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. 2017 Oct 3;21(1):84–96. doi: 10.1016/j.celrep.2017.09.019

Figure 2.

Figure 2

Neuronal Activity Induces Dab1 Phosphorylation at the Synapse through EphrinB2

(A–D) Neuronal activity induces Dab1 phosphorylation in primary hippocampal neurons. Levels of phosphorylated Dab1 were assessed by immunocytochemistry. Fluorescent images showing dendritic pDab1 staining upon stimulation with KCl for 10 min (A) to depolarize the membrane or 25 mM TEA-Cl for 10 min to induce chemical LTP (C). Quantification of relative pDab1 fluorescence intensity is shown in (B; n = 4) and (D; n = 3).

(E and F) Hippocampal neurons were stimulated with 10 mM KCl or 25mM TEA-Cl for 10 min and subjected to western blot analysis. Western blot showing increased levels of pDab1 upon KCl stimulation (E). Quantification of relative pDab1 levels (n = 4) (F).

(G and H) Induction of neuronal activity causes co-clustering of ephrinB and pDab1 at postsynaptic sites. Hippocampal neurons were treated with KCl and immunocytochemical analysis was performed. Microscopic images show dendritic pDab1 and ephrinB staining. Arrowheads indicate co-clustering of pDab1 (red) and ephrinB (green) upon KCl stimulation (G). Quantification of ephrinB2, pDab1, and ephrinB2-pDab1 colocalized clusters upon KCl stimulation (n = 5 neurons) (H).

(I and J) Co-staining of pDab1 (red) and PSD-95 (green) in response to KCl is shown by arrowheads (I). Quantification of PSD-95, pDab1, and colocalized clusters of PSD-95-pDab1 is shown (n = 5 neurons) (J).

(K and L) Dab1 phosphorylation upon KCl stimulation depends on ephrinB2. Primary hippocampal neurons were isolated from conditional neuronal specific ephrinB2 knockout embryos at E17.5. At 14 DIV, neurons were stimulated with 10 mM KCl for 10 min and immunocytochemistry for pDab1 was performed. Fluorescent images showing pDab1 in control (Nes-cre−; efnB2lox/lox) and ephrinB2 knockout (Nes-cre+; efnB2lox/lox) neurons stimulated with KCl (K). Quantification of relative pDab1 fluorescence intensity in control and ephrinB2 knockout neurons (n = 3) (L).

Scale bars represent 20 μm in (A), (C), and (K) and 5 μm in the higher magnifications in (A), (C), and (K) as well as in (G) and (I). Bar graphs show mean ± SEM (shown as error bars).∗p < 0.05, ∗∗∗p < 0.001. See also Figures S2–S4.