Ethical approval for these methods was obtained from the Auburn University Institutional Animal Care and Use Committee and all methods were performed in accordance with their guidelines and regulations. For auditory exposure, progression through the sessions is based on week number. For stationing sessions, a specific session-specified performance criterion (e.g., at least eleven-second duration of chin targeting), must be met before the trainer may advance the dog to the next session in that training phase. Otherwise, that step is repeated.
1. Auditory Exposure sessions
NOTE: These sessions constitute passive exposure and active classical counterconditioning of a positive Conditioned Emotional Response (CER) to MRI scanner noise; the scanner noise is established as a stimulus predicting the access to toy play or food rewards. Exposure sessions occur once per week for approximately 10 min.
Figure 2: Active Exposure. Active Exposure (AE) is a short-delay classical conditioning procedure. 10 s CS (i.e., scan audio presented by itself), 20 s CS + US (i.e., ball and scan audio presented together), 10 s delay (no ball, no scan audio). After this delay, the trial starts over. There are ten trials per session, with incremental volume increases over sessions. Please click here to view a larger version of this figure.
NOTE: Collect the following audio: MRI scanner baseline, shim, localizer (scout), MPRAGE, GRE Field, EPI, Multiband EPI, DTI, and RESOLVE DTI, using, for example, a smartphone's audio recording app through the open door of a 3T MRI suite during phantom scans. Determine volume level of audio playback during training sessions via a decibel meter phone app.
2. Stationing sessions
NOTE: These sessions are divided into two phases: Open environment and Mock MRI. After the chin-to-object target is learned, durations are increased on a percentile schedule of 10% increases. As new elements and pieces of equipment are added into the training context, certain criteria of the behavior (e.g., duration) are temporarily relaxed:
1) In the stationing sessions, the trainer trains a nose-touch behavior to a folded towel and then a chin rest on a folded towel. That chin rest behavior is generalized to occur in a foam chin rest and gradually built to a 5 min bout duration.
2) Simultaneously, robust down and stay behaviors are built and maintained.
3) Those behaviors are then conditioned to occur in an enclosed space (i.e., tunnel) and at a 3' elevation.
4) The dog is then acclimated to the head enclosure (mock human extremity RF coil).
5) Ear padding is introduced, and scan audio is (re)introduced in the context of the stationing behavior.
The dog will ultimately be able to perform a robust chin rest with head and body enclosed at a 3' elevation, with ear padding and scan audio playing at 90 + decibels (dB), for at least 5 min bouts. On reinforcement – some dogs are inherently more motivated by food, whereas others are more motivated by play or praise9. In "click-then-treat" (C/T), the T does not necessarily mean food treats, rather it refers to the reward procedure, whatever that may be for that particular dog at that particular stage in its training. Although food rewards lend themselves to higher rates and stiller repetitions of behavior, whatever the dog prefers can be used initially, even if it is high-motion play (e.g., ball, tug). As the chin target behavior becomes more resilient against distraction and duration, transition to using food rewards. Eventually, toy play can be saved for long-duration or chained bouts of chin rest performance.
3. Transfer
NOTE: 1) Upon reaching final criterion of the stationing behavior in the mock MRI training location (5 min down-stay and chin rest in mock bore and mock RF coil while wearing ear padding, with scanner noise playing at 80-110 dB), the dog undergoes five distinct location transfer (generalization) sessions. During these transfer sessions the dog stations to the above criteria in several indoor and outdoor locations that are as unique as possible, with different sights, sounds, and degrees of social distraction across settings (e.g., secluded grass field, quiet academic building hallway, busy academic building lobby, crowded bus stop, loud water treatment plant)8.
4. MRI
The mean number of repetitions of each session level is listed in Table 1. The complete training and testing protocol required 14 h (M = 13.55 h, range 12-16 h) and consisted of 90 sessions (range 87-93 sessions). Open environment training lasted 4.38 h (range 3-5 h), mock MRI training lasted 5.4 h (range 4.2-6.5 h), and transfer was 2.5 h divided into five 30 min sessions. Maintenance sessions at level 19 were conducted during transfer and are reflected in the complete training time above.
Session Level | Criteria | Duration | Session Repetitions (M, SE) | |
Open Environment | 1. Charge the clicker | Build an association between the ‘tic-toc’ of the clicker and the dog’s primary reward (e.g., food) while capturingattention. | 3 min | 1, 0 |
2. Capture chin target to towel | Build chin-to-towel contact to 2+ seconds. * | 5 min | 3.75, .75 | |
3. Chin-to-towel target with short duration and addition and a cue | Chin contact for 7+ seconds. * | 5 min | 8.25, 2.8 | |
4. Chin rest on towel in a down and addition of distraction | Chin contact for 11+ seconds, with and without distraction. | 5-10 min | 2.75, .25 | |
5. Chin rest on towel with distance | Chin contact for 16+ seconds, cued from progressively farther away (i.e., sitting on ground, kneeling, standing). | 5-10 min | 3.5, .87 | |
6. Chin rest on towel with increasing duration and distance | Chin contact for 26+ seconds. | 5-10 min | 5.5, 1.5 | |
7. Introduce foam chin rest, duration initially reduced | Chin contact to foam chin rest for 40+ seconds. | 5-15 min | 4.75, .75 | |
8. Chin rest in foam chin rest with increasing duration and distraction | Chin contact for 73+ seconds. | 5-15 min | 6, 1.2 | |
Mock MRI | 9. Introduce bore and elevation with reduced duration | Chin contact in bore on table for 16+ seconds. ** | 5-15 min | 2.5, .5 |
10. Elevated chin rest with increasing duration | Chin contact in bore on table for 60+ seconds. | 5-15 min | 3, 0 | |
11. Introduce mock radiofrequency (RF) coil with no elevation and reduced duration | Chin contact in RF coil on ground for 30+ seconds. | 5-15 min | 2.75, .25 | |
12. Elevated chin rest in mock RF coil | Chin contact to foam chin rest through the mock RF coil in the elevated bore for 50+ seconds. | 5-15 min | 2, 0 | |
13. Elevated chin rest in mock RF coil with increasing distraction and duration | Chin contact for 100+ seconds, with and without distraction. | 5-15 min | 2.5, .29 | |
14. Introduce ear padding, duration initially reduced | Chin contact in mock bore and RF coil (mock MRI) with ear padding for 60+ seconds. | 5-15 min | 3, .41 | |
15. Elevated chin rest in mock RF coil with ear padding and increasing duration and distraction | Chin contact for 107 seconds, with and without distractions. | 5-15 min | 2.5, .29 | |
16. Introduce scanner noise | Chin contact in mock MRI with ear padding and up to 40 dB scan audio for 107+ seconds. | 10-30 min | 2.5, .5 | |
17. Build duration to 2 minutes 30 seconds with increasing distance | Chin contact in mock MRI with ear padding and 41-70 dB scan audio for 142+ seconds, with and without distraction and distance. | 10-30 min | 2.5, .5 | |
18. Build duration to 4 minutes | Chin contact in mock MRI with ear padding and 60-90 dB scan audio for 240+ seconds, with and without distraction and distance. | 10-30 min | 2.75, .75 | |
19. Build duration to 5 minutes | Chin contact in mock MRI with ear padding and 80-110 dB scan audio for 300+ seconds, with and without distraction and distance. | 10-30 min | 10, 1.8 | |
Transfer | 20. Five distinct location transfer (generalization) sessions | During these transfer sessions the dog stations to the above criteria in several indoor and outdoor locations that are as unique as possible, with different sights, sounds, and degrees of social distraction across settings. | 30 min | 5, 0 |
Tous | Final behavior(s) | The dog performs a chin rest with head and body enclosed at a 3’ elevation, with ear padding and scan audio playing at 90 + dB, for at least five minutes. | 12-16 h (M=13.55, SE=0.94) | 87-93 sessions (M=90, SE=1.5) |
Table 1: Session levels. *See note in manuscript. **Conduct first session with mock bore on ground.
Stationing training and testing
Figure 3 shows the maximum duration of four dogs trained in the protocol for the last three sessions at the end of training and the different training locations. Performance was stable at the end of stationing training, F(2, 6) < 1, and over 5 min (M = 311 seconds, SEM = 1.9). All dogs transferred to the mock training locations with a max duration equivalent to training, F(1,3) < 1. Three of the dogs transferred to the MRI scanner and demonstrated repeated bouts of the max possible duration (206 s). The one dog that did not transfer to the MRI scanner had a larger head than the other dogs and could not comfortably fit within the coil. This discomfort likely led to the dog not willingly participating in the scans.
Figure 3: Maximum duration of four dogs trained in the protocol for the last three sessions at the end of training and the different training locations. All dogs transferred to the mock training locations and three of the dogs transferred to the MRI scanner demonstrating the maximum possible duration (206 s). Please click here to view a larger version of this figure.
Representative fMRI stimulus driven scans
In these scans, visual or odor stimuli were presented to the dog while the dog remains still. Visual stimuli were projected on a screen located in the bore of the scanner. Each scan lasted for 140 s and contained 12 different images (e.g., human and dog faces). A stimulus was presented for 5 s followed by a variable 3-11 s inter-stimulus interval (see Figure 4 for visual depiction and Thompkins et al. 2018 for additional details)10.
Figure 4: Visual stimuli. The top panel shows an example run of dog faces. The bottom panel shows an example run of human faces. Face stimuli were displayed for 5 s, with 3-11 s inter-stimulus intervals. Twelve face stimuli were shown per run. Please click here to view a larger version of this figure.
Attentional check
To determine whether dogs were attending to the visual stimuli, independent raters viewed videos of the dogs' eyes inside the bore of the MRI scanner synced up with the stimulus presentation. Based on whether the dogs' eyes were open and their pupils visible, the raters assigned an appropriate score for each stimulus (Figure 5). fMRI data was used only when there was perfect inter-rater agreement.
Figure 5: Attentional check. To ensure each dog looked at each stimulus presented during scanning, stimulus-synchronized video of the dog's eye inside the scanner was analyzed by two raters post hoc; for each trial, if the dog's eye was visibly open, the rater assigned a score of "yes" and if the dog's eye was closed, the rater assigned a score of "no." fMRI data was used only when there was perfect inter-rater agreement. This figure has been modified from Thompkins et al.10 Please click here to view a larger version of this figure.
Dog and human face processing
Figure 6 shows adjacent but different brain areas of temporal cortex in the dog brain are active for processing dog and human faces. Green regions represent areas of the brain more active for human faces contrasted with dog faces (p < 0.05, FDR (false discovery rate corrected)). Red regions represent areas of the brain more active for dog faces contrasted with human faces (p < 0.05, FDR).
Figure 6: Results of human and dog face contrasts. Regions in green represent areas that are significantly more active during processing of human faces as compared to dog faces (i.e., human face area, HFA). Regions in red represent areas that are significantly more active during processing of dog faces as compared to human faces (i.e., dog face area, DFA). This figure has been modified from Thompkins et al.10 Please click here to view a larger version of this figure.
Odor stimuli
Odor stimuli were delivered through an olfactometer (Figure 7); high (0.16 mM) and low (0.016 mM) concentrations of odorant ethyl butyrate were used to probe parametric modulation of olfactory areas by odorant concentration. Each scan lasted 200 s and contained 5 blocks of 10 s odorant stimulation, each followed by a 30 s inter-stimulus interval (see Figure 8 for visual depiction and Jia et al. 2014 for additional details)4.
Figure 7: Olfactory imaging system. Components of the dog olfactory imaging system outside the MRI room showing odorant applicator, air tank, motion parameter recording palmtop, video monitor, laptop with VT-8 software, and the entrance port to the MRI room. This figure has been modified from Jia et al.4 Please click here to view a larger version of this figure.
Figure 8: Odorant delivery. Odorant delivery was controlled by VT-8 Warner Timer software in a fMRI block design. The first row shows the odorant delivery sequence with green arrows indicating stimulus onset and red arrows indicating stimulus offset. The second row shows clearance of odorant, with green arrows indicating onset of odorant clearance and red arrows indicating offset of odorant clearance. The third row shows the fMRI block design, matching the first row, with "0" and "1" denoting odorant "off" and "on" conditions, respectively. This figure has been modified from Jia et al.4 Please click here to view a larger version of this figure.
Researchers delivered high (0.16 mM) and low (0.016mM) concentrations of an ethyl butyrate solution to six trained detection canines (Labradors) while awake and anesthetized. The parametric increases in magnitude of activation to low and high concentrations of odorant in olfactory regions (olfactory bulb, bilateral piriform lobes, cerebellum) was in accordance with Weber's Law (threefold perceived increase for a tenfold concentration increase). In addition, while the olfactory bulb, periamygdala, anterior olfactory cortex, entorhinal cortex, and piriform lobes were active in both awake and anesthetized dogs, regions implicating higher-order cognitive processing (superior, medial and orbital portions of frontal cortex) were activated mainly in awake dogs (Figure 9).
Figure 9: Group activation maps for anesthetized dogs. Three orthogonal views are shown in each subfigure. Colormap is used for activation intensity and important areas are indicated by arrows with labels (Overall FDR = 0.05, cluster threshold = 15 voxels using AlphaSim, t-contrast). A: Anterior, P: posterior, S: superior, I: inferior, L: left, R: right. Subfigure (A) corresponds to low concentration odorant (0.016 mM), subfigure (B) corresponds to high concentration odorant (0.16 mM), subfigure (C) corresponds to anesthetized dog olfactory processing, and subfigure (D) corresponds to awake dog olfactory processing. This figure has been modified from Jia et al.4 Please click here to view a larger version of this figure.
Acrylic Mock Radiofrequency Coil | Menards | TU59018594 | Mock Radiofrequency (RF) Coil: 8" diameter x 4' Concrete Form Tube. Makes four mock RF coils; cut form tube in four even lengths for four 8" diameter x 1' mock RF coils. |
Agility Tunnel | J&J Dog Supplies | TT053 | Open Agility Training Tunnel |
Bluetooth Speaker | Sharkk | SP-SK896WTR-GRY | Portable Scan Audio Playback: Waterproof Bluetooth Speaker Sharkk 2O IP67 Bluetooth Speaker Outdoor Pool Beach and Shower Portable Wireless Speaker |
Cardboard Concrete Form Tube | Menards | TU10120014 | Stationary Mock MRI Bore: Sonotube 24" diameter x 12' Standard Wall Water-Resistant Concrete Form. Makes two mock bores; cut form tube in half for two 24" diameter x 6' bores. |
Chuckit Ball | Chuckit! | 17030 | Toy Reward: Chuckit! Ultra Ball |
Decibel X | Skypaw | Decibel meter phone app | |
Exercise Mat | Foam chin rest: cut mat in half lengthwise. Roll up, and secure roll with hot glue. Cut chin-size notch in center with X-ACTO knife. Hot-glue velcro to bottom surface. | ||
Folding Table | 3' x 6' folding table | ||
Microfiber Car Wax Applicator Pad | Viking Car Care | 862400 | Viking Car Care Microfiber Applicator Pads |
Natural Balance Treat Log | Natual Balance | 236020 | Food Reward: E.g., Chicken Formula Dog Food Roll, 3.5-lb roll |
Plywood | Platform: 2"x4"x6' length of wood affixed to 3'x6' plywood board. Hot glue exercise mat on plywood board for traction. Braces: 3 4x4x4" cubes cut at 45-degree angle affixed to ends of 1"x4"x3' lengths of wood. Makes 3 braces. | ||
Sand Bags | J&J Dog Supplies | AG155 | J&J Professional Quality Sandbags x 2 |
Speaker System | Pioneer Electrics | HTD645DV | Stationary Scan Audio Playback: Pioneer HTD645DV 5 Disk DVD Home Theater System with Wireless Surround Speakers. Operating Instructions. |
Towel | standard towel |
We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for acquisition of functional and structural data. This method of training dogs for awake, unrestrained fMRI employs a generalization procedure of stationing in several dissimilar locations to facilitate transfer of the stationing behavior to the real MRI scan environment; it does so without the need for extensive training time in the MRI scan environment, which can be expensive. Further, this method splits the training of a stationing (i.e., chin rest) behavior from desensitization to the MRI environment (i.e., 100+ decibel scan audio), the latter accomplished during dedicated Auditory Exposure conditioning sessions. The complete training and testing protocol required 14 hours and resulted in immediate transfer to novel locations. We also present examples of canine fMRI data that have been acquired from visual face processing and olfactory discrimination paradigms.
We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for acquisition of functional and structural data. This method of training dogs for awake, unrestrained fMRI employs a generalization procedure of stationing in several dissimilar locations to facilitate transfer of the stationing behavior to the real MRI scan environment; it does so without the need for extensive training time in the MRI scan environment, which can be expensive. Further, this method splits the training of a stationing (i.e., chin rest) behavior from desensitization to the MRI environment (i.e., 100+ decibel scan audio), the latter accomplished during dedicated Auditory Exposure conditioning sessions. The complete training and testing protocol required 14 hours and resulted in immediate transfer to novel locations. We also present examples of canine fMRI data that have been acquired from visual face processing and olfactory discrimination paradigms.
We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for acquisition of functional and structural data. This method of training dogs for awake, unrestrained fMRI employs a generalization procedure of stationing in several dissimilar locations to facilitate transfer of the stationing behavior to the real MRI scan environment; it does so without the need for extensive training time in the MRI scan environment, which can be expensive. Further, this method splits the training of a stationing (i.e., chin rest) behavior from desensitization to the MRI environment (i.e., 100+ decibel scan audio), the latter accomplished during dedicated Auditory Exposure conditioning sessions. The complete training and testing protocol required 14 hours and resulted in immediate transfer to novel locations. We also present examples of canine fMRI data that have been acquired from visual face processing and olfactory discrimination paradigms.