Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 21;5:e14199.
doi: 10.7554/eLife.14199.

Manipulations of MeCP2 in Glutamatergic Neurons Highlight Their Contributions to Rett and Other Neurological Disorders

Affiliations
Free PMC article

Manipulations of MeCP2 in Glutamatergic Neurons Highlight Their Contributions to Rett and Other Neurological Disorders

Xiangling Meng et al. Elife. .
Free PMC article

Abstract

Many postnatal onset neurological disorders such as autism spectrum disorders (ASDs) and intellectual disability are thought to arise largely from disruption of excitatory/inhibitory homeostasis. Although mouse models of Rett syndrome (RTT), a postnatal neurological disorder caused by loss-of-function mutations in MECP2, display impaired excitatory neurotransmission, the RTT phenotype can be largely reproduced in mice simply by removing MeCP2 from inhibitory GABAergic neurons. To determine what role excitatory signaling impairment might play in RTT pathogenesis, we generated conditional mouse models with Mecp2 either removed from or expressed solely in glutamatergic neurons. MeCP2 deficiency in glutamatergic neurons leads to early lethality, obesity, tremor, altered anxiety-like behaviors, and impaired acoustic startle response, which is distinct from the phenotype of mice lacking MeCP2 only in inhibitory neurons. These findings reveal a role for excitatory signaling impairment in specific neurobehavioral abnormalities shared by RTT and other postnatal neurological disorders.

Keywords: MeCP2; Rett syndrome; glutamatergic neurons; mouse; neurological disorders; neuroscience.

Conflict of interest statement

HYZ: Senior Editor, eLife.

The other authors declare that no competing interests exist.

Figures

Figure 1.
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). DOI: http://dx.doi.org/10.7554/eLife.14199.003
Figure 2.
Figure 2.. 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. DOI: http://dx.doi.org/10.7554/eLife.14199.004
Figure 2—figure supplement 1.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.005
Figure 2—figure supplement 2.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.006
Figure 2—figure supplement 3.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.007
Figure 3.
Figure 3.. 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. DOI: http://dx.doi.org/10.7554/eLife.14199.008
Figure 3—figure supplement 1.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.009
Figure 3—figure supplement 2.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.010
Figure 4.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.011
Figure 5.
Figure 5.. 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. DOI: http://dx.doi.org/10.7554/eLife.14199.012
Figure 5—figure supplement 1.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.013
Figure 5—figure supplement 2.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.014
Figure 6.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.015
Figure 7.
Figure 7.. 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. DOI: http://dx.doi.org/10.7554/eLife.14199.016
Figure 7—figure supplement 1.
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. DOI: http://dx.doi.org/10.7554/eLife.14199.017

Similar articles

See all similar articles

Cited by 27 articles

See all "Cited by" articles

References

    1. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics. 1999;23:185–188. doi: 10.1038/13810. - DOI - PubMed
    1. Boulland JL, Qureshi T, Seal RP, Rafiki A, Gundersen V, Bergersen LH, Fremeau RT, Edwards RH, Storm-Mathisen J, Chaudhry FA. Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters. The Journal of Comparative Neurology. 2004;480:264–280. doi: 10.1002/cne.20354. - DOI - PubMed
    1. Cai T, Seymour ML, Zhang H, Pereira FA, Groves AK. Conditional deletion of Atoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti. Journal of Neuroscience. 2013;33:10110–10122. doi: 10.1523/JNEUROSCI.5606-12.2013. - DOI - PMC - PubMed
    1. Cardoza B, Clarke A, Wilcox J, Gibbon F, Smith PE, Archer H, Hryniewiecka-Jaworska A, Kerr M. Epilepsy in Rett syndrome: association between phenotype and genotype, and implications for practice. Seizure. 2011;20:646–649. doi: 10.1016/j.seizure.2011.06.010. - DOI - PubMed
    1. Chao HT, Chen H, Samaco RC, Xue M, Chahrour M, Yoo J, Neul JL, Gong S, Lu HC, Heintz N, Ekker M, Rubenstein JL, Noebels JL, Rosenmund C, Zoghbi HY. Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature. 2010;468:263–269. doi: 10.1038/nature09582. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Feedback