Manipulations of MeCP2 in glutamatergic neurons highlight their contributions to Rett and other neurological disorders
- Baylor College of Medicine, United States
- Texas Children's Hospital, United States
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
Figures
Figure 1
Mecp2 was either deleted or restored specifically in glutamatergic neurons.
(A) Vglut2-Cre expression was assayed by colocalization of the reporter tdTomato and CamKII in 6-week-old Vglut2-Cre+/-;Rosa26tdTomato male mice. Scale bar, 30 µm. (B) Quantification of images in (A) showing the percentage of tdTomato positive cells in total CamKII-immunostaining cells (black) and the percentage of CamKII-immunostaining cells in total tdTomato positive cells (red, n = 3 mice, 14 sections). (C,D) Representative images showing MeCP2 expression in the brain of Flox and CKO mice (C), as well as stop-null and male C-rescue mice (D). Scale bar, 2 mm (n = 3 mice per genotype). (E) Fluorescence images of male C-rescue cortex stained for nucleus (4′,6-diamidino-2-phenylindole, DAPI), MeCP2 and CamKII. Scale bar, 30 µm. (F) Quantification of images in (E) showing the percentage of MeCP2 positive cells in total CamKII-immunostaining cells (black), and the percentage of CamKII-immunostaining cells in total MeCP2 positive cells (red, n = 3 mice, 18 sections).
Figure 2 with 3 supplements
Reduced cortical activity was induced by conditional deletion of Mecp2 in glutamatergic neurons but rescued by conditional restoration of MeCP2.
(A) Top: Sample traces of spontaneous firing of Layer V neurons from CKO and control mice. Bottom: Average firing rate of four genotypes of mice. (B) Top: Sample traces of spontaneous firing of Layer V neurons from male C-rescue mice and its counterparts. Bottom: Quantification of spontaneous firing rate. n = 3–4 mice and 12–15 cells per genotype. Data are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by one-way ANOVA.
Figure 2—figure supplement 1
Layer V pyramidal neurons in the CKO mice received less excitatory and inhibitory input.
(A) Top: Sample traces of sEPSCs from Layer V pyramidal neurons. Bottom: Summary of sEPSCs strength calculated as total charge of responses for 120 s. (B) Top: Sample traces of sIPSCs from Layer V pyramidal neurons. Bottom: Summary of sIPSCs strength calculated as total charge of responses for 120 s. (C) In the presence of excitatory and inhibitory synaptic transmission blockers, firing rate was plotted as a function of current amplitude to determine intrinsic properties. Scale: 100 mV, 200 ms. n = 3–4 mice, >10 cells per genotype. Data are presented as mean ± SEM. *p<0.05; **p<0.01; ****p<0.0001; by one-way ANOVA.
Figure 2—figure supplement 2
CKO mice displayed reduced mEPSC frequency and normal mIPSCs.
(A) Top: Sample traces of mEPSCs. Bottom: Quantification of amplitude and frequency of mEPSCs. (B) Top: Sample traces of mIPSCs. Bottom: Average of amplitude and frequency of mIPSCs. n = 4–5 mice, >15 cells per genotype. Data are presented as mean ± SEM. *p<0.05; n.s., not significant; by one-way ANOVA.
Figure 2—figure supplement 3
Restoration of MeCP2 in glutamatergic neurons normalized reduced mEPSC frequency in stop-null mice.
(A) Top: Sample traces of mEPSCs. Bottom: Quantification of amplitude and frequency of mEPSCs. (B) Top: Sample traces of mIPSCs. Bottom: Average of amplitude and frequency of mIPSCs. n = 4–5 mice, >15 cells per genotype. Data are presented as mean ± SEM. *p<0.05; n.s., not significant; by one-way ANOVA.
Figure 3 with 2 supplements
Removal of Mecp2 from glutamatergic neurons led to early death and obesity, which were improved in C-rescue mice.
(A–B) Survival curves plotted with percentage of mice alive as a function of age (A, n = 25; B, n = 28–34). (C–D) Plot of weight as a function of age (C, n = 20; D, n = 15–22). '**' indicates a statistical significant difference between CKO or stop-null and controls, while '##' indicates a statistical significant difference between C-rescue and controls. Data are presented as mean ± SEM. **, ##, p<0.01; by two-way ANOVA.
Figure 3—figure supplement 1
CKO mice gained more weight associated with increased daily food intake.
(A) Average daily food intake from experimental day 3 to 5 (72 hr, P27-30). Values are estimated marginal means adjusted for the difference in initial body weight. (B–C) Average daily weight gain (B) and fat gain (C) from day 1 to 6 while mice were housed in the calorimetry chambers. Values are estimated marginal means adjusted for the difference in initial body weight. (D) Average daily energy expenditure during experimental day 3 to 5 from indirect calorimetry. (E) Resting metabolic rate calculated based on energy expenditure during fasting. Values for (D–E) are estimated marginal means adjusted for the difference in average fat and lean mass. (F–G) Average daily activity (F) and daily RER (G) during experimental day 3 to 5. (H) Average RER during fasting. Data are from 5–7 mice per genotype and are presented as mean ± SEM. **p<0.01; ****p<0.0001; n.s., not significant; by one-way ANOVA or ANCOVA.
Figure 3—figure supplement 2
Male C-rescue mice gained less weight associated with reduced daily food intake.
(A) Average daily food intake from experimental day 3 to 5 (72 hr, P27-30). Values are estimated marginal means adjusted for the difference in initial body weight. (B–C) Average daily weight gain (B) and fat gain (C) from day 1 to 6, while mice were housed in the calorimetry chambers. Values are estimated marginal means adjusted for the difference in initial body weight. (D) Average daily energy expenditure from day 3 to 5 in the colorimetry chambers. (E) Quantification of resting metabolic rate based on energy expenditure during fasting. Values for (D–E) are estimated marginal means adjusted for the difference in average fat and lean mass. (F–G) Quantification of average daily activity (F) and RER (G) during experimental day 3 to 5. (H) Average RER during fasting. Data are from 6–7 mice per genotype and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; n.s., not significant; by one-way ANOVA or ANCOVA.
Figure 4
Both CKO and male C-rescue mice developed abnormal EEGs.
(A) Representative EEG traces from the somatosensory and frontal cortices of 10-week-old WT, Cre, Flox, and CKO mice. Note the spike-and-wave discharge in the CKO mouse. (B) Representative EEG traces from the somatosensory and frontal cortices of 25- to 30-week-old WT, Cre, and male C-rescue mice. Note the spike-and-wave discharge in the male C-rescue mouse.
Figure 5 with 2 supplements
Loss of Mecp2 in glutamatergic neurons led to altered anxiety-like behaviors, tremor, and impaired acoustic startle response, which were normalized in male C-rescue mice.
(A–B) Time spent in the open arms of the elevated plus maze (A, n = 16–19; B, n = 9–14). (C–D) Average time spent in, and number of transitions into, the light chamber during 10min test in the light/dark box (C, n = 16–19; D, n = 13–16). (E–F) Percentage of mice displayed tremor at 8 weeks of age (E, n = 17–22; F, n = 18). (G–H) Mean of response to the 120 dB acoustic stimulus (G, n = 15; H, n = 14–18). Data are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by one-way ANOVA.
Figure 5—figure supplement 1
CKO mice have normal locomotor and hearing function.
(A) Total distance mice traveled during the 30 min open field assay (n = 16–18). (B) Mean hearing thresholds (dB SPL) were plotted as a function of stimulus frequency (n = 8–9). Data are presented as mean ± SEM. n.s., not significant; by one-way or two-way ANOVA.
Figure 5—figure supplement 2
Reversal of altered anxiety-like behaviors and impaired acoustic startle response were maintained in 20-week-old mice.
(A) Behavior of mice in the elevated plus maze, showing time spent in the open arms (n = 12–21). (B) Average time spent in, and number of transitions into, the light side during the 10 min light/dark test (n = 12–21). (C) Average response to a 120 dB stimulus (n = 12–21). Data are presented as mean ± SEM. *p<0.05; ***p<0.001; n.s., not significant; by one-way ANOVA.
Figure 6
Both CKO and male C-rescue mice suffered from ataxia but maintained normal social interaction and were free of repetitive behaviors.
(A–B) Latency to fall from the accelerating rotarod plotted as a function of trial (A, n = 15–17; B, n = 9–14). (C–D) Average time that mice spent interacting with partners when they were housed with a familiar mouse (Fam1), a novel mouse (Novel), and the same familiar mouse (Fam2) again in the partition test (C, n = 10–16; D, n = 13–15). (E–F) Number of holes explored with ≥2 sequential nose-pokes during the 10 min hole-board assay (E, n = 10–14; F, n = 11–13). Data are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; n.s., not significant; by one-way or two-way ANOVA.
Figure 7 with 1 supplement
Restoration of Mecp2 in glutamatergic neurons in stop-het mice rescued RTT-like features.
(A) Plot of weight as a function of age (n = 16). '*' indicates a statistically significant difference between stop-het and all other genotypes, while '#' indicates a statistically significant difference between female C-rescue and controls. (B) Average time spent in the open arms of the elevated plus maze (n = 16–18). (C) Mean of response to the 120 dB stimulus (n = 16–18). (D) Pre-pulse inhibition at 74 dB, 78 dB and 82 dB pre-pulses (n = 16–18). (E) Latency to fall from the accelerating rotarod is plotted as a function of trial (n = 15–16). (F) Average number of footfalls normalized by distance traveled during the 10 min foot-slip assay (n = 15–16). (G) Percentage of mice displayed tremor at 30 weeks of age (n = 20–28). Data are presented as mean ± SEM. *, # p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by one-way or two-way ANOVA.
Figure 7—figure supplement 1
Reversal of impaired acoustic startle and increased PPI were maintained at 30-week-old female C-rescue mice.
(A) Total distance traveled in the open field assay (n = 15–16). (B) Mean time spent in the open arms of the elevated plus maze (n = 15–16). (C) Average response to the 120 dB stimulus (n = 15–16). (D) Percentage of inhibition in response to three pre-pulses (n = 15–16). Data are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n.s., not significant; by one-way or two-way ANOVA.
Tables
Table 1
Comparison of stop-null, Mecp2 CKO, and male and female C-rescue mice.
| Neurological features | Stop-null | Mecp2 CKO | Male C-rescue | Female C-rescue |
|---|---|---|---|---|
| Tremor | + | + | - | - |
| Altered Anxiety | + | + | - | - |
| Impaired Acoustic Startle Response | + | + | - | - |
| Premature Lethality | + | + | Significantly delayed | -* |
| Obesity | + | + | - | - |
| Ataxia | + | + | + | - |
| Repetitive Behavior | + | - | - | NE |
| Abnormal Social Interaction | + | - | - | NE |
-
+: Presence of feature.
-
-: Absence of feature
-
*: Deficits were absent in stop-het mice.
-
NE: Mice were not examined with this particular assay.
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Manipulations of MeCP2 in glutamatergic neurons highlight their contributions to Rett and other neurological disorders
eLife 5:e14199.
https://doi.org/10.7554/eLife.14199
