Identifying Brain Networks Controlling Micturition and Continence in Mouse

Anne M.J Verstegen; Natalie Klymko; Joel C Geerling; John Mathai; Veronique G VanderHorst; Patrick M Fuller; Mark L Zeidel

doi:10.1096/fasebj.2018.32.1_supplement.734.3

Objective Lower urinary tract symptoms (LUTS) are extremely common and enormously debilitating. A significant component of LUTS is due to failure of nervous control of bladder function, or otherwise failure of neural pathways to compensate for bladder dysfunction. It remains however unclear how the brain controls bladder filling and voiding and how the micturition reflex is inhibited at times when voiding is undesirable. Prior studies have shown that the pontine micturition center (PMC, aka Barrington's nucleus) directly controls voiding. PMC neurons provide direct innervation of sacral spinal cord nuclei that control bladder contraction and sphincter relaxation. Here we identify the neurochemical identity of PMC neurons that contribute to micturition control as well as identify a network of afferent neurons, spanning several forebrain and brainstem regions, which directly modulates these PMC neurons to influence voiding behavior. Methods Micro‐injections of adeno‐associated viruses (AAVs), modified Rabies virus, Cholera Toxin b (CTb) or Diphtheria Toxin a (dtA), were placed into anatomically defined regions of the mouse brain, enabling highly selective expression of proteins in target neuron populations. A novel non‐invasive assay; micturition video thermography (MVT), was used to track voiding behavior in awake‐behaving mice. MVT was combined with telemetric measurement of bladder pressure, urethral sphincter EMG and optogenetic stimulation or inhibition. Following MVT these manipulations were repeated whilst recording cystometry, in either the awake or anesthetized state. Results Stimulating PMCCRH neurons using Gq DREADDs produced urinary frequency in awake‐behaving mice and on the anesthetized cystometrogram (CMG). Conversely, selective ablation of neurons in the PMC using dtA resulted in urinary retention, and delayed or eliminated the CMG voiding reflex. To identify the input connections controlling PMC neurons, we used CTb and modified Rabies virus. Afferents to PMCCRH neurons were found in the vlPAG, the preoptic area, the lateral hypothalamic area, and other sites. Monosynaptic connectivity was confirmed for select sites using electrophysiological recordings. To test the functional consequence of afferent neuron activity, we light‐stimulated their axon terminals within the PMC and observed micturition events within seconds (glutamatergic/excitatory afferents) or increased inter‐void intervals (GABAergic/inhibitory afferents). Finally, we recorded Ca2+‐dependent fluorescence changes in distinct neuron populations to study the timing of neuronal activity with respect to detrusor contraction and voiding. Conclusions Our results taken together, identify a network of neurons that can control urinary voiding and continence by acting on PMC neurons. We have demonstrated necessity of select afferent sites for voiding, and the effects of removing their modulatory input for continence. This information helps us with a detailed understanding of how forebrain, brainstem and spinal inputs converge to control bladder filling and voiding, and hence the neurologic mechanisms of LUTS in mice and humans. As we define the brain pathways controlling normal micturition, we will evaluate how they are altered in settings of abnormal micturition, such as brain degeneration and prostatic enlargement. Support or Funding Information National Institutes of Health (NIH); R01 DK113030‐01 This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.

Identifying Brain Networks Controlling Micturition and Continence in Mouse

View Online

Abstract

Details

Metrics