The overall goal of this video is to present and explain an efficient and easily adaptable method of fMRI in order to effectively explore the mapping of the human olfactory system without interference of natural breathing. One obstacle of using olfactory fMRI to study the brain activation responding to odor and stimulation, is the inconsistency of repetitive odor stimulation, which is caused by the asynchrony of respiration and odor delivery. As illustrated here, when the odor presentation in a fixed timing odor stimulation paradigm is not synchronized with respiration, the repetitive odor stimulation can happen at different phases of respiration.
A solution to this problem is to set up a respiration-triggered olfactory fMRI paradigm. Effective olfactory fMRI data collection is accomplished by first designing an event related respiration-triggered olfactory stimulation paradigm on the olfactometer that is resist to habituation and inconsistent inhalation patterns. The second step is to prepare odorants for the olfactory stimulation, taking care to use odorants that minimize trigeminal stimulation and habituation effects.
The third step is to set up and run the experiment in the MRI environment by assembling the olfactometer with the capability of running a respiration-triggered paradigm. The final step is to process the data using the program ONSET to define the actual onset and the duration vectors of the stimuli. Ultimately, this technique has the potential to facilitate more sensitive exploration and connections of the human olfactory system.
Here, we will demonstrate how to set up a respiration-triggered olfactory fMRI experiment using an ETT Olfactometer. To create a paradigm, assign the odorant channels to specific odorants. There are six odorant channels available in this olfactometer system.
Input and edit the sequence and timing of open and close of these channels. Push the Paradigm Setting button on the touchscreen. To input or edit the sequence of the open and close of the odorant channels, push the Edit button for the Valve Sequence.
When finished, push the Edit button again to exit the editing mode. To input or edit the timing parameters for the open and close of the odorant channels, push the Set button for Time Sequence. When finished, push the Set button again to exit the editing mode.
After finishing the Paradigm Setting, push the Return button to return to the main window. Demonstrated in this video is a simple stimulation paradigm of the same odorant in the same concentration. Each odorant presentation is interleaved with the presentation of fresh air.
In this paradigm, the same odor is presented 12 times. Each time, the odorant presentation lasts for six seconds with variable amounts of time between the odor presentations. Set the Valve Open and Valve Close durations, accordingly.
In this demonstration, one concentration of lavender oil is prepared. We use lavender oil because of its minimal trigeminal stimulation and activation of certain brain areas. The odorant concentrations can be varied depending on the odorant selected.
Choose the concentrations based upon the ideal perception threshold of the odor. By varying the concentration, we lessen the likelihood of habituation to an odorant. To prepare the concentrations, mix the lavender oil with propylene glycol.
Propylene glycol is a commonly used solvent for odorants and is stable and nearly odorless. After mixing all solutions as outlined in the paradigm design, pour the same amount of odorants into each container bottle. Then, attach the bottles to the odorant carrier in the correct order.
To set up the olfactometer in the MRI, place the odorant carrier in the magnet room next to the machine. Run the tubes through the wave guide to connect to the carrier, taking care that the order of tubes matches the order connecting to the olfactometer. For ease, we color code our tubings to match the odorant channels.
Turn the machine on. Adjust the main airflow, as necessary, as well as the individual flow rates for the carrier and flush. Then, connect the respiration belt to the olfactometer and run the belt through the wave guide.
The belt will record the subject's respiration activity, as well as the utilization of the respiration trigger. Bring the subject into the magnet room and place the face mask on the subject. When the subject is lying down, place the respiratory belt around their chest or abdomen where there is clear respiration event.
Check the respiration pattern on the screen of the olfactometer. If the waves plateau, the belt is fastened too tightly and the respiration trigger will not work properly. Adjust the belt until a complete respiration trace can be seen, as clear respiration peak signals are critical for a successful execution of a respiration-triggered paradigm.
Create a data folder on the olfactometer to store the respiration data. The synchrony of the odor presentation with respiration should be tested for accuracy by using the Paradigm Check and adjusting the Trigger Delay time, as necessary. Push the Paradigm Check button on the touchscreen and monitor if odor delivery happens during the inhalation phase of the respiration cycle.
The odor delivery period is highlighted in the color pink on the respiration trace. If odor delivery starts during the exhalation phase, to back to Paradigm Setting, adjust the Trigger Delay in the time sequence and then redo the Paradigm Check. Set the paradigm to Trigger In mode so that the execution of the odor stimulation paradigm will be triggered by MRI scanning.
Then select the Respiration Trigger Start function. The paradigm is now ready to be run. Once the odor stimulation paradigm is completed, check the respiration data saved in the olfactometer.
This file includes the respiration trace and the time stamp of each functional MRI image, respiration trigger, open and close of each odor channel, and the subject response, using a response device. It can be processed using the program ONSET to acquire the actual stimulation onset vectors and durations for fMRI data processing. ONSET can be run in the IDL Virtual Machine.
To get the odor stimulation onset vectors and durations, click Respiration Validation. Load the respiration data file that's saved from the olfactometer and set the post processing parameters for ONSET processing. Run ONSET.
The actual stimulation onset vectors, the start of each inhalation during odor delivery, will automatically be detected based on the timing of odor presentation and the respiration trace. The results, including the onset and duration vectors, can be utilized in fMRI data processing. Process the fMRI data following standard procedures.
Here is an example of data processing using SPM8. Use either the image numbers or the onset vectors and durations in seconds from the onset output, depending on the timing unit chosen for the SPM paradigm design. Generate a statistical parametric map at the individual level by fitting the stimulation paradigm using the actual onset and duration vectors to the functional data with the canonical hemodynamic response function.
Here is an example of the primary olfactory cortex activation responding to the odor stimulation. In contrast to the respiration-triggered odor stimulation paradigm, for example, a fixed timing paradigm which we can see here from the same subject, the odor delivery is not triggered by respiration activity. Since there is no control of the subject's respiration, there is significant variability in the amount of odorant that is inhaled.
Following the standard procedure to process these fMRI data with the same statistical threshold as that used for the previous respiration-triggered fMRI data, no significant activation was detected in the primary olfactory cortex. However, when a respiration trigger is implemented, odorant presentation is always synchronized with inhalation, improving the consistency and accuracy of odor stimulation. After watching this video, you should have a good understanding of the current practice of olfactory fMRI using an event related respiration-triggered paradigm.
Once the technique demonstrated in this video has been mastered, further olfactory stimulation paradigm design and analysis methods can be applied to map the functions of the human central olfactory system and to further explore the olfactory deficits in neurodegenerative diseases.
