The goal of this procedure is to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. This is accomplished by first loading the a a V that contains a designer receptor and a reporter into a syringe for infusion. The second step is to infuse a a V into the region of interest in the rat brain.
Next, the biologically inert designer drug clozapine and oxide is prepared and injected systemically into the rat. The final step is to conduct behavioral testing using a sensory preconditioning paradigm. Ultimately, immunofluorescence microscopy on the rat brain tissue is used to verify placement of the designer receptor.
This method can help answer key questions in the field of behavioral neuroscience, including but not limited to regulation of motivation, attention, and learning and memory Ensure that all institutional precautions for working with adeno-associated viruses are followed throughout the virus used in this video is classified as a biosafety level one compound. Prepare a 10%bleach solution. The decontaminating a a v waste place, a bega containing bleach solution and a sterile 10 microliter syringe.
In the workspace, place the micro centrifuge tube containing an aliquot of a a V into a standard bench vice Load, a 10 microliter syringe with at least four microliters of a A v. Ensure that there are no air bubbles in the four microliters of a a v solution. In the syringe, dispose of the empty virus tube in the beaker containing 10%bleach.
Add additional 10%bleach to the waste container, and allow the waste to sit for 30 minutes before disposing of the decontaminated liquid waste as specified by institutional guidelines. Prepare the surgical area by placing absorbent bench paper under the stereotaxic apparatus and on an adjacent space designated as a dedicated virus handling area. After inducing anesthesia was 3%isof, fluorine, and oxygen at a rate of one liter per minute.
Shaved the fur from between the eyes to slightly behind the ears. After shaving, base the rat back in the induction chamber for three minutes with the rat secured in a stereotaxic apparatus. Cover the eyes with ophthalm ointment and prepare the incision site by cleaning with Betadine.
After making a 2.4 centimeter midline incision of the skin, overlying the skull, starting approximately two millimeter cordal to the eyes, retract the skin to expose the skull and then clear membranous tissue from the skull until bgma and lambda are clearly visible. After ensuring that the skull is flat, position the drill at bgma, zero the coordinates, and then move the drill to the desired medial, lateral and anterior posterior coordinates. Drill a hole using a 0.9, 0.7 or 0.5 millimeter drill bit.
Set the infusion pump to deliver the virus at a rate of 0.2 microliters per minute. La the syringe into the infusion arm of the stereotaxic device and lower the syringe to the desired coordinate. Use the pump to deliver the desired amount of virus after the delivery of viruses complete.
Leave the syringe in place for 10 minutes to prevent backflow into the needle tracked slowly raise the syringe at a rate of about five millimeters per minute. Then move to the next coordinate and repeat the procedure until all coordinates have been infused with a a v. After closing the wound and providing postoperative analgesia according to the approved protocol, place the rat in a clean cage with bedding a lid and proper signage.
When the rat is fully recovered from anesthesia, return it to the housing room. Allow three or more weeks before beginning behavioral testing to ensure adequate expression of the designer receptor. This figure shows the three phases of the sensory preconditioning paradigm, the preconditioning phase, the conditioning phase, and the testing phase.
The designer drug, clozapine and oxide or CNO is used to silence neural activity By activating the design receptor, CNO can be injected prior to any phase of testing. Here, CNO is injected 30 minutes prior to each of the preconditioning sessions. After preparing a fresh one milligram per milliliter solution of clozapine and oxide, according to the instructions in the written portion of the protocol, inject the CNO intraperitoneal at a dose of one milligram per kilogram 30 minutes prior to behavioral testing.
Initiate the computer code for the appropriate behavioral session. After 30 minutes have elapsed, place the rats in the testing chambers and begin the first of 4 64 minute preconditioning sessions by presenting rats with 12 intermixed trials that consist of delivery of auditory and visual stimuli. On six of the trials present a tone the precondition stimulus for 10 seconds, followed immediately by a five second presentation of the flashing light stimulus on the other six trials.
Present the white noise, the unaired stimulus alone for 10 seconds. After completion of the preconditioning phase, begin the conditioning sessions, which will be conducted across five consecutive days. Present the rats with eight trials that consist of delivery of the visual stimulus, the flashing light for five seconds followed immediately by the delivery of 2 45 milligram food pellets.
This time include inter trial intervals that taver seven minutes when the conditioning sessions are complete. Perform the single 78 minute test session by presenting the rats with 12 intermixed trials that consist of delivery of the auditory stimuli alone. On six of the trials.
Present the tone stimulus, the precondition stimulus alone for 10 seconds. On the other six trials, present the white noise, the unpaired stimulus alone for 10 seconds. Upon completion of the behavioral task, immediately remove the rats from the chambers and return them to the animal colony.
Analyze the data from the conditioning and test phase according to the detailed instructions in the written portion of the protocol. After performing immunohistochemistry directed against the tagged reporter or the reporter protein, examine the stain sections by microscopy to ascertain the location and expression of the designer receptor and or reporter as expected control and experimental rats similarly learned the Pavlovian Association between the flashing light stimulus and the food. During the conditioning sessions.
Sessions, both groups were also motivated to obtain food reward during the conditioning session. In contrast, during the critical test session, experimental rats failed to discriminate between the preconditioned and the unaired auditory stimuli, whereas control rats show greater food cup behavior in response to presentations of the precondition stimulus. Thus, control but not experimental rats showed the sensory preconditioning effect.
This image shows histological verification of the location of a a v infusion into the region of interest in the rodent brain After its development. This technique paved the way for researchers in the field of neuroscience to explore the contributions of specific brain regions and or cell types to complex behavior in vivo.
