Our sense of smell allows us to detect and recognize volatile chemicals in the external environment. Ance bind to receptors in the city of olfactory sensory neurons and activate a transduction cascade that leads to the opening of cycl nucleotide gated channels followed by calcium activator chloride channels. The overall goal of this procedure is to show how to directly activate these channels by flesh Photo of cage compounds, cage compounds are molecules made physiologically inactive by a chemical cage.
A flesh of ultraviolet light breaks a cage and releases the physiologically active compound cages. Cycl, mp, or cage calcium are included in the intracellular solution, filling a patch, pipette, and diffuse to the C of olfactory sensory neurons. An ultra flash is applied to the Cary region to e engage a cycling, MP or calcium while patch clamp recordings are taken to measure the current and in the whole cell voltage clamp configuration.
Hi.Today we will show you a procedure for patch clamp recordings combined with FE policies of cage compounds. We use this procedure in our lab to study CMP gated channels and calcium activated choline channels in olfactory sensor neurons. So let's get started.
The patch clamp recording system used here includes an inverted microscope, a perfusion system, a micro manipulator, a recording electrode, and the reference electrode connected to the head stage of the patch clamp amplifier all mounted on an anti vibration table inside a Faraday cage. The patch clamp amplifier is connected to a digitizer in the computer with software for data acquisition. To generate a high intensity flash of ultraviolet light, we use excelon flash lamp.
The light flash can be triggered manually or by the computer using the same software that controls electrophysiological acquisition. The best wavelengths for photo experiments are selected inserting a UV filter into an output slot or the flash lamp. The intensity of the light flash can be modified by changing the voltage or the capacitance of the flesh lamp.
A more precise control of light intensity is obtained by placing neutral density filters into a second output slot of the flesh lamp. The light flash is directed to the neuron plated on glass bottom dishes through a quartz fiber optical light guide. The light guide is connected to the epi fluorescence attachment of the inverted microscope.
Using an adapter, a modified filter cube without excitation filter directs a UV bin to the neuron by decry mirror. The light is focused through a 100 x oil immersion objective. The area covered by the flesh is visualized by inserting the light guide into an illuminator.
The image is viewed on a monitor through a CCD camera connected to the microscope. The control of the area is drawn on the monitor to localize the UV flesh area for the experiments before the dissection, prepare three glass bottom dishes coated with conna a and poly. To prepare for the dissection fill two 50 millimeters sugar coated Petri dishes with ringer solution.
Place them on ice. Also chill. A papa insist mix 0.5 milliliter of the stop mix solution and the syringe containing about 10 milliliters of ringer solution.
Begin with the sacrificed mouse. Remove the skin of a lined the skull, cut along the jaw lines and put the head in a silver coated petri dish. Place the dish under a steroid microscope using a scalpel sagittal bi.
Dissect the head along the midline. Turn half of the head with the medial side up. Carefully remove the septum to expose the terminates.
Here we show a magnified image while removing the septum using number three forceps in the scalpel. Carefully remove the terminates lined with your fat epithelium. Transfer to another silver coated Petri dish using number 55 super fine forceps and the scalpel.
Separate pieces of the refactor epithelium from the underlying terminates. Transfer the pieces of olfactory epithelium to a tube containing one milliliter zero divalent ringer solution. Then add the papa instein.
Using micro scissors means the epithelial incubate at room temperature for three minutes gently means again with micro scissors. Wait for five additional minutes using a flame polished past pipette with a large bore transfer to a tube containing the stop solution centrifuge for five minutes, discard the supernatant. Add about one milliliter of filter ringer solution gently ate with a flame polished past pipette with a bore of about one milliliter diameter.
Place the solution containing the cells in the center of three glass. Bottom dishes allow cells to settle for about one hour at four degrees Celsius. Then add ringer solution.
Work in dim light to avoid the degradation of cage compounds from ambient light. Apply a drop of immersion oil to the 100 x objective. Place one glass bottom dish containing the dissociated oratory sensory neurons on the stage of the inverted microscope perfu.
With ringer solution. Protect the syringe containing the intracellular solution with cage compounds from light with aluminum foil and keep it refrigerated. Look for an isolated olfactory sensory neuron with intact clia.
Move the stage of the microscope to position the Cary region inside the area drawn on the monitor. To localize the UV flash area, fill a patch pipee with intracellular solution containing the cage compound. Insert the pipette into the holder of the head stage of the Apache clamp amplifier.
Using standard patch clamp techniques. Approach the somo the neuron with a patch pipette and obtain a giga seal. Then reach the whole cell mode on the computer.
Start a stimulation protocol and record the response. A typical stimulation protocol clamps the voltage at the constant value and triggers the release of a UV flesh for lysis of the cage compound. Now we will show you some representative recordings shown here is the response of an isolated mouse or fat sensory neuron to lysis of cage cycl mp.
The arrow in the top indicates the time of release of a UV flesh. The current activated by Cycl MP was recorded at minus 60 millivolt in an extracellular low calcium ringer solution. The rising phase of the current was fast and was fitted by a single exponential function.
Here we see a recording from the same neuron bathed in normal ringer solution, which contains one millimolar calcium. The rising phase of the current and minus 60 millivolt became much lower and multiphasic. This is due to the action of calcium entering the C recycling P gated channels, and activating a secondary chloride current.
In this example, we clamped the voltage at plus 60 millivolt. This is another way to reduce the influx of calcium. The rising phase of the response due to cyclone PN caging at plus 60 millivolt was well described by a single exponential indicating the presence of only one current component.
An example of responses obtained by photo releasing calcium inside the Celia is shown this current is carried by chloride ions. The voltage was minus 50 millivolt. Responses of increasing amplitudes were induced by photo release of cage calcium with UV flashes of increasing intensities.
We have just shown you how to record from dis associated factory sensory neurons when doing this procedure. It's Important to remember to be very patient. Thank you for watching And good luck with the experiment.
