Optogenetic Stimulation and Electrophysiological Recording of Synaptic Events in Rat Brain Slices

Begin with a rat brain slice containing the target neuron region connected with a light-sensitive axon from a different brain region.

Place the slice into the submerged recording chamber and secure it with a slice anchor.

Under a microscope, locate the desired layer and identify the target pyramidal neuron.

Raise the microscope objective to create a meniscus with the slice surface.

Lower a glass micropipette filled with intracellular recording solution into this meniscus.

Touch the identified pyramidal cell with the pipette tip to create an indent in the membrane.

Apply suction to form a seal.

Increase the suction until the membrane ruptures, establishing a whole-cell configuration.

Using a mounted light source, apply light pulses to the slice.

These light pulses trigger an action potential in the light-sensitive axon, releasing neurotransmitters at the synaptic cleft.

Record the pyramidal neuron's responses to these releases to analyze synaptic efficiency.

For target cell identification, place the slice into the recording chamber, and immobilize it using a slice anchor. Under low magnification, using infrared illumination, navigate to the LEC layer 5. Then, change to the high magnification water-immersion objective, and identify the pyramidal neurons.

Mark the position of the cell on the monitor with tape. Next, to form a whole-cell patch clamp, fabricate a borosilicate glass micropipette, and fill it with intracellular recording solution. Place the filled micropipette in the electrode holder, and apply positive pressure by blowing hard into a mouthpiece connected to the electrode holder side port.

Raise the microscope objective such that a meniscus forms, and insert the electrode into the meniscus until it can be seen on the microscope. Open the seal test window, and determine whether the pipette resistance is three to five megaohms. Then, touch the identified cell with a pipette tip resulting in an indentation in the cell membrane.

Next, apply negative pressure by suction at the mouthpiece, increasing pipette resistance. Continue applying negative pressure gradually until the cell membrane ruptures, resulting in whole-cell capacitance transients. To enter the current-clamp configuration, use a mounted LED directed into the microscope light path with filter cubes and appropriate optics, and apply light pulses to the slice via the 40x objective. Deliver trains of multiple light pulses at 5 Hertz, 10 Hertz, and 20 Hertz to investigate the presynaptic release properties.