- Email: [email protected]
Neuronal differentiation of EGF-propagated neurosphere cells after engraftment to the nucleus of the solitary tract
Neuroscience Letters 444 (2008) 250–253
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuronal differentiation of EGF-propagated neurosphere cells after engraftment to the nucleus of the solitary tract Masato Mitome a,∗ , Hoi Pang Low b , Karen M. Lora Rodriguez a , Masafumi Kitamoto a , Takamasa Kitamura a , William J. Schwartz b a b
Department of Pediatric Dentistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA
a r t i c l e
i n f o
Article history: Received 24 May 2008 Received in revised form 15 August 2008 Accepted 16 August 2008 Keywords: Transplantation Neural stem cells Neurogenesis GFP NeuN Gelatinous subnucleus
a b s t r a c t Neural precursor cells expanded with epidermal growth factor (EGF) exhibit multipotentiality in vitro, but they differentiate predominantly as glial phenotypes after their transplantation in vivo. Here we demonstrate that EGF-propagated precursors from the murine striatal subventricular zone can exhibit robust incorporation and neuronal differentiation within the nucleus of the solitary tract (NST) after injection into the cisterna magna of neonatal or young adult mice. About two-third of engrafted cells appeared NeuN positive in the region of the gelatinous subnucleus, a region notable for its lack of myelinated fibers. The NST may provide a useful model for understanding the physiological and metabolic regulation of postnatal neurogenesis. © 2008 Elsevier Ireland Ltd. All rights reserved.
Self-renewing, multipotent neural precursor cells have been identified in the striatal subventricular zone (SVZ) and the hippocampal (dentate) subgranular zone, and several strategies have been used to isolate and propagate uncommitted stem cells from these regions in vitro. When embryonic or adult tissues are dissociated and cultured in chemically-defined media with appropriate growth factors, populations of precursor cells proliferate as floating cellular clusters (“neurospheres”) [3]. From the murine SVZ, initially cells were expanded with epidermal growth factor (EGF) [18], and clonal analyses indicate that these EGF-propagated neurosphere cells can individually generate neurons, astrocytes, and oligodendrocytes in vitro [19]. In contrast to their multipotentiality in vitro, the in vivo differentiation of such cells after transplantation appears to be predominantly as astrocytes [4,8] – even in an environment (fetal brain) that would be conducive to neurogenesis [13,23] – and oligodendrocytes, especially in dysmyelinated hosts [1,9,14]. At least for rodent neurospheres, manipulations in addition to EGF, e.g., exposure to [7] or overexpression of [6] basic fibroblast growth factor, are necessary in order to generate mature neurons after transplantation. In the course of injecting EGF-propagated SVZ neurosphere cells into the cisterna magna of neonatal and young adult mice, we
∗ Corresponding author. Tel.: +81 88 633 7318; fax: +81 88 633 9132. E-mail address: [email protected] (M. Mitome). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.050
noticed their reproducible incorporation and neuronal differentiation within the nucleus of the solitary tract (NST). To our knowledge, this observation represents the first report that rodent precursors expanded with EGF alone can give rise to putative neurons in a region-specific manner after intrathecal injection. C57BL/6 and Swiss-Webster mice were housed in our animal facilities under defined environmental conditions (temperature, 22 ± 2 ◦ C; humidity, 60 ± 5%; 12 h:12 h light/dark cycle). Host mice for transplantation ranged in age from postnatal day P1 to P3 (where P1, the day of birth) to 4 weeks of age. Neonates were cryoanesthetized, and young adult mice were anesthetized i.p. with a solution of ketamine (125 mg/kg) and xylazine (25 mg/kg) and fixed in a stereotaxic apparatus. Animals were injected with 120,000 cells in 2 l into the cisterna magna, with the cellular suspension expelled gently via a glass micropipette inserted transcutaneously. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Tokushima University Graduate School of Dentistry and the University of Massachusetts Medical School. Donor cells were derived from E16 (where E1, the day after overnight mating) SVZ tissue microdissected from embryonic transgenic mice expressing an enhanced form of the jellyfish green fluorescent protein (GFP), as previously described [14,16]. Cells were isolated and propagated using a modification [10] of the method developed by Reynolds et al. [18]. Tissue was placed in a 1:1 mixture of Dulbecco’s modified Eagle’s medium
M. Mitome et al. / Neuroscience Letters 444 (2008) 250–253
251
Fig. 1. EGF-propagated GFP+ cells in brainstem sections from host mice examined 8 weeks after cell suspensions were injected into the cisterna magna. In coronal section of the medulla oblongata (A), GFP fluorescence is observed within the dorsal aspect of the NST (box, area enlarged in B) and in the external cuneate nucleus and spinal tract of the trigeminal nerve (single and double arrows, respectively). In horizontal section, GFP+ cells are localized primarily to the intermediate part of the NST (C). Confocal microscopy of engrafted GFP+ cells (box in B, shown in D) reveals co-localization with immunoreactive NeuN (E, merged in F). Scale bars represent 500 m (A), 50 m (B, C), or 25 m (D–F); (*) fourth ventricle.
and F-12 nutrient (DMEM/F-12; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 0.3% glucose, 23 g/ml insulin, 92 g/ml transferrin, 55 M putrescine, 27.5 nM sodium selenite, 50 U/ml penicillin–streptomycin, 20 nM progesterone, and 20 ng/ml EGF (Becton Dickinson/Collaborative Biomedical Products, Bedford, MA, USA). Tissue was triturated with a wet fire-polished Pasteur pipette to obtain a “single cell” suspension, and the cells were seeded at a density of 1.5 × 106 viable cells/20 ml in 75 cm2 flasks and maintained in a humidified incubator at 37 ◦ C and 95% atmospheric air/5% CO2 . Cultures were fed with 5 ml of fresh medium every other day and passaged weekly. For transplantation after 1 to 3 passages, cells were harvested, triturated, and the density adjusted to 6 × 104 viable cells/l in DMEM/F-12 medium. Eight weeks after transplantation, mice were deeply anesthetized with sodium pentobarbital (10 mg i.p.) or ketamine/xylazine and perfused with ice-cold heparinized phosphate-buffered saline (PBS), followed by ice-cold 4% buffered
paraformaldehyde fixative. Tissue was post-fixed for 2–5 h at 4 ◦ C, and 50 m thick coronal and/or horizontal sections were cut on a vibratome and stored at −20 ◦ C in cryoprotectant (30% sucrose, 30% ethylene glycol, 0.25 mM polyvinylpyrrolidone in PBS). For co-localizing GFP fluorescence with immunoreactive NeuN, a marker of terminally differentiated neurons, free-floating sections were rinsed and blocked with 10% normal goat serum and 0.4% Triton X-100 in Tris-buffered saline (TBS) for 30 min to 1 h followed by incubation with primary antibody (mouse, 1:1000 = 0.2 g/ml; Chemicon International, Temecula, CA, USA) diluted in 2% normal goat serum and 0.4% Triton X-100 in TBS, for 1 h at room temperature. After washing, the sections were incubated in anti-mouse Alexa Fluor 594 secondary antibody (Invitrogen / Molecular Probes, Eugene, OR, USA) in darkness for 1 h at room temperature and coverslipped in Prolong anti-fade reagent (Invitrogen / Molecular Probes). For each brain, the section with the greatest number of GFP+ cells was examined using a
252
M. Mitome et al. / Neuroscience Letters 444 (2008) 250–253
Leica True Confocal Scanning Spectrophotometer microscope, with excitation wavelengths for GFP and Alexa Fluor 594 of 488 and 568 nm, respectively. The distribution of donor-derived GFP+ cells in brainstem sections from neonatal and young adult host mice was examined 8 weeks after cell suspensions were injected into the cisterna magna; GFP+ cells were found in 12 of 15 (80%) neonatal and in 6 of 7 (86%) adult hosts. In neonates, incorporation was consistently observed within the inferior cerebellar peduncle and the spinal tract of the trigeminal nerve at the level of the lateral medulla oblongata, while incorporation within the trigeminal nucleus, cuneate and external cuneate nuclei, cochlear nucleus, and area postrema was more variable; in adults, engraftment within the peduncle and spinal tract was seen in some cases. In all these regions, cells exhibited an astroglial morphology and absent NeuN immunoreactivity. We also noted a region of reliable incorporation (in all 12 neonates and 6 adults) involving the NST; on coronal and horizontal sections, GFP+ cells were seen to be primarily localized to the dorsal and intermediate aspects of the nucleus, respectively (Fig. 1A–C), corresponding most closely to the region of the gelatinous subnucleus. Overall, the mean number of NST GFP+ cells per section at this engraftment site was 4.5 ± 1.3 (neonates) and 8.3 ± 2.9 (adults) (±S.E.M.). GFP fluorescence in the NST revealed small bipolar and triangular neuronal-like perikarya with presumptive dendrites and axons distributed in the horizontal plane (Fig. 1C). The proportion of double-labeled GFP+ /NeuN+ cells to the total number of GFP+ cells in the NST of individual mice ranged from 0% to 100% per section, but quantitative analysis of these data was complicated by considerable mouse-to-mouse variability in the total number of GFP+ cells in the NST, which ranged from 1 to 20 per section in different animals. Overall, the mean number of GFP+ /NeuN+ cells per section was 74 ± 12% (neonates) and 59 ± 16% (adults) (Fig. 1D–F). No GFP+ /NeuN+ cells were observed in any other brainstem region in which GFP+ cells engrafted. The NST receives visceral afferent signals from the cardiovascular, respiratory, gastrointestinal, and taste systems via the vagus, glossopharyngeal and facial nerves. It plays a critical role in generating and/or regulating autonomic functions [11]. Here we report a distinctive new feature of the NST, as a site supporting the neuronal differentiation of transplanted EGF-propagated neural precursor cells. About two-third of engrafted cells appeared NeuN positive in the region of the gelatinous subnucleus in neonatal and young adult hosts. In contrast, EGF-propagated cells incorporating elsewhere in the medulla or transplanted to other brain sites regularly exhibit glial phenotypes [23]. The unique characteristic(s) of the NST microenvironment that enable the preferential survival and differentiation of engrafted neuronal progenitors are unknown. The cells’ predilection for the gelatinous subnucleus, a region originally identified on Weigert preparations due to its lack of myelinated fibers [22], raises the possibility that myelin-related factors present at other engraftment sites may ordinarily act to inhibit neuronal differentiation of transplanted precursors. In addition, the dorsal vagal complex, including the NST, appears to be enriched in molecules associated with neural plasticity [15] and has been shown to be a site of ongoing, endogenous neurogenesis [2,5,17,25], suggesting its possible role as a neurogenic “niche” (see [20]). The gelatinous subnucleus of the NST receives a dense projection from gastric afferents [21], and immunoreactive c-Fos in this region is elicited by gastric stimulation, such as food intake and direct injection of chemical solutions into the stomach [24]. A relationship between ingestion, diet, and neurogenesis has already been suggested by the observation that mastication influences the survival of newly generated cells in the hippocampal dentate gyrus
of 4-week-old mice [12]. Further study of precursor cell proliferation and differentiation in the NST may provide a useful model for elucidating the physiological and metabolic regulation of postnatal neurogenesis.
Acknowledgements We thank Dr. Masaru Okabe and Dr. Anthony van den Pol for providing us with GFP-transgenic mice. This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the promotion of Science (no. 18390550) and NASA NAG9-1356.
References [1] M. Ader, J. Meng, M. Schachner, U. Bartsch, Formation of myelin after transplantation of neural precursor cells into the retina of young postnatal mice, Glia 30 (2000) 301–310. [2] S. Bauer, M. Hay, B. Amilhon, A. Jean, E. Moyse, In vivo neurogenesis in the dorsal vagal complex of the adult rat brainstem, Neuroscience 130 (2005) 75–90. [3] L.S. Campos, Neurospheres: insights into neural stem cell biology, J. Neurosci. Res. 78 (2004) 761–769. [4] M.K. Carpenter, C. Winkler, R. Fricker, D.F. Emerich, S.C. Wong, C. Greco, E.Y. Chen, Y. Chu, J.H. Kordower, A. Messing, A. Bjorklund, J.P. Hammang, Generation and transplantation of EGF-responsive neural stem cells derived from GFAPhNGF transgenic mice, Exp. Neurol. 148 (1997) 187–204. [5] C. Charrier, V. Coronas, J. Fombonne, M. Roger, A. Jean, S. Krantic, E. Moyse, Characterization of neural stem cells in the dorsal vagal complex of adult rat by in vivo proliferation labeling and in vitro neurosphere assay, Neuroscience 138 (2006) 5–16. [6] A.G. Dayer, B. Jenny, M.O. Sauvain, G. Potter, P. Salmon, E. Zgraggen, M. Kanemitsu, E. Gascon, S. Sizonenko, D. Trono, J.Z. Kiss, Expression of FGF-2 in neural progenitor cells enhances their potential for cellular brain repair in the rodent cortex, Brain 130 (2007) 2962–2976. [7] C. Eriksson, A. Bjorklund, K. Wictorin, Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions, Exp. Neurol. 184 (2003) 615–635. [8] R.A. Fricker-Gates, C. Winkler, D. Kirik, C. Rosenblad, M.K. Carpenter, A. Bjorklund, EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum, Exp. Neurol. 165 (2000) 237–247. [9] J.P. Hammang, D.R. Archer, I.D. Duncan, Myelination following transplantation of EGF-responsive neural stem cells into a myelin-deficient environment, Exp. Neurol. 147 (1997) 84–95. [10] R. Hulspas, C. Tiarks, J. Reilly, C.C. Hsieh, L. Recht, P.J. Quesenberry, In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions, Exp. Neurol. 148 (1997) 147– 156. [11] A. Jean, The nucleus tractus solitarius: neuroanatomic, neurochemical and functional aspects, Arch. Int. Physiol. Biochim. Biophys. 99 (1991) A3–A52. [12] M. Mitome, T. Hasegawa, T. Shirakawa, Mastication influences the survival of newly generated cells in mouse dentate gyrus, Neuroreport 16 (2005) 249– 252. [13] M. Mitome, H.P. Low, H.O. de la Iglesia, W.J. Schwartz, Constructing suprachiasmatic nucleus chimeras in vivo, Biol. Rhythm Res. 32 (2001) 221–232. [14] M. Mitome, H.P. Low, A. van den Pol, J.J. Nunnari, M.K. Wolf, S. Billings-Gagliardi, W.J. Schwartz, Towards the reconstruction of central nervous system white matter using neural precursor cells, Brain 124 (2001) 2147–2161. [15] E. Moyse, S. Bauer, C. Charrier, V. Coronas, S. Krantic, A. Jean, Neurogenesis and neural stem cells in the dorsal vagal complex of adult rat brain: new vistas about autonomic regulations – a review, Auton. Neurosci. 126-127 (2006) 50– 58. [16] M. Okabe, M. Ikawa, K. Kominami, T. Nakanishi, Y. Nishimune, ’Green mice’ as a source of ubiquitous green cells, FEBS Lett. 407 (1997) 313–319. [17] E. Pecchi, M. Dallaporta, C. Charrier, J. Pio, A. Jean, E. Moyse, J.D. Troadec, Glial fibrillary acidic protein (GFAP)-positive radial-like cells are present in the vicinity of proliferative progenitors in the nucleus tractus solitarius of adult rat, J. Comp. Neurol. 501 (2007) 353–368. [18] B.A. Reynolds, W. Tetzlaff, S. Weiss, A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes, J. Neurosci. 12 (1992) 4565–4574. [19] B.A. Reynolds, S. Weiss, Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell, Dev. Biol. 175 (1996) 1–13. [20] D.T. Scadden, The stem-cell niche as an entity of action, Nature 441 (2006) 1075–1079. [21] R.E. Shapiro, R.R. Miselis, The central organization of the vagus nerve innervating the stomach of the rat, J. Comp. Neurol. 238 (1985) 473–488.
M. Mitome et al. / Neuroscience Letters 444 (2008) 250–253 [22] E. Taber, The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat, J. Comp. Neurol. 116 (1961) 27–69. [23] C. Winkler, R.A. Fricker, M.A. Gates, M. Olsson, J.P. Hammang, M.K. Carpenter, A. Bjorklund, Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain, Mol. Cell Neurosci. 11 (1998) 99–116.
253
[24] T. Yamamoto, K. Sawa, c-Fos-like immunoreactivity in the brainstem following gastric loads of various chemical solutions in rats, Brain Res. 866 (2000) 135–143. [25] W. Zhang, Y. Hu, T.R. Lin, Y. Fan, M.W. Mulholland, Stimulation of neurogenesis in rat nucleus of the solitary tract by ghrelin, Peptides 26 (2005) 2280–2288.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuronal differentiation of EGF-propagated neurosphere cells after engraftment to the nucleus of the solitary tract Masato Mitome a,∗ , Hoi Pang Low b , Karen M. Lora Rodriguez a , Masafumi Kitamoto a , Takamasa Kitamura a , William J. Schwartz b a b
Department of Pediatric Dentistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA
a r t i c l e
i n f o
Article history: Received 24 May 2008 Received in revised form 15 August 2008 Accepted 16 August 2008 Keywords: Transplantation Neural stem cells Neurogenesis GFP NeuN Gelatinous subnucleus
a b s t r a c t Neural precursor cells expanded with epidermal growth factor (EGF) exhibit multipotentiality in vitro, but they differentiate predominantly as glial phenotypes after their transplantation in vivo. Here we demonstrate that EGF-propagated precursors from the murine striatal subventricular zone can exhibit robust incorporation and neuronal differentiation within the nucleus of the solitary tract (NST) after injection into the cisterna magna of neonatal or young adult mice. About two-third of engrafted cells appeared NeuN positive in the region of the gelatinous subnucleus, a region notable for its lack of myelinated fibers. The NST may provide a useful model for understanding the physiological and metabolic regulation of postnatal neurogenesis. © 2008 Elsevier Ireland Ltd. All rights reserved.
Self-renewing, multipotent neural precursor cells have been identified in the striatal subventricular zone (SVZ) and the hippocampal (dentate) subgranular zone, and several strategies have been used to isolate and propagate uncommitted stem cells from these regions in vitro. When embryonic or adult tissues are dissociated and cultured in chemically-defined media with appropriate growth factors, populations of precursor cells proliferate as floating cellular clusters (“neurospheres”) [3]. From the murine SVZ, initially cells were expanded with epidermal growth factor (EGF) [18], and clonal analyses indicate that these EGF-propagated neurosphere cells can individually generate neurons, astrocytes, and oligodendrocytes in vitro [19]. In contrast to their multipotentiality in vitro, the in vivo differentiation of such cells after transplantation appears to be predominantly as astrocytes [4,8] – even in an environment (fetal brain) that would be conducive to neurogenesis [13,23] – and oligodendrocytes, especially in dysmyelinated hosts [1,9,14]. At least for rodent neurospheres, manipulations in addition to EGF, e.g., exposure to [7] or overexpression of [6] basic fibroblast growth factor, are necessary in order to generate mature neurons after transplantation. In the course of injecting EGF-propagated SVZ neurosphere cells into the cisterna magna of neonatal and young adult mice, we
∗ Corresponding author. Tel.: +81 88 633 7318; fax: +81 88 633 9132. E-mail address: [email protected] (M. Mitome). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.050
noticed their reproducible incorporation and neuronal differentiation within the nucleus of the solitary tract (NST). To our knowledge, this observation represents the first report that rodent precursors expanded with EGF alone can give rise to putative neurons in a region-specific manner after intrathecal injection. C57BL/6 and Swiss-Webster mice were housed in our animal facilities under defined environmental conditions (temperature, 22 ± 2 ◦ C; humidity, 60 ± 5%; 12 h:12 h light/dark cycle). Host mice for transplantation ranged in age from postnatal day P1 to P3 (where P1, the day of birth) to 4 weeks of age. Neonates were cryoanesthetized, and young adult mice were anesthetized i.p. with a solution of ketamine (125 mg/kg) and xylazine (25 mg/kg) and fixed in a stereotaxic apparatus. Animals were injected with 120,000 cells in 2 l into the cisterna magna, with the cellular suspension expelled gently via a glass micropipette inserted transcutaneously. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Tokushima University Graduate School of Dentistry and the University of Massachusetts Medical School. Donor cells were derived from E16 (where E1, the day after overnight mating) SVZ tissue microdissected from embryonic transgenic mice expressing an enhanced form of the jellyfish green fluorescent protein (GFP), as previously described [14,16]. Cells were isolated and propagated using a modification [10] of the method developed by Reynolds et al. [18]. Tissue was placed in a 1:1 mixture of Dulbecco’s modified Eagle’s medium
M. Mitome et al. / Neuroscience Letters 444 (2008) 250–253
251
Fig. 1. EGF-propagated GFP+ cells in brainstem sections from host mice examined 8 weeks after cell suspensions were injected into the cisterna magna. In coronal section of the medulla oblongata (A), GFP fluorescence is observed within the dorsal aspect of the NST (box, area enlarged in B) and in the external cuneate nucleus and spinal tract of the trigeminal nerve (single and double arrows, respectively). In horizontal section, GFP+ cells are localized primarily to the intermediate part of the NST (C). Confocal microscopy of engrafted GFP+ cells (box in B, shown in D) reveals co-localization with immunoreactive NeuN (E, merged in F). Scale bars represent 500 m (A), 50 m (B, C), or 25 m (D–F); (*) fourth ventricle.
and F-12 nutrient (DMEM/F-12; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 0.3% glucose, 23 g/ml insulin, 92 g/ml transferrin, 55 M putrescine, 27.5 nM sodium selenite, 50 U/ml penicillin–streptomycin, 20 nM progesterone, and 20 ng/ml EGF (Becton Dickinson/Collaborative Biomedical Products, Bedford, MA, USA). Tissue was triturated with a wet fire-polished Pasteur pipette to obtain a “single cell” suspension, and the cells were seeded at a density of 1.5 × 106 viable cells/20 ml in 75 cm2 flasks and maintained in a humidified incubator at 37 ◦ C and 95% atmospheric air/5% CO2 . Cultures were fed with 5 ml of fresh medium every other day and passaged weekly. For transplantation after 1 to 3 passages, cells were harvested, triturated, and the density adjusted to 6 × 104 viable cells/l in DMEM/F-12 medium. Eight weeks after transplantation, mice were deeply anesthetized with sodium pentobarbital (10 mg i.p.) or ketamine/xylazine and perfused with ice-cold heparinized phosphate-buffered saline (PBS), followed by ice-cold 4% buffered
paraformaldehyde fixative. Tissue was post-fixed for 2–5 h at 4 ◦ C, and 50 m thick coronal and/or horizontal sections were cut on a vibratome and stored at −20 ◦ C in cryoprotectant (30% sucrose, 30% ethylene glycol, 0.25 mM polyvinylpyrrolidone in PBS). For co-localizing GFP fluorescence with immunoreactive NeuN, a marker of terminally differentiated neurons, free-floating sections were rinsed and blocked with 10% normal goat serum and 0.4% Triton X-100 in Tris-buffered saline (TBS) for 30 min to 1 h followed by incubation with primary antibody (mouse, 1:1000 = 0.2 g/ml; Chemicon International, Temecula, CA, USA) diluted in 2% normal goat serum and 0.4% Triton X-100 in TBS, for 1 h at room temperature. After washing, the sections were incubated in anti-mouse Alexa Fluor 594 secondary antibody (Invitrogen / Molecular Probes, Eugene, OR, USA) in darkness for 1 h at room temperature and coverslipped in Prolong anti-fade reagent (Invitrogen / Molecular Probes). For each brain, the section with the greatest number of GFP+ cells was examined using a
252
M. Mitome et al. / Neuroscience Letters 444 (2008) 250–253
Leica True Confocal Scanning Spectrophotometer microscope, with excitation wavelengths for GFP and Alexa Fluor 594 of 488 and 568 nm, respectively. The distribution of donor-derived GFP+ cells in brainstem sections from neonatal and young adult host mice was examined 8 weeks after cell suspensions were injected into the cisterna magna; GFP+ cells were found in 12 of 15 (80%) neonatal and in 6 of 7 (86%) adult hosts. In neonates, incorporation was consistently observed within the inferior cerebellar peduncle and the spinal tract of the trigeminal nerve at the level of the lateral medulla oblongata, while incorporation within the trigeminal nucleus, cuneate and external cuneate nuclei, cochlear nucleus, and area postrema was more variable; in adults, engraftment within the peduncle and spinal tract was seen in some cases. In all these regions, cells exhibited an astroglial morphology and absent NeuN immunoreactivity. We also noted a region of reliable incorporation (in all 12 neonates and 6 adults) involving the NST; on coronal and horizontal sections, GFP+ cells were seen to be primarily localized to the dorsal and intermediate aspects of the nucleus, respectively (Fig. 1A–C), corresponding most closely to the region of the gelatinous subnucleus. Overall, the mean number of NST GFP+ cells per section at this engraftment site was 4.5 ± 1.3 (neonates) and 8.3 ± 2.9 (adults) (±S.E.M.). GFP fluorescence in the NST revealed small bipolar and triangular neuronal-like perikarya with presumptive dendrites and axons distributed in the horizontal plane (Fig. 1C). The proportion of double-labeled GFP+ /NeuN+ cells to the total number of GFP+ cells in the NST of individual mice ranged from 0% to 100% per section, but quantitative analysis of these data was complicated by considerable mouse-to-mouse variability in the total number of GFP+ cells in the NST, which ranged from 1 to 20 per section in different animals. Overall, the mean number of GFP+ /NeuN+ cells per section was 74 ± 12% (neonates) and 59 ± 16% (adults) (Fig. 1D–F). No GFP+ /NeuN+ cells were observed in any other brainstem region in which GFP+ cells engrafted. The NST receives visceral afferent signals from the cardiovascular, respiratory, gastrointestinal, and taste systems via the vagus, glossopharyngeal and facial nerves. It plays a critical role in generating and/or regulating autonomic functions [11]. Here we report a distinctive new feature of the NST, as a site supporting the neuronal differentiation of transplanted EGF-propagated neural precursor cells. About two-third of engrafted cells appeared NeuN positive in the region of the gelatinous subnucleus in neonatal and young adult hosts. In contrast, EGF-propagated cells incorporating elsewhere in the medulla or transplanted to other brain sites regularly exhibit glial phenotypes [23]. The unique characteristic(s) of the NST microenvironment that enable the preferential survival and differentiation of engrafted neuronal progenitors are unknown. The cells’ predilection for the gelatinous subnucleus, a region originally identified on Weigert preparations due to its lack of myelinated fibers [22], raises the possibility that myelin-related factors present at other engraftment sites may ordinarily act to inhibit neuronal differentiation of transplanted precursors. In addition, the dorsal vagal complex, including the NST, appears to be enriched in molecules associated with neural plasticity [15] and has been shown to be a site of ongoing, endogenous neurogenesis [2,5,17,25], suggesting its possible role as a neurogenic “niche” (see [20]). The gelatinous subnucleus of the NST receives a dense projection from gastric afferents [21], and immunoreactive c-Fos in this region is elicited by gastric stimulation, such as food intake and direct injection of chemical solutions into the stomach [24]. A relationship between ingestion, diet, and neurogenesis has already been suggested by the observation that mastication influences the survival of newly generated cells in the hippocampal dentate gyrus
of 4-week-old mice [12]. Further study of precursor cell proliferation and differentiation in the NST may provide a useful model for elucidating the physiological and metabolic regulation of postnatal neurogenesis.
Acknowledgements We thank Dr. Masaru Okabe and Dr. Anthony van den Pol for providing us with GFP-transgenic mice. This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the promotion of Science (no. 18390550) and NASA NAG9-1356.
References [1] M. Ader, J. Meng, M. Schachner, U. Bartsch, Formation of myelin after transplantation of neural precursor cells into the retina of young postnatal mice, Glia 30 (2000) 301–310. [2] S. Bauer, M. Hay, B. Amilhon, A. Jean, E. Moyse, In vivo neurogenesis in the dorsal vagal complex of the adult rat brainstem, Neuroscience 130 (2005) 75–90. [3] L.S. Campos, Neurospheres: insights into neural stem cell biology, J. Neurosci. Res. 78 (2004) 761–769. [4] M.K. Carpenter, C. Winkler, R. Fricker, D.F. Emerich, S.C. Wong, C. Greco, E.Y. Chen, Y. Chu, J.H. Kordower, A. Messing, A. Bjorklund, J.P. Hammang, Generation and transplantation of EGF-responsive neural stem cells derived from GFAPhNGF transgenic mice, Exp. Neurol. 148 (1997) 187–204. [5] C. Charrier, V. Coronas, J. Fombonne, M. Roger, A. Jean, S. Krantic, E. Moyse, Characterization of neural stem cells in the dorsal vagal complex of adult rat by in vivo proliferation labeling and in vitro neurosphere assay, Neuroscience 138 (2006) 5–16. [6] A.G. Dayer, B. Jenny, M.O. Sauvain, G. Potter, P. Salmon, E. Zgraggen, M. Kanemitsu, E. Gascon, S. Sizonenko, D. Trono, J.Z. Kiss, Expression of FGF-2 in neural progenitor cells enhances their potential for cellular brain repair in the rodent cortex, Brain 130 (2007) 2962–2976. [7] C. Eriksson, A. Bjorklund, K. Wictorin, Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions, Exp. Neurol. 184 (2003) 615–635. [8] R.A. Fricker-Gates, C. Winkler, D. Kirik, C. Rosenblad, M.K. Carpenter, A. Bjorklund, EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum, Exp. Neurol. 165 (2000) 237–247. [9] J.P. Hammang, D.R. Archer, I.D. Duncan, Myelination following transplantation of EGF-responsive neural stem cells into a myelin-deficient environment, Exp. Neurol. 147 (1997) 84–95. [10] R. Hulspas, C. Tiarks, J. Reilly, C.C. Hsieh, L. Recht, P.J. Quesenberry, In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions, Exp. Neurol. 148 (1997) 147– 156. [11] A. Jean, The nucleus tractus solitarius: neuroanatomic, neurochemical and functional aspects, Arch. Int. Physiol. Biochim. Biophys. 99 (1991) A3–A52. [12] M. Mitome, T. Hasegawa, T. Shirakawa, Mastication influences the survival of newly generated cells in mouse dentate gyrus, Neuroreport 16 (2005) 249– 252. [13] M. Mitome, H.P. Low, H.O. de la Iglesia, W.J. Schwartz, Constructing suprachiasmatic nucleus chimeras in vivo, Biol. Rhythm Res. 32 (2001) 221–232. [14] M. Mitome, H.P. Low, A. van den Pol, J.J. Nunnari, M.K. Wolf, S. Billings-Gagliardi, W.J. Schwartz, Towards the reconstruction of central nervous system white matter using neural precursor cells, Brain 124 (2001) 2147–2161. [15] E. Moyse, S. Bauer, C. Charrier, V. Coronas, S. Krantic, A. Jean, Neurogenesis and neural stem cells in the dorsal vagal complex of adult rat brain: new vistas about autonomic regulations – a review, Auton. Neurosci. 126-127 (2006) 50– 58. [16] M. Okabe, M. Ikawa, K. Kominami, T. Nakanishi, Y. Nishimune, ’Green mice’ as a source of ubiquitous green cells, FEBS Lett. 407 (1997) 313–319. [17] E. Pecchi, M. Dallaporta, C. Charrier, J. Pio, A. Jean, E. Moyse, J.D. Troadec, Glial fibrillary acidic protein (GFAP)-positive radial-like cells are present in the vicinity of proliferative progenitors in the nucleus tractus solitarius of adult rat, J. Comp. Neurol. 501 (2007) 353–368. [18] B.A. Reynolds, W. Tetzlaff, S. Weiss, A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes, J. Neurosci. 12 (1992) 4565–4574. [19] B.A. Reynolds, S. Weiss, Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell, Dev. Biol. 175 (1996) 1–13. [20] D.T. Scadden, The stem-cell niche as an entity of action, Nature 441 (2006) 1075–1079. [21] R.E. Shapiro, R.R. Miselis, The central organization of the vagus nerve innervating the stomach of the rat, J. Comp. Neurol. 238 (1985) 473–488.
M. Mitome et al. / Neuroscience Letters 444 (2008) 250–253 [22] E. Taber, The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of cat, J. Comp. Neurol. 116 (1961) 27–69. [23] C. Winkler, R.A. Fricker, M.A. Gates, M. Olsson, J.P. Hammang, M.K. Carpenter, A. Bjorklund, Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain, Mol. Cell Neurosci. 11 (1998) 99–116.
253
[24] T. Yamamoto, K. Sawa, c-Fos-like immunoreactivity in the brainstem following gastric loads of various chemical solutions in rats, Brain Res. 866 (2000) 135–143. [25] W. Zhang, Y. Hu, T.R. Lin, Y. Fan, M.W. Mulholland, Stimulation of neurogenesis in rat nucleus of the solitary tract by ghrelin, Peptides 26 (2005) 2280–2288.