Patch-clamp recordings were performed as previously described16 (link),17 (link),34 (link). Briefly, borosilicate glass pipettes with 4–6 MΩ resistance were used for whole-cell recordings (in mM): 122 K-gluconate, 13 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP-Na, 0.3 EGTA (adjusted to pH 7.35 with KOH). The composition of the extracellular solution was (mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 10 μM pyruvic acid bubbled with 95% O2 and 5% CO2. Signals were amplified using an EPC10-2 amplifier (HEKA Elektronik, Lambrecht, Germany). All recordings were performed at 34 °C using a temperature control system (Bath-controller V, Luigs & Neumann, Ratingen, Germany) and slices were continuously superfused at 2–3 ml/min with the extracellular solution. Current- and voltage-clamp recordings were sampled at 10 kHz, with the Patchmaster v2x32 program (HEKA Elektronik).
Epc10 2 amplifier
Manufactured by HEKA Elektronik
Sourced in Germany
The EPC10-2 is a two-channel voltage-clamp amplifier designed for electrophysiology applications. It provides precise control and measurement of membrane potential and ionic currents in biological samples. The EPC10-2 offers high-quality signal amplification and filtering capabilities to enable accurate data acquisition during experiments.
Automatically generated - may contain errors
6 protocols using epc10 2 amplifier
1
Patch-Clamp Recordings of Neuronal Electrophysiology
2
Juxtacellular Recordings of Pyramidal Cells
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Mice were anaesthetized with urethane as described for juxtacellular recordings. A 0.5 × 0.5 mm craniotomy was performed to expose M1 (from bregma: AP +0.5 mm, ML 1.5 mm). Borosilicate glass pipettes (3.5–5 MΩ) contained (mM): K-gluconate (125), KCl (8), HEPES (10), phosphocreatine (10), ATP-Mg (4), GTP-Na (0.3) and EGTA (0.3) (adjusted to pH 7.35 with KOH). Signals were amplified using an EPC10-2 amplifier (HEKA Elektronik, Lambrecht, Germany) and sampled at 10 kHz, with Patchmaster v2x32 (HEKA Elektronik). ChR2 was activated using a 475 nm-laser diode light source (FLS-475, DIPSI, Cancale, France) with single light pulses (3–20 ms, 10 mW) at 0.33 Hz, or with 3 ms-pulse trains at 67 Hz during 5 s. The optic fiber was placed on top of the pia with an angle of 15°. Pyramidal cells were recorded at 790–1350 µm from the pia.
3
Whole-cell patch clamp for bulk endocytosis
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Cm was measured under the whole-cell configuration using an EPC10/2 amplifier controlled by Pulse software (HEKA Elektronik) as described previously (3 (link), 25 (link)). The membrane potential was clamped at −70 mV, and pipette resistance was controlled between 3 and 4 MΩ. The external solution contained, in mM, 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (H-HEPES), and 10 d -glucose, pH 7.4. The intracellular pipette solution contained, in mM, 153 CsCl, 1 MgCl2, 10 H-HEPES, and 4 Mg-ATP, pH 7.2. All recordings were performed at room temperature (22 to 25 °C). All chemicals were from Sigma unless otherwise indicated. Igor software (Wavemetrics) was used for all offline data analysis, and series conductance (Gs) and membrane conductance (Gm) were used to monitor the seal condition in the patch clamp recordings. Measurement of bulk endocytosis, which was reflected as a brief downward capacitance shift, was performed as previously described (9 (link), 25 (link)). Capacitance traces were low-pass filtered at 30 Hz, and the brief downward capacitance shift was identified when the size of the capacitance decay was >20 fF and the decay rate was >50 fF/100 ms, with unchanged Gs and Gm.
4
Patch-Clamp Recording of Neuronal Cells
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Patch-clamp recordings were performed as previously described12 (link),18 . Briefly, for whole-cell recordings in borosilicate glass pipettes of 5–7 MΩ resistance were filled with (in mM): 122 K-gluconate, 13 KCl, 10 HEPES, 10 phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP, 0.3 EGTA (adjusted to pH 7.35 with KOH). The composition of the extracellular solution was (mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 10 μM pyruvic acid bubbled with 95% O2 and 5% CO2. Signals were amplified using EPC10–2 amplifiers (HEKA Elektronik, Lambrecht, Germany). All recordings were performed at 34 °C (Bath-controller V, Luigs&Neumann, Ratingen, Germany) and slices were continuously superfused with extracellular solution, at a rate of 2 ml/min. Slices were visualized under microscope (BX51WI Olympus, Rungis, France), with a 4x/0.13 objective for the placement of the stimulating electrode and a 40x/0.80 water-immersion objective for the localization of cells for whole-cell recordings. Current- and voltage-clamp recordings were filtered at 5 kHz and sampled at 10 kHz, with the Patchmaster v2 × 32 program (HEKA Elektronik).
5
Whole-Cell Recordings of Hippocampal Neurons
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For whole-cell CA1 pyramidal neuron recordings, transverse hippocampal slices (350 µm thick) were prepared. Signals were amplified with EPC10-2 amplifiers (HEKA Elektronik). Current- and voltage-clamp recordings were sampled at 20 kHz with the Patchmaster v2 × 32 program (HEKA Elektronik). All recordings were performed at 35 °C.
6
Whole-Cell Patch-Clamp Recordings in Brain Slices
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Patch-clamp recordings were performed as previously described (Fino et al., 2010 (link); Paillé et al., 2013 (link); Cui
et al., 2015 ). For whole-cell recordings borosilicate glass pipettes of
4-6 MΩ resistance contained (in mM): 105 K-gluconate, 30 KCl, 10 HEPES, 10
phosphocreatine, 4 ATP-Mg, 0.3 GTP-Na, 0.3 EGTA (adjusted to pH 7.35 with KOH).
The composition of the extracellular solution was (mM): 125 NaCl, 2.5 KCl, 25
glucose, 25 NaHCO3, 1.25 NaH2PO4, 2
CaCl2, 1 MgCl2, 10 μM pyruvic acid bubbled with 95%
O2 and 5% CO2. Signals were amplified using EPC10-2
amplifiers (HEKA Elektronik, Lambrecht, Germany). All recordings were performed at
34°C using a temperature control system (Bath-controller V, Luigs&Neumann,
Ratingen, Germany) and slices were continuously superfused at 2–3 ml/min with the
extracellular solution. Slices were visualized on an Olympus BX51WI microscope
(Olympus, Rungis, France) using a 4x/0.13 objective for the placement of the
stimulating electrode and a 40x/0.80 water-immersion objective for localizing
cells for whole-cell recordings. Series resistance was not compensated.
Current-clamp recordings were filtered at 2.5 kHz and sampled at 5 kHz and
voltage-clamp recordings were filtered at 5 kHz and sampled at 10 kHz using the
Patchmaster v2x32 program (HEKA Elektronik).
et al., 2015
4-6 MΩ resistance contained (in mM): 105 K-gluconate, 30 KCl, 10 HEPES, 10
phosphocreatine, 4 ATP-Mg, 0.3 GTP-Na, 0.3 EGTA (adjusted to pH 7.35 with KOH).
The composition of the extracellular solution was (mM): 125 NaCl, 2.5 KCl, 25
glucose, 25 NaHCO3, 1.25 NaH2PO4, 2
CaCl2, 1 MgCl2, 10 μM pyruvic acid bubbled with 95%
O2 and 5% CO2. Signals were amplified using EPC10-2
amplifiers (HEKA Elektronik, Lambrecht, Germany). All recordings were performed at
34°C using a temperature control system (Bath-controller V, Luigs&Neumann,
Ratingen, Germany) and slices were continuously superfused at 2–3 ml/min with the
extracellular solution. Slices were visualized on an Olympus BX51WI microscope
(Olympus, Rungis, France) using a 4x/0.13 objective for the placement of the
stimulating electrode and a 40x/0.80 water-immersion objective for localizing
cells for whole-cell recordings. Series resistance was not compensated.
Current-clamp recordings were filtered at 2.5 kHz and sampled at 5 kHz and
voltage-clamp recordings were filtered at 5 kHz and sampled at 10 kHz using the
Patchmaster v2x32 program (HEKA Elektronik).
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