Expression of pituitary adenylate cyclase activating peptide in the uterine cervix, lumbosacral dorsal root ganglia and spinal cord of rats during pregnancy.

The uterine cervix is highly innervated by the sensory nerves containing neuropeptides which change during pregnancy and are regulated, in part, by estrogen. These neuropeptides act as transmitters both in the spinal cord and cervix. The present study was undertaken to determine the expression pattern of the neuropeptide pituitary adenylate cyclase activating peptide (PACAP) in the cervix and its nerves during pregnancy and the influence of estrogen on this expression using immunohistochemistry, radioimmunoassay and RT-PCR. PACAP immunoreactivity was detected in nerves in the cervix, lumbosacral (L6-S1) dorsal root ganglia (DRG) and spinal cord. PACAP immunoreactivity was highest at day 15 of pregnancy in the cervix and dorsal spinal cord, but then decreased over the last trimester of pregnancy. However, levels of PACAP mRNA increased in the L6-S1 DRG at late pregnancy relative to early pregnancy. DRG of ovariectomized rats treated with estrogen showed increased PACAP mRNA synthesis in a dose-related manner, an effect partially blocked by the estrogen receptor (ER) antagonist ICI 182,780. We postulate that synthesis of PACAP in L6-S1 DRG and utilization in the cervix and spinal cord increase over pregnancy and this synthesis is the under influence of the estrogen-ER system. Since PACAP is expressed by sensory nerves and may have roles in nociception and vascular function, collectively, these data are consistent with the hypothesis that sensory nerve-derived neuronal factors innervate the cervix and play a role in cervical ripening.

Activation and circuitry of uterine-cervix-related neurons in the lumbosacral dorsal root ganglia and spinal cord at parturition.

Stimulation of the uterine cervix at parturition activates neural circuits involving primary sensory nerves and supraspinally projecting neurons of the lumbosacral spinal cord, resulting in output of hypothalamic neurohormones. Dorsal root ganglia (DRG) and spinal neurons of these circuits are not well-characterized. The objectives of this study were to detail the activation of DRG and spinal neurons of the L6/S1 levels that are stimulated at late pregnancy, verify hypothalamic projections of activated spinal neurons, and determine whether activated neurons express estrogen receptor-alpha (ERalpha). Expression of phosphorylated cyclic-AMP response element-binding protein (PCREB) and Fos immunohistochemistry were used to "mark" activated DRG and spinal neurons, respectively. Retrograde tracing identified uterine-cervix-related and spinohypothalamic neurons. Baseline PCREB expression in the DRG increased during pregnancy and peaked during the last trimester. Some PCREB-expressing neurons contained retrograde tracer identifying them as cervix-related neurons. Fos-expressing neurons were few in spinal cords of nonpregnant and day 22 pregnant rats but were numerous in parturient animals. Some Fos-expressing neurons located in the dorsal half of the spinal cord contained retrograde tracer identifying them as spinohypothalamic neurons. Some DRG neurons expressing PCREB also expressed ERalpha, and some spinal neurons activated at parturition projected axons to the hypothalamus and expressed ERalpha. These results indicate that DRG and spinal cord neurons are activated at parturition; that those in the spinal cord are present in areas involved in autonomic and sensory processing; that some spinal neurons project axons to the hypothalamus, ostensibly part of a neuroendocrine reflex; and that sensory and spinal neurons can respond to estrogens. Moreover, some activated sensory neurons may be involved in the animal's perception of labor pain.

Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening.

Bilateral neurectomy of the pelvic nerve (BLPN) that carries uterine cervix-related sensory nerves induces dystocia, and administration of its vasoactive neuropeptides induces changes in the cervical microvasculature, resembling those that occur in the ripening cervix. This study was designed to test the hypothesis that (a) the cervix of pregnant rats expresses vascular endothelial growth factor (VEGF) and components of the angiogenic signaling pathway [VEGF receptors (Flt-1, KDR), activity of protein kinase B, Akt (phosphorylated Akt), and endothelial nitric oxide synthase (eNOS)] and von Willebrand Factor (vWF) and that these molecules undergo changes with pregnancy, and (b) bilateral pelvic neurectomy (BLPN) alters levels of VEGF concentration in the cervix. Using RT-PCR and sequencing, two VEGF isoforms, 120 and 164, were identified in the rat cervix. VEGF, VEGF receptor-1 (Flt-1), eNOS, and vWF immunoreactivities (ir) were localized in the microvasculature of cervical stroma. Their protein levels increased during pregnancy but decreased to control levels by 2 days postpartum. VEGF receptor-2 (KDR)-ir was confined to the epithelium of the endocervix. BLPN downregulated levels of VEGF by a third. Therefore, the components of the angiogenic signaling pathway are expressed in the cervix and change over pregnancy. Furthermore, angiogenic and sensory neuronal factors may be important in regulating the dynamic microvasculature in the ripening cervix and may subsequently play a role in cervical ripening and the birth process.

The role of sensory neurons in cervical ripening: effects of estrogen and neuropeptides.

Central nervous system nuclei and circuits, such as the medial preoptic, ventromedial and paraventricular nuclei of the hypothalamus, play important roles in reproduction and parturition, and are influenced by estrogen. Peripheral autonomic and sensory neurons also play important roles in pregnancy and parturition. Moreover, the steroid hormone estrogen acts directly, not only on the reproductive tract organs (uterus and cervix), but also on the central and peripheral nerves by regulating expression of various neuronal genes. The peripheral primary afferent neurons innervating the uterine cervix relay mechanical and biochemical sensory information induced by local cervical events and by passage of fetuses, to the spinal cord and supraspinal centers. Consequently, the birth process in mammals is influenced by the combined action of neurons and hormones. Peripheral sensory stimuli, induced physiologically by fetal expulsion or mechanically by vaginocervical stimulation, alter behavior, as well as autonomic and neuroendocrine systems. Recent evidence indicates that primary afferent neurons innervating the cervix, in addition to their sensory effects, likely exert local "efferent" actions on the ripening cervix near term. These efferent effects may involve estrogen-regulated production of such neuropeptides as substance P and calcitonin gene-related peptide in lumbosacral dorsal root ganglia, and their release in the cervix. Collectively, these findings suggest an interrelationship among estrogen, cervix-related sensory neurons, and local cervical events near term.

The effects of pregnancy and estrogen on the expression of calcitonin gene-related peptide (CGRP) in the uterine cervix, dorsal root ganglia and spinal cord.

Before parturition the uterine cervix undergoes a ripening process ("softens" and dilates) to allow passage of the fetus at term. The exact mechanism(s) responsible for cervical ripening are unknown, though a role for peptidergic sensory neurons is emerging. Previous work demonstrated that administration of substance P (SP) to ovariectomized rats caused events associated with cervical ripening, that production of SP in cervix-related dorsal root ganglion (DRG) is estrogen responsive, and that release of SP from neurons terminating in the cervix and spinal cord peaks prior to parturition. The present study was designed to test the hypothesis that calcitonin gene-related peptide (CGRP), a neuropeptide co-stored with SP in many sensory neurons, undergoes changes with pregnancy and hormonal environment. Immunohistochemistry, in situ hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR) and radioimmunoassay (RIA) were used to investigate CGRP in L6-S1 DRG, spinal cord and cervix during pregnancy and the role of estrogen in CGRP synthesis. CGRP-immunoreactive primary sensory neurons expressed estrogen receptors (ER-alpha and ER-beta). In the cervix, CGRP concentrations decreased, but in the L6-S1 DRG and the spinal cord segments, CGRP levels increased, with peak effects observed at day 20 of gestation. CGRP mRNA synthesis increased in DRG over pregnancy. Sensory neurons of ovariectomized rats treated with estrogen showed increased CGRP mRNA synthesis in a dose-related manner, an effect blocked by the ER antagonist ICI 182 780. From these results, we postulate that synthesis of CGRP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is the under influence of the estrogen-ER system. Collectively, these data are consistent with the hypothesis that CGRP plays a role in cervical ripening and, consequently in the birth process.

Substance P in the uterine cervix, dorsal root ganglia and spinal cord during pregnancy and the effect of estrogen on SP synthesis.

Prior to parturition the non-pliable uterine cervix undergoes a ripening process ("softens" and dilates) to allow a timely passage of the fetus at term. The exact mechanism(s) triggering and involved in cervical ripening are unknown, though evidence for a role for sensory neurons and their contained neuropeptides is emerging. Moreover, an apparent increase in neuropeptide immunoreactive nerves occurs in the cervix during pregnancy, maternal serum estrogen levels rise at term and uterine cervix-related L6-S1 dorsal root ganglia (DRG) sensory neurons express estrogen receptor (ER) and neuropeptides. Thus, we sought to test the hypothesis that the neuropeptide substance P (SP) changes biosynthesis and release over pregnancy, that estrogen, acting via the ER pathway, increases synthesis of SP in DRG, and that SP is utilized in cervical ripening at late pregnancy. Using immunohistochemistry, in situ hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR) and radioimmunoassay (RIA), we investigated coexpression of ER-alpha/beta and SP; differential expression of ER-alpha and -beta mRNA in DRG neurons; SP synthesis in DRG; and changes in SP concentration in the cervix, DRG and spinal cord over pregnancy. In addition, the effect of exogenous estrogen on SP synthesis in L6-S1 DRG of ovariectomized rats was examined. SP-immunoreactive neurons expressed ER-alpha and ER-beta. SP synthesis (expressed as beta-PPT mRNA label) was prominent in small DRG neurons. SP concentration increased in the L6-S1 DRG and spinal cord segments, with a peak at Day 20 of gestation, but decreased in the cervix during the first two trimesters, with a rise over the last trimester to Day 10 levels. SP and ER-alpha mRNA synthesis increased in DRG over pregnancy but ER-beta mRNA levels were largely unchanged. When ovariectomized rats were treated with exogenous estrogen, SP mRNA synthesis in the DRG increased in a dose-related manner, an effect blocked by ER blocker ICI 182 780. From these results, we postulate that synthesis of SP in L6-S1 DRG and utilization in the cervix increase over pregnancy and this synthesis is under influence of the estrogen-ER system, most likely ER-alpha. We postulate that SP may play a role in cervical ripening and, consequently in the birth process.

Estrogen receptors in the spinal cord, sensory ganglia, and pelvic autonomic ganglia.

Until relatively recently, most studies of the effects 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 effects 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.

Estrogen receptor-alpha and neural circuits to the spinal cord during pregnancy.

Estrogen receptor immunoreactivity and mRNAs are present in spinal cord neurons in locations that are associated with sensory and autonomic innervation of female reproductive organs. The present study was undertaken to examine the expression of estrogen receptor-alpha in the spinal cord during different stages of pregnancy and to determine whether estrogen receptor-alpha-expressing neurons are related to uterine afferent nerves bringing information to the spinal cord at parturition. Immunohistochemistry showed estrogen receptor-alpha-immunoreactive neurons in the dorsal one-half of the spinal cord, i.e., dorsal horn, dorsal intermediate gray areas (dorsal commissural nucleus), and around the central canal and sacral parasympathetic autonomic nucleus of the lumbosacral spinal cord. Neurons in these areas corresponded topographically to the distribution of central processes of visceral primary afferent neurons (e.g., containing calcitonin gene-related peptide and substance P) that innervate and activate second-order spinal cord neurons (evidenced by their expression of Fos) at parturition. Western blots showed that estrogen receptor-alpha increases in the spinal cord, with a peak at day 20 of gestation, followed by a slight decrease by 2 days postpartum. These studies show that estrogen receptor-alpha is expressed by neurons in autonomic and sensory areas of the lumbosacral spinal cord that have connections with the female reproductive system and that the level of estrogen receptor-alpha changes over the course of pregnancy, which may follow profiles of steroid hormones. Many of these neurons may be involved in processing information related to reproductive organ function, changes during pregnancy, and relays to other CNS centers.

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia.

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.

Sensory nerves and neuropeptides in uterine cervical ripening.

At the time of parturition (fetal delivery) the uterine cervix must "ripen," becoming soft, pliable, and dilated to accommodate the fetus' delivery. The fundamental processes underlying cervical ripening remain poorly understood. Knowledge that abundant autonomic and sensory nerves supply the uterine cervix, that transection of afferent nerves supplying the cervix blocks parturition, and that some of the changes in the cervix resemble those seen in inflammatory reactions suggests nerves may have a role in the cervical ripening changes. The present study utilized immunohistochemistry, plasma extravasation, and solution hybridization-nuclease protection assay to elucidate the complement of primary afferent nerves and some receptors in the rat cervix during pregnancy, and to determine if they may have roles in the ripening process at term. This study revealed an abundance of nerves associated with the cervical vasculature and myometrial smooth muscle containing immunoreactivity for substance P, calcitonin gene-related peptide, secretoneurin, and nitric oxide synthase throughout pregnancy. Many of these are small unmyelinated capsaicin-sensitive C-fibers. Substance P- (NK1-) and calcitonin gene-related peptide receptors were apparent on uterine cervix vasculature from pregnant, parturient, and postpartum rats. NK1 receptor mRNA was maximal at 20 days of pregnancy. Plasma extravasation of i.v. administered Evans Blue or Monastral Blue was most pronounced at parturition (shortly after NK1 mRNA is maximal); this was similar to plasma extravasation evoked by i.v. administration of substance P or capsaicin-treatment. This study revealed new data about the nervous system of the rat uterine cervix and that these nerves and their transmitters could very well be part of a neurogenic inflammatory process involved in cervical ripening.

Distribution and origin of corticotropin-releasing factor-immunoreactive axons in the female rat lumbosacral spinal cord.

Corticotropin-releasing factor (CRF) is a neuropeptide traditionally known for its hormonal role in the hypothalamic/pituitary/adrenal stress axis. However, CRF has been reported in axons in sites that may be considered outside of the direct stress axis, e.g., in axons in the lumbosacral spinal cord associated with the micturition response. Whether any of these CRF-immunoreactive axons interacts with uterine-related preganglionic autonomic neurons or projection neurons in the lumbosacral spinal cord is unknown. Thus, immunohistochemistry and retrograde tracing were employed to determine the presence, distribution, and origin of CRF-immunoreactive axons in the L6/S1 spinal cord of the female rat and to ascertain whether these axons are associated with uterine-related neurons. CRF-immunoreactive axons were present in the dorsal horn, medial and lateral collateral pathways, dorsal intermediate gray, laminae VlI and X, and sacral parasympathetic nucleus of the spinal cord. Nitric oxide-synthesizing, i.e., NADPH-d-positive neurons and pseudorabies virus labeled uterine-related neurons were in the sacral parasympathetic nucleus and were closely apposed by CRF-immunoreactive axons. Injection of retrograde tracers (fluorogold or fast blue) into the L6/S1 spinal cord labeled neurons in the hypothalamic paraventricular nucleus and pontine Barrington's nucleus, and some of these neurons were immunoreactive for CRF. This study demonstrates that CRF-immunoreactive axons are present in the L6/S1 spinal cord of the female rat in areas associated with sensory and autonomic processing. Some of these axons originate from the paraventricular nucleus and Barrington's nucleus and are adjacent to uterine-related neurons. These results indicate that CRF may influence neural activity related to the female reproductive system.

Estrogen receptor-alpha and beta- immunoreactivity and mRNA in neurons of sensory and autonomic ganglia and spinal cord.

Estrogen receptor-alpha immunoreactivity and mRNAs are present in neurons in locales that innervate genital organs, e.g., parasympathetic pelvic autonomic ganglia, sensory dorsal root and nodose ganglia, and autonomic areas of the lumbosacral spinal cord. With the availability of probes for the beta-isoform of the estrogen receptor, we studied this receptor in autonomic, sensory, and spinal cord neurons and compared it with the distribution of the alpha-receptor. Estrogen receptor-alpha and -beta immunoreactivity were located in the nuclei of neurons, were in subpopulations of parasympathetic neurons in pelvic ganglia, and sensory neurons of dorsal root and nodose ganglia. Both receptor subtypes were present in the lumbosacral spinal cord: in neurons of the outer laminae of the dorsal horn, lateral collateral and medial collateral pathways, sacral parasympathetic nucleus, dorsal intermediate gray, and lamina X. Similar numbers of spinal cord neurons were immunoreactive for estrogen receptor-beta and estrogen receptor-alpha. However, estrogen receptor-beta-immunoreactive neurons appeared less numerous in the outer dorsal horn, but more numerous in the deeper layers of the spinal cord than estrogen receptor-alpha neurons. Retrograde tracing from the uterus revealed "uterine-related" neurons in dorsal root and pelvic ganglia that contained estrogen receptor-alpha and -beta. In situ hybridization revealed both estrogen receptor-alpha and -beta mRNA transcripts in sensory neurons of the dorsal root and nodose ganglia, parasympathetic neurons of pelvic ganglia, and spinal cord neurons in the dorsal horn, sacral parasympathetic nucleus, and dorsal intermediate gray of L6-S1 segments. These studies show that both estrogen receptor-alpha and -beta are synthesized by autonomic and sensory neurons in parts of the nervous system that have connections with the female reproductive system. Such neurons contain neurotransmitters that have important functions in the female reproductive organs; thus, it is likely that estrogen can influence the activity of such neurons and consequently, through them, the activities of the reproductive organs.

Hypothalamic paraventricular axons projecting to the female rat lumbosacral spinal cord contain oxytocin immunoreactivity.

Oxytocin-containing axons project from the hypothalamic paraventricular nucleus to the neurohypophysis and thoracic spinal cord to ultimately influence uterine contractions and autonomic activity, respectively. Whether or not oxytocin-immunoreactive axons project to the female rat lumbosacral spinal cord to influence autonomic outflow to pelvic organs has not been investigated. Thus, the present study was designed to investigate the presence, distribution, and origin of oxytocin-immunoreactive axons in the female rat lumbosacral spinal cord. Immunohistochemistry, spinal cord transections, and axonal tracing with Fluorogold, True Blue, and pseudorabies virus were used. Oxytocin-immunoreactive nerve fibers were present in the L6/S1 segments of the spinal cord. Prominent varicose axons were evident throughout the dorsal horn, along the lateral and medial collateral pathways, in the dorsal intermediate gray area, around the central canal in lamina X, and throughout the sacral parasympathetic nucleus. Injection of retrograde tracer into the L6/S1 spinal cord labeled neurons in the hypothalamic paraventricular nucleus. Transection of the thoracic spinal cord eliminated oxytocin-immunoreactive nerve axons in the L6/S1 spinal cord. In addition, transection of the thoracic spinal cord eliminated transport of retrograde axonal tracer from the L6/S1 spinal cord to the paraventricular nucleus. Pseudorabies virus, a transneuronal retrograde tracer, injected into the uterus and cervix marked uterine-related preganglionic neuronal cell bodies in the sacral parasympathetic nucleus and uterine-related neurons in the hypothalamic paraventricular nucleus. Double immuno-labeling of viral-infected spinal cord sections showed oxytocin-immunoreactive axons closely associated with viral labeled uterine-related preganglionic cell bodies of the sacral parasympathetic nucleus. The results of this study revealed that oxytocin-immunoreactive neurons of the hypothalamic paraventricular nucleus project axons to the lumbosacral spinal cord to areas involved in sensory processing and parasympathetic outflow to the uterus.

Distribution and origin of secretoneurin-immunoreactive nerves in the female rat uterus.

Secretoneurin is a 33-amino acid peptide derived from secretogranin II. Secretoneurin immunoreactivity has been localized in the peripheral nervous system where it exerts potent chemotactic activity for monocytes and may play a role in inflammation. Secretoneurin could play a role in this process, although the presence and distribution of secretoneurin-immunoreactive neurons in the female reproductive system has not been documented. Thus, this study was undertaken to examine secretoneurin immunoreactivity in nerves of the rat uterus and uterine cervix. A moderate plexus of secretoneurin-immunoreactive nerve fibers was present in the myometrium and endometrium of the uterus as well as in the smooth muscle and endocervix of the cervix. Many of these fibers were associated with the vasculature as well as the myometrium. Secretoneurin immunoreactivity was present in small- to medium-sized neurons of dorsal root and nodose ganglia. Retrograde tracing with FluoroGold indicated that some of these sensory neurons project axons to the cervix and uterine horns. Secretoneurin-immunoreactive terminal-like structures were associated with neurons in the sacral parasympathetic nucleus of the lumbosacral spinal cord. In addition, some secretoneurin terminals were apposed to pelvic parasympathetic neurons in the paracervical ganglia that projected axons to the uterus and cervix. Double-immunostaining indicated co-existence of calcitonin gene-related peptide or substance P with secretoneurin in some sensory neurons, in some terminals of the pelvic ganglia, as well as nerve fibers in the uterine horn and cervix. Finally, fibers in the uterus and cervix were depleted of secretoneurin by capsaicin treatment. This study indicates that secretoneurin is present in the uterus in C-afferent nerve fibers whose cell bodies are located in sensory ganglia. Some of these fibers contain both secretoneurin and calcitonin gene-related peptide or substance P. These substances have functions in inflammatory reactions. Further, secretoneurin could influence postganglionic parasympathetic "uterine-related" neurons in the pelvic ganglia and preganglionic parasympathetic neurons in the lumbosacral spinal cord.

Calretinin-immunoreactive nerves in the uterus, pelvic autonomic ganglia, lumbosacral dorsal root ganglia and lumbosacral spinal cord.

Nerves containing the calcium-binding protein calretinin have been reported in several organs but not in female reproductive organs and associated ganglia. This study was undertaken to determine if nerves associated with the uterus contain calretinin and the source(s) of calretinin-synthesizing nerves in the rat (are they sensory, efferent, or both?). Calretinin-immunoreactive nerves were present in the uterine horns and cervix where they were associated with arteries, uterine smooth muscle, glands, and the epithelium. Calretinin-immunoreactive terminals were apposed to neurons in the paracervical ganglia; in addition, some postganglionic neurons in this ganglion were calretinin positive. Calretinin perikarya were present in the lumbosacral dorsal root ganglia, no-dose ganglia, and lumbosacral spinal cord. Retrograde axonal tracing, utilizing Fluorogold injected into the uterus or paracervical parasympathetic ganglia, revealed calretinin-positive/Fluorogold-labeled neurons in the dorsal root and nodose ganglia. Also, capsaicin treatment substantially reduced the calretinin-positive fibers in the uterus and pelvic ganglia, thus indicating the sensory nature of these fibers. The presence of calretinin immunoreactivity identifies a subset of nerves that are involved in innervation of the pelvic viscera and have origins from lumbosacral dorsal root ganglia and vagal nodose ganglia. Though the exact function of calretinin in these nerves is not currently known, calretinin is likely to play a role in calcium regulation and their function.

Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors.

Acetylcholine (ACh) stimulates contraction of the uterus and dilates the uterine arterial supply. Uterine cholinergic nerves arise from the paracervical ganglia and were, in the past, characterized based on acetylcholinesterase (AChE) histochemistry. However, the histochemical reaction for acetylcholinesterase provides only indirect evidence of acetylcholine location and is a nonspecific marker for cholinergic nerves. The present study: (1) reevaluated cholinergic neurons of the paracervical ganglia, (2) examined the cholinergic innervation of the uterus by using retrograde axonal tracing and antibodies against molecules specific to cholinergic neurons, choline acetyltransferase and the vesicular acetylcholine transporter, and (3) examined muscarinic receptors in the paracervical ganglia using autoradiography and a radiolabeled agonist. Most ganglionic neurons were choline acetyltransferase- and vesicular acetylcholine transporter-immunoreactive and were apposed by choline acetyltransferase/vesicular acetylcholine transporter-immunoreactive terminals. Retrograde tracing showed that some cholinergic neurons projected axons to the uterus. These nerves formed moderately dense plexuses in the myometrium, cervical smooth muscle and microarterial system of the uterine horns and cervix. Finally, the paracervical ganglia contain muscarinic receptors. These results clearly reveal the cholinergic innervation of the uterus and cervix, a source of these nerves, and demonstrate the muscarinic receptor content of the paracervical ganglia. Cholinergic nerves could play significant roles in the control of uterine myometrium and vasculature.

Vagal afferents from the uterus and cervix provide direct connections to the brainstem.

Previous anatomical studies demonstrated vagal innervation to the ovary and distal colon and suggested the vagus nerve has uterine inputs. Recent behavioral and physiological evidence indicated that the vagus nerves conduct sensory information from the uterus to the brainstem. The present study was undertaken to identify vagal sensory connections to the uterus. Retrograde tracers, Fluorogold and pseudorabies virus were injected into the uterus and cervix. DiI, an anterograde tracer, was injected into the nodose ganglia. Neurectomies involving the pelvic, hypogastric, ovarian and abdominal vagus nerves were performed, and then uterine whole-mounts examined for sensory nerves containing calcitonin gene-related peptide. Nodose ganglia and caudal brainstem sections were examined for the presence of estrogen receptor-containing neurons in "vagal locales." Labeling of uterine-related neurons in the nodose ganglia (Fluorogold and pseudorabies virus) and in the brainstem nuclei (pseudorabies virus) was obtained. DiI-labeled nerve fibers occurred near uterine horn and uterine cervical blood vessels, in the myometrium, and in paracervical ganglia. Rats with vagal, pelvic, hypogastric and ovarian neurectomies exhibited a marked decrease in calcitonin gene-related peptide-immunoreactive nerves in the uterus relative to rats with pelvic, hypogastric, and ovarian neurectomies with intact vagus nerves. Neurons in the nodose ganglia and nucleus tractus solitarius were immunoreactive for estrogen receptors. These results demonstrated: (1) the vagus nerves serve as connections between the uterus and CNS, (2) the nodose ganglia contain uterine-related vagal afferent neuron cell bodies, and (3) neurons in vagal locales contain estrogen receptors.

CNS location of uterine-related neurons revealed by trans-synaptic tracing with pseudorabies virus and their relation to estrogen receptor-immunoreactive neurons.

Retrograde, transneuronal tracing with Bartha's strain of pseudorabies virus was used in rats to identify spinal cord, brainstem and hypothalamic loci of uterine-related neurons that could function in the regulation of uterine activity. Based on the premise that estrogen might influence such uterine-related neurons, the existence of estrogen receptors in neurons in these same loci was examined. Viral injections were made into the uterine cervix, body and cervical end of the uterine horns, and the rats allowed to survive for four to six days. After four days, mainly the spinal cord, medulla and pons contained virus-infected neurons. After longer survival times, progressively higher levels of the neuraxis contained viral-labeled neurons, so that by six days hypothalamic uterine-related neurons were identified. First-order virus-infected neurons were visualized by immunohistochemistry in the pelvic paracervical parasympathetic ganglia and in inferior mesenteric sympathetic ganglia. Preganglionic and putative interneurons were labeled in the lumbosacral spinal cord and thoracic spinal cord mainly in the lateral horn area (sacral parasympathetic nucleus and intermediolateral nucleus), lateral aspect of the dorsal horn, intermediate gray, lamina X and dorsal gray commissural area. In the brainstem, labeling was most evident and consistent in the nucleus tractus solitarius, ventrolateral medulla, raphe magnus and pallidus nuclei, parapyramidal area, A5 cell group, Barrington's nucleus of the pons and periaqueductal gray of the midbrain. In the hypothalamus, virus-infected neurons were most marked in the paraventricular nucleus, with fewer in the medial preoptic area and ventromedial hypothalamic nucleus. Estrogen receptor-immunoreactive neurons were most often present among the virus-labeled uterine-related neurons of the spinal cord, nucleus tractus solitarius, ventrolateral medulla, periaqueductal gray, medial preoptic area and ventromedial hypothalamic nucleus. These results identify a multisynaptic pathway of neurons whose eventual output is involved in uterine functions, whose distribution is similar to that revealed by pseudorabies virus tracing from other visceral organs, and which are often mixed among estrogen-responsive neurons.

Localization of estrogen receptor protein and estrogen receptor messenger RNA in peripheral autonomic and sensory neurons.

The presence of estrogen receptor protein and estrogen receptor messenger RNA was revealed in peripheral ganglionic neurons of the rat. The pelvic parasympathetic autonomic ganglion and lumbosacral dorsal root sensory ganglia were examined for estrogen receptor-containing neurons because they have known projections to the uterus and uterine cervix. The vagal nodose ganglia were studied for estrogen receptor-containing neurons because they are suspected sources of influence on the uterus. Immunohistochemistry. in situ hybridization histochemistry and retrograde tracing were utilized. Immunoreactivity for estrogen receptors was evident in the nuclei of a subpopulation of neurons in the pelvic ganglia, sixth lumbar and first sacral dorsal root ganglia and nodose ganglia. Some estrogen receptor-positive neurons also contained the retrograde tracer FluoroGold that previously had been injected into the uterus and uterine cervix. Estrogen receptor messenger RNA was also evident in a subpopulation of ganglionic neurons. These data suggest that a certain population of neurons in autonomic and sensory ganglia are capable of synthesizing estrogen receptors and these receptors can serve as binding sites for estrogen. Thus, certain aspects of the structure, function and neurochemistry of some autonomic and sensory neurons may be influenced by the sex steroid estrogen.

Cytosolic estrogen receptor concentrations in the lumbosacral spinal cord fluctuate during the estrous cycle.

Estrogen responsive neurons have been anatomically identified with autoradiographic and immunohistochemical techniques and their distribution mapped in the lumbosacral spinal cord of female rats. Such neurons contain estrogen receptors (ERs). The present study was undertaken to: 1) quantify cytosolic estrogen receptor (ER) concentrations in the lumbosacral spinal cord and 2) determine if there is a relationship between cytosolic ER concentrations and fluctuations in serum estradiol (SE2) levels during the estrous cycle. Lumbosacral spinal segments were removed from intact cycling rats during the morning of proestrus, the afternoon of proestrus, and the morning of estrus, metestrus and diestrus. Trunk blood was collected at euthanasia and SE2 levels were determined using radioimmunoassay. Cytosolic ER concentrations were measured using a dextran-charcoal coated tube method. Concentrations of cytosolic ERs were low during estrus and metestrus, increased during diestrus with maximum concentrations during the afternoon of proestrus. These changes in ER concentrations paralleled SE2 levels measured in intact cycling animals; i.e., during estrus SE2 levels were low, but began to rise during metestrus, diestrus, and during the morning of proestrus with a maximum peak increase during the afternoon of proestrus. These data indicate there are fluctuations of cytosolic ER concentrations during the estrous cycle and that these changes coincide with changing SE2 concentrations suggesting that ER content is influenced by SE2.