(A) Principles of AlphaScreen technology. Donor and acceptor microbeads can be coated with target-specific antibody, proteins, or secondary reagents (streptavidin, glutathione, nickel). A signal is produced when the AlphaScreen acceptor, A, and donor, D, beads are brought into proximity by a molecular interaction occurring between the binding partners captured on the beads. Laser excitation at 680 nm causes ambient oxygen to be converted to the singlet state by photosynthesizers on the donor bead. These react with chemiluminescent agents on the Acceptor bead only when the latter is in close proximity, emitting light at 520–620 nm. Here, we illustrate a competition protocol between seric propeptide, PE, and interacting donor beads, D, coupled-biotinylated spadin (b-spadin) with antibodies anti-propeptide (anti-PE) coupled on acceptor beads, A. (B) An example of competition curve obtained with one group (n°1) of 6 mice (1.1 to 1.6) among 5 different groups (other curves are presented in the Figure S1). Values obtained are compared to the standard.
(A–E) Acute treatments: Spadin (10−4 to 10−8 M) or Fluoxetine (3 mg/kg) or Saline solutions were injected 30 min before the test in wild-type and kcnk2−/− mice (A, B, C). (A) Forced Swimming Test (FST, n=10 per group), spadin-treated mice had a shorter time of immobility comparable to those obtained with kcnk2−/− or fluoxetine-treated mice, whatever the way of spadin administration: intracerebroventricular (i.c.v., n=14 per group) (one-way ANOVA, F3,55=79.53, ***p<0.001 versus saline-treated mice), intravenous (i.v., n=8 per group except for fluoxetine and kcnk2−/− groups, n=6) (one-way ANOVA, F5,43=26.27, ***p<0.001 versus saline-treated mice) or intraperitoneal (i.p., n=10 per group except for kcnk2−/−, n=5 ) (one-way ANOVA, F3,34=40.58, *p<0.05, ***p<0.001 versus saline-treated mice). (B) Tail Suspension Test (TST, n=15 for saline and spadin groups, and n=9 for fluoxetine and kcnk2−/− groups), i.v. spadin-treated mice had a shorter immobility score comparable to those obtained with kcnk2−/− or fluoxetine-treated mice (one-way ANOVA, F3,47=11.40, **p<0.01, ***p<0.001 versus saline-treated mice). (C) Conditioned Motility Suppression Test (CMST, n=10 per group). Two-way ANOVA showed significant effects of shocks (F1,62=254.1, p<0.001), treatment (F3,62=3.87, p<0.01) and an interaction between these two factors (F3,62=8.83, p<0.001). ###p<0.01 versus non-shocked mice. In the shocked groups, spadin treatment reversed the freezing state induced by the shock training in saline-treated mice (78±7 versus 14±2 counts, respectively). This effect was stronger than those observed for kcnk2−/− or fluoxetine-treated mice (one-way ANOVA, F3,39=10,87, *p<0.05, ***p<0.001 versus saline-treated mice). Counts are the number of squares crossed plus the number of climbings. (D and E) Learned Helplessness test (LH, n=12 per group). Shocked spadin-treated mice showed shorter escape latencies than saline-treated mice. Two-way ANOVA showed significant effect for treatment (F1,110=7.93, p=0.01) and for assay (F5,110=3.56, p=0.005 , *p<0.05 in shocked groups). (D) Mean escape latencies ± SEM averaged in 6 blocks of 5 trials, and (E) mean overall latency ± SEM to escape across trials 1–30 as a function of spadin treatment. Two-way ANOVA (Shocks×Treatment) showed an interaction between these two factors (F1,44=6.9, p=0.012). ##p=0.007 for non-shocked saline-treated mice versus shocked saline-treated mice.