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Digidata 1550 digitizer

Manufactured by Molecular Devices
Sourced in United States

The Digidata 1550 is a high-performance digitizer designed for acquiring and recording electrophysiological data. It features simultaneous acquisition of up to 32 analog channels and 16 digital channels, with a maximum sampling rate of 500 kHz per channel.

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14 protocols using digidata 1550 digitizer

1

Two-Photon Imaging and Patch-Clamp Electrophysiology

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Whole-cell patch-clamp recordings of isolated flat-mount retinae were performed under current-clamp using a Multiclamp 700B amplifier, Digidata 1550 digitizer, and pClamp 10.5 data acquisition software (Molecular Devices; 10 kHz sampling). Pipettes were pulled from thick-walled borosilicate tubing (P-97; Sutter Instruments). Tip resistances were 4–8 MΩ when filled with internal solution, which contained (in mM): 120 K-gluconate, 5 NaCl, 4 KCl, 2 EGTA, 10 HEPES, 4 ATP-Mg, 7 phosphocreatine-Tris, and 0.3 GTP-Tris, pH 7.3, 270–280 mOsm). We added red fluorescent dye (Alexa Fluor 568; Invitrogen) for visual guidance during two-photon imaging and intracellular dye-filling.
Selected calcium-imaged cells were dye-filled (Alexa Fluor 568 hydrazide; Invitrogen) using fine glass pipettes (~50 MΩ resistance) guided by two-photon imaging. A current pulse (-20 nA; 100 ms) triggered cell penetration. Dye was iontophoretically injected (20 - 60 biphasic current pulses; ‐2000 pA for 500 ms and +500 pA for 400 ms) until dendrites were well filled. After all calcium imaging was completed, filled cells were documented in Z-stacks acquired in confocal (single-photon) mode.
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2

Two-Electrode Voltage Clamp Analysis of mRNA-Injected Oocytes

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One day after mRNA injection, oocytes were placed one at a time in a custom-built chamber (35 (link)), perfused with bath solution (in mm, 96 NaCl, 2 KCl, 1.8 BaCl2, and either 5 mm HEPES to pH 7.0, 7.4, 8.5, or 9.2 with NaOH/HCl or 5 mm MES to pH 5.8 with NaOH/HCl) for two-electrode voltage clamp experiments. l-Glutamate (dissolved in bath solution) was applied for ∼5 s with 1 min between subsequent applications using a ValveBank 8 perfusion system (AutoMate Scientific). Ivermectin was applied for longer until saturating current response was observed. Oocytes were clamped at −60 mV, and currents were recorded with microelectrodes filled with 3 m KCl, OC-725C amplifier (Warner Instruments), and Digidata 1550 digitizer (Molecular Devices) at 1 kHz with 200-Hz filtering. Peak current responses to l-glutamate were later analyzed in Clampfit 10 (Molecular Devices) with 10-Hz filtering for illustration. Peak current responses were plot against glutamate concentration using the four-parameter Hill equation in Prism 6 (GraphPad), and parameters were compared statistically (tests described in Table 1) using Prism 6.
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3

Electrophysiological Characterization of Locus Coeruleus

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Locus coeruleus was recognized by its proximity to the fourth ventricle bordering lateral pons and all LC neurons had a characteristic pacemaking activity. Whole cell recordings were performed at current-clamp mode using a MultiClamp 700B Amplifier (Molecular Devices). Signals were low-pass filtered at 3 kHz and digitized at 10 kHz through Digidata 1550 digitizer (Molecular Devices). Patch electrodes were fabricated from borosilicate glass capillaries (Sutter Instruments) using a P-97 micropipette puller (Sutter Instruments). Patch pipettes had a resistance of 3–8 MΩ filled with patch pipette internal solution consisting of (in mM): 125.0 KCl, 2.8 NaCl, 10.0 HEPES, 2.0 MgCl2, 2.37 ATP-Mg, 0.32 GTP-Na, 0.6 EGTA, (pH 7.23–7.28, 270–278 mOsm). Capacitance and membrane resistance were calculated from voltage response to an injection of −100 pA. For constructing averaged action potential, action potentials within pacemaking activity in each cell were detected and averaged. All electrophysiological recordings were analyzed using Clampfit software.
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4

Patch-clamp Analysis of Induced Neuronal Cells

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Whole-cell patch-clamp recordings were performed 28-35 days post-induction (PID) with miR-9/9*-124-CDM. At PID 14, cells undergoing reprogramming were transduced with pSYNAPSIN tRFP or GFP, and the next day trypsinized and plated together on top of rat primary neurons and glia isolated from perinatal pups with the exception of recordings shown in Supplementary Fig. 3h which were performed in monoculture in the absence of rate primary cells. Fluorescent reporter expression was visible within days, and remained segregated for each population. Data was acquired using pCLAMP 10 software with multiclamp 700B amplifier and Digidata 1550 digitizer (Molecular Devices). Electrode pipettes were pulled from borosilicate glass (World Precision Instruments) and typically ranged between 4–6 MΩ resistance. Solutions used to study intrinsic neuronal properties were the same as previously reported (Victor et al., Neuron 2014). Post-synaptic potentials were detected spontaneously. Data was collected in Clampex and initially analyzed in Clampfit (Molecular Devices).
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5

Electrophysiological Characterization of iPSC-Derived Neurons

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Human 18a iPSC-derived neurons were seeded on coverslips in 24-well plates (200,000 cells per well) and co-cultured with mouse cortical astrocytes (CD-1, Charles River Laboratories) for 1 month. Whole-cell patch clamp recordings were performed using a Multiclamp 700B amplifier and a Digidata 1550 Digitizer (Molecular Devices). Data were collected using pClamp 10 software (Molecular Devices, Sunnyvale, CA), sampled at 10 kHz, and filtered at 1 kHz. External solution contained (in mM): 128 NaCl, 30 glucose, 25 HEPES, 5 KCl, 2 CaCl2, and 1 MgCl2 (pH 7.3). Patch pipettes were pulled from borosilicate glass using a P-1000 Micropipette Puller (Sutter Instrument). Pipette solution contained (in mM): 147 KCl, 5 Na2-phosphocreatine, 2 EGTA, 10 HEPES, and 2 MgATP, 0.3 Na2GTP (pH 7.3). The series resistance was typically 10–20 MΩ. To record voltage-dependent Na+ and K+ currents, the membrane potential was depolarized from −60 mV to 50 mV in 10 mV increments with a holding potential of −70 mV. Leak current was subtracted using an online P/8 protocol. Spontaneous action potentials were recorded without current injection under current-clamp mode.
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6

Whole-cell Patch-clamp Recordings of ICX Neurons

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Whole-cell patch-clamp recordings were made from visually identified cells in the ICX region using borosilicate glass pipettes (2–4 MΩ). For current-clamp experiments, pipettes were filled with K-methanesulfonate-based internal solution (135 mM CH3KO3S, 10 mM HEPES, 1 mM EGTA, 4 mM Mg-ATP, 0.4 mM Na-GTP, and 10 mM phosphocreatine; 310 mOsm) containing 0.5% biocytin (Thermo Fisher). For voltage-clamp experiments, a Cs-methanesulfonate-based internal (120 mM CH3CsO3S, 15 mM CsCl, 8 mM NaCl, 10 mM TEA-Cl, 10 mM HEPES, 0.5 mM EGTA, 2 mM QX314, 4 mM Mg-ATP, and 0.3 mM Na-GTP; 310 mOsm) was used. All chemicals were from Sigma. Recordings were acquired using a Multiclamp 700B amplifier (Molecular Devices), digitized at 20 kHz with a Digidata 1550 digitizer (Molecular Devices), and low-pass filtered at 8 kHz. Evoked EPSCs were elicited in cells voltage-clamped at –70 mV in response to a brief electrical stimulus (0.2 ms, 20–50 μA) delivered through a concentric bipolar stimulating electrode (FHC). The stimulating electrode was placed locally in the ICCls. Synaptic blockers (bicuculine: 20 μM; NBQX: 10 μM; both from Sigma) were washed in during recordings, as indicated. Slices were immersion-fixed in 4% paraformaldehyde and stained with SA-488 for STED imaging.
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7

Patch-clamp Analysis of Induced Neuronal Cells

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Whole-cell patch-clamp recordings were performed 28-35 days post-induction (PID) with miR-9/9*-124-CDM. At PID 14, cells undergoing reprogramming were transduced with pSYNAPSIN tRFP or GFP, and the next day trypsinized and plated together on top of rat primary neurons and glia isolated from perinatal pups with the exception of recordings shown in Supplementary Fig. 3h which were performed in monoculture in the absence of rate primary cells. Fluorescent reporter expression was visible within days, and remained segregated for each population. Data was acquired using pCLAMP 10 software with multiclamp 700B amplifier and Digidata 1550 digitizer (Molecular Devices). Electrode pipettes were pulled from borosilicate glass (World Precision Instruments) and typically ranged between 4–6 MΩ resistance. Solutions used to study intrinsic neuronal properties were the same as previously reported (Victor et al., Neuron 2014). Post-synaptic potentials were detected spontaneously. Data was collected in Clampex and initially analyzed in Clampfit (Molecular Devices).
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8

Electrophysiological Characterization of Locus Coeruleus

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Locus coeruleus was recognized by its proximity to the fourth ventricle bordering lateral pons and all LC neurons had a characteristic pacemaking activity. Whole cell recordings were performed at current-clamp mode using a MultiClamp 700B Amplifier (Molecular Devices). Signals were low-pass filtered at 3 kHz and digitized at 10 kHz through Digidata 1550 digitizer (Molecular Devices). Patch electrodes were fabricated from borosilicate glass capillaries (Sutter Instruments) using a P-97 micropipette puller (Sutter Instruments). Patch pipettes had a resistance of 3–8 MΩ filled with patch pipette internal solution consisting of (in mM): 125.0 KCl, 2.8 NaCl, 10.0 HEPES, 2.0 MgCl2, 2.37 ATP-Mg, 0.32 GTP-Na, 0.6 EGTA, (pH 7.23–7.28, 270–278 mOsm). Capacitance and membrane resistance were calculated from voltage response to an injection of −100 pA. For constructing averaged action potential, action potentials within pacemaking activity in each cell were detected and averaged. All electrophysiological recordings were analyzed using Clampfit software.
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9

Two-Photon Imaging and Patch-Clamp Electrophysiology

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Whole-cell patch-clamp recordings of isolated flat-mount retinae were performed under current-clamp using a Multiclamp 700B amplifier, Digidata 1550 digitizer, and pClamp 10.5 data acquisition software (Molecular Devices; 10 kHz sampling). Pipettes were pulled from thick-walled borosilicate tubing (P-97; Sutter Instruments). Tip resistances were 4–8 MΩ when filled with internal solution, which contained (in mM): 120 K-gluconate, 5 NaCl, 4 KCl, 2 EGTA, 10 HEPES, 4 ATP-Mg, 7 phosphocreatine-Tris, and 0.3 GTP-Tris, pH 7.3, 270–280 mOsm). We added red fluorescent dye (Alexa Fluor 568; Invitrogen) for visual guidance during two-photon imaging and intracellular dye-filling.
Selected calcium-imaged cells were dye-filled (Alexa Fluor 568 hydrazide; Invitrogen) using fine glass pipettes (~50 MΩ resistance) guided by two-photon imaging. A current pulse (-20 nA; 100 ms) triggered cell penetration. Dye was iontophoretically injected (20 - 60 biphasic current pulses; ‐2000 pA for 500 ms and +500 pA for 400 ms) until dendrites were well filled. After all calcium imaging was completed, filled cells were documented in Z-stacks acquired in confocal (single-photon) mode.
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10

Somatosensory Cortex Pyramidal Neuron Electrophysiology

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Whole-cell recordings of pyramidal neurons in the somatosensory cortex were acquired in acute coronal brain slices. Borosilicate glass pipettes (BF100–58-10, Sutter Instrument) with resistances ranging from 5 to 8 mΩ were pulled using a laser micropipette puller (P-1000, Sutter Instrument). For sEPSCs, the pipette was filled with an internal solution containing 135 mm K-gluconate, 4 mm KCl, 2 mm NaCl, 10 mm HEPES, 4 mm EGTA, 4 mm Mg-ATP, 0.3 mm Na-GTP, pH adjusted to 7.2 with KOH (278–285 mOsmol). For spontaneous IPSCs, the pipette was filled with an internal solution containing 135 mm CsCl, 4 mm NaCl, 0.5 mm CaCl2, 10 mm HEPES, 5 mm EGTA, 2 mm Mg-ATP, 0.5 mm Na2-GTP, 10 mm QX-314, of which the pH had been adjusted to 7.2 with CsOH (278–285 mOsmol). During voltage-clamp experiments, neurons were clamped at either −70 or 0 mV to measure EPSCs or IPSCs, respectively. Whole-cell voltage-clamp recordings were performed using a MultiClamp 700B amplifier (Molecular Devices), filtered at 2 kHz, and digitized at 20 kHz using a Digidata 1550 digitizer (Molecular Devices).
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