Name: focused ultrasound induced blood-brain barrier opening for targeting brain structures and evaluating chemogenetic neuromodulation
Uploaded: 2020-12-22T12:30:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation at JoVE.com

This research was supported by Brain and Behavior Foundation, NARSAD Young Investigator Award. Several 3D printed components were originally designed by Fabien Rabusseau (Image Guided Therapy, France). Author thanks John Heath (Caltech) and Margaret Swift (Caltech) for technical help with preparing the manuscript.

No conflict of interest.

ATAC requires successful implementation of several techniques for successful neuromodulation of specific neural circuits, including accurate MRI-guided targeting, FUS-BBBO, and histological evaluation of gene expression. 3D-printable components were developed to simplify targeting of small brain structures with imaging-guided FUS-BBBO.
MRI-guided focused ultrasound (MRIgFUS) administration poses a number of challenges. First, typical MRI coil has limited space that is designed to only accommodate a specimen and not the ultrasound hardware. The larger bores of MRIs increase the cost of equipment and decrease image quality, as the signal is related to the fill factor of a coil32. Consequently, any FUS hardware placed on the top of an animal image in MRI will compromise imaging quality. Second, designing MRI-compatible devices is difficult and expensive. MRI compatible materials need to be diamagnetic, have low propensity of creating eddy currents during radiofrequency irradiation, and have low magnetic susceptibility in high magnetic fields. In any conductive material, the creation of eddy currents or its magnetic susceptibility will also negatively affect imaging quality. Finally, the available MRI-compatible materials have lower Young&#8217;s moduli and durability than the metals typically used in production of precise targeting machines e.g., stereotaxic frames. The motors used for positional adjustments need to be MRI-compatible and placed outside of the MRI bore due to their size. These motors have to be connected at a distance to the transducer inside an MRI bore using MRI-compatible materials. Issues of plastic warping, lack of sufficient space inside the bore to implement robustly sized components, and insufficient room for changing targeting positions across the entire brain have affected targeting accuracy in previous work.
To resolve these problems, a decision was made to perform imaging in MRI and FUS-BBBO administration outside of the scanner. To allow for MRI-guidance, mice were placed inside a 3D-printed restraint that had an MRI-visible targeting guide that could be used to localize the mouse brain structures both in the MRI and in the stereotax coordinate space. Since both the mouse skull and the targeting guide are firmly attached to ear bar holders (Figure 1a,b), a targeting guide can be used to correlate spatial coordinates within MRI image and zero the stereotaxic instruments. The restraint does not have moving parts and does not contain a transducer, which allowed us to make it both robust and sufficiently small to fit inside an MRI and removed signal interference from transducer&#8217;s electronics. The space inside the targeting guide has been hollowed as the 3D-printed support for some materials is visible in MRI (Figure 1c). Holes in the assembly were introduced to enable stereotax calibration (Figure 3). The ultrasound transducer was attached to an electrode holder of a stereotax, and targeting was performed as described in section 4 (Figure 1d). The transducer should be supported along its length by housing of ear bars, preventing any deviation from the level plane. The targeting in the dorso-ventral direction can be achieved using phase-shifts in an annular array.
The practical targeting precision is determined by ultrasound focusing and skull attenuation. FUS-BBBO procedure has been described in detail for rats11 and has been implemented in a number of other model organisms23,33,34 and in humans16,17. The relationship between ultrasound focus size inversely proportional to frequency, where higher frequencies can result in more precise delivery. However, the attenuation of the skull increases with frequencies35 which may lead to skull heating and damage to the cortical areas. The exact targeting strategy will depend on the brain site. The sites where a full-width half maximum pressure fits within the brain tissue allow for predictable and safe BBB opening in many brain structures such as the striatum, midbrain, and hippocampus. Regions near the base of the brain pose a specific challenge in mice. Mouse brain measures approximately 8-10 mm in dorso-ventral direction, which is comparable to the full-width half maximum size of many commercially available transducers. Consequently, targeting at the bottom of the skull can lead to ultrasound reflection from the bones and air present in ear canals, mouth, or windpipe which can lead to unpredictable patterns of high and low pressures36. Some of these pressures can cross an inertial cavitation threshold which has been shown to cause bleeding and tissue damage37. To target regions which are located near the base of the skull, it may be preferable to use intersectional ATAC7, where intersectional genetics38 is used to restrict gene expression to a smaller area then the one targeted with FUS beam. In the published example of intersectional ATAC, a transgenic animal expressing a gene editing enzyme (Cre38) in dopaminergic cells has been targeted with ultrasound in the subsection of the region containing dopaminergic cells. Finally, the cortical regions can be targeted with FUS, but the diffraction and reflection of ultrasound may occur leading to uneven pressure profiles. This protocol does not cover the targeting of cortical regions as it will be highly dependent on the used species; however, some targeting of the cortex above hippocampus7 (e.g., Figure 7) has been observed indicating that at, least in mice, it is possible.
The choice of a chemogenetic activator and dosing will depend on the specific experimental needs. A number of studies, including one of the authors&#8217; studies7, showed no significant non-specific response39,40, while higher doses (e.g., 10 mg/kg) can produce side effects, at least in some cases41. However, as with all behavioral experiments, proper controls31 are essential due to potential off-targeted activity of CNO and its metabolites42. Such controls could include administration of CNO and saline controls to animals expressing DREADDs and administration of CNO to wild-type animals or in some specific cases a comparison of ipsi- and contralateral sites of the brain that respectively do and do not express chemogenetic receptors. Additionally, recent research revealed a number of new DREADD agonists with improved specificity28,29,43. Other chemogenetic receptors5,25,44 can also be used in conjunction with ATAC procedure.
Histological evaluation of gene expression is necessary post-mortem for every animal. A small fraction of animals show poor gene expression following FUS-BBBO7. Additionally, it is necessary to show the spatial accuracy and specificity of gene expression since mis-targeting is possible. Of note, some AAVs may show retrograde or anterograde tracing capability45 and can cause transfection far from the site targeted with ultrasound despite accurate ultrasound targeting. If the expressed chemogenetic receptor is fused to or co-expresses a fluorophore, imaging of the fluorophore in tissue sections may be sufficient to evaluate localization and intensity of expression. However, many fluorescent proteins are damaged by the tissue fixation process, and immunostaining for mCherry protein that is frequently used with DREADDs yielded better signal in previous studies7. Finally, due to the density of neurons in certain parts of the brain (e.g., granular cell layer in hippocampus), using nuclearly-localized fluorophores expressed under IRES, as opposed to fusions, to perform cell-counts may be beneficial since nuclei can be easily segmented and counterstained with nuclear stains, such as DAPI or TO-PRO-3. To evaluate neuromodulation by c-Fos staining, performing nuclear counterstaining and counting c-Fos positive nuclei, rather than any fluorescence signal, is imperative. In some cases, cellular debris can show fluorescence and confound the measurements of positive cells.
Limitations of the drug and gene delivery with FUS-BBBO include lower resolution than delivery with invasive intracranial injections and the need for larger amounts of injected drugs or viral vectors. Additionally, while a direct injection into the brain results in exclusive delivery to an injected site, FUS-BBBO uses an intravenous route resulting in possible delivery to peripheral tissues. Limitations of using chemogenetics for neuromodulation include a slow timescale, which may be inadequate to some behavioral protocols which require rapid changes in intensity of neuromodulation.

Use of circuit-specific neuromodulation technologies, such as optogenetics1,2 and chemogenetics3,4,5, has advanced our understanding of psychiatric conditions as neuronal-circuit disorders. Neuronal circuits are difficult to study and even more difficult to control in treating brain disorders because they are typically defined by specific cell types, brain regions, molecular signaling pathways and timing of activation. Ideally for both research and clinical applications, such control would be exerted noninvasively, but achieving both precise and noninvasive neuromodulation is challenging. For example, while neuroactive drugs can reach the brain noninvasively, they lack spatial specificity by acting throughout the brain. On the other hand, electrical deep-brain stimulation can control specific brain regions but has difficulty controlling specific cell types and requires surgery and device placement6.
Acoustically Targeted Chemogenetics7 (ATAC) provides neuromodulation with spatial, cell-type, and temporal specificity. It combines three techniques: focused ultrasound induced blood-brain barrier opening (FUS-BBBO) for spatial targeting, use of adeno-associated viral vectors (AAVs) to noninvasively deliver genes under the control of cell-type specific promoters, and engineered chemogenetic receptors to modulate transfected neural circuits selectively via drug administration. FUS is an FDA-approved technology that takes advantage of ultrasound&#8217;s ability to focus deep within tissues, including the human brain, with millimeter spatial precision. At high power, FUS is used for noninvasive targeted ablation, including an FDA-approved treatment for essential tremor8. FUS-BBBO combines low-intensity ultrasound with systemically administered microbubbles, which oscillate in blood vessels at the ultrasound focus, resulting in localized, temporary (6-24 h) and reversible opening of the BBB9. This opening allows for the delivery of proteins9,10, small molecules11, and viral vectors7,12,13,14 to the brain without significant tissue damage in rodents10 and non-human primates15. Clinical trials are ongoing for FUS-BBBO16,17, indicating possible therapeutic applications of this technique.
Viral gene delivery using AAV is also rapidly advancing into clinical use for CNS disorders, with recent FDA and EU regulatory approvals as major milestones. Finally, chemogenetic receptors18, such as Designer Receptors Activated Exclusively by Designer Drugs (DREADDs), are widely used by neuroscientists to provide pharmacological control over neuronal excitation in transgenic or transfected animals19,20. DREADDs are G protein-coupled receptors (GPCRs) that have been genetically engineered to respond to synthetic chemogenetic molecules rather than endogenous ligands, such that systemic administration of these ligands increases or reduces the excitability of DREADD-expressing neurons. When these three technologies are combined into ATAC, they can be used for the noninvasive modulation of selected neural circuits with spatial, cell-type, and temporal precision.
Here, we expand and update a previously published protocol for FUS-BBBO11 by including methodology for accurate targeting of brain regions with FUS-BBBO in mice using simple 3D printed targeting equipment. We, also, show an application of FUS-BBBO to ATAC. We show steps necessary for the delivery of AAVs carrying chemogenetic receptors, and evaluation of gene expression and neuromodulation by histology. This technique is particularly applicable for targeting large or multiple brain regions for gene expression or neuromodulation. For example, a wide area of a cortex can be easily transduced with FUS-BBBO and modulated using chemogenetics. However, gene delivery with an alternative technique, intracranial injections, would require large number of invasive injections and craniotomies. FUS-BBBO and its application, ATAC, can be scaled to animals of different sizes, where brain regions are larger and harder to target invasively.

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focused ultrasound induced blood-brain barrier opening for targeting brain structures and evaluating chemogenetic neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

The first step of performing ATAC protocol is the targeting of the FUS-BBBO to the desired brain regions. For example, following the described protocol, the hippocampus was targeted with FUS-BBBO, and contrast agent and AAV9 carrying DREADDs were injected into the mice, followed by a FLASH 3D MRI sequence that acquires images of the mouse brain. A T1 signal enhancement was achieved at the hippocampal region (Figure 6) and in other parts of the brain (Figure 7). After several weeks, DREADDs were expressed inside the target brain region. While many DREADDs are fused to a fluorescent reporter (e.g. mCherry), the process of perfusion and fixation with formaldehyde was found to drastically reduce the fluorescence of these proteins. Immunostaining against mCherry or the DREADD led to more reliable detection of the expression (Figure 8) based on previous experience. In previous experiments, ~85% of the mice showed expression following FUS-BBBO7. A simple test for sufficient levels of expression of DREADDs is testing their functionality on a cellular level. It can be done, for example, by providing a chemogenetic ligand or a saline control, such as CNO19, deschloroclozapine28, or others29, and waiting 2 hours before a cardiac perfusion and fixation. The brain sections were then co-immunostained for c-Fos protein30, which indicates heightened activity of neurons, and for DREADD. The experiment was considered successful, if the site of the brain targeted with DREADDs showed significantly higher number of neuronal nuclei that are c-Fos positive in the group that received a chemogenetic ligand when compared to the group that received saline7 or compared to a contralateral site that was not subjected to FUS-BBBO. Of note, there is a potential for some of these ligands to activate neurons non-specifically without expression of DREADDs. For example, CNO has been shown to be metabolized into low levels of clozapine in mice, which crosses the BBB and activates DREADDs with high potency27. However, it was also shown to bind to non-specific locations. As in every experiment, it is critical to include all proper controls in chemogenetic studies31. One possible control is administration of the chemogenetic ligand to wild-type mice, without procedures, to exclude effects of the drug alone on the desired behavioral or histological assay. Another control could be inclusion of four groups: DREADD + ligand, DREADD + vehicle, EGFP + ligand, EGFP + vehicle, which will account for any potential effects of both gene delivery with FUS-BBBO, and the chemogenetic ligand.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61352/61352fig1.jpg" /> 
Figure 1: The process of MRI-guided targeting of FUS in ATAC. (a) Mouse placement with ear bars, a nose cone and a platform that can be fit inside an MRI scanner. (b) A 3D-printed guide (blue) that is visible in MRI was attached to ends of ear bar frame and then secured in place with a holder of a surface MRI coil that contains four snap-on bolts (semi-transparent blue). (c) Appearance of the 3D-printed guide in sagittal MRI (left panel), with a bottom of the virtual representation of a transducer aligned (yellow semicircle) with the bottom of the guide. Right panel shows appearance of the 3D-printed guide on MRI from coronal view. The bright circle was made of a polyjet support material that has a strong MRI contrast. The cross was formed with plastic. A yellow circle represents transducer location which was aligned concentrically with the guide inside a stereotaxic frame. (d) To target brain structures a virtual transducer was moved in z-direction above the mice to match the thickness of an ultrasound cone / housing. In this case, because of the thickness of water bath, the transducer was moved 8.2 mm above the guide for accurate targeting. Brain structures were selected using MRI imaging data, and their MRI coordinates were then written down and entered into the stereotaxic machine. <a href="https://www.jove.com/files/ftp_upload/61352/61352fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61352/61352fig2.jpg" /> 
Figure 2: Interface of the software used. <a href="https://www.jove.com/files/ftp_upload/61352/61352fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61352/61352fig3.jpg" /> 
Figure 3: Process of matching MRI coordinate space to stereotaxic instrument. (a) Three holes within a transducer holder were aligned with three holes within the MRI guide, and three conical targeting bolts were inserted without causing flex to the entire assembly. (b) Ideally, all three bolts would sit the center of the holes. (c) If there is any imprecision in alignment, not all three bolts would fit in e.g., in case of small, likely imperceptible yaw of 1&#176;, only one bolt would fit in while the opposite bolts would be stuck at the MRI guide. Alternatively, there could be visible flex of the entire assembly as bolts were forced through. (d) Enlarged view of bolt-fitting. The bolts should be placed concentrically for the best accuracy. <a href="https://www.jove.com/files/ftp_upload/61352/61352fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61352/61352fig4.jpg" /> 
Figure 4: Rapid redistribution of microbubbles within the syringe. (a) Syringe was photographed 5 s after mixing. (b) One minute later, there was a clearly visible layer showing some of the bubbles concentrate near the top of 1 mL tuberculin syringe. This example, in particular, used a solution of microbubbles. <a href="https://www.jove.com/files/ftp_upload/61352/61352fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61352/61352fig5.jpg" /> 
Figure 5: Process of placing the center of a transducer over a center of an MRI guide. (a) In the models shown in this paper, the red carrier has been designed to move 10.56 mm forward from the position shown in Figure 3b, to one shown here. (b) The blue MRI guide was removed before sonication, and an ultrasound gel was applied between the mouse and the transducer (orange) to ensure ultrasound passage. <a href="https://www.jove.com/files/ftp_upload/61352/61352fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61352/61352fig6.jpg" /> 
Figure 6: MRI visualization of the BBB opening. (a) Axial view of the BBB opening. Brighter area designated with an arrowhead shows extravasation of an MRI T1 contrast agent. (b) Coronal view of the dorsal hippocampus and the cortex above hippocampus targeted with FUS-BBBO (arrowheads). (c) Coronal view of the central hippocampus targeted with FUS-BBBO (arrowheads). <a href="https://www.jove.com/files/ftp_upload/61352/61352fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61352/61352fig7.jpg" /> 
Figure 7: Example of targeting of 4 brain sites using the three-bolt targeting system described in this paper. Areas with arrowheads showed BBB opened sites with diffusion of an MRI contrast agent. The four sites were targeted in succession, with ~150 s between each BBB opening, from the bottom to top. The image was taken within 2 min after the last BBB opening. Scale bar is 2 mm. <a href="https://www.jove.com/files/ftp_upload/61352/61352fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61352/61352fig8.jpg" /> 
Figure 8: Detection of DREADD expression.&#160; (a) Immunostaining for the fluorophore attached to DREADDs, in this case mCherry was a reliable method of detection in some studies. (b) In another representative section with DREADDs targeted to hippocampus using the same conditions as in (a), the fluorescence of mCherry by itself produced strong background and relatively weak signal. (c) As a negative control, a mouse that received systemic injection of AAV, but did not undergo FUS-BBBO, was used. No significant expression can be found by mCherry immunostaining. Scale bars are 500 mm. (Data in a, c adapted from7 with permissions, Copyright 2020 Nature-Springer). <a href="https://www.jove.com/files/ftp_upload/61352/61352fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation at JoVE.com

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