Congenic pairs of wild type and mutant RTT iPSC lines carrying the common R294X mutation were differentiated into glial fibrillary acidic protein positive (GFAP+) astrocytes as described in our previous paper (Williams et al., 2014). On average, more than 90% of the cells were GFAP +in cultures used in our study (Figure 1—figure supplement 1 and Table 1). Using the green-colored calcium-sensitive dye Fluo-4, we monitored spontaneous cytosolic calcium (Ca²⁺) dynamics in live mutant (R294X-MT) and congenic wild type control (R294X-WT) astrocytes (Videos 1–2, Figure 1A–B). Ionophore A23187 was used as a loading control (Figure 1—figure supplement 2). A significantly higher percentage of mutant astrocytes (30 ± 3% in MT vs. 18 ± 2% in WT, n = 26 randomly selected fields in each genotype, p=0.001) showed spontaneous oscillation in cytosolic Ca²⁺ levels (Figure 1C). In each astrocyte showing spontaneous oscillation, we further separated and quantified the calcium transients in the soma from those in the processes (Figure 1A). In the soma, the mutant cells had a significantly higher frequency (0.74 ± 0.02/min in MT vs. 0.41 ± 0.01/min in WT, n = 408 cells in MT and n = 280 cells in WT, p<0.001; Figure 1C) and slightly higher amplitude (Table 2) than their congenic wild type controls. Similar changes were observed in the processes of those astrocytes (Table 2 and Figure 1—figure supplement 3). Moreover, the R294X mutant astrocytes had significantly higher peak amplitude (4.60 ± 0.07 in MT vs. 4.04 ± 0.07 in WT, n = 567 cells in MT and n = 811 cells in WT, p<0.001) of ATP-induced Ca²⁺ rise than the congenic wild type control (Figure 1D–E). The increase in ATP-induced Ca²⁺ rise in human RTT mutant astrocytes was unlikely to be mediated by the P2 receptors, because no difference was detected in the expression level of any P2X or P2Y receptor family members between the wild type and mutant RTT astrocytes by RNA-seq and microarray analysis (data not shown). Similar observations of altered Ca²⁺ activities were made in human astrocytes carrying the rare RTT-causing V247fs mutation (Figure 1—figure supplement 4). Thus, both spontaneous and pharmacologically evoked cytosolic Ca²⁺ activity appeared abnormal in mutant human RTT astrocytes differentiated from patient-specific iPSCs and grown in the absence of neurons.

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To determine whether the observed abnormal cytosolic Ca²⁺ activities were caused by the loss of MeCP2 function, we infected mutant human RTT astrocytes with lentiviruses expressing either the green fluorescence protein (lenti-GFP) alone or GFP and wild type MECP2 together (lenti-MECP2/GFP). Three days after the infection, more than 90% of the cells were positive for GFP in both infection types (Figure 2A). In R294X mutant astrocytes infected with lenti-MECP2/GFP, anti-MECP2 immunoreactivity was detected in all the GFP+ cells (right, Figure 2A). Further quantification of MECP2 immunoreactivity in those cells revealed no difference from wild type astrocytes (Figure 2—figure supplement 1), suggesting the exogenous expression of MECP2 was not significant above the endogenous level. When Ca²⁺ activity was recorded (Figure 2B), the percentage of cells with spontaneous Ca²⁺ oscillation was significantly reduced by the expression of wild type MECP2, compared with expression of GFP alone, in the mutant human RTT astrocytes (20 ± 2% in MECP2/GFP vs. 36 ± 4% in GFP, n = 6 randomly selected fields in each group, p=0.006; Figure 2C). In the cells detected with spontaneous cytosolic Ca²⁺ oscillation, both the frequency (0.27 ± 0.06/min in MECP2/GFP vs. 0.59 ± 0.06/min in GFP, n = 21 cells in MECP2/GFP and n = 32 cells in GFP, p<0.001; Figure 2C) and amplitude (1.65 ± 0.08 in MECP2/GFP vs. 1.75 ± 0.03 in GFP, n = 62 events in MECP2/GFP and n = 209 events in GFP, p<0.001) were significantly reduced by the exogenous expression of wild type MECP2 as compared with expression of GFP alone. In addition, expression of wild type MECP2 significantly reduced the elevated level of pharmacologically evoked Ca²⁺ response in mutant human RTT astrocytes (3.90 ± 0.13 in MECP2/GFP vs. 4.20 ± 0.10 in GFP, n = 96 cells in MECP2/GFP and n = 123 cells in GFP, p=0.022; Figure 2D–E). Similar rescue effects were observed when the V247fs mutant astrocytes were used (Figure 2—figure supplement 2). Together, these results further support the cell autonomous nature of the abnormal Ca²⁺ activities in RTT mutant astrocytes.

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Since the phenotype of abnormal Ca²⁺ activities have not been reported in previous studies using RTT mouse models, we next examined whether they can be observed in Mecp2 null mouse astrocytes. Primary hippocampal astrocytes were isolated from newborn male Mecp2 null mice (Mecp2^-/y) and their wild type (WT) littermates, both of which also carried the Rosa-CAG-LSL-GCaMP6s allele (http://jaxmice.jax.org/strain/024106) and the hGFAP-creERT allele (http://www.jax.org/strain/012849). The astrocytes were treated with 4-OH tamoxifen for 24 hr to induce the expression of GCaMP6s, and then imaged for GCaMP6s fluorescence as the indication of cytosolic Ca²⁺ activities. Similar to our findings in human RTT astrocytes, spontaneous Ca²⁺ oscillation (Figure 3A–B) was detected in significantly higher percentage of Mecp2 null astrocytes than in wild type controls (67 ± 5% in Mecp2^-/y vs. 47 ± 6% in WT, n = 19 randomly chosen fields in both Mecp2^-/y and WT, p=0.01; Figure 3C). In cells that showed spontaneous Ca²⁺ oscillation, significantly higher frequency (2.01 ± 0.04/min in Mecp2^-/y vs. 1.64 ± 0.03/min in WT, n = 575 cells in Mecp2^-/y and n = 439 cells in WT, p<0.001; Figure 3C) and slightly higher amplitude (Table 2) were observed in the soma of Mecp2 null astrocytes than in wild type astrocytes. Since the processes were very short in primary mouse astrocyte culture, spontaneous Ca²⁺ activity in processes was not quantified separately because it closely resembled that in the soma. As for pharmacologically evoked Ca²⁺ response, significantly higher amplitude was detected in primary Mecp2 null mouse astrocytes (7.25 ± 0.23 in Mecp2^-/y vs. 6.54 ± 0.12 in WT, n = 153 cells in Mecp2^-/y and n = 278 cells in WT, p=0.04) than in wild type astrocytes (Figure 3D–E).

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Next, to explore the in situ relevance of this novel phenotype in Ca²⁺ dynamics, we studied spontaneous and agonist evoked Ca²⁺ activities in astrocytes in acute hippocampal slices prepared from 2 to 3 week-old Mecp2 null mice and their wild type littermates using Fluo4. In this series of experiments, SR101 was used to label astrocytes (Figure 4A); TTX (1 μM) was used to suppress spontaneous neuronal activity. Similar to our observation in cultured human patient-specific iPSC derived astrocytes and primary mouse astrocytes, higher frequency (0.59 ± 0.01/min in Mecp2^-/y vs. 0.47 ± 0.01/min in WT, n = 337 cells in Mecp2^-/y and n = 293 cells in WT, p<0.001) and slightly higher amplitude (Table 2) of spontaneous Ca²⁺ oscillation was observed in the soma of SR101+ cells in Mecp2 null slices than in wild type slices (Figure 4B–C). In addition, the astrocytes in Mecp2 null slices had significantly higher peak amplitude (2.75 ± 0.02 in MT vs. 2.22 ± 0.09 in WT, n = 754 cells in Mecp2^-/y and n = 238 cells in WT, p<0.001) of glutamate-induced Ca²⁺ rise than astrocytes in wild type slices (Figure 4D).

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To further rule out potential artifacts in cultured astrocytes and in acute slices, we examined spontaneous Ca²⁺ activities in astrocytes of the frontal cortex in live mice carrying the GCaMP6s sensor. We mated male Rosa-CAG-LSL-GCaMP6s mice with either female wild type (Mecp2^+/+) or Mecp2^flox/flox mice, injected adeno-associated virus (AAV) expressing mCherry and Cre recombinase under the human GFAP promoter (AAV8-hGFAP-mCherry-Cre) into the lateral ventricles of the postnatal day one male pups from these matings to activate the expression of GCaMP6s and delete Mecp2 predominantly in astrocytes. Based on the mating scheme, male pups would be of the genotype of either Mecp2^+/y;Rosa-CAG-LSL-GCaMP6s (WT) or Mecp2^flox/y; Rosa-CAG-LSL-GCaMP6s (floxed). When immunostaining with MeCP2 antibody on sections prepared from 3 months old floxed mice that receivedAAV8-hGFAP-mCherry-Cre injection at birth, 100% of the GCaMP6s (anti-GFP) positive cells in the cortex were MeCP2 negative. In contrast, GCaMP6s positive cells from WT mice were MeCP2 positive (Figure 5—figure supplement 1). When GCaMP6s fluorescence were recorded in mCherry-positive cells in the frontal cortex of 2–3 months-old live WT and floxed mice injected with AAV8-hGFAP-mCherry-Cre (MT) (Video 3–4, Figure 5A–B), higher frequency of spontaneous oscillation of GCaMP6s fluorescence was detected in both the soma (2.15 ± 0.11/min in MT vs. 1.44 ± 0.20/min in WT, n = 57 cells in MT and n = 18 cells in WT, p=0.003, Figure 5C) and processes (2.14 ± 0.04/min in MT vs. 1.69 ± 0.06/min in WT, n = 373 processes in MT and n = 110 processes in WT, p<0.001, Figure 5—figure supplement 2) of MT astrocytes. However, the amplitude of spontaneous astrocytic Ca²⁺ activity appeared to be slightly smaller in MT mice (Table 2). Taken together, the phenotype of increased frequency of spontaneous cytosolic Ca²⁺ activity was found consistently across species, as well as in vitro, in situ, and in vivo.

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To reveal the cellular and molecular mechanisms underlying the abnormal Ca²⁺ activities in the RTT astrocytes, we first examined the source of Ca²⁺. Since the abnormal spontaneous cytosolic Ca²⁺ oscillation in mutant human astrocytes persisted after Ca²⁺ was removed completely from extracellular environment (0 mM Ca²⁺ plus 2 mM EGTA, Figure 6—figure supplement 1), we measured the Ca²⁺ load in the endoplasmic reticulum (ER), the major source of intracellular Ca²⁺ storage. Treating astrocytes with thapsigargin (TG, 1 μM), an irreversible inhibitor of sarco-ER Ca²⁺ ATPase, induced a slow elevation of Ca²⁺ in the cytosol, which was mediated by the leakage of Ca²⁺ from the ER. The amplitude of the TG-induced Ca²⁺ elevation was significantly higher in the mutant human RTT astrocytes than in their congenic controls (125 ± 4%. in MT vs. 100 ± 3% in WT, n = 156 cells in MT and n = 178 cells in WT, p<0.001, Figure 6A), suggesting the mutant astrocytes had more releasable Ca²⁺ in the ER. Moreover, when the kinetics of this Ca²⁺ rise was analyzed, the leakage of Ca²⁺ from the ER in the mutant astrocytes was much faster (time constant 54 ± 1 s in MT vs. 59 ± 1 s in WT, n = 156 cells in MT and n = 178 cells in WT, p<0.001, Figure 6B–C).

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To accurately compare the baseline cytosolic Ca²⁺ level between Mecp2 mutant and wild type astrocytes, we prepared primary mouse astrocytes from Mecp2^+/- mice that carry an X chromosome-linked GFP transgene. The GFP transgene is on the same X chromosome as the wild type Mecp2 gene. Due to random X chromosome inactivation, a mixture of GFP+ and GFP- astrocytes will be present in these cultures, with the GFP+ cells expressing the wild type MeCP2 and the GFP- cells expressing no MeCP2. When the red-colored calcium-sensitive dye Rhod-2 was used to image baseline cytosolic Ca²⁺ level in the Mecp2 null (GFP-) and wild type (GFP+) astrocytes in the same culture (Figure 6D), significantly higher level of Rhod-2 fluorescence was detected in the Mecp2 null cells (154 ± 7% in GFP- vs. 100 ± 5% in GFP+, n = 30 in each group, p<0.0001; Figure 6E). As a control, astrocyte cultures were also prepared from Mecp2^+/+ female mice that carry the same X chromosome-linked GFP transgene. No significant deference was detected in baseline cytosolic Ca²⁺ level between the GFP+ and GFP- cells (107 ± 8% in GFP- vs. 100 ± 6% in GFP+, n = 31 in GFP- and n = 32 in GFP+, p=0.46; Figure 6F). These results reveal Ca²⁺ overload in the ER, the faster Ca²⁺ leak rate, and the elevated baseline cytosolic Ca²⁺ level as significant contributors to the abnormal spontaneous and pharmacologically evoked cytosolic Ca²⁺ activities in the mutant RTT astrocytes.

To further define the cellular events upstream of the abnormal Ca²⁺ homeostasis, we measured Ca²⁺ influx through the store operated calcium entry (SOCE) pathway, which is the primary pathway for reloading Ca²⁺ into the ER in astrocytes. After depleting Ca²⁺ from the ER by treating astrocytes with TG in the absence of extracellular Ca²⁺ (0 mM Ca²⁺ plus 2 mM EGTA), we switched bath solution to that containing 2 mM Ca²⁺ and measured Ca²⁺ influx. Comparing to that in the congenic control, the amplitude of Ca²⁺ influx was significantly higher in the mutant human RTT astrocytes (2.93 ± 0.07 in MT vs. 2.41 ± 0.06 in WT, n = 156 cells in MT and n = 178 cells in WT, p<0.001; Figure 6G), suggesting an abnormal increase of SOCE in the mutant cells.

In search of the molecular mechanism underlying the increased SOCE in mutant astrocyte, we performed RNA-seq and microarray experiments to compare gene transcription profiles in the mutant astrocytes and their congenic controls. Highly relevant to the observed SOCE phenotype, the expression of the transient receptor potential cation channel, subfamily C, member 4 (TRPC4), which has been previously shown to regulate SOCE in lung vascular endothelial cells (Tiruppathi et al., 2002), was found to be significantly higher in the mutant human RTT astrocytes (~27 fold increase by RNA-seq,~14 fold increase by microarray). Western blot analysis using protein lysates from mutant human RTT astrocytes and their congenic controls also confirmed the increased expression of TRPC4 (Figure 6H–I). Immunostaining with TrpC4 antibody also detected significantly higher immunoreactivity in GFAP positive astrocytes in hippocampal sections from the Mecp2 null mouse brains as compared with that in wild type mouse brains (Figure 6—figure supplement 2). Chromatin immunoprecipitation analysis revealed significant MeCP2 occupancy at the promoter of Trpc4 (Figure 6—figure supplement 3), suggesting direct regulation of Trpc4 transcription by MeCP2. To corroborate the expression level changes at the RNA and protein level for TRPC4, we performed whole cell patch clamp recording on the astrocytes to directly assess the current mediated by TRPC4 using a TRPC4-selective antagonist, ML204 (Miller et al., 2011). Significantly higher density of ML204 sensitive inward current (at −70 mV) was detected in the R294X mutant human RTT astrocytes (−0.93 ± 0.01 pA/pF in MT vs. −0.62 ± 0.08 pA/pF in WT, n = 14 cells in MT and n = 15 cells in WT, p=0.01; Figure 6J–K). Moreover, this current was significantly reduced when mutant human RTT astrocytes were infected with a lentivirus expressing an shRNA specific against TRPC4 (−0.61 ± 0.10 pA/pF in MT infected with lenti-GFP-shTRPC4 vs. −1.04 ± 0.05 pA/pF in MT infected with lenti-GFP, n = 9 cells in each condition, p=0.006, Figure 6L–M), suggesting the current is dependent on TRPC4. The I-V curve of the ML204-sensitive current can be found in Figure 6—figure supplement 4. Finally, the phenotypes of increased Ca²⁺ overload in the ER, faster Ca²⁺ leak from the ER, increased SOCE, and increased level of TrpC4 were also observed in primary astrocytes isolated from Mecp2 null mice (Figure 6—figure supplement 5).

To determine the contribution of increased TRPC4 expression to the series of Ca²⁺ related phenotypes in the Mecp2 null astrocytes, we treated these cells with ML204 for 24 hr, and then measured SOCE-mediated Ca²⁺ elevation, Ca²⁺ load in the ER, and recorded spontaneous cytosolic Ca²⁺ activities. Comparing to untreated Mecp2 null astrocytes, ML204-treated cells showed significantly reduced amplitude of SOCE mediated Ca²⁺ elevation (3.15 ± 0.12 in ML204-treated group vs. 6.61 ± 0.86 in untreated, n = 18 in ML204-treated and n = 16 in untreated, p=0.0002; Figure 7A) and lower ER Ca²⁺ load (1.86 ± 0.06 in ML204-treated vs. 2.47 ± 0.24 in untreated, n = 14 in ML204-treated and n = 15 in untreated, p=0.02; Figure 7B). More importantly, ML204 treatment significantly reduced the percentage of Mecp2 null astrocytes showing spontaneous cytosolic Ca²⁺ oscillation (23 ± 3% in ML204-treated vs. 46 ± 7% in untreated, n = 9 randomly chosen fields in ML204-treated and n = 8 randomly chosen fields in untreated, p=0.03; Figure 7C) and the frequency (0.20 ± 0.01/min in ML204-treated vs. 0.31 ± 0.01/min in untreated, n = 91 cells in ML204-treated and n = 203 cells in untreated, p<0.001; Figure 7C) and amplitude (1.64 ± 0.02 in ML204-treated vs. 1.97 ± 0.02 in untreated, n = 168 events in ML204-treated and n = 597 events in untreated, p<0.001; Figure 7C) of such oscillations.

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In addition to pharmacological manipulation, we infected mutant RTT human astrocytes with lentivirus expressing a shRNA specific against TRPC4 to reduce its level (Figure 7D–E). 72 hr after the infection, we measured SOCE-mediated Ca²⁺ elevation, ER Ca²⁺ load, and recorded spontaneous cytosolic Ca²⁺ activities. Comparing to those infected with lentivirus expressing GFP alone, the mutant astrocytes infected with lentivirus expressing shTRPC4 showed significantly reduced SOCE-mediated Ca²⁺ elevations (1.72 ± 0.05 in MT/Lenti-ShTRPC4-GFP vs. 2.02 ± 0.04 in MT/Lenti-GFP, n = 23 in MT/Lenti-shTRPC4 and n = 26 in MT/Lenti-GFP, p<0.0001; Figure 7F), lower ER Ca²⁺ load (2.37 ± 0.16 in MT/Lenti-shTRPC4-GFP vs. 2.95 ± 0.29 in MT/Lenti-GFP, n = 23 in MT/Lenti-shTRPC4 and n = 26 in MT/Lenti-GFP, p=0.04; Figure 7G), and lower frequency (0.42 ± 0.03/min in MT/Lenti-shTRPC4-GFP vs. 0.63 ± 0.03 in MT/Lenti-GFP, n = 86 cells in MT/Lenti-shTRPC4 and n = 79 cells in MT/Lenti-GFP, p<0.001, Figure 7H) of the spontaneous Ca²⁺ elevations. Together, the pharmacological and molecular manipulations demonstrated that elevated TRPC4 expression is required for the abnormal calcium homeostasis in MeCP2 deficient astrocytes.

Finally, we overexpressed TRPC4 in wild type human astrocytes (Figure 7I), and observed increased SOCE-mediated Ca²⁺ elevations (2.76. ± 0.15 in WT/GFP-TRPC4 vs. 2.36 ± 0.08 in WT/GFP, n = 52 in WT/GFP-TRPC4 and n = 55 in WT/GFP, p=0.02; Figure 7J), higher ER Ca²⁺ load (3.65 ± 0.23 in WT/GFP-TRPC4 vs. 2.62 ± 0.13 in WT/GFP, n = 52 in WT/GFP-TRPC4 and n = 55 in WT/GFP, p=0.0001; Figure 7K), more cells showing spontaneous Ca²⁺ oscillation (29 ± 5% in WT/GFP-TRPC4 vs. 16 ± 3% in WT/GFP, n = 13 in WT/GFP-TRPC4 and n = 12 in WT/GFP, p=0.03; Figure 7L), higher frequency (0.71 ± 0.04/min in WT/GFP-TRPC4 vs. 0.45 ± 0.01/min in WT/GFP, n = 74 cells in WT/GFP-TRPC4 and n = 100 cells in WT/GFP, p<0.001; Figure 7L) and amplitude (1.49 ± 0.04 in WT/GFP-TRPC4 vs. 1.22 ± 0.01 in WT/GFP, n = 523 events in WT/GFP-TRPC4 and n = 445 events in WT/GFP, p<0.001; Figure 7L) of spontaneous Ca²⁺ oscillations in those cells. These results strongly suggest that elevated TRPC4 expression is sufficient to cause abnormal calcium homeostasis in wild type astrocytes.

Previous work has shown that Ca²⁺ oscillation in astrocytes could lead to activation of eNMDARs (Fellin et al., 2004). We measured activation of eNMDAR on mouse hippocampal pyramidal neurons by whole cell patch clamp recording, and detected a significantly higher frequency of a slow inward current (SIC, Figure 8A, left) in acute hippocampal slices prepared from 2 to 3 weeks old Mecp2 null mice than those from the wild type littermate control (3.88 ± 0.46/20 min in Mecp2 null vs. 1.50 ± 0.39/20 min in WT, n = 16 in Mecp2 null and n = 12 in WT, p=0.001; Figure 8A, right). The SICs were sensitive to both D-APV (selective NMDA receptor antagonist, 50 µM, Figure 8B, left) and ifenprodil (selective NR2B-containing NMDA receptor antagonist, 10 µM, Figure 8B, right), suggesting they were mediated by NR2B-containing eNMDARs. Similar increase in SIC frequency was also detected in acute hippocampal slices prepared from 6 weeks old symptomatic Mecp2 null mice (Figure 8—figure supplement 1). Although similar phenotype was recently reported (Lo et al., 2016), the underlying cause of such increased activation of eNMDAR is not well understood.

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To determine the role of abnormal Ca²⁺ activities in the astrocytes in the increased frequency of SICs, membrane-impermeable BAPTA, along with a small dye that can pass through gap junctions, was injected into a single astrocyte in acute hippocampal slices prepared from the Mecp2 null mice. Thirty minutes after injection, many astrocytes became positive for the dye in the field of injection, indicating diffusion of the dye and BAPTA into astrocytes connected through gap junctions. Whole cell patch clamp recording on hippocampal pyramidal neurons within the field of these dye-labeled astrocytes (i.e. with the intracellular chelator BAPTA) revealed a significant reduction in the frequency of SIC (2.08 ± 0.51/20 min in Mecp2 null with BAPTA infusion into a single astrocyte vs. 5.33 ± 1.04/20 min in Mecp2 null with no treatment, n = 12 in each group, p=0.01; Figure 8C), suggesting that the abnormally high calcium activity in astrocytes is required for the excessive activation of eNMDARs on neighboring neurons.

To further determine the relationship between these two phenotypes, we examined calcium activities and activation of eNMDARs in astrocyte-specific and neuron-specific Mecp2 knockout mice. To generate cell type-specific Mecp2 knockout mice, we mated wild type males with Mecp2^flox/flox females to generate Mecp2^flox/y male pups, and injected AAV viruses (AAV8-hGFAP-mCherry-Cre, AAV8-Syn-mCherry-Cre, or AAV-mCherry) into the lateral ventricles of postnatal-day-1 old Mecp2^flox/y male pups. Consistent with previous report (Kim et al., 2013), mCherry-positive cells showed widespread presence in the forebrain (including cortex and hippocampus), suggesting robust distribution of AAV in this method. Immunohistological analysis of sections from Mecp2^flox/y male mice injected with AAV8-hGFAP-mCherry-Cre showed that 100% of mCherry-positive cells were positive for GFAP (right, Figure 8D, Table 3) and negative for MeCP2 (Figure 8—figure supplement 2), and that ~ 27% of GFAP-positive cells were positive for mCherry (Table 3), suggesting successful deletion of Mecp2 in about one third of the astrocytes in the forebrain. Similar analysis of sections from Mecp2^flox/y male mice injected with AAV8-Syn-mCherry-Cre showed that 100% of mCherry-positive cells were positive for NeuN (left, Figure 8D, Table 3) and negative for MeCP2 (Figure 8—figure supplement 2), and that ~ 74% of NeuN-positive cells were positive for mCherry (Table 3), suggesting successful deletion of Mecp2 in about more than two thirds of the neurons in the forebrain. Further double staining with either anti-GFAP and anti-MeCP2 antibodies or anti-NeuN and anti-MeCP2 antibodies on those brain sections (Figure 8—figure supplement 2) revealed that, while almost all mCherry-positive cells were indeed negative for MeCP2 in mice receiving either AAV virus, there was a negligible number (2%) of NeuN-positive cells that were negative for MeCP2 in the hippocampus of the AAV-GFAP-mCherry-Cre injected mice, and a small number (7%) of GFAP-positive cells that were negative for MeCP2 in the hippocampus of the AAV-hSyn-mCherry-Cre injected mice (Table 4). Thus, mice with predominantly astrocyte-specific deletion of Mecp2 and predominantly neuron-specific deletion of Mecp2 were generated. Mecp2^flox/y male mice injected with AAV-mCherry (no Cre recombinase) were used as wild type control for this series of experiments.

Abnormal spontaneous calcium activity in astrocytes (Figure 8E, n = 34 in AAV-mCherry group, n = 36 in AAV-Syn-mCherry-Cre group, and n = 47 in AAV-hGFAP-mCherry-Cre group; ANOVA: F = 14.11, p<0.0001) and excessive activation of extrasynaptic NMDA receptors in neighboring neurons (Figure 8F, n = 38 in AAV-mCherry group, n = 21 in AAV-Syn-mCherry-Cre group, and n = 30 in AAV-hGFAP-mCherry-Cre group; ANOVA: F = 4.23, p<0.018) were only observed in predominantly astrocyte-specific, but not in predominantly neuron-specific, Mecp2 knockout hippocampal slices. Results from this series of genetic manipulations strongly suggest that the abnormal calcium activities in astrocytes are caused by the loss of MeCP2 function in astrocytes, and can lead to excessive activation of extrasynaptic NMDA receptors on neighboring neurons (even when these neighboring neurons are wild type).

Beyond eNMDAR activation, several previous studies have suggested that increased astrocytic Ca²⁺ activity may lead to a hyperexcitable network (Gómez-Gonzalo et al., 2010; Kuchibhotla et al., 2009; Tian et al., 2005; Wetherington et al., 2008). Such a hypothesis is highly relevant to RTT, because 80–90% of RTT patients have seizures (Jian et al., 2007), and spontaneous seizures have also been reported in RTT mice (D'Cruz et al., 2010). As the first step to explore a role for abnormal astrocytic Ca²⁺ activity in leading to seizure in RTT, we adopted a widely used slice model of seizure (or epileptiform activity or network hyper-excitability) by adding the GABA_A receptor antagonist biccucullin and increasing bath Ca²⁺ concentration (Borck and Jefferys, 1999; McLeod et al., 2013; Ratté et al., 2011; Tian et al., 2005).

In this model, whole cell patch clamp recording in CA1 pyramidal neurons readily detected epileptiform activity in slices from 5 to 8 week-old wild type and Mecp2^-/y mice (Figure 8G). Comparing with wild type, epileptiform activity in Mecp2^-/y neurons had shorter latency (4.5 ± 0.4 min in Mecp2^-/y vs. 6.5 ± 0.6 min in wild type, Figure 8H), higher frequency (5.6 ± 0.4 Hz in Mecp2^-/y vs. 3.5 ± 0.5 Hz in wild type, Figure 8H), longer duration (755 ± 30 ms in Mecp2^-/y vs. 621 ± 39 ms in wild type, Figure 8H), and higher amplitude (364 ± 15 pA in Mecp2^-/y vs. 279 ± 28 pA in wild type. Figure 8H). More importantly, intracellular infusion of membrane impermeable BAPTA into a single astrocyte significantly reduced epileptiform activity in neighboring neurons (latency: 4.5 ± 0.4 min in Mecp2^-/y vs. 5.7 ± 0.4 min in Mecp2^-/y with BAPTA infusion; frequency: 5.6 ± 0.4 Hz in Mecp2^-/y vs. 3.8 ± 0.5 Hz in Mecp2^-/y with BAPTA infusion; duration: 755 ± 30 ms in Mecp2^-/y vs. 633 ± 31 ms in Mecp2^-/y with BAPTA infusion. Figure 8H). 7–10 neurons from 3 to 4 mice were analyzed from each genotype/treatment in this series of experiments. Similar results were obtained recording field potentials under the same conditions described in Figure 8G–H (data not shown). Together, these data reveal a potential link between increased astrocytic Ca²⁺ activity and increased network excitability. Since such increased network excitability may underlie seizure-a specific RTT symptom, these data further highlight the functional significance of abnormal Ca²⁺ activity in RTT.

This study used congenic pairs of wild type and mutant Rett syndrome patient specific induced pluripotent stem cell lines carrying the R294X and the V247fs mutations. The cell lines were routinely tested to ensure that the cell lines were of human origin, were not contaminated with other cell lines, and were not contaminated with other microbial species including bacteria, fungi, or mycoplasma. Human identity testing was performed using Short Tandem Repeat (STR) testing. This test method is capable of providing a unique genetic fingerprint that is traceable back to a single human donor. In addition, STR is capable of detecting other contaminating cell types, either other human cell lines or other species, at a very low level. Isoenzyme analysis could also be performed to verify species of origin for cell lines. Testing was also routinely performed to test for microbial and fungal contamination using test methods that are typically used for testing clinical-grade cell lines. In addition, testing for mycoplasma contamination was be performed using either MycoAlert (Lonza), qPCR testing, or direct/indirect culture methods. These test methods provided a very high level of assurance that cell lines were not contaminated with other mammalian, bacterial, fungal, or mycoplasma cell lines.

In addition, for congenic pairs of iPSC lines derived from the same human RTT patient, the X chromosome inactivation status and the allelic expression of MECP2 were closely monitored to ensure correct genotype identification. These methods have been described in detail in our previous studies (Ananiev et al., 2011; Williams et al., 2014).

Human astrocytes were differentiated from astroglial progenitors as previously described (Williams et al., 2014). Briefly, Terminal differentiation into astrocytes from astroglial progenitors was achieved by dissociating progenitor spheres into single cells with Accutase, and plating on coverslips pre-coated Poly-Lysine at a density of 2 × 10⁵ cells per ml in astrocytes differentiation media: DMEM/F12 with 1% N2, 1 × NEAA, 1 × pen/strep and 2 µg/ml heparin (Sigma H3149), and CNTF (BioSensis). To facilitate cells attachment, 10% FBS (Gibco) was added. The next day, the media was replaced by fresh ADM without FBS. Differentiated progenitors were then fed every other day and used for experimentation 7 days after the start of terminal differentiation. To improve cell attachment, in some experiments, dissociated astroglial progenitor cells were plated on Matrigel-coated plates. The medium used for plating was DMEM/F12 with 1% N2, 1 × NEAA, 1 × pen/strep and 2 µg/ml heparin, supplemented with 10 ng/ml BMP4, LIF, CNTF. The medium was changed once on day 3. Astrocytes were used for experiments 7 days after the start of terminal differentiation. Stereological analysis was performed to determine the percentage of GFAP positive cells in each differentiation. Astrocyte cultures with higher than 85% GFAP positive cells were used for subsequent experiments.

Mouse astrocyte cultures were prepared from postnatal day 0 (P0) male Mecp2 null and wild type littermate mice, as well as P0 female Mecp2^+/- and Mecp2^+/+ mice that carry an X chromosome-linked GFP transgene. After the brain of mice were removed and placed into ice-cold Hank's Balanced Salt Solution (HBSS), hippocampi were dissected and treated with 0.25% trypsin at 37°C for 30 min. Digestion was stopped by fetal bovine serum (FBS). Then the tissue was mechanically dissociated through a small fire-polished Pasteur pipette. After centrifuge, cells were re-suspended with DMEM, supplemented with penicillin/streptomycin and 10% FBS. Cells were plated into T-25 flasks and allowed to reach confluence. Contaminating neurons and microglia were then dislodged by shaking. The remaining adherent astrocytes were trypsinized, re-suspended in DMEM, and plated onto poly-D-lysine-coated coverslips at about 20,000 cells/ml. Cells were fed every 4 days by replacing the medium with fresh medium. The cells were used after 4 weeks in vitro. Stereological analysis was performed to determine the percentage of GFAP positive cells in each culture. Astrocyte cultures with higher than 90% GFAP positive cells were used for subsequent experiments.

For the human iPSC derived astrocytes and some mouse primary astrocytes, intracellular Ca²⁺ was indicated by Fluo-4 or Rhod-2. Cells were bulk-loaded for 15 min at 37°C in artificial cerebrospinal fluid (aCSF) containing Fluo-4/AM (12.5 μg/ml), pluronic acid (0.05%), and DMSO (0.1%). After the Ca²⁺ indicator was loaded, cells were transferred to a chamber and Ca²⁺ imaging was performed. Mouse primary astrocytes from GCaMP6s mice were treated with 4-OH tamoxifen (0.5 μM, Sigma-Aldrich Inc.) for 24 hr to induce the expression of GCaMP6s. Cells were imaged using a Nikon A1 confocal microscope at room temperature. All image data were taken in the frame-scanning mode at 1 frame every 2 s (in the experiments using GCaMP6s as the Ca²⁺ indicator, 1 frame every 1 s). Fluo-4 or GCaMP6s was excited at 488 nm. The Ca²⁺ imaging data was analyzed using custom-written programs in Python. The metadata and the image data of the raw images were read with python-bioformats. Ca²⁺ signals were presented as relative fluorescence changes (ΔF/F₀) from specified regions of interest (ROIs). ROIs were selected using an automated segmentation algorithm (Cai et al., 2016), while somata and processes were manually identified on the basis of morphology. For the traces with baseline drift, baseline correction was performed using a ‘rolling-ball’ algorithm. The peaks were detected using the algorithm developed by Matlab (findpeaks function). The frequency, amplitude and the full-width at half maximum (FWHM) were calculated and measured. Images with obvious motion were excluded for analysis. In experiments that examined spontaneous Ca²⁺ oscillations, the Ca²⁺ level was reported as F/F₀ = F_t/F₀. Calcium elevation events were detected with thresholds of 3 times of standard deviation of the baseline. The source code for Ca²⁺ events detection and automated segmentation is available in the supplementary files. Rhod-2 was used in the experiments involving viral expression of exogenous wild type MECP2, TRPC4, or shRNA against TRPC4. For the spontaneous astrocytic Ca²⁺ activity imaging, aCSF containing (in mM): 120 NaCl, 3 KCl, 15 HEPES, 1 MgCl₂, 2 CaCl₂, 20 Glucose (pH7.4) was used. For TG-induced ER Ca²⁺ release, aCSF with 0 mM Ca²⁺ and 2 mM EGTA was used, and TG was perfused to induce the astrocytic Ca²⁺ elevations. For SOCE, 2 mM Ca²⁺ containing aCSF was perfused after TG induced ER Ca²⁺ depletion. The spontaneous Ca²⁺ elevations from the soma and process of astrocytes were analyzed separately. For the analysis of Ca²⁺ imaging data in acute mouse brain slices, only Ca²⁺ elevations in soma and major process of astrocytes were included.

GCaMP6s mice (Ai96, 024106) were purchased from Jackson Laboratory. GCaMP6s and GCaMP6s;MeCP2^flox/y mouse pups were injected with AAV-GFAP-Cre-mCherry (see Materials and methods/Adeno associated virus (AAV) injection into neonatal mice). Mice were used for Ca²⁺ imaging after they are 2 months old. A cranial window (3 mm x 3 mm) was installed over the M2 frontal cortex on the left hemisphere of mutant and control mice (center coordinates: 1.5 mm anterior to Bregma, 1.0 mm lateral to midline). Mice were anesthetized with isoflurane during surgery and recovered to full awakeness (>45 min post surgery) before imaging layer I astrocyte calcium signals under the 25x water immersion lens (NA = 1.05) of a multi-photon microscope (FV1000MPE, Olympus) (Cao et al., 2013; Managò et al., 2016). The excitation wavelength of the two-photon laser was set at 900 nm, and the power of the laser emitted from the objective was set at 80 mW or lower. Image stacks had dimensions of 699 × 167 (width x length in µm) and the frame rate (frames per second) is 1.83. Three to five image recordings (655 s each) were made from each animal. The analysis of the in vivo Ca²⁺ imaging data is described in Materials and methods/In vitro Calcium imaging. The traces were low-pass filtered (cut-off frequency: 0.5 Hz) before analysis to remove noises.

Human or mouse astrocytes were infected with lentivirus encoding either GFP alone or GFP and wild type MECP2. 36 hr later, astrocytic Ca²⁺ imaging was performed. Appropriate MOI was used to achieve infection rates higher than 90% in human astrocytes and ~50% in mouse astrocytes based on the percentage of GFP positive cells.

Human astrocytes were infected with lentivirus encoding either GFP alone or GFP and TRPC4. 10 days later, astrocytic Ca²⁺ imaging was performed. The infection rate was higher than 80%, according to the percentage of GFP positive cells.

For immunohistological evaluation on brain sections, mice were killed by barbiturate overdose and perfused transcardially with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA, in PBS). Brains were removed and post-fixed overnight. Then the brains were cryoprotected in buffered 30% sucrose (wt/vol) for at least 2 d. 25 μm coronal frozen sections were prepared using a cryostat microtome. For immunocytochemistry, cultures were fixed on ice with 4% PFA for 30 min. Immunostaining was performed as previously described (Li et al., 2011). Briefly, cultures were fixed on ice with 4% PFA for 30 min. After washed three times with PBS, cultures or sections were permeablized with 1% Triton X100 (Sigma, in PBS) for 30 min, blocked with 3% normal donkey serum and 0.25% Triton X100 in PBS for 90 min at room temperature (RT), and then incubated with primary antibodies overnight at 4°C. Then the cultures were incubated with the corresponding secondary antibodies for 60 min at RT, and washed five times with PBS at RT. Primary antibody dilutions were as follows: anti-GFAP (Millipore MAB3402 RRID:AB_94844, 1:500; and Dako, Z0334 RRID:AB_10013382, 1:500), anti-MeCP2 (Abcam ab50005 RRID:AB_881466, 1:500 and ab2828 RRID:AB_2143853, 1:500; and Cell Signaling Technology Cat# 3456S, RRID:AB_10828482, 1:500), anti-NeuN (Millipore MAB377 RRID:AB_2298772, 1:100), anti-S100β (Abcam Cat# ab868, RRID:AB_306716, 1:500) anti-TRPC4 (Alomone labs ACC-018 RRID:AB_2040239, 1:100; and Abcam ab84813 RRID:AB_1860087, 1:500). Secondary antibody dilutions were as follows: Alexa Fluor 568 Donkey-anti-Rabbit antibody (Thermo Fisher Scientific A10042 RRID:AB_2534017, 1:500), Alexa Fluor 488 Donkey-anti-mouse antibody (Thermo Fisher Scientific A21202 RRID:AB_141607, 1:500), and Alexa Fluor 647 Donkey-anti-mouse antibody (Thermo Fisher Scientific A-31571 RRID:AB_162542, 1:500). DAPI was used at 3 nM for counterstaining. Images were taken using a Nikon A1 confocal microscope.

Astrocytes were collected into an Eppendorf tube and directly lysed by adding 1X LDS buffer (Life technology). Samples were sonicated to facilitate cell lysis and boiled for 5 min before loading into 10% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membrane (Whatman) using a semi-dry transfer system from BioRad. Membrane was first blocked with 5% milk solution for 1 hr and then incubated with anti-TRPC4 (Alomone labs, ACC-018 RRID:AB_2040239, 1:100) diluted in 3% BSA solution at 4°C overnight. After incubating with infrared dye-conjugated secondary antibody (Thermo Fisher Scientific Cat# 35518 RRID:AB_614942; # SA5-35571 RRID:AB_2556775;1:10,000) for 1 hr at room temperature, membrane was scanned in an Odyssey infrared imaging system.

Total RNA was extracted from R294X-WT and R294X-MT iPSC-derived astrocytes using Trizol (life technology) and cleaned up with on-column DNase treatment (Qiagen). 150 ng total RNA was used to prepare sequencing library in a Mondrian SP +Workstation according to manufacturer’s instructions (Nugen Encore SP +Complete). 100 bp single-end reads sequencing for each library was performed in an Illumina Hi-Seq 2000. Reads were mapped to the human genome build hg19 using Tophat (2.0.8) and differential gene expression analysis was done using Cuffdiff (2.0.2).

Purified RNA was sent to Affymetrix for microarray analysis. HTA-2.0 chip was used in this project. Sample processing, dye labeling, hybridization, and scanning were performed by Affymetrix. Each sample was hybridized to four chips. Spike-in RNA was supplemented to samples and used as a normalization control.

Primary astrocyte culture was prepared from the forebrain of the Mecp2-Flag mice (Li et al., 2011) and control mice. ChIP was performed as previously described (Li et al., 2011). qPCR was done using a StepOne plus (Life technology). The following primers were used in the qPCR. TRPC4-P-1 forward: 5′-GCAGAGTGAGCCTGAGTCTA-3′. TRPC4-P-1 reverse: 5′-CGTGATCTCAAGACCAAGGG −3′. TRPC4-P-2 forward: 5′-TAGTATGGTTGGAGCAGGGC-3′. TRPC4-P-2 reverse: 5′-AGCTAAGTGGTGGTCAGGAC-3′. TRPC4-P-3 forward: 5′-CACCTTGGGAACGCAACTTT-3′. TRPC4-P-3 reverse: 5′-AAAACCCGCACGAAACCAG-3′. TRPC4-P-4 forward: 5′-CCCCATCGGAACTGACCA-3′. TRPC4-P-4 reverse: 5′-AGTATCCCAGATGTGAGGCC-3′.

The hTRPC4 shRNA sequence used in our study is identical to that used in a previous study (Zagranichnaya et al., 2005). The shRNA sequence was cloned into a lentiviral construct, pLentilox3.7. Lentiviral particles were prepared as previously described (Ananiev et al., 2011). Mutant human astrocytes were infected with lentivirus encoding either GFP alone or GFP and shTRPC4. 36 hr later, astrocytic Ca²⁺ imaging was performed. Appropriate MOI was used to achieve an infection rate of higher than 90% based on the percentage of GFP positive cells.

Experiments described below were performed according to protocols approved by the IACUC at University of Wisconsin-Madison. Neonatal Mecp2^flox/y mice were anesthesized by hypothermia before the injection. The AAV solution was injected into the lateral ventricles using a 5 µl Hamilton syringe. The injection site was located three-fifths in the line defined between the lambda intersection of the skull and each eye. The needle was held perpendicular to the skull surface and inserted to a depth of ~3 mm. Then 1 µl of viral solution was injected into the ventricle. After injection, mice were transferred to a heated pad with perfused water at 38°C until they regained normal color and resumed movement. Three types of AAV were injected in this series of experiments: AAV8-hGFAP-mCherry-Cre (expression of fluorescence marker mCherry and the Cre recombinase under the astrocyte-specific hGFAP promoter), AAV8-Syn-mCherry-Cre (expression of mCherry and Cre under the neuron-specific human synapsin1 promoter), and AAV-mCherry. Stereological studies were performed to examine the infection rate of the AAV and the cell type specificity of mCherry expression and Cre-mediated deletion of MeCP2. Brain sections were immunostained with either anti-GFAP or anti-NeuN antibody and counterstained with DAPI; or combination of anti-GFAP and anti-MeCP2 antibodies; or combinations of anti-NeuN and anti-MeCP2 antibodies. Sections were viewed with a Zeiss photomicroscope at 20X. Stereology was performed with the StereoInvestigator software (MicroBrightField). The optimal number of counting sites was determined empirically based upon a Scheaffer CE value less than 0.30.

Male mice at postnatal 2–3 weeks were anesthetized and coronal brain slices (400 μm) were prepared in ice-cold modified artificial cerebrospinal fluid (aCSF) (in mM: 124 NaCl, 2.5 KCl, 0.5 CaCl₂, 5 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 15 glucose) bubbled with 95%O₂/5%CO₂. Then the slices were incubated in normal aCSF (in mM: 124 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 15 glucose) at room temperature for at least 1 hr. After incubation, the slices were bulk-loaded for 1 hr at 37°C in aCSF containing Fluo-4/AM (12.5 μg/ml), pluronic acid (0.05%), and DMSO (0.1%) saturated with 95%O₂/5%CO₂. Sulforhodamine 101 (SR101) was co-loaded as an astrocyte marker. Then slices were transferred to a chamber perfused with 95%O₂/5%CO₂ saturated aCSF. Slices were imaged using Nikon A1 confocal microscope. Fluo-4 and SR101 were excited at 488 nm and 561 nm respectively. The data acquisition protocol was the same as that used in cultured cells. The person performing the Ca²⁺ imaging experiments and the subsequent data analysis was blind to the genotypes of mice used in these experiments.

Slices were transferred to a recording chamber perfused with 95%O₂/5%CO₂ saturated aCSF. To record the extrasynaptic NMDA receptor current, hippocampal CA1 neurons were clamped at − 30 mV to relieve Mg²⁺ block. We adjusted the equilibrium potential of Cl⁻ to −30 mV by controlling the [Cl⁻] in the pipette solution. QX-314 was added to block Na⁺ channels. The intracellular solution was (in mM): 95 Cs-methanesulfonate, 34 CsCl, 10 HEPES, 10 QX-314, 2.5 MgCl2, 1 CaCl₂, 5 NaCl, and 10 EGTA, pH 7.2. To selectively inhibit the astrocytic Ca²⁺ activity, membrane-impermeable BAPTA (10 mM) was injected into the CA1 stratum radiatum astrocytes by patch clamp. To avoid a leakage of BAPTA from the pipette before the formation of whole cell patch clamp, the BAPTA solution was backfilled with the standard intracellular solution. The distinctive electrophysiological characteristics such as a linear I/V relationship was obtained to confirm the type of cells being patched.

To record store operated Ca²⁺ influx in astrocytes, we transferred the coverslip into a chamber fixed in an Olympus BX51WI upright microscope. The recording chamber was perfused with aCSF containing (in mM): 120 NaCl, 3 CsCl, 15 HEPES, 1 MgCl2, 2 CaCl2, 20 Glucose (pH7.4). The pipette solution contained (in mM): 130 Cs-gluconate, 10 CsCl, 10 HEPES, 1 EGTA, 4 Mg-ATP and 0.5 Li-GTP. After whole cell patch clamp formed, the SOCE currents (I_SOCE) were induced by switching the perfusing solution to aCSF containing 1 μM TG. ML204 (10 μM) was used to isolate ML204 sensitive currents. The membrane capacitance was also obtained to normalize the currents. To get the I-V curve of I_SOCE, a voltage ramp from 100 mV to −100 mV (for 200 ms) was applied. The raw data was acquired with a Multiclamp 700B amplifier and pClamp10.2 software (Axon Instruments, Sunnyvale, CA). All data was analyzed using Clampfit10.2. The current density at −70 mV was quantified.

For the in vitro epilepsy model, male mice at postnatal 5–8 weeks were used. The slices were transferred to a recording chamber perfused with 95%O₂/5%CO₂ saturated aCSF containing 5 mM KCl. Epileptiform activity was induced by switching perfused solution to aCSF containing the GABA_A receptor antagonist bicuculline (Bic, 3 μM; Sigma) (McLeod et al. (2013). Reduced seizure threshold and altered network oscillatory properties in a mouse model of Rett syndrome. 231, 195–205.). Epileptiform events were detected with Clampfit 10.2 (Axon Instruments) and verified visually. The intracellular solution was (in mM): 140 K-gluconate, 7.5 KCl, 10 Hepes-K, 0.5 EGTA-K, 4 Mg-ATP and Li-GTP. The currents caused by unclamped action potentials has been truncated (Fellin et al., 2006).

All data were analyzed using the SigmaPlot 13.0 (Systat Software, Inc RRID:SCR_003210). Student's t-tests or Mann–Whitney U tests were used to make comparisons between two groups unless indicated otherwise. One-way ANOVA followed by post-hoc Holm-Sidak test was performed when there are three or more groups. Ca²⁺ imaging data was analyzed by two-way ANOVA. A list of the statistical methods used in each figure can be found in Table 5. Average data are shown as the mean ±SEM. p values < 0.05 were considered statistically significant. No statistical methods were used to pre-determine sample sizes. Data were collected from randomly selected fields within images. More than three independent experiments/biological replicates (for human astrocyte data set, each independent experiment was defined as one independent differentiation; for mouse astrocyte in vitro data set, each independent experiment was defined as one isolation culture from one mouse; for mouse in situ and in vivo data set, both number of cells and number of mice were described) were included in each data set.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Xiaoji Zhang for assistance with mouse work, and Dr. Dan Bolt for assistance with statistical analysis. RL was supported by a pre-doctoral fellowship from the Stem Cell and Regenerative Medicine Center at the University of Wisconsin-Madison. This work was partially supported by R21 NS081484, R56NS100024, and R01HD064743 to QC, ZIA MH002897 to KHW and QL, and U54HD090256 to the Waisman Center.

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (G005315) of the University of Wisconsin-Madison

1. Received: November 7, 2017
2. Accepted: March 28, 2018
3. Accepted Manuscript published: March 29, 2018 (version 1)
4. Version of Record published: April 16, 2018 (version 2)

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