multiclamp 700b software, by Molecular Devices - Product details

1

Calcium Imaging of Retinal Cells

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All physiological animal procedures were conducted in accordance with US National Institutes of Health guidelines, as approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee (ASP 1361). Both male and female adult (p30 – p60) ChAT-tdTomato mice were used in the experiments (Jackson Laboratory). The mice were anaesthetized with Isoflurane (Baxter) inhalation and killed by cervical dislocation. Retinas were isolated and all subsequent procedures were performed at room temperature in Ames media (Sigma) equilibrated with 95% O₂/5% CO₂. Sharp electrodes were pulled on a P-97 Micropipette Puller (Sutter) with a resistance of 100–150 MOhms. Iontophoresis of Oregon Green 488 BAPTA-1 (OGB1, Life Technologies) into single cells was achieved by applying the buzz function in MultiClamp 700B software at 50ms pulses (Molecular Devices) while the electrode filled with OGB1 (15mM in water) was on the cell membrane. Pipettes were withdrawn as soon as cell bodies began to fill, and cells were left to recover for 20–30 min prior to imaging. To block inhibition, the GABA_A receptor antagonist SR95531 (25 μM, Tocris) was added to the extracellular medium.

Ding H., Smith R.G., Poleg-Polsky A., Diamond J.S, & Briggman K.L. (2016). Species-specific wiring for direction selectivity in the mammalian retina. Nature, 535(7610), 105-110.

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2

Dye-Filling and Morphological Tracing of Retinal Cells

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Sharp electrodes were pulled on a P-1000 micropipette puller (Sutter Instruments, Hofheim, Germany) with resistances >100 mΩ. Single cells in the inner nuclear layer were dye-filled with 10 mM Alexa Fluor 555 (Life Technologies, Waltham, USA) in a 200 mM potassium gluconate (Sigma-Aldrich, Steinheim am Albuch, Germany) solution using the “buzz” function (50 ms pulse) of the MultiClamp 700B software (Molecular Devices, Biberach, Germany). Pipettes were carefully retracted as soon as the cell began to fill. Approximately 20 minutes were allowed for the dye to diffuse throughout the cell before imaging stated. After recording, an image stack was acquired to document the cell’s morphology, which was then traced semi-automatically using the Simple Neurite Tracer plugin implemented in Fiji (http://Fiji.sc/Fihi).

Franke K., Berens P., Schubert T., Bethge M., Euler T, & Baden T. (2017). Inhibition decorrelates visual feature representations in the inner retina. Nature, 542(7642), 439-444.

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3

Calcium Imaging of Retinal Cells

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All physiological animal procedures were conducted in accordance with US National Institutes of Health guidelines, as approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee (ASP 1361). Both male and female adult (p30 – p60) ChAT-tdTomato mice were used in the experiments (Jackson Laboratory). The mice were anaesthetized with Isoflurane (Baxter) inhalation and killed by cervical dislocation. Retinas were isolated and all subsequent procedures were performed at room temperature in Ames media (Sigma) equilibrated with 95% O₂/5% CO₂. Sharp electrodes were pulled on a P-97 Micropipette Puller (Sutter) with a resistance of 100–150 MOhms. Iontophoresis of Oregon Green 488 BAPTA-1 (OGB1, Life Technologies) into single cells was achieved by applying the buzz function in MultiClamp 700B software at 50ms pulses (Molecular Devices) while the electrode filled with OGB1 (15mM in water) was on the cell membrane. Pipettes were withdrawn as soon as cell bodies began to fill, and cells were left to recover for 20–30 min prior to imaging. To block inhibition, the GABA_A receptor antagonist SR95531 (25 μM, Tocris) was added to the extracellular medium.

Ding H., Smith R.G., Poleg-Polsky A., Diamond J.S, & Briggman K.L. (2016). Species-specific wiring for direction selectivity in the mammalian retina. Nature, 535(7610), 105-110.

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4

Characterization of Claustrum Projection Neurons

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Whole-cell recordings were performed at 29–31°C using borosilicate glass recording pipettes of 3–7Ω MU resistance. For recordings performed in a current clamp configuration, recording pipettes were filled with a potassium-based solution (290–295 mOsm; pH 7.3) composed of 126 mM potassium gluconate, 4 mM KCl, 10 mM HEPES, 4 mM ATP-Mg, 0.3 mM GTP-Na and 10 mM phosphocreatine. Clampex software (Version 10.4; Molecular Devices) was used for all electrophysiological recordings. Recordings were filtered at 2 kHz and digitized at 10 kHz using MultiClamp 700B software (Molecular Devices). Claustrum projection neuron type was determined via a 5 ms depolarization step while recording in current-clamp mode to determine burst firing properties (Type I: no burst fire; Type II: burst fire). Following this protocol, membrane capacitance values were also recorded to confirm the characterization of neuron type (Type I: ~75–130 pF; Type II: ~130–200 pF).^{27 (link)} For all slice electrophysiology experiments, three 5 ms 470nm blue light pulses with 150 ms intervals were given to evoke presynaptic transmitter release while recording from fluorescently labeled claustrum projections.^{67 (link)}

Qadir H., Stewart B.W., VanRyzin J.W., Wu Q., Chen S., Seminowicz D.A, & Mathur B.N. (2022). The mouse claustrum synaptically connects cortical network motifs. Cell reports, 41(12), 111860.

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5

Dye-Filling and Morphological Tracing of Retinal Cells

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Sharp electrodes were pulled on a P-1000 micropipette puller (Sutter Instruments, Hofheim, Germany) with resistances >100 mΩ. Single cells in the inner nuclear layer were dye-filled with 10 mM Alexa Fluor 555 (Life Technologies, Waltham, USA) in a 200 mM potassium gluconate (Sigma-Aldrich, Steinheim am Albuch, Germany) solution using the “buzz” function (50 ms pulse) of the MultiClamp 700B software (Molecular Devices, Biberach, Germany). Pipettes were carefully retracted as soon as the cell began to fill. Approximately 20 minutes were allowed for the dye to diffuse throughout the cell before imaging stated. After recording, an image stack was acquired to document the cell’s morphology, which was then traced semi-automatically using the Simple Neurite Tracer plugin implemented in Fiji (http://Fiji.sc/Fihi).

Franke K., Berens P., Schubert T., Bethge M., Euler T, & Baden T. (2017). Inhibition decorrelates visual feature representations in the inner retina. Nature, 542(7642), 439-444.

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6

Characterization of Claustrum Projection Neurons

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Whole-cell recordings were performed at 29–31°C using borosilicate glass recording pipettes of 3–7Ω MU resistance. For recordings performed in a current clamp configuration, recording pipettes were filled with a potassium-based solution (290–295 mOsm; pH 7.3) composed of 126 mM potassium gluconate, 4 mM KCl, 10 mM HEPES, 4 mM ATP-Mg, 0.3 mM GTP-Na and 10 mM phosphocreatine. Clampex software (Version 10.4; Molecular Devices) was used for all electrophysiological recordings. Recordings were filtered at 2 kHz and digitized at 10 kHz using MultiClamp 700B software (Molecular Devices). Claustrum projection neuron type was determined via a 5 ms depolarization step while recording in current-clamp mode to determine burst firing properties (Type I: no burst fire; Type II: burst fire). Following this protocol, membrane capacitance values were also recorded to confirm the characterization of neuron type (Type I: ~75–130 pF; Type II: ~130–200 pF).^{27 (link)} For all slice electrophysiology experiments, three 5 ms 470nm blue light pulses with 150 ms intervals were given to evoke presynaptic transmitter release while recording from fluorescently labeled claustrum projections.^{67 (link)}

Qadir H., Stewart B.W., VanRyzin J.W., Wu Q., Chen S., Seminowicz D.A, & Mathur B.N. (2022). The mouse claustrum synaptically connects cortical network motifs. Cell reports, 41(12), 111860.

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Multiclamp 700b software

Lab products found in correlation

6 protocols using multiclamp 700b software

Calcium Imaging of Retinal Cells

Dye-Filling and Morphological Tracing of Retinal Cells

Calcium Imaging of Retinal Cells

Characterization of Claustrum Projection Neurons

Dye-Filling and Morphological Tracing of Retinal Cells

Characterization of Claustrum Projection Neurons

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