The overall aim of this procedure is to lesion a specific area of the rat brain using stereotaxic infusion of an excitor toxin. This is accomplished by first properly mounting a rat in the stereotaxic device. The second step is to locate the infusion position based on stereotaxic coordinates.
Next, the excitotoxin is infused and allowed to diffuse away from the needle tip. The final step is to remove the needle and close the incision. Ultimately, immunohistochemical counters staining of fixed tissue slices are used to show effective and correctly located lesion.
This method can help answer key questions in neuroscience field, such as functions of discrete brain areas. Visual demonstration of this method is critical because to properly mount the head so that it is level and secure requires that you know what proper position looks like. To set up for the experiment, first fill a 10 microliter Hamilton syringe with sterile water.
Then mount the filled syringe into the caddy of a six syringe programmable pump, and secure the end of the syringe plunger into the clamped holder on the pump. Insert a previously prepared 30 gauge flat cut one inch infusion needle into approximately two feet of gas sterilized PE 20 tubing. Next, use a 25 gauge needle and one milliliter syringe to prefill the tubing with sterile water.
Slide the open end of the tubing onto the Hamilton syringe. Be careful to avoid creating air bubbles in the tube. Now clamp the infusion needle end of the tubing onto the arm of a stereotaxic device using a barrel style electrode manipulator.
Then insert the infusion needle between the clamp and the grooved barrel, ensuring that the clamp is secured over a piece of tubing containing the needle within it to avoid flattening the tubing. Next, adjust the pump to a rate greater than five microliters per minute and turn on the pump. Once the pump is running, a bead of water should appear at the tip of the infusion needle.
Use this opportunity to check the joints and tubing to ensure that there are no leaks. Use a sterile swab to wipe away the water bead. Then set the pump to withdraw and suck up a two microliter air bubble.
The top of the bubble should be visible in the tubing. Now immerse the tip of the infusion needle in a 20 milligram per milliliter solution of NMDA in sterile 0.1 molar PBS and withdraw four to five microliters of the liquid. Ensure that the air bubble separates the sterile water and the NMDA solution.
Once a suitable volume of NMDA solution has been drawn up, the receptacle containing the stock solution can be removed and then the setup is ready for surgery. After mounting the rat in the stereotaxic frame, each ear bar should feel like it is resting on a solid place, not like its sinks far into the head. The penny should look symmetrical and should appear to lie flat on the ear bars.
Upward flaring of penny indicates improper placement of the ear bars. The head should not wobble in response to pressure at the neck. Use a sterile swab to apply ophthalmic ointment to the eyes.
Now apply 2%chlorhexidine solution to the scalp while moving in. Concentric circles from the planned incision site towards the edge of the shaved region. Apply 70%ethanol in the same fashion.
Repeat this process three times followed by one application of 10%Povidone iodine solution as the scalp is now sterile, it should only be touched with sterile materials. At this point, don sterile surgical gloves and places sterile drape over the animal so that the fenestration exposes the surgical site. The animal is now ready for surgery after using a scalpel fitted with a number 10 blade to make a two centimeter longitudinal incision along the midline of the scalp beginning posterior to the line of the eyes.
Use sterile cotton tips to pull the fascia overlying the skull surface to the edges of the surgical site. Use four curved sterile hemostats to clamp the fascia on each side of the anterior and posterior aspects of the incision. To create a surgical window, lay the other end of the hemostats alongside the animal.
To pull the incision open and fully expose the skull. First, locate bgma and lambda as depicted in this diagram. Then move the needle so that the tip just touches the scarlet bgma.
Note the dorsal ventral coordinate according to the venia scale. It is a good idea to familiarize oneself with reading the venia scale before beginning the procedure. A guide is provided at the end of this video.
After touching the non-sterile stereotaxic device, the surgeon is no longer sterile and should be careful not to touch the sterile surgical site or the parts of any tools that will touch the surgical site. Next, slightly raise the needle and move the needle tip straight back to lambda again. Lower the needle tip and note the dorsal ventral coordinate.
If this coordinate is within 0.1 millimeters of that recorded for broma, then the skull is considered level. If not, then the bite bar will need to be adjusted and the measurements repeated. Once the head is level in the dorsal ventral plane, return the needle torema and record the anterior, posterior and media lateral coordinates.
Calculate the target positioning of the needle based on the brain atlas coordinates of the structure of interest and move the needle to the calculated position. Mark the position of the needle and then swing the needle out of the way. Use a Dremel moto tool with a sterile dental drill bit attached to carefully drill a hole of approximately two millimeters at the site of the needle position without damaging the dura or tissue underneath.
Ream the hole to enlarge it and when finished, absorb any blood with a cotton swab. After drilling the hole, swing the needle back into position. Lower the needle into the hole until the tip touches the dura.
Record this dorsal ventral coordinate and subtract the appropriate valley required to reach the brain region of interest. Slowly lower the needle to the calculated level. Then raise the needle back up to a level way above the skull.
Use a Hamilton syringe cleaning wire to unclog the tip of the infusion needle. Now run the pump at the same rate as before. A bead of liquid should form at the end of the infusion needle.
If not, repeat the unclogging procedure and then try again. After confirming that the needle is free of blockages, slowly lower the needle back to the desired dorsal ventral level. Mark the two ends of the air bubble with black marker.
Set the infusion pump to 0.1 microliter per minute and start the infusion. The movement of the bubble indicates that the needle is not clogged and that the infusion is working. Well allow the neurotoxin to infuse into the brain for two minutes.
Then once the infusion is complete, stop the pump and wait five minutes. For the NMDH diffuse away from the needle tip. Depending on the brain region, it may be necessary to raise the needle a small amount and infuse again for a shorter time period.
This is done to ensure that the target structure is covered by the infusion. Once the infusion of neurotoxin is complete, raise the needle outta the brain slowly and swing the stereotaxic arm out of the way. After doning a fresh pair of sterile gloves, use sterile cotton tips to wipe away any excess blood on the skull.
Then remove the clamps from the facia. Then use London forceps to push the two sides of the incision together, ensuring that the inner edges of the skin meet. Do not allow the skin to curl in towards the incision.
Keep pressing the incision closed and apply wound clips along the length of the incision. This usually takes three to five wound clips. After placing the wound clips, use forceps to pinch and further secure the staples.
Finally, swap the stapled incision with 10%povidone iodine solution. Return the rat to the home cage. Monitor the recovery of the animal and administer analgesic according to institutional guidelines.
This graph shows the average weight of 17 rats that underwent stereotypes, surgery, and six rats that did not have surgery. Day zero is the day of the surgical procedure. Following surgery, the weight of rats decreased for two to three days and then increased around day five to six.
This image shows a crestal violet counter stain on a 30 micron coronal slice with the bald spot from an MDA lesion of the basal lateral amygdala. The white arrows outline the lesion. It can be compared to the contralateral side of the brain slice, which has no lesion.
This image shows a similar lesion of the central nucleus amygdala. This drawing depicts a standard stereotaxic reading to obtain the digits to the left of the decimal place. Use the markers on the right side.
The labeled digit corresponds to the tens place IE one equals 10 and two equals 20. The hash marks between the labeled digits correspond to the single units one to nine. The zero line indicates at what integer the stereotypes is set.
Here the zero line is between the 10 and 11 markers indicating that the reading is between 10 and 11. To determine the numbers to the right of the decimal place, use the markers on the left side. The hash marks on the left are numbered zero to 10 with each unlabeled hash mark representing one unit difference from those next to it, whichever of the hash marks on the left lines up best with the hash marks on the right indicates the first decimal place of the coordinate reading here.
The hash mark on the left, corresponding to number nine, lines up best with the right side hash marks, so the decimal place is nine. The final reading is 10.9 millimeters. Once mastered, this technique should take 30 minutes if performed properly.
Following this procedure. Viral vectors or sir a could be infused site specifically into the brain to answer questions like how changing gene expression in certain brain regions alter brain function.
