Accepted Manuscript

Neuroscience Forefront Review

Hippocampal estradiol synthesis and its significance for hippocampal synapticstability in male and female animals

Ricardo Vierk, Nicola Brandt, Gabriele M. Rune

PII: S0306-4522(14)00384-4DOI: http://dx.doi.org/10.1016/j.neuroscience.2014.05.003Reference: NSC 15403

To appear in: Neuroscience

Accepted Date: 4 May 2014

Please cite this article as: R. Vierk, N. Brandt, G.M. Rune, Hippocampal estradiol synthesis and its significance forhippocampal synaptic stability in male and female animals, Neuroscience (2014), doi: http://dx.doi.org/10.1016/j.neuroscience.2014.05.003

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Hippocampal estradiol synthesis and its significance for hippocampal synaptic stability in male and female animals Ricardo Vierk, Nicola Brandt, Gabriele M. Rune* Institute of Neuroanatomy, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany Running title: Estrogen-induced synaptic plasticity *Corresponding author Dr. Gabriele M. Rune Institut für Neuroanatomie Universitätsklinikum Hamburg-Eppendorf Martinistr. 52 20246 Hamburg Germany Tel: +49-7410-53575 Fax: +49-7410-5496 e-mail: rune@uke.de Key words: aromatase, estrogen, sexual dimorphism, synaptic plasticity The study was supported by the Deutsche Forschungsgemeinschaft

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Abstract Increasing evidence points to an essential role played by neuron-derived

neurosteroids, such as estrogen, on synaptic connectivity in the hippocampus.

Inhibition of local estradiol synthesis results in synapse loss specifically in

females, but not in males. Synapse loss in females, after inhibition of estradiol

synthesis in hippocampal neurons, appears to result from impairment of long-

term potentiation (LTP) and dephosphorylation of cofilin, and thereby the

destabilization of postsynaptic dendritic spines. Such clear-cut effects were

not seen in males. Cognitive deficits after inhibition of aromatase, the final

enzyme of estrogen synthesis, have been seen in women, but not in men.

Altogether, the data demonstrate distinct differences between genders in

neurosteroid-induced synaptic stability.

Introduction

Steroids are essential for brain function, from the early steps of differentiation to the

senescent brain during which they help to maintain neuronal performance and

protect against damage. The brain is equipped with all enzymes active in

steroidogenesis and is thus capable of synthesizing so-called “neurosteroids”, which

act locally to modify neural performance (Compagnone and Mellon, 2000, Shibuya et

al., 2003). Steroids, however, also enter the brain from the peripheral circulation.

Both pathways – paracrine and endocrine - are often interlinked. Thus, the precise

attribution of the individual routes to physiological processes is not easily addressed.

As to the origin of steroids in the CNS, the varying density of spines along

hippocampal dendrites of CA1 pyramidal neurons strongly supports the idea that

estrogen of ovarian origin regulates spinogenesis in the hippocampus (Woolley et al.,

1990; for review see Spencer et al., 2008). In addition, the removal of gonads has

been shown to result in reduced dendritic spine density in this brain area of males

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and of females (Gould et al., 1990, Leranth et al., 2004). A rescue of spine loss after

gonadectomy could be achieved by injections of estradiol in females but not in males.

In gonadectomized males, injections of testosterone restored reduced spine density

after removal of the testes (Leranth et al., 2004).

Our data from recent years point to hippocampus-derived estradiol as the

main and primary player in estrogen-induced synaptic plasticity in females. We

inhibited estradiol synthesis in hippocampal cultures, thereby in the absence of any

other source of estradiol, either pharmacologically, by using inhibitors of aromatase,

the final enzyme of estradiol synthesis, or by knock-down of steroidogenic acute

regulatory protein (StAR), the rate-limiting step in estrogen synthesis. Inhibition of

aromatase in hippocampal slice cultures resulted in a significant reduction in the

number of spine synapses in the CA1 hippocampal region (Kretz et al., 2004,

Prange-Kiel et al., 2006). Similarly, knock-down of StAR, the protein that transports

cholesterol through the mitochondrial membranes, where steroidogenesis

commences, induced a downregulation of pre- and postsynaptic marker proteins

(Fester et al., 2009). Systemic treatment of female mice with letrozole also

significantly reduced spine synapse density and, most importantly, this effect was

also seen in ovariectomized animals, thus after having removed the main source of

estradiol in females (Zhou et al., 2010). The latter result strongly supports the notion

that in females, estradiol originating from neurons is essential for synaptogenesis,

rather than estrogen from peripheral sources (Prange-Kiel et al., 2013).

Nevertheless, we also found that estradiol synthesis in the female

hippocampus is linked to the hypothalamic-hypophyseal axis via the regulatory role of

Gonadotropin releasing hormone (GnRH) on hippocampal estrogen synthesis. We

showed that spine synapse density increases dose-dependently upon GnRH

stimulation in hippocampal cultures from female animals; this regulation functions via

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stimulation of aromatase. Thus, in females, brain sex steroid levels should correlate

to circulating levels of sex hormones, which vary depending on the reproductive state

of the organism throughout life (Prange-Kiel et al., 2008, Prange-Kiel et al., 2013).

This was recently confirmed by Kato et al. (2013), showing that in fact estradiol

concentrations in hippocampal tissue correlate to estradiol concentrations in serum

during the estrous cycle. Consistently, it was previously shown that cyclic peripheral

concentrations of estradiol influence StAR expression in the brain (Meethal et al.,

2009), thus estradiol concentrations in serum influence steroidogenesis in the brain,

as the expression of StAR is the rate limiting step in estradiol synthesis.

Estrogen synthesis and spine synapse density in the hippocampus of males

and females

Although it has been known for years that the brain is equipped with all enzymes

active in steroidogenesis, our knowledge on differences in the capacity of

neurosteroid synthesis between genders is fragmentary. Naftolin (1971) was the first

to describe aromatase expression in the diencephalon in females. Aromatase is the

final enzyme of estradiol synthesis and catalyzes the conversion of testosterone to

estradiol. Ten years ago, we showed that the enzyme is actually functional. After a

couple of days in culture, hippocampal neurons had secreted estradiol into the

supernatant, which was determined by RIA (Prange-Kiel et al., 2003), which was

confirmed shortly thereafter by Hojo et al. (2004). As to differences between genders,

immunoreactivity of aromatase was seen in hippocampal neurons of male animals as

well as in those of female animals. Laser scanning microscopy and subsequent

image analysis did not reveal any difference in the expression of the protein between

genders (Fester et al., 2012). Although protein expression does not allow for any

conclusion on the activity of the enzyme, the amount of estradiol measured in the

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supernatant of cultivated “female” and “male” neurons was similar as well (Fester et

al., 2012). In contrast to these in vitro findings we found clear-cut differences

between genders, when we measured the content of estradiol in male and female

hippocampal tissue by mass spectrometry. In hippocampal tissue of male animals

and ovariectomized animals, the amount of estradiol was extremely low and below

the sensitivity of our system. In contrast, in hippocampal tissue of females the

amount of estradiol was easily measured. The differences between males and

females in the amount of estradiol in hippocampal tissue corresponded to the

differences between genders in estradiol concentrations in plasma (Fester, 2012).

Robust functional sex differences have been identified in the hypothalamic

circuitry regulating reproductive function. Increasing levels of estradiol synthesized by

granulosa cells in the ovaries and released into the blood stream trigger the release

of gonadotropin releasing hormone (GnRH) from neurons scattered throughout the

hypothalamus, which in turn induce the mid-cycle surge in luteinizing hormone (LH)

that is essential for ovulation. In contrast, in male rats the pattern of LH release upon

GnRH stimulation is tonic and acyclic, thus leading to a steady state of testosterone,

which always exerts a negative feed back on GnRH release (for review see Gillies

and McArthur (2010). The lack of cyclic variations of GnRH/LH in males should result

in differences between genders. In fact, we found that systemic inhibition of

aromatase in mice had no effect on spine synapse density in males (Vierk et al.,

2012). In males, an increase in synapse density was even found in tendency. In this

context, it is of note that concentrations of testosterone, being the direct substrate for

the enzyme aromatase, should increase in tissue as well as in serum after inhibition

of aromatase activity. According to data by Leranth and coworkers (2004), showing

that spine loss in response to gonadectomy was rescued by testosterone, aromatase

inhibition in hippocampal neurons should actually result in an increase in synapse

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density in males. The failure of letrozole to increase spine density in males may be

explained by insufficient activity of 5alpha reductase, which converts testosterone to

dehydrotestosterone, the active metabolite with a higher affinity to androgen

receptors. The role of hippocampus-derived testosterone in hippocampal cultures

that originate from male animals are currently under investigation in our laboratory.

Does spine synapse loss after inhibition of neuronal estradiol synthesis result

from the effects of estradiol on the spine cytoskeleton?

Both the function and stability of dendritic spines depend on an intact actin

cytoskeleton (Honkura et al., 2008, Dent et al., 2010, Hotulainen and Hoogenraad,

2010). Our results suggest that aromatase activity in hippocampal neurons is

required for the stabilization of postsynaptic spines. In stable spines, disassembly of

F-actin is prevented as soon as cofilin, an actin associated protein, becomes

phosphorylated. In hippocampal slice cultures of female animals, we found

pronounced dephosphorylation of cofilin, together with synapse loss, after 7 days of

letrozole treatment. In mice treated with letrozole, we also found enhanced

dephosphorylation of cofilin as early as after 6 hours of treatment. Accordingly,

intracellularly synthesized estradiol obviously induces phosphorylation of cofilin,

presumably via activating LIM-1 kinase, as shown by (Kramár et al., 2009b). Cofilin is

itself regulated; it becomes inactive when phosphorylated by LIM-1 kinase, thus

allowing polymerization to proceed (Bramham, 2008).

Our findings in hippocampal slice cultures confirm the studies by Kramár et al.

(Kramár et al., 2009a), insofar as we achieved opposite effects after inhibition of

estradiol synthesis in hippocampal neurons, thus, most importantly, in the absence of

any other source of estradiol. Kramár et al. (Kramár et al., 2009a) showed that

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application of estradiol to acute hippocampal slices increases phalloidin-positive

punctae, indicating enhanced F-actin polymerization, and consequently increases

phosphorylation of cofilin via activation of LIM-1 kinase, which altogether stabilizes

spines. The time course of letrozole effects on spinogenesis and cofilin activity

suggests that spine synapse loss in response to letrozole is mediated by its effects

on the spine cytoskeleton. Dephosphorylation of cofilin due to aromatase inhibition in

female mice was seen as early as after 6 hours of treatment, whereas a significant

spine synapse loss was not seen before 48 hours of treatment had elapsed.

Obviously, destabilizing the spine cytoskeleton precedes and finally results in spine

synapse loss. This result is consistent with the finding that LIM-1 kinase knock-out

mice produce spines that lack actin and have small synapses (Meng et al., 2002).

The essential role of the actin cytoskeleton, which is regulated by numerous proteins,

in the plasticity and maintenance of spines, has been extensively shown (for review

see (Tada et al., 2007, Bramham, 2008, Kasai et al., 2010).

It is of note in this context, that in females, blocking cofilin phosphorylation by

letrozole is likely to prevent the formation of large actin-rich spines, or it reduces

spine head diameter of pre-existing spines, respectively. The proportion of large,

mushroom-shaped, actin-rich spines was particularly reduced, while thin spines were

less affected in hippocampal tissue of mice treated with letrozole; mushroom spines,

in turn, are considered to be the memory spines (Bourne and Harris, 2007, Tada et

al., 2007, Kasai et al., 2010).

Regarding the spine phenotype, the findings after application of estradiol to

acute slices, as reported in the literature, are discrepant. For instance, Kawato and

coworkers (2007) found an increase in the number of spines, but no alteration in

head diameter, thus estradiol only increases the number of thin, immature spines.

Using an almost identical experimental paradigm, Kramár et al. (2009a) found an

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increase in the number of phalloidin-labeled spine-like profiles. The latter finding,

together with the findings by Kawato’s research group, would suggest that estradiol

primarily increases F-actin in thin spines, whereas mushroom spines are not affected,

although mushroom spines particularly depend on the actin cytoskeleton.

Furthermore, estradiol was found to rapidly enhance long-term depression (LTD)

(Mukai et al., 2007), which is known to cause shrinkage of spines (Nagerl et al., 2004,

Okamoto et al., 2004), whereas Kramár et al. (2009a) found LTP to be enhanced in a

non-genomic rapid manner, which stimulates spine formation (Yuste and Bonhoeffer,

2001, Matsuzaki et al., 2004). Along these lines, in ovariectomized mice, but not in

rats, estradiol injections failed to increase spine number; only the number of large

spines was increased (Li et al., 2004). We used rats and mice for our experiments

with consistent results. In addition, aromatase inhibition in female mice, even in

ovariectomized animals, significantly reduces the number of spine synapses (Zhou et

al., 2010). We found a clear time course of events that started 6 hours after

aromatase inhibition was induced, either in female mice and/or in “female” rat

hippocampal slice cultures, and which reached its maximum after 7 days.

In summary, application of estradiol at non-physiological concentrations, such

as 1 nM estradiol in male rats (Mukai et al., 2007, Kramár et al., 2009a), and

inhibition of local estradiol synthesis do not automatically cause opposite effects.

Since we have frequently shown that hippocampus-derived estradiol, rather than

estradiol originating from other sources, influences synaptic plasticity, namely due to

the regulatory role of GnRH on hippocampal estradiol synthesis (Kretz et al., 2004,

Zhou et al., 2007, Prange-Kiel et al., 2008, Prange-Kiel et al., 2009, Zhou et al.,

2010), we presume that inhibition of local estradiol synthesis is the more adequate

approach to study the role of estradiol in synaptic plasticity, at least in females.

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Despite the intriguing idea that the influence of estradiol on spine formation in

the hippocampus is mediated via the effect of estradiol on the spine cytoskeleton, it

remains an open question as to why dephosphorylation of cofilin in response to

letrozole does not result in spine synapse loss in males. Although the time course

and the degree of downregulation of p-cofilin were similar in male and female mice, a

similar decrease in the synapse density seen in females was not observed in males.

Obviously, another additional mechanism protects “male” synapses from regression.

Long-Term Potentiation, cofilin, and estradiol

Our findings on spine synapse density after inhibition of neuronal estradiol synthesis

and the question as to whether this letrozole-induced synapse loss is of any

biological significance prompted us to study the effects of letrozole on long-term

potentiation (LTP) as a cellular, electrophysiological parameter for memory. In

addition, the questions as to whether the paradigm that “LTP induces spines” (Engert

and Bonhoeffer, 1999, Yuste and Bonhoeffer, 2001, 2004) is also valid vice versa,

and the paradigm “LTP is paralleled by increased phosphorylation of cofilin”

(Fukazawa et al., 2003), were addressed in the study by Vierk et al. (Vierk et al.,

2012).

It has frequently been shown that estradiol enhances LTP in the hippocampus

(for review see Spencer et al., 2008). The effects of estradiol on spine density and

LTP, however, were shown in most studies either by using ovariectomized animals,

which were treated systemically with estradiol (Gould et al., 1990, Warren et al., 1995,

Cordoba Montoya and Carrer, 1997, Smith and McMahon, 2005, 2006, Kramár et al.,

2009a), or estradiol was applied to acute slices of mostly male rats (Foy et al., 1999,

Ito et al., 1999, Mukai et al., 2007, Kramár et al., 2009a), or by using hippocampal

slice cultures indifferently prepared from male and neonatal females (Murphy and

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Segal, 1996, Kretz et al., 2004, Mendez et al., 2011). The vast majority of results

from these studies show that exogenous application of estradiol is primarily effective

after having removed estradiol from the environment before the treatment, i.e. after

ovariectomy, and by using an in vitro experimental approach. These experimental

approaches leave the question open as to whether in females, estradiol actually

causes major alterations of non-affected pre-existing networks (Mendez et al., 2011)

or simply restores effects after estradiol depletion. In males, the biological

significance of applying estradiol at highly supraphysiological doses is a further

obvious question.

We studied LTP and synaptic density in acute slices of animals treated by

daily injections of letrozole over 7 days, and hippocampal slice cultures treated with

letrozole for 7 days. In both cases the effect was controlled by measurements of

estradiol concentrations in serum and in the supernatant resp. After 7 days of

treatment LTP could no longer be induced in females and this was paralleled by

significant synapse loss; in males, however, LTP was reduced by 20% with no loss of

synapses after treatment (Fig. 1).

Regarding time-dependency, impairment of LTP was found as early as 6

hours after a single injection of letrozole. Dephosphorylation of cofilin, was also

already apparent after 6 hours had elapsed. Spine synapse loss, however, was not

seen prior to 48 hours after the last injection, which made us assume that synapse

loss results from impaired LTP (Fig. 1;Vierk et al., 2012)) according to the paradigm

that LTP induces spines (Engert and Bonhoeffer, 1999).

In addition, in our hands effects were not seen when we applied letrozole

directly to acute slices for 3–4 hours after stable recording of LTPs for 90 min

(unpublished observations). This finding is consistent with findings by Grassi and

coworkers (Grassi et al., 2011). They found, however, that the potentiation after high

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frequency stimulation but not the maintenance of LTP was markedly reduced in the

presence of letrozole in males compared to that induced under control conditions.

This effect was seen as early as after 1 min and progressed further up to 40 min. An

effect after 1 min and 10 min would argue for a so-called rapid membrane-bound

estrogenic effect. Given this background, it is interesting that the effects of letrozole

could be mimicked by using ICI 182780. This compound has been used to

discriminate between rapid and genomic effects (Kramár et al., 2009a). Kramár et al.

describe that the rapid effects of estradiol on LTP were not affected by ICI 182780. In

addition, ICI 182780, according to the suppliers, upregulates membrane bound

receptors, which should support rapid estradiol signalling. Furthermore, it would be

interesting to know whether a substantial reduction in tissue estradiol levels is

already achieved after 1 min of letrozole treatment, which could account for the

markedly reduced synaptic potentiation. In a recent study, Tanaka and Sokabe

(Tanaka and Sokabe, 2012) provided evidence that continuous synthesis of

neurosteroids is required for normal transmission in the hippocampus. They used

aminoglutethimide (AG), which blocks steroid biosynthesis from the beginning.

Application of AG to acute slices of male animals decreased fEPSPs by 20-30% in

the dentate gyrus, an effect that was mimicked by letrozole. Similar to our findings in

the hippocampus of male animals treated with letrozole, and after direct application

as shown by Tanaka and Sokabe (Tanaka and Sokabe, 2012) LTP was also

reduced by 20% in response to letrozole treatment in acute slices of the male medial

rat vestibular nuclei (Grassi et al., 2009). Whether 20% reduction in LTP is of any

functional significance in terms of behavioural deficits remains an open question.

Our results are consistent with studies showing that estradiol induces the

magnitude of LTP at CA3-CA1 Schaffer collaterals in the hippocampus (Foy et al.,

1999, Kim et al., 2006). The results further confirm the relationship of spines and

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LTP: impairment of LTP in female letrozole-treated mice precedes spine synapse

loss, while in males no major effect on LTP was seen, paralleled by unchanged spine

synapse number. LTP and phosphorylation of cofilin appeared merely to be

phenomena independent from one another and are not necessarily linked. When LTP

is maximally downregulated after letrozole treatment of female mice, p-cofilin

expression is upregulated (Vierk et al., 2012). Along these lines, p-cofilin is

downregulated, together with minor changes in LTP in male hippocampi, after

treatment with letrozole.

Cognitive effects of aromatase inhibition in females and males

Regardless of discrepancies and inconsistencies of the data in the literature, overall,

the effects of estradiol on synapse density and LTP suggest that estrogen is involved

in the regulation of cognitive function.

Aromatase inhibitors, such as letrozole, are commonly used in the therapy of

postmenopausal women suffering from hormone-dependent breast cancer (Geisler et

al., 2002, Puddefoot et al., 2002). Clinical pilot studies have demonstrated that

aromatase-inhibitors affect cognition and memory deficits in women treated with

these inhibitors (Shilling et al., 2001, Dowsett et al., 2005, Shilling et al., 2005). Due

to the small charge of the molecule, letrozole is easily transported across the blood-

brain barrier after systemic application and exerts an inhibitory influence on

hippocampal estrogen synthesis, as it does in other regions of the body (Zhou et al.,

2010). Impaired LTP (as a cellular correlate of memory), together with spine synapse

loss, both parallel dephosphorylation of cofilin in spines after estradiol depletion. This

could be the underlying mechanisms for memory deficits in women treated with

aromatase inhibitors for therapeutical reasons (Lamprecht and LeDoux, 2004). This

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appears to be of greater importance, as letrozole also induces synapse loss in

ovariectomized animals (Zhou et al., 2010). Ovariectomy is frequently regarded as an

experimental paradigm for menopause in women. Menopausal women, as compared

to women during their reproductive phase, are preferentially treated with aromatase

inhibitors, since this does not interfere with their reproductive function.

In men, it has been shown that testosterone-induced improvement of verbal memory

in older men depends on aromatization of testosterone to estradiol, while the

improvement of spatial memory in response to testosterone treatment was

independent of conversion of estradiol (Cherrier et al., 2005). Prepubertal boys

suffering from growth disorders are increasingly being treated with aromatase

inhibitors. Aromatase inhibition is considered to be a potential new treatment modality

for idiopathic short stature. In tests for cognitive impairment, blockade of estrogen

synthesis with an aromatase inhibitor did influence cognitive performance in boys

who had been treated with letrozole for two years (Hero et al., 2010).

Studies that focus on synaptic plasticity and cognitive abilities related to sexual

hormones primarily target the effects of testosterone treatment in men and in male

animals (Bimonte-Nelson et al., 2003, Sherwin, 2003, Frye et al., 2004, Gibbs, 2005,

Romeo et al., 2005, Martin et al., 2008, Cost et al., 2012) and of estrogen treatment

in women and in female animals (Gibbs, 2005, Gasbarri et al., 2012, Inagaki et al.,

2012, Nissen et al., 2012, Ryan et al., 2012, Sherwin, 2012, Stelly et al., 2012,

Velazquez-Zamora et al., 2012, da Rocha et al., 2013, McClure et al., 2013). Studies

on the role of estrogen in male animals are more frequent than studies on the effect

of testosterone in female animals (Benice and Raber, 2009, Cost et al., 2012).

Data of estrogen-regulated cognitive abilities in females originate

predominantly from studies on estrogen replacement therapy of pre- and

postmenopausal women. Many excellent reports point to a beneficial function of

14

estradiol with respect to memory (Sherwin, 1988, Phillips and Sherwin, 1992, Duka et

al., 2000, Maki et al., 2001) and risk of dementia (Yaffe et al., 1998, Nelson et al.,

2002). Other studies failed to confirm these results (Espeland et al., 2004). They

found that hormone therapy had an adverse effect on cognition for women over the

age of 65. Similar discrepancies are also found in men and male animals with respect

to the influence of testosterone on cognition. Testosterone was postulated to

decrease CA plasticity in vivo in gonadectomized male rats (Harley et al., 2000),

while others report that testosterone replacement reverses altered synaptic

transmission after gonadectomy in males (Sakata et al., 2000). Gibbs’s (Gibbs, 2005)

data revealed a dissociation between the effects of testosterone and estradiol in

cognitive performance in male rats. Female rats outperform males on an object

recognition task when circulating levels of ovarian steroids are elevated (Cost et al.,

2012). In males, in turn, testicular androgens are important for maximal levels of

working memory (McConnell et al., 2012).

In spite of the tremendous amount of data on the role of sex steroid hormones in

cognition, the inconsistencies in the literature, which may be due to different

methodological approaches, point to the necessity for further studies in order to

understand the effects of sex steroid hormones. This could lead to new therapeutical

strategies for age-related memory decline (for review see Sherwin and Henry, Maki

and Sundermann, Sherwin and Frick (Sherwin and Henry, 2008, Frick, 2009, Maki

and Sundermann, 2009, Frick, 2012, Sherwin, 2012)). Since GnRH regulates

estradiol synthesis in the hippocampus (Prange-Kiel et al., 2008), ovariectomy results

in loss of aromatase activity in the hippocampus (Fester et al., 2012), and estradiol

content in the hippocampus varies with the estrus cycle (Kato et al., 2013), synthesis

in the ovaries is synchronized with hippocampal estradiol synthesis. Thus, one would

assume that the aging hippocampal neuron synthesizes less estradiol than the young

15

neuron, which, however, has not yet been shown. If this turns out to be true, attempts

to understand, and as a consequence to manipulate the local, intraneuronal

regulation of aromatase could deliver another therapeutical approach. For instance,

aromatase activity in neurons was shown to be regulated by neuronal activity (Hojo et

al., 2004) and becomes inactive by phosphorylation via calcium transients and

calcium-dependent kinases (Balthazart et al., 2006, Fester et al., 2012). Local

stimulation of aromatase activity in neurons would avoid undesired side effects of

estrogen replacement therapy.

Conclusion

Hippocampal neurons synthesize estradiol, which maintains LTP and synapses in

females but not in males. In females, inhibition of estradiol synthesis results in

impairment of LTP, dephosphorylation of cofilin and final synapse loss. These effects

were not seen in males. The essential role of local estrogen on the stability and

maintenance of connectivity in the hippocampus is consistent with age-related

cognitive decline in women. In male animals the regulation of synaptic stability by

local sexual steroids remains to be clarified.

16

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Legend to the figure

Fig. 1 LTP in acute hippocampal slices of mice treated with letrozole.

Results of female animals treated with vehicle and animals treated with letrozole. (A)

In slices of animals treated with vehicle (6 hrs), the average course of the fEPSP

slopes had significantly increased after 60 min to 247%±9% (n=8), whereas in

animals treated with letrozole (6 hrs) the slopes of fEPSPs increased only to

175%±4% (n=7). (B) After 1 day of vehicle treatment the slopes of fEPSPs increased

to 226%±8% (n=8), and after letrozole treatment for 1 day the fEPSP slopes had

changed only to 154%±5% (n=7). (C) After 7 days of vehicle injection, the average

slope of the fEPSPs increased to 213%±3% (n=6); in contrast, letrozole injections

after 7 days resulted in virtually unchanged fEPSP slopes 60 min after TBS,

107%±2% (n=7). Mean±SEM; time point 0 min represents TBS-stimulation; fEPSP

slopes before and 60-70 min after TBS were quantified and are indicated by grey

bars. (D) Quantitative examination of changes in the average course of the fEPSP

after letrozole treatment for 6 hrs, 1 day and 7 days illustrated a highly significant

reduction in LTP by 49%±4%, 57%±5% and 94%±1% respectively in females. (d:

day; n=number of acute slices of 3 animals; mean±SEM) (E) Quantitative

examination of changes in the average course of fEPSP slopes after letrozole

treatment in males. Letrozole treatment of male animals resulted in reduced LTP by

17%±5% after 1 day and 20%±2% after 7 days of treatment. (d: day; mean±SEM) (d:

day; n=number of acute slices of 3 animals; mean±SEM) (Vierk et al., 2012)

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Highlights aromatase; estrogen; sexual dimorphism; synaptic plasticity