Cells were studied using a MultiClamp 700A (Axon Instruments) in whole cell patch configuration. Current-voltage relationships for L-type Ca2+ current were studied and normalized to cell capacitance, as previously described [19] (link).
Multiclamp 700a
Manufactured by Molecular Devices
Sourced in United States
The Multiclamp 700A is a versatile patch-clamp amplifier designed for high-quality, low-noise intracellular and extracellular electrophysiology recordings. It features two independent amplifier channels, allowing simultaneous recordings from multiple cells or preparations. The Multiclamp 700A provides high-resolution data acquisition, advanced signal processing, and seamless integration with a wide range of electrophysiology software and hardware.
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C57 mice, by Jackson ImmunoResearch (2 mentions)
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Axopatch 200b amplifier, by Molecular Devices (2 mentions)
Borosilicate glass pipette, by Harvard Apparatus (1 mentions)
Micromanipulator, by Sutter Instruments (1 mentions)
Flaming brown micropipette puller, by Sutter Instruments (1 mentions)
54 protocols using multiclamp 700a
1
Patch Clamping L-type Ca2+ Currents
2
Whole-cell Recordings of preBötC Neurons
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Whole-cell recordings were made from inspiratory-modulated preBötC neurons (Rekling et al., 1996 (link)). A horizontal puller (P-97, Sutter Instruments, Novato, CA, United States) was used to pull whole-cell recording pipettes (3–4.5 MΩ) from filamented borosilicate glass (Clark/WPI, 1.2 mm O.D.). Patch pipettes were filled with intracellular solution containing (in mM) 140 K+-gluconate, 5 NaCl, 0.1 EGTA, 10 HEPES, 1 MgCl2, 1 glucose, and 1 Mg2ATP. Values of membrane potential (Vm) have not been corrected for the liquid junction potential of −14.1 mV (calculated using Clampex, pCLAMP Suite) that is associated with these solutions. Osmolarity was adjusted to 290–300 mOsm with sucrose, and pH was adjusted to 7.2–7.3 using KOH. Whole-cell signals (membrane current or voltage) were amplified and filtered with a patch-clamp amplifier (2–5 kHz low-pass filter, MultiClamp 700A, Axon Instruments) and sampled at 10 kHz via an analog-to-digital converter (Digidata 1322 A/D, Molecular Devices). Series resistance (Rs) was estimated and tracked throughout the experiment under voltage-clamp conditions. Rs measured 14.0 ± 1.4 MΩ (range: 12–16 MΩ). We excluded recordings in which Rs varied by more than 20%.
3
Whole-cell Recordings from Mouse Hippocampal Slices
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Slices from the RSC or subiculum were prepared from mice as described previously 74 . In brief, the animals (n = 12 VGlut2-Cre mice; 11 males, 1 female; age: 2-3 months) were deeply anesthetized with isoflurane, decapitated and the brains removed. Tissue blocks were then mounted on a vibratome (VT1200S, Leica Biosystems, Wetzlar, Germany), and slices were cut at 300 µm nominal thickness. The slices were stored in an interface chamber where they were stored for 1-5 h before being transferred to the recording chamber where they were perfused at a rate of 3-4 ml/minute. The recording chamber was mounted on an upright microscope equipped for IR-DIC microscopy. Whole-cell recordings were performed using a Multiclamp 700A (Axon Instruments, CA, USA) using glass microelectrodes filled with 120 mM K-gluconate, 10 mM Hepes, 3 mM Mg-ATP, 10 mM KCl, 5 mM EGTA, 2 mM MgSO4, 0.3 mM Na-GTP, 14 mM phosphocreatine and 2mg/mL biocytin. The resistances of the electrodes ranged between 3 and 5 MΩ. Access resistance (< 20 MΩ) was continuously monitored during the recording and was not allowed to fluctuate by more than 20%. Data were analyzed online using Igor Pro and offline using Matlab (Matworks) and were not corrected for liquid junction potential. Light-evoked responses were detected if the peak of the postsynaptic potential averaged over >10 repetitions crossed ± 0.4 mV.
4
Electrophysiological Field Potential Recordings
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The electrophysiological field potential (FP) recordings were performed at 31°C. For FP recordings glass microelectrodes (1 to 4 MΩ) were filled with ACSF solution and were inserted in the CA3 stratum pyramidale. Signals were recorded with Multiclamp 700A amplifiers (Axon Instruments, Foster City, CA, United States), low-pass filtered at 2 kHz and digitized at 10 or 20 kHz (Digidata 1320A, Axon Instruments). Recordings were analyzed after high pass filtering at 1 or 2 Hz.
5
Patch-Clamp Recording of MVN Neurons
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MVN neurons identified visually by location, size and fluorescence were recorded using borosilicate glass pipettes of external diameter 1.2 mm/internal diameter 0.69 mm (Harvard Apparatus) pulled from Flaming/Brown micropipette puller (Model P-97, Sutter Instrument) and filled with high chloride internal solution (in mM): 140 CsCl, 10 HEPES, 1 EGTA, 2 MgCl2, 2 Na2ATP, and 1 Na2GTP (adjusted to pH 7.2, 290 mOsm). The advancement of the pipette was manually operated through the micromanipulator (Sutter Instrument).
Signals were amplified using MultiClamp700A (Axon Instruments) and acquired through a 16-bit data acquisition system (DIGIDATA 1322A; Axon Instruments). During whole-cell patch-clamp recording, membrane potentials were corrected for the liquid junction potential (10 mV), and the change of series resistance was sustained within 15%. Only recordings with series resistance smaller than 15 MΩ were included for subsequent analysis. Cell recording was discarded if the leaking currents went beyond 200 pA. The signals of the recording were digitized at 10 kHz and filtered at 3 kHz by the Multiclamp 700A amplifier, DIGIDATA 1322A analog/digital interface board and pCLAMP 10.2 software (Axon Instruments). Data were captured by Clampex 10.2/Multiclamp Commander 1 (Axon Instruments) package.
Signals were amplified using MultiClamp700A (Axon Instruments) and acquired through a 16-bit data acquisition system (DIGIDATA 1322A; Axon Instruments). During whole-cell patch-clamp recording, membrane potentials were corrected for the liquid junction potential (10 mV), and the change of series resistance was sustained within 15%. Only recordings with series resistance smaller than 15 MΩ were included for subsequent analysis. Cell recording was discarded if the leaking currents went beyond 200 pA. The signals of the recording were digitized at 10 kHz and filtered at 3 kHz by the Multiclamp 700A amplifier, DIGIDATA 1322A analog/digital interface board and pCLAMP 10.2 software (Axon Instruments). Data were captured by Clampex 10.2/Multiclamp Commander 1 (Axon Instruments) package.
6
Larval Neuromuscular Physiology
7
Cerebellar Slice Electrophysiology
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Cerebellar slices were prepared as previously described (Liu and Cull-Candy, 2000 (link); Dubois et al., 2016 (link)). Briefly, the cerebellum was isolated and horizontal slices (400 μm) were cut using a vibratome (Leica VT1200) in ice cold artificial CSF (containing in mM: 81.2 NaCl, 2.4 KCl, 23.4 NaHCO3, 1.4 NaH2PO4, 6.7 MgCl2, 0.5 CaCl2, 23.3 glucose, 69.9 sucrose, and pH 7.4). Slices were then maintained in aCSF (in mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 glucose, and pH 7.4) saturated with 95% O2, 5% CO2 at room temperature for at least 30 min before recording.
Whole cell patch clamp recordings were obtained at near physiological temperature (35–37°C) from cerebellar stellate cells in an O2/CO2-saturated aCSF. Stellate cells were identified by their location in the outer two thirds of the molecular layer and by the presence of spontaneous action potentials in the cell-attached mode. Analog signals were filtered at 6 kHz and digitized at 20 kHz (Multiclamp 700A, Axon Instruments). Series resistance was monitored throughout the recordings. Recordings were terminated if series resistance changed by more than 20%.
Whole cell patch clamp recordings were obtained at near physiological temperature (35–37°C) from cerebellar stellate cells in an O2/CO2-saturated aCSF. Stellate cells were identified by their location in the outer two thirds of the molecular layer and by the presence of spontaneous action potentials in the cell-attached mode. Analog signals were filtered at 6 kHz and digitized at 20 kHz (Multiclamp 700A, Axon Instruments). Series resistance was monitored throughout the recordings. Recordings were terminated if series resistance changed by more than 20%.
8
Validating Photovoltaic Potential Measurements
9
Preparation and Electrophysiological Recording of Cerebellar Slices
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Cerebellar slices were prepared as previously described73 (link),74 (link). Briefly, P18 to P110 male mice were decapitated and the cerebellum was isolated. Sagittal slices (300 µm) were cut from the cerebellar vermis using a vibratome (Leica VT1200) in an ice-cold slicing solution (containing in mM: 81.2 NaCl, 2.4 KCl, 23.4 NaHCO3, 1.4 NaH2PO4, 6.7 MgCl2, 0.5 CaCl2, 23.3 glucose, 69.9 sucrose, pH 7.4). Slices were then maintained in aCSF (in mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 glucose, pH 7.4) saturated with 95% O2, 5% CO2 at room temperature for at least 30 min before recording. All experiments were carried out at near physiological temperature (33–37 °C). Unless otherwise noted, all recordings were obtained in lobules V and VI of the cerebellar vermis with patch pipettes (3–6 MOhm for Purkinje cells, 5–10 MOhm for interneurons) pulled from borosilicate capillary glass (Harvard Apparatus, Holliston, MA) with a Narishige PP-830 puller. EPSCs and IPSCs were recorded in the presence of GABAA-R blockers (100 µM PTX + 5 µM SR-95531), and a non-NMDAR inhibitor (5 µM NBQX), respectively. TTX (0.5 µM) was included during recordings of miniature events. Analog signals were filtered at 6 kHz and digitized at 20 kHz (Multiclamp 700A, Axon Instruments). Data were analyzed using Clampex 10.2.0.12 (Axon Instruments).
10
Voltage-Clamp Recording of GABAA Currents
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Electrophysiological recordings were performed at 31 °C. Signals were recorded with Multiclamp700A amplifiers (Axon Instruments, Foster City, CA, USA), low-pass filtered at 2 kHz and digitized at 20 kHz (Digidata1320A, Axon Instruments). For single cell recording CA1 pyramidal cells were identified visually. Pipettes (4 to 5 MΩ) were filled with a solution containing (in mM) 130 CsMeSO3, 10 NaCl, 0.05 CaCl2, 2 ATP (magnesium salt), 1 EGTA and 10 HEPES (pH set to 7.3 with 1N CsOH). To suppress escape action currents 5 mM QX 314 (Tocris, Bristol, UK) was added. Cells were voltage-clamped at 0 mV (corrected for a calculated junction potential of +15 mV) to record GABAergic (outward) currents. Input resistance was 150.7 ± 14.0 MΩ. If signs of seal deterioration or cell closure occurred (>20% change in the access resistance) the recordings were discarded. Synaptic recordings were made for 10 to 20 min in control conditions following 10 to 20 min of 100 μM SNAP-5114 application and 10 to 20 min washout.
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