Emilie Macé

Deciphering brain-wide circuits requires large animal cohorts, yet computational bottlenecks in atlas registration and whole-volume cell detection reduce precision, reproducibility, and sample yield. We present LightSuite, an open-source framework enabling scalable, multimodal registration and cellular mapping across the central nervous system (CNS). LightSuite combines damage-resilient 3D point-cloud initialization with user-steerable non-rigid registration, and GPU-accelerated local background normalization for rapid, illumination-invariant cell detection. Applying LightSuite to Fos-dependent activity labeling in mice, we extracted reproducible, layer-resolved profiles of active cell density, soma-size distributions and per-animal labeling-efficiency factors. LightSuite computationally untwists and registers cleared spinal cords to map myeloarchitecture, reconstructs serial wide-field histology to map surface GluA1 distributions, resolves optic fiber tracks, and aligns in vivo functional ultrasound hemodynamics into common atlas coordinates. LightSuite establishes an integrated, accessible platform for cohort-level structural, molecular, and functional CNS mapping.

Arousal fluctuations during wakefulness have a major impact on physiology and behavior, including perception and task performance. Arousal is also known to be a strong modulator of neural activity, but the brain-wide spatiotemporal structure of this modulation is not fully characterized. We used functional ultrasound imaging to record brain-wide hemodynamics - a proxy for neural activity - in head-fixed mice during spontaneous and sensory-evoked arousal fluctuations, tracked via pupil diameter. Both conditions recruited a common brain-wide hemodynamic wave that followed a subcortex-to-cortex gradient. We then tested whether noradrenaline, widely associated with arousal, was necessary or sufficient to drive this wave. Sustained bidirectional optogenetic manipulations of locus coeruleus activity affected brain-wide vascular signal amplitude but, surprisingly, left arousal-linked dynamics largely intact. Together, these results identify a common spatiotemporal motif of arousal that appears independent of noradrenergic tone.

Animals exhibit behavior in the absence of external stimuli or explicit tasks. Is the initiation of such spontaneous behavior shaped by internal brain states in a predictable manner? If so, does it engage specific brain circuits independent of behavioral form? Here, we studied the initiation of uninstructed behaviors of head-fixed mice in two contexts: a virtual burrow and a running wheel. Across both contexts, mice spent most of the time in quiet wakefulness and spontaneously initiated bouts of egress (exiting the burrow), running, or grooming. We employed functional ultrasound imaging (fUS) to record whole-brain activity and to identify whether the initiation of spontaneous behavior could be predicted from hemodynamic signals. We first identified distinct hemodynamic patterns associated with each behavior and subsequently performed time-resolved decoding to predict behavioral transitions from fUS data. We found that whole-brain hemodynamic signals could decode spontaneous egress and running around 10 seconds before their onset, a timescale that cannot be accounted for by preceding behavioral changes alone. Furthermore, we found a network of regions, including the medial septum (MS), that decreased their signal several seconds before the onset of egress and running. Mimicking this decrease by inhibiting neurons in the MS via optogenetics increased the probability of egress, running, and grooming. Through this unbiased approach, our work sheds light on a whole-brain transition-prone state that precedes uninstructed behavior transitions.

Animals use visual objects to guide navigation-related behaviors. However, visual object–preferring areas have yet to be described in the mouse brain, limiting our understanding of how visual objects affect spatial navigation system processing. Using functional ultrasound imaging, we identified brain areas that were preferentially activated by images of objects compared with their scrambled versions. Whereas visual cortex did not show a preference, areas associated with spatial navigation were preferentially activated by visual objects. Electrophysiological recordings in postsubiculum, the cortical head direction (HD) system hub, confirmed a preference for visual objects in both HD cells and fast-spiking interneurons. In freely moving animals, visual objects increased firing rates of HD cells aligned with a visual object but decreased activity in HD cells coding for other directions.

Optogenetic control is used to manipulate the activity of specific cell types in vivo for a variety of biological and clinical applications. Here we report ChReef, an improved variant of the channelrhodopsin ChRmine. ChReef offers minimal photocurrent desensitization, a unitary conductance of 80 fS and closing kinetics of 30 ms, which together enable reliable optogenetic control of cells at low light levels with good temporal fidelity and sustained stimulation. We demonstrate efficient and reliable red-light pacing and depolarization block of ChReef-expressing cardiomyocyte clusters. We used adeno-associated-virus-based gene transfer to express ChReef in retinal ganglion cells, where it restores visual function in blind mice with light sources as weak as an iPad screen. Toward optogenetic hearing restoration, ChReef enables stimulation of the auditory pathway in rodents and non-human primates with nanojoule thresholds, enabling efficient and frequency-specific stimulation by LED-based optical cochlear implants.

Animals use visual objects to guide navigation-related behaviors, from hunting prey, to escaping predators, to exploring the world. However, little is known about where visual objects are encoded in the mouse brain or how they impact processing in the spatial navigation system. Using functional ultrasound imaging in mice, we conducted a brain-wide screen and identified brain areas that were preferentially activated by images of objects compared to scrambled versions of the same images. While visual cortical areas did not show a significant preference, regions associated with spatial navigation were preferentially activated by visual objects. Electrophysiological recordings in postsubiculum, the primary cortical area of the head direction (HD) system, further confirmed a preference for visual objects, which was present in both HD cells and fast-spiking interneurons. Finally, we found that visual objects dynamically modulated HD cells, selectively increasing firing rates of HD cells aligned with a visual landmark’s direction, while decreasing activity in HD cells coding for other directions. These results reveal that visual objects refine population-level coding of head direction.

Neuroscientists studying the neural correlates of mouse behavior often lack access to the brain-wide activity patterns elicited during a specific task of interest. Fortunately, large-scale imaging is becoming increasingly accessible thanks to modalities such as Ca²⁺ imaging and functional ultrasound (fUS). However, these and other techniques often involve challenging cranial window procedures and are difficult to combine with other neuroscience tools. We address this need with an open-source 3D-printable cranial implant—the COMBO (ChrOnic Multimodal imaging and Behavioral Observation) window. The COMBO window enables chronic imaging of large portions of the brain in head-fixed mice while preserving orofacial movements. We validate the COMBO window stability using both brain-wide fUS and multisite two-photon imaging. Moreover, we demonstrate how the COMBO window facilitates the combination of optogenetics, fUS, and electrophysiology in the same animals to study the effects of circuit perturbations at both the brain-wide and single-neuron level. Overall, the COMBO window provides a versatile solution for performing multimodal brain recordings in head-fixed mice.

Neuroscientists studying the neural correlates of mouse behavior often lack access to the brain-wide activity patterns elicited during a specific task of interest. Fortunately, large-scale imaging is becoming increasingly accessible thanks to modalities such as Ca ²⁺ imaging and functional ultrasound (fUS). However, these and other techniques often involve challenging cranial window procedures, and are difficult to combine with other neuroscience tools. We address this need with an open-source 3D-printable cranial implant - the COMBO ( C hr O nic M ultimodal imaging and B ehavioral O bservation) window. The COMBO window enables chronic imaging of large portions of the brain in head-fixed mice while preserving orofacial movements. We validate the COMBO window stability using both brain-wide fUS and multi-site two-photon imaging. Moreover, we demonstrate how the COMBO window facilitates the combination of optogenetics, fUS and electrophysiology in the same animals to study the effects of circuit perturbations at both the brain-wide and single-neuron level. Overall, the COMBO window provides a versatile solution for performing multimodal brain recordings in 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.

SUMMARY: Imaging of large-scale circuit dynamics is crucial to gain a better understanding of brain function, but most techniques have a limited depth of field. Here we describe vfUSI, a platform for brain-wide volumetric functional ultrasound imaging of hemodynamic activity in awake head-fixed mice. We combined high-frequency 1024-channel 2D-array transducer with advanced multiplexing and high-performance computing for real-time 3D Power Doppler imaging at high spatiotemporal resolution (220×280×175-μm ³ voxel size, up to 6 Hz). In addition, we developed a standardized software pipeline for registration and segmentation based on the Allen Mouse Common Coordinate Framework, allowing for temporal analysis in 268 individual brain regions. We demonstrate the high sensitivity of vfUSI in multiple experimental situations where stimulus-evoked activity can be recorded using a minimal number of trials. We also mapped neural circuits in vivo across the whole brain during optogenetic activation of specific cell-types. Moreover, we revealed the sequential activation of sensory-motor regions during a grasping water droplet task. vfUSI will become a key neuroimaging technology because it combines ease of use, reliability, and affordability.