Androgen-sensitive preganglionic neurons innervate the male rat pelvic ganglion

Androgen-sensitive preganglionic neurons

Pergamon PII: S0306-4522(99)00237-7

Neuroscience Vol. 93, No. 3, pp. 1147–1157,1147 1999 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00

ANDROGEN-SENSITIVE PREGANGLIONIC NEURONS INNERVATE THE MALE RAT PELVIC GANGLION T. W. WATKINS and J. R. KEAST* Department of Physiology and Pharmacology, The University of Queensland, Brisbane, Queensland 4072, Australia

Abstract—In adult male rats many pelvic autonomic ganglion cells change in structure and function after androgen deprivation. In this study we have investigated whether preganglionic neurons in the lumbar and sacral spinal cord that innervate these ganglion cells are also androgen-sensitive. Numerous spinal neurons retrogradely labelled from the pelvic ganglion possessed androgen receptor immunoreactivity and this was diminished by castration or enhanced by additional testosterone exposure. These comprised 27–77% of all preganglionic neurons innervating the pelvic ganglion, depending on the spinal level and whether animals were administered testosterone prior to sacrifice or not. When adult animals were castrated, no change occurred in the soma size or number of primary dendrites in these lumbar or sacral preganglionic neurons. Mean dendrite length was also determined in lumbar preganglionic neurons supplying the pelvic ganglion, but was not affected by castration. However, the total volume of lumbar preganglionic terminal varicosities supplying each noradrenergic pelvic ganglion cell decreased in parallel with the volume of the target neuron. These studies show that many preganglionic autonomic neurons involved in pelvic reflexes are androgen-sensitive, but that androgens selectively influence particular neuronal compartments. The prevalence of androgen receptors in these neurons suggests that testosterone may directly influence gene expression of preganglionic neurons. Together these studies suggest that testosterone (or a metabolite) has widespread actions on pelvic reflex circuits during adulthood and that under conditions of diminished circulating androgens a variety of reflex activities may not function optimally. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: autonomic, spinal cord, testosterone, pelvic viscera, sympathetic.

Gonadal steroids exert potent effects on the growth and maintenance of many neurons and wide-ranging changes occur in the nervous system after long-term steroid deprivation or after chronic androgen or estrogen exposure. These include changes in soma size, dendritic field, synapse number, transmitter synthesis and receptor expression. 5,12,24 While most studies have focused upon areas of the CNS involved in control of reproductive processes, it is becoming increasingly evident that gonadal steroids can also influence other parts of the nervous system. 24 Relatively few investigations have been made of steroid action in peripheral neurons and the pelvic ganglia are one of the few known targets of androgens in the autonomic system. These ganglia innervate the internal reproductive organs, lower urinary tract and rectum, and consist of a mixture of sympathetic and parasympathetic neurons. 9,25,26,27,51 In male rats, the noradrenergic pelvic neurons are particularly androgen-sensitive. Their soma size, tyrosine hydroxylase (TH) expression and catecholamine storage change dramatically during puberty or following castration. 6,8,15,16,38–41,57 More recently it has been shown that these changes occur not only in neurons supplying reproductive organs, but also in those innervating the rectum and urinary bladder. 29 In contrast, the cholinergic neurons are less androgen-sensitive, and so far structural changes have only been documented in those neurons supplying reproductive organs. 29 Many of the pelvic neurons also express androgen receptors (ARs), suggesting that at least some of these steroid actions may occur directly on neurons.

In parallel with these morphological and chemical changes, various nerve-evoked responses in reproductive organs (e.g., penile erection, vas deferens contraction) are compromised under conditions of low circulating androgens, such as after castration or in aged animals. 11,13,18–20,31,33,43,49,60 This is consistent with increasing prevalence of impotence during aging or after anti-androgen administration in humans. 14,54 However, while some of these problems are undoubtedly due to changes in the organs and their motor supply, there is no reason to assume that androgen action is limited to these sites. Complete identification of the range of targets for testosterone within autonomic circuits is essential for understanding how reproductive reflexes may fail when circulating androgens are diminished. In this study we have focussed on the preganglionic neurons that innervate pelvic ganglion cells in the male rat. In rats, the major pelvic ganglia contain both sympathetic and parasympathetic neurons, with similar numbers of neurons receiving lumbar or sacral inputs. 27,59 Typically each pelvic neuron receives one or two “strong” (suprathreshold) cholinergic inputs from one or other spinal level and as most of the pelvic neurons are adendritic, these synapses are primarily axosomatic. 59 We have addressed two main questions in order to determine whether any or all of these preganglionic neurons are testosterone-sensitive, in parallel with their targets, the pelvic ganglion cells. First, do any of these neurons express ARs? Second, does the structure of these neurons change after castration in adults, as occurs for many pelvic ganglion cells? To address the first question we have retrogradely labelled lumbar and sacral preganglionic neurons from the pelvic ganglion and applied specific, characterized antisera against the AR 22,29,50 to spinal cord sections. Immunostaining was further examined under conditions of androgen deprivation

*To whom correspondence should be addressed. Abbreviations: AR, androgen receptor; CTB, cholera toxin B subunit; DCN, dorsal commissural nucleus; FB, Fast Blue; IR, immunoreactive; PBS, phosphate-buffered saline; TH, tyrosine hydroxylase. 1147

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(castration) and exogenous testosterone treatment, to compare with known effects of these conditions on other central ARbearing neurons. 10,22,29,35,42,58,62 To address the second question we have measured various morphological features of preganglionic neurons innervating pelvic neurons, in both intact and castrated male rats. Soma size and number of primary dendrites were measured in both lumbar and sacral preganglionic neurons. Two additional features were measured in a randomly selected smaller sample of lumbar neurons, the mean dendrite length and the total volume of terminal varicosities supplying an individual noradrenergic pelvic ganglion cell. These ganglion cells change the most dramatically of all pelvic neuron populations after castration 29 and the total volume of preganglionic terminals supplying a randomly selected group of these neurons was quantified in optical sections through entire neurons using the confocal microscope.

EXPERIMENTAL PROCEDURES

General surgical procedures A total of 34 adult male Wistar rats (outbred, central animal breeding house, University of Queensland; nine weeks-of-age; 280^20 g) were used for these experiments. This was the minimum number of animals necessary to adequately address the experimental aims. All efforts were made to minimize animal discomfort in these experiments. All surgical procedures were carried out aseptically under anaesthesia induced with sodium pentobarbitone (45 mg/kg, i.p), and all animals recovered from surgery uneventfully. Fourteen animals were castrated at five weeks-of-age (150^10 g) and allowed the following four weeks to recover. This is the time required for androgen deprivation to maximally influence morphology of postganglionic neurons. 16,29 For studies of ARs in the spinal cord, five animals were treated with testosterone enanthate (10 mg/kg; Jurox, Riverstone, Australia), administered three days (s.c.), one day (s.c.) and 1 h (i.m.) prior to perfusion fixation and tissue removal. In six of the castrated animals, seven of the intact animals and all of the testosterone-treated animals, the retrograde tracer dye, Fast Blue (FB), was injected into both pelvic ganglia (see below) to label somata of preganglionic neurons in the spinal cord that project to them. To more effectively label dendrites of preganglionic neurons, cholera toxin B subunit (CTB) was instead injected into the pelvic ganglion of another four intact and four castrated animals. FB was injected at the time of castration, whereas CTB was injected one week later; FB or CTB were injected in control (intact) animals of comparable age, with the same transport times allowed as in castrated animals. Tissues were then removed under anaesthesia for immunohistochemical and morphometric analyses (see below).

Injection of retrograde tracer and cholera toxin B subunit into pelvic ganglia The abdominal cavity was exposed in rats, anaesthetized as described above. Cotton buds were used to gently clear away the overlying connective and adipose tissue from each pelvic ganglion. The connective tissue attaching the ganglion to the underlying prostate gland was also carefully removed through blunt dissection. A small piece of plastic was then inserted into the space between the ganglion and the prostate gland to prevent tracer leakage onto the underlying tissue. Bilateral injections of FB (2% in distilled water; EMS Polyloy, Gross-Umstadt, Germany) or CTB (1% in distilled water; List Biological Laboratories, Campbell, CA) were made into each ganglion using a glass micropipette attached by flexible tubing to a glass syringe filled with silicon oil, as described previously. 32 At least four tracer injections were made into various parts of each ganglion (total volume 1–2 ml for FB, 0.5–1 ml for CTB). On completion of tracer injections the plastic film was carefully removed and the ganglia washed liberally with saline. Abdominal musculature and skin were separately sutured with silk thread.

Tissue fixation and removal Anaesthetized rats were intracardially perfused with normal saline containing 1000–2000 I.U. heparin and 5 mg sodium nitrite, followed immediately by approximately 400 ml freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.0. The spinal cord (
Androgen-sensitive preganglionic neurons

contained a visible nucleus were analysed for the presence or absence of AR immunoreactivity. Neurons in the sacral parasympathetic nucleus (intermediolateral columns) and in separate populations of the lumbar sympathetic nuclei (dorsal commissural nucleus [DCN], intermediolateral and intercalating columns) were analysed to determine the proportion of preganglionic neurons that contained ARs.

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animals. Where two groups of data were compared (e.g., control vs castration), paired or unpaired Student’s t-tests were used, as appropriate. One-way analyses were performed for all analyses except varicosity volumes. Multiple groups (e.g., soma sizes of three spinal neuron groups) were compared using one-way ANOVA, and differences between groups then identified with Bonferroni Multiple Comparisons Test. For all tests P,0.05 was regarded as significant.

Measurement of soma profile areas of preganglionic neurons FB-filled neurons in horizontal spinal cord sections (30 mm) were viewed with fluorescence and images were captured using a CCD camera (Model 200E, Videoscope International, Herndon, VA). Images were digitized (LG-3 frame grabber, Scion Corporation, Frederick, MD) and analysed on a Macintosh computer using NIHImage (Version 1.6; public domain software developed at the U.S. National Institutes of Health). Measurement of soma profile area was performed only in neurons that were entirely contained within the section, and were measured at the level where their nucleus was in focus. Most neurons fluoresced with similar intensity and their profile area could be determined by thresholding (with camera gain kept constant). However a very small number of neurons contained very large amounts of dye and fluoresced intensely. These could not be measured without first decreasing the camera gain. Their soma profile areas were then measured by manually tracing around them using the cursor. Measurement of dendritic fields of preganglionic neurons The dendritic field could not be assessed in the majority of CTBfilled neurons, where one or more dendrites could be traced to the upper or lower edges of the section. We assumed that these were severed by the sectioning procedure. We therefore restricted our analysis to those neurons for which we could be confident that the entire dendritic field was within the section—that is, where labelled dendrites terminated before reaching the upper or lower edges of the section. Images of complete dendrites were then captured and digitised as described above, and their length determined by using the computer mouse to trace along them. Volume measurements of preganglionic terminals and postganglionic somata Immunolabelled preganglionic terminals (synaptophysin) and noradrenergic ganglion somata (TH) were visualized using dual channel fluorescent imaging on a Biorad MRC-600 confocal microscope, using the K1/K2 filters. Each 50 mm ganglion section was viewed through a ×63 oil immersion objective mounted on a Zeiss Axioskop and scanned at wavelengths 488–568 nm (K1 excitation) and 560 nm (K2 long pass filter). Neutral density, gain and black level remained constant for each section and there were no apparent differences in synaptophysin or TH immunofluorescence intensity between ganglia or animal groups. The z-step (i.e. depth between each scanned image) was set at 1.08 mm. For each ganglion, 9–35 neurons were analysed by obtaining entire z-series through complete neurons, captured using Comos software (Bio-Rad Microscience, Hemel Hempstead, U.K.). Neurons chosen for analysis were restricted to those in which the associated terminals could be easily distinguished from those supplying adjacent somata. Each z-series was first visualized using NIH-image software (Version 1.6). The soma was defined by determining an appropriate threshold value that showed the entire cell body. This threshold value was then kept constant for analysis of all cell soma z-series from the same microscope slide. Rat pelvic neurons typically possess few or no dendrites, 59 but a minority do have short (,5 mm) processes, appearing “fluffy” (Fig. 5b1, c1). These and any dendrites were deleted manually before soma volume calculations. Similarly, z-series of synaptophysin terminals were thresholded using values that remained constant for all scanned terminals from the same slide. These z-series were then used for separate analysis of soma and terminal volumes, and subsequent 3D reconstruction using Biorad Microvoxel software (INDEC systems, Capitola, CA). All three-dimensional reconstructions were also rotated and viewed using NIH-image to obtain qualitative information on terminal distribution, such as clustering. Statistical analyses All data have been expressed as mean^S.E.M., where nˆnumber of

RESULTS

General features of retrogradely-labelled preganglionic neurons Bilateral injection of FB into the major pelvic ganglia resulted in a large number of labelled lumbar and sacral preganglionic neurons (Fig. 1). Neuron somata and proximal primary processes were entirely filled with FB, but dendrites were not well labelled. There were no observable differences between control and castrated groups in intensity and distribution of retrogradely-labelled FB neurons in any of the spinal regions (Fig. 1a–d). The distribution of neurons labelled by CTB was identical (compare Figs 1 and 3), and all of the numerical analyses of somata (i.e. size and androgen receptor distribution) were made only in FB neurons. In contrast, CTB labelling allowed better delineation of the dendritic processes originating from individual neurons, so was used for analyses of dendrite number and length. Most FB- and CTB-labelled neurons were located in levels L1–L2 (sympathetic) and L6–S1 (parasympathetic), and their distributions agreed with those described previously. 2,17,21,44,45 In the lumbar spinal cord, neurons were found in two locations, the single central nucleus (DCN, 269^74 FB neurons, nˆ9 animals; pooled data from control and testosteronetreated animals) and lateral columns (consisting of intermediolateral and intercalating columns; 103^46, nˆ9; Fig. 1a, b). The smaller number of retrogradely-labelled neurons in the lateral region is consistent with previous studies. 17,44 The sacral segments of the spinal cord consistently contained greater numbers of retrogradely-labelled neurons (694^261, nˆ9), all located in intermediolateral columns (Fig. 1c, d). Nuclei could be clearly discerned in neurons at all levels (Fig. 1e, f). Effects of castration on soma size of preganglionic neurons The soma size of FB-labelled preganglionic neurons in the spinal cord differed significantly between spinal regions, with sacral neurons having a smaller profile area than either group of lumbar neurons (Table 1A). This is in agreement with previous observations. 17,45 There was no significant difference between lumbar lateral and DCN neurons. Castration had no effect on the soma profile area of any group of preganglionic neurons (Table 1A). Androgen receptor immunoreactivity in preganglionic neurons Numerous neurons within spinal cord sections exhibited AR immunoreactivity (IR) and this was located exclusively within their nucleus (Fig. 2). Immunoreactivity was abolished by preabsorption of the antiserum with the peptide against which the antibody was raised, indicating that labelling was likely to represent genuine AR. Many neurons retrogradely labelled with FB from the pelvic ganglion also demonstrated this staining, and the proportion of FB neurons with AR-IR and the intensity of their immunostaining were highly

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Fig. 1. Preganglionic neurons in the rat spinal cord retrogradely filled from the major pelvic ganglion. All micrographs show FB-labelled neurons in frozen horizontal sections (30 mm) of spinal cord and are oriented as follows: left, medial; right, lateral; top, rostral; bottom, caudal. Brightness and number of retrogradely-filled neurons were indistinguishable between control (b, c, e, f) and castrated (a, d) animals. (a) Lumbar cord, castrated animal, showing the three main regions containing labelled neurons, the DCN, intercalating column (IC) and intermediolateral column (IML). (b) Lumbar cord, DCN, control animal. (c) Sacral cord, control. (d) Sacral cord, castrated animal. (e, f) Show filled neurons at higher magnification in (e) lumbar DCN and (f) sacral cord. Scale bars: (a)ˆ300 mm; (b–d)ˆ200 mm; (e–f)ˆ50 mm.

Table 1. Soma size (A) and androgen receptor immunoreactivity (B) of retrogradely-labelled preganglionic neurons supplying the pelvic ganglion

A Control Castrate B Control Testosterone

Lumbar (lateral)

Lumbar (DCN)

488.5^68.2* 484.0^43.9

404.3^15.8 352.8^15.7

19.6^5.0%** 48.2^6.8%

42.3^12.3% 67.8^3.9%

Sacral

197.1^15.8 206.7^7.8 67.3^5.3% 85.8^4.0%

*Mean areas (mm 2) of nucleated soma profiles of retrogradely-filled preganglionic neurons are shown for each spinal cord region (mean^S.E.M.). In both control and castrated animals, sacral preganglionic neurons possessed significantly smaller soma profiles than either lumbar lateral (P,0.01) or lumbar DCN (P,0.05) neurons, but there was no significant size difference between the two lumbar groups. Castration had no effect on soma profile area in any region (nˆ3 animals in each group; number of neurons assessed were lumbar lateral, 35–204; lumbar DCN, 220–318; sacral, 189–796). **Proportion of retrogradely-labelled preganglionic neurons expressing AR immunoreactivity (mean^S.E.M.). In both control and testosterone-treated animals, more sacral neurons expressed the receptor compared with lumbar lateral cord (P,0.001). Testosterone treatment (10 mg/kg, s.c.) increased the proportion of immunoreactive neurons in all areas, although this only reached statistical significance in both regions of lumbar cord (P,0.05). Data were obtained from four control and five testosterone-treated animals; number of neurons assessed: lumbar lateral, 50–180; lumbar DCN, 200–400; sacral, 200–900. Lateral regions of lumbar cord include both intercalated and intermediolateral columns.

regulated by circulating androgens. In intact untreated adults many FB neurons possessed AR-IR nuclei, and this was significantly enhanced by testosterone administration prior to tissue removal (Fig. 2, compare a, b with d, e; Table 1B). Conversely, castration four weeks before tissue removal abolished nuclear AR-IR in FB neurons (Fig. 2c, f). However faint nuclear staining was still present in rare neurons scattered sparsely throughout the spinal cord, none of which were in regions containing retrogradely-filled preganglionic neurons. Both lumbar and sacral FB neurons possessed AR-IR (Fig. 2g, h; Table 1B). In each animal and irrespective of whether testosterone was administered or not, the sacral parasympathetic nucleus contained the greatest proportion of preganglionic neurons expressing nuclear ARs. A higher density and intensity of immunolabelled neurons was noted in the more rostral regions of the parasympathetic nucleus, possibly corresponding to a specific population of neurons innervating a common target. In comparison the lateral columns and DCN in the lumbar nuclei contained smaller numbers of neurons expressing nuclear ARs (Table 1B). Effects of castration on dendritic field of preganglionic neurons Both lumbar and sacral preganglionic neurons were well labelled by CTB, clearly demonstrating their soma and dendritic field (Fig. 3). Typically, dark reaction product filled

Androgen-sensitive preganglionic neurons

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Fig. 2. AR-IR in rat preganglionic neurons. All micrographs are taken from spinal cord regions containing numerous FB-labelled neurons. (a–f) Demonstrate the testosterone sensitivity of AR-IR, with bright staining seen in numerous neuronal nuclei after testosterone treatment (a, lumbar; b, sacral). Some bright nuclei are also seen in intact, untreated animals (d, lumbar; e, sacral), although they are less numerous than after testosterone treatment. No AR-IR nuclei are visible in castrated animals (c, lumbar; f, sacral). (g, h) Show FB-labelled preganglionic neurons in lumbar (g1) and sacral (h1) cord of testosterone-treated animals, many of which express AR-IR (g2, h2; examples of immunoreactive nuclei indicated with matching arrows). Scale bars: (a–f)ˆ100 mm; (g1, g2, h1, h2)ˆ50 mm.

the cytoplasm, and the dendrites were evenly labelled along their length. No difference in the intensity or distribution of the labelling was evident between the control and castrate groups (Fig. 3). Analysis of the number of

primary neurites per neuron showed that most lumbar and sacral preganglionic neurons possessed two or three primary neurites (Table 2). Typically sacral neurons possessed fewer primary neurites than lumbar neurons (P,0.02). This was

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Fig. 3. Distribution of CTB subunit in preganglionic neurons of intact and castrated rats. All micrographs are of horizontal spinal cord sections, following CTB injection into the pelvic ganglia. Caudal is oriented left, and rostral right. No obvious differences between control (a–d) and castrated (e–h) animals are evident. (a, b) Lumbar cord, DCN, control. (c, d) Sacral cord, control. (e, f) Lumbar cord, DCN, castrated animal. (g, h) Sacral cord, castrated animal. Scale bars: (a, c, e, g)ˆ500 mm; (b, d, f, h)ˆ200 mm.

also evident when comparing neurites with four or more processes, which comprised 28–38% of lumbar neurons, but only 12% of sacral neurons (Table 2). When the length of individual lumbar DCN preganglionic neurons was measured, no difference between control and castrated animals was found (Fig. 4; control 390.0^14.1 mm, castrate

374.6^23.0; nˆ3 animals, 19–23 dendrites measured in each animal). Effects of castration on preganglionic terminal varicosities These analyses were restricted to terminals supplying

Androgen-sensitive preganglionic neurons

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Table 2. Effects of castration on number of primary dendrites in lumbar and sacral preganglionic neurons Spinal level

Lumbar (DCN) Control Castrate Lumbar (lateral) Control Castrate Sacral Control Castrate

Primary processes

Cells

2

3

4

.5

Mean

25.8^4.2* 25.4^3.0

46.5^2.9 43.8^0.4

23.8^3.1 23.9^1.9

4.0^1.3 6.9^1.6

3.1^0.1 3.1^0.1

485 688

23.8^2.8 21.8^3.4

38.2^3.2 40.6^1.9

25.2^2.5 28.5^1.9

9.8^1.3 9.0^2.2

3.2^0.1 3.2^0.1

266 316

46.2^1.8 48.7^4.0

41.7^1.5 38.2^1.9

10.8^1.1 11.2^2.1

1.4^0.6 2.0^0.6

2.7^0.1 2.7^0.1

766 749

*Data are expressed as the proportion of all labelled neurons in this region possessing this number of dendrites. Data represent mean^S.E.M. from four animals of each group; 48–201 neurons were analysed in each area in each animal. Sacral neurons possessed slightly fewer primary processes than either type of lumbar neuron (P,0.02). There were no statistically significant effects of castration on any aspect of dendrite number.

Fig. 4. Effects of castration on mean dendrite length of lumbar preganglionic neurons. Histogram shows size distribution of dendrites from lumbar preganglionic neurons labelled with CTB subunit, in both control (black bars) and castrated animals (white bars). All neurons were in the DCN. Only dendrites that were entirely within the section were measured. Between 18 and 23 dendrites were measured in each of three control and three castrated animals. There was no obvious effect of castration on mean dendrite length.

volumes, it was also possible to investigate the effects of castration on the ratio of these two values, to provide an indication of any change in the proportion of the soma area in contact with the terminal varicosities. However the calculations demonstrated no change in this ratio (controlˆ15.1^2.9, castrateˆ14.0^3.1; Pˆ0.804). A number of interesting qualitative observations were also made from the three-dimensional analyses. First, varicosities supplying TH-positive neurons were distributed evenly over their somata and quite commonly varicosities lay in deep invaginations of the plasma membrane (Fig. 5d). Second, while most varicosities could be clearly distinguished from each other, some were clustered together very closely, merging into large lobulated swellings (Fig. 5e–h). This prohibited calculation of the number of varicosities per neuron. Finally, on rare occasions terminal varicosities clustered around the axonal hillock (Fig. 5f). All of these features were present in both control and castrated animals. DISCUSSION

noradrenergic (i.e. TH-positive) neurons. TH neurons occurred in clusters throughout ganglia of control and castrated animals (Fig. 5a1, c1). These neurons were noticeably smaller in castrated animals, as previously described. 29,40,41 Earlier reports demonstrated changes in nucleated soma profile area, and the present study also showed a substantial decrease in TH soma volume (controlˆ13,941^3,431 mm 3, castrateˆ6,704^1,101 mm 3; nˆ4 animals per group, 9–35 cells analysed per animal; P,0.05). Terminal varicosities in pelvic ganglion sections immunolabelled for synaptophysin exhibited features comparable to those described previously. 27 Varicosities were very brightly labelled but intervaricose segments and preterminal axons were completely negative (Fig. 5a2–c2). The brightness of synaptophysin staining did not differ noticeably between control and castrate animals. Three-dimensional reconstructions of merged z-series were also used for calculating the total volume of synaptophysinpositive varicosities supplying TH-positive neurons. In castrated animals the total volume of varicosities per THpositive neuron was significantly less than in control animals (controlˆ1,070^145 mm 3, castrateˆ582^29 mm 3; nˆ4 animals for each group, 9–35 neurons analysed per animal; P,0.02). From the values obtained for terminal and soma

This study is the first to show that preganglionic autonomic neurons controlling the pelvic viscera are targets for circulating testosterone (or a metabolite) in male rats. Many of these neurons express ARs, suggesting that testosterone may directly affect their properties. In particular, androgen deprivation causes a substantial decrease in the total volume of sympathetic preganglionic terminal varicosities supplying individual noradrenergic pelvic ganglion cells, suggesting that normally in adults testosterone (or a metabolite) is necessary to maintain these structures. Our study also showed that the decrease in varicosity volume occurred in parallel with a decrease in volume of the target neuron, such that the ratio of the two volumes was unchanged by castration. The lack of change in soma size or dendrite number and length of the same preganglionic neurons indicates that testosterone can exert effects on particular neuronal compartments, rather than broadly maintaining all neuronal structures. Together these results demonstrate that circulating steroids can influence various components of an autonomic reflex and may have potent effects on numerous neuronal functions in the periphery. While our studies are the first to implicate steroids in maintaining the structure of preganglionic neurons in adults, there are other indicators that steroids influence the development of autonomic spinal circuitry. For example, a

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Fig. 5. Effects of castration on varicose axon terminals supplying noradrenergic pelvic neurons. Noradrenergic neurons were identified by TH immunoreactivity (a1, b1, c1, d1), while preganglionic terminal varicosities were demonstrated with synaptophysin (SYN) immunoreactivity (a2, b2, c2, d2). (a) Shows a cluster of noradrenergic neurons in a control ganglion, each surrounded by numerous synaptophysin varicosities. An example of a noradrenergic neuron and its associated varicosities is shown at higher magnification in (b), while a group of noradrenergic neurons from a castrated animal are shown in (c). (d) Shows a pair of confocal micrographs (1.08 mm optical section) taken from a control animal, demonstrating the location of some varicose terminals in invaginations of the plasma membrane (arrows); the position of the nucleus is also indicated (N). (e–g) Show three-dimensional reconstructions of z-series taken with the confocal microscope, to demonstrate the distribution of synaptophysin varicosities on TH neurons of control (e, f) and castrated (g, h) animals. Axons and adjacent neurons have been removed digitally. In some neurons numerous varicosities cluster around the axon hillock (f, lower left of micrograph). Scale bars: (a)ˆ100 mm; (b, c)ˆ50 mm; (d)ˆ20 mm; (e–h)ˆ20 mm.

Androgen-sensitive preganglionic neurons

number of features of the lumbosacral spinal circuitry are sexually dimorphic, 3 such as the number of lumbar preganglionic neurons and the distribution of various types of peptide-containing afferents supplying lumbar and sacral preganglionic neurons. 45–48 The physiological consequences of castration-induced morphological change in adult preganglionic neurons may be considerable. For example, a decrease in volume of preganglionic axon terminal varicosities within the ganglion may indicate that synaptic transmission in the peripheral ganglion is compromised. This particular synapse is usually referred to as a “relay station”, as virtually all of the neurons receive suprathreshold lumbar or sacral inputs. 59 If the morphological changes were coupled with, for example, changes in transmitter release mechanisms, the high safety factor of this synapse could be severely compromised under conditions of low or absent circulating androgens. This may lead to failure of some autonomic reflexes. An effect of castration on varicosity volume was clearly observed for terminals supplying noradrenergic neurons, and it is important to also investigate the innervation of cholinergic pelvic neurons after castration. However, these will be a mixture of sympathetic and parasympathetic neurons, 27 so the results may be difficult to interpret. It was not possible in our study to determine whether the number of varicosities or size of individual varicosities contributed to the decrease in total volume, and ultrastructural studies examining this issue would be valuable. The present study points to the ganglionic synapse as being another important site where testosterone is essential for maintaining optimal structure (and possibly function) of neurons. However, we also showed that the changes in preganglionic terminal volume occurred in parallel with changes in volume of their target neurons. This precise matching of pre- and postganglionic neurons makes it difficult to predict the net effect on ganglionic function. A second way in which androgens have been reported to influence neuronal structure is by maintaining their dendritic field and synaptic inputs. This has been reported for motoneurons supplying the bulbocavernosus muscle, and numerous other central neurons. 5,24,36,52 However, despite the significant effect of castration on axon terminals of sympathetic (lumbar) preganglionic neurons, no effect on their dendrite number or length was evident. More subtle aspects of dendrite structure, such as branching pattern and synaptic inputs, were not examined in the present study. The present study also analysed only a small proportion of the labelled dendrites. Nevertheless, androgens clearly have less dramatic effects on soma size and dendritic field in preganglionic neurons than in spinal motor neurons. Whether this translates to a difference in function of synapses on these neurons is not known. The present study is the first to directly visualize ARs in preganglionic autonomic neurons and to map their expression in various spinal regions. These receptors may be important both in establishing sexual dimorphism of these regions and in maintaining their structure and function in adults. Their location in both sympathetic and parasympathetic neurons suggests that testosterone may influence a broad range of autonomic reflexes. However, as only a proportion of neurons in each region are stained, there may be some autonomic functions that are resistant to changes in hormone exposure. It would be interesting to determine which functional classes of sympathetic and parasympathetic preganglionic neurons are androgen-sensitive and androgen-resistant.

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ARs have been localized previously in many other parts of the nervous system, 1,24,56 including a number of spinal somatic motor nuclei (e.g., the spinal nucleus of the bulbocavernosus, dorsolateral nucleus and retrodorsolateral nucleus 7,10,23,35,53). Studies on steroid receptor expression in spinal autonomic neurons are very limited, although there has been a recent report of estrogen receptors in preganglionic neurons supplying the female rat pelvic ganglia; 61 this has also been indicated by studies of radiolabelled estrogen accumulation in the same spinal nuclei. 30 Similarly, there have been some reports of testosterone accumulation by neurons in autonomic spinal nuclei, 53,55 although these observations have been described only very briefly in studies more focussed on somatic motor pathways. A comparison of spinal regions showed that a greater proportion of parasympathetic neurons possessed the AR (60–70%), compared with sympathetic neurons (20–40%). This contrasts with AR expression in the targets of these preganglionic neurons, the pelvic ganglia, which in male rats express nuclear AR in only a small minority of their cholinergic neurons. 29 It is not known whether they are innervated by AR-bearing preganglionic neurons. No nuclear ARs have been observed in the major targets of lumbar preganglionic neurons, the noradrenergic pelvic ganglion cells, although these possess an unusual cytoplasmic staining (with the same AR antibody) that cannot be translocated to the nucleus with additional testosterone exposure. 29 Together, these observations suggest that testosterone can selectively influence different components of sympathetic and parasympathetic pathways. The ARs visualized in rat preganglionic neurons are highly regulated by circulating androgens, as indicated by the changes in their expression after testosterone administration or castration. This has been demonstrated in other populations of neuronal ARs, both in the CNS and in peripheral ganglia. 10,22,29,35,58,62 It is not known whether this represents upregulation of receptor expression by testosterone, facilitated receptor translocation into the nucleus or a greater affinity of the antibody for the ligand-bound receptor. Our study found no evidence for AR within the cytoplasm of preganglionic neurons or in other regions of spinal cord sections. They were absent from tissues of control, castrated and testosterone-treated animals. In contrast, a number of previous studies using the same AR antisera in spinal cord sections have claimed that after castration there is an increased expression of cytoplasmic AR in motoneurons of the spinal nucleus of the bulbocavernosus. 10,35,62 The most likely explanation for this discrepancy is a lower sensitivity of our detection method in comparison with some of the previous studies (e.g., which have used various diaminobenzidine procedures). This may have prohibited visualization of cytoplasmic labelling in our studies, where we may have expected to see it in some preganglionic neurons from castrated animals. However, in the present study, the necessity to combine AR immunostaining with other markers determined our choice of fluorescence. A critical issue in understanding gonadal steroid actions on these autonomic neurons is to determine the nature of the active substance. For testosterone, there are three options in addition to testosterone itself acting on neuronal androgen receptors. The first is that it may be metabolised to dihydrotestosterone by 5a-reductase and that this metabolite then activates ARs; indeed, considerable levels of reductases are

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present in all levels of the rat spinal cord. 34 Second, it is possible that testosterone is aromatized to estradiol-17b, which may be either the key active molecule or supplement the actions of testosterone. This occurs in many parts of the nervous system 4,24,37 and estrogen receptors have been localised in preganglionic neurons in female rats. 61 Although similar studies have not yet been conducted on male rats, estradiol is not predicted to be a major contributor to androgen function in the spinal cord, as aromatase levels are low in both male and female rat spinal cord. 34 The third is an indirect mechanism, not mediated by steroid hormone receptors in the target neurons. This is the case for spinal motor neurons (in the spinal nucleus of the bulbocavernosus), where testosterone indirectly regulates their dendritic arborization by inducing the release of a neurotrophic factor from target musculature, but appears to directly affect their soma size via neuronal androgen receptors. 52 Androgen-regulated neurotrophic factor synthesis from autonomic ganglion cells has not yet been investigated to determine whether a similar mechanism exists for preganglionic neurons.

CONCLUSIONS

This study has shown that circulating androgens target preganglionic autonomic neurons supplying the pelvic ganglia. The presence of AR in these neurons suggests that at least some of the hormone effects may be due to direct activation of neuronal gene expression. The study has also shown that testosterone has discrete effects on particular neuronal compartments, all of which could impact significantly on the successful activation of these autonomic circuits. This demonstrates the necessity of investigating a range of neuronal features when localizing steroid actions in the nervous system.

Acknowledgements—We are indebted to Dr Gail Prins (University of Illinois) for generously supplying the antibody against the androgen receptor, and the peptide against which this antibody was raised. We are also very grateful to Mr Colin Macqueen for assistance with the confocal microscopy. This work was supported by the National Health and Medical Research Council of Australia.

REFERENCES

1. Attardi B. and Ohno S. (1976) Androgen and estrogen receptors in the developing mouse brain. Endocrinology 99, 1279–1290. 2. Barber R. P., Phelps P. E. and Vaughn J. E. (1991) Generation patterns of immunocytochemically identified cholinergic neurons at autonomic levels of the rat spinal cord. J. comp. Neurol. 311, 509–519. 3. Baron R., Ja¨nig W. and McLachlan E. M. (1985) The afferent and sympathetic components of the lumbar spinal outflow to the colon and pelvic organs in the cat. I. The hypogastric nerve. J. comp. Neurol. 238, 135–146. 4. Beyer C., Green S. J., Barker P. J. and Huskisson N. S. (1994) Aromatase-immunoreactivity is localised specifically in neurones in the developing mouse hypothalamus and cortex. Brain Res. 638, 203–210. 5. Breedlove S. M. (1992) Sexual dimorphism in the vertebrate nervous system. J. Neurosci. 12, 4133–4142. 6. Bustamante D., Lara H. and Belmar J. (1989) Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats. Biol. Reprod. 40, 541–548. 7. Cain M. P., Kramer S. A., Tindall D. J. and Husmann D. A. (1994) Expression of androgen receptor protein within the lumbar spinal cord during ontological development and following antiandrogen induced cryptorchidism. J. Urol. 152, 766–769. 8. Chamness S. L., Ricker D. D., Crone J. K., Dembeck C. L., Maguire M. P., Burnett A. L. and Chang T. S. K. (1995) The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat. Fert. Steril. 63, 1101–1107. 9. Dail W. G. (1993) Autonomic innervation of male genitalia. In Nervous Control of the Urogenital System. (ed. Maggi C. A.), pp. 69–102, Harwood Academic Publishers, Chur, Switzerland. 10. Freeman L. M., Padgett B. A., Prins G. S. and Breedlove S. M. (1995) Distribution of androgen receptor immunoreactivity in the spinal cord of wild-type, androgen-insensitive and gonadectomized male rats. J. Neurobiol. 27, 51–59. 11. Garban H., Marquez D., Cai L., Rajfer J. and Gonzalez-Cadavid N. F. (1995) Restoration of normal adult penile erectile response in aged rats by longterm treatment with androgens. Biol. Reprod. 53, 1365–1372. 12. Garcı´a-Segura L. M., Chowen J. A., Pa´rducz A. and Naftolin N. (1994) Gonadal hormones as promoters of structural synaptic plasticity: cellular mechanisms. Prog. Neurobiol. 44, 279–307. 13. Giuliano F., Rampin O., Schirar A., Jardin A. and Rousseau J.-P. (1993) Autonomic control of penile erection: modulation by testosterone in the rat. J. Neuroendocrinol. 5, 677–683. 14. Gray A., Feldman H. A., McKinlay J. B. and Longcope C. (1991) Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts male aging study. J. clin. Endocrinol. Metab. 73, 1016–1025. 15. Hamill R. W. and Guernsey L. A. (1983) Hormonal regulation of sympathetic neuron development: the effects of neonatal castration. Devl Brain Res. 11, 303–307. 16. Hamill R. W. and Schroeder B. (1990) Hormonal regulation of adult sympathetic neurons: the effects of castration on neuropeptide Y, norepinephrine, and tyrosine hydroxylase activity. J. Neurobiol. 21, 731–742. 17. Hancock M. B. and Peveto C. A. (1979) A preganglionic autonomic nucleus in the dorsal gray commissure of the lumbar spinal cord of the rat. J. comp. Neurol. 183, 65–72. 18. Hart B. J. (1967) Testosterone regulation of sexual reflexes in spinal male rats. Science 155, 1283–1284. 19. Heaton J. P. W. and Varrin S. J. (1994) Effects of castration and exogenous testosterone supplementation in an animal model of penile erection. J. Urol. 151, 797–800. 20. Holmquist F., Persson K., Bødker A. and Andersson K.-E. (1994) Some pre- and postjunctional effects of castration in rabbit isolated corpus cavernosum and urethra. J. Urol. 152, 1011–1016. 21. Hosoya Y., Nadelhaft I., Wang D. and Kohno K. (1994) Thoracolumbar sympathetic preganglionic neurons in the dorsal commissural nucleus of the male rat: an immunohistochemical study using retrograde labeling of cholera toxin subunit B. Expl Brain Res. 98, 21–30. 22. Iqbal J., Swanson J. J., Prins G. S. and Jacobson C. D. (1995) Androgen receptor-like immunoreactivity in the Brazilian opossum brain and pituitary: distribution and effects of castration and testosterone replacement in the adult male. Brain Res. 703, 1–18. 23. Jordan C. L., Padgett B., Hershey J., Prins G. and Arnold A. (1997) Ontogeny of androgen receptor immunoreactivity in lumbar motorneurons and in the sexually dimorphic levator ani muscle of male rats. J. comp. Neurol. 379, 88–98. 24. Kawata M. (1995) Roles of steroid hormones and their receptors in structural organization in the nervous system. Neurosci. Res. 24, 1–46. 25. Keast J. R. (1994) Neuropeptide-containing axon terminals in the male rat major pelvic ganglian are primarily of sacral origin. J. Auton. Nerv. Syst. 47, 151–158. 26. Keast J. R. (1995) Pelvic ganglia. In Autonomic Ganglia, (ed. McLachlan E. M), pp. 445–479, Harwood Academic Publishers, Luxembourg. 27. Keast J. R. (1995) Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion. Neuroscience 66, 655–662.

Androgen-sensitive preganglionic neurons

1157

28. Keast J. R. and de Groat W. C. (1989) Immunohistochemical characterization of pelvic neurons which project to the bladder, colon, or penis in rats. J. comp. Neurol. 288, 387–400. 29. Keast J. R. and Saunders R. J. (1998) Testosterone has potent, selective effects on the morphology of pelvic autonomic neurons which control the bladder, lower bowel and internal reproductive organs. Neuroscience 85, 543–546. 30. Keefer D. A., Stumpf W. E. and Sar M. (1973) Estrogen—topographical localisation of estrogen-concentrating cells in the rat spinal cord following 3 H-estradiol administration. Proc. Soc. exp. Biol. Med. 143, 414–417. 31. Lara H., Galleguillos X., Arrau J. and Belmar J. (1985) Effect of castration and testosterone on norepinephrine storage and on the release of [ 3H]norepihnephrine from rat vas deferens. Neurochem. Int. 7, 667–674. 32. Luckensmeyer G. B. and Keast J. R. (1995) Distribution and morphological characterisation of viscerofugal projections from the large intestine to the inferior mesenteric and pelvic ganglia in the male rat. Neuroscience 66, 663–671. 33. MacDonald A. and McGrath J. C. (1980) The effects of castration on neurotransmission in the rat vas deferens. Br. J. Pharmac. 69, 49–58. 34. MacLusky N. J., Clark C. R., Shanabrough M. and Naftolin F. (1987) Metabolism and binding of androgens in the spinal cord of the rat. Brain Res. 422, 83–91. 35. Matsumoto A., Arai Y. and Prins G. S. (1996) Androgenic regulation of androgen receptor immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus of male rats. J. Neuroendocrinol. 8, 553–559. 36. Matsumoto A., Micevych P. E. and Arnold A. P. (1988) Androgen regulates synaptic input to motoneurons of the adult rat spinal cord. J. Neurosci. 8, 4168–4176. 37. McEwen B. S., Davis P. G., Parsons B. and Pfaff D. W. (1977) Aromatization: important for sexual differentiation of the neonatal rat brain. Horm. Behav. 9, 249–263. 38. Melvin J. E. and Hamill R. W. (1987) The major pelvic ganglion: androgen control of postnatal development. J. Neurosci. 7, 1607–1612. 39. Melvin J. E. and Hamill R. W. (1989) Hypogastric ganglion perinatal development: evidence for androgen specificity via androgen receptors. Brain Res. 485, 11–19. 40. Melvin J. E., McNeill T. H. and Hamill R. W. (1988) Biochemical and morphological effects of castration on the postorganizational development of the hypogastric ganglion. Devl Brain Res. 38, 131–139. 41. Melvin J. E., McNeill T. H., Hervonen A. and Hamill R. W. (1989) Organizational role of testosterone on the biochemical and morphological development of the hypogastric ganglion. Brain Res. 485, 1–10. 42. Menard C. S. and Harlan R. E. (1993) Up-regulation of androgen receptor immunoreactivity in the rat brain by androgenic-anabolic steroids. Brain Res. 622, 226–236. 43. Mills T. M., Wiedmeier V. T. and Stopper V. S. (1992) Androgen maintenance of erectile function in the rat penis. Biol. Reprod. 46, 342–348. 44. Nadelhaft I. and Booth A. M. (1984) The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study. J. comp. Neurol. 226, 238–245. 45. Nadelhaft I. and McKenna K. E. (1987) Sexual dimorphism in sympathetic preganglionic neurons of the rat hypogastric nerve. J. comp. Neurol. 256, 308–315. 46. Newton B. W. (1992) A sexually dimorphic population of galanin-like neurons in the rat lumbar spinal cord: functional implications. Neurosci. Lett. 137, 119–122. 47. Newton B. W. and Hamill R. W. (1988) Neuropeptide Y immunoreactivity is preferentially located in rat lumbar sexually dimorphic nuclei. Neurosci. Lett. 94, 10–16. 48. Newton B. W., Unger J. and Hamill R. W. (1990) Calcitonin gene-related peptide and somatostatin immunoreactivities in the rat lumbar spinal cord: sexually dimorphic aspects. Neuroscience 37, 471–489. 49. Penson D. F., Ng C., Cai L., Rajfer J. and Gonza´lez-Cadavid N. F. (1996) Androgen and pituitary control of penile nitric oxide synthase and erectile function in the rat. Biol. Reprod. 55, 567–574. 50. Prins G. S., Birch L. and Greene G. L. (1991) Androgen receptor localization in different cell types of the adult rat prostate. Endocrinology 129, 3187–3199. 51. Purinton P. T., Fletcher T. F. and Bradley W. E. (1973) Gross and light microscopic features of the pelvic plexus in the rat. Anat. Rec. 175, 697–706. 52. Rand M. N. and Breedlove S. M. (1995) Androgen alters the dendritic arbors of SNB motoneurons by acting upon their target muscles. J. Neurosci. 15, 4408–4416. 53. Sar M. and Stumpf W. E. (1977) Androgen concentration in motor neurons of cranial nerves and spinal cord. Science 197, 77–79. 54. Schroder F. H. (1991) Endocrine therapy: where do we stand and where are we going? Cancer Surv. 11, 177–194. 55. Sheridan P. J. and Weaker F. J. (1981) The primate spinal cord is a target for gonadal steroids. J. Neuropath. exp. Neurol. 40, 447–453. 56. Simerly R. B., Chang C., Muramatsu M. and Swanson L. W. (1990) Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J. comp. Neurol. 294, 76–95. 57. Sjo¨strand N. O. and Swedin G. (1976) Influence of age, growth, castration and testosterone treatment on the noradrenaline levels of the ductus deferens and the auxiliary male reproductive glands in the rat. Acta physiol. scand. 98, 323–338. 58. Smith G. T. (1996) Use of PG-21 immunocytochemistry to detect androgen receptors in the songbird brain. J. Histochem. Cytochem. 44, 1075–1080. 59. Tabatabai M., Booth A. M. and de Groat W. C. (1986) Morphological and electrophysiological properties of pelvic ganglion cells in the rat. Brain Res. 382, 61–70. 60. Wakade A. R. and Kirkepar S. M. (1973) “Trophic” influence on the sympathetic nerves of the vas deferens and seminal vesicle of the guinea pig. J. Pharmac. exp. Ther. 186, 528–536. 61. Williams S. J. and Papka R. E. (1996) Estrogen receptor-immunoreactive neurons are present in the female rat lumbosacral spinal cord. J. Neurosci. Res. 46, 492–501. 62. Wood R. I. and Newman S. W. (1993) Intracellular partitioning of androgen receptor immunoreactivity in the brain of the male Syrian hamster: effects of castration and steroid replacement. J. Neurobiol. 24, 925–938. (Accepted 15 April 1999)