Estrogen Receptors in the Spinal Cord, Sensory Ganglia, and Pelvic Autonomic Ganglia

Estrogen Receptors in the Spinal Cord, Sensory Ganglia, and Pelvic Autonomic Ganglia R. E. Papka and C. N. Mowa Department of Neurobiology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272

Until relatively recently, most studies of the eVects of estradiol in the nervous system focused on hypothalamic, limbic, and other brain centers involved in reproductive hormone output, feedback, and behaviors. Almost no studies addressed estradiol eVects at the spinal cord or peripheral nervous system level. Prior to the mid-1960s–1970s, few studies examined neural components of reproductive endocrine organs (e.g., ovary or testis) or the genital organs (e.g., uterus or penis) because available data supported endocrine regulation of these structures. Over the last two decades interest in and studies on the innervation of the genital organs have burgeoned. Because of the responsiveness of genital organs to sex steroid hormones, these neural studies seeded interest in whether or not autonomic and sensory neurons that innervate these organs, along with their attendant spinal cord circuits, also are responsive to sex hormones. From the mid-1980s there has been a steady growth of interest in, and studies of the neuroanatomy, neurochemistry, neural connectivity, and neural functional aspects in reproductive organs and the response of these parameters to sex steroids. Thus, with the growth of probes and techniques, has come studies of anatomy, neurochemistry, and circuitry of sex hormone-responsive neurons and circuits in the spinal cord and peripheral nervous system. This review focuses on estrogen receptors in sensory, autonomic, and spinal cord neurons in locales that are associated with innervation of female reproductive organs. KEY WORDS: Estrogen, Estrogen receptor, Dorsal root ganglia, Pelvic autonomic ganglia, Pregnancy, Neurotransmitters. ß 2003 Elsevier Inc.

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Copyright 2003, Elsevier Inc. All rights reserved.

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I. Introduction In the past 10 years, there have been considerable changes in the way we look at and think about sex steroid hormones and their receptors. Until the 1990s, most attention to these hormones and receptors was directed to their sites of production in endocrine organs and their eVects on target tissues associated with the reproductive organs and reproduction-associated centers in the central nervous system (CNS) (PfaV, 1968; PfaV and Keiner, 1973; Stumpf, 1968, 1970; Stumpf and Sar, 1975; Stumpf et al., 1975; Do¨rner, 1980; Blaustein and Olster, 1989; Koch and Ehret, 1989). However, in the late 1970s information began to accumulate about the neural innervation to female reproductive organs (Papka and Traurig, 1993; Traurig and Papka, 1993) and neural connections between these organs and the CNS (mainly the spinal cord). This sparked interest in whether nerves innervating the reproductive organs were responsive to circulating sex steroids such as estrogen, and if such nerves expressed estrogen receptors (ER). Early important papers reported estrogen-sensitive neurons in the spinal cord (Keefer et al., 1973; PfaV and Keiner, 1973; Morrell et al., 1982) and autonomic ganglia (Wright and Smolen, 1983, 1985, 1987; Schleicher et al., 1985) but virtually no data on sensory ganglia prior to 1994. Seminal studies on sex steroids in the spinal cord and peripheral neurons utilized binding of radiolabeled estradiol (Keefer et al., 1973; PfaV and Keiner, 1973; Stumpf et al., 1975; Morrell et al., 1982) to identify sites for estrogen binding. These binding sites were presumed to reflect sites where ERs existed. In spinal neurons, the binding appeared to be mainly nuclear (Keefer et al., 1973; Morrell et al., 1982) as was the case for radiolabeled estradiol in numerous brain areas. With the generation and availability of well-characterized antibodies against ER protein, this nuclear localization of ERs has been substantiated in spinal neurons of rats (Amandusson et al., 1995, 1996; Williams and Papka, 1996; Papka et al., 2001, 2002) (Fig. 1) and cats (VanderHorst et al., 1997, 2001) and in ganglionic neurons of the rat peripheral nervous system (PNS) (Papka et al., 1997, 2001; Zoubina and Smith, 2002; Shinohara et al., 2000). In situ hybridization histochemistry studies confirmed the presence of mRNA for ERs in spinal cord neurons (Simerly et al., 1990; Shughrue et al., 1997; Papka et al., 2001) and ganglionic neurons of the PNS (Sohrabji et al., 1994b; Papka et al., 1997, 2001; Taleghany et al., 1999) confirming that there is local ER synthesis and translation to ER protein. In the mid-1990s, a second isoform of the ER was cloned (Kuiper et al., 1996) and was designated as ER-b, while the classic form of the ER was designated as ER-a (Greene et al., 1986; Koike et al., 1987) (the reader is referred to the review by Nilsson and Gustafsson, 2002, for complete details on

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the developments of ER-b). Activation of nuclear ER-a and -b produces, eVects resulting from initiating genomic elements that tend to be expressed over a period of time (Fig. 2). Current data also support the existence of other mechanisms of estrogens actions in both neurons and nonneuronal cells that involve membrane actions; possibly there are membrane receptors for estrogens, which are responsible for rapid and immediate eVects of the sex steroid (Razandi et al., 1999; Vasudevan et al., 2001; ToranAllerand et al., 2002; Levin, 2002) (Fig. 2). However, classic ER-a protein has been demonstrated by immunohistochemistry in sites other than the nucleus including axons in brainstem neurons (Blaustein 1992; Blaustein and Turcotte, 1989) and in hippocampal nerve terminals (Towart et al., 2002). Questions about whether such features as these operate in the spinal cord and PNS will be fruitful and valuable areas for study in the future. The aim of this review is to consolidate information from studies concerning the presence of ERs in spinal cord neurons, sensory and autonomic ganglionic neurons, provide some data about the neurochemical environs of such neurons, and build a picture of the role estrogen may play in the these neurons and their circuits. We will make specific comments about the connections between neurons in these locales, but do not want to suggest that ERs are present only in neurons involved in reproductive organ innervation. Certainly, neurons involved in reproductive behaviors (somatic) as well as others divorced from reproduction may be influenced by estrogen (e.g., urinary system—Beach, 1974; Tansy and Kaufman, 1966; Blakeman et al., 2000; Johnson and Berkley, 2002).

II. Estrogen and Estrogen Receptors Considerable evidence indicated that sex steroids influence many aspects of neuronal structure and function in the brain. Numerous studies have examined ER-containing neurons and the role of estrogen in such neuroa natomical areas as the midbrain, preoptic area, lateral septum, medial anterior and ventromedial nuclei of the hypothalamus, amygdala, limbic system, and hippocampus (PfaV and Keiner, 1973; Stumpf et al., 1975; Pelletier et al., 1988; Simerly et al., 1990; McEwen, 1992; Dellovade et al., 1992; Li et al., 1993; X. Li et al., 1997; Delville and Blaustein, 1993; Lehman et al., 1993). These areas play a role in mediating reproductive and sexual behavior. Other examples of estrogen eVects include stimulation of development and neurite outgrowth (Santagati et al., 1995; Toran-Allerand, 1976; Reisert et al., 1987; Islamov et al., 2002) and synaptic plasticity (Wright and Smolen, 1985;

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FIGS. 1–12 (1) Example of nuclear localization of ER-a (arrows) in neurons of the lumbosacral DRG. A similar nuclear presence of ER immunoreactivity was evident in spinal cord and pelvic autonomic neurons for ER-a and ER-b (not shown). (2) Schematic diagram illustrating a potential model for mechanism of action of ERs. The steroid (E on the left side of figure) diVuses into the cell and binds to intracellular ERs, the liganded receptor translocates to the nucleus, dimerizes, and interacts with the hormone response element of the DNA to activate transcription, translation, and ultimately a cellular response (e.g., protein synthesis). This classic genomic action of estrogen action is generally slow. Recent data have shown that steroids (E on the right side of the figure) have rapid actions and probably work through a membrane receptor and second messenger systems. (3) Photomicrograph of the right dorsolateral quadrant of the

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lumbosacral spinal cord illustrating part of the distribution of ERs in the cord. ERimmunoreactive neurons (arrows) are noted in the lateral part of the dorsal horn (DH), in the sacral parasympathetic nucleus (SPN), and in the lateral funiculus in or near the lateral spinal nucleus (LSN). (4) Photomicrograph of the left dorsolateral quadrant of the lumbosacral spinal cord illustrating part of the distribution of ER in the cord. This tissue was double immunostained for ER-a (green nuclei) and substance P (SP) (red fibers and terminals). ER-immunoreactive neurons (green arrows) are noted in the lateral part of the dorsal horn (DH), lateral collateral pathway (LCP), lateral funiculus in or near the lateral spinal nucleus (LSN), and in the sacral parasympathetic nucleus (SPN). In all areas ER-positive neurons are among, and closely apposed by, SP-immunoreactive fibers (red arrows). (5) ER-a neurons (arrows) in the lateral funiculus near the LSN and in the LCP. (6) Photomicrograph of the left dorsolateral quadrant of the lumbosacral spinal cord illustrating part of the distribution of Fos-immunoreactive neurons. Fos was expressed due to synaptic activation of postsynaptic neurons, presumably by stimulation of primary aVerent neurons by pups passing through the uterine cervix at parturition. Fos-immunoreactive neuronal nuclei (arrows) are present in the same areas in which ER-immunoreactive neurons are localized. For example, in the lateral part of the dorsal horn, in the lateral collateral pathway (LCP), lateral funiculus in or near the lateral spinal nucleus (LSN), and sacral parasympathetic nucleus (SPN). (7) Photomicrograph illustrating many NPYimmunoreactive fibers and terminals (arrows) in the lateral dorsal horn (DH) and in the lateral funiculus in and around the lateral spinal nucleus (LSN). (8) L6–S1 spinal cord section from a parturient rat showing colabeling for Fos immunoreactivity and ER-a immunoreactivity. Fosir neuron (green nucleus), ER-a-ir neuron (red nucleus), and double-labeled Fos þ ER-aimmunoreactive neurons (yellow-orange nucleus) are evident in the sacral parasympathetic nucleus (SPN). This suggests that presumed uterine-related neurons are activated at parturition (Fos positive) and can respond to estrogen (ER immunoreactive). (9) Schematic diagram illustrating the spinal cord, uterine cervix, sensory ganglia, and parasympathetic ganglia and their connections. Represented are hypothesized circuits and connections of neurons related to the uterine cervix that may express ERs (red dots). Primary aVerent neurons in dorsal root ganglia (DRG and nodose ganglia) are stimulated by contractions and the fetus creating cervical pressure. These stimuli, via central processes of primary aVerent neurons, activate spinal neurons (which can be identified through Fos immunostaining) that project axons to higher centers (left spinal cord neuron) or neurons that project preganglionic autonomic axons (right spinal cord neuron) to pelvic paracervical ganglia (PG). PG neurons innervate target tissues in the cervix. Consequently, at parturition, spinal cord circuits honed by rising estrogen levels could be activated, and through additional connections, neuroendocrine reflex circuits, stress circuits, and descending influences to the spinal cord could be stimulated to ensure an optimal delivery of young. In addition, autonomic and primary sensory neurons with axons in the cervix, also honed by estrogen, could also be activated to release transmitters locally in target tissues. DH, dorsal horn; DIG, dorsal intermediate gray (dorsal commissural area); LCP, lateral collateral pathway; LSN, area of the lateral spinal nucleus in the lateral funiculus; SPN, sacral parasympathetic nucleus; X, lamina X around the central canal. (10) Photomicrograph of the lateral collateral pathway (LCP) in the cord. This tissue was double immunostained for ER-a (red nuclei) and brain-derived neurotrophic factor (BDNF) (green fibers and terminals). ER-immunoreactive neurons are noted in the LCP where BDNF terminals (yellow and arrow) closely appose the neurons. (11) Photomicrograph of the lateral collateral pathway (LCP) merging with the sacral parasympathetic nucleus (SPN). This tissue was double immunostained for ER-a (green nuclei) and pituitary adenylate cyclase activating peptide (PACAP) (red fibers and terminals). ER-immunoreactive neurons are noted in the LCP and in the SPN where they are closely apposed by PACAP-immunoreactive terminals (arrows). (12) Photomicrograph of the area of the lateral collateral pathway (LCP). This tissue was double immunostained for ER-a (green nuclei) and neuropeptide tyrosine (NPY) (red fibers and terminals). ER-immunoreactive neurons are noted in the LCP where they are closely apposed by NPY-immunoreactive terminals (arrows).

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Langub et al., 1994; McEwen et al., 1991; McEwen, 1996), acting as a growth or survival factor (Matsumoto et al., 1988; Honjo et al., 1992; Gollapudi and Oblinger, 1999a,b; Patrone et al., 1999), increases the binding aYnity of oxytocin receptors (Caldwell et al., 1994), induces and accelerates neurotransmitter expression (Okamura et al., 1994a,b; Yuri and Kawata, 1994; Gangula et al., 2000a; Mowa et al., 2003a,b), and ERs may function as ligand-dependent nuclear transcription factors, to name a few. Few data are available about estrogen-responsive neurons in the lumbosacral spinal cord even though spinal neurons are involved in sexual behaviors (PfaV et al., 1994) and innervation of female genital organs (Schrøder, 1983, 1984; McKenna and Nadelhaft, 1986; Peters et al., 1987; Papka and Traurig, 1993; Papka et al., 1995a). Most current data indicate that estrogen exerts biological actions through interacting with nuclear ERs that belong to a superfamily of nuclear receptors. The steroid diVuses into the cell, binds to the ER, and the liganded ER translocates to the nucleus, where it dimerizes and activates an estrogen response element (ERE) of/on the target DNA leading to the modulation of the transcription of estrogen-regulated genes (Fig. 2) (Murdoch and Gorski, 1991). Two forms of the ER have been identified—the long established classic ER-a (Green et al., 1986; Greene et al., 1986; Koike et al., 1987) and the more recently cloned ER-b (Kuiper et al., 1996; Mosselman et al., 1996). Compared to ER-a, ER-b has relatively conserved DNA and ligandbinding domains but both receptors have similar aYnities and specificity for binding estradiol (Mosselman et al., 1996; Kuiper et al., 1997). Expression of these two receptor subtypes has been documented in multiple species and in various tissues, including in the brain, although the tissue distribution and level of expression vary (Mosselman et al., 1996; Kuiper et al., 1997; Shughrue et al., 1997, 1998). Moreover, gene transcription stimulated by ER-a and ER-b is similar, though ER-b has a lower degree of activation (Mosselman et al., 1996). ER-a and ER-b share many functional characteristics but molecular mechanisms regulating their transcriptional activity and tissue location are diVerent (Mosselman et al., 1996; Kuiper et al., 1997; Couse and Korach, 1998). In the cranial levels of the CNS, ER-a and ER-b show diVerential distributions (Shughrue et al., 1997), although in the spinal cord their distribution is similar (Papka et al., 2001; Shughrue and Merchenthaler, 2001) (Diagram 1). Evidence is accumulating that shows diVerent roles for ER-a and -b in estrogen-responsive tissues. Moreover, ER-a, the ‘‘classic’’ estrogen receptor, plays a critical role in the physiology of the female reproductive tract, development of secondary sex characteristics, reproductive behavior, and neuronal function (Giordano et al., 1991; Morrell et al., 1995; Wagner and Morrell, 1995, 1996; Shughrue et al., 1997; Nilsson and Gustafsson, 2002), however, the physiological role of ER-b is

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DIAGRAM 1 Top: Schematic illustration of the general distribution of ERs in the L6–S1 segments of the lumbosacral spinal cord of the female rat. The distribution of ER-a-immunoreactive neurons is shown on the left side and ER-b on the right side of the diagram. DH, dorsal horn; DIG, dorsal intermediate gray (dorsal commissural area); LCP, lateral collateral pathway; LSN, area of the lateral spinal nucleus in the lateral funiculus; SPN, sacral parasympathetic nucleus; X, lamina X around the central canal. Bottom: Schematic diagram of the dorsal one-half of the lumbosacral spinal cord. Shaded areas and rectangle with numbers correspond to the approximate sites that photographs in Figs. 3–8 and 10–12 were taken.

less well understood (Nilsson and Gustafsson, 2002). For example, ER-b is more expressed in the brain than ER-a (Kuiper et al., 1996). ER-b may function in the binding of phytoestrogens, which raises the question of whether ER-b is the ‘‘phytoestrogen receptor’’ (Kuiper et al., 1997, 1998). In addition, because ER-a and ER-b can form homodimers as well as heterodimers, it may well be that there are interactions between ER-a and b in more diverse functions. This is highlighted by the fact that there are specific actions of estrogen that can be attributed to one receptor but not the other.

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III. Estrogen Receptors in the Nervous System A. Estrogen Receptors in Spinal Cord As indicated previously, the initial reports of ERs in the spinal cord utilized binding of radiolabeled estradiol; moreover, examination of spinal cord tissue in such studies was sometimes secondary to a focus on the hypothalamus and limbic structures. PfaV (1968) first reported estradiol-concentrating cells in the cervical spinal cord; this report was followed by the studies of Keefer et al. (1973), PfaV and Keiner (1973), and later Morrell et al. (1982). Consequently, binding of radiolabeled estradiol in the spinal cord provided the first evidence for ERs in spinal cord neurons (Keefer et al., 1973; Morrell et al., 1982) and showed, in lumbar levels L4–6 and sacral segments, that most labeling was in the dorsal horn (mainly lamina II), midline region, and lamina X, some in the medial portions of lower dorsal horn laminae, but markedly in the lateral portions of laminae III–V and VII. No estradiol-concentrating cells were evident in the ventral horn or white matter. This pattern is consistent with that shown by immunohistochemistry (vida infra). The capacity for synthesis of ERs by spinal cord neurons was demonstrated by Simerly et al. (1990) using in situ hybridization techniques to enhance signals for ER mRNA. Though the emphasis of that study was the detection of the ER mRNA signal largely in the forebrain and brainstem, ER mRNA expression patterns were mapped throughout the CNS. Simerly et al. (1990) pointed out high hybridization signal in areas involved in visceral sensory processing including the dorsal horn (lamina II), central gray areas, and sacral parasympathetic neurons of the spinal cord (and in the brainstem — the nucleus tractus solitarius and lateral parabrachial nuclei). Based on these data (and immunohistochemical data outlined below), it appears that neurons involved in visceral sensation and autonomic functions are sensitive to sex steroids. Also, like Morrell et al. (1982), Simerly et al. (1990) did not detect labeling in somatomotor ventral horn neurons. However, a later study by VanderHorst et al. (1997) did localize ER-immunoreactive neurons in the ventral horn (including some that projected axons to brainstem levels). Amandusson et al. (1995) and Williams and Papka (1996) used immunohistochemistry and antibodies against ER protein to obtain greater cellular resolution and demonstrate nuclear localization of ER immunoreactivity in spinal cord neurons. These studies predated the cloning of ER-b, and, it is quite certain based on the characterization of the antibodies employed, that they demonstrated the classic ER-a isoform of the ER. Amandusson et al. (1995) reported labeling for ER in the cervical, thoracic, and lumbar levels with little in the sacral cord levels, whereas Williams and Papka (1996) restricted their study to the L6–S1 segments. Like the receptor binding and

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in situ hybridization studies, immunohistochemistry showed significant numbers of labeled neurons in sensory dorsal horns (DH) (Amandusson et al., 1995; Williams and Papka, 1996), largely Rexed’s laminae II, in the central gray and around the central canal (lamina X). In addition, abundant ER-a-immunoreactive nuclei were evident in the autonomic and projection neurons of the sacral parasympathetic nucleus (Williams and Papka, 1996), in neurons interspersed along the lateral collateral pathway (LCP), and in the dorsal intermediate gray (DIG) dorsal to the central canal (Williams and Papka, 1996; Papka et al., 2001) (Diagram 1 and Fig. 3). An interesting pattern of ER-expressing neurons is evident in the lateral part of the DH, in the lateral funiculus (LF), interspersed among the fibers of the LCP (Figs. 3–5, 10, and 12), and in the lateral spinal nucleus (LSN) (Williams and Papka, 1996; Papka et al., 2001)(Figs. 3–5). Such neurons are interspersed among primary aVerent fibers, many of which carry visceral sensory information (Neuhuber, 1982), that are destined for connection with neurons in the SPN (Figs. 4 and 11). Some ER-positive neurons are also among other fibers in the lateral funiculus near the LSN. This could be an important distribution of ER-expressing neurons as the LSN is considered to be a collection of neurons involved in processing of nociceptive information (Jiang et al., 1999). Moreover, neurons here express receptors for substance P (Battaglia and Rustioni, 1992; Ding et al., 1995; J. L. Li et al., 1997; Marshall et al., 1996), neuropeptide Y (Zhang et al., 1995), and cannabinoids (Salio et al., 2002), and coexpress receptors for substance P and glutamate (Benoliel et al., 2000). In addition, neurons in the LSN express calcitonin gene-related peptide (Conrath et al., 1989) and vasoactive intestinal peptide (Fuji et al., 1983; Leah et al., 1988; Sasek et al., 1991), are shrouded by terminals containing substance P, dynorphin 1–8, and met-enkephalin (Giesler and Elde, 1985), and are in a pathway for central projection of primary aVerent fibers (Menetrey et al., 1980; Neuhuber, 1982). Some of these neurons project axons to supraspinal sites including the hypothalamus (Burstein et al., 1987), thalamus and periaqueductal gray (Battaglia and Rustioni, 1992; Harmann et al., 1988), nucleus tractus solitarius (Esteves et al., 1993), as well as spinal sites (Jansen and Loewy, 1997). A rostrocaudal decrease in numbers of ER-immunoreactive neurons was reported from cervical cord segments to sacral cord segments with lack of ERimmunoreactive neurons caudal to S1 (Amandusson et al., 1995). Moreover, a crude estimate predicted about 300,000 ER-immunoreactive neurons in the superficial dorsal horn of the spinal cord and nucleus caudalis of the medullary spinal trigeminal tract (Amandusson et al., 1995). In sum, the distribution of ER-expressing neurons is largely in areas occupied by sensory processing neurons (superficial dorsal horn, neck of the dorsal horn/lateral spinal nucleus, and lamina X), autonomic neurons (SPN), projection neurons (dorsal intermediate gray, lamina X, and dorsal SPN—Willis and Coggeshall 1991; Hamilton et al., 1995). Moreover, the fact that many ERa-containing neurons

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in the dorsal horn coexpress preproenkephalin mRNA (Amandusson et al., 1996) strengthens the idea that estrogen could influence sensory processing at the level of enkephalinergic neurons of the spinal dorsal horn. Recent data (Shughrue et al., 1997; Papka et al., 2001; Shughrue and Merchenthaler, 2001) have shown ER-b immunoreactivity and mRNA in female rat spinal cord neurons. In large measure the distribution of ER-b neurons parallels that of neurons expressing ER-a with the following exceptions: there was an impression of more ER-b neurons in the deeper laminae of the spinal cord than was evident for ER-a neurons (Papka et al., 2001) and some ER-b neurons were evident in the ventral horn (Shughrue et al., 1997). In nearly all respects, in situ hybridization histochemistry has shown that neurons expressing the mRNA for ER-a and ER-b paralleled the pattern of neurons expressing the respective translated ER proteins (Papka et al., 2001; Shughrue et al., 1997). Analysis of the rostrocaudal distribution of ER-b-immunoreactive neurons revealed a similar distribution at cervical, thoracic, and lumbar levels (Shughrue and Merchenthaler, 2001). The above data were obtained from intact and ovariectomized rats. 1. Changes in Estrogen Receptors in the Spinal Cord over Pregnancy Serum estrogen levels change over the estrous cycle and peak during proestrous; there is a coincidental trend toward increasing concentrations of ERs with increasing estrogen levels during the cycle (Williams et al., 1997). Blood levels of estrogen also gradually rise over the course of pregnancy with a peak at about Day 20 of gestation and a concomitant decrease in progesterone levels (Bridges, 1984; Lye et al., 1993; Shaikh, 1971). This raises an important question: ‘‘are there changes of ERs over the course of pregnancy’’? Western blot analysis detected a single band of approximately 66 kDa in the spinal cord with a trend toward increasing levels of ER-a protein from Day 10 of pregnancy to the highest level of expression at Day 20 of gestation and a decrease thereafter (Papka et al., 2002). Parallel data indicated that there was a corresponding increase in numbers of neurons expressing ER-a in the dorsal one-half of the cord with a peak at Day 20–22 of pregnancy (number of neurons/40-mm-thick section ¼ approximately 12 on Day 10, 19 on Day 15, 21 on Day 20, 29 at parturition, and 24 at 2 days postpartum) (Papka et al., 2002). Because ERs are critical for mediating the actions of estrogen in target cells and the total amount of ER available to bind estrogen is a crucial factor in regulating estrogen eYcacy at the target cell and organ, these data suggest that increased biosynthesis of ER in target tissues enhances estrogen’s action during pregnancy and labor. Consequently, rising estrogen levels during pregnancy could influence, via the ER signaling pathway, the physiology, metabolism, and/or neurochemistry of sensory and autonomic spinal neurons. For example, sex steroids modulate neurochemical systems

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in the brain (DeVries, 1990) and especially the synthesis and expression of neurotransmitters in the hypothalamus (Akaishi and Sakuma, 1985; Alexander and Leeman, 1994; Gabriel et al., 1990; Yuri and Kawata, 1994). Serum estrogen rises over the course of pregnancy peaking at about Days 20–22 (parturition) (Bridges, 1984; Shaikh, 1971; Lye et al., 1993) as does ER, ER mRNA, and estrogen sensitivity of certain hypothalamic neurons (Morrell et al., 1995; Wagner and Morell, 1995, 1996; Giordano et al., 1991). Moreover, these changes in receptor levels and ligand might be a mechanism by which neurons become more sensitive to rising levels of estrogen near parturition. It follows that ER-containing (estrogenresponsive) spinal neurons might also undergo neurochemical, ER, and/or functional changes with rising estrogen levels near parturition since this is a time when it would be advantageous for the sensitivity and activity of spinal neurons to be heightened. Along this line, certain neurotransmitters show a cyclic (with estrogen level) variability in the spinal cord, e.g., oxytocin and vasopression (Miaskowski et al., 1987) and galanin (Newton, 1992). Newton (1992) also showed ‘‘heavier’’ immunoreactivity for galanin in lumbosacral spinal cord autonomic areas in proestrous rats vs. rats in metestrous or diestrous. The physiological eVect(s) of the transmitter variations is not fully understood, but this is an important area for future research. However, some important functional implications have been evolving over some years with regard to sex steroids, pregnancy, estrous cycle, and antinociception that have come largely from Gintzler’s laboratory. For example, pain thresholds are elevated during gestation (gestational analgesia) and following the hormonal simulation (manipulating levels of estrogen and progesterone) of pregnancy and these are opioid mediated through spinal k and d opioid receptors (Dawson-Basoa and Gintzler, 1996, 1998). 2. Neural Connection to Pelvic Reproductive Organs: Uterus and Cervix Several axonal tracing studies (Inyama et al., 1986; Papka et al., 1995a; Nadelhaft and Vera, 2001; Nadelhaft et al., 2002) showed that pelvic organs have sensory and autonomic connections with the thoracolumbar and lumbosacral spinal cord. Marked attention has been directed to the lumbosacral levels and particularly the L6–S1 segments, because of interest in urinary bladder and reproductive organ reflexes, as well as innervation of the uterine cervix related to parturition. Consequently, sizable data regarding ERs in the female rat spinal cord and dorsal root ganglia (DRG) have been derived from studies at the lumbosacral levels. Neurons expressing ER-a and ER-b in the L6–S1 spinal cord of ovariectomized rats (Williams and Papka, 1996; Papka et al., 2001; Shughrue and Merchenthaler, 2001) and pregnant rats (Papka et al., 2002) are topograph-

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ically distributed in areas densely supplied with central processes of primary aVerent neurons (Papka et al., 2001, 2002). This pattern of distribution of ER neurons was very similar to the pattern of neurons expressing the protein Fos (expression of the protein product Fos of the immediate early gene c-fos is commonly used as a marker of synaptically activated neurons) after vaginocervical–mechanical stimulation (VCS) or parturition stimulation and representing, in part, ‘‘uterine-cervical-related’’ neurons (Chinapen et al., 1992; Williams and Papka, 1996; Puder and Papka, 2001, 2002a,b) (Figs. 6, 8, and 9). This information led to the postulate that some Fosexpressing uterine–cervical neurons of the spinal cord express ER and can respond to circulating estrogens. Indeed, based on double immunostaining experiments, some uterine–cervical neurons express, at least, ER-a (Papka et al., 2002) (Fig. 8). These uterine–cervical-related neurons (expressing Fos) and containing ER-a were detected in the DH, DIG, LSN, LCP, and SPN (Fig. 8). Moreover, many of the central terminals of primary aVerent neurons located in the DRG send axons closely associated with ER-a-expressing neuron in the DH, DIG, LSN, LCP, and SPN (Papka et al., 2002) (vide infra). Because ER-a plays a critical role in the physiology of the female reproductive tract and in reproductively important nuclei and circuits in the CNS (Giordano et al., 1991; Morrell et al., 1995; Wagner and Morrell, 1995, 1996; Shughrue et al., 1997; Nilsson and Gustafsson, 2002), one could extrapolate from this information and suggest that sensory neurons innervating the uterine cervix have central processes in the lumbosacral spinal cord that relay with neurons for first level integration of uterine information and these involve steroid hormone-responsive neurons (Figs. 8 and 9). In addition, some pre- and postganglionic autonomic neurons and sensory DRG neurons that innervate the uterus express ER (Williams and Papka, 1996; Papka et al., 2001). Moreover, VanderHorst et al. (1997) combined retrograde tracing with immunohistochemistry to examine whether spinal cord neurons that project axons to brainstem areas important for reproductive behavior also expressed ERs. They found a few lumbosacral spinal neurons (mainly in the medial ventral horn) that project axons to the periaqueductal gray. Though double-labeled neurons were not numerous, it is possible that estrogen can influence reproductive behaviors by working through this system. In a subsequent study, VanderHorst et al. (2001) again utilized retrograde tracing from the urinary bladder of the cat in combination with immunohistochemistry to demonstrate that some bladder preganglionic autonomic neurons also express ER-a [also sensory DRG neurons innervating the urinary bladder express ER-a and ER-b (Bennett et al., 2003)]. These details corroborate information indicating that estrogen influences the function of systems more than just the reproductive tract, e.g., the urinary system as well (Beach, 1974; Tansy and Kaufman, 1966; Blakeman et al., 2000; Johnson and Berkley, 2002). This information indicates that caution needs

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to be exercised when making interpretations about neurons expressing ERs and their role(s), specifically in reproductive circuitries. 3. Afferent Input to Estrogen Receptor-Expressing Neurons in the Spinal Cord Some of the ER-expressing neurons in the spinal cord, especially those in the DH, DIG, SPN, and LSN, are in ideal locations to receive input derived from the central terminals of primary aVerent neurons in lumbosacral DRG. Indeed, double immunostaining revealed that many sensory nerve fibers expressing various ‘‘sensory’’ neurotransmitters and receptors are apposed to ER-positive neurons. Thus, fibers and varicosities containing the neuropeptides substance P (SP) (Fig. 4), calcitonin gene-related peptide (CGRP) (Papka et al., 2002), secretoneurin (SN), pituitary adenylate cyclase-activating polypeptide (PACAP) (Fig. 11), neuropeptide Y (NPY) (Fig. 12), neurotrophin brainderived neurotrophic factor (BDNF) (Fig. 10), vanilloid receptor (VR1), and purine receptor P2X3 were closely apposed to ER-a-containing neurons in the DH (Fig. 4), DIG, SPN (Fig. 11), LCP (Figs. 4 and 10–12), and LSN (Fig. 4). This suggests that there is potentially a significant diversity of input to estrogenresponsive neurons in the spinal cord (moreover, many of the DRG neurons themselves also express ERs—see below). In this regard, the neuropeptides released from such inputs certainly have the potential for modifying the activity (or the phenotypic expression of neurochemicals) in ER-expressing spinal neurons. NPY and BDNF may exist in central terminals of aVerent neurons that change their phenotypic expression in conditions that induce a plastic response (Carr and Undem, 2001; Galeazza et al., 1995; Hunter et al., 2000). SP and CGRP are expressed in sensory neurons involved in relaying visceral information from the internal organs to the spinal cord, e.g., pain and/or distension (Traurig et al., 1988; Papka et al., 1995b; Gebhart, 1995; Cervero, 1994) (as well as being released peripherally—see below). PACAP (Narita et al., 1996) and BDNF (Kerr et al., 1999; Thompson et al., 1999) have been suggested to play role(s) in sensory processing such as pain as well. 4. Connection to Supraspinal Sites Spinal cord neurons can be involved either in local circuits where sensory information is processed or in transmitting information rostrally to supraspinal sites in the brainstem and diencephalon. Consequently, it is important to characterize neural circuits linking the reproductive organs, spinal cord, and supraspinal sites, e.g., hypothalamus and brainstem, which are functionally important in physiological situations such as parturition. Sensory information from the uterine cervix (derived from mechanical vaginocervical stimulation, as a model, and from a fetal pup traversing the cervix at parturition as a physiological stimulus) enters the lumbosacral spinal cord

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and activates neurons (as evidenced by expression of the protein product Fos of the immediate early gene c-fos). The anatomical distribution of Fos-immunoreactive neurons, demonstrated by immunohistochemical methods (Chinapen et al., 1992; Williams and Papka, 1996; Papka et al., 2002; Puder and Papka, 2001, 2002a,b, 2003), is in a pattern that is reminiscent of the distribution of ERs—in the DH, LSN, LCP, SPN, DIG, and lamina X. Further, combining Fos staining with retrograde tracing [using the tracer Fluorogold (FG)] from the hypothalamus indicated that uterine– cervical-related neurons are present in the lumbosacral spinal cord and project axons to the hypothalamus (Puder and Papka, 2001, 2003). Neurons double labeled for both Fos and FG were scattered throughout the same areas as the neurons immunoreactive for either Fos or ER (Puder and Papka, 2003). Interestingly, there appeared to be a consistent labeling and concentration of hypothalamus-projecting (FG-labeled), Fos-positive neurons in lamina X, the dorsal part of the SPN and extending into the adjacent lateral funiculus and dorsally to the lateral aspect of the dorsal horn in that area described as the LSN (Gwyn and Waldron, 1968, 1969). These are areas occupied, in part, by neurons processing and projecting visceral information to higher centers (lamina X: Wall et al., 2002; Nahin et al., 1983; Honda, 1985; LSN: Leah et al., 1988; Menetrey et al., 1980; SPN: Giesler et al., 1994; Hamilton et al., 1995). Furthermore, the area of the LSN (lateral dorsal horn and adjacent LCP and lateral funiculus) has ER-a- and ER-b-expressing neurons (Papka et al., 2001) and primary aVerent nerves course through here (Neuhuber, 1982; McNeill et al., 1988; Papka et al., 2001, 2002). Thus, this topographic overlap of uterine-responsive neurons, spinohypothalamic neurons, ER-expressing neurons, and central branches of primary aVerent nerves is suggestive of probable processing of uterine-related information and projection to supraspinal levels (note: it should be recognized that there is some controversy as to whether sensory fibers occur and what sensory processing occurs in the LSN: Menetrey et al., 1982; CliVer et al., 1988; Giesler and Elde, 1985; Conrath et al., 1989). Additionally, some neurons that were double labeled for Fos and ER-a were present in the LCP and SPN (Papka et al., 2002) (Fig. 8). These data suggest that some uterine–cervical-related spinal neurons could be responsive to estrogen, and project axons to the parturition-important hypothalamus (presumably the paraventricular nucleus). 5. Estrogen Receptors in the Male Spinal Cord Most attention to ERs has been directed to the spinal cord of female rats, primarily because of interest in the possible links between estrogen, spinal neurons and reproductive behaviors, and innervation of genital organs. Few studies have addressed ERs in the spinal cord of males. Simerly et al. (1990) used both male and female rats in their in situ hybridization study of ER and

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androgen (AR) receptor mRNAs, but did not find any obvious sexual dimorphic diVerences with ER mRNA. One recent study focused on developmental expression of ER-a and reported some interesting results. This report showed a pattern of distribution of ER-a-expressing neurons similar to that in adult female rats, but with a few exceptions (Burke et al., 2000). In the lumbosacral spinal cord ER-a immunoreactivity was observed initially, and transiently, in ependymal cells. Moreover, ER-a immunoreactivity was described in nerve fibers and perikaryal cytoplasm, unlike the situation in the adult female rat spinal cord. Such immunoreactive fibers were abundant in the outer dorsal horn laminae (I and II), around the central canal and in the SPN, DIG of the sacral cord, and sympathetic nuclei (IML). With early postnatal, and continued development of the cord, the distribution of ER-a-immunoreactive fibers remained dense in the central autonomic area (lamina X around the central canal), but decreased in the SPN and IML. In the L6–S2 cord segments, neuronal nuclei were evident in many sites similar to those seen in adult female rats (Amandusson et al., 1995; Williams and Papka, 1996; Papka et al., 2001). These included the SPN, some in the outer dorsal horn, and at the border of the gray–white matter at the base of the dorsal horn. Those fibers in the dorsal horn were eliminated by dorsal rhizotomy indicating their origin from central processes of DRG neurons (Burke et al., 2000). Moreover, as early as embryonic Day 21, ER-immunoreactive neurons formed a subset of neurons in the DRG and are presumably the source of fibers in the superficial dorsal horn (Burke et al., 2000). Thus, ERs are not only present in neurons of the spinal cord, as in the adult, but also in the central processes of nerve fibers. The fibers were largely in the superficial dorsal horn and autonomic areas; these findings lead to the suggestion that, developmentally, estrogen may play a role in maturation of spinal circuitry (Burke et al., 2000).

B. Estrogen Receptors in Sensory Neurons With regard to the peripheral nervous system, it has been suggested as long as seven decades ago that hormones could aVect peripheral sensory systems (Nissen, 1929). Though numerous studies showed hormonal eVects on reproductive behaviors such as lordosis, these were mainly focused to CNS circuits. Direct data were not forthcoming until the late 1960s and early 1970s when classic studies by Komisaruk et al. (1972) and Kow and PfaV (1973/74) reported that estrogen treatment of ovariectomized rats enlarged the peripheral sensory fields of the genital nerves such as the pudendal nerves. Subsequent studies showed physiological variations across the estrous cycle of the sensitivity to stimulation of uterine and vaginal nerves (Adler et al., 1977; Berkley et al., 1990; Robbins et al., 1990, 1992; Bradshaw et al., 1999). For example, uterine and vaginal nerves had lower thresholds to stimulation

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when serum estrogen levels were high during proestrous and early estrous. Such studies opened the gates for morphological studies of ER expression in sensory neurons that would strongly implicate estrogen in controlling the structural and functional attributes of certain nerves. Initial studies of ERs in sensory neurons reported ER mRNA in all neurons of DRG (Sohrabji et al., 1994b), though others suggested that ER and ER mRNA were localized to a select population of DRG neurons (Oblinger et al., 1994; Papka et al., 1997; Bennett et al., 2003) and vagal neurons in the nodose ganglia (NG) (Collins et al., 1999; Papka et al., 2001). Thus, one could deduce that the expression of ER in these sensory neurons would imply that estrogen could influence activity/neurochemistry of these neurons and their peripheral innervation apparatus in visceral organs (Fig. 9) (Papka and Traurig, 1993; Traurig and Papka, 1993; Bennett et al., 2003; Mowa et al., 2003a,b). Indeed, not only does the sex steroid estrogen influence peripheral sensory ganglion neurons and axons by increasing the size of the genital receptive field and lowering thresholds to stimulation, but estrogen reportedly aVects sensory processing (Amandusson et al., 1995; Gandelman, 1983) and alters the expression of neurotrophin receptors (Sohrabji et al., 1994a,b) and apoptosis-regulating genes (Gollapudi and Oblinger, 1999a,b; Patrone et al., 1999). A subpopulation of sensory neurons in the L6–S1 DRG (about 30%) and vagal nodose ganglia are immunoreactive for ER-a and ER-b and express ER mRNAs (Papka et al., 1997, 2001; Papka and Storey-Workley, 2002; Bennett et al., 2003). In addition, subpopulations of neurons in the L6–S1 DRG project axons to the uterus, cervix, and vagina (Inyama et al., 1986; Papka et al., 1995b, 2001) or urinary bladder (Bennett et al., 2003), and increasing evidence suggests that the vagus nerve also has connections to the reproductive organs (Fig. 9) (Burden et al., 1981; Ortega-Villalobos et al., 1990; Cueva-Rolon et al., 1996; Komisaruk et al., 1996; Collins et al., 1999). Thus, because some ER-containing ganglionic neurons project axons to reproductive endorgans and urinary bladder and are responsive to the sex steroid estrogen, we speculate that estrogen might directly influence the sensory (and autonomic, see below) neural input to the uterus, cervix, and bladder, as well as the physiological responses of these fibers to stimuli. However, at this time we cannot state that only those sensory neurons that project to genital–urinary organs contain ER as opposed to those that innervate the distal colon or other sites. Studies from several laboratories indicate that sensory neurons are capable of responding to circulating estrogens and some of their properties and metabolism may be influenced by the sex steroid. In addition, substantial data indicate that estrogen increases during pregnancy (Bridges, 1984; Lye et al., 1993; Shaikh, 1971). Steroid hormone treatment (acute) promotes the synthesis of neuropeptides in DRG neurons including CGRP (Gangula et al.,

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2000a,b; Lanlua et al., 2001; Mowa et al., 2002, 2003b) as well as substance P (SP) (Mowa et al. 2002, 2003a). Mowa et al. (2002, 2003a,b) also showed that ERs are expressed with the neuropeptides CGRP and SP in DRG neurons. Moreover, levels of the mRNA and protein for these neuropeptides increase over pregnancy, possibly in conjunction with rising estrogen levels (Mowa et al., 2002, 2003a,b). Treatment with the ER antagonist ICI 182 780 blocks the estrogen-stimulated up-regulation of SP (Mowa et al., 2002, 2003a) and CGRP synthesis (Mowa et al., 2002, 2003b). It is interesting that acute (hours to a few days) estrogen stimulation increases CGRP immunoreactivity (Gangula et al., 2000a,b; Lanlua et al., 2001; Mowa et al., 2002, 2003b) and SP immunoreactivity (Mowa et al., 2002, 2003a). On the other hand, it appears that long-term (30–90 days) estrogen treatment decreases b-PPT (substance P) mRNA in DRG (Liuzzi et al., 1999b) and numbers of CGRP-immunoreactive neurons in DRG (Yang et al., 1998). The a and b isoforms of ER can be expressed separately or coexpressed in the same DRG neurons (Papka et al., 2001; Papka and Storey-Workley, 2002; Bennett et al., 2003). Emerging data from our laboratory (Papka et al., 2000; Mowa et al., 2002, 2003a,b) and others (Bennett et al., 2003) indicate that some SP- and some CGRP-immunoreactive neurons express both ER-a and others express ER-b (whether the same SP or CGRP neuron expresses both isoforms has not be reported). Moreover, there is a diverse neurochemical phenotype of neurons, in addition to those expressing CGRP and/or SP, in sensory ganglia, which also express ERs, and these are only beginning to be unraveled—e.g., neurokinin 1 receptor (SP receptor) þ ER (Fig. 13), secretoneurin (SN) þ ER (Fig. 14), nitric oxide synthetase (NOS) þ ER, brain-derived neurotrophic factor (BDNF) þ ER, and pituitary adenylate cyclase-activating peptide (PACAP) þ ER. Many of the neurons expressing other phenotypes such as galanin, somatostatin, and cholecystokinin have not yet been explored in terms of their content of ERs. This is an area of importance for future studies. On the basis of (1) estrogen’s stimulation of an increase (in a dose-dependent manner) in the synthesis of SP and CGRP in DRG, an eVect blocked by an ER antagonist (Mowa et al., 2002, 2003a,b), and (2) synthesis of SP and CGRP tended to increase over the course of pregnancy in concert with the rising levels of estrogen (Mowa et al., 2002, 2003a,b), we suggest that estradiol may be influencing the genome of sensory neurons of DRG via the ER-signaling pathway. Thus, this substantiates that estradiol can aVect peripheral sensory mechanisms. It should be noted that the eVects of estradiol could be at the level of the spinal cord (some dorsal horn neurons are ER-ir) (Amundusson et al., 1995; Williams and Papka, 1996; Papka et al., 2001, 2002), DRG or nerve ending (influencing sensitivity or number of endings), and the eVects could be cyclic with varying levels of estrogen (Adham and Schenk, 1969; Adler et al., 1977; Berkley et al., 1988; Robbins

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FIGS. 13–18 (13) Photomicrograph of the L6–S1 dorsal root ganglia (DRG) double immunostained for ER-a (red nuclei) and substance P neurokinin 1 receptor (NK1) (green cytoplasm). Some neurons express NK1 receptor (green arrow), some express ER-a (red arrow), and some express both NK1 þ ER-a (yellow arrow). ER-a- and NK1-immunoreactive neurons were mainly medium sized (about 20–40 mm diameter) neurons. (14) Photomicrograph of the L6–S1 dorsal root ganglia (DRG) double immunostained for ER-a (green nuclei) and secretoneurin (SN) (red cytoplasm). Some neurons express SN (red arrow) and some express both SN þ ER-a (yellow arrow). The double-labeled neurons appeared to be mainly in the

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et al., 1992; Brauer et al., 1995; Marshall, 1981). Furthermore, the majority of ER-ir DRG neurons are small- to medium-size cells; small-sized and some medium-sized neurons are sensitive to the sensory neurotoxin capsaicin (Papka and Traurig, 1989; Traurig et al., 1988). In addition, many of these neurons express a receptor for capsaicin (vanilloid receptor-1, VR-1) (Papka et al., 2001; Bennett et al., 2003), and are representative of visceral aVerent nociceptive neurons. Thus, these neurons could carry sensory information from pelvic organs such as pain, neuroendocrine reflexes, and bladder distention, and may well be responsive to sex steroids. Furthermore, estrogen also influences other aspects of neuronal structure and function besides transmitters. Long-term estrogen treatment up-regulates neurofilament gene expression (Scoville et al., 1997). In cultured cells, ER-a activation increased length and number of neurites, whereas activation of ER-b modulates only neurite elongation (Patrone et al., 2000). Sohrabji et al. (1994b) showed that ER mRNA and ER protein (indicative of binding sites) were present in adult rat DRG and that estrogen modulates the nerve growth factor (NGF) receptor in these neurons. Consequently, estrogen could be involved in mediating NGF-dependent regulation of neuronal plasticity and repair (Scoville et al., 1997; Liuzzi et al., 1999a). Moreover, 17 b-estradiol, working through ERs, increased the survival of cultured DRG neurons and ER-a-transfected PC12 cells deprived of NGF by upregulating antiapoptotic molecules (Patrone et al., 1999; Gollapudi and Oblinger, 1999a,b). Thus, estrogen and ERs are important players in the development and survival of NGF-dependent neurons. In this regard, many peripheral sensory (and autonomic) neurons are NGF dependent (Barde, 1989; Mendell, 1994) and some of these nerves in the uterus degenerate with pregnancy and regenerate postpartum (Alm et al., 1986, 1988a,b; Fried et al., 1985; Marshall, 1981; Owman, 1981; Stjernquist et al.,

small to medium size range (>20–40 mm diameter). (15) Photomicrograph of the pelvic paracervical ganglion (PG) double immunostained for ER-b (green nuclei) and nitric oxide synthase (NOS) (red cytoplasm). Some neurons express ER-b (green arrows) and some express both ER-b þ NOS (yellow arrows). (16) Photomicrograph of the pelvic paracervical ganglion (PG) double immunostained for ER-b (green nuclei) and choline acetyltransferase (ChAT) (red cytoplasm). Some neurons express ER-b (green arrow) and some express both ER-b þ ChAT (yellow arrows). (17) Photomicrograph of the L6–S1 dorsal root ganglia (DRG) immunostained for the androgen receptor (AR) (green nuclei). A subpopulation of neurons is AR immunoreactive (arrow) and appears to be mainly small neurons (i.e., >20 mm). (18) Photomicrograph of the lumbosacral spinal cord illustrating part of the distribution of progesterone receptor (PR)-expressing neurons (arrows). (a) PR-immunoreactive neurons in the dorsal intermediate gray (DIG); (b) PR-immunoreactive neurons in the sacral parasympathetic nucleus (SPN); and (c) PR-immunoreactive neurons in the dorsal horn (DH). Preliminary studies suggest the distribution of PR-expressing neurons is similar to that of ER-expressing neurons.

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1985; Thorbert et al., 1978). Because current data indicate that some uterinerelated neurons contain ER (Papka et al., 2001), it is feasible that estrogen could bind directly to and influence the NGF-dependent plasticity of such neurons (also, see below regarding autonomic nerves). Furthermore, the presence of ER mRNA in sensory (and autonomic) neurons suggests that the receptor is synthesized by such neurons. Because neurons in sensory (and autonomic) ganglia contain the message for ER synthesis, as well as ER protein, it is likely that estrogen can shape functional aspects of the neurons such as transmitter synthesis, receptor production, plasticity, or receptivity. As indicated previously, a and b isoforms of ER exist, are expressed in neurons in general and in sensory DRG neurons in particular (Patrone et al., 2000; Papka et al., 2001; Bennett et al., 2003), and may even be coexpressed in the same DRG neuron (Papka and Storey-Workley, 2002; Bennett et al., 2003). Furthermore, ER-a and ER-b appear to be regulated diVerentially in DRG neurons (Taleghany et al., 1999) with long-term estrogen treatment of ovariectomized rats down-regulating levels of ER-a mRNA, but upregulating levels of ER-b mRNA. Such information opens up a wide range of possibilities regarding the function of ER-a and ER-b in sensory neurons. Along this line, Shughrue et al. (1997) revealed diVerential distributions of ER-a and ER-b mRNAs in the rat CNS suggesting there is region-specific expression of either or both forms of the ER. This diVerential regional distribution of ER-a and -b in the CNS may help explain how estrogenic compounds have specific regulatory properties and elicit diVerent functional eVects (Patisaul et al., 1999; Shughrue et al., 1997, 1998). In addition, ER-a and ER-b can form heterodimers as well as homodimers (Fig. 2), and the activity of such heterodimer complexes may diVer from ER-a or ER-b homodimers (Crowley et al., 1997). These data add another dimension to the level of influence of estrogenic compounds in nerve tissue since their activity may depend upon a cell’s content of ER-a, ER-b, or both subtypes of ER. Extrapolating these ideas and information from the CNS studies (and about ERs in general) to the sensory neurons in the PNS, it is possible that sensory neurons expressing diVerent isoforms and combination of isoforms of the ER could project to diVerent organs, participate in diVerent modalities or changes in sensitivity of peripheral nerves and receptive fields (Komisaruk et al., 1972; Kow and PfaV, 1973/74), or have diVerent neurochemistries and diVerent functions. Recent data support this concept with regard to sensory neurons and cervical ripening. Small- and medium-sized sensory neurons in L6–S1 DRG express one or both ER isoforms (Papka et al., 2001; Papka and Storey-Workley, 2002). Mowa et al. (2002, 2003a) revealed that expression of ER-a mRNA is up-regulated during pregnancy, whereas ER-b remains largely unchanged implying that ER-a may play a dominant role during pregnancy and possibly mediate up-regulation of synthesis of

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the neuropeptides SP and CGRP. Moreover, 17b-estradiol stimulated SP and CGRP mRNAs synthesis in ovariectomized rats, an eVect blocked by pretreatment with an ER antagonist. This suggests that SP and CGRP synthesis in the L6–S1 DRG during pregnancy may be regulated by the estrogen–ER signaling pathway and predominantly ER-a (Mowa et al., 2002, 2003a,b). These types of data are only beginning to emerge and this is an area important for future attention.

C. Estrogen Receptors in Autonomic Neurons Data are only beginning to accumulate about ERs and estrogen eVects in peripheral autonomic ganglionic neurons. The pelvic paracervical parasympathetic ganglion (PG) (of Frankenhauser) has garnered attention in this area because of its involvement in the innervation of pelvic organs, including the female reproductive tract. The superior cervical sympathetic ganglion (SCG) has also been utilized for studies of eVects of estrogen on development, as it is a convenient model ganglion. Based on findings from several studies, neurons in the PG are responsive to circulating estrogen: (1) the volume of neurons in the PG (and the ganglion itself) varies with the estrous cycle in the female guinea pig, decreases after ovariectomy, and is restored by estrogen treatment (Coujard, 1951); (2) [3H]estradiol binds to a subpopulation (20–30%) of autonomic neurons in the mouse (PG) (uterine cervical genital ganglion) (Schleicher et al., 1985); (3) ER protein and ER mRNA have been detected in female rat PG (Papka et al., 1997, 2001); and (4) chronic estrogen treatment stimulates an increased density of cholinergic nerves in the rat uterus (Richeri et al., 2002). Moreover, Papka et al. (2001) demonstrated that both ER-a and ER-b isoforms are expressed in the female rat PG neurons and it is established that subpopulations of neurons in the PG project axons to the uterus, cervix, and vagina (Fig. 9) (Papka et al., 1995b). On the other hand, Makela et al. (2000) examined the male counterpart of the PG (which lies adjacent to the prostate gland). They found only ER-b mRNA expression in the neurons of the prostatic pelvic plexus of male rats, as well as the prostate gland and lower urinary tract. Consequently, Makela et al. (2000) suggested that based on diVerential expression of ER-a and ER-b in the lower urinary tract and accessory sex glands that each subtype may have a specific function. With regard to the sympathetic nervous system, a comprehensive study of ER-a and -b showed about 29% of sympathetic neurons at large (in the SCG, paravertebral chain ganglia, prevertebral ganglia ¼ celiac, superior mesenteric, and suprarenal) express ER-a whereas about 92% express ER-b (Zoubina and Smith, 2002). Some of the aforementioned sympathetic ganglia

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are not involved with innervation of reproductive organs, e.g., SCG, but are still responsive to estrogen (Wright and Smolen, 1983, 1985, 1987) (see below). However, Zoubina and Smith (2002) showed with retrograde tracing that of sympathetic neurons that innervate the uterus, about three times more than in the general population express ER-a and a small amount more express ER-b. On the other hand, a considerably higher proportion of autonomic neurons that project axons to the lower urinary tract and male accessory sex organs express ER-b than ER-a (Makela et al., 2000; Zoubina and Smith, 2003). Thus, it would be of interest, and important, to investigate if there is a pattern of predominance of ER-b expression in neurons innervating urinary structures vs. a predominance of ER-a expression in neurons projecting axons to reproductive organs. In any case, based on the fact that both parasympathetic and sympathetic neurons innervate reproductive organs and lower urinary tract structures and express ERs, it could be surmised that estrogen could influence structural and functional aspects of these neurons, such as activity and transmitter expression. Indeed, a series of studies by Brauer’s group and others showed chronic estrogen exposure reduces the density of sympathetic noradrenergic nerves in the uterus, but increases the density of parasympathetic cholinergic nerves, and the presence of high levels of estrogen (i.e., at late pregnancy and at estrous phase of the estrous cycle) reduces uterine levels of noradrenaline (Brauer et al., 1992, 1995, 1998; Zoubina et al., 1998; Haase et al., 1997; Cha´ vez-Genero et al., 2002; Richeri et al., 2002). These eVects of estrogen can occur, through ERs, either at the level of the ganglia or the uterus itself (Krizsan-Agbas and Smith, 2002). That estrogen and ERs are involved here is supported by the fact that in ER-a knockout mice there is a noradrenergic hyperinnervation of the uterus (Zoubina and Smith, 2001). Of interest also are the findings that (1) estrogen reduces uterine horn myometrial noradrenaline and noradrenergic nerves but increases uterine cervical noradrenaline (Brauer et al., 1995) and (2) prenatal exposure of pregnant animals to a synthetic estrogen (diethylstilbestrol) reduces, in the oVspring, the sympathetic innervation of the ovary, reduces the volume of the celiac ganglion, and reduces the number of total neurons and those expressing ER-a (Shinohara et al., 1998, 2000). As mentioned above, chronic estrogen treatment increases the volume of the PG and the density of uterine cholinergic nerves. Taken together, the aforementioned information suggests that the eVects of estrogen on the PNS occur through receptor-mediated mechanisms and that there may be diVerential eVects of estrogen in parasympathetic vs. sympathetic neurons—estrogen having enhancing eVects on parasympathetic nerves, but reducing the volume and numbers of sympathetic nerves that have connections with reproductive organs.

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Additional important data have been derived from the SCG as a model sympathetic ganglion in the sense that estrogen serves to rescue neurons destined for developmental degeneration and increases the numbers of neurons and synapses in the ganglion (Wright and Smolen, 1983, 1985). Many of the eVects outlined above could involve the action of neurotrophins since estrogen has been shown to influence the expression of neurotrophins (Bjorling et al., 2002) or neurotrophin receptors in neurons (Toran-Allerand, 1996; Sohrabji et al., 1994a,b). Taken together, current data indicate that some ER-containing ganglionic neurons project their axons to reproductive end organs and that some ganglionic neurons are responsive to the sex steroid estrogen and estrogen might well directly influence the neural input to the uterus and cervix. Moreover, autonomic neurons projecting to nonreproductive organs, e.g., accessory sex organs and lower urinary tract, also express ERs, particularly ER-b (Makela et al., 2000; Zoubina and Smith, 2003). Further, the pelvic autonomic ganglia of the male and female rat are sexually dimorphic in neuron number (Greenwood et al., 1985) and phenotype (Dail et al., 1975; Papka et al., 1987). The major pelvic ganglion (MPG) in the male, which is equivalent to the female PG, is dependent on androgens for development organization and transmitter synthetic enzymes (tyrosine hydroxlyase and choline acetyltransferase) (Melvin and Hamill, 1987, 1989) and the innervation density of target organs of these neurons is androgen sensitive (Keast, 1999, 2000; Keast et al., 2002). Furthermore, it is conceivable also that ER-b has a more marked eVect on the MPG than ER-a. In addition, Patrone et al. (2000) suggested that estradiol induces diVerential neuronal phenotypes, based on its activation of either ER-a or ER-b, triggering diVerent intracellular signals leading to activation of diVerent transcription factors resulting in distinct physiological and metabolic responses in neurons. As indicated above, the female parasympathetic PG responds to estrogen, in terms of size of neurons (Coujard, 1951), numbers of cells, and possibly transmitter content. On the other hand, prenatal exposure to diethylstilbestrol (a potent synthetic estrogen) reduces the volume of the sympathetic celiac ganglion and the density of sympathetic innervation of the ovary (Shinohara et al., 1998, 2000). In addition, estrogen accelerates expression of putative transmitters in other nonautonomic neurons such as neuropeptides substance P, calcitonin gene-related peptide (Mowa et al., 2002, 2003a,b), and metenkephalin (Yuri and Kawata, 1994) as well as nitric oxide (Okamura et al., 1994a,b). Peptide-containing, cholinergic, and nitric oxide-containing neurons are numerous in the PG (Papka and McNeill, 1992; Papka et al., 1995b) and some of these express ERs. For example, ER-b þ NOS (Fig. 15) and, ER-b þ choline acetyltransferase (ChAT) (Fig. 16). Future studies will

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detail the influence of sex steroids on these and other specific neurochemical parameters.

D. Interactions between Spinal Autonomic, Ganglionic Autonomic, and Sensory Neurons There are connections (synaptic and nonsynaptic) between preganglionic autonomic neurons from the spinal cord and postganglionic neurons in peripheral ganglia and between sensory DRG neurons and postganglionic neurons, which, when taken together, could form the basis for circuits for innervation of pelvic organs (Papka and McNeill, 1993; Papka and Traurig, 1993). Subpopulations of neurons in each of these areas express ERs (a and b) and the transmitter neurochemistry expressed by somas in each area is reflected in the terminals in each ganglion and end organ. Thus, with all of the morphological and neurochemical players in place, one could hypothesize about the existence of an estrogen-sensitive neural circuitry involving the CNS, peripheral ganglia, and genitourinary target organs (Fig. 9) such that activity and plasticity in such circuits could be influenced through hormonal involvement on developmental, cyclic, gestational, or pathological bases. These are areas that are fertile for future studies.

IV. Future Directions Areas that are important and fertile for future studies include the presence and roles of estrogen on the spinal, autonomic, and primary sensory neurons, e.g., roles in development, response to injury, and influence on neurochemical and physiological parameters. Moreover, the presence of receptors for other sex steroids and roles of those steroids on the spinal, autonomic, and primary sensory neurons are of particular interest. It is well established that progesterone and progesterone receptors are important components of reproductive circuitries in the diencephalon. Recent studies indicate the presence of progesterone receptor-expressing neurons in the brainstem (Haywood et al., 1999; Kastrup et al., 1999; Francis et al., 2002) and spinal cord (Labombarda et al., 2000; Monks et al., 2001). Analgesic roles for progesterone in the spinal cord have been proposed (Dawson-Basoa and Gintzler, 1996; Ren et al., 2000). Our preliminary studies show progesterone receptor-expressing neurons in the L6–S1 spinal cord have a distribution similar to that of ERs (Fig. 18). Additional evolving data show androgen receptors expressed in neurons of the female rat spinal cord and lumbosacral DRG (Fig. 17). Thus, the distribution of neurons, and their neurochemistry

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and circuitry, which express progesterone receptors and androgen receptors in the female rat spinal cord and DRG, is ripe for future study. Unraveling the role(s) that any or all of the sex steroids have in autonomic and sensory processing and especially pain and genitourinary reflexes would be of considerable interest and value. Moreover, the roles of these steroids in the development, maintenance, and plasticity of autonomic and sensory nerves and circuits are only beginning to be addressed.

Acknowledgments The original work described in this review was supported by NIH Grants NS-22526 and NS-33081. The expert technical assistance and scientific input of Sharon Usip, Jen Hafemeister, and Megan Storey-Workley are very much appreciated.

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