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Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development
Neuroscience Letters 454 (2009) 16–21
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development c ˜ M.B. Levanti a,1 , I. Esteban b,1 , E. Ciriaco a , P. Pérez-Pinera , R. Cabo d,e , O. García-Suarez c,h , b f b,g c,∗ B. Pardo , I. Silos-Santiago , J. Cobo , J.A. Vega a
Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Università di Messina, Messina, Italy Departamentos de Cirugía y Especialidades Médico-Quirúrgicas, Universidad de Oviedo, Oviedo, Spain Departamento de Morfologia y Biología Celular, Universidad de Oviedo, Oviedo, Spain d Departmento de Anatomía y Radiología, Universidad de Valladolid-CSIC, Valladolid, Spain e Instituto de Biología y Genética Molecular, Universidad de Valladolid-CSIC, Valladolid, Spain f Department of Pharmacology, Vertex Pharmaceuticals, Inc., San Diego, CA, USA g Instituto Asturiano de Odontología, Oviedo, Spain h ADITAS, Oviedo, Spain b c
a r t i c l e
i n f o
Article history: Received 26 January 2009 Received in revised form 23 February 2009 Accepted 24 February 2009 Keywords: Enteric nervous system Enteric glial cells Neurotrophins TrkB TrkB deficient mice
a b s t r a c t The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS. © 2009 Elsevier Ireland Ltd. All rights reserved.
The intrinsic innervation of the gastrointestinal tract, known as the enteric nervous system (ENS), consists of large numbers of phenotypically distinct neurons and glial cells organized in complex interconnected plexuses located between the smooth muscle layers of the intestinal wall. In vertebrates the ENS is derived from vagal, sacral and truncal neural crest cells, which migrate and populate the gut during embryonic development [11]. Differentiation of these neural crest cells into ENS cells depends on several neurotrophic factors, including some members of the neurotrophin (NTs) family, particularly NT-3 [3]. NTs signal throughout the Trk family of tyrosine kinase receptors (TrkA, TrkB and TrkC) and/p75NTR , a member of the superfamily of the transforming growth factor receptor (see [24]). The role of NTs and their receptors in the development and maintenance of neurons derived from the neural crest, in particular those derived from the dorsal root and sympathetic ganglia is now well established [12]. It has been shown that NTs regulate neurotransmitter and neu-
∗ Corresponding author at: Dpto. Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, C/Julian Clavería s/n. 33006 Oviedo, Spain. E-mail address: [email protected] (J.A. Vega). 1 Both authors contributed equally to this study. 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.02.051
ropeptide synthesis in the ENS throughout adult life and that NTs influence neuronal morphology [6], synaptic functions [27], as well as gastric motility [14], however, the roles of NTs in the ENS remain largely unknown [3]. Messenger RNA encoding TrkA and TrkC has been detected in mammalian ENS (see for references [7]) and, using immunohistochemistry, TrkA, TrkB, and TrkC have been detected in the ENS of mammals [7], avis [9], lower vertebrates [18,19], and invertebrates [17]. These data strongly suggest that NTs and their receptors may be important in the maintenance of the ENS. This hypothesis is supported by findings that abnormalities can be found during postnatal life of TrkC and NT-3 deficient mice although the ENS is apparently normal at birth [4]. Similarly, it has been found that enteric glial cells of different adult mammals, including human, express TrkB [7], and that BDNF is produced by different cell types in the gut [2,16]. The present study was designed to investigate the role of TrkB in the development and maintenance of the ENS. We studied using immunohistochemistry and Western blot the expression pattern of TrkB in the ENS of mice at ages ranging from embryonic life to 6 months of age, since functional innervation is already present during fetal life but full maturity of ENS is reached only during the first postnatal month [21]. Furthermore, we analyzed the structure
M.B. Levanti et al. / Neuroscience Letters 454 (2009) 16–21
of the ENS in 15-day-old TrkB-deficient mice using immunohistochemistry and electron microscopy. The intestine of C57B1/6 mice of different ages (16 and 20 days of estimated embryonic life, 1 day and 15 days, 1 month and 6 months of postnatal life (five animals per age group), was removed under deep chloral hydrate anesthesia (350 mg/kg, i.p.). Two intestine samples 1 cm long from the jejune-ileum and colon were washed with saline and immediately processed for Western blot, or placed in Bouin’s fixative for 12 h and routinely processed for paraffin embedding. The experiments were performed in accordance with the European Communities Council Directive (86/609/EEC) for the Care and Use of Laboratory Animals. The jejune-ileum and colon of 15 days old mice carrying a targeted mutation in the trkB gene, that results in non-functional gene product (kindly provided by Dr. I. Silos-Santiago), were also analyzed. These animals were bred out over the C57B1/6 background, and genotyped using polymerase chain reaction. Wild type (TrkB +/+; n = 5), heterozygous (TrkB +/−; n = 4) and homozygous (TrkB −/−; n = 5) mice were included in the study. The animals were deeply anaesthetized with ether and perfused transcardially with a cold solution of 4% paraformaldehyde in 0.1 M phosphate buffered (pH 7.4). Two-centimeter long segments of small and large intestine were removed, placed immediately into the same perfusion fixative at 4 ◦ C, and embedded in paraffin (1.5 cm, approximately) or EPON-embedding (0.5 cm, approximately). Western blot analysis was performed using fresh intestine samples and were processed as described in detail elsewhere (see [8]). The antibody used to detect TrkB (794; sc-12; Santa Cruz Biotechnology; Santa Cruz, CA, USA), raised in rabbit, binds to the intracellular tyrosine-kinase domain of TrkB (residues 794–808), and was used diluted 1:2000. For immunohistochemistry, 10 m thick tissue sections were obtained from paraffin-embedded samples and processed for TrkB detection with the EnVision antibody complex kit (Dako, Copenhagen, Denmark), following manufacturer’s recommendations. Anti-TrkB antibody (Santa Cruz Biotechnology), was used diluted 1:500 in blocking buffer, was applied to the sections and incubated for 30 min. Tissue sections processed identically were incubated with preabsorbed sera (5 g of the antigen in 1 ml of the primary antibody working solution; control peptide cat# sc12P from Santa Cruz Biotechnology) instead of the primary antibody as negative control. Cross-reactivity of the primary antibody with TrkA or TrkC was avoided incubating sections with TrkB antibody preabsorbed with control peptides for these proteins (cat# sc118P and cat# sc117P from Santa Cruz Biotechnology). To identify the cell type(s) expressing TrkB the patterns expression of PGP 9.5 (Biogenesis, Poole, England, UK; used diluted 1:1000), glial fibrillary acidic protein (G-A-5, BoehringerMannheim, Mannheim, Germany; used diluted 1 g/ml), and S100 protein (Dako, Denmark; diluted 1:1000) were studied in parallel using immunohistochemistry performed with the same detection kit. Intestine sections form 15-day and 1-month old mice were processed for simultaneous detection of TrkB and S100 protein. Double staining was performed with a sequential method on 10 m deparaffined and rehydrated sections. Non-specific binding sites were blocked for 30 min with a solution of 1% bovine serum albumin in TBS. The sections were then incubated overnight at 4 ◦ C in a humid chamber with a 1:1 mixture of anti-TrkB and anti-S100 protein antibodies (1:100 in the blocking solution, and purchased prediluted, respectively) (DPC, Los Angeles, CA, USA). After rinsing with TBS, the sections were incubated for 1 h with FITC-conjugated sheep anti-rat IgG (Serotec, Oxford, UK), diluted 1:50 in TBS containing 5% mouse serum (Serotec), then rinsed again and incubated for 1 h with a Texas red-conjugated donkey anti-rabbit antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK) diluted 1:50
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in TBS. Both incubation steps were performed at room temperature in a dark humid chamber. Double staining was detected using a Leika DMR-XA automatic fluorescence microscope coupled to a Fluorescence Adquisition Software Leika Qfluoro (Servicio de Proceso de Imágenes, Universidad de Oviedo). Samples of small intestine and colon of wild type and TrkB deficient mice were processed for transmission electron microscopy as follows: tissue samples were fixed in 4% paraformaldehyde, washed in 0.1 M PBS, pH 7.5, postfixed in 1% osmium tetroxide, and routinely processed for EPON embedding. Semi-thin sections (1 m) were obtained, stained with toluidin blue or cresyl violet, and used for structural analysis. The ultrathin sections (400 Å) were stained with uranyl acetate and lead citrate, examined, and photographed using an electron microscope JEOL-JEM-T8. To investigate whether absence TrkB expression results in a loss of enteric neurons, the number of neurons in the myenteric plexus was counted on semithin sections 200 m apart of the jejune-ileum and colon samples prepared from wild type and TrkB deficient mice. This analysis was designed to estimate loss of enteric neurons, and was not intended to establish the density of neurons in the myenteric plexus. To first address whether TrkB is expressed in the murine intestine, we analyzed using Western blot the levels of expression of TrkB in samples obtained from mice whose age ranged from 16 day of embryonic life to 6 months after birth. The results showed that, at all ages studied, a single protein with an estimated molecular weight of ∼145 kDa was detected by anti-TrkB antibody (Fig. 1). Although this study was not quantitative no significant variations in the levels of expression of TrkB were observed between mice at different ages. The results support the conclusion that full-length TrkB is expressed in murine intestine. To seek confirmation for the previous result that TrkB is expressed in murine intestine and to identify the cell type(s) that express TrkB, we used immunohistochemistry to stain tissue sections prepared from intestine obtained from mice at postnatal day 1, 15, 30, and 180. TrkB expression was detected at all ages tested and it was observed in cells inside the muscular layer of the intestinal wall that formed clusters of irregular rings surrounding non-reactive structures or forming cords that connected these clusters to one another (Fig. 2a–d). The TrkB positive cells within the intestine, based on their localization and morphology, were identified as glial cells from the ENS. Additionally, TrkB expression was found in nerve fibers crossing the muscular layer of the intestine or located underneath the mucosa. The density of TrkB immunoreactive cells was always higher in the myenteric plexus than in the submucous plexus. We also observed that within the epithelium scattered cells expressed TrkB; these cells were identified as endocrine cells based on their morphology, localization, and density (data not shown). The pattern of TrkB expression, the identity of the cells that express TrkB, and their localization were identical at all ages studied. To test the hypothesis that TrkB expressing cells in the ENS are glial cells in the ENS, we stained consecutive sections of intestine using anti-TrkB antibodies (Fig. 3a) and antibodies raised against neuron-derived antigens (PGP 9.5) (Fig. 3b) and the glial cellderived antigens S100 protein (Fig. 3c) and GFAP (Fig. 3d). The cells stained with anti-TrkB antibody were similar in morphology, density, and distribution within the ganglia to cells expressing GFAP or S100 protein, and clearly differed from neurons expressing PGP 9.5. These results support the hypothesis that the glial cells in the ENS are the cells that express TrkB. Furthermore, using double immunofluorescence, we observed that TrkB and GFAP are expressed by the same cells (Fig. 4), although TrkB is also expressed in other cell population that do not express GFAP, thus supporting the conclusion that glial cells of the enteric nervous system is one of the cell types that express TrkB.
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Figs. 1–5. Detection of TrkB expression using Western blot in intestine samples obtained from mice at days 16 and 20 of embryonic development (E16, E20), and 1 day (1Pd), 2 weeks (2w), 1 month (1m), and 6 months (6m) old mice. The anti-TrkB antibodies used recognize a sequence within the catalytic domain of TrkB, and detected a protein ∼145 kDa, which was identified as full-length TrkB. Positive controls were performed using brain (B) homogenates of 15-day-old mice. (2) Cells displaying TrkB immunoreactivity in all ages sampled formed from 1 day to 6-month-old mice localized in ganglia and nerve bundles of the enteric nervous system. They were identified as enteric glial cells. Scale bar = 1.5 mm for (a); 60 m for (b–d). (3) Serial sections of intestine processed for the detection of TrkB (a), and the neuronal (PGP 9.5, b) or glial markers (GFAP and S100 protein, b and d, respectively) demonstrated that cells expressing TrkB are glial cells and not neurons. Scale bar = 40 m. (4) Immunohistochemical localization of TrkB (green) and GFAP (red) in the ENS (colon) of a mouse 1-month old. Both proteins were localised in the enteric glial cells, some times co-localized. Nevertheless, the expression of TrkB was more widely diffused than those of GFAP. Scale bar = 40 m. (5) TrkB immunoreactivity was absent from the enteric nervous system TrkB deficient mice (a). In these animals the expression of GFAP (b) was apparently diminished with respect to the age-matched littermates, and PGP 9.5 was unchanged. m: mucous; ml: muscular layer. Scale bar = 40 m.
M.B. Levanti et al. / Neuroscience Letters 454 (2009) 16–21
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Figs. 6–7. Structure of the colon wall, including the ENS (arrows), in system in wild-type (a) and TrkB−/− (b) mice 15-day-old. Scale bar = 40 m. Table in this figure shows the number of neurons in the enteric nervous system in the three groups of animals, quantified as indicated in text. (7) Ultrastructural aspect of the colonic enteric nervous system in wild-type (a and b), and TrkB−/− (c–f) mice 15-day-old. In TrkB mutated animals there was a progressive disruption of the cytoplasm of the glial cells, as well as of the neuropil, whereas the neurons resulted unaffected by the mutation. gc: glial cells; n: nucleos of neurons; np: neuropilo. Original magnifications: 4000× for a, c, d–f; 6000× for b.
The findings that glial cells within ENS express TrkB suggest that they may depend on the activation of TrkB for survival or maintenance of their phenotype. To test this hypothesis, we analyzed the expression of some neuronal and glial markers in the ENS of
TrkB+/− and TrkB−/− mice. We did not observe significant differences in the structure of the ENS between heterozygous and wild type mice (data not shown). Observation of sections from TrkB−/− mice intestine sections stained with anti-TrkB antibodies confirmed
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M.B. Levanti et al. / Neuroscience Letters 454 (2009) 16–21
that they do not express TrkB in the ENS or intestine walls (Fig. 5a). The pattern of expression of PGP 9.5 in intestine of TrkB−/− mice was similar to that found in TrkB+/+ mice (Fig. 5b). However, GFAP was distributed forming lineal cords or disrupted rings (Fig. 5c), and, more importantly, expression of S100 protein was consistently lost (data not shown). The structure of the ENS of TrkB+/+, TrkB+/−, and TrkB−/− mice was compared using semithin sections (Fig. 6a and b) and using electron microscopy (Fig. 7). There is not a statistically significant difference in the number of myenteric neurons among these three groups of mice (table in Fig. 6). The composition of myenteric ganglia was found to be variable in number of neurons and glial cells, which were distinguished based on their location within the ganglia, size, shape of the nucleus, and distribution of chromatin and cytoplasmic organelles (Fig. 7a); unmyelinated axons were abundant, as well as synaptic buttons which were located on the surface of the ganglion, adjacent to the basal lamina. The synaptic buttons contain predominantly large agranular vesicles, but also many vesicles of variable size dense and electron lucent (Fig. 7b). Detailed analysis of the ENS from TrkB−/− mice reveals several abnormalities involving glial cells. The neuronal somata were apparently normal, but the nerve fibers forming the neuropil were disordered and disrupted; they contained cell debris and electron-dense bodies. The glial cell processes were destroyed. These defects were more evident in the TrkB−/− (Fig. 7c–f) that in TrkB+/− mice (data not shown). The neural crest-derived sensory and sympathetic neurons depend on different NTs for development and maintenance during adult life [12]. Recent evidences suggested that ENS cells, which also develop from the neural crest cells (see [1]), depend on NTs for development and survival [3]. The present study provides further evidence supporting the view that NTs signaling is necessary to maintain the normal structure of the ENS, in particular to support viability of TrkB-expressing enteric glial cells. We demonstrated that glial cells within the murine ENS express full-length TrkB during all lifespan, and that loss of TrkB expression results in deterioration of normal structure of the ENS. The expression and importance of TrkB in the development of the ENS has been controversial. Pioneer studies in mice failed to demonstrate trkB, although subsequent studies detected its expression both during embryonic development and adulthood [2,3,7]. There have been also disagreement regarding which cells type(s) express NTs and their receptors. Whereas some authors failed to detect TrkB immunoreactivity in the adult human ENS, others detected TrkB expression in both neurons and glial cells. It is currently accepted that, except in human tissue, TrkB expression is restricted to glial cells in adult mammals [7,13]. The present data in mouse are consistent with previous studies in other mammalian species. Based on the molecular weight of the TrkB identified using Western blot with antibodies that bind to the catalytic tyrosine kinase domain, we conclude that ENS express full-length TrkB, that, potentially, may initiate signaling pathways upon ligand-induced activation. TrkB ligands include BDNF and NT-4, and, in certain contexts, NT-3. BDNF is released in the intestine of vertebrates [15,16,20] and it stimulates intestinal motility [2,5]. NT-3 signaling is necessary for normal development, differentiation and maintenance of enteric neurons and glial cells both in vitro [3,26] and in vivo [22], and also inhibits gastric motility [14]. Therefore, we hypothesize that BDNF and/or NT-3 might support enteric glial cells during adulthood signaling through TrkB. To the best of our knowledge, the role of NT-4 during development and maintenance of the ENS remains unknown. The abnormalities observed in glial cells of the ENS in animals lacking TrkB, i.e. loss of some characteristic antigens and structural disarrangement, also provide indirect evidence to support the
hypothesis that TrkB is necessary for the normal development and maintenance of the glial cell phenotype in the ENS. Previous studies of the morphology of the ENS in knockout mice lacking NTs or NT receptor expression failed to identify morphological abnormalities (see [12]). Subsequent studies, in which the authors stained the tissues with neuronal or glial markers using immunohistochemistry, questioned those original findings [10], and described loss of enteric neurons in TrkC and NT-3 deficient mice [4]. It is important to note that immunohistochemistry alone is suboptimal to precisely characterize the structure of the ENS. Our results show that using neuronal and glial markers enteric neurons can be detected in the ENS of TrkB−/− mice, although we observed a significant decrease in the levels of GFAP expression and complete loss of S100 protein expression. These results suggest there are important differences in glial cells from the ENS in TrkB−/− mice compared to TrkB wild type mice. It was shown before that loss of glial cells in the ENS correlates with intestinal inflammation [28], disruption of the epithelial barrier [25], and alterations in the rate of neuronal survival [23]. However, the functional consequences of the alterations found in the enteric glia of TrkB−/− mice are unknown. Acknowledgements Authors thanks to Ms. M.L. López-Robles, and Ms Marta Alonso Guervos (Servcio de Análisis de Imágenes de la Universidad de Oviedo) for technical support. References [1] R.B. Anderson, D.F. Newgreen, H.M. Young, Neural crest and the development of the enteric nervous system, Adv. Exp. Med. Biol. 589 (2006) 181–196. [2] W. Boesmans, P. Gomes, J. Janssens, J. Tack, P. Vanden Berghe, Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system, Gut 57 (2008) 314–322. [3] A. Chalazonitis, Neurotrophin–3 in the development of the enteric nervous system, Prog. Brain Res. 146 (2004) 243–263. [4] A. Chalazonitis, T.D. Pham, T.P. Rothman, P.S. DiStefano, M. Bothwell, J. Flair-Flynn, L. Tessarollo, M.D. Neurotrophin-3 is required for the survivaldifferentiation of subsets of developing enteric neurons, J. Neurosci. 21 (2001) 2636–5620. [5] C.M. Coulis, C. Lee, V. Nardone, R.D. Prokipcak, Inhibition of c-myc expression in cells by targeting an RNA-interaction using antisense oligonucleotides, Mol. Pharmacol. 57 (2000) 485–494. [6] R. De Giorgio, J. Arakawa, C.J. Whitemore, C. Sternini, Neurotrophin-3 and neurotrophin receptor immunoreactivity and peptidergic enteric neurons, Peptides 21 (2000) 1421–1426. [7] I. Esteban, B. Levanti, O. García-Suárez, G. Germanà, E. Ciriaco, F.J. Naves, J.A. Vega, A neuronal sub-population in the mamalian enteric nervous system TrkA and TrkC neurotrophin receptor-like proteins, Anat. Rec. 251 (1998) 360–370. [8] O. García-Suárez, T. González-Martínez, M. Perez-Perez, A. Germana, M.A. Blanco-Gélaz, D.F. Monjil, E. Ciriaco, I. Silos-Santiago, J.A. Vega, Expression of the neurotrophin receptor TrkB in the mouse liver, Anat. Embryol. (Berl.) 211 (2006) 465–473. [9] A. Germanà, M.B. Levanti, D.F. Monjil, E. Ciriaco, M. Del Valle, J.A. Vega, G. Germanà, Immunohistochemical detection of TrkB in the enteric nervous system of the small intestine in pigeon (Columba livia), Eur. J. Histochem. 48 (2004) 373–376. [10] M.D. Gershon, Genes and lineages in the formation of the enteric nervous system, Curr. Opin. Neurobiol. 7 (1997) 101–109. [11] D. Grundry, M. Schelmann, Enteric nervous system, Curr. Opin. Gastroenterol. 21 (2005) 176–182. ˜ [12] M. Kirstein, I. Farinas, Sensing life: regulation of sensory neuron survival by neurotrophins, Cell. Mol. Life Sci. 59 (11) (2002) 1787–1802. [13] M. Kondyli, J. Varakis, M. Assimakopoulou, Expression of p75NTR and Trk neurotrophin receptors in the enteric nervous system of human adults, Anat. Sci. Int. 80 (4) (2005) 223–228. [14] M.A. Küper, T. Meile, T.T. Zittel, A. Konigsrainer, J. Glatzle, Effects of neurotrophin 3 on gastric and colonic motility in awake rats, Neurogastroenterol. Motil. 19 (2007) 983–989. [15] M. Lommatzsch, A. Braun, A. Mannsfeldt, V.A. Botchkarev, N.V. Botchkareva, R. Paus, A. Fiscer, G.R. Lewin, H. Renz, Abundant production of brain-derived neurotrophic factor by adult visceral epithelium. Implications for paracrine and target-derived Neurotrophic functions, Am. J. Pathol. 155 (1999) 1183–1193. [16] M. Lommatzsch, D. Quarcoo, O. Schulte-Herbrüggen, H. Weber, J.C. Virchow, H. Renz, A. Braun, Neurotrophins in murine viscera: a dynamic pattern from birth to adulthood, Int. J. Dev. Neurosci. 23 (2005) 495–500.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development c ˜ M.B. Levanti a,1 , I. Esteban b,1 , E. Ciriaco a , P. Pérez-Pinera , R. Cabo d,e , O. García-Suarez c,h , b f b,g c,∗ B. Pardo , I. Silos-Santiago , J. Cobo , J.A. Vega a
Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali, Università di Messina, Messina, Italy Departamentos de Cirugía y Especialidades Médico-Quirúrgicas, Universidad de Oviedo, Oviedo, Spain Departamento de Morfologia y Biología Celular, Universidad de Oviedo, Oviedo, Spain d Departmento de Anatomía y Radiología, Universidad de Valladolid-CSIC, Valladolid, Spain e Instituto de Biología y Genética Molecular, Universidad de Valladolid-CSIC, Valladolid, Spain f Department of Pharmacology, Vertex Pharmaceuticals, Inc., San Diego, CA, USA g Instituto Asturiano de Odontología, Oviedo, Spain h ADITAS, Oviedo, Spain b c
a r t i c l e
i n f o
Article history: Received 26 January 2009 Received in revised form 23 February 2009 Accepted 24 February 2009 Keywords: Enteric nervous system Enteric glial cells Neurotrophins TrkB TrkB deficient mice
a b s t r a c t The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS. © 2009 Elsevier Ireland Ltd. All rights reserved.
The intrinsic innervation of the gastrointestinal tract, known as the enteric nervous system (ENS), consists of large numbers of phenotypically distinct neurons and glial cells organized in complex interconnected plexuses located between the smooth muscle layers of the intestinal wall. In vertebrates the ENS is derived from vagal, sacral and truncal neural crest cells, which migrate and populate the gut during embryonic development [11]. Differentiation of these neural crest cells into ENS cells depends on several neurotrophic factors, including some members of the neurotrophin (NTs) family, particularly NT-3 [3]. NTs signal throughout the Trk family of tyrosine kinase receptors (TrkA, TrkB and TrkC) and/p75NTR , a member of the superfamily of the transforming growth factor receptor (see [24]). The role of NTs and their receptors in the development and maintenance of neurons derived from the neural crest, in particular those derived from the dorsal root and sympathetic ganglia is now well established [12]. It has been shown that NTs regulate neurotransmitter and neu-
∗ Corresponding author at: Dpto. Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, C/Julian Clavería s/n. 33006 Oviedo, Spain. E-mail address: [email protected] (J.A. Vega). 1 Both authors contributed equally to this study. 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.02.051
ropeptide synthesis in the ENS throughout adult life and that NTs influence neuronal morphology [6], synaptic functions [27], as well as gastric motility [14], however, the roles of NTs in the ENS remain largely unknown [3]. Messenger RNA encoding TrkA and TrkC has been detected in mammalian ENS (see for references [7]) and, using immunohistochemistry, TrkA, TrkB, and TrkC have been detected in the ENS of mammals [7], avis [9], lower vertebrates [18,19], and invertebrates [17]. These data strongly suggest that NTs and their receptors may be important in the maintenance of the ENS. This hypothesis is supported by findings that abnormalities can be found during postnatal life of TrkC and NT-3 deficient mice although the ENS is apparently normal at birth [4]. Similarly, it has been found that enteric glial cells of different adult mammals, including human, express TrkB [7], and that BDNF is produced by different cell types in the gut [2,16]. The present study was designed to investigate the role of TrkB in the development and maintenance of the ENS. We studied using immunohistochemistry and Western blot the expression pattern of TrkB in the ENS of mice at ages ranging from embryonic life to 6 months of age, since functional innervation is already present during fetal life but full maturity of ENS is reached only during the first postnatal month [21]. Furthermore, we analyzed the structure
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of the ENS in 15-day-old TrkB-deficient mice using immunohistochemistry and electron microscopy. The intestine of C57B1/6 mice of different ages (16 and 20 days of estimated embryonic life, 1 day and 15 days, 1 month and 6 months of postnatal life (five animals per age group), was removed under deep chloral hydrate anesthesia (350 mg/kg, i.p.). Two intestine samples 1 cm long from the jejune-ileum and colon were washed with saline and immediately processed for Western blot, or placed in Bouin’s fixative for 12 h and routinely processed for paraffin embedding. The experiments were performed in accordance with the European Communities Council Directive (86/609/EEC) for the Care and Use of Laboratory Animals. The jejune-ileum and colon of 15 days old mice carrying a targeted mutation in the trkB gene, that results in non-functional gene product (kindly provided by Dr. I. Silos-Santiago), were also analyzed. These animals were bred out over the C57B1/6 background, and genotyped using polymerase chain reaction. Wild type (TrkB +/+; n = 5), heterozygous (TrkB +/−; n = 4) and homozygous (TrkB −/−; n = 5) mice were included in the study. The animals were deeply anaesthetized with ether and perfused transcardially with a cold solution of 4% paraformaldehyde in 0.1 M phosphate buffered (pH 7.4). Two-centimeter long segments of small and large intestine were removed, placed immediately into the same perfusion fixative at 4 ◦ C, and embedded in paraffin (1.5 cm, approximately) or EPON-embedding (0.5 cm, approximately). Western blot analysis was performed using fresh intestine samples and were processed as described in detail elsewhere (see [8]). The antibody used to detect TrkB (794; sc-12; Santa Cruz Biotechnology; Santa Cruz, CA, USA), raised in rabbit, binds to the intracellular tyrosine-kinase domain of TrkB (residues 794–808), and was used diluted 1:2000. For immunohistochemistry, 10 m thick tissue sections were obtained from paraffin-embedded samples and processed for TrkB detection with the EnVision antibody complex kit (Dako, Copenhagen, Denmark), following manufacturer’s recommendations. Anti-TrkB antibody (Santa Cruz Biotechnology), was used diluted 1:500 in blocking buffer, was applied to the sections and incubated for 30 min. Tissue sections processed identically were incubated with preabsorbed sera (5 g of the antigen in 1 ml of the primary antibody working solution; control peptide cat# sc12P from Santa Cruz Biotechnology) instead of the primary antibody as negative control. Cross-reactivity of the primary antibody with TrkA or TrkC was avoided incubating sections with TrkB antibody preabsorbed with control peptides for these proteins (cat# sc118P and cat# sc117P from Santa Cruz Biotechnology). To identify the cell type(s) expressing TrkB the patterns expression of PGP 9.5 (Biogenesis, Poole, England, UK; used diluted 1:1000), glial fibrillary acidic protein (G-A-5, BoehringerMannheim, Mannheim, Germany; used diluted 1 g/ml), and S100 protein (Dako, Denmark; diluted 1:1000) were studied in parallel using immunohistochemistry performed with the same detection kit. Intestine sections form 15-day and 1-month old mice were processed for simultaneous detection of TrkB and S100 protein. Double staining was performed with a sequential method on 10 m deparaffined and rehydrated sections. Non-specific binding sites were blocked for 30 min with a solution of 1% bovine serum albumin in TBS. The sections were then incubated overnight at 4 ◦ C in a humid chamber with a 1:1 mixture of anti-TrkB and anti-S100 protein antibodies (1:100 in the blocking solution, and purchased prediluted, respectively) (DPC, Los Angeles, CA, USA). After rinsing with TBS, the sections were incubated for 1 h with FITC-conjugated sheep anti-rat IgG (Serotec, Oxford, UK), diluted 1:50 in TBS containing 5% mouse serum (Serotec), then rinsed again and incubated for 1 h with a Texas red-conjugated donkey anti-rabbit antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK) diluted 1:50
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in TBS. Both incubation steps were performed at room temperature in a dark humid chamber. Double staining was detected using a Leika DMR-XA automatic fluorescence microscope coupled to a Fluorescence Adquisition Software Leika Qfluoro (Servicio de Proceso de Imágenes, Universidad de Oviedo). Samples of small intestine and colon of wild type and TrkB deficient mice were processed for transmission electron microscopy as follows: tissue samples were fixed in 4% paraformaldehyde, washed in 0.1 M PBS, pH 7.5, postfixed in 1% osmium tetroxide, and routinely processed for EPON embedding. Semi-thin sections (1 m) were obtained, stained with toluidin blue or cresyl violet, and used for structural analysis. The ultrathin sections (400 Å) were stained with uranyl acetate and lead citrate, examined, and photographed using an electron microscope JEOL-JEM-T8. To investigate whether absence TrkB expression results in a loss of enteric neurons, the number of neurons in the myenteric plexus was counted on semithin sections 200 m apart of the jejune-ileum and colon samples prepared from wild type and TrkB deficient mice. This analysis was designed to estimate loss of enteric neurons, and was not intended to establish the density of neurons in the myenteric plexus. To first address whether TrkB is expressed in the murine intestine, we analyzed using Western blot the levels of expression of TrkB in samples obtained from mice whose age ranged from 16 day of embryonic life to 6 months after birth. The results showed that, at all ages studied, a single protein with an estimated molecular weight of ∼145 kDa was detected by anti-TrkB antibody (Fig. 1). Although this study was not quantitative no significant variations in the levels of expression of TrkB were observed between mice at different ages. The results support the conclusion that full-length TrkB is expressed in murine intestine. To seek confirmation for the previous result that TrkB is expressed in murine intestine and to identify the cell type(s) that express TrkB, we used immunohistochemistry to stain tissue sections prepared from intestine obtained from mice at postnatal day 1, 15, 30, and 180. TrkB expression was detected at all ages tested and it was observed in cells inside the muscular layer of the intestinal wall that formed clusters of irregular rings surrounding non-reactive structures or forming cords that connected these clusters to one another (Fig. 2a–d). The TrkB positive cells within the intestine, based on their localization and morphology, were identified as glial cells from the ENS. Additionally, TrkB expression was found in nerve fibers crossing the muscular layer of the intestine or located underneath the mucosa. The density of TrkB immunoreactive cells was always higher in the myenteric plexus than in the submucous plexus. We also observed that within the epithelium scattered cells expressed TrkB; these cells were identified as endocrine cells based on their morphology, localization, and density (data not shown). The pattern of TrkB expression, the identity of the cells that express TrkB, and their localization were identical at all ages studied. To test the hypothesis that TrkB expressing cells in the ENS are glial cells in the ENS, we stained consecutive sections of intestine using anti-TrkB antibodies (Fig. 3a) and antibodies raised against neuron-derived antigens (PGP 9.5) (Fig. 3b) and the glial cellderived antigens S100 protein (Fig. 3c) and GFAP (Fig. 3d). The cells stained with anti-TrkB antibody were similar in morphology, density, and distribution within the ganglia to cells expressing GFAP or S100 protein, and clearly differed from neurons expressing PGP 9.5. These results support the hypothesis that the glial cells in the ENS are the cells that express TrkB. Furthermore, using double immunofluorescence, we observed that TrkB and GFAP are expressed by the same cells (Fig. 4), although TrkB is also expressed in other cell population that do not express GFAP, thus supporting the conclusion that glial cells of the enteric nervous system is one of the cell types that express TrkB.
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Figs. 1–5. Detection of TrkB expression using Western blot in intestine samples obtained from mice at days 16 and 20 of embryonic development (E16, E20), and 1 day (1Pd), 2 weeks (2w), 1 month (1m), and 6 months (6m) old mice. The anti-TrkB antibodies used recognize a sequence within the catalytic domain of TrkB, and detected a protein ∼145 kDa, which was identified as full-length TrkB. Positive controls were performed using brain (B) homogenates of 15-day-old mice. (2) Cells displaying TrkB immunoreactivity in all ages sampled formed from 1 day to 6-month-old mice localized in ganglia and nerve bundles of the enteric nervous system. They were identified as enteric glial cells. Scale bar = 1.5 mm for (a); 60 m for (b–d). (3) Serial sections of intestine processed for the detection of TrkB (a), and the neuronal (PGP 9.5, b) or glial markers (GFAP and S100 protein, b and d, respectively) demonstrated that cells expressing TrkB are glial cells and not neurons. Scale bar = 40 m. (4) Immunohistochemical localization of TrkB (green) and GFAP (red) in the ENS (colon) of a mouse 1-month old. Both proteins were localised in the enteric glial cells, some times co-localized. Nevertheless, the expression of TrkB was more widely diffused than those of GFAP. Scale bar = 40 m. (5) TrkB immunoreactivity was absent from the enteric nervous system TrkB deficient mice (a). In these animals the expression of GFAP (b) was apparently diminished with respect to the age-matched littermates, and PGP 9.5 was unchanged. m: mucous; ml: muscular layer. Scale bar = 40 m.
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Figs. 6–7. Structure of the colon wall, including the ENS (arrows), in system in wild-type (a) and TrkB−/− (b) mice 15-day-old. Scale bar = 40 m. Table in this figure shows the number of neurons in the enteric nervous system in the three groups of animals, quantified as indicated in text. (7) Ultrastructural aspect of the colonic enteric nervous system in wild-type (a and b), and TrkB−/− (c–f) mice 15-day-old. In TrkB mutated animals there was a progressive disruption of the cytoplasm of the glial cells, as well as of the neuropil, whereas the neurons resulted unaffected by the mutation. gc: glial cells; n: nucleos of neurons; np: neuropilo. Original magnifications: 4000× for a, c, d–f; 6000× for b.
The findings that glial cells within ENS express TrkB suggest that they may depend on the activation of TrkB for survival or maintenance of their phenotype. To test this hypothesis, we analyzed the expression of some neuronal and glial markers in the ENS of
TrkB+/− and TrkB−/− mice. We did not observe significant differences in the structure of the ENS between heterozygous and wild type mice (data not shown). Observation of sections from TrkB−/− mice intestine sections stained with anti-TrkB antibodies confirmed
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that they do not express TrkB in the ENS or intestine walls (Fig. 5a). The pattern of expression of PGP 9.5 in intestine of TrkB−/− mice was similar to that found in TrkB+/+ mice (Fig. 5b). However, GFAP was distributed forming lineal cords or disrupted rings (Fig. 5c), and, more importantly, expression of S100 protein was consistently lost (data not shown). The structure of the ENS of TrkB+/+, TrkB+/−, and TrkB−/− mice was compared using semithin sections (Fig. 6a and b) and using electron microscopy (Fig. 7). There is not a statistically significant difference in the number of myenteric neurons among these three groups of mice (table in Fig. 6). The composition of myenteric ganglia was found to be variable in number of neurons and glial cells, which were distinguished based on their location within the ganglia, size, shape of the nucleus, and distribution of chromatin and cytoplasmic organelles (Fig. 7a); unmyelinated axons were abundant, as well as synaptic buttons which were located on the surface of the ganglion, adjacent to the basal lamina. The synaptic buttons contain predominantly large agranular vesicles, but also many vesicles of variable size dense and electron lucent (Fig. 7b). Detailed analysis of the ENS from TrkB−/− mice reveals several abnormalities involving glial cells. The neuronal somata were apparently normal, but the nerve fibers forming the neuropil were disordered and disrupted; they contained cell debris and electron-dense bodies. The glial cell processes were destroyed. These defects were more evident in the TrkB−/− (Fig. 7c–f) that in TrkB+/− mice (data not shown). The neural crest-derived sensory and sympathetic neurons depend on different NTs for development and maintenance during adult life [12]. Recent evidences suggested that ENS cells, which also develop from the neural crest cells (see [1]), depend on NTs for development and survival [3]. The present study provides further evidence supporting the view that NTs signaling is necessary to maintain the normal structure of the ENS, in particular to support viability of TrkB-expressing enteric glial cells. We demonstrated that glial cells within the murine ENS express full-length TrkB during all lifespan, and that loss of TrkB expression results in deterioration of normal structure of the ENS. The expression and importance of TrkB in the development of the ENS has been controversial. Pioneer studies in mice failed to demonstrate trkB, although subsequent studies detected its expression both during embryonic development and adulthood [2,3,7]. There have been also disagreement regarding which cells type(s) express NTs and their receptors. Whereas some authors failed to detect TrkB immunoreactivity in the adult human ENS, others detected TrkB expression in both neurons and glial cells. It is currently accepted that, except in human tissue, TrkB expression is restricted to glial cells in adult mammals [7,13]. The present data in mouse are consistent with previous studies in other mammalian species. Based on the molecular weight of the TrkB identified using Western blot with antibodies that bind to the catalytic tyrosine kinase domain, we conclude that ENS express full-length TrkB, that, potentially, may initiate signaling pathways upon ligand-induced activation. TrkB ligands include BDNF and NT-4, and, in certain contexts, NT-3. BDNF is released in the intestine of vertebrates [15,16,20] and it stimulates intestinal motility [2,5]. NT-3 signaling is necessary for normal development, differentiation and maintenance of enteric neurons and glial cells both in vitro [3,26] and in vivo [22], and also inhibits gastric motility [14]. Therefore, we hypothesize that BDNF and/or NT-3 might support enteric glial cells during adulthood signaling through TrkB. To the best of our knowledge, the role of NT-4 during development and maintenance of the ENS remains unknown. The abnormalities observed in glial cells of the ENS in animals lacking TrkB, i.e. loss of some characteristic antigens and structural disarrangement, also provide indirect evidence to support the
hypothesis that TrkB is necessary for the normal development and maintenance of the glial cell phenotype in the ENS. Previous studies of the morphology of the ENS in knockout mice lacking NTs or NT receptor expression failed to identify morphological abnormalities (see [12]). Subsequent studies, in which the authors stained the tissues with neuronal or glial markers using immunohistochemistry, questioned those original findings [10], and described loss of enteric neurons in TrkC and NT-3 deficient mice [4]. It is important to note that immunohistochemistry alone is suboptimal to precisely characterize the structure of the ENS. Our results show that using neuronal and glial markers enteric neurons can be detected in the ENS of TrkB−/− mice, although we observed a significant decrease in the levels of GFAP expression and complete loss of S100 protein expression. These results suggest there are important differences in glial cells from the ENS in TrkB−/− mice compared to TrkB wild type mice. It was shown before that loss of glial cells in the ENS correlates with intestinal inflammation [28], disruption of the epithelial barrier [25], and alterations in the rate of neuronal survival [23]. However, the functional consequences of the alterations found in the enteric glia of TrkB−/− mice are unknown. Acknowledgements Authors thanks to Ms. M.L. López-Robles, and Ms Marta Alonso Guervos (Servcio de Análisis de Imágenes de la Universidad de Oviedo) for technical support. References [1] R.B. Anderson, D.F. Newgreen, H.M. Young, Neural crest and the development of the enteric nervous system, Adv. Exp. Med. Biol. 589 (2006) 181–196. [2] W. Boesmans, P. Gomes, J. Janssens, J. Tack, P. Vanden Berghe, Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system, Gut 57 (2008) 314–322. [3] A. Chalazonitis, Neurotrophin–3 in the development of the enteric nervous system, Prog. Brain Res. 146 (2004) 243–263. [4] A. Chalazonitis, T.D. Pham, T.P. Rothman, P.S. DiStefano, M. Bothwell, J. Flair-Flynn, L. Tessarollo, M.D. Neurotrophin-3 is required for the survivaldifferentiation of subsets of developing enteric neurons, J. Neurosci. 21 (2001) 2636–5620. [5] C.M. Coulis, C. Lee, V. Nardone, R.D. Prokipcak, Inhibition of c-myc expression in cells by targeting an RNA-interaction using antisense oligonucleotides, Mol. Pharmacol. 57 (2000) 485–494. [6] R. De Giorgio, J. Arakawa, C.J. Whitemore, C. Sternini, Neurotrophin-3 and neurotrophin receptor immunoreactivity and peptidergic enteric neurons, Peptides 21 (2000) 1421–1426. [7] I. Esteban, B. Levanti, O. García-Suárez, G. Germanà, E. Ciriaco, F.J. Naves, J.A. Vega, A neuronal sub-population in the mamalian enteric nervous system TrkA and TrkC neurotrophin receptor-like proteins, Anat. Rec. 251 (1998) 360–370. [8] O. García-Suárez, T. González-Martínez, M. Perez-Perez, A. Germana, M.A. Blanco-Gélaz, D.F. Monjil, E. Ciriaco, I. Silos-Santiago, J.A. Vega, Expression of the neurotrophin receptor TrkB in the mouse liver, Anat. Embryol. (Berl.) 211 (2006) 465–473. [9] A. Germanà, M.B. Levanti, D.F. Monjil, E. Ciriaco, M. Del Valle, J.A. Vega, G. Germanà, Immunohistochemical detection of TrkB in the enteric nervous system of the small intestine in pigeon (Columba livia), Eur. J. Histochem. 48 (2004) 373–376. [10] M.D. Gershon, Genes and lineages in the formation of the enteric nervous system, Curr. Opin. Neurobiol. 7 (1997) 101–109. [11] D. Grundry, M. Schelmann, Enteric nervous system, Curr. Opin. Gastroenterol. 21 (2005) 176–182. ˜ [12] M. Kirstein, I. Farinas, Sensing life: regulation of sensory neuron survival by neurotrophins, Cell. Mol. Life Sci. 59 (11) (2002) 1787–1802. [13] M. Kondyli, J. Varakis, M. Assimakopoulou, Expression of p75NTR and Trk neurotrophin receptors in the enteric nervous system of human adults, Anat. Sci. Int. 80 (4) (2005) 223–228. [14] M.A. Küper, T. Meile, T.T. Zittel, A. Konigsrainer, J. Glatzle, Effects of neurotrophin 3 on gastric and colonic motility in awake rats, Neurogastroenterol. Motil. 19 (2007) 983–989. [15] M. Lommatzsch, A. Braun, A. Mannsfeldt, V.A. Botchkarev, N.V. Botchkareva, R. Paus, A. Fiscer, G.R. Lewin, H. Renz, Abundant production of brain-derived neurotrophic factor by adult visceral epithelium. Implications for paracrine and target-derived Neurotrophic functions, Am. J. Pathol. 155 (1999) 1183–1193. [16] M. Lommatzsch, D. Quarcoo, O. Schulte-Herbrüggen, H. Weber, J.C. Virchow, H. Renz, A. Braun, Neurotrophins in murine viscera: a dynamic pattern from birth to adulthood, Int. J. Dev. Neurosci. 23 (2005) 495–500.
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