The overall goal of this procedure is to functionally image the intact auditory cortex of the cat using high field functional magnetic resonance imaging. This is accomplished by first preparing the sound equipment for stimulus presentation. The second step is to prepare the cat for scanning by anesthetizing, then positioning it in an MRI compatible sled.
Next, anatomical and functional images are collected across the whole brain during stimulus presentation. The final step is to process the images to quantify the level of brain activation during stimulus presentation. Ultimately, functional imaging is used to show the extent of activation during sound presentation in the intact cat auditory cortex.
This method can be used to help answer key questions in auditory function, such as the existence of a hierarchy in auditory cortex. The main advantage of this technique over existing methods such as optical imaging, is that FMRI is non-invasive and can measure functional cortical activity within the intact skull. This method can provide insight into auditory function and can be applied to other sensory modalities such as vision and somatic sensation.
Demonstrating the procedure will be Pam Nixon, a registered veterinary technician, and Amy Hall, a graduate student in our laboratory. All animal procedures as described in this protocol, must receive approval from your institution's animal use committee prior to the experiment. To begin preparation for this protocol, first connect the computer, which will be used to present the auditory stimulus to the external sound card via A USB cable.
Then attach the cables connecting the output ports of the external sound card to the input ports of the stereo power amplifier. Next, attach cables connecting the output ports of the stereo power amplifier to the input ports of the transformer box of the FMRI compatible earphone system. Use shielded coaxial cables with BNC connections to connect the transformer box to the penetration panel outside the scanner room.
Then connect the earphone cable assembly to the corresponding BNC ports on the penetration panel. Inside the scanner room, connect the foam ear tips to the earphones. Then connect the earphones to the cable assembly.
Run a test auditory stimulus to confirm that sound is being transmitted from the computer to the earphones. Disconnect the earphones and insert the foam eart securely into the cat's ears During the animal preparation phase. First, set up the syringe pump to be used for maintaining anesthesia while imaging combined 60 milliliters of saline and 0.07 milliliters of ketamine in a 60 milliliter syringe and place it in the syringe pump.
Then administer a sedative mixture of atropine sulfate and a SUP promazine via a subcutaneous injection. After 20 minutes, administer ketamine and d meato hydrochloride via an intramuscular injection to induce anesthesia. Once the cat has lost its writing reflex, apply ophthalmic ointment to the eyes to prevent dryness during the procedure.
Also, place an indwelling catheter in the medial saphenous vein for intravenous delivery of ketamine. During the procedure, again, test for successful anesthetic induction by pinching a toe on the four paw and observing whether the cat withdraws its paw. Once the petal reflex disappears, suppress the gag reflex by spraying lidocaine onto the pharyngeal walls.
Then intubate the cat with a 4.0 or 4.5 endotracheal tube. Maintain anesthesia throughout the imaging session with a constant infusion rate of ketamine and inhaled isof fluorine delivered in 100%oxygen at one to 1.5 liters per minute. Next layer insulating plastic bubble wrap on the floor of the MRI compatible sled.
Then place warm wax filled heating pads on top of the bubble wrap. Place another layer of plastic bubble wrap on top of the heating pads. Then place the cat in a sternal position within the insulating bubble wrap in the MRI compatible sled.
Once the cat is positioned, adjust the head to gain access to the ears. Roll the foam ear tips into the smallest diameter possible. Then insert each ear tip deep into the ear canal.
Once inserted, the foam ear tips should expand to fill the space within the ear canals. Adjust the cat until its head is properly positioned within the three channel radio frequency coil, stabilize the head with acoustic dampening memory foam. Be sure to place foam around the ears to provide additional attenuation of the scanner noise.
Now wrap the cat's body in a blanket of insulating plastic bubble wrap. Then secure and transport the sled to the scanner bed. Connect the infusion lines anesthetic delivery tubes and monitoring equipment to the cat.
Also, connect the earphones to the earphone cable assembly attached to the penetration panel. Start the ketamine infusion at the base flow rate of 0.6 milligrams per kilogram per hour. Then increase the flow rate as required Based on the depth of anesthesia, set the initial isof fluorine dose to 0.5%then decrease to 0.4%once the anatomical scans have been collected.
Monitor and record the cat's blood oxygen saturation and title CO2 levels heart rate respiration and rectal temperature throughout the experiment. Using MRI compatible monitoring equipment positioned at an appropriate distance from the scanner. Bore steady increases in heart rate and respiration are usually associated with imminent recovery from anesthesia.
Begin by collecting anatomical reference scans of the cat's brain in an axial slice orientation using a flash imaging sequence with the imaging parameters seen on screen here. The duration of the anatomical scan should be approximately six minutes. Next, set up for functional imaging using a segmented interleaved echo planar acquisition with the imaging parameters seen on screen here.
Start the image volume acquisition while presenting a broadband white noise stimulus. In a block design, the auditory stimulus should be played for 30 seconds and alternated with a 32nd silent baseline condition. Repeat this step until acoustically evoked bold activities observed in the auditory cortex.
The duration of each functional run using a block design should be approximately 4.5 minutes for 90 volumes. Present the stimulus for the desired number of functional runs. After the MRI session is completed, remove the cat from the sled.
Continue to provide supplemental heating with heating pads and towels until the animal fully recovers. Once the gag reflex returns, remove the endotracheal tube. Monitor the CAT until the writing reflex is restored.
Then return the animal to the facility. Assess the animal the day after the procedure to ensure no adverse effects from the experiment. After the experiment, select appropriate FMRI analysis software such as SPM or FSL to process the acquired functional volumes.
Here we can see the bold activation in two animals in response to a 32nd broadband noise stimulus presented in a block. Design the T statistic maps of the broadband noise versus baseline contrast. In two image slice planes reveal contiguous clusters of bilateral acoustically evoked activity in the auditory cortex.
These plots show modulations in the bold signal during a single functional run at the highlighted voxels. And here we can see the event related mean time courses of the bold response normalized to the mean bold baseline signal. In these examples, the bold signal exhibits a significant increase relative to the baseline three to six seconds after the stimulus onset.
While attempting this procedure, it's important to closely monitor the cat's physiological parameters to ensure a consistent level of sedation throughout the procedure. After watching this video, you should have a good understanding of how to conduct a functional magnetic resonance imaging experiment to examine auditory cortical function in an anesthetized preparation. Don't forget that working with MRI scanners can be potentially hazardous.
While performing this procedure, always ensure that personnel have been appropriately screened, that the experimental setup is MRI compatible, and that monitoring devices are MRI safe or placed at a safe distance from the magnet.
