Transcranial Electrical Brain Stimulation in Alert Rodents

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

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The overall goal of this surgical procedure is to allow for reliable and repeated transcranial electrical brain stimulation in alert rodents. This method can help answer key questions in the field of neuromodulation and neuroplasticity by targeted electrical stimulation of brain networks to improve motor function or deficits. The main advantage of this technique is that it allows for repeated non-invasive electrical brain stimulation sessions in alert rodents without inducing discomfort and the minimal interference of his behavioral measurements.

Individuals new to this method might struggle because finding the correct path for the chest to head tunneling, as well as bone preparation before electrode socket placement is often difficult to perform for inexperienced investigators. Visual demonstration of this method is critical as the surgical steps for electrode socket placement as well as the electrode preparation and placement for stimulation will be crucial for success of the experiments. Our Doctoral Candidate, Stephanie Haschka, will assist in performing the NEBS procedures in alert rats.

Presenter}Begin by placing the rodent on its chest on the operation table. Disinfect the shaved scalp with a swab soaked in antiseptic agent. Let it air dry.

Then after checking the depth of anesthesia, cut the skin with a scalpel in one line from the rostral eye level to the mid-ear level. Next turn the rat to a supine position so that the chest is exposed and disinfect the chest. Stretch the lateral skin of the right chest with two fingers and make a straight sagittal cut in the cranial orientation.

Form a subcutaneous pouch by atraumatically disconnecting the skin from the left major pectoral muscle, do so by repeatedly opening the small scissors. Next, turn the animal on its right side, tunnel the cable path from the left occipital corner of the open head skin along the neck to exit into the pectoral pouch. By penetrating the superficial fascia using homeostatic forceps.

Carefully open the homeostatic forceps to grab the end of the electrode cable attached to the platinum electrode without allowing sharp wires to stray. Pull the cable through the tunnel until the electrode enters the pouch oriented with the cautering point towards the rodent's left hind limb. Then turn the rodent back to the prone position.

Next, fix the platinum plate with a sterile synthetic braided non-absorbable suture to the pectoral fascia at the two opposing corner holes. Similarly attach the cable to the fascia by a loose knot forming a slight loop before the entrance of the tissue tunnel. Finally, close the skin with three to four cutaneous sutures depending on the size of the cut.

Begin by scraping off the periosteum, the connective tissue on the skull to the sides with the scalpel, and thoroughly wipe off with cotton swabs. Fixate the connective tissue at the four corners of the cut with bulldog clamps, then let them hang laterally to keep the surgery field open. Next, apply 0.9%saline to clean the bone surface and tissue with cotton swabs.

Then clean the bone surface with 3%Hydrogen Peroxide. Use a cotton swab to remove any residual of the periosteum as they become visible. As fixation screws will improve sedative adherence choose a drill bit fitting the screw size.

Place two burholes on two different bone plates by pre-drilling with a hand drill, and then by slight particle pressure with a bone drill. In cases of an implanted electrode as shown in this video bur a third hole located in the right posterior parietal bone for future fixation of the tunneled cable. Place the plastic screws in the burholes and screw until the first friction is felt.

Then perform three additional 180 degree screw turns. Use forceps to check for stability of the screw and add one more turn if not tight enough. Turn on cautering iron and pre-heat for approximately five minutes.

Wind the cable exiting the tissue tunnel occipitally around the right parietal screw, and then cut it leaving approximately one centimeter of cable behind the winding. Carefully strip the insulation at the end of the cable with a scalpel. Then fix the winded cable to screw and bone with cyanoacrylate glue.

Next, apply a small amount of the lead free tin cauter to the connector and to the bare wires of the counter-electrode cable, and connect both by briefly pressing both pre-cautered parts together while touching the cautering tip until the tin cauter melts. Remove the cautering tip immediately to avoid excessive metal heating of the cable with subsequent tissue damage. For stimulation of the motor cortex use a four millimeter diameter socket.

Pick up the custom-made tES electrode socket with thin serrated tip forceps, and apply a thin layer of cyanoacrylate glue to the bottom rim of the socket. The inter-medial border of the socket should end directly at the sagittal suture and the coddle inner border should end of bregma. Then press the socket briefly onto the bone until the glue hardens.

And use a light to ensure the bone within the area of the socket does not have reflective glue on it. After the socket is in place, and the future stimulation area is free of glue, seal the lateral border of the socket to the neighboring tissue with a small drop of cyanoacrylate glue to avoid a fluid bridge that could lead to shunting of current at this location. Then cover all screws with cyanoacrylate glue.

Next, mix the two components to acrylic cement in a small silicon tube or glass. As soon as it becomes viscous, apply it with a dental spatula to seal the remaining borders of the socket to the bone. Finally, cover the whole skull, screws, counter-electrode cable, and up to 1/3 of the socket with dental acrylic cement.

After all of the bone is covered and the cement has hardened remove the bulldog clamps and reposition the skin. Begin by filling the tES electrode socket half 0.9%saline and remove air bubbles. Before cathodal tEs sessions, check chlorination and if necessary re-chlorinate the silver, silver chloride electrode.

Before anodal tES sessions, remove possible excessive silver chloride deposits from previous stimulations with sandpaper to allow good conductivity during stimulation. Then screw in the tES electrode cap. Connect the cables to the two connectors on the head.

Then place the rodent into the experimental cage with the cables connected to a swivel above the cage that allows for free movement. Turn on the stimulator and adjust the stimulation parameters. Finally, after the end of the stimulation, disconnect the cables, unscrew the electrode cap on the head, and clean and dry the socket with a cotton swab.

Results indicated that microglia activation assessed by morphological alteration occurred first at 31.8 amps per square meter, while the first signs of neuro-degeneration were detected at 47.8 amps per square meter. Samples one through four were rated as positive. The anesthesia affected the magnitude of response to DCS as the percentage of brain slices with fluoro-JC positive degenerating neurons in alert rats was higher at 47.8 amps per square meter.

Further, behavioral or molecular effects at high intensities are expected to be mechanistically different compared to low intensity effects. Once mastered, this technique including chest electrode placement can be done in 40 to 45 minutes if it is performed properly. A shorter duration of 10 to 15 minutes is usually needed if no counter-electrode is surgically placed.

After watching this video you should have a good understanding of how to place a permanent electrode set up for the different NEBS simulation techniques and how to apply an eTS in alert rodents. Don't forget that working with electrical currents can be harmful for the rodent if electrodes are not prepared sufficiently prior to stimulation. Precautions such as re-chlorination of the electrode before cathodal stimulation or removal of the debris before anodal stimulation should always be taken before performing this procedure.

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Journal (Neuroscience)

Journal (Neuroscience)

Transcranial Electrical Brain Stimulation in Alert Rodents

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