의식 쥐에 주입 및 무선 막전위의 기록 및 Visual 사건 관련 전위

The overall goal of this procedure is to measure conscious ERG and VEP signals following surgical implantation of wireless telemetry probes. This technique can help answer key questions in the region of neuroscience field, such as how neurons behave without the limitation of general anesthesia under both healthy conditions and diseased state. The main advantage of this technique is that it allows simultaneous recording of the electro physiology for the retina and for the visual cortex in conscious animal.

To begin this procedure prepare the electrodes under a microscope. To do so untwist a double stranded, stainless steel electrode with two fine tipped pliers. Trim one of the stainless steel strands at approximately one centimeter from the tip, leaving a single longer, straight strand to shape the ring electrode.

Then, fold the single stainless steel strand back into it self and twist to form a smooth ring at the tip of the electrode. Next shape an ERG active electrode by twisting the base of the loop to form a circle with a diameter of point two to point five millimeters. For the two ERG inactive and one VEP inactive electrodes make the loop diameter to point eight millimeters.

In this example, prepare one active ERG and three inactive electrodes. After that, hook the circular VEP active electrode around a stainless steel screw so the electrode rests against the screw head. Then, hook the three inactive electrodes around a second stainless steel screw.

The ground electrode will go directly into the peritoneal cavity and there is no need to make a loop at the tip. After shaving and disinfecting the surgical site of an anesthetized animal, make a 10 millimeter incision on the head along he mid line between the ears with a surgical scalpel. Then, make a five millimeter incision on the abdomen through the skin layer, along the mid line below the sternum.

After that, tunnel a cannula of five millimeter diameter subcutaneously, from the abdomen incision to the head incision. Feed the electrode wires of the tansmitter through the cannula from the abdomen to the head. Leave the reference electrode with the transmitter base.

Then, cover all the electrode tips with aseptic gauze. Next, secure the rat's head to a stereotaxic platform. Extend the forehead incision to 30 millimeters with surgical scissors.

Then, expose a surgical area by retracting the skin with two sutures. Scrape off the periosteum overlying the skull to expose the bregma, lambda and mid line sutures. Subsequently, drill two holes through the skull at the VEP active and ERG, VEP inactive stereotaxic coordinates.

Attach the active and inactive electrodes with stainless steel screws to the skull to about one millimeter in depth. To implant the ERG active electrodes, use a suture to temporarily retract the upper eye lid. Then, insert a 16 to 21 gauge cannula subcutaneously from behind the eye through the superior conjunctival fornix to the forehead.

Subsequently, remove the guiding needle. Next, feed the active electrode through plastic catheter from the forehead towards the eye. Then, remove the plastic catheter.

Use a temporary suture, which is threaded through the electrode loop to prevent the electrode from retracting back into the tunnel. After that, make a point five millimeter incision on the superior conjunctiva one millimeter behind the limbus. Use blunt dissection to expose the underlying sclera.

Then, implant a suture immediately behind the limbus at half scleral thickness and be careful not to puncture the eyeball. Subsequently, remove the temporary suture from the ERG active electrode. Anchor the ERG active electrode to the half scleral thickness suture by tying three consecutive knots and ensure the electrode tip is situated close to the limbus for good signal to noise ratio.

If necessary pull back the excess electrode lead so the active electrode doesn't impinge on the cornea. Then, close the conjuntival flap using one to two interrupted sutures to completely cover the ERG electrode. Afterward, remove the eyelid retracting suture.

Repeat the procedure for the contralateral eye. Now, apply cyanoacrylate gel over the skull to secure all the stainless steel screws and electrode wires. Ensure the ERG active electrodes are not tightly pulled before being secured to enable eye movements.

Next, close the incision using interrupted sutures. Then, rotate the rodent to expose the abdominal area. Extend the abdominal dermal incision to 40 millimeters along the linea alba with surgical scissors.

After that, make a 35 millimeter incision through the inner muscle wall, to expose the inner abdominal cavity. Using two sutures, attach the transmitter body to the animal's inner abdominal wall at the right and avoid contacting the liver. Fold the ground electrode by securing it in a loop with two sutures and place it, free floating, in the abdominal cavity along with the other electrode leads.

Subsequently, close the peritoneum using a continuous suture. Then close the skin incision using interrupted sutures. For recording dark adapted signals animals are dark adapted for 12 hours and all procedures are preformed in a dark room with the aide of dim red light.

For illustration purposes recordings will be conducted under normal room lighting conditions here. Before recording, apply topical anesthesia and dilating drops to the cornea of the rat. Next, guide the conscious animal into a custom made, clear restrainer.

Place the rat in front of the Ganzfeld bowl with it's eyes aligned with the opening of the bowl. Turn on the indwelling transmitter by passing a magnet within five centimeters of it. Verify that the transmitter is on by checking the LED status light on the receiver base.

Close the Faraday cage which house's the receiver pad to improve the signal to noise ratio. Then, collect signals over a range of luminous energies. This figure shows the ERG wave forms in conscious rats on day seven post surgery.

ERG responses were recorded to range of stimulus energy with the response to the dim flash shown at the bottom and the brightest flash at the top. On day 28 post surgery, these wave forms remained similar to those on day seven. Over four weeks there was no significant time effect for photo receptor amplitude and sensitivity.

Rod bi polar cell amplitude and sensitivity. Cone bi polar cell amplitude. And implicit time.

Similarly, VEP wave forms appear to be comparable seven and 28 days post surgery. With amplitude and timing parameters showing no significant time effect. These results indicated robust ERG and VEP signal stability.

Once mastered, the surgical component of the technique will be completed in one and half to two hours. The conscious recording component, if preformed properly can be completed in 45 minutes. Following this procedure, other methods like pharmacological or disease manipulation can also be preformed.

This will answer additional questions in drug discovery as well as chronicle longitudinal experiments. Additionally this procedure can also be preformed in other animals if required. After watching this video, you should have a good understanding of how to surgically implant and subsequently measure conscious ERG and VEP telemetry signals.