Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

All experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the California Institute of Technology, where data were originally obtained by J.O.S.

1. Design and 3D-printing of animal harness and image guidance hardware

1. Use the files from the Szablowski lab website at: https://www.szablowskilab.org/downloads for 3D-printing of the components.
2. Ensure that the printing material has low susceptibility in MRI but has an MRI-visible support. See the details of materials used in the materials and reagents section.
3. Account for the material degradation with multiple uses by testing the material repeatedly and observing the sites of wear that should be reinforced. Ensure the printed walls are at least 2 mm thick.
4. Use high precision 3D printers to improve the targeting precision.
5. Counter the gravity and other forces to avoid deviation of plastic 3D printed components by supporting the components along their length and increasing thickness of the 3D printed walls if any bending is observed.
6. Account for precision in multiple axes including anterior/posterior, medial/lateral, dorsal/ventral, as well as yaw, pitch, and tilt.
7. Test the accuracy of targeting by performing FUS-BBBO and recording the deviation from targeted position.
8. If using motorized stereotaxic systems, evaluate for the effects of dynamic movement on the material elasticity by recording the FUS-BBBO targeting procedure on video, and correct any deviations by thickening the 3D printed material walls.

2. Ultrasound system description

1. Use an ultrasound system with an eight-element annular array transducer (diameter= 25 mm, natural focal point = 20 mm; aperture (F) = 0.8)) and couple housing to the head with degassed ultrasound gel by applying gel to the shaven mouse head.
   NOTE: The center frequency of a transducer used in a previous study⁷ was 1.5 MHz, pulse duration was 10 ms, and pulse repetition frequency was 1 Hz over 120 s. The pressures were calibrated using optical fiber hydrophone and maintained between 0.36-0.45 MPa. Assume 18% acoustic attenuation through the skull²¹ for 1.5 MHz and parietal bone. The range of conditions appropriate for a safe BBB opening and AAV delivery have been described elsewhere in detail^7,14,22.

3. Animal preparation

1. Anesthetize one mouse using isoflurane inhalation at 2% with medical-grade air. Check the depth of anesthesia by a touch pinch to confirm lack of response. Then, apply ophthalmic ointment to prevent corneal drying using a single-use sterile q-tip to prevent cross-contamination of the ointment tube.
   NOTE: Typical procedure of FUS-BBBO can range between 30 minutes to 2 hours, and anesthesia must be maintained throughout.
2. After the mouse is anesthetized, wash a clean catheter with heparinized saline (10 U/ml).
   NOTE: An appropriate catheter for a 25-35 g mouse has a 30 G needle and PE10 tubing.
3. Subsequently disinfect mouse tail with 70% ethanol pad. Place the tail-vein catheter in a lateral tail vein and secure it with a tissue glue. Observe a backflow of blood from the tail vein into the catheter to confirm its placement.
4. Shave the mouse head and then use depilation cream after the tissue glue has dried in order to reduce the possibility of air bubbles being trapped under ultrasonic gel during insonation.
5. Place the mouse in a 3D-printed MRI carriage, mounting the front teeth on a bite bar and head inside a nosecone (Figure 1a).
6. Inject up to 10 µL lidocaine subcutaneously at the site contacting the blunted ear bars. Then, secure the blunted bars to the skull and apply safe amounts of pressure, taking care not to apply pressure to the windpipe as it impedes breathing. Observe the breathing for 30 s to confirm that the animal is breathing freely at a rate of 1/s.
7. Connect the targeting guide to ear bars, check breathing as in step 3.6 (Figure 1b), and continue monitoring breathing visually throughout the procedure every minute. A breathing rate elevated above 1/s is one of the indications of loss of anesthesia. Continue monitoring the toe-pinch response every 5 minutes when mice are not in the MRI scanner or if the breathing rate is elevated above 1/s.
8. Transfer the MRI carriage into an MRI holder and then inside the bore of a magnet.
   NOTE: The design of the hardware is optimized for a 72 mm coil inside a 7T MRI.
9. Acquire an MRI sequence to localize the mouse in the scanner.
10. Select the 3D fast low angle shot (FLASH) sequence to acquire entirety of the brain, using the following parameters, according to specific instructions of the instrument manufacturer. Echo time: 3.9 ms, Repetition time: 15 ms, Excitation pulse angle: 15°, Matrix size: 130 x 130 x 114, Resolution: 350 x 200 x 200 µm per voxel, Averages: 1, Acquisition time: 3 min 42s
11. Transfer files from the MRI system onto a computer controlling FUS system.
12. Open the imaging sequence in the software to perform MRI-guided targeting, where the image should appear as in Figure 1c.

4. MRI-guided targeting

NOTE: With the use of custom-designed targeting guides, it is not necessary to place ultrasound transducer within an MRI, nor it is necessary to incise the skin to perform targeting by zeroing stereotax on bregma and lambda lines. Follow the steps below to perform the targeting process.

1. Place the carriage within a stereotaxic instrument. Secure it in place using a metal block with a double-sided tape and by pressing the carriage against two support posts of the stereotaxic instrument.
2. Transfer MRI images to a computer with a running FUS guidance software by selecting files in data manager, right-clicking to bring menu options, and selecting ‘Transfer immediately’.
3. Open the FUS guidance software and load image by clicking ‘Open sequence’ and loading all files of the imaging sequence.
4. Reformat the image to three axes by pressing right-click and ‘Reformat’.
5. Localize the transducer to the circular targeting guide (Figure 1c) by right-clicking.
6. In sagittal view, adjust the vertical position of a virtual transducer to account for the thickness of water bath and the transducer housing (in this case – 8.2 mm upwards, Figure 1d).
7. Point the area(s) to be targeted in trajectory planner and note the coordinates in a spreadsheet (in this case – midbrain, as in Figure 1d).
8. Dial in the desired depth of targeting (z-value in electronic trajectory (Figure 2) and note coordinates in a spreadsheet.
9. Target each of the points by pressing ‘Send trajectory’ and ‘Execute’ (Figure 2).
   NOTE: This is done when a mouse carrier is placed inside the motors. However, the same targeting can be achieved with high accuracy on a stereotaxic instrument.
10. To correlate the coordinates of an MRI with the stereotaxic frame, place the custom-mounted transducer over a targeting guide and translate until each of the three targeting bolts (Figure 3, element A) can go through both the transducer holder (Figure 3, element B) and targeting guide (Figure 3, element C). Ascertain that the bolts are not under tension or tilting.
11. Translate the transducer 10.56 mm forward in anterior/posterior direction until it is located in the same place where a center of a targeting guide appears on an MRI.
12. Determine the distance from a center of the virtual transducer (Figure 3a, element A) to the targeted region (Figure 3a, element B), and move the transducer to these coordinates using stereotaxic frame.
13. Proceed to the preparation of injection solution.

5. Injection solution preparation

NOTE: The microbubble solutions are very sensitive to pressure. Consequently, vigorous mixing or rapid injection through thin needles can collapse the microbubbles and reduce efficacy of BBB opening. Additionally, microbubbles are lighter than water and can float to the top of a tube, catheter, or syringe (Figure 4) e.g., in an automatic injector. It is strongly recommended to resuspend microbubble solution immediately before every injection.

1. Withdraw 0.8 mL saline using a syringe into 1.5 mL tube.
2. Using a syringe, add 0.1 mL of MRI contrast agent into the same 1.5 mL tube and mix.
3. Bring the non-activated microbubble^23,24 solution to room temperature.
4. Right before the insonation, activate microbubbles for 45 s in a microbubble activation device.
5. Slowly (over ~3 s) withdraw 0.1 mL of microbubbles using a 1 mL tuberculin syringe and 21 G needle from the middle depth of a liquid.
6. Add 0.08 mL of microbubbles into the solution of contrast agent and saline prepared in step 5.3. Mix by tapping with hand for 15 s.
7. With the final concentration of AAVs being 0.5-2 x 10¹⁰ viral particles per gram of body weight (VP/g), inject the cargo for delivery (in this case AAV9) through 30 G needle into tail vein catheter, in case of ATAC – AAVs carrying chemogenetic receptors, or a negative control, such as AAVs carrying GFP under the same promoter.
8. Mix microbubbles by hand for 15 s again to avoid floatation (Figure 4).
9. Immediately afterwards, aspirate 200 µL of the microbubble solution through a syringe without a needle attached. Lack of a needle will reduce the shear forces on microbubbles.
10. Invert the syringe and mix by pressing the plunger up and down.
11. Attach the 30 G needle and, while still inverted, slowly push out the microbubbles until droplets appear at the end of a needle.

6. Insonation procedure

1. Set the parameters for insonation: 10 ms pulse duration, 120 repeats, every s, and 0.30-0.45 MPa pressure at the skull.
2. Remove the targeting guide, and apply degassed ultrasound gel to the mouse head, making sure to form no bubbles.
3. Lower the transducer and place it directly on flat the ear bar holder, and dial in the coordinates into the stereotaxic instruments (Figure 5).
4. Inject the solution of AAV (0.5-2 x 10¹⁰ VP/g).
5. Mix microbubbles and the MRI contrast agent solution for 15 s and inject in 80 µL per 30 g mouse.
6. Immediately apply ultrasound for 120 s by pressing ‘Send’ and ‘Execute’.
7. If more than one site is targeted, move the transducer to that site and adjust the depth targeting following numbers in the spreadsheet from steps 4.7-4.9. Then repeat steps 6.5-6.6 for every insonated site.

7. MRI evaluation of BBB opening

NOTE: The MRI evaluation of the BBB opening has been described in detail elsewhere¹¹. The location of BBB opening can be visualized as brighter areas in mice that received an injection of a T1-weighted Gd contrast agent.

1. After the ultrasound application, record an MRI sequence as in step 3.10.
2. Remove the mouse from the MRI scanner, and place it in a recovery cage to allow recovery from anesthesia. Monitor the mice daily for signs of distress, weight loss, or other humane endpoints. Consult veterinary staff and the institutional IACUC guidelines to proceed with any treatment should unexpected adverse events occur.

8. DREADD stimulation with a chemogenetic ligand

1. Choose a chemogenetic receptor. For DREADDs, choose the hM3Dq receptor for neuronal activation via Gq coupled pathways¹⁹, the hM4Di receptor for inhibition of neuronal activity through Gi/o coupled pathways²⁰, or the KORD receptor for activation of neurons via Gs coupled pathways using Salvinorin-B ligand²⁵.
2. Dissolve clozapine-n-oxide (CNO) in sterile saline at concentration of 1 mg/mL. Store the aliquoted CNO at -20 °C.
3. Administer CNO¹⁹, or another chemogenetic ligand^26,27,28,  through intraperitoneal route at concentration between 0.3 – 10 mg/kg.
4. If effects of CNO on behavior are to be recorded, begin recording the behavioral activity within 15-45 min after the drug administration to achieve the maximum activation of DREADDs¹⁹.
5. If analysis of neuronal activation is desired, use mice for histological evaluation after 60-120 min post injection.
6. Proceed to euthanasia through cardiac perfusion (see section 9).

9. Histological evaluation of gene expression and chemogenetic activation

NOTE: Once the experimental endpoint (e.g., end of behavioral study, time required for gene expression) is achieved, it is critical to confirm the location and presence of the gene expression.

1. After activation with a chemogenetic ligand, perform cardiac perfusion to preserve tissues.
   1. Anesthetize the mouse with Ketamine (100 mg/kg) /Xylazine (10 mg/kg) mixture in sterile saline through an IP injection. Confirm the anesthesia with a toe pinch and ensure the breathing rate has been lowered to approximately 1/s. Provide thermal support through the heating pad until the confirmation of euthanasia.
   2. Prepare 10% neutral buffered formalin (NBF) and PBS with 10 units of heparin per mL.
      NOTE: Solutions should be at 4 °C.
   3. Pour each buffer into separate 50 mL tubes and connect, and prime a peristaltic pump connected to a 25 G butterfly catheter with PBS/heparin solution.
   4. Attach the limbs to an absorbent blue pad with tape and ensure that the animal is placed in supine position on the pad. Disinfect the fur to avoid cross-contamination of peripheral organs in case they need to be collected.
   5. Open the peritoneal cavity by a transverse incision, exposing the diaphragm.
   6. Open the ribcage through two cuts of surgical scissors along the anterior / posterior axis.
   7. Expose the heart and place needle in a left chamber (right side of the heart as viewed supine) and place in the butterfly catheter in step 9.1.3.
   8. Make a small incision in the right ventricle to allow blood outflow.
   9. Turn on the peristaltic pump to begin flushing out the blood with PBS/heparin.
      NOTE: If this step is not performed adequately, the blood will clot during fixation with formalin and prevent appropriate perfusion.
   10. After all blood is flushed out and clear PBS starts coming out of the right ventricle, switch the inlet of a peristaltic pump to an NBF solution and begin perfusion for 25 mL per mouse.
   11. Extract the brain, place in at least 4 mL of NBF and post-fix for 24 h.
2. Section the brain using coronal sections on a vibratome using 50 µm section thickness.
3. Place each section into a well of a 24 well plate storing the sections throughout the brain.
4. Evaluate the expression of the chemogenetic receptors fused to fluorescent proteins (e.g., mCherry or mCitrine), under a fluorescent microscope to identify the sections showing expression to confirm the location. The expression is likely to be dim.
5. Perform immunostaining against the fluorophore using the following protocol:
   1. Place 3 sections in 0.5 mL of solution containing 10% serum of a host of a secondary antibody and incubate for 30 min.
   2. Transfer the sections into a solution of a primary antibody at 1:250 – 1:1,000 dilution, using 1:500 as a starting point.
   3. Incubate the sections with primary antibody overnight at 4 °C in a microplate sealed with a paraffin film.
   4. Wash the sections with PBS, 3x for 5 min at a time.
   5. Add 0.5 mL per well of the secondary antibody solution in 10% serum.
   6. Incubate for 4 h in room temperature.
   7. Wash the sections with PBS, 3x for 5 min at a time.
   8. Mount on slides with an aqueous mounting medium containing a nuclear stain (e.g., DAPI).
6. Evaluate the localization and spread using a confocal microscope by performing a tile scan of an entire section.
7. Evaluate intensity of expression by measuring fluorescence pixel intensity in targeted brain regions and compare against an intracranially injected control.
8. Alternatively, evaluate percent of positive neurons by counting cells stained against chemogenetic receptors, as compared to DAPI-positive cells, or cell-specific markers.
9. Evaluate the tissue damage by performing hematoxylin staining on 50 µm section and imaging for the loss of cells, accumulation of cell debris, and other signs of gross damage.
10. To evaluate the specificity of cell-targeting, perform double immunostaining as described below for the chemogenetic receptor and a cell-specific marker. Then, perform cell-positive counts as in step 9.8.
    1. Place 3 sections in 0.5 mL of a solution containing 10% serum of a host of a secondary antibody and incubate for 30 min.
    2. Transfer the sections into a solution of a primary antibody against a fluorescent marker of a chemogenetic receptors at 1:250 – 1:1,000 dilution, using 1:500 as a starting point. Add a second primary antibody from a different host species that is targeted against a cell-specific marker of interest (e.g., CamkIIa).
    3. Incubate the sections with primary antibody overnight at 4 °C in a microplate sealed with paraffin film.
    4. Wash the sections with PBS, 3x for 5 min at a time.
    5. Add 0.5 mL per well of secondary antibody solution in 10% serum of the host-species of both secondary antibodies.
       NOTE: Each antibody should have a distinct fluorophore and should be reactive against primary antibodies in step 9.10.2.
    6. Incubate for 4 h in room temperature.
    7. Wash the sections with PBS, 3x for 5 min at a time.
    8. Mount on slides and fix with an aqueous mounting medium containing a nuclear stain.

10. Evaluate neuronal activation with immunostaining for c-Fos

1. Perform c-Fos staining as in point 9.5 of this protocol using a c-Fos primary antibody and a secondary antibody with a fluorescent tag distinct from the nuclear stain.
2. Count the percent of cells that are positive for both c-Fos and nuclear stain in the area targeted by a chemogenetic receptor.
3. Analyze the percentage of c-Fos positive nuclei in group of mice expressing chemogenetic receptors and treated with a chemogenetic ligand or vehicle control and in group of wild-type mice that are treated with a chemogenetic ligand or vehicle control.