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

Autonomic Neuroscience: Basic and Clinical 105 (2003) 90 – 100 www.elsevier.com/locate/autneu

Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder ˚ ke Gustafsson b, Janet R. Keast a,* Haley L. Bennett a, Jan-A a

Prince of Wales Medical Research Institute, University of New South Wales, Sydney NSW 2052, Australia b Department of Medical Nutrition, Centre for Biotechnology, Karolinska Institute, Stockholm, Sweden Received 9 December 2002; received in revised form 11 February 2003; accepted 20 February 2003

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 (ERa or ERh) 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 ERa and ERh. ER-positive neurons were mainly small – medium size (18 – 25-Am 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 ERa and ERh suggests a broad range of potential mechanisms by which estrogens may exert their genomic effects in this system. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Visceral pain; Micturition reflex; Autonomic reflex; Neuropeptide; Tachykinin; Capsaicin

1. 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 estrogensensitive 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 * Corresponding author. Prince of Wales Medical Research Institute, Barker Street, Randwick NSW 2031, Australia. Tel.: +61-2-9382-2943; fax: +61-2-9382-2722. E-mail address: [email protected] (J.R. Keast).

(Williams and Papka, 1996; Papka et al., 1997, 1998, 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, 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;

1566-0702/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-0702(03)00044-4

H.L. Bennett et al. / Autonomic Neuroscience: Basic and Clinical 105 (2003) 90–100

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 (ERa and ERh) 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 generelated 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 ERexpression in bladder afferents was likely to be similar to other populations of pelvic visceral afferents.

2. Materials and methods 2.1. 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; Fluorochrome, Englewood, CO, USA) under anaesthesia (60 mg/kg ketamine and 10 mg/kg and xylazine i.p.). The bladder was exposed via an abdominal incision and bilateral injections of dyes were made into either the trigone or detrusor region, using a different dye in each region. The choice of dye for each bladder region alternated between animals. For each dye, three to five injections of approximately 5 Al in total were made, each covering an area approximately 1 mm in diameter. The injections were performed using a glass micropipette attached via flexible tubing to a glass syringe filled with liquid paraffin. At the injection site, the micropipette was held in place for a few seconds to ensure the diffusion of dye into the tissue and to minimize leakage from the site. The tissue was then rinsed with 0.9% saline. Abdominal musculature and skin were separately sutured with silk thread and

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the animal was allowed to recover for 2 –8 weeks before tissue removal. Previous injection studies of pelvic organs have shown that it is not possible to restrict dye to either the muscle or mucosal layers, as dye spreads from the injection site into surrounding layers (Keast et al., 1989). Therefore, the study was unable to distinguish between ER-expressing neurons projecting to muscle vs. mucosa. All tissues were removed at estrous as determined by standard cytological assessment of vaginal swabs. All animals exhibited regular cycles at the time study and estrous was chosen because it has unequivocal cytological features. Animals were anaesthetised with sodium pentobarbitone (48 mg/kg i.p.) and perfused transcardially with physiological saline followed by freshly made 4% paraformaldehyde solution (pH 7.4). Dorsal root ganglia (DRG) from L1 – S1 were removed bilaterally and postfixed in the same solution overnight (4 jC) before washing with phosphate-buffered saline (PBS, pH 7.4). Ganglia from L1, L2, L6 and S1 were used for analysis of bladder afferent neurons, as these levels have been shown previously to contain the majority of neurons projecting to this organ (Keast and de Groat, 1989). Ganglia from other levels were also sectioned to verify absence of retrograde tracer dye. 2.2. Immunohistochemical processing Prior to sectioning on a cryostat, tissues were placed in 30% sucrose in PBS for cryoprotection and then embedded in mounting medium (Tissue-Tek; Sakura Finetek, USA). For each animal, the two ganglia from each spinal region (upper lumbar [L1, L2] or lower lumbar/sacral [L6, S1]) from one side of the spinal cord were embedded together in one block. Sequential 14-Am sections were placed alternatively on sets of three gelatin-subbed slides, one of each set of three being stained with a different antibody combination (see below), to ensure that there would be no double-counting of stained neurons. Sections were allowed to air-dry then washed in PBS for 10 min to remove remaining mounting medium. The characteristics of primary and secondary antisera used are shown in Table 1. Antisera combinations were ERa or ERh with SP, CGRP or VR1, and ERa with ERh. Studies were performed using two different antisera raised against VR1, raised against either the C-terminus or the N-terminus. Early double-labelling studies showed that both VR1 antisera gave the same results, so later studies were performed mostly with the N-terminal antibody, which gave the slightly more consistent and better quality immunolabelling. Sections were incubated in a droplet of primary antibody for 18 – 24 h, followed by a PBS wash then a 2-h incubation with secondary antibody. After another PBS wash, a third incubation of 1 h with a conjugated fluorophore was performed if a biotin-labelled secondary was used. All antibody incubations were performed in a dark humid environment at room temperature. Slides were coverslipped with bicarbonate-buffered glycerol (pH 8.6) and observed under an Olympus BX51 fluorescence microscope. Filters

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Table 1 Antisera characteristics Antigen

Host species

Supplier

Dilution

Primary antibodies ERa

Rabbit

1:250

ERh

Chicken

CGRP

Goat

VR1 C terminal

Guinea pig

VR1 N terminal SP

Rabbit Rabbit

Affinity Bioreagents, Golden, CO, USA J-A Gustafsson, Karolinska Institute, Stockholm Biogenesis, Poole, UK Chemicon, Temecula, CA, USA Neuromics Incstar, Stillwater, MN, USA

Secondary antibodies Rabbit IgG, Donkey Cy3 conjugate Rabbit IgG, biotin conjugate Chicken IgG, Cy3 conjugate Chicken IgG, Cy2 conjugate Guinea pig IgG, biotin conjugate Goat IgG, biotin conjugate Fluorophores Streptavidin-FITC

1:500

1:1000 1:8000 1:8000 1:1000

1:1500

Horse

Jackson Immuno Research, West Grove, PA, USA Vector Laboratories, Burlingame, CA, USA Jackson ImmunoResearch Jackson ImmunoResearch Jackson ImmunoResearch Vector Laboratories

N/A

Vector Laboratories

1:100

Goat Donkey Donkey Donkey

1:200 1:1000 1:200 1:200 1:200

2001). In addition, we demonstrated specificity of both antibodies in tissues where ERa and ERh have been previously described to have distinct localisation patterns. These patterns were replicated in our study. These tissues were the rat ventral prostate gland and uterine horn (Ma¨kela¨ et al., 2000; Wang et al., 2000), which were harvested from animals perfused for a separate study. Each ER type was located only in the appropriate cellular targets (Fig. 1g –j), which would not be the case if there was significant crossreactivity with the inappropriate receptor type. Tests of secondary antibody species specificity were also conducted for those used in ERa and ERh coexpression analysis (anti-rabbit and anti-chicken). To confirm that each only recognised the correct species, sections stained with ERa antibody were incubated with anti-chicken antiserum, and sections stained with ERh with anti-rabbit antiserum. As no positive staining was seen for either incubation, the possibility of cross-reaction was eliminated. All ERa/ERh coexpression was therefore considered genuine. An absorption test was performed for the VR1 Nterminal antibody by preincubation with its neutralizing peptide (Neuromics, MN, USA), followed by immunohistochemical techniques described above. Preabsorption abolished all staining, indicating the specificity of the VR1 Nterminal antibody (Fig. 1e,f). An absorption test was not performed for the VR1 C-terminal antibody as a neutralizing peptide was unavailable. However, staining patterns for the two VR1 antibodies appeared identical and the specificity of the VR1 C-terminal antibody was assumed. 2.4. Analysis of immunostained and dye-filled neurons

possessing different spectral characteristics were used to visualize the various fluorophores. Retrogradely labelled neurons were visualized using the U-excitation (Wide band) filter (U-MWU2), sections stained with Cy3 using the IGexcitation (Wide band) filter (U-MWIG2), and sections stained with FITC or Cy2 using the IB-excitation, colour separation (Narrow band) filter (U-MNIBA2). 2.3. Tests of antibody specificity The specificity of the ER antibodies was primarily determined by absorption tests. This involves the preabsorption of antibody with an excess of the purified neutralizing peptide against which the ERa antibody was raised (corresponding to the N-terminal residues 1 – 21; Affinity Bioreagents). An antibody/peptide solution at equal weight per unit volume (1 Ag/ml) was incubated for 2 h or overnight (4 jC) to allow for antibody/peptide binding, followed by the immunohistochemical techniques described above. All positive staining of ERa was abolished (Fig. 1a,b) but ERh staining was unchanged (Fig. 1c,d), demonstrating that nonspecific binding was not occurring. Similarly, ERh antibody preadsorbed with ERh protein gave a negative result, and the inability of this antibody to recognise rat ERa has been documented previously (Salmi et al.,

Coexistence of both ERs or ER with SP, CGRP or VR1 was determined by counting all nucleated profiles of immunoreactive neurons from a random selection of sections for each ganglion, ignoring the presence or absence of retrograde tracer dye. This was to demonstrate the overall distribution of ER in different chemical classes of neurons throughout the ganglia, irrespective of target organ. All positive nucleated profiles in two nonconsecutive sections for each spinal level group (upper lumbar and lower lumbar/sacral) were counted. All counts were performed under the 40  objective. Neuronal cell bodies in dorsal root ganglia are seen as large round profiles (most with a diameter in the range 20– 50 Am), and these are structurally completely distinct from glial cells, which are much smaller and very elongated cells. We only quantified neuronal staining and there was no difficulty in telling this apart from glial structures. The coexistence of two substances was determined in neurons for the combinations: ERa/ERh; ERa/CGRP; ERh/CGRP; ERa/VR1; ERh/VR1; ERh/SP. ERa/SP coexistence could not be assessed because both antibodies were raised in the same species. Because our early results showed almost complete coexistence between the two ERs (see below), and because this was also reflected in our coexpression patterns with peptides and VR1, for simplicity, we

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Fig. 1. Specificity testing of VR1 and ER antisera. Panels a – f show sections of lumbar dorsal root ganglia and g – j sections of pelvic organs. ERa immunostaining occurs in nuclei (a) and is abolished by preincubation of the antibody with the peptide against which the antibody was raised (b). ERh immunostaining also occurs in nuclei (c) but is unaffected by preincubation with the ERa peptide (d). Immunoreactivity for VR1 is cytoplasmic (e) and abolished by absorption with appropriate peptide (N-terminal part of VR1) (f). Ventral prostate gland shows no ERa-positive cells (g) but ERh-positive nuclei are prevalent in the glandular epithelium (h). Numerous ERa-positive nuclei are seen in the stroma (left arrow) and luminal epithelium (right arrow) of the uterus (i). Some of the ERa-positive nuclei in the stroma also contain ERh (j; arrow), but all glandular cell nuclei are negative (although they have some autofluorescence in their cytoplasm). Calibration bar in a represents 20 Am (a – f) or 25 Am (g – j).

have presented the data pooled from both ERa and ERh observations. Further analysis was performed on coexpression patterns within neurons containing retrograde tracer, to determine if they formed a unique subpopulation of neurons, i.e. with a different ‘‘chemical code’’ to the overall population of lumbosacral ganglion neurons. In addition, this would show if neurons projecting to the two different bladder regions have different chemical properties or ER expression patterns. Neurons containing dye were categorized according to the site of dye injection (detrusor or trigone) to establish a relative distribution of bladder afferent neurons across L1, L2, L6 and S1. The proportion of dye-labelled cells immunoreactive for one or more substances was also determined. ER-positive neurons were further categorized according to soma size. This was performed after coexistence studies had been completed and had demonstrated an almost complete coexpression of ERa and ERh. For this reason, only measurements of ERh-positive neurons were made and this was conducted in sections of L1 ganglia. A SPOTcooled CCD camera (RT Slider model, Diagnostic Instruments) attached to the Olympus BX51 fluorescence microscope captured and digitised the field of view. The edge of each soma was manually outlined and the area calculated by the Image Pro-Plus program. Only cells with a visible nucleus and a clearly defined soma outline were measured.

An average of 80 –100 cells within L1 ganglia from each of three animals was measured. All data are expressed as mean F S.E., where n = number of animals. Statistical comparisons of relevant groups were performed by using paired or unpaired Student’s t-tests, as appropriate. For each test, P < 0.05 was considered statistically significant. Results obtained separately for ganglia at each spinal level were pooled into upper lumbar (L1/L2) and lower lumbar/sacral (L6/S1) ganglia. This was due to the known chemical and physiological similarities between these ganglia (Keast and Stephensen, 2000). Photomicrographs were taken with a SPOT cooled CCD camera (see above) and figures constructed using the Photoshop program, manipulating contrast and brightness to best represent the immunostaining as seen under the microscope.

3. Results 3.1. General properties of ERa- and ERb-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

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intensity of ERa or ERh-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 that the nucleus was always dimmer or the same intensity as the cytoplasm. However, in all ER-stained preparations, neurons with nuclei brighter than the cytoplasm were observed and these were regarded as immunoreactive cells. Cytoplasmic staining was observed in some neurons but was inconsistent between preparations and so was not assessed further. On rare occasions, very high levels of retrograde tracer dye in neurons led to ‘‘bleedthrough’’ of fluorescence, but this was readily distinguished from nuclear ER staining or Golgi-like neuropeptide staining. Such neurons were not assessed in sections stained for VR1 to avoid mistakenly attributing VR1 staining to these neurons. Some glia, particularly in nerve roots associated with the ganglia, were weakly stained for ERh.

Many neurons showed ER-immunoreactivity. These were not quantified but appeared to comprise about one-third to one-half of all the neurons in a given section. Sections immunostained with both ER antibodies demonstrated virtually complete coexistence of the two receptors (Fig. 2a– d). In L1/L2 ganglia, all ERa neurons contained ERh and 97.8 F 1.3% ERh neurons contained ERa (50 – 100 neurons assessed per antibody in each of four animals). Similarly, in L6/S1 ganglia, 99.8 F 0.3% ERa-positive neurons contained ERh and 96.0 F 1.8% ERh neurons contained ERh. In many ER-positive neurons, there was a considerable difference in relative immunostaining intensity between the two receptor types (Fig. 2a,b). Nevertheless, because of the almost complete overlap of staining patterns, ER-positive neurons were viewed as a single population, for the purposes of expressing the bladder innervation data (a number of animals were also assessed with each ER antibody separately to validate this). This decision was also made on the basis of the similar results

Fig. 2. Coexistence of ER with neuropeptides or VR1. Many lumbosacral neurons express both ERa (a,c) and ERh (b,d). Top panels show examples of ERpositive neurons where the relative intensity of staining for each ER type varies considerably; arrowhead shows a rare neuron with ERa but no ERh. Second row panels show examples of neurons coexpressing both ER types, and in all cases, ERa is at higher levels than ERh; arrowheads show examples where ERh appears to be absent. There is a high, but incomplete level of coexpression of ER (c, ERh) and CGRP (d). Coexistence of ER (e, ERh) and VR1 (f) is also common. Matching arrows for each pair indicate examples of coexpression. Calibration bar represents 40 Am (a,b), 30 Am (c,d) or 25 Am (e – h).

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obtained in peptide and VR1 coexpression studies carried out separately with each antibody (Table 2). ER-immunoreactivity was observed in sensory neurons of all sizes in L1 ganglia (Fig. 3). The majority of ER-positive neurons fell in to the small (soma area < 1000 Am2; diameter < 18 Am) and medium (1000 –2000 Am2; diameter 18 –25 Am) categories. A few larger ER-positive neurons (>2000 Am2; diameter >25 Am) were also observed. Nuclei of small– medium neurons typically fluoresced with the greatest intensity, while nuclear staining of larger neurons was usually less bright. Somata in ganglia from other spinal levels were not measured but in L2, L6 and S1 the general features (smaller neurons, relative brightness of staining in small vs. large neurons) appeared to be qualitatively similar to L1. 3.2. Coexpression of ERs with SP, CGRP and VR1 Neurons with ER-immunoreactivity were categorized according to their expression of the neuronal markers SP, CGRP or VR1 (Table 2). No difference was noted between the upper lumbar and lower lumbar/sacral ganglia for any of the ER/peptide or ER/VR1 coexpression patterns. The results below therefore refer collectively to lumbosacral (L1, L2, L6, S1) dorsal root ganglia. Between 20% and 30% of ER-positive neurons contained CGRP, whereas 50 –60% of CGRP-positive neurons also expressed ER (Table 2, Fig. 2e,f). As mentioned earlier, ERa and ERh show almost complete coexpression, and this is also reflected in the similar ERa/CGRP and ERh/CGRP data. Similarly, 20 – 25% of ER-positive neurons contained SP and 55 – 60% of SP-positive neurons expressed ER (Table 2). A population of small, particularly brightly stained SP-positive neurons, did not express ER. Throughout lumbosacral DRGs, 30 – 40% of ER-positive neurons contained VR1, irrespective of which ER antibody Table 2 Coexpression of ER and neuronal markers in lumbosacral DRG neurons Marker substance

Spinal level

%ERa with marker

%Marker with ERa

%ERh with marker

%Marker with ERh

CGRP

L1/L2 L6/S1 L1/L2 L6/S1 L1/L2 L6/S1

27.3 F 5.3 23.3 F 6.1 N/A N/A 42.7 F 2.0 36.0 F 9.2

63.5 F 6.4 59.3 F 5.0 N/A N/A 29.3 F 2.0 21.3 F 6.9

30.0 F 2.5 22.8 F 2.8 25.0 F 2.3 21.0 F 1.0 39.3 F 7.2 32.0 F 5.4

61.8 F 6.3 51.5 F 7.2 65.0 F 9.6 57.7 F 13.0 50.5 F 11.0 44.5 F 10.0

SP VR1

The percentages of ERa- or ERh-positive neurons that contain the neuronal markers, CGRP, SP or VR1, and of each marker-positive neuron that contains ERa or ERh are shown. All data are expressed as mean F S.E. (n = 4 animals per observation, except for ERa/VR1 data and ERh/SP data, where n = 3). Between 50 and 100 cells were counted for each substance combination. VR1 data was obtained using two antibodies; ERa/VR1 combinations used the VR1 C-terminal antibody and ERh/VR1 combinations used the VR1 N-terminal antibody. No data was obtained (N/A) for ERa/SP combinations due to these antibodies being raised in the same species. Paired t-tests were performed to compare L1/L2 and L6/S1 data for each marker. No significant difference was found between the spinal levels ( P>0.05).

Fig. 3. Sizes of ERh-positive neuron somata in L1 DRG. Profiles of immunostained somata were measured and grouped into bins of 250 Am2. Bin labels indicate the smallest size neuron found in that bin. Data were pooled from three animals, with 80 – 100 neurons counted from each animal (total 270 neurons).

was used (Table 2, Fig. 2g,h). However, there was some discrepancy between the two antibodies when the proportion of VR1-positive neurons containing ER was calculated. This ranged from 20% to 30% (for ERa) and from 45% to 50% (for ERh). It is possible that this difference is not of biological significance because the ERh/VR1 data showed quite a high degree of inter-animal variability not present with other ER antibody combinations (perhaps indicating some interaction between the ERh and VR1 antisera). A population of very small, particularly intensely stained VR1-positive neurons did not express ER. 3.3. General properties of bladder-projecting dye-labelled neurons Retrograde dye injections into the trigone and detrusor regions resulted in numerous labelled cells within lumbosacral DRGs. As described previously (Keast et al., 1989), both Fast Blue and Fluorogold dyes demonstrated a range of intensities of labelling, probably due to variation in the proximity or proportion of a neuronal terminal field to the injection site. Also consistent with this previous report, there was no obvious difference in the types or numbers of neurons labelled with each dye, or evidence of neuronal Table 3 Distribution of bladder afferent neurons in dorsal root ganglia of each spinal level Bladder region

L1

L2

L6

S1

Trigone (%) Detrusor (%)

18.0 F 6.5 16.1 F 4.7

18.8 F 3.5 18.9 F 5.5

22.0 F 6.5 33.7 F 5.9

47.0 F 10.7 36.9 F 6.1

Data describe the proportion of all retrogradely labelled neurons located at each spinal level (mean F S.E., n = 4 animals).

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Fig. 4. Immunohistochemical features of bladder-projecting sensory neurons. Panels a,d,g show Fast Blue neurons labelled from the urinary bladder. Top row shows bladder neurons (a) labelled for both ERa (b) and CGRP (c). Bottom row shows a bladder neuron (d) immunoreactive for ERh (e) and SP (f). Matching arrows for each pair indicate examples of coexpression. Calibration bar represents 25 Am in all panels.

death (or loss of a particular chemical class of neuron) after longer periods of dye labelling. Neurons with very faint dye labelling were excluded from the cell counts. Rare neurons appeared to be double-labelled (i.e. both Fast Blue and Fluorogold present in the same neuron) and were excluded from analysis. The proportion of neurons innervating each

bladder region that originate from the various spinal levels is shown in Table 3 and was similar to that previously reported (Applebaum et al., 1980; Sharkey et al., 1983; Keast and de Groat, 1992; Vera and Nadelhaft, 1992). The majority (f 65%) of neurons labelled from the trigone or detrusor were located in L6/S1 ganglia.

Table 4 Coexpression of ERs with neuropeptides or VR1 in bladder-projecting neurons Marker

Spinal level

Bladder region

Total ER + neurons

Total marker + neurons

ER + marker +

ER + only

Marker + only

CGRP

L1/L2

trigone detrusor trigone detrusor trigone detrusor trigone detrusor trigone detrusor trigone detrusor

65.3 F 7.8 70.0 F 5.4a 49.3 F 6.2 52.4 F 5.0a 77.0 F 7.0 73.3 F 9.5 56.7 F 4.3 61.3 F 11.1 57.2 F 11.2 72.6 F 6.8 52.4 F 4.8 57.8 F 7.2

82.2 F 5.0b 68.7 F 10.4 54.0 F 7.1b 49.0 F 6.7 46.0 F 0e 16.7 F 4.1e 30.0 F 4.7 27.7 F 8.4 63.4 F 5.1 53.4 F 11.7 58.9 F 5.0 59.2 F 4.6

58.2 F 5.1c 47.3 F 4.6d 31.6 F 5.2c 27.1 F 4.6d 32.5 F 2.5f 8.3 F 2.8f 16.3 F 1.5 13.7 F 6.7 32.0 F 4.9 32.6 F 11.4 26.7 F 3.4 30.8 F 5.5

12.5 F 4.1 22.7 F 8.9 17.7 F 5.2 25.3 F 5.2 44.5 F 4.5 65.0 F 7.0 40.3 F 3.7 47.7 F 9.3 25.2 F 6.6 40.0 F 9.5 25.7 F 4.5 27.0 F 5.6

29.3 F 7.2 21.3 F 6.7 22.4 F 5.9 21.9 F 5.1 13.5 F 2.5 8.3 F 2.2 13.7 F 5.2 14.0 F 4.9 31.4 F 8.7 20.8 F 10.3 32.1 F 4.4 28.3 F 4.8

L6/S1 SP

L1/L2 L6/S1

VR1

L1/L2 L6/S1

From each animal, ganglion sections containing bladder-projecting neurons were immunostained for ER/SP, ER/CGRP or ER/VR1. Here only the properties of bladder-projecting (i.e. dye-labelled) neurons are described. Results are expressed as the percentage of bladder-projecting neurons (for each bladder region) that express one or both markers. Data from ERa and ERh were pooled because of the very high level of coexpression of the two receptor subtypes and similarity of data obtained with each ER antibody (not shown). The total population of ER-expressing dye-labelled neurons (as a proportion of all dye-labelled neurons) for each bladder region and spinal level is shown under ‘‘Total ER+ neurons’’. These neurons have been further classified as neurons containing only ER (‘‘ER+ only’’), or ER with a marker of interest (i.e., SP, CGRP, VR1; ‘‘ER+ Marker + ’’). Likewise, the total population of marker-expressing dye-labelled neurons is shown under ‘‘Total Marker+ neurons’’, and of this group those containing only the marker (but no ER) shown as ‘‘Marker+ only’’. Groups that differ significantly from each other are indicated: a, P = 0.036; b, P = 0.009; c, P = 0.014; d, P = 0.010; e, P = 0.02; f, P = 0.001. Number of animals for each observation is 4 (CGRP, VR1) or 3 (SP).

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3.4. Expression of ERs, peptides and VR1 in bladderprojecting neurons Bladder afferent neurons very commonly expressed ER (Fig. 4), and the ER-positive neurons comprised a number of chemical subclasses, including putative nociceptors (as indicated by VR1 immunostaining). Half or more (50 – 77%) of bladder afferent neurons express ER, regardless of whether they innervate detrusor or trigone (Table 4). The majority of bladder afferent neurons contained CGRP, as described previously (Keast and de Groat, 1992), and most of these coexpressed ER (Table 4, Fig 4a – c). Conversely, most ER-positive bladder neurons contained CGRP. SP was present in about one-third of bladder afferent neurons, and more than half of these also contained ER (Table 4, Fig 4d– f). In contrast to the ER/CGRP distribution, only a minority of ER-positive bladder neurons contained SP. About half of the bladder afferent neurons expressed VR1 (Table 4) and of these, about half were ER-positive. Conversely, only a minority of ER-positive bladder afferent neurons contained VR1. As predicted from the complete coexistence of ERa and ERh in the entire population of DRG neurons, there was virtually complete coexistence of the two ER types in bladder-projecting neurons. When coexistence with neuropeptides or VR1 was examined further, no statistically significant difference was found between the trigone and detrusor innervation at any spinal level, with two exceptions. These were the proportion of neurons containing SP and the proportion with both SP and ER, in L1/L2 ganglia. Both values were higher for the trigone compared with the detrusor. The patterns of ER/ CGRP coexistence also showed some differences between upper lumbar and lower lumbar/sacral ganglia for both bladder regions (Table 4). In summary, there was a slightly higher prevalence of ER-positive neurons and CGRP-positive neurons in the upper lumbar ganglia, as well as an increased prevalence of neurons containing both markers.

4. 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 innervating the urinary bladder. The majority of all lumbosacral bladder sensory neurons express both ERs and it is therefore highly likely that circulating estrogens influence their activity. This may occur in a number of ways, including regulation of expression of neuropeptides (Liuzzi et al., 1999; Gangula et al., 2000) and neurotrophin receptors (Sohrabji et al., 1994;

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Liuzzi et al., 1999; Lanlua et al., 2001). A direct effect on excitability and ion channel expression is also possible but has not been examined. Clearly, a more thorough assessment of bladder afferent activation needs to be performed after estrogen treatment or deprivation, or on isolated primary afferent neurons. The latter is particularly valuable as studies in whole animals can be complicated by estrogens influencing a range of cell types, including the urothelium (Teng et al., 2001), smooth muscle (Diep and Constantinou, 1999; Ratz et al., 1999; Sa´nchez-Ortiz et al., 2001) and possibly other neurons in the bladder innervation circuitry (central or peripheral autonomic pathways). A particularly interesting observation in the current study was the expression of ERs in bladder afferent neurons that express the marker of capsaicin-sensitive neurons, VR1. While these neurons are not exclusively involved in nociception and probably also play a role in normal micturition (Sharkey et al., 1983; Santicioli et al., 1985; Lecci and Maggi, 2001), they are activated in chronic inflammatory states such as interstitial cystitis (Vizzard, 2000; 2001). Moreover, there is growing evidence for an effect of circulating estrogens on the prevalence and severity of these conditions (Wesselmann et al., 1997; Bjorling and Wang, 2001; Johnson and Berkley, 2002). It is possible that these effects of estrogens are mediated by a direct action on gene expression in the bladder primary afferent neurons, to alter their excitability and intracellular signalling. Our study also showed that ERs were expressed broadly in the VR1-positive neurons of lumbosacral dorsal root ganglia, with the likelihood that many of these VR1/ER-positive neurons do not innervate the bladder. The link proposed between estrogens and urinary tract pain and inflammation may therefore be extended to other forms of pelvic visceral pain and there is indeed evidence that many of these change in severity and frequency with the menstrual cycle or after menopause (Wesselmann et al., 1997; Bjorling and Wang, 2001). Studies in rats have also demonstrated uterine and vaginal afferent sensitivity changes during the estrous cycle (Robbins et al., 1992). Our studies of VR1 distribution parallel those published recently elsewhere using the same antisera, with expression occurring exclusively in small – medium neurons, and in many neurons also synthesising one or more neuropeptides (substance P or CGRP) (Guo et al., 1999). A recent study specifically on bladder afferent innervation in rats has shown that VR1-positive axons (also containing SP or CGRP) are located in both the mucosa and the muscle (Avelino et al., 2002), and our retrograde labelling data shows that these are likely to be similarly prevalent in the detrusor and trigone regions. Considering together the expression of ERs in VR1/neuropeptide neurons, with the ability of estrogens to alter peptide expression, this may be a mechanism by which nociceptive function could be altered, either at the peripheral level (to affect excitability of terminals within organs, or the effects of an axon reflex) or centrally, within the dorsal horn. It is also possible that estrogens directly influence VR1 signalling or expression.

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Our immunohistochemical procedures demonstrated unique cellular distributions of each substance of interest, consistent with the previous reports. Neurons with CGRPand SP-immunoreactivity showed cytoplasmic staining that was often clumped due to the localisation of the peptides to punctate structures within the soma, such as the Golgi apparatus (Keast and de Groat, 1992; Guo et al., 1999). There was no neuropeptide staining in glia. Neurons containing VR1 showed diffuse cytoplasmic staining with both the Cand N-terminal antibodies. The receptor was expressed in small to medium neurons, as previously described (Guo et al., 1999; Caterina and Julius, 2001), with brightest staining in the small neurons. Glial staining was observed around some large and medium sized neurons that did not express VR1. Together, our staining for different combinations of substances indicates that there are likely to be a number of populations of ER-positive bladder afferent neurons, and that the ER/CGRP population is more numerous than the ER/SP or ER/VR1 populations. From previous descriptions of neuropeptide and VR1 coexistence (Keast and de Groat, 1992; Guo et al., 1999), it is also likely that some ER neurons contain all three marker substances. The distribution of ERs in different chemical classes of bladder-projecting neurons did not appear to differ markedly from the total population of lumbosacral neurons, suggesting that the sensory supplies of other pelvic organs may have similar features. Further, we found no evidence for different chemical properties or ER distribution in sensory neurons projecting to each bladder region. The trigone region is thought to contain the major supply of afferent terminals involved in triggering the micturition reflex, but it is possible that sensory fibres innervating the bladder neck were also labelled with the trigone injections, and some of these are thought to detect urine flow (de Groat et al., 1993; Lincoln and Burnstock, 1993). The detrusor region has a sparser nerve supply that is not thought to be as critical for the micturition reflex but may play a role in responses to inflammation and tissue trauma (de Groat et al., 1993; Lincoln and Burnstock, 1993). We did not find a major difference in ER expression between detrusor and trigone afferents so would predict that circulating estrogens influence a range of bladder afferent functions. Another novel aspect of our study was the almost complete coexpression of ERa and ERh in lumbosacral dorsal root ganglia. This contrasts with studies of ER distribution in the central nervous system, which generally show different expression patterns for each ER class (Shughrue et al., 1998a,b). We performed various studies to confirm that each antibody was only recognising the appropriate receptor class so we are confident that this coexpression occurs. In particular, we showed that the pattern of expression of each ER type previously documented by other groups in other non-neuronal tissues was demonstrated by the antibodies used in this study. The high levels of ERa and ERh coexpression seen in our study contrast with previous studies from two other groups, using different antibodies or methods. Importantly, aspects of

the results from these two groups are also in disagreement with each other, especially regarding ERh distribution. Taleghany et al. (1999) showed that while many small dorsal root ganglion neurons express ERa (as in our study), all dorsal root ganglion neurons were positive for ERh mRNA (Taleghany et al., 1999). Their choice of rat strain differed from ours (Sprague– Dawley cf. Wistar in the current study), but this is probably unlikely to be the basis of their much higher levels of ERh-positive neurons. It is possible that all dorsal root ganglion neurons do synthesise ERh message but only a minority produce the ERh protein. A more likely explanation is a problem with ERh probe specificity, especially since neither the Papka group (see below) or our own work has shown ERh protein expression by more than a minority of neurons. More recently, using immunohistochemical methods, Papka and co-workers (Papka et al., 2001; Papka and Storey-Workley, 2002) have reported ER distribution in dorsal root ganglia, with most ER-positive neurons being of small to medium size. Interestingly, their most recent doublelabelling study showed similar proportions of ER-positive neurons as our study (f 20% ERa and f 25% ERh), but only about 5% neurons with coexisting ERs. Whether lower level of ER coexpression in their study compared to ours reflects a difference in animal strain or reagents is unknown. It is possible that none of the ERh antibodies used by Papka’s group or ours is cross-reacting with ERa, but instead that each ERh antibody recognises different types of ERh splice variants (Pettersson and Gustafsson, 2001). If one ERh variant is commonly expressed in ERa-positive neurons but another is expressed mainly in ERa-negative neurons, then this would account for the discrepancy between Papka’s results and our own. The only other difference between these two studies is that ours used tissue from cycling animals in estrous, whereas the previous study used ovariectomised animals. We consider this unlikely to be responsible for the differing coexpression patterns, but nevertheless should be studied further. ERa and ERh coexistence has many implications regarding the mechanisms of estrogen effects on transcription in sensory neurons. Firstly, in vitro and intact cell studies have demonstrated that ERa and ERh heterodimers can form (Cowley et al., 1997; Pace et al., 1997; Pettersson et al., 1997), so three alternative signalling pathways could exist in cells with ER coexistence, through homodimers of either ERa or ERh, or through heterodimers (Kuiper et al., 1998). It has been proposed that in cells with a higher level of expression of one receptor, homodimers of the most highly expressed receptor and heterodimers will be preferentially formed over homodimers of the receptor with the lower expression level (Cowley et al., 1997). As levels of ER protein have been shown to be up- or down-regulated by circulating estrogen levels, depending on the receptor isoform and tissue type (Sohrabji et al., 1994; Ing and Ott, 1999), ultimately dimer formation will be under the modulation of estrogen in this model. Another important consideration is the effect of heterodimers on transcriptional activity (Pettersson

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and Gustafsson, 2001). Homodimers bind to estrogen response elements in the promoter region of target genes and ERa and ERh have different transcriptional responses. It has been shown that heterodimers can also bind to these elements, with a transcriptional activity between that of the two types of homodimers (Cowley et al., 1997). However, it is speculated that a novel response element may exist that is exclusively recognised by the heterodimer (Kuiper et al., 1998). This implies that a previously unrecognised collection of genes may be regulated by estrogen by a previously unknown mechanism. It will be an exciting challenge to determine not only the effects of estrogens on sensory neuron activity, but also the cellular mechanism by which this occurs. This is restricted at present due to a paucity of alpha- or betaspecific pharmacological tools. However, some insights may emerge from studies of sensory neurons in ERa or ERh knockout animals. 4.1. Conclusions The lower urinary tract has not been commonly studied as a target for circulating estrogens, although it has been shown that ERa and ERh are expressed within the bladder wall of male rats (Salmi et al., 2001). This supports previous functional studies that have shown changes in transmitter receptor expression or contractile properties in tissues removed during different stages of the estrous cycle (Diep and Constantinou, 1999; Ratz et al., 1999; Salmi et al., 2001). We have shown that various components of the sensory nerve supply of the bladder are also likely to be targets of circulating estrogens, and the way in which estrogens impact on the cellular function of neurons responsible for micturition reflex activation, or nociceptor activation, should be investigated. ERs have also been found in many lumbosacral preganglionic neurons and paracervical (autonomic) ganglion cells in female rats, including many neurons in these locations, which innervate the uterus. We propose that many of these ERexpressing neurons in the autonomic circuitry also supply the urinary bladder, and together form an ‘‘estrogen-sensitive circuit’’. It is essential that the role that estrogens play to influence activation of this circuit is pursued more vigorously. Acknowledgements 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). References Applebaum, A.E., Vance, W.H., Coggeshall, R.E., 1980. Segmental localization of sensory cells that innervate the bladder. J. Comp. Neurol. 192, 203 – 209.

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