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

All procedures and animal handling complied with NSFC ethics guidelines and approved protocol requirements of the Institutional Animal Care and Use Committee of the Guangdong Institute of Intelligence Science and Technology.

1. Transducer preparation

1. Prepare a piezoelectric plate with an inner diameter of 3 mm, an outer diameter of 7 mm, and a center frequency of 500 kHz.
   NOTE: The outer diameter can be adjusted based on the specific brain region being targeted and should be maximized while maintaining stimulation accuracy and without exceeding the boundary of the mouse skull.
2. Attach the wire to the two sides of the piezoelectric plate using epoxy silver paste (Figure 1). After the epoxy silver paste has solidified, use a multimeter to measure the resistance at both ends of the wire to ensure that it is approximately 0.
3. Apply a layer of double-sided tape onto a clean glass sheet surface. Adhere to the piezoelectric plate and copper ring, with a height of 8 mm, an outer diameter of 8 mm, and an inner diameter of 7.6 mm, tightly to the glass sheet.
   NOTE: The inner diameter of the copper ring is determined by the size of the piezoelectric plate to ensure the plate is covered by the copper ring.
4. Securely insert the polypropylene pipe with an outer diameter of 3 mm into the center of the piezoelectric plate and firmly adhere it to the glass sheet (Figure 1).
5. Prepare an appropriate amount of epoxy resin glue and vacuum it. Extract the epoxy using a disposable syringe and slowly inject it into the copper ring. Wait about 10 h until the epoxy has solidified (Figure 1).
6. Solder the loose ends of two wires onto the bayonet nut connector using an electronic soldering iron. Remove the glass sheet. Clean the surface of the transducer with alcohol (Figure 1).

2. Reporting parameters for FUN

1. Place the hydrophone and transducer in a water tank filled with deionized water (Figure 2A). Ensure that the central beam (Z-axis) of the positioning system is aligned with the transducer axis. This alignment can be achieved by firstly, discovering a field maximum in the focal plane through 2D scanning; secondly, identifying a field maximum in another plane with a clear maximum; thirdly, comparing the X and Y coordinates of the two maxima and then iteratively adjusting the position and/or orientation of the transducer if needed²⁸.
2. Adjust the tip of the hydrophone with the surface of the transducer at a distance of 1 mm, keeping this distance constant while positioning the hydrophone in the middle of the right edge of the transducer. Initiate the scanning program to capture the free acoustic field in the XZ plane (Figure 2B).
   NOTE: The construction method of the hydrophone can be found at https://github.com/HQArrayLab/Hydrophone_system_control.
3. Move the hydrophone along the Z-axis to determine the depths associated with the spatial peak pressure. In this experiment, the spatial peak pressure appears at a distance of 3.4 mm from the surface of the transducer; maintain this distance when moving 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 (Figure 2B).
4. Place the transducer on the skull of a mouse that underwent surgery as described in step 4. Acquire the transcranial acoustic field on the XZ plane and XY plane (Figure 2D) through hydrophone scanning as described in 2.1-2.3.
5. Read the pressure amplitudes at the focal point, which is the spatial peak region in the free acoustic field and transcranial acoustic field. The pressure amplitude at the focal point in the free acoustic field is 730k Pa, and in the transcranial acoustic field is 580k Pa. Read the focal dimensions at -3 dB (Figure 2C,E), and the position on the XY and XZ planes within the transcranial acoustic field to assess whether the acoustic field of this transducer can cover the target brain areas.
6. Compute the Mechanical Index (MI), which is constrained by the FDA guidance document to be below 1.9 in order to mitigate cavitation. The calculation of MI is given by the equation:
        (1)
   where p_r,.3 represents the peak-rarefactional pressure in MPa adjusted by an attenuation coefficient of 0.3 dB cm^-1 MHz^-1, and f₀ is the operating frequency in MHz. The transcranial-field peak rarefactional pressure measured is 580 kPa, , 3.4 mm from the transducer, the f₀ is 500 kHz, so the derated p_r,.3 is 576.6 kPa. The MI is 0.82.
7. Calculate the spatial-peak pulse-average intensity (I_sppa), which is required to be below 190 W/cm² in the plane direction according to the FDA guidance document. The calculation of intensity is given by the equation:
        (2)
   where p_sp (t) is the time-varying acoustic pressure at the spatial-peak location, Z is the characteristic acoustic impedance of the medium (approximately 1.5 x 10⁶ Rayls for soft tissue), and PD is pulse duration. In the case of the square envelope, this reduces to the equation:
        (3)
   where A is the spatial-peak pressure amplitude. The A measured at the ultrasound focused location is 580 kPa, and the Z of the brain is about 1.58 x 10⁶ Rayls, so the I_sppa of the square envelope is 10.65 W/cm² and the I_sppa of the sinusoidal envelope is 10.65 W/cm².
8. Calculate the spatial-peak time-average intensity (I_spta), which is constrained by the FDA guidance document to be below 430 mW/cm² in the plane direction. The calculation of intensity is given by the equation:
        (4)
   where T is the time period over which the average is calculated. In the case of the square envelope, this reduces to the equation:
        (5)
   where DC_{pulse train} is the duty cycle of pulse. Here, the DC pulse train is 1% because continuous waves were used, so the I_spta is equal to the spatial-peak pulse-average intensity, 106.5 mW/cm² for a square envelope. The MI, I_sppa, and I_spta can be calculated using the software (Figure 3A). A MATLAB-based code for easy use can be found at https://github.com/HQArrayLab/Ultrasound_Parameter_Caculation.
9. Report the pulse timing parameters, including A_max, pulse duration, pulse repetition interval, pulse train duration, and envelope (Figure 3B).

3. Preparing the animal for surgery

1. Weigh 8-week-old male GCaMP6s transgenic mice, with an approximate weight of about 20 g. Prepare a solution containing ketamine at 10 mg/mL and xylazine at 2 mg/mL in sterile saline. Administer the ketamine/xylazine solution by intraperitoneal injection at a dosage of 100 mg/kg ketamine and 20 mg/kg xylazine using a 26G needle and 1 mL disposable syringe. Commence surgical preparation once the animal is unresponsive to painful stimuli, such as toe pinch.
2. Use a fader to trim the hair on the animal's head and disinfect the area with 70% ethanol and povidone-iodine prior to the surgical procedure.
3. Place the mouse in a prone position on the stereotaxic frame and ensure that the skull is level. Place a protective ophthalmic ointment over the animal's eyes to maintain moisture.

4. Surgical procedure

1. Make an incision along the sagittal suture, starting from the occipital bone to the beginning of the nasal bone. Use surgical scissors to remove the skin covering both hemispheres.
2. Use sterile saline to cleanse the skull and eliminate any remaining periosteum.
3. Apply 3% hydrogen peroxide to the exposed cranium using a cotton swab for approximately 2 s-3 s to create micropores. Rinse with sterile saline thoroughly and ensure the area is completely dry.
4. Create a 0.6 mm diameter burr hole craniotomy using a sterile, autoclaved drill bit above the brain area location as determined by the stereotaxic atlas aligned to bregma and lambda. Wash away any debris with sterile saline and ensure thorough drying. Be careful not to damage any tissue.
5. Insert the fiber optical ferrule (implant) into the probe holder and connect it to the stereotaxic arm.
6. Align the implant directly above the region of interest using the stereotaxic arm. When inserting the optical fiber into the brain tissue, advance the fiber slowly at a rate of approximately 2 mm/min.
7. Mix the dental cement to achieve a viscosity that allows for easy application across the cranium. Use a sterile toothpick to spread a thin layer of dental cement over the cranium and onto the lower part of the implant. Allow it to dry completely.
8. Detach the probe holder carefully. Prepare a polypropylene pipe with a height of 3 mm, an outer diameter of 3 mm, and an inner diameter of 2.6 mm, and then cut the pipe throughout its length.
9. Attach the pipe to the bottom of the implant using tweezers. Pour the dental cement powder into the pipe ensuring sufficient length above the implant for recording the optical fiber signal. Add the required liquid and allow a few minutes for the dental cement to solidify.
10. Locate the opening of the pipe and carefully clamp it to remove the pipe using tweezers. Prepare the dental cement mixture for application, ensuring an even and thin layer is spread across the cranium. Cover as much surface area on the cranium as possible with dental cement. Wait a few minutes for the dental cemental to solidify.
    NOTE: Do not let the dental cement come into contact with the skin of the mouse.
11. Drill three holes (1 mm diameter) into the 3D-printed ring, evenly divided on the horizon, with a height of 7 mm, an outer diameter of 10 mm, and an inner diameter of 8.4 mm. Secure the screws (1 mm length) into their respective holes.
12. Insert the top of the implant into the hole of the pre-manufactured transducer. Make sure the inner wall of the 3D-printed ring is smooth, and then place it around the transducer positioned on the mouse skull. Ensure that the transducer is centered within the ring.
13. Apply dental cement to the junction between the ring and the skull, then wait a few minutes for dental cement to solidify. Avoid placing the dental cement on the connection between the transducer and the skull.
14. Carefully remove the transducer and securely tighten the screws.Transfer the mouse to a warm cage and ensure it is monitored until fully recovered before returning it to its original cage.
15. After surgery, administer subcutaneous Carprofen (2 mg/kg) for analgesia and continue every 24 h for 3 days to manage inflammation and pain. Monitor the animals daily for any signs of distress, abnormal weight loss, pain, or infection. Normally, by the 3^rd day post-surgery, all mice should be exhibiting normal behavior. If any signs of distress or illness are observed in a mouse after the 3^rd day, follow institutional guidelines for euthanasia.

5. Stimulation and signal recording

1. At 7 days after surgery, turn on the oxygen supply to the gas anesthesia machine and adjust the oxygen flow regulator to set the gas flow to 300-500 mL/min.
2. Place the mouse in the induction chamber and close the anesthetic gas delivery to the mask. Rotate the vaporizer dial to adjust the appropriate anesthetic concentration (2%- 2.5%).
3. After the mouse is anesthetized, place it on the stereotaxic frame with an anesthetic mask. Close the induction line to let the anesthetic gas flow into the anesthetic mask. Adjust the appropriate maintenance anesthetic concentration (1%-1.5%).
4. Clean the top surface of the implant with alcohol, and then insert the fiber optic patch cord into the center of the prepared transducer.
5. Inject water into the space between the implant and the 3D-printed ring using a 26G needle and 1 mL disposable syringe to moisten the skull. Use paper towels to absorb excess water.
6. Inject a coupling agent into the space between the implant and the 3D-printed ring using a 26G needle and 1 mL disposable syringe to facilitate the easy propagation of ultrasound from the transducer to the brain.
7. Connect the implant to the fiber optic patch cord. Carefully insert the transducer into the area which is filled with a coupling agent and securely tighten the screws.
8. 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, enabling freedom of movement for the mouse.
   NOTE: The fiber optic patch cord has a length of 2m and a diameter of 1.25 mm. The light intensity for the 405 channel is 20 µW, and for the 470 channel is 40 µW.
9. Activate both the ultrasonic excitation device and the optical fiber recording system in order to synchronize ultrasonic neural modulation with optical fiber signal recording.