In today’s aging society, which has become a global concern, the management of lower urinary tract dysfunction (LUTD) is increasingly important. LUTD becomes more prominent with aging and causes various lower urinary tract symptoms (LUTS), leading to reduced quality of life (QoL). Age-related LUTS are often refractory to treatment, likely due to the still poorly understood pathophysiology of age-related LUTD. We hypothesized that age-related functional changes in the Barrington's nucleus, a pontine center for micturition, are a key mechanism underlying age-associated LUTD. To test this, we conducted a comprehensive functional analysis of the Barrington's nucleus in aged and young mice.

In this study, we focused on CRH-positive neurons (Barr-CRH) within the Barrington’s nucleus, which represent the majority of neurons in this region. Using stereotaxic surgery under isoflurane anesthesia, we injected Cre-dependent adeno-associated viruses (AAVs) into the Barrington’s nucleus of male CRH-ires-Cre mice from aged (100–107 weeks) and young (10–12 weeks) groups. These AAVs selectively expressed either the light-sensitive opsin Channelrhodopsin-2 (ChR2) or the neurotoxin DTA (diphtheria toxin A fragment) in Barr-CRH neurons (Figure1A).

Four weeks after ChR2 injection, cystometric bladder pressure recordings were performed under urethane anesthesia. Barr-CRH neurons were optogenetically stimulated with blue LED light (465 nm, 20 ms pulses, 20 Hz, train duration 5sec). For DTA-injected mice, a void spot assay (VSA) was conducted to assess spontaneous voiding behavior, followed by cystometry 4 weeks after injection. VSA was performed during the daytime for 4 hours without water loading. Finally, in situ hybridization (ISH) was performed on perfusion-fixed brains from both DTA-injected and untreated mice to quantify CRH-positive cells in the Barrington's nucleus of aged and young mice.

In aged mice injected with ChR2, immunohistology confirmed that ChR2 was specifically introduced into the Barrington’s nucleus (Figure1B). Cytometric analysis revealed significantly increased maximum voiding pressure, baseline pressure, bladder capacity, and both the frequency and amplitude of non-voiding contractions (NVCs) compared to young mice (Table1, Figure1C). Upon optogenetic stimulation of Barr-CRH neurons, the evoked bladder contraction response rate was significantly higher in the aged group (young: 42.3% [29.1–54.8] vs. aged: 85.2% [78.2–91.1], P =0.0022, Figure1D). 

Prior to DTA injection, there was no difference in voiding behavior between aged and young mice in the VSA. However, following DTA injection, only aged mice showed a significant reduction in voiding frequency and volume 2 weeks after injection (Figure2A). At 4 weeks post-injection, bladder cystometry revealed marked loss of normal micturition patterns or urinary retention only in aged mice (Figure2B).

ISH analysis confirmed a reduction in CRH mRNA-positive cells in both aged and young mice following DTA injection. In contrast, among untreated mice, no significant difference was observed in the number of CRH-positive neurons between age groups (Figure2C).

The enhanced response rate to optogenetic stimulation of Barr-CRH neurons suggests increased excitability of Barr-CRH neurons themselves in aged mice or increased responsiveness of downstream bladder pathways. Furthermore, ablation of Barr-CRH neurons with DTA significantly impaired micturition reflexes only in aged mice, indicating a greater reliance on Barr-CRH in aged micturition circuits compared to young mice. As ISH revealed no difference in the number of CRH-positive neurons between groups, the increased dependence on Barr-CRH in aged mice may be attributed to qualitative functional changes rather than quantitative differences.

Compared to young mice, aged mice appear to exhibit a strong dependency on Barr-CRH neurons for normal micturition. This finding suggests that functional alterations in Barr-CRH may contribute to age-related lower urinary tract symptoms (LUTS).