MeCP2 controls excitatory synaptic strength by regulating glutamatergic synapse number

Abstract

MeCP2 is a transcriptional repressor critical for normal neurological function. Prior studies demonstrated that either loss or doubling of MeCP2 results in postnatal neurodevelopmental disorders. To understand the impact of MeCP2 expression on neuronal function, we studied the synaptic properties of individual neurons from mice that either lack or express twice the normal levels of MeCP2. Hippocampal glutamatergic neurons that lack MeCP2 display a 46% reduction in synaptic response, whereas neurons with doubling of MeCP2 exhibit a 2-fold enhancement in synaptic response. Further analysis shows that these changes were primarily due to the number of synapses formed. These results reveal that MeCP2 is a key rate-limiting factor in regulating glutamatergic synapse formation in early postnatal development and that changes in excitatory synaptic strength may underlie global network alterations in neurological disorders due to altered MeCP2 levels.

Figure 1. Loss or doubling of MeCP2 alter the magnitude of evoked EPSCs and frequency of spontaneous mEPSC events

(A) Mean action potential evoked EPSC trace from data shown in (B, C). Data collected at an earlier time point for Mecp2^Tg1 neurons to reduce voltage clamp errors. (B, C) Bar graphs show EPSC amplitude. (D) Representative mEPSC traces with upper trace in the presence of 3 mM glutamate receptor blocker, kynurenic acid (Kyn). (E, F) Bar graphs show mEPSC frequency. Number of neurons analyzed (n) shown in the bars. Data shown as mean ± SEM. ** P<0.01, *** P<0.001

Figure 2. Loss or doubling of MeCP2 alter the magnitude of the RRP, without altering short-term plasticity and synaptic release probability

(A, B) Average current response to 4 s application of sucrose from data shown in (C, D). (C–F) Bar graphs show RRP size (C, D) and P_vr (E, F). Number of neurons analyzed (n) shown in the bars. (G, H) Normalized EPSC amplitude with train stimulation. (I, J) Normalized NMDA-EPSC amplitude with 5 µM MK801. Data shown as mean ± SEM. ** P<0.01, *** P<0.001

Figure 3. MeCP2 regulates glutamatergic synapse numbers

(A, B) Representative images of autaptic neurons show colocalization between MAP2 (green) and VGLUT1 (A, blue) or PSD95 (B, blue). Scale bar equals 10 µm. (C) Bar graph shows synaptic density of each marker normalized to WT (dotted line). (D) Representative images showing colocalization of VGLUT1 (blue) and PSD95 (red), arrowheads, with MAP2 (green). Scale bar equals 5 µm. (E–G) Bar graphs show fraction of colocalization for PSD95 (E) and VGLUT1 (F), and synaptic density of VGLUT1-PSD95 colocalized puncta (G). Number of neurons analyzed (n) shown in the bars is the same for WT and corresponding group. Data shown as mean ± SEM normalized to WT (dotted line). * P<0.05, ** P<0.01, *** P<0.001

Figure 4. Physiological and morphological phenotypes are restored in Mecp2^Null;Tg1

(A) Bar graph shows EPSC amplitude, RRP charge and P_vr (WT, n=65) and mEPSC frequency, amplitude, and decay (WT, n=48) normalized to WT (dotted line). (B) Representative images showing colocalization of VGLUT1 (blue) and PSD95 (red), arrowheads, with MAP2 (green). Scale bar equals 5 µm. (C–E) Bar graphs show fraction of colocalization for PSD95 (C) and VGLUT1 (D), and synaptic density of VGLUT1-PSD95 colocalized puncta (E). Number of neurons (n) shown in the bars. Data shown as mean ± SEM.

Figure 5. Loss of and doubling of MeCP2 levels lead to in vivo alterations in synapse numbers in stratum radiation of hippocampal CA1

(A, B) Representative images of hippocampal CA1 show MAP2 (red) and VGLUT1 (blue) at 2 weeks of age. Scale bar equals 50 µm. Or = stratum oriens. Pyr = stratum pyramidale. Rad = stratum radiatum. (C, D) Graphs show VGLUT1 intensity in Rad. (E) Bar graph shows VGLUT1 intensity normalized to WT (dotted line). (F, G) Graphs show MAP2 intensity in Rad. (H) Bar graph shows MAP2 intensity normalized to WT (dotted line). Number of sections analyzed (n) for bar graphs is shown in corresponding graphs. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001