Differential regulation of bladder pain and voiding function by sensory afferent populations revealed by selective optogenetic activation

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

Urothelial bladder afferents selectively project to L6/S1 levels and are more peptidergic than those projecting to the T13/L1 levels in female rats

This neuroanatomical study in four, adult, Sprague-Dawley female rats quantified the number of Urothelial (labeled by intravesical DiI dye administration) and Non-Urothelial (labeled by intraparenchymal injection of Fast blue dye) bladder primary afferent neurons (bPANs) located in the T13, L1, L6 and S1 dorsal root ganglia. Additional immunohistochemical labeling using antibodies to detect either Substance P or CGRP further characterized the bPAN samples as peptidergic or non-peptidergic. Cell counts indicated that Urothelial bPANs were more common at the L6/S1 levels and more likely to be identified as peptidergic when compared with bPANs characterized at T13/L1 levels and with Non-Urothelial bPANs. These studies provide additional evidence that at least two distinct neuronal populations, with differing localization of sensory terminals, differing peptide content, and differing projections to the central nervous system, are responsible for bladder sensation

Optimization of a pain model: effects of body temperature and anesthesia on bladder nociception in mice.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating urological condition that is resistant to treatment and poorly understood. To determine novel molecular treatment targets and to elucidate the contribution of the nervous system to IC/BPS, many rodent bladder pain models have been developed. In this study we evaluated the effects of anesthesia induction and temperature variation in a mouse model of bladder pain known as urinary bladder distension (UBD). In this model compressed air is used to distend the bladder to distinct pressures while electrodes record the reflexive visceromotor response (VMR) from the overlying abdominal muscle. Two isoflurane induction models are commonly used before UBD: a short method lasting approximately 30 minutes and a long method lasting approximately 90 minutes. Animals were anesthetized with one of the methods then put through three sets of graded bladder distensions. Distensions performed following the short anesthesia protocol were significantly different from one another despite identical testing parameters; this same effect was not observed when the long anesthesia protocol was used. In order to determine the effect of temperature on VMRs, animals were put through three graded distension sets at 37.5 (normal mouse body temperature), 35.5, and 33.5°C. Distensions performed at 33.5 and 35.5°C were significantly lower than those performed at 37.5°C. Additionally, Western blot analysis revealed significantly smaller increases in spinal levels of phosphorylated extracellular-signal regulated kinase 2 (pERK2) following bladder distension in animals whose body temperature was maintained at 33.5°C as opposed to 37.5°C. These results highlight the significance of the dynamic effects of anesthesia on pain-like changes and the importance of close monitoring of temperature while performing UBD. For successful interpretation of VMRs and translation to human disease, body temperature should be maintained at 37.5°C and isoflurane induction should gradually decrease over the course of 90 minutes

Visceromotor responses (VMR) from urinary bladder distension (UBD).

<p>(A) Schematic of UBD setup. Compressed air is delivered into bladder via urethral catheter. During distensions, electrodes in abdominal muscle record EMG. Temperature is maintained throughout the procedure using a battery operated heating pad and overhead lamp. (B) Example EMG traces during UBD. As pressure increases, electrical output from abdominal muscles increases congruently. (C) Example VMR from one complete set of distensions. During each set, the bladder is distended three times at each pressure. VMRs are normalized with 20 s pre-distension interval then averaged for each pressure.</p

Animal's body temperature affects VMR.

<p>Following long anesthesia, animals (n = 6) were placed through three sets of graded distensions at 37.5, 33.5, and 35.5°C. Two-way ANOVA yielded a significant main effect of temperature (***p<0.0001), but not of pressure (p = 0.4536). Bonferroni's posttest revealed differences between 37.5 and 33.5°C at 60 mmHg and 75 mmHg (***p<0.001), as well as differences between 37.5 and 35.5°C at 60 mmHg (***p<0.001) and 75 mmHg (*p<0.05), and differences between 35.5 and 33.5°C at 75 mmHg (*p<0.05).</p