The overall goal of this procedure is to simultaneously measure the electroretinogam and visual evoked potential in a rat. This method can help answer key questions in the field of fissure neuroscience. Such as whether the effect of aging or diseases affect certain components in the visual pathway.
The main advantage of this technique is that it allows comprehensive assessment of the retinal function and its upstream affect on the visual evoked cortical function. To begin this procedure, pre-fashion the custom made ERG inactive electrodes by cutting a 70 millimeter length of silver wire and forming a loop of eight millimeters in diameter to encircle the rat eye. Prepare a uniform circle and adjust the size of the loop by shaping it on a one millimeter pipe head tip.
This is important because a ring that is too small may inadvertently raise inarticular pressure, where as an oversized ring can increase the variability of signals. Next, pre-fashion the custom made VEP inactive electrode by cutting a 70 millimeter length of silver. And forming an ellipse of eight millimeters in diameter to hook on to the rat incisors.
Then, pre-fashion the custom made ERG active electrodes by cutting a 30 millimeter length of silver wire and form a small loop of about one to two millimeters in diameter which is used to gently contact the rat cornea with minimal corneal abrasions. Afterward, securely attach the electrodes to the electrode leads by entwining the silver with the exposed inner wire. Insulate excess exposed metal with masking tape to reduce photo-voltaic artifacts.
For the ERG active electrodes, more layers of masking tape can be applied to achieve a thicker and more rigid wrapping so that the electrode is easier to be held with a micro-manipulator. On each ERG inactive electrode, stick a small piece of hook and loop fastener to the masking tape to enable stable attachment to the rodent neck strap. Then, for each VEP active electrode, attach an alligator clip to the inner wire of the electrode lead.
Immediately before recordings, electro plate the exposed surfaces of the silver wires with chloride to improve signal conduction. To do so, connect the cathode to the negative terminal of the battery and immerse the other end into the saline. Subsequently, immerse the silver tip on the anode wire into normal saline and connect the other end of this electrode wire, to the positive terminal of a nine volt battery.
Disconnect them after 20 seconds and the silver tip of the electrode wire should be coated evenly in white. Repeat the chloriding procedure for all the silver ERG and VEP electrodes. In this procedure, disinfect the shaved area of an anesthetized animal with 10%povidone iodine three times.
Make a sagittal incision along the mid-line on the head with a scalpel and excise a 20 millimeter diameter circle of dermal tissue to expose the cranial bone. Next, remove the underlying periosteum by scraping and drying with gauze, to expose the coronal and sagittal cranial sutures. Drill two holes through the skull on both hemispheres.
Screw in stainless steel screws into the two premade holes up to a depth of one millimeter to allow firm anchorage. Then, prepare the surgical area for dental amalgam by drying the cranial bone with gauze and retracting the skin with two sutures. Subsequently, spread the dental amalgam over the exposed skull to secure the screw electrodes in place and ensure about 1.5 millimeters of the screws are exposed for recording.
After that, remove the retraction sutures. For scotopic recordings, all the procedures are performed in a dark room. For illustration purposes, electrode positioning is conducted under normal room lighting here.
The animal was dark adapted fore 12 hours and anesthetized with ketamine and xylazine. After pupil dilatation and topical anesthesia, hook the inactive VEP electrode around the bottom incisors. Then, place the animal on the ERG platform in front of the Ganzfeld bowl situated in the Faraday cage.
Secure the animal to the platform with a strip of hook and loop fastener placed firmly but not tightly around the nape. Next, position the ERG inactive electrodes by encircling the sclaral ring non invasively around the eyes equator. Approximately three meters behind the limbus.
Stabilize this by attaching the electrode to the hook and loop fastener strip around the nape. Repeat the procedure for contralateral eye. Now, fasten the VEP active electrodes by attaching alligator clips to the stainless steel screws, pre-implanted on the skull.
Prior to the ERG active electrode placement, place a small drop of 1%carboxymethylcellulose sodium on the electrode to improve signal quality. Position the ERG active electrodes to lightly touch the central corneal surface using a micro manipulator attached to the custom built stereotaxic arm. Ensure the carboxymethylcellulose sodium only contacts the cornea and not the sclara, by wiping excess fluid away.
Then insert two to five millimeters of the stainless steel ground needle electrode, subcutaneously into the tail. Slide the platform closer to the Ganzfeld bowl and ensure the animals eyes align with the opening of the bowl to enable even illumination of both retinas. Subsequently, close the Faraday cage to reduce extraneous noise.
Using the above protocol, a stack of ERG wave forms can be recorded in response to a range of stimuli with the dimmest flash shown at the bottom and the brightest at the top. At very dim light levels, scotopic threshold responses can be recorded. With increasing stimulus brightness, the wave forms become dominated by the B wave and then the negative going A wave emerge at the brightest stimuli.
The rod photoreceptor function can be assaid by using a P3 to model the A wave. This figure shows that P2 amplitude grows with increasing stimulus energy. Rod bipolar cell function can be assaid by modeling the intensity response theories of the rod P2 with a naka rushtin function.
Retinal ganglion cell function is assaid at dim luminous energies and quantified by the pSTR peak amplitude and timing. Cone bipolar cell function is elicited with a paired flash paradime and quantified by cone P2 peak amplitude and timing. Amplitude analysis of the VEP is taken as peak the troph and troph to peak amplitudes which gives the timing of these responses.
The amplitude of the VEP wave forms increasing with increasing stimulus energy. When it's mastered, this technique can be done within about 30 to 45 minutes excluding the time for preparing the electrode and placing the electrode and adakadaptation. While attempting this procedure, it is particularly important to pay attention to how accurately you place the electrodes and allow enough time for adakadaptation.
Following this procedure, other procedures such as disease modification or therapies can be performed to answer specific questions. After watching this video, you should have a good understanding of how to prepare and place the electrodes as well as how to record and analyze the ERG and the VEP wave forms.
