Impact of estrogen receptor agonists and model of menopause on enzymes involved in brain metabolism, acetyl-CoA production and cholinergic function.

Our goal is to understand how loss of circulating estrogens and estrogen replacement affect brain physiology and function, particularly in brain regions involved in cognitive processes. We recently conducted a large metabolomics study characterizing the effects of rodent models of menopause and treatment with estrogen receptor (ER) agonists on neurochemical targets in hippocampus, frontal cortex, and striatum. Here we characterize effects on levels of several key enzymes involved in glucose utilization and energy production, specifically phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate dehydrogenase. We also evaluated effects on levels of ß-actin and α-tubulin, choline acetyltransferase (ChAT) activity, and levels of ATP citrate lyase. All experiments were conducted in young adult rats. Experiment 1 compared the effects of ovariectomy (OVX), a model of surgical menopause, and 4-vinylcyclohexene diepoxide (VCD)-treatments, a model of transitional menopause, with tissues collected at proestrus and at diestrus. Experiment 2 used a separate cohort of rats to evaluate the same targets in OVX and VCD-treated rats treated with estradiol or with selective ER agonists. Differences in the expression of metabolic enzymes between cycling animals and models of surgical and transitional menopause were detected. These differences were model-, region- and time- dependent, and were modulated by selective ER agonists. Collectively, the findings demonstrate that loss of ovarian function and ER agonist treatments have differing effects in OVX vs. VCD-treated rats. Differences may help to explain differences in the effects of estrogen treatments on brain function and cognition in women who have experienced surgical vs. transitional menopause.

Use of the REVERT® total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states.

Western blot is routinely used to quantify differences in the levels of target proteins in tissues. Standard methods typically use measurements of housekeeping proteins to control for variations in loading and protein transfer. This is problematic, however, when housekeeping proteins also are affected by experimental conditions such as injury, disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative and perhaps superior method for conducting Western blot analysis of brain tissue homogenates from rats with distinct physiologically relevant gonadal hormone states. Tissues were collected from the hippocampus, frontal cortex, and striatum of young adult female rats that either were ovariectomized to model surgical menopause, or were treated with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause. Tissues also were collected from rats with a normal estrous cycle killed at proestrus when estradiol levels are high, and at diestrus when estradiol levels are low. Western blot detection of α-tubulin, ß-actin, and GAPDH was performed and were compared for sensitivity and reliability with a fluorescent total protein stain (REVERT®). Results show that the total protein stain was much less variable across samples and had a greater linear range than α-tubulin, ß-actin, or GAPDH. The stain was stable and easy to use, and did not interfere with the immunodetection or multiplexed detection of the housekeeping proteins. In addition, we show that normalization of our data to total protein, but not to GAPDH, revealed significant differences in α-tubulin expression in the hippocampus as a function of treatment, and that gel-to-gel consistency in measuring differences between paired samples run on multiple gels was significantly better when data were normalized to total protein than when normalized to GAPDH. These results demonstrate that the REVERT® total protein stain can be used in Western blot analysis of brain tissue homogenates to control for variations in loading and protein transfer, and provides significant advantages over the use of housekeeping proteins for quantifying changes in the levels of multiple target proteins.

Role of GPR30 in mediating estradiol effects on acetylcholine release in the hippocampus.

We have hypothesized that estradiol enhances basal forebrain cholinergic function and cognitive performance, at least in part, via activation of the novel estrogen receptor GPR30. Here we evaluated the effects of estradiol, G-1 (a selective GPR30 agonist), and tamoxifen (TAM; an ERα/ERß antagonist that also acts as a GPR30 agonist), on acetylcholine (ACh) release in the hippocampus, as well as the ability to block the effects of 17ß-estradiol (E) or TAM with the GPR30 antagonist G-15. Note that G-1 was included to evaluate the effects of selectively activating GPR30, whereas TAM was included to differentiate effects of E associated with activation of GPR30 vs. ERα or ERß. The study was designed to test effects on potassium-stimulated release, as well as on ACh release stimulated by feeding. Effects of feeding were included because the tasks we used previously to demonstrate beneficial effects of E on cognitive performance were motivated by food reward, and we hypothesized that E may enhance performance by increasing ACh release in association with that reward. Ovariectomized rats were treated for 1week, and ACh release was evaluated using in vivo microdialysis. In addition, rats were fed at the same time daily for several days and were fasted overnight prior to microdialysis. For each rat, ACh release was evaluated under basal conditions, in response to feeding, and in response to elevated potassium. Both feeding and elevated potassium increased ACh release in the hippocampus. In response to feeding, E, G-1, and TAM all significantly increased the percent change in release. The effects of E and TAM were blocked by G-15, and the effects of combining E+TAM did not differ significantly from the effects of E or TAM alone. In response to elevated potassium, E, and TAM significantly increased the percent change in ACh release. G-1 produced a slightly lesser effect. The effect of TAM was reduced by G-15, but the effect of E was not. These findings suggest that activation of GPR30 is both necessary and sufficient to account for the effects of E on ACh release associated with feeding. In contrast, activation of GPR30 appears to be sufficient, but may not be necessary for increased release associated with elevated potassium. The changes associated with feeding are consistent with the effects of E, G-1 and G-15 on acquisition of a spatial learning task previously described. These data confirm and extend previous reports, and support a hypothesis wherein E treatment can improve learning on specific tasks by activating GPR30 and enhancing ACh release in association with food reward.

The effect of the steroid sulfatase inhibitor (p-O-sulfamoyl)-tetradecanoyl tyramine (DU-14) on learning and memory in rats with selective lesion of septal-hippocampal cholinergic tract.

Dehydroepiandrosterone sulfate (DHEAS), is an excitatory neurosteroid synthesized within the CNS that modulates brain function. Effects associated with augmented DHEAS include learning and memory enhancement. Inhibitors of the steroid sulfatase enzyme increase brain DHEAS levels and can also facilitate learning and memory. This study investigated the effect of steroid sulfatase inhibition on learning and memory in rats with selective cholinergic lesion of the septo-hippocampal tract using passive avoidance and delayed matching to position T-maze (DMP) paradigms. The selective cholinergic immunotoxin 192 IgG-saporin (SAP) was infused into the medial septum of animals and then tested using a step-through passive avoidance paradigm or DMP paradigm. Peripheral administration of the steroid sulfatase inhibitor, DU-14, increased step-through latency following footshock in rats with SAP lesion compared to both vehicle treated control and lesioned animals (p<0.05). However, in the DMP task, steroid sulfatase inhibition impaired acquisition in lesioned rats while having no effect on intact animals. These results suggest that steroid sulfatase inhibition facilitates memory associated with contextual fear, but impairs acquisition of spatial memory tasks in rats with selective lesion of the septo-hippocampal tract.

Chronic treatment with a GPR30 antagonist impairs acquisition of a spatial learning task in young female rats.

We hypothesize that the beneficial effects of estradiol on cognitive performance may be mediated through GPR30, a putative membrane target of estrogens. Recently we showed that administration of a selective GPR30 agonist (G-1) to ovariectomized rats enhanced acquisition of a delayed matching-to-position (DMP) T-maze task and increased potassium-stimulated acetylcholine release in the hippocampus, similar to estradiol (E2) (Hammond et al., 2009). The present study tested whether treating with a selective GPR30 antagonist (G-15) would impair spatial learning in gonadally intact rats and in ovariectomized (OVX) rats treated with E2. As predicted, G-15 dose-dependently impaired DMP acquisition both in gonadally intact rats and in OVX rats treated with E2. G-15 specifically reduced the rate of acquisition, and this effect was associated with an increased predisposition to adopt a persistent turn. In contrast, G-15 alone at the highest dose had no significant effect on DMP acquisition in OVX controls. The effects were task dependent, as similar effects of G-15 were not observed in gonadally intact rats tested on an operant discrimination/reversal learning task motivated by the same food reward. This suggests that the effects on DMP acquisition were not due to effects on motivation for food. Effects of G-15 on DMP acquisition were similar to previously published work showing significant impairment produced by selective cholinergic denervation of the hippocampus. These data suggest that GPR30 can play an important role in mediating the effects of estradiol on spatial learning, possibly by mediating estradiol effects on basal forebrain cholinergic function.

Galanthamine plus estradiol treatment enhances cognitive performance in aged ovariectomized rats.

We hypothesize that beneficial effects of estradiol on cognitive performance diminish with age and time following menopause due to a progressive decline in basal forebrain cholinergic function. This study tested whether galanthamine, a cholinesterase inhibitor used to treat memory impairment associated with Alzheimer's disease, could enhance or restore estradiol effects on cognitive performance in aged rats that had been ovariectomized in middle-age. Rats were ovariectomized at 16-17 months of age. At 21-22 months of age rats began receiving daily injections of galanthamine (5mg/day) or vehicle. After one week, half of each group also received 17ß-estradiol administered subcutaneously. Rats were then trained on a delayed matching to position (DMP) T-maze task, followed by an operant stimulus discrimination/reversal learning task. Treatment with galanthamine+estradiol significantly enhanced the rate of DMP acquisition and improved short-term delay-dependent spatial memory performance. Treatment with galanthamine or estradiol alone was without significant effect. Effects were task-specific in that galanthamine+estradiol treatment did not significantly improve performance on the stimulus discrimination/reversal learning task. In fact, estradiol was associated with a significant increase in incorrect responses on this task after reversal of the stimulus contingency. In addition, treatments did not significantly affect hippocampal choline acetyltransferase activity or acetylcholine release. This may be an effect of age, or possibly is related to compensatory changes associated with long-term cholinesterase inhibitor treatment. The data suggest that treating with a cholinesterase inhibitor can enhance the effects of estradiol on acquisition of a DMP task by old rats following a long period of hormone deprivation. This could be of particular benefit to older women who have not used hormone therapy for many years and are beginning to show signs of mild cognitive impairment. Potential mechanisms for these effects are discussed.

Donepezil plus estradiol treatment enhances learning and delay-dependent memory performance by young ovariectomized rats with partial loss of septal cholinergic neurons.

Effects of estrogen therapy on cognitive performance appear to diminish with age and time following the loss of ovarian function. We hypothesize that this is due to a reduction in basal forebrain cholinergic function and that treatment with a cholinergic enhancer can reverse the effect. This study tested whether combining the cholinesterase inhibitor donepezil with estradiol treatment can enhance/restore estradiol effects on cognitive performance in young ovariectomized rats with selective lesions of septal cholinergic neurons. 192IgG-saporin was injected directly into the medial septum to produce selective cholinergic lesions. Rats were then treated with donepezil (Don, daily injections of 3mg/kg/day, i.p.) or vehicle, and then with 17ß-estradiol (E2, administered by silastic capsule implanted s.c.) or an empty capsule. Rats were trained on a delayed matching-to-position (DMP) T-maze task which previous studies have shown is sensitive to ovariectomy and estrogen replacement. Results show that neither estradiol nor donepezil alone significantly enhanced acquisition of the DMP task in rats with cholinergic lesions. Combination therapy was effective, however, depending on the severity of the lesion. Don+E2 significantly enhanced acquisition of the task in rats with partial lesions (<50% loss of cholinergic neurons), but not in rats with severe lesions. This effect was due largely to a reduction in perseverative behavior. Don+E2 also improved working memory in rats with partial lesions, as evidenced by significantly better performance than controls during increased intertrial delays. These findings suggest that even partial loss of septal cholinergic neurons can reduce effects of estrogen therapy on cognitive performance, and demonstrate that combining a cholinesterase inhibitor with estrogen therapy can help to restore beneficial effects on performance. We propose that combination therapy may have similar beneficial effects in women, particularly in older women who have not used estrogen therapy for many years and are beginning to show signs of cognitive impairment or early Alzheimer's disease.

GPR30 co-localizes with cholinergic neurons in the basal forebrain and enhances potassium-stimulated acetylcholine release in the hippocampus.

GPR30 is a novel, membrane-bound, G-protein coupled estrogen receptor (Filardo et al., 2002; Prossnitz et al., 2008). We hypothesize that GPR30 may mediate effects of estradiol (E2) on basal forebrain cholinergic neurons and cognitive performance. Recently we showed that G-1, a selective GPR30 agonist, enhances the rate of acquisition on a delayed matching-to-position (DMP) T-maze task (Hammond et al., 2009). In the present study, we examined the distribution of GPR30 in the rat forebrain, and the effects of G-1 on potassium-stimulated acetylcholine release in the hippocampus. GPR30-like immunoreactivity was detected in many regions of the forebrain including the hippocampus, frontal cortex, medial septum/diagonal band of Broca, nucleus basalis magnocellularis and striatum. GPR30 mRNA also was detected, with higher levels in the hippocampus and cortex than in the septum and striatum. Co-localization studies revealed that the majority (63-99%) of cholinergic neurons in the forebrain expressed GPR30-like immunoreactivity. A far lower percentage (0.4-42%) of GABAergic (parvalbumin-containing) cells also contained GPR30. Sustained administration of either G-1 or E2 (5 µg/day) to ovariectomized rats produced a nearly 3-fold increase in potassium-stimulated acetylcholine release in the hippocampus relative to vehicle-treated controls. These data demonstrate that GPR30 is expressed by cholinergic neurons in the basal forebrain, and suggest that activation of GPR30 enhances cholinergic function in the hippocampus similar to E2. This may account for the effects of G-1 on DMP acquisition previously reported.

GPR30 is positioned to mediate estrogen effects on basal forebrain cholinergic neurons and cognitive performance.

Beneficial effects of estrogen therapy on cognitive performance diminish with age and time following the loss of ovarian function. This has led to the 'Window of Opportunity' hypothesis, which states that estrogen therapy must be administered within a limited period of time following menopause in order to be effective. Effects of estrogen therapy on cognitive performance are due, at least in part, to the effects on cholinergic afferents innervating the hippocampus and cortex, and it has been suggested that the loss of estrogen effect with age and time following menopause is due to a substantial reduction in the function of these projections. The mechanisms that underlie the effects are not clear. GPR30 is a novel G-protein coupled estrogen receptor that is expressed in the brain and other tissues. Our recent studies show that GPR30 is expressed in areas of the brain important for spatial learning, memory, and attention. In addition, GPR30 in expressed by the vast majority of cholinergic neurons in the basal forebrain, and appears to be an important regulator of basal forebrain cholinergic function. We hypothesize that GPR30 plays an important role in mediating direct effects of estradiol on basal forebrain cholinergic neurons, with corresponding effects on cognitive performance. Hence, GPR30 may be an important target for developing new therapies that can enhance or restore estrogen effects on cognitive performance in older women. Here we briefly review the cholinergic hypothesis and summarize our findings to date showing effects of a GPR30 agonist and antagonist on basal forebrain cholinergic function and cognitive performance.

Studies of the localisation of kisspeptin within the pituitary of the rhesus monkey (Macaca mulatta) and the effect of kisspeptin on the release of non-gonadotropic pituitary hormones.

Kisspeptin neurones in the arcuate nucleus play a pivotal role in the regulation of hypothalamic gonadotrophin-releasing hormone (GnRH) secretion in higher primates. To examine whether kisspeptin also influences the function of the primate pituitary directly, two experiments were performed in adult male rhesus monkeys. First, the distribution of kisspeptin-containing cells in the pituitary was described using fluorescence immunohistochemistry. Second, the secretion of non-gonadotrophin adenohypophysial hormones [growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH)] and cortisol in response to i.v. kisspeptin administration was examined. Eight animals were deeply anaesthetised and transcardially perfused with 4% paraformaldehyde. Fluorescence immunohistochemistry was performed on 25-microm thick free-floating pituitary sections to localise immunopositive kisspeptin cells and to examine their relationship with immunostaining for luteinising hormone (LH), follicle-stimulating hormone, GH, prolactin, alpha-melanocyte-stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH) and GnRH. Kisspeptin cells were found in the intermediate lobe of all animals and, in four monkeys, this neuropeptide was also observed in cells scattered in the periphery of the anterior lobe. Kisspeptin colocalised with alpha-MSH-immunopositive cells in the intermediate lobe and, in 50% of the monkeys, with ACTH-immuunopositive cells in the periphery of the adenohypophysis. There was no evidence for colocalisation of kisspeptin with gonadotrophs, somatotrophs or lactotrophs. Beaded kisspeptin axons were observed in the neural lobe. In addition, assay of plasma samples that had been collected for a previous study documenting kisspeptin-10-induced LH release in male monkeys revealed that kisspeptin administration failed to influence circulating concentrations of GH, prolactin, TSH and cortisol. Release of all four of these non-gonadotrophic hormones, however, was stimulated markedly by NMDA, which is considered to act centrally. Although the morphological findings obtained in the present study are consistent with the notion that kisspeptin may act directly at the level of the pituitary, the nature of such an action remains to be defined.

Chronic treatment with estrogen receptor agonists restores acquisition of a spatial learning task in young ovariectomized rats.

Previous work has shown that continuous estradiol replacement in young ovariectomized rats enhances acquisition of a delayed matching-to-position (DMP) T-maze task over that of ovariectomized controls. The mechanism by which estradiol confers this benefit has not been fully elucidated. This study examined the role of selective estrogen receptor agonists of ERalpha, ERbeta, and GPR30 in the enhancement of spatial learning on a DMP task by comparing continuous estradiol replacement with continuous administration of PPT (an agonist of ERalpha), DPN (an agonist of ERbeta), or G-1 (an agonist of GPR30) relative to gonadally intact and ovariectomized vehicle-treated controls. It was found that ovariectomy impaired acquisition on this task, whereas all ER selective agonists restored the rate of acquisition to that of gonadally intact controls. These data suggest that estradiol can work through any of several estrogen receptors to enhance the rate of acquisition on this task.

Donepezil treatment restores the ability of estradiol to enhance cognitive performance in aged rats: evidence for the cholinergic basis of the critical period hypothesis.

Recent studies suggest that the ability of estradiol to enhance cognitive performance diminishes with age and/or time following loss of ovarian function. We hypothesize that this is due, in part, to a decrease in basal forebrain cholinergic function. This study tested whether donepezil, a cholinesterase inhibitor, could restore estradiol effects on cognitive performance in aged rats that had been ovariectomized as young adults. Rats were ovariectomized at 3 months of age, and then trained on a delayed matching to position (DMP) T-maze task, followed by a configural association (CA) operant condition task, beginning at 12-17 or 22-27 months of age. Three weeks prior to testing, rats started to receive either donepezil or vehicle. After one week, half of each group also began receiving estradiol. Acclimation and testing began seven days later and treatment continued throughout testing. Estradiol alone significantly enhanced DMP acquisition in middle-aged rats, but not in aged rats. Donepezil alone had no effect on DMP acquisition in either age group; however, donepezil treatment restored the ability of estradiol to enhance DMP acquisition in aged rats. This effect was due largely to a reduction in the predisposition to adopt a persistent turn strategy during acquisition. These same treatments did not affect acquisition of the CA task in middle-aged rats, but did have small but significant effects on response time in aged rats. The data are consistent with the idea that estrogen effects on cognitive performance are task specific, and that deficits in basal forebrain cholinergic function are responsible for the loss of estradiol effect on DMP acquisition in aged ovariectomized rats. In addition, the data suggest that enhancing cholinergic function pharmacologically can restore the ability of estradiol to enhance acquisition of the DMP task in very old rats following long periods of hormone deprivation. Whether donepezil has similar restorative effects on other estrogen-sensitive tasks needs to be explored.

Cholinergic lesions produce task-selective effects on delayed matching to position and configural association learning related to response pattern and strategy.

192IgG-saporin (SAP) was used to selectively destroy cholinergic neurons in the rostral basal forebrain (e.g., medial septum (MS) and vertical limb of the diagonal band of Broca (VDB)) and/or the caudal basal forebrain (e.g., nucleus basalis magnocellularis (NBM)) of ovariectomized Sprague-Dawley rats. The effects of these lesions on two different cognitive tasks, a delayed matching to position (DMP) T-maze task, and a configural association (CA) operant conditioning task, were evaluated and compared. Injecting SAP into either the MS or NBM significantly impaired acquisition of the DMP task. Analysis showed that the effects were due largely to an affect on response patterns adopted by the rats during training, as opposed to an effect on working memory performance. Notably, the impairment in DMP acquisition did not correlate with the degree of cholinergic denervation of the hippocampus. Despite the deficit, most animals eventually learned the task and reached criterion; however by the end of training, controls and animals that received SAP into either the MS or NBM appeared more likely to use an allocentric place strategy to solve the task, whereas animals that received SAP into both the MS and NBM were more likely to use an egocentric response strategy. Cholinergic lesions also produced a small but significant affect on acquisition of the CA task, but only with respect to response time, and only in the SAP-NBM-treated animals. SAP-NBM lesions also produced small but significant impairments in both the number of responses and response time during the acquisition of simple associations, possibly reflecting an effect on alertness or attention. Notably, the effects on CA acquisition were small, and like the effects on DMP acquisition did not correlate with the degree of cholinergic denervation of the hippocampus. We conclude that selective basal forebrain cholinergic lesions produce learning deficits that are task specific, and that cholinergic denervation of either the frontal cortex or hippocampus can affect response patterns and strategy in ways that affect learning, without necessarily reflecting deficits in working memory performance.

Effects of long-term hormone treatment and of tibolone on monoamines and monoamine metabolites in the brains of ovariectomised, Cynomologous monkeys.

The effects of long-term hormone treatment on monoamines and monoamine metabolites in different regions of the primate brain were examined and compared. Ovariectomised Cynomologous monkeys received daily oral administration of either conjugated equine oestrogens (CEE), CEE + medroxyprogesterone acetate (MPA), or a low or high dose of tibolone, for a period of 2 years. Tissue punches collected from frozen sections through various regions of the forebrain, midbrain, and hindbrain were assayed for levels of dopamine, dihydroxyphenylacetic acid (DOPAC), serotonin, 5-hydroxyindole acetic acid (5-HIAA), and norepinephrine by high-performance liquid chromatography. Few differences between hormone-treated animals and ovariectomised controls were observed. No statistically significant effects of CEE relative to controls were detected in any of the seven brain regions analysed. Animals treated with CEE + MPA showed significant reductions in 5-HIAA in the dorsal raphe nucleus, a significant reduction in dopamine in the hypothalamus, and a significant reduction in serotonin (5-HT) levels in area 8AD of the frontal cortex. Similar to CEE, no significant effects of tibolone relative to controls were detected; however, animals treated with high-dose tibolone showed a decrease in 5-HT levels in the frontal cortex that approached significance and was similar to the effect of CEE + MPA. Collectively, the findings suggest that long-term oral administration of these compounds has relatively few effects on the levels of dopamine, serotonin, and their primary metabolites in the primate brain. This differs from the significant effects on serotonergic and dopaminergic systems detected following parenteral treatment with oestradiol and progesterone, and likely reflects differences between the effects of treating with CEE + MPA versus oestradiol and progesterone on brain monoaminergic systems.

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity.

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.

Effects of ageing and long-term hormone replacement on cholinergic neurones in the medial septum and nucleus basalis magnocellularis of ovariectomized rats.

Ovariectomized aged rats, some of which received long-term hormone replacement with oestrogen or oestrogen plus progesterone, were evaluated for the number and size of basal forebrain cholinergic neurones, as well as relative levels of choline acetyltransferase (ChAT) and trkA mRNA, in order to determine whether effects on basal forebrain cholinergic cell survival and function correspond with differences in cognitive performance previously described. The results show that ageing combined with long-term loss of ovarian function produced substantial reductions in the levels of ChAT and trkA mRNA in the medial septum and nucleus basalis magnocellularis, relative to much younger ovariectomized controls. In contrast, no significant effects on the number or size of the cholinergic cells were detected, indicating that loss of ovarian function does not cause a loss of cholinergic neurones with age. Long-term hormone replacement had no apparent effect on the number of ChAT-positive neurones detected, and did not prevent the reductions in ChAT and trkA mRNA associated with ovariectomy and ageing. Collectively, the data suggest that ageing combined with long-term loss of ovarian function has a severe negative impact on basal forebrain cholinergic function, but not on cholinergic cell survival per se.

Effects of long-term hormone replacement and of tibolone on choline acetyltransferase and acetylcholinesterase activities in the brains of ovariectomized, cynomologus monkeys.

We examined long-term effects of low and high doses of tibolone, conjugated equine estrogens, and conjugated equine estrogens plus medroxyprogesterone acetate on choline acetyltransferase and acetylcholinesterase activities within different regions of the brain in cynomologus monkeys. All treatments were administered for 2 years. None of the treatments produced significant increases in either choline acetyltransferase or acetylcholinesterase in any of eight brain regions analyzed. In contrast, treatment with conjugated equine estrogens plus medroxyprogesterone acetate, but not conjugated equine estrogens alone, produced significant reductions in both choline acetyltransferase and acetylcholinesterase in the medial septum/diagonal band of Broca compared with untreated controls. Treatment with tibolone also resulted in significant reductions in both choline acetyltransferase and acetylcholinesterase in the medial septum/diagonal band of Broca, and this effect was dose-related. These findings are the first to report the effects of long-term therapies used by postmenopausal women on cholinergic measures in the primate brain. The findings are consistent with recent reports in rats, and suggest that any positive effects of long-term estrogen or hormone replacement therapy on cognitive processes are probably not due to significant effects on choline acetyltransferase or acetylcholinesterase activities.

Effects of gonadal hormone replacement on measures of basal forebrain cholinergic function.

The effects of different hormone replacement regimens on basal forebrain cholinergic function were examined by measuring changes in choline acetyltransferase activity and high affinity choline uptake in adult, ovariectomized, rats. Increases in choline acetyltransferase activity were detected in the frontal cortex (20. 1%) and olfactory bulbs (30.4%) following two weeks, but not four weeks, of repeated treatment with estrogen plus progesterone. Increases in high affinity choline uptake were detected in the frontal cortex (39.5-55.1%), hippocampus (34.9-48.9%), and olfactory bulbs (29.9%) after two weeks, but not four weeks, of either continuous estrogen administration, repeated progesterone administration, or repeated treatment with estrogen plus progesterone. Repeated administration of estradiol (2-25 microg/250 g body weight) for two or four weeks, and continuous estrogen administration for four weeks and six months, produced no significant changes in choline acetyltransferase activity or high affinity choline uptake in the hippocampus, frontal cortex or olfactory bulbs. Continuous estrogen administration for 13 months produced a significant decrease in high affinity choline uptake across all regions with the largest effect (-28.1%) detected in the hippocampus. The findings demonstrate that short-term treatment with estrogen and/or progesterone can significantly enhance cholinergic function within specific targets of the basal forebrain cholinergic projections. Most important is the fact that the effects varied considerably according to the manner and regimen of hormone replacement and did not persist with prolonged treatment. These findings could have important implications for the effective use of hormone replacement strategies in the prevention and treatment of Alzheimer's disease and age-related cognitive decline in women.

Estrogen replacement does not prevent the loss of choline acetyltransferase-positive cells in the basal forebrain following either neurochemical or mechanical lesions.

Recent studies have shown that estrogen replacement can enhance the functional status of basal forebrain cholinergic neurons. Studies have also shown that estrogen has neuroprotective effects both in vitro and in vivo on a variety of cells and against a variety of insults. The present study examined the ability of estrogen replacement to protect basal forebrain cholinergic neurons from the effects of neurochemical and mechanical injury. Ovariectomized Sprague-Dawley rats received either estrogen replacement or sham surgery, and then received either a unilateral injection of ibotenic acid into the nucleus basalis magnocellularis, or unilateral transection of the fimbria fornix. Cholinergic neurons in the medial septum and nucleus basalis were detected and quantified using immunohistochemical techniques. The data show that neither 3 weeks nor 13 weeks of continuous estrogen replacement prevented the loss of choline acetyltransferase (ChAT)-containing cells in the nucleus basalis following a unilateral injection of ibotenic acid. Likewise, estrogen replacement did not prevent a decrease in ChAT-positive cells detected in the medial septum following unilateral transection of the fimbria fornix. Notably, increased numbers of ChAT-positive cells were detected in the contralateral nucleus basalis, and in the ipsilateral and contralateral medial septum, at 2 weeks following a unilateral injection of ibotenic acid into the nucleus basalis; however, these effects were not related to hormone treatment. These data suggest that estrogen replacement does not protect cholinergic neurons in the medial septum and nucleus basalis from the effects of excitotoxic or mechanical injury.

Oestrogen and the cholinergic hypothesis: implications for oestrogen replacement therapy in postmenopausal women.

Cholinergic deficits in the basal forebrain, hippocampus and cortex are thought to contribute to the risk and severity of cognitive decline associated with ageing and Alzheimer's disease. Work in our laboratory has demonstrated that in rats, basal forebrain cholinergic neurons are affected by physiological fluctuations in circulating oestrogen and progesterone, and that long-term loss of ovarian function produces decreases in cholinergic parameters and nerve growth factor receptor (trkA) mRNA beyond the effects of normal ageing. Conversely, short-term treatment with oestrogen or oestrogen plus progesterone produces increases in cholinergic parameters and trkA, as well as increases in potassium-stimulated acetylcholine release, that are consistent with an increase in basal forebrain cholinergic function. These findings are consistent with recent studies showing the ability of oestrogen and progesterone replacement to enhance spatial memory and reduce performance deficits associated with hippocampal cholinergic impairment. We hypothesize that similar effects of the ovarian hormones on basal forebrain cholinergic neurons in humans may contribute to the effects of hormone replacement on cognitive processes that have recently been described, and to the ability of oestrogen replacement to reduce the risk and severity of Alzheimer's-related dementia in postmenopausal women.