Abstract
Bladder-innervating primary sensory neurons mediate reflex-driven bladder function under normal conditions, and contribute to debilitating bladder pain and/or overactivity in pathological states. The goal of this study was to examine the respective roles of defined subtypes of afferent neurons in bladder sensation and function in vivo via direct optogenetic activation. To accomplish this goal, we generated transgenic lines that express a Channelrhodopsin-2-eYFP fusion protein (ChR2-eYFP) in two distinct populations of sensory neurons: TRPV1-lineage neurons (Trpv1Cre;Ai32, the majority of nociceptors) and Nav1.8+ neurons (Scn10aCre;Ai32, nociceptors and some mechanosensitive fibers). In spinal cord, eYFP+ fibers in Trpv1Cre;Ai32 mice were observed predominantly in dorsal horn (DH) laminae I-II, while in Scn10aCre;Ai32 mice they extended throughout the DH, including a dense projection to lamina X. Fiber density correlated with number of retrogradely-labeled eYFP+ dorsal root ganglion neurons (82.2% Scn10aCre;Ai32 vs. 62% Trpv1Cre;Ai32) and degree of DH excitatory synaptic transmission. Photostimulation of peripheral afferent terminals significantly increased visceromotor responses to noxious bladder distension (30–50 mmHg) in both transgenic lines, and to non-noxious distension (20 mmHg) in Scn10aCre;Ai32 mice. Depolarization of ChR2+ afferents in Scn10aCre;Ai32 mice produced low- and high-amplitude bladder contractions respectively in 53% and 27% of stimulation trials, and frequency of high-amplitude contractions increased to 60% after engagement of low threshold (LT) mechanoreceptors by bladder filling. In Trpv1Cre;Ai32 mice, low-amplitude contractions occurred in 27% of trials before bladder filling, which was pre-requisite for light-evoked high-amplitude contractions (observed in 53.3% of trials). Potential explanations for these observations include physiological differences in the thresholds of stimulated fibers and their connectivity to spinal circuits.
Highlights
The urinary bladder is innervated by primary sensory neurons with somata in the dorsal root ganglia (DRG) giving rise to lightly-myelinated (Aδ) or unmyelinated (C) axons, the majority of which are polymodal mechanosensors (Sengupta and Gebhart, 1994a; Su et al, 1997; Shea et al, 2000)
In Trpv1Cre;Ai32 mice (Figure 1I), the heaviest distribution of eYFP+ terminals was in the most superficial layers of the dorsal horn (DH) and to a lesser degree in lamina X with sparse projections to parasympathetic preganglionic neurons in the sacral parasympathetic nucleus (SPN; recent articles argue that these cells have more in common with neurons in the sympathetic intermediolateral cell column (IML) found at T1-L2 spinal cord levels (Espinosa-Medina et al, 2016, 2017))
We demonstrate successful application of optogenetics to in vivo modulation of bladder sensation as it relates to both nociception and voiding
Summary
The urinary bladder is innervated by primary sensory neurons with somata in the dorsal root ganglia (DRG) giving rise to lightly-myelinated (Aδ) or unmyelinated (C) axons, the majority of which are polymodal mechanosensors (Sengupta and Gebhart, 1994a; Su et al, 1997; Shea et al, 2000). Additional studies that have manipulated the expression or function of Nav1.8, a TTX-resistant voltage-gated sodium channel expressed by a combination of myelinated and unmyelinated afferents including putative C-low-threshold mechanoreceptors (CLTMs; Shields et al, 2012) have demonstrated changes in both normal and pathological bladder sensation (Yoshimura et al, 2001; Laird et al, 2002). It is unclear whether changes in bladder sensation observed in these studies are due to the loss of specific channel activity vs altered function in the afferent populations in which they are expressed
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