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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. Cerebral blood volume dynamics in awake head-fixed mice are tracked via functional ultrasound imaging through a chronic cranial window, giving a high-resolution measure of brain-wide activity.

A miniaturized ultrasound probe enables functional brain imaging in freely behaving rats. The large field of view and deep penetration makes this technique complementary to optical imaging approaches. 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.

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.

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 7days without loss of quality. Then, we investigated the spatiotemporal evolution of the CBV hemodynamic response function (HRF) in response to sensory-evoked electrical stimulus (1mA) ranging from 1 (200μs) 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. [Display omitted] •High spatio-temporal resolution (100μm/400ms) ultrasound brain imaging•Observation of brain hemodynamics in the entire depth of the brain•Chronic functional imaging using a novel thin skull surgery•A really sensitive method able to detect a single evoked stimulus (200μs)

The brain is composed of a dense and ramified vascular network of arteries, veins and capillaries of various sizes. One way to assess the risk of cerebrovascular pathologies is to use computational models to predict the physiological effects of reduced blood supply and correlate these responses with observations of brain damage. Therefore, it is crucial to establish a detailed 3D organization of the brain vasculature, which could be used to develop more accurate in silico models. To this end, we have adapted our functional ultrasound imaging platform, previously designed for recording large scale activity, to enable rapid and reproducible acquisition, segmentation and reconstruction of the cortical vasculature. For the first time, it allows us to digitize the cortical ∼ 100 - μ m3 spatial resolution. Unlike most available strategies, our approach can be performed in vivo within minutes. Moreover, it is easy to implement since it requires neither exogenous contrast agents nor long post-processing time. Therefore, we performed a cortex-wide reconstruction of the vasculature and its quantitative analysis, including i) classification of descending arteries versus ascending veins in more than 1500 vessels/animal and ii) rapid estimation of their length. Importantly, we confirmed the relevance of our approach in a model of cortical stroke, which allows rapid visualization of the ischemic lesion. This development contributes to extending the capabilities of ultrasound neuroimaging to better understand cerebrovascular pathologies such as stroke, vascular cognitive impairment and brain tumors, and is highly scalable for the clinic.

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.

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. Increasing electrode count of flexible electrode arrays in a sustainable way: Incorporating metal oxide thin‐film transistors into flexible micro‐electrocorticography (µECoG) electrode arrays enables multiplexing of the recording electrodes, overcoming the wiring bottleneck faced by current µECoG technologies. By combining the arrays with a dedicated silicon readout chip, 256 electrodes can be recorded simultaneously in time‐division multiplexing mode employing only 16 channels.

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.

The brain is composed of a dense and ramified vascular network of arteries, veins and capillaries of various sizes. One way to assess the risk of cerebrovascular pathologies is to use computational models to predict the physiological effects of reduced blood supply and correlate these responses with observations of brain damage. Therefore, it is crucial to establish a detailed 3D organization of the brain vasculature, which could be used to develop more accurate in silico models. To this end, we have adapted our functional ultrasound imaging platform, previously designed for recording large scale activity, to enable rapid and reproducible acquisition, segmentation and reconstruction of the cortical vasculature. For the first time, it allows us to digitize the cortical $$\\sim 100$$ ∼100- $$\\mu $$ μm3 spatial resolution. Unlike most available strategies, our approach can be performed in vivo within minutes. Moreover, it is easy to implement since it requires neither exogenous contrast agents nor long post-processing time. Therefore, we performed a cortex-wide reconstruction of the vasculature and its quantitative analysis, including i) classification of descending arteries versus ascending veins in more than 1500 vessels/animal and ii) rapid estimation of their length. Importantly, we confirmed the relevance of our approach in a model of cortical stroke, which allows rapid visualization of the ischemic lesion. This development contributes to extending the capabilities of ultrasound neuroimaging to better understand cerebrovascular pathologies such as stroke, vascular cognitive impairment and brain tumors, and is highly scalable for the clinic.

Over the last decade, functional ultrasound (fUS) has risen as a critical tool in functional neuroimaging, leveraging hemodynamic changes to infer neural activity indirectly. Recent studies have established a strong correlation between neural spike rates (SR) and functional ultrasound signals. However, understanding their spatial distribution and variability across different brain areas is required to thoroughly interpret fUS signals. In this regard, we conducted simultaneous fUS imaging and Neuropixels recordings during stimulus-evoked activity in awake mice within three regions the visual pathway. Our findings indicate that the temporal dynamics of fUS and SR signals are linearly correlated, though the correlation coefficients vary among visual regions. Conversely, the spatial correlation between the two signals remains consistent across all regions with a spread of approximately 300 micrometers. Finally, we introduce a model that integrates the spatial and temporal components of the fUS signal, allowing for a more accurate interpretation of fUS images.