Regional Targeting of Bladder and Urethra Afferents in the Lumbosacral Spinal Cord of Male and Female Rats: A Multiscale Analysis.

Sensorimotor circuits of the lumbosacral spinal cord are required for lower urinary tract (LUT) regulation as well as being engaged in pelvic pain states. To date, no molecular markers have been identified to enable specific visualization of LUT afferents, which are embedded within spinal cord segments that also subserve somatic functions. Moreover, previous studies have not fully investigated the patterning within or across spinal segments, compared afferent innervation of the bladder and urethra, or explored possible structural sex differences in these pathways. We have addressed these questions in adult Sprague Dawley rats, using intramural microinjection of the tract tracer, B subunit of cholera toxin (CTB). Afferent distribution was analyzed within individual sections and 3D reconstructions from sections across four spinal cord segments (L5-S2), and in cleared intact spinal cord viewed with light sheet microscopy. Simultaneous mapping of preganglionic neurons showed their location throughout S1 but restricted to the caudal half of L6. Afferents from both LUT regions extended from L5 to S2, even where preganglionic motor pathways were absent. In L6 and S1, most afferents were associated with the sacral preganglionic nucleus (SPN) and sacral dorsal commissural nucleus (SDCom), with very few in the superficial laminae of the dorsal horn. Spinal innervation patterns by bladder and urethra afferents were remarkably similar, likewise the patterning in male and female rats. In conclusion, microscale to macroscale mapping has identified distinct features of LUT afferent projections to the lumbosacral cord and provided a new anatomic approach for future studies on plasticity, injury responses, and modeling of these pathways.

Superficial peritoneal endometriotic lesions are histologically diverse and rarely demonstrate menstrual cycle synchronicity with matched eutopic endometrium.

STUDY QUESTION: Do menstrual cycle-dependent changes occur in the histological appearance of superficial peritoneal endometriotic lesions, and are they equivalent to those observed in the eutopic endometrium? SUMMARY ANSWER: Only a small subset of superficial peritoneal endometriotic lesions exhibits some histological features in phase with menstrual cycle-related changes observed in eutopic endometrium. WHAT IS KNOWN ALREADY: Endometriotic lesions are frequently described as implants that follow menstrual cycle-related changes in morphology, as per the eutopic endometrium. This concept has been widely accepted despite the lack of conclusive published evidence. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study of 42 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Patients were a subset selected from a larger endometriosis study being conducted at the Royal Women's Hospital, Melbourne since 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological features of epithelium, stroma and gland morphology were examined in haematoxylin and eosin stained sections of superficial peritoneal endometriotic lesions and matched eutopic endometrium (menstrual: n = 4, proliferative: n = 11, secretory: n = 17, hormone-treated: n = 10). At least two biopsies (average = 4, range = 2-8 biopsies) and a matched endometrial sample were analysed for each patient and results were presented per endometriotic gland profile (n = 1051). Data were analysed using mixed effects logistic regression to account for multiple patients and multiple endometriotic biopsies, each with multiple endometriotic gland profiles. This model also enabled analysis of endometriotic lesions versus eutopic endometrium. MAIN RESULTS AND THE ROLE OF CHANCE: There was considerable inter- and intra-patient variability in the morphology of superficial peritoneal endometriotic lesions. Menstrual cycle-associated changes were only observed for some features in a subset of endometriotic gland profiles. The proportion of endometriotic gland profiles with epithelial mitoses significantly increased in the proliferative phase (18% of gland profiles) relative to the menstrual phase (0% of endometriotic gland profiles) (odds ratios (OR) 9.30; 95% confidence intervals (CI) = 3.71-23.32; P < 0.001). Fewer blood-filled gland lumens were observed in the secretory phase (45% of endometriotic gland profiles) compared to the menstrual phase (67% of endometriotic gland profiles) (OR, 0.30; 95% CI = 0.11-0.79; P = 0.015). The features of the eutopic endometrium analysed in this study did not reflect the results in matched endometriotic lesions (P > 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study focused on features observed in sections of superficial peritoneal lesions and these may differ from features of deep infiltrating endometriosis or ovarian endometriomas. Cycle phases were limited to menstrual, proliferative and secretory phases to allow appropriate statistical modelling. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights heterogeneity in the histological characteristics of superficial peritoneal lesions. It challenges the assumption that lesion morphology consistently reflects menstrual cycle-associated changes. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported in part by National Health and Medical Research Council (NHMRC) project grants GNT1012245, GNT1105321 and GNT1026033 (P.A.W.R., J.E.G. and S.J.H.-C.). There are no competing interests.

Identification of a Sacral, Visceral Sensory Transcriptome in Embryonic and Adult Mice.

Visceral sensory neurons encode distinct sensations from healthy organs and initiate pain states that are resistant to common analgesics. Transcriptome analysis is transforming our understanding of sensory neuron subtypes but has generally focused on somatic sensory neurons or the total population of neurons in which visceral neurons form the minority. Our aim was to define transcripts specifically expressed by sacral visceral sensory neurons, as a step towards understanding the unique biology of these neurons and potentially leading to identification of new analgesic targets for pelvic visceral pain. Our strategy was to identify genes differentially expressed between sacral dorsal root ganglia (DRG) that include somatic neurons and sacral visceral neurons, and adjacent lumbar DRG that comprise exclusively of somatic sensory neurons. This was performed in adult and E18.5 male and female mice. By developing a method to restrict analyses to nociceptive Trpv1 neurons, a larger group of genes were detected as differentially expressed between spinal levels. We identified many novel genes that had not previously been associated with pelvic visceral sensation or nociception. Limited sex differences were detected across the transcriptome of sensory ganglia, but more were revealed in sacral levels and especially in Trpv1 nociceptive neurons. These data will facilitate development of new tools to modify mature and developing sensory neurons and nociceptive pathways.

Semaphorin 3A inhibits growth of adult sympathetic and parasympathetic neurones via distinct cyclic nucleotide signalling pathways.

BACKGROUND AND PURPOSE: Semaphorin 3A (Sema3A) is an important secreted repulsive guidance factor for many developing neurones. Sema3A continues to be expressed in adulthood, and expression of its receptor, neuropilin-1 (Nrp-1), can be altered by nerve injury. Autonomic neurones innervating the pelvic viscera are particularly susceptible to damage during pelvic surgical procedures, and failure to regenerate or aberrant growth of sympathetic and parasympathetic nerves lead to organ dysfunction. However, it is not known if adult pelvic neurones are potential targets for Sema3A. EXPERIMENTAL APPROACH: The effects of Sema3A and activation or inhibition of cyclic nucleotide signalling were assessed in adult rat pelvic ganglion neurones in culture using a growth cone collapse assay. KEY RESULTS: Sema3A caused growth cone collapse in both parasympathetic and sympathetic neurones expressing Nrp-1. However, the effect of Sema3A was mediated by distinct cyclic nucleotide signalling pathways in each neurone type. In parasympathetic neurones, cAMP and downstream activation of protein kinase A were required for growth cone collapse. In sympathetic neurones, cGMP was required for Sema3A-induced collapse; cAMP can also cause collapse but was not required. Sema3A-mediated, cGMP-dependent collapse in sympathetic neurones may require activation of cyclic nucleotide-gated ion channels (CNGCs). CONCLUSIONS AND IMPLICATIONS: We propose that Sema3A is an important guidance factor for adult pelvic autonomic neurones, and that manipulation of their distinct signalling mechanisms could potentially promote functional selective regeneration or attenuate aberrant growth. To our knowledge, this is also the first study to implicate CNGCs in regulating growth cone dynamics of adult neurones.

Conditioning lesions enhance growth state only in sensory neurons lacking calcitonin gene-related peptide and isolectin B4-binding.

A conditioning lesion improves regeneration of central and peripheral axons of dorsal root ganglion (DRG) neurons after a subsequent injury by enhancing intrinsic growth capacity. This enhanced growth state is also observed in cultured DRG neurons, which support a more sparsely and rapidly elongating mode of growth after a prior conditioning lesion in vivo. Here we examined differences in the capacity or requirements of specific types of sensory neurons for regenerative growth, which has important consequences for development of strategies to improve recovery after injury. We showed that after partial or complete injury of the sciatic nerve in mice, an elongating mode of growth in vitro was activated only in DRG neurons that did not express calcitonin gene-related peptide (CGRP) or bind Bandeiraea simplicifolia I-isolectin B4 (IB4). We also directly examined the response of conditioned sensory neurons to nerve growth factor (NGF), which does not enhance growth in injured peripheral nerves in vivo. We showed that after partial injury, NGF stimulated a highly branched and linearly restricted rather than elongating mode of growth. After complete injury, the function of NGF was impaired, which immunohistochemical studies of DRG indicated was at least partly due to downregulation of the NGF receptor, tropomyosin-related kinase A (TrkA). These results suggest that, regardless of the type of conditioning lesion, each type of DRG neuron has a distinct intrinsic capacity or requirement for the activation of rapidly elongating growth, which does not appear to be influenced by NGF.

Androgen and estrogen receptor-mediated mechanisms of testosterone action in male rat pelvic autonomic ganglia.

Although male reproductive function is primarily androgen dependent, many studies suggest that estrogens have direct actions on the male reproductive organs. Pelvic autonomic neurons provide the motor control of the internal reproductive organs and the penis and various properties of these neurons are affected by endogenous androgens. However, the possible role of estrogens at this site has not been examined. Here we have investigated the significance of estrogens produced by aromatization of testosterone (T) in the physiological actions of androgens on adult male rat pelvic ganglion neurons. Reverse transcriptase polymerase chain reaction (RT-PCR) studies showed that aromatase and both estrogen receptors (ERalpha and ERbeta) are expressed in these ganglia. Western blotting also showed that aromatase is expressed in male pelvic ganglia. Using immunohistochemical visualization, ERalpha was predominantly expressed by nitric oxide synthase (NOS)-positive parasympathetic pelvic ganglion neurons. In vivo studies showed that the decrease in pelvic ganglion soma size caused by gonadectomy could be prevented by administration of T or dihydrotestosterone (DHT), but not 17beta-estradiol (E2), showing that this maintenance action of testosterone is mediated entirely by androgenic mechanisms. However, in vitro studies of cultured pelvic ganglion neurons revealed that T, DHT and E each stimulated the growth of longer and more complex neurites in both noradrenergic and cholinergic NOS-expressing neurons. The effects of T were attenuated by either androgen or estrogen receptor antagonists, or by inhibition of aromatase. Together these studies demonstrate that estrogens are likely to be synthesized in the male pelvic ganglia, produced from T by local aromatase. The effects of androgens on axonal growth are likely to be at least partly mediated by estrogenic mechanisms, which may be important for understanding disease-, aging- and injury-induced plasticity in this part of the nervous system.

Postnatal androgen deprivation dissociates the development of smooth muscle innervation from functional neurotransmission in mouse vas deferens.

The pelvic autonomic nervous system is a target for circulating androgens in adults, with androgen exposure or deprivation affecting the structure and function of urogenital tract innervation. However, the critical period for androgen exposure to initially establish pelvic autonomic neuromuscular transmission has not been determined. We have examined the sympathetic innervation of the vas deferens in hypogonadal (hpg) mice that are deprived of androgens after birth but undergo normal prenatal sexual differentiation and remain androgen responsive throughout life. In vasa deferentia from hpg mice, purinergic excitatory junction potentials and contractions could not be elicited by electrical stimulation and P2X(1) purinoceptors could not be demonstrated by immunofluorescence. Moreover, a novel inhibitory nitrergic transmission developed. Administering testosterone to adult hpg mice restored purinergic excitatory transmission and P2X(1) purinoceptor immunofluorescence, and nitrergic inhibitory transmission was lost. Despite the deficit in excitatory neurotransmission in hpg mice, their vasa deferentia were innervated by numerous noradrenergic axons and pelvic ganglia appeared normal. In addition, noradrenergic contractions could be elicited by electrical stimulation. This study has revealed that postnatal androgen exposure has a profound effect on the development of excitatory transmission in vas deferens smooth muscle, primarily by a postjunctional action, but is not essential for development of the structural innervation of this organ. Our results also indicate that there is no postnatal critical period for androgen exposure to establish neuroeffector transmission and that postnatal androgen exposure can be delayed until adulthood, with little consequence for establishment of normal sympathetic neurotransmission.

Nerve growth factor, glial cell line-derived neurotrophic factor and neurturin prevent semaphorin 3A-mediated growth cone collapse in adult sensory neurons.

Developmentally, semaphorin 3A (sema3A) is an important chemorepellent that guides centrally projecting axons of dorsal root ganglion (DRG) neurons. Sema3A-mediated growth cone collapse can be prevented by cyclic GMP (cGMP) and nerve growth factor (NGF) in embryonic neurons. Sema3A may also play a role in directing regrowth of injured axons in adults, and interactions with neurotrophic factors near the injury site may determine the extent and targeting of both regenerative and aberrant growth. The aim of this study was to determine whether NGF, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) modulate sema3A-mediated growth cone collapse in cultured adult rat DRG neurons. Sema3A caused a significant increase in growth cone collapse, which was completely prevented by prior treatment with NGF, GDNF or NTN. Immunocytochemical experiments showed that sema3A-sensitive neurons were heterogeneous in their expression of neurotrophic factor receptors and responses to neurotrophic factors, raising the possibility of novel, convergent signaling mechanisms between these substances. Increasing cGMP levels caused growth cone collapse, whereas sema3A-mediated collapse was prevented by inhibition of guanylate cyclase or by increasing cyclic AMP levels. In conclusion, sema3A signaling pathways in adult neurons differ to those described in embryonic neurons. Three different neurotrophic factors each completely prevent sema3A-mediated collapse, raising the possibility of novel converging signaling pathways. These studies also show that there is considerable potential for neurotrophic factors to regulate sema3A actions in the adult nervous system. This may provide insights into the mechanisms underling misdirected growth and targeting of sensory fibers within the spinal cord after injury, that is thought to contribute to development of autonomic dysreflexia and neuropathic pain.

Localization of immunoreactivity for deleted in colorectal cancer (DCC), the receptor for the guidance factor netrin-1, in ventral tier dopamine projection pathways in adult rodents.

DCC (deleted in colorectal cancer)-the receptor of the netrin-1 neuronal guidance factor-is expressed and is active in the central nervous system (CNS) during development, but is down-regulated during maturation. The substantia nigra contains the highest level of netrin-1 mRNA in the adult rodent brain, and corresponding mRNA for DCC has also been detected in this region but has not been localized to any particular neuron type. In this study, an antibody raised against DCC was used to determine if the protein was expressed by adult dopamine neurons, and identify their distribution and projections. Significant DCC-immunoreactivity was detected in midbrain, where it was localized to ventrally displaced A9 dopamine neurons in the substantia nigra, and ventromedial A10 dopamine neurons predominantly situated in and around the interfascicular nucleus. Strong immunoreactivity was not detected in dopamine neurons found elsewhere, or in non-dopamine-containing neurons in the midbrain. Terminal fields selectively labeled with DCC antibody corresponded to known nigrostriatal projections to the dorsolateral striatal patches and dorsomedial shell of the accumbens, and were also detected in prefrontal cortex, septum, lateral habenular and ventral pallidum. The unique distribution of DCC-immunoreactivity in adult ventral midbrain dopamine neurons suggests that netrin-1/DCC signaling could function in plasticity and remodeling previously identified in dopamine projection pathways. In particular, a recent report that DCC is regulated through the ubiquitin-proteosome system via Siah/Sina proteins, is consistent with a potential involvement in genetic and sporadic forms of Parkinson's disease.

Rapid actions of estradiol on cyclic amp response-element binding protein phosphorylation in dorsal root ganglion neurons.

Actions of gonadal steroids have not been widely investigated in the peripheral nervous system, although many dorsal root ganglion (DRG) and autonomic pelvic ganglion (PG) neurons express estrogen receptors (ERs). We have studied the effects of 17beta-estradiol exposure on cultured DRG and PG neurons from adult rats. Western blotting analysis of DRG extracts detected phosphorylation of ERK1 and ERK2 (extracellular signal-regulated kinases) that peaked 10 min after exposure to 17beta-estradiol. These extracts contain both neurons and glia; therefore, to determine if this response occurred in DRG neurons, we developed an immunocytochemical method to specifically measure activation in individual neurons. These measurements showed that estradiol increased phosphorylation of CREB (cyclic AMP response-element binding protein), which was consistently blocked by the ERK pathway inhibitor PD98059 but not by the inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002. 17beta-Estradiol activation of CREB in DRG neurons was reduced by the ER antagonist, ICI182780. In contrast, in PG neurons estradiol did not affect CREB phosphorylation, highlighting a difference in E2 responses in different populations of peripheral neurons. This study has shown that estrogens can rapidly activate signaling pathways associated with CREB-mediated transcriptional regulation in sensory neurons. As these pathways also mediate many effects of neurotrophic factors, changes in estrogen levels (e.g. during puberty, pregnancy or menopause) could have broad-ranging genomic and non-genomic actions on urogenital pain sensation and reflex pathways.

Remodelling of connections in pelvic ganglia after hypogastric nerve crush.

Pelvic ganglia innervate the urogenital organs and contain both sympathetic and parasympathetic neurons. Previous studies have shown that within days of cutting either the lumbar or sacral preganglionic axons that innervate pelvic ganglia, many axon collaterals grow and appear to form specific connections with denervated pelvic neurons. Here we have examined the longer term consequences of partial deafferentation by studying pelvic ganglia up to 7 weeks after hypogastric nerve (HGN) crush, a lesion which also allows faster regeneration of spinal axons. Noradrenergic neurons were denervated by HGN crush, as demonstrated by loss of varicosities immunostained for the synaptic proteins, synaptophysin and synapsin. A week after HGN crush, axon collaterals grew from parasympathetic pelvic ganglion neurons, shown by the presence of numerous varicose fibers immunostained for vasoactive intestinal peptide (VIP). These VIP fibers were poorly stained or unstained for synaptophysin, even after 7 weeks. At early post-operative times the VIP fibers grew irregularly; however, with longer post-operative times they appeared to target particular VIP-negative, noradrenergic neurons. Our results also indicate that some lumbar preganglionic axons regenerated during the post-operative period, although this only affected a minority of sympathetic neurons. These reinnervated sympathetic neurons were not associated with VIP fibers, suggesting that the new intrinsic connections may have precluded regeneration or targeting of preganglionic axons. Together these results demonstrate that there is considerable remodelling within pelvic ganglia after partial deafferentation. This occurs under conditions where spinal preganglionic axons can regenerate. New intra-ganglionic connectivity may be permanent and may impact on this regeneration.

Neurturin signalling via GFRalpha2 is essential for innervation of glandular but not muscle targets of sacral parasympathetic ganglion neurons.

Neurturin, a member of the glial cell-derived neurotrophic factor familys of ligands, is important for development of many cranial parasympathetic ganglion neurons. We have investigated the sacral component of the parasympathetic nervous system in mice with gene deletions for neurturin or its preferred receptor, GFRalpha2. Disruption of neurturin signalling decreased cholinergic VIP innervation to the mucosa of the reproductive organs, but not to the smooth muscle layers of these organs or to the urinary bladder. Thus, neurturin and its receptor are involved in parasympathetic innervation of a select group of pelvic visceral tissues. In contrast, noradrenergic innervation was not affected by the gene ablations. The epithelium of reproductive organs from knockout animals was atrophied, indicating that cholinergic innervation may be important for the maintenance of normal structure. Cholinergic neurons express GFRalpha2 on their terminals and somata, indicating they can respond to neurotrophic support, and their somata are smaller when neurturin signalling is disrupted. Colocalisation studies showed that many peripheral glia express GFRalpha2 although its role in these cells is yet to be determined. Our results indicate that neurturin, acting through GFRalpha2, is essential for parasympathetic innervation of the mucosae of reproductive organs, as well as for maintenance of a broader group of sacral parasympathetic neurons.

Postnatal maturational changes in rat pelvic autonomic ganglion cells: a mixture of steroid-dependent and -independent effects.

Androgens have potent effects on the maturation and maintenance of a number of neural pathways involved in reproductive behaviors in males. Most studies in this area have focused on central pathways, but androgen receptors are expressed by many peripheral neurons innervating reproductive organs, and previous studies have demonstrated structural and chemical changes in these neurons at puberty and after castration. We have performed the first electrophysiological comparison of pelvic autonomic ganglion neurons in male rats before and after puberty and following pre- or postpubertal castration. Studies were performed in vitro on intact ganglia with hypogastric and pelvic nerves attached to allow synaptic activation of sympathetic or parasympathetic neurons, respectively. Pelvic ganglion neurons underwent many changes in their passive and active membrane properties over the pubertal period, and some of these changes were dependent on exposure to circulating androgens. The most pronounced steroid-dependent effects were on membrane capacitance (soma size) in sympathetic neurons and duration of the action potential afterhyperpolarization in tonic neurons. Our study also showed that rat pelvic ganglion cells and their synaptic inputs were more diverse than previously reported. In conclusion, this study demonstrated that rat pelvic ganglion neurons undergo considerable postnatal changes in their electrophysiological properties. The steroid dependence of some of these changes indicates that circulating androgens may influence reproductive behaviors at many locations within the nervous system not just in the brain and spinal cord.

Maturational and maintenance effects of testosterone on terminal axon density and neuropeptide expression in the rat vas deferens.

Testosterone causes growth of many pelvic ganglion cells at puberty and their maintenance during adulthood. Here we have focused on two populations of pelvic ganglion cells that project to the rat vas deferens: noradrenergic neurons that innervate the smooth muscle and synthesize neuropeptide Y, and cholinergic neurons that primarily innervate the mucosa and contain vasoactive intestinal peptide. We have assessed the muscle innervation after pre- or postpubertal castration, using immunohistochemistry to determine axon density and radioimmunoassay to quantify levels of neuropeptides in tissue extracts. Our results show that androgen deprivation in each period causes substantial effects. Noradrenergic axons in the muscle increase in density after castration, partly due to organ size being smaller than age-matched controls. However, when corrected for target size, there is an overall decrease in total number of axons. This implies that androgen exposure at puberty has a direct effect on neurons to ensure that the adult pattern of innervation is attained, and that this is not simply by matching terminal field to target size. Similar effects of pre- and postpubertal castration imply that continued exposure to testosterone is necessary to maintain normal target innervation. Castration in both time periods increased the density of axons containing vasoactive intestinal peptide, however the effects of castration on the total number of these axons in the muscle were more variable. The concentration of vasoactive intestinal peptide increased substantially following either pre- or postpubertal castration although absolute amounts per vas deferens were decreased. Effects on neuropeptide Y concentration were less pronounced but the total amount per vas deferens was decreased after pre- or postpubertal castration. Our study shows that the action of testosterone (or a metabolite) on a pelvic ganglion cell soma is likely to reflect a change in its terminal field, but that these effects are not mediated simply by testosterone influencing the size of its target organ.

Testosterone and nerve growth factor have distinct but interacting effects on structure and neurotransmitter expression of adult pelvic ganglion cells in vitro.

Circulating testosterone has potent effects on the structure and function of many pelvic ganglion cells in adult rats in vivo. However not all androgen-sensitive pelvic neurones possess androgen receptors and testosterone effects may therefore be indirect, by an action on the target organs. Here we have examined if testosterone influences neuronal structure in vitro in pelvic ganglion cells cultured from adult male rats. We have also used multiple label immunofluorescence to monitor the expression of transmitter-synthesising enzymes and peptides under various culture conditions. Testosterone was a more potent stimulant of noradrenergic soma growth in culture than nerve growth factor. Whereas nerve growth factor increased the number, branching and length of neurites, testosterone stimulated growth of a small number of very short processes, each of which bore numerous short protrusions. Testosterone also impeded the longer neurite growth induced by nerve growth factor. Many pelvic ganglion cells altered their expression of transmitters/neuropeptides under different culture conditions. In particular, under control conditions or during nerve growth factor treatment, vasoactive intestinal peptide was up-regulated in noradrenergic and cholinergic neurones; testosterone impeded this up-regulation in noradrenergic neurones. Choline acetyltransferase immunoreactivity could only be visualised when nerve growth factor was present in the cultures, and cholinergic neurones showed less neurite outgrowth than noradrenergic neurones under all culture conditions. Nerve growth factor did not stimulate levels of this enzyme as strongly if testosterone was present. This study has shown that testosterone has potent effects on the structure of many pelvic ganglion cells in vitro. It is possible that these effects are mediated indirectly, e.g. by stimulating glial-derived substances, however our results suggest that the effects are not mediated by nerve growth factor. The results also show that testosterone influences some of the actions of nerve growth factor, suggesting that there may be complex interactions between steroid signalling and neurotrophic factors in maintaining neuronal structure and function in vivo.

Differential regulation of trkA and p75 in noradrenergic pelvic autonomic ganglion cells after deafferentation of their cholinergic neighbours.

In rats, following lesion of lumbar or sacral preganglionic axons, many pelvic ganglion cells undergo axogenesis to form baskets of terminals around select populations of nearby ganglion cells. The aim of the current study was to address mechanisms underlying initiation of this sprouting, focusing on a possible role for nerve growth factor (NGF). Immunohistochemical localization of NGF receptors (trkA and p75) showed that virtually all noradrenergic and a minority of cholinergic pelvic neurons expressed both receptors. Terminals immunoreactive for each substance were found in pelvic viscera. In pelvic ganglia, many glial cells expressed p75 but not trkA, and very few lumbar or sacral preganglionic neurons expressed either receptor. Lumbar and/or sacral preganglionic inputs were removed from ganglion cells by cutting the hypogastric, pelvic or both nerves, and tissues analysed 8 days later. Levels of receptor expression in noradrenergic pelvic ganglion cells were estimated by calculating the proportion that were receptor-immunopositive, and quantifying the intensity of trkA or p75 immunofluorescence. No lesion had a significant effect on trkA expression, however, a marked decrease in p75 occurred after cutting pelvic nerves, i.e. after deafferentation of neighbouring cholinergic neurons. These injuries appeared to cause little overall change in glial p75 expression. This study shows that manipulations that trigger sprouting from noradrenergic pelvic neurons cause downregulation of p75 but not trkA. Interestingly, this is occurring while some of their target organs are synthesizing high levels of NGF. This contrasts with other NGF-sensitive cells, in which one or both receptor types are upregulated by increased exposure to the ligand. The current study is also the first to show a change in p75 expression in neurons that are neither deafferented nor axotomized.

Glutamate and aspartate immunoreactivity in dorsal root ganglion cells supplying visceral and somatic targets and evidence for peripheral axonal transport.

Glutamate (Glu) is released by primary sensory neurons at their central synapses, although immunohistochemical studies have shown that only a proportion of these cell bodies are Glu-immunoreactive. Antisera raised against Glu or aspartate (Asp) were used to investigate whether neurons that store high levels of these substances have a unique neuropeptide content or target projection. In male rats, many lumbar and sacral dorsal root ganglion cells and their associated glia show high levels of Glu or Asp immunoreactivity, and fewer than half of these also express substance P or calcitonin gene-related peptide. Conversely, only a minority of peptide-containing neurons store high levels of excitatory amino acids. When neurons that were labelled retrogradely from somatic (skin, gastrocnemius muscle) or visceral (bladder, rectum) targets were immunostained for peptides or amino acids, there was some variation in the peptide expression of their sensory nerve supply, but there was very little or no difference in the prevalence of Glu- or Asp-immunoreactive neurons. In vitro studies on isolated lumbar dorsal root ganglia showed that, after crushing nerve roots, Glu and Asp were transported in both central and peripheral directions, similar to substance P. These studies showed that primary afferent neurons store different levels of Glu and Asp in their somata but that this is not correlated with their target tissue or peptide content. This suggests that both visceral and somatic sensory neurons may vary considerably in the way they release, store, or metabolise amino acids. Peripheral and central transport of amino acids suggests that, in some neurons, reuptake at the synapse may need to be supplemented by amino acids that are produced or accumulated in the soma.

Effects of testosterone on pelvic autonomic pathways: progress and pitfalls.

Testosterone has potent effects on reproductive behavior, many of which are due to actions on brain nuclei and spinal motoneurons controlling perineal muscles. The autonomic circuits involved in penile erection, ejaculation and emission, have been less commonly considered as targets for circulating androgens. This review demonstrates that many components of pelvic autonomic reflex pathways, including preganglionic neurons, autonomic ganglion cells and primary afferent neurons, are likely to be influenced by testosterone. The steroid appears to play an important role in maintaining neuronal morphology, transmitter synthesis and receptor expression throughout adulthood. Surprisingly, the effects of testosterone are not limited to neurons involved in reproductive reflexes. The challenge is now to determine the range of neuronal features influenced by androgens, and the mechanisms by which these occur. Studies of androgen receptor location indicate that in many autonomic neurons gene expression may be directly influenced by androgens, but a mismatch between receptor distribution and androgen action shows that in some cells other mechanisms must exist. It is also possible that androgens are metabolised to estrogens by some peripheral neurons. Irrespective of the mechanism, it is time to acknowledge that testosterone is an important "maintenance factor" for autonomic neurons.

Transmitter profile and spinal inputs of pelvic ganglion cells projecting with preganglionic axons along the hypogastric and pelvic nerves of the male rat.

Pelvic autonomic ganglion cells receive spinal preganglionic inputs via the hypogastric (lumbar) or pelvic (sacral) nerves. Damage to these nerves stimulates axogenesis (sprouting) from pelvic ganglion cells and two possible triggers are deafferentation (decentralisation) or, if some ganglion cells project centrally in these nerves, axotomy. We have used a combination of retrograde tracing and immunohistochemistry in male rats to identify the number of pelvic ganglion cells that project centrally along these nerves, their transmitter type and the spinal level of their preganglionic inputs. Only a small number (<1%) of pelvic ganglion cells project along these nerves; 29-65 project in each hypogastric nerve and 41-71 in each pelvic nerve. These neurons comprise of both cholinergic and noradrenergic classes and the majority receive preganglionic inputs from the nerve in which they also project. These results suggest that damage of the hypogastric and pelvic nerves close to the pelvic ganglion is unlikely to cause axotomy of many pelvic ganglion cells. Therefore deafferentation rather than axotomy is likely to be the primary trigger of axogenesis occurring in pelvic ganglia after these lesions.