Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice
Transcrição
Take the severed head of a genetically engineered mouse. The olfactory epithelium in its nose contains olfactory sensory neurons, or OSNs, with fluorophore-tagged odor receptors.
Remove the skin and dissect the head to isolate the septum with attached olfactory epithelium.
Separate and secure the epithelium in a recording chamber.
Using a fluorescence microscope, detect the labeled receptors to target the OSNs' dendritic knob.
Bring a recording pipette with an intracellular solution containing nystatin, a membrane-perforating antibiotic.
Apply positive pressure to avoid pipette clogging. Insert the pipette into the epithelium and approach an OSN.
Switch to negative pressure, forming a seal with the cell membrane. Apply current to maintain the membrane at the resting potential.
Nystatin perforates the membrane, allowing measurement of intracellular ionic currents.
Position a pipette containing an odor stimulant near the OSN. Release the stimulant, which binds to the odor receptors, triggering ion channel opening to allow ion influx. Record the resulting electrophysiological signals.
