The Alvar Gaaud Procedure is to apply transcranial direct current stimulation in mice. This achieved by generating low intense currents from a direct current generator, and sending it down directly to the animal through electrodes. tDCS has been investigated as a non-drug therapeutical alternative for main psychiatric disorders in humans, such as depression, schizophrenia, Alzheimer disease, AHDH, and autism.
Moreover, tDCS is a unique technique due to its low cost, ease of use, and non-invasive profile. However, the biological effects of tDCS are not entirely understood, and there is no consensus regarding stimulation parameters, such as current intensity, duration, and tile of brain areas. Therefor, the use of minimum orders is essential for a thorough study of such mechanisms, which will lead to a better understanding of the clinical efficacy of tDCS through the acquisition and analysis of behavior cellular and molecular data.
There are currently two electrode setups for tDCS, referred as anodal and cathodal stimulation. In the anodal stimulation, currents are delivered directly to the animal's head, through the animal's body, and into the cathode positioned on the animal's thorax. While in the cathodal stimulation the current enters through the animal's thorax, travels up to its head and into the cathode.
In both situations a current generator controls the current intensity and stimulation duration. While producing a contact quality and inferring feedback. There are many different setups for active positioning.
Therefor, all three dimensional axis should be taken into consideration. In this protocol a head electrode was implanted one millimeter into rooted bregma, onto the side to middle line of the skull, and the body electrode was positioned onto the animal's chest. Due to a short period simulation, it was recommended to use a fast action and short term anesthesia, such as vaporized isoflurane.
This procedure is comprised of two critical steps. Electrode placement and transcranial direct current stimulation. Surgical instruments were sterilized with pre-maintenance at 440 degrees Celsius.
Cotton swabs were autoclaved at 20 pounds per square inch at 121 degrees Celsius for 20 minutes. Turn the thermal platform controller to 37 degrees Celsius. Weigh the animal, and calculate the appropriate dose for anesthesia induction.
Use a mixture of ketamine and xylazine at a dose of 100 milligrams per kilogram of ketamine, and 8 milligrams per kilogram of xylazine. Needle size 31G. The animal should fall asleep within 2 to 3 minutes.
Use an electrical shaver or razor to shave down the surgical site. Place the animal onto the stereotaxic apparatus over the pre-warmed heating plate. Hold the animal's head and insert the tip ear bars into each of the animal's ears, to fix it to the sterotaxic platform.
Verify there is no lateral head shifting, and little vertical movement by slowly shifting the animal's head. Gently slide the anesthesia mask over the mouse's nose and fix in place by tightening the screw. Apply eye ointment to the animal's eyes to prevent corneal drying during the surgery.
Use a cotton swab to prepare the surgical site with three alternating scrubs of povidone iodine, or 2%chlorohexidine and 70%ethinol. Use a pair of tweezers to verify anesthesia adopt by lightly squeezing the animal's toes, and verifying the laws of animal pedo-withdrawal general pinch reflex. Make an incision about three millimeters posterior to the animal's ear line, and stop in the eye line.
The incision site must have approximately one centimeter in length to be large enough to receive the implant. Gently scrape the cranium with a bone scraper to improve glue and cement adherence. This must be done light handed with intention of creating micro-scratches.
Carefully position surgical hooks to the loose skin to maintain an open surgical field and free it of obstructions, such as skin an fur. Use a sterile cotton swab to gently dry the animal's scalp. Use a dissection microscope to visualize the top of the animal's cranium.
Attach a needle to the sterotaxic holder and locate the bregma. Position the needle directly above the animal's head is lightly touching the bregma. Use the bregma as a reference to adjust the coordinates of the area of interest.
Fix the implant on the stereotaxic holder. Position the implant over the animal's head and lower it slowly onto the region of interest. Use a needle to spread one drop, approximately 35 microliters, of superglue onto the implant space.
Slowly move the holder downwards until it touches the skull. Be sure the implant space is entirely in contact with the surface. Prepare the surgical cement according to the manufacturer's instructions.
After precise positioning, apply three thin, even layers of cement across the cranium, and onto the lower portion of the implant. Apply drop per drop by using an application brush. Layers must form U-shaped structure for further structural support of the implant.
Leave the implant's screw thread clean of cement to allow a smooth, unobstructed connection. Allow each layer to dry for approximately four minutes. After drying, carefully remove the holder until it is completely detached from the implant.
Always use extreme caution when handling the implant, since it might be accidentally destructed from the animal's skull. Hydrate the animal's skin and incision site with a saline soaked cotton swab. Cut the skin over the base of the implant.
Use a pair of tweezers to bring the tissue together, and close the incision with one drop of tissue surgical glue per point 2 centimeters of tissue. Infiltrate 1 to 2%lidocaine in the incision site and underlying tissues. Hydrate the animal with 500 microliters of lactate ringer simultaneously.
Place the mouse into a pre-warmed 37 degree Celsius clean single house cage. Build a small dish of wet food pellets in the cage, for easy access to food in the following hours. Register the animal's post surgical weight.
The animal must be administered with ketoprofen. 5 milligrams per kilogram simultaneously after the surgery and over the next two days. Make sure that tDCS stimulator is fully charged.
Attach the anode and cathode cables to the tDCS stimulator and make them available near the simulation site. Attach the pin-type electrode to the sterotaxic holder. Set the thermal platform to 37 degrees Celsius.
Turn on the oxygen flowmeter on the inhalation anesthesia system to 1 Liter per minute. Place the mouse into the anesthesia induction chamber. Turn on the isoflurane vaporizer to 3%Allow the animal to undergo isoflurane effects for four minutes.
While the animal is in the induction chamber, use a sterile syringe to fill the body electrode with 0.9%saline solution. Remove the animal from the induction chamber and position its chest over the body electrode. Gently slide the anesthesia mask over the mouse's nose and fix in place.
Lower the isoflurane output to 1.5%Fill the implant and the pin-type electrode with saline. Carefully attach them together. Adjust the stimulation time and current intensity according to your protocol.
Verify the contact quality on the tDCS system later. Start the stimulation. Observe the current ramping up for 20 seconds to the selected value.
And maintaining itself stead for the established time. Then, at the end of the section ramping down again. Activate the shin button for controlise.
Observe the current ramping up for 20 seconds to the selected value. And then down one for the rest of the stimulation period, with a final ramp to the selected value, at the end, with a consecutive ramped down. One the stimulation section is complete, carefully transfer the animal to a pre-warmed 37 degree Celsius cage for 10 minutes.
This protocol uses tDCS to stimulate the mouse's cerebral cortex one millimeter anterior to the bregma. This graph shows the statisticalized and gene expression after tDCS stimulation protocol. Using a 0.35 milliamperes current intensity for 10 minutes per day.
The tCDS implant presented self viable from day one through five with no significant difference among the days in contact quality. There are a variety of tCDS stimulation protocols in model brains. Protocols must be chosen according to the particularities of your experiment is basing itself on.
Area of stimulation, current intensity, session duration, and electrode positioning. In this particular protocol, we aimed at modulating the motor cortex through our known tDCS with the current of 350 microamperes for 10 minutes. After watching this video you will be able to perform tDCS in mice.
When someone practices surgery may take up to four minutes per animal. It is essential to take into consideration the animal care guidelines when utilizing the mice during the operation and following the post-surgical care to the animals so that the animals stay healthy during the study. We also recommend waiting five to seven days after implant placement to perform the experiments, since the animal's physiological response to trauma may interfere with the biological outcomes.
