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
I. M.
842
MIJSWL VI ul.
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-
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
I. M.
842
MIJSWL VI ul.
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-
