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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			<td height="21" style="height:21px;width:265px;">21-gauge needles (BD)</td>
			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:161px;">14826C</td>
			<td style="width:275px;"></td>
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			<td height="21" style="height:21px;width:265px;">25-gauge butterfly catheter</td>
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			<td>725966</td>
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			<td>902406</td>
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			<td height="21" style="height:21px;width:265px;">Anti-c-Fos antibody</td>
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			<td height="21" style="height:21px;width:265px;">Anti-mCherry antibody</td>
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			<td>includes the RF coils</td>
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			<td height="63" style="height:63px;width:265px;">Custom designed 3D printed mouse harnesses and MRIgFUS targeting components</td>
			<td style="width:173px;">ImageGuidedTherapy, Szablowski lab</td>
			<td style="width:161px;">custom</td>
			<td>download from szablowskilab.org/downloads</td>
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			<td height="21" style="height:21px;width:265px;">Custom MRIgFUS machine</td>
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			<td height="21" style="height:21px;width:265px;">Definity microbubbles</td>
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			<td>5067714</td>
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			<td height="21" style="height:21px;width:265px;">Depilation cr&#232;me</td>
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			<td></td>
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			<td style="width:173px;">Imasonic Inc.</td>
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			<td height="42" style="height:42px;width:265px;">Ethanol Pads/Alcohol Swabs (70%) (BD)</td>
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			<td height="21" style="height:21px;width:265px;">Phosphate-buffered saline (PBS)</td>
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			<td>524650-1EA</td>
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			<td height="42" style="height:42px;width:265px;">Secondary antibody, Donkey-anti rabbit</td>
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			<td>14823434</td>
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			<td style="width:173px;">Stratasys</td>
			<td style="width:161px;">RGD810</td>
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			<td style="width:161px;">VMIX</td>
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			<td height="21" style="height:21px;width:265px;">Vibrating microtome</td>
			<td style="width:173px;">Compresstome</td>
			<td style="width:161px;">VF-300</td>
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			<td height="21" style="height:21px;width:265px;">Xylazine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td>X1251-1G</td>
			<td></td>
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	</tbody>
</table>

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

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

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

focused-ultrasound-induced-blood-brain-barrier-opening-for-targeting

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JoVE Journal

Neuroscience

3.4K Views.  Rice University. This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays

The neuromodulatory effects of focused ultrasound (FUS) have been demonstrated in animal models, and FUS has been used successfully to treat movement and psychiatric disorders in humans. However, despite the success of FUS, the mechanism underlying its effects&#160;on neurons remains poorly understood, making treatment optimization by tuning FUS parameters difficult. To address this gap in knowledge, we studied human neurons in vitro using neurons cultured from human-induced pluripotent stem cells (HiPSCs). Using HiPSCs allows for the study of human-specific neuronal behaviors in both physiologic and pathologic states. This report presents a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of FUS on HiPSC neurons. By varying the FUS parameters and manipulating the HiPSC neurons through pharmaceutical and genetic modifications, researchers can evaluate the neural responses and elucidate the neuro-modulatory effects of FUS on HiPSC neurons. This research could have significant implications for the development of safe and effective FUS-based therapies for a range of neurological and psychiatric disorders.

Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays

Here, we present a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of focused ultrasound on human-induced pluripotent stem cell (HiPSC) neurons.

The neuromodulatory effects of focused ultrasound (FUS) have been demonstrated in animal models, and FUS has been used successfully to treat movement and ...

High-Precision TUS: Planning and Execution with Acoustic and Thermal Simulations

Pipeline for Planning and Execution of Transcranial Ultrasound Neuromodulation Experiments in Humans

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique capable of manipulating both cortical and subcortical structures with high precision. Conducting experiments involving humans necessitates careful planning of acoustic and thermal simulations. This planning is essential to adjust for bone interference with the ultrasound beam's shape and trajectory and to ensure TUS parameters meet safety requirements. T1- and T2-weighted, along with zero-time echo (ZTE) magnetic resonance imaging (MRI) scans with 1 mm isotropic resolution, are acquired (alternatively computed tomography x-ray (CT) scans) for skull reconstruction and simulations. Target and trajectory mapping are performed using a neuronavigational platform. SimNIBS is used for the initial segmentation of the skull, skin, and brain tissues. Simulation of TUS is carried over with the BabelBrain tool, which uses the ZTE scan to produce synthetic CT images of the skull to be converted into acoustic properties. We use a phased array ultrasound transducer with electrical steering capabilities. Z-steering is adjusted to ensure that the target depth is reached. Other transducer configurations are also supported in the planning tool. Thermal simulations are run to ensure temperature and mechanical index requirements are within the acoustic guidelines for TUS in human subjects as recommended by the FDA. During TUS delivery sessions, a mechanical arm assists in the movement of the transducer to the required location using a frameless stereotactic localization system.

Author Spotlight: Advancing Human Brain Modulation &#8211; Optimized Protocols for Transcranial Ultrasound Stimulation Experiments

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique that requires careful planning of acoustic and thermal simulations. The methodology describes an image processing and ultrasound simulation pipeline for efficient, user-friendly, streamlined planning for human TUS experimentation.

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique capable of manipulating both cortical and subcortical ...

Integrated Protocol for FUN and Fiber Photometry in Free-Moving Mice

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

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is compatible with most of the existing modalities for monitoring brain functions in vivo. Integration with brain function recording modalities not only enables us to address orders and disorders of specific brain functions with closed-loop feedback but also provides us with mechanistic insights about FUN itself. Here, we provide a modified, simple, dependable, and robust protocol for the simultaneous application of FUN and fiber photometry GCaMP6s fluorescence recording in free-moving mice. This involves the fabrication of a well-sized single transducer and its temporary placement on the mice, along with the secure fixation of a fiber optical implant to facilitate the smooth passage of the transducer. The combination of FUN and fiber photometry provides for the optical recording of neural circuitry responses upon FUN in real-time in deep brain regions. To demonstrate the efficiency of this protocol, Thy1-GCaMP6s mice were used as an example to record the neuroactivity in the anterior thalamic nucleus during FUN while the mice are freely moving. We believe that this protocol can promote the widespread use of FUN in both the neuroscience field and the biomedical ultrasound field.

Author Spotlight: Integration of Fiber Photometry and Focused Ultrasound Neuromodulation for Investigating Neural Modulation in Freely Moving Mice

The protocol includes transducer manufacturing, parameters reporting, surgical procedure, and signal recording for the entire operational workflow of concurrent focused ultrasound neuromodulation and fiber photometry recording in free-moving mice.

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is ...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...