In adults, the switch between urine storage and voiding is under direct control of the central nervous system. Afferent information from the bladder is sent to the periaqueductal gray (PAG), and from there is relayed to higher subcortical and cortical areas that will ultimately decide when it is acceptable to void. Neurons in several brain regions, including in the PAG, connect to the pontine micturition control center (Barrington’s nucleus; Bar), activation of which leads to voiding (1). If voiding is socially inappropriate, the threshold for micturition is increased and the urge to void reduced (2). We hypothesize that activation of Bar, which is essential for volitional micturition, is critically controlled by neurons in the PAG. To test this hypothesis, we mapped the different neuronal populations in the PAG related to lower urinary tract (LUT) function and tested the role of GABAergic PAG neurons for maintaining urinary continence.

In transgenic mice, adeno-associated viruses (AAVs) were injected into the lumbosacral spinal cord, PAG, or Bar. To determine the function of specific neuronal populations, we used chemogenetic and optogenetic approaches to selectively manipulate their activity and monitored changes in calcium signals as an indicator of neuronal activity. The opsins and calcium sensor-AAVs were injected unilaterally with an optical fiber placed over transduced cells or their axon terminals, while the Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were delivered bilaterally. Micturition behavior was recorded in freely behaving animals using a thermal camera. For the chemogenetic experiments, animals with DREADDs were assessed after administration of vehicle (saline, i.p.) or a DREADDs agonist (Compound 21, 1mg/kg i.p.). Additionally, photometry and optogenetic experimental mice were fit with a bladder catheter to infuse saline and measure intravesical pressure (cystometrogram, CMG).

We traced the axonal projections of excitatory neurons in the sacral spinal cord to the lateral and ventrolateral (vl) compartments of the PAG and observed activation of these neurons during bladder filling. In contrast, excitatory Bar-projecting neurons in vlPAG, as well as neurons in Bar, became active approximately 20 seconds before voiding. 
To further explore the mechanisms that regulate Bar and suppress its neuronal activation to maintain continence, we focused on the population of inhibitory neurons in vlPAG that project to Bar. Activity in these Vgat-positive GABAergic neurons also increased ~20 seconds before voiding and then dropped sharply with the void onset (Figure 1A). Chemogenetic inhibition of the vlPAGVgat neurons significantly increased the number of “leak events” (n=9, 3.8 vs. 18.7, p=0.0078), and optogenetic activation of Bar-projecting GABAergic vlPAG neurons prolonged the inter-void interval by 150-250% on CMG recordings (Figure 1B).

Increasing activity in excitatory spinal neurons during bladder filling reflects increasing afferent signaling to the PAG. Neuronal populations in the vlPAG and Bar become active specifically under conditions of high bladder pressure and are likely subject to tonic inhibitory control from higher brain areas (3).
Our findings show that GABAergic neurons in vlPAG exert strong control over Bar: loss of this inhibitory signal produces an incontinent behavioral phenotype, whereas increased inhibitory input to Bar is sufficient to delay voiding.
Together, these results suggest that removal of PAG-mediated inhibition allows activation of Bar (Figure 1A), thereby tightly regulating the timing of micturition initiation for efficient voiding.

The PAG gates the activity of Bar neurons, which connect with spinal circuitry to promote bladder contraction and EUS relaxation. Damage to the networks that control Bar activation can result in incontinence symptoms.

Verstegen AMJ, Marshall KL. From bladder to brain: How you know when it's time to go. Curr Opin Neurobiol. 2025 Dec;95:103114. PMID: 41075343.
Zare A, Jahanshahi A, Rahnama'i MS, Schipper S, van Koeveringe GA. The Role of the Periaqueductal Gray Matter in Lower Urinary Tract Function. Mol Neurobiol. 2019 Feb;56(2):920-934. PMID: 29804231.
de Rijk MM, Fernández Chadily S, Knops A, Schoutens Y, Verstegen AMJ. The periaqueductal gray and its role in the neural control of lower urinary tract function. Auton Neurosci. 2026 Mar 10;265:103413. PMID: 41819782.

Continence 19S (2026) 102494
DOI: 10.1016/j.cont.2026.102494