Longitudinal recording of neuronal function using two photon fluorescence microscopy in adult rats co-expressing genetically encoded calcium indicators and channelrhodopsin-2

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Summary of Proposal The hundred billion neurons that make up the brain are organized in a highly complex and interconnected network. Supporting the high energetic needs of these neurons is an intricate, hierarchical network of vessels. Superimposed on this anatomical complexity, is great diversity in the interaction between neuronal and vascular networks. For the system to function, an increase in neuronal activity must be accompanied by an increase in blood flow in the surrounding vessels. Although central to healthy brain functioning, the neurovascular coupling is still incompletely understood. Hitherto, neurovascular coupling studies have been impeded by the methodological difficulty of stimulating individual neurons and recording their activity and its effects on neighboring vessels. Recent methodological developments, however, have greatly improved optical measurements of neuronal activity and direct optical stimulation of neurons. These novel techniques have the potential to enable non-invasive cellular scale examinations of neuronal activity in vivo and thus offer an unprecedented opportunity to gain mechanistic understanding of neurovascular coupling at the cellular level. The goal of this proposal is to make use of these state-of-the-art optical techniques to examine the interaction between individual neurons and surrounding brain microvessels while using our established imaging and computational analysis techniques to capture, in mathematical terms, the architecture of local neuronal and vascular networks. Building upon our prior studies, we will employ two photon fluorescence microscopy, which affords cellular scale resolution, in live, anesthetized rats prepared with a cranial window over the brain region involved in processing of somatosensory stimuli, thus allowing us to evaluate neuronal and vascular responses to simple peripheral stimuli akin to the median nerve stimulation in humans. To allow non-invasive stimulation of individual neurons and recording of the neuronal activity, we will inject viral constructs, designed to infect specific subpopulations of neurons, directly into the animals’ brains. Once infected by these viruses, neurons will express light activatable ion channels in their membranes and calcium concentration sensitive fluorescent dyes in their cytosol, thus allowing us to use light to stimulate or inhibit the infected neurons as well as record their activity optically. In the first part of the project, we will stimulate individual infected neurons with light and measure the effect of their increased activity on the surrounding vessels. In the second part of the project, we will stimulate the animals peripherally while shining light onto specific infected neurons in the somatosensory cortex so as to inhibit their activity and measure the effects of the individual neuron’s inhibition on the vascular response to peripheral stimulation. Combined, these studies will give us a mechanistic understanding of the link between neurons and brain microvessels, thus providing the foundation for understanding the complex changes in neuronal and vascular networks that give rise to “brain plasticity” and that are fundamental for maintenance of healthy brain function. Moreover, the insight obtained in these studies will enable quantitative interpretation of functional MRI, which makes inferences about neuronal activity based on measurements of vascular state. Given the widespread use of functional MRI for studying human brain function, a quantitative model of functional MRI signal will have a broad impact on neuroscience research and will greatly enhance our ability to examine human brain function.