Estimulação elétrica transcraniana crônica e gravação Intracortical em ratos

The overall goal of this procedure is to show a step-by-step protocol to fabricate and implant chronic transcranial stimulation electrodes. This method can help to answer key questions in the neuro-stimulation field such as the long term effects of temporally targeted diffused transcranial electrical stimulation. The main advantage of this technique is that the small size of the implant makes this method suitable to be combined with regular electro-physiological optogenetic and imaging techniques in freely moving animals.

The implications of this technique is centered toward the therapy of epilepsy, depression and another neuro-psychological disorders because it is capable to promptly influence large scale brain networks. To make a stimulation electrode, cut six 10 centimeter long pieces of miniature hookup wires. Remove two centimeters of the cladding on one end, and one centimeter on the other end.

Next, twist two cables together and solder them with the shorter peeled side. Afterward, acquire a strip of three tape packaging of any integrated circuit with SOT-353 case. Stitch the twisted wires through the three holes of the package.

The longer peeled segment of the wires should be in the cavities of the package. Position the cable so that the end of the insulation is in line with the edge of the holes. Then, apply dental cement on the holes to fix the wires.

Once the cement is solidified, flip the package to input a thin layer of super glue into the hole to form a water-type seal towards the porous cement. Avoid gluing the peeled wires. After that, twist the cables together on the shorter peeled side, and solder them.

Grab the longer peeled segment of the wires with the tip of fine tweezers. Roll them around, and wrap them into the cavities. Cut excess cable if necessary.

To make a recording triplet electrode, cut a one centimeter long piece of stainless steel tube using a high-speed rotary saw. Remove the fin from the ends of the tube with a sharp needle and ensure that the tube is free of debris and completely permeable. Bend the gold-plated board spacer pin with the minimum length of three centimeters to form a J-shaped holder.

Cut the longer leg of the J-shape and solder it together to get back the original shape to make a detachable joint between the long linear part, which will be held by the stereotaxic device, and the U-shape, which will hold the electrode and serve as an anchoring point. Subsequently, glue the tube and the shorter leg of the J-holder together. Glue some additional one centimeter long bars if necessary for stability.

Next, cut three pieces of 2.5 centimeter long electrode wires with stainless steel ultra-fine scissors. Make sure that it is a clear and sharp cut and the insulation is intact at the circular surface of the electrode wire. Also, make sure that the cut tip is not bent due to the force applied.

Bend one or two millimeters on one end of each of the wires at different angles to make them distinguishable. Afterward, fill the tube with the wires and choose an appropriate spacing of the electrodes for the experiment. Note which wire corresponds to which depth for maintaining a proper channel order.

Once the wires align right next to each other and are parallel with the tube, fix the electrode wires by placing a single drop of liquid super glue at both ends of the tube with a sharp needle. Make sure that the glue is flowing into the tube due to the capillary effect but not towards the recording sites at the protruding ends. Prepare an electrode interface board with a proper micro-connector compatible with a recording system to be used.

Then, introduce the bent ends of the wires into the holes of the electrode interface board corresponding to the desired recording channel. When all the wires are in position, push the gold pins into the holes with tweezers. In this procedure, remove most of the hair from the animal scalp with a hair clipper.

Next, apply depilatory cream on the scalp. Spread it evenly on the surface, and wait a few minutes. Use a spatula to gently remove the cream and the remaining hair.

Rinse the skin with disinfectant. Inject 1 to 2%lidocaine subcutaneously to numb the skin. Then, apply a single drop of vet ointment on each eye.

Make a thorough and a long sagittal incision along the midline with a scalpel from the forehead to the neck. Subsequently, dissect the tissues including the periosteum from the skull. Then using a chisel or tooth tweezers, clean the area between the cristae of the two temporal bones.

Keep the skull exposed by retracting the dissected skin using four bulldogs. Gently place the fine tweezers between the steep edge of the temporal bone and the muscles and separate them. Make jiggling movements to expose as much of the large surface of the temporal bone as possible, preferably from the edge of the occipital bone to the plane of the coronal sutures without damaging the muscles.

Following that, place the retractors bi-temporally to keep the temporal bones exposed. Rinse the surface of the skull with one to two milliliters of 3%hydrogen peroxide. Then wash it with one to two milliliters of water very carefully and leave the surface of the temporal bones to mop up the moisture with ocular sticks.

Next, test if the stimulation electrodes fit on to the cleaned vertical skull surface. The upper edge of the stimulation electrode should be in line with the edge of the crista of the temporal bone. Readjust the bulldogs and the retractors, or shape the stimulation electrodes with scissors, if necessary.

Following that, fill the cavities of the stimulation electrodes with electro-conductive gel. And put a thin layer of glue on to the rim of the electrodes. Place the stimulation electrode on the dry surface of the temporal bone with one accurate movement and hold it firmly in place for one minute with fine tweezers.

Make sure that no moisture is in contact with the glue, and mop up with ocular sticks if necessary. Make sure that the stimulation electrode is evenly glued to the dry temporal bone. Mop out the moisture frequently, and retract the surrounding tissues carefully.

Ask for assistance if necessary. After that, apply dental cement over the edges of the stimulation electrode while the leaking moisture of the tissues is continuously dried up with ocular sticks. Cover the whole stimulation electrode with cement.

After the cement has completely hardened, repeat these steps on the contra-lateral side. Subsequently, using a 10%smaller diameter drill head compared to the screw diameter, drill some holes all over the skull for anchoring screws. Drive miniatures screws into the holes, and apply dental cement over them.

Then, continue with implantation of the recording electrodes. Solder the connectors and anchor them to the construct at the end. Afterward, wash the exposed tissues abundantly with disinfectant.

Inject 1 to 2%lidocaine subcutaneously. Then, to breed wound edges, enclose them with simple interrupted sutures around the connector implant. Subsequently, disinfect the wound with povidone iodine.

Here, we show the representative results of a closed loop epileptic seizure detection and intervention system, applied on Long-Evans rats, expressing spontaneous seizures. This train is known to show the behavioral symptoms in electrographic patters of absence epilepsy, called spike and wave discharge. Shown here are the LFP traces of seizure intervention, demonstrating the stable performance of the temporally targeted stimuli in interrupting seizures for 16 weeks.

This demonstrates the robustness and reliability of the implanted stimulation electrodes. To put these results in context, here is the recording of an aborted experiment where secondary tissue grew between the temporal bone and electrode surface, due to the improper sealing and cementing of the electrodes. After watching this video, you should have a good understanding of how to implant transcranial stimulation electrodes.

Following this procedure, other methods like implantation of recording electrodes or cranial windows can be performed in order to record different modalities during stimulation.