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. 2018 Sep 12;38(37):7935-7951.
doi: 10.1523/JNEUROSCI.0801-18.2018. Epub 2018 Aug 1.

Memory-Related Synaptic Plasticity Is Sexually Dimorphic in Rodent Hippocampus

Weisheng Wang  1 , Aliza A Le  1 , Bowen Hou  1 , Julie C Lauterborn  1 , Conor D Cox  1 , Ellis R Levin  2   3 , Gary Lynch  4   5 , Christine M Gall  4   6
Affiliations

Memory-Related Synaptic Plasticity Is Sexually Dimorphic in Rodent Hippocampus

Weisheng Wang et al. J Neurosci. .

Abstract

Men are generally superior to women in remembering spatial relationships, whereas the reverse holds for semantic information, but the neurobiological bases for these differences are not understood. Here we describe striking sexual dimorphism in synaptic mechanisms of memory encoding in hippocampal field CA1, a region critical for spatial learning. Studies of acute hippocampal slices from adult rats and mice show that for excitatory Schaffer-commissural projections, the memory-related long-term potentiation (LTP) effect depends upon endogenous estrogen and membrane estrogen receptor α (ERα) in females but not in males; there was no evident involvement of nuclear ERα in females, or of ERβ or GPER1 (G-protein-coupled estrogen receptor 1) in either sex. Quantitative immunofluorescence showed that stimulation-induced activation of two LTP-related kinases (Src, ERK1/2), and of postsynaptic TrkB, required ERα in females only, and that postsynaptic ERα levels are higher in females than in males. Several downstream signaling events involved in LTP were comparable between the sexes. In contrast to endogenous estrogen effects, infused estradiol facilitated LTP and synaptic signaling in females via both ERα and ERβ. The estrogen dependence of LTP in females was associated with a higher threshold for both inducing potentiation and acquiring spatial information. These results indicate that the observed sexual dimorphism in hippocampal LTP reflects differences in synaptic kinase activation, including both a weaker association with NMDA receptors and a greater ERα-mediated kinase activation in response to locally produced estrogen in females. We propose that male/female differences in mechanisms and threshold for field CA1 LTP contribute to differences in encoding specific types of memories.SIGNIFICANCE STATEMENT There is good evidence for male/female differences in memory-related cognitive function, but the neurobiological basis for this sexual dimorphism is not understood. Here we describe sex differences in synaptic function in a brain area that is critical for learning spatial cues. Our results show that female rodents have higher synaptic levels of estrogen receptor α (ERα) and, in contrast to males, require membrane ERα for the activation of signaling kinases that support long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning. The additional requirement of estrogen signaling in females resulted in a higher threshold for both LTP and hippocampal field CA1-dependent spatial learning. These results describe a synaptic basis for sexual dimorphism in encoding spatial information.

Keywords: LTP; TrkB; estrogen; estrogen receptor alpha; long-term potentiation; object location memory.

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Figures

Figure 1.
Figure 1.
Electrode and image sample field placement in CA1 SR. A, B, Images show at low-magnification, DAPI-labeled cellular nuclei (A) and immunofluorescence (B) for PSD-95 in the same CA1 field of a hippocampal slice to illustrate the position of the stimulating electrodes (arrows indicate areas of visible electrode damage) and of image z-stack collection (rectangle drawn to the scale of the sample field). In this case, the electrodes were lowered deep within the tissue to create visible damage for illustration purposes. SP, Stratum Pyramidale; SR, Stratum Radiatum. Scale bar: A (for A, B), 200 μm.
Figure 2.
Figure 2.
Local estrogen promotes LTP via membrane ERα in female hippocampus. LTP was induced by TBS of S-C projections, and fEPSPs were recorded from CA1 SR; in this and subsequent illustrations, periods of reagent infusion are indicated by a horizontal gray line on the fEPSP plot. A, Left, In vivo formestane pretreatment severely impaired LTP in slices from female rats (p = 0.0026, t(10) = 3.98 for veh vs formestane during the last 5 min of recordings; n = 6/group). Right, E2 (1 nm) perfusion initiated 30 min before TBS rescued LTP in slices from formestane-pretreated females (p = 0.017, t(17) = 2.65; formestane, n = 6; formestane + E2, n = 13). B, ERα antagonist MPP blocked LTP in female slices (left; p < 0.0001, t(26) = 5.55; n = 14/group) but not male slices (right; p = 0.87, t(16) = 0.16, n = 9/group). C, D, Neither ERβ antagonist PHTPP (C; p = 0.70, t(10) = 0.39; n = 6/group) nor GPER1 antagonist G15 (D; p = 0.84, t(17) = 0.21; veh, n = 10; G15, n = 9) influenced S-C LTP in females. E, The size (area) of the first theta burst response was not different among female, female + MPP, or male slices (p = 0.20, F(2,26) = 1.70, one-way ANOVA; male, n = 7; other groups, n = 10). F, MPP did not influence the theta burst response enhancement between the first and second burst in females (p = 0.77, F(1,18) = 0.09, two-way ANOVA; n = 10/group). G, The fiber volley-to-fEPSP amplitude relationship (I/O curve) for female NOER mice was comparable to that of female WTs (p = 0.61, F(9,90) = 0.81, two-way ANOVA; n = 6/group) but S-C LTP failed to stabilize (p = 0.0006, t(14) = 4.39 vs WT; WT, n = 6; NOER, n = 10). H, For female MOER mice, both the I/O curve (p = 0.21, F(8,144) = 1.37, two-way RM ANOVA; WT, n = 10; MOER, n = 12) and S-C LTP (inset bar graph; LTP expressed as percentage baseline, p = 0.99, t(20) = 0.01; WT, n = 10; MOER, n = 12) were comparable to measures from female WTs. I, For male NOER mice, both the I/O curve (p = 0.93, F(7,77) = 0.36, two-way ANOVA; WT, n = 7; NOER, n = 6) and S-C LTP (inset bar graph, p = 0.77, t(23) = 0.30; WT, n = 9; NOER, n = 16) were comparable to measures from male WTs.
Figure 3.
Figure 3.
TBS-driven increases in synaptic pSrc and pERK are ERα dependent in females. Fluorescence deconvolution tomography was used to assess the effects of S-C stimulation on synaptic immunolabeling at 3–4 min post-TBS. Line graphs show immunolabeling density frequency distributions for the phosphoprotein at all double-labeled (phosphoprotein-IR + PSD-95-IR) synapses (see Materials and Methods); treatment effects on frequency distributions were assessed using two-way RM ANOVA. Bar graphs show the proportion of double-labeled synapses containing dense immunolabeling for the phosphoprotein (≥90 density units) with group mean values normalized to the mean for control (LFS) slices. A, Deconvolved images show punctate localization of PSD-95-IR (green) in combination with that for pSrc (red, left) and pERK1/2 (red, right); yellow indicates double-labeled elements (arrows). Scale bar: large image, 10 μm; inset, 2 μm. B, In slices from males, TBS caused a greater rightward skew in the pSrc density frequency distribution (thus, an increase in the proportion of synapses with dense pSrc immunoreactivity) compared with the curve for slices receiving LFS (p < 0.0001, F(19,323) = 5.348; LFS, n = 9; TBS, n = 10; TBS + MPP, n = 10); this effect was not influenced by ERα antagonist MPP (p = 0.917, F(19,342) = 0.585). Bar graph shows that in males TBS increased the numbers of PSD-95-IR synapses with dense pSrc immunolabeling relative to measures from slices receiving LFS and that this effect was not altered by MPP (p = 0.0135, F(2,28) = 5.11, Bonferroni's test for post hoc comparisons; LFS vs TBS, *p < 0.05; LFS vs TBS + MPP, *p < 0.05; TBS vs TBS + MPP, n.s.). C, In females, TBS also caused a greater rightward skew in the pSrc immunolabeling density frequency distribution relative to that for LFS slices (left; p < 0.0001, F(19,342) = 24.56; n = 10/group); MPP substantially reduced this effect (p < 0.0001, F(19,342) = 5.981). The bar graph shows that in females the proportion of PSDs with dense pSrc-IR was increased by TBS and that this increase was substantially reduced by MPP (p = 0.0002, F(2,29) = 12.33; post-test: LFS vs TBS, p < 0.001; TBS vs TBS + MPP, #p < 0.02; LFS vs TBS + MPP, n.s.). D, TBS caused a greater rightward skew in the density frequency distribution for synaptic pERK-IR in female slices (p < 0.0001, F(19,361) = 34.62; LFS, n = 10; TBS, n = 11; TBS + MPP, n = 8); MPP substantially reduced this effect (p < 0.0001, F(19,323) = 10.01); bar graph shows that the TBS-driven increase in the numbers of densely pERK-IR synapses was similar in magnitude to that for pSrc and attenuated by MPP (p < 0.0001, F(2,28) = 19.68; LFS vs TBS, ***p < 0.0001; TBS vs TBS + MPP, ##p < 0.01).
Figure 4.
Figure 4.
E2 infusion increases synaptic ERK1/2 and Src phosphorylation. A, E2 perfusion alone increased the percentage of synapses with dense concentrations of synaptic pERK1/2-IR in female slices relative to veh treatment; the effect was reduced by MPP (p < 0.0001, F(2,35) = 26.21; post hoc tests: veh vs E2, ***p < 0.0001; E2 vs E2 + MPP, ###p < 0.001; n = 10/group; quantitative FDT analysis). B, Additional experiments confirmed that E2 increased synaptic pERK1/2-IR and further showed that the ERβ antagonist PHTPP reduced the effect (p = 0.0002, F(2,28) = 12.27; post hoc tests: veh vs E2, *p < 0.05; E2 vs E2 + PHTPP, #p < 0.05; veh, n = 10; E2 and E2 + PHTPP. n = 11). C, E2 increased the rightward skew in the density frequency distribution for pSrc-IR colocalized with PSD-95 (vs vehicle, p < 0.0001, F(19,418) = 33.42); MPP blocked most of this effect (p < 0.0001, F(19,418) = 12.32; n = 12/group). D, E2 treatment of female slices produced the predicted, MPP-sensitive increase in the proportion of doubled-labeled synapses with dense pSrc-IR relative to vehicle controls (p = 0.0002, F(2,29) = 12.33; Bonferroni's post-test: veh vs E2, ***p < 0.0001; E2 vs E2 + MPP, #p < 0.05; n = 12/group). E, In male slices, E2 infusion increased the percentage of synapses with dense pERK1/2-IR, but the increase was not affected by MPP (p < 0.0001, F(2,29) = 37.51; post hoc tests: veh vs E2, ***p < 0.001; E2 vs E2 + MPP, #p > 0.05; n = 10/group). F, Separate experiments replicated the E2-induced increase in synaptic pERK1/2 in male slices and determined that this was suppressed in the presence of PHTPP (p = 0.0002, F(2,26) = 12.27; post hoc tests: veh vs E2, ***p = 0.0001; E2 vs E2 + PHTPP, ##p = 0.01; veh and E2, n = 10; E2 + PHTPP, n = 9).
Figure 5.
Figure 5.
ERα is present at higher concentrations in female than in male CA1 synapses. A, Top left, Deconvolved two-photon microscopic images of immunofluorescent labeling were used to construct a 3D montage of the CA1 sample field (shown); one can see that a subpopulation of PSD-95-IR contacts (green) also contains ERα immunoreactivity (double labeling appears yellow). Scale bar, 2 μm. Top right, Image of a single double-labeled PSD shows the spatial relationship of areas occupied by PSD-95 (green) and ERα (red) immunoreactivities and the extent of overlap (merge, yellow). Scale bar, 0.1 μm. Bottom, Image shows the montage from the same z-stack illustrated in the top left but with the top of that panel rotated away from the viewer to show double labeling of the same puncta (arrows) from a different 3D viewpoint. B, The density frequency distribution for ERα-IR (colocalized with PSD-95) shows a greater rightward skew in females relative to males (p < 0.0001, F(19,646) = 17.28; males, n = 12; female, n = 24). C, Bar graph shows the percentage of double-labeled synapses with high concentrations of ERα immunolabeling (density units of ≥90) normalized to the mean male value shows that there were far more dense ERα-IR synapses in females than in males (***p = 0.0001, t(34) = 4.40). D, E, Density frequency distributions for all synapse-sized clusters of ERβ (D) and GPER1 (E) immunoreactivities colocalized with PSD-95 in the CA1 SR sample field (n = 12/group, females in diestrus). For ERβ-IR (D), there was a significant interaction between sex and immunolabeling density (p = 0.0005, F(21,462) = 2.4) because of a slightly greater proportion of synapses with low-density ERβ-IR (density units 83–88) in males than in females. There was no effect of sex on the numbers of densely ERβ-IR synapses (density units, ≥90; p = 0.12; t(22) = 1.6). For GPER1-IR, there were no group differences (GPER1: p = 0.89; F(20,440) = 0.33). F, Deconvolved epifluorescence images shows that ERβ and GPER1 (red) are both localized to synapse-sized puncta in CA1 SR and that some of those are colocalized with PSD-95 (green; doubles appear yellow). Scale bar, 2 μm.
Figure 6.
Figure 6.
Female S-C LTP depends on an RGD-binding β1 integrin. A, Image shows dual immunolabeling for Act-β1 integrin (red) and PSD-95 (green) in CA1 SR; inset shows the area surrounding a double-labeled PSD (arrow) at higher magnification. Scale bars: A, D, large image, 10 μm; A, D, inset, 2 μm. B, Plot shows the immunolabeling density frequency distributions for Act-β1-IR colocalized with PSD-95 in CA1 SR in female slices that received LFS or TBS of S-C projections; with TBS, compared with LFS, there was a greater rightward skew in the Act-β1 density frequency distribution, indicating an increase in the proportion of synapses with dense Act-β1-IR (p < 0.0001; F(21,882) = 7.91; n = 22/group). C, The percentage of double-labeled PSDs with dense Act-β1-IR (>90 units on B), normalized to the mean of the LFS control group, showed that TBS increased the numbers of Act-β1-enriched synapses (**p = 0.009, t(42) = 2.75). D, Deconvolved image shows colocalization of pFAK (red) and PSD-95 (green) immunoreactivities; double labeling appears yellow (arrows); inset shows the area surrounding a double-labeled PSD (arrow) at higher magnification. E, TBS increased the rightward skew in the density frequency distribution for synaptic (PSD-95 colocalized) pFAK-IR (p < 0.0001, F(19,323) = 6.294) that was largely eliminated by ERα antagonist MPP (p < 0.0001, F(19,285) = 7.514; LFS, n = 10; TBS, n = 9; TBS + MPP, n = 8). F, The percentage of double-labeled synapses with high concentrations of pFAK (>90 units), normalized to LFS control slices, was increased by TBS; MPP blocked this pFAK increase (p = 0.0037, F(2,26) = 7.13; Bonferroni's post-test: LFS vs TBS, **p < 0.01; TBS vs TBS + MPP, #p < 0.05). G, The fiber volley amplitude vs fEPSP amplitude relationship (I/O curve) for female wild-type and cKO mice were comparable (p = 1.0, F(7,126) = 0.01; n = 9/group). H, Plot of fEPSP slopes (expressed as a percentage of the mean baseline response) shows that in female mice TBS (applied at 20 min) induced robust LTP in wild types, whereas, in β1 cKOs, potentiation declined toward baseline over 60 min. I, The percentage of LTP (expressed as the percentage at baseline) measured at 55–60 min post-TBS was greatly reduced in female β1 cKO mice relative to WT mice. A similarly pronounced suppression of female LTP was produced by the RGD-binding β1 integrin blocker echistatin (ECH) infused for 40 min before TBS (p < 0.0001, F(3,27) = 24.01; post hoc tests: WT vs cKO and veh vs ECH, ***p < 0.001; n = 9/group).
Figure 7.
Figure 7.
Synaptic TrkB activation depends on ERα and β1 integrin, and is required for LTP in females. A, Deconvolved image shows punctate localization of pTrkB Y515 (red) and PSD-95 (green); yellow indicates double labeling (arrow). Scale bar, 2 μm. Line graph shows that TBS, compared with LFS, caused a greater rightward skew in the density frequency distribution for pTrkB-IR colocalized with PSD-95 (p < 0.0001, F(19,342) = 10.44), and MPP substantially reduced this effect (p < 0.0001, F(19,342) = 10.66; n = 10/group). Right, The percentage of double-labeled synapses with dense pTrkB-IR (≥90 units) was elevated after TBS in vehicle-treated, but not in MPP-treated, female slices (group means normalized to the LFS group mean; p = 0.0025, F(2,29) = 7.55; post hoc tests: LFS vs TBS, *p < 0.05; TBS vs TBS + MPP, ##p < 0.01). B, In slices from male rats, TBS increased both (left) the rightward skew in the synaptic pTrkB-IR density frequency distribution (vs LFS, p < 0.0001; F(19,342) = 6.794) that was not influenced by MPP (p = 0.939; F(19,342) = 0.549, n = 10/group) and (right) the percentage of PSD-95-IR synapses associated with dense pTrkB-IR (≥90 units), also not influenced by MPP (p = 0.009, F(2,29) = 5.68; Bonferroni's post-test: **p < 0.01 vs LFS). C, TrkB blocker ANA-12 (750 nm) disrupted the stabilization of CA1 LTP in female slices (p < 0.0001, t(10) = 8.36; n = 6/group). D, S-C TBS produced a marked increase in the percentage of PSDs associated with dense pTrkB-IR in wild-type mice but not in β1 integrin cKOs (p < 0.0001, F(3,36) = 25.55; post hoc tests: LFS vs TBS for wild types, ***p < 0.0001; LFS, n = 9; TBS, n = 8; LFS vs TBS for β1 cKOs, n.s.; n = 10/group). E, Image shows pCofilin-IR colocalized with PSD-95 in female CA1 SR. Right, In females, S-C TBS increased the rightward skew in the density frequency distribution for synaptic pCofilin (relative to LFS; p < 0.0001, F(19,399) = 7.69; LFS, n = 12; TBS, n = 11). F, The selective ROCK inhibitor H1152 (100 nm, 160 min) blocked S-C LTP in female slices (p = 0.0013, t(9) = 4.57; veh,, n = 6; H1152, n = 5).
Figure 8.
Figure 8.
Sex differences in thresholds for LTP and spatial learning. A, Five pairs of two theta bursts produced significant S-C LTP in male but not in female CA1 (p = 0.0005, t(11) = 4.86; female, n = 7; male, n = 6). B, The S-C fEPSP slope immediately after each burst pair increased steadily across the first three pairs for both sexes but diverged for the last two pairs (p = 0.045, F(4,44) = 2.666). C, Five pairs of two theta bursts increased the percentage of CA1 SR synapses with dense pERK1/2-IR in male but not female slices (values normalized to the mean of their respective LFS groups; one-way ANOVA: p < 0.0001, F(3,56) = 11.28; post hoc tests: male TBS vs each other group, ***p ≤ 0.0001; 14–16 slices/group). D, Four sets of TBS triplets (three bursts, 200 ms between bursts, 2 min between triplets) produced significant LTP in female slices (p = 0.0025, t(11) = 3.91 vs 2 in burst group; n = 6/group). E, The fEPSP slope immediately after each burst triplet steadily increased (∼150% to 209% of baseline). F, E2 (1 nm), perfused for 10 min before collecting baseline responses and continued 30 min more, increased LTP magnitude in females (five pairs of theta bursts, LTP expressed as the percentage of baseline); this enhancement was significantly attenuated by either ERα antagonist MPP or ERβ antagonist PHTPP applied 30 min before and during E2 application (p = 0.0001, F(3,25) = 9.52, one-way ANOVA; post hoc tests: con vs E2, ***p = 0.0001; E2 vs E2 + MPP, ##p = 0.003; E2 vs E2 + PHTPP, #p < 0.02; con vs E2 + MPP or E2 + PHTPP, p > 0.35). G, For OLM training, mice explored the chamber containing two identical objects (A1 and A2); for testing 24 h later, they returned to the chamber with one of the objects moved to a novel location. H, With 5 min of training, OLM was greater for proestrus (Pro) than non-proestrus (Non-pro) mice (signified by DI; ***p < 0.0001, t(18) = 7.27; Pro, n = 8; Non-pro, n = 12). I, MPP or PHTPP (0.6 mg/kg) blocked OLM in proestrus mice (p < 0.0001, F(2,17) = 28.94; post hoc tests: veh vs MPP, ***p < 0.001; veh vs PHTPP, ***p < 0.001; veh, n = 6; MPP, n = 5; PHTPP, n = 7). J, Location of CA1 fields for measures of pERK-IR. K, After 5 min of OLM training in proestrus, the numbers of dense pERK1/2-IR PSDs were increased in mice that sampled the objects relative to those that explored an empty chamber; this effect was blocked by MPP (p = 0.003, F(2,23) = 7.78; Control vs Learn, **p < 0.01; Learn vs Learn + MPP, #p < 0.05; n = 8/group; normalized to control mean). L, The 5 min of OLM training did not affect the numbers of dense pERK1/2-IR PSDs in non-proestrus mice (p = 0.44, t(23) = 0.79; Control, n = 13; Learn, n = 12).
Figure 9.
Figure 9.
A two-factor hypothesis for sexual dimorphism at hippocampal synapses. A, Prior studies showed that, in male hippocampus, infused E2 acts via ERβ to stimulate modulatory receptors (β1 integrins, TrkB) leading to actin signaling and transiently enhanced baseline synaptic responses. Experiments described here demonstrate that E2 engages two NMDAR-associated kinases (ERK1/2, Src) that are upstream from these events; in males, this effect is mediated by ERβ with no detectable contribution from ERα. B, E2 application also activates the two kinases in females, but the response to exogenous E2 is mediated by both ERα and ERβ. It is proposed that this sex difference reflects the greater concentration of ERα in female synapses (first dimorphic feature). C, Induction of LTP in males activates the two NMDAR-related kinases and downstream signaling events that stabilize the potentiated state. These downstream steps, and LTP itself, do not depend on local estrogen, ERα, or ERβ. D, LTP induction in females also activates ERK1/2 and Src but, in contrast to males, the effect is dependent upon ERα. We propose that the functional links between NMDARs and the kinases are weaker in females than in males (second dimorphic feature), and so kinase activation requires a boost from released estrogen and stimulation of ERα. The dependency upon local estrogen is accompanied by a higher threshold for LTP in females, an effect that can be offset by exogenous estradiol acting via mechanisms described in B.
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