Hemodynamic neuroimaging methods like functional magnetic resonance imaging (fMRI) have revolution- ized neuroscience by allowing researchers to characterize spatiotemporal features of brain-wide activity in hu- mans and animals. A major disadvantage of such approaches, however, is their lack of specificity for well-defined cellular and molecular sources; this limits their ability to yield explanatory insights into neural function. To address this problem, we recently developed an unprecedented family of genetically encodable molecular probes, called NOSTICs, that transduce intracellular calcium activity into artificial hemodynamic responses, permitting spatially comprehensive neuroimaging of genetically targeted cells and circuit elements. Hemogenetic signals arising from the NOSTICs may be differentiated from endogenous blood flow changes by pharmacological means and can be detected by any hemodynamic imaging modality. Our preliminary experiments indicate that cell-specific activity of even sparse neuronal populations can be identified using hemogenetic fMRI. These capabilities will enable hemogenetic imaging to confront some of the most outstanding problems in neuroscience, such as de- scribing functional properties of discrete cell populations on a brain-wide scale, defining input-output relation- ships among interacting brain regions and neural circuit components, and relating behavior and activity to plas- ticity and gene expression changes that occur throughout the brain. In this project, we will use hemogenetic imaging to address each of these broad problems in the context of sensory function in rodents, while at the same time refining the technology and laying a foundation for its wider application to many research topics and model systems in neuroscience. In Aim 1, we propose to use the technology for investigation of network-level processing in the somatosen- sory system. Anticipated results will inform a first-of-its-kind model of multiregional stimulus processing that con- stitutes a data-driven alternative to traditional correlative functional connectivity measures. We will use this model to examine the importance of feedback relationships and to help explain the phenomena of sensory adaptation and salience encoding at the network level. In Aim 2, we will exploit this capability by applying NOSTIC probes for genetically targeted fMRI of excitatory and inhibitory neural subtypes during forepaw stimulation and resting state dynamics in rats, addressing hypotheses about the functional roles of the different cell types. In addition, we will apply ultrahigh resolution fMRI to examine the relationships between single vessel-level hemodynamics and the cell type-specific distributions of NOSTIC expression, enabling a rich analysis that simultaneously in- forms interpretation of conventional fMRI results and rigorously characterizes performance of the hemogenetic technique itself. In Aim 3, we propose to improve the NOSTIC reporters themselves. Improvements we plan will enhance the detectability of hemogenetic signals and give rise to hemogenetic gene reporters that will be useful for mapping neural connectivity and plasticity in future applications.

Brain function depends on the integration of multiple neural structures and processes that currently cannot be studied at a whole-brain level in living organisms. To address this problem, we introduce a novel technology called hemogenetic imaging, which will enable the biology of genetically-targeted cells to be comprehensively studied by functional magnetic resonance imaging (fMRI) or related techniques. Here we propose to apply hemo- genetic fMRI to study brain-wide aspects of sensory processing, resting state dynamics, and neurovascular cou- pling. We will also improve the technology itself, setting the stage for many future applications.