The proposed studies seek P20 funding to bring together a team of scientists to begin to define the CNS neural network controlling urine storage and voiding in awake mice. We will use state of the art optogenetic tools to activate or silence specific neuronal populations in the Pontine Micturition Center (PMC) and determine the effects of these interventions on urine storage and voiding in conscious mice. Our preliminary data, generated using mice developed by our collaborators, in which Cre recombinase is expressed selectively in distinct neuronal subpopulations, identified 4 subpopulations of PMC neurons, which we will stimulate or inhibit by optogenetic and DREADDs (designer receptors exclusively activated by designer drug) methods. By coupling these neural optogenetics and neural mapping techniques with capabilities we already have within our group to examine urine storage and voiding in conscious animals (spontaneous voiding and conscious cystometrograms (CMGs)), we can now define the functional roles of specific brainstem neuron subpopulations in controlling storage and voiding in the awake mouse.
Aim 1 will use selective expression of placental alkaline phosphatase or synaptophysin coupled to green fluorescent protein to map the axonal projections of each of the PMC neuronal subpopulations. We will then stimulate or silence individual PMC neuronal populations using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) or optogenetic methods (Channel 2 rhodopsin or a halorhodopsin (NpHR), and measure the impact of the stimulation or silencing on spontaneous voiding and conscious cystometrograms (CMGs).
This aim will identify the specific subpopulations of PMC neurons which directly control urine storage and voiding and determine their roles.
In Aim 2 we will perform monosynaptic circuit tracing using modified rabies virus, to identify the upstream neurons which innervate the PMC neuronal subpopulations which, in Aim 1 have been shown to control urine storage and voiding. These studies address directly several of the major goals of the RFA: 1. They are a "resource development project" which will develop new tools to advance the field. 2. They seek to "develop and validate optogenetic tools to dissect the causal and precise circuitry underlying bladder function." 3. They seek to "develop a large scale map of neuronal connectivity between urological organs and/or the brain." In addition, as envisioned in the RFA, the proposed studies and the P20 mechanism bring to bear on urological diseases techniques which have not been used before and bring to the field the expertise of investigators who, up to now, have been focused not on urological diseases. These studies will set the stage for a multiyear program project or U award, for detailed mapping of the mouse CNS control mechanisms for regulating urine storage and voiding. Achievement of these aims will advance considerably our understanding of LUTS and related disorders in humans and will provide tools to scientists who seek to test novel therapeutics in model animals.
Disorders of voiding, including incontinence, overactive bladder and lower urinary tract symptoms and related disorders, as reported in patients, grows rapidly as people age, and can reach up to 30% of elderly people. Our overall goal is to develop robust mouse models of these human disorders, permitting studies of its mechanism. By developing tools which permit functional mapping of the CNS neural circuits that control urine storage and voiding, these studies will lead to profound insights into the pathogenesis and treatment of LUTS and related disorders.