Sr101

Reagents for ACSF and internal solutions, biocytin, NBQX, BaCl₂, and picrotoxin were obtained from Sigma-Aldrich. CNQX, AP-5, DHK, DL-TBOA, TTX, ouabain, and D-serine were obtained from Tocris. L-glutamic acid from BioTrend and SR-101 from Invitrogen. NBQX, CNQX, and DL-TBOA were dissolved in DMSO. picrotoxin was dissolved in EtOH. AP-5, D-serine, TTX, ouabain, and DHK were dissolved in ddH₂O.
In both patch-clamp and two-photon imaging experiments, following baseline recordings, drugs were applied in the external solution for at least 15 min prior to recordings. For the double pharmacology imaging experiments (Fig. 9) following baseline recordings, DHK (300 μM) was first applied and recordings were acquired 20 min later. Subsequently, DHK (300 μM) and DL-TBOA (68 μM) were applied for 20 min before recording. In a number of recordings in which we applied much higher doses of DL-TBOA (300 µM), we observed a change in baseline iGluSnFr fluorescence (similar to ref. ^{18 (link)}), a reduced amplitude of the evoked responses and cellular swelling often accompanied by a lateral or Z drift. These experiments had to be excluded, which explains the low n value for this set of experiments (Supplementary Fig. 9).

OGB-1-labelled GCL cells were targeted with electrodes (5–15 MΩ) subsequent to two-photon recordings. Single cells in the GCL were dye-filled with SR101 (Invitrogen) using the buzz function (100-ms pulse) of the MultiClamp 700B software (Molecular Devices). Pipettes were carefully retracted as soon as the cell began to fill. Approximately 20 min were allowed for the dye to diffuse throughout the cell before imaging started. After recording, an image stack was acquired to document the cell’s morphology, which was then traced semi-automatically using the Simple Neurite Tracer plugin implemented in Fiji. In cases of any warping of the IPL we used the original image stack to correct the traced cells using custom-written scripts in IGOR Pro (for details, see ref. ^{37 (link)}).

We used viral injections to express Chronos (Klapoetke et al., 2014 (link)) in PV-Cre animals. Mice were anesthetized (isoflurane 1–1.5%) and the cranial window implant removed. We used a stereotaxic injection system (QSI, Stoelting Inc) to deliver 500 nL of a 10:1 mixture of AAV1-hSyn-FLEX-Chronos-GFP (UNC Vector Core Stock) and 100 µM sulforhodamine (SR101, Invitrogen, for visualization) at a depth of 200–400 µm. We made 3 to 4 injections spaced 0.5–1.0 mm apart to cover the visual cortex. After the injections a new cranial window was affixed. Viral expression was monitored over the course of days by imaging GFP fluorescence, and allowed to develop for greater than 4 weeks before electrophysiological recordings.

SR101 was purchased from Invitrogen (New York, NY). All other chemicals and salts used in intracellular and extracellular solutions were purchased from Sigma-Aldrich. 100 μM BaCl₂ and the 100 μM meclofenamic acid (MFA) were dissolved directly in aCSF.

SR101 was purchased from Invitrogen (New York, NY, USA). All other used chemicals and salts were purchased from Sigma-Aldrich (St. Louis, MO, USA). Meclofenamic acid (MFA) was directly dissolved in aCSF before experiment. 100 mM picrotoxin (PTX) were dissolved in dimethyl sulfoxide (DMSO) and stored in a −20 °C freezer prior to use. The stock solutions were diluted to the final experimental concentration just before each experiment.

Non-transfected astrocytes and astrocytes transfected with pAQP4b-EGFP or pAQP4d-EGFP were loaded with cytosolic sulphorhodamine 101 dye (SR101; 30 μM; Invitrogen, Carlsbad, CA, USA) for 15 min at 37 °C in 95% air/5% CO₂. The cells were then stimulated under either isoosmotic (300 mOsm) or hypoosmotic (200 mOsm) conditions and recorded with a laser confocal microscope (LSM 780, Zeiss, Jena, Germany) using an oil-immersion objective (40×/NA 1.3). Transfected cells were confirmed by Ar laser excitation (488 nm) and emitted light was filtered with a bandpass filter at 493–553 nm. SR101 was excited by a diode-pumped solid-state laser (561 nm) and the emission light was filtered with a bandpass filter at 567–649 nm. Time-series images were recorded every 2 s for 120 s (30 s before and 90 s after hypoosmotic stimulation). Changes in the fluorescence intensity of the cytosolic fluorescent dye were used as an indicator of changes in the cell volume [19 (link),26 (link),30 (link)]. Recordings were analysed in ZEN 2010 software (Zeiss, Jena, Germany) using the mean region of interest tool, which calculates the average fluorescence intensity, as in our previous study [19 (link)]. Detailed analysis and fitting of the data were performed in SigmaPlot 11.0 (SYSTAT, San Jose, CA, USA).