Basic Intro to Whole Cell Patch Clamp Electrophysiology

Mix regular ACSF and sucrose ACSF solutions with essential salts, ions, sodium pyruvate, and L-ascorbic acid to maintain neuronal health. Verify pH is between 7.3-7.4 and osmolarity is approximately 10 mOsm higher than the internal solution.

Formulate internal solution containing cesium (potassium channel blocker), EGTA (calcium chelator), HEPES buffer, energy sources, QX314 bromide (sodium channel blocker), and picrotoxin (GABA-A antagonist). Ensure osmolarity is approximately 10 mOsm lower than external ACSF.

Remove the brain by incising the skull and extracting it carefully into ice-chilled carbogen-bubbled ACSF. Remove the cerebellum and prefrontal cortex, split hemispheres along the midline, and angle-cut a section of dorsal cerebrum.

Mount prepared hemispheres onto the vibratome chuck and carefully obtain 400-micron slices. Transfer slices to an incubation chamber with carbogen-bubbled ACSF, starting at 32°C and slowly cooling to room temperature over 45 minutes to one hour.

Start the ACSF perfusion at 2-3 mL/min with carbogen bubbling and turn on all hardware including pumps, amplifiers, and heating boxes set to 32°C. Initialize the MultiClamp and ClampFit software for live data acquisition.

Fabricate a custom pipette filler by heating and stretching a pipette tip to create a fine diameter, then assemble with syringe and filter. Pull glass capillaries using a pipette puller to achieve 3-8 MΩ tip resistance and fill with intracellular solution avoiding air bubbles.

Attach the filled recording pipette to the electrode holder, place a brain slice in the chamber, and secure with a weight. Switch from 5× to 40× objective lens to visualize the tissue surface and identify healthy, viable pyramidal cells with circular morphology for patching.