Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animals

1. Use genetically engineered OR-IRES-tauGFP (OR = Olfactory receptor, IRES = Internal ribosome entry site, tauGFP = tau-green fluorescent protein) mice available at the Jackson Laboratory. These mice were developed in Dr. Peter Mombaerts' laboratory in order to analyze axon targeting and development of the olfactory system. For example, the MOR23-IRES-tauGFP line, stock number 006643, bears the official strain name B6;129P2-Olfr16tm2Mom/MomJ; similarly, the SR1-IRES-tauGFP line, stock number 6717 bears the official name B6;129P2-Olfr124tm1Mom/MomJ.
2. Regarding the age of the animals: for a better outcome of the protocol, use animals between 2 and 4 weeks of age. In this age group, the dissection is easier (softer bones, firmer olfactory epithelium) and the dendritic knobs are bigger compared to older animals.

2. Preparation of Electrodes and Solutions

1. For the stimulating pipettes: purchase pre-pulled stimulating pipettes. Otherwise, manually prepare them.
   1. Using a flame, bend six 1 mm glass pipettes at about 1 cm from the tip. Insert these six pipettes plus a straight 7th in 1.5 cm heat-shrink tubing strengthened by an eyelet.
   2. Heat shrinks the tubing to maintain the barrels attached together. Attach an additional heat-shrink tubing to the other extremity of the barrels. Pull this stimulating pipette with a multi-barrel puller.
   3. Add some white liquid glue around the eyelet to strengthen the bent tips. Let dry overnight (O/N).
2. Prepare 1 or 2 L of normal Ringer's extracellular solution (in mM: NaCl 124, KCl 3, MgSO₄ 1.3, CaCl₂ 2, NaHCO₃ 26, NaH₂PO₄ 1.25, glucose 15; pH 7.6 and 305 mOsm). Keep at 4 °C until use.
3. Prepare intracellular stock solution (in mM): KCl 70, KOH 53, methanesulfonic acid 30, ethylene glycol tetraacetic acid (EGTA) 5, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, sucrose 70; pH 7.2 (KOH) and 310 mOsm. Keep at 4 °C until use. Good for several weeks.
4. Prepare intracellular recording solution with nystatin extemporaneously (at the last minute) before experiment.
   1. Weigh 3 mg of nystatin, add 50 µl of dimethyl sulfoxide (DMSO); vortex 20 sec then sonicate 2-3 min until entirely diluted.
   2. Add 20 µl of DMSO-nystatin solution in 5 ml of intracellular stock solution. Vortex 20 sec, then sonicate 3 min. Keep this solution at 4 °C and protect from direct light, nystatin is light sensitive. This solution can be used for a few hours. Replace every day.
   3. Once the olfactory epithelium preparation is under the microscope, take some of this solution in a 1 ml syringe with a flame-elongated yellow tip to fill the electrodes; protect from direct light. Once at room temperature (RT), the nystatin solution is not stable; replace the solution in the 1 ml syringe every hour or keep it in ice.
5. Pull recording electrodes with a puller to obtain long neck and small tip (~2 µm) with a resistance of 15-20 MΩ with the internal nystatin solution.
6. Prepare odorant solution at 0.5 M in DMSO under fume hood; aliquot and keep at -20 °C until use. Dilute odorant in Ringer's solution until the desired concentration. Fill the stimulating pipette.

3. Preparation of Olfactory Epithelium

Note: OR-IRES-tauGFP mice express the tauGFP under the control of the OR promoter. In these mice, all neurons expressing the OR of interest are labeled with GFP. This protocol is adapted for ORs expressed in all zones. However, dissections and recordings are easier for ORs expressed in the dorsal zone.

1. Anesthetize the animal by injecting a mix of ketamine and xylazine (150 mg/kg and 10 mg/kg body weight, respectively). Decapitation can be performed with sharp scissors for young mice or with a properly maintained rodent guillotine for older mice.
   1. Using ring dissecting scissors make a longitudinal medial incision through the dorsal skin. Remove the skin by pulling it apart. Using the scissors, cut the lower jaws at the jaw joint. Remove the upper front teeth by a coronal cut parallel to the teeth.
   2. Make a coronal cut of the head behind the eyes and keep only the anterior part of the head. Dip it in an ice-cold Ringer solution for the dissection under the scope.
2. Dissect under the scope: in the ventral side, make a longitudinal cut along the upper jaw/the teeth. Cut the dorsal bones longitudinally, following the dorsolateral side of the nasal cavity. Remove most bones and palate; transfer the septum and the epithelium attached to it in oxygenated Ringer at RT.
3. For the final dissection: Right before starting the recording session, peel away the epithelium from the underlying septum with forceps. Detach the epithelium with forceps and with two 4-5 mm scissor cuts (use micro-vannas scissors) at the anterior part of the septum where the adhesion is stronger.
   1. Carefully remove the vomeronasal organ by cutting it out along its dorsal connection to the septal epithelium. Transfer the epithelium to a recording chamber with the mucus layer facing up; keep it flat in the chamber with a harp.
4. Install chamber under an upright microscope equipped with fluorescence optics and a sensitive camera. Visualize the preparation on the computer screen at high magnification through a 40X water-immersion objective (numerical aperture 0.8) and an extra 2X or 4X magnification achieved by a magnifying lens. Perfuse continuously with oxygenated Ringer at RT (1-2 ml/min).

4. Recording Session

1. Search for fluorescent cells: excite the preparation at 480 nm for EGFP, which emits light in the 530-550 nm range; target one dendritic knob which is reliably visible in fluorescence and under bright field.
2. Fill the electrode with intracellular solution with nystatin; remove bubbles by gently tapping on the electrode.
3. Insert electrode on electrode holder, apply positive pressure in the pipette; Resistance should be 15-20 MΩ.
4. Bring electrode close to the cell; once resistance reaches ~40 MΩ, release positive pressure and apply slight negative pressure to reach a gigaseal.
5. Once seal is reached, set the membrane potential at about -75 mV;
6. Once the cell is opened, proceed with stimulation protocols, pharmacological treatments. To measure the response to a single odorant stimulation, record 200-500 msec of spontaneous activity, stimulate for 500 msec and measure the response of the cell for up to 10 sec. For pharmacological treatments, perfuse the pharmacological agents at the desired concentration.