In this original investigation, we aimed to determine whether GABAergic periaqueductal gray (PAG) neurons are essential for the maintenance of continence during the storage phase. The PAG is a key brainstem nucleus which receives afferent information from the lower urinary tract (LUT) and facilitates communication with higher cortical and sub-cortical brain areas regarding LUT control. Through the PAG, the pontine micturition center (PMC, also known as Barrington’s nucleus) is activated, which, via spinal cord regions involved in LUT control, facilitates the switch from storage to voiding of urine. The excitation of the PMC by the PAG is known to be dependent on glutamatergic signaling. Very little research has been conducted on the possible role of GABAergic neurons in PAG for preventing activation of PMC. However, one study proposes that the dorsolateral rostral PAG may be a site that contributes to the suppression of micturition [1]. In the current study, we used chemogenetic manipulation of GABAergic PAG neurons in mice to assess whether these neurons directly influence continence and voiding behavior. We hypothesize that chemogenetic silencing of GABAergic PAG neurons decreases the inhibition of PMC and will therefore lead to increased voiding frequency and an incontinent behavioral phenotype.

We performed bilateral injections of Cre-dependent inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) (AAV8- hSyn-FLEX-hM4Di-mCherry, 40nl per hemisphere) in the PAG of Vgat-ires-Cre/+ mice (n=3). After 4 weeks (for optimal protein expression), we performed micturition video thermography (MVT) [2] recordings during which mice were volume loaded subcutaneously with 1ml Dextrose 5% in water, and voiding behavior was recorded in a behavioral cage on filter paper for a 2 hour long period using a thermal camera positioned directly above the animals. Animals were run twice after intraperitoneal injections with vehicle (NaCl 0.9%), and twice after intraperitoneal injections with a DREADDs agonist (Compound 21 (C21), 1mg/kg concentration) on sequential days. Number of continent voids and number of leaks were analyzed and quantified from the MVT recordings, and statistically compared between vehicle and C21 runs.

The number of voids per 2 hours did not statistically differ between vehicle and C21 runs (p ≥ 0.05) (Figure 1A), while the number of leakage episodes per 2 hours significantly increased after administration of C21 compared to vehicle (p=0.031) (Figure 1B). This indicates that chemogenetic inhibition of GABAergic PAG neurons induces an increase in urinary leakage events, while not significantly impacting continent voiding frequencies. See Figure 1C and 1D for representative examples of MVT video frames. Projections from GABAergic PAG neurons to the PMC were clearly visible.

In the absence of activity of GABAergic PAG neurons, mice exhibit an incontinent behavioral phenotype. These results suggest that activity of these neurons is necessary to maintain continence and prevent the involuntary loss of urine. The absence of alterations in voiding frequency underscores the specificity of involvement of GABAergic PAG neurons in the regulation of continence dynamics. The observed direct projections from GABAergic PAG neurons to PMC indicate that these PAG neurons may contribute to the maintenance of continence through direct inhibition of PMC neurons.

To our knowledge, this is the first time the effects of cell-type-specific chemogenetic manipulation on urine storage have been investigated. The findings presented here highlight the specialized role of GABAergic PAG neurons in the regulation of continence dynamics and provide valuable insights into the neural circuitry governing LUT control. The specificity of their effects for maintaining continence, coupled with their anatomical connectivity to key brain regions involved in bladder function, underscores their significance as potential targets for therapeutic interventions aimed at managing urinary incontinence. Further investigations into the precise mechanisms by which GABAergic PAG neurons modulate PMC activity will be essential for elucidating the pathophysiology of urinary dysfunction and developing targeted therapies to alleviate symptoms associated with urinary incontinence.

Numata, A., Iwata, T., Iuchi, H., Taniguchi, N., Kita, M., Wada, N., ... & Kakizaki, H. (2008). Micturition-suppressing region in the periaqueductal gray of the mesencephalon of the cat. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 294(6), R1996-R2000.
Verstegen, A. M., Tish, M. M., Szczepanik, L. P., Zeidel, M. L., & Geerling, J. C. (2020). Micturition video thermography in awake, behaving mice. Journal of neuroscience methods, 331, 108449.