The oxygen initial dip in the brain of anesthetized and awake mice.

An ongoing controversy in brain metabolism is whether increases in neural activity cause a local and rapid decrease in oxygen concentration (i.e., the "initial dip") preceding functional hyperemia. This initial dip has been suggested to cause a transient increase in vascular deoxyhemoglobin with several imaging techniques and stimulation paradigms, but not consistently. Here, we investigate contributors to this initial dip in a distinct neuronal network, an olfactory bulb (OB) glomerulus most sensitive to a specific odorant (ethyl tiglate [ET]) and a site of strong activation and energy consumption upon ET stimulation. Combining two-photon fluorescence and phosphorescence lifetime microscopy, and calcium, blood flow, and pO2 measurements, we characterized this initial dip in pO2 in mice chronically implanted with a glass cranial window, during both awake and anesthetized conditions. In anesthetized mice, a transient dip in vascular pO2 was detected in this glomerulus when functional hyperemia was slightly delayed, but its amplitude was minute (0.3 SD of resting baseline). This vascular pO2 dip was not observed in other glomeruli responding nonspecifically to ET, and it was poorly influenced by resting pO2. In awake mice, the dip in pO2 was absent in capillaries as well as, surprisingly, in the neuropil. These high-resolution pO2 measurements demonstrate that in awake mice recovered from brain surgery, neurovascular coupling was too fast and efficient to reveal an initial dip in pO2.

Diversity of neurovascular coupling dynamics along vascular arbors in layer II/III somatosensory cortex.

The spatial-temporal sequence of cerebral blood flow (CBF), cerebral blood volume (CBV) and blood velocity changes triggered by neuronal activation is critical for understanding functional brain imaging. This sequence follows a stereotypic pattern of changes across different zones of the vasculature in the olfactory bulb, the first relay of olfaction. However, in the cerebral cortex, where most human brain mapping studies are performed, the timing of activity evoked vascular events remains controversial. Here we utilized a single whisker stimulation model to map out functional hyperemia along vascular arbours from layer II/III to the surface of primary somatosensory cortex, in anesthetized and awake Thy1-GCaMP6 mice. We demonstrate that sensory stimulation triggers an increase in blood velocity within the mid-capillary bed and a dilation of upstream large capillaries, and the penetrating and pial arterioles. We report that under physiological stimulation, response onset times are highly variable across compartments of different vascular arbours. Furthermore, generating transfer functions (TFs) between neuronal Ca2+ and vascular dynamics across different brain states demonstrates that anesthesia decelerates neurovascular coupling (NVC). This spatial-temporal pattern of vascular events demonstrates functional diversity not only between different brain regions but also at the level of different vascular arbours within supragranular layers of the cerebral cortex.

Iliski, a software for robust calculation of transfer functions.

Understanding the relationships between biological processes is paramount to unravel pathophysiological mechanisms. These relationships can be modeled with Transfer Functions (TFs), with no need of a priori hypotheses as to the shape of the transfer function. Here we present Iliski, a software dedicated to TFs computation between two signals. It includes different pre-treatment routines and TF computation processes: deconvolution, deterministic and non-deterministic optimization algorithms that are adapted to disparate datasets. We apply Iliski to data on neurovascular coupling, an ensemble of cellular mechanisms that link neuronal activity to local changes of blood flow, highlighting the software benefits and caveats in the computation and evaluation of TFs. We also propose a workflow that will help users to choose the best computation according to the dataset. Iliski is available under the open-source license CC BY 4.0 on GitHub (https://github.com/alike-aydin/Iliski) and can be used on the most common operating systems, either within the MATLAB environment, or as a standalone application.

Transfer functions linking neural calcium to single voxel functional ultrasound signal.

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.