiological and electron microscope studies have shown that rodent neocortical circuits mature gradually between the first and sixth week postnatal (Blue and Parnavelas, 1983
; Bähr and Wolff, 1985
; Luhmann and Prince, 1991
; Agmon and ODowd, 1992
; Agmon et al., 1993
, 1996
; Micheva and Beaulieu, 1996
; White et al., 1997
; DeFelipe et al., 1997
; Wells et al., 2000
). Thus, the changes in the afferent information that reaches the sensorimotor cortex induced by selected muscle atrophy and the altered use of hindlimb muscles in microgravity may affect cortical synaptic organization. Therefore, we examined the maturation of synaptic circuits of the neocortex of neonatal rats that developed for 16 days in a low Earth orbit (Neurolab mission, STS-90).
[Neurolab was a NASA research mission dedicated mainly to study how the nervous system responds in microgravity, a fundamental question for future long-duration space flights. Neurolab was born when the US President declared the 1990s the Decade of the Brain, and NASA proposed the Neurolab mission as its contribution to this dictate. Other International Space Agencies also participated in the Neurolab mission. The seven-member crew were not only involved in various experiments with animals (rats, mice, fish, snails and crickets) aboard the Space Shuttle Columbia, but they were also themselves the subjects of a number of sophisticated biomedical examinations. The Shuttle was launched on 17 April and landed on 4 May 1998 at Kennedy Space Center in Cape Canaveral (Florida, USA). The Shuttle reached an altitude of ~320 km above the planets surface and traveled at a speed of ~7.5 km/s. Since the shuttle orbited the Earth every 92 min, during the 16 day spaceflight there were 16 sunsets and 16 sunrises every 24 h. Therefore, a total of 256 complete orbits around the Earth were undertaken. Crew members were Scott D. Altman, Jay C. Buckey, Richard M. Linnehan, Kathryn P. Hire, James A. Pawelczyk, Richard A. Searfoss and Dafydd Rhys Williams.]
We focused our ultrastructural analysis on the hindlimb region of the cortex as extensive overlapping of the motor and sensory maps occurs in this region (Hall and Lindholm, 1974; Jones and Porter, 1980
; Donoghue, 1995
). We discuss the relationship between the effects observed and the motor strategies adopted by neonates in microgravity as well as the persistence of microgravity-induced alterations in the motor system after returning to terrestrial gravity.
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Materials and Methods |
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Ultrastructural Analysis
Since fresh tissue from the different regions of the nervous system or from other organs from these rats was to be used in other studies, animals were anesthetized with Nembutal and decapitated. The brains were removed rapidly and three blocks of tissue per hemisphere were obtained from each rat. These blocks were fixed by immersion in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4, for 36 h at 4°C. Serial 50 µm Vibratome sections were cut from the blocks containing the hindlimb representation of the somatosensory cortex [between Bregma 0.26 mm and Bregma 2.12 mm; Paxinos and Watson (Paxinos and Watson, 1997)]. Some sections were stained with thionin to identify the hindlimb cortex through the presence of a prominent granular cell layer (layer IV) and of large pyramidal cells in layer V, similar in size to those found in the adjacent agranular (motor) cortex (Jones and Porter, 1980
) (Fig. 1A,B
). Cortical layers were identified as layers I, II/III, IV, Va, Vb and VI (Zilles and Wree, 1995
). Serial adjacent sections to those Nissl-stained sections including the hindlimb cortex were processed for electron microscopy, as follows. Sections were post-fixed in 2% glutaraldehyde in PB for 1 h and in 1% osmium tetroxide, dehydrated and embedded in Araldite. These plastic-embedded sections were resectioned serially into semithin (2 µm thick) sections (Fig. 1C,D
) with a Reichert ultramicro-tome. The semithin sections were stained with 1% toluidine blue in 1% borax and examined by light microscopy to identify the cortical layers. Semithin sections containing the hindlimb cortex were photographed and then resectioned into serial ultrathin sections with a silvergray interference color (DeFelipe and Fairén, 1993
). The ultrathin sections were collected on Formvar-coated single-slot grids, stained with uranyl acetate and lead citrate, and examined in a Jeol-1200 EX electron microscope.
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Synaptic density per unit area (NA) was estimated from 10 electron microscope samples of neuropil from each layer (layers I, II/III, IV, Va, Vb and VI) from each animal. These samples were non-overlapping electron micrographs taken at an initial magnification of 10 000 and printed at a final magnification of 30 000 (DeFelipe et al., 1999). All synaptic profiles were counted in each print within an unbiased counting frame (Gundersen, 1977
) which represented 35 µm2 of tissue. Synaptic profiles that touched the exclusion lines were not counted. These counts were used to determine the numerical density of synapses per unit volume of neuropil, using the formula NV = NA/d (Weibel, 1979
), where NA is the number of synaptic profiles per unit area and d the average cross-sectional length of synaptic junctions [see DeFelipe et al. (DeFelipe et al., 1999
)]. The cross-sectional lengths of synaptic junctions (length of the paired membrane densities at each junction) of all asymmetrical and symmetrical synaptic profiles, as well as the lengths of the postsynaptic densities in the case of oblique and en face synaptic profiles, were measured in the prints using a magnetic tablet (SummaSketch III) and the Scion Image image analysis program (Scion corporation, Frederick, MD). Statistical comparisons of the means were carried out with an unpaired Students t-test. All these studies were performed with the aid of the SPSS statistical package (SPSS Science, Chicago, IL).
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Results |
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We have examined the effects of microgravity on the maturation of synaptic circuits of the neocortex of neonatal rats that developed for 16 days in a low Earth orbit (Neurolab mission, STS-90). After landing, we first analyzed the body weight of the FLT and AGC animals and found no significant difference between the two groups of animals (mean ± SEM) (97.9 ± 3.2 and 103.7 ± 3.2 g, respectively). These observations suggest that feeding during the spaceflight was normal and that no apparent nutritional problems might have affected our colony of animals.
Light Microscopy and Ultrastructural Analysis
We first analyzed Nissl-stained cortical sections of the hindlimb region of the cortex obtained from the animals using light microscopy. No differences in the cytoarchitectonic (Table 1) or cytological characteristics of neurons were detected between FLT and AGC animals (Fig. 1C,D
).
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Changes in Synaptic Cross-sectional Lengths in FLT Rats
To determine whether the synaptic cross-sectional lengths and the densities of synapses were affected by spaceflight, we compared these parameters between FLT and AGC animals on the day of landing (R+0) and after 4 months of re-adaptation to terrestrial gravity (R+129 or R+135). At R+0, the cross-sectional lengths of asymmetrical synapses of FLT rats were longer in all layers except layers I and VI (Fig. 3). The increase in length of asymmetrical synapses was significantly longer in layers II/III and Va. No significant differences were found in the lengths of symmetrical synapses between the two groups of animals. However, on R+0 the symmetrical synaptic profiles were shorter in FLT animals in all layers, except in layer IV where they were longer than in AGC animals.
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Changes in Synaptic Density in FLT Rats
A comparison of the synaptic density for all synapses between FLT and AGC animals on R+0 also revealed differences (Fig. 4). Lower synaptic densities were observed in the cortex of FLT animals except in layers I and VI, although these differences were only statistically significant in layers II/III, IV and Va. The greatest difference was found in layer II/III, where 344 million fewer synapses per mm3 were found, a decrease of 15.6%. After the period of re-adaptation, however, significant differences in the density of synapses were found in layers IV and Vb.
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In summary, exposure to microgravity for 16 days produced laminar-specific changes in asymmetric synapses but not in symmetric synapses. Both the length and density of asymmetric synapses in certain layers was initially affected and while some changes disappeared after 4 months of re-adaptation to terrestrial gravity, new alterations also appeared (late changes).
Changes in Synaptic Cross-sectional Lengths and Synaptic Density from P30 to Adult in AGC and FLT Rats
From P30 to adult (~5 months old), AGC animals showed significant increases in synaptic cross-sectional lengths in all cortical layers (P < 0.001; Fig. 5). These increases varied from a minimum of 12% in layer VI to a maximum of 22% in layer II/III. In FLT animals there were also significant increases (P < 0.001) in synaptic cross-sectional lengths during the recovery period from P30 to adult in all cortical layers except in layer IV, where the synaptic lengths did not change significantly (Fig. 5
). The increase in length in FLT animals was less marked than in AGC animals, only varying between 6% (layer Va) and 13% (layer II/III).
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Thus, during normal late development and maturation of cortex, there were significant increases in synaptic cross-sectional lengths and reductions in synaptic densities in all layers of AGC animals. The increase in synaptic cross-sectional length, however, was less marked in all layers after spaceflight, and especially in layer IV, where synaptic length did not change at all. The decrease in synaptic density was also less accentuated, especially in layers IV and II/III.
Differences in Locomotion in FLT and AGC Animals
Since changes in cortical circuitry were most probably related to use-dependent changes in locomotion and hindlimb posture in microgravity, we analyzed the videotapes taken on flight day 6 (FD6) and FD11. On both occasions, the animals used their forelimbs, but not their hindlimbs for propulsion. Sometimes the hindlimbs floated behind the animals, but usually the animals grasped the surface with their toes to maintain a normal horizontal posture. Another clear difference in locomotion between FLT and AGC animals was the lack of weight-bearing undertaken in microgravity. Thus, both the muscle action and afferent signals from the hindlimbs clearly differed in FLT and AGC animals. One example of the changes was the tactic used to reverse direction while progressing along a rod. FLT animals lifted both hindlimbs at the same time during 26% of the movements, while AGC animals never did this.
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Discussion |
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During normal late development and maturation of the cortex in AGC animals (from P30 to 5 months old), synaptic lengths increase and synaptic densities decrease in all layers. Whilst these two phenomena were maintained in the late development and maturation after spaceflight, they were less accentuated, across all cortical layers when considering synaptic cross-sectional lengths and in layers II/III, IV and Va for synaptic density. These results suggest that the development of synaptic circuits in certain layers of the hindlimb neocortex is sensitive to microgravity during the second and third postnatal weeks. The changes that we observed probably occurred during the 16 days of microgravity and not during the relatively short recovery period after landing. In addition, after 4 months of re-adaptation to terrestrial gravity some recovery could be seen, suggesting that some of the changes were transient. Moreover, new changes appeared after re-adaptation, indicating that spaceflight induces more long-term effects on development.
Synaptic size seems to play an important role in the functional properties of synapses (Lisman and Harris, 1993; Nusser et al., 1997
; Mackenzie et al., 1999
; Takumi et al., 1999
; Kubota and Kawaguchi, 2000
; Lüscher et al., 2000
; Lee and Sheng, 2000
). For example, larger synapses appear to express a greater number of postsynaptic receptors (Mackenzie et al., 1999
). Thus, the changes in synapse density and size found after spaceflight may reflect functional alterations of the cortical circuits involved. As only asymmetrical synapses were significantly affected by the changes, and this type of synapse is known to be excitatory (Houser et al., 1984
; White, 1989
; Peters et al., 1991
; DeFelipe and Fariñas, 1992
; Peters and Palay, 1996
; Conti and Weinberg, 1999
), we conclude that microgravity affects excitatory synaptic circuits in a laminar-specific manner. However, our data do not permit us to determine which of the possible sources of these synapses were involved. For example, the higher synaptic density found in layer IV of FLT rats after re-adaptation could be due to an increase in the number of axon terminals originating from layer IV spiny stellate cells, from pyramidal cells, from thalamocortical afferents, or from all of these cells [being the major sources of excitatory asymmetrical synapses (White, 1989
; DeFelipe and Fariñas, 1992
; Jones, 2000
; Amitai, 2001
)].
The variation in cortical circuitry between FLT and AGC animals is likely to be related to the differences observed in the use of the hindlimbs between these two groups of animals during the 16 day period. Movement in FLT animals may be termed modified quadrupedal locomotion (Sulica et al., 1999), in microgravity a unique gait predominates where the major propulsive power is provided by the forelimbs. In this modified quadrupedal locomotion the hindlimbs are sometimes placed anterior to the forelimbs, as in a gallop, to help propel the animal forward. In no case are the hindlimbs used to bear weight as in AGC animals. Thus, both the motor and sensory aspects of locomotion are changed in microgravity. In the presence of terrestrial gravity, extensor antigravity muscles play a key role in posture and locomotion. This is not the case in microgravity, where flexor muscle activity predominates. The need for weight-bearing during development is well documented at the muscle level. Indeed, in age-matched animals on the same spaceflight, expression of the type I MHC gene in antigravity skeletal muscles was markedly reduced (Adams et al., 2000
; Ikemoto et al., 2001
). The impact of these structural changes on sensorymotor control of locomotion and postural reflexes after landing is currently being analyzed. The surface righting reaction does not occur in microgravity (Harding et al., 1999
) and preliminary results indicate that surface righting does not mature post-flight. Rather, FLT animals right themselves using the same immature tactics seen at launch. In contrast, righting in AGC animals is typical of adult rats (Harding et al., 1999
).
Synaptic plasticity in the cerebral cortex (neoformation and/ or loss of synapses, and the ultrastructural changes in synapses) has been associated with a number of factors that include learning motor skills, complex environment exposure, and recovery from cortical injury (Cragg, 1974; Greenough and Chang, 1988
; Calverley and Jones, 1990
; Horner, 1993
; Rakic et al., 1994
; Rioult-Pedotti et al., 1998
; Klintsova and Greenough, 1999
; Harris, 1999
; Lüscher et al., 2000
; Trachtenberg and Stryker, 2001
; Lüscher and Frerking, 2001
). Therefore, changes in cortical circuits during spaceflight may reflect abnormal afferent information reaching the sensorimotor cortex, and thus may represent an anatomical substrate for some aspects of these behavioral alterations. For example, the higher density of synapses seen in layer IV after recovery may be due to the neoformation of excitatory synapses resulting from an enhanced afferent input associated with weight-bearing. Another possibility to explain the changes observed is that during spaceflight, the animals move in three dimensions instead of the two dimensions on Earth. Thus, some of the changes found could result from the enriched environment that microgravity represents.
Furthermore, modifications in the volume and distribution of extracellular fluids and plasma occur during spaceflight that in turn induce a number of hormonal, physiological and biochemical modifications that are also involved in the regulation of extra- and intracellular volumes (e.g. see García-Ovejero et al. (García-Ovejero et al., 2001) and references contained therein). Therefore, the volume of cortical tissue in microgravity may differ when compared with AGC animals. However, we did not find any significant change in the width of cortical layers between the two groups of animals (Table 1
). Thus, it is unlikely that the re-distribution of fluids significantly affected our estimations of synaptic density in either of the two groups of animals. Nevertheless, we cannot discard the possibility that other hormonal and/or physiological adjustments to micro-gravity (White and Averner, 2001
) may have influenced the density of synapses. In future experiments it will be important to include the appropriate controls for synaptogenesis during spaceflight to ascertain the extent to which changes in synaptic density are induced by altered proprioceptive synaptic activity or what is the influence of the hormonal/physiological adjustments.
The decrease in the density of synapses observed in both AGC and FLT animals between P30 and 45 months might be related to the normal loss of synapses during puberty and potentially throughout adulthood [reviewed in Rakic et al. (Rakic et al., 1994); see also (DeFelipe et al., 1997
; Poe et al., 2001
)]. During this period, there were significant increases in synaptic cross-sectional lengths (1222% increase) and reductions in synaptic densities (1133% decrease) in all layers in AGC animals. While following spaceflight these two processes also occurred, they were clearly less accentuated, particularly in layers II/III, IV and Va (see Fig. 5
). Therefore, the mechanism of postnatal synapse elimination was characterized by a selective loss of synapses in a layer-specific manner [see also (Rakic et al., 1994
; DeFelipe et al., 1997
; Poe et al., 2001
] in both groups of animals. This mechanism, however, appears to be altered in certain layers of the FLT animals where the decrease was less pronounced, giving rise to the differences in synaptic density found between adult AGC and FLT rats. Cell death in developing rodent neocortex mostly takes place during the first postnatal week (Ferrer et al., 1994
; Spreafico et al., 1995
; Poe et al., 2001
) and is relatively slight. As the rats of the present study were already age P14 at launch, the decrease in synaptic density was likely due to a pruning of synaptic connections, rather than cell death.
Finally, further studies are necessary to examine whether the changes that we have described are selective for those cortical areas related to somatosensory and motor processing. Moreover, to find out whether they are more prominent during certain critical periods of development, as well as to determine to what extent synaptic plasticity occurs in the mature cerebral cortex in an environment of microgravity [(Walton et al., 1992); for reviews, see (Horner, 1993
; Constantine-Paton and Cline, 1998
; Klintsova and Greenough, 1999
; Berardi et al., 2000
)]. In addition, the changes observed were induced after a relatively short time in space and, therefore, it is likely that longer periods in space (White and Averner, 2001
) would induce more significant structural changes. As the neocortex is the site of higher brain functions, the synaptic plasticity induced by microgravity may be of particular relevance for future prolonged human spaceflights.
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Acknowledgments |
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