We developed an integrated experimental framework which extends the brain exploration capabilities of functional ultrasound imaging to awake/mobile animals. In addition to hemodynamic data, this method further allows parallel access to EEG recordings of neuronal activity. This approach is illustrated with two proofs of concept: first, a behavioral study, concerning theta rhythm activation in a maze running task and, second, a disease-related study concerning spontaneous epileptic seizures.In vivo brain activity recordings provide a unique contribution to neuroscience to unravel the underlying mechanisms of complex behaviors and pathologies. Ideally we would like to capture instantly both neuronal activity and metabolic events, which form two major facets of global brain equilibrium, in the most natural conditions, that is when the subject is awake and freely moving. There is an increasing awareness today that major issues of neurophysiology need to be addressed with such global approaches. Notably, the basic mechanisms involved in functional network dynamics and pathologies including epilepsy can be deciphered only if the electrographic and metabolic components are sensed concomitantly [Logothetis 2008, Sada 2015. More generally, the multiple feedbacks between electrographic and metabolic signals represent the rule, rather than the exception. They have been tackled separately only by lack of appropriate methods for accessing the global picture, prompting the development of multimodal strategies.In practice there is a compromise between the size of imaging field, time resolution, sensitivity, separability of processing and metabolism, and physical constraint on the animal.Corresponding author: IC, ivan.cohen@upmc.fr. Author contributions LAS designed the surgical procedure and prosthetic skull, and performed and analyzed epilepsy experiments; AB designed intrahippocampal recording procedure and performed and analyzed navigation experiments; ET and TD developed the "burst mode" ultrasound recording sequence; MP and JLG developed the "continuous mode" ultrasound recording sequence; JLG and MT designed and supervised the ultrasound scanner and probe; IC designed and supervised the experiments, designed the probe holder, programmed acquisition and analysis software; IC, LAS, AB wrote the paper.

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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.

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.

Ultrafast imaging using plane or diverging waves has recently enabled new ultrasound imaging modes with improved sensitivity and very high frame rates. Some of these new imaging modalities include shear wave elastography, ultrafast Doppler, ultrafast contrast-enhanced imaging and functional ultrasound imaging. Even though ultrafast imaging already encounters clinical success, increasing even more its penetration depth and signal-to-noise ratio for dedicated applications would be valuable. Ultrafast imaging relies on the coherent compounding of backscattered echoes resulting from successive tilted plane waves emissions; this produces high-resolution ultrasound images with a trade-off between final frame rate, contrast and resolution. In this work, we introduce multiplane wave imaging, a new method that strongly improves ultrafast images signal-to-noise ratio by virtually increasing the emission signal amplitude without compromising the frame rate. This method relies on the successive transmissions of multiple plane waves with differently coded amplitudes and emission angles in a single transmit event. Data from each single plane wave of increased amplitude can then be obtained, by recombining the received data of successive events with the proper coefficients. The benefits of multiplane wave for B-mode, shear wave elastography and ultrafast Doppler imaging are experimentally demonstrated. Multiplane wave with 4 plane waves emissions yields a 5.8  ±  0.5 dB increase in signal-to-noise ratio and approximately 10 mm in penetration in a calibrated ultrasound phantom (0.7 d MHz(-1) cm(-1)). In shear wave elastography, the same multiplane wave configuration yields a 2.07  ±  0.05 fold reduction of the particle velocity standard deviation and a two-fold reduction of the shear wave velocity maps standard deviation. In functional ultrasound imaging, the mapping of cerebral blood volume results in a 3 to 6 dB increase of the contrast-to-noise ratio in deep structures of the rodent brain.

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.

Acute spinal cord injury (SCI) leads to severe damage to the microvascular network. The process of spontaneous repair is accompanied by formation of new blood vessels; their functionality, however, presumably very important for functional recovery, has never been clearly established, as most studies so far used fixed tissues. Here, combining ultrafast Doppler imaging and ultrasound localization microscopy (ULM) on the same animals, we proceeded at a detailed analysis of structural and functional vascular alterations associated with the establishment of chronic SCI, both at macroscopic and microscopic scales. Using a standardized animal model of SCI, our results demonstrate striking hemodynamic alterations in several subparts of the spinal cord: a reduced blood velocity in the lesion site, and an asymmetrical hypoperfusion caudal but not rostral to the lesion. In addition, the worsening of many evaluated parameters at later time points suggests that the neoformed vascular network is not yet fully operational, and reveals ULM as an efficient in vivo readout for spinal cord vascular alterations. Finally, we show statistical correlations between the diverse biomarkers of vascular dysfunction and SCI severity. The imaging modality developed here will allow evaluating recovery of vascular function over time in pre-clinical models of SCI. Also, used on SCI patients in combination with other quantitative markers of neural tissue damage, it may help classifying lesion severity and predict possible treatment outcomes in patients.

Theta and gamma rhythms coordinate large cell assemblies during locomotion. Their spread across temporal and spatial scales makes them challenging to observe. Additionally, the metabolic cost of these oscillations and their contribution to neuroimaging signals remains elusive. To finely characterize neurovascular interactions in running rats, we monitored brain hemodynamics with functional ultrasound and hippocampal local field potentials in running rats. Theta rhythm and running speed were strongly coupled to brain hemodynamics in multiple structures, with delays ranging from 0.8 seconds to 1.8 seconds. Surprisingly, hemodynamics was also strongly modulated across trials within the same recording session: cortical hemodynamics sharply decreased after 5-10 runs, while hippocampal hemodynamics strongly and linearly potentiated, particularly in the CA regions. This effect occurred while running speed and theta activity remained constant, and was accompanied by increased power in hippocampal high-frequency oscillations (100-150 Hz). Our findings reveal distinct vascular subnetworks modulated across fast and slow timescales and suggest strong adaptation processes despite stereotyped behavior. IntroductionFrom the early days of electroencephalography (EEG), brain rhythms have been observed in a wide range of models and used as specific markers to characterize specific behaviors such as locomotion, sleep states, attention or cognitive control 1,2 . Neural oscillations support timely communication between coherent distant brain areas by providing windows of opportunity for efficient spike synchrony 3,4 and their disruption often is a hallmark of pathological conditions like epilepsy, schizophrenia, or Parkinson's disease 5 . Over the past decade, studies have reported that numerous brain rhythms are global processes that are not stationary, but instead circulate across brain regions. During locomotion in rodents, theta waves travel along the septotemporal axis of the hippocampus 6,7 , slow waves during NREM sleep travel from anterior towards posterior sites 8 and sleep spindles in humans rotate along a temporal, parietal and frontal cortical sites 9 . Because it is challenging to capture neural activity globally, this poses a problem from an experimental standpoint that high-density recordings can only partially solve, let alone the intrinsic caveats of electrophysiology.Theta rhythm (6-12 Hz) is extensively studied in behavioral neuroscience, because it is a fundamental model to understand neural synchronization during complex behavior. It is observable in many brain structures (hippocampus, entorhinal cortex, subiculum, striatum and thalamus) and species (bats, cats, rabbits, dogs, rodents, monkeys) 10 when an animal engages in walking, running, whisking and foraging behaviors or enters rapid-eye-movement sleep 11 . Among the multiple functions attributed to theta rhythm, it is critical for sensorimotor integration 12 , contextual information encoding 13 , hippocampal-cortical communication 14 and memory consolidation...