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Oestrogens, androgens and progestogens are most
commonly associated with their roles in reproduction,
despite their involvement in a broad range of physio-
logical and neural functions. One particular realm in
which these sex steroid hormones exert wide-ranging
effects is that of learning and memory. In particular,
considerable effort has been devoted to understanding
how 17β-oestradiol (E2), the most potent and prevalent
circulating oestrogen, regulates the function of the hip-
pocampus, a bilateral medial temporal lobe structure
with extensive connections to numerous cortical and
subcortical brain regions. Although the hippocampus is
not the only brain region important for memory, damage
to or dysfunction of this structure leads to deficits in the
formation and retention of numerous types of memories,
including those used for spatial navigation, recognizing
objects and conspecifics, and recalling fear-associated
contexts. However, the roles of sex steroids in regulat-
ing the function of mnemonic brain regions such as the
hippocampus have rarely been considered by scientists
other than neuroendocrinologists.
In the early 1990s, a series of articles describing the
effects of oestrogens on neuronal morphology and
plasticity in the hippocampus were paradigm shifting
for the field of neuroendocrinology1–5. These publi-
cations demonstrated that dendritic spine density on
CA1 pyramidal neurons in the hippocampus fluctuates
across the reproductive (oestrous) cycle of female rats,
and is increased in bilaterally ovariectomized female rats
by exogenous E2 treatment3. Other contemporaneous
work showed that E2 potentiates hippocampal excit-
atory synaptic plasticity5–7 and protects hippocampal
neurons from drug-induced excitoxicity8. Collectively,
this body of work unlocked a new research frontier that
has broadened understanding of how E2 regulates hip-
pocampal function to modulate memory. Since their
publication, these findings have inspired much research
examining the actions of E2 and other sex steroid hor-
mones in non-reproductive, procognitive brain regions,
including the hippocampus and prefrontal cortex (PFC).
A subset of this newer research has unveiled very rapid
actions of E2 on molecular and cellular mechanisms in
the hippocampus, PFC, amygdala and other regions that
facilitate memory-consolidation processes.
Although the myriad effects of oestrogens such as
E2 on hippocampal function have been intensely studied
by neuroendocrinologists for nearly three decades, oes-
trogens have yet to be widely accepted in neuroscience
as general neuromodulators of cognitive processes. As
oestrogens exert numerous effects in multiple cognitive
brain areas in both sexes9–13, elucidating how these hor-
mones regulate memory could result in better mental
health outcomes in people of both sexes by identifying
potential therapeutic targets for neurodegenerative dis-
eases, mood disorders and other conditions in which
memory dysfunction features prominently. Effective
treatments for memory loss are sorely lacking, highlight-
ing an urgent need to characterize the neural mecha-
nisms underlying memory formation, including those
mediated by oestrogens. Moreover, such information
Retention
Storage of acquired and
consolidated information that
enables subsequent recall or
retrieval of the information.
Ovariectomized
Ovariectomy involves surgical
removal of the ovaries to
eliminate ovarian hormone
cycling. Subjects that have
undergone ovariectomy are
considered ovariectomized.
Consolidation
Process through which learned
information is encoded and
stored to form a memory that
can be recalled at a later time.
Oestradiol as a neuromodulator
of learning and memory
LisaR.Taxier , KellieS.Gross and KarynM.Frick ✉
Abstract | Although hormones such as glucocorticoids have been broadly accepted in recent
decades as general neuromodulators of memory processes, sex steroid hormones such as the
potent oestrogen 17β-oestradiol have been less well recognized by the scientific community in
this capacity. The predominance of females in studies of oestradiol and memory and the general
(but erroneous) perception that oestrogens are ‘female’ hormones have probably prevented
oestradiol from being more widely considered as a key memory modulator in both sexes. Indeed,
although considerable evidence supports a crucial role for oestradiol in regulating learning and
memory in females, a growing body of literature indicates a similar role in males. This Review
discusses the mechanisms of oestradiol signalling and provides an overview of the effects of
oestradiol on spatial, object recognition, social and fear memories. Although the primary focus
is on data collected in females, effects of oestradiol on memory in males will be discussed, as will
sex differences in the molecular mechanisms that regulate oestrogenic modulation of memory,
which may have important implications for the development of future cognitive therapeutics.
Department of Psychology,
University of Wisconsin-
Milwaukee, Milwaukee,
WI, USA.
✉e-mail: frickk@uwm.edu
https://doi.org/10.1038/
s41583-020-0362-7
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will greatly inform our general understanding of how
memories are formed. Thus, more broadly considering
oestrogens and related sex steroid hormones as impor-
tant neuromodulators of memory formation will provide
fundamental insights into the neurobiology of memory
and new avenues for drug development.
The goal of this Review is to highlight the impor-
tance of E2 as a critical modulator of synaptic plas-
ticity and memory circuitry. Although other sex
steroid hormones can regulate memory, the influence of
E2 on learning and me mory is the most extensive ly doc-
umented, and thus E2 is the focus of this Review. The
discussion will centre on the effects of E2 in rats and
mice because their size and short lifespans make them
convenient mammalian systems in which to explore
molecular mechanisms of E2 action in the brain, and
thus the preponderance of data are from these species.
However, non-human primate studies (reviewed in11–13)
have also provided insights into oestrogenic regulation
of memory and neuronal morphology that corroborate
findings from rodent studies, highlighting the trans-
lational potential of this work. Here we first outline
molecular mechanisms underlying E2 signalling in the
rodent brain, including both classical and non-classical
signalling. Next, we describe several forms of learning
and memory modulated by E2 in rodent s, and the mech-
anisms thus far identified that underlie this mediation.
Although findings from female rodents are the primary
focus, data from males are also discussed, as are sex
differences in the neural mechanisms through which
E2 influences memory. Last, we conclude by discuss-
ing the broad health implications of considering E2 as
a neuromodulator, and speculate about the next steps
necessary to further advance knowledge of oestro-
genic regulation of memory. We hope readers will be
convinced of the importance of E2 as a general neuro-
modulator that, like stress hormones and growth fac-
tors, should be considered in mainstream models of the
neurobiological mechanisms underlying memory.
E2 signalling
Unlike neurotransmitters, which are stored in vesicles
after synthesis for later release, steroid hormones are
generated in response to a stimulus and released imme-
diately. Sex steroid biosynthesis begins with the catabo-
lism of cholesterol to progesterone, which is then broken
down into the androgens testosterone and androstenedi-
one, which are subsequently converted to the oestrogens
E2 and oestrone, respectively14,15. In this synthesis path-
way, E2 is generated from testosterone via the enzyme
aromatase. The primary sources of sex steroids in both
sexes are the gonads, which synthesize progestogens,
androgens and oestrogens in differing sex-dependent
amounts (with more androgens in males, and more
progestogens and oestrogens in females). Hormones
synthesized in the gonads can exert paracrine effects
on adjacent cells in the gonads, or endocrine effects on
distant tissues via the bloodstream16. However, other tis-
sues synthesize sex steroid hormones as well14, including
adipose tissue, the adrenal glands and numerous brain
areas, including the hippocampus16–19 (BOX1). In the
brain, steroids including E2 exert rapid paracrine or
synaptocrine effects on neighbouring cells20,21.
In female mammals, circulating E2 is synthesized
by the ovaries, which release fluctuating levels into the
bloodstream as part of the reproductive cycle. During
the human menstrual cycle and rodent oestrous cycle,
oestrogen levels rise to promote follicle maturation in
advance of ovulation, peak to stimulate ovulation and
then return to the baseline as the degenerating folli-
cle secretes progesterone in preparation for implanta-
tion of a fertilized egg. In mice and rats, the oestrous
cycle consists of four approximately 12–24-h-long
stages: proestrus, oestrus, metoestrus and dioestrus22,23.
E2 levels surge during proestrus and remain high
through early oestrus, after which they plunge in late
oestrus and remain low throughout metoestrus, ris-
ing again during late dioestrus. These fluctuations
are reflected in the hippocampus, where E2 levels are
substantially higher during proestrus than during
other phases24, as is CA1 dendritic spine density2 and
neurogenesis25. Hippocampal E2 levels in ovariectomized
rats are similar to those of ovary-intact rats in dioestrus
and metoestrus24, reflecting hippocampal E2 synthesis in
the absence of the ovaries. As discussed in BOX1, denovo
hippocampal E2 synthesi s is activ ity depen dent and cr u-
cial for rapid synaptic plasticity and memory formation.
Oestrogen receptor (ER) expression is also influenced by
the oestrous cycle26.
Box 1 | A role for hippocampally synthesized oestrogens in memory
Although the gonads are a primary source of oestrogens in both sexes, oestrogens are
synthesized in numerous tissues, including the brain. Of most relevance to learning and
memory, the enzyme aromatase, which converts testosterone into 17β-oestradiol (E2),
is widely expressed in the brain and has been shown to produce E2 locall y in region s
such as the hypothalamus and hippocampus17,18,200,237,238. In the hippocampus,
neuron-derived denovo E2 supports multiple aspects of synaptic plasticity, including
synaptogenesis and long-term potentiation19,204,239,240. In hippocampal cultures from
female rats, pharmacologically blocking denovo E2 synthesi s with th e aromatas e inhibi tor
letrozole results in reduced spine density, decreased expression of synaptic proteins and
impaired long-term potentiation19,204,239.
Neural E2 synthesis seems to be regulated by neuronal activity. For example,
activation of NMDA receptors in cultured hippocampal neurons or in hippocampal
slices increased E2 synthesi s200,203, and exposure to a learning stimulus in ovariectomized
mice increased denovo E2 synthe sis in th e hippoca mpus, an e ffect blo cked by l etrozole226.
Invivo, systemic letrozole treatment decreased CA1 dendritic spine density and levels
of hippocampal synaptic proteins in both ovary-intact and ovariectomized females240,
demonstrating that neuron-derived E2 contributes to hippocampal synaptic plasticity
regardle ss of other so urces of the hor mone.
The central importance of denovo hippocampal E2 synthesis in both sexes is
illustrated by studies in which letrozole infusion into the dorsal hippocampus of
gonadectomized male or female mice impaired consolidation of spatial and object
recogni tion memory215,226. Similarly, hippocampal implants of the aromatase inhibitor
1,4,6-androstatriene-3,17-dione impaired spatial memory in male zebra finches241,
suggesting that the importance of hippocampal E2 synthesis is conserved across
species. Supporting this conclusion are recent data showing oral letrozole treatment
impaired spatial working memory and reduced hippocampal intrinsic excitability
in male and female marmosets228.
Aromatase outside the hippocampus also seems to be important for memory
consolidation, as infusion of letrozole into the perirhinal cortex of gonad-intact male
mice impaired short-term and long-term memory in an object placement memory task224.
In humans, letrozole is an approved treatment for oestrogen receptor (ER)-positive breast
cancer; however, women taking letrozole may experience a number of adverse side
effects that affect memory242. For example, women taking letrozole for ER-positive breast
cancer exhibited episodic memory deficits, supporting the idea that brain-synthesized
oestrogens are crucial in memory processes227.
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Classical and non-classical signalling
Early research demonstrated that sex steroid recep-
tors act as transcription factors, dimerizing and
translocating to the nucleus on ligand activation to
bind hormone response elements and regulate gene
transcription27. E2 binds two ER subtypes, ERα and ERβ,
to accomplish this ‘classical’ mechanism of steroid hor-
mone action, resulting in slow transcriptional changes
that become evident within hours to days28. However,
data from the 1960s revealed that E2 could increase
cellular concentrations of cyclic AMP (cAMP) in uter-
ine tissue within seconds29, a time course too rapid for
classical genomic signalling. Similarly rapid effects on
the order of seconds to minutes were later found in the
brain, where E2 altered neuronal activity in the preoptic
area of the hypothalamus30. More recent studies have
characterized myriad ‘non-classical’, membrane-initiated
mechanisms through which oestrogen rapidly triggers
cell signalling and epigenetic processes to produce
downstream alterations in gene expression, local protein
synthesis, actin polymerization, synaptic physiology and
dendritic spine morphology31–35.
Non-classical oestrogen signalling is initiated at
the cell membrane and influences cellular processes
through the activation of downstream second messen-
ger pathways. The identities of the ERs responsible for
this signalling have been controversial, as the early char-
acterization of ERα and ERβ as purely nuclear recep-
tors seemed to exclude a role in membrane-initiated
signalling. However, technological advances have since
permitted observation of these receptors at the cell
membrane36–40, and studies using overexpression or dele-
tion of ERα and ERβ demonstrated that these classical
receptors are indeed responsible for many of the defin-
ing effects of rapid E2 action, including increased cAMP
response-element binding protein (CREB) phospho-
rylation and activation of extracellular signal-regulated
kinase (ERK)–mitogen-activated protein kinase
(MAPK) signalling38,41,42. Evidence of the classical ERs
(that is, ERα and ERβ) in dendritic spines and axon ter-
minals of neurons43–45 suggests that these receptors could
be directly involved in synaptic signalling.
How classical ERs initiate non-classical oestrogen
signalling is still not thoroughly understood. The field
continues to struggle with technical limitations, such as
insufficient antibody validation
46
, in studying the subcellu-
lar localization of ERs, and many questions remain about
the nature of the interaction between classical ERs and the
cell membrane. The ERα and ERβ structures do not con-
tain prototypical transmembrane domains
47,48
, indicating
that these receptors are probably not fully inserted into
the membrane. However, ERs undergo post-translational
lipidation
49–51
and associate with caveolins
52,53
. Both of
these factors contribute to the ability of ERs to localize to
and associate with the cell membrane
50,53–55
.
In addition, how ERα and ERβ, which do not have
inherent kinase activity, can interact with and activate
downstream second messengers is unclear. One expla-
nation is that ERs localized to the membrane co-opt
the signalling machinery of other membrane receptors.
At the membrane, ERα and ERβ can physically inter-
act with and activate metabotropic glutamate receptors
(mGluRs) independently of glutamate release, resulting
in rapid ERK and CREB phosphorylation56,57. Functional
coupling of ERs to mGluRs is observed throughout the
nervous system and seems to contribute to the effects
of E2 on memory consolidation, motivated behaviour
and sexual receptivity57–59. ERs also interact with receptor
tyrosine kinases at the membrane, activating signalling
at insulin-like growth factor receptor 1 and the neuro-
trophin receptor TRKB through both rapid and gene
expression-dependent mechanisms60–63. Activation of
these receptors subsequently engages neuroprotec-
tive and plasticity-related signalling cascades62–66. The
integration of ERs into a signalling complex with other
receptors, G proteins and kinases at the cell membrane
is facilitated by multiple scaffold and adaptor proteins.
These include: caveolins, which facilitate ER interaction
with mGluRs as well as membrane association50,53,55;
p130CAS, which couples ERs to kinases such as SRC
and phosphoinositide 3-kinase (PI3K)67; and striatin,
which binds caveolins and G proteins at the membrane68.
Non-classical E2 signalling also occurs through
recently identified membrane-localized ERs. Although
multiple putative ERs exist, the most well-defined and
most studied is G protein-coupled ER (GPER; also
known as GPR30). Characterized in the early years of
the first decade of the twenty-first century, GPER is
a seven-transmembrane-domain G protein-coupled
receptor activated by oestrogens69,70. It most frequently
interacts with Gαs subunits, leading to increased levels
of cAMP and activation of the ERK, protein kinase A
(PKA) and PI3K signalling pathways69–72. GPER is highly
expressed in the brain, including in forebrain regions
such as the cortex, hippocampus and hypothalamus73,74,
and pharmacological targeting of GPER has implicated
it in mediating oestrogen effects on cognitive75–78, social79
and reproductive80,81 behaviours. Given the considerable
overlap of GPER and classical ER expression in regions
such as the hippocampus and PFC, as well as similar-
ities in the outcomes of their activation, an intriguing
question is whether these ER subtypes use parallel or
distinct mechanisms to modulate neuronal function and
behaviour. Current evidence suggests that the answer to
this question is complex, with classical ERs and GPER
often producing similar or complementary outcomes but
through distinct molecular mechanisms78,82.
Intracellular cascades
E2 activation of membrane-localized receptors triggers
numerous intracellular signalling events that ultimately
influence synaptic plasticity in cognitive brain regions.
These downstream cellular and molecular mechanisms
have been best elucidated in the hippocampus (FIG.1).
Activation of membrane-localized ERs in cultured hip-
pocampal neurons leads to rapid calcium influx83,84 and
activation of phospholipase C and adenylyl cyclase sig-
nalling via G protein activation56,85. In turn, these events
activate small GTPases such as RHOA35,82 and a host of
kinase pathways, including the ERK–MAPK56,84,86–89,
PI3K–AKT62,89–92, PKA93–95, protein kinase C (PKC)56,96
and JUN amino-terminal kinase (JNK)78 pathways. The
activation of these second messenger cascades influences
processes that regulate synaptic structure and function,
Hormone response
elements
Short DNA sequence within
the promoter region of a
gene that binds a hormone
receptor complex to enable
gene transcription.
Caveolins
Integral membrane proteins
that form functional
microdomains of receptors
and their associated signalling
proteins at the plasma
membrane.
Sexual receptivity
A positive state of responsivity
towards the initiation of
sexual behaviour by another
individual. Often indicated
by a species-specific
mating posture.
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such as local protein synthesis, actin polymerization, ion
channel dynamics and gene expression.
E2 activation of ERK and AKT stimulates local protein
translation by activating mechanistic target of rapamycin
(mTOR) signalling and promoting the phosphorylation
of translational initiation factors such as 4E-binding
protein 1 (4E-BP1)32,82,97,98, and this new protein syn-
thesis is required for E2-induced spinogenesis in the
hippocampus99. Recruitment of actin-remodelling path-
ways also contributes to E2-induced structural plasticity:
E2 activation of RHOA elicits phosphorylation of LIM
domain kinase (LIMK) and its substrate, the actin-binding
protein cofilin, to promote actin polymerization35,100–103.
LIMK–cofilin activation and enhanced actin polym-
erization contribute to E2 effects on spine structure
and synaptic transmission35,100,103. Moreover, although
membrane-initiated E2 signalling is often referred to as
‘non-genomic’ to differentiate it from the classical effects
of E2, the intracellular signalling cascades initiated by sur-
face ERs ultimately influence gene expression. E2 activa-
tion of ERK signalling rapidly induces phosphorylation of
the transcription factor CREB56,83,88,104 and produces epi-
genetic modifications such as histone acetylation31,105,106 —
both effects that can increase expression of genes with
neurotrophic effects, such as Bdnf106.
Synaptic function
E2-induced changes in intracellular signalling ulti-
mately alter synaptic function. In the hippocampus,
the general result of E2 exposure is a rapid increase in
excitatory neurotransmission. Application of E2 to hip-
pocampal slices increases intrinsic excitability of CA1
pyramidal neurons107,108, enhances baseline excitatory
neurotransmission5,6,35,109–111 and enhances long-term
potentiation (LTP) at CA3–CA1 synapses35,109,111–114.
The effects of E2 on synaptic function correlate with
increases in dendritic spine density5,110,113,114 and res ult pr i-
marily from regulation of ionotropic glutamate receptors.
In hippocampal neurons, E2 in fluences the phosphor yla-
tion and subcellular trafficking of both AMPA receptors
(AMPARs) and NMDA receptors (NMDARs)112,115–120.
AMPAR-mediated excitatory postsynaptic potentials
can be positively modulated by E2 (REFS6,35); however,
many of the effects of E2 on synaptic plasticity rely on
NMDAR-dependent mechanisms5,109,110,120,121. In particu-
lar, NMDARs containing the NR2B subunit are required
for E2 enhancement of LTP120,121. E2 can also increase
hippocampal excitability by suppressing inhibitory
GABAergic signalling; however, this mechanism differs
by sex122,123.
In sum, considerable data support the view that the
rapid cell-signalling mechanisms resulting from acti-
vation of surface-localized ERs enhance the multiple
forms of synaptic plasticity thought to be cellular sub-
strates for learning and memory. As we discuss in the
next section, the activation of intracellular signalling and
the resulting effects on neuronal structure and function
are necessary for the beneficial effects of E2 on memory
in female rodents.
E2 modulation of memory in females
The numerous classical and non-classical effects of E2 on
brain function provide abundant opportunities for this
hormone to influence learning and memory. Copious
data supporting a modulatory influence of E2 on hip-
pocampal plasticity have led most rodent researchers
to focus on forms of learning and memory mediated
by the hippocampus, such as spatial and object-based
memories. However, oestrogens also regulate other
types of learning and memory, including social learn-
ing, social discrimination and fear memory mediated by
the amygdala, perirhinal cortex, PFC and other regions.
Thus, the following sections will focus not only on the
hippocampus but also on these brain regions as well.
Studies of ER-null mice
114,124–126
or using viral vectors to
modulate ERα expression
127
have suggested key roles for
ERα and ERβ in various forms of memory. However, phar-
macological manipulations are the most common method
of interrogating the roles of E
2
and ERs in memory pro-
cesses (TABLE1). Although the compounds used afford
better temporal specificity than genetic manipulations,
they lack absolute specificity for one ER over the other.
However, most can be used at dos es that promote prefer-
ential binding to a single ER. Ongoing synthesis of more
potent and selective ER compounds will undoubtedly
help pinpoint discrete functions of specific ERs
128
.
Most studies examining the neuromodulatory role
of E2 have been conducted in bilaterally ovariectomized
Local protein synthesis
↑ mTOR activation
↑ p4E-BP1 Kinase activity
↑ MEK–ERK
↑ PI3K–AKT
↑ PKA
↑ PKC
↑ JNK (GPER only)
Gene expression
↑ pCREB
↑ Histone acetylation
↑ DNA methylation
Cytoskeletal regulation
↑ pLIMK–pcofilin
↑ Actin polymerization
↑ Dendritic spine density
Ion channel
dynamics
↑ Phosphorylation
↑ Trafficking to
synapses
Local protein synthesis
↑ mTOR activation
↑ p4E-BP1
ER
E2
E2
AMPAR or
NMDAR
mGluR
RTK
GPER
Fig. 1 | Membrane-initiated oestrogen signalling and downstream intracellular
events. I ntracellu lar proce sses are in itiated b y 17β-oestradiol (E2) binding to G protein-
coupled oestrogen receptor (GPER), or functional interaction between the canonical
oestrogen receptors (ERα and ER β) and other receptors located at the membrane (such as
metabotropic glutamate receptors (mGluRs)). Several kinase cascades, key for the memory-
enhancing effects of E2, rapidly increase activity in response to membrane-initiated
signalling events. ERα and ERβ seem to trigger similar kinases, whereas GPER activates
distinct signalling pathways such as the JUN amino-terminal kinase (JNK) pathway. In turn,
kinase activity facilitates additional regulatory processes, including protein synthesis,
ion channel phosphorylation and trafficking, gene expression and cytoskeletal regulation.
AMPAR, AMPA receptor; ERK, extracellular signal-regulated kinase; MEK, mitogen-
activated protein kinase/extracellular signal-regulated kinase; mTOR, mechanistic
target of rapamycin; NMDAR, NMDA receptor; p4E-BP1, phosphorylated 4E-binding
protein 1; pCREB, phosphorylated cyclic AMP response element-binding protein;
PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C; pLIMK,
phosphorylated LIM domain kinase; RTK, receptor tyrosine kinase.
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female rodents, although some reports have examined
memory in naturally cycling females or in males (with or
without gonadectomy). Although the widespread use of
ovariectomized females has limitations that require cau-
tious considerations about extrapolating effects to the
natural cycle, this model has permitted more systematic
investigations of oestrogen actions than is possible in the
presence of daily ovarian hormone fluctuations. Much of
this work has used young rodents (2–3 months old), but
a rich literature also exists on the mnemonic effects of
E2 in middle-aged rodents (for example, 14–19 months
old) and aged rodents (older than 20 months)129,130
(BOX2). Besides animal age, there are several other
experimental variables that influence behavioural and
mechanistic outcomes. The timing of administration
relative to testing, the length of administration (for
example, long (that is, over several days) or short (that
is, a single dose)) and the dose can differ across studies,
making direct comparisons across studies challenging.
Moreover, when E2 is administered systemically, the site
of E2 action is often unknown. Indeed, given that E2 is
also synthesized in the brain (BOX1), differentiating the
effects of brain-derived E2 from systemically adminis-
tered E2, or from gonadal E2 in ovary-intact rodents,
poses a unique challenge. In addition, the age at ovariec-
tomy and the duration between ovariectomy and first
E2 treatment have also been demonstrated to affect oes-
trogenic regulation of memory129,130. The age at testing
and the duration of oestrogen deprivation are particu-
larly important, as ER expression in the rodent hip-
pocampus declines not only with age but also several
months after bilateral ovariectomy131,132. Combined,
these data highlight the importance of experimental
parameters when one is designing studies or interpreting
data. The following sections mirror the preponderance
of studies using young ovariectomized rodents (but see
BOX2 and reviews129,133–135 for additional information
relevant to ageing female and male rodents). As a com-
prehensive synthesis of all types of memory modulated
by E2 is beyond the scope of this Review, the following
text focuses on select forms of memory and behavioural
tasks that have proved especially useful to pinpoint the
neural mechanisms underlying E2-facilitated memory.
Spatial and object memory
The tasks most commonly used to assess hippocampal-
dependent spatial and object memory include the Morris
water maze (MWM), the radial arm maze (RAM), and
object recognition (OR) and object placement (OP; also
known as object location) tasks (FIG.2). In the MWM and
RAM, animals use extramaze cues to traverse a round
or wheel-shaped maze to escape from water or locate a
food or water reward. Memory in these tasks is typically
measured across multiple days, enabling within-subject
and between-subject performance to be assessed over
time. Whereas both the MWM and the RAM involve
explicit motivational stimuli to compel performance, the
OR and OP tasks rely on the animal’s inherent proclivity
for novelty. That is, mice and rats will spend more time
investigating a novel object, or a familiar object in a novel
location, than a previously investigated object in a famil-
iar location. One advantage of these single-trial learning
tasks is that they permit learning assessment while mini-
mizing appetitive or aversive confounders. Moreover, the
rapidity with which object tasks are learned allows
the effects of short-term hormone administration to
be associated with specific neural events and discrete
phases of memory formation (including acquisition,
consolidation and retention).
Morris water maze. Studies using the MWM to test oes-
trogenic regulation of spatial reference memory present
conflicting data on the role of E
2
in this form of memory.
For example, long-term administration of systemic E
2
to
ovariectomized rats or high E
2
levels during the rat oes-
trous cycle can impair spatial learning and memory
136–138
,
whereas high E
2
levels in cycling mice were associated with
enhanced spatial learning
139
, and ovariectomized rats sys-
temically administered E
2
72 a nd 48 h before testing exhib-
ited better memory for a hidden platform
140
. Moreover,
high plasma E
2
levels in ovary-intact female meadow
voles correlated with a longer latency to locate the escape
platform during acquisition trials, whereas lower plasma
E
2
lev els wer e asso ciated with b etter learni ng
141
. Given the
varied reported effects of E
2
level on spatial MWM learn-
ing in different rodent studies, the E
2
dose and species may
determine how E
2
affects spatial memory in this task.
Although few studies have examined the short-term
effects of intracranially administered E2 on MWM per-
formance, existing research suggests beneficial effects.
For example, E2 infusion into the dorsal hippocampus of
young ovariectomized rats immediately, but not 3 h, after
spatial MWM training facilitates retention 24 h later,
Gonadectomy
Surgical removal of the
gonads (ovaries or testes);
because ‘ovariectomy’ is the
preferred term for females,
‘gonadectomy’ is most
commonly used for males.
Acquisition
A process through which
information is learned through
physical or sensory interaction
with environmental stimuli.
Spatial reference memory
Memory for locations that
do not change over time
(for example, the layout of
buildings on a college campus).
Used for navigating through
an environment.
Ta bl e 1 | Compounds used to investigate the role of oestrogen receptors in memory
Target Action Compounds Effects on memory
ERαAgonist PPT ↑ Objec t recognit ion57,154,173
↑ Spati al memory57,173,224
↑ Socia l recognit ion173,175
Antagonist MPP dihydrochloride ↓ Spati al memory225
ERβAgonist DPN; WAY200070
(benzoxazole); ERB 041;
ISP358-2
↑ Objec t recognit ion57
↑ Spati al memory57,224
↑ Socia l recognit ion175
↑ Fear gen eralizati on198
Antagonist PHTPP ↓ Objec t recognit ion225
↓ Spati al memory225
ERα and
ERβAntagonist ICI 182,780 (fulvestrant) ↓ Objec t recognit ion87
↓ Spati al memory224
↓ Fear gen eralizati on198
GPER Agonist G1 ↑ Objec t recognit ion78,103
↑ Spati al memory76,78,103
↑ Socia l recognit ion175
Antagonist G15 ↓ Objec t recognit ion78
↓ Spati al memory78,224
Aromatase Inhibitor Letrozole; anastrozole; ATD ↓ Objec t recognit ion215,226
↓ Spat ial me mory215,224,226–228
ATD, 1,4,6-androstatriene-3,17-dione; DPN, diarylpropionnitrile; ERα, oestrogen
receptor-α; ERβ, oestrogen receptor-β; GPER, G protein-coupled oestrogen receptor;
MPP, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) phenol]-1H-pyrazole
dihydrochloride; PHTPP, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo [1,5-a]pyrimidin-3-yl]
phenol; PPT, propyl pyrazole triol.
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indicating that E2 specifically enhances spatial memory
consolidation142.
Radial arm maze. The RAM allows researchers to dis-
tinguish the effects of E2 on spatial reference memory
and spatial working memory (FIG.2). Broadly, long-term
systemic administration of E2 in ovariectomized rats
enhances spatial working memory in the RAM, an effect
that manifests itself most prominently after several days
of E2 admini stration143–145. Consistent with this, long-term
systemic E2 treatment in rats rescued ovariectomy-
induced deficits in working memory but not reference
memory146. Similarly, intracerebroventricular E2 infu-
sion reduced working memory errors in ovariectomized
rats147. Therefore, E2 appears to facilitate spatial working
memory but not spatial reference memory in the RAM
among young ovariectomized rodents.
Many brain regions, including the PFC, are required
to coordinate the effects of E2 in the RAM, although the
effects of E2 in these brain regions differ by dose. For
example, spatial working memory in ovariectomized rats
was facilitated by infusion of a high, but not a low, dose
of E2 into the PFC, and infusion of a low, but not a high,
dose into the dorsal hippocampus148. Thus, the effects of
E2 on spatial working memory in the RAM may depend
on both the dose and the brain region148.
Single-trial learning. It is difficult to disaggregate the
rapid non-classical effects of E2 from its long -term classi-
cal effects in tasks such as the MWM and RAM, because
learning in these tasks requires multiple trials across sev-
eral days. Moreover, both tasks involve motivational com-
ponents (for example, escape stress and thirst or hunger)
to compel performance, which may alter how the brain
responds to E2. By contrast, single-trial learning tasks
such as OR and OP tasks afford more precise assessment
of the neural mechanisms through which E2 facilitates
memory formation. As illustrated below, the findings
from studies using these tasks to assess the effects of E2 on
spatial and recognition memory are considerably more
consistent than those from maze studies.
As in the MWM and RAM, memory in the OR and
OP tasks is negatively affected by ovariectomy149,150.
However, unlike in the MWM and RAM, exogenous
E2 consistently improves spatial and OR memory in
the object tasks. For example, systemic administration
of E2 immediately after training enhances memory
consolidation in the OR and OP tasks among young
ovariectomized rats and mice151–153. However, systemic
administration of E2 does not enhance memory when
injected 2 h after training152, suggesting that the win-
dow in which E2 exerts procognitive effects on OR and
OP memory consolidation is time limited and probably
dependent on non-classical mechanisms.
E2 infuse d imme diately, but n ot 3 h, after training into
the dorsal hippocampus similarly enhances memory
consolidation in the OR and OP tasks, indicating that
the dorsal hippocampus has a crucial role in mediating the
rapid effects of E2 on memory consolidation57,87,154,155.
The perirhinal cortex also plays a part, as E2 infused
immediately after training into the perirhinal cortex
of ovariectomized rats enhanced their preference for
a novel object over a familiar object compared with
vehicle-treated controls156. However, perirhinal E2 infu-
sions also impaired object memory consolidation in a
delayed non-match-to-sample task, complicating the role
of this brain region in mediating the influence of E2 on
OR memory156,157. Overall, these data suggest that E2 gen-
erally enhances OR and OP memory consolidation in
young ovariectomized rodents when it is administered
either before training or immediately after training.
Molecular mechanisms. The memory-enhancing effects
of E2 in the hippocampus involve numerous cellular
and molecular events in female rodents. Several cell-
signalling cascades are rapidly activated in response to E2,
Box 2 | The effects of oestradiol in ageing rodents
Ageing female rodents are frequently used to examine the effects of oestrogen
deprivation and replacement during the transition to reproductive senescence (see
REFS133,134,243 for recent reviews). In humans, menopause onset coincides with follicular
depletion, whereas oestropause, the menopausal parallel in rodents, is driven by
alterations in the function of the hypothalamus–pituitary–gonad axis244,245. Given this
difference, researchers should be cautious when interpreting studies using rodent
models of reproductive ageing. Nevertheless, the ageing rodent model has proved useful
in understanding the effects of oestrogen deprivation, the timing of hormone therapy
administration and age-associated alterations in responsivity to 17β-oestradiol (E2).
Broadly, E2 deprivation as a consequence of ageing or artificial long-term ovarian
hormone depletion lowers sensitivity to the memory-enhancing effects of E2. In spatial
memory tasks such as the Morris water maze (MWM), systemic E2 administration typically
enhances memory in middle-aged (14–19-month-old) but not aged (20-month-old
or older) ovariectomized female rodents246–249. However, E2 can still facilitate memory
in aged females under certain experimental conditions. Long-term, high doses of
E2 elicit ed better memory in the MWM am ong aged o variect omized fe male rats250 an d
aged female mice251. Similarly, 3 weeks of E2 administration enhanced object memory
in aged ovariectomized female mice252.
Age and duration of oestrogen deprivation also modulate the effects of systemic E2
treatment on spatial reference and working memory. For example, long-term systemic
E2 treatment enhanced spatial working memory in middle-aged ovariectomized rats
in a water-motivated radial arm maze (RAM) task253. However, daily systemic injections
of E2 given to ageing female mice ovariectomized at 17.5 months did not affect spatial
reference or working memory in a water-motivated RAM task, whereas intermittent
E2 treatment mimicking normal hormonal cycling impaired spatial reference and working
memory254. As with the MWM, long-term E2 treatment enhanced spatial reference memory
in aged ovariectomized rats, but only when rats were primed with additional short-term
injections of E2 (REF.255). These data suggest not only an age-related loss of responsivity
to the memory-enhancing effects of E2 but als o a detrim ental eff ect of E2 on memory in
aged rodents. Supporting the idea that the duration of hormone loss affects responsivity
to E2, systemic E2 implants administered to 12-month and 17-month-old mice immediately
after ovariectomy enhanced spatial working memory in a RAM256, whereas such
E2 treatment given to 17-month-old mice 5 months after ovariectomy no longer
enhanced spatial working memory. These data support the well-accepted notion that
the ageing brain is less responsive to E2 after long peri ods of oes trogen dep rivation .
As in the maze tasks, ageing female rodents or rodents subjected to delayed oestrogen
replacement after ovariectomy exhibit diminished sensitivity to the beneficial effects
of E2 in obje ct memory consolid ation. For example, rats treate d with sys temic E2 9 or
15 months after ovariectomy exhibited intact memory for a previously seen object91.
However, increasing the treatment delay to 19 months after ovariectomy eliminated the
memory-facilitating effects of E2 (REF.162), consistent with other findings that E2 typically
enhances object recognition and object placement memory in middle-aged, but not
aged, female rodents248,257. Combined, these data suggest that E2 is less efficacious
when given to aged rodents, especially when postovariectomy E2 treatm ent is del ayed.
Several potential mechanisms may underlie decreased sensitivity to E2 in older rodents.
For ex ample, E2-induced hippocampal transcription is reduced in aged female
rodents258, and several studies suggest that the expression of oestrogen receptor-α and
oestrogen receptor-β dimini shes with age131,259,260. The precise mechanisms underlying
decreased responsivity to E2 in adv anced age remain to b e determi ned.
Spatial working memory
Memory for locations that
change over time (for example,
the locations of your keys
or your car in your campus
car park).
Delayed non-match-to-
sample task
Test of memory for items that
differ from an initial stimulus
array, assessed at some delay
after the original stimulus
presentation.
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including the ERK, mTOR and PI3K–AKT pathways;
activity in these pathways is required for E2 to enhance
memory consolidation in the OR and OP tasks among
ovariectomized mice57,87,91,97,158. Within the dorsal hip-
pocampus, these signalling cascades are initiated by E2
binding to hippocampal ERα and ERβ but not to
GPER57,78. Indeed, although GPER activation in the
dorsal hippocampus enhances OR and OP memory
consolidation in ovariectomized females, these effects
are independent of E2 signalling78.
a Morris water maze
c Object recognition and object placement
b Radial arm maze
Standard working memory Working or reference memory
Training Testing Training Context test
Delay
d Social discrimination e Fear conditioning
Training Testing
Delay
Training Testing Tone test Extinction
Delay
(repeated exposure
to context without
CS presentation)
Fig. 2 | Behavioural approaches to studying oestrogenic effects on
memory. a | The Morris water maze tests a rodent’s ability to navigate in
space and remember the location of spatial cues in the environment229,230.
Rodents are placed in a large round pool of water made opaque with
non-toxic paint or powdered milk, and must use extramaze spatial cues to
navigate to a platform hidden just below the surface of the water229,230.
Measures of performance include time to reach the platform, distance swum
and swim speed. During probe trials in which the hidden platform is
inaccessible to subjects for a portion of or throughout the trial, memory for
the platform location can be assessed by measuring the number of times that
animals cross the platform location and/or the time spent close to the
location of the platform. b | The radial arm maze also tests spatial navigation
abilities. Food-restricted or water-restricted subjects traverse a wheel-
shaped maze in which they must retrieve food or water rewards from the
ends of 8 or 12 long arms that radiate from a round central platform231,232.
Rewards are not replaced, so animals should visit each arm only once. In the
standard version, all arms are baited to measure working memory, which is
memory for information that changes from trial to trial. In a common
variation, half of the arms are baited to measure working memory and half
are never baited to measure reference memory, or memory for information
that does not change from trial to trial. c | Object recognition and object
placement tasks typically consist of a training trial, during which animals
explore objects in an open field for 5–20 minutes, followed by a delay
(minutes to days) and a single testing phase, during which a new object is
introduced (object recognition) or a training object is moved (object
placement)233,234. Because rodents are drawn to novelty, animals that
remember the identity and location of the training objects will spend more
time than chance with a novel or moved object during testing. d | Social
recognition can be tested using a habituation–dishabituation task or a
social discrimination task. In habituation–dishabituation, subjects are
repeatedly exposed to the same stimulus conspecific, leading to a decrease in
investigative behaviour (habituation) as the stimulus animal becomes known.
Animals are then exposed to a novel conspecific, and if social recognition
memory is intact, investigative behaviour returns (dishabituation). In social
discrimination, animals undergo a test trial where they are simultaneously
exposed to a familiar conspecific, seen previously in habituation trials, and a
novel conspecific. Increased investigative behaviour of the novel conspecific
is indicative of intact social recognition memory. e | Fear memory is most
commonly studied with use of classical conditioning paradigms that pair a
stimulus (a cue or context) with a foot shock, generating a learned association
that leads to fear responses (such as freezing) on subsequent presentations of
the conditioned stimulus (CS). Cued and contextual fear conditioning differ
in their underlying neurocircuitry, with cued fear being independent of
hippocampal function and contextual fear requiring hippocampal input235.
Extinction can be tested with repeated presentation of the context in the
absence of CS presentation236. Figure adapted from REF.236, Springer.
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The ability of E2 to activate dorsal hippocampal cell
signalling is closely tied to its ability to mediate epige-
netic processes associated with facilitated memory. ERK
activation is necessary for E2 to increase acetylation of
histone H3 within the dorsal hippocampus of ovariect-
omized mice31,106. Dorsal hippocampal E2 infusion also
decreases levels of histone deacetylases 2 and 3 in the
hippocampus of ovariectomized mice, thereby pro-
viding a mechanism for increased H3 acetylation in
the E2-treated hippocampus31,106. Importantly, dorsal
hippocampal infusion of a histone acetyltransferase
inhibitor prevents E2 from enhancing OR memory con-
solidation in ovariectomized mice, demonstrating a cru-
cial role for histone acetylation in the mnemonic effects
of E2 (REFS31,105).
In ovariectomized females, systemic E2 (REF.159)
and intrahippocampally administered E2 increase the
dendritic spine density of CA1 pyramidal neurons,
an effect dependent on ERK and mTOR signalling99.
Consistent with these data, elevated E2 levels robustly
influence hippocampal synaptic plasticity by facilitating
LTP121,160,161, which correlates with enhanced memory in
hippocampus-dependent tasks120,162.
Although much remains to be learned, researchers
are well on their way towards characterizing the neuro-
biological mechanisms through which E2 modulates
spatial and object memory consolidation.
Social cognition
The term ‘social cognition’ encompasses various pro-
cesses, but perhaps the most well studied in animals
is social recognition. Memory for conspecifics and past
social interactions allows the formation of meaningful
relationships and selection of appropriate behavioural
responses in future social interactions, which are cru-
cial abilities in social species163. Social discrimination
is heavily influenced by neuroendocrine mechanisms;
however, much of this research has focused on the
neuropeptides oxytocin and vasopressin, so only more
recently have sex steroid hormones become appreciated
as modulators of social memory164.
Ovary-intact female mice trained on a habituation–
dishabituation task (FIG.2) during proestrus show
better discrimination of a novel conspecific 24 h later rel-
ative to females trained in dioestrus, suggesting that ele-
vated E2 levels during training may improve long-term
social recognition165. Other studies specifically testing
the effects of exogenous E2 on social recognition in
ovariectomized rodents have found that systemically
administered E2 given before training improves social
memory in a habituation–dishabituation task166–168.
Many of these studies use long-term E2 replacement
before training and then test memory at extended time-
points, suggesting that classical actions of E2 are involved.
Indeed, E2 action at ERα increases transcription of the
oxytocin receptor in the medial amygdala169, a region
where oxytocin signalling is crucial for social recogni-
tion memory170. However, rapid membrane-initiated
E2 signalling also contributes substantially to these
effects. In a modified social discrimination task where
acquisition and recall occur within 40 min of drug
administration, systemic administration of E2 enhanced
social recognition memory in ovariectomized rats33.
The relatively short period between training and testing
makes it unlikely that classical E2 mechanisms have a role
in this effect, thereby implicating membrane-initiated
signalling as the key mediator. Furthermore, rapid
E2-induced CA1 plasticity correlates with this memory
enhancement; within 40 min of systemic or intracranial
administration, E2 increased CA1 dendritic spine density
in ovariectomized mice, which was surprisingly asso-
ciated with reduced AMPAR-mediated signalling33,119.
This change in excitatory signalling may reflect the
generation of new silent synapses.
The effects of E2 on social recognition memory are
mediated by both classical receptors and GPER. ERα
plays a particularly important role, with several studies
reporting that ERα-null or ERα-deficient female rodents
show impaired social recognition memory165,169,171,172.
Systemic injection of the ERα-selective agonist propyl
pyrazole triol (PPT) improved memory among ovariecto-
mized mice in a social discrimination task within 40 min,
suggesting that rapid action of membrane-localized ERα
is important for this effect173. The GPER agonist G1
induces similarly rapid improvements in social recog-
nition memory when systemically injected in ovariecto-
mized mice77. However, whether ERα and GPER initiate
similar cellular and molecular mechanisms to facilitate
social recognition memory is unclear. The facilita-
tive effects of ERα and GPER on social recognition do
not seem to extend fully to ERβ. Social recognition
memory in ERβ-null female mice is either unaffected
or only modestly impaired165,169,171, and treatment with
the ERβ-selective agonist diarylpropionitrile (DPN) in
ovariectomized mice either impairs or does not improve
social recognition memory173.
Similarly to spatial and OR memory, the hippocam-
pus is a critical locus of E2 action for social recognition
memory. Infusions of E2, PPT or G1 directly into the
dorsal hippocampus of ovariectomized mice induce a
rapid enhancement of social recognition memory that is
correlated with increased CA1 spine density and mod-
ulation of excitatory neurotransmission119,174. However,
E2 also modulates the function of other brain regions
crucial for social recognition memory. For example,
knockdown of ERα in the medial amygdala of ovariecto-
mized female rats abolishes social recognition memory172,
and infusion of ERα and GPER agonists into the medial
amygdala of ovariectomized mice rapidly enhances social
recognition175. An integrated understanding of how
E2 works ac ross multiple brain regi ons, re ceptor subtyp es
and signalling mechanisms to influence social cogni-
tion and recognition behaviour remains an area ripe for
future research.
Fear memory
The study of fear conditioning and its underlying cir-
cuitry and mechanisms is a cornerstone of the learning
and memory field. Nevertheless, despite its prominence,
researchers have until relatively recently largely ignored
how sex steroid hormones such as E2 affect fear learning.
Existing work reveals an interesting, and at times con-
tradictory, role for E2 in modulating both the acquisition
and the extinction of fear memory.
Silent synapses
Immature synapses containing
few AMPA receptors, which
could allow greater synaptic
potentiation and learning
facilitation on interaction
with a training stimulus.
Extinction
Process whereby a learned
association between two
stimuli (for example, shock
occurs in context A) becomes
unlearned through repetitive
exposure to one stimulus
(context A) without the
other (shock).
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Fear acquisition. Studies examining how E2 modulates
fear acquisition report both enhancing and impairing
effects. For example, long-term systemic E2 administra-
tion enhanced cued and context-dependent fear condi-
tioning in ovariectomized mice176,177 and fear-potentiated
startle in ovariectomized rats178, suggesting that
E2 enhances acquisition of fear learning. However, other
findings reveal impaired contextual fear conditioning
(CFC) in proestrus rats relative to males or rats in
oestrus179. Similarly, ovary-intact or ovariectomized rats
given E2 for 2 days before CFC froze less than did males
or untreated ovariectomized rats180, suggesting E2 may
impair fear learning.
These seemingly contradictory findings probably
result from differences in experimental approaches, with
the dose and timing of E2 administration as important
variables. High-dose systemic E2 administration days or
weeks before CFC enhanced acquisition in ovariecto-
mized mice, with lower doses of E2 having no effect 181,182.
By contrast, when E2 was injected into ovariectomized
rats 30 min before CFC training, high doses impaired
acquisition, whereas a low dose enhanced learning183.
These results suggest important differences in how
E2 dose and treatment duration interact to impact fear
learning, but further research is needed to more clearly
define these relationships.
Fear extinction. Research on the role of E2 in modulat-
ing fear extinction paints a clearer picture than does the
fear acquisition literature, with evidence from passive
avoidance tasks184, conditioned taste aversion185 and both
cued and conditioned fear conditioning186–188 supporting
the conclusion that E2 improves extinction learning. In
ovary-intact female rats, fear extinction training during
proestrus produces more rapid learning across trials186
and reduced freezing during extinction recall testing187
compared with extinction during metoestrus. E2 seems
to be both necessary and sufficient for these effects, as
systemic E2 treatment given to metoestrus or ovariect-
omized rats enhances extinction learning186,187, whereas
systemic blockade of ERs in proestrus rats impairs
extinction recall187. Similarly, reducing circulating
E2 levels via hormon al cont racept ives impai rs extinc tion
recall in cycling female rats189.
As with fear acquisition, the influence of E2 on
extinction is dose dependent. In cycling female rats,
E2 exhi bits an invert ed-U d ose–resp onse effe ct on exti nc-
tion learning, such that rats with very low (untreated
metoestrus) or very high (proestrus or metoestrus given
a high dose of E2) levels of E2 exhibited poor extinction
recall, and rats with moderate E2 levels (untreated proes-
trus or metoestrus given a low dose of E2) extinguished
recall well190.
ERβ is an important mediator of the effects of E2 on
fear extinction. Systemic injection of the ERβ-selective
agonist DPN before extinction training improves
extinction learning in ovariectomized or metoestrus
rats, whereas the ERα-selective agonist PPT has no
effect186,188. Infusion of DPN into the dorsal hippocam-
pus of ovariectomized rats enhanced contextual fear
extinction learning, suggesting that this brain region is
an important locus for ERβ activity in this paradigm186.
However, E2 has more pervasive actions in the neuro-
circuitry of fear extinction beyond the hippocampus.
The amygdala, a central region in fear extinction, shows
widespread expression of ER subtypes16 and structural
and synaptic plasticity following E2 treatment191,192. The
infralimbic cortex sends excitatory projections that regu-
late amygdala subregions during extinction193, and is also
sensitive to oestrogens194. E2 see ms to modulate th e acti v-
ity and connectivity of these regions during extinction
recall; studies using FOS as a marker of neuronal activ-
ity report that metoestrus rats treated with E2 displayed
less amygdalar activation188,195 and greater infralimbic
cortex activation188 following extinction recall than did
untreated controls. Both the oestrous-cycle phase and
exogenous E2 treatment influence the activity of infral-
imbic cortex–amygdala projections in female rats195,196,
suggesting that E2 increases infralimbic cortex-driven
activation of an inhibitory circuitry in the amygdala that
reduces fear responses.
Fear generalization. In addition to fear acquisition
and extinction, E2 also influences fear generalization.
Ovariectomized rats systemically treated with E2 show
increased generalization of fear to neutral contexts rel-
ative to untreated controls197. Similarly to extinction,
this effect seems to depend primarily on hippocampal
ERβ signalling, as infusion of the ERβ-selective agonist
DPN, but not ERα-selective PPT, into the hippocampus
of ovariectomized rats increases fear generalization198.
That E2 can enhance both fear expression (via acqui-
sition and generalization) as well as extinction may at
first seem contradictory, but is not surprising given
that these processes share certain underlying molecular
mechanisms. Moreover, fear acquisition, generalization
and extinction all require new learning. Thus, the effects
of E2 on all aspects of fear learning highlight its actions as
a general promoter of learning and memory.
To ge t h er w it h fi n d in g s f r om t h e s p at i al , ob j e ct a nd
social memory tasks discussed earlier, the fear memory
data suggest that E2 is uniquely positioned as a general
neuromodulator that broadly facilitates new learning of
many sorts.
E2 modulation of memory in males
Although oestrogens are often (incorrectly) considered
‘female’ hormones, accumulating research findings
indicate a primary role for E2 in enhancing cognition
in male rodents. Indeed, E2 levels in the male rat hip-
pocampus are higher than in cycling females199,200.
Emerging findings, reviewed below, imply that E2 may
influence memory via distinct molecular mechanisms
in males and females. Why might these differences
matter if the beneficial effects on memory are simi-
lar in both sexes? From a drug discovery standpoint,
these data suggest the potential need to develop sex-
specific treatments for memory dysfunction in humans.
Tre at me nt s tha t t ar get me ch an is ms t hr ou gh wh ic h
E2 modulates memory in one sex (for example, ERK in
females) but not in the other are bound to fail in half
the population. Additional research to determine which
oestrogenic mechanisms are sex specific and which are
common to both sexes will substantially advance the
Contextual fear conditioning
Model of fear learning in
which repeated exposure to
foot shocks in one context
eventually elicits fear
(freezing) of the context
in the absence of shock.
FOS
A transcription factor
encoded by an immediate
early gene that is activated
rapidly and transiently in
response to neuronal activity,
leading to expression of
memory-related genes.
Generalization
Process whereby a stimulus–
response association learned
in one context (for example,
a stimulus induces fear)
becomes transferred to
another, similar context.
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development of effective memory therapeutics for both
men and women.
Sex differences in hippocampal E2 signall ing
In the hippocampus, E2 acts as a neuromodulator in both
males and females, but an emerging body of literature has
found notable sex differences in how E2 influences hip-
pocampal neurotransmission and the molecular under-
pinnings of these effects. In both sexes, E2 potentiates
excitatory neurotransmission in the hippocampus6,35,111,
and although some molecules and pathways, such as
SRC, ERK–MAPK, calcium/calmodulin-dependent pro-
tein kinase II and TRKB signalling, contribute to these
effects in both sexes35,201,202, others are sex specific. For
example, potentiation of excitatory postsynaptic currents
and LTP in the hippocampus depend on PKA in female
rats but not in males202. The sexes also differ in the ER
subtypes that mediate neural excitation, as hippocampal
glutamatergic neurotransmission is regulated presynap-
tically by ERβ in females and ERα in males, and postsyn-
aptically by GPER in females and ERβ in males111. Other
work has similarly shown differential contributions of
ERα versus ERβ in facilitating LTP between males and
females35,201. Differing roles of ER subtypes in modulat-
ing excitatory signalling may arise from sex differences
in subcellular ER localization, which in turn could
lead to sex-specific recruitment of signalling kinases
by E2 (REFS167,201).
Unlike excitator y signalling , inhibitor y h ippocam-
pal neurotransmission seems to be modulated by E2
only in females and through a sex-specific mechanism.
In the ovariectomized female rat hippocampus, ERα
activates postsynaptic mGluR1 signalling to stimulate
endocannabinoid release, which inhibits presynaptic
GABAergic terminals122. The functional coupling of
ERs with mGluR subtypes was shown to occur only in
females56, and further investigation found that although
ER–mGluR complexes can exist in both sexes, E2 acti vates
mGluR-dependent signalling only in females123.
A similar sex difference has been found with locally
synthesized E2 in the hippocampus. Although male and
female rodents both express aromatase and synthesize E2
in the hippocampus17,200,203, systemic aromatase inhibi-
tion reduces hippocampal synapse number and severely
impairs LTP induction in female mice, but has no effect
on synapse number and only modestly reduces LTP
in male mice204. Similarly, aromatase-null female mice
were shown to have reduced hippocampal spine density
compared with wild-type female mice, whereas spine
density was unaffected in aromatase-null male mice205.
However, in a forebrain-specific aromatase-null mouse,
both males and females exhibited reduced hippocampal
spine density206. Understanding the cellular and molecu-
lar consequences of local oestrogen synthesis in the male
hippocampus will require further investigation207.
Oestrogenic modulation of cognition in males
Despite differences in the mechanisms of E2 signalling
in cognition-related regions of the brain, E2 exerts sim-
ilar mnemonic benefits in males and females. Systemic
injection or dorsal hippocampal infusion of E2 immedi-
ately after training enhances memory consolidation in
the OR and OP tasks in gonad-intact and gonadecto-
mized male rats and mice208,209. Notably, sex differences
in the mechanisms that drive the beneficial effects of E2
on these tasks have been found: in contrast to its cen-
tral importance to the memory-enhancing effects of
E2 in ovariectomized mice, dorsal hippocampal ERK
phosphorylation is not necessary for E2 to facilitate
spatial and object memory consolidation in male mice,
regardless of gonadal status209.
In the MWM, gonad-intact adult male rats and aged
male mice systemically treated with E2 immediately after
training exhibited enhanced spatial memory compared
with vehicle-treated males210,211. Gonad-intact adult
males receiving E2 long term also made fewer working
memory errors in the RAM than did vehicle-treated
controls212.
Local synthesis of oestrogens is also important for
memory processes in male rodents, as aromatase-null
male mice exhibit deficits in social recognition213.
Furthermore, short-term oral aromatase inhibition
increases errors in a working memory task in male
rats214, and short-term intrahippocampal aromatase
inhibition prevents object and spatial memory consoli-
dation in gonadectomized male mice215. Therefore, the
data thus far suggest that hippocampally synthesized E2
is important for memory formation in male rodents, as
has been observed in female rodents. Although much
more remains to be learned, these selected findings sup-
port a beneficial effect of E2 on memory formation in
male rodents.
Conclusions and future directions
This Review has illustrated some of the myriad ways
in which E2, acting in several brain regions via mul-
tiple ERs, can facilitate numerous forms of learning
and memory (FIG.3). Three decades of research has
demonstrated that E2 is a potent regulator of the neural
mechanisms that are critically important for memory
formation, including cell signalling, gene expression,
protein synthesis, extrinsic and intrinsic excitability,
dendritic spine morphology and neurogenesis. Thus,
researchers studying the neurobiology of learning and
memory should recognize oestrogens as essential neuro-
modulators akin to other well-accepted modulatory
factors such as glucocorticoids and growth factors.
Widespread acceptance of oestrogenic neuromod -
ulation may have been historically slow to take hold
because of the strong association between oestrogens
and female reproduction, which has led to the erroneous
perceptions that oestrogens are ‘female’ hormones and
that cyclic oestrogen fluctuations in females confound
experimental outcomes due to increased variability.
In fact, oestrogens are important modulators of mem-
ory and neural function in both sexes216,217. Depending
on the behaviour being assessed, ovary-intact females
do not necessarily exhibit more behavioural variability
than males218, which argues for greater consideration of
the modulatory influence of oestrogens in females and in
males. Future progress on this issue may stem from the
US National Institutes of Health’s 2016 policy requiring
vertebrate animal researchers to consider sex as a biolog-
ical variable219–221, as more explicit comparisons between
www.nature.com/nrn
REVIEWS
the sexes could lead to new insights into hormonal regu-
lation of cognition. Although some investigators were
initially resistant to this policy, attitudes towards the
requirement are improving222. Greater appreciation of
oestrogens as neuromodulators that exert wide-ranging
effects in all individuals — not just females — is crucial
for the advancement of both basic and clinical science.
To further advance knowledge of oestrogenic
modulation in both sexes, future studies should use
newer technologies, including single-cell sequencing,
‘om i cs’ -l e ve l an al ys es an d ta rg et ed ge ne ti c ma ni pu la -
tions, to pinpoint crucial molecules and cellular pro-
cesses that underlie oestrogenic facilitation of learning
and memory. For example, a multiplexed CRISPR–Cas9
gene-editing approach could be used to simultaneously
target multiple oestrogen-responsive genes implicated
in neurodegenerative disease to understand their role in
memory function223. Future research must also evolve
from a focus on oestrogenic effects in individual brain
regions to addressing how oestrogens concurrently
influence multiple brain areas within memory cir-
cuits. Recent work using multiplexed chemogenetic
silencing has shown that the memory-enhancing effects
of E2 in the dorsal hippocampus requires concurrent
activity of the dorsal hippocampus and PFC155. Thus,
determining how brain regions interact synergisti-
cally to support oestrogen-mediated memory pro-
cesses is a crucial next step for the field. As part of this
approach, researchers should also consider cell-type
specificity, as memory-modulating effects of E2 on cell
types such as inhibitory neurons and glial cells have been
overshadowed by a predominant focus on excitatory
neurons.
By leveraging new technologies and asking circuit-
level and cell type-specific questions in both males
and females, scientists will discover fundamental new
insights into the ways in which E2-induced modulation
of brain function influences memory formation. Given
the dearth of effective treatments for memory dysfunc-
tion in various disorders, this information could provide
valuable new avenues for therapeutic development that
benefits both sexes.
Published online xx xx xxxx
Memory process ER subtype Site of oestrogenic action Behavioural output
Spatial and object
memory
• ERα
• ERβ
• GPER
Social memory • ERα
• GPER
↑ Spatial reference memory
↓ Spatial reference memory
↑ Spatial working memory
↑ Object recognition and
object placement memory
Fear memory • ERβ↑ Fear aquisition
↓ Fear aquisition
↑ Fear extinction learning
and recall
↑ Fear generalization
↑ Social recognition memory
PRC
HPC
HPC
MeA
PFC
HPC
PFC
Amy
Fig. 3 | Summary of oestrogenic actions on memory processes. This schematic shows the receptors and brain regions
involved in the effects of 17β-oestradiol (E2) on memory processes and the behavioural output of the actions of E2 in
young female rodents. Spatial reference memory142, working memory148 and ob ject memo ry57,87,154,155,173 are facilitated
by E2 (but see the discussion in the main text), and are largely dependent on the hippocampus (HPC) and prefrontal cortex
(PFC). The perirhinal cortex (PRC) is also involved in E2-mediated object recognition memory156,157. Social recognition
memory is facilitated by oestrogen receptor-α (ERα) and G protein-coupled oestrogen receptor (GPER) signalling in the
HPC and medial amygdala (MeA)119,172–174. Fear acquisition, fear extinction learning and recall, and fear generalization are
regulat ed by ERβ in th e PFC, amy gdala (Am y) and HPC186,188,195,196,198.
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Acknowledgements
The Frick laboratory is supported by the US National
Institutes of Health (R01MH107886, 2R15GM118304-02,
F31MH118822 and F32MH118782), the Alzheimer’s
Association (SAGA-17-419092), the University of Wisconsin
System, the University of Wisconsin-Milwaukee Research
Foundation, the University of Wisconsin-Milwaukee Office
of Undergraduate Research and the University of
Wisconsin-Milwaukee College of Letters and Science.
Author contributions
The authors contributed equally to all aspects of the article.
Competing interests
K.M.F. is a co-founder and Chief Scientific Officer of Estrigenix
Therapeutics Inc., and is listed as an inventor of a pending
patent held by the University of Wisconsin-Milwaukee,
Marquette University and Concordia University Wisconsin
entitled “Substituted (4′-hydroxyphenyl)cycloalkane and
(4′-hydroxyphenyl)cycloalkene compounds and uses thereof
as selective agonists of the estrogen receptor beta isoform
for enhanced memory consolidation”, inventors W. A.
Donaldson, D. S. Sem and K.M.F. (WO2018183800A1). The
other authors declare no competing interests.
Peer review inf ormat ion
Nature Reviews Neuroscience thanks H. Bimonte-Nelson, who
co-reviewed with V. Bernaud, and the other, anonymous,
reviewer(s) for their con tribution to the peer revi ew of this work.
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