P 1000 puller

To perform patch-clamp experiments in whole-cell configuration, both WT-iAstro and 3Tg-iAstro were plated separately in 35 mm dishes 24 h prior experiments. Cells were plated at low density to allow recordings from isolated astrocytes. They were transferred from culture medium to an extracellular solution containing (in mM): 138 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose and 10 HEPES at pH 7.25 adjusted with NaOH. Borosilicate patch pipettes were pulled with a P-1000 puller (Sutter Instruments, USA) and were filled with a solution containing (in mM): 140 KCl, 2 NaCl, 5 EGTA, 0.5 CaCl₂ and 10 HEPES at pH 7.25 adjusted with KOH. Pipette tip resistance containing this solution was between 3 and 5 MΩ. Experiments were performed using an EPC7 Plus amplifier (HEKA Elektronik, Germany) in voltage-clamp configuration (holding potential, –80 mV). Access resistance (8–12 MΩ) was compensated (80–90%) and experiments were performed at RT and in a static bath. Data were acquired at 5 kHz and filtered at 1 kHz using a 7-pole Bessel filter and digitized with a low noise data acquisition system, Digidata 1440A (Molecular Devices, USA). Data were recorded and analyzed in pClamp 10 (Molecular Devices, Crisel Instruments, Italy). Data were initially processed with Microsoft Excel. Plots, bar diagrams and figure preparations were finalized with GraphPad Prism (GraphPad Software, La Jolla, CA).

Whole cell patch clamp experiments were performed at 22°C and 37°C with an EPC9 patch-clamp amplifier (HEKA Elektronik) and Patchmaster software (Heka Electronic). Temperature was maintained with a TC-10 Temperature controller (Dagan). Glass pipettes were pulled with a P-1000 puller (Sutter Instruments), dipped in wax, and polished to a resistance of 1.0–1.5 MΩ. Intracellular solution contained 130 mM CsF, 10 mM NaCl, 10 mM EGTA, and 10 mM HEPES. Extracellular solution contained 140 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES. Both solutions were titrated to a pH 7.4 with CsOH.

Volume regulated anion channel currents were recorded in the standard whole‐cell configuration at 15–25°C using EPC 10 USB patch‐clamp amplifier and PATCHMASTER NEXT software (HEKA, GER). The sampling rate was 20 kHz and digitally filtered at 2 kHz. Series resistance compensation was set to 60%–80%. The electrodes were pulled by SUTTER P‐1000 puller, and the resistance was maintained at 3–5 MΩ. In the patch‐clamp experiments, bathing solution perfusion was delivered by eight‐channel perfusion valve control system (Warner Instruments).
For VRAC currents whole‐cell recording, the isotonic extracellular solution contained (in mM) 150 NaCl, 6 KCl, 1 MgCl₂, 1.5 CaCl₂, 10 glucose, and 10 HEPES (pH 7.4; 320 mOsm). To elicit VRAC currents, cells were exposed to a hypotonic solution containing (in mM) 105 NaCl, 6 CsCl, 1 MgCl₂, 1.5 CaCl₂, 10 glucose, 10 HEPES (pH 7.4; 240 mOsm). Capillary glass electrodes were filled with solution containing 140 mM CsCl, 1 mM MgCl₂, 5 mM EGTA, 4 mM Na₂ATP, and 10 mM HEPES (pH 7.2 with NaOH, 290 mOsm).
The standard test protocol consisted of a 600 ms step to −80 mV followed by a 2600 ms ramp from −100 to +100 mV from a holding potential of −30 mV, applied at 1500 ms intervals. The full voltage protocols consisted of a 2000 ms step protocol from −120 to +120 mV in 20 mV increment from a holding potential of −80 mV applied every 5000 ms.

Whole-cell patch-clamp recordings were made from the bipolar cell or ganglion cell somas in the retinal preparations by viewing them with an upright microscope (Slicescope Pro 2000, Scientifica, UK) equipped with a CCD camera (Retiga-2000R, Q-Imaging, Canada). The light-evoked postsynaptic potentials and currents (L-EPSPs and L-EPSCs) were recorded at the resting membrane potential and the equilibrium potential for chloride ions (E_Cl; −60 mV), respectively. All recordings were performed at 30–34°C. The liquid junction potentials were corrected after each recording. Whole-cell recordings from bipolar cells usually lasted 20–30 min without significant rundown (Ichinose et al., 2014 (link)). The electrodes were pulled from borosilicate glass (1B150F-4; WPI, FL, USA) with a P1000 Puller (Sutter Instruments, Novato, CA, USA) and had resistances of 8–12 MΩ. Clampex and MultiClamp 700B (Molecular Devices, San Jose, CA, USA) were used to generate the waveforms, acquire the data, and control light stimuli by a light-emitting diode (LED; Cool LED, UK). The data were digitized and stored on a personal computer using Axon Digidata 1440A (Molecular Devices). The responses were filtered at 1 kHz with the four-pole Bessel filter on the MultiClamp 700B and sampled at 2–5 kHz.

Brain slices were prepared as described above for calcium imaging experiments; similar ACSF solutions were also used. Thalamic neurons were visualized using a Zeiss Axio Examiner.A1 microscope (Zeiss Microscopy, Thornwood, NY, USA) and an sCMOS camera (ORCA-Flash4.0, Hamamatsu). Recording pipettes were pulled on a P1000 puller (Sutter Instruments) from thin-walled borosilicate capillary glass (Sutter Instruments, Novato, CA, USA). Pipettes (2–3 MΩ tip resistance) were filled with (in mM) 100 K-gluconate, 9 MgCl₂, 13 KCl, 0.07 CaCl₂, 10 HEPES, 10 EGTA, 2 Na₂ATP, 0.5 NaGTP, pH adjusted to 7.3 with KOH, and osmolality adjusted to 275 mOsm. Recordings were performed in the whole cell patch clamp configuration. Data were acquired in pClamp software (Molecular Devices, San Jose, CA, USA) using a Multiclamp 700B amplifier (Molecular Devices), low-pass filtered at 2 kHz, and digitized at 10 kHz (Digidata 1,440 A, Molecular Devices). Access resistance was monitored by repeatedly applying a –5 mV hyperpolarizing voltage step and converting the resultant capacitive transient response into resistance (Ulrich and Huguenard, 1997 (link)). A good recording consisted of an access resistance less than 20 MΩ that changed by less than 20% over the course of the recording; recordings that did not meet these criteria were discarded.