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GDNF induces synaptic vesicle markers in enteric neurons
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GDNF induces synaptic vesicle markers in enteric neurons M. Böttner a,∗,1 , J. Harde a,1 , M. Barrenschee a , I. Hellwig a , I. Vogel b , M. Ebsen c , T. Wedel a a
Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany Department of Surgery, Städtisches Krankenhaus Kiel, Kiel, Germany c Department of Pathology, Städtisches Krankenhaus Kiel, Kiel, Germany b
a r t i c l e
i n f o
Article history: Received 27 May 2013 Received in revised form 18 August 2013 Accepted 29 August 2013 Available online xxx Keywords: Enteric nervous system Synaptic vesicle Synaptic plasticity Cell culture mRNA Localization
a b s t r a c t Regulation of intestinal motility depends on an intact synaptic vesicle apparatus. Thus, we investigated the expression of the synaptic vesicle markers synaptophysin and synaptobrevin in the human enteric nervous system (ENS) and their regulation by glial cell line-derived neurotrophic factor (GDNF) in cultured enteric neurons. Full-thickness specimens of the human colon were assessed for expression of synaptophysin and synaptobrevin and neuronal localization was assessed by dual-label immunocytochemistry with PGP 9.5. Effects of GDNF on both synaptic markers were monitored in enteric nerve cell cultures and the presence of varicosities was determined by applying electron microscopy to the cultures. Human colonic specimens showed immunoreactivity for synaptophysin and synaptobrevin in both myenteric and submucosal ganglia as well as in nerve fibers. Both synaptic vesicle markers co-localized with the neuronal marker PGP 9.5 and exhibited granular accumulation patterns in the human and rat ENS. In cultured rat myenteric neurons GDNF treatment promoted expression of both synaptic vesicle markers and the formation of neuronal varicosities. The regulation of synaptophysin and synaptobrevin in enteric neurons by GDNF argues for the induction of functional neuronal networks in culture characterized by an increase of synaptogenesis. © 2013 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society.
1. Introduction The ENS contains more than 150 million nerve cells and constitutes an integrative neuronal network composed of intramural ganglia and interconnecting nerve fibers arranged in two major nerve plexuses, the submucosal plexus and myenteric plexus (Wedel et al., 1999). Mediation of gastrointestinal (GI) motility as well as resorption, secretion and immune functions are regulated by intrinsic reflex circuits orchestrated by the ENS. Intestinal peristalsis requires the precise interaction of all key-components regulating intestinal motility, i.e. the enteric
Abbreviations: CM, circular muscle layer; BDNF, brain derived neurotrophic factor; CNS, central nervous system; ENS, enteric nervous system; GDNF, glial cell line-derived neurotrophic factor; GFR␣1, GDNF family receptor; GI, gastrointestinal; LM, longitudinal muscle layer; MP, myenteric plexus; Muc, mucosa; N, neuronal soma; qPCR, quantitative PCR; RET, rearranged during transfection; SNAP 25, synaptosome-associated protein 25 kDa; SNARE, soluble N-ethylmaleimidesensitive-factor attachment receptor; SMP, submucosal plexus; VAMP, vesicle associated membrane protein. ∗ Corresponding author at: Institute of Anatomy, Christian-Albrechts-University of Kiel, Otto-Hahn-Platz 8, D-24118 Kiel, Germany. Tel.: +49 431 880 2430; fax: +49 431 880 2699. E-mail address: [email protected] (M. Böttner). 1 These authors contributed equally to the study.
nervous system (“initiators”), the enteric musculature (“effectors”) and enteric neurotransmitters and their receptors (“mediators”). Besides these components, a crucial role for interstitial cells of Cajal in regulating intestinal motility was demonstrated by analysis of mice in which selective depletion of myenteric interstitial cells of Cajal resulted in subsequent absence of peristaltic waves (Nakagawa et al., 2005). Intact intestinal neurotransmission, however, depends on a functional vesicle apparatus composed of specific transporters such as vesicular monoamine transporter (VMAT) (Nirenberg et al., 1995) and vesicular acetylcholine transporter (VAcht) (Weihe et al., 1996), membrane proteins including synaptophysin (Jahn et al., 1985; Wiedenmann and Franke, 1985) and synaptotagmin (Perin et al., 1991) as well as vesicle fusion-mediating proteins of the SNARE complex such as synaptobrevin (Baumert et al., 1989; Trimble et al., 1988) and synaptosomal associated protein (SNAP25) (Oyler et al., 1989). The physiological importance of the key components of the vesicle apparatus and their delicate interplay becomes evident under pathophysiological conditions as described for the central nervous system (CNS). Recent research work in several neurodegenerative disorders of the CNS has emphasized the importance of synaptic dysfunction as the initial event preceeding subsequent neurodegeneration (Burgoyne and Morgan, 2011). This pathogenetic concept has given rise to the term “synaptopathies” applying for Alzheimer’s and Parkinson’s disease as well as prion
0168-0102/$ – see front matter © 2013 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society. http://dx.doi.org/10.1016/j.neures.2013.08.012
Please cite this article in press as: Böttner, M., et al., GDNF induces synaptic vesicle markers in enteric neurons. Neurosci. Res. (2013), http://dx.doi.org/10.1016/j.neures.2013.08.012
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diseases, schizophrenia and autism (Brose et al., 2010). In contrast to the CNS in which many constituents of the synaptic vesicle apparatus have been extensively characterized, research on the ENS demands identification of the respective proteins, characterization of factors and drugs affecting their regulation, addressing the pathophysiology with regard to a loss of function and identifying their role in enteric neuropathies. Survival, differentiation and maintenance of enteric neurons are strongly influenced by neurotrophic factors. Glial cell line-derived neurotrophic factor (GDNF) is a key neurotrophin for the ENS isolated originally from the supernatant of the glial cell line B49 and characterized by its ability to promote the survival of cultured dopaminergic neurons (Lin et al., 1993). GDNF is a member of the TGF- superfamily of growth factors which regulate numerous functions in the development and differentiation of the nervous system (Böttner et al., 2000). The GDNF-induced signal transduction is mediated via the glycosyl phosphaditylinositol-anchored receptor GDNF family receptor ␣1 (GFR␣1) and then rearranged during transfection (RET) receptor tyrosine kinase (Airaksinen et al., 1999). The impact of the GDNF system on the ENS became evident, when gene-ablated animal models were analyzed for ENS defects. Deletion of GDNF leads to total intestinal aganglionosis, i.e. the complete loss of enteric neurons in the small and large intestines (Moore et al., 1996). The importance of GDNF in regulating components of the enteric synaptic vesicle apparatus, however, remains to be determined. The SNARE-complex is indispensable for membrane fusion and neurotransmitter release. Synaptobrevin is an important component of the SNARE-complex, belonging to the family of vesicle-associated membrane proteins (VAMP). The function of synaptophysin is still not completely clarified, but several studies could show that synaptophysin binds synaptobrevin suggesting a regulatory role in SNARE assembly and vesicle fusion (Edelmann et al., 1995; Valtorta et al., 2004). As synaptophysin and synaptobrevin exert different functions within the synaptic vesicle apparatus, the concomitant detection of both markers gives more precise evidence for a functionally intact vesicle apparatus, particularly in enteric nerve cell cultures. Thus, it was the aim of the study to characterize the synaptic proteins synaptophysin and synaptobrevin in the human ENS and in cultured myenteric neurons. We therefore verified the neuronal expression and analyzed the co-localization of both proteins in the human colon, determined their mRNA levels and localization in myenteric plexus cultures in response to GDNF treatment and established a cell culture model of an enteric neuronal network that comprises a functional vesicle apparatus as evidenced by the presence of synaptophysin and synaptobrevin, as well as of neuronal varicosities, i.e. the sides of neurotransmitter release in the ENS.
2. Materials and methods 2.1. Human gastrointestinal tissue Colonic full-thickness specimens obtained from human rectosigmoid segments were used for immunocytochemical studies. Segments of sigmoid colon were obtained from patients (n = 3, mean age 58.0 years, 1 female, 2 males) who underwent anterior rectosigmoid resection for non-obstructive colorectal carcinoma. Patients reported normal bowel habits and showed no evidence of anorectal out-let obstruction. After surgical removal specimens were harvested at safe distance (>5 cm) from the tumor site and transferred immediately to the laboratory for further tissue processing. The study of human tissue received approval from the Ethics Committee of the Faculty of Medicine, Christian-Albrecht’s
University in Kiel, Germany (B 299/07) and has been carried out in accordance with “The Code of Ethics of the World Medical Association”. 2.2. Tissue processing for immunocytochemical studies Full-thickness tissue blocks were trimmed to a size of 30 mm × 10 mm, pinned out flat and tension-free on a cork plate and fixated for 24 h with 4% paraformaldehyde in PBS. After dehydration the tissue blocks were transferred into paraffin wax and cut in 6 m sections. The cutting surface for the sections was the 30 mm border orientated perpendicular to the gut axis, so that myocytes of the circular muscle layer were cut along their longitudinal axis. 2.3. Immunohistochemical studies 2.3.1. Immunohistochemical detection of synaptophysin and synaptobrevin Visualization of synaptophysin and synaptobrevin was achieved by immunohistochemistry using corresponding primary antibodies (rabbit anti-synaptophysin antibody, 1:1000, Biozol; mouse anti-synaptobrevin, 1:200, Thermoscientific). Immunohistochemistry was performed as described previously (Böttner et al., 2012). Briefly, after pretreatment of the sections with citrate buffer (pH 6.0, microwaves 750 W) for 10 min, samples were incubated overnight with primary antibodies diluted in antibody diluent (Zymed, Invitrogen, CA) followed by incubation with biotinylated secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG, 1:400) for 45 min and treatment with an avidin–biotin-complex (Vectastain ABC Standard, Vector Laboratories, Burlingame, CA) conjugated with horseradish peroxidase for 45 min. Antibody binding was visualized with 3,3 -diaminobenzidine (DAB, DakoCytomation) followed by staining with hematoxylin. Analysis was carried out with a light optical microscope (Nikon 6000, Nikon, Tokyo) coupled to a digital camera (Digital Sight, Nikon, Tokyo). Data acquisition was performed with NIS-Elements BR 3.2 software (Nikon, Tokyo). The pictures depicted show one representative out of three experiments. 2.3.2. Dual-label immunocytochemistry with neuronal marker To demonstrate neuronal localization of synaptophysin and synaptobrevin, co-staining with the pan-neuronal marker PGP 9.5 was performed. After pre-treatment with citrate buffer (pH 6.0, 750 W microwaves for 2 × 5 min) sections were incubated overnight with mouse anti-synaptophysin-antibodies (1:200, Millipore, Merck, Darmstadt, Germany) or mouse anti-synaptobrevinantibodies (1:100, Thermoscientific, Ulm, Germany) diluted in antibody diluent (Zymed, Invitrogen, CA). Afterwards, sections were incubated for 2 h at room temperature with a goat anti-mouse AlexaFluor488 antibody (1:250, Invitrogen, Karlsruhe, Germany). To visualize a neuronal localization co-incubation with rabbit PGP 9.5-antibody (1:2000, UCL, Isle of Wight, GB) was performed followed by incubation with goat anti-rabbit AlexaFluor546 antibody (1:250, Invitrogen, Karlsruhe, Germany) for 2 h at room temperature. Finally, DAPI (Roche, Mannheim, Germany) was used to visualize cellular nuclei. Analysis was carried out with a fluorescence microscope (Axiovert 200M, Zeiss, Jena, Germany) coupled to a digital camera (Axiocam, Zeiss, Jena, Germany). Data acquisition was performed using Axiovision software (Zeiss, Jena, Germany). 2.4. Enteric nerve cells culture Preparation of myenteric ganglionic cells was performed according to a method described previously (Schäfer et al., 1997). Briefly, after removing the small intestine from newborn Wistar rats (postnatal day 2–3), the tunica muscularis was stripped from
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the mucosa, followed by incubation for 2 h at 37 ◦ C in Ca2+ and Mg2+ -free Hanks’ Balanced Salt Solution (HBSS, Gibco Life Technologies, Germany) with antibiotics containing 1 mg/ml collagenase (SIGMA, Munich, Germany). Afterwards, fragments of myenteric plexus were collected under stereomicroscopic control and incubated for 15 min at 37 ◦ C in trypsin/EDTA (0.125 mg/ml, Gibco, Life Technologies, Germany) to dissociate the plexus. The procedure was stopped by replacing trypsin/EDTA with fetal calf serum (FCS, Gibco, Life Technologies, Germany). The cells were triturated, counted and seeded in a density of 100,000 cells/ml on poly-d-Lysin-(SIGMA)/Laminin- (SIGMA, Munich, Germany) coated coverslips for immunocytochemistry studies or 12-wellplates for gene expression studies. Cells were incubated in defined medium consisting of Neurobasal A (Gibco, Life Technologies, Germany) and B27 supplement (Gibco, Life Technologies, Germany). Additionally, GDNF (Peprotech, Hamburg, Germany) was added to a final concentration of 2, 10, or 50 ng/ml. For gene expression analysis cells were cultured for 1 week, for immunocytochemical analysis culture time was 3 weeks. Medium was changed every second day.
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2.8. Transmission electron microscopy of enteric nerve cell cultures Cultured cells were fixed in 3% glutaraldehyde in PBS for 30 min and post-fixed in 2% osmiumoxide for 2 × 15 min, followed by dehydration and embedding in araldite overnight. Ultra-thin sections (40–50 nm) were cut (Ultracut, Reichert-Jung, Germany) and contrasted with uranyl acetate for 15 min and with lead citrate for 7 min. Analysis was carried out with a transmission electron microscope (EM 900, Zeiss) coupled to a digital imaging system (Slow Scan CCD-Camera, type 7888-IV, TRS).
2.9. Statistical analysis The effects of the GDNF treatment on rat enteric nerve cell cultures regarding gene expression studies compared to controls were analyzed by student’s t-test (PrizmTM, GraphPad, Sand Diego, CA, USA) followed by post hoc test according to the false-discovery rate procedure, using R 2.8.0 (R-Core-Team, 2012). Differences were considered significant if p < 0.05.
2.5. RNA extraction, reverse transcription and quantitative PCR Extraction of RNA from enteric nerve cell cultures was performed using a Nucleospin XS kit (Macherey and Nagel, Düren, Germany) according to the manufacturer’s guidelines. Reverse transcription and qPCR were performed as described before (Böttner et al., 2010). Forward and reverse primers as well as probes are listed below. rat synaptophysin – forward primer: 5 -gcagtgggtctttgccatctt-3 , reverse primer: 5 -tgagggcactctccgtcttg-3 , probe: 5 -cctttgctacgtgtggcagctaca-3 ; rat synaptobrevin – forward primer: 5 -cagcaaacccaggcacaagt3 , reverse primer: 5 -cgtcagctcggtcatccaa-3 , probe: 5 -atgcgcgtgaatgtggacaaggt-3 ; rat HPRT – forward primer: 5 -cgccagcttcctcctcaga-3 , reverse primer: 5 -ggtcataacctggttcatcact-3 , probe: 5 -ttttcccgcgagccgaccgg-3 .
3. Results 3.1. Localization of synaptophysin in the human colon To assess the distribution of synaptophysin in the human colon, immunohistochemistry using DAB as a chromogene was applied to colonic full-thickness sections. Synaptophysin immunoreactivity was detectable in all parts of the ENS, including submucosal (Fig. 1A and B) and myenteric ganglia (Fig. 1C and D) as well as in nerve fibers of the circular (Fig. 1E) and longitudinal muscle layer (Fig. 1F). Immunoreactivity in neuronal somata appeared to be less intense compared to the surrounding neuropil. At higher magnifications a small-sized granular staining pattern could be observed in the ganglionic neuropil and in the intramuscular nerve fibers (Fig. 1B, D, E, and F).
3.2. Localization of synaptobrevin in the human colon 2.6. Immunocytochemistry of enteric nerve cell cultures Cells were fixed for 30 min with 4% paraformaldehyde, permeabilized for 10 min with methanol and treated for 10 min with 3% H2 O2 . Afterwards, incubation with a rabbit antisynaptophysin antibody (1:1000, Biozol, Echingen, Germany) and a mouse anti-synaptobrevin antibody (1:100, Thermoscientific) for 1 h was performed, followed by incubation with the secondary antibodies, goat anti-rabbit-AlexaFlour546 (1:250, Invitrogen, Karlsruhe, Germany) and goat-anti-mouse-AlexaFluor488 (1:250, Invitrogen, Karlsruhe, Germany). Finally, cells were counterstained with DAPI (Roche, Mannheim, Germany) to visualize cell nuclei. Analysis was carried out with a fluorescent microscope (Axiovert 200M, Zeiss, Göttingen, Germany) coupled to a digital camera (Axiocam, Zeiss, Göttingen, Germany). Data acquisition was performed with Axiovision software (Zeiss, Göttingen, Germany). 2.7. Scanning electron microscopy of enteric nerve cells cultures Fixation of 3 weeks cultured enteric neurons was performed by using 3% glutaraldehyde in PBS for 30 min, followed by postfixation for 20 min in 2% osmiumoxide and dehydration. After critical point drying (CPD 030, Balzers), probes were sputtered with gold (Ion Tech Ltd.). Processed cultured cells were examined using a scanning electron microscope (Phillips XL 20, Phillips).
Immunohistochemistry was also applied to colonic fullthickness sections to assess the distribution of synaptobrevin in the human colon. Similar to the distribution of synaptophysin, synaptobrevin immunoreactivity was detectable in all parts of the ENS, including submucosal (Fig. 2A and B) and myenteric ganglia (Fig. 2C and D) as well as in nerve fibers of the circular (Fig. 2E) and longitudinal muscle layer (Fig. 2F). The small-sized granular staining pattern for synaptophysin at higher magnifications could also be observed for synaptobrevin in the ganglionic neuropil and in the intramuscular nerve fibers (Fig. 2B, D, E, and F). Consistently, neuronal cytoplasmatic stain appeared less intense than signals in the neuropil.
3.3. Neuronal localization of synaptophysin To further prove a neuronal localization of synaptophysin, duallabel immunocytochemistry with the pan-neuronal marker PGP 9.5 was performed (Fig. 3). Co-localization of synaptophysin and PGP 9.5 was detectable indicating a specific neuronal expression of synaptophysin in the ENS (Fig. 3C and F). The previously observed granular staining pattern was more intense, particularly in the neuropil around neuronal somata (Fig. 3A and C). In addition, enteric nerve fibers exhibited co-localization of synaptophysin and PGP 9.5 (Fig. 3F).
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Fig. 1. Localization of synaptophysin in the human colon. Submucosal (arrows in A and B) and myenteric ganglia (arrows in C and D) as well as intramuscular nerve fibers (arrows in E and F) are readily identified by robust synaptophysin immunoreactive signals (brown color). Neuronal somata (arrows in B and D) mostly show weaker synaptophysin immunoreactivity than the surrounding ganglionic neuropil. At higher magnifications a small-sized granular staining pattern is discernible within ganglia and nerve fibers (B, D, E, and F). Muc, mucosa; SM, submucosa; CM, circular muscle layer; LM, longitudinal muscle layer; SMP, submucosal plexus; MP, myenteric plexus; N, neuronal somata; NF, nerve fibers. Hematoxylin counterstain. Scale bars = 100 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.4. Neuronal localization of synaptobrevin To prove neuronal localization of synaptobrevin in the ENS, we performed co-labeling with the pan-neuronal marker PGP9.5 that stains neuronal somata and processes (Fig. 4). As observed for synaptophysin, co-localization with PGP9.5 demonstrated immunoreactivity of synaptobrevin in neurons (Fig. 4A–C). A particular granular staining pattern of synaptobrevin was detectable in the ganglionic neuropil surrounding neuronal somata (Fig. 4A and C) as well as in enteric nerve fibers (Fig. 4D and F). 3.5. Effects of GDNF on synaptophysin and synaptobrevin mRNA expression in cultured enteric neurons To investigate the effect of the neurotrophic factor GDNF on vesicle protein expression, a cell culture model of rat postnatal dissociated myenteric plexus exposed to increasing concentrations of GDNF was implemented. mRNA expression of synaptophysin and synaptobrevin was measured by quantitative RT-PCR (Fig. 5). After GDNF-treatment for 1 week, mRNA expression of synaptophysin (Fig. 5A) as well as mRNA expression of synaptobrevin (Fig. 5B) was significantly increased compared to untreated controls. mRNA expression of synaptophysin in GDNF-treated cultures
was nearly 4-fold up-regulated compared to controls (Fig. 5A). mRNA expression of synaptobrevin in cultures treated with 50 ng GDNF/ml was about 6-fold higher than in untreated controls (Fig. 5B). 3.6. Localization of synaptophysin and synaptobrevin in cultured enteric neurons The topographical expression pattern of synaptophysin and synaptobrevin in cultured myenteric neurons was assessed by performing a co-staining of enteric nerve cell cultures treated with 50 ng GDNF/ml for 3 weeks (Fig. 6). During the culture period, cells treated with GDNF form a neuronal network consisting of ganglionic aggregates interconnected by nerve fiber strands, whereas control cultures degenerate over time (data not shown). Immunoreactivity of synaptophysin (Fig. 6A) as well as immunoreactivity of synaptobrevin (Fig. 6B) was detectable in cultured myenteric neurons including neuronal ganglia and interganglionic nerve fiber strands. Co-localization of both synaptic proteins (yellow) was observed in cultured myenteric neurons by merging the images, particularly in granular accumulations (Fig. 6C) presumably resembling varicosities, i.e. the side of neurotransmitter release in the ENS.
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Fig. 2. Localization of synaptobrevin in the human colon. Synaptobrevin immunoreactivity (brown color) is detected in submucosal (arrows in A and B) and myenteric ganglia (arrows in C and D) as well as intramuscular nerve fibers (arrows in E and F). Immunoreactive signals are stronger in the ganglionic neuropil compared with neuronal somata (arrows in B and D). A granular staining pattern is visible at higher magnifications within ganglia and nerve fibers (B, D, E, and F). Muc, mucosa; SM, submucosa; CM, circular muscle layer; LM, longitudinal muscle layer; SMP, submucosal plexus; MP, myenteric plexus; N, neuronal somata; NF, nerve fibers. Hematoxylin counterstain. Scale bars = 100 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.7. Electron microscopy of neuronal varicosities in myenteric plexus cultures To verify the presence of varicosities in the cultured myenteric neurons, scanning electron microscopy of cultures treated for three weeks with 50 ng/ml was performed. As demonstrated by the overview of a three week culture, the cells form an extensive neuronal network characterized by the presence of ganglionic aggregates and nerve fiber strands (Fig. 7A). High power magnification reveals abundant presence of varicosities on nerve fiber strands (Fig. 7B). Supporting transmission electron microscopy underlines the functionality of these varicosities by revealing the presence of large dense core as well as small and large electrolucent synaptic vesicles arguing for the presence of peptidergic and cholinergic neurotransmitters present in the putative sites of neurotransmitter release (Fig. 7C and D). 4. Discussion The present study shows four important findings: (1) Synaptophysin and synaptobrevin are localized in submucosal and myenteric ganglia and nerve fiber strands of the human ENS. (2) Co-localization experiments reveal the presence of both proteins in enteric neurons. (3) Myenteric plexus cultures treated with GDNF
show an up-regulation of synaptophysin and synaptobrevin mRNA and both proteins co-localize in cultured neurons. (4) In response to GDNF treatment enteric nerve cell cultures form an extensive neuronal network comprising neuronal varicosities filled with heterogeneous synaptic vesicles. 4.1. Localization of synaptophysin in the human ENS Communication between the billions of neurons in the brain requires synaptic transmission. Release of neurotransmitters, an important part of synaptic transmission, is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals (Sudhof, 1995, 2004). Synaptophysin is the most abundant integral synaptic vesicle protein in the CNS and is therefore often used to quantify synapses (Calhoun et al., 1996). The function of synaptophysin is still not completely clarified, but several studies could show that synaptophysin binds synaptobrevin suggesting a regulatory role in SNARE assembly and vesicle fusion (Edelmann et al., 1995; Valtorta et al., 2004). We show an abundant granular expression of synaptophysin in nerve fibers of the muscle layers and in the neuropil around neuronal somata of both the myenteric and the submucosal plexus, i.e. in enteric nerve fibers and in enteric ganglia. Additionally, we could describe weak immunoreactivity in ganglionic neuronal
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Fig. 3. Co-staining of synaptophysin and PGP 9.5 in the human colon. Synaptophysin immunoreactivity (green) is detectable in enteric nerve cells visualized by PGP 9.5 (red) of enteric ganglia of the myenteric plexus (A–C) as well as in enteric nerve fibers of the circular muscle layer (arrows in D–F). Immunoreactive signals of synaptophysin are stronger in neuropil and enteric nerve fibers compared to neuronal somata (A and D). Synaptophysin shows a granular staining pattern in the neuropil, particularly around neuronal somata (A and C), and in enteric nerve fibers (D and F). MP, myenteric plexus; N, neuronal somata; NF, nerve fibers, blue color: DAPI staining of nuclei. Scale bars = 50 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
somata. The neuronal localization was verified by performing dual-label immunohistochemistry with the pan-neuronal marker PGP 9.5, an established marker for enteric ganglia (Krammer et al., 1993). Analysis of the distribution pattern of synaptophysin
in healthy intestine is of special importance regarding altered expression pattern under pathological conditions as demonstrated for pelviureteral junction obstruction (Demirbilek et al., 2006).
Fig. 4. Co-staining of synaptobrevin and PGP 9.5 in the human colon. Synaptobrevin immunoreactivity (green) is detectable in enteric nerve cells visualized by PGP 9.5 (red) of enteric ganglia in the myenteric plexus (A–C) as well as in enteric nerve fibers between muscle cells of the circular muscle layer (arrows in D–F). Immunoreactive signals of synaptobrevin are mostly granular and stronger in neuropil somata (A and C) and enteric nerve fibers (D and F), than in neuronal somata (A and C). Granular staining is particularly intense in neuropil around neuronal somata (A and C). MP, myenteric plexus; N, neuronal somata; NF, nerve fibers, blue color: DAPI staining of nuclei. Scale bars = 50 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. mRNA expression of synaptophysin and synaptobrevin in myenteric plexus cultures in response to GDNF treatment. GDNF increases mRNA levels of synaptophysin and synaptobrevin. Cells were cultured for one week and expression levels of the target genes were normalized to expression of the house-keeping gene HPRT. Data are shown as mean ± SEM, synaptophysin mRNA expression: n = 18–21, synaptobrevin mRNA expression: n = 12–13, *p < 0.05 vs. control.
4.2. Localization of synaptobrevin in the human ENS The SNARE-complex is indispensable for membrane fusion and neurotransmitter release. Synaptobrevin is an important component of the SNARE-complex, belonging to the family of vesicle-associated membrane proteins (VAMP). We detected granula of synaptobrevin in the nerve fibers of the muscle layers as well as in the neuropil around the neuronal somata of both myenteric and submucosal ganglia. Also weak immunofluorescence was observed in neuronal somata of the ganglia marking the functionally active synaptic apparatus in the human ENS. 4.3. GDNF induces synaptophysin and synaptobrevin expression in myenteric plexus cultures To assess the regulatory effect of GDNF on the expression of synaptophysin and synaptobrevin, we used an in vitro model of enteric neurons by culturing rat postnatal day-2 myenteric neurons. Cultured enteric neurons display formation of neuronal networks composed of ganglia with interconnecting nerve fiber strands when cultured in the presence of GDNF. Regarding neuronal network formation, a study reported by Takaki et al. (2006) described the in vitro formation of enteric neural network structures in gut-like organs differentiated from embryonic stem cells.
As demonstrated in this study, addition of brain-derived neurotrophic factor (BDNF) during the formation of embryonic bodies led to the in vitro formation of enteric neural ganglia with interconnecting nerve fibers in gut-like structures and the generation of distinct peristalsis-like movements in the differentiated gut tissue. In contrast, application of GDNF or neurotrophin-3 did not result in enteric ganglia formation indicating that BDNF and GDNF in vivo might act in a temporally sequential fashion on enteric neuronal precursors to induce formation of neuronal ganglia and consequently functional networks. To address whether the neuronal networks formed in our culture system are functionally active as assessed by the presence of markers of synaptic plasticity, we performed a qPCR for synaptophysin and synaptobrevin of cultured myenteric neurons treated with GDNF for one week. The mRNA expression of both synaptophysin as well as synaptobrevin was significantly increased suggesting that GDNF has also positive effects on synaptogenesis and neuronal plasticity in the ENS. The GDNF-induced increase in synaptophysin expression might be due to activation of the PI-3-kinase/Akt pathway as it has been demonstrated that GDNF activates the PI-3-kinase pathway in primary enteric neurons (Srinivasan et al., 2005) and induces phosphorylation of Akt in enteric neuronal cell lines (Anitha et al., 2008) and that in addition activation of PI-3 kinase promotes synaptogenesis in primary
Fig. 6. Co-localization of synaptophysin and synaptobrevin in myenteric plexus cultures. Rat enteric nerve cells were cultured for 3 weeks with 50 ng GDNF/ml (A–C). Synaptophysin immunoreactivity (red, A) and synaptobrevin immunoreactivity (green, B) are detectable in cultured myenteric neurons including neuronal somata as well as nerve fibers. Co-localization of both proteins is detectable (yellow) in the merged image (C), particularly in granula. The granular staining pattern is detectable in neuronal ganglia (arrows in C) as well as in the interganglionic nerve fiber strands (arrowhead in C). Blue color: DAPI staining of nuclei. Scale bars = 100 m (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 7. Scanning and transmission electron microscopy of cultured myenteric neurons. Rat enteric nerve cells were cultured for 3 weeks with 50 ng GDNF/ml (A–D). Scanning electron microscopy (A and B) shows multiple round thickenings along enteric nerve fiber strands at higher magnifications (arrow in B). Transmission electron microscopy (C and D) demonstrates multiple high (arrow in D) and low dense (arrowhead in D) vesicles in the thickenings most likely representing neuronal varicosities filled with synaptic vesicles. Scale bars = 200 m (A), 2 m (B), 1 m (C), and 500 nm (D).
cultures of rat hippocampal neurons as well as in CA1 hippocampal neurons in vivo (Cuesto et al., 2011). After extending the culture time to a period of three weeks, neuronal localization of both vesicle markers, particularly in neuronal varicosities could be observed. These data are supported by a recent report describing enhanced synaptic communication by modulating potassium currents and response to serotonin of cultured myenteric neurons after GDNF treatment (Zeng et al., 2009). Supporting data are reported from cultured CNS neurons, as enhanced synaptic efficacy of cultured dopaminergic neurons after GDNF treatment by counting synapsin-1-positive puncta per neuron was shown (Bourque and Trudeau, 2000). Furthermore, it was demonstrated that synapsin-1 and synaptophysin antibodies labeled the same population of putative nerve terminals. Data on synaptobrevin expression following GDNF treatment, however, are contradictory. Whereas one study could not observe changes in synaptobrevin protein expression in cultured mesencephalic neurons after GDNF or BDNF treatment by performing Western blot analysis (Feng et al., 1999), another report described increased number and size of synaptobrevin positive clusters in xenopus nerve-muscle cultures after GDNF and neurturin treatment suggesting not only an enhancement of synaptic vesicle clustering, but also implying an increase in the number of release sites by GDNF treatment (Wang et al., 2002). These data are in accordance with our observation of increased synaptobrevin mRNA expression after GDNF treatment indicating a growth-factor induced rise in synaptic plasticity. 4.4. Presence of synaptic vesicles in varicosities The granular staining pattern of both synaptic proteins shown in the human tissue was reproducible in the in vitro model of cultured rat myenteric neurons. However, a culture period of the three weeks was necessary to visualize immunoreactivity in granula most likely representing synaptic varicosities indicating that this period was required for the synaptic apparatus to mature. Additionally, we could show co-localization of synaptophysin
and synaptobrevin in ganglia and interganglionic fiber strands of cultured enteric neurons arguing for the presence of a functional synaptic apparatus, as not only the vesicle marker synaptophysin was detectable but also the vesicle-associated SNARE protein necessary for mediating membrane fusion of the synaptic vesicles. Other investigations described an interplay of synaptophysin and the SNARE-protein synaptobrevin by applying immunoprecipitation, suggesting a physiological role for synaptophysin in control membrane fusion and exocytosis (Edelmann et al., 1995; Valtorta et al., 2004). By assessing the ultrastructure of the granula we could demonstrate vesicles in variable electron densities accumulating in thickenings representing synaptic varicosities, the sites of neurotransmitter release in the ENS. Low dense core vesicles represent most likely small synaptic vesicles (SSV), whereas the larger vesicles constitute large dense core vesicles (LDCV) (Bruns and Jahn, 1995). 5. Conclusion Taken together, our data indicate that the cultured enteric neurons form a functional neuronal network that comprises not only fiber strands and ganglia, but also a functional synaptic vesicle apparatus similar to the situation in humans. Components of the synaptic vesicle apparatus would provide the necessary equipment for functional neuron-to-neuron communication. Thus, this culture model might be an ideal tool to assess components increasing synaptic plasticity and the mechanisms underlying this process or also the respective confounding factors. Due to the fact that GDNF knockout mice exhibit complete intestinal aganglionosis resembling the pathology observed in Hirschsprung’s disease (Moore et al., 1996; Pichel et al., 1996), the underlying mechanism might be initially established by a loss of synaptic communication. Furthermore, components of the synaptic vesicle apparatus might be promising candidates to characterize the initial steps in the manifestation of human intestinal motility disorders, as these pathologies are frequently associated with
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neuronal loss and an underlying synaptopathy may be the initial event in the neurodegenerative process. Funding The study was supported by research grants from the Deutsche Forschungsgemeinschaft (DFG, WE 2366/4-2). The funding source had no role in conduction of the experiments and preparation of the article. Author contributions M. Böttner was responsible for study design, data analysis and writing of the paper; J. Harde, M. Barrenschee, I. Hellwig were responsible for acquisition and interpretation of data; I. Vogel and M. Ebsen contributed to the acquisition of the human material; and Thilo Wedel designed the study, critically revised the manuscript and wrote the grants financing the study. Acknowledgements The authors thank Karin Stengel, Inka Geurink, Miriam Lemmer, Frank Lichte, Bettina Facompré, and Clemens Franke (Institute of Anatomy, Christian-Albrechts-University of Kiel) for their excellent technical assistance. References Airaksinen, M.S., Titievsky, A., Saarma, M., 1999. GDNF family neurotrophic factor signaling: four masters, one servant? Molecular and Cellular Neurosciences 13, 313–325. Anitha, M., Joseph, I., Ding, X., Torre, E.R., Sawchuk, M.A., Mwangi, S., Hochman, S., Sitaraman, S.V., Anania, F., Srinivasan, S., 2008. Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function. Gastroenterology 134, 1424–1435. Baumert, M., Maycox, P.R., Navone, F., De Camilli, P., Jahn, R., 1989. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. The EMBO Journal 8, 379–384. Böttner, M., Bar, F., Von Koschitzky, H., Tafazzoli, K., Roblick, U.J., Bruch, H.P., Wedel, T., 2010. Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine. Neurogastroenterology and Motility 22, 168–172, e152. Böttner, M., Krieglstein, K., Unsicker, K., 2000. The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions. Journal of Neurochemistry 75, 2227–2240. Böttner, M., Zorenkov, D., Hellwig, I., Barrenschee, M., Harde, J., Fricke, T., Deuschl, G., Egberts, J.H., Becker, T., Fritscher-Ravens, A., et al., 2012. Expression pattern and localization of alpha-synuclein in the human enteric nervous system. Neurobiology of Disease 48, 474–480. Bourque, M.J., Trudeau, L.E., 2000. GDNF enhances the synaptic efficacy of dopaminergic neurons in culture. The European Journal of Neuroscience 12, 3172–3180. Brose, N., O’Connor, V., Skehel, P., 2010. Synaptopathy: dysfunction of synaptic function? Biochemical Society Transactions 38, 443–444. Bruns, D., Jahn, R., 1995. Real-time measurement of transmitter release from single synaptic vesicles. Nature 377, 62–65. Burgoyne, R.D., Morgan, A., 2011. Chaperoning the SNAREs: a role in preventing neurodegeneration? Nature Cell Biology 13, 8–9. Calhoun, M.E., Jucker, M., Martin, L.J., Thinakaran, G., Price, D.L., Mouton, P.R., 1996. Comparative evaluation of synaptophysin-based methods for quantification of synapses. Journal of Neurocytology 25, 821–828. Cuesto, G., Enriquez-Barreto, L., Carames, C., Cantarero, M., Gasull, X., Sandi, C., Ferrus, A., Acebes, A., Morales, M., 2011. Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons. Journal of Neuroscience 31, 2721–2733. Demirbilek, S., Edali, M.N., Gurunluoglu, K., Turkmen, E., Tas, E., Karaman, A., Akin, M., Aksoy, R.T., Celbis, O., Uzun, I., 2006. Glial cell line-derived neurotrophic factor and synaptophysin expression in pelviureteral junction obstruction. Urology 67, 400–405.
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GDNF induces synaptic vesicle markers in enteric neurons M. Böttner a,∗,1 , J. Harde a,1 , M. Barrenschee a , I. Hellwig a , I. Vogel b , M. Ebsen c , T. Wedel a a
Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany Department of Surgery, Städtisches Krankenhaus Kiel, Kiel, Germany c Department of Pathology, Städtisches Krankenhaus Kiel, Kiel, Germany b
a r t i c l e
i n f o
Article history: Received 27 May 2013 Received in revised form 18 August 2013 Accepted 29 August 2013 Available online xxx Keywords: Enteric nervous system Synaptic vesicle Synaptic plasticity Cell culture mRNA Localization
a b s t r a c t Regulation of intestinal motility depends on an intact synaptic vesicle apparatus. Thus, we investigated the expression of the synaptic vesicle markers synaptophysin and synaptobrevin in the human enteric nervous system (ENS) and their regulation by glial cell line-derived neurotrophic factor (GDNF) in cultured enteric neurons. Full-thickness specimens of the human colon were assessed for expression of synaptophysin and synaptobrevin and neuronal localization was assessed by dual-label immunocytochemistry with PGP 9.5. Effects of GDNF on both synaptic markers were monitored in enteric nerve cell cultures and the presence of varicosities was determined by applying electron microscopy to the cultures. Human colonic specimens showed immunoreactivity for synaptophysin and synaptobrevin in both myenteric and submucosal ganglia as well as in nerve fibers. Both synaptic vesicle markers co-localized with the neuronal marker PGP 9.5 and exhibited granular accumulation patterns in the human and rat ENS. In cultured rat myenteric neurons GDNF treatment promoted expression of both synaptic vesicle markers and the formation of neuronal varicosities. The regulation of synaptophysin and synaptobrevin in enteric neurons by GDNF argues for the induction of functional neuronal networks in culture characterized by an increase of synaptogenesis. © 2013 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society.
1. Introduction The ENS contains more than 150 million nerve cells and constitutes an integrative neuronal network composed of intramural ganglia and interconnecting nerve fibers arranged in two major nerve plexuses, the submucosal plexus and myenteric plexus (Wedel et al., 1999). Mediation of gastrointestinal (GI) motility as well as resorption, secretion and immune functions are regulated by intrinsic reflex circuits orchestrated by the ENS. Intestinal peristalsis requires the precise interaction of all key-components regulating intestinal motility, i.e. the enteric
Abbreviations: CM, circular muscle layer; BDNF, brain derived neurotrophic factor; CNS, central nervous system; ENS, enteric nervous system; GDNF, glial cell line-derived neurotrophic factor; GFR␣1, GDNF family receptor; GI, gastrointestinal; LM, longitudinal muscle layer; MP, myenteric plexus; Muc, mucosa; N, neuronal soma; qPCR, quantitative PCR; RET, rearranged during transfection; SNAP 25, synaptosome-associated protein 25 kDa; SNARE, soluble N-ethylmaleimidesensitive-factor attachment receptor; SMP, submucosal plexus; VAMP, vesicle associated membrane protein. ∗ Corresponding author at: Institute of Anatomy, Christian-Albrechts-University of Kiel, Otto-Hahn-Platz 8, D-24118 Kiel, Germany. Tel.: +49 431 880 2430; fax: +49 431 880 2699. E-mail address: [email protected] (M. Böttner). 1 These authors contributed equally to the study.
nervous system (“initiators”), the enteric musculature (“effectors”) and enteric neurotransmitters and their receptors (“mediators”). Besides these components, a crucial role for interstitial cells of Cajal in regulating intestinal motility was demonstrated by analysis of mice in which selective depletion of myenteric interstitial cells of Cajal resulted in subsequent absence of peristaltic waves (Nakagawa et al., 2005). Intact intestinal neurotransmission, however, depends on a functional vesicle apparatus composed of specific transporters such as vesicular monoamine transporter (VMAT) (Nirenberg et al., 1995) and vesicular acetylcholine transporter (VAcht) (Weihe et al., 1996), membrane proteins including synaptophysin (Jahn et al., 1985; Wiedenmann and Franke, 1985) and synaptotagmin (Perin et al., 1991) as well as vesicle fusion-mediating proteins of the SNARE complex such as synaptobrevin (Baumert et al., 1989; Trimble et al., 1988) and synaptosomal associated protein (SNAP25) (Oyler et al., 1989). The physiological importance of the key components of the vesicle apparatus and their delicate interplay becomes evident under pathophysiological conditions as described for the central nervous system (CNS). Recent research work in several neurodegenerative disorders of the CNS has emphasized the importance of synaptic dysfunction as the initial event preceeding subsequent neurodegeneration (Burgoyne and Morgan, 2011). This pathogenetic concept has given rise to the term “synaptopathies” applying for Alzheimer’s and Parkinson’s disease as well as prion
0168-0102/$ – see front matter © 2013 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society. http://dx.doi.org/10.1016/j.neures.2013.08.012
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diseases, schizophrenia and autism (Brose et al., 2010). In contrast to the CNS in which many constituents of the synaptic vesicle apparatus have been extensively characterized, research on the ENS demands identification of the respective proteins, characterization of factors and drugs affecting their regulation, addressing the pathophysiology with regard to a loss of function and identifying their role in enteric neuropathies. Survival, differentiation and maintenance of enteric neurons are strongly influenced by neurotrophic factors. Glial cell line-derived neurotrophic factor (GDNF) is a key neurotrophin for the ENS isolated originally from the supernatant of the glial cell line B49 and characterized by its ability to promote the survival of cultured dopaminergic neurons (Lin et al., 1993). GDNF is a member of the TGF- superfamily of growth factors which regulate numerous functions in the development and differentiation of the nervous system (Böttner et al., 2000). The GDNF-induced signal transduction is mediated via the glycosyl phosphaditylinositol-anchored receptor GDNF family receptor ␣1 (GFR␣1) and then rearranged during transfection (RET) receptor tyrosine kinase (Airaksinen et al., 1999). The impact of the GDNF system on the ENS became evident, when gene-ablated animal models were analyzed for ENS defects. Deletion of GDNF leads to total intestinal aganglionosis, i.e. the complete loss of enteric neurons in the small and large intestines (Moore et al., 1996). The importance of GDNF in regulating components of the enteric synaptic vesicle apparatus, however, remains to be determined. The SNARE-complex is indispensable for membrane fusion and neurotransmitter release. Synaptobrevin is an important component of the SNARE-complex, belonging to the family of vesicle-associated membrane proteins (VAMP). The function of synaptophysin is still not completely clarified, but several studies could show that synaptophysin binds synaptobrevin suggesting a regulatory role in SNARE assembly and vesicle fusion (Edelmann et al., 1995; Valtorta et al., 2004). As synaptophysin and synaptobrevin exert different functions within the synaptic vesicle apparatus, the concomitant detection of both markers gives more precise evidence for a functionally intact vesicle apparatus, particularly in enteric nerve cell cultures. Thus, it was the aim of the study to characterize the synaptic proteins synaptophysin and synaptobrevin in the human ENS and in cultured myenteric neurons. We therefore verified the neuronal expression and analyzed the co-localization of both proteins in the human colon, determined their mRNA levels and localization in myenteric plexus cultures in response to GDNF treatment and established a cell culture model of an enteric neuronal network that comprises a functional vesicle apparatus as evidenced by the presence of synaptophysin and synaptobrevin, as well as of neuronal varicosities, i.e. the sides of neurotransmitter release in the ENS.
2. Materials and methods 2.1. Human gastrointestinal tissue Colonic full-thickness specimens obtained from human rectosigmoid segments were used for immunocytochemical studies. Segments of sigmoid colon were obtained from patients (n = 3, mean age 58.0 years, 1 female, 2 males) who underwent anterior rectosigmoid resection for non-obstructive colorectal carcinoma. Patients reported normal bowel habits and showed no evidence of anorectal out-let obstruction. After surgical removal specimens were harvested at safe distance (>5 cm) from the tumor site and transferred immediately to the laboratory for further tissue processing. The study of human tissue received approval from the Ethics Committee of the Faculty of Medicine, Christian-Albrecht’s
University in Kiel, Germany (B 299/07) and has been carried out in accordance with “The Code of Ethics of the World Medical Association”. 2.2. Tissue processing for immunocytochemical studies Full-thickness tissue blocks were trimmed to a size of 30 mm × 10 mm, pinned out flat and tension-free on a cork plate and fixated for 24 h with 4% paraformaldehyde in PBS. After dehydration the tissue blocks were transferred into paraffin wax and cut in 6 m sections. The cutting surface for the sections was the 30 mm border orientated perpendicular to the gut axis, so that myocytes of the circular muscle layer were cut along their longitudinal axis. 2.3. Immunohistochemical studies 2.3.1. Immunohistochemical detection of synaptophysin and synaptobrevin Visualization of synaptophysin and synaptobrevin was achieved by immunohistochemistry using corresponding primary antibodies (rabbit anti-synaptophysin antibody, 1:1000, Biozol; mouse anti-synaptobrevin, 1:200, Thermoscientific). Immunohistochemistry was performed as described previously (Böttner et al., 2012). Briefly, after pretreatment of the sections with citrate buffer (pH 6.0, microwaves 750 W) for 10 min, samples were incubated overnight with primary antibodies diluted in antibody diluent (Zymed, Invitrogen, CA) followed by incubation with biotinylated secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG, 1:400) for 45 min and treatment with an avidin–biotin-complex (Vectastain ABC Standard, Vector Laboratories, Burlingame, CA) conjugated with horseradish peroxidase for 45 min. Antibody binding was visualized with 3,3 -diaminobenzidine (DAB, DakoCytomation) followed by staining with hematoxylin. Analysis was carried out with a light optical microscope (Nikon 6000, Nikon, Tokyo) coupled to a digital camera (Digital Sight, Nikon, Tokyo). Data acquisition was performed with NIS-Elements BR 3.2 software (Nikon, Tokyo). The pictures depicted show one representative out of three experiments. 2.3.2. Dual-label immunocytochemistry with neuronal marker To demonstrate neuronal localization of synaptophysin and synaptobrevin, co-staining with the pan-neuronal marker PGP 9.5 was performed. After pre-treatment with citrate buffer (pH 6.0, 750 W microwaves for 2 × 5 min) sections were incubated overnight with mouse anti-synaptophysin-antibodies (1:200, Millipore, Merck, Darmstadt, Germany) or mouse anti-synaptobrevinantibodies (1:100, Thermoscientific, Ulm, Germany) diluted in antibody diluent (Zymed, Invitrogen, CA). Afterwards, sections were incubated for 2 h at room temperature with a goat anti-mouse AlexaFluor488 antibody (1:250, Invitrogen, Karlsruhe, Germany). To visualize a neuronal localization co-incubation with rabbit PGP 9.5-antibody (1:2000, UCL, Isle of Wight, GB) was performed followed by incubation with goat anti-rabbit AlexaFluor546 antibody (1:250, Invitrogen, Karlsruhe, Germany) for 2 h at room temperature. Finally, DAPI (Roche, Mannheim, Germany) was used to visualize cellular nuclei. Analysis was carried out with a fluorescence microscope (Axiovert 200M, Zeiss, Jena, Germany) coupled to a digital camera (Axiocam, Zeiss, Jena, Germany). Data acquisition was performed using Axiovision software (Zeiss, Jena, Germany). 2.4. Enteric nerve cells culture Preparation of myenteric ganglionic cells was performed according to a method described previously (Schäfer et al., 1997). Briefly, after removing the small intestine from newborn Wistar rats (postnatal day 2–3), the tunica muscularis was stripped from
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the mucosa, followed by incubation for 2 h at 37 ◦ C in Ca2+ and Mg2+ -free Hanks’ Balanced Salt Solution (HBSS, Gibco Life Technologies, Germany) with antibiotics containing 1 mg/ml collagenase (SIGMA, Munich, Germany). Afterwards, fragments of myenteric plexus were collected under stereomicroscopic control and incubated for 15 min at 37 ◦ C in trypsin/EDTA (0.125 mg/ml, Gibco, Life Technologies, Germany) to dissociate the plexus. The procedure was stopped by replacing trypsin/EDTA with fetal calf serum (FCS, Gibco, Life Technologies, Germany). The cells were triturated, counted and seeded in a density of 100,000 cells/ml on poly-d-Lysin-(SIGMA)/Laminin- (SIGMA, Munich, Germany) coated coverslips for immunocytochemistry studies or 12-wellplates for gene expression studies. Cells were incubated in defined medium consisting of Neurobasal A (Gibco, Life Technologies, Germany) and B27 supplement (Gibco, Life Technologies, Germany). Additionally, GDNF (Peprotech, Hamburg, Germany) was added to a final concentration of 2, 10, or 50 ng/ml. For gene expression analysis cells were cultured for 1 week, for immunocytochemical analysis culture time was 3 weeks. Medium was changed every second day.
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2.8. Transmission electron microscopy of enteric nerve cell cultures Cultured cells were fixed in 3% glutaraldehyde in PBS for 30 min and post-fixed in 2% osmiumoxide for 2 × 15 min, followed by dehydration and embedding in araldite overnight. Ultra-thin sections (40–50 nm) were cut (Ultracut, Reichert-Jung, Germany) and contrasted with uranyl acetate for 15 min and with lead citrate for 7 min. Analysis was carried out with a transmission electron microscope (EM 900, Zeiss) coupled to a digital imaging system (Slow Scan CCD-Camera, type 7888-IV, TRS).
2.9. Statistical analysis The effects of the GDNF treatment on rat enteric nerve cell cultures regarding gene expression studies compared to controls were analyzed by student’s t-test (PrizmTM, GraphPad, Sand Diego, CA, USA) followed by post hoc test according to the false-discovery rate procedure, using R 2.8.0 (R-Core-Team, 2012). Differences were considered significant if p < 0.05.
2.5. RNA extraction, reverse transcription and quantitative PCR Extraction of RNA from enteric nerve cell cultures was performed using a Nucleospin XS kit (Macherey and Nagel, Düren, Germany) according to the manufacturer’s guidelines. Reverse transcription and qPCR were performed as described before (Böttner et al., 2010). Forward and reverse primers as well as probes are listed below. rat synaptophysin – forward primer: 5 -gcagtgggtctttgccatctt-3 , reverse primer: 5 -tgagggcactctccgtcttg-3 , probe: 5 -cctttgctacgtgtggcagctaca-3 ; rat synaptobrevin – forward primer: 5 -cagcaaacccaggcacaagt3 , reverse primer: 5 -cgtcagctcggtcatccaa-3 , probe: 5 -atgcgcgtgaatgtggacaaggt-3 ; rat HPRT – forward primer: 5 -cgccagcttcctcctcaga-3 , reverse primer: 5 -ggtcataacctggttcatcact-3 , probe: 5 -ttttcccgcgagccgaccgg-3 .
3. Results 3.1. Localization of synaptophysin in the human colon To assess the distribution of synaptophysin in the human colon, immunohistochemistry using DAB as a chromogene was applied to colonic full-thickness sections. Synaptophysin immunoreactivity was detectable in all parts of the ENS, including submucosal (Fig. 1A and B) and myenteric ganglia (Fig. 1C and D) as well as in nerve fibers of the circular (Fig. 1E) and longitudinal muscle layer (Fig. 1F). Immunoreactivity in neuronal somata appeared to be less intense compared to the surrounding neuropil. At higher magnifications a small-sized granular staining pattern could be observed in the ganglionic neuropil and in the intramuscular nerve fibers (Fig. 1B, D, E, and F).
3.2. Localization of synaptobrevin in the human colon 2.6. Immunocytochemistry of enteric nerve cell cultures Cells were fixed for 30 min with 4% paraformaldehyde, permeabilized for 10 min with methanol and treated for 10 min with 3% H2 O2 . Afterwards, incubation with a rabbit antisynaptophysin antibody (1:1000, Biozol, Echingen, Germany) and a mouse anti-synaptobrevin antibody (1:100, Thermoscientific) for 1 h was performed, followed by incubation with the secondary antibodies, goat anti-rabbit-AlexaFlour546 (1:250, Invitrogen, Karlsruhe, Germany) and goat-anti-mouse-AlexaFluor488 (1:250, Invitrogen, Karlsruhe, Germany). Finally, cells were counterstained with DAPI (Roche, Mannheim, Germany) to visualize cell nuclei. Analysis was carried out with a fluorescent microscope (Axiovert 200M, Zeiss, Göttingen, Germany) coupled to a digital camera (Axiocam, Zeiss, Göttingen, Germany). Data acquisition was performed with Axiovision software (Zeiss, Göttingen, Germany). 2.7. Scanning electron microscopy of enteric nerve cells cultures Fixation of 3 weeks cultured enteric neurons was performed by using 3% glutaraldehyde in PBS for 30 min, followed by postfixation for 20 min in 2% osmiumoxide and dehydration. After critical point drying (CPD 030, Balzers), probes were sputtered with gold (Ion Tech Ltd.). Processed cultured cells were examined using a scanning electron microscope (Phillips XL 20, Phillips).
Immunohistochemistry was also applied to colonic fullthickness sections to assess the distribution of synaptobrevin in the human colon. Similar to the distribution of synaptophysin, synaptobrevin immunoreactivity was detectable in all parts of the ENS, including submucosal (Fig. 2A and B) and myenteric ganglia (Fig. 2C and D) as well as in nerve fibers of the circular (Fig. 2E) and longitudinal muscle layer (Fig. 2F). The small-sized granular staining pattern for synaptophysin at higher magnifications could also be observed for synaptobrevin in the ganglionic neuropil and in the intramuscular nerve fibers (Fig. 2B, D, E, and F). Consistently, neuronal cytoplasmatic stain appeared less intense than signals in the neuropil.
3.3. Neuronal localization of synaptophysin To further prove a neuronal localization of synaptophysin, duallabel immunocytochemistry with the pan-neuronal marker PGP 9.5 was performed (Fig. 3). Co-localization of synaptophysin and PGP 9.5 was detectable indicating a specific neuronal expression of synaptophysin in the ENS (Fig. 3C and F). The previously observed granular staining pattern was more intense, particularly in the neuropil around neuronal somata (Fig. 3A and C). In addition, enteric nerve fibers exhibited co-localization of synaptophysin and PGP 9.5 (Fig. 3F).
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Fig. 1. Localization of synaptophysin in the human colon. Submucosal (arrows in A and B) and myenteric ganglia (arrows in C and D) as well as intramuscular nerve fibers (arrows in E and F) are readily identified by robust synaptophysin immunoreactive signals (brown color). Neuronal somata (arrows in B and D) mostly show weaker synaptophysin immunoreactivity than the surrounding ganglionic neuropil. At higher magnifications a small-sized granular staining pattern is discernible within ganglia and nerve fibers (B, D, E, and F). Muc, mucosa; SM, submucosa; CM, circular muscle layer; LM, longitudinal muscle layer; SMP, submucosal plexus; MP, myenteric plexus; N, neuronal somata; NF, nerve fibers. Hematoxylin counterstain. Scale bars = 100 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.4. Neuronal localization of synaptobrevin To prove neuronal localization of synaptobrevin in the ENS, we performed co-labeling with the pan-neuronal marker PGP9.5 that stains neuronal somata and processes (Fig. 4). As observed for synaptophysin, co-localization with PGP9.5 demonstrated immunoreactivity of synaptobrevin in neurons (Fig. 4A–C). A particular granular staining pattern of synaptobrevin was detectable in the ganglionic neuropil surrounding neuronal somata (Fig. 4A and C) as well as in enteric nerve fibers (Fig. 4D and F). 3.5. Effects of GDNF on synaptophysin and synaptobrevin mRNA expression in cultured enteric neurons To investigate the effect of the neurotrophic factor GDNF on vesicle protein expression, a cell culture model of rat postnatal dissociated myenteric plexus exposed to increasing concentrations of GDNF was implemented. mRNA expression of synaptophysin and synaptobrevin was measured by quantitative RT-PCR (Fig. 5). After GDNF-treatment for 1 week, mRNA expression of synaptophysin (Fig. 5A) as well as mRNA expression of synaptobrevin (Fig. 5B) was significantly increased compared to untreated controls. mRNA expression of synaptophysin in GDNF-treated cultures
was nearly 4-fold up-regulated compared to controls (Fig. 5A). mRNA expression of synaptobrevin in cultures treated with 50 ng GDNF/ml was about 6-fold higher than in untreated controls (Fig. 5B). 3.6. Localization of synaptophysin and synaptobrevin in cultured enteric neurons The topographical expression pattern of synaptophysin and synaptobrevin in cultured myenteric neurons was assessed by performing a co-staining of enteric nerve cell cultures treated with 50 ng GDNF/ml for 3 weeks (Fig. 6). During the culture period, cells treated with GDNF form a neuronal network consisting of ganglionic aggregates interconnected by nerve fiber strands, whereas control cultures degenerate over time (data not shown). Immunoreactivity of synaptophysin (Fig. 6A) as well as immunoreactivity of synaptobrevin (Fig. 6B) was detectable in cultured myenteric neurons including neuronal ganglia and interganglionic nerve fiber strands. Co-localization of both synaptic proteins (yellow) was observed in cultured myenteric neurons by merging the images, particularly in granular accumulations (Fig. 6C) presumably resembling varicosities, i.e. the side of neurotransmitter release in the ENS.
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Fig. 2. Localization of synaptobrevin in the human colon. Synaptobrevin immunoreactivity (brown color) is detected in submucosal (arrows in A and B) and myenteric ganglia (arrows in C and D) as well as intramuscular nerve fibers (arrows in E and F). Immunoreactive signals are stronger in the ganglionic neuropil compared with neuronal somata (arrows in B and D). A granular staining pattern is visible at higher magnifications within ganglia and nerve fibers (B, D, E, and F). Muc, mucosa; SM, submucosa; CM, circular muscle layer; LM, longitudinal muscle layer; SMP, submucosal plexus; MP, myenteric plexus; N, neuronal somata; NF, nerve fibers. Hematoxylin counterstain. Scale bars = 100 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.7. Electron microscopy of neuronal varicosities in myenteric plexus cultures To verify the presence of varicosities in the cultured myenteric neurons, scanning electron microscopy of cultures treated for three weeks with 50 ng/ml was performed. As demonstrated by the overview of a three week culture, the cells form an extensive neuronal network characterized by the presence of ganglionic aggregates and nerve fiber strands (Fig. 7A). High power magnification reveals abundant presence of varicosities on nerve fiber strands (Fig. 7B). Supporting transmission electron microscopy underlines the functionality of these varicosities by revealing the presence of large dense core as well as small and large electrolucent synaptic vesicles arguing for the presence of peptidergic and cholinergic neurotransmitters present in the putative sites of neurotransmitter release (Fig. 7C and D). 4. Discussion The present study shows four important findings: (1) Synaptophysin and synaptobrevin are localized in submucosal and myenteric ganglia and nerve fiber strands of the human ENS. (2) Co-localization experiments reveal the presence of both proteins in enteric neurons. (3) Myenteric plexus cultures treated with GDNF
show an up-regulation of synaptophysin and synaptobrevin mRNA and both proteins co-localize in cultured neurons. (4) In response to GDNF treatment enteric nerve cell cultures form an extensive neuronal network comprising neuronal varicosities filled with heterogeneous synaptic vesicles. 4.1. Localization of synaptophysin in the human ENS Communication between the billions of neurons in the brain requires synaptic transmission. Release of neurotransmitters, an important part of synaptic transmission, is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals (Sudhof, 1995, 2004). Synaptophysin is the most abundant integral synaptic vesicle protein in the CNS and is therefore often used to quantify synapses (Calhoun et al., 1996). The function of synaptophysin is still not completely clarified, but several studies could show that synaptophysin binds synaptobrevin suggesting a regulatory role in SNARE assembly and vesicle fusion (Edelmann et al., 1995; Valtorta et al., 2004). We show an abundant granular expression of synaptophysin in nerve fibers of the muscle layers and in the neuropil around neuronal somata of both the myenteric and the submucosal plexus, i.e. in enteric nerve fibers and in enteric ganglia. Additionally, we could describe weak immunoreactivity in ganglionic neuronal
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Fig. 3. Co-staining of synaptophysin and PGP 9.5 in the human colon. Synaptophysin immunoreactivity (green) is detectable in enteric nerve cells visualized by PGP 9.5 (red) of enteric ganglia of the myenteric plexus (A–C) as well as in enteric nerve fibers of the circular muscle layer (arrows in D–F). Immunoreactive signals of synaptophysin are stronger in neuropil and enteric nerve fibers compared to neuronal somata (A and D). Synaptophysin shows a granular staining pattern in the neuropil, particularly around neuronal somata (A and C), and in enteric nerve fibers (D and F). MP, myenteric plexus; N, neuronal somata; NF, nerve fibers, blue color: DAPI staining of nuclei. Scale bars = 50 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
somata. The neuronal localization was verified by performing dual-label immunohistochemistry with the pan-neuronal marker PGP 9.5, an established marker for enteric ganglia (Krammer et al., 1993). Analysis of the distribution pattern of synaptophysin
in healthy intestine is of special importance regarding altered expression pattern under pathological conditions as demonstrated for pelviureteral junction obstruction (Demirbilek et al., 2006).
Fig. 4. Co-staining of synaptobrevin and PGP 9.5 in the human colon. Synaptobrevin immunoreactivity (green) is detectable in enteric nerve cells visualized by PGP 9.5 (red) of enteric ganglia in the myenteric plexus (A–C) as well as in enteric nerve fibers between muscle cells of the circular muscle layer (arrows in D–F). Immunoreactive signals of synaptobrevin are mostly granular and stronger in neuropil somata (A and C) and enteric nerve fibers (D and F), than in neuronal somata (A and C). Granular staining is particularly intense in neuropil around neuronal somata (A and C). MP, myenteric plexus; N, neuronal somata; NF, nerve fibers, blue color: DAPI staining of nuclei. Scale bars = 50 m (A–F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. mRNA expression of synaptophysin and synaptobrevin in myenteric plexus cultures in response to GDNF treatment. GDNF increases mRNA levels of synaptophysin and synaptobrevin. Cells were cultured for one week and expression levels of the target genes were normalized to expression of the house-keeping gene HPRT. Data are shown as mean ± SEM, synaptophysin mRNA expression: n = 18–21, synaptobrevin mRNA expression: n = 12–13, *p < 0.05 vs. control.
4.2. Localization of synaptobrevin in the human ENS The SNARE-complex is indispensable for membrane fusion and neurotransmitter release. Synaptobrevin is an important component of the SNARE-complex, belonging to the family of vesicle-associated membrane proteins (VAMP). We detected granula of synaptobrevin in the nerve fibers of the muscle layers as well as in the neuropil around the neuronal somata of both myenteric and submucosal ganglia. Also weak immunofluorescence was observed in neuronal somata of the ganglia marking the functionally active synaptic apparatus in the human ENS. 4.3. GDNF induces synaptophysin and synaptobrevin expression in myenteric plexus cultures To assess the regulatory effect of GDNF on the expression of synaptophysin and synaptobrevin, we used an in vitro model of enteric neurons by culturing rat postnatal day-2 myenteric neurons. Cultured enteric neurons display formation of neuronal networks composed of ganglia with interconnecting nerve fiber strands when cultured in the presence of GDNF. Regarding neuronal network formation, a study reported by Takaki et al. (2006) described the in vitro formation of enteric neural network structures in gut-like organs differentiated from embryonic stem cells.
As demonstrated in this study, addition of brain-derived neurotrophic factor (BDNF) during the formation of embryonic bodies led to the in vitro formation of enteric neural ganglia with interconnecting nerve fibers in gut-like structures and the generation of distinct peristalsis-like movements in the differentiated gut tissue. In contrast, application of GDNF or neurotrophin-3 did not result in enteric ganglia formation indicating that BDNF and GDNF in vivo might act in a temporally sequential fashion on enteric neuronal precursors to induce formation of neuronal ganglia and consequently functional networks. To address whether the neuronal networks formed in our culture system are functionally active as assessed by the presence of markers of synaptic plasticity, we performed a qPCR for synaptophysin and synaptobrevin of cultured myenteric neurons treated with GDNF for one week. The mRNA expression of both synaptophysin as well as synaptobrevin was significantly increased suggesting that GDNF has also positive effects on synaptogenesis and neuronal plasticity in the ENS. The GDNF-induced increase in synaptophysin expression might be due to activation of the PI-3-kinase/Akt pathway as it has been demonstrated that GDNF activates the PI-3-kinase pathway in primary enteric neurons (Srinivasan et al., 2005) and induces phosphorylation of Akt in enteric neuronal cell lines (Anitha et al., 2008) and that in addition activation of PI-3 kinase promotes synaptogenesis in primary
Fig. 6. Co-localization of synaptophysin and synaptobrevin in myenteric plexus cultures. Rat enteric nerve cells were cultured for 3 weeks with 50 ng GDNF/ml (A–C). Synaptophysin immunoreactivity (red, A) and synaptobrevin immunoreactivity (green, B) are detectable in cultured myenteric neurons including neuronal somata as well as nerve fibers. Co-localization of both proteins is detectable (yellow) in the merged image (C), particularly in granula. The granular staining pattern is detectable in neuronal ganglia (arrows in C) as well as in the interganglionic nerve fiber strands (arrowhead in C). Blue color: DAPI staining of nuclei. Scale bars = 100 m (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 7. Scanning and transmission electron microscopy of cultured myenteric neurons. Rat enteric nerve cells were cultured for 3 weeks with 50 ng GDNF/ml (A–D). Scanning electron microscopy (A and B) shows multiple round thickenings along enteric nerve fiber strands at higher magnifications (arrow in B). Transmission electron microscopy (C and D) demonstrates multiple high (arrow in D) and low dense (arrowhead in D) vesicles in the thickenings most likely representing neuronal varicosities filled with synaptic vesicles. Scale bars = 200 m (A), 2 m (B), 1 m (C), and 500 nm (D).
cultures of rat hippocampal neurons as well as in CA1 hippocampal neurons in vivo (Cuesto et al., 2011). After extending the culture time to a period of three weeks, neuronal localization of both vesicle markers, particularly in neuronal varicosities could be observed. These data are supported by a recent report describing enhanced synaptic communication by modulating potassium currents and response to serotonin of cultured myenteric neurons after GDNF treatment (Zeng et al., 2009). Supporting data are reported from cultured CNS neurons, as enhanced synaptic efficacy of cultured dopaminergic neurons after GDNF treatment by counting synapsin-1-positive puncta per neuron was shown (Bourque and Trudeau, 2000). Furthermore, it was demonstrated that synapsin-1 and synaptophysin antibodies labeled the same population of putative nerve terminals. Data on synaptobrevin expression following GDNF treatment, however, are contradictory. Whereas one study could not observe changes in synaptobrevin protein expression in cultured mesencephalic neurons after GDNF or BDNF treatment by performing Western blot analysis (Feng et al., 1999), another report described increased number and size of synaptobrevin positive clusters in xenopus nerve-muscle cultures after GDNF and neurturin treatment suggesting not only an enhancement of synaptic vesicle clustering, but also implying an increase in the number of release sites by GDNF treatment (Wang et al., 2002). These data are in accordance with our observation of increased synaptobrevin mRNA expression after GDNF treatment indicating a growth-factor induced rise in synaptic plasticity. 4.4. Presence of synaptic vesicles in varicosities The granular staining pattern of both synaptic proteins shown in the human tissue was reproducible in the in vitro model of cultured rat myenteric neurons. However, a culture period of the three weeks was necessary to visualize immunoreactivity in granula most likely representing synaptic varicosities indicating that this period was required for the synaptic apparatus to mature. Additionally, we could show co-localization of synaptophysin
and synaptobrevin in ganglia and interganglionic fiber strands of cultured enteric neurons arguing for the presence of a functional synaptic apparatus, as not only the vesicle marker synaptophysin was detectable but also the vesicle-associated SNARE protein necessary for mediating membrane fusion of the synaptic vesicles. Other investigations described an interplay of synaptophysin and the SNARE-protein synaptobrevin by applying immunoprecipitation, suggesting a physiological role for synaptophysin in control membrane fusion and exocytosis (Edelmann et al., 1995; Valtorta et al., 2004). By assessing the ultrastructure of the granula we could demonstrate vesicles in variable electron densities accumulating in thickenings representing synaptic varicosities, the sites of neurotransmitter release in the ENS. Low dense core vesicles represent most likely small synaptic vesicles (SSV), whereas the larger vesicles constitute large dense core vesicles (LDCV) (Bruns and Jahn, 1995). 5. Conclusion Taken together, our data indicate that the cultured enteric neurons form a functional neuronal network that comprises not only fiber strands and ganglia, but also a functional synaptic vesicle apparatus similar to the situation in humans. Components of the synaptic vesicle apparatus would provide the necessary equipment for functional neuron-to-neuron communication. Thus, this culture model might be an ideal tool to assess components increasing synaptic plasticity and the mechanisms underlying this process or also the respective confounding factors. Due to the fact that GDNF knockout mice exhibit complete intestinal aganglionosis resembling the pathology observed in Hirschsprung’s disease (Moore et al., 1996; Pichel et al., 1996), the underlying mechanism might be initially established by a loss of synaptic communication. Furthermore, components of the synaptic vesicle apparatus might be promising candidates to characterize the initial steps in the manifestation of human intestinal motility disorders, as these pathologies are frequently associated with
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neuronal loss and an underlying synaptopathy may be the initial event in the neurodegenerative process. Funding The study was supported by research grants from the Deutsche Forschungsgemeinschaft (DFG, WE 2366/4-2). The funding source had no role in conduction of the experiments and preparation of the article. Author contributions M. Böttner was responsible for study design, data analysis and writing of the paper; J. Harde, M. Barrenschee, I. Hellwig were responsible for acquisition and interpretation of data; I. Vogel and M. Ebsen contributed to the acquisition of the human material; and Thilo Wedel designed the study, critically revised the manuscript and wrote the grants financing the study. Acknowledgements The authors thank Karin Stengel, Inka Geurink, Miriam Lemmer, Frank Lichte, Bettina Facompré, and Clemens Franke (Institute of Anatomy, Christian-Albrechts-University of Kiel) for their excellent technical assistance. References Airaksinen, M.S., Titievsky, A., Saarma, M., 1999. GDNF family neurotrophic factor signaling: four masters, one servant? Molecular and Cellular Neurosciences 13, 313–325. Anitha, M., Joseph, I., Ding, X., Torre, E.R., Sawchuk, M.A., Mwangi, S., Hochman, S., Sitaraman, S.V., Anania, F., Srinivasan, S., 2008. Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function. Gastroenterology 134, 1424–1435. Baumert, M., Maycox, P.R., Navone, F., De Camilli, P., Jahn, R., 1989. Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. The EMBO Journal 8, 379–384. Böttner, M., Bar, F., Von Koschitzky, H., Tafazzoli, K., Roblick, U.J., Bruch, H.P., Wedel, T., 2010. Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine. Neurogastroenterology and Motility 22, 168–172, e152. Böttner, M., Krieglstein, K., Unsicker, K., 2000. The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions. Journal of Neurochemistry 75, 2227–2240. Böttner, M., Zorenkov, D., Hellwig, I., Barrenschee, M., Harde, J., Fricke, T., Deuschl, G., Egberts, J.H., Becker, T., Fritscher-Ravens, A., et al., 2012. Expression pattern and localization of alpha-synuclein in the human enteric nervous system. Neurobiology of Disease 48, 474–480. Bourque, M.J., Trudeau, L.E., 2000. GDNF enhances the synaptic efficacy of dopaminergic neurons in culture. The European Journal of Neuroscience 12, 3172–3180. Brose, N., O’Connor, V., Skehel, P., 2010. Synaptopathy: dysfunction of synaptic function? Biochemical Society Transactions 38, 443–444. Bruns, D., Jahn, R., 1995. Real-time measurement of transmitter release from single synaptic vesicles. Nature 377, 62–65. Burgoyne, R.D., Morgan, A., 2011. Chaperoning the SNAREs: a role in preventing neurodegeneration? Nature Cell Biology 13, 8–9. Calhoun, M.E., Jucker, M., Martin, L.J., Thinakaran, G., Price, D.L., Mouton, P.R., 1996. Comparative evaluation of synaptophysin-based methods for quantification of synapses. Journal of Neurocytology 25, 821–828. Cuesto, G., Enriquez-Barreto, L., Carames, C., Cantarero, M., Gasull, X., Sandi, C., Ferrus, A., Acebes, A., Morales, M., 2011. Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons. Journal of Neuroscience 31, 2721–2733. Demirbilek, S., Edali, M.N., Gurunluoglu, K., Turkmen, E., Tas, E., Karaman, A., Akin, M., Aksoy, R.T., Celbis, O., Uzun, I., 2006. Glial cell line-derived neurotrophic factor and synaptophysin expression in pelviureteral junction obstruction. Urology 67, 400–405.
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Please cite this article in press as: Böttner, M., et al., GDNF induces synaptic vesicle markers in enteric neurons. Neurosci. Res. (2013), http://dx.doi.org/10.1016/j.neures.2013.08.012