Figure 6
Wnt5a increases the GluN2B-containing NMDAR activity. (a) Plots show the normalized Fura-2 AM fluorescence ratio (340/380) over time before and after NMDA + glycine exposure in neurons treated for 1 h with control (C) or Wnt5a (W5a) medium in the presence or absence of HRI-i and 7-NI. Graphs show the AUC of the represented plots. Bars show the mean ± SEM. N = 6 independent experiments. Control (100); Wnt5a (110); Wnt5a+HRI-I (111); HRI-i (109); Control (32); Wnt5a (42); Wnt5a+7-NI (111) number of cells (*p 0.05, ***p = 0.00009). (b) Averaged sample traces of NMDAR-EPSCs obtained from the soma of CA1 pyramidal cells voltage-clamped at + 40 mV. Top panel, control currents at 30 and 60 min to show the stability of recordings; second, the current after Wnt5a bath application (1 h) and the selective GluN2B antagonist Ro25-6981 (3 µM); third panel, current after preincubation with HRI-i (1 µM) and Wnt5a addition; fourth, current after preincubation with CHX (50 µM) and Wnt5a. (c) The dot plots show the NMDA current amplitudes measured during every treatment relative to the control current in the cell before Wnt5a or drug application. The relative values for each condition were averaged and plotted as shown (Control, Wnt5a: N = 9 cells from 5 mice; Wnt5a + Ro: N = 10 cells from 5 mice; Ro: 6 cells from 5 mice; Wnt5a + HRI-i: N = 5 cells from 5 mice; HRI-i: N = 3 cells from 3 mice; Wnt5a+CHX: N = 3 from 3 mice; CHX: N = 3 from 3 mice). Lines represent the mean ± SEM; (*p 0.05, **p < 0.001, ***p = 0.00009). (d,e) Spontaneous action potentials (APs) in current clamp configuration. The presence of Wnt5a reduces the firing threshold for AP generation (d), and at the same resting membrane potential, Wnt5a induces more APs and also the generation of regular periods of bursts of activity (e). Notably, the presence of Ro prevented this feature. (f) The recordings obtained in (b) were normalized to obtain decay kinetics (tau, ms) in every tested condition. The dot plots show the relative values of the decay measurements and the mean ± SEM (*p < 0.05; **: p < 0.001).
