Real-time imaging of brain activity in freely moving rats using functional ultrasound.

Innovative imaging methods help to investigate the complex relationship between brain activity and behavior in freely moving animals. Functional ultrasound (fUS) is an imaging modality suitable for recording cerebral blood volume (CBV) dynamics in the whole brain but has so far been used only in head-fixed and anesthetized rodents. We designed a fUS device for tethered brain imaging in freely moving rats based on a miniaturized ultrasound probe and a custom-made ultrasound scanner. We monitored CBV changes in rats during various behavioral states such as quiet rest, after whisker or visual stimulations, and in a food-reinforced operant task. We show that fUS imaging in freely moving rats could efficiently decode brain activity in real time.

Chronic assessment of cerebral hemodynamics during rat forepaw electrical stimulation using functional ultrasound imaging.

Functional ultrasound imaging is a method recently developed to assess brain activity via hemodynamics in rodents. Doppler ultrasound signals allow the measurement of cerebral blood volume (CBV) and red blood cells' (RBCs') velocity in small vessels. However, this technique originally requires performing a large craniotomy that limits its use to acute experiments only. Moreover, a detailed description of the hemodynamic changes that underlie functional ultrasound imaging has not been described but is essential for a better interpretation of neuroimaging data. To overcome the limitation of the craniotomy, we developed a dedicated thinned skull surgery for chronic imaging. This procedure did not induce brain inflammation nor neuronal death as confirmed by immunostaining. We successfully acquired both high-resolution images of the microvasculature and functional movies of the brain hemodynamics on the same animal at 0, 2, and 7 days without loss of quality. Then, we investigated the spatiotemporal evolution of the CBV hemodynamic response function (HRF) in response to sensory-evoked electrical stimulus (1 mA) ranging from 1 (200 µs) to 25 pulses (5s). Our results indicate that CBV HRF parameters such as the peak amplitude, the time to peak, the full width at half-maximum and the spatial extent of the activated area increase with stimulus duration. Functional ultrasound imaging was sensitive enough to detect hemodynamic responses evoked by only a single pulse stimulus. We also observed that the RBC velocity during activation could be separated in two distinct speed ranges with the fastest velocities located in the upper part of the cortex and slower velocities in deeper layers. For the first time, functional ultrasound imaging demonstrates its potential to image brain activity chronically in small animals and offers new insights into the spatiotemporal evolution of cerebral hemodynamics.

Brain-wide continuous functional ultrasound imaging for real-time monitoring of hemodynamics during ischemic stroke.

Ischemic stroke occurs abruptly causing sudden neurologic deficits, and therefore, very little is known about hemodynamic perturbations in the brain immediately after stroke onset. Here, functional ultrasound imaging was used to monitor variations in relative cerebral blood volume (rCBV) compared to baseline. rCBV levels were analyzed brain-wide and continuously at high spatiotemporal resolution (100 µm, 2 Hz) until 70mins after stroke onset in rats. We compared two stroke models, with either a permanent occlusion of the middle cerebral artery (MCAo) or a tandem occlusion of both the common carotid and middle cerebral arteries (CCAo + MCAo). We observed a typical hemodynamic pattern, including a quick drop of the rCBV after MCAo, followed by spontaneous reperfusion of several brain regions located in the vicinity of the ischemic core. The severity and location of the ischemia were variable within groups. On average, the severity of the ischemia was in good agreement with the lesion volume (24 hrs after stroke) for MCAo group, while larger for the CCAo + MCAo model. For both groups, we observed that infarcts extended to initially non-ischemic regions located rostrally to the ischemic core. These regions strongly colocalize with the origin of transient hemodynamic events associated with spreading depolarizations.

A deep learning classification task for brain navigation in rodents using micro-Doppler ultrasound imaging.

Positioning and navigation are essential components of neuroimaging as they improve the quality and reliability of data acquisition, leading to advances in diagnosis, treatment outcomes, and fundamental understanding of the brain. Functional ultrasound imaging is an emerging technology providing high-resolution images of the brain vasculature, allowing for the monitoring of brain activity. However, as the technology is relatively new, there is no standardized tool for inferring the position in the brain from the vascular images. In this study, we present a deep learning-based framework designed to address this challenge. Our approach uses an image classification task coupled with a regression on the resulting probabilities to determine the position of a single image. To evaluate its performance, we conducted experiments using a dataset of 51 rat brain scans. The training positions were extracted at intervals of 375 µm, resulting in a positioning error of 176 µm. Further GradCAM analysis revealed that the predictions were primarily driven by subcortical vascular structures. Finally, we assessed the robustness of our method in a cortical stroke where the brain vasculature is severely impaired. Remarkably, no specific increase in the number of misclassifications was observed, confirming the method's reliability in challenging conditions. Overall, our framework provides accurate and flexible positioning, not relying on a pre-registered reference but rather on conserved vascular patterns.

Multiplexed Surface Electrode Arrays Based on Metal Oxide Thin-Film Electronics for High-Resolution Cortical Mapping.

Electrode grids are used in neuroscience research and clinical practice to record electrical activity from the surface of the brain. However, existing passive electrocorticography (ECoG) technologies are unable to offer both high spatial resolution and wide cortical coverage, while ensuring a compact acquisition system. The electrode count and density are restricted by the fact that each electrode must be individually wired. This work presents an active micro-electrocorticography (µECoG) implant that tackles this limitation by incorporating metal oxide thin-film transistors (TFTs) into a flexible electrode array, allowing to address multiple electrodes through a single shared readout line. By combining the array with an incremental-ΔΣ readout integrated circuit (ROIC), the system is capable of recording from up to 256 electrodes virtually simultaneously, thanks to the implemented 16:1 time-division multiplexing scheme, offering lower noise levels than existing active µECoG arrays. In vivo validation is demonstrated acutely in mice by recording spontaneous activity and somatosensory evoked potentials over a cortical surface of ≈8×8 mm2 . The proposed neural interface overcomes the wiring bottleneck limiting ECoG arrays, holding promise as a powerful tool for improved mapping of the cerebral cortex and as an enabling technology for future brain-machine interfaces.

Functional ultrasound imaging of stroke in awake rats.

Anesthesia is a major confounding factor in preclinical stroke research as stroke rarely occurs in sedated patients. Moreover, anesthesia affects both brain functions and the stroke outcome acting as neurotoxic or protective agents. So far, no approaches were well suited to induce stroke while imaging hemodynamics along with simultaneous large-scale recording of brain functions in awake animals. For this reason, the first critical hours following the stroke insult and associated functional alteration remain poorly understood. Here, we present a strategy to investigate both stroke hemodynamics and stroke-induced functional alterations without the confounding effect of anesthesia, i.e., under awake condition. Functional ultrasound (fUS) imaging was used to continuously monitor variations in cerebral blood volume (CBV) in +65 brain regions/hemispheres for up to 3 hr after stroke onset. The focal cortical ischemia was induced using a chemo-thrombotic agent suited for permanent middle cerebral artery occlusion in awake rats and followed by ipsi- and contralesional whiskers stimulation to investigate on the dynamic of the thalamocortical functions. Early (0-3 hr) and delayed (day 5) fUS recording enabled to characterize the features of the ischemia (location, CBV loss), spreading depolarizations (occurrence, amplitude) and functional alteration of the somatosensory thalamocortical circuits. Post-stroke thalamocortical functions were affected at both early and later time points (0-3 hr and 5 days) after stroke. Overall, our procedure facilitates early, continuous, and chronic assessments of hemodynamics and cerebral functions. When integrated with stroke studies or other pathological analyses, this approach seeks to enhance our comprehension of physiopathologies towards the development of pertinent therapeutic interventions.

Quantitative Hemodynamic Measurements in Cortical Vessels Using Functional Ultrasound Imaging.

Red blood cell velocity (RBCv), cerebral blood flow (CBF), and volume (CBV) are three key parameters when describing brain hemodynamics. Functional ultrasound imaging is a Doppler-based method allowing for real-time measurement of relative CBV at high spatiotemporal resolution (100 × 110 × 300 µm3, up to 10 Hz) and large scale. Nevertheless, the measure of RBCv and CBF in small cortical vessels with functional ultrasound imaging remains challenging because of their orientation and size, which impairs the ability to perform precise measurements. We designed a directional flow filter to overpass these limitations allowing us to measure RBCv in single vessels using a standard functional ultrasound imaging system without contrast agents (e.g., microbubbles). This method allows to quickly extract the number of vessels in the cortex that was estimated to be approximately 650/cm3 in adult rats, with a 55-45% ratio for penetrating arterioles versus ascending venules. Then, we analyzed the changes in RBCv in these vessels during forepaw stimulation. We observed that ∼40 vessels located in the primary somatosensory forelimb cortex display a significant increase of the RBCv (median ΔRBCv ∼15%, maximal ΔRBCv ∼60%). As expected, we show that RBCv was higher for penetrating arterioles located in the center than in the periphery of the activated area. The proposed approach extends the capabilities of functional ultrasound imaging, which may contribute to a better understanding of the neurovascular coupling at the brain-wide scale.

Location Matters: Navigating Regional Heterogeneity of the Neurovascular Unit.

The neurovascular unit (NVU) of the brain is composed of multiple cell types that act synergistically to modify blood flow to locally match the energy demand of neural activity, as well as to maintain the integrity of the blood-brain barrier (BBB). It is becoming increasingly recognized that the functional specialization, as well as the cellular composition of the NVU varies spatially. This heterogeneity is encountered as variations in vascular and perivascular cells along the arteriole-capillary-venule axis, as well as through differences in NVU composition throughout anatomical regions of the brain. Given the wide variations in metabolic demands between brain regions, especially those of gray vs. white matter, the spatial heterogeneity of the NVU is critical to brain function. Here we review recent evidence demonstrating regional specialization of the NVU between brain regions, by focusing on the heterogeneity of its individual cellular components and briefly discussing novel approaches to investigate NVU diversity.

Whole-brain functional ultrasound imaging in awake head-fixed mice.

Most brain functions engage a network of distributed regions. Full investigation of these functions thus requires assessment of whole brains; however, whole-brain functional imaging of behaving animals remains challenging. This protocol describes how to follow brain-wide activity in awake head-fixed mice using functional ultrasound imaging, a method that tracks cerebral blood volume dynamics. We describe how to set up a functional ultrasound imaging system with a provided acquisition software (miniScan), establish a chronic cranial window (timing surgery: ~3-4 h) and image brain-wide activity associated with a stimulus at high resolution (100 × 110 × 300 µm and 10 Hz per brain slice, which takes ~45 min per imaging session). We include codes that enable data to be registered to a reference atlas, production of 3D activity maps, extraction of the activity traces of ~250 brain regions and, finally, combination of data from multiple sessions (timing analysis averages ~2 h). This protocol enables neuroscientists to observe global brain processes in mice.

A Platform for Brain-wide Volumetric Functional Ultrasound Imaging and Analysis of Circuit Dynamics in Awake Mice.

Imaging large-scale circuit dynamics is crucial to understanding brain function, but most techniques have a limited depth of field. Here, we describe volumetric functional ultrasound imaging (vfUSI), a platform for brain-wide vfUSI of hemodynamic activity in awake head-fixed mice. We combined a high-frequency 1,024-channel 2D-array transducer with advanced multiplexing and high-performance computing for real-time 3D power Doppler imaging at a high spatiotemporal resolution (220 × 280 × 175 µm3, up to 6 Hz). We developed a standardized software pipeline for registration, segmentation, and temporal analysis in 268 individual brain regions based on the Allen Mouse Common Coordinate Framework. We demonstrated the high sensitivity of vfUSI under multiple experimental conditions, and we successfully imaged stimulus-evoked activity when only a few trials were averaged. We also mapped neural circuits in vivo across the whole brain during optogenetic activation of specific cell types. Moreover, we identified the sequential activation of sensory-motor networks during a grasping water-droplet task.

Evidence from functional ultrasound imaging of enhanced contralesional microvascular response to somatosensory stimulation in acute middle cerebral artery occlusion/reperfusion in rats: A marker of ultra-early network reorganization?

Following middle cerebral artery (MCA) stroke, enhanced contralesional evoked responses have been consistently reported both in man and rodents as part of plastic processes thought to influence motor recovery. How early this marker of large-scale network reorganization develops has however been little addressed, yet has clinical relevance for rehabilitation strategies targeting plasticity. Previous work in mice has reported enhanced contralesional responses to unaffected-side forepaw stimulation as early as 45 min after MCA small branch occlusion. Using functional ultrasound imaging (fUSi) in anesthetized rats subjected to distal temporary MCA occlusion (MCAo), we assessed here (i) whether enhanced contralesional responses also occurred with unaffected-side whisker pad stimulation, and if so, how early after MCAo; and (ii) the time course of this abnormal response during occlusion and after reperfusion. We replicate in a more proximal MCA occlusion model the earlier findings of ultra-early enhanced contralesional evoked responses. In addition, we document this phenomenon within minutes after MCAo, and its persistence throughout the entire 90-min occlusion as well as 90-min reperfusion periods studied. These findings suggest that plastic processes may start within minutes following MCAo in rodents. If replicated in man, they might have implications regarding how early plasticity-enhancing therapies can be initiated after stroke.

Understanding the neurovascular unit at multiple scales: Advantages and limitations of multi-photon and functional ultrasound imaging.

Developing efficient brain imaging technologies by combining a high spatiotemporal resolution and a large penetration depth is a key step for better understanding the neurovascular interface that emerges as a main pathway to neurodegeneration in many pathologies such as dementia. This review focuses on the advances in two complementary techniques: multi-photon laser scanning microscopy (MPLSM) and functional ultrasound imaging (fUSi). MPLSM has become the gold standard for in vivo imaging of cellular dynamics and morphology, together with cerebral blood flow. fUSi is an innovative imaging modality based on Doppler ultrasound, capable of recording vascular brain activity over large scales (i.e., tens of cubic millimeters) at unprecedented spatial and temporal resolution for such volumes (up to 10µm pixel size at 10kHz). By merging these two technologies, researchers may have access to a more detailed view of the various processes taking place at the neurovascular interface. MPLSM and fUSi are also good candidates for addressing the major challenge of real-time delivery, monitoring, and in vivo evaluation of drugs in neuronal tissue.

Mapping the dynamics of brain perfusion using functional ultrasound in a rat model of transient middle cerebral artery occlusion.

Following middle cerebral artery occlusion, tissue outcome ranges from normal to infarcted depending on depth and duration of hypoperfusion as well as occurrence and efficiency of reperfusion. However, the precise time course of these changes in relation to tissue and behavioral outcome remains unsettled. To address these issues, a three-dimensional wide field-of-view and real-time quantitative functional imaging technique able to map perfusion in the rodent brain would be desirable. Here, we applied functional ultrasound imaging, a novel approach to map relative cerebral blood volume without contrast agent, in a rat model of brief proximal transient middle cerebral artery occlusion to assess perfusion in penetrating arterioles and venules acutely and over six days thanks to a thinned-skull preparation. Functional ultrasound imaging efficiently mapped the acute changes in relative cerebral blood volume during occlusion and following reperfusion with high spatial resolution (100 µm), notably documenting marked focal decreases during occlusion, and was able to chart the fine dynamics of tissue reperfusion (rate: one frame/5 s) in the individual rat. No behavioral and only mild post-mortem immunofluorescence changes were observed. Our study suggests functional ultrasound is a particularly well-adapted imaging technique to study cerebral perfusion in acute experimental stroke longitudinally from the hyper-acute up to the chronic stage in the same subject.