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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Jan 17;177(3):642–655. doi: 10.1111/bph.14880

Activation of oestrogen receptor α induces a novel form of LTP at hippocampal temporoammonic‐CA1 synapses

Leigh Clements 1, Jenni Harvey 1,✉
PMCID: PMC7012968  PMID: 31637699

Abstract

Background and Purpose

17β estradiol (E2) rapidly regulates excitatory synaptic transmission at the classical Schaffer collateral (SC) input to hippocampal CA1 neurons. However, the impact of E2 on excitatory synaptic transmission at the distinct temporoammonic (TA) input to CA1 neurons and the oestrogen receptors involved is less clear.

Experimental Approach

Extracellular recordings were used to monitor excitatory synaptic transmission in hippocampal slices from juvenile male (P11‐24) Sprague Dawley rats. Immunocytochemistry combined with confocal microscopy was used to monitor the surface expression of the AMPA receptor (AMPAR) subunit, GluA1 in hippocampal neurons cultured from neonatal (P0‐3) rats.

Key Results

Here, we show that E2 induces a novel form of LTP at TA‐CA1 synapses, an effect mirrored by the ERα agonist, PPT, and blocked by an ERα antagonist. ERα‐induced LTP is NMDA receptor (NMDAR)‐dependent and involves a postsynaptic expression mechanism that requires PI 3‐kinase signalling and synaptic insertion of GluA2‐lacking AMPARs. ERα‐induced LTP has overlapping expression mechanisms with classical Hebbian LTP, as HFS‐induced LTP occluded PPT‐induced LTP and vice versa. In addition, activity‐dependent LTP was blocked by the ERα antagonist, suggesting that ERα activation is involved in NMDA‐LTP at TA‐CA1 synapses.

Conclusion and Implications

ERα induces a novel form of LTP at juvenile male hippocampal TA‐CA1 synapses. As TA‐CA1 synapses are implicated in episodic memory processes and are an early target for neurodegeneration, these findings have important implications for the role of oestrogens in CNS health and neurodegenerative disease.

Abbreviations

aCSF

artificial CSF

AD

Alzheimer's disease

AMPAR

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors

D‐AP5

D‐2‐amino‐5‐phosphonopentanoate

DPN

diarylpropionitrile

E2

17β estradiol

ERα

oestrogen receptor α

ERβ

oestrogen receptor β

HFS

high‐frequency stimulation

MPP

1,3‐bis(4‐hydroxyphenyl)‐4‐methyl‐5‐[4‐(2‐piperidinyl‐ethoxy)phenol]‐1H‐pyrazole dihydrochloride

PHTPP

4‐[2‐Phenyl‐5,7‐bis(trifluoromethyl)pyrazolo[1,5‐a]pyrimidin‐3‐yl]phenol

PhTx

philanthotoxin

PI 3‐kinase

phosphoinositide 3‐kinase

PPR

paired‐pulse facilitation ratio

PPT

4,4′,4″‐(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol

SC

Schaffer collateral

SLM

stratum lacunosum‐moleculare

TA

temporoammonic

What is already known

  • Oestrogens regulate excitatory synaptic transmission at hippocampal CA1 synapses.

  • The ER subtypes mediating the regulatory actions of oestrogens at TA‐CA1 synapses are not clear.

What this study adds

  • ERα activation induces a novel form of LTP at juvenile male TA‐CA1 synapses.

  • ERα‐induced LTP is NMDAR dependent and involves AMPAR trafficking to synapses.

What is the clinical significance

  • TA‐CA1 synapses are an early target for degeneration in Alzheimer's disease.

  • ERα regulation of TA‐CA1 synapses has important implications for CNS health and disease.

1. INTRODUCTION

Numerous studies indicate that oestrogens influence hippocampal synaptic function, as 17β estradiol (E2) rapidly potentiates excitatory synaptic transmission at classical Schaffer collateral (SC)‐CA1 synapses (Teyler, Vardaris, Lewis, & Rawitch, 1980). The effects of E2 are primarily mediated by activation of two oestrogen receptors, oestrogen receptor α (ERα) and oestrogen receptor β (ERβ) that are differentially expressed in the CNS and have distinct functional roles. Several studies support a role for ERβ in mediating E2 effects at SC‐CA1 synapses, as ERβ enhances synaptic plasticity and promotes AMPA receptor (AMPAR) trafficking to synapses (Liu et al., 2008). ERβ also mediates E2‐induced rapid changes in dendritic morphology (Srivastava, Woolfrey, Liu, Brandon, & Penzes, 2010). However, ERα also enhances excitatory synaptic transmission at SC‐CA1 synapses (Oberlander & Woolley, 2016).

In addition to the SC input, CA1 pyramidal neurons are innervated by the temporoammonic (TA) pathway that arises in layer III of the entorhinal cortex and forms a direct connection to the stratum lacunosum‐moleculare (SLM) region of CA1 neurons. Excitatory synaptic transmission at TA‐CA1 synapses is regulated by many neuromodulators, including leptin (Luo, McGregor, Irving, & Harvey, 2015) and dopamine (Otmakhova & Lisman, 1999). TA‐CA1 synapses play a role in hippocampus‐dependent memory processes, including spatial novelty detection and memory consolidation (Remondes & Schuman, 2004; Vago & Kesner, 2008). The TA input to CA1 neurons is also implicated in episodic memory as it integrates information from cortical regions and regulates place cell activity (Stokes, Kyle, & Ekstrom, 2015). This input is also linked to CNS disease as the TA pathway is an early site for tau pathology in Alzheimer's disease (AD; Buxbaum et al., 1998). However, the impact of E2 on excitatory synaptic transmission at TA‐CA1 synapses is unclear. Although E2 regulates synaptic transmission at adult TA‐CA1 synapses (Smith, Smith, Bredemann, & McMahon, 2016), the acute actions of E2 at juvenile TA‐CA1 synapses are unknown.

Here, E2 induced a persistent increase in excitatory synaptic transmission (LTP) at juvenile male TA‐CA1 synapses, an effect mirrored by the ERα agonist PPT. LTP induced by E2 and PPT was blocked by ERα but not ERβ antagonists, suggesting a role for ERα. ERα‐induced LTP involves a postsynaptic expression mechanism and activation of GluN2B‐containing NMDA receptors (NMDARs) as well as PI 3‐kinase signalling. Synaptic insertion of GluA2‐lacking α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) underlies ERα‐induced LTP in slices and PPT increased trafficking of GluA1 to synapses in hippocampal neurons. ERα‐induced LTP shares similar expression mechanisms to activity‐dependent LTP as PPT‐induced LTP was occluded by activity‐dependent LTP and vice versa. Activity‐dependent LTP was blocked by the ERα antagonist, indicating that ERα activation underlies NMDA‐LTP at this synapse. These findings suggest an important role for ERα at TA‐CA1 synapses, which has important implications for the role of oestrogens in health and disease.

2. MATERIALS AND METHODS

2.1. Electrophysiology

All experiments on animals were performed according to U.K. laws (Scientific Procedures Act 1986). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and the recommendations made by the British Journal of Pharmacology. All experimental procedures on animals were approved by the Animal Welfare Ethical Review Committee at the University of Dundee. Parasaggital hippocampal slices (350 μm) were prepared from P11‐P24 old male Sprague Dawley (RRID:RGD_5508397) rats as before (Luo et al., 2015). Animals were killed by cervical dislocation in accordance with Schedule 1 of the U.K. Scientific Procedures Act 1986. Brains were rapidly removed and placed in ice‐cold artificial CSF (aCSF; bubbled with 95% O2 and 5% CO2) containing the following (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4, and 10 d‐glucose. Once prepared, slices were allowed to recover at room temperature in oxygenated aCSF for 1 hr before transferring to a submerged chamber maintained at room temperature and perfused with oxygenated aCSF. In all slices, the dentate gyrus and CA3 region were removed to prevent indirect stimulation of the SC input.

Standard extracellular recordings of local field EPSPs (fEPSPs) were used to monitor excitatory synaptic transmission at TA‐CA1 synapses. Recording pipettes contained aCSF (3–5 MΩ) and were placed in SLM to record TA‐CA1 responses. The direct TA pathway was stimulated at 0.033 Hz, using a stimulus intensity that evoked a peak amplitude ∼50% of the maximum. Synaptic field potentials were low‐pass filtered at 2 kHz and digitally sampled at 10 kHz. The slope of the evoked fEPSPs was expressed relative to the preconditioning baseline. Data were monitored online and analysed offline using the WinLTP 2.20 program (RRID:SCR_008590; Anderson & Collingridge, 2007). Recording of data was not blinded to the operator, as each electrophysiology experiment required an awareness by the experimenter of the running protocol. In all electrophysiology experiments, fEPSP baseline recordings were only considered stable when both peak amplitude and slope value measurements did not deviate more than 10% from each other. If baseline recordings were not stable, no drug treatment was initiated. The magnitude of LTP induced by ERs was calculated 50–55 min after addition of the selective agonists and expressed as a percentage of baseline ± SEM. Activity‐dependent LTP was induced by delivery of a high‐frequency stimulation (HFS; 100 Hz·s−1) paradigm and, as reported previously, blockade of GABAergic inhibition was not required (Luo et al., 2015). All data are expressed as means ± SEM and statistical analyses were performed using Student's paired t‐test (two‐tailed; 95% confidence interval) or repeated‐measures ANOVA for comparison of means within subject or one‐way ANOVA with Tukey's post hoc test for comparisons between multiple groups. P < .05 was considered significant. Data analysis was not blinded but was double checked by more than one researcher. All electrophysiology experiments were performed from n ≥ 5 slices for each group. Each n value represents an individual slice taken from a separate animal, which is considered to be sufficient for the evaluation of statistical difference in our recordings.

2.1.1. Hippocampal cell culture

Hippocampal cultures were prepared as previously (McGregor, Clements, Farah, Irving, & Harvey, 2018). Neonatal Sprague Dawley rats (1–3 days old) were killed by cervical dislocation in accordance with Schedule 1 of U.K. Animals Scientific Procedures Act (1986). Hippocampi were removed and following washing in HEPES buffered saline (HBS) comprising (in mM): 135 NaCl; 5 KCl; 1 CaCl2; 1 MgCl2; 10 HEPES; 25 d‐glucose (pH 7.4), were treated with papain (1.5 mg·ml−1; Sigma‐Aldrich, UK) for 20 min at 37°C. Dissociated cells were plated onto sterile dishes (35‐mm diameter; Greiner Bio‐One Ltd., UK) treated with poly‐d‐lysine (20 μg·ml−1; 1–2 hr) at a density of 5 × 105 cells ml−1. Cultures were maintained in Neurobasal‐A medium (Thermofisher Scientific, UK) in a humidified atmosphere of 95% O2 and 5% CO2 at 37°C for up to 2 weeks.

2.1.2. Immunocytochemistry

Immunocytochemistry was performed on 7‐ to 15‐day‐old cultured hippocampal neurons and all immunocytochemical procedures and analyses comply with the recommendations of Alexander et al. (2018). The Immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology. Before labelling, neurons were washed with HBS containing glycine (0.01 mM) and treated with PPT for 15 min at 21–23°C. For antagonist experiments, neurons were pretreated with inhibitors for 30 min prior to PPT. To label surface GluA1, living neurons were incubated with an antibody against the N‐terminal region of GluA1 (1:100; Moult et al., 2010) at 4°C. Neurons were then fixed with 4% paraformaldehyde for 5 min, and surface GluA1 immunostaining was visualised by addition of an anti‐sheep Alexa 488‐conjugated secondary antibody (1:250; Life Technologies, UK) for 30 min. In a subset of experiments, neurons were permeabilised with 0.1% Triton X‐100 (5 min) after fixation. A second primary antibody was then applied to compare GluA1 surface immunostaining relative to PSD‐95 (mouse anti‐PSD‐95; 1:500; Thermo Fisher, UK). An Alexa 568‐conjugated anti‐mouse secondary antibody (1:200; ThermoFisher, UK) was used to visualise PSD‐95 labelling as before (McGregor, Irving, & Harvey, 2017). No labelling was observed after incubation with secondary antibodies.

2.1.3. Confocal analysis

A confocal imaging system (Zeiss LSM 510) was used for image acquisition, and 488‐ and 543‐nm laser lines were used to excite the Alexa 488 and 555 or 568 fluorophores respectively. Images were obtained in a single‐tracking mode or multi‐tracking mode for dual labelling experiments using a 15‐s scan speed. Intensity of staining was determined offline using Lasersharp software (Carl Zeiss). Analysis lines (50 μm) were drawn along randomly selected dendritic regions and mean fluorescence intensity for GluA1 was calculated for each dendrite (McGregor et al., 2018). For synaptic co‐localisation experiments, surface GluA1 immunolabelling was compared with dendritic PSD‐95 immunostaining and the number of GluA1‐positive sites that co‐localised with PSD‐95‐positive sites were expressed as % of PSD‐95‐positive sites (McGregor et al., 2018). Data were obtained from at least three dendrites from a minimum of four randomly selected neurons for each treatment and from at least three different cultures from different animals. Within a given experiment, all conditions were kept constant. Data were normalised relative to mean fluorescence intensity in control neurons. All data are expressed as means ± SEM, and statistical analyses were performed using one‐way ANOVA for comparisons between multiple groups. P < 0.05 was considered significant with n representing the number of analysed dendrites.

2.2. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). No power analysis, randomisation or blinding of the samples was performed under recording. The exact group size (n) for each experimental group/condition signifies independent values, not replicates. Normality of all data was verified using the Kolmogorov–Smirnov test. To allow for comparison between individual data sets, all data have been normalised. In electrophysiology experiments, data were normalised to the mean slope value of the initial baseline recording and expressed as percentage means ± SEM. In immunocytochemistry experiments, data were normalised to mean intensity value of control neurons and expressed as percentage means ± SEM. In all studies, statistical differences were evaluated when n ≥ 5, and a Tukey's multiple comparison test was used following one‐way ANOVA to compare all pairwise, whereas a Dunnett's multiple comparison test was used following one‐way ANOVA to compare every group with the control group. Post hoc tests were conducted only if F was significant and there was no variance inhomogeneity.

2.3. Drugs

All drugs were dissolved in aCSF and applied in the bath at the desired final concentration (see Table 1). Drugs used were dopamine, 17β‐estradiol (E2), 4,4′,4″‐(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT), diarylpropionitrile (DPN), 1,3‐bis(4‐hydroxyphenyl)‐4‐methyl‐5‐[4‐(2‐piperidinyl‐ethoxy)phenol]‐1H‐pyrazole dihydrochloride (MPP), 4‐[2‐Phenyl‐5,7‐bis (trifluoromethyl)pyrazolo[1,5‐a]pyrimidin‐3‐yl]phenol (PHTPP), D‐2‐amino‐5‐phosphonopentanoate (D‐AP5), wortmannin, LY294002, PD98059, U0126, philanthotoxin (PhTx), letrozole, NVP‐AAM077, ifenprodil, picrotoxin, and CGP55845.

Table 1.

Summary of pharmacological tools used

Drug Concentration Biological activity
17β‐estradiol 1 μM Non‐selective, oestrogen receptor agonist
CGP55845 100 nM GABABR antagonist
D‐AP5 50 μM Competitive NMDAR antagonist
DPN 1 nM, 10 nM, 25 nM ERβ agonist
Dopamine 100 μM Neurotransmitter/hormone
Ifenprodil 3 μM GluN2B‐NMDAR antagonist
Letrozole 100 nM Aromatase inhibitor
LY294002 10 μM PI3‐Kinase inhibitor
MPP 1 μM ERα antagonist
NVP‐AAM077 100 nM GluN2A‐NMDAR antagonist
PD98059 10 μM ERK inhibitor
Philanthotoxin 1 μM GluA2‐lacking AMPAR antagonist
PHTPP 1 μM ERβ antagonist
Picrotoxin 50 μM GABAAR antagonist
PPT 25 nM, 50 nM ERα agonist
U0126 10 μM ERK inhibitor
Wortmannin 50 nM PI3‐Kinase inhibitor
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2.4. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/2020 (Alexander et al., 2019).

3. RESULTS

3.1. E2 rapidly modulates excitatory synaptic transmission at TA‐CA1 synapses

Oestrogens modulate excitatory synaptic transmission at SC‐CA1 synapses (Oberlander & Woolley, 2016; Smejkalova & Woolley, 2010), but the effects of oestrogens at TA‐CA1 synapses are less clear. To evaluate if oestrogens influence TA‐CA1 synapses, the effects of E2 were assessed in slices from juvenile male rats. Application of 1‐μM E2 (15 min) caused an increase in excitatory synaptic transmission (LTP) (Figure 1a), that was sustained following E2 washout (up to 1 hr). As E2 activates different ERs, the role of ERα and ERβ were addressed. To verify the role of ERα, the effects of the ERα antagonist, 1,3‐Bis(4‐hydroxyphenyl)‐4‐methyl‐5‐[4‐(2‐piperidinyl‐ethoxy)phenol]‐1H‐pyrazole dihydrochloride (MPP) were assessed. Addition of 1‐μM MPP (60 min) had no effect on synaptic transmission (96 ± 1.7% of baseline; n = 5), but in MPP‐treated slices, the ability of E2 to induce LTP was blocked (Figure 1b). To clarify involvement of ERβ, the effects of the ERβ antagonist, 4‐[2‐Phenyl‐5,7‐bis (trifluoromethyl) pyrazolo[1,5‐a]pyrimidin‐3‐yl]phenol (PHTPP) were examined. Addition of PHTPP (1 μM; 60 min) had no effect on synaptic transmission (103 ± 2.3 of baseline; n = 5) and in PHTPP‐treated slices, E2 increased synaptic transmission (n = 5; data not shown). These data indicate that E2 induces LTP at TA‐CA1 synapses via ERα activation. As dopamine selectively depresses excitatory synaptic transmission at TA‐CA1synapses (Otmakhova & Lisman, 1999), 100‐μM dopamine was applied at the end of experiments to confirm TA stimulation (Luo et al., 2015).

Figure 1.

Figure 1

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ERα activation induces LTP at juvenile TA‐CA1 synapses. (a–e) Pooled data showing the effects of E2 (1 μM; 15 min; a,b), PPT (25–50 nM; c,d) and DPN (10 nM; e) on excitatory synaptic transmission at TA‐CA1 synapses in juvenile hippocampal slices. At the end of experiments, application of dopamine (100 μM; 5 min) inhibits synaptic transmission confirming TA stimulation. In this and subsequent figures, each point is the average of four successive responses, and representative fEPSPs are shown above each plot and for the time indicated. Application of E2 caused a persistent increase in synaptic transmission (a) that was blocked by selective inhibition of ERα with MPP (b). (c) Addition of the ERα agonist PPT induced a persistent increase in synaptic efficacy, an effect that was blocked by the ERα antagonist MPP (d). (e) In contrast, the ERβ agonist DPN has no effect on excitatory synaptic transmission. (f) Bar chart of pooled data illustrating the effects of PPT alone and in the presence of MPP or the ERβ antagonist PHTPP on synaptic transmission. ERα activation induced a novel form of LTP at TA‐CA1 synapses

3.2. PPT induces LTP at TA‐CA1 synapses via activation of ERα

ERα and ERβ are differentially distributed throughout the hippocampus and divergent effects of ERs at SC‐CA1 synapses have been reported (Oberlander & Woolley, 2016). As our data suggest that LTP is induced by ERα activation, we examined the role of ERα using the ERα‐selective agonist, 4,4′,4″‐(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT). Application of 25‐nM PPT increased synaptic transmission (Figure 1c), an effect sustained after washout for up to 60 min. Similarly, 50‐nM PPT caused a persistent increase in synaptic transmission (140 ± 13.7% of baseline; n = 5.) These data indicate that PPT induces a novel form of LTP at TA‐CA1 synapses.

To further verify ERα involvement, the effects of the ERα antagonist MPP were assessed. Application of 1‐μM MPP (60 min) had no effect on synaptic transmission. However, in the presence of MPP, PPT (25 nM) failed to increase synaptic transmission (Figure 1d,f) and this effect was significantly different to PPT‐induced LTP (to 131 ± 5.3% of baseline; n = 5).

As ERβ also regulates SC‐CA1 synapses (Kramár et al., 2009; Oberlander & Woolley, 2016; Wang et al., 2016), the effects of the ERβ agonist DPN were compared. Application of 10‐nM DPN (15 min) had no effect on synaptic transmission (Figure 1e). Addition of other concentrations of DPN also had no effect as synaptic transmission which was 101 ± 1.1% of baseline in the presence of 1‐ or 25‐nM DPN respectively. To explore the role of ERβ further, the effects of the ERβ antagonist PHTPP were examined. PHTPP (1 μM) had no effect on basal synaptic transmission. Moreover in PHPTT‐treated slices, PPT (25 nM) evoked a significant increase in synaptic transmission (Figure 1f). These data indicate that the ability of PPT to induce LTP involves activation of ERα.

3.3. PPT‐induced LTP has a postsynaptic locus of expression

As ERα is highly expressed within presynaptic terminals and at postsynaptic densities (McEwen & Milner, 2017; Waters et al., 2009), activation of ERα at either locus may contribute to PPT‐induced LTP. To address the locus of PPT‐induced LTP, the paired‐pulse facilitation ratio (PPR) was analysed as changes in PPR typically reflect alterations in release probability. Paired‐pulse facilitation was induced by delivering two pulses at a 50‐ms interval. In slices where PPT (25 nM) increased synaptic transmission (Figure 2a), no significant change in the PPR was observed (Figure 2b), indicating a likely postsynaptic expression mechanism. As a control, the effects of dopamine were compared as dopamine depresses excitatory synaptic transmission at TA‐CA1 synapses via a presynaptic mechanism (Otmakhova & Lisman, 1999). Application of dopamine (100 μM) resulted in depression of synaptic transmission which was accompanied by a significant increase in PPR (Figure 2b). Overall, these data indicate that PPT‐induced LTP at TA‐CA1 synapses involves a postsynaptic expression mechanism.

Figure 2.

Figure 2

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PPT‐induced LTP is NMDAR‐dependent and involves a postsynaptic expression mechanism. (a) Plot of pooled data illustrating the effects of 25‐nM PPT (15 min) on synaptic transmission at TA‐CA1 synapses. (b) Corresponding plot of the pooled paired‐pulse ratio (PPR) against time for experiments shown in (a). The effects of PPT were not accompanied by any change in PPR, indicating a postsynaptic mechanism. (c–e) Plots of pooled data illustrating the effects of PPT (25 nM; 15 min) on synaptic transmission in hippocampal slices. NMDAR activation was involved as PPT failed to induce LTP in the presence of the competitive NMDAR antagonist D‐AP5 (50 μM; c). (d,e) The ability of PPT to induce LTP was blocked following inhibition of GluN2B subunits with ifenprodil (d) but was unaffected by the GluN2A inhibitor, NVP‐AAM0077 (e). (f) Bar chart of pooled data illustrating the relative effects of PPT on synaptic transmission in control conditions and after treatment with D‐AP5, ifenprodil, or NVP‐AAM0077. PPT‐induced LTP involves activation of GluN2B subunits

3.4. NMDAR activation is required for PPT‐induced LTP

As synaptic activation of NMDARs is crucial for activity‐dependent hippocampal synaptic plasticity (Bliss & Collingridge, 1993) and E2 induces an NMDAR‐dependent form of LTP at adult TA‐CA1 synapses (Smith et al., 2016), the role of NMDARs was examined using the competitive NMDAR antagonist, D‐AP5. Application of 50‐μM D‐AP5 (60 min) had no effect on basal synaptic transmission. However, in the presence of D‐AP5, PPT (25 nM) failed to alter synaptic transmission (Figure 2c) and this effect was significantly different to the magnitude of PPT‐induced LTP. These data suggest that activation of NMDARs is required for PPT‐induced LTP.

Different GluN2 subunits determine the biophysical and pharmacological properties of NMDARs and have differential roles in synaptic plasticity (Paoletti, Bellone, & Zhou, 2013). As E2‐induced LTP at adult TA‐CA1 synapses involves recruitment of GluN2B subunits (Smith et al., 2016), the role of different NMDAR subunits was examined using selective antagonists. Application of ifenprodil (GluN2B antagonist; 3 μM) or NVP‐AAM077 (putative GluN2A antagonist; 100 nM) had no effect on basal synaptic transmission (n = 5) for each respectively. In control slices, PPT (25 nM) increased synaptic transmission (to 139 ± 8.8% of baseline; n = 5). In interleaved slices treated with NVP‐AAM007, a significant increase in synaptic transmission (Figure 2e), was induced by PPT. However, in slices treated with ifenprodil, PPT failed to significantly alter synaptic transmission (Figure 2d), suggesting that GluN2B‐containing NMDARs are required for ERα‐induced LTP.

As stimulating the TA pathway can activate γ‐aminobutyric acid (GABA)ergic interneurons, which synapse onto CA1 pyramidal cells (Dvorak‐Carbone & Schuman, 1999), GABAergic inhibition may play a role in ERα‐induced LTP. To explore this, the effects of the GABAA and GABAB receptor antagonists, picrotoxin (50 μM) and CGP55845 (100 nM) were examined. In slices, co‐application of picrotoxin and CGP55845 had no effect on basal synaptic transmission (100 ± 3.3% of baseline, n = 5). In control slices, application of PPT induced LTP as synaptic transmission increased (to 146 ± 6.0% of baseline; n = 5). However, the ability of PPT to induce LTP was unaffected when GABAergic inhibition was blocked, as PPT significantly increased synaptic transmission (to 133 ± 11% of baseline; n = 5; data not shown), in picrotoxin‐ and CGP55845‐treated slices. These data indicate that ERα‐induced LTP at TA‐CA1 synapses is independent of GABAA and GABAB receptors.

3.5. Insertion of GluA2‐lacking AMPARs underlies PPT‐induced LTP

NMDAR activation promotes AMPAR trafficking and insertion of GluA2‐lacking AMPARs into SC‐CA1 synapses during LTP (Collingridge, Isaac, & Wang, 2004). To verify the role of AMPAR trafficking, the effects of PhTx, an inhibitor of GluA2‐lacking AMPARs, were assessed. Application of 1‐μM PhTx (60 min) had no effect on basal synaptic transmission (n = 5). In control slices, application of PPT increased synaptic transmission (Figure 3a), but, in slices incubated with PhTx (1 μM; 90 min), PPT failed to alter synaptic transmission (Figure 3b). Application of PhTx immediately after PPT application reversed PPT‐induced LTP such that the initial PPT‐induced increase in synaptic transmission was returned to baseline levels after Phtx (Figure 3c). Conversely, addition of PhTx, 45 min after PPT, had no effect on PPT‐induced LTP (Figure 3d), indicating that insertion of GluA2‐lacking AMPARs is key for induction, but not maintenance, of ERα‐induced LTP.

Figure 3.

Figure 3

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Synaptic insertion of GluA2‐lacking AMPARs underlies ERα‐induced LTP. (a–d) Plots of pooled data illustrating effects on synaptic transmission in hippocampal slices. (a) LTP was evoked by addition of 25‐nM PPT, but prior exposure to Phtx (1 μM; b) prevented PPT‐induced LTP. (c) Application of Phtx immediately after addition of PPT resulted in reversal of PPT‐induced. (d) Addition of Phtx, 45 min after PPT failed to reverse PPT‐induced LTP. (e) Representative confocal images of surface GluA1 labelling in control hippocampal neurons and after PPT, MPP or PPT plus MPP (DIV7‐13). (f) Pooled data illustrating the relative effects on GluA1 surface labelling in control conditions and after addition of PPT, MPP, or PPT plus MPP. ERα activation increases GluA1 surface expression (DIV7–13). (g) Pooled data showing the effects on % GluA1/PSD‐95 co‐localisation. The ERα agonist, PPT, increased GluA1 expression at hippocampal synapses (DIV7‐13)

Our data suggest that insertion of GluA1‐containing AMPARs into TA‐CA1 synapses underlies ERα‐induced LTP. To directly verify the role of AMPAR trafficking, the effects of PPT on the cell‐surface expression of AMPARs were assessed using an antibody against the N‐terminal domain of GluA1 in cultured hippocampal neurons (McGregor et al., 2017; Moult et al., 2010). Application of 25‐nM PPT (15 min) resulted in a significant increase in GluA1 surface immunostaining (n = 48). To verify involvement of ERα, the effects of the ERα antagonist MPP were evaluated. Treatment with 1‐μM MPP (15 min) had no effect on GluA1 surface expression ( (Figure 3e,f). However, in MPP‐treated neurons, the ability of PPT (25 nM) to alter AMPAR trafficking was inhibited, as GluA1 surface expression was not significantly different to control, in neurons treated with PPT plus MPP ( (Figure 3e,f). These data indicate that ERα activation increases GluA1 surface expression in hippocampal neurons.

As excitatory synaptic strength depends on AMPAR density at synapses, we determined if ERα activation alters the synaptic expression of GluA1, using dual labelling techniques to compare surface GluA1 relative to synapses (anti‐PSD‐95 antibody) in cultured hippocampal neurons (McGregor et al., 2018). Treatment with PPT (25 nM) increased surface GluA1 labelling (to 183 ± 9.6% of control; n = 36) and increased surface GluA1 that co‐localised with PSD‐95 (Figure 3g). These data indicate that activation of ERα increases the synaptic expression of GluA1 in hippocampal neurons.

As NMDAR activation promotes the synaptic insertion of AMPAR during LTP (Collingridge et al., 2004) and PPT‐induced LTP at TA‐CA1 synapses is NMDAR dependent, the effects of the NMDAR antagonist D‐AP5, were examined. Treatment with D‐AP5 (50 μM) had no effect on surface GluA1 labelling (100 ± 4.3% of control; n = 36). In control neurons, application of 25‐nM PPT increased GluA1 surface staining (to 174 ± 7.0% of control; n = 36), but in D‐AP5‐treated neurons, the ability of PPT to enhance GluA1 surface labelling was significantly reduced as PPT failed to enhance GluA1 surface expression in the presence of D‐AP5 (to 84 ± 2.9% of control; n = 36). These data indicate that the ERα‐driven increase in AMPAR trafficking is NMDAR‐dependent.

3.6. PPT‐induced LTP involves PI 3‐kinase signalling

As rapid activation of oestrogen receptors stimulates various downstream signalling cascades, including ERK and PI 3‐kinase (D'Astous, Mendez, Morissette, Garcia‐Segura, & Di Paolo, 2006; Smith et al., 2016; Titolo, Mayer, Dhillon, Cai, & Belsham, 2008), the role of these signalling cascades was investigated. To assess the role of PI 3‐kinase, two distinct inhibitors of PI 3‐kinase were used. Application of either wortmannin (50 nM) or LY294002 (10 μM) for 60 min had no effect on basal synaptic transmission. In interleaved slices, application of PPT (25 nM) increased synaptic transmission (Figure 4a). However, in the presence of either PI 3‐kinase inhibitor, PPT failed to induce LTP as no significant increase in synaptic transmission was observed after treatment with wortmannin (Figure 4d) or LY294002 ( (Figure 4b). Conversely, the magnitude of PPT‐induced LTP was not altered following inhibition of ERK with PD98059 or UO126. Application of either ERK inhibitor had no effect on synaptic transmission (n = 5 for each) and PPT significantly increased synaptic transmission in slices treated with PD98059 (Figure 4c), or U0126 (Figure 4d). Together, these data indicate that PPT‐induced LTP at TA‐CA1 synapses involves PI 3‐kinase signalling.

Figure 4.

Figure 4

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ERα‐induced LTP involves PI 3‐kinase signalling. (a–c) Pooled data illustrating the effects on excitatory synaptic transmission in slices. The ability of PPT to induce LTP (a) was blocked after PI3K inhibition with LY294002 (b) but was unaffected in the presence of the ERK inhibitor, PD98059 (c). (d) Pooled data illustrating the effects of PPT on synaptic transmission in control conditions and after incubation with inhibitors of PI3‐kinase (LY294002; wortmannin) or ERK (PD98059; U0126). PPT‐induced LTP involves a PI 3‐kinase‐driven process. (e) Representative confocal images of surface GluA1 labelling in control neurons and after PPT, PPT plus LY294002 or PPT plus wortmannin (DIV8‐15). (f) Pooled data illustrating the relative effects on GluA1 surface labelling in control conditions, after addition of PPT and in the combined presence of PPT plus LY294002 or PPT plus wortmannin (DIV8‐15). The ability of ERα to increase GluA1 surface expression involves PI 3‐kinase signalling

To verify that similar signalling pathways underlie ERα regulation of AMPAR trafficking, the role of PI 3‐kinase signalling was explored in hippocampal neurons. In control neurons, treatment with PPT (25 nM; 15 min) increased GluA1 surface staining (Figure 4e,f). In parallel studies, addition of LY294002 or wortmannin had no effect on surface GluA1 immunostaining (n = 36 for each) (Figure 4e,f). However, in neurons treated with LY294002 or wortmannin, the ability of PPT to increase GluA1 surface expression was inhibited ( (Figure 4e,f), indicating that PI 3‐kinase signalling is required for PPT‐driven alterations in AMPAR trafficking.

3.7. PPT‐induced LTP occludes HFS‐induced LTP at TA‐CA1 synapses

LTP is induced at TA‐CA1 synapses by HFS and HFS‐induced LTP displays parallels to LTP induced by ERα at TA‐CA1 synapses, as both are NMDAR‐dependent (Aksoy‐Aksel & Manahan‐Vaughan, 2015), expressed postsynaptically and require synaptic insertion of GluA2‐lacking AMPARs (Luo et al., 2015). Thus, to determine whether PPT‐induced LTP and HFS‐induced LTP share similar expression mechanisms, occlusion experiments were performed. Initially, an HFS (100 Hz; 1 s) paradigm was delivered to induce LTP and then PPT (25 nM) was applied, 30 min after LTP induction. HFS induced LTP (Figure 5b), an effect not increased further by subsequent addition of PPT (n = 5). In parallel studies, PPT (25 nM) was initially applied to induce LTP and this was followed by HFS, 35 min after PPT washout. PPT significantly increased synaptic transmission (n = 5), but the magnitude of PPT‐induced LTP was not altered following HFS (Figure 5a). These data indicate that ERα‐induced LTP and activity‐dependent LTP at TA‐CA1 synapses share similar mechanisms of expression.

Figure 5.

Figure 5

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ERα activation is involved in activity‐dependent LTP at TA‐CA1 synapses. (a–e) Plots of pooled data illustrating effects on synaptic transmission at TA‐CA1 synapses. (a) Application of PPT induces LTP, however subsequent delivery of HFS (shown by arrow) failed to increase synaptic strength further. (b) LTP was evoked by HFS, however subsequent addition of PPT failed to alter synaptic transmission. ERα‐induced LTP and activity‐dependent LTP at TA‐CA1 synapses share similar expression mechanisms. (c) In control slices, delivery of HFS induced LTP. (d) In interleaved slices treated with MPP, delivery of HFS failed to induce LTP. (e) HFS fails to induce LTP in slices treated with letrozole. (f) Bar chart of pooled data illustrating the effects of HFS on synaptic transmission in control conditions (black bar) and following treatment with either MPP, PHTPP, or letrozole (grey bars)

3.8. ERα activation plays a role in activity‐dependent LTP

HFS‐induced LTP in young rodents is markedly reduced in the presence of ER antagonist ICI 182 780. Letrozole, which blocks endogenous E2 production by inhibiting P450 aromatase, blocks TBS‐induced LTP (Pettorossi et al., 2013), suggesting that ERs play a role in activity‐dependent synaptic plasticity. To assess if ERs are involved in HFS‐induced LTP at TA‐CA1 synapses, the effects of selective ERα and ERβ antagonists were assessed. Application of either MPP (1 μM) or PHTPP (1 μM) had no effect on basal synaptic transmission (n = 5 for each). In control slices, delivery of HFS increased synaptic transmission (Figure 5c), but in slices treated with MPP (1 μM), HFS failed to induce LTP (Figure 5d) after HFS. Conversely, in slices treated with PHTPP (1 μM), robust LTP was observed after HFS(Figure 5e) and this effect was not significantly different (P > .05) to the magnitude of HFS‐induced LTP in interleaved slices (Figure 5f). These data indicate that activation of ERα, but not ERβ, is required for NMDA‐dependent LTP at TA‐CA1 synapses.

Oestrogens can be produced de novo in neurons as P450 aromatase; the enzyme involved in E2 synthesis is highly expressed in the brain (Hojo et al., 2003). Thus, the involvement of newly synthesised E2 in activity‐dependent synaptic plasticity was examined, using the P450 aromatase inhibitor letrozole (Fester et al., 2016). Application of letrozole (100 nM; 60 min) had no effect on basal synaptic transmission (n = 5). In control slices, delivery of HFS significantly increased synaptic transmission (Figure 5f). However, in slices treated with letrozole (100 nM) for 20 min, subsequent delivery of HFS failed to alter synaptic transmission (Figure 5e,f), an effect significantly different from HFS‐induced LTP in control slices. Additionally, in slices treated with letrozole (100 nM) for 20 min prior to PPT addition, the ability of PPT to induced LTP was not altered, such that application of 25‐nM PPT evoked a significant increase in synaptic transmission (n = 5). These data suggest that endogenous production of E2 is required for activity‐dependent LTP but does not contribute to PPT‐induced LTP at TA‐CA1 synapses.

4. DISCUSSION

It is known that E2 potentiates excitatory synaptic transmission at SC‐CA1 synapses (Smejkalova & Woolley, 2010; Smith & McMahon, 2005; Smith, Vedder, & McMahon, 2009). Previous studies indicate that E2 is capable of regulating adult female TA‐CA1 synapses (Smith et al., 2016), however the acute effects of E2 on juvenile male TA‐CA1 synapses are unclear. Here, we provide the first compelling evidence that E2 activation of ERα induces LTP at TA‐CA1 synapses, an effect mirrored by ERα but not ERβ agonists. ERα‐induced LTP is NMDAR‐dependent and involves a postsynaptic expression mechanism that requires PI 3‐kinase signalling and synaptic insertion of GluA2‐lacking AMPARs (Figure 6). ERα‐induced LTP has overlapping expression mechanisms with classical Hebbian LTP, as HFS‐induced LTP occluded PPT‐induced LTP and vice versa. Activation of ERα was also involved in NMDA‐dependent LTP as antagonism of ERα inhibited HFS‐induced LTP at TA‐CA1 synapses. Endogenous production of E2 is also required for NMDA‐dependent LTP as blockade of E2 synthesis with the aromatase inhibitor, letrozole, blocked HFS‐induced LTP at TA‐CA1 synapses.

Figure 6.

Figure 6

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Activation of ERα induces a novel form of NMDA‐dependent LTP at juvenile TA‐CA1 synapses. Schematic representation of the key cellular mechanisms involved in the novel form of LTP induced by ERα at juvenile TA‐CA1 synapses. Activation of ERα by selective agonists or endogenous E2 results in stimulation of PI 3‐kinase signalling. This in turn increases the synaptic activation of GluN2B‐containing NMDA receptors which ultimately promote insertion of GluA2‐lacking AMPA receptors into hippocampal TA‐CA1 synapses

Here, we show that the ERα agonist, PPT, induces a persistent increase in synaptic efficacy at juvenile male TA‐CA1 synapses. Conversely, the ERβ agonist DPN was without effect. The effects of PPT on synaptic efficacy involve ERα as PPT‐induced LTP was blocked by an ERα antagonist indicating that ERα activation mediates LTP induced by PPT. E2 also induced LTP at TA‐CA1 synapses, and this effect was blocked by MPP. These findings contrast with previous studies as ERβ enhances excitatory synaptic efficacy at SC‐CA1 synapses (Kramár et al., 2009; Oberlander & Woolley, 2016), suggesting that the two inputs to CA1 neurons are differentially regulated by ERs. Recent evidence has identified sex differences in the ER subtype mediating E2‐induced synaptic potentiation (Oberlander & Woolley, 2016). Thus, in addition to distinct ERs regulating synaptic efficacy at the two CA1 synapses, different ERs have distinct regulatory roles in male and female. As our studies were performed in male, an ERα‐independent process may regulate TA‐CA1 synaptic efficacy in female, although this remains to be determined.

Our data indicate that a postsynaptic expression mechanism underlies PPT‐induced LTP as no alteration in PPR was observed after PPT treatment. This contrasts with the ERα‐driven increases in excitatory synaptic transmission at male SC‐CA1 synapses as this involves a presynaptic mechanism (Oberlander & Woolley, 2016). However, a postsynaptic mechanism underlies the ERβ increase in synaptic transmission at male SC‐CA1 synapses (Oberlander & Woolley, 2016). Thus, clear differences also exist in the locus and mechanisms of action of different ERs between SC‐CA1 and TA‐CA1 synapses.

NMDAR activation is critical for LTP induction at SC‐CA1 (Bliss & Collingridge, 1993) and TA‐CA1 (Luo et al., 2015) synapses. NMDAR activation was also required for PPT‐induced LTP as D‐AP5 blocked the effects of PPT on synaptic efficacy. NMDARs composed of different subunits are implicated in different forms of hippocampal synaptic plasticity (Bartlett et al., 2007; Liu et al., 2004) and GluN2B subunits are required for HFS‐induced LTP at TA‐CA1 synapses (McGregor et al., 2018). Similarly, PPT‐induced LTP is prevented following inhibition of GluN2B subunits, not GluN2A subunits, indicating that activation of GluN2B‐containing NMDARs is required for PPT‐induced LTP. In accordance with these findings, GluN2B subunits are also implicated in E2‐mediated regulation of synaptic function at adult female TA‐CA1 synapses (Smith et al., 2016).

Synaptic activation of NMDARs is crucial for LTP induction and synaptic insertion of AMPARs during LTP (Collingridge et al., 2004). In support of a role for AMPAR trafficking, PPT‐induced LTP was blocked by inhibiting GluA2‐lacking AMPARs with Phtx. However, Phtx failed to reverse established PPT‐induced LTP, suggesting that PPT insertion of GluA2‐lacking AMPARs underlies induction but not maintenance of LTP. Previous studies have identified that ERβ regulates GluA1 trafficking (Liu et al., 2008; Srivastava et al., 2010), as ERβ increases hippocampal GluA1 phosphorylation and surface expression (Liu et al., 2008). Reductions in GluA1 surface expression have also been detected following ERβ activation (Srivastava et al., 2010). Conversely, we show that PPT increased synaptic density of GluA1 and in agreement with others (Man et al., 2003) PI 3‐kinase signalling is involved. E2‐driven trafficking of AMPARs involves actin polymerisation (Kramár et al., 2009). As PI 3‐kinase phosphorylates PIP2 into PIP3 and elevated PIP3 levels stimulates actin polymerisation (Dotti, Esteban, & Ledesma, 2014), ERα activation may regulate GluA1 trafficking via PI 3‐kinase‐driven changes in actin dynamics.

Although ERα activation stimulates ERK signalling in hippocampal neurons (Pereira, Bastos, de Souza, Ribeiro, & Pereira, 2014), we found no evidence to support a role for ERK as inhibition of ERK failed to inhibit PPT‐induced LTP. The role of an ERK‐independent pathway in ERα‐induced LTP contrasts with ERK involvement in E2 regulation of SC‐CA1 synapses (Jain, Zhe Huang, & Woolley, 2019; Kumar, Bean, Rani, Jackson, & Foster, 2015; Zadran et al., 2009) and adult female TA‐CA1 synapses (Smith et al., 2016).

The novel form of LTP induced by ERα displays parallels to activity‐dependent hippocampal LTP as synaptic activation of NMDARs and synaptic insertion of GluA2‐lacking AMPARs are required for both. Moreover, ERα‐induced LTP occluded synaptically induced LTP and vice versa, indicating that similar expression mechanisms are involved. Treatment with the ERα antagonist MPP, also prevented HFS‐induced LTP suggesting that ERα activation is crucial for LTP induction at TA‐CA1 synapses. Moreover, inhibition of aromatase activity also blocked HFS‐induced LTP, indicating that endogenous production of E2 is required for activity‐dependent LTP at TA‐CA1 synapses. These findings are consistent with previous studies demonstrating that rodents treated with letrozole display impairments in hippocampal LTP (Grassi et al., 2011; Kretz et al., 2004).

4.1. Physiological significance

Numerous studies support a potential cognitive enhancing role for oestrogens, as variations in oestrogen levels are associated with altered memory function, whereas spatial memory deficits observed in ovariectomised animals are reversed by oestrogen (Foster, Sharrow, Kumar, & Masse, 2003; Frick & Kim, 2018; Holmes, Wide, & Galea, 2002). Treatment with E2 also improves retention in hippocampal memory tasks (Gibbs & Johnson, 2008). Several studies have identified a role for ERβ in memory, as ERβ knockout mice exhibit spatial memory impairments (Liu et al., 2008), whereas ERβ agonists enhance memory performance (Rhodes & Frye, 2006; Rissman, Heck, Leonard, Shupnik, & Gustafsson, 2002). ERα activation is also linked to memory as Erα knockout mice display impairments in hippocampus‐dependent memory (Fugger, Cunningham, Rissman, & Foster, 1998), whereas lentiviral expression of ERα rescues memory impairments in Erα knockout mice (Foster, Rani, Kumar, Cui, & Semple‐Rowland, 2008) and intra‐hippocampal infusions of PPT enhance memory (Boulware, Heisler, & Frick, 2013; Phan, Lancaster, Armstrong, MacLusky, & Choleris, 2011). Increased hippocampal expression of ERα in aged animals is also reported to have beneficial effects on hippocampus‐dependent memory (Bean et al., 2015). As TA‐CA1 synapses are implicated in spatial novelty and formation of episodic memories (Remondes & Schuman, 2004; Stokes et al., 2015), the ability of PPT to induce TA‐CA1 LTP may be important for memory consolidation and episodic memory.

The TA pathway degenerates in early AD (Buxbaum et al., 1998) and deficits in synaptic plasticity at TA‐CA1 synapses occur in rodent models of AD (Booth et al., 2016). The incidence of AD is higher in women and the hypo‐estrogenic state of postmenopausal women is related to AD risk (Barnes et al., 2005). Oestrogens also influence AD pathology, with alterations in ApoE gene expression detected after oestrogen (Srivastava, Bhasin, & Srivastava, 1996) and decreased ERα levels in AD patients (Hu et al., 2003). Consequently, as oestrogens are linked to AD pathology, the ability of ERα to regulate TA‐CA1 synaptic efficacy has important implications for their role in neurodegenerative disorders.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

L.C. performed all the studies and analysed all the data. J.H. designed and supervised the study and drafted the manuscript. All authors took part in correcting the proofs and approved the final manuscript.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Clements L, Harvey J. Activation of oestrogen receptor α induces a novel form of LTP at hippocampal temporoammonic‐CA1 synapses. Br J Pharmacol. 2020;177:642–655. 10.1111/bph.14880

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