The recording chamber was continuously perfused with ACSF at a rate of 1–2 ml/min, at room temperature. The slices used were those containing a ventral rootlet of sufficient length to be mounted on a suction stimulation electrode: a glass pipette with a tip size adapted to the diameter of the rootlet (40–170 µm) and filled with ACSF. Patch pipettes had an initial open-tip resistance of 3–6 MΩ. The internal solution contained (in mM): 140 K-gluconate, 6 KCl, 10 HEPES, 1 EGTA, 0.1 CaCl2, 4 Mg-ATP, 0.3 Na2GTP. The pH was adjusted to 7.3 with KOH, and the osmolarity to 285–295 mOsm. An AxoClamp 700B (Molecular Device, Sunnydale, CA) amplifier was used for data acquisition. Whole-cell recordings were filtered at 3 kHz, digitized at 10 kHz using a CED 1401 and monitored using the Signal 5 software (Cambridge Electronic Design Limited). Bridge resistance was compensated in current-clamp mode. Liquid junction potential was not corrected in order to readily compare with previous studies.
Axoclamp 700b
Manufactured by Molecular Devices
Sourced in United States
The Axoclamp 700B is a versatile electrophysiology amplifier designed for high-performance intracellular and extracellular recordings. It provides precise control of current and voltage, enabling researchers to conduct a wide range of electrophysiological experiments.
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6 protocols using axoclamp 700b
1
Whole-Cell Patch-Clamp Recordings in Ventral Roots
2
Compound Action Potentials in Mouse Brain Slices
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Compound action potentials (CAPs) in the external capsule were recorded [17 (link)]. Briefly, mice were sacrificed under deep anesthesia to rapidly collect brain samples. Coronal brain slices of 350-μm thickness were obtained using a vibratome (1200 s, Leica). These slices were then transferred to artificial cerebrospinal fluid (aCSF) consisting 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM NaH2PO4, 24 mM NaHCO3, 1.3 mM MgSO4, and 10 mM D-glucose. Subsequently, the slices were incubated in oxygenated aCSF with a mixture of 95% O2 + 5% CO2 at 32 °C for 0.5 h, followed by recovery at room temperature for 1 h. CAPs were induced using monophasic square waves (0.1 ms) with a concentric stimulating electrode and a glass microelectrode (5 ~ 8 MΩ). A stimulus generator (STG 4002, Multichannel) was employed for this purpose. The resulting signals were amplified using an Axoclamp 700B (Molecular Devices) and digitized using an Axon Digidata 1440A (Molecular Devices).
3
Electrophysiological Recording of Compound Action Potentials
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Compound action potentials (CAPs) in the external capsule were recorded as previously described [20 (link)]. Mice were anesthetized with isoflurane and were decapitated to remove the brains rapidly. A 350-μm brain slices coronal were made using a vibratome (1200s, Leica). Slices were transferred in artificial cerebrospinal fluid (aCSF) saturated with 95% O2+5% CO2 mixture at 32°C for 0.5 h and at room temperature for 1 h subsequently for recovery. The aCSF contained 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM NaH2PO4, 24 mM NaHCO3, 1.3 mM MgSO4, and 10 mM d -glucose. A concentric stimulating electrode and a glass microelectrode (5~8 MΩ) were used for stimulating and recording. CAPs were induced by monophase square waves (0.1 ms duration) with the stimulus generator (STG 4002, Multichannel). Signals evoked were amplified by Axoclamp 700B (Molecular Devices) and digitized Axon Digidata 1440A (Molecular Devices).
4
VTA Neuronal Activity Recordings
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Mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p) and perfused intracardially with ice-cold sucrose solution (in mM: 260 sucrose, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 D-glucose, saturated with 95% O2/5% CO2). Coronal midbrain slices (200 μm) containing the VTA were cut by a vibratome (VT1200S; Leica, Germany) in ice-cold sucrose solution and then placed in 37°C oxygenated ACSF (in mM: 130 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 D-glucose) for 30 min. The slices were then left to recover at room temperature for about 90 min, transferred to the recording chamber and perfused with oxygenated ACSF. Patch pipettes (3–5 MΩ, pulled from borosilicate glass capillaries by a four-stage horizontal puller, P97, Sutter Instruments, USA) were filled with ACSF. The spontaneous firing activity was recorded in the cell-attached mode; electrical signals were amplified and filtered by the Axoclamp 700B preamplifier and recorded by the Digidata 1440A AD converter (Molecular Devices, USA); the data were analyzed by Clampfit 10.3 (Molecular Devices, USA).
5
Patch-clamp recording of hippocampal neurons
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Whole-cell recordings were taken from pyramidal neurons in the CA1 cell layer. Borosilicate pipettes (2 to 6 MΩ) were filled with an internal solution containing (in mM): 8 NaCl, 130 CsMeSO4, 10 HEPES, 0.5 EGTA, 4 MgATP, 0.3 NaGTP, and 5 QX-314. Recordings were accepted for analysis with an uncompensated series resistance of <2.5 times the pipette resistance. Recordings were not corrected for series resistance due to the small current amplitudes. During mEPSC and mIPSC recordings, 100 µM D-AP5 was added to the perfused aCSF to block NMDA receptor-mediated currents. mEPSCs were recorded at a membrane potential of −70 mV and for mIPSCs, membrane potential was held at 0 mV. Recordings were amplified using an AxoClamp 700B (Molecular Devices) for whole-cell voltage-clamp recordings. Data were acquired using WinLTP software at a sampling rate of 10 KHz, and filtered at 6 KHz, before being analyzed offline using ClampFit 9.2. mEPSCs and mIPSCs were identified when the rise time was faster than the decay time and had a peak amplitude >6 mV.
6
Measuring Compound Action Potentials in Brain Slices
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CAPs in the CC were measured as described previously [36 (link)]. The brain tissue was cut into coronal slices and placed in pregassed (95% O2/5% CO2) artificial cerebrospinal fluid (aCSF: 126 mmol/L NaCl, 2.5 mmol/L KCl, 1 mmol/L Na2H2PO4, 2.5 mmol/L CaCl2, 26 mmol/L NaHCO3, 1.3 mmol/L MgCl2, and 10 mol/L glucose; pH 7.4) for 1 h at room temperature. Slices were perfused with aCSF at a constant rate (3 ~ 4 mL/min) at 22 °C. A bipolar tungsten-stimulating electrode was positioned across the CC 0.9 mm lateral to the midline. A glass extracellular recording pipette was placed in the external capsule. Only recordings at 0.48 mm from the stimulating electrode were reported in the current study. Both electrodes were placed 50 ~ 100 mm below the surface of the slice, with adjustments to optimize the signal. The CAP was amplified (× 1 k) and recorded using an Axoclamp 700B (Molecular Devices, Inc., San Jose, CA, USA) and then analyzed using pCLAMP 10.0 software (Molecular Devices, Inc.). Input–output curves were generated by varying the intensity of the stimuli from 0.25 to 2 mA in 0.25-mA increments. The average waveforms of four successive sweeps in two slices per animal were analyzed. The amplitude of the N1 component of the CAP (representing myelinated fibers) was quantified as the difference from the first positive peak to the first negative peak.
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