Intracellular recordings from STG somata were made in the desheathed STG with 10–30 MΩ sharp glass microelectrodes filled with either an internal solution: 10 mM MgCl2, 400 mM potassium gluconate, 10 mM HEPES buffer, 15 mM NaSO4, 20 mM NaCl (Hooper et al., 2015 (link)) or 0.6M K2SO4 with 20mM KCl. Intracellular signals were amplified with an Axoclamp 900A amplifier (Molecular Devices, San Jose). Extracellular nerve recordings were made by building wells around nerves using a mixture of Vaseline and 10% mineral oil and placing stainless-steel pin electrodes within the wells to monitor spiking activity. Extracellular nerve recordings were amplified using model 3500 extracellular amplifiers (A-M Systems). Data were acquired using a Digidata 1440 digitizer (Molecular Devices, San Jose) and pClamp data acquisition software (Molecular Devices, San Jose, version 10.5). For identification of Pyloric Dilator (PD) neurons, somatic intracellular recordings were matched to extracellular action potentials on the pyloric dilator nerve (pdn) and/or the lateral ventricular nerve (lvn).
Digidata 1440 digitizer
Manufactured by Molecular Devices
Sourced in Uruguay
The Digidata 1440 digitizer is a high-performance data acquisition device designed for a wide range of electrophysiology applications. It features multiple analog and digital inputs to capture data from various experimental setups. The Digidata 1440 digitizer is capable of converting analog signals into digital data for further processing and analysis.
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Lab products found in correlation
Model 3500 extracellular amplifier, by A-M Systems (3 mentions)
Pclamp data acquisition software, by Molecular Devices (3 mentions)
Pclamp 10, by Molecular Devices (2 mentions)
Axopatch 700b amplifier, by Molecular Devices (2 mentions)
Clampfit software, by Molecular Devices (2 mentions)
Model 3800 stimulator, by A-M Systems (2 mentions)
Axopatch 200b amplifier, by Molecular Devices (2 mentions)
Axoclamp 900a amplifier, by Molecular Devices (1 mentions)
Axon multiclamp 700b amplifier, by Molecular Devices (1 mentions)
Borosilicate glass pipette, by Warner Instruments (1 mentions)
Axopatch 200b, by Molecular Devices (1 mentions)
Multiclamp 700b, by Molecular Devices (1 mentions)
P 97 flaming brown micropipette puller, by Sutter Instruments (1 mentions)
Pc running pclamp, by Molecular Devices (1 mentions)
Clampex 10, by Molecular Devices (1 mentions)
Sutter p97, by Sutter Instruments (1 mentions)
9 protocols using digidata 1440 digitizer
1
Intracellular Recordings from Stomatogastric Ganglion Neurons
2
DREADD-Mediated Neuronal Recordings
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Following at least 1 h of recovery, the coronal brain slices were transferred to a recording chamber circulated with carbonated ACSF in room temperature (∼23°C) at a flow rate of 2 ml/min. A borosilicate glass pipette (tip resistance between 3 and 6 MΩ) was filled with a solution composed of 130 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 4 mM Mg-ATP, 0.2 mM EGTA, and 0.5 mM Na-GTP at an osmolarity of 290 mOsm (pH 7.25). CNO-evoked currents were recorded in voltage-clamp mode with membrane potential held at −70 mV, and CNO-evoked spikes were recorded in current-clamp mode with a holding potential at around −55 mV. DREADD-mCherry-expressing cells were identified by mCherry expression, and cells were stimulated using 10 µM CNO. Data, acquired with an Axon Multiclamp 700B amplifier and a Digidata 1440 digitizer (Molecular Devices), were analyzed using Axon Clampfit. Recordings with access resistance >25 MΩ or with changes in access resistance >15% were discarded.
3
Whole-cell patch-clamp recordings of neurons
4
Electrophysiological Characterization of Neuronal Cultures
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Coverslips were transferred to a recording chamber perfused at a rate of 2 ml/min at 32°C with ACSF containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.3 adjusted with NaOH). Recordings were performed with an Axopatch 700b amplifier equipped with Digidata 1440 digitizer and pClamp10 software (Molecular Devices). Whole cell voltage and current clamp recordings were performed with borosilicate glass pipettes (4–6 MΩ) backfilled with a solution containing 135 mM K-gluconate, 20 mM KCl, 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na2GTP, and 0.5 mM EGTA. Traces were analyzed offline using Clampfit software (Molecular Devices). Whole-cell voltage clamp recording were performed at a holding potential of −70 mV in neurons cultured over E18 rat astrocytes. Action potentials were induced by a series of 100 mS, 10 pA depolarizing current injections under current clamp configuration. Data are presented as means ± the standard error of the mean. For statistical analysis, a Student’s t test was performed using GraphPad Prism 6 unless otherwise noticed.
5
Extracellular Nerve Recordings and Stimulation
6
Electrophysiological Characterization of Neuronal Cultures
7
Preparation and Electrophysiology of Giant E. coli Spheroplasts
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Giant spheroplasts were prepared using established protocols56 (link), with several important modifications. Briefly, a culture of E. coli BL21 DE3 ΔmscL containing the construct of interest was treated with cephalexin for 1.5 hours and induced with 1 mM IPTG for 30 minutes. Spheroplasts were then prepared by lysozyme treatment at room temperature for 18–20 min. The spheroplast suspension was centrifuged through 7 mL column of 1 M sucrose at 4 °C and re-suspended in 300 µL 1 M sucrose. Aliquots were stored at −80 °C.
Patch-clamp experiments were carried out using pipette buffer (200 mM KCl, 90 mM MgCl2, 5 mM CaCl2, 5 mM HEPES, pH 7.4) and bath buffer (200 mM KCl, 90 mM MgCl2, 5 mM CaCl2, 5 mM HEPES, pH 7.4, 450 mM Sucrose). Excised inside-out patches from spheroplast membranes clamped at −20 mV membrane potential were treated with 5-second symmetric triangle pressure ramps of amplitudes from −50 to −290 mm Hg, using pipettes with bubble number of about 4.5, as previously described57 (link). A high-speed pressure clamp system, HSPC-1 (ALA Scientific), was utilized in the experiments. Data were acquired with an Axopatch 200B amplifier and a Digidata 1440 digitizer (Molecular Devices) at 20 kHz, filtered at 5 kHz, and further analyzed with the pCLAMP 10.6 software suite (Molecular Devices). Unitary conductances of MscL channel variants were corrected for pipette access resistance.
Patch-clamp experiments were carried out using pipette buffer (200 mM KCl, 90 mM MgCl2, 5 mM CaCl2, 5 mM HEPES, pH 7.4) and bath buffer (200 mM KCl, 90 mM MgCl2, 5 mM CaCl2, 5 mM HEPES, pH 7.4, 450 mM Sucrose). Excised inside-out patches from spheroplast membranes clamped at −20 mV membrane potential were treated with 5-second symmetric triangle pressure ramps of amplitudes from −50 to −290 mm Hg, using pipettes with bubble number of about 4.5, as previously described57 (link). A high-speed pressure clamp system, HSPC-1 (ALA Scientific), was utilized in the experiments. Data were acquired with an Axopatch 200B amplifier and a Digidata 1440 digitizer (Molecular Devices) at 20 kHz, filtered at 5 kHz, and further analyzed with the pCLAMP 10.6 software suite (Molecular Devices). Unitary conductances of MscL channel variants were corrected for pipette access resistance.
8
Patch-clamp recording of ion channels
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Patch pipettes were manufactured from soda lime capillary glass (Thermo Fisher Scientific) using a Sutter P-97 (Sutter Instrument) puller. When filled with standard recording solutions, pipettes had a tip resistance of 1–3 MΩ. Recordings were filtered at 5 kHz and sampled at 10 kHz, with manual capacitance compensation and series resistance compensation at 80%, and stored directly on a computer hard drive using an Axopatch 200B amplifier, Digidata 1440 digitizer, and Clampex 10 software (Molecular Devices). Zero current levels are denoted by dashed lines in representative current panels. The external (bath) solution had the following composition (in mM): 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, adjusted to pH 7.3 with NaOH. The internal (pipette) solution had the following composition (in mM): 135 KCl, 5 EGTA, and 10 HEPES, adjusted to pH 7.2 using KOH. Chemicals were purchased from Sigma-Aldrich or Thermo Fisher Scientific. Patch-clamp experiments were conducted at room temperature (22 ± 1°C).
9
Extracellular Nerve Recordings and Stimulation
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Extracellular nerve recordings were made by building wells around nerves using a mixture of Vaseline and 10% mineral oil and placing stainless‐steel pin electrodes within the wells to monitor spiking activity. Extracellular nerve recordings were amplified using model 3500 extracellular amplifiers (A‐M Systems). Data were acquired using a Digidata 1440 digitizer (Molecular Devices) and pClamp data acquisition software (Molecular Devices, version 10.5). We stimulated nerves innervating the CG while simultaneously recording the rhythm with an extracellular electrode. For each nerve, stainless steel pin electrodes were placed on either side of the nerve and sealed using Vaseline. Stimuli were delivered using a model 3800 stimulator (A‐M Systems). Stimuli were 0.5 ms pulses in 1 s trains with a within‐stimulus rate of 20 Hz.
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