Cerebral oxygenation during locomotion is modulated by respiration.

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.

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation.

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2-3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.

Unbiased Analysis Method for Measurement of Red Blood Cell Size and Velocity With Laser Scanning Microscopy.

Two-photon laser scanning microscopy is widely used to measure blood hemodynamics in brain blood vessels. Still, the algorithms used so far to extract red blood cell (RBC) size and velocity from line-scan acquisitions have ignored the extent to which scanning speed influences the measurements. Here, we used a theoretical approach that takes into account the velocity and direction of both scanning mirrors and RBCs during acquisition to provide an algorithm that measures the real RBC size and velocity. We validate our approach in brain vessels of anesthetized mice, and demonstrate that it corrects online measurement errors that can reach several 10s of percent as well as data previously acquired. To conclude, our analysis allows unbiased comparisons of blood hemodynamic parameters from brain capillaries and large vessels in control and pathological animal models.

Mesoscopic and microscopic imaging of sensory responses in the same animal.

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.

Mapping oxygen concentration in the awake mouse brain.

Although critical for brain function, the physiological values of cerebral oxygen concentration have remained elusive because high-resolution measurements have only been performed during anesthesia, which affects two major parameters modulating tissue oxygenation: neuronal activity and blood flow. Using measurements of capillary erythrocyte-associated transients, fluctuations of oxygen partial pressure (Po2) associated with individual erythrocytes, to infer Po2 in the nearby neuropil, we report the first non-invasive micron-scale mapping of cerebral Po2 in awake, resting mice. Interstitial Po2 has similar values in the olfactory bulb glomerular layer and the somatosensory cortex, whereas there are large capillary hematocrit and erythrocyte flux differences. Awake tissue Po2 is about half that under isoflurane anesthesia, and within the cortex, vascular and interstitial Po2 values display layer-specific differences which dramatically contrast with those recorded under anesthesia. Our findings emphasize the importance of measuring energy parameters non-invasively in physiological conditions to precisely quantify and model brain metabolism.