Hypothesis / aims of study
This is the first study to identify PMC neuron subpopulations critical for micturition control, and to reveal neuron-specific representation of the temporal sequence underlying PMC neuron activation.
A significant component of lower urinary tract symptoms (LUTS) is due to failure of nervous control of bladder function or failure of neural pathways to compensate for bladder dysfunction. How the brain controls bladder filling and voiding remains however poorly understood. Glutamatergic neurons in the pontine micturition center (PMCVglut2) and subsets of these, expressing corticotropin releasing hormone (PMCCrh) or estrogen receptor (PMCEsr), provide descending control of detrusor and sphincter function [1, 2].
Our hypotheses are 1) The population of Vglut2, but not Crh-expressing neurons in PMC is necessary for control of bladder function, and 2) neurons in select brain sites upstream of PMC; the lateral hypothalamic area (LHA), the periaqueductal grey (PAG) and the medial preoptic area (MPOA), cause activation or inhibition of this brain micturition control center.
Study design, materials and methods
Micro-injections of adeno-associated viruses (AAVs) were placed into anatomically defined regions of the mouse brain or spinal cord, enabling highly selective expression of proteins in target neuron populations.
We used a novel non-invasive void-spot assay; video thermography, to record mouse behavior in awake-behaving mice and combined it with optogenetic and chemogenetic stimulation of neurons, conditional neuron ablation, brain site-specific gene knockout, and monitoring neural population activity.
Experimental manipulations were also performed in conjunction with urodynamic recordings of bladder pressure in behaving mice and under anesthesia. Lastly, we applied electrophysiological techniques to characterize the functional contribution of neurons that provide afferent regulation of the PMC.
While injury or inhibition of the PMC region prevents voiding and results in urinary retention, we found that mice with complete bilateral genetic lesion of the PMCCrh neuron population still exhibited relatively normal voiding behaviors. Furthermore, optogenetic stimulation of this cell group produced only modest responses, in sharp contrast to light stimulation of PMCVglut2 neurons which yielded reliable, immediate and strong bladder contractions.
We did observe an increase in baseline bladder pressure and a decreased void contraction amplitude while chemogenetically activating the Crh-expressing neurons.
In parallel studies we explore how bladder filling is sensed in the brain and study how bladder afferent sensory signals to the brain eventually regulate PMC neuron activity. We focused on axonal projections from neurons in the LCP (lateral collateral pathway), PAG and LHA regions because PAG receives abundant afferent input from the sacral level of the spinal cord, and LHA likely coordinates bladder function with other homeostatic mechanisms. Firstly, using Channelrhodopsin assisted circuit mapping (CRACM) we demonstrated synaptic connections between PAG or LHA neurons and postsynaptic PMC neurons. Secondly, optogenetic stimulation of PAGVglut2 -> PMC and LHAVglut2 -> PMC terminals led to prompt detrusor contraction and voiding. Thirdly, through recording of Ca2+-dependent fluorescence changes, -indicative of neural activity- in distinct neuron populations, we found that activity in axon terminals to the PMC preceded increase in bladder pressure during conscious CMGs.
Interpretation of results
Our deletion and gene knockout studies and in vivo monitoring of neural activity, suggest that PMCCrh neurons are not driving detrusor activity. PMCVglut2 neurons on the contrary, appear to function as an on-off switch for micturition as their spontaneous activity precedes and is correlated with detrusor activity, and activating these neurons immediately triggers voiding. While Crh remains a useful marker for a subpopulation of PMCVglut2 neurons, they represent only a portion of the neurons involved, and neither Crh peptide expression nor the neurons that express it are necessary to control bladder function. Activity in Crh neurons can possibly contribute to bladder control by increasing detrusor tone and therefore may be an appropriate therapeutic target for suppressing detrusor activity, as would be desired in the setting of an overactive bladder.
We identified a network of afferent neurons, spanning several brain regions that directly modulates different PMC neuron populations to initiate voiding behavior. Pharmacological studies have previously suggested that modulation of midbrain circuitry can alter the bladder volume threshold for micturition  and that the PAG may thereby ‘gate’ activity of PMC neurons in control of micturition. In our effort to tease out how PMC-afferent neurons direct the PMC to regulate bladder function and EUS activity, we demonstrated functional and facilitory roles of PAG and hypothalamic afferents for voiding and modulating continence.
Taken together, and using a novel, non-invasive assay for analyzing micturition behavior in awake, behaving mice, this study begins to unravel the molecular identity of PMC neurons with functional roles and ‘PMC-regulating’ neurons, in controlling urinary voiding and continence. We will further explore if spinal input to the PAG can inhibit the micturition reflex and, if so, which PAG cell populations are postsynaptic targets of this spinal input.
This information helps us understand how forebrain, brainstem and spinal inputs converge to control bladder filling and voiding, and allows more detailed studies of 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.