Project - Abstract Millions of people suffer from urinary incontinence, urgency, frequency and bladder pain; conditions which are often grouped under the catchall of lower urinary tract symptoms (LUTS). Little is known of the underlying mechanisms that cause these symptoms, but disruptions to brain function as seen in dementia, stroke and brain injury often result in LUTS. Neural circuit defects may in fact contribute to or cause many benign urological disorders. Neural control of urine storage and voiding is highly complex and is coordinated in specific regions of the brain. Modern transgenic ?tools? enable specific neuron populations (such as glutamatergic neurons in a brain region like the ventrolateral periaqueductal gray, PAGVLGLUT neurons) to be mapped, monitored, and manipulated in vivo. While these approaches represent an enormous advance in our ability to understand how particular brain circuits control organ function, we still lack the ability to identify and work with specific subpopulations of neurons which have discrete functions. For example, although PAGVLGLUT neurons share a major neurotransmitter and are located in a discrete brain area, this group of neurons is made up of many different subpopulations, each of which has a different set of inputs, a distinct set of projections, and of course a different function. What is needed is a method to identify each of these subpopulations, and to develop approaches to target them selectively, so that we can tease apart their discrete functions as well as their connections. Single cell RNA-seq (scRNA-seq) involves dissociation of brain regions to single cells, followed by sequencing of the RNA expression pattern of each cell. This approach provides an unbiased method for defining neuron subpopulations by means of their transcriptional profiles within a heterogenous tissue. Members of our research team have used this approach both to create an atlas of neuron subpopulations from the arcuate hypothalamic nucleus and median eminence, and to deduce/validate the function of these cell types. In the proposed studies, we will create an atlas of neuron subpopulations in the pontine micturition center- locus coeruleus (PMC-LC) and the PAGVL regions, which are known to regulate bladder function. Follow- up studies will identify neural subpopulations that form the bladder-controlling neurocircuit, and those that alter significantly their RNA expression profiles in response to forced profound polyuria. Our results will guide strategies to access and manipulate genetically these discrete, functionally important neuron subpopulations. The atlas and its mapping and functional annotations will be published and will be reported with regular updates on the P20 website. The proposed studies will provide the urological research community with tools of unprecedented specificity, permitting vastly improved mapping of neural circuits controlling normal voiding, and direct means of determining how disease states alter neural function so as to cause bladder dysfunction.
