Alberto L. Vazquez, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Changes in the cerebral metabolic rate of oxygen consumption (CMR02) produced by neuronal activity are directly related to the energetics of brain tissue function. These changes contribute to the blood oxygenation level dependent (BOLD) fMRI signal and have been partly responsible for the extensive use of BOLD fMRI in multi-disciplinary studies of brain function. Determining the magnitude and dynamics of CMR02 responses from BOLD fMRI data has been of great interest not only to unravel the underpinning of the BOLD response, but also in the understanding of brain function. The goals of this proposal are to determine the dynamic changes in CMR02 elicited by neural activation using fMRI data and investigate whether the assumption of instantaneous oxygen transport and consumption is valid for this purpose. To this end, non-invasive and invasive measurements of CBF, cerebral blood volume (CBV), tissue oxygen tension (PO2tiss). optical imaging of intrinsic signals (OIS) and BOLD measurements will be acquired using MRI (9.4 Tesla scanner) and non-MRI methodologies in a well-established rat somato-sensory model. As a result of this proposed project, a method to determine the dynamic changes in CMR02 using fMRI data will be developed and validated. These methods can be used in the clinical setting to assess tissue viability and function in the brain as well as other organs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The objective of the proposed work is to determine and characterize the spatio-temporal limits of the metabolic and hemodynamic responses that result from evoked neural activity. For this purpose a genetically enhanced mouse model expressing the light-sensitive protein Channelrhodopsin-2 will be used whereby the neuronal activity can be controlled through the modulation of the diameter and intensity of a light stimulus delivered directly onto the surface of the brain (photo-stimulus). First, the minimum neuronal activity that elicits a blood flow response will be determined using two-photon microscopy with sub-micrometer resolution. The size and strength of the neuronal activity will be characterized using a calcium indicator. Then, an intra-vascular fluorescent dye will be used to measure corresponding changes in blood velocity and vessel diameter in capillaries. Second, the consistency of these findings and the effect of resolution will be investigated by measuring the functional cerebral blood flow, metabolic and overall hemodynamic responses using larger- scale optical imaging with resolution of tens of micrometers. Specifically, the following optical methods will be used to measure blood flow, metabolic and overall hemodynamic responses to neural activity: laser speckle imaging, intrinsic auto-fluorescence imaging and optical imaging of intrinsic signal, respectively. The latter is a direct analog of blood oxygenation level dependent functional magnetic resonance imaging (BOLD fMRI). As a result of this project, the spatio-temporal limits of the metabolic and hemodynamic responses will be uncovered with unparalleled resolution. In addition, sufficient knowledge will be obtained to elucidate the theoretical and practical requirements of current brain research tools like fMRI to detect activities encoded in the hemodynamic response that may be thought to be undetectable. This work will have a tremendous impact in brain imaging studies of function in animals and humans, including clinical and basic science research. To perform the proposed studies I will undergo training in two-photon imaging of brain function and complementary optical imaging methods, and in electrophysiological recording of neuronal activity. A unique combination of local and national experts will provide and oversee the necessary training. This proposal is vital in fulfilling my ultimate career goal of attaining an independent and successful research program that focuses on imaging normal and pathological brain function. PUBLIC HEALTH RELEVANCE: The hemodynamic response induced by neural activity is, and has been, the principal means of studying brain function in animals and humans. Methods like functional magnetic resonance imaging are now essential in basic science, developmental, cognitive and clinical studies of normal and impaired brain function. This proposal will reveal the fundamental limits of hemodynamic-based methods to image and detect brain function.

PROJECT SUMMARY/ABSTRACT Most approaches to assess brain function and connectivity, including functional magnetic resonance imaging (fMRI) and optical imaging of intrinsic signals (OIS), rely on activity-evoked changes in cerebral blood flow, blood volume and/or blood oxygenation as indirect measures of neural activity. While these hemodynamic methods are immensely valuable to map brain function and connectivity in humans and animals, fundamental gaps remain in our understanding of neuro-vascular mechanisms due to its complexity and methodological difficulties. This gap in knowledge greatly impedes our understanding of the driving neuronal processes behind BOLD fMRI findings of brain function, dysfunction and development. The goal of this proposal is to determine the role of excitatory and inhibitory neuronal activity, including glutamatergic and GABAergic synaptic activity, in vascular regulation and their representation in hemodynamic signals. To activate specific neuronal sub-populations in vivo, novel optogenetic mouse models expressing Channelrhodopsin-2 (ChR2) in cortical excitatory or inhibitory neurons will be used. These optogenetic models will be combined with an innovative approach to concurrently record neural and vascular signals under pharmacological conditions designed to modulate glutamatergic and GABAergic synaptic transmission. Neural activity will be measured by electrophysiology and two-photon calcium imaging. Vascular signals of blood flow will be obtained by laser Doppler flowmetry, while OIS will be used to image changes in cerebral blood volume and blood oxygenation (directly analogous to fMRI). These data will then be used to model and integrate excitatory and inhibitory activity contributions to hemodynamic signals. This work will have a tremendous impact by advancing our understanding of relevant neuro-vascular mechanisms as well as establish a more concrete framework that can allow for a deeper understanding of the neuronal processes behind hemodynamic alterations in brain development and dysfunction.

PROJECT SUMMARY/ABSTRACT Neuronal activity actively modulates local cerebral vasculature. This neuro-vascular interaction is the foundation of imaging studies of human brain function in health and disease. These studies are routinely performed while subjects perform tasks (evoked activity) or lie resting (task-free or resting-state activity), and they assume that imaging signals loyally reflect local neuronal activity. However, the neuro-vascular signaling mechanism is complex and instances of uncoupling have been reported, limiting the interpretability of these studies. Recent reports have shown that different types of neurons can regulate local blood supply stronger than others, especially inhibitory neurons. These findings underscore the need to understand the vaso-regulatory roles of different neuronal populations, especially considering that neurological disorders have been associated with dysfunction of specific inhibitory neuron sub-populations. The goal of this proposal is to determine the role of different sub-populations of inhibitory neurons on the regulation of local blood flow during evoked stimulation as well as during resting-state activity periods. Experiments will be performed using unique transgenic mouse models. In addition, we will determine whether these findings generalize over different cortical regions and explore the translatability of these findings to human subjects by comparing the distribution of inhibitory neurons that strongly regulate local blood flow in targeted regions of mouse and human brains. Our group has extensive multi-modal expertise in neuro-vascular (and neuro-metabolic) physiology, including the models and techniques proposed, and we are uniquely positioned to successfully complete the aims of this project. We will achieve these goals through three aims: (Aim 1) Determine which inhibitory neuron sub-types strongly regulate local blood flow changes evoked by optogenetic stimulation in different cortical regions; (Aim 2) Determine whether the same sub-population of inhibitory neurons regulate local blood flow changes during ongoing awake activity periods; and (Aim 3) Determine whether the sub-populations of inhibitory neurons identified in Aims 1 and 2 are similarly distributed across the targeted regions of mouse and human brains. Since inhibitory neurons shape network activity, these studies will detail the impact of specific inhibitory neuronal sub-type function and dysfunction on local blood supply and hemodynamic-based imaging signals, expanding the interpretability and clinical utility of human brain imaging studies in health and disease.

Abstract Neurovascular coupling (NVC) is the temporal relationship between neural activity and cerebral blood flow (CBF). This neural-evoked hemodynamic response is fundamental to local cerebrovascular homeostasis and is disrupted in cerebrovascular diseases, such as stroke, cerebral amyloid angiopathy, traumatic brain injury, as well as Alzheimer's Disease. The neurons that express neuronal nitric oxide synthase (Nos1) are ideal candidates for the regulation of NVC since nitric oxide (NO) is a very potent vasodilator. Our group has recently developed a Tacr1CreER allele that enables the visualization and manipulation of these neurons. We now have exciting preliminary data supporting the hypothesis that Tacr1 neurons mediate vasodilation. Here, we propose to test this idea through a set of experiments that will: determine the relationship between Tacr1 neurons and blood vessels; examine causality in the regulation of NVC by Tacr1 neurons; and investigate the underlying circuitry. These experiments include correlative studies that will establish whether the structure (place) and function (activity) of Tacr1 neurons positions them to regulate CBF. We will also use optogenetic approaches and laser Doppler flowmetry (LDF) to record CBF in awake behaving mice to test whether Tacr1 neurons necessary and sufficient for vasodilation. Finally, we will use a combination of optogenetic manipulation, GCaMP6f-, and 2P-imaging to elucidate the underlying circuitry of NVC. Overall, our proposal will address a critical gap in knowledge with respect to the specific neural mechanisms that underlie the BOLD signal, which is a widely used, but poorly understood research and clinical tool. Moreover, this insight into NVC is fundamental to our understanding of the pathogenesis of common cerebrovascular diseases and the advancement of pharmacotherapeutics targeting cerebral perfusion.