Dynamic Expression of Serotonin Receptor 5-HT3A in Developing Sensory Innervation of the Lower Urinary Tract

Abstract

Sensory afferent signaling is required for normal function of the lower urinary tract (LUT). Despite the wide prevalence of bladder dysfunction and pelvic pain syndromes, few effective treatment options are available. Serotonin receptor 5-HT3A is a known mediator of visceral afferent signaling and has been implicated in bladder function. However, basic expression patterns for this gene and others among developing bladder sensory afferents that could be used to inform regenerative efforts aimed at treating deficiencies in pelvic innervation are lacking. To gain greater insight into the molecular characteristics of bladder sensory innervation, we conducted a thorough characterization of Htr3a expression in developing and adult bladder-projecting lumbosacral dorsal root ganglia (DRG) neurons. Using a transgenic Htr3a-EGFP reporter mouse line, we identified 5-HT3A expression at 10 days post coitus (dpc) in neural crest derivatives and in 12 dpc lumbosacral DRG. Using immunohistochemical co-localization we observed Htr3a-EGFP expression in developing lumbosacral DRG that partially coincides with neuropeptides CGRP and Substance P and capsaicin receptor TRPV1. A majority of Htr3a-EGFP+ DRG neurons also express a marker of myelinated Aδ neurons, NF200. There was no co-localization of 5-HT3A with the TRPV4 receptor. We employed retrograde tracing in adult Htr3a-EGFP mice to quantify the contribution of 5-HT3A+ DRG neurons to bladder afferent innervation. We found that 5-HT3A is expressed in a substantial proportion of retrograde traced DRG neurons in both rostral (L1, L2) and caudal (L6, S1) axial levels that supply bladder innervation. Most bladder-projecting Htr3a-EGFP+ neurons that co-express CGRP, Substance P, or TRPV1 are found in L1, L2 DRG, whereas Htr3a-EGFP+, NF200+ bladder-projecting neurons are from the L6, S1 axial levels. Our findings contribute much needed information regarding the development of LUT innervation and highlight the 5-HT3A serotonin receptor as a candidate for future studies of neurally mediated bladder control.

Serotonin Receptor 5-HT3A Affects Development of Bladder Innervation and Urinary Bladder Function

“…In our previous study examining expression of Htr3a in developing dorsal root ganglia (Ritter and Southard-Smith, 2017 ), we also observed transgene fluorescence in the autonomic pelvic ganglia during fetal stages. To further characterize the expression of this serotonin receptor in developing autonomic LUT innervation, we evaluated the distribution and cell type specificity of Htr3a in Htr3a- EGFP transgenic tissues (Tg( Htr3a- EGFP)DH30Gsat/Mmnc, hereafter referred to as Htr3a- EGFP) by immunohistochemistry and confocal microscopy.…”

“…The sensation of bladder filling and muscle stretch is primarily detected by sensory afferent neurons in the dorsal root ganglia (de Groat and Yoshimura, 2015 ). During our prior analysis of Htr3a expression in the developing dorsal root ganglia, we documented that many bladder-innervating sensory neurons expressing Htr3a also express neuropeptide Calcitonin Gene Related Peptide (CGRP) and mechanosensory marker Neurofilament 200 (NF200) (Ritter and Southard-Smith, 2017 ). We were interested to see if these populations would be affected by loss of Htr3a .…”

“…It is also possible that expression of CGRP in neuronal fibers within the bladder changes over postnatal development. Our prior study examining gene expression profiles in developing dorsal root ganglia indicates that CGRP expression is somewhat dynamic through development (Ritter and Southard-Smith, 2017 ), but additional work is needed to explore these possibilities further.…”

“…In an effort to characterize patterns of gene expression in the development of LUT sensory innervation, we previously examined expression of the serotonin receptor 5-HT3A (encoded by the Htr3a gene) in developing dorsal root ganglia (Ritter and Southard-Smith, 2017 ). In these analyses we also observed pronounced expression of Htr3a in fetal mouse pelvic ganglia that supply autonomic innervation to the bladder.…”

Functional Clustering of Neuronal Signals with FMM Mixture Models

“…The function in the nervous system of the different kinds of GABAergic neurons is radically different, so the distinction of their signals is crucial. In VGA, signals from three types of neurons are differentiated: Pvalb-positive neurons, key in learning and memory processes, as detailed in [13]; Vip-positive neurons, related to circadian rhythms [24]; and Htr3a-positive neurons, related to bladder dysfunction [34]. In addition, the methodology can be adapted to cluster multichannel spike recordings [49], combining the information of FMM models for each channel, or with multivariate FMM models similar to those introduced in [39] to analyze ECG multilead signals.…”