This article details a method for recording intracellular calcium levels in neurons using whole-cell patch-clamp techniques. The process involves preparing a mouse brain slice, loading it with a calcium indicator, and utilizing a recording pipette to establish a tight seal on target neurons.
Secure a mouse brain slice in a recording chamber, perfused with oxygenated aCSF after prolonged incubation.
The cells in the slice are loaded with a calcium indicator, facilitating intracellular calcium measurements.
Take a recording pipette comprising an electrode in an electrolyte solution.
Identify a target neuron. Position the recording pipette with positive pressure on the cell membrane, creating a dimple.
Apply negative pressure to create a tight seal.
Further, apply negative pressure pulses, rupturing the membrane and establishing a whole-cell configuration .
Perfuse aCSF with a high potassium concentration, which depolarizes the neuron's membrane.
Depolarization opens sodium channels, triggering an action potential, which in turn activates voltage-gated calcium channels and allows calcium influx.
Calcium ions bind to the indicator, enhancing fluorescence, which can be visualized under suitable illumination.
The increase in intracellular fluorescence, caused by calcium influx following potassium application, indicates neuronal viability after prolonged incubation.
For recording, place tissue in a submerged recording chamber under a microscope, and perfuse it with oxygenated aCSF at a flow rate of 4 to 5 milliliters per minute. Hold the tissue in by using a custom-made harp. Next, prepare some recording pipettes using a micropipette puller to achieve a final resistance of 5 to 6 mega ohms.
Fill the pipette with 3 to 4 microliters of internal solution, and then place the solution on ice. Then, visualize the cells using a CCD camera under IR-DIC. Position the pipette on the cell membrane using a micromanipulator. Maintain positive pressure through a suction port on the pipette holder. Once the pipette is on the cell, apply gentle negative pressure to the pipette to achieve a gigaohm seal.
Then, rupture the cell membrane with brief negative pressure. Subsequently, start whole-cell current or voltage clamp recording. For a single excitation wavelength of flow 4, filter the excitation light through a 460- to 490-nanometer bandpass filter and emitted light through a 515- to 550-nanometer bandpass filter.
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