The goal of this study is to understand the influence of microgravity on the development, maturation, and maintenance of the neuromuscular system of terrestrial mammals, including humans. The proposed studies will use rats as a model and will explore the broad hypothesis stating that gravity-associated weightbearing is required postnatally for normal neuromuscular development of motoneurons, neuromuscular junctions, and muscle fiber types of the antigravity soleus muscle, but not for that of the extensor digitorum longus, a nonweightbearing muscle. Rat pups (3-5 days old) will be exposed to microgravity for 14-21 days. One group of flight animals will be killed inflight near the end of the mission, and another group will be returned to terrestrial gravity for 1 month before tissues are processed. Parallel groups of ground controls will be conducted on normal and hindlimb suspended unloaded (HSU) rats. Three predictions will be tested in these studies. Firstly, that the neuromuscular system of the soleus will not develop normally in the rat pups exposed to microgravity whereas the EDL development will proceed uncompromised. This analysis will establish whether microgravity disrupts maturation of motoneurons, whether there will be normal elimination of multiple innervation of muscle fibers, and whether normal differentiation of muscle fiber types in the soleus and EDL will occur. Secondly, that the effects on neuromuscular development of unloading by hindlimb suspension (in ground-based study) and microgravity may be similar. But they may not be identical since there are added variables in the hindlimb suspension study that should not be a problem in space flight (isolation from mother and disruption of suckling, body temperature changes, etc). Thirdly, the aberrant soleus neuromuscular system of rat pups raised in microgravity will not be restored to normality by late exposure to weight bearing whereas the EDL will continue normal maturation. Animals returned to earth and processed 1 month after gravity exposure will be analyzed. Overall analysis in these studies will involve processing of the lumbar spinal cords and soleus and EDL muscles using histochemistry, immuno-cytochemistry, in situ hybridization, gel electrophoresis, and electron microscopy. Retrograde labelling will be employed to identify soleus and EDL motoneurons. Analysis of the neuromuscular system development will include assessing maturation of the motoneurons of the two muscle groups (using somal size, cytochrome oxidase (CO) activity, CO mRNA levels, and choline acetyltransferase (CHAT) activity), elimination of polyneuronal innervation of motor endplates, and differentiation of muscle fiber types (using myosin protein isoforms, and messages, myofibrillar ATPase activity, and CO activity and message). It is predicted that microgravity will cause persistence of neonatal attributes and/or the development of anomalies in the soleus but not the EDL, and returning animals to terrestrial gravity is not predicted to reverse induced abnormalities. It is anticipated that these results will raise important implications for rearing normal animals (including humans) in the microgravity environment of space, but additionally will further our understanding of the importance of weightbearing activity for motor system development of individuals on Earth.
