Cells in layer 2/3 were visualized with an Olympus BX61WI microscope coupled with a 40× water immersion lens (Olympus), infrared-DIC optics and CCD camera (Qimaging). Slices were screened for cell bodies containing tdTomato using a custom fluorescence filter. Glass pipettes (4 −7 MΩ) were pulled with a Sutter Instruments P1000 puller. Data was collected and acquired with a MultiClamp 700B amplifier and a Digidata 1440A system (Molecular Devices), with WinWCP software (Strathclyde). For all cells, response to current steps, input resistance, and access resistance was measured before drug application and after washout (>30min) to verify the health of each cell. Only cells without significant changes in current-step responses were used for further analysis (Extended Data Fig 2 ). Firing rate and changes in membrane potential were analyzed using Clampfit software.
Digidata 1440a system
Manufactured by Molecular Devices
The Digidata 1440A system is a data acquisition device designed for electrophysiology applications. It features high-speed, high-resolution analog-to-digital conversion and provides a flexible interface for connecting various electrophysiology instruments. The Digidata 1440A system is capable of digitizing and recording multiple analog and digital signals simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Pclamp 10, by Molecular Devices (5 mentions)
Axopatch 200b amplifier, by Molecular Devices (3 mentions)
Water immersion lens, by Olympus (2 mentions)
P 1000 puller, by Sutter Instruments (2 mentions)
Bx61wi microscope, by Olympus (2 mentions)
Multiclamp 700b amplifier, by Molecular Devices (2 mentions)
Eclipse ti u inverted microscope, by Nikon (2 mentions)
Clampfit 10, by Molecular Devices (2 mentions)
Axon multiclamp 700b amplifier, by Molecular Devices (1 mentions)
Vt1200, by Leica (1 mentions)
Submerged type recording chamber, by Warner Instruments (1 mentions)
Multiclamp 700b, by Molecular Devices (1 mentions)
Ds qi1 monochrome digital camera, by Nikon (1 mentions)
Axon axopatch 200b amplifier, by Molecular Devices (1 mentions)
Axopatch multiclamp 700b amplifier, by Molecular Devices (1 mentions)
10 protocols using digidata 1440a system
1
Visualizing Layer 2/3 Neurons
2
Electrophysiological Profiling of Rat Piriform Cortex
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For electrophysiological studies, slices of aPC were prepared from male and female Wistar rat pups at age P5–P8 and P14–P17 as described previously (Pardo et al. 2018 (link)). Whole-cell patch-clamp recordings were made on pyramidal cells located in the L2/3 of aPC under the mode of current-clamp with axon Multiclamp 700B amplifier (Molecular Devices). For current-clamp recording (Fig. 1 A), the intracellular solution contained (in mM): potassium gluconate (120), KCl (10), MgCl2 (1), CaCl2 (0.025), EGTA (0.2), Na2-ATP (2), Na2-GTP (0.2), HEPES (10), titrated to pH 7.2 with KOH, and 290 mOsmol L−1. Whole-cell recording pipettes had a tip resistance of 3–4 MΩ. Data were digitalized at 10 kHz with Digidata 1440-A System (Molecular Devices), filtered at 1 kHz (−3 dB, eight-pole Bessel) and analyzed offline with pClamp 10.6 software (Molecular Devices). The membrane potential was held at −65 mV for all neurons. Cells were excluded if they did not meet the following criteria: a stable resting membrane potential more negative than −55 mV, action potential crossing 0 mV. For measuring intrinsic properties of cells, a series of depolarizing and hyperpolarizing currents, 500 msec long, square-pulse current steps were injected (−180 pA to +300 pA) with intervals of 500 msec, steps of 20 pA.
3
Whole-cell Voltage Clamp of Eag1 K+ Currents
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Whole-cell voltage clamp recordings of Eag1 K+ currents in HEK293T cells were performed 24 h posttransfection. Electrode solution contains (in mM) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. Bath solution comprises (in mM) 140 NaCl, 5 KCl, 1 CaCl2, and 10 HEPES, pH 7.2. Data were acquired with the Axopatch 200B amplifier (Molecular Devices) and digitized with the Digidata 1440A system and the pCLAMP 10.2 software (Molecular Devices). Cells with large currents in which voltage clamp errors might appear were excluded from data analyses. Passive membrane properties were compensated by using the -P/4 leak subtraction method. Data were sampled at 10 kHz and filtered at 1 kHz. All recordings were performed at room temperature (20–22 °C).
4
Whole-cell Patch Clamp Analysis of iPSC-derived Spheroids
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Whole-cell patch clamp was used to record from mature iPSK3-derived dorsal and ventral spheroids cultured on glass covered slips. Cover slips were washed three times with extracellular recording solution containing (in mM) 136 NaCl, 4 KCl, 2 MgCl, 10 HEPES, and 1 EGTA (312 mOsm, pH 7.39) and were incubated in this solution at room temperature during recording. Glass electrodes (resistance 1–5 MΩ) were filled with intracellular solution containing 130 mM KCl, 10 mM HEPES, and 5 mM EGTA (292 mOsm, pH 7.20). Cells were visualized under phase contrast with a Nikon Eclipse Ti-U inverted microscope and attached DS-Qi1 monochrome digital camera. Recordings were made with an Axopatch 200B amplifier (Molecular Devices) and digitized with a Digidata 1440A system (Molecular Devices). Ionic currents were recorded under a voltage clamp protocol (−60 mV to 135 mV in 15 mV steps, 250 ms in duration). Action potentials were recorded under a current clamp protocol (−100 pA to 200 pA in 20 pA steps, 800 ms in duration). Spontaneous post-synaptic currents were recorded under continuous voltage clamp at −80 mV for 2 min. Signals were filtered at 1 kHz and sampled at 10 kHz. Data was collected and analyzed using pCLAMP 10 software (Molecular Devices).
5
Acute Brain Slice Electrophysiology Procedure
6
Patch-Clamp Electrophysiology of iPSC-Derived Spheroids
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Whole-cell patch clamp was used to record from iPSK3-derived spheroids cultured on glass covered slips. Cover slips were washed three times with extracellular recording solution containing (in mM) 136 NaCl, 4 KCl, 2 MgCl, 10 HEPES, and 1 EGTA (312 mOsm, pH 7.39) and were incubated in this solution at room temperature during recording. Glass electrodes (resistance 1–5 MΩ) were filled with intracellular solution containing 130 mM KCl, 10 mM HEPES, and 5 mM EGTA (292 mOsm, pH 7.20). Cells were visualized under phase contrast with a Nikon Eclipse Ti-U inverted microscope and attached DS-Qi1 monochrome digital camera. Recordings were made with an Axopatch 200B amplifier (Molecular Devices) and digitized with a Digidata 1440 A system (Molecular Devices). Ionic currents were recorded under a voltage clamp protocol (−60 mV to 135 mV in 15 mV steps, 250 ms in duration). Action potentials were recorded under a current clamp protocol (−100 pA to 200 pA in 20 pA steps, 800 ms in duration). Spontaneous post-synaptic currents were recorded under continuous voltage clamp at −80 mV for 2 min. Signals were filtered at 1 kHz and sampled at 10 kHz. Data was collected and analyzed using pCLAMP 10 software (Molecular Devices).
7
Visualizing Layer 2/3 Neurons
8
Miniature EPSC Recording in Cultured Hippocampal Neurons
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Cultured hippocampal neurons at 18-19 DIV were used to record miniature EPSCs (mEPSCs) by whole-cell patch clamps. Neurons were incubated in extracellular solution, i.e. artificial cerebrospinal fluid (ACSF) solution containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 26.2 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM CaCl2, 11 mM glucose, 0.001 mM tetrodotoxin, and 0.04 mM bicuculline. The intracellular solution contained 131 mM K-gluconate, 8 mM NaCl, 20 mM KCl, 10 mM HEPES, 2 mM Mg-ATP, 2 mM EGTA and 0.3 mM Na3GTP. Neurons were voltage-clamped at − 70 mV for recording with an Axon Axopatch 200B amplifier (Molecular Devices) and Digidata 1440A system (Molecular Devices), and then filtered at 1 kHz. Raw data were analyzed using Clampfit 10.7 software (Molecular Devices) and Mini Analysis (Syanptosoft). Statistical analyses of amplitude and frequency were performed using Prism.
9
Optogenetic Activation of Oxytocin Neurons
10
Patch-clamp recording of retinal mIPSCs
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The retinal slices were transferred to a chamber, covered with nylon mesh, and continuously perfused with oxygenated (95% O2 and 5% CO2) ACSF at a rate of 2–3 ml/min. To record mIPSCs, patch pipettes were filled with solution containing the following (in mM): CsCl 150, HEPES 10, EGTA 1, CaCl2 0.1, MgCl2 1, GTP-Na 0.4, and ATP-Mg 4 (pH 7.2 adjusted with CsOH, 275–290 mOsm/l). The neurons were voltage-clamped at −70 mV using an Axopatch-Multiclamp 700B Amplifier (Molecular Devices, Foster City, CA, USA). The sampling frequency was set at 10 kHz, and the filter frequency was 1 kHz. The signals were digitized using a Digidata 1440 A system (Molecular Devices). Data analysis was performed using Clampfit 10.2 (Molecular Devices), Mini Analysis (Synaptosoft), and Origin 8.0 software.
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