Title: Evaluating endothelial function during neurovascular coupling in awake behaving mice using advanced imaging technologies
Description: Local neuronal activity in the brain results in increased blood flow and is called neurovascular coupling. Such blood flow changes result in the blood-oxygen level dependent (BOLD) fluctuations detectable by functional magnetic resonance imaging (fMRI). The hemodynamic response is also an essential component of brain health and is impaired in various models of cognitive dysfunction. However, we still do not understand why functional hyperemia in the brain is important. To understand this question, various groups have studied brain metabolic activity as well as the mechanisms underlying neurovascular coupling. Over the years, several cell types have been proposed to contribute to functional hyperemia in the brain, including neurons, astrocytes and pericytes. However, the picture remains incomplete – controversies abound regarding the exact role of astrocytes, and pericytes in neurovascular coupling. Our lab has studies the mechanisms of neurovascular coupling from a mesoscopic perspective, as vasodilation in the rodent cortex involves capillaries and diving arterioles in the brain parenchyma as well as surface vasculature in the brain.

We proposed that the vascular endothelium itself might provide a continuous conduit for transmitting vasodilatory signals initiated at the capillary level due to local neuronal activity. Given that systemic endothelial dysfunction could contribute to decreased neurovascular function, this hypothesis raised important concerns regarding endothelial vulnerabilities in common diseases like hypertension and diabetes and its role in diminished cognitive function and neurodegeneration. Based on findings from vascular research in other organ systems, we hypothesized that two distinct mechanisms of endothelium-derived vasodilation significantly contribute to neurovascular coupling the brain. These two mechanisms were expected to consist of fast long-range endothelium-derived hyperpolarization (EDH) dependent vasodilation (conducted vasodilation) and slower, more localized endothelium calcium-wave dependent vasodilation (propagated vasodilation). Together, we expected these mechanisms to shape the spatio-temporal evolution of hemodynamic responses in the brain.

This dual mechanism of endothelial control of the hyperemic response in the brain might explain the complex spatiotemporal properties and non-linearities of the fMRI blood oxygen level dependent (BOLD) signal. My initial experiments were conducted in anesthetized rats, where I pharmacologically inhibited endothelial dependent vasodilation during functional hyperemia in the somatosensory cortex under a hind-paw electrical stimulus paradigm. While the results gleaned from these experiments were very revealing, it was important to consider the effect of the pharmacological manipulations on neuronal activity in the brain. In addition, neurovascular coupling and overall brain blood flow in anesthetized animals is dramatically altered when compared to awake animals. In order to accomplish these goals, I built a wide-field optical imaging system that could simultaneously measure fluorescence-based neuronal activity and reflectance-based hemodynamic activity in awake head-restrained mice. I then used non-blood brain barrier permeable pharmacology to study endothelial mechanisms of neurovascular coupling in awake Thy1-GCaMP6f mice, which express the calcium fluorophore in a subset of excitatory neurons in the cortex.

I found that using this pharmacology I could dissect out the hypothesized two spatiotemporally distinct components of whisker-stimulus evoked neurovascular coupling in awake mice. With simultaneous recording of the neuronal activity driving this blood flow, I was able to build a mathematical model for neurovascular coupling that accounted for these two mechanisms by allowing for the superposition of a time-invariant, constant hemodynamic response with a hemodynamic response obtained by convolving the underlying neuronal response with a hemodynamic response function (HRF). I was able to linearize these apparent non-linearities in the hemodynamic response by studying the properties of deconvolved HRFs for stimuli of different durations before and after pharmacological manipulation of endothelial activity. Two important considerations remain. Firstly, our wide-field, mesoscopic view of the brain prevents observations of endothelial function (hyperpolarization and calcium activity) and the propagation dynamics of dilation best observed at the microscopic level.

To accomplish this task, ongoing experiments currently use our high-speed volumetric imaging technology (SCAPE – Swept Confocally Aligned Planar Excitation microscopy) to study stimulus-evoked vascular dynamics in mouse lines expressing GFP and GCaMP8 in endothelial cells. Secondly, our longitudinal imaging of these animals is ideal for studying the acute and long-term effects of endothelial dysfunction on cognitive function. This requires adequate study of changes in mouse behavior during manipulations of endothelial function longitudinally in awake mice. Future experiments should involve the development and implementation of appropriate task-based behavior experiments, and analysis methods for more carefully exploring changes in neuronal activity in the mouse brain during stimulus and non-stimulus dependent activity.