Simultaneous Focused Ultrasound Neuromodulation and Fiber Photometry Recording in Free-Moving Mouse

Our laboratory is dedicated to points application of ultrasound based technologies to study brain function and address brain disorders. Our current research primarily revolves around two key errors:functional ultrasound imaging and focused ultrasound neuromodulation. Focused on ultrasound neuromodulation is considered a potential approach for brain intervention in humans, however, its mechanisms are unclear.

Recently, the integration of fiber optometry and focus on ultrasound neuromodulation has been approved to be useful for screen effective parameters for modulating specific neuro types in various developable regions. This protocol aims both the researchers to detect ultrasound modulated neuroactivity in freely-moving mice without anesthesia or stereotactic frame fixation by using minimally invasive fiber optometry technology to capture calcium dynamic as a measure of neuroactivity beneath the implant. It mitigates potential confounding effects of culture sound vibration of the probe itself.

This protocol is suitable for studying perception, coordination, and behavior. It enables the investigation of involved matching enzymes of ultrasonic neuromodulation and the development of novel parameter sites for treating brain disorders. To begin, prepare a piezoelectric plate.

Using epoxy silver paste, attach the wire to both sides of the piezoelectric plate. Once the epoxy paste has solidified, use a multimeter to measure the resistance at both ends of the wire to ensure it is approximately zero. Then apply a layer of double-sided tape onto a clean glass sheet surface.

Adhere the piezoelectric plate and copper ring tightly to the glass sheet. Securely insert the polypropylene pipe with an outer diameter of three millimeters into the center of the piezoelectric plate and firmly adhere it to the glass sheet. Now, vacuum the prepared epoxy resin glue.

Using a disposable syringe, extract the epoxy and slowly inject it into the copper ring. Using an electronic soldering iron, solder the loose ends of two wires onto the bayonet nut connector. After removing the glass sheet, clean the surface of the transducer with alcohol.

To begin, place the hydrophone and transducer in a water tank filled with deionized water. Discover a field maximum in the focal plane through 2D scanning. Then identify a field maximum in another plane with a clear maximum.

After comparing the X and Y coordinates of the two maxima, adjust the position and orientation of the transducer if needed. Adjust the hydrophone tip to be one millimeter from the transducer surface, positioning it in the middle of the right edge. Then initiate the scanning program to capture the free acoustic field in the XC plane.

Move the hydrophone along the Z-axis to determine the depths associated with the spatial peak pressure. Afterward, move the hydrophone to the bottom right corner of the transducer at the XY plane. Power up and initiate the scanning program to capture the free acoustic field in the XY plane.

After placing the transducer on the skull of the prepared mouse, acquire the XC in XY plane's transcranial acoustic field through hydrophone scanning. Read the pressure amplitudes. Read the focal dimensions at 3 decibels and the position on the XY and XC planes within the transcranial acoustic field, Report the pulse timing parameters, including Amax, pulse duration, pulse repetition interval, pulse train duration, and envelope.

To begin, use a fader to trim the hair on the anesthetized animal's head and disinfect the area with 70%ethanol and povidone iodine. Place the mouse in a prone position on the stereotaxic frame. Make an incision along the sagittal suture, starting from the occipital bone to the beginning of the nasal bone.

Once the skin covering the hemispheres is removed, use sterile saline to cleanse the skull and eliminate any remaining periosteum. Using a cotton swab, apply 3%hydrogen peroxide to the exposed cranium for approximately two to three seconds to create micropores. Afterward, thoroughly rinse with sterile saline and make sure the area is completely dry.

Next, create a 0.6 millimeter diameter bur hole craniotomy. Wash away any debris with sterile saline. Insert the fiber optical ferrule into the probe holder and connect it to the stereotaxic arm.

Use the stereotaxic arm to align the implant directly above the region of interest and insert it into the region of interest. Now use a sterile toothpick to spread a thin layer of prepared dental cement over the cranium and onto the lower part of the implant. Carefully detach the probe holder.

Prepare a polypropylene pipe and cut it throughout its length. Using tweezers, attach the pipe to the bottom of the implant. After pouring the dental cement powder into the pipe, add the required liquid and allow a few minutes for the dental cement to solidify.

Locate the pipe opening and carefully clamp it to remove it using tweezers. After preparing the dental cement mixture for application, ensure an even and thin layer is spread across the cranium. Then drill three holes into the 3D printed ring.

Secure the screws into their respective holes. Insert the top of the implant into the hole of the pre-manufactured transducer. Ensure the inner wall of the 3D printed ring is smooth.

Then place it around the transducer positioned on the mouse skull. Apply dental cement to the junction between the ring and the skull. Then wait a few minutes for the dental cement to solidify.

Carefully remove the transducer and securely tighten the screws. After positioning the anesthetized mouse on the stereotaxic frame, clean the top surface of the implant with alcohol. Inject water and a coupling agent into the space between the implant and the 3D printed ring.

Insert the fiber optic patch cord into the center of the prepared transducer. Then connect the implant to the fiber optic patch cord. Carefully insert the transducer into the area.

Next, place the mouse in an open field and allow it to wake up. Attach the transducer to the ultrasonic excitation system and connect the fiber optic patch cord to the optical fiber recording system. Activate both the ultrasonic excitation device and the optical fiber recording system.

The optical fiber signals were recorded under focused ultrasound neuromodulation with the envelopes being square and sinusoidal respectively. The square signal lasted 300 milliseconds, while the continuous sinusoidal signal lasted 471 milliseconds.