Chordin expression in the adult rat brain

Chordin expression in the adult rat brain

Neuroscience 258 (2014) 16–33 CHORDIN EXPRESSION IN THE ADULT RAT BRAIN I S. MIKAWA AND K. SATO * single transmembrane, and the intracellular serine...

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Neuroscience 258 (2014) 16–33

CHORDIN EXPRESSION IN THE ADULT RAT BRAIN I S. MIKAWA AND K. SATO *

single transmembrane, and the intracellular serine/ threonine kinase domain, including bone morphogenetic protein receptor type I (BMPRIA, BMPRIB) and type II (BMPRII) (Bragdon et al., 2011). On BMP binding, the type I BMPRs activate the receptor-activated Smads (R-Smads; Smad1/5/8) which oligomerize with commonmediator Smad (Co-Smad; Smad4). The Smad complex then translocates to the nucleus and acts as a transcription regulator (Moustakas and Heldin, 2009). Although many of the biological effects of BMPs have been related to the Smad-dependent pathways, Smadindependent pathways have been also reported (Massague, 2003). Functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators such as chordin, noggin, follistatin, neurogenesin-1, which are reported to bind BMPs and prevent their interaction with their receptors (Cho and Blitz, 1998; Ueki et al., 2003). Chordin contains four cysteine-rich domains which bind BMPs, inhibiting BMP binding to their receptors (Zakin and De Robertis, 2010). Chordin plays major roles in dorsoventral axis formation and in the induction, maintenance, and differentiation of neural tissues during gastrulation, and is secreted by the Spemann organizer of Xenopus and zebrafish, and by the node of chick and mouse embryos (Sasai and De Robertis, 1997; MillerBertoglio et al., 1997; Streit et al., 1998). When chordin is secreted from the organizer, it acts by interfering in BMP signaling, allowing dorsally derived tissues, such as neuroectoderm and somatic muscle, to develop (MillerBertoglio et al., 1997). About half of the chordin knockout mice resulted in stillborn animals, which have normal early development and neural induction, but at later stages of embryogenesis, they showed defects in inner and outer ear development and abnormalities in pharyngeal and cardiovascular organization (Bachiller et al., 2000). In the adult CNS, some papers report the contributions of chordin to synaptic plasticity and lineage plasticity. Since about half of the chordin knockout mice survive and exhibit a normal gross morphology of the CNS (Bachiller et al., 2003), Sun et al. (2007) have reported that these adult chordin null mice exhibited a significant increase in presynaptic transmitter release from hippocampal neurons, resulting in enhanced paired-pulse facilitation and long-term potentiation (LTP). In addition, Jablonska et al. (2010) have shown that, in the subventricular zone (SVZ), chordin up-regulated by demyelination redirected neuroblasts from neuronal to glial fates, generating new oligodendrocytes in the corpus callosum. However, the other functions of chordin in the adult CNS are largely unknown.

Department of Anatomy & Neuroscience, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashiku, Hamamatsu, Shizuoka 431-3192, Japan

Abstract—Bone morphogenetic proteins (BMPs) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators. Chordin is an extracellular BMP antagonist that binds BMP-2, 4, and 7 with high affinity and thus interferes with binding to BMP receptors. Although chordin expression has been well described in the early development of the CNS, little information is available for its expression in the adult CNS. We, thus, investigated chordin expression in the adult rat CNS using immunohistochemistry. Chordin was intensely expressed in most neurons, and their dendrites and axons. In addition, abundant chordin expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes and ependymal cells also express chordin protein. These data indicate that chordin is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that chordin plays pivotal roles also in the adult brain. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: axon, neuropil, immunohistochemistry.

INTRODUCTION Chordin is an extracellular bone morphogenetic protein (BMP) antagonist that binds BMPs and thus interferes with binding to BMP receptors. BMPs were initially detected by their ability to direct ectopic bone formation, are now shown to play an important role in multiple biological events (Bragdon et al., 2011). The activities of the BMPs are mediated by a heterodimeric complex of type I and type II BMP serine/threonine kinase receptors, composed of a short extracellular domain, a I Grant sponsor: the Ministry of Education, Science and Culture of Japan. *Corresponding author. Tel/fax: +81-53-435-2582. E-mail address: [email protected] (K. Sato). Abbreviations: BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; BSA, bovine serum albumin; DCX, doublecortin; GFAP, glial fibrillary acidic protein; IR, like immunoreactivity; LTD, long-term depression; LTP, long-term potentiation; MAP2, microtubule-associated protein-2; NeuN, neuronal nuclei; PB, phosphate buffer; SVZ, subventricular zone.

0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.11.006 16

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Chordin expression has been well described in the early development of CNS (Streit et al., 1998; Bachiller et al., 2000; Anderson et al., 2002). However, little information is available for chordin expression in the adult CNS. Although some reports delineate its expression in the adult CNS (Pappano et al., 1998; Scott et al., 2000), the reports mainly deal with its expressions in restricted areas. Furthermore, BMP ligands, such as BMP2 and 4, have been also reported to be abundantly expressed in the adult rat brain (Mikawa et al., 2006; Sato et al., 2010). It is, thus, necessary to perform more wide and detailed investigations of the expression pattern of chordin in the adult rat brain. In the present study, we show that chordin is more widely expressed than previously reported, and that chordin is expressed in neurons, astrocytes, and ependymal cells.

EXPERIMENTAL PROCEDURES Animals and section preparation Under deep diethylether anesthesia, brain samples were isolated from male Wistar rats (7 weeks old; Japan SLC Inc., Shizuoka, Japan). For immunohistochemistry, the rats were perfused transcardially with saline followed by 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde and 0.2% picric acid. The brains were removed rapidly, and then postfixed in the same fixative for 2 h at 4 °C. All brains were immersed in 10%, 20%, 25% buffered sucrose each, overnight at 4 °C, respectively. Frozen sections (20 lm in thickness) were cut on a cryostat. All experiments conformed to the Guidelines for Animal Experimentation at the Hamamatsu University School of Medicine on the ethical use of animals. Immunohistochemistry For immunoperoxidase staining, the sections were treated with 10% normal goat serum, 2% bovine serum albumin (BSA) and 0.2% Triton X-100 in 0.1 M PB for 2 h at room temperature, and incubated further in rabbit anti-chordin (diluted 1:50, the final concentration, 2 lg/ml; ABGENT Inc., San Diego, CA, USA) overnight at 4 °C. After being washed with 0.1 M PB, sections were incubated in goat anti-rabbit IgG with peroxidase complex (no dilution, ready-to-use; EnVision System, K4002; DAKO, Tokyo, Japan) for 2 h at room temperature. After being washed with 0.1 M PB, immunoreaction was visualized with 3,30 -diaminobenzidine (Wako, Osaka, Japan). For pre-absorption control studies, synthetic chordin peptide (926–955 amino acids from the C-terminal region of human chordin) was added to the antibody solution (20 lg/ml), incubated overnight at 4 °C, and further processed for immunoperoxidase staining as described above. For double immunofluorescence, sections were treated with 10% normal goat serum, 2% BSA and 0.2% Triton X-100 in 0.1 M PB for 2 h at room temperature, and incubated further in rabbit anti-chordin antibody (diluted 1:10; the final concentration, 10 lg/ml; ABGENT

Inc.) together with mouse anti-NeuN antibody (diluted 1:100; the final concentration, 10 lg/ml; Millipore, Temecula, CA, USA), or mouse anti-MAP2 antibody (diluted 1:150; the initial concentration is not available; Millipore), or mouse anti-GFAP antibody (diluted 1:1,000; the initial concentration is not available; Millipore), or mouse anti-oligodendrocytes antibody (diluted 1:10,000; the initial concentration is not available; Millipore), or mouse anti-Nestin antibody (diluted 1:100; the final concentration, 10 lg/ml; Millipore, Temecula, CA, USA), or mouse antidoublecortin (DCX) antibody (diluted 1:500; the final concentration, 2 lg/ml; Abcam, Cambridge, UK), or mouse anti-S100b antibody (diluted 1:10,000; the final concentration, 1 lg/ml; GeneTex, Inc., Ivine, CA, USA). After being washed with 0.1 M PB, sections were incubated in both Alexa Fluor 488 goat anti-mouse IgG (diluted 1:1,000; the final concentration, 2 lg/ml; Molecular Probes, Inc., Eugene, USA) and Alexa Fluor 594 goat anti-rabbit IgG (diluted 1:250; the final concentration, 8 lg/ml; Molecular Probes, Inc.). After being washed with 0.1 M PB, sections were mounted using ProLong Gold Antifade Reagents with DAPI (Life Technologies, Carlsbad, CA, USA). For double immunofluorescence with anti-chordin antibody and mouse Fluoro anti-pan neuronal marker antibody cocktail, sections were treated with 10% normal goat serum, 2% BSA and 0.2% Triton X-100 in 0.1 M PB for 2 h at room temperature, and incubated in rabbit antichordin antibody (diluted 1:10) overnight at 4 °C. After being washed with 0.1 M PB, sections were further incubated in mouse Fluoro anti-pan neuronal marker antibody cocktail (diluted 1:100; the initial concentration is not available; Millipore) and Alexa Fluor 594 goat antirabbit IgG (diluted 1:250) for 1.5 h at room temperature. After being washed with 0.1 M PB, sections were mounted using ProLong Gold Antifade Reagents with DAPI (Life Technologies). Bright-field images were obtained using a microscope (Eclipse 80i; Nikon, Tokyo, Japan) equipped with a CCD camera (DS-Ri; Nikon). Fluorescence images were obtained using a microscope (Eclipse E-600; Nikon) equipped with a CCD camera (DS-Ri). They were further processed in image analysis software (Photoshop; Adobe, Tokyo, Japan).

RESULTS We first investigated whether the antibody (rabbit antichordin) can specifically recognize chordin protein using a pre-absorption test. As shown in Fig. 1, pre-absorption of the antibody with a chordin peptide completely abolished immunostainings in the cerebral cortex (Fig. 1A, B) and cerebellum (Fig. 1C, D), indicating that the antibody specifically recognizes chordin protein. General expression patterns Fig. 2 shows the overview of chordin expressions in the adult rat brain. Chordin-like immunoreactivity (IR) was

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Fig. 1. Specificity of anti-chordin antibody. Photopmicrographs of pre-absorption test; control (A, C), pre-absorbed (B, D). Note that chordin-IR in the cerebral cortex and cerebellum was completely abolished by pre-absorption test. Scale bar = 40 lm.

observed throughout the brain. Abundant chordin-IR was seen in the olfactory bulb (Fig. 2A), basal ganglia (Fig. 2B–D), cerebral cortex (Fig. 2B–G), hippocampus (Fig. 2D–F), thalamus (Fig. 2D–F), hypothalamic regions (Fig. 2C–F), midbrain (Fig. 2G, H), cerebellum (Fig. 2I), brainstem (Fig. 2I, J), and spinal cord (Fig. 2K). The relative intensity of chordin-IR in major regions of the rat CNS is summarized in Table 1. Telencephalon

Olfactory bulb. Chordin-IR was abundantly detected throughout the olfactory bulb, with very strong expression in the glomerular layer and granular layer (Fig. 3A). In the olfactory nerve layer, many astrocytelike cells were stained (arrows in Fig. 3A, C). In the glomerular layer, periglomerular neurons were very strongly stained (white arrows in Fig. 3C) and glomeruli also showed moderate neuropil staining (asterisks in Fig. 3C). In the external plexiform layer, tufted cells were stained strongly (white arrows in Fig. 3B). In addition, strong chordin-IR was observed in apical dendrites of mitral/tufted (arrowheads in Fig. 3B), and moderate neuropil staining was also observed (Fig. 3B). In the mitral cell layer, the cell bodies of mitral cells were strongly labeled (black arrows in Fig. 3B). In the granular layer, granular neurons were very strongly stained (arrows in Fig. 3D), and strong neuropil staining was also observed (Fig. 3D).

Septum and nuclei of the diagonal band of Broca. In the lateral and medial septal nuclei and diagonal band of Broca, many neurons were strongly stained, in addition, weak neuropil staining was also observed (Fig. 3E). Piriform cortex. In the layer I, chordin-IR-positive cells were scattered, in addition moderate neuropil staining was observed (Fig. 3F). In the layers II and III, many pyramidal neurons were intensely stained (Fig. 3F), and their apical dendrites were also intensely stained (arrowheads in Fig. 3G). Bed nucleus of the stria terminalis. Interestingly, very strong chordin-IR-positive cells and very strong neuropil staining were seen in the bed nucleus of the stria terminalis (Fig. 3J). Corpus callosum and fornix. In the corpus callosum and dorsal hippocampal commissure, chordin-IRpositive cells were scattered (Fig. 3H). In addition, neuropil staining was also observed. Interestingly, the intensity of neuropil staining was different between them. The corpus callosum showed relatively stronger chordin-IR than the dorsal hippocampal commissure (Fig. 3H). Closer observation revealed that the chordinIR-positive cells showed astrocyte-like shapes (arrows in Fig. 3I). Interestingly, the intensity of neuropil staining was also different among the subareas in the fornix. The

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Fig. 2. Chordin expression in the whole rat brain. 12, hypoglossal nucleus; Acb, accumbens nucleus; Amy, amygdala; AP, area postrema; cc, corpus callosum; Cer, cerebellum; CG, central gray; Cor, cerebral cortex; CPu, caudate putamen; DC, dorsal cochlear nucleus; DH, dorsal horn; fi, fimbria of the hippocampus; GP, globus pallidus; Hi, hippocampus; IC, inferior colliculus; ic, internal capsule; Int, interposed cerebellar nucleus; IO, inferior olive; LG, lateral geniculate nucleus; LH, lateral hypothalamic area; MG, medial geniculate nucleus; MV, medial vestibular nucleus; OB, olfactory bulb; Pir, piriform cortex; Pn, pontine nucleus; SC, superior colliculus; SN, substantia nigra; Sp5, spinal trigeminal nucleus; Th, thalamus, VH, ventral horn; VMH, ventromedial hypothalamic nucleus. Scale bar = 0.5 mm for A, J, K; 1 mm for B–I.

medial part of the fornix exhibited stronger chordin-IR than the lateral part (white dotted-line in Fig. 3J). Subfornical organ. In the subfornical organ, very strong chordin-IR-positive cells were scattered (Fig. 3K). In addition, strong neuropil staining was also observed (Fig. 3K). Cerebral cortex. Abundant chordin-IR was detected in the layers I–VI of the cerebral cortex (Fig. 4A). Strong chordin-IR-positive cells and moderate neuropil staining were observed throughout the cerebral cortex (Fig. 4B– D). In the layer V, the cell bodies of pyramidal neurons were intensely stained (arrows in Fig. 4D). Interestingly, the apical dendrites of pyramidal neurons were also strongly stained (arrowheads in Fig. 4B–D). Hippocampus. Chordin-IR was observed throughout the hippocampus (Fig. 4E). Pyramidal cells of the Ammon’s horn showed strong chordin-IR (Fig. 4F, G), in

addition, the apical dendrites of pyramidal cells were also strongly stained (arrowheads in Fig. 4G). In the dentate gyrus, strong chordin-IR was detected in granule cells and moderate neuropil staining was observed in the stratum moleculare and polymorphic layer (Fig. 4H, I). The basal ganglia. The olfactory tubercule contained strongly positive neurons (Fig. 5A). Interestingly, the islands of Calleja showed very strong chordin-IR (Fig. 5A). Closer observation showed very-strongly stained neurons and strongly stained neuropils (Fig. 5B). The caudate putamen showed strong chordin-IR (Fig. 5C) and the globus pallidus exhibited moderate chordin-IR (Fig. 5C). In the caudate putamen, strong positive neurons and moderate neuropil staining were observed in the gray matter (Fig. 5D). Closer observation showed that in fiber tracts in the caudate putamen weak chordin-IR was seen (asterisks in Fig. 5D). In the globus pallidus, moderate positive

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Table 1. Distribution and intensity of chordin-IR in the rat CNS Area I. Telencephalon Olafactory bulb Olfactory nerve layer Glomerular layer External plexiform layer Mitral cell layer Granular layer Subependymal layer Cerebral cortex Hippocampal formation Amygdala Central amygdaloid nucleus Lateral amygdaloid nucleus Basolateral amygdaloid nucleus Medial amygdaloid nucleus Bed nucleus of the stria terminalis Basal ganglia Caudate putamen Globus pallidus Ilands of Calleja Corpus callosum Dorsal hippocampal commissure Subfornical organ II. Diencephalon Thalamus Reticular nucleus Medial habenular nucleus Lateral habenular nucleus Ventroposterior nucleus Dorsal lateral geniculate nucleus Ventral lateral geniculate nucleus Other nuclei Hypothalamus Supraoptic nucleus Suprachiasmatic nucleus Medial eminence Arcuate nucleus Ventromedial hypothalamic nucleus Supraoptic decussation Optic tract III. Midbrain Red nucleus Substantia nigra Interpeduncular nucleus

Intensity

++ ++++ +++ +++ ++++ + +++ +++ +++ ++ ++ +++ ++++ +++ ++ ++++ ++ + ++++

+++ ++++ ++ +++ ++ +++ ++ +++ +++ +++ ++ ++ + +++ +++ +++ ++++

Area IV. Pons and medulla Motor system Oculomotor nucleus Trigeminal motor nucleus Facial nucleus Hypoglossal nucleus General somatosensory system Trigeminal mesencephalic nucleus Trigeminal spinal nucleus General visceromotor system Dorsal nucleus of the vagus General viscerosensory system The nucleus of the solitary tract Special somatosensory system Auditory system Dorsal cochlear nucleus Ventral cochlear nucleus Inferior colliculus Superficial part The other parts Vestibular system Medial vestibular nucleus Lateral vestibular nucleus Vestibulocochlear nerve Visual system Superior colliculus Superficial part The other parts Pontine dorsal tegmental nucleus Pontine nucleus Area postrema Locus ceruleus Inferior olive V. Cerebellum Cortex Molecular layer Purkinje cell layer Granule cell layer Cerebellar nuclei VI. Spinal cord Dorsal horm Ventral horn

Intensity

+++ +++ +++ +++ +++ +++ +++ +++

+++ ++ +++ ++ ++ + ++

+++ ++ ++++ +++ +++ ++++ +++

+++ ++++ +++ +++ ++++ +++

Relative intensities were estimated by visual comparison of immunostained slide: +, low; ++, moderate; +++, strong; ++++, very strong.

neurons with stained dendrites and weak neuropil staining were seen in the gray matter (Fig. 5E). Closer observation showed that in fiber tracts in the globus pallidus weak chordin-IR was also seen (asterisks in Fig. 5E). These appearances show that axons in the fibers express chordin proteins. The amygdala. The central amygdaloid nucleus, medial amygdaloid nucleus showed strong chordin-IR (Fig. 5F), while chordin-IR was moderate in the lateral and basolateral amygdaloid nuclei (Fig. 5H). Optic tract and supraoptic decussation. Interestingly, there was a significant difference in chordin expression between two fiber tracts, optic tract and supraoptic decussation (Fig. 5G). Chordin-IR was strongly detected

in the optic tract, while weak in the supraoptic decussation. Diencephalon Chordin-IR-positive neurons and neuropil staining were detected in all nuclei in the thalamus (Figs. 2D–F and 6A). Interestingly, in the medial habenular nucleus, we found very strong chordin-IR-positive neurons and neuropil (Fig. 6B), while chordin-IR was moderate in the lateral habenular nucleus (Fig. 6B). In the reticular thalamic nucleus, we observed strong positive neurons and moderate neuropil staining (Fig. 6A, C). In the ventroposterior thalamic nucleus, neurons showed strong staining, and moderate neuropil staining was also observed (Fig. 6D). In the lateral geniculate nucleus, the

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Fig. 3. Chordin expression in the olfactory bulb (A–D), medial septal nucleus and diagonal band (E), piriform cortex (F, G), corpus callosum (H, I), fornix (J) and subformical organ (K). Note that astrocyte-like cells in the olfactory nerve layer and corpus callosum were intensely stained (black arrows in A, C and I), and that tufted cells in the external plexiform layer (white arrows in B) and the mitral cells (black arrows in B) were intensely stained, and that apical dendrites of mitral/tufted cells were intensely stained (arrowheads in B), and that pyramidal neuron and their apical dendrites were intensely stained (arrowheads in G), and that glomeruli were also moderately stained (asterisks in C), and periglomerular cells were also intensely stained (white arrows in C). I–III, layers I–III of the piriform cortex; BST, bed nucleus of the stria terminalis; cc, corpus callosum; DB, diagonal band; dh, dorsal hippocampal commissure; EPl, external plexiform layer; f, fornix; Gl, glomerular layer; Gr, granular layer; LS, lateral septal nucleus; MS, medial septal nucleus; ON, olfactory nerve layer; Pir, piriform cortex; SE, subependymal layer; SFO, subfornical organ. Scale bar = 400 lm for E; 160 lm for A, H, J, K; 80 lm for F; 40 lm for B–D, G, I.

ventral part showed strong chordin-IR, while the dorsal part exhibited moderate staining for chordin (Fig. 6E). Closer observation showed that strong positive neurons, strong positive line-like structures, and moderate neuropil staining were observed in the ventral part (Fig. 6F).

In the hypothalamic area, chordin-IR-positive neurons and neuropil staining were detected in all nuclei (Fig. 2C– F). The ventromedial hypothalamic nucleus and arcuate nucleus showed moderate chordin-IR (Fig. 6G, H). Strong chordin-IR was observed in the medial

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Fig. 4. Chordin expression in the cerebral cortex (A–D) and hippocampus (E–I). Note that the apical dendrites of pyramidal cells in the cerebral cortex (arrowheads in B–D) and the Ammon’s horn (arrowheads in G) were intensely stained, and that pyramidal neurons in the layer V were also strongly stained (arrows in D). I–VI, layers I–VI of the cerebral cortex; cc, corpus callosum; CA1, field CA1 of Ammon’s horn; DG, dentate gyrus; Gr, granular layer; Mo, stratum moleculare; Or, stratum oriens; Po, polymorphological layer; Py, stratum pyramidale; Ra, stratum radiatum. Scale bar = 400 lm for E; 160 lm for A; 80 lm for F, H; 40 lm for B–D, G, I.

eminence, supraoptic nucleus, and suprachiasmatic nucleus (Fig. 6H–J). Midbrain The interpeduncular nucleus exhibited very strong chordin-IR (Fig. 7A). The central gray showed moderate chordin-IR (Fig. 7A). In the red nucleus, large-sized neurons with many neurites showed strong chordin-IR, while neuropil staining was weak (arrows in Fig. 7B).

The substantia nigra also showed strong staining (Fig. 7A). In the pars compacta of the substantia nigra, strongly-stained neurons and moderate neuropil staining were seen (arrows in Fig. 7C). Pons and medulla Motor system. The nuclei belonging to the general somatomotor and brachiomotor system, such as the

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Fig. 5. Chordin expression in the olfactory tubercule (A, B), basal ganglia (C–E), amygdala (F), optic tract and supraoptic decussation (G). Note that the islands of Calleja were intensely stained (A, B), and that fiber tracts in the caudate putamen and globus pallidus were weakly stained (asterisks in D and E). Amy, amygdala; BL, basolateral amygdaloid nucleus; Ce, central amygdaloid nucleus; CPu, caudate putamen; GP, globus pallidus; ICj, islands of Calleja; La, lateral amygdaloid nucleus; Me, medial amygdaloid nucleus; opt, optic tract; sox, supraoptic commissure; Tu, olfactory tubercule. Scale bar = 400 lm for C, F; 160 lm for A, G; 40 lm for B, D, E.

oculomotor nucleus (Fig. 7D), motor trigeminal nucleus, facial nucleus (Fig. 7E) and hypoglossal nucleus, showed strong chordin-IR in neuronal cell bodies and their dendrites (arrows in Fig. 7D, E). Interestingly, in the facial nerve chordin-IR was observed in axon fibers (arrowheads in Fig. 7F). General somatosensory system. In the mesencephalic trigeminal nucleus, large primary afferent neurons were stained strongly (arrows in Fig. 7G). The spinal trigeminal nucleus (Fig. 7H) was also strongly stained. Interestingly, the spinal trigeminal tract also contained many chordinIR-positive axons (arrowheads in Fig. 7I). General visceromotor and general viscerosensory systems. The dorsal nucleus of the vagus contained

many strong chordin-IR-positive neurons and also showed strong neuropil staining (Fig. 8A, B). In the nucleus of the solitary tract, many strong chordin-IRpositive neurons and strong neuropil staining were observed (Fig. 8A, B). Special somatosensory system. Auditory system: The dorsal cochlear nucleus showed strong chordin-IR in the neuropil and neuronal cell bodies (Fig. 8C, D), while in the ventral cochlear nucleus, although intensely-labeled neurons were scattered, neuropil staining was weak (Fig. 8C, E). In the inferior colliculus, the superficial part showed strong chordin-IR in the neuropil (asterisk in Fig. 8F), in addition, moderately-stained neurons and weak neuropil staining were observed in the other parts (Fig. 8F).

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Fig. 6. Chordin expression in the thalamus (A–E) and hypothalamus (F–J). Arc, arcuate nucleus; DLG, dorsal lateral geniculate nucleus; LHb, lateral habenular nucleus; ME, medial eminence; MHb, medial habenular nucleus; Po, posterior thalamic nucleus; Rt, reticular thalamic nucleus; Sch, suprachiasmatic nucleus; SO, supraoptic nucleus; VLG, ventral lateral geniculate nucleus; VMH, ventromedial hypothalamic nucleus; VP, ventroposterior thalamic nucleus. Scale bar = 400 lm for A; 160 lm for B, E, H; 80 lm for G, I, J; 40 lm for C, D, F.

Vestibular system: In the medial vestibular nucleus, intensely-stained small neurons and moderate neuropil staining were observed (Fig. 8G). In the lateral vestibular nucleus, although large-sized neurons exhibited moderate chordin-IR, neuropil staining was weak (Fig. 8G). Interestingly, vestibulocochlear nerve contained strongly-stained axons (arrowheads in Fig. 8H). Visual system: In the superior colliculus, the superficial part showed strong chordin-IR in neuropil (asterisk in Fig. 8I), in addition, moderately-stained

neurons and moderate neuropil staining were observed in the other parts (Fig. 8I). Other lower brain stem areas. In the pontine dorsal tegmental nucleus, very strong chordin-IR was observed (Fig. 9A). In the pontine nucleus, neurons were strongly stained (arrows in Fig. 9C) and moderate neuropil staining was also observed (Fig. 9B, C). In addition, in the medial cerebellar peduncle, where axons of pontine projecting neurons exist, strong chordin-IR was detected

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Fig. 7. Chordin expression in the midbrain (A–C), motor system (D–F) and general somatosensory system (G–I). Note that neurons in the red nucleus, pars compacta of the substantia nigra, oculomotor nucleus, motor trigeminal nucleus, facial nucleus and mesencephalic trigeminal nucleus were strongly stained (arrows in B–E and G) and that axons in the facial nerve and spinal trigeminal tract showed strong chordin-IR (arrowheads in F and I). 3, occulomotor nucleus; 7, facial nucleus; 7n, facial nerve; CG, central gray; Me5, mesencephalic trigeminal nucleus; R, red nucleus; SN, substantia nigra; sp5, spinal trigeminal tract; Sp5, spinal trigeminal nucleus. Scale bar = 400 lm for A; 40 lm for B–I.

in axons (arrowheads in Fig. 9D). In the area postrema, strong chordin-IR was detected (Fig. 8A). In the locus ceruleus, neurons were very strongly stained and very strong neuropil staining was also seen (arrows in Fig. 9E). In the inferior olive, neurons were strongly stained (arrows in Fig. 9G), and moderate neuropil staining was also observed (Fig. 9F, G). Interestingly, in the inferior cerebellar peduncle, many chordin-IRpositive axons were also observed (arrowheads in Fig. 9H).

Cerebellum In the cerebellum, very strong staining was observed in the Purkinje cell layer. Cell bodies of Purkinje neurons were very strongly stained (Fig. 9I). In addition, small Bergmann glia-like cells also showed very strong chordin-IR. The molecular cell layer contained many strongly-positive neurons, in addition, strong neuropil staining was also observed. Granular cells also exhibited strong chordin-IR (Fig. 9I). In the cerebellar

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Fig. 8. Chordin expression in the general visceral system (A, B) and special somatosensory system (C–I). 8n, vestibulocochlear nerve; 10, dorsal nucleus of the vagus; 12, hypoglossal nucleus; AP, area postrema; DC, dorsal cochlear nucleus; IC, inferior colliculus; LVe, lateral vestibular nucleus; MVe, medial vestibular nucleus; SC, superior colliculus; Sol, nucleus of the solitary tract; VC, ventral cochlear nucleus. Note that the superficial layer of the inferior colliculus and superior colliculus showed strong chordin-IR (asterisks in F and I). Scale bar = 400 lm for I, 160 lm for A, C, F, G; 80 lm for B; 40 lm for D, E, H.

nuclei, strong neural cell body staining and moderate neuropil staining were observed (Fig. 9J). Spinal cord Intense chordin-IR was observed in the gray matter (Fig. 10A). In the dorsal horn, very strong neuropil and neuronal cell body staining was observed in the layers I and II (Fig. 10B). In the ventral horn, large-sized motor

neurons exhibited strong chordin-IR in the somata (arrows in Fig. 10C) and dendrites (arrowheads in Fig. 10C). In the white matter, we observed all fiber tracts contain chordin-IR-positive structures. In the posterior column, the ascending cuneate fasciculus, gracile fasciculus, and the descending pyramidal tract exhibited abundant chordin-IR (Fig. 10D). Closer observation showed that in the cuneate fasciculus and pyramidal tract, besides strongly-stained astrocyte-like

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Fig. 9. Chordin expression in the other lower brain stem areas (A–H) and cerebellum (I, J). Note that the neurons in the pontine nucleus, locus ceruleus and inferior olive were intensely stained (arrows in C, E and G), and that axons of medial cerebellar peduncle and inferior cerebellar peduncle exhibited intense chordin-IR (arrowheads in D and H). CN, cerebellar nuclei; Gr, granular layer; icp, inferior cerebellar peduncle; IO, inferior olive; LC, locus ceruleus; mcp, medial cerebellar peduncle; Mol, molecular layer; PDTg, pontine dorsal tegmental nucleus; Pn, pontine nucleus, Pur, Purkinje cell layer. Scale bar = 160 lm for A, B, F; 80 lm for E; 40 lm for C, D, G–J.

cells (arrows in Fig. 10E, G), many axons express abundant chordin proteins (arrowheads in Fig. 10F, H). Other areas In the SVZ, very strong chordin-IR was observed in ependymal cells, cell bodies and neuropil (asterisks in Fig. 10I). In addition, choroidal plexus also expressed abundant chordin proteins (Fig. 10L).

Double fluorescence immunohistochemistry First, to further confirm that neurons exactly express chordin protein, we performed double fluorescence immunohistochemistry using antibodies that recognize three different neuronal-specific markers (Fig. 11A–I). Fig. 11A–C exhibits that chordin-IR-positive neurons in the cerebral cortex were simultaneously NeuN (neuronal nuclei)-IR positive (arrows in Fig. 11A–C), indicating that

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Fig. 10. Chordin expression in the spinal cord (A–H), subventricular zone (I) and choroidal plexus (J). Note that the cell bodies of motor neurons in the ventral horn (arrows in C) and their dendrites (arrowheads in C) showed intense chordin-IR, and that astrocyte-like cells in the cuneate fasciculus and pyramidal tract (arrows in E and G) were strongly stained, and that axons in the cuneate fasciculus and pyramidal tract also exhibited intense chordin-IR (arrowheads in F and H), and that the subventricular zone also showed intense chordin-IR (asterisks in I). Ch, choroidal plexus; cu, cuneate fasciculus; DH, dorsal horn; gr, gracile fasciculus; py, pyramidal tract; SVZ, subventricular zone, VH, ventral horn. Scale bar = 800 lm for A; 160 lm for D; 40 lm for B, C, E, G, I, J; 16 lm for F, H.

neuronal cell bodies express chordin proteins. In addition, Fig. 11D–F shows that chordin-IR-positive apical dendrites of CA1 pyramidal neurons in the hippocampus were simultaneously MAP2 (microtubule-associated protein-2)-IR positive (arrows in Fig. 11D–F), indicating that dendrites express chordin proteins. Furthermore, to confirm that axons exactly express chordin protein, we performed double fluorescence immunohistochemistry using anti-pan-neuronal marker antibody cocktail in the facial nerve. Fig. 11G–I exhibits that chordin-IR-positive structures were simultaneously pan-neuronal marker-IR positive (arrows in Fig. 11G–I), indicating that axons express chordin proteins. Second, to further confirm that chordin-IR-positive cells in the white matter are astrocytes, we performed double fluorescence immunohistochemistry using antiGFAP (glial fibrillary acidic protein) antibody. Fig. 11J–L shows that chordin-IR-positive cells in the corpus callosum were simultaneously GFAP-IR positive (arrows in Fig. 11J–L), indicating that astrocytes express chordin proteins. We also performed fluorescence immunohistochemistry using anti-oligodendrocytes antibody. However, we could not detect chordin proteins in oligodendrocytes (Fig. 11M–O). Third, to further confirm that chordin-IR small cells around Purkinje neurons in the cerebellum are Bergmann glia, we performed double fluorescence immunohistochemistry using anti-GFAP antibody.

Fig. 12A–D shows that chordin-IR-positive cells around Purkinje cells (asterisk in Fig. 12A–D) were simultaneously GFAP-IR positive (arrows in Fig. 12A– D), indicating that Bergmann glias express chordin proteins. Finally, to further investigate the detailed chordin expression profiles in the SVZ, we performed double fluorescence immunohistochemistry using anti-nestin (a marker for neural stem cells), anti-DCX (doublecortin; a marker for neural progenitor cells), and S100b (a marker for ependymal cells and glial cells). Fig. 12E–G exhibits that chordin-IR-positive cells were simultaneously nestin-IR positive (arrows in Fig. 12E–G), indicating that neural stem cells express chordin proteins. In addition, Fig. 12H–J shows that chordin-IR-positive cells were simultaneously doublecotin-IR positive (arrows in Fig. 12H–J), indicating that neural progenitor cells also express chordin proteins. Furthermore, Fig. 12K–M exhibits that chordin-IR-positive cells around the lateral ventricle were simultaneously S100b-IR positive (arrows in Fig. 12K–M), indicating that ependymal cells also express chordin proteins.

DISCUSSION To date, chordin expression in the adult brain has been investigated in the restricted regions (Pappano et al., 1998; Scott et al., 2000). In the present study, we first

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Fig. 11. Double immunostaining study showing that chordin-IR cells in the cerebral cortex (arrows in A) were also positive for NeuN (arrows in B and C), and that the apical dendrites of CA1 pyramidal neurons in the hippocampus (arrows in D) are also positive for MAP2 (arrows in E and F), and that axons in the facial nerve (arrows in G) were also positive for pan-neuronal marker (arrows in H and I), and that cells in the corpus callosum (arrows in J) were also positive for GFAP (arrows in K and L). However, double immunostaining study shows that chordin-IR-positive cell bodies (white arrows in M and O) and processes (yellow arrow in M and O) in the cerebral cortex were negative for the oligodendroglial marker (white and yellow arrowheads in N and O). 7n, facial nerve; CA1, field CA1 of Ammon’s horn; cc, corpus callosum; Cor, cerebral cortex; Oligo, oligodendroglial marker; pan-NM, pan-neuronal marker. Scale bar = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

show that chordin is widely expressed throughout the adult CNS. In addition, besides abundant chordin expression in neurons, we exhibited chordin expression in astrocytes and ependymal cells. These results

suggest that chordin is abundantly produced by neurons, astrocytes and ependymal cells throughout the adult CNS. Since, BMP ligands, such as BMP2 and 4, have been also reported to be abundantly expressed in

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Fig. 12. Double immunostaining study showing that chordin-IR small cells around Purkinje neurons (asterisk in A–D) in the cerebellum were also positive for GFAP (arrows in A–D), indicating that Bergmann glias express chordin proteins. And double immunostainning study showing that chordin-IR cells in the SVZ (E–M) were also positive for nestin (arrows in E–G), DCX (arrows in H–J) and S100b (arrows in KM), respectively, indicating that neural stem cells, neural progenitor cells and ependymal cells express chordin proteins. Cer, cerebellum; DAPI, 40 , 6-diamidino-2phenylindole (a blue fluorescent nucleic acid stain); SVZ, subventricular zone. Scale bar = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the adult rat brain (Mikawa et al., 2006; Sato et al., 2010), chordin might play pivotal roles as an antagonist regulating BMP signaling in the adult brain.

Is chordin functional in the CNS? Interestingly, Allen brain atlas clearly shows that chordin mRNA is also widely expressed throughout the adult

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mouse CNS, supporting the present data. Since chordin plays its antagonistic role by binding BMPs, and by inhibiting BMP binding to their receptors (Zakin and De Robertis, 2010), if chordin exactly works, BMPs and BMP receptors should be expressed in the CNS. BMP ligands, such as BMP2 and 4, have been reported to be abundantly expressed in the adult rat brain (Mikawa et al., 2006; Sato et al., 2010). In addition, BMPRIA, IB and II are also reported to be widely expressed throughout the rat CNS (Miyagi et al., 2011). Furthermore, in the present study, we found that chordin-IR shows not only cytoplasmic and nuclear localization but also neuropil localization, strongly suggesting that chordin is produced in cells and released into the extracellular space, where chordin and BMPs are thought to make a complex. Finally, the functional involvement of chordin in the adult brain has been reported in the hippocampus (Sun et al., 2007) and in the SVZ (Jablonska et al., 2010). Taken together, we believe that chordin is actually functional in the adult rat CNS. Chordin expressions in dendrites Interestingly, we observed intense chordin protein expressions in apical dendrites of mitral cells in the olfactory bulb, apical dendrites of pyramidal neurons in the cerebral cortex, and hippocampus. In addition, we found many chordin-IR-positive dendrites throughout the CNS. What are the functions of chordin in dendrites? The involvement of BMP signalings in the process of dendritic formation and maturation is well documented (Lein et al., 1995; Le Roux et al., 1999; Withers et al., 2000). Withers et al. (2000) showed that on cultures of hippocampal neurons BMP7 acts as a growth factor selective for dendritic development. Addition of BMP7 to cultured hippocampal neurons results in a rapid and profound acceleration of dendritic growth and enhances synaptogenesis. In addition, enhancement of dendritic growth due to BMP7 has been demonstrated for sympathetic (Lein et al., 1995) and cerebral cortical neurons (Le Roux et al., 1999). In addition, Althini et al. (2004) have recently reported that BMP4 potentiates neurotrophin 3 and neurturin-induced neurite outgrowth of peripheral neurons from the E9 chicken embryo. Furthermore, BMP2 is also reported to promote mitral/ tufted cell dendritic outgrowth (Tran et al., 2008). Taken together, BMP2, 4 and 7 are deeply involved in dendritic growth and synaptogenesis. Interestingly, as chordin is known to show high affinity against BMP2, 4 and 7 (Bragdon et al., 2011), chordin released from dendrites may block BMP signals, resulting in the regulation of dendrite morphology and synaptic homeostasis. Chordin expressions in axons Interestingly, we observed that chordin protein is expressed in axons of many kinds of fiber tracts, with quite different intensities, such as in the fornix, corpus callosum, dorsal hippocampal commissure, optic tract, facial nerve, vestibule-cochlear nerve, medial and inferior cerebellar peduncles, pyramidal tracts, and

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cuneate fasciculus. Our observations revealed, almost all axons are positive for chordin expression, indicating that this phenomenon is common in the adult rat CNS. What are the physiological roles of chordin protein expressed in fiber tracts? Retrograde BMP signaling is a conserved mechanism that directs neuronal terminal differentiation and synaptic efficacy (da Silva and Wang, 2011). For example, in Drosophila, McCabe et al. (2003) have reported that the BMP homolog Glass bottom boat (Gbb) acts as a muscle-derived retrograde signal that promotes synaptic growth and neurotransmitter release. In addition, Eade and Allan (2009) reported that persistent retrograde BMP signaling is important to induce and to subsequently maintain the expression of a stably expressed phenotypic marker in a subset of mature drosophila neurons. In mammalian species, target-derived retrograde BMP signaling has been also reported to regulate trigeminal sensory neuron identities (Hodge et al., 2007), and to determine the number of neurons in the trigeminal ganglion (Guha et al., 2004). This finding strongly suggests that in the mammalian brain, almost all neurons are involved in this kind of target-derived retrograde BMP signal. In such a case, chordin protein may be transported from the cell body to the axon terminal, where chordin is released and regulates BMP signaling. Recently, we reported that BMP 2 and 4, binding proteins to chordin, are abundantly expressed in the adult rat CNS, supporting this theory (Mikawa et al., 2006; Sato et al., 2010). Chordin in the neuropil In the present study, intense chordin expression was observed in the neuropil of the gray matter throughout the adult rat brain. For example, we found intense chordin-IR in the neuropil of the molecular layer of the cerebellum (Fig. 9I), the dorsal cochlear nucleus (Fig. 8C, D), and the superficial layer of the superior colliculus (Fig. 8I). In the molecular layer of the cerebellum, parallel fibers originated from granule cells and climbing fibers originated from the inferior olive nucleus make synapses with the dendrites of Purkinje cells, where highly plastic phenomena, such as longterm depression (LTD) and LTP, are reported (Ito, 2001). The structure of the superficial layer of the dorsal cochlear nucleus resembles that of the molecular layer of the cerebellum. The dorsal cochlear nucleus integrates acoustic information with multimodal sensory inputs from widespread areas of the brain. Multimodal inputs are brought to spiny dendrites of fusiform and cartwheel cells in the molecular layer by parallel fibers through synapses that are subject to LTP and LTD (Fujino and Oertel, 2003). In the superficial layer of the superior colliculus, also similar to the molecular layer of the cerebral cortex, many ascending dendrites of neurons situated in the deeper zones make synapses with marginal and horizontal cells, and this layer has also been reported to possess high plasticity (GiraldiGuimaraes and Mendez-Otero, 2005). These data suggest that chordin in these regions might be involved in keeping plasticity by regulating important phenomena, such as LTP and LTD. Interestingly, Sun et al. (2007)

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have reported presynaptic contributions of chordin to hippocampal plasticity, further supporting this possibility. Chordin in ependymal cells and astrocytes In the present study, we found that ependymal cells express chordin protein. In the adult CNS, Lim et al. (2000) have reported that in the adult SVZ BMP4 expressed in the SVZ astrocytes potently inhibits neurogenesis, and its antagonist noggin secreted from the ependymal cells makes a niche for adult neurogenesis. In the present study, we found chordin is also expressed in the SVZ, suggesting that chordin is also involved in the regulation of neurogenesis. Interestingly, Jablonska et al. (2010) have reported chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination, supporting this possibility. In addition, we found that astrocytes express chordin protein. What are the functions of chordin released from astrocytes? One possibility might be that chordin secreted from astrocytes control local microenvironmental conditions like in the abovementioned SVZ. Another possibility might be that chordin secreted from astrocytes affects astrocytes themselves. For example, BMP4 promotes astrogliogenesis and inhibits oligodendrocyte preprogenitors or precursors from becoming immature oligodendrocytes (Gross et al., 1996; Mabie et al., 1997). In addition, Gomes et al. (2003) have reported that transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment. These data raise the possibility that chordin controls BMP signaling to regulate the functions of astrocytes.

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(Accepted 3 November 2013) (Available online 11 November 2013)