Structure–function–behavior relationship in estrogen-induced synaptic plasticity

    Structure-function-behavior relationship in estrogen-induced synaptic plasticity R.Vierk, J. Bayer, S. Freitag, M. Muhia, K. Kutsche, T. Wolbers, M. Kneussel, T. Sommer, G.M. Rune PII: DOI: Reference:

S0018-506X(15)00089-6 doi: 10.1016/j.yhbeh.2015.05.008 YHBEH 3879

To appear in:

Hormones and Behavior

Please cite this article as: R.Vierk, Bayer, J., Freitag, S., Muhia, M., Kutsche, K., Wolbers, T., Kneussel, M., Sommer, T., Rune, G.M., Structure-function-behavior relationship in estrogen-induced synaptic plasticity, Hormones and Behavior (2015), doi: 10.1016/j.yhbeh.2015.05.008

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Structure-function-behavior relationship in estrogen-induced

T

synaptic plasticity

1 Institute

SC R

IP

Vierk R. 1#, Bayer J. 2#, Freitag S. 3, Muhia M. 3, Kutsche K. 4, Wolbers T. 5, Kneussel M. 3, Sommer T. 2*, Rune G.M. 1* of Neuroanatomy, University Medical Center Hamburg-Eppendorf, Martinistr. 52,

20246 Hamburg, Germany

Institute for Systems Neuroscience, University Medical Center Hamburg-Eppendorf,

Martinistr. 52, 20246 Hamburg, Germany

Department of Molecular Neurogenetics, Center for Molecular Neurobiology (ZMNH),

MA

3

NU

2

University Medical Center Hamburg-Eppendorf, Falkenried 94, 20151 Hamburg 4

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Martinistr.

Center for Behavioral Brain Sciences, Leipziger Str. 44, 39120 Magdeburg, Germany

TE

5

D

52, 20246 Hamburg, Germany

CE P

# These authors contributed equally to the manuscript

*Corresponding author: G. M. Rune, Institute of Neuroanatomy,

AC

University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany Tel +49-(0)-40741053575, email: [email protected]

*T. Sommer, Department of Systems Neuroscience, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany Tel +49-(0)-40741054763 email: [email protected]

1

ACCEPTED MANUSCRIPT Abstract In estrogen-induced synaptic plasticity, a correlation of structure, function and

T

behavior in the hippocampus has been widely established. 17ß-estradiol has been

IP

shown to increase dendritic spine density on hippocampal neurons and is accompanied by enhanced long-term potentiation and improved performance of animals in

SC R

hippocampus-dependent memory tests. After inhibition of aromatase, the final enzyme of estradiol synthesis, with letrozole we consistently found a strong and significant impairment of long-term potentiation (LTP) in female mice as early as after six hours

NU

of treatment. LTP impairment was followed by loss of hippocampal spine synapses in the hippocampal CA1 area. Interestingly, these effects were not found in male animals.

MA

In the Morris water maze test, chronic administration of letrozole did not alter spatial learning and memory in either female or in male mice. In humans, analogous effects of estradiol on hippocampal morphology and physiology were observed using

D

neuroimaging techniques. However, similar to our findings in mice, an effect of

CE P

TE

estradiol on memory performance has not been consistently identified/observed.

Keywords: aromatase, CYP19A1, letrozole, long-term potentiation, Morris water maze,

AC

spatial memory task, verbal source memory task, MRI, voxel-based-morphometry

Introduction

Subsequent to the pioneer work carried out by Gould and Woolley (Gould et al., 1990; Woolley et al., 1990), showing that estradiol induces the formation of spines in the female hippocampus, a number of studies have demonstrated a role for estradiol in synaptic plasticity and cognition in the adult, as well as during development (Daniel, 2013; McCarthy, 2011; Spencer et al., 2008). In addition to the role of estradiol on spine density, it has also been shown that estradiol enhances hippocampal long-term potentiation (LTP), a cellular model for learning and memory (for review see Spencer et al., 2008)). Estradiol increases the magnitude of LTP at hippocampal CA3-CA1 synapses in acute hippocampal slices (Foy et al., 1999; 2

ACCEPTED MANUSCRIPT Kramár et al., 2009; Smith and McMahon, 2005). Both phenomena, increased spine density and enhanced LTP, appear to be related to the memory-enhancing effects of sex steroids (Maki et al., 2001; Phillips and Sherwin, 1992b). As far as the responsiveness of hippocampal

T

tissue to estradiol is concerned, the expression of estrogen receptors (ERs) in the

IP

hippocampus has also been acknowledged (Foster, 2012; Shughrue et al., 1997). Both ER subtypes, ER and ER, are expressed in the hippocampus of male and female animals, with

SC R

differences in expression existing between rats, mice and primates (Milner et al., 2001). ER is the predominant receptor expressed in the rat hippocampus (Milner et al., 2001; Shughrue et al., 1997), whereas ER appears to be the predominant receptor in the hippocampus of

NU

mice (Mitra et al., 2003). Here we report on our findings, which question the postulated close correlation of an estradiol-induced increase in hippocampal synapse density, enhancement of

MA

LTP and memory in laboratory animals and humans. We primarily tested the effects after inhibition of aromatase, the final enzyme of estrogen synthesis, encoded by CYP19A1, in mice and used magnetic resonance imaging (MRI) to correlate morphology of the

D

hippocampus with variations in peripheral estradiol levels in humans. MRI was also used to

TE

explore the correlation of a genotype of a CYP19A1 variant associated with higher serum

AC

Mice and rats

CE P

estradiol levels with hippocampal morphology and memory performance.

Sex neurosteroids are synthesized in the hippocampus and act in a paracrine manner Sex steroids mainly originate from the gonads and enter the central nervous system via peripheral circulation. The vertebrate brain, however, is also fully equipped with all enzymes for steroid biosynthesis, and thus the brain is capable of synthesizing sex steroids that exert local effects on the circuitry and the animal’s performance (Compagnone and Mellon, 2000; Shibuya et al., 2003). Sex steroids, which are synthesized in the brain and function in a paracrine manner, are defined as sex neurosteroids (SN). In final SN synthesis, testosterone is either irreversibly converted to estradiol by the activity of aromatase, or testosterone is irreversibly metabolized to dihydrotestosterone (DHT) by the activity of 5-reductase. The expression of aromatase has long been known; it was shown for the first time in the diencephalon by Naftolin et al. (Naftolin et al., 1971). Over 10 years ago, we and others demonstrated that the enzyme is indeed functional (Hojo et al., 2004; Prange-Kiel et al., 2003). Dissociated hippocampal neurons synthesize and secrete considerable amounts of 17ß3

ACCEPTED MANUSCRIPT estradiol, which could be measured in the supernatant. Moreover, the expression of estrogen receptors in response to letrozole, a potent aromatase inhibitor, in the neurons pointed to an auto/paracrine mode of action, since ERβ was upregulated and ERα downregulated after

T

inhibition of aromatase, while application of 17ß-estradiol to the cultures induced opposite

IP

effects (Prange-Kiel et al., 2003). The auto/paracrine mode of action was also demonstrated by the region-specific downregulation of synaptic proteins in hippocampal slice cultures after

SC R

treatment with letrozole (Prange-Kiel et al., 2006). Using mass spectrometry, we found higher amounts of 17ß-estradiol in hippocampal tissue of proestrus female animals, compared to the

NU

hippocampal tissue of male animals (Fester et al., 2012).

Estradiol effects on synaptic plasticity in the hippocampus

MA

Synapse density. Recent findings in the aromatase knock-out mouse (ArKO), where synapse density is reduced in the hippocampus of female animals, but not of male animals (Zhou et al., 2014), re-confirm the paradigm of estradiol-induced spine and spine synapse formation at

D

hippocampal CA1 dendrites (Spencer et al., 2008). This phenomenon appears to be region-

TE

dependent; a similar reduction in synapse density was found neither in the neo-cortex nor in the cerebellum (Fig. 1). Varying density of spines along the dendrites of hippocampal CA1

CE P

pyramidal neurons during the estrus cycle of females strongly support the idea that estrogen of ovarian origin regulates spinogenesis in the hippocampus (Woolley et al., 1990). In addition, the removal of the gonads has been shown to result in reduced hippocampal dendritic spine density in males and females (Gould et al., 1990), and it was possible to rescue

AC

spine loss after ovariectomy by injections of 17ß-estradiol. In order to approach estradiol-induced synaptic plasticity, we inhibited estradiol synthesis in hippocampal slice cultures by treatment of the cultures with letrozole. In these cultures, in which synaptic connectivity is preserved over weeks, we found, consistent with the studies mentioned above, a significant reduction of estradiol release by 60% and a significant reduction in the number of spine synapses after 48 hours of treatment with letrozole. This effect was also observed when we treated female animals systemically with letrozole (10 μg/g body weight i.p.). At this dose estradiol in serum could not be measured, since the concentrations were below the detection level of our RIA. Since we did not determine the estrus stage of the control animals and synaptic density is highest during proestrus, the difference between control and treated animals would very likely have been greater if we had had taken exclusively proestrus animals. The effects of letrozole, however, were even seen in the hippocampus of ovariectomized animals (Fig. 1; Zhou et al., 2010). In addition, dendritic 4

ACCEPTED MANUSCRIPT spine density of enhanced green fluorescent protein (EGFP)-transfected dissociated hippocampal neurons was consistently reduced after pharmacological inhibition of aromatase (Vierk et al., 2012). These results point to a role of hippocampus-derived estradiol in synaptic

T

plasticity, not exclusively, as previously believed, to a role of estradiol originating from the

IP

gonads. In addition, estrus cyclicity of synapse density in the hippocampus (Woolley et al., 1990) very likely results from cyclic estradiol synthesis in the hippocampus, since

SC R

gonadotropin releasing hormone, which is released from the hypothalamus in a cyclic fashion, regulates estradiol synthesis in the hippocampus in a dose-dependent manner (Prange-Kiel et al., 2008; Prange-Kiel et al., 2013). Interestingly enough, the effects of letrozole were sex-

cultures originating from male animals.

NU

specific and were found neither in male animals (Vierk et al., 2012), nor in hippocampal

MA

Long-term potentiation. Numerous studies have shown that estrogens enhance LTP in the hippocampus (Spencer et al., 2008). The effects of estrogens on LTP, however, were mostly shown in experiments by testing either ovariectomized animals, which were treated

D

systemically with estradiol (Cordoba Montoya and Carrer, 1997; Kramár et al., 2009; Smith

TE

and McMahon, 2005) or testing after estrogens were applied to acute slices of mostly male rats (Foy et al., 1999; Ito et al., 1999; Kramár et al., 2009). The majority of these studies

CE P

illustrate the potential effects of exogenous application of estrogens to females after removal of estrogen by ovariectomy before treatment, and to male animals, where estrogen in the tissue is almost undetectable and serum concentrations are much lower. The findings that estrogens enhance LTP in the hippocampus on the one hand, and that LTP induces spine

AC

formation (Yuste and Bonhoeffer, 2001) on the other, prompted us to study the consequences of estrogen synthesis inhibition on synaptic long-term potentiation (LTP), as a cellular model for learning and memory. Systemic daily injections of high doses of letrozole over various time periods resulted in decreased synaptic potentiation in female, but not in male mice (Vierk et al., 2012). In acute slices from female animals treated with letrozole at a high dose of 10μg/g body weight, LTP was significantly reduced by 50% as early as 6 hours after treatment. After one week of letrozole application, synaptic potentiation was completely abolished and could not be induced by TBS. In males, however, LTP was merely reduced by 20 percent after 24 hours and remained at this level, even after one week had elapsed. At a lower dose of 0.4μg/g body weight, LTP was less, but also significantly impaired, and the difference between males and female persisted (see below). Interestingly, the impairment in synaptic potentiation after inhibition of estrogen synthesis using high doses of letrozole preceded spine synapse loss in the hippocampus of female mice, suggesting that according to 5

ACCEPTED MANUSCRIPT the paradigm – LTP induces dendritic spines – LTP impairment accounts for synapse loss. In this context, it needs to be mentioned that inhibition of aromatase should result in increased levels of testosterone and dehydrotestosterone (DHT) respectively. The role of testosterone

T

and DHT on synapse density and LTP in the hippocampus of male and female animals,

IP

however, has not been studied yet. According to the data by Leranth et al. (Leranth et al., 2004), it is tempting to speculate that testosterone and DHT possibly control synaptic

SC R

plasticity in the hippocampus of males, while estradiol controls hippocampal synaptic plasticity in females.

NU

Correlation of LTP and behavior

Long-term potentiation. The aromatase inhibitor letrozole, which we used for our

MA

experiments, is commonly used in the therapy of breast cancer. Letrozole is a reversible, nonsteroidal aromatase inhibitor. The effects we obtained in animals, as described above, were reversed after 32 hours (Prange-Kiel and Rune, 2006), and letrozole is more potent in

D

reducing estradiol synthesis compared to fadrozole and anastrozole (Prange-Kiel et al., 2013).

TE

Aromatase inhibitors are suspected of inducing subtle hippocampus-dependent memory deficits in women, as shown in our recent functional magnetic resonance imaging (fMRI)

CE P

study (Bayer et al., 2015) and clinical pilot experiments (Jenkins et al., 2008; Collins et al., 2009; Hurria et al., 2014). Given this background, we aimed to investigate putative associations between inhibition of hippocampal aromatase, hippocampal LTP, and hippocampus-dependent behavior. To this end, we applied letrozole at a dose similar to the

AC

dose received by women suffering from breast cancer. We dissolved letrozole in DMSO, which by itself had no effect on the magnitude of LTP, as shown in Fig. 2. Application of therapeutical doses of letrozole over the same period of time resulted in a significant reduction in synaptic potentiation of roughly 39% ± 6% (Fig. 2, 198% vs. 159.5% fEPSP slope compared to 100% baseline) in female mice treated with letrozole compared to untreated female mice. In males, however, the reduction in LTP after aromatase-inhibition was less than 17% ± 3% as compared to control males (Fig. 2, 198% vs. 182% fEPSP slope compared to 100% baseline). This experiment confirmed our previous results regarding sexdependent impairment of synaptic potentiation in female and male mice in response to letrozole. Most importantly, the results show that LTP is reduced in a dose-dependent manner upon inhibition of estradiol synthesis in female animals, but not in male animals.

Behavior. Our findings of dose-dependent and sex-specific LTP impairment after inhibition of 6

ACCEPTED MANUSCRIPT aromatase prompted us to examine whether these cellular effects may also exert sex-specific effects in vivo, at the behavioral level. Given that the hippocampus plays a key role in spatial learning and memory, we addressed our hypothesis by testing male and female mice

T

(C57/Bl6) for reference memory in the Morris water maze (MWM) task. The MWM

IP

comprises one of many test batteries that are available for the examination of hippocampusrelated memory in rodent animal models. These tests consist of place learning in a reward-

SC R

motivated plus maze, a baited radial maze test for working and reference memory, active avoidance paradigms, object placement learning and water maze navigation. Here, the subjects were initially handled and tested in the Open Field test for locomotor

NU

activity. In addition, prior to MWM reference memory acquisition, the animals were habituated to water and tested in the cued non-spatial version of the task to minimize stress

MA

responses commonly associated with initial introduction to the MWM pool. Following performance in the cued test, 3 equal groups consisting of 8-9 mice per sex were created. All groups were counterbalanced for body weight and litter. Daily intraperitoneal injections (i.p.

D

0.4 g/g body weight) of letrozole, DMSO and sham injections commenced 1 week prior to

TE

the first day of MWM training and were continued until the last day of MWM. Mice were trained on 5 consecutive days (4 trials/day) with an inter-trial interval of 15-20 min (see Fig.

CE P

3A). A trial ended when the mouse successfully climbed onto the platform, or after 60s, during which it was gently guided by the experimenter to the platform, had elapsed. The mouse was allowed to remain on the platform for 10s before being removed from the pool. On the 5th training day, the mice received a 90s probe trial on the last acquisition trial to

AC

examine successful acquisition of the task. 24 hrs later (day 6), a second probe trial was performed to examine memory of the training quadrant. Given our a priori hypothesis of a sex-dependent effect of aromatase inhibition, data from the male and female groups were analyzed separately. Differences among the three treatment groups were examined using an analysis of variance (ANOVA) with the between-subjects factors (Treatment) and Days as repeated measures. Aromatase inhibition did not alter reference memory acquisition in either the female or male mice. Indeed, all treatment groups successfully acquired the task across training as evidenced from the significant decline in the path length and latency measures (Fig. 3B, C). Similarly, the mice demonstrated significant preference for the training quadrant in the probe trials, indicating intact reference memory in all treatment groups. This outcome was in apparent contrast to our data on synaptic potentiation. Hippocampal LTP recordings were performed on a sub-set of mice at the end of the MWM experiment. LTP 7

ACCEPTED MANUSCRIPT deficits emerged in both the male and female mice, but this effect was markedly stronger in females compared to male mice (Fig. 2). Our behavioral data are however, consistent with a study by Aydin and coworkers (Aydin et al., 2008) conducted on female rats that were treated

T

with letrozole for 6 weeks. When tested in the MWM, the animals were unaffected during the

IP

training period, and surprisingly, performed better than controls in the subsequent probe trials. In summary, a close correlation was found with synapse density and LTP but not with the

SC R

results from the MWM experiment.

It is notable that the MWM was designed to examine only one specific aspect of memory, i.e. reference memory. The memory deficits reported in women treated with aromatase inhibitors

NU

are rather subtle (Bayer et al., 2015), and a memory task requiring long-term training such as the WMW may fail to capture such subtle deficits. Thus, it would be worthwhile examining

MA

the functional significance of aromatase inhibition across different types of memory using, for instance, tasks designed to address recognition and emotional memory, as in studies in

AC

CE P

TE

D

humans.

8

ACCEPTED MANUSCRIPT Humans With non-invasive neuroimaging techniques such as MRI, it is also possible to explore the effects of estradiol in the human brain and to relate subsequent findings to mouse studies. In

T

the following sections, we will briefly review structural MRI, fMRI and magnet resonance

IP

spectroscopy studies on estradiol’s effect on hippocampal morphology and physiology that have been conducted by ourselves and others. Motivated by our results with respect to

SC R

aromatase inhibition on synapse density, LTP and behavior in mice, we subsequently focused on the effects of estradiol on human memory. In particular, we report on results on the association of a polymorphism in the gene coding for aromatase (CYP19A1) with

NU

hippocampal morphology, but not memory performance, in men.

MA

Effects of estradiol on hippocampal morphology in female and male humans In humans, it is possible to investigate effects of estradiol on hippocampal morphology, i.e. hippocampal volume and gray matter density, with structural MRI. Although MRI allows only

D

conclusions about hippocampus morphology on the mesoscopic level, this method is feasible

TE

for the detection of morphological changes across the estrus cycle in rodents that have been extensively described on the cellular level (Qiu et al., 2013; Woolley, 1998). In women, MRI

CE P

data show that menopause is associated with a decline in hippocampal volume that cannot be attributed to age-related changes (Goto et al., 2011). In addition, postmenopausal women using hormone therapies (HT) to treat menopause-related symptoms have larger hippocampi compared to age-matched non-users and men (Erickson et al., 2010; Lord et al., 2008).

AC

Changes in hippocampal volume have also been described across the menstrual cycle and correlate with fluctuations in hormone levels (Protopopescu et al., 2008). The neuroprotective effects of estradiol were explored using magnet resonance spectroscopy, a method that is able to quantify cellular markers of membrane metabolism. HT users had reduced concentrations of these markers, suggesting a positive effect of HT on neuronal integrity (Ernst et al., 2002; Robertson et al., 2001) Taken together, evidence from various experimental settings suggests that estradiol affects not only hippocampal morphology in female rodents, but also in women. In particular, both acute, i.e. menstrual cycle, as well as chronic, e.g. menopause, effects of estradiol have been observed. However, it should be emphasized that the interpretation of these findings is complicated by the fact that neither menopause nor HTs, nor the menstrual cycle lead to changes in only estradiol, but also in other neuroactive steroid levels. Using a genetic approach, we have recently studied whether estradiol affects hippocampal morphology in men (Bayer et al., 2013). In particular, we used the synonymous sequence 9

ACCEPTED MANUSCRIPT variant c.240A>G [p.(=) single nucleotide polymorphism (SNP) rs700518] in the gene coding for aromatase, CYP19A1 that has been repeatedly associated with inter-individual differences in serum estradiol levels in men (Eriksson et al., 2009; Olivo-Marston et al., 2010; Peter et al.,

T

2008). For example, Peter et al. (2008) reported a decrease of 9.59% in the group with the GA

IP

genotype and of 11.93% in the group with GG genotype in estradiol serum level in comparison with the group with AA genotype. To explore whether the observed associations

SC R

between the CYP19A1 genotypes and serum estradiol levels are also associated with differences in hippocampal volume, we grouped individuals of two independent samples of 77 and 84 healthy young men according to their genotype in this SNP.

NU

To assess brain morphology, we employed voxel-based morphometry (VBM) of the MR images of the participants. VBM is a whole-brain, semi-automatic and unbiased technique for

MA

characterizing local differences in gray matter, which is as sensitive as manual segmentation, to detect hippocampal volume differences (Bergouignan et al., 2009). Consistent with higher serum estradiol levels, individuals in the group with AA genotype had greater hippocampal

D

volumes compared to those in the groups with GA and GG genotype (Fig. 5A and B) (Bayer

TE

et al., 2013). This genotype-dependent VBM difference was restricted to the bilateral posterior hippocampus and, importantly, replicated in both independent samples.

CE P

Such mesoscopic differences in VBM can be explained by different cellular mechanisms, such as structural changes of spines and dendrites, neurogenesis, gliogenesis, and synaptogenesis (Zatorre et al., 2012). The interpretation of the observed MRI signal difference in terms of the underlying cellular substrate may be guided by the literature on

AC

rodents. We have previously shown that estradiol affects hippocampal spine density only in female, but not in male, rodents (Vierk et al., 2012; Zhou, 2014). Others describe a similar sexual dimorphic effect of estradiol only in females on fiber outgrowth and sprouting responses (Barker and Galea, 2008; Leranth et al., 2003; Morse et al., 1986; Spritzer and Galea, 2007; Woolley and McEwen, 1993). In contrast, neuroprotective effects of estradiol in the hippocampus have been observed in both sexes after both acute and chronic treatment (Azcoitia et al., 2001; McCullough and Hurn, 2003; Veiga et al., 2005, Arevalo et al., 2014). The genotype-dependent VBM difference in men is therefore consistent with greater neuroprotection stimulated by the chronically higher estradiol serum levels in men with the AA genotype. In summary, MRI data suggest that estradiol similarly affects hippocampal morphology both in rodents and humans.

1 0

ACCEPTED MANUSCRIPT Estradiol effects on hippocampal physiology The effects of estradiol and the inhibition of aromatase on LTP that we and others have described in rodents cannot be explored in humans. Currently available non-invasive used

to

examine

synaptic

changes

(LTP),

such

as

electro-

or-

T

procedures

IP

magnetoencephalograms (EEG and MEG), are restricted to the cortex (Clapp et al., 2012). The functional MRI (fMRI) signal, i.e. the blood oxygen level dependent (BOLD) effect,

SC R

reflects synaptic processes and is therefore currently the best proxy to study changes in synaptic plasticity and efficiency in humans (Lee et al., 2010; Logothetis et al., 2001). Using fMRI (and positron emission tomography), positive effects of HT on hippocampal activity

NU

during memory tasks has been observed in some of the studies that explored HT-related brain activity differences (Gleason et al., 2006; Maki et al., 2011; Maki and Resnick, 2000). We

MA

have recently shown that hippocampal activity during emotional memory encoding varies across the menstrual cycle (Fig. 6; (Bayer et al., 2014). Consistent with this finding, others have reported that hippocampal activity fluctuates with the menstrual cycle during processing

D

of emotional pictures (Goldstein et al., 2010). Similar to the morphological MRI findings,

TE

however, these differences in activity cannot be attributed unambiguously to the actions of estradiol because other neuroactive steroids, e.g. progesterone, also fluctuate across the

CE P

menstrual cycle.

To connect more closely to our studies in rodents, in which we inhibited aromatase instead of applying estradiol, we conducted a longitudinal study in postmenopausal women taking aromatase inhibitors as breast cancer relapse prophylaxis (Bayer et al., 2015). In particular,

AC

we scanned patients using fMRI prior to, and 3-6 months after continuous aromatase inhibition and compared brain activity during successful memory formation with a control group. Hippocampal activity was reduced after aromatase inhibition, although this effect reached only trend level significance. Crucially important, we observed a parallel increase in prefrontal activity that putatively compensated for the hippocampal decline as has been described in aging and Alzheimer’s disease (Bäckman et al., 1999; Cabeza et al., 2002; Gould et al., 2006; Grady et al., 2002; Gutchess et al., 2005). In summary, there is early evidence that estradiol levels affect synaptic activity in the human hippocampus, although these effects have yet to be explored in men.

Effects of estradiol on hippocampal-dependent memory in women In rodents, we observed a puzzling dissociation between estradiol’s partly sex-specific effects 11

ACCEPTED MANUSCRIPT on hippocampal morphology and LTPs on the one hand, and behavior on the other. We therefore then addressed the issue as to whether, and how consistently, the reviewed effects of estradiol on human hippocampal morphology and physiology translate to hippocampus-

T

dependent memory. Effects of HT or estradiol treatment in postmenopausal women on

IP

memory performance are inconsistent, i.e. effects on verbal memory were observed in only some of the studies. A recent review including 27 studies on the effects of HT on verbal

SC R

memory reported that 26% of the tests yielded negative effects, 37% null effects and only 37% positive effects (Hogervorst and Bandelow, 2010). Interestingly, another MRI study that reported larger hippocampal volumes in women who initiated hormone treatment at

NU

menopause did not observe differences in spatial memory performance (Erickson et al., 2010). The pharmacological administration of estradiol in younger women led to a slight

MA

improvement in verbal memory in some studies (Bartholomeusz et al., 2008; Phillips and Sherwin, 1992a; Sherwin, 1998; Sherwin and Tulandi, 1996). Natural fluctuations in estradiol across the menstrual cycle are associated with fluctuations in verbal, emotional and spatial

D

memory performance in some studies (Ertman et al., 2011; Solis-Ortiz and Corsi-Cabrera,

TE

2008) in particular, menstrual cycle-dependent changes in verbal memory occurred simultaneously to changes in hippocampal volume (Protopopescu et al., 2008). However,

CE P

others have failed to find such a correlation between fluctuations in hormone levels and memory (Phillips and Sherwin, 1992b; Resnick et al., 1998). In our fMRI study on menstrual cycle-related changes in hippocampal activity, we additionally assessed potential fluctuations in recognition memory performance (Bayer et al., 2014). Recognition is based on two mnestic

AC

processes, whereby only one is – similar to free and cued recall – hippocampus-dependent. In particular, the hippocampus is critically involved in retrieving an item together with associated contextual information (recollection). Recognizing an item without any associated information (familiarity) can be based purely on the parahippocampal cortices (Brown et al., 2010; Eichenbaum et al., 2012). Tests that assess both forms of recognition memory are more sensitive to subtle changes in memory performance and allow for a more specific inference to the underlying neural substrates. Therefore, in our study we used such a process-specific recognition test to assess fluctuations in emotional memory performance (Bayer et al., 2014). Indeed, we observed subtle changes specifically in the hippocampus-dependent, recollectionbased recognition of negative pictures across the menstrual cycle. Estradiol’s effect on women’s memory was also investigated using aromatase inhibition, with relatively inconsistent outcomes (Bender et al., 2006; Breckenridge et al., 2012; Collins et al., 2009; Hedayati et al., 2012; Jenkins et al., 2008; Lejbak et al., 2010; Schilder et al., 2010; 1 2

ACCEPTED MANUSCRIPT Shilling et al., 2003). In our recent fMRI study on the neural effects of aromatase inhibition we again employed process-specific memory tests to explore hippocampus-dependent memory changes (Bayer et al., 2015). Indeed, estradiol depletion decreased hippocampus-

T

dependent recollection in two independent recognition paradigms, although only with trend

IP

level significance in one of the two tests. In addition, memory performance in a standard neuropsychological test was impaired only after a delay, a test known to be hippocampus

SC R

sensitive.

In summary, an effect of differences in estradiol levels on women’s memory was only observed in some of the studies, suggesting a rather subtle influence. The use of sensitive

NU

memory tests that specifically target hippocampus-dependent memory, as in our menstrual cycle and aromatase inhibition studies, increases the likelihood of detecting a possible

MA

relationship (Bayer et al., 2015). Our findings on aromatase inhibition further suggest that other brain regions, i.e. the prefrontal cortex, can partially compensate for reduced hippocampal efficiency. Such compensation on the neuronal level might mask estradiol’s

TE

D

effect on observable behavior, i.e. memory performance, in some of the studies.

Effects of estradiol on hippocampus-dependent memory in male humans

CE P

To explore the effects of estradiol on hippocampus-dependent memory in men, we extended our previous finding, i.e. the association between genetic differences in estradiol levels and hippocampal volume (Bayer et al., 2013). Specifically, we tested memory in an independent sample of 195 healthy young men using three different tasks that are all known to rely on the

AC

hippocampus: spatial learning, emotional memory and verbal source memory. The behavioral data were acquired in the course of a larger project, and genotyping was done as described previously (Bayer et al., 2013). Memory in the three tasks was assessed on the same day, but also on the following day after overnight consolidation. The emotional memory task was similar to the one used in the aforementioned fMRI study (Bayer et al., 2014), but memory for half of the pictures was tested on the same day, the other half on the next day. In particular, old pictures were shown randomly intermixed with new pictures, and volunteers indicated for each, on a 6-point scale, their confidence that this was an old or a new picture. Memory accuracy was assessed by means of d-prime. The contribution of hippocampus-, i.e. recollection, and non-hippocampus-dependent, i.e. familiarity, processes to recognition was estimated by fitting the dual process model to the single-subject confidence ratings using maximum likelihood estimation (Yonelinas, 2002). To test for differences between the three genotype groups, a repeated-measures ANOVA with the 1 3

ACCEPTED MANUSCRIPT within-subject factors valence (positive, neutral, negative) and day (day 1, day 2) and the between-subject factor genotype group was calculated separately for all three memory parameters (Table 1). As expected, memory was better on day 1 than day 2, and was better for

T

emotional than for neutral pictures (statistics not shown for brevity’s sake). Crucially

neither d-prime, nor recollection or familiarity (all ps > 53).

IP

important, genotype did not show any main effect or interaction with day or valence for

SC R

The verbal source memory task was modified according to a previously published study (Cansino et al., 2002). The stimulus material consisted of 200 nouns that were presented during encoding in one of the four quadrants of the screen. During the memory tests on day 1

NU

and 2, each time, half of the old words intermixed with 50 lures were presented in a pseudorandomized order in the middle of the screen. Volunteers indicated for each word whether it

MA

was 'old', and then in which quadrant of the screen it was presented, during encoding. Only the second step, i.e. memory for the word position during encoding, is hippocampusdependent (Cansino et al., 2002). Accuracy of item memory was assessed by means of d-

D

prime, the percentage of correctly remembered word positions relative to the number of words

TE

that were correctly identified as old, served as a measure of associative spatial memory. To examine differences between the genotype groups, a repeated-measures ANOVA with the

CE P

within-subject factor day and the between-subject factor group was calculated separately for the item memory accuracy d-prime and word location. Again, volunteers had better memory on day 1 than 2, but the main affects as well as interactions of genotype were clearly nonsignificant (figure 5C, all ps > .48).

AC

Spatial learning was assessed using a short version of the yellow cab task (Newman et al., 2007). Volunteers had to navigate a taxi from a first-person perspective through a virtual town using a joystick. Each trial began with a search phase, in which volunteers had to pick up virtual passengers who were randomly placed in the town. The passenger needed then to be driven to a specific target store as quickly as possible. The virtual town was identical on both days and each store served three times as target location on each day, so that volunteers could acquire spatial knowledge. Spatial memory performance was measured as the time from pickup to successful delivery ('delivery time') as well as the ratio between the length of the executed delivery path and the optimal delivery path ('path ratio'). Genotype-dependent differences were explored using a repeated-measures ANOVA with the within-subject factor day and the between-subject factor genotype group with the dependent variables delivery times and path ratios. Analyses showed faster delivery times and more efficient delivery pathways on day 2 compared with day 1. Again, the main effects and interactions of genotype 1 4

ACCEPTED MANUSCRIPT were far from being significant (figure 5C, ps>.51). Taken together, we found that none of the memory variables varied with genotype, although we employed several sensitive paradigms to assess specific hippocampus-dependent tasks. In other words, the genotype associated with

T

higher estradiol levels is associated with greater hippocampi, but not better memory

IP

performance. Although this discrepancy seems to be surprising at first sight, a dissociation between an influence of estradiol on hippocampal volume but not memory has also been

SC R

reported for postmenopausal women on HT (Erickson et al., 2010). Interestingly, both studies explored the effects of chronic, i.e. genotype- associated and HT-based differences in estradiol levels and observed a correlation with mesoscopic hippocampal morphology, but not memory

NU

performance. This dissociation suggests that the chronic effects of estradiol on hippocampal morphology might not be relevant for synaptic plasticity. More generally, the observed

in women described in many studies.

MA

discrepancy is also in accordance with the inconsistency of the effect of estradiol on memory

In summary, estradiol´s effects in humans have so far only been investigated using quasi-

D

experimental designs (i.e. not placebo-controlled and double-blind, randomized) that are often

TE

confounded by, for instance, differences in other neuroactive steroids between experimental groups. However, evidence accumulates that estradiol affects human hippocampal

CE P

morphology and physiology. In particular, long-term, i.e. HT, as well as short-term, e.g. menstrual cycle, fluctuations in hormone concentrations affect women‘s hippocampal morphology and physiology. Changes on the behavioral level seem to be rather subtle,

AC

resulting in inconsistencies across studies (Luine, 2008). In addition, non-hippocampal memory tests, as well as compensation by other brain regions for hippocampal decline, might explain these inconsistencies. In men, we have recently shown for the first time that putative chronic differences in estradiol levels, associated with genotype differences, lead to differences in hippocampal volume, which is consistent with animal studies on neuroprotection. On the behavioral level, this difference was also not reflected by differences in memory performance. Remarkably, women receiving aromatase inhibitors for therapeutical reasons actually suffer from subtle memory deficits (Bayer et al., 2015). Thus, further studies are needed concerning the dose of letrozole and duration of treatment. Additional memory tests are required to study putative effects of aromatase inhibition on various types of memory, given that possibly only some of the cellular effects translate into hippocampus-dependent memory differences.

1 5

ACCEPTED MANUSCRIPT Acknowledgements: Sommer: This work was supported by the Deutsche Forschungsgemeinschaft (Grant No: SO 952/6-1, KN556/6-1, and Ru 436/6-1) and by the Landesexzellenzcluster 12/09 "neurodapt!".

T

We wish to thank H. Ehmke (Department of Cellular and Integrative Physiology) for

SC R

assistance. We wish to thank Liz Grundy for linguistic help.

IP

assistance with the electrophysiological setup and I. Jantke and V. Kolbe for skillful technical

References

AC

CE P

TE

D

MA

NU

Arevalo, M.A., Azcoitia I., Garcia-Segura L.M., 2015. The neuroprotective actions of estradiol and estrogen receptors Nat Neurosci Rev 16, 17-29 Aydin, M., Yilmaz, B., Alcin, E., Nedzvetsky, V.S., Sahin, Z., Tuzcu, M., 2008. Effects of letrozole on hippocampal and cortical catecholaminergic neurotransmitter levels, neural cell adhesion molecule expression and spatial learning and memory in female rats. Neuroscience 151, 186-194. Azcoitia, I., Sierra, A., Veiga, S., Honda, S., Harada, N., Garcia-Segura, L.M., 2001. Brain aromatase is neuroprotective. J Neurobiol 47, 318-329. Bäckman, L., Andersson, J.L., Nyberg, L., Winblad, B., Nordberg, A., Almkvist, O., 1999. Brain regions associated with episodic retrieval in normal aging and Alzheimer's disease. Neurology 52, 1861-1870. Barker, J.M., Galea, L.A., 2008. Repeated estradiol administration alters different aspects of neurogenesis and cell death in the hippocampus of female, but not male, rats. Neuroscience 152, 888-902. Bartholomeusz, C.F., Wesnes, K.A., Kulkarni, J., Vitetta, L., Croft, R.J., Nathan, P.J., 2008. Estradiol treatment and its interaction with the cholinergic system: effects on cognitive function in healthy young women. Horm. Behav. 54, 684-693. Bayer, J., Rune, G., Kutsche, K., Schwarze, U., Kalisch, R., Buchel, C., Sommer, T., 2013. Estrogen and the male hippocampus: genetic variation in the aromatase gene predicting serum estrogen is associated with hippocampal gray matter volume in men. Hippocampus 23, 117121. Bayer, J., Rune, G., Schultz, H., Tobia, M.J., Mebes, I., Katzler, O., Sommer, T., 2015. The effect of estrogen synthesis inhibition on hippocampal memory. Psychoneuroendocrinology 56, 213-225. Bayer, J., Schultz, H., Gamer, M., Sommer, T., 2014. Menstrual-cycle dependent fluctuations in ovarian hormones affect emotional memory. Neurobiology of Learning and Memory 110, 55-63. Bender, C.M., Sereika, S.M., Berga, S.L., Vogel, V.G., Brufsky, A.M., Paraska, K.K., Ryan, C.M., 2006. Cognitive impairment associated with adjuvant therapy in breast cancer. Psychooncology 15, 422-430. Bergouignan, L., Chupin, M., Czechowska, Y., Kinkingnehun, S., Lemogne, C., Le Bastard, G., Lepage, M., Garnero, L., Colliot, O., Fossati, P., 2009. Can voxel based morphometry, manual segmentation and automated segmentation equally detect hippocampal volume differences in acute depression? Neuroimage 45, 29-37. Breckenridge, L.M., Bruns, G.L., Todd, B.L., Feuerstein, M., 2012. Cognitive limitations associated with tamoxifen and aromatase inhibitors in employed breast cancer survivors. Psycho-Oncology 21, 43-53. 1 6

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Brown, T.I., Ross, R.S., Keller, J.B., Hasselmo, M.E., Stern, C.E., 2010. Which way was I going? Contextual retrieval supports the disambiguation of well learned overlapping navigational routes. J Neurosci 30, 7414-7422. Cabeza, R., Anderson, N.D., Locantore, J.K., McIntosh, A.R., 2002. Aging gracefully: compensatory brain activity in high-performing older adults. NeuroImage 17, 1394-1402. Cansino, S., Maquet, P., Dolan, R.J., Rugg, M.D., 2002. Brain activity underlying encoding and retrieval of source memory. Cereb Cortex 12, 1048-1056. Clapp, W.C., Hamm, J.P., Kirk, I.J., Teyler, T.J., 2012. Translating long-term potentiation from animals to humans: a novel method for noninvasive assessment of cortical plasticity. Biol Psychiatry 71, 496-502. Collins, B., Mackenzie, J., Stewart, A., Bielajew, C., Verma, S., 2009. Cognitive effects of chemotherapy in post-menopausal breast cancer patients 1 year after treatment. Psychooncology 18, 134-143. Compagnone, N.A., Mellon, S.H., 2000. Neurosteroids: biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol 21, 1-56. Cordoba Montoya, D.A., Carrer, H.F., 1997. Estrogen facilitates induction of long term potentiation in the hippocampus of awake rats. Brain Res 778, 430-438. Daniel, J.M., 2013. Estrogens, estrogen receptors, and female cognitive aging: the impact of timing. Horm Behav 63, 231-237. Eichenbaum, H., Sauvage, M., Fortin, N., Komorowski, R., Lipton, P., 2012. Towards a functional organization of episodic memory in the medial temporal lobe. Neuroscience and biobehavioral reviews 36, 1597-1608. Erickson, K.I., Voss, M.W., Prakash, R.S., Chaddock, L., Kramer, A.F., 2010. A crosssectional study of hormone treatment and hippocampal volume in postmenopausal women: Evidence for a limited window of opportunity. Neuropsychology 24, 68-76. Eriksson, A.L., Lorentzon, M., Vandenput, L., Labrie, F., Lindersson, M., Syvanen, A.C., Orwoll, E.S., Cummings, S.R., Zmuda, J.M., Ljunggren, O., Karlsson, M.K., Mellstrom, D., Ohlsson, C., 2009. Genetic variations in sex steroid-related genes as predictors of serum estrogen levels in men. J Clin Endocrinol Metab 94, 1033-1041. Ernst, T., Chang, L., Cooray, D., Salvador, C., Jovicich, J., Walot, I., Boone, K., Chlebowski, R., 2002. The effects of tamoxifen and estrogen on brain metabolism in elderly women. J Natl Cancer Inst 94, 592-597. Ertman, N., Andreano, J.M., Cahill, L., 2011. Progesterone at encoding predicts subsequent emotional memory. Learn Mem 18, 759-763. Fester, L., Prange-Kiel, J., Zhou, L., Blittersdorf, B.V., Bohm, J., Jarry, H., Schumacher, M., Rune, G.M., 2012. Estrogen-regulated synaptogenesis in the hippocampus: sexual dimorphism in vivo but not in vitro. J Steroid Biochem Mol Biol 131, 24-29. Foster, T.C., 2012. Role of estrogen receptor alpha and beta expression and signaling on cognitive function during aging. Hippocampus. Foy, M.R., Xu, J., Xie, X., Brinton, R.D., Thompson, R.F., Berger, T.W., 1999. 17betaestradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation. J Neurophysiol 81, 925-929. Gleason, C.E., Schmitz, T.W., Hess, T., Koscik, R.L., Trivedi, M.A., Ries, M.L., Carlsson, C.M., Sager, M.A., Asthana, S., Johnson, S.C., 2006. Hormone effects on fMRI and cognitive measures of encoding: importance of hormone preparation. Neurology 67, 2039-2041. Goldstein, J.M., Jerram, M., Abbs, B., Whitfield-Gabrieli, S., Makris, N., 2010. Sex differences in stress response circuitry activation dependent on female hormonal cycle. J Neurosci 30, 431-438. Goto, M., Abe, O., Miyati, T., Inano, S., Hayashi, N., Aoki, S., Mori, H., Kabasawa, H., Ino, K., Yano, K., Iida, K., Mima, K., Ohtomo, K., 2011. 3 Tesla MRI detects accelerated hippocampal volume reduction in postmenopausal women. Journal of magnetic resonance 1 7

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

imaging: JMRI 33, 48-53. Gould, E., Woolley, C.S., Frankfurt, M., McEwen, B.S., 1990. Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. J Neurosci 10, 12861291. Gould, R.L., Arroyo, B., Brown, R.G., Owen, A.M., Bullmore, E.T., Howard, R.J., 2006. Brain mechanisms of successful compensation during learning in Alzheimer disease. Neurology 67, 1011-1017. Grady, C.L., Bernstein, L.J., Beig, S., Siegenthaler, A.L., 2002. The effects of encoding task on age-related differences in the functional neuroanatomy of face memory. Psychology and aging 17, 7-23. Gutchess, A.H., Welsh, R.C., Hedden, T., Bangert, A., Minear, M., Liu, L.L., Park, D.C., 2005. Aging and the neural correlates of successful picture encoding: frontal activations compensate for decreased medial-temporal activity. Journal of cognitive neuroscience 17, 8496. Hedayati, E., Alinaghizadeh, H., Schedin, A., Nyman, H., Albertsson, M., 2012. Effects of adjuvant treatment on cognitive function in women with early breast cancer. Eur J Oncol Nurs 16, 315-322. Hogervorst, E., Bandelow, S., 2010. Sex steroids to maintain cognitive function in women after the menopause: a meta-analyses of treatment trials. Maturitas 66, 56-71. Hojo, Y., Hattori, T., Enami, T., Furukawa, A., Suzuki, K., Ishii, H.T., Mukai, H., Morrison, J.H., Janssen, W.G.M., Kominami, S., Harada, N., Kimoto, T., Kawato, S., 2004. Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017 alpha and P450 aromatase localized in neurons. Proc. Natl. Acad. Sci. U. S. A. 101, 865-870. Ito, K., Skinkle, K.L., Hicks, T.P., 1999. Age-dependent, steroid-specific effects of oestrogen on long-term potentiation in rat hippocampal slices. J Physiol 515 ( Pt 1), 209-220. Jenkins, V., Shilling, V., Deutsch, G., Bloomfield, D., Morris, R., Allan, S., Bishop, H., Hodson, N., Mitra, S., Sadler, G., Shah, E., Stein, R., Whitehead, S., Winstanley, J., 2006. A 3-year prospective study of the effects of adjuvant treatments on cognition in women with early stage breast cancer. Br J Cancer 94, 828-834. Hurria, A., Patel, S.K., Mortimer, J., Luu, T., Somlo, G., Katheria, V., Ramani, R., Hansen, K., Feng, T., Chuang, C., Geist, C.L., Silverman, D.H.S., 2014. The effect of aromatase inhibition of cognitive function of older patients with breast cancer. Clinical Breast Cancer 14, 132-140 Jenkins, V.A., Ambroisine, L.M., Atkins, L., Cuzick, J., Howell, A., Fallowfield, L.J., 2008. Effects of anastrozole on cognitive performance in postmenopausal women: a randomised, double-blind chemoprevention trial (IBIS II). The Lancet. Oncology 9, 953-961. Kramár, E.A., Chen, L.Y., Brandon, N.J., Rex, C.S., Liu, F., Gall, C.M., Lynch, G., 2009. Cytoskeletal changes underlie estrogen's acute effects on synaptic transmission and plasticity. J Neurosci 29, 12982-12993. Lee, J.H., Durand, R., Gradinaru, V., Zhang, F., Goshen, I., Kim, D.S., Fenno, L.E., Ramakrishnan, C., Deisseroth, K., 2010. Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature 465, 788-792. Lejbak, L., Vrbancic, M., Crossley, M., 2010. Endocrine therapy is associated with low performance on some estrogen-sensitive cognitive tasks in postmenopausal women with breast cancer. Journal of Clinical and Experimental Neuropsychology 32, 836-846. Leranth, C., Hajszan, T., MacLusky, N.J., 2004. Androgens increase spine synapse density in the CA1 hippocampal subfield of ovariectomized female rats. J Neurosci 24, 495-499. Leranth, C., Petnehazy, O., MacLusky, N.J., 2003. Gonadal hormones affect spine synaptic density in the CA1 hippocampal subfield of male rats. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 23, 1588-1592. Logothetis, N.K., Pauls, J., Augath, M., Trinath, T., Oeltermann, A., 2001. Neurophysiological investigation of the basis of the fMRI signal. Nature 412, 150-157. 1 8

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Lord, C., Buss, C., Lupien, S.J., Pruessner, J.C., 2008. Hippocampal volumes are larger in postmenopausal women using estrogen therapy compared to past users, never users and men: A possible window of opportunity effect. Neurobiology of Aging 29, 95-101. Luine, V.N., 2008. Sex steroids and cognitive function. J Neuroendocrinol 20, 866-872. Maki, P.M., Dennerstein, L., Clark, M., Guthrie, J., LaMontagne, P., Fornelli, D., Little, D., Henderson, V.W., Resnick, S.M., 2011. Perimenopausal use of hormone therapy is associated with enhanced memory and hippocampal function later in life. Brain Res 1379, 232-243. Maki, P.M., Resnick, S.M., 2000. Longitudinal effects of estrogen replacement therapy on PET cerebral blood flow and cognition. Neurobiol Aging 21, 373-383. Maki, P.M., Zonderman, A.B., Resnick, S.M., 2001. Enhanced verbal memory in nondemented elderly women receiving hormone-replacement therapy. Am J Psychiatry 158, 227-233. McCarthy, M.M., 2011. A lumpers versus splitters approach to sexual differentiation of the brain. Front Neuroendocrinol 32, 114-123. McCullough, L.D., Hurn, P.D., 2003. Estrogen and ischemic neuroprotection: an integrated view. Trends in endocrinology and metabolism: TEM 14, 228-235. Milner, T.A., McEwen, B.S., Hayashi, S., Li, C.J., Reagan, L.P., Alves, S.E., 2001. Ultrastructural evidence that hippocampal alpha estrogen receptors are located at extranuclear sites. J Comp Neurol 429, 355-371. Mitra, S.W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H.A., Hayashi, S., Pfaff, D.W., Ogawa, S., Rohrer, S.P., Schaeffer, J.M., McEwen, B.S., Alves, S.E., 2003. Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha. Endocrinology 144, 2055-2067. Morse, J.K., Scheff, S.W., DeKosky, S.T., 1986. Gonadal steroids influence axon sprouting in the hippocampal dentate gyrus: a sexually dimorphic response. Exp. Neurol. 94, 649-658. Naftolin, F., Ryan, K.J., Petro, Z., 1971. Aromatization of androstenedione by the diencephalon. J Clin Endocrinol Metab 33, 368-370. Newman, E.L., Caplan, J.B., Kirschen, M.P., Korolev, I.O., Sekuler, R., Kahana, M.J., 2007. Learning your way around town: how virtual taxicab drivers learn to use both layout and landmark information. Cognition 104, 231-253. Olivo-Marston, S.E., Mechanic, L.E., Mollerup, S., Bowman, E.D., Remaley, A.T., Forman, M.R., Skaug, V., Zheng, Y.L., Haugen, A., Harris, C.C., 2010. Serum estrogen and tumorpositive estrogen receptor-alpha are strong prognostic classifiers of non-small-cell lung cancer survival in both men and women. Carcinogenesis 31, 1778-1786. Peter, I., Kelley-Hedgepeth, A., Fox, C.S., Cupples, L.A., Huggins, G.S., Housman, D.E., Karas, R.H., Mendelsohn, M.E., Levy, D., Murabito, J.M., 2008. Variation in estrogen-related genes associated with cardiovascular phenotypes and circulating estradiol, testosterone, and dehydroepiandrosterone sulfate levels. The Journal of Clinical Endocrinology and Metabolism 93, 2779-2785. Phillips, S.M., Sherwin, B.B., 1992a. Effects of estrogen on memory function in surgically menopausal women. Psychoneuroendocrinology 17, 485-495. Phillips, S.M., Sherwin, B.B., 1992b. Variations in memory function and sex steroid hormones across the menstrual cycle. Psychoneuroendocrinology 17, 497-506. Prange-Kiel, J., Fester, L., Zhou, L., Lauke, H., Carretero, J., Rune, G.M., 2006. Inhibition of hippocampal estrogen synthesis causes region-specific downregulation of synaptic protein expression in hippocampal neurons. Hippocampus 16, 464-471. Prange-Kiel, J., Jarry, H., Schoen, M., Kohlmann, P., Lohse, C., Zhou, L., Rune, G.M., 2008. Gonadotropin-releasing hormone regulates spine density via its regulatory role in hippocampal estrogen synthesis. J Cell Biol 180, 417-426. Prange-Kiel, J., Rune, G.M., 2006. Direct and indirect effects of estrogen on rat hippocampus. Neuroscience 138, 765-772. 1 9

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Prange-Kiel, J., Schmutterer, T., Fester, L., Zhou, L., Imholz, P., Brandt, N., Vierk, R., Jarry, H., Rune, G.M., 2013. Endocrine regulation of estrogen synthesis in the hippocampus? Progress in Histochemistry and Cytochemistry 48, 49-64. Prange-Kiel, J., Wehrenberg, U., Jarry, H., Rune, G.M., 2003. Para/autocrine regulation of estrogen receptors in hippocampal neurons. Hippocampus 13, 226-234. Protopopescu, X., Butler, T., Pan, H., Root, J., Altemus, M., Polanecsky, M., McEwen, B., Silbersweig, D., Stern, E., 2008. Hippocampal structural changes across the menstrual cycle. Hippocampus 18, 985-988. Qiu, L.R., Germann, J., Spring, S., Alm, C., Vousden, D.A., Palmert, M.R., Lerch, J.P., 2013. Hippocampal volumes differ across the mouse estrous cycle, can change within 24hours, and associate with cognitive strategies. NeuroImage 83, 593-598. Resnick, A., Perry, W., Parry, B., Mostofi, N., Udell, C., 1998. Neuropsychological performance across the menstrual cycle in women with and without Premenstrual Dysphoric Disorder. Psychiatry research 77, 147-158. Robertson, D.M., van Amelsvoort, T., Daly, E., Simmons, A., Whitehead, M., Morris, R.G., Murphy, K.C., Murphy, D.G., 2001. Effects of estrogen replacement therapy on human brain aging: an in vivo 1H MRS study. Neurology 57, 2114-2117. Schilder, C.M., Seynaeve, C., Beex, L.V., Boogerd, W., Linn, S.C., Gundy, C.M., Huizenga, H.M., Nortier, J.W., van de Velde, C.J., van Dam, F.S., Schagen, S.B., 2010. Effects of tamoxifen and exemestane on cognitive functioning of postmenopausal patients with breast cancer: results from the neuropsychological side study of the tamoxifen and exemestane adjuvant multinational trial. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 28, 1294-1300. Sherwin, B.B., 1998. Estrogen and cognitive functioning in women. Proc Soc Exp Biol Med 217, 17-22. Sherwin, B.B., Tulandi, T., 1996. "Add-back" estrogen reverses cognitive deficits induced by a gonadotropin-releasing hormone agonist in women with leiomyomata uteri. J Clin Endocrinol Metab 81, 2545-2549. Shibuya, K., Takata, N., Hojo, Y., Furukawa, A., Yasumatsu, N., Kimoto, T., Enami, T., Suzuki, K., Tanabe, N., Ishii, H., Mukai, H., Takahashi, T., Hattori, T.A., Kawato, S., 2003. Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction. Biochim Biophys Acta 1619, 301-316. Shilling, V., Jenkins, V., Fallowfield, L., Howell, T., 2003. The effects of hormone therapy on cognition in breast cancer. J Steroid Biochem Mol Biol 86, 405-412. Shughrue, P.J., Lane, M.V., Merchenthaler, I., 1997. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. The Journal of comparative neurology 388, 507-525. Smith, C.C., McMahon, L.L., 2005. Estrogen-induced increase in the magnitude of long-term potentiation occurs only when the ratio of NMDA transmission to AMPA transmission is increased. J Neurosci 25, 7780-7791. Solis-Ortiz, S., Corsi-Cabrera, M., 2008. Sustained attention is favored by progesterone during early luteal phase and visuo-spatial memory by estrogens during ovulatory phase in young women. Psychoneuroendocrinology 33, 989-998. Spencer, J.L., Waters, E.M., Romeo, R.D., Wood, G.E., Milner, T.A., McEwen, B.S., 2008. Uncovering the mechanisms of estrogen effects on hippocampal function. Front Neuroendocrinol 29, 219-237. Spritzer, M.D., Galea, L.A., 2007. Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats. Developmental neurobiology 67, 1321-1333. Veiga, S., Azcoitia, I., Garcia-Segura, L.M., 2005. Extragonadal synthesis of estradiol is protective against kainic acid excitotoxic damage to the hippocampus. Neuroreport 16, 15992 0

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

1603. Vierk, R., Glassmeier, G., Zhou, L., Brandt, N., Fester, L., Dudzinski, D., Wilkars, W., Bender, R.A., Lewerenz, M., Gloger, S., Graser, L., Schwarz, J., Rune, G.M., 2012. Aromatase Inhibition Abolishes LTP Generation in Female But Not in Male Mice. J. Neurosci. 32, 8116-8126. Woolley, C.S., 1998. Estrogen-Mediated Structural and Functional Synaptic Plasticity in the Female Rat Hippocampus. Horm. Behav. 34, 140-148. Woolley, C.S., Gould, E., Frankfurt, M., McEwen, B.S., 1990. Naturally-Occurring Fluctuation in Dendritic Spine Density on Adult Hippocampal Pyramidal Neurons. J. Neurosci. 10, 4035-4039. Woolley, C.S., McEwen, B.S., 1993. Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. The Journal of Comparative Neurology 336, 293-306. Yonelinas, A.P., 2002. The nature of recollection and familiarity: a review of 30 years of research. Journal of Memory and Language 46, 441-517. Yuste, R., Bonhoeffer, T., 2001. Morphological changes in dendritic spines associated with long-term synaptic plasticity. Annu Rev Neurosci 24, 1071-1089. Zatorre, R.J., Fields, R.D., Johansen-Berg, H., 2012. Plasticity in gray and white: neuroimaging changes in brain structure during learning. Nat Neurosci 15, 528-536. Zhou, L., Fester, L., Haghshenas, S., de Vrese, X., von Hacht, R., Gloger, S., Brandt, N., Bader, M., Vollmer, G., Rune, G.M., 2014. Oestradiol-Induced Synapse Formation in the Female Hippocampus: Roles of Oestrogen Receptor Subtypes. J Neuroendocrinol 26, 439447. Zhou, L., Fester, L., von Blittersdorff, B., Hassu, B., Nogens, H., Prange-Kiel, J., Jarry, H., Wegscheider, K., Rune, G.M., 2010. Aromatase inhibitors induce spine synapse loss in the hippocampus of ovariectomized mice. Endocrinology 151, 1153-1160. Zhou, L., Fester, L., Haghshenas, S., de Vrese, X., v. Hacht, R., Gloger, S., Brandt, N., Bader, M., Vollmer, G., Rune, G.M. , 2014. Oestradiol-induced synapse formation in the female hippocampus: roles of estrogen receptor subtypes. J Neuroendocrinol.

2 1

ACCEPTED MANUSCRIPT Legends to the figures

Table 1

T

Results of the three memory tasks (mean (M) and standard error (SE)) in humans grouped

IP

according to the genotype of the c.240A>G variant in CYP19A1 (AA, GG or AG genotype).

SC R

The recognition sensitivity index d-prime is in standard deviation units. The parameter estimates recollection and familiarity, as derived from the dual process model, are in arbitrary

NU

units.

Fig. 1

Spine synapse density. (A) Stereological evaluation of spine synapse density in stratum

MA

radiatum of the CA1 region in the hippocampus. In cycling females as well as in ovariectomized (OVX) animals, letrozole results in a significant decrease in spine synapse density after 7 days and 4 weeks of treatment. (B) Stereological evaluation of spine synapse

D

density in prefrontal and cerebellar cortex. Spine synapse density does not differ from the

TE

controls after 4 weeks of treatment with letrozole in cycling animals. All groups n=6, mean ±

Fig. 2

CE P

SEM; control: cycling, intact animals.

Changes in the average course of fEPSP slopes in female and male mice before and after

AC

letrozole treatment. (A) Quantitative evaluation of changes in the average course of fEPSP slopes after letrozole treatment in females and in males. In control animals, theta-burst stimulation (TBS) resulted in 198% ± 4% LTP in female animals and 198% ± 8% in male animals 60 min after stimulation, compared to baseline. Animals treated with DMSO showed similar potentiation 60 min after TBS in both sexes (female 207% ± 3%; male, 199% ± 5%). Letrozole treatment of female animals resulted in 160% ± 10% LTP 60 min after high frequency stimulation and 182% ± 5% in male animals treated with letrozole. (B) Changes in average fEPSP slopes over time before and after theta-burst stimulation (arrow) in letrozoletreated (Letro), DMSO-treated (DMSO) and sham-treated (Sham) female and male mice, shown in (A). Mean ± SEM; n = number of acute slices of N = different animals.

Fig. 3 (A) Experimental design of Morris water maze experiment to examine reference memory. Mice received daily i.p. injections of respective drugs 1 week prior to commencement of the 2 2

ACCEPTED MANUSCRIPT MWM experiment. Three groups were tested: animals injected with letrozole (Letro), control animals injected with DMSO and control sham injected animals (handled similar to the injected groups, but without injection). All treatment groups showed significant improvement

T

in performance with increased acquisition training. (B. i-iii) Male mice [main effect of days:

IP

Path length (F 4, 84) = 12.80, P < 0.0001); Latency (F 4, 84) = 13.13, P < 0.0001). The effect of drug did not reach statistical significance, indicating that aromatase inhibition does not lead to

SC R

apparent changes in acquisition in the MWM [Treatment: Path length (P = 0.44); Latency (P = 0.55)]. (C. i-iii) Female mice [main effect of days: Path length (F

4, 92)

= 19.53, P < 0.0001);

Latency (F 4, 92) = 17.93, P < 0.0001)]. Similarly, letrozole did not induce significant changes

NU

in performance [Treatment: Path length (P = 0.14); Latency (P = 0.11)]. Swim speed during acquisition were comparable for all treatment groups, indicating that letrozole treatment does

MA

not lead to sensorimotor changes. Data expressed as mean ± SEM; N = 8-9 mice per group and sex.

D

Fig. 4

TE

Probe trials performed to examine reference memory. All treatment groups spent significantly more time in the target training quadrant, thus demonstrating successful acquisition of the

CE P

task. (A) Proportion to time (%) spent in the target quadrant in the male mice [main effect of quadrant: Probe 1 (F

3, 63

= 26.64, P < 0.0001); Probe 2 (F

3, 63

= 18.70, P < 0.0001). Again,

there were no differences in swim speed. (B) Percentage time spent in the target quadrant in female mice [main effect of quadrant: Probe 1 (F

3, 69

= 21.69, P < 0.0001); Probe 2 (F

3, 69

=

AC

10.38, P < 0.0001). Data expressed as mean ± sem. Dashed line depicts chance level performance (25%). Fig. 5 Effect of a single nucleotide polymorphism c.240A>G in the gene coding for aromatase (CYP19A1) that has been consistently associated with differences in serum estradiol levels in men (individuals with the AA genotype have higher estradiol levels; (Peter et al., 2008) on hippocampal morphology and hippocampus-dependent memory. (A) Glass brain and overlay on structural MR image (t-values color-coded) of the conjunction where AA carriers of two independent samples of healthy young men show greater posterior bilateral hippocampi. (B) Genotype-dependent grey matter distribution differences in the left and right posterior hippocampus (Bayer et al., 2013). (C) Performance of a third independent sample of 195 young men in two hippocampus-dependent memory tasks on day 1 and 2. Left panel proportion of correct source memory in a verbal source memory task, right panel path ratio in 2 3

ACCEPTED MANUSCRIPT the spatial learning task (48 volunteers were homozygous for the A allele, 67 were homozygous for the G allele and 80 were heterozygous).

T

Fig. 6

IP

Greater hippocampal activity (t-values color-coded) during encoding of positive pictures in

AC

CE P

TE

D

MA

NU

SC R

the luteal compared to early follicular phase of the menstrual cycle (Bayer et al., 2014).

2 4

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 1

2 5

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 2

2 6

Figure 3

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

2 7

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Figure 4

2 8

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Figure 5

2 9

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 6

3 0

ACCEPTED MANUSCRIPT Table 1 AA M

AG SE

M

GG SE

M

SE

0.558 0.374 0.479

TE

AC

familiarity negative neutral positive

CE P

recollection negative neutral positive

D

dprime negative neutral positive

day 1 dprime word position (percentage correct) day 2 dprime word position (percentage correct) day 1 path ratio (executed path/optimal path) time (ms) day 2 path ratio (executed path/optimal path) time (ms)

2.676 2.105 2.418

0.124 0.120 0.111

2.593 2.044 2.637

0.121 0.100 0.116

0.042 0.041 0.046

0.519 0.383 0.419

0.033 0.031 0.034

0.497 0.390 0.430

0.039 0.034 0.038

2.322 1.498 2.143

0.359 0.177 0.288

2.244 1.720 2.057

0.276 0.228 0.229

2.137 1.271 2.019

0.297 0.089 0.234

1.790 1.185 1.520

0.122 0.078 0.120

1.800 1.259 1.567

0.081 0.086 0.094

1.858 1.288 1.568

0.100 0.084 0.078

0.272 0.147 0.220

0.030 0.022 0.029

0.240 0.132 0.193

0.025 0.018 0.022

0.252 0.164 0.198

0.024 0.020 0.022

1.433 0.897 1.032

0.212 0.084 0.094

1.230 0.843 1.216

0.072 0.069 0.102

1.329 0.834 1.037

0.128 0.065 0.058

0.073 0.024

1.961 0.558

0.058 0.018

1.928 0.549

0.063 0.020

0.861 0.051 0.294 0.014 spatial memory task

0.835 0.302

0.045 0.013

0.883 0.306

0.046 0.015

1.793

0.051

1.802

0.054

25.793 1,124.736

28.279

MA

familiarity negative neutral positive day 2

IP

recollection negative neutral positive

0.131 0.125 0.133

SC R

2.662 2.183 2.673

NU

day 1 dprime negative neutral positive

T

emotional memory task

verbal source memory task 1.974 0.519

1.747 1,105.763

0.057

32.751 1,111.915

1.313

0.054

1.318

0.038

1.344

0.059

863.853

30.521

864.942

21.692

857.070

28.752

3 1

ACCEPTED MANUSCRIPT Highlights

AC

CE P

TE

D

MA

NU

SC R

IP

T

LTP and synapse density depend on hippocampus-derived sex steroids Local estradiol synthesis is essential for synaptic plasticity in female hippocampus Estrogens effect hippocampal morphology in women

3 2