Estrogenic modulation of brain activity:implications for schizophreniaand Parkinson's disease

Abstract

Evidence suggests the estrogens may play a role in various mental and neurodegenerative diseases. We review the evidence implicating estradiol in schizophrenia and Parkinson's disease. Epidemiologic and clinical studies on the effects of estrogens in schizophrenia are surveyed, and animal studies and in vitro models of the modulatory effects of estrogens on neurotransmitters associated with schizophrenia (i.e., dopamine, serotonin, glutamate) are reviewed. Epidemiologic and clinical data suggesting a role for estrogens in Parkinson's disease and in vivo and in vitro models demonstrating neuroprotective effects of estrogens are then examined. Despite the numerous animal studies on the effects of estrogens in the brain, clinical data are sparse and often contradictory. Compounds with more specific and potent estrogenic activity in the brain are required to further research efforts in this area. Possible candidates are the selective estrogen receptor modulators (SERMs), whose agonist or antagonist properties depend on the target tissue. The effects of various SERMs in the brain are reviewed, and our novel findings on the effects of SERMs on 5-HT_2A receptors in the rat cortex and nucleus accumbens are presented. We suggest that drugs with estrogenic activity in the brain may have therapeutic potential, either by modulating brain neurotransmission or through neuroprotective activity.

Introduction

The sex steroid hormones (estrogens and androgens) have been shown to exert profound effects on brain differentiation, neural plasticity and central neurotransmission during development.^1,2 Accumulating evidence supports a modulatory role of these steroids in the brain³ and, more recently, their prime importance in the normal maintenance of brain function during aging.⁴ Beneficial effects of sex steroids in several mental diseases, such as schizophrenia, depression, premenstrual syndrome, postnatal depression and Gilles de la Tourette's syndrome, have also been reported.^5,6 Estradiol may also be beneficial in neurodegenerative diseases such as Alzheimer's (AD).^7,8,9,10 With the increased life expectancy and the average age of menopause remaining constant, women can now expect to live up to one half of their adult lives after menopause.¹¹ Women represent more than half the population, and more than 60 million women in the world now use oral contraceptives.¹² The recent determination that long-term use of oral contraceptives is safe, the lower steroid concentrations now in use and their reported safety for women over the age of 35 years of age¹² support the possible use of sex steroids to improve drug treatments and the search for new applications as drugs for central nervous system (CNS) disorders.

The importance of estrogen in the development and maintenance of the female reproductive system has led to the pharmaceutical development of a variety of steroidal and nonsteroidal compounds that interact with the estrogen receptor as contraceptives and are used to treat breast cancer, uterine dysfunction and other reproductive disorders. A role of these estrogen receptor-directed drugs in nontarget tissues such as the skeleton, the cardiovascular system and the CNS has also been recognized.^13,14 Hormone replacement therapy, when given after menopause, is reported to be beneficial for osteoporosis, coronary artery disease, depression¹⁵ and AD.^7,9 However, hormone replacement therapy also has less desirable effects, such as increasing risk of breast and endometrial cancer.¹⁶

Given that the number of women at midlife and beyond who may seek professional advice regarding hormone replacement therapy is expected to double over the next 2 decades,^17,18 finding new drugs that effectively reduce the symptoms associated with menopause and do not increase the cancer risk, is of great interest. Tamoxifen, a mixed estrogen agonist-antagonist, currently used in the prevention and treatment of breast cancer, is a first-generation selective estrogen receptor modulator (SERM); it acts as an estrogen antagonist in mammary tissue but mimics the effects of estrogen in other tissues.¹⁹ Raloxifene, a second-generation SERM, acts as an estrogen antagonist in mammary and uterine tissues and an agonist in bone and cholesterol metabolism.¹⁹ The activities of tamoxifen and raloxifene have been reviewed with respect to their effects in nontraditional target tissues (i.e., skeletal and cardiovascular systems),²⁰ but there is currently little relevant biological data available to support the use of these compounds for CNS diseases.

We review the effects of estrogens on mental and neurodegenerative diseases, with a focus on schizophrenia and PD because of their prevalence and the documented effects of steroids in these diseases. We review data from clinical studies and molecular investigations on the effects of estrogens in the brain and present novel findings with SERMs. We suggest drugs with estrogenic activity in the brain may have therapeutic potential, either by modulating brain neurotransmission or through neuroprotective activity.

Estrogens and schizophrenia

Schizophrenia is a mental illness characterized by episodic positive symptoms such as delusions, hallucinations, paranoia and psychosis and may include persistent negative symptoms such as flattened affect, impaired attention, social withdrawal and cognitive impairment.^21,22 Epidemiologic and clinical evidence suggests an influence of estrogens on the vulnerability threshold for schizophrenia.^23,24 Although early studies suggested the incidence of schizophrenia in men and women was about equal,^25,26 more recent studies indicate incidence rates are higher in men.^27,28

It has been suggested that estrogen may act as a protective factor in women; the age of onset of schizophrenia is significantly later in women than in men, with a second peak of onset larger and later in women after 40–45 years of age.^22,29 As well, levels of psychopathology have been observed to fluctuate with phases of the menstrual cycle.^30,31 In women with schizophrenia, relapse rates are higher when estrogen levels are low during the menstrual cycle, whereas relapse is low when estrogen levels are high.^32,33 Higher rates of relapse in women with schizophrenia are also observed during the postpartum period (low estrogens),³⁴ whereas relapse is low during pregnancy (high estrogens).³³

Sex differences in the clinical expression of schizophrenia have also been observed. For example, women with schizophrenia have a higher prevalence of auditory hallucinations than men.^35,36,37 Women with schizophrenia have been described as “hallucinatory, illusionary,” with symptoms mimicking affective disorders, whereas men tend to become “dull, autistic” and have an amotivational syndrome with loss of organization and regulation.^38,39,40 Women appear to require lower doses of neuroleptics than men in the acute phase of illness and in the maintenance stage⁴¹ and respond better to both psychosocial and pharmacological treatments.⁴² In a long-term follow-up study, women tended, more than men, to deteriorate in the perimenopausal period when there is a marked fall in estrogen levels.⁴³ Overall, men with schizophrenia have an earlier age of onset, are admitted to hospital earlier and demonstrate a more typical picture and poorer prognosis than women.⁴⁴

There have been few treatment studies investigating the effects of estrogen in patients with schizophrenia. One study assessed the response of women with acute psychotic symptoms to either an 8-week therapy of estradiol and a neuroleptic or the neuroleptic treatment alone.⁴⁵ The results suggested that estradiol may have antipsychotic properties or act as a catalyst for neuroleptic responsiveness in women with schizophrenia. A case study also reported a beneficial effect of estradiol on psychotic symptoms in a woman with recurrent psychotic and affective symptoms.⁴⁶ It has been suggested that the beneficial effects of estrogen in schizophrenia may be associated with a modulation of dopaminergic activity.²⁴ After menopause, women require higher doses of neuroleptics, the risk for dyskinesias increases and more severe forms of dyskinesias are more common.⁴⁷ Some clinical studies report beneficial effects of estradiol^{48,49,50,51,52} in women with tardive dyskinesia, whereas others do not.^53,54,55

Despite abundant clinical and epidemiologic literature on the beneficial effect of endogenous estrogens, there are few studies of estrogen therapy in schizophrenia and contradictory findings for tardive dyskinesia. Although the effects of estrogens on cognitive impairment in schizophrenia have not been systematically reported, improvement has been reported with hormone replacement therapy in normal women.⁵⁶ This warrants further studies with drugs with estrogen-like potency that are more selective for the brain.

Estrogenic modulation of neurotransmitters implicated in schizophrenia

Schizophrenia has long been attributed to abnormal hyperactivity of central dopaminergic mechanisms on the basis of the observation that drugs effective in treating this illness share the common feature of blocking dopamine (DA) receptors.^57,58 With the advance of brain imaging techniques, direct evidence suggestive of dysfunctional dopaminergic transmission in schizophrenia has emerged. This includes measures of striatal [¹⁸F]fluorodopa or [¹¹C]dopa accumulation in patients with schizophrenia^59,60,61,62 and synaptic dopamine concentrations measured indirectly by a decrease in binding potential of [¹¹C]raclopride or [¹²³I]IBZM after amphetamine challenge.^63,64,65 A recent single-photon emission computed tomography study demonstrated increased baseline occupancy of D₂ receptors by dopamine in schizophrenia.⁶⁶ This finding provides direct evidence of increased stimulation of D₂ receptors by dopamine in schizophrenia, consistent with phasic activity of dopaminergic neurons.

In experimental animals, sex differences in brain dopaminergic activity, as well as estrous cycle variations, acute effects of a physiological dose of estrogens and chronic effects of high doses of this hormone have been reported.^6,67 Estradiol may modulate dopaminergic activity at various steps of DA transmission (i.e., DA release and metabolism, pre- and postsynaptic DA receptors and transporter).⁶ Both pro- and antidopaminergic effects of estrogens have been documented in animals. These opposing activities may be related to factors such as sex-specific responses, dose of estrogen, short- versus long-term administration and time after administration.^6,67 A single low dose of estradiol and estrous cycle variations support a stimulatory role of estrogen on dopaminergic activity. This stimulatory effect on DA release, metabolism, receptor, transporter and behaviour is rapid and well documented in the striatum and nucleus accumbens of adult rats. It is stereospecific and, in vitro, shown to be antagonized by the pure estrogen receptor antagonist ICI 182,780 but not by tamoxifen, which has mixed agonist and antagonist activity at this receptor.⁶⁷ Changes in DA receptors and transporter after estradiol are not paralleled by changes of their expression.^6,68 The effects are rapid, as fast as 2 minutes for the stereospecific effect of estradiol on DA release in the nucleus accumbens.⁶⁹ Furthermore, estrogen was shown within seconds to decrease L-type Ca²⁺ currents in striatal neurons, and this was also observed with estrogen conjugated with bovine serum albumin to prevent entry in the cells.⁷⁰ Morphology of rat striatal membranes, as measured by freeze fracture, varies during the estrous cycle, and this is correlated with striatal estradiol and progesterone concentrations.⁷¹ The collective evidence suggests that it is doubtful that these rapid effects of estrogen in the striatum are mediated by genome-activating estrogen receptors, but likely occur at the membrane surface.^6,72,73 Additional support for nongenomic mechanisms comes from a study demonstrating that long-term administration of high doses of estradiol increases the density of DA receptors and transporters.⁶ It has been suggested that the hormonal modulation of the striatum may have evolved to facilitate reproductive success in female rats by enhancing pacing behaviour.⁶⁷ In humans, the hormonal modulation of the striatum may have relevance in addiction and mental disorders associated with abnormalities in this brain region.

The efficacy of atypical antipsychotics such as clozapine and risperidone, which are mainly serotonergic (5-HT) 2A receptor antagonists,⁷⁴ has led to theories of serotonergic involvement in schizophrenia. This system, in addition to the DA system, may be important for an optimal antipsychotic response.⁷⁵ Several studies have reported a decrease in 5-HT_2A receptor density in the frontal cortex of patients with schizophrenia,^76,77,78 although others report no change.^79,80 In a study of postmenopausal women using positron emission tomography, estradiol and progesterone administration increased 5-HT_2A receptor binding potential in widespread areas of the cerebral cortex.⁸¹ Studies in rats have also shown that estradiol modulates brain serotonergic activity, increasing 5-HT_2A receptors and the serotonin transporter.⁵ Short- and long-term estradiol treatments increase the density of 5-HT_2A receptors, mainly in the anterior cingulate and anterior frontal cortices.^82,83

An alternative explanation for the pathogenesis of schizophrenia, the glutamate dysfunction hypothesis, originated from the observation that phencyclidine, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor, produces a psychotomimetic state in normal humans that is similar to schizophrenic psychosis.⁸⁴ Ketamine and MK-801, 2 other noncompetitive antagonists of NMDA receptors, also produce schizophrenic symptoms^85,86,87 and exacerbate psychosis in patients with schizophrenia.⁸⁸ Several glutamate-related neurochemical abnormalities are reported in postmortem tissue of patients with schizophrenia⁸⁹ that provide evidence that schizophrenia is associated with reduced glutamate release in the hippocampus and frontal cortex.

Recently, we demonstrated that estradiol has regionally specific effects on glutamate receptors in the rat brain. Estradiol treatment increases NMDA receptors in the CA1 region and dentate gyrus of the hippocampus and decreases these receptors in the frontal cortex and dorsal part of the caudate putamen.⁹⁰ In addition, estradiol has no effect on AMPA receptor binding density in the hippocampus, but it decreases receptor density in the frontal cortex, caudate putamen and nucleus accumbens.⁹⁰ These results confirm and extend those previously published on the effect of short-term treatment with estradiol on rat hippocampal NMDA and AMPA receptors.^91,92,93 In schizophrenia, disruption of glutamate-mediated transmission involving the NMDA receptor is suspected to occur in the intrahippocampal circuitry, interrupting information flow within hippocampus and diminishing excitatory neuronal activity in its glutamatergic efferent pathway.⁸⁹ Recently, this concept has been supported by the development of mice with reduced NMDA expression that display behaviours related to schizophrenia.⁹⁴ These behavioural alterations are similar to those observed in pharmacologically induced animal models of schizophrenia and can be improved with haloperidol or clozapine treatment.⁹⁴

Estrogens and Parkinson's disease

Parkinson's disease (PD), the second most common neurodegenerative disorder, is mainly characterized by the progressive and selective depletion of DA synthesizing neurons in the substantia nigra pars compacta.⁹⁵ The regulation of dopaminergic neurotransmission in the basal ganglia by estrogens is now well established.^6,67,96 DA synthesis and release, DA receptor, DA uptake site, as well as catechol-o-methyl transferase (COMT) expression have been shown to be regulated by ovarian steroids.^6,67,97

The influence of sex steroids on movement disorders, either during the menstrual cycle or after hormone therapy in postmenopausal women, has been investigated.^6,96,98,99 Estrogens, in particular, have been shown to modulate symptoms of PD and levodopa-induced dyskinesias.^{6,100,101,102} Many of these studies are based on the effect estrogen replacement therapy has on the response of parkinsonian patients to standard levodopa therapy and generally support an antidopaminergic effect of estrogens on parkinsonian symptoms.¹⁰¹ However, a recent study suggests that estrogen replacement therapy might be beneficial to women with early PD before the initiation of levodopa.¹⁰³ Estrogen was reported to improve motor disability in postmenopausal women with PD with motor fluctuations.¹⁰⁴ However, a slight antiparkinsonian effect¹⁰⁵ and no effect¹⁰⁶ of 17β-estradiol treatment has been reported in clinical trials. Hence, in humans, the issue of whether estrogens stimulate or inhibit the dopaminergic system is still open. Animal studies also support both pro- and antidopaminergic activities of estrogens; it is possible that these effects might be dissociated with SERMs. The effect of newer SERMs such as raloxifene on parkinsonism clearly deserves investigation.

In addition to the modulatory effect of estrogens on dopaminergic activity, evidence also suggests estrogens may possess neuroprotective activity in PD. Although numerous studies report a greater prevalence and incidence of PD in men than in women,^{107,108,109,110,111,112,113,114,115} no such sex difference has also been reported.¹¹⁶ As well, sex differences have been reported for the evolution of symptoms and response to levodopa treatment.¹¹⁷ Moreover, estrogen replacement therapy may decrease the risk of developing dementia.¹¹³

Current concepts of the cause of PD suggest a role for both genetic and environmental influences.^{118,119,120,121} Common to a variety of potential causes of nigral cell degeneration in PD is the involvement of oxidative stress, exitotoxicity and metabolic compromise.^118,122 Potential neuroprotective agents such as ovarian steroids may act at multiple levels to exert their effects, both by genomic action on transcription of genes related to cell survival and antiapoptotic proteins and nongenomic actions such as a NMDA receptor antagonists, antioxidants, interaction with the MAPK signalling pathway and the phosphatidylinositol 3-kinase cascade.^123,124

Estrogens may be neurotoxic during development and neuroprotective in aging.¹²⁵ In primates, estrogen was shown to be essential to maintain nigrostriatal neurons,¹²⁶ intact females having a higher density of dopamine cells than males and ovariectomized females. Estradiol replacement early after ovariectomy prevented the neuronal loss.¹²⁶

The potential of 17β-estradiol to alter neuronal survival may depend on the estrogen receptor subtype present, with the α subtype having a neuroprotective effect and the β subtype mediating the induction of apoptosis in neuronal cells.¹²⁷ Nielsen and colleagues¹²⁷ also observed that in cells expressing both estrogen receptor subtypes, estradiol was neuroprotective, and that estrogen-induced apoptosis (through β subtype) required the expression of Fas- and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevented this effect. These authors note that the microenvironments in the brain play an important role in the response to estradiol and may partly explain the different responses of developing and aging brains.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that selectively destroys dopaminergic neurons of the substantia nigra pars compacta in humans and experimental animals.^128,129,130 MPTP is therefore a useful drug to investigate the neurodegeneration process associated with idiopathic PD.¹³⁰ Sex differences are observed in mice bearing MPTP-induced nigrostriatal lesions; the neurotoxic effect of MPTP and methamphetamine is greater in male than in female mice.^131,132 Studies show that estrogen pretreatment prevents the dramatic depletion of striatal DA induced by MPTP^{132,133,134,135,136} and methamphetamine.¹³⁷

Neuroprotection of dopaminergic neurons against degeneration is a possible explanation of the effect of estrogens on striatal DA.¹³⁸ Changes in the metabolism of residual neurons to increase available DA in the striatum may alternatively explain these data. For example, 17β-estradiol, but not 17α-estradiol, rapidly induces a dose-dependent increase in striatal tyrosine hydroxylase activity in ovariectomized rats.¹³⁹ Reduction of COMT activity may also be a mechanism by which estrogen increases DA striatal content in MPTP-treated mice.⁹⁷

Other data also suggest a neuroprotective action of estrogens. The glial fibrillary acidic protein (GFAP) is localized in the astrocyte and used as a marker of neuronal damage.¹³² Miller et al¹³² observed that estrogen replacement reduces the GFAP elevation induced by MPTP, and this may imply actual neuroprotection.¹³² Low endogenous levels of estrogens in intact female mice appear to be sufficient to provide the neuroprotection observed.¹³²

The effect of estradiol may be indirect and involve other substances, such as the neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), whose secretion is amplified by estrogens.^140,141 For instance, the proto-oncogene Bcl-2 protects neurons from MPTP neurotoxicity, perhaps by mechanisms which involve inhibition of apoptosis and antioxidant activity.^142,143 Estrogens increase the number of Bcl-2 immunoreactive cells in rat brain, and this may be linked to its possible neuroprotective activity.¹⁴⁴

The beneficial effects of estrogens that were observed with 17β-estradiol and not with 17α-estradiol suggest a stereospecific effect, possibly via the classical intracellular estrogen receptor.¹³⁵ Expression of mRNA transcripts for both α and β estrogen receptors was detected in the substantia nigra,¹⁴⁵ whereas immunohistochemical and binding studies have not confirmed the presence of any estrogen receptor protein in this structure.¹⁴⁶ Classical estrogen receptors are thought to be located within the cells, but recent evidence suggests that the existence of membrane-bound estrogen receptors should not be ruled out.^70,147,148 A stereospecific effect of 17β-estradiol (and a membrane impermeable estrogen – bovine serum albumin construct) to stimulate neurite growth of midbrain dopaminergic neurons, which is inhibited by antagonists of cAMP/protein kinase A and calcium signalling pathways but not by the estrogen receptor antagonist ICI 182,780, was recently reported.¹⁴⁹ These results support nongenomic membrane effects of estradiol.

Cell culture studies reveal neuroprotective action of 17β-estradiol and 17α-estradiol against various insults.^{150,151,152,153} Tamoxifen, used for its estrogen receptor antagonist activity, did not block estradiol's protection from oxidative stress in mesencephalic dopaminergic neurons.¹⁵² By contrast, the same authors noted that the estrogen antagonist ICI 182,780 did oppose the antiapoptotic effect of estradiol caused by bleomycin sulfate or buthionine sulfoximine.¹⁵³

Estrogen receptor-independent mechanisms, such as antioxidant properties, have been proposed for the possible action of ovarian steroids in these in vitro models. Oxidative stress may contribute to dopaminergic cell death in PD,¹¹⁸ but clinical studies based on antioxidative strategies have generally been inconclusive.¹⁵⁴ The concentration of estrogens used in these in vitro studies are in the micromolar range and are higher than those generally found in the brain.⁷¹ At these higher concentrations, the stereospecificity of the mechanism of action of estrogens may be lost.

It has been suggested that estrogen may act on dopaminergic neurons by hampering DA uptake and release.^{155,156,157,158} More specifically, studies show that estradiol inhibits striatal DA uptake by decreasing the affinity of the transporter for DA.¹⁵⁵ This has also been proposed as a mechanism whereby MPTP-induced dopaminergic cell destruction is prevented because MPP+, the active form of MPTP, enters dopaminergic cells via the DA uptake site before initiating the cell death process.^130,155,156 This is not supported by our recent findings where DA concentrations are spared, whereas DA transporter-specific binding and expression remains unchanged after estrogen treatment in MPTP mice versus saline-treated MPTP animals.¹³⁵

Well-designed large clinical trials might help to determine the neuroprotective activity of estrogens and other estrogen receptor-directed drugs such as SERMs. However, neuroprotection is difficult to assess in clinical studies in which a symptomatic effect is hard to distinguish from a real neuroprotective effect.¹⁵⁴ The need for reliable biological markers for nigrostriatal degeneration is evident, and brain imaging tools, such as single photon emission computed tomography and PET, may provide a satisfactory solution.¹⁵⁹

Selective estrogen receptor modulators

Clinical studies on the effect of tamoxifen in patients with schizophrenia are lacking, and animal studies on the effects of tamoxifen in brain areas associated with mental and neurodegenerative diseases are much less abundant than they are for estradiol. Tamoxifen, but not estradiol, is a weak competitive inhibitor of the DA antagonist site of D₂ receptors in striatal membranes.^160,161 Ferretti et al¹⁶² found, in animals treated with estradiol, tamoxifen antagonizes both increases in [³H]spiperone binding to D₂ receptors and stereotyped behaviour induced by apomorphine. Tamoxifen was also reported to mimic some of estradiol's activities on dopaminergic systems. In the pituitary, tamoxifen increases adenohypophysal DA receptors labelled with [³H]spiperone.¹⁶² McDermott et al^{163,164,165,166} investigated the interactive effects of tamoxifen and estrogen upon the nigrostriatal dopaminergic system. Their findings suggest that tamoxifen, like estradiol, can alter DA output through direct, nongenomic effects on striatal neurons. In addition, an indirect effect of tamoxifen on dopaminergic activity was demonstrated when tamoxifen reduced nigral glutamic acid decarboxylase activity.¹⁶⁷ Tamoxifen also increases striatal extracellular levels of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in freely moving male rats,¹⁶⁸ and was found to increase synaptic density in the hippocampus of ovariectomized rats.¹⁶⁹ It was also shown to block the effect of estradiol on dendritic spines of hippocampal neurons.¹⁷⁰

An early safety assessment of raloxifene's effects on cognition and mood in postmenopausal women participating in a randomized, double-blind osteoporosis treatment trial was reported.¹⁷¹ After 12 months of treatment, no clinically significant effect of raloxifene on cognitive performance was observed.¹⁷¹ The penetration of raloxifene in blood–brain barrier protected regions is low and may render the detection of any brain effect difficult at the dosage currently used in practice.¹⁷² In rats, Wu et al¹⁷² report that systemic administration of raloxifene reverses the ovariectomy-induced reduction in choline acetyltransferase activity in the hippocampus. CI-628, a nonsteroidal estrogen antagonist which readily crosses the blood–brain barrier, prevented estrogen induction of spines on CA1 pyramidal neurons, without having any agonist effects of its own.¹⁷³ In contrast, CI-628 has an estrogen-like agonist effect on brain monoamine oxidase (MAO) (reduction of activity) and choline acetyltransferase (increase of activity).¹⁷⁴ The raloxifene analog LY117018 has an estrogen-like action on neuroendocrine opiatergic pathways and on allopregnanolone concentrations when administered alone in ovariectomized rats, whereas it exerts an antiestrogen effect in fertile or ovariectomized rats treated with 17β-estradiol.^175,176

We previously reported that estradiol increases 5-HT_2A receptor density and expression in rat brain⁸³ and then sought the specificity of this hormonal modulation. Raloxifene mimics the effect of estradiol to increase 5-HT_2A receptor binding and its mRNA levels. (Fig. 1). Of the 18 brain regions investigated, this was specific to the anterior cingulate (Fig. 2) and anterior frontal cortices (Fig. 3), the cortical nucleus of the amygdala, the nucleus accumbens (Fig. 4) and the striatum.¹⁷⁷ More specifically, in the cortical areas, the hormonal effect was observed in the anterior, but not the posterior part, as shown for lamina IV of the cingulate cortex for 5-HT_2A mRNA levels in rats after ovariectomy and chronic estradiol or raloxifene treatment (Fig. 2). In the anterior frontal cortex, the effect of ovariectomy and estradiol or raloxifene treatment in rats on 5-HT_2A mRNA levels was observed in lamina II and IV (Fig. 3). The effect of ovariectomy and hormone treatment in rats on [³H]ketanserine-specific binding to 5-HT_2A receptors was more pronounced in the shell than the core of the nucleus accumbens (Fig. 4), the shell being more related to the mesolimbic system.¹⁷⁸

Fig. 1: Examples of in situ hybridization of 5-HT_2A receptors mRNA in the rat brain showing the effect in frontal cortex of ovariectomy and a 2-week treatment of 17β-estradiol (1 silastic implant) or raloxifene (250 μg). Coronal brain sections (30 μm thick) of frontal cortex (4.7–3.7 mm from bregma²¹¹) are shown. Fr = frontal cortex, Cg1= cingulate cortex, PrL = prelimbic cortex, Pir = piriform cortex, TT = Taenia tecta.

Fig. 2: Uterine weight and in situ hybridization of brain 5-HT_2A receptors in the anterior and posterior cingulate cortex of intact rats that received vehicle (once daily, subcutaneously) for 2 weeks, as well as ovariectomized (OVX) rats treated for 2 weeks with vehicle (once daily, subcutaneously) or with 17β-estradiol (1 silastic implant), tamoxifen (250 μg) or raloxifene (250 μg). Mean in situ hybridization results are expressed as percentage of relative optical density control values (and standard errors of the means [SEM]) of 4–6 rats per group. Control values in relative optical density were 0.42 (SEM 0.02) (upper panel) and 0.33 (SEM 0.02) (lower panel). **p < 0.005 v. intact/saline; †p < 0.05 and ††p < 0.01 v. OVX/saline.

Fig. 3: In situ hybridization of 5-HT_2A receptors in lamina II, IV and V of the anterior frontal cortex of intact rats that received vehicle (once daily, subcutaneously) for 2 weeks, as well as ovariectomized (OVX) rats treated for 2 weeks with vehicle (once daily, subcutaneously) or with 17β-estradiol (1 silastic implant), tamoxifen (250 μg) or raloxifene (250 μg). Results are expressed as percentage of control values and SEMs of 4 rats per group. Control values in relative optical density were 0.27 (SEM 0.02) for lamina II, 0.33 (SEM 0.01) for lamina IV and 0.23 (SEM 0.01) for lamina V. *p < 0.05 and **p < 0.005 v. intact/saline; †p < 0.05 v. OVX/saline.

Fig. 4: [³H]Ketanserin-specific binding to 5-HT_2A receptors in the core and shell of the nucleus accumbens of intact rats that received vehicle (once daily, subcutaneously) for 2 weeks, as well as ovariectomized (OVX) rats treated for 2 weeks with vehicle (once daily, subcutaneously) or with 17β-estradiol (1 silastic implant), tamoxifen (250 μg) or raloxifene (250 μg). Results are means and SEMs of 6 rats per group. *p < 0.05 and **p < 0.005 v. intact/saline; †p < 0.05 and ††p < 0.01 v. OVX/saline.

In general, tamoxifen did not significantly increase 5-HT_2A receptor expression, except in one part of the anterior cingulate cortex and binding in the striatum.¹⁷⁷ Considering the short- versus the long-term treatment and the dose used, this is in overall agreement with the study of Sumner et al.¹⁷⁹ This suggests that raloxifene, more than tamoxifen, acts as an estrogen agonist at brain 5-HT_2A receptors and that it is possible to have brain estrogenic agonist activity dissociated from agonist activity in the periphery. Indeed, whereas raloxifene displays as estradiol activity to increase 5-HT_2A receptor density and expression, this effect can be dissociated from the activity to stimulate the uterus.

Tamoxifen and raloxifene also modulate brain NMDA and AMPA receptors. These SERMs increase NMDA-specific binding in the hippocampus but decrease binding in the frontal cortex, striatum and nucleus accumbens.¹⁸⁰ As with estradiol treatment, AMPA receptor-specific binding is decreased by tamoxifen and raloxifene.¹⁸¹ Using specific ligands for binding autoradiography of subunits of NMDA receptors and specific probes for subunits measured by in situ hybridization, it was shown that estradiol and SERMs modulate NR1 and NR2B subunits, whereas the NR1/2A subunit remains unchanged.¹⁸⁰

Clinical data are not yet available for a possible neuroprotective activity of SERMs. At this point, preclinical as well as clinical experiments are needed to assess the potential neuroprotective effects of the available SERMs and make comparisons with what is known with estradiol. Tamoxifen may stimulate DA liberation in the striatum of rats through direct, nongenomic action.^{163,164,165,182} An in vivo microdialysis study revealed that tamoxifen administration increased DA and DOPAC in the striatum of freely moving rats.¹⁶⁸ The effect of a 40-day treatment of ovariectomized rats with tamoxifen, either alone or in combination with estradiol, showed that estradiol amplified amphetamine-stimulated DA release, and this effect was blocked by tamoxifen.¹⁶⁶ This suggests that, in the dopaminergic nigrostriatal system, tamoxifen may act as an estradiol agonist for short-term nongenomic effects, but may block estradiol in regards to long-term activity. In addition, the neuroprotective effect of estradiol from glutamate neurotoxicity was blocked by tamoxifen in primary cortical neurons,¹⁸³ but it was not affected by tamoxifen in mesencephalic dopaminergic neurons in culture.¹⁵² Studies in mice show that tamoxifen does not protect from MPTP- or methamphetamine-induced striatal DA decrease and it may block the protective effect of estrogens.^184,185 On the other hand, we have shown that raloxifene prevents the MPTP-induced striatal depletion of DA and its metabolites DOPAC and homovanillic acid.¹³⁶ In NGF-primed PC12 cells expressing both estrogen α and β receptors, raloxifene-induced neurite outgrowth, and the combined treatment of estradiol and raloxifene produced a statistically greater effect than either agent alone.¹⁸⁶ These findings on dopaminergic toxicity and those reported above for 5-HT_2A receptors suggest that raloxifene has more brain estrogenic agonist activity than tamoxifen, the latter compound displaying both agonist and antagonist activities in the CNS. For brain NMDA and AMPA receptors, both tamoxifen and raloxifene have similar estrogenic activity.

Discussion

Effects of estrogens on the brain are reported in numerous epidemiological, clinical as well as animal and in vitro cell culture studies. Some possible explanations for the contradictory or discordant findings of these studies are discussed below.

In studies on sex differences in mental and neurodegenerative diseases, estrogens and androgens are often the focus and the other major ovarian steroid progesterone receives less attention. Progesterone may also play a role in the sex differences observed, however. Progestatives alone are in use as contraceptives,^187,188 although it is less common than contraceptives combining estrogens and progestatives.¹⁸⁷ Animal studies with progesterone often use an estrogen priming,^{73,189,190,191} although clear evidence of an effect of progesterone alone on neurotransmitter activity is observed in several animal models.^192,193,194 In hormone replacement therapies in humans, estrogens are often associated with progestatives to avoid sustained uterine stimulation. The brain effects observed are likely due to both estrogens and progesterone. In animal studies, progesterone can potentiate,^73,195 leave unchanged¹⁹⁶ or antagonize^197,198,199 the effect of estrogens on various neurotransmitter activities. In human studies, an adverse effect of progestins on mood, including increasing depression scores, has been reported.^{200,201,202,203} With the availability of SERMs that do not stimulate the uterus, it will be possible to probe estrogenic activity in the brain without the need of a progestative in the treatment. Nevertheless, the effect of progestatives on the brain is beyond the scope of this paper.

A review of the literature shows multiple activities of estrogens on brain neurotransmitters involving various genomic and nongenomic mechanisms. For example, genomic mechanisms involving an estrogen receptor either of the α or β subtype likely involve a stereospecific activity, 17α-estradiol being much less active than the active natural 17β-estradiol; this is noted in the neuroprotection of DA neurons in mice from MPTP toxicity.^135,136 In contrast, nongenomic mechanisms involving the antioxidant activity of estradiol in protection against various sources of neurotoxicity in cell cultures^150,204,205 are associated with the presence of a hydroxyl group in the C3 position. In assessing estrogenic activity through an estrogen receptor, a standard pharmacological approach is to probe if an estrogen receptor antagonist opposes the estrogenic effect. This is somewhat difficult for in vivo experiments at present because of the lack of pure antiestrogen compounds devoid of estrogenic activity for the brain. Reasons for this include the fact that agonist or antagonist effects in the brain are not yet possible to predict, several existing antagonists do not cross the blood–brain barrier (some of them were designed not to do so) and the design of such compounds may be of less therapeutic interest than would be the case for SERMs.¹³

Dose-related biphasic effects of estradiol are observed in animal studies.^70,206,207 Some effects are lost at higher concentrations,^206,207 whereas others require high doses.^151,152,204 This could reflect an adaptation of the brain to fluctuating levels of estradiol during the menstrual cycle and the very high levels of the steroid during pregnancy.²⁰⁸ These dose-related effects could be taken as an advantage to possibly dissociate some activities in the brain. It is not known how SERMs will behave in this respect or if biphasic dose-related effects will be found, even for those compounds devoid of antagonistic activity in the target tissue.

For the CNS, the ideal estrogen-like compound would have activity in the brain and none in the periphery. Is it possible to find such a compound? SERMs have gone some way in that direction, and 17α-estradiol has shown activity similar to 17β-estradiol in models in vitro,^151,209 although it is inactive in others in vivo.^135,136 Estrogen receptor β-selective ligands are also likely to be fairly specific to brain since this receptor is localized in brain regions associated with learning and memory and is much less abundant in the uterus and pituitary than the α subtype.²¹⁰ Although a variety of synthetic and naturally occurring estrogenic compounds (including phytoestrogens and environmental estrogenic compounds) were found to have similar affinity for the α and β subtypes, some phytoestrogens, such as genistein and coumestrol, have higher binding affinity for estrogen receptor β.²¹⁰ At this point in human studies, it is important to learn as much as possible about the brain effect of SERMs and other estrogenic compounds (e.g., phytoestrogens, xenoestrogen, DHEA) that are given for traditional indications and to pursue animal studies with such compounds as well.

Clinical data should be gathered in patients with schizophrenia and PD who are taking raloxifene for osteoporosis or tamoxifen for breast cancer. Research on brain hormonal effects is not simple but is quite relevant. Indeed, schizophrenia generates considerable health costs and the incidence of neurodegenerative diseases such as Alzheimer's disease and PD is increasing with the aging population. Hence, research on the effects of estrogenic-like compounds in the brain has important social and financial implications.