This method can help answering key questions in the field of neuroendocrinology. Such as how do pituitary cells communicate, respond to releasing factor from the brain? The main advantage of this technique is that compared to this first primary cell culture this procedure allows the investigation of cells in a more intact environment.
Although this method can provide insight into the electrical properties of luteinizing hormone producing cells in Medaka. With only minor adjustments, it can also be used for other cell types or for other fish species. While holding a euthanized Teleost fish with ethanol sterilized strong forceps under a dissecting microscope, sever the two optic nerves behind the eyes with sharp forceps and break the skull bones from the posterior to the anterior side.
Use the forceps to peel off the skull and to sever the spinal cord. Gently grasping the spinal cord with the forceps flip the brain from the posterior to the anterior orientation, and place the organ in a dish filled with ice cold, calcium free, extra cellular medium. Next fill a one point five to two cubed centimeter metal hold with liquid agarose and place the mold on ice.
Just before the agarose solidifies, use the forceps to gently pick up the brain by the spinal cord, dry the forceps with a piece of lab paper, and place the brain into the agarose. Then quickly mix the agarose to dilute any traces of extra cellular medium left on the tissue and adjust the orientation of the brain within the mold as necessary. When the agarose has solidified, use a scalpel blade to trim a square block of agarose around the tissue and use surgical glue to attach the sample onto a vibratome specimen holder.
Fill the cuvette of the vibratome holder with ice cold, calcium free extra cellular medium and place the holder into the vibratome. Using a high frequency and a low speed, cut the agarose block to make 150 micrometer parasagittal sections, placing the selected section into the patch-clamp recording chambers containing three milliliters of ice cold, calcium free extra cellular solution. It is important to move quickly once the brain has been added to the agarose, as the tissue will dry out if left in the molded agarose for too long and has limited access to air before it is sectioned.
When all of the sections have been obtained, place a grid harp onto the tissue section and replace the calcium free extra cellular medium with calcium containing extra cellular medium supplemented with bovine serum albumen. Then let the tissue rest for 10 minutes. To set up the patch-clamp, place the recording chamber with the brain pituitary slice onto the patch-clamp recording microscope stage, and use the 10X objective to locate the pituitary gland.
Place the grounding electrode through the 2%agar bridge into the extra cellular medium bath, and use the 40X objective to locate a healthy cell. Set the amplifier to voltage clamp mode and open the seal test window. Select the bath configuration and use a five millivolt pulse to monitor the pipette resistance.
Backfill the tip of the patch pipette with antifungal free intra cellular medium before using a microfiller syringe to add intra cellular solution supplemented with Amphotericin B to the posterior section of the patch pipette. Use a one milliliter syringe to add a slightly positive air pressure into the patch pipette to avoid contamination of the pipette tip and assess the patch pipette resistance. If no particles are attached to the tip of the pipette, use the micromanipulators to guide the patch pipette down to the cell.
Readjusting the pipette when it is a few microns above the cell so that the tip of the pipette will touch the cell a third of the distance from the middle of the cell. To touch the cell, release the pressure and apply gentle suction to make a seal. It's essential to apply the gigaseal quickly as positive pressure within the patch pipette will result in antifungal solution leakage after about one minute damaging the cells.
When the seal is in place, immediately switch to the patch window in the patch-clamp software and adjust the holding potential to between negative 50 and negative 60 millivolts. Zero out the fast capacitance made up by the glass pipette and switch to the cell window to begin monitoring the access resistance, zeroing out the membrane capacitance in the amplifier software after sufficient access. Switch to the current clamp and adjust the correct value of the fast capacitive currents of the pipette in the I-Clamp window, and check the resulting membrane potential.
Then with the cell firmly attached to a tissue with a membrane potential below negative 40 millivolts, begin the experimental recordings according to the manufacturer's protocol. Action potentials can be triggered in a subset of pituitary cells using a five to nine picoampere current injections from a resting potential between negative 50 and negative 60 millivolts. These action potentials have a fast rundown and after about four to six minutes, triggered action potentials are no longer achievable.
Switching to the perforated patch configuration using Amphotericin B, spontaneous action potentials can be observed in about 50%of the recorded cells and the action potentials can be triggered in 95%of these cells with no observable rundown, even after up to one hour of recordings. Gonadotropin releasing hormone one, puff ejected onto cells in the perforated patch configuration reveals a biphasic response, during which the cells are hyper-polarized in the first phase of the response resulting in calcium release from internal stores followed by de-polarization of the cells and increased action potential firing frequency from one to two hertz to three hertz in the second phase. While attempting this procedure, it is important to remember that all of the solutions should be adapted to the physiology of the experimental organism that you're using, in this case, Medaka, and you need to have particular attention to pH as this value varies across species living at different temperatures.
