Functional ultrasound is a new neuroimaging modality that allows the mapping of cerebral blood volume in the living rodent brain. Using ultra fast plane wave imaging, we can measure whole brain hemodynamic responses with unmatched special temporary resolution and sensitivity. This protocol explains how to perform transcranial functional ultrasound imaging in mice, both for anesthetized and awake animal experiments.
Compared to other whole brain functional imaging techniques such as FMRI, functional ultrasound provides high portability, ease of use and allows experiments in awake and freely moving subjects, avoiding anesthesia bias and enabling behavioral studies. Until recently, FUS imaging was only used in collaboration with ultrasound expert. Now, this technology is available to the wide neuroscience community, thanks to commercially available scanners and dedicated software for preclinical brain imaging, making FUS pretty easy to use without any background in ultrasound.
For an anesthetized imaging session, apply eye ointment to the mouse eyes to avoid any corneal damage and shave the mouse head using a tremor. Apply some depilatory cream and rinse after a couple of minutes. Repeat this until the hair is completely removed.
Insert subcutaneous pins in the limbs for electrocardiogram recording and place centrifuged ultrasound gel on the head. For awake mice experiments, a preliminary surgery is required for head fixation. Place the anesthetized mouse and a stereotactic frame on a 37 degrees Celsius heating pad.
Apply protective gel for the eyes and subcutaneously administer lidocaine under the scalp using a 26 gauge needle, then wait a few minutes. Make an incision following the sagittal suture from behind the occipital bone to the beginning of the nasal bone, then use surgical scissors to excise the skin over both hemispheres. Clean the skull with one percent iodine solution and remove any remaining periosteum.
Using the head plate as a template, mark two holes in the skull to position the anchoring screws. Position the head plate with the screws and use dental cement to fix the screws and the head plate in the front and back of the frame to maintain good grip of the implant. Remove the animal from the stereotactic frame after the cement is dry and reverse the anesthesia via subcutaneous injection of one milligram per kilogram of atipamezole.
Administer prophylactic meloxicam for post-operative pain. Place a magnetic 3D printed cap over the head plate for protection and allow the mouse to recover for four to six days before the beginning of habituation to the mobile home cage. Place the animal in a recovery cage on a heating pad for a few hours, then return the mouse to its home cage with litter mates.
On day four and five post recovery, repeatedly clamp the mouse to the mobile home cage and gradually increase the head fixed time, starting from five minutes and up to 30 minutes. Apply some saline and ultrasound gel to the imaging window to habituate the mouse. Repeat this process on day six post recovery.
Start the software and create an experiment session. Go to the move probe menu to adjust the position of the ultrasound probe using the navigation keyboard. Start the live view acquisition and adjust probe position if needed via real time imaging of the animal cerebral blood volume, or CBV.
Align the brain at the center of the image, then optimize the imaging parameters to capture the highest signal to noise ratio. Open the Angio 3D option of the acquisition software. On the preset panel, adjust the first slice, last slice and step size scanning parameters in order to scan the whole brain and start the acquisition.
Leave the acquisition software open and start the software for data analysis and visualization, then load the Angio 3D scan. Navigate through the acquisition volume using the three views panel and select the coronal scan direction, anteroposterior or postural anterior. Go to the brain registration panel and load the mouse reference template for the registration process.
Register the scan on the Allen Mouse Common Coordinates Framework using the fully automatic or the manual registration modes. Check the result by looking at the superposition of the Angio 3D scan and the reference template, or by looking at the superposition of the scan and the Allen Reference Atlas using the Atlas manager panel. Save the registration as a BPS file.
In the ICO studio software, make sure that the angiographic scan and its BPS file are loaded. Go to the brain navigation panel. In the Atlas Manager panel, navigate through the mouse Allen Brain Atlas with the parent child tree navigator.
Find the anatomical targeted regions and select them to superimpose them to the scan in the three views. Visualize the targeted regions in the three view panel and choose an imaging plane that overlaps the targeted regions for the experiment by manually setting two markers on the coronal position that includes the region of interest. Click on Brain Positioning System, or BPS, to extract the resulting motor coordinates which correspond to the probe position to image the targeted plane.
Check the preview of the image which is computed from the angio scan. In the ICO scan software, enter the probe positioning panel and click on enter BPS coordinates, then apply the extracted coordinates, causing the probe to move and align on the targeted imaging plane. Perform a live view acquisition and check that the current imaging plane corresponds to the prediction.
Predefine the stimulation sequence, including time of stimulation, interest stimulation time, and number of repetitions. Run a 3D FUS sequence by defining the total time of acquisition, the number of positions, and the dead time between positions. For automatic stimulation synchronized with the acquisition system through TTL input, select the trig in option before starting the acquisition.
Open the acquisition in ICO studio software and enter the activation map menu, then fill the activation pattern field with a start and end times and compute the activation map. Adjust the display parameters for visualization and export the activation map as a H5 file for offline analysis. Run a 3D FUS sequence by defining the total time of acquisition, the number of imaging plane positions, and the dead time between positions.
Save the acquisition and load it in the ICO studio software. If necessary, load the BPS file and the Allen Mouse Brain Coordination Framework. In the Atlas Manager, select regions of the Atlas as regions of interest.
Enter the functional conductivity menu and select the desired regions and the ROI manager. Visualize the results as connectivity matrix or seed based correlation map, then select and adjust the bandwidth filters as desired and export correlation results for statistical analysis. This protocol was used for 3D quantification of cerebral hemodynamic variations transcranially in the mouse brain.
Whisker stimulation was selected as an example of sensory stimulation evoked response. Significant activation was determined with a resolution of a general linear model, or GLM, using default mouse hemodynamic response function. The total trial time was 760 seconds, with a 60 second baseline, an 80 second stimulation, and a 60 second recovery time repeated five times.
Using a voxel wise time course of the contralateral primary somatosensory cortex, the barrel field region, or S1BF, revealed a 15 to 20%increase of the CBV compared to baseline. The same paradigm was applied in a head fixed behaving mouse in the mobile home cage using the awake preset of ICO scan. The activation map after a multiple whisker stimulation experiment is shown here.
Significant activation was determined with the resolution of a GLM using a default mouse hemodynamic response function. The temporal correlations have normalized low-frequency spontaneous CBV fluctuations between 3D brain regions in a ketamine xylazine anesthetized mouse are shown here. Seed based analysis and the dorsal hippocampus revealed the significant interhemispheric conductivity between the right and left hippocampus, as well as deeper retro hippocampal regions and piriform courtesies.
A seed region selected in the S1BF also resulted in a symmetrical correlation pattern. The critical point for successful experiments is the animal preparation, in particular the level of anesthesia for experiments involving anesthetized animals and the protection of a skull in experiments in awake animals. If US enabled us to study important brain functions in awake animals dealing with fundamental questions on sleep, learning or behavior, but also the pharmacological modulation of functional connectivity for drug discovery.
