Deficiency in Endothelin Receptor B Reduces Proliferation of Neuronal Progenitors and Increases Apoptosis in Postnatal Rat Cerebellum

Abstract

Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2′-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm2) in the external germinal layer of sl/sl rats (Mean ± SEM, 977 ± 388) was significantly reduced compared to +/+ (4915 ± 631) and +/sl (2304 ± 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 ± 0.5/mm2) was significantly more increased than +/+ rats (4 ± 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.