Multiclamp 700b patch clamp amplifier

Micropipettes were fabricated from quartz glass capillaries (QF100-50–7.5, Sutter Instrument, Novato, CA) using a P-2000 micropipette laser puller (World Precision Instruments, Sarasota, FL). An Ag/AgCl wire was inserted in the micropipette to act as the working electrode and another Ag/AgCl wire was immersed in a 1 × PBS bath (Oxoid Ltd, Thermo Fisher, UK), acting as the reference electrode. Current–voltage measurements are performed using a MultiClamp 700B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA). The signal was filtered using a Digidata 1550B digitizer, with a low-pass filter at 10 kHz, and signal recording was performed using the pClamp 10 software (Molecular Devices), at a rate of 100 kHz. For electrochemical analysis experiments, the micropipettes were filled with 1 × PBS. For electrowetting experiments, the micropipettes were filled with a solution of 10 mM tetrahexylammonium tetrakis(4-chlorophenyl)borate (THATPBCl) salt in 1,2-dichloroethane (1,2-DCE).

After approximately 1 h of perfusion with ACSF, extracellular recordings of brain slice preparations were performed, with the experimenter blinded to treatment. Extracellular voltage‐clamp recordings at a holding potential of 0 mV were used, because the recording technique adopted enables the capacitor aspect of the membrane patch to provide a low impedance pathway through the patch for fast events such as action potentials (APs). Consequently, when a current is generated, the current is leaked across the seal rather than the patch under the loose patch configuration (Perkins 2006). Spontaneous AP currents were recorded using a Multiclamp‐700B patch‐clamp amplifier (Molecular Devices, Sunnyvale, CA, USA). Microelectrodes were fabricated from glass capillaries (O.D.: 1.5 mm, I.D.: 0.86 mm; BF150‐86‐10; Sutter Instrument Company, Novato, CA, USA) on a P‐97 puller (Sutter Instrument Company). The tip resistance of each electrode was 3–7 MΩ when filled with 0.9% NaCl. A depolarizing rectangular voltage pulse of 10 mV was applied to the electrode and negative pressure gently applied to complete a loose patch with an average seal resistance of 49.5 ± 2.4 MΩ (n = 65). Membrane currents were filtered at 2 kHz and acquired at a sampling frequency of 10 kHz. Data acquisition was performed using a Digidata 1440A interface with pClamp software version 10.2 (Molecular Devices).

To measure responses to FMRFamide, PSEM^89S, GABA, and muscimol, HCs were recorded with the perforated patch configuration using gramicidin as the perforating agent. The bathing solution contained the following: 120 mm NaCl, 2.5 mm KCl, 1.2 mm MgSO₄, 2.2 mm CaCl₂, and 3.0 mm HEPES, pH 7.7. A stock solution of gramicidin in ethanol (5 mg/ml) was prepared and diluted into the pipet solution (130 mm KCl, 3 mm NaCl, and 10 mm HEPES, pH 7.4) at a ratio of 6 μl/ml. Pipets were pulled to a resistance of ∼10–12 MΩ using a vertical puller (PC-100, Narishige Scientific Instruments). Following seal formation, the presence of a membrane potential in the current clamp recording configuration confirmed successful perforation, usually within the first minute. PSEM^89S (500 μm), GABA (1 mm), and muscimol (100 μm) were applied with 100-ms pulses of positive pressure (1–2 psi) from a second pipet positioned ∼100 μm from the cell. FMRFamide (30 μm) was bath applied for 5 min before measuring changes in membrane potential. FMRFamide was purchased from Sigma-Aldrich. Other agonists were purchased from Tocris Biosciences. Recordings were made using the MultiClamp 700b patch clamp amplifier, and pClamp software (Molecular Devices). All reported values were corrected for junction potentials, calculated using pClamp software.

After approximately 1 h of perfusion with ACSF, blind extracellular recordings of the brain slice preparation were performed. Extracellular voltage‐clamp recording at a holding potential of 0 mV is able to avoid disrupting the intracellular milieu, and provides a low impedance pathway through the patch for fast events occurring in a few ms, such as action potentials (APs) (Perkins 2006 (link)). Spontaneous AP currents were recorded using a Multiclamp‐700B patch‐clamp amplifier (Molecular Devices, Sunnyvale, CA). Microelectrodes were fabricated from glass capillaries (O.D.: 1.5 mm, I.D.: 0.86 mm) (BF150‐86‐10, Sutter Instrument Company, Novato, CA) on a P‐97 puller (Sutter Instrument Company). The tip resistance of each electrode was 3–9 MΩ when filled with 0.9% NaCl. A depolarizing rectangular voltage pulse of 10 mV was applied to the electrode and negative pressure was gently applied to complete a loose patch. Seal resistance was 40–200 MΩ and was periodically monitored during recordings. Membrane currents were filtered at 2 kHz and acquired at a sampling frequency of 10 kHz. Data acquisition was performed using a Digidata 1440A interface and pClamp software version 10.2 (Molecular Devices).

Ts or Eu mice were anesthetized with 15% urethane (1.5 g/kg in physiological solution) and placed on a stereotaxic apparatus. The body temperature was constantly monitored and kept at 37°C with a heating blanket. To ensure a deep and constant level of anesthesia, vibrissae movement, eyelid reflex, response to tail, and toe pinching were visually controlled before and during the surgery. A local lidocaine injection was performed over the cranial area of interest and, after a few minutes, a longitudinal incision was performed to expose the skull. Two small cranial windows (<1 mm diameter) were opened at at 2.5 mm from bregma and ±0.5 mm lateral to sagittal sinus (corresponding to the frontal lobe) carefully avoiding any damage to the main vessels while keeping the surface of the brain moist with the normal HEPES-buffered artificial cerebrospinal fluid. Pipettes used to record LFP had 1–2 MΩ resistance while those used for juxtacellular patch-clamp recordings typically had 5–7 MΩ resistance. LFP and patch electrodes were pulled from borosilicate glass capillaries. Signals were amplified with a Multiclamp 700B patch-clamp amplifier (Molecular Devices), sampled at 20 KHz and filtered online at 10 KHz. Signals were digitized with a Digidata 1440A and acquired, using the pClamp 10 software package (Molecular Devices).

For mCherry⁺ cells, whole-cell recordings were performed using Multiclamp 700B patch-clamp amplifier (Molecular Devices) as described before^{13 (link)}, and the chamber was constantly perfused with a bath solution containing the following (in mM): 125 NaCl, 3 KCl, 2 CaCl₂, 2 MgSO₄, 1.25 NaH₂PO₄, 1.3 Na⁺-ascorbate, 0.6 Na⁺-pyruvate, 26 NaHCO₃, and 11 glucose, at pH 7.4, 290−310 mOsm, saturated with 95% O₂ and 5% CO₂. Patch pipettes were pulled from borosilicate glass (3–5 MΩ) and filled with a pipette solution consisting of (in mM): 130 K-gluconate, 20 KCl, 10 HEPES, 0.2 EGTA, 4 Mg₂ATP, 0.3 Na₂GTP, and 10 Na₂-phosphocreatine, at pH 7.3 (290−310 mOsm). For the morphological reconstruction experiment, biocytin was included in the internal solution. To evoke currents, step voltages (500 ms, 10 mV step) from −110 to 60 mV were applied in the voltage-clamp mode. To evoke membrane potential deflections, step currents (500 ms duration) were injected in the current-clamp mode. Data were collected using pClamp 10 software (molecular Devices) digitized at 20–100 kHz, and analyzed with Clampfit 10.