The overall goal of this procedure is to record odorant induced responses from mouse olfactory receptor neurons. This is accomplished by first dissecting out the olfactory epithelium and isolating the mouse olfactory receptor neurons. The second step is to place the isolated olfactory receptor neurons in a recording chamber mounted on an inverted microscope.
Micro manipulators hold the recording electrode and a solution changer. Next, suck an identified olfactory receptor neuron into the tip of a suction pipette electrode. The final step is to place the pipette with the sucked cell in its tip in front of a solution changer to expose the neuron to odorants.
Ultimately, the suction pipette recording method is used to investigate olfactory receptor neuron function. The main advantage of this technique over existing methods like whole cell voltage clamp technique is that it allows for long recording durations using fast and precise solution changes. Visual demonstration of this method is critical as the shape of the recording pipette is important, and placing the socket cells stably in the tip of the pipette is difficult to learn.
Begin this procedure by pulling a micro pipette with an UNFI lamented bo silicate glass capillary in a micro pipette puller with a long taper. Then transfer the pipette to a custom made micro pipette holder mounted on an upright microscope. The microscope has a redle in the eyepiece and a movable diamond knife mounted on the stage.
Next, observe the tip of the pipette under the 20 x objective. Use the redle as a guide. Gently scribe the pipette at a 90 degree angle using the custom made diamond knife at the outer diameter of the pipette of 10 micrometers.
Move the diamond knife further towards the tip. Gradually apply pressure on it. The pipette tip should break cleanly at the point where it was scribed.
Then under a 40 x objective fire polish the tip of the pipette to an inner diameter of five micrometers using an electrically heated filament. Once finished, the micro pipette is ready to be used for recording currents from the mouse orns. In the next step, sacrifice a mouse and remove its head.
Then peel away the skin overlying the skull, subsequently bisect the head along the midline. Next, transfer the two Hemi headss to a dissecting stereo microscope. Pull off the nasal septum and remove the olfactory turbinates.
Place the tissue in a Petri dish with glucose containing mammalian ringer solution. After that, peel the olfactory epithelium off the underlying cartilage from two turbinates. Transfer the tissue into an einor tube containing 250 microliters of ringer.
Then store the remaining tissue at four degrees Celsius for later. Use vortex the EOR tube containing the olfactory epithelium twice briefly for one second at medium speed. This step leads to the mechanical dissociation of the NS from the epithelium.
Next place the ringer solution containing the dissociated orns in the recording chamber mounted on the microscope, remove the large pieces of tissue in the suspension using a pair of fine forceps. Let the ORN settle for 20 minutes before beginning the continuous perfusion with ringer solution and proceeding to the recordings in this procedure, fill the pipette with ringer solution. Place the pipette in the pipette holder and attach the holder to the patch clamp amplifier head stage.
Next, connect the oil line to the side port of the pipette holder. Then lower the pipette into the recording chamber. Regulate the height of the oil reservoir to establish a slightly positive pressure at the pipette tip.
This can be done by observing the cell debris moving away from or towards the pipette. Try not to contaminate the tip of the pipette with cell debris. Next, scan the recording chamber for an isolated ORN, which can be recognized by its typical bipolar morphology using a magnification of 20 to 40 x.
Then move the recording electrode to close proximity of the ORN cell body. Gently suck on the airline connected to the oil reservoir so that the cell body enters into the tip of the pipette. Continue to apply suction carefully and slowly until the entire cell body is drawn into the tip of the suction pipette, leaving the dendrite and cilia exposed to the bath solution.
Move the suction pipette from the section containing the settled orns to the recording section of the chamber. Carefully place the suction pipette in front of the three barreled tube for solution exchange. The suction pipette should be placed close enough to the opening of the three barreled tube so that the ORN is exposed to laminar flow and thus avoiding mixing turbulence To achieve solution exchange, move the boundary between the parallel streams of flowing solution from the three barrel tube of the solution changer across the tip of the suction pipette to monitor the quality and reliability of solution exchange.
The junction current evoked by moving the cell between solutions of different ionic composition can be measured. The time course of the solution exchange is typically around 20 milliseconds. When the olfactory receptor neuron is stepped into an odorant solution, an odorant evoked response can be elicited here.
An ORN was exposed to a hundred micro molars eugenol for one second as indicated by the bar above the recording. In the upper panel, the receptor current was filtered at a bandwidth of zero to 50 hertz to display the receptor current Only the lower panel shows the same recording filtered at the wide bandwidth of zero to 5, 000 hertz to also display the action potentials. Here are the dose responses of a eugenol responsive ORN Eugenol was used at the concentrations ranging from 0.3 micromolar to a hundred micromolar.
The stimulus duration was one second, and traces were filtered at zero to 50 hertz. A progressive increase in odorant concentration led to a larger and faster rising response, which also terminated more slowly. Once odorant exposure was terminated, ORNS can display an oscillatory response pattern during long stimulations at intermediate odorant concentrations.
Here, an acetophenone responsive ORN was stimulated for eight seconds After a fast and transient peak response at the onset of stimulation. A series of smaller recurring responses are observed While attempting this procedure. It is important to remember to handle olfactory receptor neurons carefully and to be very patient.
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