Figure 5

Ephrin A5 stimulation increases gephyrin cluster density via EphA7.

(a,b) Hippocampal cultures were treated with preclustered ephrin A5-Fc for 24 hrs. Cells were co-stained for MAP2 (red) and gephyrin (yellow). (a’, b’) The same micrographs as in (a,b), showing gephyrin signals only. All images were rendered using Imaris software. Scale bars, 10 μm. (c) After ephrin A5-Fc treatment, gephyrin and PSD95 cluster densities were quantified on MAP2-positive dendrites. n = 30; ***p < 0.001. (d) Prior to ephrin A5-Fc stimulation, hippocampal neurons were transfected with control siRNA or siRNA specific for EphA7 as well as pEGFP-N1 for the identification of transfected cells. n = 30; ***p < 0.0001. (e,f) Determination of relative gephyrin expression levels after ephrin A5-Fc treatment. Protein lysates of cortical neurons were treated for 30 min with BDNF or preclustered ephrin A5-Fc, submitted to SDS-PAGE and subsequent Western Blotting. Gephyrin expression was quantified in comparison to the actin signal as a loading control. n = 3. (g) Untreated or ephrin A5-Fc-stimulated hippocampal neurons were further treated with inhibitors for PI3K (Ly294002), MEK (U0126), or mTOR (rapamycin) for 24 hrs. n = 135; **p < 0.01; ***p < 0.0001; ANOVA, Fisher-PLSD; error bars, S.E.M.