Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

The overall goal of this procedure is to accurately manufacture a cannula and micro injectors as well as properly perform microinjection. This is accomplished by first manufacturing a cannula to an appropriate length. The second step is to cut and assemble micro injectors.

Next, prepare micro injectors, tubing, and a pump for microinjection. The final step is to complete microinjection and allow a diffusion period to pass. Ultimately, microinjection is used to accurately inject reagents into the desired brain region Demonstrating proper handling.

During microinjection will be Ryan Poland, a student in my laboratory. Generally, individuals new to this method will struggle because particular care must be taken and all steps require great attention in order to ensure proper cannula placement and microinjection location. To prepare the cannula first, draw a 15 millimeter reference line on the tape using a fine tipped pen.

Then use it as a guide for marking the 15 millimeter sections sequentially on the tubing with a sharp permanent marker. Next notch the cannula tubing by touching it to the slow spinning cutoff disc of the rotary tool. At the marked points, rotate the cannula 180 degrees and notch the opposite side of the tubing.

Do not cut through the tubing completely because it may lead to occlusion. Afterward, sharply bend the tubing in order to break it into 15 millimeter sections. Then hold each end of the cannula perpendicular to the cutoff disc between the thumb and forefinger.

Rotate the cannula tubing while touching its tip to the surface of the cutoff disc to generate a blunt tip. Subsequently, ream both ends of the cannula with a 26 gauge beveled needle to ensure that all burrs have been removed and that the cannula is unobstructed. To prepare the obturator clamp one cannula at approximately half length with medium weight straight hemostats, lay the locked hemostat on the bench so that the cannula is perpendicular to the bench.

Then feed one end of the ator wire into the cannula until it is reached the bench. Bend the wire by pinching with fingers until a 30 degree angle is achieved while maintaining contact between the obturator and the bench. After that, remove the wire from the cannula and cut off the excess portion at 2.5 millimeters from the bend with small diagonal cutters.

In this procedure, hold one end of the micro injector tubing perpendicularly with a locked straight hemostat. Twist a hemostat while keeping tension on the tubing and coil it tightly around the hemostat at least one time. Next, measure 32 millimeters from the opposite loose end of the micro injector tubing and grasp this point perpendicularly with a second hemostat.

Bend the tubing back and forth while keeping the tension until it breaks. Then inspect the micro injector tubing to ensure that both ends are straight and that the inside diameter is unobstructed. Draw a five millimeter reference line on the tape measure and mark five millimeter sections of cannula tubing sequentially using a permanent marker.

Next, touch the cannula tubing to the slow spinning cutoff disc at marked points in order to notch the tubing. Then sharply bend the micro injector collar tubing in order to break it into five millimeter sections. Slide one collar onto each micro injector tube at about two centimeters from the end.

Apply a small amount of super glue to the micro injector tube at five millimeters from one end. Next, slide the collar over the super glue bead so that it is one millimeter from the end of the microinjection tube. After that cover both ends of the collar with glue.

Then attach a 26 gauge needle to the one milliliter syringe filled with sterile water. And slide the eight centimeter piece of micro injector plastic tubing onto the needle, making sure not to puncture the tube after that slide the other end of the plastic tubing over the collar end of the dried micro injector. Push the water filled syringe plunger to test for patent C.In this step, bend the completed micro injector at 15 millimeters from the end opposite to the collar by 95 degrees.

Next, cut the plastic tubing to 70 centimeters and slide it over the micro injector collar. To prepare the pump for microinjection, set the diameter of the microinjection needle desired infusion rate and the target injection volume on the tube. Then set the pump mode to volume so that a precise volume is delivered automatically.

Next, adjust the pump backstop to allow the syringe to be filled to the injection volume plus an additional 0.2 microliters. Now place the tip of the gast tight syringe into the sterile water contained in a small whey boat and gently flutter the plunger To lubricate the internal wire evenly. Pull the plunger to the backstop on the pump to fill the microinjection needle with water.

Next slide. The PE 20 tubing that is connected to the micro injector onto a 26 gauge needle attached to a one milliliter sterile water filled syringe. Spray sterile water through the micro injector and inspect the spray pattern and distance.

Then place a sterilized micro injector onto a sterile field. Slide the tubing off the needle, cut the tubing below the area damaged by the needle while keeping it upright to prevent water from dripping out. Next, push the micro injector syringe plunger slightly forward to create a droplet on the end of the needle and slide the water-filled PE 20 tubing onto the needle.

After that, push the plunger completely forward to prepare for sample loading and ensure that no trace ethanol remains in the micro injector. Check up the setting for leaks by touching the water droplet that is formed at the tip of the micro injector with a clean lab wipe a few times. Then pull the syringe plunger back 0.2 microliters to create a bubble between the sterile water and the PE 20 tubing and the solution to be injected.

After that, place the micro injector into the reagent to be injected and slowly pull the syringe plunger to the backstop. To prepare the animal for injection, hold the rat's chest against the experimenter's chest. Clean the area around the cannula with a cotton applicator soaked in Betadine.

Swab the head cap three times with Betadine and then swab with ethanol two times. Then place the filled micro injector into the cannula. Press start on the micro injection pump and monitor the bubble movement.

When the injection is complete, remove the micro injector from the cannula and replace with obturator. This figure shows the transfection of the nucleus accumbens astrocytes region by micro injected virus, and this figure shows the reduction of the motivation of rats to self-administer ethanol by activating a transgene that was over expressed in nucleus accumbens. Core astrocytes sites activation of dread by systemic administration of CNO significantly reduced the motivation of rats to self-administer ethanol after abstinence compared to vehicle CNO, which had no effect in an equally trained cohort that was expressing GFP instead of the dread it Once mastered.

This technique can allow for customized brain microinjection implements, and further flexibility of experimental procedures. While attempting this procedure, it's important to remember that precise microinjection location is imperative to achieve the desired effect.