Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder

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Abstract

We have investigated whether bladder afferent neurons are likely to be targets for circulating estrogens by mapping estrogen receptor (ER) distribution in lumbosacral dorsal root ganglia (DRG) of adult female rats. Sensory neurons innervating either the detrusor or trigone regions were identified by application of fluorescent retrograde tracer dyes to the bladder wall. Labelled neurons were classified by their immunoreactivity for either type of ER (ERα or ERβ) and further compared with subpopulations of neurons containing substance P, calcitonin gene-related peptide and vanilloid receptor (a marker of polymodal nociceptors). Both ER types were expressed in numerous sensory neurons of either upper lumbar (L1/L2) or lower lumbar/sacral (L6/S1) ganglia and there was almost complete coexpression of ERα and ERβ. ER-positive neurons were mainly small–medium size (18–25-μm diameter), indicating that they may be nociceptors and/or supply visceral targets. Most bladder-projecting neurons expressed ERs and the majority of these also expressed neuropeptides or vanilloid receptor. Afferent neurons supplying detrusor and trigone regions had similar immunohistochemical features. About a third of the bladder-projecting neurons expressed both ER and vanilloid receptor, suggesting a mechanism by which estrogens could influence bladder pain. The prevalence of different chemical classes of ER-positive bladder-projecting neurons was reflected throughout the entire population of neurons in dorsal root ganglia of these spinal levels, suggesting that neurons supplying other pelvic visceral targets may have similar chemical profiles. These results suggest that many functional classes of sensory neurons innervating the lower urinary tract are likely to be targets for circulating estrogens, including many nociceptor neurons. The coexistence of ERα and ERβ suggests a broad range of potential mechanisms by which estrogens may exert their genomic effects in this system.

Introduction

Estrogens have powerful effects on numerous brain regions, and appear to regulate a diverse array of behaviours in female animals. The best characterised of these relate closely to reproduction, such as the lordosis response and suckling, but there is growing recognition of estrogen-sensitive areas in the central nervous system that appear to have little or nothing to do with reproductive behaviours Woolley, 1999, Wise et al., 2001, Pfaff et al., 2002.

Estrogen receptors (ERs) have also recently been localised in various components of pelvic autonomic reflex circuits Williams and Papka, 1996, Papka et al., 1997, Papka et al., 1998, Papka et al., 2001, and both physiological and anatomical studies have indicated that estrogens may influence some sensory and autonomic nerves innervating the pelvic viscera Sato et al., 1989, Brauer et al., 1992, Brauer et al., 1999, Bradshaw et al., 1999, Zoubina et al., 2001, Zoubina and Smith, 2001. The focus of most studies has been innervation of the uterus, however, there is a growing body of data that suggest that estrogen effects on the pelvic viscera are unlikely to be restricted to the reproductive organs. For example, changes in estrogen exposure can influence bladder innervation or activity Sato et al., 1989, Shimonovitz et al., 1997, Keast and Saunders, 1998, Diep and Constantinou, 1999, Ratz et al., 1999, Blakeman et al., 2000, Johnson and Berkley, 2002. Hormonal status also appears to influence development or severity of various pelvic visceral pain states, including the chronic inflammatory bladder condition, interstitial cystitis Berkley, 1997, Wesselmann et al., 1997, Sant and Theoharides, 1999, Hanno and Sant, 2001. We propose that the micturition reflex may therefore be an estrogen-sensitive nerve circuit.

The first aim of our study was to determine whether sensory neurons supplying different functional regions of the bladder wall are likely to be targets for estrogens by localising estrogen receptors (ERα and ERβ) in lumbosacral dorsal root ganglion neurons that project to either the bladder trigone or detrusor of the adult female rat. These have been identified by prior application of small volumes of fluorescent retrograde tracer dyes to the wall of each bladder region. Our second aim was to determine which particular functional or chemical class(es) of bladder afferent neurons are likely to be influenced by estrogens, using well-characterised markers of bladder afferent neurons in conjunction with ER immunostaining. Specifically, coexpression of ER with substance P (SP), calcitonin gene-related peptide (CGRP), or a marker of polymodal nociceptor neurons, VR-1 (vanilloid receptor-1), was assessed. These patterns of coexpression in bladder afferent neurons were further compared with the entire population of lumbosacral visceral afferent neurons to determine if bladder neurons were likely to form a chemically unique functional subclass of ER-expressing neurons, or if the pattern of ER-expression in bladder afferents was likely to be similar to other populations of pelvic visceral afferents.

Section snippets

General surgical and tissue sampling procedures

All procedures on animals followed the “Australian code of practice for the care and use of animals for scientific purposes” (NHMRC) and UNSW ethical guidelines. Ten adult female outbred Wistar rats (180–320 g) were used.

Bladder-projecting neurons were identified as described previously (Keast et al., 1989) by injection of fluorescent retrograde tracer dyes into the bladder wall, Fast Blue (2% in distilled water; Sigma Aldrich, Castle Hill, Australia) and Fluorogold (4% in distilled water;

General properties of ERα- and ERβ-immunoreactive neurons

ER-immunoreactivity is typically indicated by a bright nucleus (Fig. 1a,c,d), as the majority of receptors are localised to the nuclear compartment in the presence of circulating estrogens (Hager et al., 2000). In the current study, the intensity of ERα or ERβ-immunoreactive nuclei varied considerably between neurons (Fig. 2a–d). In control sections (sections where primary antibody against ER was omitted or sections treated with pre-absorbed antibody), no nuclear staining was observed, such

Discussion

Changes in estrogen exposure can influence bladder innervation or activity Sato et al., 1989, Shimonovitz et al., 1997, Keast and Saunders, 1998, Diep and Constantinou, 1999, Ratz et al., 1999, Blakeman et al., 2000, Johnson and Berkley, 2002. An effect of circulating estrogens on micturition threshold, particularly when the bladder is inflamed, has also been suggested. (Johnson and Berkley, 2002). This is the first study to identify estrogen receptor expression in primary afferent neurons

Acknowledgments

This work was supported by the National Health and Medical Research Council (Australia) by Project Grant #990034 and Fellowship Grant #157253 (JRK) and The Swedish Cancer Fund (JAG).

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