Motor Cortex, Primary

Adult mice (P42–P56) were deeply anesthetized with isofluorane and transcardially perfused with 10 ml 1× Dulbecco's phosphate-buffered saline (DPBS, Life Technologies), followed by 50 ml 4% paraformaldehyde in 0.1 M phosphate buffer. After perfusion, the brains were removed and post-fixed overnight at 4°C. The brains were embedded in 5% agarose in DPBS, and cut into 50 µm thick coronal sections with a vibratome (Leica VT 1200S). Since DPBS contains a saturating concentration of calcium (0.9 mM) GCaMP brightness will be maximal. Every other section was dehydrated with DPBS and coverslipped with Vectashield mounting medium (H-1400, Vector laboratories). The coverslipped sections were imaged using a slide scanner (Nanozoomer, Hamamatsu). Confocal images (LSM 710, Zeiss) were collected for selected brain regions (Fig. 1 and 2, Fig. S1 and S3) [26] , using an 20× 0.8 NA objective and standard GFP imaging filters. Individual images were tiled and stitched using commercial software (Zeiss).
For a subset of mouse lines (GP4.3, GP4.12, GP5.5, GP5.11, and GP5.17) we visualized neurons using NeuN to measure the fraction of neurons expressing GCaMP. Staining was performed on sections that were not used for quantification of expression. Sections were blocked with 2% BSA and 0.4% Triton X-100 solution for 1 hour at room temperature to prevent nonspecific antibody binding, followed by incubation overnight at 4°C with mouse anti-NeuN primary antibody (1∶500; Millipore, MAB 377) and incubation with Alexa594-conjugated goat-anti-mouse secondary antibody (1∶ 500; Life Technologies, A11032) for 4 hours at room temperature. Sections were mounted on microscope slides with Vectashield mounting medium (H-1400, Vector laboratories).
We analyzed primary motor cortex (M1), primary somatosensory cortex (S1), primary visual cortex (V1) and hippocampus (CA1, CA3, and Dentate Gyrus, DG) using confocal microscopy. For sample images in each area we identified all labeled cells, segmented their somata, and calculated the somatic GCaMP fluorescence brightness for each cell. For cortical regions, cells were grouped into layer 2/3 (L2/3) and layer 5 (L5) cells. We also counted the fraction of GCaMP labeled cells (green channel) as a fraction of the NeuN stained cells (red channel). To compensate for variations of imaging conditions across time (e.g. changes in the excitation light source intensity), images of a fluorescence standard, 3.8 µm fluorescent beads (Ultra Rainbow Fluorescent Particles, Bangs Laboratories), were acquired. The average bead brightness was used to normalize the GCaMP signal.
In addition we performed a coarse analysis of expression levels across numerous brain regions (Table 1; Data S1).

Sequencing read mapping, quality filtering and the summary of DNA methylation level for each cytosine was performed as previously described with minor modifications^{5 (link)}. Non-clonal mapped reads were filtered for MAPQ > 10 using samtools view -bq10 option. MethylC-seq data generated from pan-excitatory mouse cortical neurons were used for the comparison with snmC-seq and snmC-seq2 datasets (GSM1541958, GSM1541959)^{10 (link)}. Methylome profiles generated by snmC-seq or snmC-seq2 for Layer 2/3 excitatory neurons were aggregated for the comparison with MethylC-seq data. Layer 2/3 excitatory neurons in the snmC-seq dataset was annotated in our previous study^{5 (link)}.
To identify layer 2/3 excitatory neurons in snmC-seq2 dataset generated for mouse primary motor cortex, we computed CH methylation ratio of each non-overlapping 100 kb bins across the genome, defined as the number of methylated basecalls divided by the number of total basecalls in the bin with CH context. We only included 18 893 bins with >100 total basecalls in more than 97.5% cells in the downstream analysis. Bin-level methylation ratios were divided by the global mCH level of each cell analyzed. We performed principal component analysis on the cell-by-bin methylation ratio matrix and use the top 150 principal components for hierarchical clustering. The expression level of a gene is known to show anti-correlation with gene body mCH level^{10 (link),11 (link)}, we therefore assigned the clusters to putative cell types based on the gene body mCH level of the established marker genes. Layer 2/3 excitatory neurons were identified as the cell cluster with low mCH level at Cux1 (marker of layer 2/3 and L4) and Satb2 (marker of excitatory neurons) and high mCH level at Rorb (marker of L4 and L5a). The identified L23 neurons are robust to different number of principal components used for clustering.
Preseq was used to estimate library complexity using forward reads with Preseq gc_extrap function with options -e 5e + 09 -s 1e + 07^{12 (link)}. Library complexity values shown in this study were estimated for the sequencing depth of 50 million read pairs.

To apply tDCS, a commercial CE-certified device named DC-Stimulator (NeuroConn GmbH, Germany) was used. For each subject, we started by locating the primary motor cortex (M1) on the more-affected side through targeting the abductor pollicis brevis (APB) hot spot at rest with transcranial magnetic stimulation (TMS) with a device called Magstim Rapid 2 (Magstim Co., UK). A pair of sponge electrodes (6.5 cm*6.5 cm) moistened with 0.9% NaCl solution were placed regarding different tDCS setups, as shown in Figure 3A (assuming the MAS is on subject's left side): in the anodal setup, the anode was placed over the left M1 hotspot and the cathode was placed over the right supraorbital region. An opposite electrode placement setup was used in the cathodal setup. For bilateral setup, we placed the anode and the cathode over the right and the left M1 hotspot symmetrically. In all aforementioned active stimulations, a direct current of 1.5 mA was delivered constantly to the skull over 20 min with a ramp-up and ramp-down of 20 s. The parameters of the active stimulations were chosen in accordance with the most up-to-date safety guidelines for tDCS (Bikson et al., 2016 (link)). In sham tDCS, the electrode placement was the same as in the bilateral setup, however, in the 20-min protocol the direct current only lasted for a short time, followed by a serial pulse train of 110 uA (Figure 3B) without any therapeutic effect (Palm et al., 2013 (link)). In either active or sham setup of tDCS, the subject was seated comfortably on a chair in a quiet state and waited until the end of the intervention.

ROIs were manually outlined around each anatomical subregion according to the Allen Brain Atlas, and using visible anatomical landmarks. Due to differences in fluorescent labeling intensity, each region was thresholded individually to isolate labeled blood vessels from the background, so each region measured from 0.023 to 4.67 mm² within each hemisphere/section. Area fraction of blood vessels above the threshold was measured for each ROI. In total, mean vascular density was calculated from multiple subregions for 17 ROI (olfactory area - OLF; cingulate cortex - CA; retrosplenial cortex - RSP; primary visual - V1; primary somatosensory - S1; primary motor area - M1; auditory - AUD; hippocampus - HIP; perirhinal - PERI; insular - INS; thalamus - TH; habenula - HAB; hypothalamus - HY; caudate putamen - CP; white matter - WM; pericisternal - PCS; ependymal around lateral ventricles - EPD) in 6 KO and 6 WT animals. Further statistical comparison was performed assuming inhomogeneous signal distribution properties between different ROI (similarly as for DWI). Hence, considering independent measurements of vascular densities among ROI analyzed and due to small group size, nonparametric Mann-Whitney U-test was employed to compare the vessel densities from KO and WT animals ROI-wise.
For ROI-wise correlation analysis, a mean value of AQP4 expressions as well as vascular densities at ROI was calculated from all respective animals strain-wise.

Participants were seated in a comfortable armchair in a dimly illuminated, electrically shielded, and sound-proof room. Since fasting levels might have an impact on food related inhibitory performance (35 (link)), the experimental procedure consisted of a single session that was programmed at least 2 h after the last meal. Participants first performed the Go/NoGo task whilst TMS was delivered to the right primary motor cortex. At the end of the session, they were asked to rate the palatability of the food images presented during the task and to fill out questionnaires (see below for details).