Sex Differences in Molecular Signaling at Inhibitory Synapses in the Hippocampus

Mutual occlusion of E2- and TBF-induced suppression of inhibition. A, Individual traces and time course of IPSC suppression in a representative experiment in which E2 (100 nm) was applied before delivering TBF stimulation. E2 occluded TBF-induced iLTD. Each point in the time course is an individual sweep, and SR 95531 (SR; 2 μm) applied at the end of the experiment blocked IPSCs (also in E). B, Group IPSC amplitude data for experiments with E2-responsive IPSCs (n = 6). Connected open symbols are individual cells; filled symbols are mean ± SEM for all cells (also in C, D, F–H). C, Group PPR data for the same cells as in B; *p < 0.05, paired t test. D, Group IPSC amplitude data for E2- and TBF-nonresponsive experiments (n = 6). E, Individual traces and time course of IPSC suppression in a representative experiment in which TBF was delivered before E2. TBF occluded E2-induced IPSC suppression. F, Group IPSC amplitude data for all TBF-responsive experiments (n = 6). G, Group PPR data for the same cells as in F; **p < 0.01, paired t test. H, Group IPSC amplitude data for TBF- and E2-nonresponsive experiments (n = 7). Not shown are two cells that failed to respond to TBF but did respond to E2 (see Results). Calibration: A, 50 pA, 25 ms (also applies to E).

E2-induced suppression of inhibition requires PLC. A, Individual traces and time course of IPSC suppression in a representative experiment in which E2 (100 nm) was applied first in the presence of U73122 (U; PLC inhibitor, 10 μm) and then again after U73122 washout to confirm E2 responsiveness of IPSCs. U73122 blocked E2-induced IPSC suppression. Each point in the time course is an individual sweep, and SR 95531 (SR; 2 μm) applied at the end of the experiment blocked IPSCs (also in E). B, Group IPSC amplitude data for experiments with E2-responsive IPSCs (n = 7). Connected open symbols are individual cells; filled symbols are mean ± SEM for all cells (also in C, F, G). C, Group PPR data for the same cells as in B; **p < 0.01, paired t test. D, Normalized IPSC amplitude for E2-responsive (n = 7, same as in B and C) and E2-nonresponsive (n = 5) experiments. E, Individual traces and time course of IPSC suppression in a representative experiment in which E2 was applied in the presence of U73343 (an inactive analog of U73122, 10 μm). E2 decreased IPSC amplitude in the presence of U73343. F, Group IPSC amplitude data for all E2-responsive experiments (n = 7). G, Group PPR data for the same cells as in F; **p < 0.01, paired t test. H, Normalized IPSC amplitude for E2-responsive (n = 7, same as in F and G) and E2-nonresponsive (n = 5) experiments in the presence of U73343. Calibration: A, 50 pA, 25 ms; E, 25 pA, 25 ms.

Sex difference in E2-induced generation of IP3. A, Fold change in intracellular IP₃ levels measured in hippocampal slices from male rats treated with vehicle (veh) or DHPG (100 μm) for 0, 5, 10, 15, 20, 30, or 60 s. Connected open symbols are independent experiments (n = 3); filled symbols are mean ± SEM for all experiments (also in B–D). DHPG increased IP₃ levels by 9.7-fold in males peaking at 15 s and returning to baseline by 30 s. B, Fold change in intracellular IP₃ levels measured in hippocampal slices from female rats treated exactly as in A (n = 3). Similar to males, DHPG increased IP₃ levels by 9.8-fold in females, peaking at 15 s and returning to baseline by 30 s. C, Fold change in intracellular IP₃ levels measured in hippocampal slices from male and female rats treated with vehicle or E2 (100 nm) for 0, 10, 15, 30, 60, or 90 s (n = 3 in each sex). E2 increased IP₃ levels in both sexes but to a much greater extent in females (7.3-fold) than males (2.6-fold). Two-way ANOVA with repeated measures showed significant effects of treatment (p < 0.001), sex (p < 0.001), and a treatment × sex interaction (p < 0.001). D, Fold change in intracellular IP₃ levels measured in hippocampal slices from female rats pretreated with vehicle or JNJ 16259685 (JNJ; mGluR1 antagonist, 0.2 μm) each followed by the addition of vehicle or E2 (100 nm) for 0, 10, 15, 30, 60, or 90 s (n = 2). JNJ 16259685 blocked the E2-induced increase in IP₃ (p < 0.05 one-way repeated-measures ANOVA, followed by Tukey's post hoc test).

E2 acutely promotes ERα–mGluR1 interaction in females but not males. A, Hippocampal slices from male and female rats were treated with either E2 (100 nm) or vehicle (veh) for 10 min. Membrane fractions were prepared from these slices and subjected to IP using anti-mGluR1a. Representative Western blots (WB) of input and IP samples from males and females on the same gel including a no-lysate control are shown. Blots were probed for ERα, PSD-95 (negative control), and mGluR1a. B, Mean ± SEM of ERα and mGluR1 levels in males and females (n = 5 independent experiments). E2 increased the levels of ERα associated with mGluR1a in females, but not in males, without affecting mGluR1a levels in either sex; *p < 0.05, Student's t test. C, ERα/mGluR1 ratio for the same five experiments as in B (see Materials and Methods). Connected open symbols are individual experiments; filled symbols are mean ± SEM for all experiments. E2 increased ERα/mGluR1 ratio in females with no effect in males; *p < 0.05, paired t test. D, Hippocampal slices from female rats were treated either with E2 (100 nm) or vehicle for 10 min. Membrane fractions were prepared from these slices and subjected to IP using anti-mGluR5. Representative Western blots of input and IP samples probed for ERα and mGluR5 are shown. No interaction was detected between ERα and mGluR5 in either of the IP samples. E, Representative electron micrographs of three serial sections (E1–E3) showing ERα immunoreactivity (ERα-IR) located in patches along the CA1 pyramidal cell somatic plasma membrane. Note endoplasmic reticulum associated with ERα–IR and an axonal varicosity apposed to the soma near ERα–IR. Scale bar: E3, 500 nm (applies to all panels).

E2 acutely promotes mGluR1–IP₃R interaction in females but not males. A, Hippocampal slices from male and female rats were treated either with E2 (100 nm) or vehicle (veh) for 10 min. Membrane fractions were prepared from these slices and subjected to IP using anti-mGluR1a. Representative Western blots (WB) of input and IP samples from males and females on the same gel are shown. The blots were probed for IP₃R, Homer-1b/c and mGluR1. B, Mean ± SEM of IP₃R and mGluR1 levels in males and females (n = 6 independent experiments). E2 increased the levels of IP₃R associated with mGluR1a in females, but not in males, without affecting mGluR1a levels in either sex; **p < 0.01, Student's t test. C, IP₃R/mGluR1 ratio for the same six experiments as in B (see Materials and Methods). Connected open symbols are individual experiments (n = 6); filled symbols are mean ± SEM for all experiments. E2 increased the IP₃R/mGluR1 ratio in females with no effect in males; **p < 0.01, paired t test. D, Individual traces and time course of IPSC suppression in a representative experiment in which E2 (100 nm) was applied first in the presence of Xest (IP₃R inhibitor, 2 μm) and then again after Xest washout to confirm E2 responsiveness of IPSCs. Dotted line shows average IPSC amplitude during 2 min before the second E2 application. Xest blocked E2-induced IPSC suppression. Each point in the time course is an individual sweep, and SR 95531 (SR; 2 μm) applied at the end of the experiment blocked IPSCs. E, Group IPSC amplitude data for all experiments with E2-responsive IPSCs (n = 4). Connected open symbols are individual cells; filled symbols are mean ± SEM for all cells. Calibration: D, 25 pA, 25 ms.

A subset of CA1 pyramidal cells expresses mGluR1a mRNA. A, A schematic mGluR1a mRNA sequence showing three regions that were targeted in single-cell nested PCR experiments: the first region is located within the N-terminal extracellular domain common in all splice variants, the second region is located after the seven transmembrane domain (7TMD) and includes the splicing region, and the third region is located in the C-terminal domain that contains the recognition site for the mGluR1a antibody used in co-IP experiments. In the second real-time PCR step, five separate pairs of nested PCR primers targeted fragments of various lengths within the first PCR amplicons (mGluR1 pan, mGluR1a-1, -2, -3, -4). B, Specificity of all primers was tested on whole hippocampal mRNA isolated from a female rat. Representative gel image showing unique amplicons of appropriate sizes for each gene tested. C, Single-cell nested PCR results showing mRNA expression profiles for all putative CA1 pyramidal neurons and GABAergic neurons derived from eight female rats. To be considered as a putative CA1 pyramidal neuron, a cell was required to be positive for GAPDH and HPRT (housekeeping genes) and negative for ERBB4, vGAT, GAD65 (GABAergic markers), and GFAP (astrocyte marker). To be considered mGluR1a positive, a cell was required to be positive for all five mGluR1a fragments. Of 28 putative CA1 pyramidal neurons, 14 (50%) were mGluR1a positive and 24 (86%) were mGluR5 positive.

E2-induced suppression of inhibition is Ca²⁺ dependent and requires postsynaptic eCB release. A, Individual traces and time course of a representative experiment in which E2-induced suppression of inhibition was tested with AM404 (AM; eCB transporter inhibitor, 2 μm) applied postsynaptically via the recording pipette. Each point in the time course is an individual sweep, and SR 95531 (SR; 2 μm) applied at the end of the experiment blocked IPSCs (also in D, G, H). E2-induced suppression of inhibition was rare in AM404-loaded cells. B, Normalized IPSC amplitude for E2-responsive and -nonresponsive experiments with and without AM404 in the recording pipette. Of 19 cells recorded with AM404, only three showed E2-induced IPSC suppression compared with 29 of 52 cells recorded without AM404; p < 0.01, χ² test. C, DSI was normal in the same AM404-loaded cells as in B, confirming that AM404 did not affect 2-AG-mediated modulation of inhibition. Points are mean ± SEM for all experiments. D, Individual traces and time course of a representative experiment in which BAPTA (Ca²⁺ chelator, 20 mm) was applied postsynaptically via the recording pipette. E, Group IPSC amplitude data for all experiments with BAPTA (n = 17). Connected open symbols are individual cells; filled symbols are mean ± SEM for all cells. E2-induced suppression of inhibition was rare in BAPTA-loaded cells. F, Normalized IPSC amplitude for E2-responsive and -nonresponsive experiments with and without BAPTA in the recording pipette. Of 17 cells recorded with BAPTA, only one showed E2-induced IPSC suppression, compared with 29 of 52 cells recorded without BAPTA; p < 0.001, χ² test. G, Individual traces and time course of a representative experiment in which URB597 (URB; FAAH inhibitor, 1 μm) was applied to a male slice. URB597 had no overall effect on IPSC amplitude in males. H, Individual traces and time course of a representative experiment in which URB597 was applied to a female slice. URB597 often decreased IPSC amplitude in females. I, Normalized IPSC amplitude for all cells recorded from both sexes in the presence of URB597 showing a significant sex difference in response rate. Of 17 cells recorded in males, only one showed a URB597-induced decrease in IPSC amplitude, compared with 10 of 18 cells recorded in females; p < 0.01, χ² test. J, Group PPR data for all URB597-responsive cells in females (n = 10); **p < 0.01, paired t test. Calibration: 25 pA, 25 ms.