Effects of age on messenger RNA expression of glucocorticoid, thyroid hormone, androgen, and estrogen receptors in postmortem human hippocampus

BRAIN RESEARCH ELSEVIER

Brain Research 700 (1995) 245-253

Research report

Effects of age on messenger RNA expression of glucocorticoid, thyroid hormone, androgen, and estrogen receptors in postmortem human hippocampus Hideo Tohgi a, *, Kimiaki Utsugisawa a, Munehisa Yamagata a, Masahiro Yoshimura b a Department of Neurology lwate Medical University Morioka, Morioka, Japan b Department of Neuropathology, Tokyo Metropolitan Medical Examiner's Office, Tokyo, Japan Accepted 11 July 1995

Abstract

We studied messenger RNA (mRNA) expressions of receptors for glucocorticoid (GR), thyroid hormone (TR), androgen (AR), and estrogen (ER) and their changes with age in the hippocampal subregions in postmortem human brain. In situ hybridization was done with biotin-labeled antisense synthetic oligonucleotide probes. About 80% or more of the pyramidal neurons in the hippocampal subregions expressed mRNAs for individual receptors in the brains of subjects younger than 65. The ratio of mRNA-containing neuron density to total neuron density significantly decreased with age for GR in CA1 and CA3, and for AR in CA1. Non-significant trends in the reduction with age in the ratio of ER mRNA-containing neurons in CA1 and the ratio of GR mRNA-containing neurons in the hilus also were found. Age-related reductions in nuclear receptor protein mRNA expression in neurons in the hippocampal subfields may be important in the impairments of cognition, emotion, and responses to acute stress in the aged. Keywords: Hippocampus; Aging; Glucocorticoid receptor; Thyroid hormone receptor; Androgen receptor; Estrogen receptor; In situ hybridization histochemistry

1. Introduction

The hippocampus has a crucial role in learning and memory [51] and in the regulation of vegetative function in the brain [28]. It is one of the brain regions in which the neuropathological changes of Alzheimer's disease (AD) first develop, and are most conspicuous in the advanced stage [3,8]. Potential age-related changes in the number of neurons and their receptors in the hippocampus also may in part be related to the cognitive impairments associated with aging. Of the many neurotransmitters and neuromodulators involved in hippocampal functions, glucocorticoid, thyroid hormone, androgen, and estrogen have important roles. They can penetrate the blood brain barrier and have profound effects on behavior, cognitive functions, and neuroendocrine responses (see for review [10]). These hormones act on ligand-dependent nuclear transcripton factors

* Corresponding author. Fax: (81) (196) 54 9860. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 9 7 1 - X

that alter specific gene expression; therefore, they are members of the nuclear receptor superfamily [5,33]. In situ hybridization histochemistry has shown the wide distribution of messenger RNA (mRNA)-containing neurons in rat brain for the glucocorticoid receptor (GR) [1,2], thyroid hormone receptor (TR) [9], androgen receptor (AR), and estrogen receptor (ER) [49], particularly in the septohippocampal area. These results in general are similar, but not identical, to immunohistochemical findings for GR [12,19], TR [15], and ER [18,44], as well as to the distribution of 3H-labeled androgen-containing neurons for AR [45]. The specific glucocorticoid receptors in the central nervous system (CNS), first demonstrated by McEwen et al. (1969) [37], were classified as classic glucocorticoid receptor (GR, type II), characterized by its affinity for dexamethasone, and the type I receptor with a preference for corticosterone over dexamethasone, which biochemically is similar to renal mineralocorticosteroid receptor (MR) [16,41]. The cis/trans contransfection assay has shown that the MR has a much higher affinity for glucocoticoid than GR, so that MR only partially increases its promoter

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H. Tohgi et aL/Brain Research 700 (1995) 245-253

activity for normal circulating levels of glucocoticoid, whereas GR completes full activation for higher glucocorticoid levels [2,14,16]. Thyroid receptors are encoded by the cellular homologue of the viral erbA oncogene (c-erb A), and multiple c-erbA cDNAs have been identified and classified as ot and /3 subtypes. Alternative splicings of the ot gene gives rise to the functional receptor r T R a l and the non-thyroid hormone-binding isotype r T R a 2 in the rat [34,39]. The /3 gene encodes two functional receptors, rTR/31, and the pituitary specific receptor rTR/32 [25]. The sequence of the intron/exon junctions of the coding region of the human androgen receptor has been determined [35], and the complementary DNA for the human estrogen receptor cloned [20]. GR and MR mRNA expression have been shown in postmortem human hippocampus [46]. To our knowledge, no information is available on TR, AR, and ER mRNA expression in the human brain, and no study has evaluated age-related changes in nuclear receptors in the human hippocampus. Using in situ hibridization, we studied the ratio of the densities of neurons having GR, TR-/3, AR, and ER mRNAs to the total neuron density, as well as their changes with age in postmortem human hippocampus. Previous studies used radiolabeled DNA probes, but we used biotin-labeled oligonucleotide probes because their high sensitivity and specificity allow us to detection of the respective mRNAs without the use of radioactive substances.

2. Materials and methods 2.1. Specimens

Brains were obtained postmortem from patients (12 men and 3 women: 35-90 years) who had no history of brain disease or of psychotropic or hormone medication. The subjects had lived normal healthy lives, except for such chronic diseases as diabetes and hypertension that are common in old age. They died suddenly outside medical institutions and death was not preceded by an acute illness, underwent autopsy to determine the cause of death. Their premortem physical and mental conditions, obtained through family and collateral interviews, were assessed by pathological examination. The autopsy was performed within 12 h after death. The brains were cut coronally at the level of the mammillary body. Blocks from the head of the hippocampus were fixed in 4% paraformaldehyde (in PBS treated with 0.1% diethylpyrocarbonate [DEPC])for 6 h, rapidly frozen at - 4 5 ° C in liquid nitrogen, and stored at - 7 0 ° C . Serial cryostat sections (15 /zm) were cut and mounted on heat-dried silane slides.

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2.2. Oligonucleotide probes

In situ hybridization was done with the antisense probes for GR, TR-/3, AR, and ER (Oncogene Science, Institute, NY, USA). These probes are 40 base single-stranded synthetic oligonucleotides with sequences of the antisense orientation. The sequences of the GR probe are derived from sequences corresponding to the N-terminus of hGR [26]; those of the TR-/3 probe (complementary to both rTR/31 and rTR/3 2 cDNAs) from upstream regions corresponding to the translated sequences of TR-/3 [53]; those of the AR probe from 5'-untranslated and translated sequences within 'exon A' as described by Lubahn et al. (1989) [35]; and those of the ER probe from translated sequences near the N-terminus of the ER [20]. The probes were biotin-labeled at the 3' end. 2.3. In situ hybridization

Sections were dehydrated in increasing concentrations of ethanol at 4°C then dried, after which they were treated with prewarmed 40 ~ g / m l proteinase K in PBS for 5 min at 37°C, and washed with PBS. They next were immersed in 4% paraformaldehyde in PBS, washed in PBS, again dehydrated in increasing concentrations of ethanol, then dried. Hybridization was done with buffer containing 50% formamide, 10% dextran sulfate, 4 X SSC, 2 X Denhardt's solution, and 400 / z / m l salmon sperm DNA. The antisense probes at final concentrations of 0.5 /xg/ml were heated at 98°C for 10 min then quickly cooled on ice. The hybridization mixture (50 /.d/slide) and 200 mM of vanadyl ribonucleoside complex (5 /~l/slide) were pipetted onto the sections, and the sections hybridized at 50°C for 12 h. The slides then were washed in 0.2 x SSC (3 m i n x 3 at room temperature, and 30 min X 2 at 50°C) and treated with streptoavidin-alkaline phosphatase conjugate. In situ signals were developed in N B T / B C I P solution. Neurons that were distinctly more intensely colored than the backgrounds were considered to have an mRNA for a specific probe (Fig. la for GR). Nuclei were stained with methyl green, and the immediately adjacent sections stained with hematoxylin and eosin (HE). 2.4. In situ hybridization controls

To ensure that alkaline phosphatase oligonucleotide was not binding nonspecifically to tissue sections or slides, sections were pretreated with ribonuclease (RNase; 40 /x/ml, Pharmacia Biotech) for 30 min. To compete out the labelled nucleotides non-labelled oligonucleotides were used. These slides were then processed as described in the

Fig. 1. Expression of glucocorticoid mRNA in CA2 (a). No specific hybridization was observed after pretreatment with RNase (b) or non-labelled oligonucleotide (c).

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in situ hybridization protocol. No specific hybridization was observed in the slides following the pretreatment with either RNase (Fig. lb for GR) or non-labelled oligonucleotides (Fig. lc for GR). 2.5. Neuron numbers

Table 1 Densities (neuron number/mm 2) of total neurons and neurons expressing glucocoticoid receptor (GR), thyroid hormone /3 receptor (TR-/3), androgen receptor (AR), and estrogen receptor (ER) mRNAs. The ratios of neurons expressing GR, TR-/3, AR and ER mRNAs to the total neuron density (%) are shown in parentheses, n = 6 for < 65 years; n - 9 for > = 65 years. CA1

CA1, CA2, CA3, and the hilus regions of the hippocampus were photographed at × 100 magnification. The density of the neurons (number/mm 2) containing individual mRNAs (i.e. GR) and that of the total numbers of neurons as cut through the nucleus in the adjacent HE sections (T) were counted, and the ratio ( G R / T × 100%) calculated.

CA2

CA3

Hilus

159+33 145_+22

169+39 148_+26

49+5 50+6

157+36 (93_+5%) 126_+22 (87_+6%)

40_+8 (85+5%)% 41_+9 (80+6%) *%

138_+33 (89 _+ 10%) 130_+25 (88_+11%)

37_+4 (78 _+8%) % 40+7 (81_+6%)

154_+25 (94_+5%) 135_+24 (92_+4%)

38_+4 (80_+6%)% 37_+10 (77_+6%)%

150_+34 (91 _+5%) 134_+27 (90 _+6%)

43_+4 (88 _+9%) 41_+11 (81 + 13%)

Total neurons

< 65 >65

154-t-33 136+19

GR mRNA-containing neurons

< 65 >_65

136+34 (88+9%) 92+25 (70_+11%) *%

148+34 (92+3%) 125_+22 (89_+7%)

TR mRNA-containing neurons

2.6. Statistics

Intergroup differences in means were evaluated with paired or unpaired t-tests. Correlations between age and histological parameters were tested with product-moment correlation coefficients. P < 0.05 was considered significant, and 0.05 < P < 0.10 a non-significant trend.

<65 >65

107_+24 (78 + 9%) 102_+32 (83+10%)

137_+28 (85 ± 7%) 126+18 (87+5%)

AR mRNA-containing neurons

<65 >_65

127_+22 (83_+7%) 98_+21 (72_+11%) * t

143_+20 (91_+9%) 130_+24 (90_+7%)

ER mRNA-containing neurons

<65

3. Results >_65

Table 1 shows the total neuron density, the GR, TR-/3, AR, and ER mRNA-containing neuron densities, and their ratios to the total neuron density in the hippocampal subfields for subjects < 65 and those > 65 years old. Total neuron density in the CA1, CA2, and CA3 regions was slightly decreased in the age group > 65 years compared with the age group < 65 years, but the differences were not significant. Fig. 2 compares representative in situ hybridization histologies for the receptor mRNAs of a young adult and an elderly person. In brains from subjects < 65 years, the GR, AR, TR-/3, and ER mRNAs were expressed in about 80% or more of the pyramidal neurons in all the hippocampal subregions studied (Fig. 2, Table 1); therefore, a large proportion of neurons coexpressed these receptor mRNAs. The ratios of neurons expressing GR, TR, and AR mRNAs were significantly lower in the hilus than in CA2 and CA3 in subjects < 65 years (Table 1). In subjects > 65 years old, the GR and AR mRNA-expressing neuron ratios were significantly lower in CA1 and the hilus than in CA2. The difference in the means for the younger and older age groups was significant only for the rate of GR and AR mRNA-containing neurons in CA1 (Table 1). In part this was due to moderate reductions with

137_+36 (89 _+ 13%) 109_+27 (87 _+9%)

144-+30 (92 _+3%) 127_+21 (91 + 4%)

* P < 0.05 compared with subjects < 65 years "~P < 0.05 compared with CA2 and CA3

increasing age in the ratios of some receptor mRNA-containing neurons in some subregions in subjects < 65 years. We then calculated the correlation coefficients for age and the total neuron density and the ratios of neurons expressing individual receptor mRNAs in the hippocampal subregions. The ratio of neurons that express GR mRNA was significantly decreased in CA1 (Fig. 3a) and CA3 (Fig. 3b), and a nonsignificant trend to decrease in the hilus with advancing age was seen (Fig. 3c). The ratio of neurons expressing AR mRNA was significantly decreased with age only in CA1 (Fig. 3d), and the ratio of ER mRNA-containing neurons also showed a nonsignificant trend to decrease with age only in CA1 (Fig. 2e). The ratio of neurons having GR, AR, and ER mRNAs did not remarkably decrease in the group < 65 years, but there was considerable individual variation in the > 65 year old group, resulting in significant decreases with age (Fig. 3a to e). The total neuron density and the ratio of TR-fl mRNA-containing neurons were not significantly corre-

Fig. 2. Representative in situ hybridization histologies showing glucocorticoid receptor (GR) (a,b), thyroid hormone receptor /3 (TR-/3) (c,d), androgen receptor (AR) (e,f), and estrogen receptor (ER) (g,h) mRNA-containing neurons in the CA1 subfield. Left column (a,c,e, and g), a young adult; right column (b,d,f, and h) an elderly person. The GR, AR, and ER mRNA-containing neurons, but not the TR-fl mRNA-containing neurons, are decreased in the elderly person as compared to the young adult. × 100 (bar: 100 /zm)

H. Tohgi et a l . / Brain Research 700 (1995) 245-253

c

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11, Tohgi et al. / Brain Research 700 (1995) 245-253

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r = I 0152 ( p < 0 05)

8O!o

,o 50 60 30 40

4"0 50 6"0 7"0 8"0 90 160

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GR-hilus

;o,o80 ,o 90

years

,o ] 0

AR-CA1

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r=-0.49

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(p =0.09)

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Fig. 3. Ratios o f neurons c o n t a i n i n g glucocorticoid receptor (GR), androg e n receptor ( A R ) a n d estrogen receptor (ER) m R N A s to the total neurons in the h i p p o c a m p a l subfields plotted as a function of age (years).

Regression lines are shown for significant correlations (P < 0.05).

lated with age in any hippocampal subfield (data not shown).

4. Discussion Previous reports on changes in hippocampal neuron number and density during normal aging have not always been consistent, but the overall findings show that neurons decrease with age and the ratio of neuron loss is considered very similar in all the subfields (see for review [13]). In one stereological study, however, the neuron number was significantly reduced with age only in the hilus [55], whereas it was significantly decreased in CA1 in AD brains [54]. Although our purpose was not the study of change with age in the total neuron number, we found a slight (8-12%) nonsignificant decrease in the total neuron density in the CA1, CA2, and CA3 regions, but none in the hilus. These results cannot, however, be directly compared with previous findings in which the neuron number in the entire volume of the subregions was studied because we evaluated only two-dimensional neuron density in particular sections as a reference for obtaining the ratio of neurons expressing hormone receptor mRNAs. We used biotin-labeled probes and classified neurons as those expressing or not expressing mRNAs for the individ-

ual receptors studied. The neurons considered as not expressing an mRNA may, however, contain small amount of that mRNA, and there may be wide variation in the amounts of mRNA present in neurons considered to express an mRNA. The percent of neurons expressing an mRNA, therefore, is by no means the actual percent of neurons having or not having the mRNA, rather it is an indicator of the percent of neurons having an mRNA of an amount higher than a certain level. Our study has shown that GR, TR-/3, AR, and ER mRNAs are expressed in 80% or more of human hippocampal neurons, indicative of the importance of nuclear receptor proteins in the normal functioning of these neurons. With advancing age, the percent of GR mRNA-containing neurons decreased in CA1, CA3, and the hilus, the percents of AR and ER mRNA-expressing neurons also decreasing in CA1. Therefore, age-related reductions were most conspicuous in CA1 in hippocampal subregions and involved all the steroid receptor mRNAs studied. This may reflect the fact that the CA1 region is particularly vulnerable to various noxious conditions including epilepsy [50], ischemia, and glutamate cytotoxicity [6]. Our study, however, did not determine whether GR, AR, and ER mRNAcontaining neurons are selectively susceptible to aging, or whether the receptor mRNA contents decrease in individual neurons. The selective involvement of CA1 neurons relative to the other subregions is important in relation to senescent intellectual decline because persistent amnesia has been reported in a patient with bilateral lesions confined to CA1 [56]. GR was reduced in the hippocampus in old rats in comparison to in younger rats [42], but the reduction was, unlike our findings in human brains, more pronounced in CA3 than in CA1, suggesting a species difference in selective vulnerability to aging within the hippocampal subfields. Whereas glucocorticoid type 1 receptors (MR) show diurnal rhythm and regulate circadian rhythmicity, type 2 receptors (GR) respond only at high hormone levels and regulate stress responses. The time of day at death, therefore, may not substantially influence GR mRNA expression. The reduction in neuronal GR mRNA expression may be due to the aging of neurons, but such chronic stresses as glucose intolerance and elevated blood pressure, which are frequent in old age, also may down-regulate the GR mRNA. GR reduction may impair negative feedback regulation of ACTH secretion [36], thereby increasing the circulating glucocorticoid levels in the aftermath of acute stress and resulting in increases in the damping effects of glucocorticoid on such biological defense responses as the inflammatory response, central and peripheral catecholamine release, and norepinephrine-evoked excitability in the hippocampus [30]. If, however, elevated glucocoticoid levels are prolonged, they may accelerate neuronal aging and degeneration. This has particular implications for the elderly because they respond to various stresses with greater and more prolonged secretion of cortisol as

H. Tohgi et al. / Brain Research 700 (1995) 245-253

compared to younger subjects, although their basal 24-h urinary secretion of glucocorticoid metabolites generally is reduced [21]. Previous experimental studies have shown adverse effects of glucocorticoid on neuron survival. Glucocorticoid induces neuronal loss similar to that associated with aging [43]; conversely, pharmacological intervention to decrease life-long glucocorticoid exposure retards senescent neuronal loss [32,38]. It impairs the ability of neurons to survive such metabolic insults such as hypoxia-ischemia [31], excitotoxins, antimetabolites, and oxygen radical generators. It also impairs the ability of cultured hyppocampal neurons to survive the excitotoxin kainic acid [40], which ability seems to be mediated by the GR (type 2), not the MR (type 1). Moreover, behaviorally adrenalectomized rats performed better than aged controls on a maze reversal task, resembling young animals rather than aged controls in their morphological variables for brain aging [32]. Glucocorticoid-induced cell loss may occur in the aged under repeated or prolonged exposure to stresses, in whom GR is reduced but not completely lost. It also is possible that the reduction in GR may impair glucocorticoid-dependent modulation of neurotransmitter receptors because corticosterone decreases [3H]5-HT binding in rat CA1 [7]. Thyroid disease, both hyperthyroidism and hypothyroidism, is a major cause of metabolic dementia in old people [17]. The reduction in blood tri-iodothyronine (T3) levels in the elderly, however, is slight and within the normal range unless there is systemic disease [21,24]. The lack of age-relate.d changes in the percent of TR-fl mRNA-containing neurons in the hippocampus suggests that the susceptibilit3/ of the senescent brains of aged persons to thyroid dysfunctions is not explained by receptor alterations, at least not in the hippocampus. Although the circulating levels of androgen and estrogen differ for men and women, we could not study gender differences in the percentages of AR and ER mRNA-containing neurons because of the small number of women. A previous study found no gender differences in the overall distributions of AR or ER mRNA-containing neurons in the rat [49]. The possibility that more detailed quantitative studies on larger numbers of subjects would reveal a slight gender difference, however, can not be ruled out. The reductions in AR and ER mRNA-containing neurons in CA1 in the elderly, in conjunction with postmenopausal reductions in estrogen and androgen levels in women [52] and a slight reduction in androgen levels in men [22,23], may have not only neuroendocrinological consequences, but also a role in cognitive decline with age. Testosterone and visu0spatial ability is related in young men [11], and testosterone treatment enhances performance by elderly men on the Block Design subtest of the Wechsler Adult Intelligence Scales (WAIS) [29]. Estrogen acts as a growth factor for CNS neurons [27]. Estrogen-androgen administration improves memory function in surgically induced menopausal women [47,48], but estrogen replacement ther-

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apy does not in postmenopausal old women (65-95 years) [4]. The reduction in AR and ER mRNA-containing neurons in CA1 may limit the potential effects of androgen/estrogen replacement treatment aimed at improving cognitive functions in old people. In conclusion, in situ hybridization using biotin-labeled oligonucleotide probes has shown that about 80% or more neurons in the hippocampal subfields have mRNAs for GR, TR-[3, AR, and ER. The ratio of mRNA-containing neuron density to total neuron density decreased significantly with age for GR in CA1 and CA3, and for AR in CA1, and showed nonsignificant trends to decrease with age for GR in the hilus and for ER in CA1. These age-related reductions in nuclear receptor proteins in part may be related to cognitive and emotional dysfunctions and impaired responses to stresses in the elderly.

References [1] Aronsson, M.,o Fuxe, K., Dong, Y., Agnati, L.F., Okret, S. and Gustafsson, J-A., Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization, Proc. Natl. Acad. Sei., 85 (1988) 9331-9335. [2] Arriza, J.L. and Simerly, R.B., Swanson, L.W. and Evans, R.M., The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response, Neuron, 1 (1987) 887-900. [3] Ball, M.J., Topographic distribution of neurofibrillary tangles and granulovacuolar degeneration in hippocampal cortex of ageing and demented patients. A quantitative study, Interdiscipl. Topics Gerontol., 25 (1988) 16-37. [4] Barrett-Connor, E. and Kritz-Silverstein, D., Estrogen replacement therapy and cognitive function in older women, J. Am. Med. Assoc., 269 (1993) 2637-2641. [5] Beato, M., Gene regulation by steroid hormones, Cell, 56 (1989) 335-344. [6] Benveniste, H., Drejer, J., Shousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. [7] Biegon, A., Rainbow, T.C. and McEwen, B.S., Corticosterone modulation of neurotransmitter receptors in rat hippocampus: a quantitative autoradiographic study, Brain Res., 332 (1985) 309-314. [8] Braak, H. and Braak, E., Neuropathological stageing of Alzheimerrelated changes, Acta. Neuropathol., 82 (1991) 239-259. [9] Bradley, D.J., Young III, W.S. and Weinberger, C., Differential expression of a and /3 thyroid hormone receptor genes in rat brain and pituitary, Proc. Natl. Acad. Sci., 86 (1989) 7250-7254. [10] Brown, R., An Introduction to Neuroendocrinology, Cambridge University Press, Cambridge, 1994, pp. 147-190. [11] Christiansen, K. and Knussmann, R., Sex hormones and cognitive functioning in Men, Neuropsychobiology, 18 (1987) 27-36. [12] Cintra, A., Fuxe, K., H~irfstrand, A., Agnati, L.F., WikstrSm, A-C., Okret, S., Vale, W. and Gustafsson, J-,~., Presence of glucocorticoid receptor immunoreactivity in corticotrophin-releasing factor and in growth hormone-releasing factor immunoreactive neurons of the rat di- and telencephalon, Neurosci. Lett., 77 (1987) 25-30. [I3] Coleman, P.D. and Flood, D.G., Neuron numbers and dendritic extent in normal aging and Alzheimer's disease, Neurobiol. Aging, 8 (1987) 521-545. [14] de Kloet, E.R., Ratka, A., Reul, J.M.H.M., Sutanto, W. and vanEekelen, J.A.M., Corticosteroid receptor types in brain: regulation and putative function, Ann. NYAcad. Sci., 512 (1987) 351-361.

252

H. Tohgi et al. / Brain Research 700 (1995) 245-253

[15] Dratman, M.B., Crutchfield, F.L., Futaesaku, Y., Goldberger, M.E. and Murray, M., [125I] triiodothyronine in the rat brain: evidence for neural localization and axonal transport derived from thaw-mount film autoradiography, J. Comp. Neurol., 260 (1987) 392-408 [16] Evans, R.M. and Arriza, J.L., A molecular framework for the actions of glucocorticoid hormones in the nervous system, Neuron, 2 (1989) 1105-1112. [17] Feldmann, E. and Plum, F.P., Metabolic dementia. In P.J. Whitehouse (Ed.), Dementia, F.A. Davis, Philadelphia, 1993, pp. 307-336. [18] Fuxe, K., Cintra, A., H~irfstrand, A., Agnati, L.F., Kalia, M., Zoli, M., Wikstr6m, A-C., Okret, S., Aronsson, M. and Gustafsson, J-,~., Central glucocorticoid receptor immunoreactive neurons: new insights into the endocrine regulation of the brain, Ann. NYAcad. Sci., 512 (1987) 362-393. [19] Fuxe, K., Wikstr~3m, A-C., Okret, S., Agnati, L.F., H~irfstrand, A., Yu, Z-Y., Granholm, L., Zoli, M., Vale, W. and Gustafsson, J-A., Mapping of glucocorticoid receptor immunoreative neuro~ts in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor, Endocrinology, 117 (1985) 1803-1812. [20] Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J-M., Argos, PI and Chambon, P., Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A, Nature, 320 (1986) 134-139. [21] Harman, S.M. and Blackman, M.R., The hypothalamic-pituitary axes. In J.G. Evans and T.F. Williams (Eds.), Oxford Textbook of Geriatric Medicine, Oxford University Press, Oxford, 1992, pp. 159-166. [22] Harman, S.M. and Tsitouras, P.D., Reproductive hormones in aging men. I. Measurement of sex steroids, basal luteinizing hormone, and Leydig cell response to human chorionic gonadotropin, J. Clin. Endocrinol. Metab., 51 (1980) 35-40. [23] Harman, S.M., Tsitouras, P.D., Costa, P.T. and Blackman, M.R., Reproductive hormones in aging men. II. Basal pituitary gonadotropins and gonadotropin responses to luteinizing hormone-releasing hormone, J. Clin. Endocrinol. Metab., 54 (1982) 547-551. [24] Harman, S.M., Wehmann, R.E. and Blackman, M.R., Pituitary thyroid hormone economy in healthy aging men: basal indices of thyroid function and thyrotropin responses to constant infusions of thyrotropin releasing hormone, J. Clin. EndocrinoL Metab., 58 (1984) 320-326. [25] Hodin, R.A., Lazar, M.A., Wintman, B.I., Darling, D.S., Koenig, R.J., Larsen, P.R., Moore, D.D. and Chin, W.W., Identification of a thyroid hormone receptor that is pituitary-specific, Science, 244 (1989) 76-79. [26] Hollenberg, S.M., Weinberger, C., Ong, E.S., Cerelli, G., Oro, A., Lebo, R., Thompson, E.B., Rosenfeld, M.G. and Evans, R.M., Primary structure and expression of a functional human glucocorticoid receptor cDNA, Nature, 318 (1985) 635-641. [27] Honjo, H., Tamura, T., Matsumoto, Y., Kawata, M., Ogino, Y., Tanaka, K., Yamamoto, T., Ueda, S. and Okada, H., Estrogen as a growth factor to central nervous cells. Estrogen treatment promotes development of acetylcholinesterase-positive basal forebrain neurons transplanted in the anterior eye chamber, J. Steroid Biochem. Mot. BioL, 41 (1992) 633-635. [28] Jacobs, M.S., McFarland, W.L. and Morgane, P.J., The anatomy of the Bettlenose Dolphin (Tursiops truncatus). Rhinic lobe (Rhinencephalon): the archicortex, Brain Res. Bull., 4 Suppl. 1 (1979) 1-108. [29] Janowsky, J.S., Oviatt, S.K., Carpenter, J.S. and Orwoll, E.S., Testosterone administration enhances spatial cognition in older men, Soc. Neurosci. Abstr., 16 (1991) 340-352. [30] Jofils, M. and de Kloet, E.R., Effects of glucocorticoids and norepinephrine on the excitability in the hippocampus, Science, 245 (1989) 1502-1505. [31] Koide, T., Wieloch, T. and Siesjo, B., Chronic dexamcthasone pretreatment aggrevates ischemic neuronal necrosis, J. Cereb. Blood Flow Metab., 6 (1986) 395-404. [32] Landfield, P.W., Baskin, R.K. and Pitier, T.A., Bi'ain aging corre-

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50] [51] [52]

lates: retardation by hormonal-pharmacological treatments, Science, 214 (1981) 581-584. Laudet, V., H~inni, C., Coil, J., Catzeflis, F. and St6helin, D., Evolution of the nuclear receptor gene superfamily, EMBO J., 11 (1992) 1003-1013. Lazar, M.A., Hodin, R.A., Darling, D.S. and Chin, W.W., Identification of a rat c-erbA alpha-related protein which binds deoxyribonucleic acid but does not bind thyroid hormone, Mot. Endocrinol., 2 (1988) 893-901. Lubahn, D.B., Brown, T.R., Simental, J.A., Higgs, H.N., Migeon, C.J., Wilson, E.M. and French, F.S., Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity, Proc. Natl. Acad. Sci. USA, 86 (1989) 9534-9538. McEwen, B., Chao, H., Spencer, R., Brinton, R., Macisaac, L. and Harrelson, A., Corticosteroid receptors in brain: relationship of receptors to effects in stress and aging, Ann. NY Acad. Sci., 512 (1987) 394-401. McEwen, B.S., Weiss, J.M. and Schwartz, L.S., Uptake of corticosterone by rat brain and its concentration by certain limbic structures, Brain Res., 16 (1969) 227-241. Meaney, M.J., Altken, D.H., Berkel, C.V., Bhatnagar, S. and Sapolsky, R.M., Effects of neonatal handling on age-related impairments associated with the hippocampus, Science, 239 (1988) 766-768. Mitsuhashi, T., Tennyson, G.E. and Nikodem, V.M., Alternative splicing generates messages encoding rat c-erbA proteins that do not bind thyroid hormone, Proc. Natl. Acad. Sci. USA, 85 (1988) 5804-5808. Packan, D.R. and Sapolsky, R.M., Glucocorticoid endangerment of the hippocampus: tissue, steroid and receptor specificity, Neuroendocrinology, 51 (1990) 613-618. Reul, J.M.H.M. and de Kloet, E.R., Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985)2505-2511. Sapolsky, R.M., Krey, L. and McEwen, B.S., Corticosterone receptors decline in a site-specific manner in the aged rat brain, Brain Res., 289 (1983) 235-240. Sapolsky, R.M., Krey, L.C. and McEwen, B.S., Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging, J. Neurosci., 5 (1985) 1222-1227. Sar, M. and Parikh, I., Immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibodies, J. Steroid Biochem., 24 (1986) 497-503. Sar, M. and Stumpf, W.E., Distribution of androgen-concentrating neurons in rat brain. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 120-133. Seckl, J.R., Dickson, K.L., Yates, C. and Fink, G., Distribution of glucocorticoid and mineralocorticoid receptor messenger RNA expression in human postmortem hippocampus, Brain Res., 561 (1991) 332-337. Sherwin, B.B., Estrogen and/or androgen replacement therapy and cognitive functioning in surgically menopausal women, Psychoneurol. Endocrinol., 13 (1988) 345-357. Sherwin, B.B. and Gelfand, M.M., The role of androgens in the maintenance of sexual functioning in oophorectomized women, Psychosom. Med., 49 (1987) 297-357. Simcrly, R.B., Chang, C., Muramatsu, M. and Swanson, L.W., Distribution of androgen and estrogen receptor mRNA-contatining cells in the rat brain: an in situ hybridization study, J. Comp. Neurol., 294 (1990) 76-95. Sommer, W., Erkrankung des Ammonshorns als aetiologisches Moment der Epilepsie, Arch. Psychiatry, 10 (1980) 631-675. Squire, L.R., Mechanisms of memory, Science, 232 (1986) 16121619. Vermeulen, A., The hormonal activity of the postmenopausal ovary, J. Clin. Endocrinol. Metab., 42 (1976) 247-253.

H. Tohgi et al. / Brain Research 700 (1995) 245-253 [53] Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J. and Evans, R.M., The c-erb-A gene encodes a thyroid hormone receptor, Nature, 324 (1986) 641-646. [54] West, M.J. and Gurdersen, H.J.G., Unbiased stereological estimation of the number of neurons in the human hippocampus, J. Comp. Neurol., 296 (1990) 1-22.

253

[55] West, M.J., Regionally specific loss of neurons in the aging human hippocampus, Neurobiol. Aging, 14 (1993) 287-293. [56] Zola-Morgan, S., Squire, L.R. and Amaral, D.G., Human amnesia and the medial temporal region: enduring memory impairment following a bilateral lesion limited to field CA1 of the hippocampus, J. Neurosci., 6 (1986) 2950-2967.