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Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord
Journal of Chemical Neuroanatomy 16 (1999) 135 – 145
Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord Kirsten Pru¨fer a, Timothy D. Veenstra b, Gustav F. Jirikowski c,*, Rajiv Kumar b b
a NIH, NIDDK, LCBB, 8 Center Dr. Bldg.8 /Room 422, Bethesda, MD 20892, USA Nephrology Research Unit, Department of Medicine and Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA c Institut fu¨r Anatomie II, Klinikum der FSU, Friedrich-Schiller Uni6ersita¨t Jena, D-07740 Jena, Teichgraben 7, Germany
Received 22 June 1998; received in revised form 6 January 1999; accepted 6 January 1999
Abstract A complete mapping study on the 1,25-dihydroxyvitamin D3 receptor immunoreactivity within the rat central nervous system was performed with a monoclonal and a polyclonal antibody. Specific immunostaining was observed within both nuclear and cytoplasmic compartments of a variety of cells in the cerebellum, mesopontine area, diencephalon, cortex, spinal cord, and limbic system. Both monoclonal and polyclonal antibodies provided similar staining patterns. The monoclonal antibody stained distinct domains within the nuclei of all and the cytoplasm of specific neuronal cell types, like motor neurons, Purkinje cells, and pyramidal cells of the cortex more clearly than the polyclonal antibody. The expression of vitamin D3 receptor in the rat central nervous system was confirmed by in situ hybridisation. The widespread distribution of vitamin D3 receptor in distinct portions of the sensory, motor, and limbic brain systems suggests multiple functional properties of 1,25-dihydroxyvitamin D3 in the central nervous system. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Steroids; Brain; Immunohistochemistry; Calcitriol; VDR
1. Introduction 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to play an important role in calcium homeostasis (Kumar, 1984; DeLuca, 1988; Kumar, 1992). 1,25(OH)2D3 primarily interacts with the intracellular 1,25(OH)2D3 receptor (VDR) to regulate the transcription of several genes (Kumar, 1984; DeLuca, 1988; Kumar, 1992; Hannah and Norman, 1994; Carlberg, 1995). There is increasing evidence that the VDR is present in tissues not normally associated with mineral metabolism (Walters et al., 1992; Walters, 1992; Johnson et al., 1995, 1996a; Pru¨fer et al., 1997), however, the function of 1,25(OH)2D3 in many of these tissues is unclear to date.
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The VDR has been detected in developing dorsal root ganglia in vivo and in dorsal root ganglion cells maintained in culture (Johnson et al., 1996b), as well as in several other areas within the central nervous system of the developing rat (Veenstra et al. 1998), suggesting that 1,25(OH)2D3 plays a so far undetermined role in cellular development within the nervous system. 1,25(OH)2D3 has been shown to increase the expression of nerve growth factor (NGF), a neurotrophic agent which promotes the survival and differentiation of specific neuronal cells (Ayer-Lelievre et al., 1988), in neuroblastoma cells (Veenstra et al., 1997), glial cells (Neveu et al., 1994), fibroblasts (Wion et al. 1991) as well as rat brain in vivo (Saporito et al., 1993). 1,25(OH)2D3 has been shown to affect cholinergic activity in several discrete areas of the rat brain via elevation of choline acetyltransferase activity (Sonnenberg et al., 1986), to alter cerebral dopamine levels
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(Baksi and Hyghes, 1982), to influence expression of hypothalamic serotonin (Privette et al., 1991), and to regulate the expression of the tyrosine hydroxylase gene in mice (Puchacz et al., 1991). Furthermore, various neuronal and behavioral functions have been linked to 1,25(OH)2D3, including central control of reproduction (Stumpf, 1988, 1995). Light-induced production of 1,25(OH)2D3 is hypothesized to be associated with the therapeutic action of light therapy in the treatment of depression (Stumpf and Privette, 1989). It is of interest that treatment of vitamin D-dependent rickets with supplements of 1,25(OH)2D3, calcium, and phosphorous improves motor limb ability and hypotonia before serum ion normalisation (Mize et al., 1988). A topographical assessment of 1,25(OH)2D3 targets in the brain has been performed in various species by in vivo autoradiography using [3H]1,25(OH)2D3 (Stumpf and O’Brien, 1987; Stumpf et al., 1992; Musiol et al., 1992; Bidmon and Stumpf, 1994; Stumpf, 1995; Bidmon and Stumpf, 1996). Autoradiographic studies, however, do not unequivocably show the presence of the VDR, but only accumulations of the radiolabelled ligand. In the present study, we employed immunocytochemistry and in situ hybridisation in order to assess the topographical distribution of VDR immunoreactivity throughout the rat brain and spinal cord.
2. Materials and methods Three male and three female Sprague – Dawley rats, kept under normal conditions with an artificial 12 h day –night rhythm and with free access to food and water, were killed by CO2 inhalation. The animals were immediately perfused with Bouin’s fixative. Brain and spinal cord were postfixed for 24 h in the above fixative and embedded in paraffin. Ten micrometer thick serial sections were stained. After deparaffinisation, immunohistochemistry was performed with a rat anti VDR monoclonal antibody, clone 9A7 (Chemicon) and a polyclonal rabbit antibody to the VDR (antibody 2152). Characterisation and specificity of the polyclonal antibody has been previously described (Kumar et al. 1994). The monoclonal antibody has been well characterized by the manufacturer. Both antibodies have been shown to not crossreact with the estrogen or glucocorticoid receptors. Endogenous peroxidase activity was blocked and sections were placed in 10 mM citrate, pH 6.0, and heated twice for 2 min in a 780 W microwave oven set on high. Sections were treated with 5% normal goat serum in PBS-Tween (phosphate buffered saline, pH 7.4, containing 0.1% Tween 20) followed by incubation with a 1:300 dilution of rat anti VDR antibody (over night at room temperature) or a 1:1000 dilution
of rabbit anti-VDR antibody (60 min), respectively. After several washes with PBS the sections were treated with a 1:200 dilution of biotinylated rabbit anti-rat IgG or goat-anti-rabbit IgG, respectively (Vector, Burlingame, CA) followed by a 1:500 dilution of peroxidase-labeled streptavidin (Dako, Carpintine, CA). Diaminobenzidine and hydrogen peroxide were used for color development. For control purposes, monoclonal antibody that had been preadsorbed with an excess of VDR was used instead of the first antibody, nonimmune serum was used instead of the polyclonal VDR antibody. The atlas by Ko¨nig and Klippel (1967) was used for verification of anatomical structures. [35S]UTP-labeled RNA probes were prepared from a 966bp cDNA encoding the human vitamin D receptor ligand binding domain subcloned into pCRII. [35S]UTP was purchased from NEN Life Sciences Products Boston, MA and Invitrogen, Carlsbad, CA. The VDR cDNA clone, isolated from human intestine, was obtained from Dr J.W. Pike (Ligand Pharmaceuticals, San Diego, CA; Baker et al., 1988). Based on the GenBank sequence accession numbers AF026260 (human) and J04147 (rat) the homology between the hVDR and rat VDR corresponding nucleotide sequences is 87%. After linearizing the plasmid with Hind or XbaI, anti-sense and sense RNA probes were transcribed with T7- and SP6-RNA polymerases, respectively, according to the protocols provided by Promega Corporation (Madison, WI). The probes were hybridized to paraffin embedded rat brain sections which had been deparaffined in xylene and rehydrated through a series of graded ethanol. Probes were hybridized to the sections. Washing conditions after hybridisation were according to the manufacture’s protocols (Novagen SureSite II System, Novagen, Madison, WI). For autoradiography slides were coated with Nucleartrack emulsion (KODAK NTB 3) and exposed at 4°C for 2 weeks. Sections were counterstained with 0.5% (w/v) methyl green pyronene.
3. Results The distribution of VDR like immunoreactivity in the rat brain is shown in Fig. 1. The monoclonal and the polyclonal antibody stained identical areas within the brain. VDR like immunoreactivity was observed in nuclei and cytoplasm of cells with both antibodies. Neuronal and glial cell types were not distinguished. However, with the monoclonal antibody VDR immunoreactivity appeared distinctly compartmentalised within the nuclei. Both nuclear and strong cytoplasmic staining occured preferentially in specific neuronal cell types like Purkinje cells, motor neurons, and pyramidal cells. In all brain areas single fibre bundles showed
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Fig. 1. Distribution of the VDR like immunoreactive neurons in the rat brain. Frontal sections in rostro-caudal sequence, showing the approximate localisation of VDR positive neurons (dots). Abbreviations and outlines of structures, according to the Atlas by Ko¨nig and Klippel.
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Fig. 1. (Continued)
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Fig. 1. (Continued)
VDR like immunoreactivity with both the polyclonal and the monoclonal antibody. In the pontine-midbrain area VDR like immunoreactivity was localised in single neurons within the lateral, medial, and spinal vestibular nucl. (Fig. 1m – s), the periolivary nucl. (oli, old), the cochlear nucl., the cuneate nucl. (cul), the locus coeruleus, the laterall paragigantocellular nucl. (pgl), the nucl. of the solitary tract (sol), the hypoglossal nucl. (XII), the nucl. spinalis nervi trigemini (VS, Vspip), the raphe nucl., the pretectal area (p), the midbrain and pontine periventricular gray (gc). Scattered neurons of the intermediate gray
layer of the superior colliculus (sgm) and the nucl. brachium inferior colliculus were VDR like immunoreactive as well as neurons of the medial (ccgm, mcgm) and lateral geniculate nucll. (dcgl), the magnocellular red nucl. (ru), the substantia nigra (sn), and the nucl. Edinger–Westphal. In the diencephalon (Fig. 1f–p), moderate VDR like staining was seen within the posterior thalamic (tpo), centromedial thalamic (cm), the anterior medial (tam) and ventral (tv), the lateral habenular (lh), the lateral (tl, tlp), the reuniens (re), the paratenial (pt), the parafascicular (pf), paraventricular thalamic nucll. (pvs,
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Fig. 2. (A) Cells of the frontopolar cortex show distinct VDR immunoreactive patterns within the nuclei (arrow. Scalebar= 10 mm); (B) immunocytochemical control of the same area shows unstained nuclei (arrow. Scalebar = 10 mm). (C) VDR like immunoreactivity in the frontopolar cortex, obtained with the monoclonal antibody (Scalebar = 100 mm); (D) in the temporal cortex VDR like immunoreactivity occured in all layers (Bar= 100 mm); (E) hippocampal pyramidal neurons show both stained nuclear epitops (closed arrow) and weak cytoplasmic VDR like immunoreactivity (open arrow. Scalebar = 10 mm); (F) the hippocampus (CA1 and CA3) and dentate gyrus (GD) shows widespread VDR like immunoreactivity (Scalebar = 100 mm).
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Fig. 3.
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pvr), as well as the ventrolateral and the ventromedial (tvm, tvd), the ventroposterior (tvp), lateral, and medial thalamic nucll. (tml, tmm). In the hypothalamus (Fig. 1c – i), the preoptic area (pom), the arcuatic (ar), the suprachiasmatic (sc), the retrochiasmatic, the parvocellular part of the paraventricular, periventricular (hpv), the ventromedial (hvm), the dorsomedial hypothalamic (hd) nucll., and the zona incerta (zi) were moderately stained. In the magnocellular supraoptic (so) and paraventricular nucll. weak cytoplasmic staining was noticeable in few cells. The cortex showed widespread distribution of VDR like immunoreactive neurons. Immunoreactivity for the receptor was intense in the frontopolar (Fig. 2A, C), somatosensory, and motoric areas of the parietal and frontal cortex not confined to specific layers. Neurons of the entorhinal/rhinal and the piriform cortex were VDR immunoreactive as well as the lateral and medial parts of the anterior olfactory nucl. and the olfactory tubercle. VDR like staining was observed within the visual cortex (Fig. 1p, q) as well as the temporal (Fig. 1p, q; Fig. 2D), the orbital and the cingular cortex (Fig. 1m, n). In the striatum, scattered cells were positive for the VDR with the highest density of immunopositive cells in close vicinity of the ventricle. Single neurons of the islands of Calleja were VDR like immunoreactive as well. In the limbic system (Fig. 1a – r) the presence of the VDR was detected within a band from the accumbens nucl. (a), the diagonal band of Broca (td), the bed nucl. of the stria terminalis (st), and the lateral septum (sl) extending into the anterior (AA), central (ac), and cortical amygdala (aco). However, no staining was observed within the basal lateral and intercalated amygdaloid areas. Pyramidal neurons of the hippocampal regions CA1, CA2, CA3, and CA4 were generally VDR positive (HI, Fig. 2E, F), as well as neurons of the gyrus dentatus (GD) and the subiculum (S). Within the cerebellum, Purkinje cells of the IV, V, VI, VIII, and IX vermian areas, as well as the paraflocculus showed both cytoplasmic and nuclear immunostaining. Golgi and granular cells of all cerebellar areas showed VDR like nuclear immunostaining (Fig. 3A, B). In the spinal cord VDR like immunoreactivity was observable as both nuclear and cytoplasmic staining in the anterior horn cells (Fig. 3C). The choroid plexus epithelium contained mostly nuclear VDR staining (Fig. 3D).
Control incubations, either performed with the antibody preabsorbed with an excess of antigen (monoclonal antibody) or with preimmune serum instead of the polyclonal antibody did not provide staining (Fig. 2B). In situ hybridisation performed with the antisense VDR probe showed accumulated hybridisation product in the same locations that contained positive immunostaining (Fig. 3E). Controls, performed with the respective sense probe were devoid of radiolabelling (Fig. 3F).
4. Discussion Few studies exist on the topographical distribution of VDR like immunoreactivity in the rat brain. We found that VDR immunoreactivity is distributed throughout the brain. Two VDR antibodies, a monoclonal antibody and a polyclonal antibody gave similar results. A previous study with RT-PCR on RNA extracts from the cerebellum, spinal cord, thalamus and whole brain showed the presence of VDR encoding transcripts, indicating VDR expression in these areas (Veenstra et al., 1998). The monoclonal antibody stained distinct regions within the nuclei, possibly showing accumulations of the nuclear receptor protein. Although VDR is predominantly a nuclear protein, a significant amount of immunostaining was also found in the cytoplasm of certain cells. This may reflect de novo synthesised receptor protein prior to the transport into the nuclear matrix (Berger et al. 1988; Barsony et al. 1997). Autoradiographic localisation of VDR hybridisation product revealed a similar distribution as observed with VDR immunocytochemistry. However, due to the nature of [35S] autoradiography a single cell resolution of hybridisation product could not be obtained. Brain VDR is known to be expressed on a comparatively low level, resulting in relatively low abundant mRNA. Therefore nonradioactive approaches for in situ hybridisation proved to be unsuccessful in the current study. The VDR is found in the olfactory, visual and auditory sensory systems. The olfactory system shows widespread VDR immunoreactivity extending from the anterior olfactory nucl. to the olfactory tubercle, the septum, the prepiriform cortex, the diagonal band of Broca, the hippocampus and subiculum, the corticobasolateral amygdala and the prefrontal and entorhinal cortex, as well as the thalamic parts, nucll. medialis
Fig. 3. (A) VDR immunoreactivity in the cerebellum is visible in Purkinje cells (P), granular cells (G) and in Golgi cells (arrowhead) (Scalebar = 100 mm); (B) Purkinje cells (P) show both nuclear and cytoplasmic immunostaining. Granular cells (G) show weakly stained nuclei (Scalebar =10 mm); (C) motoneuron in the spinal cord with both scattered nuclear staining (closed arrow) and cytoplasmic (open arrow) VDR immunoreactivity (Scalebar = 10 mm); (D) nuclear VDR like immunoreactivity in epithelial cells of the choroid plexus (arrows. Scalebar= 100 mm). All shown immunostaining was performed with the monoclonal antibody. (E) Autoradiogram after in situ hybridisation with a [35S] UTP-labeled VDR RNA anti-sense probe reveals accumulation of silvergrains in a section of the cerebellar layers. Purkinje cells (P), granular cells (G) (Scalebar=50 mm). The respective sense control (F) is devoid of hybridisation signal.
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thalami and lateralis habenulae. The midbrain periaqueductal gray and the raphe nucl. send projections to the nucl. olfactorius anterior. The diagonal band of Broca, as a projection to the magnocellular forebrain, is the source of cholinergic innervation of the olfactory bulb. The occurrence of VDR like immunoreactivity in this whole system strongly suggests an important influence of 1,25(OH)2D3 on the olfactory system of rats. VDR like immunoreactivity in the temporal (auditory) cortex, the inferior colliculus, the nucl. cochlearis, the nucll. periolivaris and the medial geniculate nucl. suggest that this sensory system is influenced by 1,25(OH)2D3 as well. VDR like immunoreactivity was noted in neurons of the visual system. As shown by Johnson et al. (1995), the inner nuclear and plexiforme layers of the retina and the ganglion cells contain VDR immunoreactivity. VDR staining was present in the lateral geniculate nucleus, which receives inputs from both retinae as part of the network to the primary and secondary visual cortex. The area pretectalis is responsible for the control of eye movement, the parasympathic nucl. Edinger–Westphal regulates the inner eye muscles, the lateral thalamic nuclei receive information from several vision-related structures. All these areas contain scattered VDR like immunopositive neurons as well, suggesting an influence of 1,25(OH)2D3 on the visual system. Our findings further suggest that also the somatosensory system is a target of 1.25(OH)2D3. A large number of VDR like immunoreactive neurons were observed in the somatosensory areas of the parietal and frontal cortex. VDR like immunoreactive neurons were also seen within the ventroposterior thalamic nucl., which is known to act as the relay to the primary somatosensory cortex. The nucl. spinalis nervii trigemini, responsible for the facial nociception, contains VDR like immunoreactive neurons as well as do the hypoglossal nuclei. In contrast to results shown by Clemens et al. (1988), we found VDR like immunoreactivity within a fraction of the Purkinje cells in the vestibular part of the cerebellum. The finding that the VDR like immunoreactivity is located in the nucl. vestibularis, which extends its efferences to this part of the cerebellar Purkinje cells and in the thalamic part of the vestibular system nucl. ventrolateralis, suggests that the vestibular system is also a target of 1,25(OH)2D3. Motor neurons also express the VDR. The pyramidal neurons in the motor areas of the frontal and parietal cortex are nearly all VDR immunoreactive. The striatum, the thalamic nucll. ventrolateralis and ventromedialis, and the anterior horn cells of the spinal cord are known to be parts of the motoric system. Also these neurons show occasionally VDR immunoreactivity.
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Previous observations of widespread distribution of [3H](1,25(OH)2D3 accumulation in the limbic system (Stumpf, 1988; Stumpf and Privette, 1989; Musiol et al., 1992) were confirmed. The precise function of 1,25(OH)2D3 in the brain is unknown so far. We observed VDR immunoreactivity in the prefrontal cortex, in the thalamic midline nucll., the mediodorsal and paraventricular nucll., and the gigantocellular reticular nucleus. These areas are responsible for the integration of inputs from sensoric, motoric and limbic brain systems, suggested is a regulatory influence of 1,25(OH)2D3 on brain functions controlled by these areas. The occurence of VDR in the hypothalamic nuclei suggests that 1,25(OH)2D3 exerts its effects on neuroendocrine brain systems as supported by the observation that cells of the hypophysis are VDR immunoreactive as well (Pru¨fer et al., 1997; Pru¨fer and Jirikowski, 1997). The presence of VDR in the hypothalamic neuroendocrine nuclei would support the hypothesis of Stumpf (1988), Stumpf and Denny, 1989) about the influence of 1,25(OH)2D3 on vegetative functions regulated by hypothalamic systems. VDR in the choroid plexus may be in part responsible for the calcium homeostasis of the cerebrospinal fluid (Walters et al., 1992). Interestingly, the plasma membrane calcium pump which is regulated by 1,25(OH)2D3, is also found in the choroid plexus (Borke et al., 1989; Wasserman et al., 1992). Most previous studies examining 1,25(OH)2D3 binding sites in the brain are based on autoradiographic assessment of [3H]1,25(OH)2D3 incorporation in vivo (Stumpf and O’Brien, 1987). Autoradiography reveals the site at which radiolabeled metabolites accumulate. It does not necessarily visualize the actual receptor protein while immunocytochemistry detects both active and inactive as well as recently synthesized receptor in the cytoplasm. Nonetheless, most of our results are generally in agreement with the findings of Stumpf and O’Brien (1987), concerning the [3H]1,25(OH)2D3 binding sites in the rat brain and of related brain structures examined in other species (Musiol et al., 1992; Stumpf et al., 1992; Bidmon and Stumpf, 1994, 1996). Brain areas which were not found to accumulate [3H]1,25(OH)2D3 like some thalamic nuclei, showed mostly weak VDR like immunoreactivity. The distribution of the VDR like immunostaining within the brain overlaps to a great extent with the known distribution of estradiol- (Cintra et al., 1986) glucocorticoid- (Fuxe et al., 1985), progesterone(Kawata, 1995) and androgen-receptors (Clancy et al., 1992) suggesting a possible functional interaction. It is possible that these receptors regulate limbic, sensory and motory brain actions through their concerted effects. The functions of the VDR in mammalian brain remain to be investigated.
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The present findings of VDR expression in many different brain areas suggest a great functional diversity of 1,25(OH)2D3 in the mammalian central nervous system.
Acknowledgements Supported by NIGH grant DK 25409 to RK, by DFG grant Ji 10/4-2 and by the Thu¨ringer Ministerium fu¨r Wissenschaft, Forschung und Kultur (Project B30196095).
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Veenstra, T.D., Pru¨fer, K., Koenigsberger, C., Brimijoin, S.W., Kumar, R., 1998. Immunolocalisation of the 1,25-dihydroxyvitamin D3 receptor in the central nervous system of the developing rat embryo. Dev. Brain Res. 100. Walters, M.R., Fischette, C.T., Fetzer, C., May, B., Riggle, P.C., Tibaldo-Bongiorno, M., Christakos, S., 1992. Specific 1,25-dihydroxyvitamin D3 binding sites in choroid plexus. Eur. J. Pharmacol. 213, 309 – 311. Walters, M.R., 1992. Newly identified actions of the vitamin D endocrine system. Endocr. Rev. 13, 719 – 764. Wasserman, R.H., Smith, C.A., Brindak, M.E., de Talamoni, N., Fullmer, C.S., Penniston, J.T., Kumar, R., 1992. Vitamin D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine. Gastroenterology 102, 886 – 894. Wion, D., MacGrogan, D., Neveu, I., Jehan, F., Houlgatte, R., Brachet, P., 1991. 1,25-Dihydroxyvitamin D3 is a potent inducer of nerve growth factor synthesis. J. Neurosci. Res. 28, 110–114.
Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord Kirsten Pru¨fer a, Timothy D. Veenstra b, Gustav F. Jirikowski c,*, Rajiv Kumar b b
a NIH, NIDDK, LCBB, 8 Center Dr. Bldg.8 /Room 422, Bethesda, MD 20892, USA Nephrology Research Unit, Department of Medicine and Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA c Institut fu¨r Anatomie II, Klinikum der FSU, Friedrich-Schiller Uni6ersita¨t Jena, D-07740 Jena, Teichgraben 7, Germany
Received 22 June 1998; received in revised form 6 January 1999; accepted 6 January 1999
Abstract A complete mapping study on the 1,25-dihydroxyvitamin D3 receptor immunoreactivity within the rat central nervous system was performed with a monoclonal and a polyclonal antibody. Specific immunostaining was observed within both nuclear and cytoplasmic compartments of a variety of cells in the cerebellum, mesopontine area, diencephalon, cortex, spinal cord, and limbic system. Both monoclonal and polyclonal antibodies provided similar staining patterns. The monoclonal antibody stained distinct domains within the nuclei of all and the cytoplasm of specific neuronal cell types, like motor neurons, Purkinje cells, and pyramidal cells of the cortex more clearly than the polyclonal antibody. The expression of vitamin D3 receptor in the rat central nervous system was confirmed by in situ hybridisation. The widespread distribution of vitamin D3 receptor in distinct portions of the sensory, motor, and limbic brain systems suggests multiple functional properties of 1,25-dihydroxyvitamin D3 in the central nervous system. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Steroids; Brain; Immunohistochemistry; Calcitriol; VDR
1. Introduction 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to play an important role in calcium homeostasis (Kumar, 1984; DeLuca, 1988; Kumar, 1992). 1,25(OH)2D3 primarily interacts with the intracellular 1,25(OH)2D3 receptor (VDR) to regulate the transcription of several genes (Kumar, 1984; DeLuca, 1988; Kumar, 1992; Hannah and Norman, 1994; Carlberg, 1995). There is increasing evidence that the VDR is present in tissues not normally associated with mineral metabolism (Walters et al., 1992; Walters, 1992; Johnson et al., 1995, 1996a; Pru¨fer et al., 1997), however, the function of 1,25(OH)2D3 in many of these tissues is unclear to date.
* Corresponding author. Tel.: +49-3641-938553; fax: +49-3641938552; e-mail: [email protected]
The VDR has been detected in developing dorsal root ganglia in vivo and in dorsal root ganglion cells maintained in culture (Johnson et al., 1996b), as well as in several other areas within the central nervous system of the developing rat (Veenstra et al. 1998), suggesting that 1,25(OH)2D3 plays a so far undetermined role in cellular development within the nervous system. 1,25(OH)2D3 has been shown to increase the expression of nerve growth factor (NGF), a neurotrophic agent which promotes the survival and differentiation of specific neuronal cells (Ayer-Lelievre et al., 1988), in neuroblastoma cells (Veenstra et al., 1997), glial cells (Neveu et al., 1994), fibroblasts (Wion et al. 1991) as well as rat brain in vivo (Saporito et al., 1993). 1,25(OH)2D3 has been shown to affect cholinergic activity in several discrete areas of the rat brain via elevation of choline acetyltransferase activity (Sonnenberg et al., 1986), to alter cerebral dopamine levels
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(Baksi and Hyghes, 1982), to influence expression of hypothalamic serotonin (Privette et al., 1991), and to regulate the expression of the tyrosine hydroxylase gene in mice (Puchacz et al., 1991). Furthermore, various neuronal and behavioral functions have been linked to 1,25(OH)2D3, including central control of reproduction (Stumpf, 1988, 1995). Light-induced production of 1,25(OH)2D3 is hypothesized to be associated with the therapeutic action of light therapy in the treatment of depression (Stumpf and Privette, 1989). It is of interest that treatment of vitamin D-dependent rickets with supplements of 1,25(OH)2D3, calcium, and phosphorous improves motor limb ability and hypotonia before serum ion normalisation (Mize et al., 1988). A topographical assessment of 1,25(OH)2D3 targets in the brain has been performed in various species by in vivo autoradiography using [3H]1,25(OH)2D3 (Stumpf and O’Brien, 1987; Stumpf et al., 1992; Musiol et al., 1992; Bidmon and Stumpf, 1994; Stumpf, 1995; Bidmon and Stumpf, 1996). Autoradiographic studies, however, do not unequivocably show the presence of the VDR, but only accumulations of the radiolabelled ligand. In the present study, we employed immunocytochemistry and in situ hybridisation in order to assess the topographical distribution of VDR immunoreactivity throughout the rat brain and spinal cord.
2. Materials and methods Three male and three female Sprague – Dawley rats, kept under normal conditions with an artificial 12 h day –night rhythm and with free access to food and water, were killed by CO2 inhalation. The animals were immediately perfused with Bouin’s fixative. Brain and spinal cord were postfixed for 24 h in the above fixative and embedded in paraffin. Ten micrometer thick serial sections were stained. After deparaffinisation, immunohistochemistry was performed with a rat anti VDR monoclonal antibody, clone 9A7 (Chemicon) and a polyclonal rabbit antibody to the VDR (antibody 2152). Characterisation and specificity of the polyclonal antibody has been previously described (Kumar et al. 1994). The monoclonal antibody has been well characterized by the manufacturer. Both antibodies have been shown to not crossreact with the estrogen or glucocorticoid receptors. Endogenous peroxidase activity was blocked and sections were placed in 10 mM citrate, pH 6.0, and heated twice for 2 min in a 780 W microwave oven set on high. Sections were treated with 5% normal goat serum in PBS-Tween (phosphate buffered saline, pH 7.4, containing 0.1% Tween 20) followed by incubation with a 1:300 dilution of rat anti VDR antibody (over night at room temperature) or a 1:1000 dilution
of rabbit anti-VDR antibody (60 min), respectively. After several washes with PBS the sections were treated with a 1:200 dilution of biotinylated rabbit anti-rat IgG or goat-anti-rabbit IgG, respectively (Vector, Burlingame, CA) followed by a 1:500 dilution of peroxidase-labeled streptavidin (Dako, Carpintine, CA). Diaminobenzidine and hydrogen peroxide were used for color development. For control purposes, monoclonal antibody that had been preadsorbed with an excess of VDR was used instead of the first antibody, nonimmune serum was used instead of the polyclonal VDR antibody. The atlas by Ko¨nig and Klippel (1967) was used for verification of anatomical structures. [35S]UTP-labeled RNA probes were prepared from a 966bp cDNA encoding the human vitamin D receptor ligand binding domain subcloned into pCRII. [35S]UTP was purchased from NEN Life Sciences Products Boston, MA and Invitrogen, Carlsbad, CA. The VDR cDNA clone, isolated from human intestine, was obtained from Dr J.W. Pike (Ligand Pharmaceuticals, San Diego, CA; Baker et al., 1988). Based on the GenBank sequence accession numbers AF026260 (human) and J04147 (rat) the homology between the hVDR and rat VDR corresponding nucleotide sequences is 87%. After linearizing the plasmid with Hind or XbaI, anti-sense and sense RNA probes were transcribed with T7- and SP6-RNA polymerases, respectively, according to the protocols provided by Promega Corporation (Madison, WI). The probes were hybridized to paraffin embedded rat brain sections which had been deparaffined in xylene and rehydrated through a series of graded ethanol. Probes were hybridized to the sections. Washing conditions after hybridisation were according to the manufacture’s protocols (Novagen SureSite II System, Novagen, Madison, WI). For autoradiography slides were coated with Nucleartrack emulsion (KODAK NTB 3) and exposed at 4°C for 2 weeks. Sections were counterstained with 0.5% (w/v) methyl green pyronene.
3. Results The distribution of VDR like immunoreactivity in the rat brain is shown in Fig. 1. The monoclonal and the polyclonal antibody stained identical areas within the brain. VDR like immunoreactivity was observed in nuclei and cytoplasm of cells with both antibodies. Neuronal and glial cell types were not distinguished. However, with the monoclonal antibody VDR immunoreactivity appeared distinctly compartmentalised within the nuclei. Both nuclear and strong cytoplasmic staining occured preferentially in specific neuronal cell types like Purkinje cells, motor neurons, and pyramidal cells. In all brain areas single fibre bundles showed
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Fig. 1. Distribution of the VDR like immunoreactive neurons in the rat brain. Frontal sections in rostro-caudal sequence, showing the approximate localisation of VDR positive neurons (dots). Abbreviations and outlines of structures, according to the Atlas by Ko¨nig and Klippel.
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Fig. 1. (Continued)
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Fig. 1. (Continued)
VDR like immunoreactivity with both the polyclonal and the monoclonal antibody. In the pontine-midbrain area VDR like immunoreactivity was localised in single neurons within the lateral, medial, and spinal vestibular nucl. (Fig. 1m – s), the periolivary nucl. (oli, old), the cochlear nucl., the cuneate nucl. (cul), the locus coeruleus, the laterall paragigantocellular nucl. (pgl), the nucl. of the solitary tract (sol), the hypoglossal nucl. (XII), the nucl. spinalis nervi trigemini (VS, Vspip), the raphe nucl., the pretectal area (p), the midbrain and pontine periventricular gray (gc). Scattered neurons of the intermediate gray
layer of the superior colliculus (sgm) and the nucl. brachium inferior colliculus were VDR like immunoreactive as well as neurons of the medial (ccgm, mcgm) and lateral geniculate nucll. (dcgl), the magnocellular red nucl. (ru), the substantia nigra (sn), and the nucl. Edinger–Westphal. In the diencephalon (Fig. 1f–p), moderate VDR like staining was seen within the posterior thalamic (tpo), centromedial thalamic (cm), the anterior medial (tam) and ventral (tv), the lateral habenular (lh), the lateral (tl, tlp), the reuniens (re), the paratenial (pt), the parafascicular (pf), paraventricular thalamic nucll. (pvs,
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Fig. 2. (A) Cells of the frontopolar cortex show distinct VDR immunoreactive patterns within the nuclei (arrow. Scalebar= 10 mm); (B) immunocytochemical control of the same area shows unstained nuclei (arrow. Scalebar = 10 mm). (C) VDR like immunoreactivity in the frontopolar cortex, obtained with the monoclonal antibody (Scalebar = 100 mm); (D) in the temporal cortex VDR like immunoreactivity occured in all layers (Bar= 100 mm); (E) hippocampal pyramidal neurons show both stained nuclear epitops (closed arrow) and weak cytoplasmic VDR like immunoreactivity (open arrow. Scalebar = 10 mm); (F) the hippocampus (CA1 and CA3) and dentate gyrus (GD) shows widespread VDR like immunoreactivity (Scalebar = 100 mm).
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Fig. 3.
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pvr), as well as the ventrolateral and the ventromedial (tvm, tvd), the ventroposterior (tvp), lateral, and medial thalamic nucll. (tml, tmm). In the hypothalamus (Fig. 1c – i), the preoptic area (pom), the arcuatic (ar), the suprachiasmatic (sc), the retrochiasmatic, the parvocellular part of the paraventricular, periventricular (hpv), the ventromedial (hvm), the dorsomedial hypothalamic (hd) nucll., and the zona incerta (zi) were moderately stained. In the magnocellular supraoptic (so) and paraventricular nucll. weak cytoplasmic staining was noticeable in few cells. The cortex showed widespread distribution of VDR like immunoreactive neurons. Immunoreactivity for the receptor was intense in the frontopolar (Fig. 2A, C), somatosensory, and motoric areas of the parietal and frontal cortex not confined to specific layers. Neurons of the entorhinal/rhinal and the piriform cortex were VDR immunoreactive as well as the lateral and medial parts of the anterior olfactory nucl. and the olfactory tubercle. VDR like staining was observed within the visual cortex (Fig. 1p, q) as well as the temporal (Fig. 1p, q; Fig. 2D), the orbital and the cingular cortex (Fig. 1m, n). In the striatum, scattered cells were positive for the VDR with the highest density of immunopositive cells in close vicinity of the ventricle. Single neurons of the islands of Calleja were VDR like immunoreactive as well. In the limbic system (Fig. 1a – r) the presence of the VDR was detected within a band from the accumbens nucl. (a), the diagonal band of Broca (td), the bed nucl. of the stria terminalis (st), and the lateral septum (sl) extending into the anterior (AA), central (ac), and cortical amygdala (aco). However, no staining was observed within the basal lateral and intercalated amygdaloid areas. Pyramidal neurons of the hippocampal regions CA1, CA2, CA3, and CA4 were generally VDR positive (HI, Fig. 2E, F), as well as neurons of the gyrus dentatus (GD) and the subiculum (S). Within the cerebellum, Purkinje cells of the IV, V, VI, VIII, and IX vermian areas, as well as the paraflocculus showed both cytoplasmic and nuclear immunostaining. Golgi and granular cells of all cerebellar areas showed VDR like nuclear immunostaining (Fig. 3A, B). In the spinal cord VDR like immunoreactivity was observable as both nuclear and cytoplasmic staining in the anterior horn cells (Fig. 3C). The choroid plexus epithelium contained mostly nuclear VDR staining (Fig. 3D).
Control incubations, either performed with the antibody preabsorbed with an excess of antigen (monoclonal antibody) or with preimmune serum instead of the polyclonal antibody did not provide staining (Fig. 2B). In situ hybridisation performed with the antisense VDR probe showed accumulated hybridisation product in the same locations that contained positive immunostaining (Fig. 3E). Controls, performed with the respective sense probe were devoid of radiolabelling (Fig. 3F).
4. Discussion Few studies exist on the topographical distribution of VDR like immunoreactivity in the rat brain. We found that VDR immunoreactivity is distributed throughout the brain. Two VDR antibodies, a monoclonal antibody and a polyclonal antibody gave similar results. A previous study with RT-PCR on RNA extracts from the cerebellum, spinal cord, thalamus and whole brain showed the presence of VDR encoding transcripts, indicating VDR expression in these areas (Veenstra et al., 1998). The monoclonal antibody stained distinct regions within the nuclei, possibly showing accumulations of the nuclear receptor protein. Although VDR is predominantly a nuclear protein, a significant amount of immunostaining was also found in the cytoplasm of certain cells. This may reflect de novo synthesised receptor protein prior to the transport into the nuclear matrix (Berger et al. 1988; Barsony et al. 1997). Autoradiographic localisation of VDR hybridisation product revealed a similar distribution as observed with VDR immunocytochemistry. However, due to the nature of [35S] autoradiography a single cell resolution of hybridisation product could not be obtained. Brain VDR is known to be expressed on a comparatively low level, resulting in relatively low abundant mRNA. Therefore nonradioactive approaches for in situ hybridisation proved to be unsuccessful in the current study. The VDR is found in the olfactory, visual and auditory sensory systems. The olfactory system shows widespread VDR immunoreactivity extending from the anterior olfactory nucl. to the olfactory tubercle, the septum, the prepiriform cortex, the diagonal band of Broca, the hippocampus and subiculum, the corticobasolateral amygdala and the prefrontal and entorhinal cortex, as well as the thalamic parts, nucll. medialis
Fig. 3. (A) VDR immunoreactivity in the cerebellum is visible in Purkinje cells (P), granular cells (G) and in Golgi cells (arrowhead) (Scalebar = 100 mm); (B) Purkinje cells (P) show both nuclear and cytoplasmic immunostaining. Granular cells (G) show weakly stained nuclei (Scalebar =10 mm); (C) motoneuron in the spinal cord with both scattered nuclear staining (closed arrow) and cytoplasmic (open arrow) VDR immunoreactivity (Scalebar = 10 mm); (D) nuclear VDR like immunoreactivity in epithelial cells of the choroid plexus (arrows. Scalebar= 100 mm). All shown immunostaining was performed with the monoclonal antibody. (E) Autoradiogram after in situ hybridisation with a [35S] UTP-labeled VDR RNA anti-sense probe reveals accumulation of silvergrains in a section of the cerebellar layers. Purkinje cells (P), granular cells (G) (Scalebar=50 mm). The respective sense control (F) is devoid of hybridisation signal.
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thalami and lateralis habenulae. The midbrain periaqueductal gray and the raphe nucl. send projections to the nucl. olfactorius anterior. The diagonal band of Broca, as a projection to the magnocellular forebrain, is the source of cholinergic innervation of the olfactory bulb. The occurrence of VDR like immunoreactivity in this whole system strongly suggests an important influence of 1,25(OH)2D3 on the olfactory system of rats. VDR like immunoreactivity in the temporal (auditory) cortex, the inferior colliculus, the nucl. cochlearis, the nucll. periolivaris and the medial geniculate nucl. suggest that this sensory system is influenced by 1,25(OH)2D3 as well. VDR like immunoreactivity was noted in neurons of the visual system. As shown by Johnson et al. (1995), the inner nuclear and plexiforme layers of the retina and the ganglion cells contain VDR immunoreactivity. VDR staining was present in the lateral geniculate nucleus, which receives inputs from both retinae as part of the network to the primary and secondary visual cortex. The area pretectalis is responsible for the control of eye movement, the parasympathic nucl. Edinger–Westphal regulates the inner eye muscles, the lateral thalamic nuclei receive information from several vision-related structures. All these areas contain scattered VDR like immunopositive neurons as well, suggesting an influence of 1,25(OH)2D3 on the visual system. Our findings further suggest that also the somatosensory system is a target of 1.25(OH)2D3. A large number of VDR like immunoreactive neurons were observed in the somatosensory areas of the parietal and frontal cortex. VDR like immunoreactive neurons were also seen within the ventroposterior thalamic nucl., which is known to act as the relay to the primary somatosensory cortex. The nucl. spinalis nervii trigemini, responsible for the facial nociception, contains VDR like immunoreactive neurons as well as do the hypoglossal nuclei. In contrast to results shown by Clemens et al. (1988), we found VDR like immunoreactivity within a fraction of the Purkinje cells in the vestibular part of the cerebellum. The finding that the VDR like immunoreactivity is located in the nucl. vestibularis, which extends its efferences to this part of the cerebellar Purkinje cells and in the thalamic part of the vestibular system nucl. ventrolateralis, suggests that the vestibular system is also a target of 1,25(OH)2D3. Motor neurons also express the VDR. The pyramidal neurons in the motor areas of the frontal and parietal cortex are nearly all VDR immunoreactive. The striatum, the thalamic nucll. ventrolateralis and ventromedialis, and the anterior horn cells of the spinal cord are known to be parts of the motoric system. Also these neurons show occasionally VDR immunoreactivity.
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Previous observations of widespread distribution of [3H](1,25(OH)2D3 accumulation in the limbic system (Stumpf, 1988; Stumpf and Privette, 1989; Musiol et al., 1992) were confirmed. The precise function of 1,25(OH)2D3 in the brain is unknown so far. We observed VDR immunoreactivity in the prefrontal cortex, in the thalamic midline nucll., the mediodorsal and paraventricular nucll., and the gigantocellular reticular nucleus. These areas are responsible for the integration of inputs from sensoric, motoric and limbic brain systems, suggested is a regulatory influence of 1,25(OH)2D3 on brain functions controlled by these areas. The occurence of VDR in the hypothalamic nuclei suggests that 1,25(OH)2D3 exerts its effects on neuroendocrine brain systems as supported by the observation that cells of the hypophysis are VDR immunoreactive as well (Pru¨fer et al., 1997; Pru¨fer and Jirikowski, 1997). The presence of VDR in the hypothalamic neuroendocrine nuclei would support the hypothesis of Stumpf (1988), Stumpf and Denny, 1989) about the influence of 1,25(OH)2D3 on vegetative functions regulated by hypothalamic systems. VDR in the choroid plexus may be in part responsible for the calcium homeostasis of the cerebrospinal fluid (Walters et al., 1992). Interestingly, the plasma membrane calcium pump which is regulated by 1,25(OH)2D3, is also found in the choroid plexus (Borke et al., 1989; Wasserman et al., 1992). Most previous studies examining 1,25(OH)2D3 binding sites in the brain are based on autoradiographic assessment of [3H]1,25(OH)2D3 incorporation in vivo (Stumpf and O’Brien, 1987). Autoradiography reveals the site at which radiolabeled metabolites accumulate. It does not necessarily visualize the actual receptor protein while immunocytochemistry detects both active and inactive as well as recently synthesized receptor in the cytoplasm. Nonetheless, most of our results are generally in agreement with the findings of Stumpf and O’Brien (1987), concerning the [3H]1,25(OH)2D3 binding sites in the rat brain and of related brain structures examined in other species (Musiol et al., 1992; Stumpf et al., 1992; Bidmon and Stumpf, 1994, 1996). Brain areas which were not found to accumulate [3H]1,25(OH)2D3 like some thalamic nuclei, showed mostly weak VDR like immunoreactivity. The distribution of the VDR like immunostaining within the brain overlaps to a great extent with the known distribution of estradiol- (Cintra et al., 1986) glucocorticoid- (Fuxe et al., 1985), progesterone(Kawata, 1995) and androgen-receptors (Clancy et al., 1992) suggesting a possible functional interaction. It is possible that these receptors regulate limbic, sensory and motory brain actions through their concerted effects. The functions of the VDR in mammalian brain remain to be investigated.
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The present findings of VDR expression in many different brain areas suggest a great functional diversity of 1,25(OH)2D3 in the mammalian central nervous system.
Acknowledgements Supported by NIGH grant DK 25409 to RK, by DFG grant Ji 10/4-2 and by the Thu¨ringer Ministerium fu¨r Wissenschaft, Forschung und Kultur (Project B30196095).
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