הכנת עכבר ער להקלטת תגובות עצביות והזרקת קליעים נותבים

The overall aim of this procedure is to record responses from single neurons in an awake mouse. This is accomplished by first anesthetizing the mouse with isof fluorine inhalation, placing the mouse in a rodent stereotaxic frame and exposing the skull. The second step is to locate the brain region of interest using stereotaxic coordinates and mark this region for a subsequent craniotomy.

Next, a custom made head post is mounted onto the skull and the head post is placed in a mounting bar that is attached to a micro manipulator on the stereotaxic apparatus. The final step is to perform a craniotomy to allow an electrode to be inserted into the brain. Once the mouse recovers from the surgery, the mouse is restrained in a foam block.

The head post is placed back into the mounting bar and attached to a custom made stereotaxic apparatus, and a micro pipette electrode can be inserted into the brain. This procedure allows isolation of single units. Strong responses to stimuli can be recorded from the same brain structure over multiple days, and traces can be used to examine ascending or descending projections to physiologically identified regions or without affecting the responses with anesthetics.

The main advantage of this technique over existing methods is that response properties of neurons are not influenced by anesthetics. Demonstrating this procedure will be Zachary Mako, a graduate student from my laboratory To prepare the head restraint, first, assemble a custom built head post by drilling a hole into a 15 millimeter, three over 32 inch stainless steel rod. Then insert a 20 millimeter stainless steel roll pin perpendicularly into the hole.

To form a cross tap one end of the vertical rod to accept a number 1 72 screw as shown here. Next, ready the mounting bar. This piece of apparatus consists of a longer brass or aluminum bar of around 90 millimeters long and five to 10 millimeters wide, and a shorter 15 millimeter length bar that is attached to the end of the long bar at 45 degrees.

The shorter bar has one groove cut into the bottom and a shorter groove across the width fit the head post into these grooves with the cross piece nestling into the shorter groove. The mounting block is two pieces of aluminum fitted together with screws to form a block of approximately 30 millimeters by 30 millimeters by 25 millimeters with a cutout to hold the mounting bar, insert the mounting bar into the cutout in the mounting block parallel to the table surface. Attach the mounting block to a micro manipulator so that the position of the head post can be accurately placed along the midline and a brima.

Finally cut a 0.01 inch diameter tungsten rod to approximately five millimeters in length with approximately one millimeter bent at 45 degrees. This rod serves as the ground pin. After placing the animal in an induction chamber with 5%ice of fluorine, ensure that the animal is fully anesthetized by performing a toe pinch and checking for the absence of a reaction.

Once fully anesthetized, place the mouse in the rodent stereotactic frame fitted with a mouse adapter by first placing the teeth inside the hole of the bite bar and gently tightening the nose clamp until it is snug. To ensure that the animal remains anesthetized. Place a face mask made from a piece of latex glove over the mouse's nose and maintain 5%isof fluorine until the respiration rate is approximately one breath per second.

After which time, reduce the isof fluorine to 1.5 to 2%Now secure the head by gently placing the ear barss into each ear canal. While being careful not to puncture the eardrum, fix the ear barss so that they are snug, but do not penetrate too deeply into the ear canals. Place a gel heating pad underneath the mouse.

Then gently apply ophthalmic ointment to prevent the eyes from drying out. Use using a small shaver or fine point scissors. Remove hair from between the ears up to the eyes.

Now clean and sterilize the scalp with chlorhexidine or Betadine scrub, followed by an alcohol rinse repeated three times. Make an incision along the midline from the back of the head to the eyes. Scrape the periosteum to the edges of the incision with a scalpel if required.

Carefully retract the musculature at the back of the neck with forceps while allowing the muscle to tear along the sles to minimize bleeding. Apply gel foam underneath the scalp tissue to minimize moisture and prevent the skin from moving over the skull. Then moisten a cotton tipped applicator with isop profile alcohol and dry the skull surface to enhance the visualization of skull landmarks.

Once the animal is prepared, insert a fine point. Probe such as a sharpened metal rod into the electrode holder and attach it to the stereotaxic micro manipulator. Move the probe back and forth between bgma and Lambda, ensuring that the lateral medial coordinate for each location differs by no more than 50 microns.

Gently reposition the head using the ear bars as needed. Verify that the dorsal ventral height at Bgma and Lambda differs by no more than 50 microns. Adjust the height of the nose clamp to level the pitch of the skull if necessary, the height of the ear bar might also need to be adjusted.

Use an atlas of the mouse brain to identify the coordinates of the target of interest relative to bgma. Use India ink to mark the skull above the target where a craniotomy will be made. First, clean the skull by spreading dental etch gel over the surface and scrubbing for around 10 seconds.

Then wash with water and dry completely. Next, apply the adhesive primer for the dental cement to the skull surface. Using the applicator, then use a new applicator to spread the adhesive cure the adhesive with one cycle of a UV light gun.

Identify a location distal to the target site. For the installation of the ground pin, use the tip of a number 65 mini blade scalpel to carefully bore a small hole in the skull. At this point, the hole should be wide enough for the short end of the ground pin.

Insert the ground pin so that it rests upon the meninges. Ensure that the pin is oriented so that it is pointing away from bgma and the head post attachment site. Now, move the micro manipulator so that the head post is positioned over bgma just touching the skull.

The base of the head post should lie flat on the skull. Then use dissection probes to apply a small amount of dental cement around the head post and ground pin. Press the cement down to ensure good contact with the skull.

Then cure with three to four cycles from a UV light gun. Finally, apply a second round of cement to build up the base around the head Post and cover the grooves of the head post and cure us. Before first locate the craniotomy reference mark, which will be partially obscured by the primer.

Then use a number 65 mini blade scalpel to mark a three millimeter by three millimeter square around this site. Apply a drop of lidocaine solution to reduce sensitivity. Now make multiple scores on each side of the square, being careful not to cut through the bone and into the brain.

Once the bone flap becomes loose, use the tip of the scalpel to pry it up, leaving the dura mater intact. Then cover the area with bone wax. Once the surgery is complete, turn off the iso fluorine and release the mouse from the stereotaxic frame.

Apply lidocaine to the exposed skin, followed by neo spore anointment, administer post-surgical analgesia as indicated by veterinarian, and return the mouse to the home cage. After 24 hours, the mouse may be used for electrophysiological recordings. This figure demonstrates that successful installation of the head post allows the experimenter to record single and multi-unit responses from an awake mouse over multiple days.

The restraint system allows for stable electrophysiological recordings of single neurons in the brain. Excellent isolation of single units and strong responses to stimuli can be recorded from the same brain structure over multiple days. For example, in the mouse inferior colliculus over a period of two days, the left panel shows recordings obtained from day one and the right panel on day two to assess descending projections from the auditory midbrain to the auditory brainstem, biotinylated, dextra and amine visualized using Dino Benadine as the chromogen can be injected into the mouse inferior colliculus as seen here after electrophysiological identifying neuronal response properties at the site of injection and after tissue processing.

Anterograde labeling in the contralateral dorsal cochlear nucleus can be visualized and plotted as seen here as demonstrated here. Photo micrographs at higher resolution can also be obtained to view anter greatly labeled axons and terminals. After watching this video, you should have a good understanding of how to record response properties from individual neurons and inject dyes or tracers for neuro anatomical studies in an awake mouse.

This preparation eliminates the influence of anesthetics on neural response properties and allows for experiments to be performed over multiple days with the same animal. The key to the procedure is ensuring the head post is securely mounted onto the skull and the mouse is well restrained. While this video has illustrated how to use this procedure for recording from individual neurons in the auditory system, other areas of the brain can be studied using this technique.