Studying the Excitability of Fluorescent Neurons using a Whole-Cell Patch Clamp
Trascrizione
Take an immobilized transfected mouse coronal slice in a recording chamber perfused with aCSF.
The slice contains a sparse pyramidal neuron population expressing a target enzyme and a fluorescent protein.
Assemble a recording pipette comprising an intracellular solution and an electrode connected to an amplifier for measuring neuronal signals.
Microscopically identify the target tissue area and locate a fluorescent neuron.
Apply positive pressure to the pipette to prevent clogging and advance it to the target neuron.
The positive pressure causes a slight indentation on the neuronal membrane upon contact.
Release the pressure, forming a tight seal between the membrane and the tip.
Set the holding potential at a constant negative value to stabilize the neuron.
Apply brief negative pressure to rupture the membrane, connecting the cytoplasm to the pipette interior and establishing a whole-cell configuration.
Switch to current-clamp mode and apply positive current pulses, generating action potentials indicative of neuronal excitability.
