This article describes a method for electrophysiological recording from sensory neurons in the vomeronasal organ (VNO) of transgenic mice. The technique involves preparing a coronal slice of the VNO and utilizing whole-cell patch clamp to study the neurons' responses to chemical stimuli.
Take a coronal slice of the chemosensory vomeronasal organ from a transgenic mouse expressing fluorescently labeled sensory neurons.
The sensory epithelium contains these labeled neurons, which are exposed to the lumen connected to the nasal cavity for sensing chemical cues.
Secure the slice in an imaging chamber using a slice anchor.
Transfer the chamber to a recording setup and flow oxygenated extracellular solution to maintain tissue viability.
Position a perfusion pipette near the slice to introduce chemical stimuli and a recording pipette over the sensory neurons.
Using a fluorescence microscope, identify the labeled neurons.
Apply positive pressure to prevent pipette clogging, and approach a neuron until a dimple forms on the membrane.
Switch to negative pressure, drawing the membrane into the pipette.
Apply suction to rupture the membrane and establish continuity with the cytoplasm, called whole-cell patch clamp, allowing the introduction of chemical stimuli and recording the resulting electrophysiological signals.
Transfer a VNO slice to the imaging chamber, and fix the slice position using stainless steel anchor wired with 0.1-millimeter thick synthetic fibers. Do not cover the tissue slice with any of the synthetic fiber threads. Next, transfer the imaging chamber to the recording setup, and continuously superfuse the slice with oxygenated S2 at room temperature.
Adjust the suction capillary to the surface of the solution to create a constant exchange of bath solution. Then, adjust the 8-in-1 multi-barrel perfusion pencil above and close to the non-sensory part of the VNO slice that contains the blood vessel. Afterward, connect the reference electrode and bath solution using the L-shaped agar bridge filled with 150 millimolar potassium chloride.
Subsequently, fill the patch pipette with pipette solution S4. Mount the pipette over the silver chloride-coated electrode connected to the head stage without scraping off the coating, and attach firmly. Then, apply slight positive pressure to the patch pipette before entering the bath. Monitor the pipette, and make sure that it is between four megaohms and 10 megaohms.
Visualize the VNO slice with a CCD camera using infrared-optimized DIC, and identify FPR-rs3-i-Venus-expressing cells, or similarly labeled neurons using fluorescence illumination, and an appropriate filter cube. Approach the cell body using handwheels for maximum sensitivity. Due to the positive pressure, a small dent on the cell soma membrane becomes visible, once the pipette tip is in close proximity.
Then, release the positive pressure and apply slight negative pressure to suck in the cell membrane, in order to gain a high resistance seal of 1 to 20 giga-ohms. Subsequently, apply short and gentle suction to disrupt the cell membrane and establish the whole-cell configuration.
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