Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat

The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non-noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non-noradrenergic sympathetic neurons of the pelvic ganglia.     

Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin

The neurotrophic factors that influence the development and function of the parasympathetic branch of the autonomic nervous system are obscure. Recently, neurturin has been found to provide trophic support to neurons of the cranial parasympathetic ganglion. Here we show that GDNF signaling via the RET/GFR(alpha)1 complex is crucial for the development of cranial parasympathetic ganglia including the submandibular, sphenopalatine and otic ganglia. GDNF is required early for proliferation and/or migration of the neuronal precursors for the sphenopalatine and otic ganglia. Neurturin exerts its effect later and is required for further development and maintenance of these neurons. This switch in ligand dependency during development is at least partly governed by the altered expression of GFR(&agr;) receptors, as evidenced by the predominant expression of GFR(&agr;)2 in these neurons after ganglion formation.     

Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study

Summary In rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.     

Embryonic sympathoblasts transiently express TrkB <Emphasis Type="Italic">in vivo</Emphasis> and proliferate in response to brain-derived neurotrophic factor <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Nerve growth factor and neurotrophin-3 are involved in the development of sympathetic neurons; however, whether brain derived neurotrophic factor also plays a role is not known. The purpose of this study was to determine whether BDNF and its receptor, TrkB, are expressed during the development of paravertebral sympathetic ganglia in vivo and to determine the effect of BDNF in vitro.     

Differential Age-Dependent Trophic Responses of Nodose,Sensory, and Sympathetic Neurons to Neurotrophins and GDNF: Potencies for Neurite Extension in Explant Culture

The age-dependent trophic responses of sympathetic, sensory, and nodose neurons to the neuro-trophins NGF, BDNF, and NT-3 and to glial cell line-derived neurotrophic factor (GDNF) were examined by an explant culture system. Superior cervical ganglia (SCG), dorsal root ganglia (DRG), and nodose ganglia (NG) were removed from rat embryos (E18), neonatals ( 1 day old), young adults (3–6 months old), and aged adults (>24 months old). The ganglia were cultured with and without each neurotrophic factor; the neurite extension and neurite density were then assessed. The SCG from rats of all ages were significantly influenced by NGF, NT-3, and GDNF; the effects of NT-3 and GDNF were reduced after maturation. The DRG from embryos and neonates were influenced by all neurotrophic factors; however, the effects of BDNF and NT-3 disappeared after maturation. The GDNF showed little effect on adult DRG and no effect on aged DRG. The effect of NGF was preserved over all ages of DRG. The NG from embryonic rats were significantly responsive to BDNF and GDNF; their effects decreased in the neonatal NG, but a minimum effect remained in the aged NG. These results indicate that age-dependent profiles of trophic effects differ extensively among the lineages of the peripheral nervous system and also among the individual neurotrophic factors.     

Neurturin is a neurotrophic factor for penile parasympathetic neurons in adult rat

Neurturin (NRTN), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. Recent gene ablation studies have shown that NRTN is a neurotrophic factor for many cranial parasympathetic and enteric neurons, whereas its significance for the sacral parasympathetic neurons has not been studied. NRTN signals via a receptor complex composed of the high-affinity binding receptor component GFRalpha2 and the transmembrane tyrosine kinase Ret. The aim of this study was to determine whether NRTN could be an endogenous trophic factor for penis-projecting parasympathetic neurons. NRTN mRNA was expressed in smooth muscle of penile blood vessels and corpus cavernosum in adult rat as well as in several intrapelvic organs, whereas GFRalpha2 and Ret mRNAs were expressed in virtually all cell bodies of the penile neurons, originating in the major pelvic ganglia. (125)I-NRTN injected into the shaft of the penis was retrogradely transported into the major pelvic and dorsal root ganglia. Mice lacking the GFRalpha2 receptor component had significantly less nitric oxide synthase-containing nerve fibers in the dorsal penile and cavernous nerves. In conclusion, these data suggest that NRTN acts as a target-derived survival and/or neuritogenic factor for penile erection-inducing postganglionic neurons.     

Distribution of NADPH-diaphorase activity in rat paravertebral,prevertebral and pelvic sympathetic ganglia

Summary Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive baskets of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.     

胶质细胞源性神经营养因子对疼痛的调节作用

胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)属转化生长因子β超家族成员,其成熟蛋白由134个氨基酸残基组成,而GDNF受体广泛分布于外周和中枢神经系统。GDNF不仅可以促进多巴胺能神经元、运动神经元的存活,对交感、副交感以及感觉神经元具有营养作用,还能够影响神经元的发育、分化并对非神经系统的发育也具有重要作用。近年来随着人们对疼痛认识的深入,疼痛的机制也不再限于神经元功能的改变,还受胶质细胞活化、多种营养因子、细胞因子及相应受体、离子通道等多方面因素的影响。为此,本文就近年来GDNF参与疼痛调节的相关研究进展做一简要综述。     

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

Background & Aims

Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons.

Methods

Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored.

Results

mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression.

Conclusions

Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system.     

In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction.

The study of mammalian neural crest development has been limited by the lack of an accessible system for in vivo transplantation of these cells. We have developed a novel transplantation system to study lineage restriction in the rodent neural crest. Migratory rat neural crest cells (NCCs), transplanted into chicken embryos, can differentiate into sensory, sympathetic, and parasympathetic neurons, as shown by the expression of neuronal subtype-specific and pan-neuronal markers, as well as into Schwann cells and satellite glia. In contrast, an immunopurified population of enteric neural precursors (ENPs) from the fetal gut can also generate neurons in all of these ganglia, but only expresses appropriate neuronal subtype markers in Remak's and associated pelvic parasympathetic ganglia. ENPs also appear restricted in the kinds of glia they can generate in comparison to NCCs. Thus ENPs have parasympathetic and presumably enteric capacities, but not sympathetic or sensory capacities. These results identify a new autonomic lineage restriction in the neural crest, and suggest that this restriction preceeds the choice between neuronal and glial fates.     

GDNF is a chemoattractant for enteric neural cells

In situ hybridization revealed that GDNF mRNA in the mid- and hindgut mesenchyme of embryonic mice was minimal at E10.5 but was rapidly elevated at all gut regions after E11, but with a slight delay (0.5 days) in the hindgut. GDNF mRNA expression was minimal in the mesentery and in the pharyngeal and pelvic mesenchyme adjacent to the gut. To examine the effect of GDNF on enteric neural crest-derived cells, segments of E11.5 mouse hindgut containing crest-derived cells only at the rostral ends were attached to filter paper supports and grown in catenary organ culture. With GDNF (100 ng/ml) in the culture medium, threefold fewer neurons developed in the gut explants and fivefold more neurons were present on the filter paper outside the gut explants, compared to controls. Thus, in controls, crest-derived cells colonized the entire explant and differentiated into neurons, whereas in the presence of exogenous GDNF, most crest-derived cells migrated out of the gut explant. This is consistent with GDNF acting as a chemoattractant. To test this idea, explants of esophagus, midgut, superior cervical ganglia, paravertebral sympathetic chain ganglia, or dorsal root ganglia from E11.5-E12.5 mice were grown on collagen gels with a GDNF-impregnated agarose bead on one side and a control bead on the opposite side. Migrating neural cells and neurites from the esophagus and midgut accumulated around the GDNF-impregnated beads, but neural cells in other tissues showed little or no chemotactic response to GDNF, although all showed GDNF-receptor (Ret and GFRalpha1) immunoreactivity. We conclude that GDNF may promote the migration of crest cells throughout the gastrointestinal tract, prevent them from straying out of the gut (into the mesentery and pharyngeal and pelvic tissues), and promote directed axon outgrowth.     

A rapid and dynamic regulation of GDNF-family ligands and receptors correlate with the developmental dependency of cutaneous sensory innervation.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are members of the transforming growth factor-beta family and have been shown to elicit neurotrophic effects upon several classes of neurons including dopaminergic neurons, motoneurons, parasympathetic, sympathetic as well as primary sensory neurons. However, there is little information available on their roles in cutaneous innervation. Herein, we have studied the regulation of gdnf, ntn and the GDNF family receptors and examined their role in the development of facial cutaneous innervation in GDNF mutant mice. A dynamic spatial and temporal regulation of gdnf, ntn and their ligand binding receptors within the follicle-sinus complex correlate with development of distinct subclasses of sensory nerve endings. Furthermore, development of NGF-dependent myelinated mechanoreceptors, i.e. reticular and transverse lanceolate endings also require GDNF during ending formation and maintenance. In addition, ligand and receptor association seems to be intricately linked to a local Schwann cell-axon interaction essential for sensory terminal formation. Our results suggests that functionally specified nerve endings depend on different GDNF family members and that in contrast to neurotrophins, this family of neurotrophic factors may be acting at local sites of terminal Schwann cell-axon growth cone interactions and that they collaborate with neurotrophins by supporting the same populations of neurons but at different times in development.     

Glial cell line-derived neurotrophic factor is expressed in penis of adult rat and retrogradely transported in penile parasympathetic and sensory nerves

Glial cell line-derived neurotrophic factor (GDNF), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. In the present study, expression of GDNF mRNA in the shaft of adult rat penis is demonstrated. In situ hybridization revealed GDNF mRNA expression in cells lying in the narrow zone between the tunica albuginea and the cavernous tissue. Most subtunical cells exhibited immunoreactivity for vimentin and S100 beta, but they did not stain for smooth muscle alpha actin or PGP9.5. This suggests that the GDNF mRNA-expressing cells may have a mesenchymal origin. Also retrograde axonal transport of intracavernously injected 125I-labeled GDNF in penile parasympathetic and sensory neurons is shown. The transport was inhibited by excess unlabeled GDNF, whereas excess cytochrome c had no effect. This is in agreement with the view that the transport was mediated by binding to specific receptors located on axon terminals. In addition, this study demonstrates expression of GDNF family receptor-alpha 3 (GFR alpha 3) mRNA in most adrenergic, but only in a minor part (5.3%) of the penis-projecting adult rat major pelvic ganglion neurons, as well as in almost half (45.6%) of the penile S1 dorsal root ganglion neurons. In conclusion, the present data suggest that GDNF may act as a neurotrophic factor for subpopulations of adult rat penile parasympathetic and sensory neurons.     

胶质源性神经营养因子对不同组织细胞的影响

GDNF来自于小胶质神经元,首先作为中脑多巴胺能神经元的复活因子被发现,可促进细胞存活,并有增加多巴胺神经元细胞大小及轴突长度的作用。GDNF通过与锚定蛋白细胞表面受体糖基磷脂酰肌醇的相互作用来调节细胞活性。GDNF家族a-1受体,通过跨膜酪氨酸受体或者神经元细胞黏附分子,来促进细胞存活,神经突生长,以及突触发育。后续的研究提示,无论未成年还是成体大脑,GDNF对多种神经细胞都有复活的作用,并与一些周围神经复活、迁移、分化相关。不同的脑缺血实验模型均证实了外源性GDNF对于病灶部位及全脑的神经保护作用,包括局部应用营养因子,利用病毒载体运载GDNF基因以及移植表达GDNF的细胞。近来研究还证实,GDNF不仅对多巴胺能神经元,中枢和周围神经系统的运动、感觉神经元,以及自主神经元有营养和保护作用,对于非神经系统也有不同调节作用。本文将重点讨论这些GDNF作用的不同策略以及机制。     

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.     

Inhibition of Neuronal Apoptosis and Axonal Regression Ameliorates Sympathetic Atrophy and Hemodynamic Alterations in Portal Hypertensive Rats

Background and Aim

A neuronal pathway participates in the development of portal hypertension: blockade of afferent sensory nerves in portal vein ligated (PVL) rats simultaneously prevents brain cardiovascular regularory nuclei activation, neuromodulator overexpression in superior mesenteric ganglia, sympathetic atrophy of mesenteric innervation and hemodynamic alterations. Here we investigated in PVL rats alterations in neuromodulators and signaling pathways leading to axonal regression or apoptosis in the superior mesenteric ganglia and tested the effects of the stimulation of neuronal proliferation/survival by using a tyrosine kinase receptor A agonist, gambogic amide.

Results

The neuronal pathway was confirmed by an increased neuronal afferent activity at the vagal nodose ganglia and the presence of semaphorin3A in sympathetic pre-ganglionic neurons at the intermediolateral nucleus of the spinal cord of PVL rats. Expression of the active form of tyrosine kinase receptor A (phosphorylated), leading to proliferation and survival signaling, showed a significant reduction in PVL comparing to sham rats. In contrast, the apoptotic and axonal retraction pathways were stimulated in PVL, demonstrated by a significant overexpression of semaphorin 3A and its receptor neuropilin1, together with increases of cleaved caspase7, inactive poly(ADP-ribose) polymerase and Rho kinase expression. Finally, the administration of gambogic amide in PVL rats showed an amelioration of hemodynamic alterations and sympathetic atrophy, through the activation of survival pathways together with the inhibition of apoptotic cascades and Rho kinase mediated axonal regression.

Conclusion

The adrenergic alteration and sympathetic atrophy in mesenteric vessels during portal hypertension is caused by alterations on neuromodulation leading to post-ganglionic sympathetic regression and apoptosis and contributing to splanchnic vasodilation.     

GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.