We also analyzed the cell cycle of transfected astrocytes and found that the cell population in the G1 phase was higher than that in the control (Figure 4(h)). exert some regulatory effects on astrocyte development. Mice deficient for glycogen synthase show perinatal lethality,17 which indicates that glycogen is an important energy source for neuronal development. However, ontogenic changes in glycogen localization in astrocytic cells and their functional significance have not yet been examined in detail. In the present study, we investigated how glycogen is involved in perinatal forebrain development. We found that large amounts of glycogen accumulated in glutamate aspartate transporter (GLAST)+ cells located in the subventricular zone (SVZ) as well as the developing rostral migratory stream (RMS). We also demonstrated that glycogen levels decreased soon after birth. We concomitantly observed the increase of glycogen phosphorylase along the RMS. The inhibition of glycogen breakdown in primary cultured SVZ astrocytes and reduced astrocytic cell proliferation. The knockdown of brain glycogen phosphorylase induced the expression of p21 and p27, both of which act as cell cycle inhibitors. Furthermore, the inhibited breakdown of glycogen decreased the phosphorylation of retinoblastoma protein (pRB), indicating that cell cycle arrest occurred when glycogen-derived energy was not available. These results suggest that glycogen serves as an energy store MK-2894 sodium salt for maintaining astrocyte cell proliferation in the postnatal telencephalon. Materials and methods Animals Pregnant ICR mice were obtained CACNA1H from SLC (Shizuoka, Japan) and were housed under a 12?h light/dark cycle and had ad libitum access to water and foods. Regarding histochemical and biochemical analyses, pregnant or newborn mice were anesthetized using pentobarbital (100?mg/kg, intraperitoneal injection). Embryos from three pregnant mice at each stage were histochemically examined (see Figures 1 and ?and3).3). Four mothers with their newborn pups were used in primary culture experiments (see Figures 4 and ?and6).6). In an analysis of glycogen phosphorylase functions, 12 newborn pups from two dams were examined at each experimental time point (see Figure5(a) and (?(o))o)) and 8 newborn pups from two dams were examined in Figure 5(p) and (?(r)r) so that each treatment group include individuals from multiple litters. The unintended death of newborn mice occurred due to a failure to recover from anesthesia in the experiment shown in Figure 5. The percentage of unintended deaths was less than 5%. In order to label S-phase cells, 5-ethynyl-2-deoxyuridine (EdU, Invitrogen, Carlsbad, USA) or 5-bromo-2-deoxyuridine (BrdU, Wako, Osaka, Japan) was injected intraperitoneally one hour before sampling (2?mg/kg). All animal procedures were treated in compliance with the Guidelines for Proper Conduct of Animal Experiment and Related activities (Ministry of Education, Culture, Sports, Science and Technology of Japan) and were approved by the Animal Committee of Kyoto Prefectural University of Medicine. Reporting of this work complies with ARRIVE guidelines. Open in a separate window Figure 1. Localization of glycogen in the embryonic telencephalon. (aCc): Coronal sections were pretreated with dimedone and stained using periodic acid Schiff reagent. CX indicates the cerebral cortex and Str, the striatum. The bar indicates 100?m. (d) In an E18.5 sagittal section, glycogen was observed along the rostral migratory stream (RMS). The bar indicates 100?m. (eCg) The dorsal/ventral boundary region of aCc (subventricular zone, MK-2894 sodium salt SVZ) was magnified. (h) The pretreatment of sections with amylase completely abolished the staining of PAS+ glycogen. The bar indicates 50?m. (i) Sections at E18.5 were stained by glycogen (magenta) with III-tubulin (green). (jCk) Sections were stained by glycogen (k and n) with III-tubulin (j) or GLAST (m). The SVZ area corresponding to the dashed box in (i) is shown. Merged images are MK-2894 sodium salt shown in (l) and (o), respectively. The GLAST+/Glyc+ cell in (o, arrow) was merged with Hoechst staining (blue) and shown in the inset. The bar indicates 50?m. Open in a separate window Figure 3. Expression of in the SVZ and its postnatal increase. (a) A RT-PCR analysis was performed using cDNA prepared from the E18.5 olfactory bulb (Ob), rostral.