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.

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.

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.

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.

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.

Unusual autonomic ganglia: connections, chemistry, and plasticity of pelvic ganglia.

The pelvic ganglia provide the majority of the autonomic nerve supply to reproductive organs, urinary bladder, and lower bowel. Of all autonomic ganglia, they are probably the least understood because in many species their anatomy is particularly complex. Furthermore, they are unusual autonomic ganglia in many ways, including their connections, structure, chemistry, and hormone sensitivity. This review will compare and contrast the normal structure and function of pelvic ganglia with other types of autonomic ganglia (sympathetic, parasympathetic, and enteric). Two aspects of plasticity in the pelvic pathways will also be discussed. First, the influence of gonadal steroids on the maturation and maintenance of pelvic reflex circuits will be considered. Second, the consequences of nerve injury will be discussed, particularly in the context of the pelvic ganglia receiving distributed spinal inputs. The review demonstrates that in many ways the pelvic ganglia differ substantially from other autonomic ganglia. Pelvic ganglia may also provide a useful system in which to study many fundamental neurobiological questions of broader relevance.

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.

Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation.

The biogenesis of follicles from aggregates of precursor cells is an important morphogenetic process in thyroid embryology. It necessitates the creation of a polarized cell phenotype, assembly of specialized cell-cell junctions, and generation of follicular lumena. In this study we sought to investigate the relationship between cell polarization and lumen formation by studying the cell surface events that occurred when freshly isolated adult porcine thyroid cells reorganized to form follicles in primary culture. Follicular reorganization entailed the initial formation of solid three-dimensional cell aggregates and the subsequent appearance of lumena within aggregates. During the initial stage of cell aggregation, the adhesion molecule, E-cadherin, became expressed at all surfaces involved in cell-cell contact. Aggregation was inhibited by monoclonal antibodies that block cadherin function, indicating directly that E-cadherin is a dominant initial cell-cell adhesion molecule. Cell aggregation was also associated with the recruitment to the cell surface of ZO-1, a tight junction-associated protein, and Na+/K(+)-adenosine triphosphatase. These proteins were initially found throughout regions of cell-cell contact and only subsequently redistributed to their mature locations in tight junctions and the basolateral cell surface, respectively. In contrast, components associated with the apical membrane were first detected within large intracellular vacuoles, which subsequently fused with the cell surface between maturing tight junctions to yield the apical membrane domain and nascent follicular lumena. Follicle formation occurred independently of basal lamina assembly and TSH, although maintenance of follicular architecture required the presence of this hormone. These findings indicate that cultured follicles form in two distinct stages: 1) initial aggregation mediated by E-cadherin and associated with recruitment of components of both tight junctions and the basolateral membrane domain, and 2) subsequent formation of a specialized apical membrane domain by coordinated fusion of intracellular vacuoles at sites of the cell surface where tight junctions are maturing. We propose that follicular morphogenesis may arise as a consequence of epithelial cell polarization within coherent three-dimensional cell aggregates.

Innervation of the monotreme gastrointestinal tract: a study of peptide and catecholamine distribution.

The distribution of neurons and endocrine cells containing various peptides or catecholamines was examined in the digestive tracts of the echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus). Comparisons were made with published studies in other species in order to obtain a broader view of the phylogenetic distribution and possible functions of gut peptides and catecholamines. Further comparisons between the echidna and platypus were made in light of their different dietary features and gut histology. The distribution of neurons and axons containing catecholamines or various peptides resembled that in other species (such as the frequent appearance of axons containing substance P and vasoactive intestinal peptide in the intestinal mucosa, and axons containing substance P or enkephalins in the circular muscle). In both species, the stomach histologically resembles the esophagus, being aglandular and lined with stratified squamous epithelium. Innervation of these two organs was similar but not identical, with a greater array of peptides found in the gastric muscle. The intestinal mucosa was densely innervated in both species. The platypus small intestine is unusual in having a thick and deeply folded mucosa (but no villi), in which the superficial epithelium is absent or incomplete at many sites; many axons travel close to these luminal surfaces. Many (putative noradrenergic) axons associated with blood vessels contained neuro-peptide Y, but there was no evidence for intrinsic catecholamine-containing neurons. Somatostatin and cholecystokinin were present in some endocrine cells, but unlike many mammals, absent in neuronal tissue. These studies have shown that there are many strong similarities between monotremes and other mammals in the distribution and array of peptides found within nervous and endocrine tissues of the digestive tract. However, numerous small differences of the echidna and platypus innervation may be correlated with their different digestive structures.

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.

Characterisation of the adventitial rectal ganglia in the male rat by their immunohistochemical features and projections.

In recent years, considerable progress has been made in characterising the neural circuitry of the pelvic plexus, particularly in the male rat. However, the small ganglia on the adventitial surface of the rectum remain largely unstudied. We have used immunohistochemistry and retrograde tracing techniques to determine the content and projections of these neurons. The adventitial ganglia contain 600-1,000 neurons. All of these are immunoreactive for choline acetyltransferase, 44% are immunoreactive for calbindin, and 35% are immunoreactive for vasoactive intestinal peptide. Very few (1-5%) adventitial neurons contain tyrosine hydroxylase or neuropeptide Y. In contrast, most adventitial neurons are surrounded by varicose axons that do contain tyrosine hydroxylase or neuropeptide Y. Retrograde tracing studies showed that the primary targets of adventitial neurons within the bowel are the internal anal sphincter and the circular muscle directly adjacent to the sphincter. However, more adventitial neurons project out of the gut wall than to targets within the bowel. These are most likely to be viscerofugal and rectospinal neurons. Combining the immunohistochemical and tracing observations, these studies suggest that the rat adventitial ganglia do not represent an additional source of pelvic (autonomic postganglionic) neurons but, instead, that they are comprised primarily of viscerofugal and rectospinal neurons. This is very different from the adventitial rectal ganglia of the cat, which represent merely an extension of the pelvic plexus.

Immunohistochemical characterization of pelvic neurons which project to the bladder, colon, or penis in rats.

Retrograde-tracing and immunohistochemical techniques were used in combination to investigate the types of putative transmitters in pelvic neurons that project to the bladder, colon or penis of rats. In addition, populations of axon varicosities associated with these neurons were characterized. Subpopulations of neurons in colchicine-treated major pelvic ganglia and accessory ganglia of male rats contained immunoreactivity (IR) for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), or enkephalin (ENK), while types of immunoreactivity found in major groups of varicose axons were ENK, cholecystokinin (CCK), and somatostatin (SOM). Substance P (SP)-IR varicose axons were much less common. Bladder and colon neurons were similar in a number of ways. Many neurons contained NPY-IR (greater than or equal to 50%), fewer contained TH-IR (25-30%), and even fewer contained ENK-IR (5-15%) or VIP-IR (5-10%); many neurons were associated with baskets of ENK-IR varicosities (50-65%) and fewer neurons were surrounded by CCK- or SOM-IR varicosities (30-35%). Colon neurons differed from penis neurons in having a slightly larger proportion that contained ENK-IR (10-15%, compared with 1-3%). Penis neurons were markedly different from the other two groups in additional ways. More than 90% of them contained VIP-IR, whereas only 5-7% contained NPY-IR and none were immunoreactive for TH. Furthermore, although the proportion of penile neurons associated with many ENK-IR varicosities was similar to the bladder and colon neurons (45-50%), they were rarely seen close to CCK- or SOM-IR varicose axons. These studies describe similarities and differences in the histochemical properties of neurons which project to the bladder, colon, or penis and of the varicose axons associated with those neurons. This gives further insights into the possible transmitter mechanisms involved in the regulation of different pelvic functions.

Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats.

Many visceral afferent neurons contain peptides, which have been proposed as histochemical markers for nerve pathways of particular targets or as transmitter candidates. The former possibility was investigated in the present study. Primary afferent neurons which project to the urinary bladder, distal colon or penis of rats, and the colon of cats were labelled with retrogradely transported fluorescent dyes (Fast Blue, True Blue, or Fluoro Gold). One to six weeks after dye injection into the organs, lumbosacral dorsal root ganglia were removed, treated with colchicine, and processed for immunohistochemical identification of five peptides. Dye-labelled neurons were distributed in an organ-specific manner in the lower lumbosacral ganglia, where colon afferent neurons were almost exclusively found in S1 ganglia, penis neurons primarily in L6, and bladder neurons at both levels. Substance P- (SP), calcitonin gene-related peptide-(CGRP), vasoactive intestinal peptide- (VIP), enkephalin- (ENK), and somatostatin- (SOM) immunoreactivity (IR) were detected in neurons in all lumbosacral ganglia but only some of these peptides were present in a large percentage of labelled neurons. The numbers of peptide-containing neurons innervating each organ were CGRP greater than SP greater than VIP greater than ENK greater than SOM; however some differences were observed in the relative proportions of these neuronal populations between upper lumbar and lower lumbosacral ganglia and between different organs. The major difference seen at the upper lumbar level was amongst the SP-IR neurons, which were common (25-30%) amongst bladder and colon afferent neurons but absent in penis neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of certain peptide-containing nerve fibres and endocrine cells in the gastrointestinal mucosa in five mammalian species.

The distribution of mucosal nerve fibres containing vasoactive intestinal peptide (VIP), substance P, somatostatin, neuropeptide Y (NPY), and enkephalinlike immunoreactivity was mapped by conventional immunohistochemical techniques throughout the mucosa of the esophagus, stomach, small and large intestines, and gall bladder. In addition, the distributions of endocrine cells immunoreactive for three peptides localized by these antisera (namely somatostatin, pancreatic polypeptide, and substance P) were recorded. Tissues from guinea pigs, rats, dogs, marmosets, and humans were studied. It was hoped that this information would enable possible target tissues and functional roles for the peptides to be identified. In the mucosa, peptide nerve fibres were found throughout the lamina propria, including some which were close to the epithelium and others associated with small blood vessels. Although there was a general similarity of peptide nerve distribution between regions and species, many small variations were observed. VIP and substance P fibres were the most prevalent nerve type; NPY fibres were also usually quite common. The distribution of somatostatin fibres was extremely variable between regions and species, and enkephalin fibres were usually rare. Endocrine cells of open (flask- or pyramid-shaped) and closed (rounded) types were seen; basal cytoplasmic processes (of variable length) were seen on many cells immunoreactive for somatostatin or pancreatic polypeptide. Epithelial cells immunoreactive for substance P were seen in the dog, marmoset, and human. The distributions and shapes of endocrine cells varied widely between areas and species. These studies provide a basis for the correlation of nerve distribution with pharmacological and physiological studies.

Peptide neurons in the canine small intestine.

The distributions of peptide-containing nerve fibers and cell bodies in the canine small intestine were determined with antibodies raised against seven peptides: enkephalin, gastrin-releasing peptide (GRP), neuropeptide Y, neurotensin, somatostatin, substance P, and vasoactive intestinal peptide (VIP). Immunoreactive nerve cell bodies and fibers were found for each peptide except neurotensin. In the muscle layers there were numerous substance P, VIP, and enkephalin fibers, fewer neuropeptide Y fibers, and very few GRP or somatostatin fibers. The mucosa contained many VIP and substance P fibers, moderate numbers of neuropeptide Y, somatostatin, and GRP fibers and rare enkephalin fibers. Nerve cell bodies reactive for each of the six neural peptides were located in both the myenteric and submucous plexuses. The distributions of nerve cell bodies and processes in the canine small intestine show many similarities with other mammals, for example, in the distributions of VIP, substance P, neuropeptide Y, and somatostatin nerves. There are some major differences, such as the presence in dogs of numerous submucosal nerve cell bodies with enkephalinlike immunoreactivity and of GRP-like immunoreactivity in submucous nerve cell bodies and mucosal fibers.

Three electrophysiological classes of guinea pig sympathetic postganglionic neurone have distinct morphologies.

Sympathetic postganglionic neurones can be differentiated electrophysiologically into three classes (phasic, Ph; tonic, T; and long-afterhyperpolarising, LAH) based on their potassium channel expression and consequent differences in excitability. We tested whether neuronal morphology differs between these classes. Neurones in coeliac, inferior mesenteric, and lower lumbar paravertebral ganglia of guinea pigs were filled with biocytin during in vitro experiments in which electrical properties were recorded. The dimensions of somata and dendrites were measured in approximately equal numbers of stained neurones of each class. The three electrophysiological classes were distinct in terms of soma shape, soma size (Ph < T = LAH), total dendritic length (LAH < Ph < T) and average length of dendrites (LAH < Ph < T) (P < 0.0001, multivariate analysis of variance). The mean number of primary dendrites also differed (LAH 13, Ph 16, T 20). The majority of dendrites did not branch, the ratios of terminations to primary dendrites being 1.36 (LAH), 1.63 (Ph) and 1.81 (T). Overall, LAH neurones, with medium-sized somata but the smallest dendritic trees, were more distinct morphologically than Ph and T neurones. The morphological differences between classes were not dependent on differences in location. Further, there was no apparent relation between morphology and the pattern of synaptic input each class receives. The results indicate that three distinct groups of sympathetic postganglionic neurone exist in adult guinea pigs, although more than three functions are subserved by these neurones.

Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion.

Pelvic ganglia contain a mixture of sympathetic and parasympathetic neurons which are innervated by either lumbar (sympathetic) or sacral (parasympathetic) preganglionic axons, respectively. However, until recently no stain for these axon terminals has been available. In the present study of the male rat major pelvic ganglion, a ganglion which supplies axons to the lower urinary and digestive tracts and internal reproductive organs, the total population of preganglionic axon terminals was immunostained using an antiserum against synaptophysin, a protein associated with all small synaptic vesicles (such as the presumptive cholinergic vesicles present in all preganglionic terminals). Selective bilateral lesions of either the hypogastric or pelvic nerves, which carry the sympathetic and parasympathetic preganglionic axons, respectively, were carried out and three to seven days later ganglia were examined immunohistochemically for the distribution of residual synaptophysin-positive terminals. Neurons remaining innervated following hypogastric nerve lesion were therefore classified as parasympathetic and those innervated after pelvic nerve section were defined as sympathetic. These two cell groups are present in approximately equal proportions. Double-staining immunofluorescence to identify which transmitters or peptides are present in either sympathetic or parasympathetic neurons showed that the majority (approximately 75%) of sympathetic neurons are presumed to be noradrenergic (i.e. contain tyrosine hydroxylase immunoreactivity) and are also immunoreactive for neuropeptide Y; the remainder contain vasoactive intestinal peptide-immunoreactivity but not tyrosine hydroxylase and may be cholinergic. Parasympathetic neurons were virtually all non-noradrenergic (tyrosine hydroxylase negative) and were also of two histochemical types, with some neurons containing neuropeptide Y- and others containing vasoactive intestinal peptide-immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Distribution and morphological characterization of viscerofugal projections from the large intestine to the inferior mesenteric and pelvic ganglia of the male rat.

Viscerofugal neurons are enteric neurons in the myenteric plexus of the stomach and intestine that project to the prevertebral ganglia as the afferent limb of intestino-intestinal reflexes. This study characterizes viscerofugal projections to the inferior mesenteric ganglion and investigates the possibility of similar projections to the major pelvic ganglia in the male rat. The colon and rectum were examined for retrogradely labelled neurons following the injection of retrograde tracer into the inferior mesenteric or major pelvic ganglia, or following the application of tracer to the caudal end of the cut intermesenteric nerves, or either end of the cut hypogastric nerves. All labelled viscerofugal neurons were found in the myenteric plexus and were often grouped near the mesenteric attachment. The number of viscerofugal neurons projecting to the inferior mesenteric ganglion via the lumbar colonic nerves increases along the length of the large intestine with the maximum number of viscerofugal neurons found in the rectum. Some viscerofugal neurons from the distal colon and rectum reach the inferior mesenteric ganglion via the hypogastric nerves. A similar number and distribution of viscerofugal neurons project via the inferior mesenteric ganglion into the intermesenteric nerves as terminate in the inferior mesenteric ganglion. Very few viscerofugal neurons project to the neurons of the major pelvic ganglia via the rectal nerves, and no viscerofugal neurons project caudally in the hypogastric nerves to these ganglia. The majority of labelled neurons resembled Dogiel type I morphology. Thus the inferior mesenteric ganglion receives a substantial innervation from viscerofugal neurons of the large intestine, with the greatest supply from the distal colon and rectum.(ABSTRACT TRUNCATED AT 250 WORDS)