This work was supported by the European Research Council (ERC) Advanced Grant N&#176; 339244-FUSIMAGINE, the National Agency for Research funding &#39;Pinch&#39; (ANR-18-CE37-005), the Inserm Research Technology Accelerator in Biomedical Ultrasound, the ElfUS technical core of the IPNP, Inserm U1266, the European research program FUSIMICE of the Human Brain Project, and EMBO Short-Term Fellowship 8439 to Andrea Kliewer.

<ol>
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functional ultrasound imaging in freely moving awake mice and anesthetized young rats without contrast agent.</a> Ultrasound in Medicine and Biology. 43 (8), 1679-1689 (2017).</li><li>Claron, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+functional+ultrasound+imaging+of+the+spinal+cord+reveals+in+depth+spatiotemporal+responses+of+spinal+nociceptive+circuits+in+both+normal+and+inflammatory+state.">Large scale functional ultrasound imaging of the spinal cord reveals in depth spatiotemporal responses of spinal nociceptive circuits in both normal and inflammatory state.</a> Pain. , (2020).</li><li>Aydin, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+functions+linking+neural+calcium+to+single+voxel+functional+ultrasound+signal.">Transfer functions linking neural calcium to single voxel functional ultrasound signal.</a> Nature Communications. 11 (1), 2954 (2020).</li><li>Bimbard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-scale+mapping+along+the+auditory+hierarchy+using+high-resolution+functional+ultrasound+in+the+awake+ferret.">Multi-scale mapping along the auditory hierarchy using high-resolution functional ultrasound in the awake ferret.</a> eLife. 7, 35028 (2018).</li><li>Boido, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscopic+and+microscopic+imaging+of+sensory+responses+in+the+same+animal.">Mesoscopic and microscopic imaging of sensory responses in the same animal.</a> Nature Communications. 10 (1), 1110 (2019).</li><li>Kislin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flat-floored+air-lifted+platform:+A+new+method+for+combining+behavior+with+microscopy+or+electrophysiology+on+awake+freely+moving+rodents.">Flat-floored air-lifted platform: A new method for combining behavior with microscopy or electrophysiology on awake freely moving rodents.</a> Journal of Visualized Experiments. (88), e51869 (2014).</li><li>Juczewski, K., Koussa, J. A., Kesner, A. J., Lee, J. O., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+behavioral+correlates+in+the+head-fixed+method:+stress+measurements,+habituation+dynamics,+locomotion,+and+motor-skill+learning+in+mice.">Stress and behavioral correlates in the head-fixed method: stress measurements, habituation dynamics, locomotion, and motor-skill learning in mice.</a> Scientific Reports. 10 (1), 12245 (2020).</li></ol>

Jeremy Ferrier and Bruno-F&#233;lix Osmanski are employees of Iconeus. Thomas Deffieux, Zsolt Lenkei, Bruno-F&#233;lix Osmanski,&#160;and Mickael Tanter are co-founders and shareholders of Iconeus.

Whole brain imaging methods are crucial tools to better understand brain physiology and pathology. The method described here allows the precise quantification of hemodynamic signals in the living brain directly at the bench. The unmatched sensitivity and spatio-temporal resolution of functional ultrasound is particularly well suited for the mouse physiology. Functional responses and resting-state networks can be mapped within short acquisition times, longitudinally and without having to average trials or subjects to obtain a reliable measure. The relevant combination of high sensitivity ultrasonic linear probes and fast motorized setups enables one to perform transcranial volumetric fUS imaging in mice within reasonable acquisition times. This protocol can be performed either on anesthetized or awake mice using a mobile home cage.
Whisker stimulation, the sensory stimulus used as an illustrating example in this manuscript, is a standard functional activation paradigm in rodents and a reliable read-out to study sensory processing, neurovascular coupling and their alterations5,6,10, 11. While coarse manual brushing of the whiskers may be preferred for its ease of use, this method lacks spatial and temporal precision. The use of an automatic stimulator, such as the one described here triggered with the fUS imaging scanner, allows for a better control of several parameters including the time of onset, the amplitude displacement, the frequency as well as the angle of the Q-tip/comb, resulting in a better inter-animal reproducibility. Additionally, a more precise timing of stimulation enables the modeling of the Hemodynamic Response Function (HRF) by determining the time to onset and time to peak parameters12,13. To ensure better precision on the number of whiskers deflected during the stimulation (and thus the area of the activated region), more sophisticated stimulators can be adapted to this protocol. Many other stimuli such as light8, sound14 or odor presentation15 can be implemented using the same protocol.
The compatibility of functional ultrasound with awake and behaving animals is an important advantage compared to other neuroimaging techniques, enabling functional activation mapping without the anesthesia bias. Using an air-lifted mobile homecage is a good alternative to other existing head-fixed apparatus such as linear or spherical treadmills. While being firmly head-fixed, the motion of the homecage gives the mouse the illusion to navigate the environment, allowing a wide-range of behavioral testings to be coupled to fUS imaging16. However, the habituation procedure to head-fixing constitutes an important step to decrease stress, especially for experiments where it can be considered a confounding factor. The procedure detailed here (6-days of handling and habituation to head fixation) gives robust results for sensory stimulation and resting-state functional connectivity. However, it might be necessary to extend the habituation period for more refined behavioral tests17.

Over the last two decades, neuroimaging has become an important tool for studying brain function and organization, enabling researchers to make important discoveries in the field of neuroscience. Today, functional magnetic resonance imaging (fMRI) has become the gold standard clinical neuroimaging technique to assess task or drug-evoked brain activation and to map functional connectivity at rest. While human fMRI has high reliability and sensibility, mouse fMRI remains technically challenging for numerous reasons1. First, fMRI has a poor spatial and temporal resolution. The small size of the mouse brain necessitates the use of strong magnetic fields using expensive scanners to achieve reasonable spatial resolution. Second, maintaining stable physiological parameters within the narrow range allowing efficient neuro-vascular coupling is very difficult in anesthetized mice. Finally, the blood oxygen level dependent (BOLD) signal on which fMRI studies rely has relatively poor sensitivity, leading to low signal-to-noise ratio when applied to mice and often requires repeated stimulus presentation over long acquisition to detect small variations. The mouse being the most widely used animal model in biomedical preclinical research, these limitations are partly responsible for the translational gap in neuropsychiatry, hindering new promising therapeutic targets on the bench to be transposed into effective treatments on bedside.
Functional ultrasound (fUS) is a recently developed neuroimaging technique based on ultrafast doppler2. By directly sampling cerebral blood volume, this technique allows probing brain activity in real-time through the neurovascular coupling. Compared to other neuroimaging techniques, fUS yields a spatial resolution of 100 &#181;m and a temporal resolution in the tens of milliseconds. This technique allows whole-brain imaging of complete coronal sections of the mouse brain, completely non-invasively. Furthermore, it is fully compatible with conscious and behaving animals3,4,5. One of the main current limitations of fUS is its 2D feature, allowing to record a single coronal plane at the same time. While volumetric 3D fUS using 2D matrix array transducers has already been successfully demonstrated in rats6 and confirmed in mice7, its current lack of sensitivity requires a full craniotomy as well as averaging an important number of trials to detect a slight change of activity. Alternatively, linear transducers can be stepped across multiple positions and perform functional imaging plane by plane to cover the whole brain. However, this technique requires numerous experimental paradigm repetitions and as such long acquisition times (3-4 hours for the mouse brain)8,9.
In the present work, we describe a robust experimental platform including a commercially available functional ultrasound scanner and a fast plane-switching linear transducer with procedures to acquire 3D fUS data in anesthetized and awake mice, allowing volumetric and transcranial functional mapping of the mouse brain, non-invasively, without contrast-agent and within short acquisition times. We illustrate this feature by mapping somatosensory cortex activation following whisker stimulation as well as resting-state functional connectivity. Aside from animal preparation and data collection, we also describe the procedure for visualization, atlas registration and analysis of real-time fUS signals.

<table><tbody><tr><td>BD Plastipak 1 mL syringes</td><td>Dutscher, France</td><td>303172</td><td></td></tr><tr><td>BD Microlance 26 Gauge needles</td><td>Dutscher, France</td><td>303800</td><td></td></tr><tr><td>Animal Temperature Controller (heating Plate coupled with a rectal probe)</td><td>Physitemp</td><td>TCAT-2DF</td><td></td></tr><tr><td>Arduino</td><td>Arduino</td><td>Arduino Uno-Rev3</td><td></td></tr><tr><td>Atipamezole</td><td>Orion Pharma, France</td><td>Antisedan&amp;reg;</td><td>5 mg/ml injectable solution</td></tr><tr><td>Dental Ciment</td><td>Sun M&amp;eacute;dical, Shiga, japan</td><td>Superbond C&amp;amp;B</td><td></td></tr><tr><td>Depilatory cream</td><td>Klorane</td><td>N/A</td><td></td></tr><tr><td>Eye Ointment</td><td>TVM, UK</td><td>Ocry-gel</td><td></td></tr><tr><td>Hair trimmer</td><td>Wella Profesionnals</td><td>N/A</td><td></td></tr><tr><td>Head plates</td><td>Neurotar, Finland</td><td>Model 14</td><td></td></tr><tr><td>Iconeus One standard package for fUS</td><td>Iconeus, France</td><td>Iconeus One</td><td></td></tr><tr><td>IcoScan acquisition software (v1.0)</td><td>Iconeus, France</td><td>IcoScan</td><td></td></tr><tr><td>IcoStudio analysis software (v1.0)</td><td>Iconeus, France</td><td>IcoStudio</td><td></td></tr><tr><td>Isoflurane Anesthesia station</td><td>Minerve, Esternay, France</td><td></td><td></td></tr><tr><td>Ketamine</td><td>Virbac, France</td><td>Ketamine1000</td><td>100 mg/ml injectable solution</td></tr><tr><td>Lidocaine</td><td>Vetoquinol</td><td>Lurocaine&amp;reg;</td><td>20 mg/ml injectable solution</td></tr><tr><td>Medetomidine</td><td>Orion Pharma, France</td><td>Domitor&amp;reg;</td><td>1 mg/ml injectable solution</td></tr><tr><td>Meloxicam</td><td>Boehringer lingelheim</td><td>Metacam&amp;reg;</td><td>0.5 mg/ml injectable solution</td></tr><tr><td>Mobile HomeCage Large with tracking capability</td><td>Neurotar, Finland</td><td>MHC-L-T-V4</td><td></td></tr><tr><td>Monitoring of ECG and breathing rate</td><td>AD Systems, (USA) and LabChart software</td><td></td><td></td></tr><tr><td>Servomotor</td><td>Feetech</td><td>FT90B</td><td></td></tr><tr><td>Stereotaxic frame</td><td>David Kopf (Tujunga, USA)</td><td>900-WA</td><td>Using Mouse Adaptor&amp;nbsp; (Ref: 922) and Non-Rupture Ear Bars (ref: 922)</td></tr><tr><td>Surgical glue</td><td>3M, USA</td><td>Vetbond</td><td></td></tr><tr><td>Syringe Pump</td><td>KD Scientific, USA</td><td>Legato&amp;reg; 130, Cat# 788130</td><td></td></tr><tr><td>Ultrasound gel</td><td>DREXCO medical, France</td><td>Medi&#039;Gel</td><td></td></tr><tr><td>Xylazine 2%</td><td>Bayer, France</td><td>Rompun&amp;reg;</td><td>20 mg/ml injectable solution</td></tr></tbody></table>

whole-brain 3d activation and functional connectivity mapping in mice using transcranial functional ultrasound imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the 3D quantification of cerebral hemodynamic variations transcranially in the mouse brain, at rest or in response to sensory stimulation. Whisker stimulation, a standard paradigm to map brain functional activation in rodents, has been selected as an example of sensory stimulation-evoked response. Figure 4 shows a representative activation map in response to mechanical whisker stimulation in an anesthetized mouse obtained using transcranial fUS imaging. The total trial time was 760 s, with a 60 s baseline (before and after the stimulation), an 80 s stimulation and a 60 s recovery time, repeated 5x. Significant activation was determined with the resolution of a general linear model (GLM) using a default mouse hemodynamic response function (HRF). The activated regions (Z scores with p-value &#62;0.0000006 after stringent Bonferroni correction for multiple comparison) are displayed as color-coded values overlaid onto the Allen common coordinate framework template. Voxel-wise time course of the contralateral primary somatosensory cortex, barrel field region (S1BF) revealed a 15-20% increase of the CBV compared to baseline.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62267/62267fig4v2.jpg" /> 
Figure 4: Transcranial activation Maps and rCBV time course following whiskers stimulation in ketamine/xylazine anesthetized mouse.&#160;A. Activation map showing significantly activated voxels following mechanical stimulation of the right whiskers (80 s ON, 60 s OFF ,5x) under ketamine/xylazine anesthesia. Maps were obtained by computing Z-scores based on general linear model analysis (GLM) with Bonferroni correction for multiple comparison. Z-scores (color coded) are overlaid on the Allen brain 3D template (after registration with the brain positioning system) and displayed in three-views: coronal (left), sagittal (middle) and axial (right). Anatomical regions from the Allen mouse brain common coordinate framework are displayed for reference. Activated voxels are well located inside the left S1BF cortex. Scale bar: 1 mm. Each sample volume was scanned over 2.8 mm (corresponding to 7 slices in the elevation direction) in 3.85 s allowing to record 20 volumic samples during each functional response. B. 3D rendering of whisker stimulation-evoked relative cerebral blood volume (rCBV) increase compared to baseline level. The anatomical delineation of the S1BF is indicated in blue. C. Time course of CBV variations in the left S1BF (blue) and the corresponding stimulus applied (red).&#160;<a href="https://www.jove.com/files/ftp_upload/62267/62267fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The same paradigm has been applied in a head-fixed behaving mouse in the mobile homecage using the awake preset of IcoScan. Figure 5 presents the activation map after a multiple whisker stimulation experiment using the experimental setup described in Figure 2. A few posterior and caudal whiskers were stimulated with the following pattern: 30 s baseline followed by five consecutive trials of 30 s ON (4 Hz) and 30 s OFF (Figure 5C). Stimulation was delivered using a servo-motor driven by an Arduino UNO card triggering the image acquisition sequence for synchronization. Significant activation was determined with the resolution of a general linear model (GLM) using a default mouse hemodynamic response function (HRF). Multiple comparison correction was performed with the Bonferroni method. Conventional alpha level of 0.05 was normalized by the total number of voxels in the acquisition volume, resulting in a final stringent threshold of 0.000003.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62267/62267fig5v2.jpg" /> 
Figure 5: Activation Maps and rCBV time course following whiskers stimulation in awake behaving mouse.&#160;A. Activation map showing significantly activated voxels following mechanical stimulation of the right whiskers (30 s ON, 30 s OFF, 5x) in an awake mouse in the mobile homecage. Maps were obtained by computing Z-scores based on general linear model analysis (GLM) with Bonferroni correction for multiple comparison (normalization by the total number of voxels). Z-scores (color coded) are overlaid on the Allen brain 3D template (after registration with the Brain Positioning System) and displayed in three-views: coronal (left), sagittal (middle) and axial (right). Anatomical regions from the Allen Mouse Brain Common Coordinate Framework are displayed for reference. Activated voxels are well located inside the left S1BF cortex. Scales bars, 1 mm. Each sample volume was scanned over 1.6 mm (corresponding to 3 slices in the elevation direction) in 3.85 s allowing to record 17 volumic samples during each functional response. B. 3D rendering of whisker stimulation-evoked relative Cerebral Blood Volume (rCBV) increase compared to baseline level. The anatomical delineation of the S1BF is indicated in blue. C. Illustration of the mouse in the mobile homecage during the right whisker stimulation experiment, during which five 30 s trials were performed for a total acquisition time of 330 s. D. Instantaneous relative CBV time course extracted inside the activated area (blue), with the corresponding stimulus superimposed (red).&#160;<a href="https://www.jove.com/files/ftp_upload/62267/62267fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 6&#160;shows the temporal correlations of normalized low-frequency (&#60;0.2 Hz) spontaneous CBV fluctuations between 3D brain regions (identified from registration to the Allen common coordinate framework) in a ketamine-xylazine anesthetized mouse. Total acquisition time was 20 min (1200 s). Atlas-supervised analysis revealed strong interhemispheric connectivity patterns, with resulting correlation coefficient values up to 0.8. Seed-based analysis in the dorsal hippocampus revealed a significant interhemispheric connectivity between right and left hippocampus as well as deep retro-hippocampal regions and piriform cortices. A seed region selected in the S1BF also resulted in a symmetrical (cortico-cortical) correlation pattern, as previously described.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62267/62267fig6v2.jpg" /> 
Figure 6: Transcranial volumetric resting-state functional connectivity of the mouse brain under ketamine/xylazine anesthesia assessed on a 20 min 3D fUS acquisition.&#160;A. Correlation matrix based on 3D regions of the Allen common coordinate framework registered on the transcranial functional acquisition. The matrix is obtained by computing the normalized Pearson&#39;s correlation of spontaneous low-frequency fluctuations (&#60;0.1 Hz) of the average time signals from all the voxels included in each identified ROI after slice timing correction. Each sampled volume was scanned over 1.6 mm in the elevation direction (corresponding to 4 slices) acquired over 2.2 s. B. Seed-based analysis projected onto a 3D template. The seed was selected within the right dorsal hippocampus at &#946; - 2.1 mm. Correlation map is obtained by computing the Pearson Correlation coefficient between the temporal signals of the seed and each voxel of the whole acquisition after slice timing correction. C. 3D correlation map based on seed-based analysis with seed region selected within the S1BF at &#946; - 2.1 mm. Scale bars: 1 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62267/62267fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging at JoVE.com

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

whole-brain-3d-activation-functional-connectivity-mapping-mice-using

Research

JoVE Journal

Neuroscience

9.9K Views.  PSL Research University. Functionele echografie is een nieuwe neuroimaging-modaliteit die het mogelijk maakt om het cerebrale bloedvolume in het levende knaagdierbrein in kaart te brengen. Met behulp van ultrasnelle vliegtuiggolfbeeldvorming kunnen we hemodynamische reacties van de hele hersenen meten met een ongeëvenaarde speciale tijdelijke resolutie en gevoeligheid. Dit protocol legt uit hoe transcraniële functionele echografie bij muizen kan worden uitgevoerd, zowel voor verdoofde als wakkere dierproeven.

Video: Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Medicine

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 ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Bioengineering

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Biology

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.

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 ...

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 ...

Enhancing Drug Delivery to the Brain: MRI-Guided Ultrasound Techniques in Rats

Monitoring Blood-Brain Barrier Opening in Rats with a Preclinical Focused Ultrasound System

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic macromolecular agents to the brain. The integration of MR-guided low-intensity pulsed focused ultrasound (FUS) with microbubble pre-injection is a promising technique for non-invasive and non-toxic BBB modulation. MRI can offer superior soft-tissue contrast and various quantitative assessments, such as vascular permeability, perfusion, and the spatial-temporal distribution of MRI contrast agents. Notably, contrast-enhanced MRI techniques with gadolinium-based MR contrast agents have been shown to be the gold standard for detecting BBB openings. This study outlines a comprehensive methodology involving MRI protocols and animal procedures for monitoring BBB opening in a rat model. The rat model provides the added benefit of jugular vein catheter utilization, which facilitates rapid medication administration. A stereotactic-guided preclinical FUS transducer facilitates the refinement and streamlining of animal procedures and MRI protocols. The resulting methods are characterized by reproducibility and simplicity, eliminating the need for specialized surgical expertise. This research endeavors to contribute to the optimization of preclinical procedures with rat models and encourage further investigation into the modulation of the BBB to enhance therapeutic interventions in neurological disorders.

This study describes a combined magnetic resonance imaging (MRI) and low-intensity pulsed focused ultrasound (FUS) protocol, utilizing living rats with jugular vein catheterization to monitor blood-brain barrier (BBB) opening.

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic ...

Functional Imaging of Cerebral Blood Flow Using Transcranial Doppler Ultrasound

Source:&#160;<a style='text-decoration:none;' href='https://dx.doi.org/10.3791/62048-v'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Hage, B. D.&#160;et. al.,<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2021)</a> &#160;This video demonstrates the use of Doppler ultrasound as a functional imaging tool to measure cerebral blood flow velocity, or CBFV in real time. It highlights how frequency shifts in the middle cerebral artery during tasks like breath-holding reveal changes in CBFV, offering insights into brain activity and vascular dynamics.

Source:&#160;Hage, B. D.&#160;et. al., Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow.&#160;J. Vis. Exp. (2021)&#160;This ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

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