Our protocol enables sub-millimeter precision targeting of focused ultrasound in mice. It allows us to target ultrasound using inexpensive 3D printed parts without the need of using surgery or MRI compatible ultrasound equipment. Focused ultrasound blood-brain barrier opening can be quite tricky at first.

It is critical that the transducer is properly coupled to the animal using degassed gel or water and that the micro-bubbles are not collapsed during the injection. Demonstrating the procedure will be Richard Li, a graduate student at Caltech. After confirming a lack of response to pedal reflex in an anesthetized mouse, use 10 units per milliliter of heparinized saline to wash a clean 25 to 35 gauge catheter and use an ethanol pad to disinfect the tail of the animal.

Insert the catheter into a lateral tail vein and use tissue glue to secure the catheter in place. If the needle has been placed correctly, a backflow of blood will be observed in the catheter. When the glue has dried, remove the hair on the animal's head with depilation cream and place the mouse in a 3D printed MRI carriage, mounting the front teeth on a bite bar with the head inside a nose cone.

Secure the blunted bars to the skull with a safe amount of pressure and observe the breathing for 30 seconds to confirm that the animal is breathing freely at a rate of one breath per second. Connect the targeting guide to the ear bars and check the breathing again. Place the MRI carriage into an MRI holder and place the holder inside the bore of a magnet.

Then acquire an MRI sequence to localize the mouse in the scanner, using the 3D fast low angle shot sequence to image the entire brain. To perform an MRI guided targeting, place the carriage within a stereotaxic instrument and use a metal block with double-sided tape to secure the block in place against two support posts of the stereotaxic instrument. Transfer the MRI images to a computer with a running focused ultrasound guidance software program, and open the images.

When all the images have been loaded, right-click and click reformat to reformat the image to three axes. Right-click to localize the transducer to the circular targeting guide. In the sagittal view, adjust the vertical position of the virtual transducer to account for the thickness of the water bath and the transducer housing.

Indicate the areas to be targeted in the trajectory planner and note the coordinates in a spreadsheet. Dial-in the desired depth of targeting and note the coordinates as demonstrated earlier. Then click send trajectory and execute to target each of the points.

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 can go through both the transducer holder and the targeting guide. Confirm that the bolts are not under tension or tilting and translate the transducer 10.56 millimeters forward in the anterior-posterior direction until the transducer is located at the point where the center of a targeting guide appears on the MRI. Then determine the distance from the center of the virtual transducer to the targeted region and use the frame to move the transducer to these coordinates.

To prepare the injection solution, load 800 microliters of saline and 100 microliters of MRI contrast agent into a 1.5 milliliter tube with mixing. Next, bring a nonactivated microbubble solution to room temperature, before activating the micro-bubbles for 45 seconds in a microbubble activation device. After the activation, slowly load 100 microliters of micro-bubbles from the middle of the solution into a one milliliter tuberculin syringe equipped with a 21 gauge needle, and add 80 microliters of the micro-bubbles into the contrast agent solution.

Mix the prepared solution by gently inverting the 1.5 milliliter tube for 15 seconds to avoid flotation. At the end of the mixing, load 200 microliters of the microbubble solution into a syringe without a needle and invert the syringe. Depress and retract the plunger to mix and attach a 30 gauge needle to the syringe while it is still inverted.

Then slowly push out the microbubble solution until droplets appear at the end of the needle. As soon as the micro-bubbles are ready, set the pulse duration for the insonation to 10 milliseconds, with 120 repeats every second, with 0.3 to 0.45 mega pascals of pressure at the skull. Remove the targeting guide and apply degassed ultrasound gel to the mouse head, taking care to avoid bubbles.

Lower the transducer until it is placed directly on the flat part of the ear bar holder and dial the coordinates into the stereotaxic instruments. Dilute the adeno-associated virus of interest to a 0.5 to 2 times 10 to the 10th viral particle per gram of body weight concentration, and load the virus particles into a one milliliter syringe equipped with a 30 gauge needle. Inject the solution into the tail vein catheter and inject 80 microliters of the microbubble solution into the mouse.

To evaluate the opening of the blood-brain barrier by MRI, record a fast low angle shot sequence as demonstrated. For DREADD stimulation, dissolve clozapine N-oxide in sterile saline at a one milligram per milliliter concentration, and inject it intraperitoneally. 15 to 45 minutes after delivery, begin recording the behavioral activity of the animal.

For neuronal activation analysis, use brain tissue. Following the protocol as demonstrated, a T1 signal enhancement can be achieved at the hippocampal region and in other parts of the brain. As observed, immunostaining against mCherry leads to a more reliable detection of DREADD expression In this procedure, remember to handle the micro-bubbles gently and to ensure that the animal is stable during the MRI imaging and the insonation.

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