NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2 - PubMed (original) (raw)

NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2

Severine Durand et al. Neuron. 2012.

Abstract

Brain function is shaped by postnatal experience and vulnerable to disruption of Methyl-CpG-binding protein, Mecp2, in multiple neurodevelopmental disorders. How Mecp2 contributes to the experience-dependent refinement of specific cortical circuits and their impairment remains unknown. We analyzed vision in gene-targeted mice and observed an initial normal development in the absence of Mecp2. Visual acuity then rapidly regressed after postnatal day P35-40 and cortical circuits largely fell silent by P55-60. Enhanced inhibitory gating and an excess of parvalbumin-positive, perisomatic input preceded the loss of vision. Both cortical function and inhibitory hyperconnectivity were strikingly rescued independent of Mecp2 by early sensory deprivation or genetic deletion of the excitatory NMDA receptor subunit, NR2A. Thus, vision is a sensitive biomarker of progressive cortical dysfunction and may guide novel, circuit-based therapies for Mecp2 deficiency.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Reduced Visual Acuity and Neuronal Activity of Visual Cortical Neurons in Adult Mecp2 Knockout (KO) Mice

(A) Average acuity assessed by optomotor task (OPT; p < 0.0001, t test) or visual-evoked potential (VEP, p < 0.05, t test) in wild-type (WT; white, n = 16 and 8) or Mecp2 KO mice (black, n = 15 and 5). (B) Visual acuity emerges normally but then regresses drastically after 40 days of age in Mecp2 KO mice. Acuity development over time for WT (○; n = 5–16) and KO (•, n = 5–15) mice. (C) Visual acuity significantly decreases in Mecp2loxstop mutant mice after the age of P55 as the RTT phenotype emerges (6–8 mice each, left, hatched bars, p < 0.01, Mann-Whitney test). Mecp2 heterozygote females also exhibit a progressive loss of visual acuity starting around P80 and reaching a minimum around P240 (6-8 mice each, p < 0.01, Mann-Whitney test). (D) Representative Mecp2 WT and KO spike trains and corresponding PSTH in response to two oriented gratings or a uniform gray stimulus (8 presentations each). (E) Spontaneous and evoked neuronal activity are significantly reduced in the absence of Mecp2. Mecp2 WT: white bars, n = 47 cells; Mecp2 KO: black bars, n = 58 cells (p < 0.005, Mann-Whitney test). (F) Signal-to-noise ratio (SNR) cumulative distribution is significantly increased in Mecp2 KO compared to WT visual cortical neurons (KS test, p < 0.01). All data are presented as mean ± standard error. See also Figures S1–S3.

Figure 2

Figure 2. Reversible Parvalbumin (PV)-Circuit Hyperconnectivity and Acuity Defects in Mecp2 KO Mice

(A) Upper panels: Mean pixel intensity of PV immunofluorescence is increased in light-reared (LR) Mecp2 KO mouse visual cortex compared to WT levels and restored to normal levels in dark-reared (DR) Mecp2 KO. Cortical layers are indicated on the left side of upper left panel. wm = white matter. Scale bar, 100 μm. Lower panels: Presynaptic PV-puncta per cell perimeter are restored to WT levels by DR. Scale bar, 10 μm. (B) Changes in mean intensity (upper) and PV-cell innervation of pyramidal cell somata (lower) are reversibly increased in Mecp2 KO mouse compared to WT levels (WT versus Mecp2 KO, p < 0.0005; *p < 0.05, **p < 0.01, Mann-Whitney test). Error bars, mean ± SEM. (C) Single-unit recordings from DR WT and KO adult mice revealed a significant increase in the level of spontaneous activity. Spontaneous but not evoked activity was restored to LR WT level in DR Mecp2 KO mice. Red dotted lines indicate the WT adult level of spontaneous and evoked activity. (D) Measurements made just before and after 30 days in the dark (DR) reveal little acuity loss (n = 4, p > 0.05). Visual acuity change for LR Mecp2 KO mice or those placed in the dark from P30 (0.40 ± 0.01 and 0.39 ± 0.002 cpd) to P60 (0.22 ± 0.02 and 0.33 ± 0.10 cpd), respectively. (E) Acuity comparison at P55-60 between Mecp2 KO (n = 15) and dark-reared (DR) KO mice from different postnatal ages, P0 (n = 6), P14–15 (n = 6), P20 (n = 4), or P24–30 (n = 8). Shaded region indicates range of normal WT acuity. Error bars, mean ± SEM.

Figure 3

Figure 3. Enhanced Inhibitory Gating in Mecp2 KO Mouse Visual Cortex

(A) PV-immunofluorescence is elevated at P15 in Mecp2 KO animals (3 mice each, p < 0.01, Mann-Whitney test), and this difference persists into adulthood (see also Figure 2). The density of perisomatic PV-boutons upon pyramidal cell somata is significantly increased in the absence of Mecp2 starting already at P15 and throughout life (upper right, 3–4 mice each, versus WT; *p < 0.01, **p < 0.001, Mann-Whitney test). WT mice also exhibit a significant increase in PV-puncta across development (3–4 mice each, p < 0.01, Mann-Whitney test).The level of GAD65 within PV puncta is significantly decreased starting from P30 in Mecp2 KO mice compared to WT (lower right, 3–4 mice each, versus WT; *p < 0.01, **p < 0.001, Mann-Whitney test). Scale bar, 5 μm. (B) Propagation of neuronal activity through layer 4 at threshold stimulation is reduced in upper layers in the Mecp2 KO mouse. Schematic of recording area indicating position of the stimulating electrode (black arrowhead) in the white matter (WM) and ROIs (squares) for analysis in upper and lower layers “on beam.” Pseudocolor peak response frame from VSDI movies of WT slices 15 ms after half maximal WM stimulus, revealing strong WT response propagation to the upper layers. The upper layer response in KO slices is suppressed at threshold stimulus intensity (graph arrow). Scale bar, 250 μm. Upper/lower layer response ratio as a function of WM stimulus intensity. All results expressed as mean ± SEM (4–5 mice each); *p < 0.001, t test. See also Figure S2.

Figure 4

Figure 4. Late Mecp2 Deletion in PV Cells Induces Expression but Not Hyperconnectivity or Loss of Visual Function

(A) Double staining for PV (green) and Mecp2 (red) shows that the majority of PV-neurons retain Mecp2 expression at P22; whereas by P90, only 8% of PV cells still express Mecp2 in Mecp2lox/y/PV-Cre−/+ (c-KO) mice compared to Mecp2+/y/PV-Cre−/+ littermates (c-WT). Scale bar, 35μm. (B) Loss of Mecp2 expression is paralleled by an increase in PV immunoreactivity (left) but not in PV puncta density (right) in Mecp2 c-KO compared to control littermates at P90 (3 mice each, p < 0.001, Mann-Whitney test). (C) SNR cumulative distribution is no different between late Mecp2 c-KO (solid green) and control (c-WT; dashed green) (63 and 53 cells, respectively; p = 0.88, KS test). (D) Visual acuity is not affected in adult c-KO mice (5 mice each, p = 0.8, Mann-Whitney test). All data are presented as mean ± standard error. See also Figure S4.

Figure 5

Figure 5. Rescue of PV-Cell Hyperconnectivity by NR2A Regulation

(A) NR2A/2B ratio in WT mice is significantly decreased by DR in visual cortex homogenates (quantified by qPCR). Mecp2 KO mice exhibit an increased ratio compared to WT which is reduced by DR to reach normal WT levels (LR WT versus LR KO: p < 0.05; LR WT versus DR WT: p < 0.05; LR KO versus DR KO: p < 0.005, one-way ANOVA). (B) Left: Mecp2 KO/NR2A Het mice appear indistinguishable from WT or Mecp2 WT/NR2A Het mice. Double mutants exhibit regular breathing, absence of tremor and do not show hindlimb clasping phenotype. Right: Double mutant mice (blue diamond) exhibit a higher weight than Mecp2 KO mice (black circle) and in the same range as WT animals (white circle); ***p < 0.001, t test. (C) Density of PV-positive puncta upon pyramidal cells is not significantly different between Mecp2 KO/NR2A Het and Mecp2 WT/NR2A Het across development (3–5 mice each; p = 0.9, Mann-Whitney test). Similarly, there is no difference in GAD65 intensity in PV-positive puncta between double mutant and control littermates across development (3–5 mice each; p = 0.8, Mann-Whitney test). Scale bars, 100 (upper) and 10 μm (lower). All data are presented as mean ± standard error. See also Figures S2–S4.

Figure 6

Figure 6. Rescue of Spontaneous Activity and Visual Acuity by NR2A Regulation

(A) Examples of orientation tuning curve (insets) for high and low OSI visual cortical neurons in Mecp2 WT mice. Solid black, KO; dashed, WT; solid blue, KO/Het. (B) Mean OSI in Mecp2 KO/NR2A Het mice is significantly decreased compared to Mecp2 WT mice (ANOVA test, Mecp2 KO/NR2A Het, n = 55 and Mecp2 WT/NR2A Het, n = 80 cells p = 0.89 and p < 0.0001 versus Mecp2 KO). (C) Signal-to-noise ratio (SNR) cumulative distribution is not significantly different between Mecp2 KO/NR2A Het and Mecp2 WT/NR2A Het cells (KS test, p = 0.88, n = 55 and 80 cells, respectively; p > 0.05 and p < 0.0001 versus Mecp2 KO). Inset, both spontaneous and evoked activity are at WT levels following NR2A deletion (p > 0.05, Mann-Whitney test). (D) Optomotor visual acuity comparison between Mecp2 KO/NR2A Het (blue column) and Mecp2 WT/NR2A Het (dotted column) reveals intact vision in double mutants (versus Mecp2 KO alone, p < 0.005, n = 5 mice each, Mann-Whitney test). Error bars, mean ± SEM.

Figure 7

Figure 7. Cortical Circuits Are Developmentally Disrupted in the Absence of Mecp2

Parvalbumin (PV) intensity and puncta density onto pyramidal cells reveal a PV-circuit hyperconnectivity as early as P15 that increases throughout life, while GAD65 is not downregulated until adulthood. Circuit-based therapeutic strategies independent of Mecp2 may now be relevant. An effective strategy to prevent the delayed loss of cortical functions (such as vision or language) should focus on early interventions to dampen PV hyperconnectivity (A) rather than on acute enhancement of inhibition once regressive symptoms have emerged (B). See also Figure S4.

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