Figure 2. Immature spine morphology and reduced excitability of layer 2 excitatory neurons following Dnmt3a conditional knockout (cKO).

(A) Example dendritic segments of layer 2 pyramidal neurons in the prelimbic region labeled with DiI and visualized using a 63× objective coupled to an Airyscan confocal microscope. Arrowheads show filopodia, which were more abundant in Dnmt3a cKO mice. (B) The density of membrane protrusions was unchanged in the Dnmt3a cKO (Wilcoxon test, n.s., not significant). (C) Membrane protrusions were significantly longer and narrower in the Dnmt3a cKO (KS test, p<0.001). (D) More spines were classified as immature filopodia, and fewer as mature mushroom-shaped spines with large postsynaptic densities in the Dnmt3a cKO (Wilcoxon test, **, p=0.0015; *, p=0.046 and 0.011 for thin and mushroom, respectively). (E) Example whole-cell patch-clamp recordings from prelimbic layer 2 pyramidal neurons following 60 pA current injections. (F) The median rheobase (i.e. the minimal current necessary to elicit an action potential) was significantly higher in the Dnmt3a cKO (t-test, **, p=0.0042). (G) Action potential frequency vs. injected current (mean ± SEM) showed reduced excitability in Dnmt3a cKO (Wilcoxon test, *, p<0.05). (H) and (I) Dnmt3a cKO neurons were slightly hyperpolarized at V_rest when compared to control (Wilcoxon test, *, p=0.049) and had lower membrane resistance (Wilcoxon test, *, p=0.023).

Figure 2—figure supplement 1. The membrane protrusions in the Dnmt3a conditional knockout (cKO) showed longer dendritic spines and narrower heads.

Related to Figure 2C. Here, each line/box represents data from one dendrite fragment. (A) and (C) The cumulative distribution of the length (A) and width (C) of membrane protrusions in the Dnmt3a cKO (orange), as compared to control mice (black). (B) and (D) Distribution of the length (B) and width (D) of the protrusions. The shaded colors in the box represent the individual animals from which the dendrite fragment originated (5 control mice and 4 cKO mice). The middle horizontal bar represents the median; the lower and upper hinges correspond to the first and third quartiles, and the whisker extends from the hinge to the value no further than 1.5 * IQR from the hinge, where IQR is the interquartile range. The Wilcoxon test was done with the medians of the boxes (16 control vs. 15 cKO).

Figure 2—figure supplement 2. The conditional ablation of Dnmt3a in pyramidal neurons did not significantly alter the membrane potential threshold for action potential generation, the mean amplitude, or frequency of miniature excitatory postsynaptic events.

(A) Action potentials were initiated at a similar membrane potential in both genotypes (Wilcoxon test, n.s., not significant). (B) While miniature excitatory postsynaptic currents (mEPSCs) amplitude was not significantly different between genotypes (Wilcoxon test, n.s., not significant), it was slightly, yet significantly, more variable in the Dnmt3a conditional knockout (cKO) (F-test, p=0.0032). (C) mEPSCs frequency was not significantly changed. In all boxplots, the middle horizontal bar represents the median; the lower and upper hinges correspond to the first and third quartiles, and the whisker extends from the hinge to the value no further than 1.5 * IQR from the hinge, where IQR is the interquartile range. The values of individual experiments are represented by dots superimposed on the boxplots.