Immunohistochemical studies and 2-dimensional gel electrophoretic analysis on the cerebellum from the staggerer mutant mouse

$33 DISTRIBUTION OF PROSTAGLANDIN D SYNTHETASE IN CEREBRAL NEOCORTICAL NEURONS: AN IMMUNOHISTOCHEMICALSTUDY IN NEONATAL RATS TAKESHI KANEKO*, YOSHIHIRO URADE*, NOBORU FUJIMOTO*, ¥ASUYOSHI WATANABE*, AKIRA KONISHI, NOBORU MIZUNO and OSAMU HAYAISHI, Hayaishi Bioinformation Transfer Project, Research Development Coorperation of Japan, Kyoto 601, Japan, and Department of Anatomy ( I s t D i v i s i o n ) , Faculty of Medicine, and College of Medical Technology, Kyoto U n i v e r s i t y , Kyoto 606, Japan. Prostaglandin D (PGD) synthetase, which catalyzes the isomerization from PGH to PGD, was found to e x i s t in cerebral neocortical neurons in r a t s . The enzyme a c t i v i t y was immunohistochemically demonstrated with r a b b i t antibody raised against p u r i f i e d PGD synthetase. In i-week-old r a t s , intense immunoreactivity was seen in many pyramidal neurons. Weak immunoreactivity was also observed in some s t e l l a t e neurons in layers I I Vl, but no neuronal c e l l bodies in layer I showed immunoreactivity. In 2-week-old r a t s , both pyramidal and s t e l l a t e neurons in layers I I VI showed intense immunoreactivity. Immunoreactive pyramidal neurons in 2-week-old r a t s , however, were m o r e sparse than in i-week-old r a t s ; although the immunorectivity in each neuron in 2-week-old rats was more intense than that in I-week-old rats. In 3 week-old-rats, the immunoreactivity was c l e a r l y recognized in m u l t i p o l a r neurons in layer I. The immunoreactivity of neurons in layers I I - Vl almost disappeared by the end of the 4th postnatal week; whereas that of m u l t i p o l a r neurons in layer I remained even in adult rats. In accordance with immunohistochemica] f i n d i n g s , biochemical data showed that the PGD synthetase a c t i v i t y in the te]encephalon was highest in i - to 2-week-old rats. The t r a n s i e n t high a c t i v i t y of PGD synthetase in many neocortical neurons in the postnatal period suggests that PGD may be involved in the process of neuronal maturation. IM/MUNOHISTOCHEMICAL STUDIES AND 2-DIMENSIONAL GEL ELECTROPHORETIC ANALYSIS ON THE CEREBELLUM FROM THE STAGGERER MUTANT MOUSE. KATSUHIKO MIKOSHIBA, HIDEYUKI OKANO, MASATOSHI FUJISHIRO and YASUZO TSUKADA Department of Physlology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160, Japan. Staggerer mutant mouse is autosomal recessive and is characterized by severe cerebellar hypoplasia accompanying typical cerebellar symptoms. Morphological analysis of the staggerer cerebellum by hematoxylin eosin staining showed that the Purkinje cells were randomly distributed. It appears that the migration of external granule cells were retarded in the staggerer. Analysis of the chimera produced from the staggerer and wild type control mice, revealed that Purkinje cells were primarily affected by the mutation. Immunohistochemical staining of the control cerebellum by GFAP (glial fibrillary acidic protein) antiserum revealed that the fibers were clearly observed and were vertically radiating from the Bergmann cell bodies. In the staggerer, the length of the fibers was short, and the GFAP reaction was stronger than the control; and GFAP reactivity was strong at the granular layer. Antiserum against MBP (myelin basic protein) was used to trace the pathway of the myelinated fibers which are mainly composed of mossy fibers. Most of the fibers were radiating from the white matter to the granular layer and each fibers could be clearly traced in the control. However, in the staggerer, radiation of the myelinated fibers could not be observed, instead, degenerative figures of myelinated fibers could be observed in the staggerer. SDS-polyacrylamide gel electrophoresis revealed that P400 protein, characteristic of the Purkinje cells, was missing and could not he found by the autoradiogram of the labelling by 14C-leucine after injection into the staggerer cerebellum. Two dimensional gel electrophoresis by the method of O'Farrell revealed that many proteins increased or decreased in the staggerer compared to the control.