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.
Functional ultrasound (fUS) is a neuroimaging method that uses ultrasound to track changes in cerebral blood volume as an indirect readout of neuronal activity at high spatiotemporal resolution. fUS ...is capable of imaging head-fixed or freely behaving rodents and of producing volumetric images of the entire mouse brain. It has been applied to many species, including primates and humans. Now that fUS is reaching maturity, it is being adopted by the neuroscience community. However, the nature of the fUS signal and the different implementations of fUS are not necessarily accessible to nonspecialists. This review aims to introduce these ultrasound concepts to all neuroscientists. We explain the physical basis of the fUS signal and the principles of the method, present the state of the art of its hardware implementation, and give concrete examples of current applications in neuroscience. Finally, we suggest areas for improvement during the next few years.
The in vivo assessment of the biomechanical properties of the skeletal muscle is a complex issue because the muscle is an anisotropic, viscoelastic and dynamic medium. In this article, these ...mechanical properties are characterized for the brachialis muscle in vivo using a noninvasive ultrasound-based technique. This supersonic shear imaging technique combines an ultra-fast ultrasonic system and the remote generation of transient mechanical forces into tissue via the radiation force of focused ultrasonic beams. Such an ultrasonic radiation force is induced deep within the muscle by a conventional ultrasonic probe and the resulting shear waves are then imaged with the same probe (5 MHz) at an ultra-fast framerate (up to 5000 frames/s). Local tissue velocity maps are obtained with a conventional speckle tracking technique and provide a full movie of the shear wave propagation through the entire muscle. Shear wave group velocities are then estimated using a time of flight algorithm. This approach provides a complete set of quantitative and in vivo parameters describing the muscle's mechanical properties as a function of active voluntary contraction as well as passive extension of healthy volunteers. Anisotropic properties are also estimated by tilting the probe head with respects to the main muscular fibers direction. Finally, the dispersion of the shear waves is studied for these different configurations and shear modulus and shear viscosity are quantitatively assessed assuming the viscoelastic Voigt's model.
Functional ultrasound (fUS) is a hemodynamic-based functional neuroimaging technique, primarily used in animal models, that combines a high spatiotemporal resolution, a large field of view, and ...compatibility with behavior. These assets make fUS especially suited to interrogating brain activity at the systems level. In this review, we describe the technical capabilities offered by fUS and discuss how this technique can contribute to the field of functional connectomics. First, fUS can be used to study intrinsic functional connectivity, namely patterns of correlated activity between brain regions. In this area, fUS has made the most impact by following connectivity changes in disease models, across behavioral states, or dynamically. Second, fUS can also be used to map brain-wide pathways associated with an external event. For example, fUS has helped obtain finer descriptions of several sensory systems, and uncover new pathways implicated in specific behaviors. Additionally, combining fUS with direct circuit manipulations such as optogenetics is an attractive way to map the brain-wide connections of defined neuronal populations. Finally, technological improvements and the application of new analytical tools promise to boost fUS capabilities. As brain coverage and the range of behavioral contexts that can be addressed with fUS keep on increasing, we believe that fUS-guided connectomics will only expand in the future. In this regard, we consider the incorporation of fUS into multimodal studies combining diverse techniques and behavioral tasks to be the most promising research avenue.
In the early visual system, cells of the same type perform the same computation in different places of the visual field. How these cells code together a complex visual scene is unclear. A common ...assumption is that cells of a single-type extract a single-stimulus feature to form a feature map, but this has rarely been observed directly. Using large-scale recordings in the rat retina, we show that a homogeneous population of fast OFF ganglion cells simultaneously encodes two radically different features of a visual scene. Cells close to a moving object code quasilinearly for its position, while distant cells remain largely invariant to the object's position and, instead, respond nonlinearly to changes in the object's speed. We develop a quantitative model that accounts for this effect and identify a disinhibitory circuit that mediates it. Ganglion cells of a single type thus do not code for one, but two features simultaneously. This richer, flexible neural map might also be present in other sensory systems.
General anesthetics induce loss of consciousness, a global change in behavior. However, a corresponding global change in activity in the context of defined cortical cell types has not been ...identified. Here, we show that spontaneous activity of mouse layer 5 pyramidal neurons, but of no other cortical cell type, becomes consistently synchronized in vivo by different general anesthetics. This heightened neuronal synchrony is aperiodic, present across large distances, and absent in cortical neurons presynaptic to layer 5 pyramidal neurons. During the transition to and from anesthesia, changes in synchrony in layer 5 coincide with the loss and recovery of consciousness. Activity within both apical and basal dendrites is synchronous, but only basal dendrites’ activity is temporally locked to somatic activity. Given that layer 5 is a major cortical output, our results suggest that brain-wide synchrony in layer 5 pyramidal neurons may contribute to the loss of consciousness during general anesthesia.
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•Activity of layer 5 PNs synchronizes globally in different anesthetics•Other mouse cortical cell types show no consistent increase in synchrony•Changes in layer 5 synchrony coincide with the loss and recovery of consciousness•Basal, but not apical, layer 5 dendrites are in synchrony with somas
Bharioke, Munz et al. discovered that different general anesthetics consistently synchronize spontaneous activity of mouse layer 5 pyramidal neurons, but of no other cortical cell type, in vivo. Strikingly, changes in synchrony across layer 5 pyramidal neurons, during transitions to and from anesthesia, coincide with loss and recovery of consciousness.
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 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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated ...virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell–specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.
The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore ...retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca2+-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus CMV) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.
Vision restoration using microbial opsins has substantial clinical potential; however, it requires high-level expression of a foreign protein in the patient’s eyes. Our study shows the feasibility of obtaining safe and functional expression in primates using a cell-specific promoter and provides the basis for further clinical development of this optogenetic strategy.
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 μm
, 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/cm
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.
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