Takano et al. 10.1073/pnas.0506382102. |
Supporting Materials and Methods
Cell Cultures.
All experiments except for Cx43 knockout study was done with rat astrocytes. Cortical astrocyte cultures were isolated from P1 Sprague–Dawley rat pups as described in ref. 1. Primary culture once it reached confluence was plated onto cover glasses, then grown to confluence and used within 1 to 2 weeks. Heterozygotes of the Cx43 knockout line were obtained from The Jackson Laboratory. Pregnant females were killed at 18–20 days of gestation, and the embryonic brains were cultured as described in ref. 2. To identify Cx43 null homozygotes, heterozygotes, and wild-type, PCR for amplifying tail-blood genomic DNA flanking the null deletion was used, as per The Jackson Laboratory’s protocol.Cell Volume Measurements in Vitro.
For confocal measurements, astrocytes grown on coverslips were loaded with calcein/acetoxymethyl ester (AM) (5 mM for 30 min). The vertical cross-sectional areas of the cells were visualized by using repetitive x-z line confocal scanning at 488 nm excitation and a ×100/1.3 oil-immersion lens (Olympus) (3). A total of 90 z-line scans with a step distance of 0.175 mm was collected every 30 s. Acquisition of each image takes 5–10 s. The magnitude of fluorescence quenching was estimated by the control run without any stimulation to 1.71 ± 1.04%. The final numbers were adjusted so that only the changes by the stimulation were shown. The cross-sectional areas of collected astrocyte images were measured by image1 software (Universal Imaging). Hypotonic stimulation was generated by diluting the recording buffer with distilled H2O. Buffer osmolarity was measured and adjusted with sucrose in all experiments by using a Vapro vapor pressure osmometer (Wescor). The fluorescence dilution technique involved the use of astrocytes grown on coverslips and loaded with fura-2/AM (5 mM for 30 min) (4). The cells were excited at 358 ± 5 nm (isosbestic point), and the emission (>510 nm) was collected by using a SITS camera (Dage Instruments). No detectable fluorescence quenching was observed. Fluorescent intensity was measured for each cell in the field before and after the stimulation. The volume of astrocytes in suspension was analyzed with a Coulter counter (Multisizer, Coulter Electronics, Hialeah, FL). Astrocytes were dissociated and allowed to recover for 1 h in serum-free DMEM/F12 medium before measurements. For each measurement, data were collected from 10,000 cells (roughly 10 s). The diameter of the aperture tube orifice was 100 mm. Cell volumes were calculated by using 9.89-mm diameter beads as standards.Glutamate Measurements.
An enzymatic fluorescence detection assay for monitoring glutamate was used (5, 6). Astrocytes grown on coverslips were placed inside a quartz cuvette and placed in LS50B spectroluminometer (PerkinElmer, Boston). The cuvette contained buffer (138 mM NaCl/1.5 mM K2HPO4/2 mM CaCl2/2 mM MgCl2/10 mM Hepes/10 mM glucose, pH 7.3) with 1 mM NADP+ and 40 units of glutamate dehydrogenase (Sigma). The cuvette and the buffer were kept at 37°C with constant stirring. After being placed in the cuvette, the cells were incubated at least 10 min before the recording to ensure a stable baseline. Antagonists were introduced to the buffer 5–10 min before the stimulation, unless otherwise noted. Fluorescence of NADPH was excited at 340 nm and collected at 450 nm. For analysis by high-performance liquid chromatography (HPLC), confluent culture of astrocytes grown on coverslips were mounted in a perfusion chamber (chamber volume: 27 ml) and perfused with a speed of 100 ml/min with the same buffer as above. The perfusion chamber and the perfusion buffer were kept at 37°C. Perfusion was run for 30 min before sample collection started. Fractions were collected at a 90-sec interval. The amino acid content was analyzed after reaction with o-phthaldialdehyde by using fluorometric detection (7).Slice Preparation and 2-Photon Laser Scanning Imaging of Ca2+.
Acutely isolated cortical or hippocampus slices were prepared from 14- to 18-day-old Sprague–Dawley rats as described in ref. 6. The laser-scanning microscope (Fluoview, Olympus) was attached to Mai Tai laser (SpectraPhysics) and an upright microscope (BX61WI, Olympus). Slices were loaded with the Ca2+ indicator, fluo-4/AM (10 mM for 1.5 h, Molecular Probes). Fluo-4 was excited at 800 nm, and light emission was collected at 525 nm.
Electrophysiology.
The recording chamber was placed on the stage of an Olympus BX51 upright microscope (Olympus Optical) equipped with DIC optics, and astrocytes in culture or acute slices were visualized with a ×40 water immersion lens. Whole-cell recordings were obtained by using an Axopatch 200B amplifier (Axon Instruments) as described in ref. 8. The pipette solution for whole-cell recording in cultured astrocytes contained 123 mM Cs-gluconate, 1 mM MgSO4, 10 mM Hepes, 1 mM ATP, 0.2 mM GTP (pH 7.3) with KOH. In selected experiments, Cs-gluconate was substituted by 100 or 123 mM Cs-glutamate. The extracellular solution contained 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM Hepes (pH 7.4). In selected experiments, NaCl was replaced by equimolar NMDG-Cl, Na-gluconate, choline-Cl, or 250 mM sucrose. The pipette solution for whole-cell recording in slices contained 50 mM K-glutamate, 73 mM K-gluconate, 10 mM KCl, 1 mM MgSO4, 10 mM Hepes, 1 mM ATP, 0.2 mM GTP (pH 7.3) with KOH, and the standard slice solution contained 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 26 mM NaHCO3 (pH 7.4). ATP (final concentration: 100 mM) was added to the bath solution or slice solution. Experiments with a holding current more than –200 pA or a change in input resistance >20% of control were rejected. Recording signals were filtered through an eight-pole Bessel low-pass filter with a 2 kHz cutoff frequency and sampled by a pclamp 8.2 program (Axon Instruments) with an interval of 50 ms. Data were further processed with origin 5.1 (Microcal Software, Northampton, MA) and coreldraw 9.0 (Corel) programs.1. Arcuino, G., Lin, J. H., Takano, T., Liu, C., Jiang, L., Gao, Q., Kang, J. & Nedergaard, M. (2002) Proc. Natl. Acad. Sci. USA 99, 9840–9845.
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