Axoclamp 700b amplifier

CAPs in the CC and external capsule (EC) were measured as we described previously [28 (link)]. Briefly, fresh brains were rapidly harvested from euthanized mice, and 350-µm thick coronal brain slices were cut − 1.06 mm posterior from bregma using a Leica VT1200 S vibratome. Slices were placed in pre-gassed (95% O₂/5% CO₂) artificial cerebrospinal fluid (aCSF; NaCl 130 mmol/L, KCl 3.5 mmol/L, Na₂HPO₄ 1.25 mmol/L, MgSO₄ 1.5 mmol/L, CaCl₂ 2 mmol/L, NaHCO₃ 24 mmol/L, glucose 10 mmol/L; pH 7.4) for 1 h at 20 °C, followed by incubation in a recording chamber where they were submerged and perfused at 3–4 mL/min with aCSF at 20 °C. A concentric bipolar electrode was placed into the CC approximately 0.9 mm lateral to the midline. A glass extracellular recording pipette (5–8 MΩ tip resistance when filled with aCSF) was placed into the EC, 0.75 mm and 1 mm from the stimulating electrode. The input stimuli ranged from 0 to 2 mA (100-µs duration; delivered at 0.05 Hz). The evoked CAPs were recorded by a Molecular Devices Axoclamp 700B amplifier and analyzed with pCLAMP 10 software. The average waveforms of four successive sweeps were quantified. The recording shows two positive peaks and two negative peaks conventionally referred to as N1 and N2, reflecting the responses from myelinated and unmyelinated fibers, respectively [4 (link)].

One hour or later (max 4 h), one slice was transferred to a custom made recording chamber superfused with oxygenated ACSF (3–5 ml/min). LA neurons were visualized with an Olympus BX51WI (Center Valley, PA) microscope, equipped with infrared differential contrast optics. Under visual guidance, we obtained whole-cell recordings of LA neurons using pipettes (3–6 MΩ) pulled from borosilicate glass capillaries and filled with a solution containing (in mM): 145 potassium gluconate, 5 NaCl, 10 HEPES, 0.5 EGTA, 4 MgATP, 0.3 Na₂GTP, for current clamp recordings and 120 Cs methanesulfonate, 8 NaCl, 15 CsCl, 10 TEACl, 10 HEPES, 0.5 EGTA, 10 QX-314, 4 MgATP and 0.3 Na₂GTP for voltage clamp recordings. The intracellular solutions were adjusted to a pH around 7.25 ± 0.03 and the osmolarity was adjusted to 290 mOsm ± 5. In cases where the recording technique required a morphological analysis of the neurons, biocytin (0.2%) was added into the intracellular solution. Current-clamp recordings were obtained with an Axoclamp 700B amplifier and digitized at 10 kHz with a Digidata 1440A interface (Molecular Devices, Palo Alto, CA). Data acquisition ensued 5–10 min after whole cell access. During experiments, access resistances were monitored before the onset of each electrophysiological protocol. Cases where the access resistances exceeded 20 MΩ were discarded from analysis.

Receptor desensitization was recorded with whole-cell configuration of the transfected HEK 293 T cells. The external solution was as follows (in mM): 140 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose and 10 HEPES (pH 7.4). Patch pipettes (resistance 3 to 5 MΩ) were filled with a solution containing the following (in mM): 130 KF, 33 KOH, 2 MgCl2, 1 CaCl2, 11 EGTA and 10 HEPES (pH 7.4). Glutamate (10 mM) diluted into the external solution was applied to lifted HEK cells with whole-cell configuration for 500 ms with a theta glass pipette mounted on a piezoelectric bimorph every 5 s [34 (link)]. Glutamate-induced currents were recorded with holding potential of − 70 mV. All the currents were collected with an Axoclamp 700B amplifier and Digidata 1440A (MolecularDevices, Sunnyvale, CA, USA), filtered at 2 kHz and digitized at 100 kHz. The current data were analyzed using Clampfit software.

Membrane currents and potentials were recorded using an Axoclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) filtered at 3 kHz and digitized at 10 kHz using National Instruments acquisition boards and ScanImage (available at: scanimage.org) written in MATLAB (Mathworks, Natick, MA). Electrophysiology and imaging data were analyzed offline using Igor Pro (Wavemetrics, Lake Oswego, OR), ImageJ (NIH, Bethesda, MD) and GraphPad Prism (GraphPad Software, La Jolla, CA). In figures, voltage-clamp traces represent the average waveform of 3–6 acquisitions. Peak current amplitudes were calculated by averaging over a 1 ms window around the peak. For pharmacological analyses, 3–7 consecutive acquisitions (20 s inter-stimulus interval) were averaged following a 3-min wash-in period for NBQX and CPP or a 4-min wash-in period for MEC, MLA, and DHβE. For TTX and 4AP conditions, current averages were composed of the acquisitions following full block or first-recovery of ChR2 evoked currents, respectively. Data (reported in text and figures as mean ± sem) were compared statistically using the Mann–Whitney test. p values smaller than 0.05 were considered statistically significant.

Slices were initially visualized under epifluorescence illumination with a high-sensitivity digital frame transfer camera (Cooke SensiCam) mounted on an Olympus BX50-WI epifluorescence microscope with a × 40 long working distance water-immersion lens. Once a GFP+ interneuron was identified, visualization was switched to infrared–differential interference contrast microscopy for the actual patching of the neuron. Micropipettes for whole-cell recording were constructed from 1.2 mm outer diameter borosilicate pipettes on a Narishige PP-83 vertical puller. The standard internal solution for whole-cell current-clamp recording was as follows (in mM): 130 K-gluconate, 10 KCl, 2 MgCl₂, 10 HEPES, 4 Na₂ATP, 0.4 Na₂GTP, pH 7.3. These pipettes had a DC impedance of 3–5 MΩ. Membrane currents and potentials were recorded using an Axoclamp 700B amplifier (Molecular Devices). Recordings were digitized at 20–40 kHz with a CED Micro 1401 Mk II and a PC running Signal, version 5 (Cambridge Electronic Design). Optogenetic stimulation in vitro consisted of 2–5 ms duration blue light pulses delivered using a high-power (750 mW) LED. Sweeps were run at 20 s intervals.

Coverslips with live neurons were transferred into the recording chamber containing standard extracellular solution (in mM: 140 NaCl, 3 KCl, 2 CaCl₂, 1.25 MgCl₂, 1.25 NaH₂PO₄, 20 D-glucose, and 10 HEPES). Whole-cell recordings were established using DIC optics on an upright microscope (Olympus BX51WI with LUMPLFLN40XW objective). Recording pipettes had resistances ranging from 3 to 6 MΩ and series resistances were <20 MΩ. Data were measured and acquired with an Axoclamp 700B amplifier and Digidata 1440 (Molecular Devices). Internal electrode solutions contained, in mM, 135 K gluconate, 2 MgCl₂, 2 MgATP, 0.5 NaGTP, 0.5 EGTA, 10 HEPES, and 10 phosphocreatine (pH 7.35). Cells were driven by current injection to test for the presence of action potentials.

Twenty-four hours after transfection, a whole-cell recording was performed at room temperature using an Axoclamp 700B amplifier and a Digidata 1550B (Molecular Devices, CA, USA). When filled with pipette solution, the resistances of glass micropipettes were 3.0 to 5.0 MΩ. The extracellular solution contained the following (in mM): 145 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 Glucose, adjusted pH 7.4 with NaOH; the pipette solution contained the following (in mM): 145 KCl, 1 MgCl₂, 5 EGTA, 10 HEPES, and 5 ATP-Mg, adjusted pH 7.4 with KOH. The membrane potentials were held at −90 mV, then depolarized for 2 s from −90 to +60 mV with a 10-mV increment. The tail current elicited at −120 mV was measured to obtain a conductance-voltage (G-V) curve. Pynegabine (HN37), XEN1101, and retigabine (RTG) were provided by Dr. Fa-jun Nan (National Center for Drug Screening, Chinese Academy of Sciences, Shanghai, China). ICA-069673 was purchased from MCE (MedChemExpress, Shanghai, China).