Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Take an immobilized transfected mouse brain slice in an electrophysiology recording chamber perfused with oxygenated aCSF.

This slice contains thalamic neurons' axon terminals expressing light-sensitive ion channels fused to a fluorescent protein.

Visualize the fluorescent thalamic neuron axons and select a pyramidal neuron.

Advance a recording pipette containing an electrode in an intracellular solution toward the selected neuron with slight positive pressure.

The positive pressure creates a dimple on the neuronal membrane upon contact.

Release the pressure to form a high-resistance seal.

Set the membrane potential to a constant negative value.

Apply a negative pressure pulse to rupture the membrane, achieving a whole-cell configuration.

Illuminate the axon terminals, activating the light-sensitive channels and depolarizing the neuronal membranes, triggering action potentials.

Excitatory neurotransmitters are released, binding to receptors on the recorded neuron, causing cation influx.

Record the resulting excitatory post-synaptic currents, indicating a direct synaptic connection between the neurons.

To perform whole-cell patch clamp recording, gently transfer a brain slice containing the hippocampal complex to the recording chamber. Continuously perfuse the recording chamber with 34 degrees Celsius ACSF bubbled with carbogen at 2 to 3 milliliters per minute. Briefly examine chronos GFP expression in axon terminals in the region of interest with blue LED illumination at 4x magnification.

Change to a 63x immersion objective and adjust the focus. Check for axons expressing Chronos GFP and choose a pyramidal neuron for patch recording. Next, fill a pipette with potassium gluconate-based internal solution. Mount it in the pipette holder on the head stage. Patch the cell in voltage clamp configuration.

Approach the identified neuron and delicately press the pipette tip onto the soma. The positive pressure should produce a dimple on the membrane surface. Then, release the pressure to create a giga ohm seal. Once sealed, set the holding voltage to minus 65 millivolts.

Break the membrane with a sharp pulse of negative pressure. Record in current or voltage clamp postsynaptic responses to 475 nanometer LED whole field stimulation of afferent fibers expressing chronos. Stimulate with trains of 10 stimulations of 2 millisecond duration at 20 hertz.