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The quest to decode the complex supraspinal mechanisms that integrate cutaneous thermal information in the central system is still ongoing. The dorsal horn of the spinal cord is the first hub that encodes thermal input which is then transmitted to brain regions via the spinothalamic and thalamocortical pathways. So far, our knowledge about the strength of the interplay between the brain regions during thermal processing is limited. To address this question, we imaged the brains of adult awake male mice in resting state using functional ultrasound imaging during plantar exposure to constant and varying temperatures. Our study reveals for the first time the following: (1) a dichotomy in the response of the somatomotor-cingulate cortices and the hypothalamus, which was never described before, due to the lack of appropriate tools to study such regions with both good spatial and temporal resolutions. (2) We infer that cingulate areas may be involved in the affective responses to temperature changes. (3) Colder temperatures (ramped down) reinforce the disconnection between the somatomotor-cingulate and hypothalamus networks. (4) Finally, we also confirm the existence in the mouse brain of a brain mode characterized by low cognitive strength present more frequently at resting neutral temperature. The present study points toward the existence of a common hub between somatomotor and cingulate regions, whereas hypothalamus functions are related to a secondary network., (Copyright © 2024 Koorliyil et al.)

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.

During locomotion, theta and gamma rhythms are essential to ensure timely communication between brain structures. However, their metabolic cost and contribution to neuroimaging signals remain elusive. To finely characterize neurovascular interactions during locomotion, we simultaneously recorded mesoscale brain hemodynamics using functional ultrasound (fUS) and local field potentials (LFP) in numerous brain structures of freely-running overtrained rats. Locomotion events were reliably followed by a surge in blood flow in a sequence involving the retrosplenial cortex, dorsal thalamus, dentate gyrus and CA regions successively, with delays ranging from 0.8 to 1.6 seconds after peak speed. Conversely, primary motor cortex was suppressed and subsequently recruited during reward uptake. Surprisingly, brain hemodynamics were strongly modulated across trials within the same recording session; cortical blood flow sharply decreased after 10-20 runs, while hippocampal responses strongly and linearly increased, particularly in the CA regions. This effect occurred while running speed and theta activity remained constant and was accompanied by an increase in the power of hippocampal, but not cortical, high-frequency oscillations (100-150 Hz). Our findings reveal distinct vascular subnetworks modulated across fast and slow timescales and suggest strong hemodynamic adaptation, despite the repetition of a stereotyped behavior.

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia. Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain., Competing Interests: Declaration of Competing Interest T.D., M.T. and Z.L are co-founders and shareholders of Iconeus company commercializing ultrasound neuroimaging scanners., (Copyright © 2020. Published by Elsevier Inc.)

The default mode network (DMN) has been defined in functional brain imaging studies as a set of highly connected brain areas, which are active during wakeful rest and inactivated during task-based stimulation. DMN function is characteristically impaired in major neuropsychiatric diseases, emphasizing its interest for translational research. However, in the mouse, a major preclinical rodent model, there is still no functional imaging evidence supporting DMN deactivation and deconnection during high-demanding cognitive/sensory tasks. Here we have developed functional ultrasound (fUS) imaging to properly visualize both activation levels and functional connectivity patterns, in head-restrained awake and behaving mice, and investigated their modulation during a sensory-task, whisker stimulation. We identified reproducible and highly symmetric resting-state networks, with overall connectivity strength directly proportional to the wakefulness level of the animal. We show that unilateral whisker stimulation leads to the expected activation of the contralateral barrel cortex in lightly sedated mice, while interhemispheric inhibition reduces activity in the ipsilateral barrel cortex. Whisker stimulation also leads to elevated bilateral connectivity in the hippocampus. Importantly, in addition to functional changes in these major hubs of tactile information processing, whisker stimulation during genuine awake resting-state periods leads to highly specific reductions both in activation and interhemispheric correlation within the restrosplenial cortex, a major hub of the DMN. These results validate an imaging technique for the study of activation and connectivity in the lightly sedated awake mouse brain and provide evidence supporting an evolutionary preserved function of the DMN, putatively improving translational relevance of preclinical models of neuropsychiatric diseases., Competing Interests: Competing interest statement: T.D., M.T., and Z.L. are founders, shareholders, and scientific consultants for Iconeus., (Copyright © 2020 the Author(s). Published by PNAS.)

We extended the capabilities of functional ultrasound to whole-brain four-dimensional (4D) neuroimaging. Our multiplane-wave transmission scheme on matrix arrays at thousands of frames per second provides volumetric recordings of cerebral blood volume changes at high spatiotemporal resolution. We illustrated the approach in rats while providing multiple sensory stimuli, for 4D functional connectivity and during instantaneous tracking of epileptiform events.

In the last decade, ultrasound imaging has gained new capabilities and produced new insights in the field of neuroscience. The development of new concepts, such as ultrafast ultrasound, has enhanced Doppler sensitivity by orders of magnitude and has paved the way for ultrasonic functional neuroimaging. In this review, we position ultrasound in the field of neuroimaging and discuss how it complements current tools available to neurobiologists and clinicians., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Functional ultrasound (fUS) imaging by ultrasensitive Doppler detection of blood volume was previously reported to measure adult rat brain activation and functional connectivity with unmatched spatiotemporal sampling (100 μm, 1 ms), but skull-induced attenuation of ultrasonic waves imposed skull surgery or contrast agent use. Also, fUS feasibility remains to be validated in mice, a major pre-clinical model organism. In the study described here, we performed full-depth ultrasensitive Doppler imaging and 3-D Doppler tomography of the entire mouse brain under anesthesia, non-invasively through the intact skull and skin, without contrast agents. Similar results were obtained in anesthetized young rats up to postnatal day 35, thus enabling longitudinal studies on postnatal brain development. Using a newly developed ultralight ultrasonic probe and an optimized ultrasonic sequence, we also performed minimally invasive full-transcranial fUS imaging of brain vasculature and whisker stimulation-induced barrel cortex activation in awake and freely moving mice, validating transcranial fUS for brain imaging, without anesthesia-induced bias, for behavioral studies., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

4D ultrasound microvascular imaging was demonstrated by applying ultrafast Doppler tomography (UFD-T) to the imaging of brain hemodynamics in rodents. In vivo real-time imaging of the rat brain was performed using ultrasonic plane wave transmissions at very high frame rates (18,000 frames per second). Such ultrafast frame rates allow for highly sensitive and wide-field-of-view 2D Doppler imaging of blood vessels far beyond conventional ultrasonography. Voxel anisotropy (100 μm × 100 μm × 500 μm) was corrected for by using a tomographic approach, which consisted of ultrafast acquisitions repeated for different imaging plane orientations over multiple cardiac cycles. UFT-D allows for 4D dynamic microvascular imaging of deep-seated vasculature (up to 20 mm) with a very high 4D resolution (respectively 100 μm × 100 μm × 100 μm and 10 ms) and high sensitivity to flow in small vessels (>1 mm/s) for a whole-brain imaging technique without requiring any contrast agent. 4D ultrasound microvascular imaging in vivo could become a valuable tool for the study of brain hemodynamics, such as cerebral flow autoregulation or vascular remodeling after ischemic stroke recovery, and, more generally, tumor vasculature response to therapeutic treatment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We developed an integrated experimental framework that extends the brain exploration capabilities of functional ultrasound imaging to awake and mobile rats. In addition to acquiring hemodynamic data, this method further allows parallel access to electroencephalography (EEG) recordings of neuronal activity. We illustrate this approach with two proofs of concept: a behavioral study on theta rhythm activation in a maze running task and a disease-related study on spontaneous epileptic seizures.

In vivo feasibility of using low-intensity focused ultrasound (FUS) to transiently modulate the function of regional brain tissue has been recently tested in anesthetized lagomorphs [1] and rodents [2-4]. Hypothetically, ultrasonic stimulation of the brain possesses several advantages [5]: it does not necessitate surgery or genetic alteration but could ultimately confer spatial resolutions superior to other noninvasive methods. Here, we gauged the ability of noninvasive FUS to causally modulate high-level cognitive behavior. Therefore, we examined how FUS might interfere with prefrontal activity in two awake macaque rhesus monkeys that had been trained to perform an antisaccade (AS) task. We show that ultrasound significantly modulated AS latencies. Such effects proved to be dependent on FUS hemifield of stimulation (relative latency increases most for ipsilateral AS). These results are interpreted in terms of a modulation of saccade inhibition to the contralateral visual field due to the disruption of processing across the frontal eye fields. Our study demonstrates for the first time the feasibility of using FUS stimulation to causally modulate behavior in the awake nonhuman primate brain. This result supports the use of this approach to study brain function. Neurostimulation with ultrasound could be used for exploratory and therapeutic purposes noninvasively, with potentially unprecedented spatial resolution., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Low-intensity focused ultrasound has been shown to stimulate the brain noninvasively and without noticeable tissue damage. Such a noninvasive and localized neurostimulation is expected to have a major impact in neuroscience in the coming years. This emerging field will require many animal experiments to fully understand the link between ultrasound and stimulation. The primary goal of this paper is to investigate transcranial ultrasonic neurostimulation at low frequency (320 kHz) on anesthetized rats for different acoustic pressures and estimate the in situ pressure field distribution and the corresponding motor threshold, if any. The corresponding acoustic pressure distribution inside the brain, which cannot be measured in vivo, is investigated based on numerical simulations of the ultrasound propagation inside the head cavity, reproducing at best the experiments conducted in the first part, both in terms of transducer and head geometry and in terms of acoustic parameters., Methods: In this study, 37 ultrasonic neurostimulation sessions were achieved in rats (N=8) using a 320 kHz transducer. The corresponding beam profile in the entire head was simulated in order to investigate the in situ pressure and intensity level as well as the spatial pressure distribution, thanks to a rat microcomputed tomography scan (CT)-based 3D finite differences time domain solver., Results: Ultrasound pulse evoked a motor response in more than 60% of the experimental sessions. In those sessions, the stimulation was always present, repeatable with a pressure threshold under which no motor response occurred. This average acoustic pressure threshold was found to be 0.68±0.1 MPa (corresponding mechanical index, MI=1.2 and spatial peak, pulse averaged intensity, Isppa=7.5 W cm(-2)), as calibrated in free water. A slight variation was observed between deep anesthesia stage (0.77±0.04 MPa) and light anesthesia stage (0.61±0.03 MPa), assessed from the pedal reflex. Several kinds of motor responses were observed: movements of the tail, the hind legs, the forelimbs, the eye, and even a single whisker were induced separately. Numerical simulations of an equivalent experiment with identical acoustic parameters showed that the acoustic field was spread over the whole rat brain with the presence of several secondary pressure peaks. Due to reverberations, a 1.8-fold increase of the spatial peak, temporal peak acoustic pressure (Psptp) (±0.4 standard deviation), a 3.6-fold increase (±1.8) for the spatial peak, temporal peak acoustic intensity (Isptp), and 2.3 for the spatial peak, pulse averaged acoustic intensity (Isppa), were found compared to simulations of the beam in free water. Applying such corrections due to reverberations on the experimental results would yield a higher estimation for the average acoustic pressure threshold for motor neurostimulation at 320 KHz at 1.2±0.3 MPa (MI=2.2±0.5 and Isppa=17.5±7.5 W cm(-2))., Conclusions: Transcranial ultrasonic stimulation is pressure- and anesthesia-dependent in the rat model. Numerical simulations have shown that the acoustic pattern can be complex inside the rat head and that special care must be taken for small animal studies relating acoustic parameters to neurostimulation effects, especially at a low frequency.

Over 4 million U.S. men and women suffer from Alzheimer's disease; 1 million from Parkinson's disease; 350,000 from multiple sclerosis (MS); and 20,000 from amyotrophic lateral sclerosis (ALS). Worldwide, these four diseases account for more than 20 million patients. In addition, aging greatly increases the risk of neurodegenerative disease. Although great progress has been made in recent years toward understanding of these diseases, few effective treatments and no cures are currently available. This is mainly due to the impermeability of the blood-brain barrier (BBB) that allows only 5% of the 7000 small-molecule drugs available to treat only a tiny fraction of these diseases. On the other hand, safe and localized opening of the BBB has been proven to present a significant challenge. Of the methods used for BBB disruption shown to be effective, Focused Ultrasound (FUS), in conjunction with microbubbles, is the only technique that can induce localized BBB opening noninvasively and regionally. FUS may thus have a huge impact in trans-BBB brain drug delivery. The primary objective in this paper is to elucidate the interactions between ultrasound, microbubbles and the local microenvironment during BBB opening with FUS, which are responsible for inducing the BBB disruption. The mechanism of the BBB opening in vivo is monitored through the MRI and passive cavitation detection (PCD), and the safety of BBB disruption is assessed using H&E histology at distinct pressures, pulse lengths and microbubble diameters. It is hereby shown that the BBB can be disrupted safely and transiently under specific acoustic pressures (under 0.45 MPa) and microbubble (diameter under 8 μm) conditions.

In diffusion MRI, standard approaches for fibertract identification are based on algorithms that generate lines of coherent diffusion, currently known as tractography. A tract is then identified as a set of such lines selected on some criteria. In the present study, we investigate whether fibertract identification can be formulated as a segmentation task that recognizes a fibertract as a region where diffusion is intense and coherent. Indeed, we show that it is possible to segment efficiently well-known fibertracts with classical image processing methods provided that the problem is formulated in a five-dimensional space of position and orientation. As an example, we choose to adapt to this newly defined high-dimensional non-Euclidean space, called position orientation space, an algorithm based on the hidden Markov random field framework. Structures such as the cerebellar peduncles, corticospinal tract, association bundles can be identified and represented in three dimensions by a back projection technique similar to maximum intensity projection. Potential advantages and drawbacks as compared to classical tractography are discussed; for example, it appears that our formulation handles naturally crossing tracts and is not biased by human intervention.