G-protein coupled estrogen receptor (GPER1) activation promotes synaptic insertion of AMPA receptors and induction of chemical LTP at hippocampal temporoammonic-CA1 synapses

E2-induced cLTP is blocked by a GPER1 antagonist

We have shown previously that acute exposure to E2 (1 µM; 15 min) induces a novel form of cLTP at juvenile TA-CA1 synapses [19]. As there is growing evidence that E2 is an agonist at GPER1, we assessed the role of GPER1 in E2-mediated cLTP at this synapse, using the GPER1 selective antagonist, G15 [21]. Application of G15 (200 nM, 60 min) had no effect on basal excitatory synaptic transmission (101 ± 2.9% of baseline; n = 4; F[1,3] = 0.188; p > 0.05). In agreement with previous work [19], addition of E2 (1 µM) for 15 min resulted in a rapid potentiation of synaptic transmission (to 145 ± 9.5% of baseline, n = 4; F[1,3] = 22.399; p < 0.01; Fig. 1A). In a subset of G15-treated slices (n = 5 out of 8 slices), application of E2 (1 µM; 15 min) failed to evoke a significant increase in excitatory synaptic transmission (96 ± 1.7% of baseline; n = 5; F[1,4] = 1.909; p > 0.05; Fig. 1B), suggesting possible involvement of GPER1 in mediating the actions of E2 at juvenile TA-CA1 synapses.

Fig. 1figure 1

GPER1 activation results in a persistent increase in excitatory synaptic transmission. Ai, Bi. Representative experiment showing effects of estrogen (1 µM; 15 min) on excitatory synaptic transmission at TA-CA1 synapses in juvenile male hippocampal slices. Application of E2 resulted in a persistent increase in excitatory synaptic transmission (Ai) that was blocked by the GPER1 antagonist, G15 (Bi). At the end of experiments, addition of dopamine (100 µM; 5 min) inhibits synaptic transmission, confirming stimulation of TA input. AiiBii, Plots of pooled data demonstrating that E2 increases synaptic efficacy (Aii) and this effect is blocked by G15 (Bii). 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. C, D Plots of pooled data demonstrating that addition of the GPER1 agonist, G1 (10 nM; 15 min) resulted in a persistent increase in synaptic transmission at TA-CA1 synapses (C) but was without effect at SC-CA1 synapses (D). Note the lack of effect of dopamine at SC-CA1 synapses. E, F Pooled data showing that the effects of G1 are due to activation of GPER1 as G1 effects were blocked by the selective GPER1 antagonists, G15 (200 nM; E) and G36 (1 µM; F)

The GPER1 agonist, G1 induces LTP at TA-CA1 synapses

Previous work has identified that E2 modulates excitatory synaptic transmission at SC-CA1 synapses via activation of GPER1 [48], and our data suggests that activation of GPER1 is involved in cLTP induced by E2 at TA-CA1 synapses. G1 has been shown to selectively bind to GPER1 at low nM concentrations, without significantly interacting with ERα or ERβ [12, 21]. Therefore, to assess the effects of GPER1 activation on excitatory synaptic transmission at juvenile TA-CA1 synapses, various concentrations (range 1–10 nM) of the GPER1 agonist, G1 were examined. Addition of 1 nM G1 (15 min) failed to significantly alter basal synaptic transmission (96 ± 4.7% of baseline; n = 5; F[1,4] = 0.758; p > 0.05; not shown). However, application of 10 nM G1 (15 min) induced a persistent increase in excitatory synaptic transmission (to 129 ± 6.4% of baseline; n = 5; F[1,4] = 11.432; p < 0.01; Fig. 1C). These data indicate that at higher concentrations, G1 induces a novel form of cLTP at juvenile TA-CA1 synapses.

As E2 also potently influences synaptic transmission at SC-CA1 synapses via activation of GPER1 [38], in parallel studies we compared the effects of G1 at juvenile male SC-CA1 synapses. In contrast to the actions at TA-CA1 synapses, addition of 10 nM or 1 µM G1 (15 min), had no effect on basal excitatory synaptic transmission at SC-CA1 synapses, as the magnitude of synaptic transmission was unaltered in the presence of either 10 nM G1 (102 ± 2.1% of baseline, n = 5; F[1,4] = 0.136; p > 0.05) or 1 µM G1 (101 ± 4.3% of baseline; n = 5; F[1,4] = 0.165; p > 0.05; Fig. 1D). These data suggest that the ability of G1 to influence excitatory synaptic transmission is restricted to TA-CA1 synapses, in juvenile male hippocampus.

To further verify involvement of GPER1 at TA-CA1 synapses, the effects of two GPER1-selective antagonists, G15 and G36, were assessed. Application of either G15 (200 nM; 60 min) or G36 (1 µM; 60 min) had no effect on basal synaptic transmission (101 ± 2.9% of baseline; n = 4; F[1,3] = 0.188; p > 0.05, and 94 ± 2.4% of baseline; n = 4; F[1,3] = 0.661; p > 0.05; respectively). In control slices, addition of G1 (10 nM; 15 min) caused a significant increase in synaptic transmission (to 132 ± 7.4% of baseline; n = 5; F[1,4] = 13.484; p < 0.001). However, in slices treated with G15, addition of G1 (10 nM; 15 min) failed to alter synaptic transmission (98 ± 3.9% baseline; n = 5; F[1,4] = 0.166; p > 0.05; Fig. 1E). Similarly, in G36-treated slices, addition of G1 (10 nM; 15 min) failed to alter the magnitude of excitatory synaptic transmission (98 ± 3.6% of baseline; n = 4; F[1,3] = 0.279; p > 0.05; Fig. 1F). Together, these data indicate that the ability of G1 to induce cLTP at juvenile TA-CA1 synapses, requires activation of GPER1.

GPER1-induced LTP has a postsynaptic expression mechanism

In hippocampal neurons, GPER1 is expressed postsynaptically and it interacts with postsynaptic proteins, PSD-95 and SAP97 [1, 67]. GPER1 is also reported to modulate excitatory synaptic transmission at SC-CA1 synapses via a postsynaptic mechanism [48]. However, as GPER1 is also expressed at presynaptic sites [5], it is feasible that GPER1 located at either pre- or postsynaptic sites contributes to LTP induced at TA-CA1 synapses. Thus, to examine the locus of G1-induced cLTP, the effects on paired-pulse facilitation ratio (PPR were assessed, as changes in PPR likely indicate presynaptic alterations in neurotransmitter release probability. Application of G1 (10 nM, 15 min induced a significant increase in excitatory synaptic transmission (139 ± 9.2% of baseline; n = 4; F[1,3] = 9.893; p < 0.01; Fig. 2A); an effect not accompanied by any significant change in PPR (from 1.60 ± 0.1% to 1.66 ± 0.1% of baseline; n = 4; F[1,3] = 0.012; p > 0.05; Fig. 2B), suggesting likely involvement of a postsynaptic mechanism. At TA-CA1 synapses, dopamine depresses synaptic transmission via a presynaptic mechanism [49]. In accordance with this, application of dopamine not only depressed synaptic transmission, but this effect was accompanied by significant changes in PPR (from 1.6 ± 0.1% to 2.5 ± 0.2%, n = 4; F[1,3] = 19.600; p < 0.001; Fig. 2B). These data indicate that GPER1-induced cLTP at TA-CA1 synapses is likely to involve a postsynaptic expression mechanism.

Fig. 2figure 2

G1-induced cLTP involves a postsynaptic expression mechanism, and is dependent on NMDA, but not GABA, receptors. A Plot of pooled data illustrating the effects of 10 nM G1 (15 min) on synaptic transmission at TA-CA1 synapses. B Corresponding plot of the pooled paired pulse ratio (PPR) against time for the experiments shown in A. The effects of G1 were not associated with any change in PPR, indicating a postsynaptic expression mechanism. CF Plots of pooled data illustrating the effects of G1 (10 nM; 15 min) on synaptic transmission in juvenile hippocampal slices. Activation of NMDA receptors was involved as G1 failed to increase synaptic efficacy in the presence of the NMDA receptor antagonist, D-AP5 (50 µM; C). D, E The ability of G1 to induce cLTP was blocked by inhibition of GluN2A subunits with NVP-AAM0077 (D), but was not altered by the GluN2B antagonist, ifenprodil (E). F G1-induced cLTP was independent of GABAergic inhibition as the effects of G1 were unaffected in the presence of GABAA and GABAB receptor antagonists, picrotoxin and CGP55845, respectively

Activation of GluN2A-containing NMDA receptors is required for GPER1-induced LTP

Synaptic activation of NMDA receptors is required for activity-dependent LTP at many central synapses and for E2-induced LTP at SC-CA1 synapses [59, 60]. Similarly, at adult TA-CA1 synapses, E2-induced LTP involves NMDA receptors [61] whereas ERα induces a novel form of NMDA-dependent form of LTP at juvenile TA-CA1 synapses [19]. Therefore, to assess the potential involvement of NMDA receptors, the effects of the competitive NMDA receptor antagonist, D-AP5 were evaluated. Treatment of slices with D-AP5 (50 µM, 60 min) had no significant effect on basal synaptic transmission (102 ± 0.9% of baseline; n = 5; F[1,4] = 1.217; p > 0.05). In control slices, application of G1 (10 nM; 15 min) induced robust cLTP, as the magnitude of excitatory synaptic transmission was significantly increased (to 144 ± 5.4% of baseline; n = 5; F[1,4] = 25.725; p < 0.001). In contrast, with D-AP5-treated slices, no significant change in excitatory synaptic transmission was detected following addition of G1 (95 ± 3.3% of baseline; n = 5; F[1,4] = 0.017; p > 0.05; Fig. 2C). These data indicate that NMDA receptor activation is required for GPER1-mediated cLTP at juvenile TA-CA1 synapses.

GluN2 subunits control the biophysical and pharmacological characteristics of NMDA receptors [50], and distinct GluN2 subunits are implicated in activity-dependent synaptic plasticity at different stages of development and at distinct synapses [9, 44]. Previous studies have identified a role for GluN2B in ERα-induced LTP at juvenile TA-CA1 synapses [19], and in E2-induced LTP at adult TA-CA1 synapses [61]. Thus, the role of distinct GluN2 subunits in GPER1-induced cLTP was examined using subunit-selective NMDA receptor antagonists. Application of the putative GluN2A antagonist, NVP-AAM077 (100 nM, 60 min) or GluN2B antagonist, ifenprodil (3 µM; 60 min) had no effect on basal synaptic transmission (NVP-AAM077: 99 ± 3.7% of baseline; n = 5; F[1,4] = 0.386; p > 0.05; Ifenprodil: 99 ± 1.1% of baseline; n = 5; F[1,4] = 0.137; p > 0.05), respectively. In control slices, G1 (10 nM; 15 min) induced robust cLTP as synaptic transmission was significantly increased (to 144 ± 8.4% of baseline; n = 5; F[1,4] = 17.136; p < 0.01). However, in NVP-treated slices (100 nM; 90 min), application of G1 (10 nM; 15 min) failed to alter synaptic transmission (92 ± 5.0% of baseline; n = 4; F[1,3] = 0.449; p > 0.05; Fig. 2D). In contrast, with slices treated with ifenprodil (3 µM; 90 min), a significant increase in excitatory synaptic transmission was observed after addition of G1 (to 144 ± 5.2% of baseline; n = 4; F[1,3] = 36.040; p < 0.001; Fig. 2E). Collectively, these data indicate that GPER1-mediated cLTP at TA-CA1 synapses involves activation of GluN2A-containing NMDA receptors.

GPER1-mediated LTP occurs independently of GABAergic inhibition

As GPER1 has been detected on hippocampal GABAergic interneurons [7] and stimulation of the TA input can activate GABAergic interneurons [22], it is feasible that GABAergic synaptic transmission plays a role. To address this possibility, the effects of blocking GABAA and GABAB receptors using selective antagonists, namely picrotoxin and CGP55845, were evaluated. Co-application of picrotoxin (50 µM; 60 min) and CGP55845 (100 nM; 60 min) had no effect on basal synaptic transmission (100 ± 3.3% of baseline, n = 5; F[1,4] = 0.792; p > 0.05). In line with previous findings, addition of G1 (10 nM; 15 min) evoked a significant increase in excitatory synaptic transmission (to 139 ± 4.35% of baseline, n = 5; F[1,4] = 62.099; p < 0.001). Similarly, in slices treated with both GABA receptor antagonists, G1 induced an increase in excitatory synaptic transmission (to 150 ± 13.6% of baseline, n = 5; F[1,4] = 17.544; p < 0.001; Fig. 2F); an effect not significantly different to the magnitude of G1-induced cLTP in control slices (n = 5; F[1,4] = 0.688; p > 0.05). These data indicate that GPER1-induced cLTP at TA-CA1 synapses is likely to be independent of GABA receptors.

G1-induced LTP involves the insertion of GluA2-lacking AMPA receptors

Activity dependent changes in excitatory synaptic strength involves trafficking of AMPA receptors to and from synapses [20], and synaptic insertion of GluA2-lacking AMPA receptors contributes to NMDA-dependent LTP evoked at juvenile SC-CA1 synapses [52]. Synaptic insertion of GluA2-lacking AMPA receptors is also implicated in activity-dependent LTP [41] and ERα-induced LTP [19] at TA-CA1 synapses. Therefore, the role of GluA2-lacking AMPA receptors in G1-induced cLTP was examined using philanthotoxin to inhibit GluA2-lacking AMPA receptors. In control slices, philanthotoxin (1 µM, 60 min had no effect on basal synaptic transmission (97 ± 1.2% of baseline n = 5; F[1,4] = 0.556; p > 0.05). Addition of G1 alone (10 nM; 15 min) evoked a significant increase in excitatory synaptic transmission (to 138 ± 9.0% of baseline; n = 4; F[1,3] = 16.772; p < 0.001; Fig. 3A). However, in slices pre-treated with philanthotoxin (1 µM; 90 min), G1 failed to induce cLTP (103 ± 6.0% of baseline; n = 5; F[1,4] = 16.772; p > 0.05; Fig. 3B). In contrast, application of philanthotoxin, 20 min after the addition of G1, did not influence the magnitude of G1-induced cLTP (146 ± 8.8% of baseline; n = 6; F[1,5] = 24.865; p < 0.001). Similarly, application of philanthotoxin, 60 min after G1, had no effect on the magnitude of G1-induced cLTP (131 ± 9.4% of baseline; n = 4; F[1,3] = 7.816; p < 0.01; Fig. 3C). Together these data suggest that insertion of GluA2-lacking AMPA receptors is involved in the induction phase of GPER1-induced cLTP at TA-CA1 synapses.

Fig. 3figure 3

GPER1-induced cLTP involves synaptic insertion of GluA2-lacking AMPA receptors. AC Plots of pooled data illustrating the effects on synaptic transmission at TA-CA1 synapses in juvenile hippocampal slices. A Application of G1 resulted in induction of cLTP, but prior exposure to philanthotoxin (Phtx; 1 µM; B) blocked G1-induced cLTP. C Application of philanthotoxin, 30 min after G1 failed to reverse the increase in synaptic efficacy induced by G1. Di Representative confocal images of surface GluA1 labelling in control hippocampal neurons (8–12 DIV) and after addition of G1, G15 and G1 plus G15. Dii Pooled data illustrating the relative effects on surface GluA1 immunolabelling in control neurons and after G1, G15, and G1 plus G15. G1 increases surface GluA1 expression via activation of GPER1. Ei Representative confocal images of surface GluA1 (green) and PSD-95 (red) labelling in control and G1 treated (DIV 8–13) neurons. G1 increases surface GluA1 that co-localises with PSD-95, indicating increased postsynaptic levels of GluA1. Eii, iii Pooled data indicating relative intensity of GluA1 (Eii) and % GluA1-PSD-95 co-localisation (Eiii) in control conditions and after G1 application. G1 increased GluA1 expression at hippocampal synapses

G1 increases synaptic insertion of GluA1-containing AMPA receptors

As our data suggest that GPER1 activation influences AMPA receptor trafficking to synapses, immunocytochemical techniques were performed to directly assay the cell surface density of AMPA receptors using an antibody against the AMPA receptor subunit, GluA1 on hippocampal neurons [47]. Exposure of hippocampal neurons to G1 (10 nM) for 15 min increased GluA1 surface immunolabelling (to 136 ± 2.2% of control, n = 36; F[1,71] = 64.780; p < 0.001; Fig. 3D). To verify the involvement of GPER1, the effects of the GPER1 antagonist, G15 were evaluated. Treatment with G15 (200 nM; 15 min) alone, had no significant effect on GluA1 surface staining per se (94 ± 4.7% of control; n = 36; F[1,71] = 1.048; p > 0.05; Fig. 3D). However, in G15-treated neurons, the effects of G1 were abolished as no significant change in GluA1 surface immunolabelling was detected (105 ± 2.9% of control; n = 36; F[1,72] = 0.708; p > 0.05; Fig. 3D). These data indicate that the G1-induced increase in GluA1 surface expression requires GPER1 activation.

As AMPA receptor density at synapses is a key determinant of excitatory synaptic strength, we examined the effects of G1 on synaptic AMPA receptors by comparing the degree of co-localisation between surface GluA1 and the synaptic marker, PSD-95 [45]. Exposure of neurons to G1 (10 nM, 15 min), significantly increased GluA1 surface expression (to 165 ± 8.6% of control; n = 36; F[1,71] = 43.358; p < 0.001; Fig. 3E); an effect accompanied by an increase in % co-localisation between surface GluA1 and PSD-95 from 46 ± 1.3% to 76 ± 1.3% (n = 36; F[1,71] = 263.005; p < 0.001; Fig. 3E). Following G1 treatment, PSD-95 labelling was not significantly different to control neurons (104 ± 6.5% of control; n = 36; F[1,71] = 0.263; p > 0.05), indicating that G1 does not alter synapse density per se. These data indicate that GPER1 activation increases the density of GluA1-containing AMPA receptors at hippocampal synapses.

GPER1-driven AMPA receptor insertion requires NMDA receptor activation

As NMDA receptor activation can drive the movement of AMPA receptors to synapses [20], the role of NMDA receptors was explored using the competitive NMDA receptor antagonist, D-AP5. Treatment of neurons with G1 (10 nM, 15 min) significantly increased GluA1 surface immunolabelling (to 206 ± 7.6% of control; n = 36; F[1,71] = 140.971; p < 0.001; Fig. 4A, B). Application of D-AP5 (50 μM; 15 min) alone had no effect on GluA1 surface staining (100 ± 5.4% of control; n = 36; F[1,71] = 0.013; p > 0.05), however, in D-AP5-treated neurons, G1 (10 nM; 15 min) failed to significantly alter GluA1 surface expression (106 ± 4.5% of control; n = 36; F[1,71] = 0.860; p > 0.05; Fig. 4A, B). These data indicate that NMDA receptor activation is required for GPER1-driven increase in AMPA receptor trafficking.

Fig. 4figure 4

ERK signalling underlies GPER1-induced cLTP and synaptic insertion of GluA1. A Representative confocal images of surface GluA1 labelling in hippocampal neurons (DIV 8–12) in control conditions and after G1, D-AP5, and G1 plus D-AP5. NMDA receptor activation is required for G1 induced trafficking of GluA1-containing AMPA receptors. B Pooled data showing the relative effects on surface GluA1 labelling in control neurons and after exposure to G1, D-AP5 and G1 plus D-AP5. C, D Pooled data illustrating the effects on excitatory synaptic transmission in hippocampal slices. The ability of G1 to induce cLTP was blocked after ERK inhibition with PD98059 (C) but was unaffected in the presence of the PI3K inhibitor, LY294002 (D). E Representative confocal images of surface GluA1 labelling in hippocampal neurons (DIV 7–13) in control conditions, after G1, and in the combined presence of G1 and either PD98059 or U0126. F Pooled data showing the relative effects on surface GluA1 labelling in control neurons and after addition of G1, PD98059, U0126 or in the combined presence of G1 plus either PD98059 or LY294002. The G1-induced increase in GluA1 expression requires ERK signalling

GPER1-induced LTP involves ERK signalling but is independent of PI3K

As GPER1 can couple to a wide variety of signalling pathways, including ERK and PI 3K [23, 56], we investigated whether GPER1-induced cLTP at TA-CA1 synapses might involve one or more of these signalling pathways. To determine a potential role of ERK, the effects of two different inhibitors of MAPK activation, PD98059 and UO126, were investigated. Application of either PD98059 (10 µM, 60 min) or U0126 (10 µM; 60 min) had no effect on basal synaptic transmission (100 ± 1.0% of baseline; n = 5; F[1,4] = 2.319; p > 0.05; and 99 ± 1.4% of baseline; n = 5; F[1,4] = 0.143; p > 0.05; respectively). As before, application of G1 (10 nM; 15 min) readily induced LTP as synaptic transmission was significantly increased (to 147 ± 12.0% of baseline; n = 5; F[1,4] = 12.688; p < 0.001). However, in slices treated with either PD98059 or U0126, G1 failed to significantly increase synaptic transmission (PD98059; 95 ± 4.0% of baseline; n = 4; F[1,3] = 1.862; p > 0.05; Fig. 4C: U0126; 99 ± 1.4% of baseline; n = 4; F[1,3] = 0.143; p > 0.05), suggesting involvement of ERK signalling in the effects of G1.

To explore the potential involvement of PI3K signalling, the effects of the PI3K inhibitor LY294002, were also examined. Application of LY294002 (10 µM; 60 min) had no significant effect on basal synaptic transmission (101 ± 4.8% of baseline; n = 5; F[1,4] = 3.845; p > 0.05). Moreover, in interleaved slices treated with LY294002 (10 µM; 90 min), subsequent addition of G1 resulted in a persistent increase in synaptic transmission (to 133 ± 11.3% of baseline; n = 5; F[1,4] = 33.041; p < 0.01; Fig. 4D). Together these data indicate that GPER1-induced cLTP involves stimulation of ERK, but not PI3K, signalling.

ERK signalling is required for GPER1-driven insertion of AMPA receptors in hippocampal neurons

To verify if ERK signalling also underlies GPER1-driven AMPA receptor trafficking, the effects of two ERK inhibitors were examined. Treatment of hippocampal neurons with either PD98059 (10 µM; 15 min) or UO126 (10 µM; 15 min) had no effect on surface GluA1 staining per se (PD98059: 108 ± 5.3% of control; n = 36; F[1,71] = 1.989; p > 0.05; U0126: 105 ± 4.39% of control; n = 36; F[1,71] = 0.837; p > 0.05; Fig. 4F). In control neurons treated with G1 (10 nM; 15 min), GluA1 surface immunolabelling was significantly increased (to 178 ± 8.3% of control; n = 36; F[1,71] = 67.275; p < 0.001; Fig. 4E, F). However, in PD98059-treated neurons, G1 failed to alter GluA1 immunolabelling (107 ± 3.3% of control; n = 36; F[1,71] = 1.715; p > 0.05; Fig. 4E, F). Similarly, in neurons treated with UO126 the ability of G1 (10 nM; 15 min) to increase surface GluA1 immunolabelling was inhibited (108 ± 4.7% of control; n = 36; F[1,71] = 1.522; p > 0.05; Fig. 4E, F). Together, these data indicate that ERK signalling mediates GPER1-driven changes in AMPA receptor trafficking.

GPER1-induced LTP occludes HFS-induced LTP at TA-CA1 synapses

Several studies have determined that activity-dependent LTP at TA-CA1 synapses is NMDA receptor dependent [2, 27, 54, 55] and requires ERK signalling and synaptic insertion of GluA2-lacking AMPA receptors [41]. Thus, analogous cellular mechanisms underlie activity-dependent LTP and GPER1-mediated cLTP at TA-CA1 synapses and consequently both processes may share similar expression mechanisms. To verify if overlapping expression mechanisms play a role, occlusion experiments were performed. In the first experiments, a HFS paradigm (100 Hz, 1 s) was applied to induce LTP and 30 min later slices were treated with G1 (10 nM; 15 min). Delivery of HFS readily increased the magnitude of synaptic transmission (to 135 ± 9.3% of baseline; n = 4; F[1,3] = 18.003; p < 0.001), but subsequent application of G1 had no further effect on synaptic transmission (133 ± 8.1% of baseline; n = 4; F[1,3] = 14.782; p < 0.001; Fig. 5A), indicating that HFS-induced LTP occludes G1-induced cLTP. In parallel studies, the reverse experiment was carried out, such that G1 (10 nM; 15 min) was initially applied to slices which significantly increased excitatory synaptic transmission (to 134 ± 9.1% of baseline; n = 5; F[1,4] = 12.894; p < 0.001; Fig. 5B). Subsequent delivery of HFS failed to alter the magnitude of G1-induced cLTP (132 ± 6.8% of baseline; n = 5; F[1,4] = 18.902; p < 0.001; Fig. 5B), indicating that GPER1-induced cLTP also occludes HFS-induced LTP at TA-CA1 synapses. These data demonstrate that HFS-induced LTP and GPER1-induced cLTP at TA-CA1 synapses occlude one another and are likely to have comparable expression mechanisms.

Fig. 5figure 5

GPER1 is involved in activity-dependent LTP at TA-CA1 synapses. AD Plots of pooled data illustrating effects on synaptic transmission at juvenile TA-CA1 synapses. A Delivery of HFS (shown by arrow) resulted in the induction of LTP, however subsequent addition of G1 failed to alter synaptic transmission. B Application of G1 induced cLTP, but subsequent delivery of HFS failed to increase synaptic transmission further. Activity-dependent LTP and GPER1-induced LTP share analogous expression mechanisms. C In control slices, HFS readily induced LTP. D In interleaved slices exposed to the GPER1 antagonist, G15 (200 nM), delivery of HFS failed to induce LTP. GPER1 activation is involved in activity-dependent LTP

ER antagonists have been shown to significantly reduce or abolish hippocampal LTP [28, 30] and a specific role for ERα in activity-dependent LTP at both SC-CA1 and TA-CA1 synapses has been demonstrated [19, 66]. Given that our data indicates that GPER1 modulates synaptic efficacy at TA-CA1 synapses, it is feasible that GPER1 plays a role in activity-dependent TA-CA1 LTP. Thus, to assess this possibility, the effects of the GPER1-selective antagonist G15 on HFS-induced LTP were examined. Application of G15 alone (200 nM, 60 min) failed to alter basal synaptic transmission (n = 4; p > 0.05). In control slices, delivery of HFS paradigm (100 Hz; 1 s) resulted in LTP induction as excitatory synaptic transmission was significantly increased (to 160 ± 15.3% of baseline; n = 5; F[1,4] = 15.548; p < 0.001; Fig. 5C). However, in the presence of G15 (200 nM; 80 min), HFS failed to significantly increase synaptic transmission above basal levels (95 ± 3.2% of baseline; n = 5; F[1,4] = 1.572; p > 0.05; Fig. 5D). These data suggest that activation of GPER1 contributes to the ind

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