This article describes a method for investigating the connectivity between the amygdala and medial prefrontal cortex (mPFC) neurons in the mouse brain using optogenetics and patch-clamp techniques. The procedure involves preparing brain slices, visualizing neurons, and recording synaptic activity.
In the mouse brain, the amygdala contains neurons that form synaptic connections with the medial prefrontal cortex, or mPFC neurons.
Take an amygdala brain slice in which the mPFC neurons express a light-sensitive channel rhodopsin fused to a fluorescent protein.
Place the slice in a recording chamber and visualize it under a fluorescence microscope to locate the mPFC neurons.
Switch to a brightfield view and introduce a patch pipette containing an internal solution.
Apply positive pressure to advance the pipette toward an amygdala neuron.
Upon neuronal contact, a dimple forms.
Apply suction to form a tight seal.
Continue suction to rupture the membrane, establishing contact with the cell's interior.
Illuminate the slice to activate the mPFC neuronal channelrhodopsin, triggering positive ion influx, action potential generation, and neurotransmitter release.
The neurotransmitters bind to postsynaptic amygdala neuron receptors, causing ion influx and current generation.
Record the postsynaptic neuronal current to analyze mPFC-amygdala connectivity.
To prepare the patch microscope for optogenetic activation of fibers and cells, center the mounted light-emitting diode, or LED, onto the light delivery pathway. Use a power meter to measure the LED light intensity at the back focal plane, and at the output of each objective at a wavelength of 470 nanometers. Use a spreadsheet to calculate the light intensity in milliwatts per millimeter squared, and create a calibration curve for each objective.
Next, retrieve an acute amygdala slice from the interface chamber, and place it in the slice chamber mounted onto the microscope. Position the slice so that the slice surface facing upward in the interface chamber is also facing upward in the recording chamber. Perfuse the slice with fresh, oxygenated ACSF at a rate of 1 to 2 milliliters per minute. The temperature should be approximately 31 degrees Celsius. Turn on the fluorescent lamp, and select the appropriate filter sets for the specific fluorescent protein expressed. Use the 5x objective to obtain an overview.
Next, open or restrict the aperture in the microscope light pathway, as necessary for the experiment. To obtain a patch recording, fill a patch pipette with internal solution and mount it in the electrode holder. Apply positive pressure to the patch pipette, and slowly lower it into the bath solution. Then, under visual control, use the micromanipulator to lower the patch pipette into the slice. Approach the neuron of interest with the patch pipette from the side and top.
Release the positive pressure when the pipette reaches the surface of the cell, as indicated by a dimple visible on the cell's surface. Apply negative pressure to obtain a gigaseal. Apply further suction to rupture the membrane patch and obtain the whole-cell recording. Next, stimulate the labeled fibers with the connected LED by activating channelrhodopsin with 470-nanometer wavelength light while recording electrical responses from the cell.
For synaptic stimulation, use the digital outputs of the data acquisition software to trigger the LED. Adjust the LED stimulation intensity manually. Repeat the stimulation with an opened or restricted aperture as necessary for the next recorded cell, or in the presence of specific test substances.
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