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Neurovascular coupling (NVC), which mediates rapid increases in cerebral blood flow in response to neuronal activation, is commonly used to map brain activation or dysfunction. Here we tested the reemerging hypothesis that CO 2 generated by neuronal metabolism contributes to NVC. We combined functional ultrasound and two-photon imaging in the mouse barrel cortex to specifically examine the onsets of local changes in vessel diameter, blood flow dynamics, vascular/perivascular/intracellular pH, and intracellular calcium signals along the vascular arbor in response to a short and strong CO 2 challenge (10 s, 20%) and whisker stimulation. We report that the brief hypercapnia reversibly acidifies all cells of the arteriole wall and the periarteriolar space 3-4 s prior to the arteriole dilation. During this prolonged lag period, NVC triggered by whisker stimulation is not affected by the acidification of the entire neurovascular unit. As it also persists under condition of continuous inflow of CO 2 , we conclude that CO 2 is not involved in NVC., (© 2024. The Author(s).)

Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels. We find that transfer functions are robust across a wide range of stimulation paradigms and animals, and reveal a second vascular component of neurovascular coupling upon very strong stimulation. We propose that transfer functions can be considered as reliable quantitative reporters to follow neurovascular coupling dynamics.

Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics. Many questions about perivascular spaces remain, but what is now clear is that normal perivascular space function is important for maintaining brain health. Here, we review perivascular space anatomy, physiology and pathology, particularly as seen with MRI in humans, and consider translation from models to humans to highlight knowns, unknowns, controversies and clinical relevance.

In the brain, increased neural activity is correlated with increases of cerebral blood flow and tissue oxygenation. However, how cerebral oxygen dynamics are controlled in the behaving animal remains unclear. We investigated to what extent cerebral oxygenation varies during locomotion. We measured oxygen levels in the cortex of awake, head-fixed mice during locomotion using polarography, spectroscopy, and two-photon phosphorescence lifetime measurements of oxygen sensors. We find that locomotion significantly and globally increases cerebral oxygenation, specifically in areas involved in locomotion, as well as in the frontal cortex and the olfactory bulb. The oxygenation increase persists when neural activity and functional hyperemia are blocked, occurred both in the tissue and in arteries feeding the brain, and is tightly correlated with respiration rate and the phase of respiration cycle. Thus, breathing rate is a key modulator of cerebral oxygenation and should be monitored during hemodynamic imaging, such as in BOLD fMRI.

Imaging based on blood flow dynamics is widely used to study sensory processing. Here we investigated the extent to which local neuronal and capillary responses (two-photon microscopy) are correlated to mesoscopic responses detected with fast ultrasound (fUS) and BOLD-fMRI. Using a specialized chronic olfactory bulb preparation, we report that sequential imaging of the same mouse allows quantitative comparison of odour responses, imaged at both microscopic and mesoscopic scales. Under these conditions, functional hyperaemia occurred at the threshold of neuronal activation and fUS-CBV signals could be detected at the level of single voxels with activation maps varying according to blood velocity. Both neuronal and vascular responses increase non-linearly as a function of odour concentration, whereas both microscopic and mesoscopic vascular responses are linearly correlated to local neuronal calcium. These data establish strengths and limits of mesoscopic imaging techniques to report neural activity.

Optogenetics is increasingly used to map brain activation using techniques that rely on functional hyperaemia, such as opto-fMRI. Here we test whether light stimulation protocols similar to those commonly used in opto-fMRI or to study neurovascular coupling modulate blood flow in mice that do not express light sensitive proteins. Combining two-photon laser scanning microscopy and ultrafast functional ultrasound imaging, we report that in the naive mouse brain, light per se causes a calcium decrease in arteriolar smooth muscle cells, leading to pronounced vasodilation, without excitation of neurons and astrocytes. This photodilation is reversible, reproducible and energy-dependent, appearing at about 0.5 mJ. These results impose careful consideration on the use of photo-activation in studies involving blood flow regulation, as well as in studies requiring prolonged and repetitive stimulations to correct cellular defects in pathological models. They also suggest that light could be used to locally increase blood flow in a controlled fashion., Competing Interests: The authors declare no competing financial interests.

Stimulation of light-sensitive chemical probes has become a powerful tool for the study of dynamic signaLing processes in living tissue. Classically, this approach has been constrained by limitations of lens-based and point-scanning illumination systems. Here we describe a microscope configuration that incorporates a nematic liquid-crystal spatial light modulator to generate holographic patterns of illumination. This microscope can produce illumination spots of variable size and number, and in patterns shaped to precisely match user-defined elements in a specimen. Using holographic illumination to photolyze caged glutamate in brain slices, we show that shaped excitation on segments of neuronal dendrites and simultaneous, multispot excitation of different dendrites enables precise spatial and rapid temporal control of glutamate receptor activation. By allowing the excitation volume shape to be tailored precisely, the holographic microscope provides an extremely flexible method for activation of various photosensitive proteins and small molecules.