The ventrolateral periaqueductal gray (VLPAG) is thought to be the main PAG column for bladder control. PAG neurons (especially VLPAG neurons) and neurons in the pontine micturition center (PMC) innervating the bladder detrusor have anatomical and functional synaptic connections. The prevailing viewpoint on neural control of the bladder is that PAG neurons receive information on the decision to void made by upstream brain regions, and consequently activate the PMC through their direct projections to initiate urination reflex. However, the exact location of the PMC-projecting VLPAG neurons, their activity in response to urination, and their whole-brain inputs remain unclear. Here, we identified the distribution of VLPAG neurons that may participate in control of the bladder or project to the PMC through retrograde neural tracing. Population Ca2⁺ signals of PMC-projecting VLPAG neurons highly correlated with bladder contractions and urination as shown by in vivo recording in freely moving animals. Using a RV-based retrograde trans-synaptic tracing strategy, morphological results showed that urination-related PMC-projecting VLPAG neurons received dense inputs from multiple urination-related higher brain areas, such as the medial preoptic area, medial prefrontal cortex, and lateral hypothalamus. Thus, our findings reveal a novel insight into the VLPAG for control of bladder function and provide a potential therapeutic midbrain node for neurogenic bladder dysfunction.

Urination, a fundamental physiological process, is intricately regulated by central neural circuits. The periaqueductal gray (PAG) is a critical hub for urination control, yet the specific neuronal subtypes involved remain undefined. Here, we identified somatostatin (SST)-expressing neurons in the lateral and ventrolateral PAG (l/vlPAG) as key regulators of urination. SST⁺ neurons in the l/vlPAG exhibited activity closely aligned with urination onset in freely moving animals. Optogenetic activation of these neurons reliably triggered urination and bladder contraction, whereas their acute inhibition at urination onset halted ongoing urination. Moreover, activation of l/vlPAG SST⁺ neurons projecting to the pontine micturition center (PMC) elicited urination and bladder contraction, effects that were absent following pelvic nerve transection. These findings reveal that l/vlPAG SST⁺ neurons regulate urination through the PAG-PMC pathway, providing new insights into bladder control and potential therapeutic targets for bladder dysfunction.

Lower urinary tract dysfunction (LUTD) is a prevalent condition characterized by symptoms such as urinary frequency, urgency, incontinence, and difficulty in urination, which can significantly impair patient's quality of life and lead to severe physiological complications. Despite the availability of diverse treatment options, including pharmaceutical and behavioral therapies, these approaches are not without challenges. The objective of this study was to enhance treatment options for LUTD by developing a wireless, battery-free device for managing bladder contractions. We designed and validated a compact, fully implantable, battery-free pulse generator using the magnetic induction coupling mechanism of wireless power transmission. Weighing less than 0.2 g and with a volume of less than 0.1 cubic centimeters, this device enables precise stimulation of muscles or neurons at voltages ranging from 0 to 10 V. Wireless technology allows real-time adjustment of key stimulation parameters such as voltage, duration, frequency, pulse width, and pulse interval. Our findings demonstrate that the device effectively controlled bladder contractions in mice when used to stimulate the Major Pelvic Ganglion (MPG). Additionally, the device successfully managed micturition in mice with bilateral transection of the pudendal nerve. In conclusion, the development of this innovative wireless pulse generator provides a safer and more cost-effective alternative to conventional battery-powered neurostimulators for bladder control, addressing the limitations of such devices. We anticipate that this novel technology will play a pivotal role in the future of electrical stimulation therapies for voiding dysfunctions.

Voluntary urination—the conscious control over urine release based on internal and external cues—is essential for social functioning and physiological homeostasis, yet the neural mechanisms underlying this behavior remain poorly understood. Effective mechanistic dissection requires genetically tractable models with robust naturalistic behaviors. However, C57BL/6J mice, despite their rich genetic toolkit, exhibit weak scent-marking unless subjected to prolonged social isolation, whereas BALB/c mice display robust marking but lack genetic accessibility. To bridge this gap, we employed F1 hybrid males (C57BL/6J × BALB/c) to combine these complementary strengths and established a 10-minute odor-driven paradigm in which both group-housed and socially isolated F1 males exhibited robust scent-marking toward estrous female urine. To facilitate efficient and accurate analysis of urine deposition from this behavioral paradigm, we trained a YOLOv8-based deep learning model for automated quantification, achieving high accuracy. Furthermore, whole-brain c-Fos mapping revealed significant activation in the prefrontal cortex, brainstem, and other key regions implicated in regulating this voluntary voiding behavior. Thus, our study establishes a novel behavioral paradigm for odor-induced voluntary urination, providing a genetically accessible model system to dissect the neural circuits governing physiological voiding and neurogenic lower urinary tract dysfunction.

Urination, a vital and conserved process of emptying urine from the urinary bladder in mammals, requires precise coordination between the bladder and external urethral sphincter (EUS) that is tightly controlled by a complex neural network. However, the specific subpopulation of neurons that accounts for such coordination remains unidentified, limiting the development of target-specific therapies for certain urination disorders, e.g., detrusor-sphincter dyssynergia. Here, we find that cells expressing estrogen receptor 1 (ESR1 ⁺ ) in the pontine micturition center (PMC) initiate voiding when activated and suspend ongoing voiding when suppressed, each at 100% reliability. Transection of the pelvic nerve does not impair PMC ^ESR1+ neurons’ control of the EUS via the pudendal nerve, whereas transection of the pudendal nerve does not impair their control of the bladder via the pelvic nerve. Anatomically, PMC ^ESR1+ neurons consist of three distinct spinal-projection-based subpopulations: one targeting the sacral parasympathetic nucleus (SPN), one innervating the dorsal gray commissure (DGC), and a third that projects to both regions, thereby enforcing the coordination of bladder contraction and sphincter relaxation in a rigid temporal sequence. Thus, we identify a cell type in the brainstem that controls the bladder-urethra coordination for urination.