The overall goal of this procedure is to demonstrate the recording of LTP in a freely behaving mouse model. This is accomplished by first surgically implanting micro electrodes in the perent path and the dentate gyrus. The second step is to record the baseline evoked responses in the dentate gyrus in response to single pulse stimulation of the perent pathway.
Next LTP is induced following a izing stimulation protocol. The final step is to record post synaptic responses and compare them to baseline values. Ultimately enhanced population spike amplitude.
Measures of the evoked response are used to show successive induction of LTP. The main advantage of this technique over existing methods like the in vitro slice preparation, is that the freely behaving mouse model provides the most physiologically relevant platform for testing the effects of neuropharmacology drugs or genetic manipulation on wooden models of human neurological diseases. Begin this procedure by shaving the fur on top of the mouse's head from the area just above the eyes, so that just in front of the ears, make sure not to shave the whiskers as they are part of the mouse barrel sensory system.
Next, clean the shaved area first with isopropyl alcohol, then with Betadine, then apply a small amount of mineral oil with cotton swabs on the eyes to prevent them from drying. Transfer the mouse to the stereotaxic apparatus. Use infant rat ear cuffs instead of regular ear bars to mount the animal's head by First mounting the left ear on the left ear cuff.
Then gently ease the right ear onto the right cuff by supporting the animal's head with one hand and advancing the ear cuff with the other hand. After that, place a heating pad under the animal's body and set it to 86 degrees Fahrenheit to maintain the body temperature. Then check for bilateral rigidity By attempting to move the head side to side, the animal is properly mounted on the stereotaxic frame when the head is rigid and cannot be moved side to side, but can easily pivot up and down.
Subsequently rest the mouse's snout on the tooth bar assembly and make sure that the incisors are resting gently but securely within the tooth aperture of the tooth bar. Using a sterile scalpel, make a midline incision starting from the middle between the eyes to the middle of the ears. Make sure the scalpel is held at a 45 degree angle and enough pressure is applied to cut through the underlying fascia, but not through the skull, as that will cause excessive bleeding.
Then use cotton swabs to separate the fascia and curved hemostats to expose the skull. The skull landmarks, bgma, and lambda should be clearly visible. Ensure that the animal's skull is leveled by adjusting the tooth bar such that bgma and lambda are at the same DV position.
Next, clean the exposed skull with a small amount of sterile saline and wait two to three minutes for the skull to completely air dry before proceeding. After that, mount a needle on the left stereotaxic arm. To measure the DV positions of bgma and lambda.
Make sure that they're within 0.1 millimeter of each other. If not, adjust the nose bar of the stereotaxic frame up and down to achieve this. Then position the needle on B bgma and record its AP L-A-T-N-D-V measurements.
Be sure the needle just touches the skull and does not penetrate it. Use a mouse brain atlas to determine the coordinates of the target structures relative to lambda and bgma. In our case, they're the medial perent pathway in the angular bundle and dentate gyrus of the hippocampus.
Then use a fine point pen or pencil to mark these points on the skull. Next, mark, two more points on the contralateral side of the skull. These points which will serve as the ground should be about three millimeters from the midline and position longitudinally to each other.
After that, remove the needle marker from the left stereotaxic arm and replace it with an electric dental drill equipped with a stereotaxic mount. Position the drill over each marked point and make small bur bowls of approximately 0.5 millimeter in diameter. When drilling, use a repetitive up and down motion check frequently to see whether the drill has penetrated the skull to expose the brain surface gently but firmly drive a screw electrode into each of the contralateral holes such that they just touch the cortical surface without penetrating it.
These two screw electrodes serve as the ground and reference. Subsequently mount the stimulating and recording electrodes in the electrode holders on the left and right stereotaxic arms respectively. Afterwards, connect the stimulating electrode terminals to an electrophysiological stimulator with current isolation and the recording electrode terminal to an electrophysiological differential amplifier, and then to a digital oscilloscope for visual inspection of evoked responses.
Next, connect the ground and reference electrode terminals to the differential amplifier amplifier gently and slowly lower the stimulating electrode to the medial perran pathway and the recording electrode to the dentate gyrus in 0.5 millimeter increments, use the oscilloscope to monitor the evoked response to a single pulse stimulus. Each incremental lowering of the electrodes continue to lower the electrodes until they reach their respective target DV positions and a stereotypical signal is observed on the oscilloscope afterward. Use the dental acrylic cement to make a cap to hold the electrode terminals in place.
Any dental cement that touches the skin should be wiped off immediately. Once the dental cement is completely cured, transfer the animal from the stereotaxic frame to a clean rodent cage and use a heating lamp or a pad to maintain core temperature. Monitor the animal every hour until it regains consciousness.
An injection of fuxin may be administered as an analgesic after the animal has been allowed to recover from the surgery for five to seven days. Place it in the recording environment consisting of a faraday cage outfitted with soundproofing material and a five channel rotating commutator connecting the electrode that leads to the stimulating recording set up. Allow the animal to acclimate for one to two hours in the recording environment before connecting the electrodes to the stimulating and recording instruments.
Next, set the stimulator controls to output 400 microamps. Record the amplitude of an average of 10 evoked responses with at least 10 seconds elapsing between stimuli and repeat this procedure for 600 eight hundred one thousand one thousand two hundred and one thousand four hundred microamps. Then use the method shown in this figure to quantify the amplitude of the evoked responses.
Construct an input output curve by plotting the average amplitude of the evoked response versus stimulus intensity. Then determine the stimulus intensity which corresponds to 50%of the maximum amplitude measured, and use this intensity for the remainder of the experiment. Now, obtain a baseline by recording the average amplitude of five evoked responses every minute for 15 minutes with at least 10 seconds elapsing between stimuli.
Subsequently deliver the Titanic stimulation to the medial perent pathway. In the meantime, monitor the animal closely for signs of seizure, including wet dog shakes. If the animal has a seizure, the experiment should be terminated and all data from that animal should be excluded from the study Results.
Then continue to record the average amplitude of five evoked responses every minute for 30 minutes. Post ization after that, compare this amplitude to the baseline amplitude obtained earlier by calculating the percent change from baseline. Shown here is the diagrammatic illustration of the relative location of electrodes on the mouse skull, and these are the typical traces of the evoked responses from both pre and post ization.
This figure shows the LTP induction in the medial peripheral path dentate gyrus synapse of a freely behaving mouse Once mastered. This technique can be done in two hours if it is performed properly. While attempting this procedure, it is important to remember to check the animal frequently to ensure depth of anesthesia is maintained.
After watching this video, you should have a good understanding of how to perform stereotactic surgery to chronically implant micro electrodes, which are used subsequently to record LTP in a freely behaving mouse.
