Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain

Pergamon PII:

Neuroscience Vol. 91, No. 3, pp. 1143–1153, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00690-3

ESTROGEN MODULATES SPONTANEOUS ALTERNATION AND THE CHOLINERGIC PHENOTYPE IN THE BASAL FOREBRAIN M. M. MILLER,*†‡ k ¶ S. M. HYDER,†† R. ASSAYAG,* S. R. PANARELLA,* P. TOUSIGNANT‡** and K. B. J. FRANKLIN§ Departments of *Obstetrics and Gynecology, †Anatomy, ‡Medicine, and §Psychology, k Centre for Studies on Aging, **Clinical Epidemiology Division, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada, H3A 1A1 ††Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, TX 77225, U.S.A.

Abstract—We report that a small population of neurons expresses both choline acetyltransferase and classical estrogen receptor immunoreactivity and they are found primarily in the bed nucleus of the stria terminalis. In short-term ovariectomized ageing mice (24 months, n ˆ 5) there were 41.0 ^ 4.1% fewer of these double-labeled cells than in young (five months, n ˆ 5) short-term ovariectomized C57BL/6J mice. To study cholinergic neuron estrogen responsiveness, young mice (n ˆ 8) were ovariectomized at puberty (five weeks). After three months half of the mice (n ˆ 4) were given physiological levels of 17b estradiol for 10 days. Bed nucleus double-labeled neurons increased by 32.9% (P ⱕ 0.003) in the young mice given estrogen. In a gel shift assay, double-stranded oligonucleotides with putative estrogen response elements from the choline acetyltransferase gene were used as competitors against estrogen receptor binding to consensus estrogen response elements. A sequence with 60% homology to the vitellogenin estrogen response element was found to compete at 500- and 1 000-fold excess. Young mice (five months) with ovaries demonstrated significantly (P ⱕ 0.04) better performance in the spontaneous alternation T-maze test than did old (19 month) mice with ovaries (young ˆ 66.3 ^ 3.3% correct choices; vs old ˆ 55.0 ^ 4.0% in old mice with ovaries). Young mice (five months old), ovariectomized for one month and treated with estrogen, showed significantly more spontaneous alternation than ovariectomized controls (69.1 ^ 2.8% vs 58.3 ^ 3.9%; P ⱕ 0.04). Estrogen also increased spontaneous alternation in old, shortterm ovariectomized mice (61.5 ^ 2.7% vs 48 ^ 3.3%; P ⱕ 0.005). In either young or old ovariectomized mice, estrogen increased spontaneous alternation to levels seen in young animals with ovaries. Estrogen increases the number of choline acetyltransferase-immunoreactive and choline acetyltransferase/estrogen receptor-immunoreactive cells in old or young mice lacking estrogen, and enhances working memory in old or young mice lacking estrogen. Our data suggest that estrogen may act at the level of the choline acetyltransferase gene, but in view of the limited distribution of cholinergic cells expressing the classical estrogen receptor, it is unlikely that these cells can account for a memory enhancing effect of estrogen replacement. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholine, choline acetyltransferase, memory, ageing, mouse, estrogen receptor alpha.

Alzheimer’s disease (AD) is more common in women than men, and females are at increased risk of the cognitive decline associated with AD. 12 This increased susceptibility to cognitive decline may be due to the loss of estrogen at the menopause 39,68 (but see Ref. 9). Women with AD given estrogen replacement therapy (ERT) show improvements in tests of cognitive perfomance function compared with men with the disease. 10 In one study women at risk for AD given five years of ERT did not develop the disease, and estrogen users who developed AD did ¶To whom correspondence should be addressed. Abbreviations: AD, Alzheimer’s disease; BST, bed nucleus of the stria terminalis; ChAT, choline acetyltransferase; EDTA, ethylenediaminetetra-acetate; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; ERT, estrogen replacement therapy; GnRH, gonadotropin hormone releasing hormone; MAPK, microtubule-associated protein kinase; NGF, nerve growth factor.

so much later in life. 84 Estrogen also appears to enhance performance in elderly women who do not have dementia. One case–control study found that women on estrogen replacement had better recall of proper names. 73 Another study examined memory after ovariectomy and found that estrogentreated women showed no change in recall in a paired-associate test while performance deteriorated two months after ovariectomy in women who did not receive estrogen. 71 A recent study of long-term, lowdose ERT in female AD patients found improvement of cognitive function which disappeared upon withdrawal of estrogen. 65 Such findings have suggested that E2 treatment may have the potential to alleviate the impact of AD and enhance cognitive function in post-menopausal women, but why E2 should be effective is not clear. One hypothesis is that estrogen’s ability to enhance the function of cholinergic neurons may explain its ability to ameliorate

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memory deficits. 77,80 A decline in acetylcholine and its synthetic enzyme, choline acetyltransferase (ChAT), is a prominent biochemical feature of AD. 8,33,74 The decline is most marked in the areas innervated by the basal forebrain cholinergic cell group. These areas include the hippocampus and cingulate gyrus, innervated predominantly from the rostral portion of the basal forebrain cholinergic cell group in the medial septum and vertical limb of the diagonal band; and the neocortex and amygdala innervated from the nucleus basalis. 92 The idea that cognitive deficits in the aged are specifically due to loss of cholinergic neurons remains problematic 16,59,66 because selective destruction of basal forebrain cholinergic cells does not produce the severe deficits in spatial and working memory caused by non-specific toxins. 16,59 However, there is little doubt that cholinergic dysfunction would be deleterious for memory and cognition, and that preservation of cholinergic function would be beneficial. Septal/diagonal band cholinergic cells drive the theta rhythm which may be important for memory, particularly spatial and working memory. 34,63,93 Experiments with the selective neurotoxin IgG-saporin show that destruction of the septal/diagonal band cholinergic cells produces impairments in working memory, 4,45,83,91 although spatial reference memory appears to be preserved. 4,5 In addition, cholinergic inputs to the cortex play an important role in cortical arousal, 75 and selective lesions or inactivation of the nucleus basalis cholinergic cells may impair attentional processes. 60,61,69 Nuclear estrogen receptors (ER) have been reported in neurons of the basal forebrain region, 76 and there is evidence suggesting a link between hippocampal acetylcholine, estrogen and memory. In ovariectomized rats estrogen treatment significantly increases the expression of ChAT in this region. 23,25 Estradiol microinjected into the hippocampus following a session of trials in a spatial water maze improves performance 24 h later, and this memory enhancement is blocked by a muscarinic antagonist. 67 Conversely, systemic injections of a muscarinic antagonist disrupt the performance of ovariectomized rats in a food-rewarded T-maze alternation task but three days of treatment with 25 mg estradiol with or without progesterone restores performance to normal. 67 While there is direct evidence to support the notion that estrogen is important to the cholinergic system 84 and to cognitive function, little is known about the mechanisms of interaction of cholinergic neurons and gonadal steroids. There are now known to be two subtypes of estrogen receptors—the classical ERa, and a novel ERb. 42 We have investigated whether specific subsets of cholinergic neurons express ERa, whether there may be a direct effect of estrogen on the ChAT gene, and whether there are physiological effects of short-term estrogen treatment on memory. In this regard we used the

spontaneous alternation test as an indicator of working memory, 50,51 which has been shown to be sensitive to impairments induced by age, 43,94 high levels of amyloid-beta protein 28,52 and anticholinergic treatments. 2,6,88 EXPERIMENTAL PROCEDURES

Subjects We studied young (five months) and old (19–24 months) C57BL/6J mice (Jackson Labs, Bar Harbor, ME) that were gonadally intact or ovariectomized. In normal animals reproductive cyclicity patterns were established by daily vaginal smears. Animals were ovariectomized as previously described 57 and maintained in an isolated, pathogen-free colony with a 12-h light/dark photoperiod and free access to food and water. All animal care was according to the standards outlined by the NIH Guideline for the Care and Use of Laboratory Animals and the Canadian Council on Animal Care Standards. All protocols were approved by the McGill University and Royal Victoria Hospital Animal Care Utilization Committee. Immunocytochemical procedure Under 2,22-tribromoethanol (150 mg/kg, i.p.) anesthesia, mice were perfused and tissues processed according to published immunocytochemical protocols. 57 For studies of ChAT localization a rabbit antiserum against human placental enzyme (Boehringer–Mannheim) was used. Concentrations from 1:100 to 1:2 000 were tested in preliminary experiments, and the optimal concentration of 1:250 was selected for the studies. For immunocytochemical labelling of ERa a monoclonal rat antibody to estrogen receptors (H222Spd ) 72 was used. Antibody H222 recognizes a portion of the E domain between base pairs 467 and 528 in the human ERa gene. 1 Concentrations of antibody from 1 to 20 m g/ml were tested in preliminary experiments and the optimal concentration of 4 m g/ml was selected for use. Immunoreactivity was revealed by nitroblue tetrazolium (ChAT) or 3,3 0 -diaminobenzidine (ERa ). For details see Miller et al. 57 Preliminary studies confirmed that the numbers and distributions of immunoreactive cells in double-reacted tissues were the same as when single primary antibodies were used alone. 54 To test for non-specific immunoreactivity, some sections were incubated with non-immune serum instead of anti-ChAT followed by the anti-ER antibody reaction; with anti-ChAT followed by non-immune serum instead of anti-ER antibody; or with non-immune serum instead of either anti-ChAT or anti-ER antibodies. Coronal sections from the rostral forebrain were examined at × 400 on a Zeiss photomicroscope. Sections counted were between the first appearance of the vertical limb of the diagonal band rostrally (approximately Fig. 19 of Ref. 19) and the center of the optic chiasm caudally (approximately Fig. 33 of Ref. 19). For each section the populations of neurons which contained either ChAT alone, ER alone, or both ChAT and ER were counted by two observers who were blind to the identification of the sections. The correlation between observers was r ˆ 0.90. Immunocytochemical studies In the first experiment we tested whether there was a subset of basal forebrain ChAT-immunoreactive neurons which contain ER located within the basal forebrain, and whether there was an effect of age on these neurons. Since it is known that ChAT levels change during the estrous cycle, 23 mice were ovariectomized 10 days before animals were killed and brain tissue was taken for

ChAT and estrogen during ageing

immunocytochemical processing. Young mice (five months, n ˆ 5) were ovariectomized at the diestrous stage of the cycle; old mice (24 months, n ˆ 5) were in persistent diestrous. In a second experiment we studied young females who had never been exposed to estrogen during their postnatal lives to determine whether short-term exposure to estrogen would affect the number of ChAT-immunoreactive cells. This experiment was designed to eliminate any variable inherent in cyclical estrogen changes. Mice (n ˆ 8) were ovariectomized at puberty (five weeks of age) and were maintained for three months without gonadal steroids until the time of the experiment. Half of the animals received physiological levels of estrogen for the last 10 days. The remaining animals received capsules with vehicle alone. After 10 days of estrogen or vehicle treatment mice were killed under anesthesia and tissue sections from the basal forebrain treated to reveal ChAT and ERa . 57 Estradiol 17b was administered by subcutaneous silastic capsule delivering 13.5 ^ 2.9 pg/ml. 35 E2 capsules contained estradiol 17b mixed with Silastic adhesive (0.5 mg/ml Type A, no. 891; Dow Corning Corp., Midland, MI) packed in 10 mm of Silastic tubing (i.d. 0.04 in; o.d. 0.085 in; Dow Corning). 36 The serum concentration of E2 maintained with this procedure was determined in a parallel cohort of mice by radioimmunoassay (Radioimmunoassay Systems Laboratories, Carson, CA). Sensitivity of the assay was 1.0 pg/ml E2 and inter- and intra-assay coefficients of variation were 13.8% and 12.7%, respectively. Gel shift assay for estrogen response elements Rat uterus was used as the source of ER and doublestranded oligodeoxynucleotides. Binding reactions contained 10 mM Tris–HCl, 50 mM NaCl, 1 mM dithiothreitol and 1 mg of poly (dI-dC) for every 5 ml soluble protein. 31 Extract containing ER was incubated with either 50-fold molar excess of unlabeled vit-ERE (estrogen response element; positive control), or 500- or 1000-fold molar excess of the putative ERE (15 min, 4⬚C) before addition of radioactive vit-ERE probe (0.2 ng). Protein-DNA complexes were separated on 4% polyacrylamide gels (6.7 mM Tris–HCl, pH 7.5, 1 mM EDTA, 3.3 mM sodium acetate). Samples were electrophoresed (160 V, 90 min), gels dried and autoradiographed, and the ability of our potential ERE to compete for ER binding was determined by comparing the intensities of the migrated bands relative to vit-ERE. 31 The sequence of the choline acetyltransferase gene 58 was searched by NCBI Blast for concensus sequences to either the canonical vitellogenin ERE, or half-palindromic sites which have been shown to function as EREs in vitro. 31 A total of 18 possible half sites was identified in the 5 0 flanking region of the gene. A gel shift assay was done to determine whether any of the three most likely candidate EREs could bind ER in an in vitro system. Spontaneous alternation test Groups of mice were tested for spontaneous alternation 43,94 using a T-maze constructed of transparent Plexiglas. Because spontaneous alternation requires little training and does not require food or water deprivation it is particularly suitable for testing aged animals. The Tmaze was composed of a central pathway (28 × 9 × 20.5 cm) and two lateral arms (19 × 9 × 20.5 cm). The start box (12 × 9.4 × 20.5 cm) opened into the central pathway via an opaque Plexiglas sliding door (20.5 × 9.4 × 0.3 cm). Two sliding doors were also placed 10 cm from the end of each lateral arm to create a goal box. Testing took place in the animal colony room. The maze was placed on a steel table in an open space in the center of the room. The orientation and positions of the maze and

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other objects in the room were not changed during the experiment. A mouse was placed at the start of the Tmaze with one arm blocked off, and allowed to explore until it reached the end of the open arm (goal box). The mouse was confined in the goal box for 15 s, then removed to a holding cage and, after a timed delay (30 s or 60 s), placed back at the start of the maze where both arms were open. When the mouse reached either goal box, it was confined there for 15 s, then removed to the holding cage for 30 or 60 s and replaced at the start of the maze as before. When the mouse reached the goal box on this second freechoice trial it was confined for 15 s and then returned to its home cage. Thus each test session comprised a forcedchoice followed by two free choices. Each mouse first received two training sessions with an intertrial interval of 30 s, then on five successive days, five sessions with a 60-s intertrial interval. Spontaneous alternation studies Six groups of female C57BL/6J mice were tested. The first experiment explored whether there were age-related differences in working memory in C57BL/6J mice. Young mice (three months, n ˆ 16) having normal estrous cycles as judged by daily vaginal smears were studied at all stages of the estrous cycle. Old mice (19 months, n ˆ 16) were in persistent estrous. The second experiment examined the effect of short-term estrogen (E2) treatment on memory in young and old (25 months) ovariectomized mice which were in persistent diestrous. Young (five months, n ˆ 12) mice were either ovariectomized (one month, n ˆ 12) or ovariectomized and E2 treated (one month, n ˆ 11); similarly, old (24 months) mice were either ovariectomized (one month, n ˆ 15) or ovariectomized and E2 treated (one month, n ˆ 13). Estrogen treatment was as described above for immunocytochemical procedures. Mice were six and 19 or 25 months of age at the time of the study, respectively. Statistics The interactions of age and E2 treatment were tested by ANOVA (SigmaStat 1.0, Jandel Scientific, San Raphael, California). Cells immunoreactive for ChAT alone were much more numerous than double-labelled ChAT/ER cells and cell counts were log transformed to equate the variances. RESULTS

Immunocytochemical studies Few neurons in the basal forebrain cholinergic cell groups contained ERa immunoreactivity. There were no cells clearly identifiable as showing ERa immunoreactivity in the region of diagonal band. In the septum there were both ChAT and ERa reactive cells but not double-labeled cells. Isolated double-labeled cells were seen in the striatum. The only region in which there was a clustering of double-labeled cells was the bed nucleus of the stria terminalis (BST). An example of the double immunoreactivity seen in the BST is shown in Fig. 1. The majority of ChAT/ERa-immunoreactive cells was located just dorsal or ventral to the anterior commissure, lateral to the fornix and medial to the stria terminalis. This region corresponds to the BST medial anterior and BST medial ventral (Figs 30– 31 19). More posteriorly there was dense immunoreactivity for ERa in the medial posteromedial

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Table 1. Effect of age and estrogen on the mean (^S.E.M.) number/mm 2 of neurons immunoreactive for choline acetyltransferase only, and double labeled for choline acetyltransferase and estrogen receptor, in the bed nucleus of the stria terminalis of the C57BL/ 6J female mouse Ovariectomized 10 days

ChAT ChAT/ER Mean

Five months old

24 months old

Five months old, ovariectomized at five weeks No estrogen 10-day estrogen

55.3 ^ 6.0 13.0 ^ 0.9 26.3 ^ 1.04

28.6 ^ 1.5 7.3 ^ 0.1 14.4 ^ 1.04*

26.3 ^ 1.2 8.8 ^ 0.4 15.1 ^ 1.06

32.9 ^ 3.8 12.1 ^ 0.5 19.95 ^ 1.06**

*P ⬍ 0.003 five months old vs 24 months old; **P ⬍ 0.003 no estrogen vs 10-day estrogen.

division of the BST 19 but no ChAT-immunoreactive cells. Cell counts are shown in Table 1. In the first experiment with mice ovariectomized 10 days before, 20.7% of ChAT-immunoreactive neurons contained ERa while 1.5% of ERa neurons were ChAT-containing in the BST. The relative percentages of these cell types were similar in five-monthold and 24-month-old mice. However, compared to young (five months) short-term ovariectomized mice, old (24 months) short-term ovariectomized mice had 44.1% fewer BST neurons which contained both ChAT and ERa or ChAT only (ANOVA main effect, P ⱕ 0.003). The ratio of ChAT to ChAT/ERa -immunoreactive neurons did not change significantly with age. In the second experiment, with five-month-old mice ovariectomized prepubertally at five weeks, 10 days of estrogen treatment increased the number of neurons in the BST expressing ChAT with or without ER by 32.1% (ANOVA main effect, P ⱕ 0.003). Estrogen treatment did not significantly alter the ratio of ChAT to ChAT/ERa-labeled cells. Gel shift assay A number of putative estrogen response elements (EREs), some of which are half sites within the ChAT gene itself and its 5 0 flanking region, was identified. Oligonucleotides for these fragments from the mouse gene were prepared and gel shift assays done on the three most likely candidates based on at least 60% homology with the consensus ERE. 30 One candidate ERE, designated ChAT 3, competed for binding of ER to the vitellogenin ERE at 500- and 1000-fold excess (Fig. 2). ChAT 3 is located within the ChAT gene itself. The

sequence used for the sense strand in the competing oligo was (⫹) GATCGAGCATGTCATCGTGGCCTCCTG. Oligos designated as “ChAT 1” and “ChAT 2” failed to compete. Spontaneous alternation studies With an intertrial interval of 60 s, 19-month-old mice, which were gonadally intact but in persistent diestrous, showed significantly less spontaneous alternation than young intact mice (P ⱕ 0.03) (Table 2). Regardless of estrogen status, fivemonth-old mice showed significantly more spontaneous alternation than 24-month-old mice (ANOVA main effect, P ⬍ 0.02). In fact, for 24-month-old ovariectomized mice, alternation behavior was near chance level (50%). Regardless of age, mice with ERT for 30 days alternated significantly more than mice without estrogen (ANOVA main effect, P ⬍ 0.001). There was no interaction between the effects of age and ERT—the effects were additive. Comparing the performance estimates from the two behavioral experiments, the mean spontaneous alternation performance of young or old ovariectomized mice with ERT lay within the 95% confidence interval for the performance of five-month-old gonadally intact mice (59.0–73.6%). In contrast, the mean performance of ovariectomized mice, or of old gonadally intact mice in permanent diestrous, was below the lower limit of this interval (see Table 2). DISCUSSION

The behavioral results confirm that lack of estrogen, whether produced by ageing or by ovariectomy, is reflected in reduced alternation behavior. Alternation performance depends on recall of information that is variable from trial to trial. Spontaneous

Fig. 1. Photomicrographs of sections through the bed nucleus of the stria terminalis of the C57BL/6J mouse showing immunohistochemical localization of the classical estrogen receptor in choline acetyltransferasecontaining neurons. The alkaline phosphatase reaction is blue and indicates ChAT immunoreactivity in stellateor fusiform-shaped perikarya. The diaminobenzidine reaction is orange and indicates the presence of the E2, which is localized exclusively to the nucleus. Final magnification ˆ × 3900. (A) A large stellate-shaped neuron is present in the center of the micrograph containing immunolabeling for ChAT only. (B) Two orange neurons in the center of the field are overlapping and are exclusively ER-containing. (C) Immunolabeling for both ChAT and ER in a single fusiform shaped neuron. (D) A ChAT-containing neuron with an ER-containing nucleus (arrow) is seen in the lower left hand corner. Neurons containing ChAT alone or ER alone are below the plane of section of the micrograph. (E) Two ChAT neurons with ER containing nuclei (arrow).

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Fig. 2. Autoradiograph of a gel shift assay to identify a putative estrogen response element (ERE) in the ChAT gene. Rat uterus was used as the source of ER; double-stranded oligodeoxynucleotides with putative EREs from the ChAT gene were used as competitors against the ER binding to the consensus ERE. It can be seen that the putative ERE designated ChAT3 competed with vit-ERE at 500- and 1000-fold excess as indicated by the reduced optical density in the autoradiograph. Oligos designated as “ChAT 1” and “ChAT 2” failed to compete. ChAT 3 has two half sites homologous to a previously described functional ERE. The sequence in the competing oligo was (⫹) GATCGAGCATGTCATCGTGGCCTCCTG.

alternation is therefore classed as a test of working memory but, since the signals that distinguish the two alleys and goal boxes include both local and extra-maze cues, the test is not selective for spatial working memory. 50,51 This test has been shown to be sensitive to treatments that affect forebrain acetylcholine systems and hippocampal function in both rats and mice. 2,6,50,51,70,88,89 Ageing has previously been shown to lead to an alternation deficit in the rat, 94 and this is now shown in the C57BL/6J female mouse. Our results also show that ERT facilitates performance in aged mice, and in ovariectomized mice. In ovariectomized mice the effect of ERT is independent of age. Similar beneficial effects of ERT have been seen in delayed non-matching to sample, passive avoidance, two-way active avoidance and spatial working memory performance in

the rat. 13,64,80,90 In contrast, tests that are heavily dependent on spatial reference memory have found no improvement or even impairment in memory following estrogen treatment, 7,21,80 except in males. 47 In humans it also appears that not all memory tasks are sensitive to estrogen. Estrogen is beneficial to verbal memory in human females, while spatial memory appears to be unaffected or even impaired by estrogen. 73,77 The possibility that changes in spontaneous alternation reflect changes in the tendency to alternate as opposed to changes in working memory cannot be excluded. Nevertheless, tasks like alternation, that test non-spatial memory, may be better models to study the potential beneficial effects of ERT. The immunocytochemical results showed that, in parallel with the behavioral results, estrogen significantly increased the number of cells double labeled for ChAT and the classical estrogen receptor, ERa. Estrogen did not seem to differentially alter the numbers of ChAT- and ChAT/ERaimmunoreactive cells, since the ratio of these two cell types did not change significantly with age or estrogen treatment. It is not clear whether estrogen alters the number of neurons containing ERa, or whether there is simply an increase in the amount of immunoreactive material in each cell so that previously undetectable cells become detectable. Nevertheless, these data are in general agreement with previous biochemical data. Several studies have reported that ChAT is increased after estrogen administration in ovariectomized rats in the caudate, cortex, hippocampus and rostral forebrain. 38,48,49 Septal ChAT messenger RNA levels increase with estrogen treatment 53 and fluctuate with rodent hormone levels during estrous cycles. 23 It has also been reported that the number of nicotinic cholinergic receptors is increased in the forebrain after estrogen administration to ovariectomized females. 55,56 There is thus considerable evidence that estrogen enhances or preserves cholinergic cell function. Our results support the view that it may do so by acting through estrogen receptors to modify the production of ChAT. The DNA region which binds the estrogen receptor and either activates or suppresses gene transcription is known as the estrogen response element (ERE). The consensus ERE is a perfect palindromic sequence initially discovered in the chicken and Xenopus vitellogenin genes 41 and is referred to as the vit-ERE. Our results from the gel mobility assay detecting specific protein-DNA interactions indicate binding of the vit-ERE to one region of a set of putative EREs from the ChAT gene. This region contained a sequence with 60% homology to the vit-ERE. However, this sequence, like many other non-consensus EREs identified, binds the ER with relatively low affinity. While perfect EREs are rare, several estrogen responsive genes which contain imperfectly palindromic EREs either in

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Table 2. Working memory performance of young and old, intact or ovariectomized C57BL/6J female mice as indicated by the mean ( ^ S.E.M.) percentage of trials on which mice showed spontaneous alternation in a T-maze. Random behavior yields 50% alternation Comparison groups

Intact

Five months old 24 months old Mean across age

Five months old

19 months old

66.3 ^ 3.3

55.0 ^ 4.0*

Ovariectomized

Ovariectomized ⫹ estrogen

58.3 ^ 3.3 48.0 ^ 2.3 53.2 ^ 2.2

68.2 ^ 3.5 61.5 ^ 3.2 64.9 ^ 2.3**

*P ⬍ 0.04; **P ⬍ 0.001 (ANOVA).

isolation or in multiple arrays 3,15,37 are now being found in endogenous estrogen-responsive genes. EREs usually act as enhancers in that their influence on gene transcription is not necessarily position or orientation dependent. 30 Although imperfect EREs confer estrogen responsiveness to target genes, they are sometimes less potent activators of transcription than the consensus ERE. 62 The affinity of the ER for the ERE is an important indicator of the ability of an individual ERE to activate transcription, but is not a guaranteed predictor of transcriptional strength. 62 In a large number of cases, two consensus or widely spaced EREs can act in concert as an “estrogen responsive unit”. We do not as yet know whether the sequence we have identified interacts with any other sequences in the vicinity. The 5 0 regulatory region of the ChAT gene is complex, and at least three promoter regions have been identified. 58 The affinity of the ER for the ERE, and magnitude and orientation of ChAT gene DNA bending induced by binding of ER or other proteins which are all important in transcription of estrogenresponsive genes, 62 have not been determined. Our results suggest that there is a population of cholinergic neurons in which ChAT synthesis is stimulated via an estrogen receptor. Nevertheless, the hypothesis that the protective effect of estrogen on memory is mediated by cholinergic cells expressing the classical estrogen receptor is problematic. First, the neurological and neuropharmacological evidence implicates the cholinergic cells of the diagonal band and septum as the major cholinergic modulators of working memory. 17,24,40,44 However, we found only small numbers of double-labeled cells, and there were very few in the septum and diagonal band. The strongest concentration of ChAT/ER neurons was in the BST, which is implicated in emotionality 29 rather than memory. Furthermore, ERT seemed to increase the expression of ChAT in regions with cells lacking ERa as much as in those containing double-labeled cells. This agrees with other recent studies that have found that ERT increases the expression of ChAT mRNA in the medial septum and diagonal band nuclei of female ovariectomized rats. 24,26,53 Thus

the double-labeled ERa/ChAT cells cannot account for the increase in ChAT in the areas most likely to be involved in a memory facilitating effect of estrogen. There are mechanisms other than estrogen receptors that might explain estrogen modulation of the ChAT gene. Estrogen could act trans-synaptically via estrogen-modulated afferents. For example, gonadotropin hormone releasing hormone (GnRH) neurons do not contain ER, but estrogen is a necessary hormone for ovulation to occur. It is suggested that an intervening neuron containing an ER may modulate GnRH neurons. 27 Estrogen receptors can also be activated as transcription factors either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. 11 For example, epidermal growth factor (EGF) reproduces many of the effects of estrogen on the murine female reproductive tract. 32 Using dominant-negative Ras and microtubule-associated protein kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, it has been shown that EGF can activate ER by signaling through the MAPK pathway. This finding suggests a steroid-independent activation of ER. 11 EGF can also directly induce the phosphorylated active form of the ER, which might involve an action on cyclic GMP, with the latter then intervening as cofactor of the involved phosphokinase(s) in estrogen target tissues. 20 EGF mediates ChAT activity in forebrain cholinergic neurons in vitro indirectly via glia, and a direct glial cell response to EGF may have an important influence on the expression of cholinergic forebrain neurons. 40 Another possible intermediary signaling mechanism for ER may include nerve growth factor (NGF). ER have been co-localized with NGF receptor-containing neurons in the basal forebrain 82 and estrogen modulates NGF mRNA levels, 81 allowing the possibility that estrogen acts on cells producing NGF, making the factor more available to cells containing the receptor. Thus, indirect signalling pathways acting either within the ER-containing neuron or in adjacent neurons or glial cells might account for E2 modulation of ChAT activity. Although there are grounds to suggest that

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estrogen might modulate ChAT and memory without the mediation of a specific ER, there is evidence that basal forebrain cholinergic neurons do sequester estrogen 86 suggesting they contain an ER. Using a rabbit polyclonal antibody to rat ER (ER715) Gibbs 22 found ChAT/ER-immunoreactive cells in the septum and diagonal band of the rat. In situ hybridization also reveals ER mRNA in the diagonal band and lateral septum of the rat. 79 However, we found that in the mouse these regions do not contain ERa as indicated by immunoreactivity to the monoclonal H222 antibody. This antibody is believed to be quite specific to the unique region of the E domain of the classical ERa . 1 Other studies of ERa distribution in the brain of the mouse (rabbit polyclonal antibody to ER 409 87), the hamster (H222 antibody 46), the musk shrew (H222 antibody 14), the opossum (H222 antibody 18) and the ferret (H222 antibody 85) agree with our finding of dense immunoreactivity in the posterior BST, and also do not report cells with ERa reactivity in the medial septum, diagonal band and nucleus basalis. These findings suggest that either the rat differs from other species in the distribution of ERa , or the septum and diagonal band contain an ER that is

different from the ERa recognized by the H222 antibody. In this regard it has been recently reported that the septum and diagonal band of the rat expresses ERb mRNA for the novel ERb 42 more heavily than ERa mRNA. 78 Our data indicate that an ER may act at the level of the ChAT gene to modulate the production of ChAT. However, the number and location of the ERa/ChAT-containing cells does not seem to be appropriate to explain the memory preserving effects of estrogen. The recent discovery of the ERb receptor leaves open the possibility that estrogen may regulate ChAT and behavior via ERb , or via an action that is not mediated by estrogen receptors. Acknowledgements—The authors wish to thank Ms Lixia Zhu for the careful preparation and examination of tissue sections used in this study and Ms Ria Falvo for excellent technical assistance in the memory studies. We thank Dr G. Greene and Abbott Laboratories for the gift of antibody H222. Dr Hugh Bennett generously provided oligonucleotides used in the gel shift assays. This work was supported by NIH AG07795 (MMM), the Fraser Fund of the Royal Victoria Hospital (MMM), and the Canadian Alzheimer’s Society of Canada (MMM), NIH HD08615(SH), NSERC A6303 (KBJF).

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