Pilot study on the effects of early exposure to hypergravity on the behavioural and cerebellar development of CD-1 mice

• ¹ Fondazione Istituto Italiano di Technologia, Italy
• ² Centro di riferimento per le scienze comportamentali e la salute mentale, Istituto Superiore di Sanità, Italy
• ³ Ospedale San Martino (IRCCS), Italy
• ⁴ Dipartimento di Biologia e Biotecnologie Charles Darwin, Sapienza Università di Roma, Italy

Abstract The nervous system is known to be highly sensitive to environmental alterations. Nevertheless, changes induced by exposure to altered gravity have not been fully understood. In this study we investigated the effects of hypergravity on motor coordination and cerebellum structural development of CD-1 mice exposed to centrifugation-induced 2g hypergravity (HG) from post-natal day (PND) 1 to PND21. Not rotated mice (stationary control, SC), and mice exposed to 1g (rotational control, RC) were used as controls. Behavioural endpoints were assessed at PND7, 14 and 21, while cerebellar structure was analysed at PND7. Results showed that PND7 HG mice exhibited a delay in the Righting response, while Pole grasping and Auditory startle responses were negatively affected only in RC mice. On PND21, an impaired Dowel test performance was observed in the HG group. Histological analysis showed alterations in cerebellar granule cell migration of rotated mice, HG animals being more affected than RC ones. These data suggest that altered gravity delays cerebellar development, which results in impaired motor coordination performances. Further understanding how cerebellum responds to altered gravity could provide insights into its implications in several neurological disorders, and contribute to the development of therapeutic tools and strategies. Introduction The possibility that exposure to an altered gravitational environment may interfere with the development and maturation of the nervous system is relevant for vertebrate physiology. A number of animal studies have shown that changes in sensorimotor behaviour are associated with brain structural changes (Aizikov and Markin, 1981). The cerebellum, renowned for its role in motor coordination, is affected by exposure to hypergravity (Lalonde 2003). While it is difficult to perform extensive developmental studies in space, hypergravity paradigms using centrifuges provide a fruitful way to study the effects of gravity changes on biological organisms (Serova et al., 1993). However, a few studies have directly correlated changes in motor behaviour with cerebellar structural alterations. In this pilot study, CD-1 mice were exposed from PND1 to PND21 to centrifuge-induced gravity (see Francia et al., 2006 for methodological details and behavioural tests). Three experimental groups were designed: stationary controls (SC), rotational control (RC, 1g) and hypergravity (HG, 2g). Both sensorimotor reflexes and motor coordination were assessed at PND7, 14 and 21, while histological analyses were conducted at PND7, peak day of granule cell migration, and when several genes important for cell cycle progression and differentiation are either up- or down-regulated transiently (Kagami and Furuichi, 2001). Results At PND7, a significant delay in the emergence of the Righting reflex (returning to the feet when placed on the back) was observed in HG mice (Fig. A). By contrast, impairment in Pole grasping (grasping a toothpick with forepaws) and Startle responses (whole body startle response to a laud hand clap) were observed exclusively in RC animals (Fig. 1B, C). To assess balance skills, the Dowel test (walking on a pencil) was performed at PND14 and 21. While no major differences were observed between SC, RC and HG mice at PND14, a significant impairment was seen at PND 21 in HG mice (Fig. 1E), confirming a specific delay in motor coordination development in mice exposed to hypergravity. Histological analyses were performed on paraffin-embedded cerebellum slices. In SC mice (Fig. 2A-C), maturing granule cells are still mainly localized in the external germinal zone (EGZ), where they form a compact cellular area. Differentiating granule cells migrate to final destination, either isolated or in chains, across the Purkinje cell (PC) layer, which are organizing in the characteristic monolayer. (Fig. 2B,C). In HG mice (Fig. 2D-F), the EGZ appear to be thicker and more cell-packed compared to SC mice, with the inner layer of granule cells distributing with a different orientation compared to outer ones. This implies that the PC layer get closer to EGZ, suggesting a sort of hold up of granule cell migration. PC mono-layer organization also appears delayed. In RC mice (Fig. 2G,H), these features are intermediate between the two groups, although closer to the HG mice. Conclusions Results on mice exposed to hypergravity during the first three weeks of postnatal life indicates that this stimulus influences the behavioural profile, with a delay in the mode and time of reflex occurrences. In particular, the investigated behavioural endpoints are directly related to the ontogeny of neuromotor functions and represent sensitive indicators of the level of maturation of nervous system and cerebellum in particular. On PND7 the Auditory startle response and the Pole grasping were affected in RC mice, while only the Righting reflex was impaired in HG, confirming a specific response to hypergravity exposure. Of a note, Righting reflex requires an appropriate integration between vestibular and somatosensory inputs in order to make postural adjustments when the body is displaced from its normal vertical position. Moreover, significant changes in the ability to remain in balance in the Dowel test, emerged only in PND 21 HG mice The non-linear trend of behavioural responses, in which some effects were more marked at PND7 and PND 21, is in agreement with data previously reported (Sajdel-Sulkowska et al., 2001) and confirms the existence of specific windows of vulnerability during critical phases of development. In agreement with previous data, in which a reduction in the cerebellar mass was observed in rats chronically exposed to gravitational acceleration during the perinatal period (Sajdel-Sulkowska et al., 2005), this pilot study on CD-1 mice suggests that early exposure to hypergravity causes an increase in the EGZ thickness and a reduction in the space between this and the PC layer. This suggests a slower migration rate of granule cells towards their final destination compared to control, with possible alteration in the wiring of cerebellar circuits responsible for the vestibular system proper activity. Indeed, in the first 20 PND, the cerebellum is in its most plastic period, characterized by granule cell maturation and migration from the EGZ, in which they are tightly clustered, through the PC layer, down to their final destination. It is particularly interesting how these anatomical and morphological changes parallel the behavioral ones, confirming that perinatal exposure to hypergravity interferes with correct CNS wiring. This could end up in short-, medium- or long-term repercussions, which may be more or less pronounced, depending on the targeted system, e.g. sensorimotor, neuromotor, cognitive, emotional. Our preliminary data indicate both morphological and behavioral changes in early postnatal mice exposed to hypergravitational stimuli, which can be perceived as a ”delay” in our “gravitational system”, but that can be also interpreted as a plastic reorganization of the developing nervous system in a “challenging” environment. Further studies will be devoted to analyse later stages of cerebellar development, when its different neuronal types have fully matured and reached their final destination. Figure Legend Figure 1. Battery of behavioral tests assessing motor coordination of mice exposed to altered gravitational acceleration. Righting reflex, Pole grasping and Auditory startle tests were used at PND 7 (upper panel) and Dowel test at PND 14 and 21 (lower panel). A) Righting reflex. Hypergravity (HG) mice show a significant delay in the emergence of reflex (F (2.68) = 7.68, p <0.001 Bonferroni post-hoc correction) compared to stationary control (SC) and rotational control (RC) groups. B) After the Pole grasping test, only mice of the RC group show a significant delay in the emergence of this response compared to the other two (F (2.68) = 7.01; p <0.01). C) Similarly to Pole grasping, the RC group manifests a significant delay in the emergence of the Auditory startle response (F (2, 66) = 3.026, p <0.05). D, E) Dowel test results from PND 14 and PND 21 mice, respectively. Exposition to 2g negatively affects the ability to maintain equilibrium in PND 21 mice (F (2.48) = 10.545; p <0.02), whereas a slight, but non-significant, improvement is observed in PND 14 mice. Figure 2. Histological analysis on PND7 mouse cerebellum in control and hypergravity conditions. A-C: SC mouse group. A: At PND 7, cerebellar folia show the expected subdivision in external granular zone (EGZ) -where immature granule cells are located-, molecular layer (ML) – through which granule cell migrate-, the granule cell layer (GL) and the central white matter (WM). B,C: Higher magnification of the cortical portion of the cerebellum. Migrating granular cells (Gc) are seen crossing the ML and reach their definitive layer (GL), crossing the Purkinje cell (Pc) layer. Pc are disposing in a monolayer, a distinctive feature of maturation. D-F: HG mouse group. D: Gross organization of cerebellar folia is similar to the SC group. E,F. At higher magnification. the EGZ appear larger, and the immature Gc more packed, than in the SG mice, As a consequence, the ML is narrower compared to control groups and Pc appear distributed on multiple layers. G-H: RC mouse group. F: Gross organization of cerebellar folia is similar to the SC group. G-H. At higher magnification, the organization of the cortical layers appears to be somehow in between the SC and the HG conditions. Scale bar: low magnifications, 200 m; high magnifications, 40 m.