Fluorescence images were acquired by using a high-speed confocal laser scanning microscope (CSU22, Yokogawa, Japan), as described (Tanimura et al., 2009). Other details are described in Supplemental Experimental Procedures. Under anesthesia with chloral hydrate (350 mg/kg Cabozantinib price of body weight, i.p.), a glass pipette filled with 2–3 μl of 10% solution of DTR (3,000 molecular weight; Invitrogen, Carlsbad, CA) in PBS (pH 7.4) was inserted stereotaxically to the inferior olive. The tracer was injected by air pressure at 10 psi with 5 s intervals for 1 min. After 4 days of survival, mice were fixed by transcardial perfusion with 4% paraformaldehyde

in 0.1 M PB, and parasagittal cerebellar sections (50 μm in thickness) were cut. The DTR-labeled sections were incubated overnight with a mixture of goat calbindin antibody and guinea pig type II VGluT2 antibody (Miyazaki et al., 2003) followed by 2 hr incubation with a mixture of Alexa

488- (Invitrogen) and Cy5-labeled species-specific secondary antibodies. Images of triple labeling were taken with confocal laser-scanning microscope (FV1000, Olympus). Procedures for immunohistochemistry are described in Supplemental Experimental Procedures. To inhibit GAD activity or enhance GABAA receptor sensitivity to GABA in the cerebellum, we applied 3-MP or diazepam, respectively, by continuous infusion from Elvax implants prepared as described (Kakizawa et al., 2000, Kakizawa et al., 2003 and Kakizawa et al., 2005). For the implantation of an

Elvax piece, Epacadostat clinical trial mice at P10 or P17 were anesthetized with isoflurane, the skin over the cerebellum was cut, and the occipital bone and the dura over the cerebellar lobules 6–8 was removed. A small piece of Elvax containing drugs or vehicle was placed on the cerebellar surface and then the skin was sutured. Effects of chronic infusion of 3-MP or diazepam were examined electrophysiologically at P23–P40 within those the lobules 6–8. Throughout the text and figures, data are presented as means ± SEM. Statistical significance was assessed by Student’s t test or Mann-Whitney U test (for comparison of two independent samples), two-tailed paired-t test or Wilcoxon’s signed-rank test (for paired comparison of the same sample), depending on whether the data sets pass the normality test and equal variance test, unless otherwise mentioned in the text. For comparison of frequency distributions, Mann-Whitney U test was used. Statistical analysis was conducted with Sigma Stat 3.1 program. p value was described as p < 0.001 when the actual p value was smaller than 0.001. Differences between data sets were judged to be statistically significant if the p value was less than 0.05. We thank S. Kakizawa for helpful advice on preparation and implantation of Elvax, A. Koseki for mice genotyping and members of Kano’s lab for discussion.