Age-related reductions in the excitability of phasic dorsal root ganglion neurons innervating the urinary bladder in female rats

Highlights

• Bladder primary sensory neurons are divided into phasic and tonic neurons based on the responses to super-threshold stimuli.

• Phasic and tonic neurons have distinct electrical properties.

• No age-related change in excitability was found in tonic bladder sensory neurons.

• An age-related reduction in excitability was observed in phasic bladder sensory neurons.

Abstract

Previous studies have revealed an impairment in bladder sensory transduction in aged animals. To examine the contributions of electrical property changes of bladder primary afferents to this impairment, we compared the electrical properties of dorsal root ganglion (DRG) neurons innervating the bladder among young (3 months), middle-aged (12 months), and old (24 months) female rats. The DRG neurons were labeled using axonal tracing techniques. Whole-cell current-clamp recordings of small and medium-sized neurons were performed to assess their passive and active properties. Two patterns of firing were identified based on responses to super-threshold stimuli (1.5, 2.0, 2.5, and 3.0 × rheobase): tonic neurons fired more action potentials (APs), whereas phasic neurons fired only one AP at the onset of stimulus. Tonic neurons were smaller and had a slower rate of AP rise, longer AP duration, more depolarized voltage threshold, and greater rheobase than phasic neurons. In phasic neurons, there was an age-associated increase in voltage threshold and an increase of rheobase (P < 0.05), suggesting an age-related decrease in excitability. In addition, both middle-aged and old rats had longer AP durations and slower rates of AP rise than young rats (P < 0.05). In tonic neurons, old rats had a greater AP overshoot and greater rate of AP rise, but no age-associated changes were identified in any other electrical properties. Our results suggest that the electrical properties of tonic and phasic bladder afferents are differentially altered with aging. A decrease in excitability may contribute to age-related reductions in bladder sensory function.

Introduction

Aging dramatically affects bladder sensory function. The prevalence of overactive bladder syndrome significantly increases with age, both in women and men (Kraus et al., 2010, Yoshida, 2009). Metabolic cage studies on aged rats or mice have reported a significant increase in the frequency of voiding (Chun et al., 1989, Daly et al., 2014). However, clinical research has also revealed an age-related increase in bladder capacity in women (Collas and Malone-Lee, 1996, Pfisterer et al., 2006, Pfisterer et al., 2007, Zimmern et al., 2014). Age-related reductions in bladder volume sensitivity, which exhibit as increased inter-void intervals, have been observed in both the aged rat and mouse (Hotta et al., 1995, Lai et al., 2007, Smith et al., 2012). In support of these reports, the direct recording of pelvic nerve afferents has demonstrated either reduced or increased sensitivity to bladder volume in aged rats or mice (Aizawa et al., 2011, Daly et al., 2014, Hotta et al., 1995), which suggests an impairment in peripheral sensory transduction.

Bladder peripheral sensory pathways detect volume or distension changes during bladder filling, and transmit the information to the central nervous system. Bladder sensory innervation, which is carried in the pelvic and hypogastric nerves, consists of myelinated Aδ fibers and unmyelinated C fibers and originates from cell bodies located in the lumbosacral dorsal root ganglia (DRG). It is generally considered that Aδ bladder afferents respond to low-threshold mechanical stimuli, such as bladder distension, and play a major role in the initiation of the micturition reflex. In contrast, C-fiber afferents do not respond to mechanical stimuli and have thus been termed “silent C-fibers”; they are activated by chemical irritation or cold stimulation of the bladder, and can in turn facilitate the micturition reflex (de Groat and Yoshimura, 2009, Yoshimura and de Groat, 1999).

DRG neurons innervating the bladder have heterogeneous electrical properties, which have been extensively investigated (Dang et al., 2008, Sculptoreanu and de Groat, 2007, Yamane et al., 2007, Yoshimura and de Groat, 1999, Zhong et al., 2003). In general, they are divided into two populations based on the electrical characteristics of their action potentials (APs) (Sculptoreanu and de Groat, 2007, Yoshimura et al., 1996, Yoshimura et al., 2003, Yoshimura and de Groat, 1999). One population of bladder afferent neurons exhibits high-threshold, long-duration APs with an inflection on the repolarization phase. These neurons are small in size and have APs that are resistant to the application of tetrodotoxin (TTX), a Na⁺ channel blocker. They account for more than 70% of all bladder afferent neurons. The other population is larger in size, and exhibits low-threshold, short-duration APs that are reversibly blocked by TTX. When stimulated with long-duration depolarizing current pulses, the population of neurons with TTX-resistant spikes usually exhibits a phasic firing pattern (i.e., one or two spike generation), while the neurons with TTX-sensitive spikes have a tonic firing pattern (i.e., multiple spikes).

Studies of age-associated changes of bladder afferents are limited. Some morphological studies have suggested that there is an age-dependent reduction in the number of C-fiber bladder afferents (Mohammed and Santer, 2002, Nakayama et al., 1998). Direct recordings of the firing of pelvic nerve afferents has revealed either decreased or increased afferent activity in response to bladder filling in aged rats or mice (Daly et al., 2014, Hotta et al., 1995). Changes in bladder afferent sensitivity may reflect alterations in transducer channels, such as TRPV1 and P2X3, on bladder afferents (de Groat and Yoshimura, 2009, Wang et al., 2006, Wen et al., 2018). Moreover, changes in bladder afferent sensitivity are also likely to reflect changes in the electrical properties of bladder afferent neurons. To explore the latter possibility in the present study, we used whole-cell patch-clamp recording in combination with axonal tracing techniques, to compare the passive and active electrical properties of DRG neurons innervating the bladder among young (3 months), middle-aged (12 months), and old (24 months) female rats.