We'll be demonstrating today a recording system we developed to record in vivo from the optic chiasm of rat. These types of recordings have been very rare in rat, in part because the retinal ganglion cell axons that make up the optic nerve are very small, making it difficult to record from individual cells, and since rat is a common animal model for retinal degeneration, these types of recordings have the potential to produce a lot of useful information. The key to achieving these recordings was in the tung stain glass electrodes that we designed that have a long sharp tip that allow action potentials to be recorded from thin diameter axons with a high signal to noise ratio.
We'll walk you through the fabrication process of these electrodes and we will show you how to, how we set up the animal on the stereotactic apparatus, and then we will show you an example of recording spike trains from a single ganglion cell. Three main pieces of equipment are needed to fabricate electrodes, a standard glassblower to make the micro pipettes. A sewing machine is used to sharpen the tungsten and a standard microscope with 40 x objective.
In addition, a graphite ring is needed with attached pole and it will be placed in the concentrated solution of potassium hydroxide and sodium nitrate. This is placed under the sewing arm, and then a thin piece of tungsten wire is then taped to the sewing arm, partially immersed in solution. Leads are then applied to the graphite pole on one end into the sewing arm.
On the other two volts will be applied across the leads and the sewing machine is turned on. This dips the dunston into solution and electromagnetically sharpens it for two minutes. Once the tungsten is sharpened, it is carefully removed from the sewing arm, Then dipped into water to remove excess solution, and then placed under the microscope under 40 x objective.
The glass micro pipette has a small diameter opening about one to two microns in diameter. The sharpened tungsten is then carefully inserted into the back end of the micro pipette and pushed with a mechanical micro manipulator. The tungsten is pushed through the end of the glass very carefully.
It is important that there is a tight fit between the tungsten and the glass, and it is common to push enough so that a small amount of glass will actually break off. A small amount of crazy glue can be applied to the back end of the micro pipet to fasten the tung to the glass. Now that the electrode fabrication is complete, its impedance is measured using a metal electrode Impedance testing.
Generally we look for impedances near one mega ohm. The electrode is then put away in a case for safekeeping, as it is very fragile. Visual Stimuli are presented on a monitor that is placed on a movable platform.
Heating blanket is used to control the body temperature of the animal, and the stereotactic system allows gross movement of the micro drive, which is shown here. The metal case in the hypodermic guide needle shield, the recording electrode in act to reduce noise levels. The guide needle is inserted into the brain and a micro drive is used to slowly lower the electrode through the guide needle.
This is illustrated here where the metal case is lowered onto a crossbar that will rest on the animal's skull. The guide needle will stick out below the crossbar as shown, and the electrical contact between the metal case and the crossbar acts to reduce noise levels. The animal is then placed on the heating blanket.
The ear bars are carefully inserted. It is important at this point to make sure the head remains level. A tooth bar is then inserted to make sure the head is in all directions.
A rectal Probe is inserted using Vaseline. This will measure body temperature, which is then used to control the temperature of the heating blanket. The body temperature should remain between 36 and 37 degrees.
A single EKG lead is inserted into the skin to monitor heart rate, which is generally around 300 feet per minute. Heart rate is a good indicator of anesthetic depth. The vital signs of the animal are continuously monitored with custom software.
These include blood pressure entitled CO2 body temperature and heart rate. Skin is then removed from the top of the skull and the skull is exposed. It is important to thoroughly clean the skull in order to see the proper skull markers that are used to find the optic chiasm.
The crossbar assembly is then put into place. This is used to trace out a five millimeter circle on the skull that will then be drilled and removed. The hole will be drilled around the bgma skull Marker illustrated here.
The exposed skull is shown with a circular pencil mark around bgma, which will be drilled before the skull is drilled. The stereotaxic system must be calibrated torema, and this is done by lining up the guide needle precisely on bgma and documenting the position of the stereo. After drilling, the skull has been removed and the brain is exposed.
After the skull is drilled, the animal is paralyzed and ventilated in order to prevent eye movements. This is done with an intravenous injection of gamine. The breathing is carefully monitored as it is, and when the breathing stops, the ventilation tubing is inserted into the tracheal connector, at which point the animal is artificially respir.
The guide needle is then lowered into the brain. The recording electrode will be lowered through the gyal to about eight to 10 millimeters below the skull where action potentials from visual cells in the optic chiasm can be recorded. The electrode is slowly load into the brain.
With The micro drive, a visual cell can be heard spiking spontaneously listen to the cell, respond to the presence of a handheld wand in its receptive field. Drifting grading is then presented, and at this point, any visual stimuli of interest can be used to study the response properties of retinal ganglion cells using optic chiasm.Recording.
