BDNF western blot analysis

Forty micrograms of total protein lysate was separated on a 15% SDS protein gel with a Kaleidoscope-prestained protein standard (Bio-Rad). Blots were blocked in 5% non-fat dry milk and incubated in primary antibody (mature BDNF, Abcam, Catalog #ab108319 used at 1:5000 and secondary at 1:5000; proBDNF, Santa-Cruz Biotechnology, Catalog #sc-65514 used at 1:500 and secondary at 1:5000) for 24 hours at 4°degrees celsius. Blots were incubated in horseradish peroxidase-linked IgG-conjugated secondary antibody for 1 hour. Protein bands were visualized by chemiluminescence solution (Western Lightening, Perkin Elmer Life Sciences). Band at 15kDa was quantitated for mature BDNF, and band at 28kDa was quantitated for proBDNF. GAPDH (37 kDa; Abcam, Catalog# Ab22555 used at 1:20000 and secondary at 1:30000) was used as a loading control.

For I/O relationships, slopes for each slice were calculated and a two way ANOVA was used to assess differences between groups. For PPR, paired pulse ratios were analyzed using a one-way ANOVA. If the overall F value was significant, Student Newman-Keuls post-hoc tests were used to determine significant differences between groups. LTP was analyzed using two way repeated measures ANOVA between vehicle groups. If the interaction effect was significant, Student Newman-Keuls was used as a post-hoc test. For the BDNF experiments, a two-way ANOVA was used followed by Bonferroni post-hoc test for factors with a main effect. Power analyses were conducted on PPR data to determine whether sample sizes were appropriate to achieve sufficient statistical power. Using GPower (Franz Faul, Universitat Kiel, Germany), we determined that the n numbers for the PPR experiments were sufficient to reject the null hypothesis, Power≥0.80 for 20, 30, and 100 ms interstimulus intervals and 0.7 for the 200 ms interstimulus interval.