Supplementary Material for: Prenatal Infection Decreases Calbindin, Decreases Purkinje Cell Volume and Density and Produces Long-Term Motor Deficits in Sprague-Dawley Rats

The cerebellum is involved in the control of motor functions with Purkinje cells serving as the only output from the cerebellum. Purkinje cells are important targets for toxic substances and are vulnerable to prenatal insults. Intrauterine infection (IUI) has been shown to selectively target the developing cerebral white matter through lesioning, necrosis and inflammatory cytokine activation. Developmental and cognitive delays have been associated with animal models of IUI. The aim of this study was to determine if IUI leads to damage to Purkinje cells in the developing cerebellum and if any damage is associated with decreases in calbindin and motor behaviors in surviving pups. Pregnant rats were injected with <i>Escherichia coli</i> (1 × 10<sup>5</sup> colony-forming units) or sterile saline at gestational day 17. Beginning at postnatal day (PND) 2, the pups were subjected to a series of developmental tests to examine developmental milestones. At PND 16, some pups were sacrificed and their brains extracted and processed for histology or protein studies. Hematoxylin and eosin (HE) staining was done to examine the general morphology of the Purkinje cells and to examine Purkinje cell density, area and volume. Calbindin expression was examined in the cerebellum via immunohistochemistry and Western blot techniques. The remaining rat pups were used to examine motor coordination and balance on a rotating rotarod at the prepubertal and adult ages. Prenatal <i>E. coli</i> injection did not significantly change birth weight or delivery time, but did delay surface righting and negative geotaxis in pups. Pups in the <i>E. coli</i> group also had a decrease in the number of Purkinje cells, as well as a decrease in Purkinje cell density and volume. HE staining demonstrated a change in Purkinje cell morphology. Calbindin expression was decreased in rats from the <i>E. coli</i> group as well. Locomotor tests indicated that while there were no significant changes in gross motor activity, motor coordination and balance was impaired in both prepubertal and adult rats from the <i>E. coli</i> group. In this model of IUI, we observed changes in Purkinje cell development which were associated with alterations in cerebellum-dependent motor behaviors. The decreases in calbindin and Purkinje cells were associated with developmental delays. These data further support the importance of IUI in brain development.