arbor was obtained by the addition of lengths of all the branches constituting the terminal field of an arbor. Only the portion of the arbor located in layers II/III and IV was considered for these analyses. Very short endings less than 5 mm were omitted from the analyses. Western blotting The adequate condition of BoNT/E injection was determined using rats. BoNT/E solution (50 nM in PBS containing 2% rat serum albumin) was injected at single location in area 17 of the visual cortex (A = 6.5 mm, L = 4.0 mm from bregma; depth from cortical surface, 0.8 mm) in 6-week-old rats using a 30 G stainless steel cannula (1.5 ml; rate, 0.05 ml/min). On various days after surgery, we recorded visual cortical activity with a tungsten microelectrode under anesthesia induced with pentobarbital (50 mg/kg, i.p.) After recording, the rats were euthanized with an overdose of pentobarbital (150 mg/kg, i.p.) and cortical tissue specimens including the visual cortex (A = 6.5–7.5 mm, L = 3.8– 4.4 mm from bregma) were dissected out. The tissue specimens were homogenized with a glass-Teflon homogenizer and sonicated in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM EGTA, 1 mM EDTA, 2 mM tetrasodium pyrophosphate, 4 mM para-nitrophenyl-phosphate, 1 mM sodi- um orthovanadate, 5 mg/mL aprotinin and 1 mM PMSF in 50 mM Tris-HCl, pH 7.4). The homogenates were centrifuged at 700 g for 10 min at 4uC. The resultant pellets were dissolved in the homogenizing buffer and stored at 280uC until western blotting was performed. Protein concentration was determined with a Micro BCA Protein Assay kit (Pierce, Rockford, IL, USA). References 1. Antonini A, Stryker MP (1993) Rapid remodeling of axonal arbors in the visual cortex. Science 260: 1819–1821. 2. 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The membranes were blocked with 5% skimmilk in Tris-buffered saline (TBS) containing Tween-20 (T-TBS) and incubated with T-TBS containing a primary antibody against SNAP-25 (1:2000; mouse monoclonal antibody; Covance, Princeton, NJ, USA), Egr-1 (1:2000) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000, mouse monoclonal antibody; Chemicon Temecula, CA, USA) overnight at 4uC. They were then incubated with a peroxidase-conjugated secondary antibody (1:5000, anti- mouse IgG antibody, 1:5000, anti-rabbit IgG antibody, 1:10000, anti-mouse IgG antibody; GE Healthcare, Piscataway, NJ, USA) at room temperature for 1 hr. Signals were detected by enhanced chemiluminescence (ECL kit; GE Healthcare) and quantified using the ImageJ software (NIH, Bethesda, MD, USA). The blot densities of the proteins of interest were normalized to those of GAPDH in the same membrane. Acknowledgments We thank Drs. S. Kozaki and T. Kohda for kindly providing BoNT/E and technical comments. 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