In VitroCell. Dev.Biol.--Animal 40:159-165, May and June 2004 9 2004 Society for In VitroBiology 1071-2690/04 $18.00+0.00 EFFECTS OF SIMULATED MICROGRAVITY ON THE DEVELOPMENT AND MATURATION OF DISSOCIATED CORTICAL NEURONS ALESSIO CRESTIN1, CRISTINA ZONA, PIERLUIGI SEBASTIANI,MASSIMOPIERI, VALENTINA CARACCIOLO, LORENZO MALVEZZI-CAMPEGGI, ANNAMARIACONFALONI,ANDSILVIA DI LORETO 1 Department of Cellular Biology and Neuroscience, lstituto Superiore di Sanitdt, Rome, Italy (A. C., L. M.-C., A. C.), D@artment of Neuroscience, University Tot Vergata and I.R.C.C.S., Fondazione S. Lucia, Rome, Italy (C. Z., M. P.), and Institute of I.T.O.I.-C.N.R., p.le Collemaggio, 67100 L'Aquila, Italy (P. S., E C., S. D. L.) (Received 10 October 2003; accepted 20 April 2004) SUMMARY Although a wealth of evidence supports the hypothesis that some functions of the nervous system may be altered during exposure to microgravity, the possible changes in basic neuronal physiology are not easy to assess. Indeed, few studies have examined whether microgravity affects the development of neurons in culture. In the present study, a suspension of dissociated cortical cells fi'om rat embryos were exposed to 24 h of simulated microgravity before plating in a normal adherent culture system. Both preexposed and control cells were used after a period of 7-10 d in vitro. The vitality and the level of reactive oxygen species of cultures previously exposed did not differ from those of normal cultures. Cellular characterization by immnnostaining with a specific antibody displayed normal neuronal phenotype in control cells, whereas pretreatment in simulated microgravity revealed an increase of glial fibrillary acidic protein fluorescence in the elongated stellate glial cells. Electrophysiological recording indicated that the electrical properties of neurons preexposed were comparable with those of controls. Overall, our results indicate that a short time of simulated microgravity preexposure does not affect dramatically the ability of dissociated neural cells to develop and differentiate in an adherent culture system. Key words: simulated microgravity; neural cultures; electrophysiology; immunocytochemistry; oxidative stress. INTRODUCTION In 1994, an extensive review by Krasnov detailed the structural changes in various areas of the central and peripheral nervous sys- tems of rats after exposure to real and simulated microgravity. Al- tered gravity force can provoke changes in neurotransmitter recep- tors in rat cortices (Miller et al., 1989) and alterations of enzymatic mechanisms in central and peripheral neurons of dorsal root ganglia (Ishibara et al., 1997), which are mainly ascribed to hypoactivity. Moreover, decreased glial fibrillary acidic protein (GFAP) expres- sion was demonstrated in hippocampal astrocytes of rats exposed to microgravity (Day et al., 1998). Changes in protein production and lhe hypoactivity of neural cells may provoke a consequent loss of afferents and connections between the various nuclei of the central nervous system (CNS). Considerable attention has been directed at examining whether neural development can be affected by changes in gravity fields. Because these processes are regulated by both chemical and mechanical factors, gravity may play a crucial role as a stimulus for proper development of the nervous system. In a re- view, Unsworth et al. (1998) reported significant acceleration of the aggregation and rate of proliferation of PC12 pheochromocytoma ceils after a prolonged period of simulated microgravity exposure. Moreover, the rate of glucose consumption, as a measure of cellular metabolic activity and proliferation, was about five times higher in 1To whonl correspondence should be addressed at E-maih s.diloreto@ itoi.cnr.it space than on the ground. In fact, some aspects of differentiation, including synapse formation and myelination, may occur more read- ily in aggregate cultures than in dissociated cell cultures (Trapp et al., 1982). Furthermore, data from Gruener and Hoeger (1991) sug- gested that the process of synapse formation is sensitive to the grav- itational vector. Developmental and adult synaptic plasticity is nec- essary for determining and maintaining normal brain function, but it is also critical for the restoration of function in neural tissues after trauma or disease. Many studies suggest that mechanisms that regulate neuronal development are often the same as those that regulate plasticity and repair in the adult CNS. In fact, accumulated data support the idea that extensive plastic changes occur in the adult CNS, and that these may be regulated by similar factors that are operative during early neural development (Vasquez, 1998). Fur- thermore, in the study of the effects of microgravity, neuronal cul- ture is an underused experimental model, even if some experiments reported morphological alterations in neurons cultured in an ahered gravity force (Gruener et al., 1990, 1991). Recently, it has been reported (Uva et al., 2002) that monolayer-cultured glial cells showed morphological alterations as early as 30 rain of simulated microgravity, and even if after 20-32 h of exposition glial cells were able to reorganize and to divide, the cell death rate was elevated. The in vitro neural cells, either in monolayer or in aggregate cuhure systems, were used to study (to our knowledge) mainly the effects of a long-term exposition to altered gravitational forces. Mostly, differentiated line cultures (Gruener, 1998; Lelkes et al., 159