Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABAA receptors at inhibitory synapses

Jayanta Mukherjee; Ross A. Cardarelli; Yasmine Cantaut-Belarif; Tarek Z. Deeb; Deepak P. Srivastava; Shiva K. Tyagarajan; Menelas N. Pangalos; Antoine Triller; Jamie Maguire; Nicholas J. Brandon; Stephen J. Moss

doi:10.1073/pnas.1705075114

New Research In

Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 22, 2017 (received for review March 28, 2017)

Significance

Our knowledge of how estrogen signaling can influence inhibitory synaptic transmission is rudimentary and is addressed here. Collectively, our data suggest that estrogen modulates the dynamics of surface GABA_A receptors and hence efficacy of synaptic inhibition, via a postsynaptic mechanism that relies on disrupting the postsynaptic scaffold. This regulatory mechanism may have profound effects on the efficacy of neuronal inhibition and therefore synaptic plasticity and thus play a role in the pathophysiology of estrogen-related seizure and cognitive disorders.

Abstract

Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c a_A receptors (GABA_ARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABA_ARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABA_ARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABA_ARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABA_ARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABA_ARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.

Estrogens exert profound effects on neuronal excitability which are likely to underlie, for example, their role in seizure disorders and in regulating cognitive function (1, 2). Consistent with this, estrogen has been shown to potentiate excitatory neurotransmission, likely through effects on glutamate receptor trafficking (3 ⇓–5). In contrast, their effects on inhibitory neurotransmission mediated through GABA_A receptors (GABA_ARs) remain relatively poorly described and mechanistically obscure.

GABA_ARs are chloride-selective pentameric ligand-gated ion channels that are coassembled from a diverse array of subunits α (1–3), β (1–3), γ (1–3), δ, ε, and π with the majority of benzodiazepine-sensitive synaptic GABA_ARs being composed of α (1–3), β (1–3), and γ2 subunits (6 ⇓–8). The number of GABA_ARs at inhibitory synapses is a critical determinant of the efficacy of phasic GABAergic inhibition, a process that is orchestrated by a family of receptor-associated proteins. Central to the accumulation of GABA_ARs at inhibitory synapses is the multifunctional scaffold protein gephyrin, which is capable of oligomerization, forming a hexagonal lattice, in addition to binding both actin and microtubules (9). Gephyrin binds directly to conserved amino acid motifs within the intracellular loop domain of the GABA_AR α1–3 subunits, acting as a bridge to link these receptors to the cytoskeleton. Consistent with this concept, single-particle tracking (SPT) experiments have revealed that gephyrin selectively traps and reduces the mobility of GABA_ARs at inhibitory synapses, thereby enriching their accumulation at these subcellular specializations (10 ⇓⇓–13).

Here we have examined the role that estrogen plays in regulating the cell surface dynamics of GABA_ARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with estradiol (E2) disrupts the clustering and reduces the confinement of GABA_ARs at inhibitory synapses without altering either their total or plasma membrane protein expression. Additionally, we show that E2 drives a selective reduction in amplitude of inhibitory synaptic currents in both cultured cortical neurons and male hippocampal slices. This regulatory mechanism may have profound effects on the efficacy of neuronal inhibition and may contribute to the effects of estrogen on synaptic plasticity and disease pathology, including disorders with seizures and cognitive deficits.

Results

E2 Treatment Reduces the Number of Synaptic GABA_ARs and Gephyrin in Cultured Cortical Neurons.

To initiate our study, we examined the effect of the stable estrogen analog E2 on the stability of inhibitory synapses in cultured cortical [∼24 d in vitro (DIV)] neurons. Cultures were treated with E2 (10 nM) for 2 h and then stained with antibodies against an extracellular epitope for the α2 subunit and gephyrin and analyzed by confocal microscopy. Surface α2 subunit-containing GABA_ARs were chosen, as they are largely restricted to gephyrin-enriched inhibitory postsynaptic sites and are major mediators of phasic inhibition in the brain (14, 15). For the analysis, a cluster is considered synaptic if it is colocalized with the inhibitory scaffolding protein gephyrin. The number of synaptic α2 clusters was compared per 30 µm of dendrite. E2 significantly decreased the number of synaptic α2 puncta (Fig. 1 A and B; control = 9.7 ± 0.5 and E2 = 3.74 ± 0.32 clusters per 30 μm; P < 0.001, unpaired t test, n = 25–26 cells). We also compared the total fluorescence intensity of remaining clusters, a combined measure of the average intensity and area of an individual cluster, by normalizing this value to that seen in vehicle-treated neurons. E2 treatment significantly decreased the intensity of remaining synaptic α2 puncta to 78.6 ± 2.6% of control (Fig. 1B; P < 0.001; unpaired t test, n = 25 cells).

Fig. 1.

E2 decreases the number and size of synaptic GABA_ARs and gephyrin in cultured neurons. Cortical neurons (DIV ∼24) were treated with E2 (10 nM) or DMSO (Con) for 2 h. Neurons were fixed and stained with anti-(α2, γ2) subunit antibody and following permeabilization, with an anti-gephyrin (GPHN) antibody. Large panels are the merged image of the maximum intensity projection of a representative confocal image. Right-hand panels represent enlargements of the boxed areas consisting of individual and merged channels. (Scale bars: 20 μm.) (A) E2 reduced the clustering of synaptic α2-GABA_ARs. Images showing the clustering of α2 (red) and gephyrin (green), control (Left), E2 (Right). (B) The number of α2/gephyrin clusters per 30 μm was compared between treatments in the right-hand panel. In the left-hand panel cluster intensity was compared by normalized values to those seen in control (100%). In both panels asterisks indicate significantly different from control, P < 0.001 (n = 25 cells). (C) E2 reduced the synaptic clustering of γ2-GABA_ARs. Images showing the clustering of γ2 (green) and GPHN (red), control (Left) and E2 (Right). (D) The number of γ2/gephyrin clusters per 30 μm was compared between treatments in the right-hand panel. In the left-hand panel cluster intensity was compared by normalized values to those seen in control (100%). In both panels * indicates significantly different from control, P < 0.001 (n = 25 cells). All data are presented as mean ± SEM.

To confirm our results with the α2 subunit we also examined the effects of E2 on the synaptic accumulation of the γ2 subunit, which plays an essential role in facilitating the targeting of GABA_ARs to inhibitory synapses (16). E2 treatment significantly reduced the number of synaptic γ2 puncta (Fig. 1C; control = 14.3 ± 0.57 and E2 = 5.71 ± 0.35 clusters per 30 μm, P < 0.001, unpaired t test, 25 cells). The average fluorescence intensity of the remaining synaptic γ2 puncta was also significantly reduced to 78.2 ± 2.5% of control (Fig. 1D; P < 0.001; unpaired t test, n = 25). We also assessed the effects of E2 on inhibitory synapses containing the α1 subunit, the most abundant receptor α-subunit isoform expressed in the adult brain (17). Compared with control, E2 also reduced the number of synaptic α1 puncta (Fig. S1A).

Fig. S1.

Acute treatment with E2 disrupts synaptic clustering of α1-GABA_ARs. Primary cortical neurons (DIV ∼24 d) were treated with E2 (10 nM) for 2 h. Cells were fixed, stained with an anti-α1 GABA_AR subunit antibody (red), and subsequently permeabilized and stained with an anti-gephyrin antibody. Larger panels show a merged (α1 and gephyrin) image of a single plane confocal image. Right-hand panels represent enlargements of the boxed areas in the large panels. (Scale bars: 20 μm.)

Estrogen signals via a range of receptors including the canonical nuclear hormone receptors estrogen receptor α (ERα) and β (ERβ) and the G protein-coupled ER (GPER/GPR30) (18, 19). To begin to address which ERs mediate the effects of E2 on inhibitory synapses, we exposed cortical cultures to the ERα agonist [4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol] (PPT) (10 nM) or the ERβ agonist WAY-200070 [7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol] (070) (100 nM) for 2 h. Both of these agents induced reductions in the number of inhibitory synapses similar to those seen with E2 (Fig. S2). Compared with control (DMSO), both 070 and PPT significantly decreased the number of synaptic α2 puncta (Fig. S2B; control = 9.1 ± 0.4, 070 = 4.2 ± 0.32, PPT = 5.6 ± 0.38 clusters per 30 μm; P < 0.001, control vs. 070 and PPT, one-way ANOVA with Bonferroni’s post hoc test, n = 29–32 cells). We also compared the total fluorescence intensity of remaining clusters, a combined measure of the average intensity and area of an individual cluster, by normalizing this value to that seen in vehicle-treated neurons. The 070 and PPT treatment significantly decreased the intensity of remaining synaptic α2 puncta (070, 82 ± 2.9% of control and PPT, 79.8 ± 1.8% of control, Fig. S2B; P < 0.001; one-way ANOVA with Bonferroni’s post hoc test, n = 29–32 cells).

Fig. S2.

ER α and β agonists decrease the number and size of synaptic GABA_ARs and gephyrin puncta in cultured neurons. Cortical neurons (DIV ∼24) were treated with DMSO (Con), WAY200070 (070; 100 nM), and PPT(10 nM) for 2 h. Neurons were fixed and stained with anti-α2 GABA_AR subunit antibody and, following permeabilization, with an anti-gephyrin (GPHN) antibody. Large panels are the merged image of a representative single plane confocal image. Right-hand panels represent enlargements of the boxed areas consisting of individual and merged channels. (Scale bars: 20 μm.) (A) Both ER agonists reduced the clustering of synaptic α2-GABA_ARs. Images showing the clustering of α2 (red) and gephyrin (green), control (Left), 070 (Middle), and PPT (Right). (B) The left graph shows the reduction in the numbers of synaptic α2-GABA_AR clusters/30 μm (P < 0.001, control vs. 070 & PPT, n = 29–32 cells). The bar graph (Right) shows change in the fluorescence intensities of the α2-GABA_AR clusters compared with control. (P < 0.001, control vs. 070 & PPT, n = 29–32 cells). Data represent mean ± SEM (one-way ANOVA with Bonferroni’s post hoc test, *P < 0.05 to represent significance).

Finally, we assessed if ERs are expressed on the plasma membrane of neurons and whether they are found in the vicinity of inhibitory synapses. Due to the paucity of reliable, high-affinity antibodies to detect estrogen receptors, we exposed neurons to fluorescently labeled E2 coupled to BSA (FITC-E2) as a means of labeling cell surface populations of ERs (20). FITC-E2 labeled the plasma membrane population of ERs of live neurons, which was blocked by preincubation with E2, demonstrating the specificity of this staining (Fig. S3A). Significantly, FITC-E2 labeling was found within the same neuron and close to the puncta containing GABA_ARs, suggesting E2 can potentially influence the clustering of GABA_ARs via a local signaling mechanism (Fig. S3B).

Fig. S3.

Expression of estrogen receptors in cortical neurons and colocalization with GABA_ARs. (A) Live cortical neurons (DIV ∼24) were treated with E2-BSA-FITC (5 μg/mL) for 45 min with or without 30-min preblocking with E2 (1 μM). Cells were then washed, fixed, and imaged. A single-plane confocal image shows the binding of E2 (green) to the surface ERs (Left), as no specific staining was seen when cells were preblocked with E2 (Right). (B) Live neurons were treated with E2-BSA-FITC (5 μg/mL) for 45 min. Cells were then washed, fixed, and stained with α2-GABA_AR subunit (red) antibodies without permeabilization. The individual panel shows the expression of ERs (green) and α2-GABA_ARs (red) and a merged confocal image shows the expression of GABA_ARs with respect to the ERs in the same cell. (Scale bars: 20 μm.)

Collectively, these results reveal that acute exposure of cortical neurons to E2 disrupts the clusters of the GABA_ARs containing α1, α2, and γ2 subunits and gephyrin at the inhibitory synapses and the effects of E2 at these structures are mediated in part by ERα and ERβ.

E2 Selectively Reduces the Amplitude of Miniature Inhibitory Synaptic Currents in Cultured Cortical Neurons.

Given the results of our imaging studies, we tested if E2 leads to changes in the properties of miniature inhibitory synaptic currents (mIPSCs) in cultured cortical neurons (DIV ∼24). These events were isolated using the glutamate receptor antagonists CNQX/AP5 and in the presence of TTX. Exposure to E2 (10 nM) (n = 14–16 cells) decreased the size of postsynaptic GABA currents (Fig. 2A). A cumulative amplitude histogram revealed a leftward shift toward smaller mIPSC amplitudes in response to E2 treatment (Fig. S4A). Accordingly, the average mIPSC amplitude was significantly reduced upon exposure to E2 (Fig. 2B; control = 40.1 ± 4.2 pA, E2 = 27.4 ± 1.6 pA, P = 0.01, unpaired t test, n = 14–16). E2 exposure did not modify mIPSC rise time (control τ_rise = 4.99 ± 0.2 ms, E2 τ_rise = 4.90 ± 0.16 ms, P = 0.72, unpaired t test) or decay (control τ_decay = 21.02 ± 0.6 ms, E2 τ_decay = 20.40 ± 0.6 ms, P = 0.48, unpaired t test, n = 14–16). Likewise, E2 did not modify mIPSC frequency (Fig. 2C; control = 3.22 ± 0.33 Hz, E2 = 2.57 ± 0.26 Hz, P = 0.14, unpaired t test, n = 14–16).

Fig. 2.

E2 selectively reduces the amplitude on mIPSC in cultured cortical neurons. (A) Sample traces are shown of mIPSCs recorded from neurons (DIV ∼24 d) either treated with DMSO (Con) or E2 (10 nM) for 2 h. (B) The bar graph shows the decrease in the average mIPSC amplitude per cell (unpaired t-test; P = 0.01, n = 14–16 cells) (C) The graph shows no significant changes in the average mIPSC frequency per cell (P = 0.14; t test, n = 14–16 cells). All data are presented as mean ± SEM.

Fig. S4.

E2 selectively reduces the amplitude of mIPSC in cultured cortical neurons and amplitude of sIPSC in male hippocampal slices without affecting frequencies. (A) A cumulative frequency distribution of mIPSC amplitudes recorded from neurons (DIV ∼24) either treated with DMSO (Con) or E2 (10 nM) for 2 h revealed a leftward shift toward smaller amplitudes in response to E2 treatment. (n = 14–16) cells. (B) A cumulative frequency distribution of sIPSC amplitudes revealed a leftward shift toward smaller amplitudes in response to E2 treatment recorded in CA1 pyramidal neurons from slices from male mice treated with vehicle (Con) or E2 (10 nM) (n = 11–13 cells, three mice per group).

Collectively, our electrophysiological measurements suggest that E2 acts to selectively reduce the amplitude of mIPSCs, an effect that is consistent with its ability to reduce the size and number of synaptic GABA_ARs (Fig. 1).

E2 Does Not Modify the Cell Surface or Total Protein Expression of GABA_ARs or Gephyrin.

The number of GABA_ARs on the neuronal membrane has profound effects on the efficacy of GABAergic inhibition, a process that is critically dependent upon regulated receptor exo- and endocytosis (21, 22). Therefore, we assessed if exposure to E2 modifies the cell surface expression of GABA_ARs subunits. To isolate surface GABA_ARs, cultured cortical neurons were exposed to NHS-Biotin and after purification on streptavidin beads; cell surface and total fractions were immunoblotted with antibodies against the α1 and γ2 subunits. This revealed that, compared with control, exposure to E2 did not significantly alter the cell surface stability of α1 or γ2 subunits (Fig. 3A; α1: 98 ± 10.96%, P = 0.87 and γ2: 105.8 ± 1.8%, P = 0.08 of control, respectively, unpaired t test, n = 3). Significantly, the surface fractions were free of the cytosolic protein GAPDH, verifying the integrity of our biotinylation procedure (Fig. 3A). Likewise, E2 did not modify the total expression of GABA_AR subunits (Fig. 3B; α1 = 102.2 ± 8%, P = 0.81 and γ2: 101.4 ± 5.7%, P = 0.83 of control, respectively, unpaired t test, n = 3). Moreover, the total expression level of gephyrin was comparable in neurons treated with E2, (Fig. 3B; 107.9 ± 4.2% of control, P = 0.13, unpaired t test, n = 5).

Fig. 3.

E2 does not affect the total or surface level expression of the GABA_AR or the stability of the GABA_AR–gephyrin complex. Cortical neurons (DIV ∼24) were treated with E2 (10 nM) or DMSO (Con) for 2 h and subject to immunoblotting after biotinylation or co-IP. Indicated molecular weights are in kilodaltons. (A) Blots showing the total (Left) and the surface (Right) expression of the GABA_AR subunits, immunoblotted with anti-α1 or -γ2 GABA_AR subunit and anti-GAPDH antibodies. The bar graph shows the surface/total levels of each subunit that was normalized to the control (100%). (P > 0.05, not significant, unpaired t test, n = 3.) GAPDH serves as loading control. (B) Blots showing the total expression of gephyrin. No significant change was observed after E2 treatment (P = 0.13, n = 5). GAPDH serves as loading control. (C) Detergent-solubilized cell extracts were subject to IP with IgG or α1 antibodies. Precipitated material was then immunoblotted with anti-α1 and anti-GPHN antibodies. The ratio of GPHN/α1 immunoreactivity was determined and normalized to the control (100%). (P = 0.18, n = 3.) All data are presented as mean ± SEM.

Next, we assessed if E2 treatment modifies the association of gephyrin with GABA_ARs. We subjected neuronal lysates to immunoprecipitation (IP) with an antibody against the α1 subunit or mouse IgG as a control (10). Precipitated material was then immunoblotted with gephyrin and α1-subunit antibodies, and the ratio of gephyrin immunoreactivity was compared between control and E2 treatment. This approach revealed that E2 did not significantly alter the association of GABA_ARs with gephyrin (Fig. 3C; 121.8 ± 10.9% of control, P = 0.18, unpaired t test, n = 3).

Therefore, E2 does not act to modify either the total or cell-surface levels of synaptic GABA_ARs or the stability of the inhibitory scaffold gephyrin. Finally, exposure to E2 does not compromise the ability of gephyrin to bind GABA_ARs.

SPT Reveals That ER-Mediated Signaling Reduces the Confinement of GABA_ARs at Inhibitory Synapses.

To assess the effects of E2 on the dynamics of inhibitory synapses we used a transgenic mouse strain that expresses gephyrin fused with a monomeric red fluorescent protein (mRFP-GPHN) (23). To track the movement of individual GABA_ARs on neuronal membranes, cortical cultures were labeled with low concentrations of quantum dots (QDs) coupled to anti-α2 antibodies, allowing us to monitor the mobility of individual GABA_ARs using the SPT technique (24). As an initial control, we measured the effects of E2 on the fluorescence intensity of the synaptic α2-GABA_ARs and mRFP-GPHN. GABA_ARs that are colocalized with gephyrin were considered to be synaptic and integrated fluorescence intensities, reflecting their relative accumulation, were quantified. Consistent with our immunofluorescence experiments with rat cortical culture, exposure to E2 reduced the intensity of α2-GABA_ARs and mRFP-GPHN fluorescence in neurons (Fig. S5; α2: 75.2 ± 3.2% and mRFP-GPHN: 79.2 ± 2.6% of control P < 0.0001, unpaired t test, n = 19–20 cells).

Fig. S5.

Analyzing the clustering of mRFP-GPHN and the GABA_AR-α2 subunit in mouse cortical neurons. (A) Representative confocal images of cultured mouse cortical neurons stained for the α2-GABA_AR subunit (green) in control conditions (Con, vehicle, 2 h) or after E2 treatment (E2, 10 nM, 2 h). mRFP-GPHN (red) positive loci define the endogenously fluorescent inhibitory postsynaptic densities. Boxed areas are highlighted under principal images and show a decreased fluorescence intensity of both synaptic components after E2 treatment. (Scale bars, 30 μm.) (B) Quantification of α2 subunit-containing GABA_AR (Left) and (Scale bars, 30 μm.) mRFP-GPHN (Right) fluorescence intensities in control condition (Con) and after E2 treatment at synaptic sites. Synapses were defined as GABA_AR-α2 and mRFP-GPHN–positive loci (colocalized clusters). Data represent mean ± SEM (*P < 0.05; unpaired t test).

The lateral diffusion of neurotransmitter receptors within cell membrane hinders its stability at the synapses. Therefore, we examined the potential effect of E2 on the lateral mobility of GABA_ARs and monitored the dynamic behavior of QD-labeled endogenous α2-GABA_ARs on neuronal membranes (Fig. 4A). To discriminate between synaptic and extrasynaptic receptor dynamics, only mRFP-GPHN–positive membrane compartments were defined as synaptic (25). At synapses, E2 enhanced the mean square displacement (MSD) of QD labeled-α2-GABA_ARs, suggesting a large decrease of their confinement at synaptic sites (Fig. 4B). A reduced dwell time of α2-GABA_ARs at inhibitory synapses was also observed, suggesting an enhanced dispersal and a decreased stability of GABA_ARs within synapses [Fig. 4C; P = 0.0075, Kolmogorov–Smirnov (KS) test, control, n = 309 and E2, n = 285 synaptic trajectories]. Accordingly, the percentage of trajectories stabilized at synapses was decreased from 13.4 ± 2.4 to 7.48 ± 1.6% after E2 exposure (P = 0.035, unpaired t test, n = 3). The diffusion coefficient distribution of α2-GABA_ARs was not modified, suggesting that their diffusion properties were not affected at synaptic membrane compartments. In contrast to this, a modest but significant increase in the diffusion coefficient of α2-GABA_ARs located at extrasynaptic sites was measured upon exposure to (Fig. 4D; E2 P = 2.94E-05, KS test, n = 1,188, and 1,174 extrasynaptic trajectories for control and E2, respectively).

Fig. 4.

E2 decreases the dwell time of GABA_ARs at inhibitory synapses. Cortical neurons (DIV ∼24) from mRFP-gephyrin transgenic mice were treated with E2 (10 nM) or DMSO (Con) for 2 h. (A) Representative reconstructed traces of QD-α2GABA_ARs are shown (black). In both cases, synaptic membrane areas are indicated by the endogenous fluorescence of mRFP-gephyrin, segmented in red domains. (Scale bar: 1 µm.) (B) The graph shows the change in the MSD (square micrometers) over time of the endogenous QD-α2GABA_ARs within synaptic membrane compartments, control (black) and E2 (gray). E2 treatment resulted in the increased explored area over time at the synaptic sites. (C) The graph shows the cumulative distribution of the dwell times of QD-α2GABA_ARs on the mRFP-gephyrin, control (black) and E2 (gray). The residence time of endogenous synaptic α2-GABA_ARs is decreased after E2 treatment. (D) Distribution of QD–α2GABA_AR complexes diffusion coefficients (D, square micrometers per second) in synaptic (S, left) and extrasynaptic (E, right) membrane compartments. After E2 exposure (gray), α2GABA_ARs exhibit increased diffusion coefficient at extrasynaptic but not synaptic neuronal membranes. Box plots indicate the D value for (90, 75, 50, 25, and 10%) of the population.

Thus, our results using SPT suggest that E2 acts to decrease the dwell time of GABA_ARs at synaptic sites, which at a steady state accounts for a reduced accumulation of GABA_ARs at synapses.

E2 Modifies the Efficacy of GABAergic Inhibition in Hippocampal Slices.

To examine the significance of our findings using cultured cortical neurons we assessed the effects of E2 on the properties of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal neurons in hippocampal slices prepared from male (∼3 mo old) C57/BL6 mice. Exposure of slices to E2 (10 nM) for 2 h significantly reduced the amplitude of sIPSCs in CA1 neurons (Fig. 5A). A cumulative amplitude histogram revealed a leftward shift toward smaller sIPSC amplitudes in response to E2 treatment (Fig. S4B), Likewise, the average sIPSC amplitude was significantly decreased upon E2 treatment (Fig. 5C; control: 46.1 ± 7.7 pA, E2: 29.4 ± 2.7 pA, n = 11–13 cells, three mice per experimental group, P = 0.04, unpaired t test). However, we have not seen any significant changes in their frequencies (Fig. 5D; control: 10.1 ± 2.1 Hz, E2: 7.3 ± 1.1 Hz, n = 11–13 cells, three mice per experimental group, P = 0.21, unpaired t test). Also, exposure to E2 did not have any significant effect on the sIPSC decay (control: 7.4 ± 0.8 and E2: 7.0 ± 0.5 ms).

Fig. 5.

E2 decreases the amplitude of sIPSC in male hippocampal slices without affecting frequencies. (A) Representative traces of sIPSCs recorded in CA1 pyramidal neurons from slices from male mice treated with vehicle (Con) or E2 (10 nM). (B) The average amplitude of sIPSCs is decreased following E2 treatment compared with vehicle (P = 0.04, unpaired t test, n = 11–13 cells). (C) The bar graph shows no significant changes in average frequencies of sIPSCs in either group (P = 0.21, unpaired t test, n = 11–13 cells).

Collectively, these results further suggest a critical role for a postsynaptic mechanism in mediating the effects of E2 on GABAergic inhibition.

Discussion

Estrogen plays a key role in regulating neuronal activity and animal behavior and alterations in estrogen signaling are linked to a range of neurological and psychiatric conditions (26, 27). Estrogen regulation of excitatory neurotransmission has been heavily investigated (4, 5, 28) but how estrogen shapes inhibitory synaptic transmission is still poorly understood. It has been shown previously that E2 can suppress inhibitory synaptic transmission in young adult rat hippocampal slices—intriguingly, an effect only seen in females. Furthermore, it was shown to be mediated primarily via a presynaptic mechanism involving mGluR1 regulation of endocannabinoid signaling and subsequent regulation of GABA release from the presynaptic interneuron, which is engaged preferentially in females (29 ⇓–31). However, to date, there have been no detailed studies to define the role that E2 plays in determining the dynamics of GABA_ARs at inhibitory synapses.

To address this issue, we examined the effects of E2 on the number of inhibitory synapses in cultured cortical neurons. Exposure to E2 reduced the number of gephryin-positive inhibitory synapses. Parallel electrophysiological studies revealed that E2 induced a reduction in the amplitudes of mIPSCs, without changing their frequency, consistent with the removal of GABA_ARs from synaptic sites. Collectively, these results suggest that E2 acts to reduce the stability of inhibitory synapses and/or the number of functional GABA_ARs at these structures via a postsynaptic mechanism.

The efficacy of GABAergic inhibition and the maintenance of inhibitory synapses are in part determined by the plasma membrane stability of GABA_ARs. This process is dependent upon the rates of receptor exo- and endocytosis, which is in turn subject to dynamic modulation by neuronal activity (22, 32). Therefore, we examined the effects of E2 on the accumulation of GABA_ARs on the plasma membrane. Our results revealed that E2 did not significantly modify either the total expression levels or the cell-surface accumulation of GABA_ARs. The stability of synaptic GABA_ARs clusters is largely dependent on the integrity of a gephyrin scaffold. Multiple studies have documented compromised synaptic clustering of GABA_ARs (e.g., α2 and γ2), upon reduction of gephyrin (14, 16, 33). We have provided direct evidence that E2 does lead to the disruption of gephyrin clusters as shown by a reduction in the numbers and fluorescence intensities of individual puncta. Since we did not observe any significant change in the level of expression of gephyrin, a reduction in the fluorescence intensity is very likely due to the destabilization of the gephyrin scaffold upon E2 treatment.

To further evaluate the mechanism by which E2 modulates the stability of inhibitory synapses we examined the effects of E2 on the mobility of synaptic GABA_ARs in real time. SPT data reveal that E2 selectively increased the MSD of synaptic GABA_ARs and decreased their dwell time at these structures. We also noted a modest but significant increase in the diffusion coefficient of α2-GABA_ARs located at extrasynaptic sites. This is likely due to the interaction of α2-GABA_ARs with the scaffolding protein gephyrin outside of the synapse, as reported previously for glycine receptors, where it is estimated that 40% of receptor/gephyrin puncta are extrasynaptic (34). Therefore, our data clearly demonstrate that treatment with E2 resulted in reduced confinement of GABA_ARs at synaptic sites.

Finally, we examined the effects of E2 on the efficacy of GABAergic inhibition in hippocampal slices from male mice. In CA1 neurons, E2 decreased sIPSC amplitude but without significant changes in frequency, which is consistent with our results using cultured cortical neurons. Previously, it has been reported that the suppression of amplitude of IPSCs in hippocampal slices was only seen in females due to a sex-specific endocannabinoid-dependent presynaptic mechanism (30). These apparent discrepancies might be due to methodological differences and/or difference in species used. However, our data strongly suggests that E2 can attenuate inhibitory synaptic transmission via a postsynaptic mechanism.

The precise mechanism by which E2 influences the stability of inhibitory synapses remains to be determined, but our results suggest that the effects of E2 are in part mediated via the activation of ERα and/or ERβ. In addition to modulating transcription, these receptors can exert a rapid nongenomic effect on cells by modulating MAPK signaling, GSK3β, and the activity of small GTPases (18 ⇓–20). Interestingly, it has been shown previously that CaMKII, MAPK, or GSK3β can phosphorylate gephyrin in an activity-dependent manner and thereby regulate its stability (35 ⇓–37). Therefore, it will be of interest to determine if E2 modulates gephyrin or GABA_ARs subunit phosphorylation, a process that can have profound effects on the membrane trafficking and stability of these key components of inhibitory synapses.

In summary, our studies provide a molecular mechanism by which estrogen acts to reduce the efficacy of GABAergic inhibition by decreasing the stability of inhibitory synapses. Such modulation may have profound effects on the maintenance of neuronal excitation/inhibition balance and thus contribute to the enhancement of cognition and epilepsy in which excessive estrogen signaling is believed to be of significance.

Materials and Methods

Biochemical Measurements, Confocal Imaging, and Image Analysis.

Rat primary cortical neurons (DIV ∼24) were used throughout unless otherwise stated. See SI Materials and Methods for detailed biochemical methods, reagents, imaging, and analysis.

Electrophysiology.

Detailed methods on the electrophysiological recordings from both cultured neurons and brain slices are described in SI Materials and Methods.

SPT Experiments.

Methods of the SPT experiment and analysis have been previously described (24) and are outlined in detail in SI Materials and Methods.

SI Materials and Methods

Cell Culture, Drug Treatment, Detergent Solubilization, and Western Blotting.

Primary cortical cultured neurons were prepared as described previously from E18 rat embryos of either sex (38). For biochemical assays, cultures were solubilized in a buffer containing (in millimolar) 5 EGTA, 5 EDTA, 50 NaCl, 50 Na₃PO₄, 5 benzamidine, 10 NaF, 2 Na₃VO₄, and 2 PMSF; 10 p-nitrophenylphosphate, aprotinin, leupeptin, and pepstatin were included at a concentration of 10 μg/mL each with 2% Triton X-100, pH 7.5, at 4 °C for 2 h. Dissociated neuronal cultures were treated for 2 h with vehicle (DMSO) and E2 (10 nM) unless otherwise stated. Extracts were then used either for Western blotting or IP. Co-IP experiments were performed using either mouse monoclonal (M3-M4 loop-specific α1 antibody) or mouse IgG as a control, followed by coupling to protein G-Sepharose, as described before (39). Immunoblots were visualized using ECL followed using the BioRad Imaging Station (model universal hood III) using Image laboratory software. All blots were analyzed using ImageJ (Fiji) software.

Biotinylation.

To isolate cell-surface proteins, cortical neurons were first treated with either DMSO (control) or E2 (10 nM) for 2 h. Cells were labeled with 2 mg/mL NHS-Biotin (Pierce; Thermo Fisher Scientific) at 4 °C for 30 min. Cultures were then lysed as outlined above, and detergent-soluble extracts were exposed to avidin beads (Pierce; Thermo Fisher Scientific) (40). Cell surface and total fractions were then subjected to immunoblotting with respective GABA_AR subunit antibody. Data were presented as a surface to the total ratio or total alone by normalizing to control (100%). GAPDH was used for loading control.

Reagents and antibodies.

The 17-β-estradiol and E2-BSA-FITC (E6507) were purchased from Sigma. PPT, an ERα-specific agonist, and WAY200070, an ERβ-specific agonist, were purchased from Tocris Biosciences. Mouse monoclonal gephyrin (clone 3B11 & mAB7), polyclonal guinea pig anti-γ2 (1 μg/mL), and polyclonal rabbit anti-α2 GABA_AR subunit antibodies were purchased from Synaptic Systems. Clone mAB7 (1 μg/mL) was used to immunostain gephyrin and clone 3B11 (1 μg/mL) was used for biochemical assays. Mouse monoclonal α1-GABA_AR (1 μg/mL for Western blot) was purchased from Antibodies Incorporated. Rabbit polyclonal anti-α1 extracellular epitope (1 ⇓⇓⇓⇓⇓⇓⇓–9); (1 μg/mL) and rabbit polyclonal anti-γ2 (intracellular epitope (319-366); 1 μg/mL), GABA_ARs subunit was a kind gift from Werner Seighart laboratory, Medical University of Vienna, Vienna. Mouse monoclonal GAPDH (clone 6C5; 1:5,000), was purchased from Santa Cruz Biotechnology. Mouse True Blot secondary antibody (1:1,500) was purchased from Rockland Antibodies.

Surface Labeling of Estrogen Receptors.

Surface labeling of ERs was performed using methods described previously (20). Briefly, live (DIV 24) rat cortical neurons were incubated with E2-BSA-FITC (5 μg/mL) at 37 °C for 45 min in neurobasal media. For the negative control, cells were first preincubated with E2 (5 μM) for 30 min, followed by E2-BSA-FITC (5 μg/mL) at 37 °C. Cells were then washed three times with 1× PBS and fixed with 4% paraformaldehyde. To label surface GABA_ARs, coverslips were blocked with 5% BSA and stained with anti-α2 subunit (1 μg/mL) and subsequently stained with rabbit “Alexa Fluor” secondary antibodies (1:500; Thermo Fisher Scientific). Images were acquired using a Nikon Eclipse Ti series confocal microscope with a 60× objective (N.A. = 1.4). The parameters for image acquisition were kept constant between the two conditions.

Confocal Microscopy and Image Analysis.

All imaging experiments were performed using ∼24 DIV rat cortical cultures. Dissociated neuronal cultures were treated for 2 h with vehicle (DMSO), E2 (10 nM), PPT (10 nM), and WAY070 (100 nM) unless otherwise stated. To label surface GABA_ARs subunits, cultures were fixed, blocked with 5% BSA, stained with respective subunit antibodies (1 μg/mL), and then permeabilized with 0.1% Triton and incubated with the gephyrin antibody (1 μg/mL). Cultures were subsequently stained with respective Alexa Fluor secondary antibodies (1:500; Thermo Fisher Scientific). Images were acquired using a Nikon Eclipse Ti series confocal microscope with a 60× objective (N.A. 1.4). All parameters for acquisition were kept constant among control and drug-treated cells. All images were analyzed using MetaMorph software (Molecular Devices). For image analysis, a receptor cluster was defined as being ∼0.1 μm² or greater and two- to threefold more intense than background fluorescence using the same threshold value throughout. Synaptic clusters were defined as those colocalized with gephyrin clusters. For intensity measurements, total intensities of individual clusters were measured by using the integral morphometric analysis function. Data were normalized to control and expressed as a percentage. The mean intensity value for individual clusters for each cell was calculated. Data were normalized by dividing the mean values for an individual cell with the mean value that of control, within each experiment. Analyses are based upon the number of cells (minimally 25 cells per group), as indicated in the text from at least three independent cultures. All statistics were done using Kaleidagraph software (Synergy).

Cortical Culture Electrophysiology.

All recordings were performed on ∼24 DIV cortical rat neurons plated on glass coverslips, as described previously (41). Following a 2-h treatment with E2 (10 nM), neurons were transferred to a submerged recording chamber (RC-26G; Warner Instruments) heated to 33 °C. The composition of the recording solution (in millimolar) was 140 NaCl, 2.5 KCl, 2.5 CaCl₂, 2.5 MgCl₂, 10 Hepes, and 11 glucose (pH adjusted to 7.4 with NaOH). Neurons were visually identified by morphology and targeted for recording with borosilicate glass pipettes (World Precision Instruments) containing (in millimolar) 140 CsCl, 2 MgCl₂, 0.1 CaCl₂, 1.1 EGTA, and 10 Hepes (pH adjusted to 7.4 with CsOH). After establishing whole-cell configuration and a 5-min stabilization, mIPSCs were isolated by addition of racemic 2-amino-5-phosphonopentanoic acid (DL-APV; 50 μM), 6, 7-dinitroquinoxaline-2, 3-dione (DNQX; 10 μM), and TTX (0.3 μM) at a holding potential of −60 mV. Whole-cell capacitance and input resistance were monitored throughout the recording period. Recordings were conducted with an Axopatch 200B amplifier (Molecular Devices), low-pass-filtered at 2 kHz, and sampled at 10 kHz (Digidata 1440A; Molecular Devices). Bicuculline, DL-APV, DNQX, muscimol, and TTX were purchased from Tocris Bioscience; all other reagents were purchased from Sigma-Aldrich. mIPSCs were detected using MiniAnalysis (Synaptosoft). The first 100 events per cell were used to determine the distribution of mIPSC amplitudes per treatment. Frequency calculations were performed over a 2-min recording. Graphs and statistical tests were assembled in GraphPad Prism. Unpaired Student’s t tests or Mann–Whitney nonparametric tests were used as indicated to assess significance with α = 0.05. Data are displayed as mean ± SEM.

SPT and analysis.

Cell culture.

Primary cortical cultures were prepared from knock-in mRFP-gephyrin mice (23) at E18. Dissociated cells were plated on 18-mm glass coverslips at a density of 10⁵ cells per mL and maintained in neurobasal medium supplemented with B-27 (1×), 2 mM glutamine, and 5 μg/mL of penicillin and streptomycin at 37 °C and 5% CO₂. Cortical neurons were imaged between 21 and 26 (DIV) and cultures were treated for 2 h with vehicle (DMSO) or E2 (10 nM) before imaging.

SPT experiments and analysis.

Neurons were sequentially incubated with an anti-α2-GABA_AR antibody (224103, 1:300; Synaptic Systems), and an anti-rabbit-Fab fragment coupled with QDs emitting at 655 nm (Invitrogen, Thermo Fisher Scientific). All incubation steps and washes steps were performed at 37 °C in minimal essential medium (MEM) recording medium (phenol red-free MEM, 33 mM d-glucose, 20 mM Hepes, 2 mM glutamine, 1 mM Na-pyruvate, 1× B27). Cells were imaged within 30–40 min after staining, at 37 °C, using an IX70 inverted microscope (Olympus) equipped with a 60× oil immersion objective (N.A. 1.45). Fluorescence was detected using a xenon lamp and appropriate filters. QD movements were recorded at 13 Hz for 75 s. Analyses were performed as previously described (42). Single QDs were identified by their blinking properties. Tracking was performed with homemade software in MATLAB (MathWorks). The center of fluorescence spots was determined with a spatial accuracy of 10–20 nm after 2D-Gaussian fit. QD trajectories were reconstructed as previously detailed (34). Images of mRFP-gephyrin–positive synapses were filtered using a multidimensional image analysis (MIA) on MetaMorph software. Trajectories were defined as synaptic when colocalized with mRFP-gephyrin MIA mask. The MSD (5 μm²) was calculated using the following equation:MSD(ndt)=(N−n)−1∑i=1N−n(xi+n−xi)2+(yi+n−yi)2),where xi and yi are the coordinates of an object on frame i, N is the total number of steps in the trajectory, and ndt is the time interval over which displacement is averaged. The diffusion coefficient D (σs The micrometers per second) was calculated by fitting the first two to five points of the MSD plot versus time with the equationMSD(t)=4D2−5t+4sx2,where sx is the spot localization accuracy in one direction (34). IQR distributions rather that mean values were represented given the large dispersal of the D values (several orders of magnitude). Synaptic dwell time was calculated as the time spent at synapses over the number of exists from synapses. Stable QD receptors were defined as a population staying the whole duration of the recording at mRFP-gephyrin positive loci.

Immunostaining for RFP-gephyrin and analysis.

Neuronal cultures were fixed with 4% paraformaldehyde in 0.1 mM sodium phosphate buffer (pH 7.4) for 15 min at room temperature. Paraformaldehyde was then quenched for 15 min using 33 mM NH₄Cl. Cells were blocked with 0.25% fish gelatin (Sigma) for 30 min at least before immunostaining, using an antibody raised against extracellular epitopes of α2-GABA_AR (224103, 1:700 dilution; Synaptic Systems). Secondary antibodies were coupled to Alexa 488 and used at a 1:1,000 dilution. Imaging was performed on a spinning disk confocal microscope (Leica DM5000B; Leica Microsystems; spinning disk head CSU10; Yokogawa; and Photometrics 63× oil immersion objective N.A. 1.4) equipped with a CCD camera (Coolsnap HQ2; Princeton Instruments) driven by MetaMorph software (Molecular Devices). Endogenous mRFP-gephyrin immunostaining was binarized to determine synaptic localization masks using an MIA interface in MetaMorph as previously described (43). Integrated fluorescence intensities were then quantified cluster-by-cluster using these synaptic localizations. A cluster was defined as synaptic when containing at least a receptor immunoreactivity colocalized with endogenous mRFP gephyrin fluorescence. Quantification of the integrated intensity of colocalized puncta of receptors and scaffold was achieved using custom software in MATLAB (MathWorks) (42). The intensity of colocalized fluorescent spots was averaged for each analyzed cell. Data were normalized by dividing these values by the mean intensity in control conditions. The relative enrichment of receptors over scaffold clusters was calculated cluster by cluster.

Acute slice electrophysiological recordings.

Adult (3-mo-old) mice were anesthetized with isoflurane and decapitated and the brains were rapidly removed and placed in ice-cold normal ACSF (nACSF) containing (in millimolar): 126 NaCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, and 10 dextrose (300–310 mOsm). Coronal sections (350 μm) were prepared in ice-cold ACSF containing 3 mM kynurenic acid using a Leica vibratome. The intact coronal brain slices were stored oxygenated at 33 °C for 2 h before recording. Recordings were performed in a submerged recording chamber maintained at 33 °C (in-line heater; Warner instruments) with a high perfusion rate (4–6 mL/min) while maintaining adequate O₂ tension and physiological pH (7.3–7.4) by continually bubbling the media with a gas mixture of 95% O₂/5% CO₂. Whole-cell patch-clamp recordings were performed on CA1 pyramidal cells using an intracellular recording solution containing (in millimolar): 140 CsCl, 1 MgCl₂, 10 Hepes, 4 NaCl, 0.1 EGTA, 2 Mg-ATP, and 0.3 Na-GTP (pH 7.25, 280–290 mOsm) and electrodes with DC resistance of 5–8 MΩ. Spontaneous IPSCs at V_H = −70 mV in the whole-cell, voltage-clamp configuration were recorded in the presence of 3 mM kynurenic acid. Slices were incubated with either nACSF or E2 (10 nM) for 2 h before recording. Series resistance and whole-cell capacitance were continually monitored and compensated throughout the course of the experiment. Recordings were eliminated from data analysis if series resistance increased by >20%. For all electrophysiology experiments, data acquisition was carried out using an Axon Instruments Axopatch 200B and PowerLab hardware and software (ADInstruments) and data analysis was performed using MiniAnalysis software (Synaptosoft).

Acknowledgments

This work was supported by funding from AstraZeneca, and NIH Grants NS051195, NS081986, MH097446, DA037170-01, 1R01NS087662, MH106954 and Department of Defense Grant AR140209 (to S.J.M.) and NIH, National Institute of Neurological Disorders and Stroke Grant R01 NS073574 (to J. Maguire). S.J.M. acknowledges the support of Grant P30 NS047243 that supports the Tufts Center for Neuroscience Research. A.T. is supported by the Agence Nationale de la Recherche “Synaptune” (Programme blanc, ANR-12-BSV4-0019-01), European Research Council advanced research grant “PlasltInhib”, the program “Investissements d’Avenir” (ANR-10-LABX-54 MEMO LIFE and ANR-11-IDEX-0001-02 PSL* Research University), and the Institut National de la Santé et de la Recherche Médicale). D.P.S. is supported by Medical Research Council Grant MR/L021064/1.

Footnotes

↵¹To whom correspondence should be addressed. Email: stephen.moss{at}tufts.edu.

Author contributions: J. Mukherjee, R.A.C., Y.C.-B., T.Z.D., M.N.P., N.J.B., and S.J.M. designed research; J. Mukherjee, R.A.C., Y.C.-B., and J. Maguire performed research; S.K.T. contributed new reagents/analytic tools; J. Mukherjee, R.A.C., T.Z.D., D.P.S., A.T., J. Maguire, N.J.B., and S.J.M. analyzed data; and R.A.C., T.Z.D., D.P.S., M.N.P., A.T., J. Maguire, and N.J.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1705075114/-/DCSupplemental.

Published under the PNAS license.

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Article Classifications

Biological Sciences
Neuroscience

[1] ↵
Woolley CS
(2007) Acute effects of estrogen on neuronal physiology. Annu Rev Pharmacol Toxicol 47:657–680.
OpenUrl CrossRef PubMed

[2] Woolley CS

[3] ↵
Sato SM,
Woolley CS
(2016) Acute inhibition of neurosteroid estrogen synthesis suppresses status epilepticus in an animal model. ELife 5:e12917, and erratum (2016) 5:e19109.
OpenUrl

[4] Sato SM,

[5] Woolley CS

[6] ↵
Potier M, et al.
(2016) Temporal memory and its enhancement by estradiol requires surface dynamics of hippocampal CA1 N-methyl-D-aspartate receptors. Biol Psychiatry 79:735–745.
OpenUrl CrossRef PubMed

[7] Potier M, et al.

[8] ↵
Kramar EA, et al.
(2009) Cytoskeletal changes underlie estrogens acute effects on synaptic transmission and plasticity. J Neurosci 29:12982–12993.
OpenUrl Abstract/FREE Full Text

[9] Kramar EA, et al.

[10] ↵
Srivastava DP,
Woolfrey KM,
Penzes P
(2013) Insights into rapid modulation of neuroplasticity by brain estrogens. Pharmacol Rev 65:1318–1350.
OpenUrl Abstract/FREE Full Text

[11] Srivastava DP,

[12] Woolfrey KM,

[13] Penzes P

[14] ↵
Moss SJ,
Smart TG
(2001) Constructing inhibitory synapses. Nat Rev Neurosci 2:240–250.
OpenUrl CrossRef PubMed

[15] Moss SJ,

[16] Smart TG

[17] ↵
Sieghart W,
Sperk G
(2002) Subunit composition, distribution and function of GABA(A) receptor subtypes. Curr Top Med Chem 2:795–816.
OpenUrl CrossRef PubMed

[18] Sieghart W,

[19] Sperk G

[20] ↵
Jacob TC,
Moss SJ,
Jurd R
(2008) GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat Rev Neurosci 9:331–343.
OpenUrl CrossRef PubMed

[21] Jacob TC,

[22] Moss SJ,

[23] Jurd R

[24] ↵
Fritschy JM,
Harvey RJ,
Schwarz G
(2008) Gephyrin: Where do we stand, where do we go? Trends Neurosci 31:257–264.
OpenUrl CrossRef PubMed

[25] Fritschy JM,

[26] Harvey RJ,

[27] Schwarz G

[28] ↵
Mukherjee J, et al.
(2011) The residence time of GABAARs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin. J Neurosci 31:14677–14687.
OpenUrl Abstract/FREE Full Text

[29] Mukherjee J, et al.

[30] ↵
Tretter V, et al.
(2008) The clustering of GABAA receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin. J Neurosci 28:1356–1365.
OpenUrl Abstract/FREE Full Text

[31] Tretter V, et al.

[32] ↵
Tretter V, et al.
(2011) Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin. J Biol Chem 286:37702–37711.
OpenUrl Abstract/FREE Full Text

[33] Tretter V, et al.

[34] ↵
Specht CG, et al.
(2013) Quantitative nanoscopy of inhibitory synapses: Counting gephyrin molecules and receptor binding sites. Neuron 79:308–321.
OpenUrl CrossRef PubMed

[35] Specht CG, et al.

[36] ↵
Kneussel M, et al.
(1999) Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice. J Neurosci 19:9289–9297.
OpenUrl Abstract/FREE Full Text

[37] Kneussel M, et al.

[38] ↵
Brünig I,
Scotti E,
Sidler C,
Fritschy JM
(2002) Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro. J Comp Neurol 443:43–55.
OpenUrl CrossRef PubMed

[39] Brünig I,

[40] Scotti E,

[41] Sidler C,

[42] Fritschy JM

[43] ↵
Essrich C,
Lorez M,
Benson JA,
Fritschy JM,
Lüscher B
(1998) Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat Neurosci 1:563–571.
OpenUrl CrossRef PubMed

[44] Essrich C,

[45] Lorez M,

[46] Benson JA,

[47] Fritschy JM,

[48] Lüscher B

[49] ↵
Möhler H,
Fritschy JM,
Crestani F,
Hensch T,
Rudolph U
(2004) Specific GABA(A) circuits in brain development and therapy. Biochem Pharmacol 68:1685–1690.
OpenUrl CrossRef PubMed

[50] Möhler H,

[51] Fritschy JM,

[52] Crestani F,

[53] Hensch T,

[54] Rudolph U

[55] ↵
Nilsson S,
Koehler KF,
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New Research In

Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABA_A receptors at inhibitory synapses

Significance

Abstract

Results

E2 Treatment Reduces the Number of Synaptic GABA_ARs and Gephyrin in Cultured Cortical Neurons.

E2 Selectively Reduces the Amplitude of Miniature Inhibitory Synaptic Currents in Cultured Cortical Neurons.

E2 Does Not Modify the Cell Surface or Total Protein Expression of GABA_ARs or Gephyrin.

SPT Reveals That ER-Mediated Signaling Reduces the Confinement of GABA_ARs at Inhibitory Synapses.

E2 Modifies the Efficacy of GABAergic Inhibition in Hippocampal Slices.

Discussion

Materials and Methods

Biochemical Measurements, Confocal Imaging, and Image Analysis.

Electrophysiology.

SPT Experiments.

SI Materials and Methods

Cell Culture, Drug Treatment, Detergent Solubilization, and Western Blotting.

Biotinylation.

Reagents and antibodies.

Surface Labeling of Estrogen Receptors.

Confocal Microscopy and Image Analysis.

Cortical Culture Electrophysiology.

SPT and analysis.

Cell culture.

SPT experiments and analysis.

Immunostaining for RFP-gephyrin and analysis.

Acute slice electrophysiological recordings.

Acknowledgments

Footnotes

References

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Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABAA receptors at inhibitory synapses

Significance

Abstract

Results

E2 Treatment Reduces the Number of Synaptic GABAARs and Gephyrin in Cultured Cortical Neurons.

E2 Selectively Reduces the Amplitude of Miniature Inhibitory Synaptic Currents in Cultured Cortical Neurons.

E2 Does Not Modify the Cell Surface or Total Protein Expression of GABAARs or Gephyrin.

SPT Reveals That ER-Mediated Signaling Reduces the Confinement of GABAARs at Inhibitory Synapses.

E2 Modifies the Efficacy of GABAergic Inhibition in Hippocampal Slices.

Discussion

Materials and Methods

Biochemical Measurements, Confocal Imaging, and Image Analysis.

Electrophysiology.

SPT Experiments.

SI Materials and Methods

Cell Culture, Drug Treatment, Detergent Solubilization, and Western Blotting.

Biotinylation.

Reagents and antibodies.

Surface Labeling of Estrogen Receptors.

Confocal Microscopy and Image Analysis.

Cortical Culture Electrophysiology.

SPT and analysis.

Cell culture.

SPT experiments and analysis.

Immunostaining for RFP-gephyrin and analysis.

Acute slice electrophysiological recordings.

Acknowledgments

Footnotes

References

Citation Manager Formats

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Article Classifications

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Information

Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABA_A receptors at inhibitory synapses

E2 Treatment Reduces the Number of Synaptic GABA_ARs and Gephyrin in Cultured Cortical Neurons.

E2 Does Not Modify the Cell Surface or Total Protein Expression of GABA_ARs or Gephyrin.

SPT Reveals That ER-Mediated Signaling Reduces the Confinement of GABA_ARs at Inhibitory Synapses.