Differential effects of oestrogen on developing and mature uterine sympathetic nerves

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.     

Development of sensory neurons in the absence of NGF/TrkA signaling in vivo

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.     

Nerve Growth Factor Decreases in Sympathetic and Sensory Nerves of Rats with Chronic Heart Failure

Nerve growth factor (NGF) plays a critical role in the maintenance and survival of both sympathetic and sensory nerves. Also, NGF can regulate receptor expression and neuronal activity in the sympathetic and sensory neurons. Abnormalities in NGF regulation are observed in patients and animals with heart failure (HF). Nevertheless, the effects of chronic HF on the levels of NGF within the sympathetic and sensory nerves are not known. Thus, the ELISA method was used to assess the levels of NGF in the stellate ganglion (SG) and dorsal root ganglion (DRG) neurons of control rats and rats with chronic HF induced by myocardial infarction. Our data show for the first time that the levels of NGF were significantly decreased (P < 0.05) in the SG and DRG neurons 6–20 weeks after ligation of the coronary artery. In addition, a close relation was observed between the NGF levels and the left ventricular function. In conclusion, chronic HF impairs the expression of NGF in the sympathetic and sensory nerves. Given that sensory afferent nerves are engaged in the sympathetic nervous responses to somatic stimulation (i.e. muscle activity during exercise) via a reflex mechanism, our data indicate that NGF is likely responsible for the development of muscle reflex-mediated abnormal sympathetic responsiveness observed in chronic HF.     

Protein gene product 9.5 (PGP 9.5)

Summary The guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.     

Inhibition of non-genomic responses to oestrogen in the rat uterus by testosterone propionate

Testosterone propionate (50 mg/kg), administered together with oestradiol, inhibited the oestrogen-induced uterine eosinophilia, deep endometrial oedema and the increase in uterine wet weight, 6 h after treatment. The same dose of the androgen decreased the number of eosinophils in the blood and increased their degranulation, explaining the effect of testosterone in the uterus. The high doses of the androgen used were in the range of the doses reported by others to block selectively the oestrogen-induced increase in uterine peroxidase content but not other responses to oestrogen or the cytosolic oestrogen receptor translocation to the nucleus. The dissociation by high doses of testosterone of the oestrogen-induced uterine eosinophilia, wet weight increase and oedema from other responses to oestrogen in the absence of any measurable effect of testosterone upon cytosolic-nuclear oestrogen receptors supports the idea that uterine eosinophilia and oedema are oestrogenic responses regulated by mechanisms different from those of the genomic responses, and is in agreement with the hypothesis of the mediation of uterine oedema by eosinophils.     

Endometriosis as a neurovascular condition: estrous variations in innervation, vascularization, and growth factor content of ectopic endometrial cysts in the rat

Endometriosis is a poorly understood, estradiol-dependent condition associated with severe pelvic pains and defined by vascularized endometrial growths outside the uterus. Endometriosis is produced in cycling rats by autotransplanting pieces of uterus onto abdominal arteries where they develop into cysts. The surgery induces vaginal and abdominal muscle hyperalgesia, whose severity is greatest in proestrus and nearly absent in estrus. The cysts contain growth factors and cytokines and develop their own sympathetic and sensory C- and Adelta-fiber innervation. Here, we used quantitative immunostaining and protein array analyses to test the hypothesis that the innervation and growth factor/cytokine content of the cysts, but not uterine horn, contribute to proestrous-to-estrous changes in hyperalgesic severity. If so, these characteristics in the cysts, but not the uterine horn, should change with estrous stage. In cysts, the density of sympathetic (but not sensory) neurites and amounts of NGF and VEGF proteins (but not cytokines IL-1, IL-6, IL-10, or TNF-alpha) were greater in proestrus than estrus. These changes were accompanied by vascular changes. Both sympathetic and sensory fibers in both stages colabeled with TrkA, indicating that changes in NGF could act on both afferent and efferent fibers. In contrast with the cysts, no changes occurred in the uterine horn between proestrus and estrus. Together, these results suggest that coordinated proestrous-to-estrous changes in innervation and vascularization of the cysts contribute to similar changes in hyperalgesic severity. The findings also encourage consideration of endometriosis as a neurovascular condition.     

A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.     

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.     

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.     

Gestational changes in calcitonin gene-related peptide, nerve growth factor, and its receptors in rat dorsal root ganglia.

In dorsal root ganglia (DRG) cell cultures, levels of calcitonin gene-related peptide (CGRP) are increased in the presence of ovarian hormones and nerve growth factor (NGF). In addition, injection of ovariectomized rats with ovarian hormones led to an increase in levels of two NGF receptors, TrkA and p75(NTR), in DRG. Thus, we hypothesized that increased levels of ovarian hormones during pregnancy may elevate the synthesis of CGRP and NGF receptors in the DRG. DRG harvested from rats on specific days of pregnancy, on Day 2 postpartum, and after ovariectomy were subjected to radioimmunoassay, Western blot analysis, and NGF immunoassay to determine levels of CGRP, TrkA and p75(NTR), and NGF, respectively. CGRP levels in rat DRG were significantly higher during pregnancy than at Day 2 postpartum or in ovariectomized rats. Levels of both TrkA and p75(NTR) in DRG increased during pregnancy and remained elevated at Day 2 postpartum, but CGRP levels declined. Levels of NGF reached a statistically significant peak at Day 18 of gestation, and were not significantly reduced at Day 2 postpartum. Increased levels of ovarian steroid hormones during pregnancy may be involved in the synthesis of CGRP, however, the postpartum decreases in CGRP synthesis appear to be unrelated to NGF and its receptors.     

NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals

Target-derived NGF promotes the phenotypic maintenance of mature dorsal root ganglion (DRG) nociceptive neurons. Here, we provide in vivo and in vitro evidence for the presence within DRG neurons of endosomes containing NGF, activated TrkA, and signaling proteins of the Rap1/Erk1/2, p38MAPK, and PI3K/Akt pathways. Signaling endosomes were shown to be retrogradely transported in the isolated sciatic nerve in vitro. NGF injection in the peripheral target of DRG neurons increased the retrograde transport of p-Erk1/2, p-p38, and pAkt in these membranes. Conversely, NGF antibody injections decreased the retrograde transport of p-Erk1/2 and p-p38. Our results are evidence that signaling endosomes, with the characteristics of early endosomes, convey NGF signals from the target of nociceptive neurons to their cell bodies.     

Nitric oxide nerves in the uterus are parasympathetic,sensory, and contain neuropeptides

Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. The uterus contains abundant NO-synthesizing nerves which could be autonomic and/or sensory. This study was undertaken to determine: 1) the source(s) of NO-synthesizing nerves in the rat uterus and 2) what other neuropeptides or transmitter markers might coexist with NO in these nerves. Retrograde axonal tracing, utilizing Fluorogold injected into the uterine cervix, was employed for identifying sources of uterine-projecting neurons. NO-synthesizing nerves were visualized by staining for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and immunostaining with an antibody against neuronal/type I NO synthase (NOS). NADPH-d-positive perikarya and terminal fibers were NOS-immunoreactive (-I). Some NOS-I/NADPH-d-positive nerves in the uterus are parasympathetic and originate from neurons in the pelvic paracervical ganglia (PG) and some are sensory and originate from neurons in thoracic, lumbar, and sacral dorsal root ganglia. No evidence for NOS-I/NADPH-d-positive sympathetic nerves in the uterus was obtained. Furthermore, double immunostaining revealed that in parasympathetic neurons, NO-I/NADPH-d-reactivity coexists with vasoactive intestinal polypeptide, neuropeptide Y, and acetylcholinesterase and in sensory nerves, NOS-I/NADPH-d-reactivity coexists with calcitonin generelated peptide and substance P. In addition, tyrosine hydroxylase(TH)-I neurons of the PG do not contain NOS-I/NADPH-d-reactivity, but some TH-I neurons are apposed by NOS-I varicosities. These results suggest NO-synthesizing nerves in the uterus are autonomic and sensory, and could play significant roles, possibly in conjunction with other putative transmitter agents, in the control of uterine myometrium and vasculature.     

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target‐derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret‐expressing neurons in the DRG complete developmental cell death prior to TrkA‐expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701–717, 2018     

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.     

The effect of relaxin on in vivo uterine electromyographic activity in the conscious ovariectomized ewe is oestrogen-dependent

Electromyographic (EMG) activity of the uterus was recorded in vivo in 4 conscious ovariectomized ewes treated with oestrogen (one injection of 100 micrograms of oestradiol benzoate). Doses of 500 micrograms of purified porcine relaxin were injected intravenously 12, 16, 20, 24, 28 and 32 hrs after oestrogen treatment. Relaxin had an inhibitory effect on uterine activity between 16 and 32 hrs after oestrogen treatment. Because the period during which relaxin had a biological effect corresponded to the moment when oestrogen-induced uterine activity was maximal, it is suggested that the ability of relaxin to inhibit uterine activity in the ewe is oestrogen-dependent.     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.