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Vitamin d nuclear binding to neurons of the septal, substriatal and amygdaloid area in the siberian hamster (Phodopus sungorus) brain
0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd IBRO
Neuroscience Vol. 48, No. 4, pp. 841-848, 1992 Printed in Great Britain
VITAMIN D NUCLEAR BINDING TO NEURONS OF THE SEPTAL, SUBSTRIATAL AND AMYGDALOID AREA IN THE SIBERIAN HAMSTER (PHODOPUS SUNGORUS) BRAIN I. M. MusroL,*t W. E. ST~JMPF,*H.-J. BIDMON,*C. HEN,* A. MAYHUIOFBR~: and A. BARTKE$ *Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599-7090, U.S.A. SDepartment of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois, U.S.A. Abstract-Autoradiographic experiments were performed on brains of Siberian hamsters (Phodopus sungorus) injected with tritiated 1,25-dihydroxycholecalciferol. Nuclear labeling was prevented in the presence of excess unlabeled hormone. Strong nuclear concentration of radioactivity was observed in neurons of the nucleus basalis of Meynert, the medial septal nucleus, the nucleus of the diagonal band of Broca and the central amygdaloid group. The latter has been defined as consisting of the central nucleus of the amygdala, its extension into the sublenticular part of the substantia innominata of Reichert, and the lateral division of the bed nucleus of the stria terminalis. All these structures have been reported to be involved in memory and other cognitive processes, and to be affected by age-dependent neurodegenerative disorders such as Alxheimer’s disease. Corresponding localization of 1,25-dihydroxycholecalciferol receptor sites in these select basal forebrain nuclei of the Siberian hamster may implicate vitamin D (soltriol), the steroid hormone of sunlight, in memory processing.
Receptors for vitamin D (1,2%dihydroxycholecalciferol, 1-25-D,, soltriol) have been discovered in the pituitary,36 in the brain,37s40 and in various endocrine organs such as the B-cells of the pancreas,” reticular
cells of the thymus, 38G-cells of the stomach,” adrenal medullaI and others. These findings led to the concept of vitamin D as seasonal regulator of vital functions,4’ extending its traditional role as the calcium homeostatic steroid hormone. The present report focuses on vitamin D localization in select basal forebrain regions of the Siberian hamster, including the medial septal nucleus, the nucleus of the diagonal band of Broca, the bed nucleus of the stria terminalis, the substantia innominata and the nucleus basalis of Meynert.26 These areas largely overlap with the choline@ CH,-CH4 forebrain system of the primate brain,25 and are reported to show atrophic changes in Alzheimer’s disease.& Geriatric and seasonal fluctuations in blood levels of vitamin D and its metabolites have been investigated recent~y.9J2,30J3
The present study provides further evidence about sites of vitamin D action in the central nervous system utilizing a species strongly adapted to seasonal changes, the Siberian hamster Phodopus sungorus.
tTo whom correspondence should be addressed at: Department of Cell Biology and Anatomy, University of North Carolina, 528 Taylor Hall, Chapel Hill, NC 27599-7090, U.S.A.
EXPERIMENTAL PROCEDURES Animals
Fourteen Siberian hamsters (Phodopus sungorus), six months old and weighing 24-40 g, including seven males and seven females, were raised on a normal diet. The animals were kept under long-day conditions (L/D 16: 8, four males and three females) or transferred for two months to short-day conditions (L/D 8 : 16, three males and four females). Short-day cycle causes testis regression and weight loss in males and changes in hair color in both sexes2’ Autoradiography
During the daylight phase, animals received S.C.injections of [‘H]l,25-dihydroxycholecalciferol (1,25-D,, vitamin D, soltriol; DuPont, Boston, MA) with a specific activity of 160Ci/mM, dissolved in 20% ethanol-isotonic saline, via two pulses, each 0.21(g/lOOg body weight, with a l-h interval. For competition control, one male hamster, kept long day, and one female, kept short day, were injected with 1000 x excess of unlabeled 1,25-D, (50% each) 30 min prior to first pulse and at the same time as the lirst pulse of [‘H]l,25-D,. Three hours after the final injection, animals were anesthetized with ether and decapitated. Brains were resected, placed on tissue holders with a minced liver base, and frozen in liquid propane (- 180°C). Sections (4 pm) were then cut in a cryostat (Reichert Jung, Heidelberg, F.R.G.) and thaw-mounted onto nuclear emulsion-coated slides (Kodak NTB 3) according to the method of Stumpf.3s The slides were then exposed for three to 12 months in a light-tight desiccator box at - 15°C. For photographic development the slides were fixed in 4% paraformaldehyde-phosphate-buffered saline (pH 7.0) for 1 min. rinsed in water, developed for 1 min in Kodak developer Dl9, rinsed, fixed in Kodak fixer for 1 min, rinsed, counterstained with methylgreen-pyronin, air-dried and coverslipped with Permount. 841
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The preseptal, septal, hypothalamic and amygdaloid regions of the hamster brain were evaluated. Cells were considered labeled when the silver grain density in the nucleus exceeded the silver grain density in the cytoplasm
and adjacent neuropil by five times, and when neurons of the same nucleus appeared unlabeled in the competition controls. RESULTS
After injection of [3H] 1,25-D,, concentrated radioactivity was found in lumina of blood vessels and in nuclei of cells comprising specific neuronal groups in the basal forebrain of male and female hamsters. No gross qualitative differences in topographic labeling between animals kept long or short day, nor between sexes, were observed in the investigated brain regions. Nuclear labeling was prevented when excess unlabeled hormone was injected prior to and together with labeled hormone (Fig. 3). Examples of autoradiograms are shown in Figs 1-6. The topographic distribution of target neurons is depicted in schematic drawings (Fig. 7a-f) according to Paxinos and Watson2’ in rostrocaudal sequence. Dots represent numbers of labeled cells, and enlarged diameters are proportional to increasing cell numbers (small: l-3 labeled cells; medium: ~5, large < 10). The density of labeled neurons varies among the differnt target neuron populations, and the nuclear concentration of radioactivity varies among neurons of a select nucleus. Cells with high uptake are found adjacent to cells with intermediate or low uptake.
Cells with strong nuclear labeling arc seen !II ihc* medial septal nucleus (Figs la,b. 7a,b) and m the
vertical and horizontal divisions of the diagonal hand nucleus (Figs 2a,b, 7a-e). Numerous strongly labeled cells in the horizontal portion of the diagonal band nucleus can be followed throughout the ventral scptal and preoptic regions (Fig. 7a-e). Scattered labeled cells are found ventral and ventromedial tn the internal capsule, including cells that belong ta the bed nucleus of the stria terminalis (Figs 6a.b. 7h d). More caudally, the number of target neurons for [‘H]1,25-D, is increased, predominantly in the lateral subdivision of the bed nucleus of the stria terminalis. the substantia innominata, the nucleus basalis of Meynert, and the ventral pallidum (Figs 4a,b. 7c-
Our results demonstrate that neurons in the basal forebrain of the Siberian hamster concentrate and retain [3H]1,25-dihydroxycholecalciferol in nuclei of certain neuron groups which are anatomically and functionally related. Since these neurons are
Figs 14.
Autoradiograms of the hamster forebrain after injection of [3H]-1,25-D, showing nuclear concentration of radioactivity in neurons; 4-pm frontal sections, exposure time 11 months; stained with methylgreen-pyronin. Cell groups are depicted topographically with size of dots corresponding to labeled cell numbers. Abbreviations used in the figures
ac aca acp ACo AHA AHP al Arc AVPO B BMA BST BSTD BSTV Ce CPU EP f GP HDB ic ICj LA LH
anterior commissure anterior commissure, anterior anterior commissure, posterior anterior cortical amygdaloid nucleus anterior hypothalamic area, anterior anterior hypothalamic area, posterior ansa lenticularis arcuate hypothalamic nucleus anteroventral preoptic nucleus nucleus basalis of Meynert basomedial amygdaloid nucleus, anterior bed nucleus of stria terminalis bed nucleus of stria terminalis, dorsal bed nucleus of stria terminalis, ventral central amygdaloid nucleus caudate putamen entopeduncular nucleus fomix globus pallidus horizontal nucleus of the diagonal band of Broca internal capsule islands of Calleja lateroanterior hypothalamic nucleus lateral hypothalamic area
LOT MCI’0 MnPO MPO MS 2n opt ; Pe PS SCh SHY SI ZI so
st StHv
Tu . 3v
VDB VMH VP
nucleus of the lateral olfactory tract, layers 1 3 magnocellular preoptic nucleus median preoptic nucleus medial preoptic nucleus medial septal nucleus optic nerve optic tract optic chiasm paraventricular hypothalamic nucleus periventricular hypothalamic nucleus parastrial nucleus suprachiasmatic nucleus septohypothalamic nucleus subatantia innominata of Reichert stria medullaris nucleus stria medullaris supraoptic nucleus stria terminalis striohypothalamic nucleus olfactory tubercle third ventricle vertical nucleus of the diagonal band of Broca ventromedial hypothalamic nucleus ventral pallidum
Vitamin D receptors in hamster forebrain
Fig. 1. Low magnification autoradiogram showing the medial septdl nucleus between fornix subdivisions medial and lateral to the nucleus (a; scale bar = SOpm); its labeled cells in detail (b; scale bar = 20 pm). Fig. 2. Strong radioactive labeling occuts in cells of the horixontal portion of the diagonal band nucleus of Broca (a; scale bar = 600 pm); nuclear labeling of neurons in detail (b; scale bar = 20 ,um). Fig. 3. Neurons of the horizontal diagonal band nucleus of Broca appear unlabeied when a MOO-fold excess of unlabeled hormone is injected prior to radioactive @and. Scale bar = 20 gm.
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Fig. 4. Photomicro~aphic montage demonstratjng labeled magnocelklar neurons of the nucleus basalis of Meynert and the area of the substantia innominata in overview (a; scale bar = SOpm): arrows point out areas shown in detail (b-d, scale bar = 20 iirn).
unlabeled in animals treated with excess 1,25-D, prior to tracer injection, the results indicate specific bighaffinity binding of [3HJ1,25-D, to nuclear acceptor sites, Results from similar autoradiographic studies in rats injected with [‘H]1,25-D, or [3HJ25-hydroxycholecalciferol also indicate that the nuclear radioactivity represents 1,25-dihydroxycholecalciferol.~ Radioactivity found in lumina of blood vessels may also reflect vitamin D bound to serum vitamin D binding protein, as reported by Bouillon er aL8 The peripheral re~lation of calcium homeostasis was generally believed to comprise the full scope of the function of vitamin D, when target neurons for vitamin D were reported in forebrain and hindbrain of rats and mice.37,39s40 Subsequently, acceptor sites of vitamin D were discovered in the spinal cord of rats and mice,42 and in the brains of platyfish, clawed frog, lizard4 and zebra finch.6 SubsequentIy, choroid epithelium and ependyma of hamster brain have been
identified as vitamin D targets6 These data suggest that vitamin D (soltriol) exerts an intkzence on neuroendocrine, autonomic, sensory, somatomotor and mental processes in response to seasonally changing blood levels of 1.25-D, !3 The distribution of 1,25-D, receptor cells in the hamster forebrain corresponds to the dist~but~on of cholinergic neurons in the basal forebrain of humans and rats, Prior morphomet~c studies indicate that murine cholinergic neurons form a continuum within the basal forebrain, referred to as the “cholinergic basal nuclear complex”.‘* It reaches from the medial septal nucleus and the nucleus of the diagonal band to the substantia innominata and the basal nucleus of Meynert. overlapping with the cholinergic basal nuclear complex, the “central amygdaloid group” has been described as the extension of the central amygdaloid nucleus into the sublenticular substantia innomina~~ and the lateral subd~vjsion of the bed
Vitamin D receptors in hamster forebrain
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Fig. 5. Low magnification of the amygdaloid complex (a; bar = 600 pm); strongly labeled neurons of the central amygdaloid nucleus in detail (b; scale bar = 20 pm). Fig. 6. Labeled cells of the lateral subdivision of the bed nucleus of the stria terminalis in overview (a; scale bar = 50 pm); arrow marks area shown in detail (b; scale bar = 20 pm).
nucleus of the stria terminalis.LJ6*20The topography of vitamin D, target neurons in the basal forebrain of the Siberian hamster as shown in this study is comparable with the topography reported in the central nucleus of the amygdala and the lateral bed nucleus of the stria terminalis of rats and mk40 1,25-D, target neurons appear to be much more extensive
in the basal forebrain of the Siberian hamster, additionally involving the nucleus of the diagonal band and the ventral striatal region. Localization of vitamin D receptor sites also corresponds to the CH,-CH4 classification of cholinergic neurons in the basal forebrain of primates.25 Distinct labeling is observed in the vertical (CH,) and
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horizontal (CH,) portions of the nucleus of the diagonal band, and the nucleus basalis of Meynert (CH,). The finding of large longitudinally shaped neurons with vertical alignment, conspicuously labeled with [3H]1.25-D,, beneath and between the fornix in the medioseptal supracommissural cell population indicates a posterior outskirt of the medial septal nucleus (CH,), rather than a part of the median preoptic nucleus. This interpretation is supported by the observation that the parvocellular b supra- and subcomissural divisions of the median preoptic nucleus remain unlabeled or only sporadically exhibit weak single cell labeling. [‘H]1,25-D, nuclear labeling in the basal forebrain of the hamster suggests the existence of an integrated system. Regardless of the classification scheme employed, vitamin D labeling is coincident with the extent of the theorized continuum of the chohnergic neurons. The populations of 1,25-D, target neurons m the investigated regions appear to be interrelated and to constitute a system or circuits. Archicortical fields, including hippocampal formation, cingular and retrosplenial cortex, and paleocortical fields, including olfactory bulb, piriform and entorhinal cortex, are reported to receive inputs from the CH,-CH, area.” The CH, complex is reported to innervate neocortical fields.47 The basal nucleus of Meynert (CH,) has been described as the central site of integration within the d limbic circuits, filtering and processing limbic signals to the neocortex.47 These integration centers are labeled with [3H]1,25-D, in the hamster brain, and partially in the rat brain, including projection sites such as cingulate, piriform and entorhinal cortex.4” The cholinergic forebrain plays a distinct role in memory, and is affected in neurodegenerative disorders, such as Alzheimer’s disease.‘.5.“(.ro.3’The nucleus basalis of Meynert complex (medial septal nucleus, nucleus of the diagonal band and basal nucleus of Meynert) of Alzheimer’s patients shows the typical accumulation of amyloid and neurofibrilary tangles,4 as well as cell shrinkage and diminished cell numbers.2x46 The presence of nuclear 1,25-D, binding in these brain areas suggests an influence of this steroid hormone on memory-processing neurons. Furthermore vitamin D seems to have a supportive influence on long-term memory in rats." In elderly people, 25.hydroxyvitamin D, (25~OHD3) levels are lower than in young persons. in spite of normal blood levels of 1,25-D,. Vitamin D levels vary throughout the annual cycle in all age groups. “*J Although findings are still controversial,” vitamin D metabolism seems to be affected by aging. 1,25-D, may modulate metabolic functions of Fig. 7. Schemata a-f provide outline maps of the basal forebrain based on the atlas of the rat brain by Paxinos and neurons affected by Alzheimer’s disease. The medial Watsonm adapted for the septal substriatal and amygdaloid septal nucleus, the diagonal band nuclei and the region of the Siberian hamster. Dots represent [‘H&1,25-D, nucleus basalis of Meynert show immunoreaction labeled cells, increasing diameters according to increasing numbers of labeled cells (small: l-3 labeled cells; medium: with antisera to choline acetyltransferase and nerve growth factor receptor.i.‘“,49 nerve growth factor <5, large: < 10).
Vitamin D receptors in hamster forebrain
has a stimulating effect on cholinergic neurons of the basal forebrain.r9 nerve growth factor receptor gene expression is decreased in the basal nucleus of Meynert of Alzheimer patients.21 1,25-D, enhances the cellular pool of nerve growth factor receptormRNA in a fibroblast cell culture model,” thus raising the possibility that vitamin D has a modulatory impact on nerve growth factorstimulated processes. These vitamin D target areas in the brain also contain non-cholinergic cell types which exhibit immunoreaction, e.g. for neurotensin,24 vasopressin,‘* galanin’O~lLand 28,000 mol. wt calcium-binding protein.22s23 The nucleus basalis of Meynert shows decreased calcium binding protein-immunoreactivity22.23in patients with Alzheimer’s disease. 1,25-D, treatment results in an increase of choline acetyltrans-
847
ferase activity in the rat bed nucleus of the stria terminalis, whereas calcium binding protein levels were unchanged.” As with other brain regions, in this area, 1,25-D, may be related to a wider range of modulation of neuronal functions beyond the control of calcium homeostasis.‘J9v45 In conclusion, receptor sites for vitamin D (soltriol) in the Siberian hamster are found in those forebrain nuclei which are known to be affectd by neurodegenerative processes such as Alzheimer’s disease. Vitamin D (soltriol) may be involved in the modulation of neuroendocrine-mediated integrative and memory functions. Acknowledgements-We
gratefully appreciate the technical assistance of Mrs L. Li and Mr M. McQuown. This work was supported by US PHS grant NS 09914 fellowship from Merck Sharp & Dohme.
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Neuroscience Vol. 48, No. 4, pp. 841-848, 1992 Printed in Great Britain
VITAMIN D NUCLEAR BINDING TO NEURONS OF THE SEPTAL, SUBSTRIATAL AND AMYGDALOID AREA IN THE SIBERIAN HAMSTER (PHODOPUS SUNGORUS) BRAIN I. M. MusroL,*t W. E. ST~JMPF,*H.-J. BIDMON,*C. HEN,* A. MAYHUIOFBR~: and A. BARTKE$ *Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599-7090, U.S.A. SDepartment of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois, U.S.A. Abstract-Autoradiographic experiments were performed on brains of Siberian hamsters (Phodopus sungorus) injected with tritiated 1,25-dihydroxycholecalciferol. Nuclear labeling was prevented in the presence of excess unlabeled hormone. Strong nuclear concentration of radioactivity was observed in neurons of the nucleus basalis of Meynert, the medial septal nucleus, the nucleus of the diagonal band of Broca and the central amygdaloid group. The latter has been defined as consisting of the central nucleus of the amygdala, its extension into the sublenticular part of the substantia innominata of Reichert, and the lateral division of the bed nucleus of the stria terminalis. All these structures have been reported to be involved in memory and other cognitive processes, and to be affected by age-dependent neurodegenerative disorders such as Alxheimer’s disease. Corresponding localization of 1,25-dihydroxycholecalciferol receptor sites in these select basal forebrain nuclei of the Siberian hamster may implicate vitamin D (soltriol), the steroid hormone of sunlight, in memory processing.
Receptors for vitamin D (1,2%dihydroxycholecalciferol, 1-25-D,, soltriol) have been discovered in the pituitary,36 in the brain,37s40 and in various endocrine organs such as the B-cells of the pancreas,” reticular
cells of the thymus, 38G-cells of the stomach,” adrenal medullaI and others. These findings led to the concept of vitamin D as seasonal regulator of vital functions,4’ extending its traditional role as the calcium homeostatic steroid hormone. The present report focuses on vitamin D localization in select basal forebrain regions of the Siberian hamster, including the medial septal nucleus, the nucleus of the diagonal band of Broca, the bed nucleus of the stria terminalis, the substantia innominata and the nucleus basalis of Meynert.26 These areas largely overlap with the choline@ CH,-CH4 forebrain system of the primate brain,25 and are reported to show atrophic changes in Alzheimer’s disease.& Geriatric and seasonal fluctuations in blood levels of vitamin D and its metabolites have been investigated recent~y.9J2,30J3
The present study provides further evidence about sites of vitamin D action in the central nervous system utilizing a species strongly adapted to seasonal changes, the Siberian hamster Phodopus sungorus.
tTo whom correspondence should be addressed at: Department of Cell Biology and Anatomy, University of North Carolina, 528 Taylor Hall, Chapel Hill, NC 27599-7090, U.S.A.
EXPERIMENTAL PROCEDURES Animals
Fourteen Siberian hamsters (Phodopus sungorus), six months old and weighing 24-40 g, including seven males and seven females, were raised on a normal diet. The animals were kept under long-day conditions (L/D 16: 8, four males and three females) or transferred for two months to short-day conditions (L/D 8 : 16, three males and four females). Short-day cycle causes testis regression and weight loss in males and changes in hair color in both sexes2’ Autoradiography
During the daylight phase, animals received S.C.injections of [‘H]l,25-dihydroxycholecalciferol (1,25-D,, vitamin D, soltriol; DuPont, Boston, MA) with a specific activity of 160Ci/mM, dissolved in 20% ethanol-isotonic saline, via two pulses, each 0.21(g/lOOg body weight, with a l-h interval. For competition control, one male hamster, kept long day, and one female, kept short day, were injected with 1000 x excess of unlabeled 1,25-D, (50% each) 30 min prior to first pulse and at the same time as the lirst pulse of [‘H]l,25-D,. Three hours after the final injection, animals were anesthetized with ether and decapitated. Brains were resected, placed on tissue holders with a minced liver base, and frozen in liquid propane (- 180°C). Sections (4 pm) were then cut in a cryostat (Reichert Jung, Heidelberg, F.R.G.) and thaw-mounted onto nuclear emulsion-coated slides (Kodak NTB 3) according to the method of Stumpf.3s The slides were then exposed for three to 12 months in a light-tight desiccator box at - 15°C. For photographic development the slides were fixed in 4% paraformaldehyde-phosphate-buffered saline (pH 7.0) for 1 min. rinsed in water, developed for 1 min in Kodak developer Dl9, rinsed, fixed in Kodak fixer for 1 min, rinsed, counterstained with methylgreen-pyronin, air-dried and coverslipped with Permount. 841
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The preseptal, septal, hypothalamic and amygdaloid regions of the hamster brain were evaluated. Cells were considered labeled when the silver grain density in the nucleus exceeded the silver grain density in the cytoplasm
and adjacent neuropil by five times, and when neurons of the same nucleus appeared unlabeled in the competition controls. RESULTS
After injection of [3H] 1,25-D,, concentrated radioactivity was found in lumina of blood vessels and in nuclei of cells comprising specific neuronal groups in the basal forebrain of male and female hamsters. No gross qualitative differences in topographic labeling between animals kept long or short day, nor between sexes, were observed in the investigated brain regions. Nuclear labeling was prevented when excess unlabeled hormone was injected prior to and together with labeled hormone (Fig. 3). Examples of autoradiograms are shown in Figs 1-6. The topographic distribution of target neurons is depicted in schematic drawings (Fig. 7a-f) according to Paxinos and Watson2’ in rostrocaudal sequence. Dots represent numbers of labeled cells, and enlarged diameters are proportional to increasing cell numbers (small: l-3 labeled cells; medium: ~5, large < 10). The density of labeled neurons varies among the differnt target neuron populations, and the nuclear concentration of radioactivity varies among neurons of a select nucleus. Cells with high uptake are found adjacent to cells with intermediate or low uptake.
Cells with strong nuclear labeling arc seen !II ihc* medial septal nucleus (Figs la,b. 7a,b) and m the
vertical and horizontal divisions of the diagonal hand nucleus (Figs 2a,b, 7a-e). Numerous strongly labeled cells in the horizontal portion of the diagonal band nucleus can be followed throughout the ventral scptal and preoptic regions (Fig. 7a-e). Scattered labeled cells are found ventral and ventromedial tn the internal capsule, including cells that belong ta the bed nucleus of the stria terminalis (Figs 6a.b. 7h d). More caudally, the number of target neurons for [‘H]1,25-D, is increased, predominantly in the lateral subdivision of the bed nucleus of the stria terminalis. the substantia innominata, the nucleus basalis of Meynert, and the ventral pallidum (Figs 4a,b. 7c-
Our results demonstrate that neurons in the basal forebrain of the Siberian hamster concentrate and retain [3H]1,25-dihydroxycholecalciferol in nuclei of certain neuron groups which are anatomically and functionally related. Since these neurons are
Figs 14.
Autoradiograms of the hamster forebrain after injection of [3H]-1,25-D, showing nuclear concentration of radioactivity in neurons; 4-pm frontal sections, exposure time 11 months; stained with methylgreen-pyronin. Cell groups are depicted topographically with size of dots corresponding to labeled cell numbers. Abbreviations used in the figures
ac aca acp ACo AHA AHP al Arc AVPO B BMA BST BSTD BSTV Ce CPU EP f GP HDB ic ICj LA LH
anterior commissure anterior commissure, anterior anterior commissure, posterior anterior cortical amygdaloid nucleus anterior hypothalamic area, anterior anterior hypothalamic area, posterior ansa lenticularis arcuate hypothalamic nucleus anteroventral preoptic nucleus nucleus basalis of Meynert basomedial amygdaloid nucleus, anterior bed nucleus of stria terminalis bed nucleus of stria terminalis, dorsal bed nucleus of stria terminalis, ventral central amygdaloid nucleus caudate putamen entopeduncular nucleus fomix globus pallidus horizontal nucleus of the diagonal band of Broca internal capsule islands of Calleja lateroanterior hypothalamic nucleus lateral hypothalamic area
LOT MCI’0 MnPO MPO MS 2n opt ; Pe PS SCh SHY SI ZI so
st StHv
Tu . 3v
VDB VMH VP
nucleus of the lateral olfactory tract, layers 1 3 magnocellular preoptic nucleus median preoptic nucleus medial preoptic nucleus medial septal nucleus optic nerve optic tract optic chiasm paraventricular hypothalamic nucleus periventricular hypothalamic nucleus parastrial nucleus suprachiasmatic nucleus septohypothalamic nucleus subatantia innominata of Reichert stria medullaris nucleus stria medullaris supraoptic nucleus stria terminalis striohypothalamic nucleus olfactory tubercle third ventricle vertical nucleus of the diagonal band of Broca ventromedial hypothalamic nucleus ventral pallidum
Vitamin D receptors in hamster forebrain
Fig. 1. Low magnification autoradiogram showing the medial septdl nucleus between fornix subdivisions medial and lateral to the nucleus (a; scale bar = SOpm); its labeled cells in detail (b; scale bar = 20 pm). Fig. 2. Strong radioactive labeling occuts in cells of the horixontal portion of the diagonal band nucleus of Broca (a; scale bar = 600 pm); nuclear labeling of neurons in detail (b; scale bar = 20 ,um). Fig. 3. Neurons of the horizontal diagonal band nucleus of Broca appear unlabeied when a MOO-fold excess of unlabeled hormone is injected prior to radioactive @and. Scale bar = 20 gm.
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Fig. 4. Photomicro~aphic montage demonstratjng labeled magnocelklar neurons of the nucleus basalis of Meynert and the area of the substantia innominata in overview (a; scale bar = SOpm): arrows point out areas shown in detail (b-d, scale bar = 20 iirn).
unlabeled in animals treated with excess 1,25-D, prior to tracer injection, the results indicate specific bighaffinity binding of [3HJ1,25-D, to nuclear acceptor sites, Results from similar autoradiographic studies in rats injected with [‘H]1,25-D, or [3HJ25-hydroxycholecalciferol also indicate that the nuclear radioactivity represents 1,25-dihydroxycholecalciferol.~ Radioactivity found in lumina of blood vessels may also reflect vitamin D bound to serum vitamin D binding protein, as reported by Bouillon er aL8 The peripheral re~lation of calcium homeostasis was generally believed to comprise the full scope of the function of vitamin D, when target neurons for vitamin D were reported in forebrain and hindbrain of rats and mice.37,39s40 Subsequently, acceptor sites of vitamin D were discovered in the spinal cord of rats and mice,42 and in the brains of platyfish, clawed frog, lizard4 and zebra finch.6 SubsequentIy, choroid epithelium and ependyma of hamster brain have been
identified as vitamin D targets6 These data suggest that vitamin D (soltriol) exerts an intkzence on neuroendocrine, autonomic, sensory, somatomotor and mental processes in response to seasonally changing blood levels of 1.25-D, !3 The distribution of 1,25-D, receptor cells in the hamster forebrain corresponds to the dist~but~on of cholinergic neurons in the basal forebrain of humans and rats, Prior morphomet~c studies indicate that murine cholinergic neurons form a continuum within the basal forebrain, referred to as the “cholinergic basal nuclear complex”.‘* It reaches from the medial septal nucleus and the nucleus of the diagonal band to the substantia innominata and the basal nucleus of Meynert. overlapping with the cholinergic basal nuclear complex, the “central amygdaloid group” has been described as the extension of the central amygdaloid nucleus into the sublenticular substantia innomina~~ and the lateral subd~vjsion of the bed
Vitamin D receptors in hamster forebrain
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Fig. 5. Low magnification of the amygdaloid complex (a; bar = 600 pm); strongly labeled neurons of the central amygdaloid nucleus in detail (b; scale bar = 20 pm). Fig. 6. Labeled cells of the lateral subdivision of the bed nucleus of the stria terminalis in overview (a; scale bar = 50 pm); arrow marks area shown in detail (b; scale bar = 20 pm).
nucleus of the stria terminalis.LJ6*20The topography of vitamin D, target neurons in the basal forebrain of the Siberian hamster as shown in this study is comparable with the topography reported in the central nucleus of the amygdala and the lateral bed nucleus of the stria terminalis of rats and mk40 1,25-D, target neurons appear to be much more extensive
in the basal forebrain of the Siberian hamster, additionally involving the nucleus of the diagonal band and the ventral striatal region. Localization of vitamin D receptor sites also corresponds to the CH,-CH4 classification of cholinergic neurons in the basal forebrain of primates.25 Distinct labeling is observed in the vertical (CH,) and
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horizontal (CH,) portions of the nucleus of the diagonal band, and the nucleus basalis of Meynert (CH,). The finding of large longitudinally shaped neurons with vertical alignment, conspicuously labeled with [3H]1.25-D,, beneath and between the fornix in the medioseptal supracommissural cell population indicates a posterior outskirt of the medial septal nucleus (CH,), rather than a part of the median preoptic nucleus. This interpretation is supported by the observation that the parvocellular b supra- and subcomissural divisions of the median preoptic nucleus remain unlabeled or only sporadically exhibit weak single cell labeling. [‘H]1,25-D, nuclear labeling in the basal forebrain of the hamster suggests the existence of an integrated system. Regardless of the classification scheme employed, vitamin D labeling is coincident with the extent of the theorized continuum of the chohnergic neurons. The populations of 1,25-D, target neurons m the investigated regions appear to be interrelated and to constitute a system or circuits. Archicortical fields, including hippocampal formation, cingular and retrosplenial cortex, and paleocortical fields, including olfactory bulb, piriform and entorhinal cortex, are reported to receive inputs from the CH,-CH, area.” The CH, complex is reported to innervate neocortical fields.47 The basal nucleus of Meynert (CH,) has been described as the central site of integration within the d limbic circuits, filtering and processing limbic signals to the neocortex.47 These integration centers are labeled with [3H]1,25-D, in the hamster brain, and partially in the rat brain, including projection sites such as cingulate, piriform and entorhinal cortex.4” The cholinergic forebrain plays a distinct role in memory, and is affected in neurodegenerative disorders, such as Alzheimer’s disease.‘.5.“(.ro.3’The nucleus basalis of Meynert complex (medial septal nucleus, nucleus of the diagonal band and basal nucleus of Meynert) of Alzheimer’s patients shows the typical accumulation of amyloid and neurofibrilary tangles,4 as well as cell shrinkage and diminished cell numbers.2x46 The presence of nuclear 1,25-D, binding in these brain areas suggests an influence of this steroid hormone on memory-processing neurons. Furthermore vitamin D seems to have a supportive influence on long-term memory in rats." In elderly people, 25.hydroxyvitamin D, (25~OHD3) levels are lower than in young persons. in spite of normal blood levels of 1,25-D,. Vitamin D levels vary throughout the annual cycle in all age groups. “*J Although findings are still controversial,” vitamin D metabolism seems to be affected by aging. 1,25-D, may modulate metabolic functions of Fig. 7. Schemata a-f provide outline maps of the basal forebrain based on the atlas of the rat brain by Paxinos and neurons affected by Alzheimer’s disease. The medial Watsonm adapted for the septal substriatal and amygdaloid septal nucleus, the diagonal band nuclei and the region of the Siberian hamster. Dots represent [‘H&1,25-D, nucleus basalis of Meynert show immunoreaction labeled cells, increasing diameters according to increasing numbers of labeled cells (small: l-3 labeled cells; medium: with antisera to choline acetyltransferase and nerve growth factor receptor.i.‘“,49 nerve growth factor <5, large: < 10).
Vitamin D receptors in hamster forebrain
has a stimulating effect on cholinergic neurons of the basal forebrain.r9 nerve growth factor receptor gene expression is decreased in the basal nucleus of Meynert of Alzheimer patients.21 1,25-D, enhances the cellular pool of nerve growth factor receptormRNA in a fibroblast cell culture model,” thus raising the possibility that vitamin D has a modulatory impact on nerve growth factorstimulated processes. These vitamin D target areas in the brain also contain non-cholinergic cell types which exhibit immunoreaction, e.g. for neurotensin,24 vasopressin,‘* galanin’O~lLand 28,000 mol. wt calcium-binding protein.22s23 The nucleus basalis of Meynert shows decreased calcium binding protein-immunoreactivity22.23in patients with Alzheimer’s disease. 1,25-D, treatment results in an increase of choline acetyltrans-
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ferase activity in the rat bed nucleus of the stria terminalis, whereas calcium binding protein levels were unchanged.” As with other brain regions, in this area, 1,25-D, may be related to a wider range of modulation of neuronal functions beyond the control of calcium homeostasis.‘J9v45 In conclusion, receptor sites for vitamin D (soltriol) in the Siberian hamster are found in those forebrain nuclei which are known to be affectd by neurodegenerative processes such as Alzheimer’s disease. Vitamin D (soltriol) may be involved in the modulation of neuroendocrine-mediated integrative and memory functions. Acknowledgements-We
gratefully appreciate the technical assistance of Mrs L. Li and Mr M. McQuown. This work was supported by US PHS grant NS 09914 fellowship from Merck Sharp & Dohme.
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