Neuron, Volume 76
Supplemental Information
NMDA Receptor Regulation Prevents
Regression of Visual Cortical Function
in the Absence of Mecp2
Severine Durand, Annarita Patrizi, Kathleen B. Quast, Lea Hachigian, Roman Pavlyuk, Alka Saxena, Piero Carninci, Takao K. Hensch, and Michela Fagiolini
Supplemental Figure S1. Visual acuity evaluated by VEP was rescued in Mecp2 KO mice by dark rearing. This figure is related to Figure 1.
Supplemental Figure S2. Discrimination of putative excitatory and inhibitory neurons in WT and KO mice across cortical layers. This figure is related to Figures 1, 3 and 5.
Supplemental Figure S3. DR did not rescue motor deficits in Mecp2 KO mice. This figure is related to Figures 1 and 5.
Supplemental Figure S4. ChIP-qPCR analysis of Mecp2 revealed selective binding to PV and NR2A promoters. This figure is related to Figures 4,5 and 7.
Supplemental Table S1. Primers used for ChIP-qPCR analysis
Supplemental Table S2. Relative mRNA expression levels in LR and DR Mecp2 WT, LR and DR Mecp2 KO primary visual cortex homogenates determined by RT-qPCR and REST 2009 Software analysis (relative expression ratio, standard error, statistical significance). This figure is related to Table 1.
SUPPLEMENTAL MATERIAL
Supplemental Data
Supplemental Figure S1. Visual acuity evaluated by VEP was rescued in Mecp2 KO mice by dark rearing
(A) Representative VEPs recorded at different depths of the binocular visual cortex, contralateral to the stimulated eye, in response to sinusoidal gratings at 0.05 cpd (See Methods). Maximal VEP amplitude in response to low spatial frequency (0.05 cpd) is elicited deep in layer III / top of layer IV. At this depth, visual acuity is then recorded (Porciatti et al., 2007).
(B) Representative VEP acuity for a Mecp2 WT (open circles) and Mecp2 KO (black squares) mouse; 0.41 and 0.33 cpd, respectively. Threshold acuity is evaluated by extrapolating VEP amplitude data to 0V.
(C) Mean VEP acuity for adult WT (0.47 ± 0.02, n = 6), Mecp2 KO (0.32 ± 0.02, n = 4), DR WT (0.63 ± 0.03, n = 5) and DR Mecp2 KO (0.53 ± 0.04, n = 4). Error bars, mean ± s.e.m; * P<0.05, Mann-Whitney test.
Supplemental Figure S2. Discrimination of putative excitatory and inhibitory neurons in WT and KO mice
(A) Spike waveform parameters: spike width from initial trough minimum (a) to the following peak (b) and their ratio (b/a). These parameters were robust and sufficient to separate and distinguish two groups of waveforms.
(B) Scatter plots of waveform parameters for all units in LR Mecp2 WT, LR Mecp2 KO and DR Mecp2 KO mice. Putative inhibitory cells were isolated from putative excitatory cells based on characteristic parameters values Units having a Spike Width < 0.4 msec and ratio b/a > 0.5 were identified as putative inhibitory cells. Insert. Sample spike waveforms for excitatory (top) and inhibitory (bottom) neurons.
(C) Laminar analysis of Signal-to-Noise Ratio (SNR = Rmax-Spont/Rmax) at different depths for LR KO (black symbols, n = 58) and DR KO (grey symbols, n = 30). Triangles, putative inhibitory cells. Vertical lines, mean SNR for KO (dashed) and KO DR (solid).
Supplemental Figure S3. DR did not rescue motor deficits in Mecp2 KO mice
Left, Time spent on Rotarod without falling (5 minute session, low speed) (see Methods). Middle, LR and DR Mecp2 KO exhibited a significantly reduced velocity and Right, distance traveled (lower panel) in an open field arena compared to LR Mecp2 WT mice. 4 to 10 mice each. Error bars, mean ± s.e.m.
Supplemental Table 1. The following primer sequences were used to amplify the region of interest for ChIP-PCR.
Supplemental Figure S4. Mecp2 binds to NR2A and PV promoter regions
(A) Snapshots of UCSC Browser for the genomic locations of UCSC and RefSeq genes Grin2a (NR2A; top panel) and Pvalb (PV, bottom panel) as labeled, for the mm9 assembly. Scale is shown at the top and chromosome locations are shown in the second line. The genomic location for Grin2a also harbors an un-annotated UCSC transcript AK086290 (as labeled) and a longer non RefSeq alternate transcript for Pvalb is also seen (as labeled). The bottom line shows CpG islands in the region as green horizontal bars (light green <300 bases, dark green >300 bases). ChIP-qPCR primer locations are shown as black vertical bars above the genes and PCR amplicons are depicted as black lines connecting the primers.
(B) Mecp2 binding was enriched in the region of amplicon ‘1’ (red arrow) for Pvalb and amplicon ‘1’ (red arrow) for Grin2a in the WT visual cortex at postnatal day P15. Whereas Pvalb and Grin2a were significantly enriched in all 4 ChIP biological samples, we also confirmed binding of Mecp2 to the reported enrichment site for Grin2b (from Lee et al., 2008) in 3 out of 4 samples (therefore did not meet statistical significance). Data are mean± SEM from four independent experiments with three technical repetitions each.
Supplemental Table 2. Relative mRNA expression levels in LR and DR Mecp2 WT, LR and DR Mecp2 KO primary visual cortex homogenates determined by RT-qPCR and REST 2009 Software analysis (relative expression ratio, standard error, statistical significance).
Supplemental Experimental Procedures
All procedures were approved by the IACUC of Boston Children’s Hospital. All experiments were conducted in the Mecp2 KO mouse line generated by A. Bird and colleagues (Guy et al., 2001). Mice carrying a deletion of Mecp2 were generated by crossing Mecp2 heterozygote females with C57BL6 males. Double mutants for Mecp2 and NR2A were generated by crossing Mecp2 hererozygote females with NR2AKO males (originally generated by M. Mishina; Sakimura et al.,1995). Experiments were performed blind to the genotype of the animals and were conducted using male mice unless otherwise specified. In some experiments, flox-STOP-Mecp2 (Guy et al., 2007) or PV-Cre x flox-Mecp2 animals were used (Hippenmeyer et al., 2005; Jackson laboratories). All control animals were WT age-matched littermates of the mutant mice.
Single-cell electrophysiology in vivo
Electrophysiological recordings were performed under Nembutal (50mg/kg, i.p.) anesthesia and chlorprothixene (0.2 mg, i.m.) using standard techniques (Fagiolini and Hensch, 2000). Single-unit responses were recorded using multichannel probes (a1x16-3mm50-177, Neuronexus Technologies). The signal was amplified, thresholded and discriminated (SortClient, Plexon Technologies). To ensure single-unit isolation, the waveforms of recorded units were further examined offline (Offline Sorter, Plexon Technologies) and discriminated on the basis of their individual characteristics.
Single-units were classified as narrow (putative inhibitory cells) and broad-spiking (putative excitatory cells) based on shape properties of their average waveforms (Fig. S2). Two parameters were used for discrimination: the ratio of the height of the positive peak to the initial negative trough and the time from the initial trough minimum to the following peak maximum (Niell and Stryker, 2008; Durand et al., 2007). These two parameters were sufficient to distinguish the two groups of waveforms.
For each animal, 5-8 single units were recorded in each of 3 to 5 penetrations spaced evenly (>200μm intervals) across the medio-lateral extent of binocular primary visual cortex. Spontaneous and evoked activity were recorded in response to high contrast low spatial frequency sine wave gratings (100% contrast, 0.025 or 0.07 cpd; 2 Hz) presented on a computer monitor (mean luminance = 32 cd/m2). Twelve different orientations were presented in random order (3 sec each), interleaved by uniform gray screen of intermediate luminance (3 sec). Response firing rate to each orientation was averaged over 8 to 10 trials. Spontaneous activity was averaged over 8-10 random repeats of the gray screen and the maximum evoked response was taken as the preferred visual stimulus.
Visual Evoked Potential (VEP)
VEPs were recorded under nembutal / chlorprothixene anesthesia using standard techniques in mice (Porciatti et al., 2007; Fagiolini and Hensch, 2000 ). A tungsten electrode (1.5MΩ, FST) was inserted into V1 where the maximal VEP response is located within the visual field 20° from the vertical meridian (usually 2.9-3.0 mm from intersection between midline and lambda). To record VEPs, the electrode was advanced to a depth of 100 - 400 μm within cortex where VEPs exhibit their maximal amplitude. Signals were band-pass-filtered (0.1–100 Hz), amplified, and fed to a computer for analysis. In brief, at least 20 events were averaged in synchrony with the abrupt stimulus contrast reversal (100%, 1Hz). Transient VEPs were evaluated in the time domain by measuring the peak-to-baseline amplitude of the major negative component. Visual stimuli were horizontal sinusoidal gratings of different spatial frequencies at 90% contrast. Visual acuity was obtained by extrapolation to zero amplitude of the linear regression through the last four to five data points along a curve of VEP amplitude plotted against log spatial frequency (Fig. S1).
Visual behavioral test: Optomotor task
Behavioral threshold acuity was evaluated using published methods (Prusky et al., 2004). In brief, vertical sine wave gratings were projected as a virtual cylinder in three-dimensional coordinate space on four computer monitors arranged in a quadrangle around a testing arena (OptoMotry; CerebralMechanics). Unrestrained animals were placed on an elevated platform at the epicenter of the arena. The experimenter followed the mouse’s head with a crosshair superimposed on the video image to center the rotation of the cylinder at the mouse’s viewing position, thereby clamping the spatial frequency of the grating. If the mouse’s head tracked the cylinder rotation, it was judged that the animal could see the grating.
A process of incrementally changing the spatial frequency of the test grating was repeated until the highest spatial frequency tracked was identified as the threshold. A threshold for each direction of rotation was assessed and the highest spatial frequency tracked in either direction was recorded as the visual acuity. Each session lasted generally 15-20 minutes per mouse. To evaluate the development of visual acuity, mice were tested every 3-5 days starting after eye opening until P60.Mecp2 heterozygote females, fLox-STOP-Mecp2 (Guy et al., 2007), or PV-Cre x fLox-Mecp2 were tested at different time in adulthood. Experimenters were blind to genotype and the animal’s previously recorded thresholds. All animals were habituated before the onset of testing by gentle handling and placing them on the arena platform for a few minutes at a time.
Motor behaviors
Rotarod assay was performed as previously published (Crawley, 2008). Mice were placed on a rotarod (Noldus) that was rotating at a constant speed of 5 rpm for a maximum time of 5 minutes over 8 trials (four trials a day on two consecutive days). The latency to fall onto a platform below was recorded. Mice were habituated to the rotarod apparatus on day one and tested on day two.
Open Field assay was performed as previously published (Crawley, 2008). The open field apparatus consisted of a clear, open Plexiglass box (43.2 x 43.2x30.5cm) within a soundproof chamber under 200-lux illumination. Mutant and control mice were placed in the center of the box, habituated for 5 minutes and then recorded by digital video camera for an additional 10 minutes using the HVS video tracking system (Ethovision XT, Noldus System). The velocity and total distance walked by the mice were recorded as measures of motor activity.
Quantitative RT-PCR
Mice were briefly anesthetized using 3% isoflurane and Oxygen before cervical dislocation and rapid brain removal. After rinsing in sterile saline, the binocular visual cortex was dissected, snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. The tissue was homogenized in QIAzol Lysis Reagent with a Model 100 Sonic Dismembrator (Fisher Scientific), and the RNA extracted using the RNAeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's protocol. RNA yield and purity was assessed by OD using the NanoDrop™ 1000 instrument (Thermo Fisher).
First-strand RT was performed with random hexamers using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol, and the reaction product was used as cDNA template for quantitative PCR. Gene expression was assessed by the ∆∆CT method using a StepOnePlus PCR System (Applied Biosystems) with TaqMan Gene Expression Master Mix (Cat# 4369016) and inventoried TaqMan gene expression assays (Applied Biosystems). The following TaqMan assays were used: BDNF (Mm00432069_m1), VGAT (Mm00494138_m1), Gephyrin (Mm00556895_m1), Parvalbumin (Mm00443100_m1), Kv3.1 (Mm00657708_m1), Gad1 (Mm00725661_s1), Gad2 (Mm00484623_m1), NR2A (Mm00433802_m1), NR2B (Mm00433820_m1), Gabra1 (Mm00439040_m1), AMPA1 (Mm00433753), AMPA2 (Mm00442822_m1), Calb1 (Mm00486645_m1), Calb2 (Mm00801461_m1), relative to TaqMan endogenous controls GAPDH: (Cat# 4352932E) and ACTB: (Cat# 4352933E).
Triplicate reactions for each sample (light-reared WT and KO n=7-8 mice; DR WT and DR KO n=4 mice each) were set up according to the manufacturer’s instructions, and run in parallel for two genes-of-interest and both endogenous controls. CT values of the triplicate reactions in each run were determined and averaged after compiling all run data in StepOne™ software (Applied Biosystems) using a matching threshold level. Group-wise relative expression levels for genes-of-interest and statistical analysis were performed by importing the resulting CT values into “REST 2009 software” and normalizing the results to ACTB and GAPDH endogenous controls.
Immunofluorescence
Mice were transcardially perfused with saline, followed by 45 ml of phosphate-buffered 4% paraformaldehyde solution. Brains were extracted, post-fixed for two hours in the same fixative, and cryoprotected in ascending sucrose solution (10%, 20% and 30%) and sectioned with a criostat. Cryosections were first incubated for 30 minutes at room temperature in blocking solution (10% normal goat serum, 0.5% Triton X-100 (Sigma) in PBS), then transferred for overnight 4°C incubation into primary antibody solution (3% normal goat serum, 0.5% Triton X-100, rabbit anti-Parvalbumin (Swant, 1:2500 dilution), mouse anti-Parvalbumin (Swant 1:10000), and mouse anti-GAD65 (Developmental Studies Hybridoma Bank, 1:1000). NeuroTrace blue fluorescent Nissl (Invitrogen, 1:500 dilution) was added to the primary antibody solution for counter-stain. After rising in washing buffer (0.1% Triton X-100 in PBS), the sections were incubated for two hours at room temperature in secondary antibody solution (3% normal goat serum, 0.5% Triton-X, goat anti-mouse Alexa 488 (Invitrogen, 1:1000 dilution) alone, or goat anti-mouse Alexa 488 (Invitrogen, 1:1000 dilution) and goat Anti-rabbit Alexa 594 (Invitrogen, 1:500 dilution) in PBS). Sections were again washed in three 15-minute rounds of washing buffer, mounted on glass slides with DAPI Fluoromount-G mounting medium (Southern Biotech).