GoKawiilMice
Animal procedures were performed in accordance with National Institutes of Health guidelines with the approval of NYU Grossman School of Medicine’s Institutional Animal Care and Use Committee (IACUC). All animals were housed at 22–25 °C with 50–60% humidity. Animals had access to food and water ad libitum and were housed under a 12 h–12 h light–dark cycle. Experiments in Figs. 2 and 3 were on male 12-week-old C57BL/6J mice. For experiments in Fig. 4, the Saab laboratory crossbred mice1 expressing the tamoxifen-sensitive Cre recombinase cre-ERT2 under the control of the mouse Slc1a3 (encoding GLAST) promoter54 with mice carrying floxed Gjb6 (Gjb6fl/fl)55 and floxed Gja1 (Gja1fl/fl)56. Mice hemizygous for Slc1a3:cre-ERT2 were bred to noncarriers to generate Slc1a3:cre-ERT2+/ × Gja1fl/fl × Gjb6fl/fl experimental mice and Gja1fl/fl × Gjb6fl/fl littermate controls for in vivo experiments; in vivo experiments on this genotype were balanced for sex. When primary mouse astrocytes were isolated, Slc1a3:cre-ERT2 was kept homozygous to obtain a culture in which all astrocytes could be induced through 4-hydroxytamoxifen. All mice were on a C57BL/6 background. The primer sequences used for genotyping were as follows: for Gjb6 flox: 5′-TTCCCTATGCTGGTAGAGTGCTTGT-3′ and 5′-GCAGTAACTTATTGAAACCCTTCACCT-3′; for Gja1 flox: 5′-GGGATACAGACCCTTGGACTCC-3′ and 5′-TCACCCCAAGCTGACTCAACCG-3′; and for Slc1a3-creERT2: 5′-GAGGCACTTGGCTAGGCTCTGAGGA-3′, 5′-GAGGAGATCCTGACCGATCAGTTGG-3′ and 5′-GGTGTACGGTCAGTAAATTGGACAT-3′. The Saab laboratory previously analysed Cre reporter function1 by crossing with ROSA26-floxed-STOP-GCaMP6s (Ai96, JAX, 024106). After shipment, mice were rederived by NYU Grossman School of Medicine’s Rodent Genetic Engineering Laboratory.
Tamoxifen treatment
Tamoxifen treatment was performed as previously described11. For 3 consecutive days, both Slc1a3:cre-ERT2+/ × Gja1fl/fl × Gjb6fl/fl experimental and littermate control mice received daily (between 15:00 and 17:00) gavage of a tamoxifen solution (20 mg ml−1 dissolved in corn oil). The solution was prepared 2 h before administration, during which it was shaken at 37 °C in the dark. Any further experimental manipulations were performed at least 7 days after the last dose of tamoxifen.
Primary rat astrocyte cultures
Isolation of primary rat astrocytes by immunopanning was performed as previously described21. In brief, cortices of postnatal day 6/7 Sprague–Dawley rats (Charles River) were blunt dissected, the meninges were removed and the cortices were enzymatically dissociated at 37 °C and 10% CO 2 using papain (Worthington Biochemical, LS003126). Tissue was then triturated using a 5 ml serological pipette to generate a single cell suspension, resuspended in Dulbecco’s PBS (VWR, SH30264.FS) with BSA (BSA, Sigma-Aldrich, A4161) and DNase (Worthington Biochemical, LS002007), and filtered using a 20 µm nitex filter. The suspension was negatively panned for non-specific secondary antibody binding, endothelial cells (BSL-1, Vector Labs L-1100), microglia (CD45, BD Pharmingen, 553076) and oligodendrocyte lineage cells (O4 hybridoma, generated in house), and positively panned for astrocytes (ITGB5, Thermo Fisher Scientific, 14-0497-82). Astrocytes were removed from the positive panning plate using TrypLE (Thermo Fisher Scientific, 12-605-010) and plated at 10,000 cells per well in an eight-well chamber slide precoated with poly-l-lysine (Ibidi 80804). Astrocytes were cultured in serum-free medium containing 50% neurobasal, 50% DMEM, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 1 mM sodium pyruvate, 292 μg ml−1 l-glutamine, 1× SATO, 5 μg ml−1 N-acetylcysteine and 5 ng ml−1 HBEGF (Sigma-Aldrich, E4643-50UG). After 7 days, AAV was added to the medium change, resulting in an effective titre of 105. Cells were incubated for a further 7 days, with biotin (effective concentration, 250 µM) added to the medium 2 days before fixation.
Expansion microscopy
Expansion microscopy was performed based on published protocols20. Primary astrocytes were quickly washed in PBS, then fixed in 4% PFA in PBS at room temperature for 15 min. After three 5-min washes in PBS, the cells were permeabilized for 15 min in PBS with 0.5% Triton X-100, then blocked for 1 h in 5% BSA and 0.2% Tween-20 in PBS with shaking at room temperature. They were then incubated in primary antibody solution overnight at 4 °C (3% BSA, 0.2% Tween-20 in PBS containing mouse monoclonal anti HA-tag (Cell Signaling, 2367) directly conjugated to ATTO488-NHS (ATTO-TEC AD 488-31), rabbit polyclonal anti-Cx43 (Cell Signaling, 3512) directly conjugated to ATTO565-NHS (ATTO-TEC AD 565-31) and ATTO643-streptavidin (ATTO-TEC, AD 643-61)). Dye conjugation was performed according to the manufacturer’s protocol. Antibodies were added for an effective concentration of 2 µM for anti-Cx43 and 10 µM for anti-HA, and streptavidin (stock concentration 2 μg ml−1) was added at 1:1,000. Cells were then washed three times for 5 min in PBS and incubated for 10 min in 300 nM DAPI in PBS, then imaged on a spinning-disk confocal microscope (CrestOptics X-LIGHT V3 Confocal on Nikon Ti2) with a ×60/1.4 NA oil lens to later establish an expansion coefficient. Then, 250 µl Acryloyl-X, SE (Invitrogen, A20770) was added for overnight incubation at room temperature.
Cells were washed twice for 15 min in PBS, then incubated in 300 µl Gelation Solution per well (for 2 ml: 542 µl 4 M Na acrylate (VWR, S03880), 1 ml PROTOGEL (Thermo Fisher Scientific, 50-899-90119), 200 µl 10× PBS, 198 µl H 2 O (MQ), 30 µl 10% ammonium persulfate (Thermo Fisher Scientific, 17874), 30 µl tetramethylethylenediamine (TEMED, Thermo Fisher Scientific, 17919)) for 1 h at 37 °C. Then, 300 μl digestion buffer was added to each well and the gels were carefully outlined with a needle to facilitate detachment; once the gels detached, they were each transferred to one well of a 12-well plate for the remainder of the 4 h incubation at 37 °C (volume per gel/well of 12-well plate: 1,550 µl Tris-acetate-EDTA buffer, 100 µl 10% Triton X-100 in PBS, 320 µl 5 M NaCl with 26 µl proteinase K (Thermo Fisher Scientific, EO0491) added immediately before use). The gels were then each transferred to 15 cm Petri dishes filled with room-temperature H 2 O (MQ) and incubated for 30 min at room temperature. H 2 O (MQ) was then replaced and the gels were left overnight at room temperature to expand. The gels were then stored at 4 °C until imaging.
Single-molecule imaging and localization
... continue reading