The authors would like to acknowledge the contributions of Kyle Gilbert, who designed the custom RF coil, and Kevin Barker, who designed the MRI-compatible sled. This work was supported by the Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC), and Canada Foundation for Innovation (CFI).

The following procedure can be applied to any imaging experiment in which anesthetized cats are used. Steps which are specifically required for auditory experiments (steps 1.1-1.7, 2.8, 4.1) can be modified to accommodate other sensory stimulus protocols.
All experimental procedures received approval from the Animal Use Subcommittee of the University Council on Animal Care at the University of Western Ontario and followed the guidelines specified by the Canadian Council on Animal Care (CCAC)44. The outlined experiment requires approximately 150 min from animal preparation to recovery. The time course of the experiment is illustrated in Figure 1.
1. Stimulus Equipment Preparation
Figure 2 shows the electronic components and corresponding connections required for generating an auditory stimulus in the MRI scanner. The requirements are as follows: a computer, an external sound card, a stereo power amplifier and an fMRI-compatible earphone system.
<ol>
	<li>Connect the computer which will be used to present the auditory stimulus to the external sound card via the Universal Serial Bus (USB) cable.</li>
	<li>Attach the cables connecting the output ports of the external sound card to the input ports of the stereo power amplifier.</li>
	<li>Attach the cables connecting the output ports of the stereo power amplifier to the input ports of the transformer box of the fMRI-compatible earphone system.</li>
	<li>Connect the binaural earphones to the output ports of the transformer box.</li>
	<li>Use shielded coaxial cables with BNC connections to connect the transformer box to the penetration panel outside the scanner room.</li>
	<li>Connect the earphone cable assembly to the corresponding BNC ports on the penetration panel inside the scanner room.</li>
	<li>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 ear tips securely into the cat&#39;s ears during the animal preparation phase (step 2.7).</li>
</ol>
2. Animal Preparation
<ol>
	<li>To premedicate the cat, administer a sedative mixture of atropine sulphate (0.02 mg/kg) and acepromazine (0.02 mg/kg) via a subcutaneous (SC) injection.</li>
	<li>After 20 min, administer ketamine (4 mg/kg) and dexmedetomidine hydrochloride (0.02-0.03 mg/kg) via an intramuscular (IM) injection to induce anesthesia. Ketamine is usually combined with a sedative and muscle relaxant, in this case, dexmedetomidine hydrochloride, to reduce the tremors and muscle rigidity commonly observed when ketamine is used alone45. This anesthetic combination typically induces approximately 150 min of sedation and is frequently used in veterinary practice to induce anesthesia in small animals.</li>
	<li>Once the cat has lost its righting reflex, apply ophthalmic ointment to the eyes to prevent dryness during the procedure. Place an indwelling catheter in the medial saphenous vein for intravenous delivery of ketamine.</li>
	<li>Test for successful anesthetic induction by pinching a toe on the forepaw then observing whether the cat withdraws its paw. Once the pedal reflex disappears, suppress the gag reflex by spraying lidocaine onto the pharyngeal walls then intubate the cat with a 4.0-4.5 endotracheal tube.</li>
	<li>Maintain anesthesia throughout the imaging session with a constant rate infusion of ketamine (0.6-0.75 mg/kg/hr) and inhaled isoflurane (0.4-0.5%) delivered in 100% oxygen at 1-1.5 L/min. Combine 60 ml of saline and 0.07 ml of ketamine in a 60 ml syringe then place the syringe in the syringe pump. This step can be performed before premedicating the cat.</li>
	<li>Place warm wax-filled heating pads on the floor of the MRI-compatible sled (Figures 3a and 3c) then layer the insulating plastic bubble wrap around the interior walls of the sled.</li>
	<li>Place the cat in a sternal position within the insulating bubble wrap in the MRI-compatible sled (Figure 3c).</li>
	<li>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.</li>
	<li>Adjust the cat until its head is properly positioned within the 3-channel radio-frequency (RF) coil (Figure 3b). Stabilize the head with acoustic dampening memory foam (Figure 3d). Place foam around the ears to provide additional attenuation of the scanner noise.</li>
	<li>Wrap the cat in the blanket of insulating plastic bubble wrap then secure and transport the sled to the scanner bed.</li>
	<li>Connect the infusion lines, anesthetic delivery tubes and monitoring equipment to the cat. Connect the earphones to the earphone cable assembly attached to the penetration panel.</li>
	<li>Start the ketamine infusion at the base flow rate of 0.6 mg/kg/hr then increase the flow rate as required based on the depth of anesthesia. Set the initial isoflurane dose to 0.5% then decrease to 0.4% once the anatomical scans have been collected.</li>
	<li>Monitor and record the cat&#39;s blood oxygen saturation, end-tidal CO2 levels, heart rate, respiration and rectal temperature (if possible) throughout the experiment using MRI-compatible monitoring equipment positioned at an appropriate distance from the scanner bore. Table 1 lists the mean values and ranges of the physiological measurements for successful execution of this procedure. Steady increases in heart rate and respiration are usually associated with imminent recovery from anesthesia.</li>
	<li>After the 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 righting 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.</li>
</ol>
3. Brain Imaging
<ol>
	<li>Collect anatomical scans of the cat&#39;s brain in an axial slice orientation. Use the following imaging parameters for the anatomical reference volume: FLASH imaging sequence with TR = 750 msec, TE = 8 msec, matrix = 256 x 256, acquisition voxel size = 281 &#956;m x 281 &#956;m x 1.0 mm. The duration of the anatomical scan is approximately 6 min. Figure 4 (left panel) provides a sample anatomical image slice obtained using the specified parameters.</li>
	<li>Use the following imaging parameters for the functional volumes: segmented interleaved echo-planar acquisition (EPI) with TR = 1,000 msec, TE = 15 msec, 3 segments/plane, 21 x 1 mm slices; matrix = 96 x 96; field-of-view = 72 mm x 72 mm; acquisition voxel size = 0.75 mm x 0.75 mm x 1.0 mm; acquisition time = 3 sec/volume. Figure 4 (right panel) provides a sample functional image slice obtained using the specified parameters.</li>
</ol>
4. Stimulus Presentation
<ol>
	<li>Present a broadband white noise stimulus (0-25 kHz, 100 msec bursts with 5 msec rise/fall time, 1 presentation every 200 msec, 90-100 dB SPL) in a block design in which the auditory stimulus is played for 30 sec and alternated with a 30 sec baseline (no-stimulus) condition (Figure 5). Repeat this step until acoustically-evoked BOLD activity is observed in the auditory cortex. The duration of each functional run using a block design is approximately 4.5 min for 90 volumes.</li>
	<li>Present the stimulus in the appropriate block design configuration for the desired number of functional runs.</li>
</ol>
5. Data Analysis
<ol>
	<li>Select appropriate fMRI analysis software (e.g. SPM, FSL) to process the acquired functional volumes.</li>
	<li>Realign each functional volume to the volume acquired closest in time to the reference anatomical scan. Save the resulting motion correction values for use in step 5.6. Exclude any functional runs in which rotational head movements exceed 1&#176; or translational head movements exceed 1 mm.</li>
	<li>Coregister each volume to the reference anatomical scan.</li>
	<li>Smooth each volume with a 2 mm full-width half-maximum (FWHM) Gaussian filter.</li>
	<li>Incorporate a square-wave (boxcar) function which corresponds to the ON-OFF stimulus block design as a regressor to the general linear model (GLM).</li>
	<li>Incorporate motion correction values as regressors of no interest to account for movement-related artifacts. Apply an uncorrected statistical threshold of p = 0.001 to the GLM results to view clusters of BOLD activation. Determine the size of the smallest cluster which meets a corrected (family-wise error: FWE) threshold of p &#60; 0.05 at the cluster level. Set the cluster extent threshold to this value to view statistically significant clusters in regions of interest.</li>
	<li>Define the BOLD percent signal change (PSC) at each voxel as the difference between the mean BOLD signal during the stimulation blocks and the mean BOLD signal during the baseline blocks.</li>
</ol>

The authors declare no conflicts of interest, financial or otherwise.

In designing an fMRI experiment for an anesthetized animal model of auditory function, the following issues should be given careful consideration: (i) the impact of anesthesia on cortical responses, (ii) the effect of background scanner noise, and (iii) the optimization of the data collection phase of the experimental procedure.
While an anesthetized preparation offers the important advantage of producing a prolonged period of sedation and minimizing potential head motion during a functional i...

Current understanding of auditory processing in mammals is mainly derived from invasive electrophysiological studies in monkeys1-5, ferrets6-10, bats11-14, rodents15-19, and cats20-24. Electrophysiological techniques commonly utilize extracellular microelectrodes to record the activity of single and multiple neurons within a small area of neural tissue surrounding the electrode tip. Established functional imaging methods, such as optical imaging and functional magnetic resonance imaging (fMRI), serve as useful complements to extracellular recordings by providing a macroscopic perspective of simultaneous driven activity across multiple, spatially distinct regions of the brain. Intrinsic signal optical imaging facilitates visualization of evoked activity in the brain by measuring activity-related changes in the reflectance properties of surface tissue while fMRI utilizes the blood-oxygen level-dependent (BOLD) contrast to measure stimulus-evoked hemodynamic changes in brain regions which are active during a particular task. Optical imaging requires direct exposure of the cortical surface to measures changes in surface tissue reflectance that are related to stimulus-evoked activity25. In comparison, fMRI is noninvasive and exploits the paramagnetic properties of deoxygenated blood to measure both cortical surface26-28 and sulcus-based27,29 evoked activity within an intact skull. Strong correlations between the BOLD signal and neuronal activity in nonhuman primate visual cortex30 and in human auditory cortex31 validate fMRI as a useful tool to study sensory function. Since fMRI has been used extensively to study features of the auditory pathway such as tonotopic organization32-36, lateralization of auditory function37, patterns of cortical activation, identification of cortical regions38, effects of sound intensity on auditory response properties39,40, and characteristics of the BOLD response time course29,41 in human, monkey, and rat models, the development of a suitable functional imaging protocol to study auditory function in the cat would provide a useful complement to the functional imaging literature. While fMRI has also been used to explore various functional aspects of the visual cortex in the anesthetized cat26-28,42, few studies have used this technique to examine sensory processing in cat auditory cortex. The purpose of the present protocol is to establish an effective method of using fMRI to quantify function in the auditory cortex of the anesthetized cat. The experimental procedures outlined in this manuscript have been successfully used to describe the features of the BOLD response time course in the adult cat auditory cortex43.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Atropine sulphate injection 0.5 mg/mL</td>
 <td>Rafter 8 Products</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Acepromazine 5 mg/mL</td>
 <td>Vetoquinol Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine hydrochloride 100 mg/mL</td>
 <td>Bimeda-MTC</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dexmedetomidine hydrochloride (Dexdomitor 0.5 mg/mL)</td>
 <td>Orion Pharma</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane 99.9%</td>
 <td>Abbott Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lidocaine (Xylocaine endotracheal 10 mg/metered dose)</td>
 <td>Astra Zeneca</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lubricating opthalmic ointment (Refresh Lacri Lube)</td>
 <td>Allergan Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Saline 0.95%</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IV Catheter 22g (wings)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IV Extension Set</td>
 <td>Codan US Corp.</td>
 <td>BC 269</td>
 <td></td>
 </tr>
 <tr>
 <td>IV Administration Set 10 drips/mL</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Endotracheal tube 4.0</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pads (Snuggle Safe)</td>
 <td>Lenric C21 Ltd.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe 60 mL</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>External sound card</td>
 <td>Roland Corporation</td>
 <td>Cakewalk UA-25EX</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereo power amplifier</td>
 <td>Pyle Audio Inc.</td>
 <td>Pyle Pro PCAU11</td>
 <td></td>
 </tr>
 <tr>
 <td>MRI-compatible insert earphone system</td>
 <td>Sensimetric Corporation</td>
 <td>Model S14</td>
 <td></td>
 </tr>
 <tr>
 <td>Foam ear tips for insert earphones</td>
 <td>E-A-R Auditory Systems</td>
 <td>Earlink 3B</td>
 <td></td>
 </tr>
 <tr>
 <td>End-tidal CO2 monitor</td>
 <td>Nellcor&nbsp;</td>
 <td>N-85</td>
 <td></td>
 </tr>
 <tr>
 <td>MRI-compatible pulse oximeter</td>
 <td>Nonin Medical Inc.</td>
 <td>Model 7500</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Harvard Apparatus</td>
 <td>70-2208</td>
 <td></td>
 </tr>
 </tbody>
 </table>

functional imaging of auditory cortex in adult cats using high-field fmri

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Representative functional data were acquired in a 7T horizontal bore scanner and analyzed using the Statistical Parametric Mapping toolbox in MATLAB. Robust cortical hemodynamic responses to auditory stimulation have consistently been observed in cats using the described experimental protocol43. Figure 6 illustrates the BOLD activation in 2 animals in response to a 30 sec broadband noise stimulus presented in a block design. T-statistic maps of the broadband noise vs. baseline (no stimulus) co...

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Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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11.4K Views.  University of Western Ontario. Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.