Estrogen and Basal Forebrain Cholinergic Neurons: Implications for Brain Aging and Alzheimer's Disease-Related Cognitive Decline

Hormones and Behavior 34, 98 –111 (1998) Article No. HB981451

Estrogen and Basal Forebrain Cholinergic Neurons: Implications for Brain Aging and Alzheimer’s Disease-Related Cognitive Decline Robert B. Gibbs1 and Payal Aggarwal University of Pittsburgh School of Pharmacy, 1004 Salk Hall, Pittsburgh, Pennsylvania 15261 Received January 29, 1998; revised March 30, 1998; accepted April 8, 1998

Recent studies suggest that estrogen replacement therapy can reduce the risk and severity of Alzheimer’s disease (AD)-related dementia in postmenopausal women. Many different mechanisms by which estrogen therapy may help to reduce the risk and severity of AD-related pathophysiology have been proposed. Recent animal studies suggest that one way in which estrogen replacement may help to reduce cognitive deficits associated with aging and AD is by enhancing the functional status of cholinergic projections to the hippocampus and cortex. Here we review the evidence that estrogen is important in the maintenance of cholinergic neurons projecting to the hippocampus and cortex and that estrogen replacement can enhance the functional status of these neurons, as well as reduce cognitive deficits associated with muscarinic cholinergic impairment. Based on these studies, we conclude that, in animals, short-term treatment with physiological levels of estrogen, or estrogen and progesterone, has significant positive effects on cholinergic neurons in the medial septum and nucleus basalis magnocellularis and on their projections to the hippocampus and cortex. We hypothesize that similar effects in humans may help delay the decline in basal forebrain cholinergic function associated with aging and AD and thereby reduce the risk and severity of AD-related dementia in postmenopausal women. © 1998 Academic Press Key Words: gonadal hormones; learning and memory; dementia; acetylcholine; hippocampus; cortex.

Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by a progressive

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To whom correspondence should be addressed.

neuropathology with a corresponding loss of learning and memory and other cognitive processes. AD affects approximately 6 –10% of the population over age 65 (Ha¨fner, 1990). In the United States, 4 million Americans currently have AD, and it is estimated that 14 million Americans will have AD by the middle of the next century (The National Public Policy Program to Conquer Alzheimer’s Disease, 1995). After age 65, the prevalence of dementia and AD doubles approximately every 5 years (Jorm et al., 1987). AD is much more prevalent in women than in men, due largely to the fact that women tend to live longer than men. Recent studies suggest that the incidence of AD may also be greater in women than in men (see Henderson, 1997, for review) and that estrogen replacement can help to reduce the risk (Henderson et al., 1996; Kawas et al., 1997; Paganini-Hill and Henderson, 1996; Tang et al., 1996) and severity (Honjo et al., 1994; Ohkura et al., 1994b, 1995a) of AD-related dementia in some postmenopausal women. One of the consistent biochemical deficits associated with AD is a decrease in cholinergic innervation of the hippocampal formation and neocortex and a corresponding loss of cholinergic neurons in the medial septum (MS), diagonal band of Broca (DB), and nucleus basalis magnocellularis (NBM) (see Kasa et al., 1997 for review). These neurons are the major source of cholinergic input to the hippocampal formation and cortex (Woolf, 1991) and have been shown in numerous studies to play an important role in learning and memory (Fibiger, 1991; McEntee and Crook, 1992; Olton, 1990), as well as attentional processes (see Lawrence and Sahakian, 1995; Voytko, 1996; Wenk, 1997, for reviews). These neurons are also severely affected 0018-506X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

Estrogen, Cholinergic Neurons, and AD

by AD and are thought to contribute to the cognitive decline associated with AD-related dementia (Fibiger, 1991; Weinstock, 1995). We have hypothesized that ovarian steroid hormones play a role in the normal physiological regulation of cholinergic neurons in the MS and NBM and that the loss of ovarian function with age has adverse effects on these neurons which contribute to the increased risk for developing ADrelated dementia in women. Likewise, we hypothesize that the ability for estrogen replacement to reduce the severity of AD-related dementia in some women is due, in part, to the ability for estrogen replacement to enhance the functional status of cholinergic projections to the hippocampus and cortex. This review summarizes recent data which demonstrate that basal forebrain cholinergic neurons are significantly affected by changes in circulating levels of estrogen and progesterone and which support the idea that the effects of estrogen and progesterone on basal forebrain cholinergic neurons are relevant to the prevention and treatment of AD-related dementia in women.

BASAL FOREBRAIN CHOLINERGIC NEURONS, AD, AND ESTROGEN Cholinergic neurons in the MS, the vertical limb of the diagonal band of Broca (VDB), and the NBM are the primary source of cholinergic innervation to the hippocampal formation and cortex. Studies have demonstrated that agents which destroy these neurons, such as excitatory neurotoxins, AF64A, or the selective immunotoxin 192IgG-saporin, or manipulations which disrupt their projections such as muscarinic inhibitors and mechanical lesions, produce learning and memory and attentional deficits similar in many ways to the deficits observed with aging and AD (Chrobak et al., 1988; Dekker et al., 1991; Dornan et al., 1996; Dunnett et al., 1987; Etherington et al., 1987; Leanza et al., 1996; Nilsson et al., 1992). Conversely, agents which enhance cholinergic activity, such as muscarinic agonists, cholinesterase inhibitors, and implants of embryonic cholinergic neurons (in rats), have been shown to enhance learning and memory processes and to attenuate cognitive deficits associated with aging and AD (Bartus et al., 1983; Dunnett et al., 1982; Fine et al., 1985; Kumar and Calache, 1991; Matsuoka et al., 1991; McGurk et al., 1991; Murray and Fibiger, 1985, 1986; Nilsson et al., 1987; Ridley et al., 1992; Welner et al., 1988). Studies of the anatomical and biochemical changes associated with AD have consis-

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tently demonstrated reductions in the number of basal forebrain cholinergic neurons with corresponding reductions in choline acetyltransferase (ChAT) activity, high-affinity choline uptake (HACU), and acetylcholine (ACh) production in the hippocampus and cortex (see Kasa et al., 1997 for review). Decreased numbers of p75NTR and trkA nerve growth factor receptor expressing cells, as well as decreases in p75NTR and trkA mRNA, have also been reported and are consistent with the decreased numbers of basal forebrain cholinergic neurons (see Mufson, 1997 for review). These findings have resulted in a general consensus that the loss and/or functional impairment of basal forebrain cholinergic projections is one of the hallmarks of AD and have led to the expectation that agents which enhance basal forebrain cholinergic activity will be useful in preventing and treating ADrelated dementia. Indeed, cholinesterase inhibitors such as tacrine (Cognex) and donepezil (Aricept) have been shown to produce significant, though marginal, improvement in Alzheimer’s patients. Recent studies have shown that cholinergic neurons in the basal forebrain are significantly affected by changes in circulating levels of estrogen. In several early studies, Luine and colleagues demonstrated that continuous estrogen replacement resulted in significant dose-related increases in ChAT activity within specific regions of the rat basal forebrain, hippocampus, and frontal cortex, providing evidence for a direct estrogen effect on basal forebrain cholinergic function (Luine and McEwen, 1977; Luine, 1985; Luine et al., 1975). O’Malley et al. (1987) subsequently demonstrated significant increases in HACU and ACh synthesis in cortical synaptosomes following short-term estrogen replacement, providing further evidence for an estrogen-mediated enhancement of basal forebrain cholinergic activity. These studies have since been confirmed and extended by studies showing increases in ChAT mRNA and protein (Gibbs, 1996b, 1997; Gibbs et al., 1994), ChAT activity (Singh et al., 1993), HACU (Singh et al., 1994), and potassium-evoked ACh release (Gibbs et al., 1997) within specific regions of the basal forebrain, hippocampus, and cortex following short-term estrogen treatment. The specificity and time-course of the effects do not always agree; however, these differences may be related to different doses and regimens of estrogen replacement, as well as to differences in the way in which regions of the basal forebrain have been defined (see below). Evidence that estrogen plays a role in the normal physiological regulation of basal forebrain cholinergic neurons has also been described (Gibbs, 1996b). In this

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FIG. 1. (A and B) Relative levels of ChAT mRNA detected in the MS and NBM of rats sacrificed at different stages of the estrous cycle. Bars represent the percentage change from the arithmetic mean of the Diestrus 2 animals 6 SEM. (C and D) Relative levels of ChAT mRNA detected in the MS and NBM at different times following acute administration of estrogen or estrogen followed 48 h later with progesterone. Animals were killed 5, 24, 53, and 72 h after receiving estrogen or 5 and 24 h after receiving estrogen followed by progesterone. Bars represent the percentage change from the arithmetic mean of the ovariectomized controls 6 SEM. *P , 0.05 compared with ovariectomized controls; **P , 0.01 compared with ovariectomized controls; †P , 0.05 compared with E53. Adapted from Gibbs (1996b).

study, relative levels of ChAT mRNA in the MS were observed to fluctuate across the estrous cycle and to correspond in both timing and magnitude to changes observed following acute administration of either estrogen or estrogen and progesterone (see Fig. 1). Notably, peak levels of ChAT mRNA in the MS and NBM were observed on the morning of diestrus 1 and diestrus 2 when estrogen levels are low (Figs. 1A and 1B). This is explained, however, by the fact that following an acute administration of estradiol, significant increases in ChAT mRNA were not observed for 24 – 48 h and were then maintained for at least 24 h after estrogen levels had declined (Figs. 1C and 1D). One important observation of these studies was that progesterone, administered 48 h after estrogen, significantly enhanced the effects of estrogen by either accelerating or prolonging the effects of estrogen on the

levels of ChAT mRNA. Collectively, these studies suggest that estrogen and progesterone play a role in the normal physiological regulation of basal forebrain cholinergic neurons and that hormone replacement enhances basal forebrain cholinergic function.

PHYSIOLOGICAL AND BEHAVIORAL SIGNIFICANCE OF ESTROGEN EFFECTS ON BASAL FOREBRAIN CHOLINERGIC NEURONS Evidence that estrogen replacement produces an increase in basal forebrain cholinergic parameters has led to the speculation that estrogen replacement may help to reduce the risk and severity of AD-related

Estrogen, Cholinergic Neurons, and AD

cognitive decline by enhancing the functional status of basal forebrain cholinergic projections. Studies by Sherwin and colleagues have demonstrated significant changes in memory function across the menstrual cycle, as well as the ability for estrogen replacement to enhance specific cognitive abilities in surgically menopausal women (Phillips and Sherwin, 1992a, 1992b; Sherwin and Gelfand, 1985). These elegant studies have demonstrated that estrogen can have direct benefits on discrete cognitive abilities in women. Recent studies by Honjo et al. (1993, 1994), Ohkura et al. (1994a, 1994b, 1995), and Henderson et al. (1996) suggest that estrogen replacement may reduce the severity of dementia in some women with AD. Most of these studies were conducted over short periods of time (6 weeks– 6 months) and evidence pertaining to the effects of long-term estrogen replacement in women with AD still needs to be evaluated. Studies by Paganini-Hill et al. (1994, 1996), Tang et al. (1996), and Kawas et al. (1997) also suggest that estrogen replacement therapy can reduce the risk of developing AD in postmenopausal women. In some studies, the relative risk of developing AD was reportedly reduced by over 40% in women who had received estrogen replacement therapy prior to enrollment vs those who had never received estrogen replacement therapy. Whether any of these effects are directly related to effects of estrogen on basal forebrain cholinergic neurons is currently unknown. Animal studies, however, are beginning to provide evidence that the effects of estrogen on basal forebrain cholinergic neurons are physiologically and behaviorally relevant. Evidence that estrogen replacement may help cholinergic neurons maintain elevated levels of acetylcholine release was recently reported by Gibbs et al. (1997). In this study, ovariectomized animals received continuous estradiol replacement for 10 –11 days to correspond with conditions under which elevations in ChAT mRNA and protein had been previously observed. Basal and potassium-evoked acetylcholine release in the hippocampus and overlying cortex were then measured using in vivo microdialysis and HPLC and compared with corresponding values from ovariectomized, non-estrogen-treated controls. Results from this study revealed no significant effect of estrogen replacement on basal acetylcholine release; however, significantly greater potassium-evoked release was detected in the estrogen-treated animals, particularly after a longer period of potassium-stimulated release (see Fig. 2). These results suggest that estrogen replacement may enable the neurons to maintain elevated levels of acetylcholine release during periods of

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FIG. 2. Percentage change in ACh release 6 SEM detected after 30, 60, and 90 min of potassium-induced depolarization relative to baseline in E-treated ( ) and non-E-treated (■) animals. Group averages were calculated based on the percentage change relative to baseline for each animal. The overall mean percentage change in release induced by potassium was greater in the E-treated animals than in the non-E-treated animals (137.8 6 12.6% vs 97.5 6 12.6%, P , 0.05) and was most apparent after 90 min of potassium-stimulated release. *P , 0.05 relative to the non-E-treated control. Adapted from Gibbs et al. (1997).

increased demand while, at the same time, having relatively little impact on basal cholinergic tone. Note that the increased ability to maintain elevated levels of acetylcholine release is consistent with the increases in ChAT and HACU previously described. Ordinarily, these effects may have little impact on cholinergic function and cognitive processes in a young healthy brain. These effects may be of greater significance, however, in AD where the number of cholinergic cells is substantially reduced and physiological demands on remaining cells are correspondingly increased. Evidence that estrogen replacement can reduce behavioral effects associated with cholinergic impairment, aging, and gonadectomy has also been described. Dohanich and co-workers have recently reported that estrogen replacement prevents the effects of both systemic (Dohanich et al., 1994; Fader et al., 1998) and intrahippocampal (Fader et al., 1998) administration of scopolamine on reinforced alternation in a T-maze and that estrogen replacement prior to and/or during training improves acquisition of an 8-arm radial maze (Daniel et al., 1998). Interestingly, Packard et al. (1996) demonstrated that posttraining intrahippocampal administration of estrogen can improve performance in the Morris

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water maze, that this effect is blocked by scopolamine, suggesting that the effects are directly related to effects on cholinergic synapses in the hippocampus, and that estrogen may also be effective in males. Work by Williams and co-workers has demonstrated that early postnatal exposure to estradiol improves radial arm maze acquisition in adulthood and can alter the way in which adult animals process spatial information (Williams et al., 1990; Williams and Meck, 1991). More recently, Williams and Einstein (unpublished observations) observed that estrogen replacement to both young (3 month) and old (15 month) rats improves working memory performance on a radial arm maze particularly when the estrogen is administered short-term. Similarly, Luine and Rodriguez (1994) demonstrated that estrogen replacement producing average serum levels of ;90 pg/ml E2 produced small but significant improvements in radial arm maze performance in young and aged male rats, but not female rats, when delays between the fourth and fifth choice were introduced to increase the difficulty of the task. A similar effect in females was subsequently observed when the dose of hormone replacement was reduced to achieve diestrus, as opposed to proestrus, levels of E2 (Luine, 1997). Evidence that these behavioral effects are related to some degree to the enhancement of basal forebrain cholinergic function has also been described. Singh et al. (1994) reported that the effects of estrogen replacement on active avoidance in young and aged rats correlated with the effects on ChAT activity and highaffinity choline uptake, suggesting that the effects on active avoidance may be related to some degree to effects on basal forebrain cholinergic activity. We have recently examined the ability for estrogen replacement to attenuate impairments in passive avoidance behavior induced by scopolamine (a muscarinic antagonist) and lorazepam (a benzodiazepine) (see Gibbs et al., 1998). Animals were trained using a multiple-trial passive avoidance paradigm to distinguish effects on acquisition from effects on retention. The results indicate that estrogen replacement attenuated a small but significant scopolamine-induced impairment in passive avoidance acquisition, but did not attenuate a scopolamine-induced impairment on retention. Like the studies by Dohanich and colleagues, this result is consistent with an ability for estrogen replacement to attenuate learning deficits associated with muscarinic cholinergic impairment. In addition, estrogen replacement significantly attenuated a lorazepam-induced impairment on passive avoidance retention (lorazepam did not impair passive avoidance acquisition). This effect appeared to be related to effects of estrogen

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mediated prior to and/or during training, not during retention testing, and was not due to an effect of estrogen on lorazepam kinetics. Lorazepam, like other benzodiazepines, facilitates GABAergic effects by binding to the GABAA receptor and enhancing GABAmediated effects on chloride conductance. The fact that estrogen replacement attenuated a lorazepaminduced deficit in passive avoidance retention suggests that effects on noncholinergic systems are also likely to contribute to the effects on learning and memory that have recently been described. Notably, results from our recent studies suggest that estrogen replacement was not effective at attenuating the scopolamine-induced impairment in passive avoidance acquisition when circulating levels of estradiol were very high (.400 pg/ml) (Gibbs et al., 1998). This result is consistent with recent data showing that the effects of estrogen replacement on the number of ChAT-IR cells detected in the MS and NBM vary as a function of the dose and duration of estrogen treatment (Gibbs, 1997). In this study, animals were treated with 2, 10, 25, or 100 mg estradiol (E2) every other day for 1, 2, or 4 weeks. Treatment with 2–25 mg E2 for 1 week (producing mean serum E2 concentrations of 14.3–105.0 pg/ml) resulted in a dose-dependent increase in the number of ChAT-IR cells detected in the MS, whereas treatment with 100 mg E2 (producing a mean serum E2 concentration of 419.6 pg/ml), or with lower doses of E2 for longer periods of time, was ineffective (Table 1). Similarly, treatment with 10 mg E2 for 1 week or 2 mg E2 for 2 weeks resulted in increased numbers of ChAT-IR cells in the NBM, whereas treatment with higher doses, or with low doses for longer periods of time, was ineffective (Table 1). These data suggest that increases in cholinergic function following estrogen replacement are dosedependent and that increases are not maintained in response to uninterrupted treatment with high physiological levels of estradiol. In addition, the data correspond with our data suggesting that estrogen was effective at preventing a scopolamine-induced impairment in passive avoidance acquisition only in those animals with serum levels of estradiol that were ,200 pg/ml. Notably, recent studies by Luine (1997) and by Williams (1996) likewise report significant beneficial effects of estrogen replacement on radial arm maze performance following low-dose but not high-dose, or short-term but not long-term, estrogen treatment. These studies suggest that the dose and regimen of estrogen replacement may be important factors in determining the subsequent effects of estrogen on cognitive processes.

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TABLE 1 Percentage Change in the Average Number of ChAT-IR Cells Detected in the MS and NBM Relative to Controls after Treatment with 2–100 mg E2 Every Other Day for 1, 2, or 4 Weeks Dose of E2

1 Week

2 Weeks

4 Weeks

MS 0 2 10 25 100

0.0 6 3.8 14.4 6 6.0 21.4 6 8.6* 30.3 6 6.2* 10.1 6 6.7

0.0 6 6.8 11.2 6 8.4 4.2 6 4.9 1.9 6 4.7 22.89 6 4.3

0.0 6 21.3 6 27.9 6 24.1 6 212.6 6

5.1 4.5 2.6 4.0 5.8

NBM 0 2 10 25 100

0.0 6 5.0 0.5 6 6.9 21.6 6 5.9* 23.2 6 3.8 22.6 6 6.1

0.0 23.3 21.0 26.3 0.0

6 3.3 6 6.3* 6 6.5 6 5.7 6 8.9

0.0 6 2.6 14.1 6 5.5 9.4 6 6.9 29.5 6 11.4 0.0 6 9.7

Source. Adapted from Gibbs (1997). Note. Values represent the mean percentage change relative to corresponding non-E-treated controls 6 SEM. * P , 0.05 relative to non-E-treated controls.

MECHANISMS OF ESTROGENMEDIATED EFFECTS ON BASAL FOREBRAIN CHOLINERGIC NEURONS The mechanisms by which estrogen influences basal forebrain cholinergic neurons is still largely unclear. Toran-Allerand et al. (1992) demonstrated that the cholinergic neurons contain high-affinity estrogen binding sites indicative of estrogen receptors. More recently, Gibbs (1996a) reported the detection of estrogen receptor-like immunoreactivity in the majority of cholinergic neurons in the MS, HDB, and NBM. These studies suggest that estrogen may directly influence the cholinergic neurons via binding to intracellular receptors followed by direct steroid-mediated effects on gene transcription; however, the possibility that estrogen may affect cholinergic neurons indirectly must also be considered. Cholinergic neurons in the MS and NBM are also affected by nerve growth factor (NGF), a polypeptide growth factor produced in the hippocampus and cortex (Thoenen et al., 1987). The effects of NGF are mediated by binding to two cell-surface receptors, a low-affinity receptor (p75NTR), which binds NGF and other related neurotrophins, and a protein tyrosine kinase receptor (TrkA) that is an essential component of high-affinity NGF binding sites (Chao and Hemp-

stead, 1995). Studies have shown that binding to TrkA, activation of the receptor tyrosine kinase, and autophosphorylation of the receptor are essential for mediating biological effects of NGF (see Segal and Greenberg, 1996 for review). The role of the p75NTR receptor in mediating biological effects of NGF is more controversial; however, several studies have demonstrated significant effects of p75NTR receptor expression on the sensitivity and specificity of NGF effects in some cells (Benedetti et al., 1993; Chao and Hempstead, 1995; Davies et al., 1993; Hantzopoulos et al., 1994; Lee et al., 1994a; Verdi et al., 1994). The p75NTR receptor has also been implicated in the regulation of cell death (Courtney et al., 1997; Lee et al., 1994b) and shares structural homology with the cell death domains of TNFR-1 and FAS (Chapman, 1995). Nevertheless, NGF has been shown to promote both the survival and the function of basal forebrain cholinergic neurons and to upregulate the expression of both p75NTR and TrkA mRNA by the cholinergic neurons both during development and in adulthood (see Gibbs, 1994 for review). Consequently, the effects of estrogen on basal forebrain cholinergic neurons could result from effects of estrogen on NGF and NGF receptors. Evidence that estrogen affects both neurotrophin and neurotrophin receptor expression and that these effects are dose- and time-dependent has been described. One study by Gibbs and Pfaff (1992) demonstrated that p75NTR mRNA and protein were reduced in the MS and VDB following 2 or 4 weeks of estrogen replacement, despite the fact that the number of ChAT-IR cells detected in the MS after 1 and 2 weeks of treatment was significantly increased. Subsequent studies confirmed this result and demonstrated that, like the effects on ChAT-IR, decreases in p75NTR-IR were both dose- and time-dependent with maximal decreases observed following longer-term treatment with high levels of estradiol (Gibbs, 1997). Sohrabji et al. (1994) likewise reported a decrease in p75NTR expression in dorsal root ganglion neurons following estrogen treatment. In contrast, Sohrabji et al. (1994) and more recently McMillan et al. (1996) have reported increases in trkA mRNA within dorsal root ganglion neurons and in cholinergic neurons in the HDB and NBM following short-term estrogen treatment. However, evidence that trkA mRNA decreases in dorsal root ganglion neurons (Scoville et al., 1997), as well as in the MS and NBM (Gibbs et al., 1994), following longer-term continuous treatment has also been reported, suggesting that the elevated levels of trkA are not maintained and that both p75NTR expression and

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trkA expression decrease in response to long-term continuous estrogen replacement. Nevertheless, the fact that short-term estrogen replacement produced increases in trkA expression suggest that short-term or intermittent estrogen treatment may help to enhance basal forebrain cholinergic function by increasing the responsiveness of the cholinergic neurons to endogenous neurotrophins. Effects of estrogen on neurotrophin expression have been somewhat less consistent. Miranda et al. (1993) have demonstrated that cells in the postnatal rat hippocampus and cortex, as well as the postnatal and adult basal forebrain, coexpress estrogen receptor mRNA with neurotrophin and neurotrophin receptor mRNAs, suggesting that estrogen may directly affect neurotrophin and neurotrophin receptor expression. Consistent with this finding, Singh et al. (1993, 1995) reported significant decreases in both NGF and BDNF mRNA within specific hippocampal and cortical regions following ovariectomy and a significant increase in BDNF mRNA within specific hippocampal regions following long-term (25 weeks) estrogen treatment. Conversely, Gibbs et al. (1994) reported a significant decrease in hippocampal levels of NGF mRNA following 1–2 weeks of continuous estrogen treatment, comparable to the effects on p75NTR and trkA previously described. More recently, Gibbs and co-workers examined changes in basal forebrain levels of trkA, as well as hippocampal levels of BDNF and NGF mRNA, across the estrous cycle and in response to acute treatment with estrogen and estrogen plus progesterone (Gibbs, 1998b). Significant fluctuations in trkA mRNA were detected in the MS across the estrous cycle and corresponded with increases in trkA mRNA detected in the MS following acute treatment with estrogen or estrogen plus progesterone (Fig. 3). As with the changes in ChAT mRNA described above, a significant increase in trkA mRNA was detected in the MS 24 h after estrogen administration and persisted in response to treatment with estrogen followed by progesterone (Fig. 3B). Significant fluctuations in BDNF mRNA were likewise observed in hippocampal regions CA1 (51.7%) and CA3/4 (39.7%) across the estrous cycle (not shown) and were increased significantly in region CA1 (28.1%), CA3/4 (76.9%), and the dentate granule cell layer (73.4%) of animals sacrificed 53 h after receiving estrogen and 5 h after receiving progesterone (Fig. 4). The increases in BDNF mRNA may be due to increased levels of excitation resulting from an estrogen-mediated decrease in GABA production in the hippocampus (Murphy et al., 1998). Decreased GABA

Gibbs and Aggarwal

FIG. 3. (A) Relative levels of trkA mRNA detected in the MS of rats sacrificed at different stages of the estrous cycle. Bars represent the percentage change from the arithmetic mean of the Diestrus 2 animals 6 SEM. (B) Relative levels of trkA mRNA detected in the MS at different times following acute administration of estrogen or estrogen followed 48 h later with progesterone. Animals were killed 5, 24, 53, and 72 h after receiving estrogen or 5 and 24 h after receiving estrogen followed by progesterone. Bars represent the percentage change from the arithmetic mean of the ovariectomized controls 6 SEM. *P , 0.05 compared with ovariectomized controls. Adapted from Gibbs (1998b).

production has, in turn, been correlated with increased dendritic spine density on hippocampal neurons in vitro, suggesting that decreased GABA production (and possibly increases in BDNF) may be responsible for the estrogen-induced increases in dendritic spine density and synapse number in the hippocampus which have been described (Gould et al., 1990; McEwen, 1996; Woolley and McEwen, 1992). Unlike the effects on ChAT and trkA mRNA described above, levels of BDNF mRNA within specific cell layers of the hippocampus were not increased significantly in response to the acute administration of es-

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these studies suggest that short-term treatment with estrogen plus progesterone produces increased levels of trkA mRNA in the basal forebrain and BDNF mRNA (but not NGF mRNA) in the hippocampus and are consistent with effects on trkA and BDNF mRNA reported by others (see above).

EFFECTS ON BASAL FOREBRAIN CHOLINERGIC NEURONS ASSOCIATED WITH LONG-TERM LOSS OF OVARIAN FUNCTION

FIG. 4. Relative levels of BDNF mRNA detected in the dentate granule cell layer (GCL), region CA1, and region CA3/4 of the hippocampus at different times following acute administration of estrogen or estrogen followed 48 h later with progesterone. Animals were killed 5, 24, 53, and 72 h after receiving estrogen or 5 and 24 h after receiving estrogen followed by progesterone. Bars represent the percentage change from the arithmetic mean of the ovariectomized controls 6 SEM. Note that relative levels of BDNF mRNA were significantly elevated in all three regions in E 1 P-treated animals killed 53 h after receiving estrogen and 5 h after receiving progesterone. *P , 0.05, **P , 0.005. Adapted from Gibbs (1998b).

trogen alone, and increased levels detected after combined treatment with E 1 P were not maintained after hormone levels had declined. No significant fluctuations in NGF mRNA were detected in the hippocampus across the estrous cycle, and no change in hippocampal levels of NGF mRNA was detected in response to acute hormone treatment. Collectively,

Many of the studies described above have focused on the effects of estrogen replacement in young, ovariectomized animals. These studies have demonstrated significant effects of estrogen replacement on basal forebrain cholinergic function, neurotrophin and neurotrophin receptor expression, and behavior, all of which provide important clues to potential mechanisms by which estrogen replacement might help to reduce the severity of AD-related dementia in postmenopausal women. Fewer studies have been conducted with aging animals or have attempted to assess the extent to which the loss of ovarian function with age contributes to brain aging and age-related cognitive decline. Gibbs (1998a) recently completed a study in which the effects of aging on cholinergic neurons were compared between male and female rats and between ovariectomized animals and age-matched, gonadally intact controls, in order to determine whether the loss of ovarian function contributes to a loss and/or impairment of basal forebrain cholinergic neurons beyond the effects of normal aging. Parameters which were measured included the number and size of ChAT-IR profiles and relative levels of ChAT and trkA mRNA in the MS and NBM. No significant changes in the number or size of ChAT-IR profiles were detected in the MS or NBM between 13 and 25 months of age or in response to ovariectomy. A significant decrease in relative levels of trkA mRNA was detected, however, in the MS of gonadally intact females, but not males, between 13 and 25 months of age (Fig. 5). In addition, significant decreases in ChAT and trkA mRNA were detected in the MS and NBM of animals sacrificed 6 months, but not 3 months, following ovariectomy relative to age-matched gonadally intact controls (Fig. 6). Short-term (3 days) treatment with E2 partially restored levels of ChAT mRNA in the MS, but not to the extent predicted by the effects of estrogen replacement

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FIG. 5. Changes in relative cellular levels of trkA mRNA detected in the MS of gonadally intact male (■) and female ( ) animals sacrificed at 13, 19, and 25 months of age. Bars represent the percentage change 6 SEM from the mean of the corresponding (same sex) 13-month-old animals. Note the decrease in trkA mRNA detected in the MS of females, but not males. *P , 0.05. Adapted from Gibbs (1998a).

Gibbs and Aggarwal

in young adults. The results from this study demonstrate that (a) trkA expression decreases in the MS of females, but not males, between 13 and 25 months of age and (b) long-term loss of ovarian function results in decreased levels of both ChAT and trkA mRNA in the MS and NBM beyond the effects of normal aging. These findings suggest that ovarian hormones play a role in maintaining normal levels of ChAT and trkA expression in the MS and NBM and that long-term hormone deprivation combined with aging produces significant adverse effects on basal forebrain cholinergic neurons. The physiological significance of the decreases in ChAT and trkA mRNA detected in the MS and NBM following the long-term loss of ovarian function still needs to be determined. We hypothesize that the decrease in trkA mRNA reflects a decrease in TrkA recep-

FIG. 6. Graphs summarizing changes in relative cellular levels of ChAT and trkA mRNA detected in the MS (A and B) and NBM (C and D) following ovariectomy. Animals were sacrificed 3 and 6 months following ovariectomy and compared to gonadally intact, age-matched controls. In addition, some animals which had been ovariectomized for 6 months received estrogen replacement for 3 days prior to being sacrificed. Bars represent the percentage change 6 SEM from the mean of the corresponding controls. Note the decreases in ChAT and trkA mRNA detected in the MS and NBM at 6 months, but not at 3 months, following ovariectomy. Note also that short-term estrogen replacement partially restored levels of ChAT mRNA in the MS (A). *P , 0.05. Adapted from Gibbs (1998a).

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tors on the cholinergic neurons and a decrease in the responsiveness of the cholinergic cells to endogenous NGF. It is notable that decreases in both ChAT (Perry et al., 1997, 1978; Whitehouse et al., 1982) and trkA (Mufson et al., 1996, 1997) have been observed in association with AD and are thought to contribute to the corresponding decline in cholinergic activity and cognitive function. Loy and co-workers have likewise demonstrated a significant decrease in NGF transport and p75NTR expression with age in rats, with a corresponding spatial memory impairment (Koh et al., 1989; Koh and Loy, 1988). One possibility is that decreases in NGF receptors lead to a reduction in trophic support for the cholinergic neurons, making them more susceptible to the effects of injury and disease. This would suggest that the decreases in cholinergic function, as well as the cholinergic cell loss, that are associated with AD may be exacerbated by decreases in trkA. We conclude, therefore, that the long-term loss of ovarian function has a negative impact on basal forebrain cholinergic neurons which, in turn, may contribute to the risk and severity of cognitive decline associated with aging and AD in postmenopausal women. The physiological significance and behavioral significance of these effects, and the extent to which they can be prevented or reversed by acute and/or chronic hormone replacement, are currently under study.

CONCLUSIONS The studies presented provide considerable evidence that estrogen plays a role in the normal physiological regulation of basal forebrain cholinergic neurons and that estrogen replacement can significantly enhance the functional status of cholinergic projections to the hippocampal formation and cortex. The increases in ChAT mRNA and protein, trkA mRNA, ChAT activity, and HACU are consistent with the estrogen-mediated increase in potassium-evoked ACh release recently described and with the ability for estrogen replacement to attenuate scopolamine-induced impairments in spatial and aversive tasks. Furthermore, recent studies showing that the long-term loss of ovarian function results in significant decreases in both ChAT and trkA mRNA in the MS and NBM beyond the effects of normal aging suggest that ovarian hormones play a role in maintaining normal levels of cholinergic function. These data also suggest that the loss of ovarian function with age has deleterious effects on basal forebrain cholinergic neurons which, over time, may contribute to the loss of cholinergic neurons associated with aging and AD. Collectively,

these findings suggest that, in rats, treatment with physiological levels of estrogen, or estrogen plus progesterone, can help to enhance and maintain the functional status of cholinergic neurons projecting to the hippocampus and cortex. We hypothesize that similar effects in humans could help to delay the decline in basal forebrain cholinergic function associated with aging and AD and thereby reduce the risk and severity of AD in postmenopausal women. The fact, however, that treatment with cholinesterase inhibitors is only marginally effective at attenuating AD-related cognitive decline, combined with the more robust positive effects of estrogen recently described, suggests that other estrogen-mediated effects must also play a role. Recent studies have demonstrated significant neuroprotective effects of estrogen and estrogen-related compounds on different cell types in vitro (Behl et al., 1997; Green et al., 1997a, 1997b), including the ability to protect against the toxic effects of b-amyloid (Gridley et al., 1997; Keller et al., 1997) and mutant presenilin-1 (Mattson et al., 1997) and to reduce cell death resulting from ischemia (Alkayed et al., 1998; Shi et al., 1997; Simpkins et al., 1997). Many of these effects appear to be related to protection against oxidative stress and/or increases in glucose transport and utilization (Bishop and Simpkins, 1992). Other potentially beneficial effects of estrogen include alterations in b-amyloid and lipoprotein metabolism (ApplebaumBowden et al., 1989; Jaffe et al., 1994; Kushwaha et al., 1991; Muesing et al., 1992; Xu et al., 1998), increased cerebral blood flow (Belfort et al., 1995; Ohkura et al., 1995b), anti-inflammatory effects (Bauer et al., 1992), antioxidant effects (Mooradian, 1993; Niki and Nakano, 1990), and neurotrophic effects (Gould, et al., 1990; McEwen, 1996; Woolley and McEwen, 1992). None of these mechanisms are mutually exclusive and it is likely that many of the effects are interrelated. The extent to which each contributes to the ability for estrogen replacement to reduce the risk and severity of AD-related dementia in women will need to be determined.

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