Altered cerebellar development in mice lacking pituitary adenylate cyclase-activating polypeptide

Previous studies have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects during neurodevelopment. In particular, the occurrence of PACAP and its receptors in the cerebellum during pre- and postnatal periods suggests that it could play a crucial role in ontogenesis of this structure. To test this hypothesis, we compared the histogenesis of cerebellar cortex in wild-type and PACAP-knockout (PACAP(-/-)) mice at postnatal days (P)4 and 7. Morphometric analysis of PACAP(-/-) mice revealed a significant reduction in the thickness of the external granule cell layer at P4 and of the internal granule cell layer at P7. Expression of nestin, a neural precursor marker, and synaptophysin, a mature neuronal marker, was quantified by realtime PCR and Western blot. No modification of nestin expression was noticed between wild-type and PACAP-/- mice, but a substantial decrease in synaptophysin expression was observed inPACAP(-/-) mice at P4 and P7. lmmunohistochemistry revealed a reduction in synaptophysin labelling in the molecular and internal granule cell layers of PACAP(-/-) mice at P7. Caspase-3 activation was significantly increased in PACAP(-/-) mice at P4 and P7. Autoradiographic studies revealed no difference in PACAP binding site distributions and PACAP was effective at stimulating cAMP production in both wild-type and PACAP(-/-) cultured granule cells. This study demonstrates that disruption of the PACAP gene induces alteration of the immature cerebellum. Neuronal differentiation of granule cells was delayed whereas cell death that naturally occurs during ontogeny was increased in PACAP(-/-) mice. These data provide the first evidence of a physiological role for PACAP during cerebellar development.