Estrogen receptor alpha and beta specific agonists regulate expression of synaptic proteins in rat hippocampus

Abstract

Changes in hippocampal CA1 dendritic spine density and synaptic number across the estrous cycle in female rats correlate with increased hippocampal-dependent cognitive performance in a manner that is dependent on estrogen receptors (ERs). Two isoforms of the estrogen receptor, alpha and beta are present in the rat hippocampus and distinct effects on cognitive behavior have been described for each receptor. The present study generated a profile of synaptic proteins altered by administration of estradiol benzoate, the ERalpha selective agonist PPT (1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole) and the ERbeta selective agonist DPN (2,3-bis (4-hydroxyphenyl) propionitrile) alone and in combination in comparison to vehicle in the CA1 region of the dorsal hippocampus. In the stratum radiatum, estradiol, DPN, and PPT increased PSD-95 and AMPA-type glutamate receptor subunit GluR1. Only DPN administration regulated expression of AMPA receptor subunits GluR2 and GluR3, increasing and decreasing levels respectively. DPN also increased GluR2 expression in the other lamina of the CA1. These results support previous reports that estradiol and isoform specific agonists differentially activate ERalpha and ERbeta to regulate protein expression. The distinct effects of DPN and PPT administration on synaptic proteins suggest that the desired therapeutic outcome of estrogen may be accomplished by using specific estrogen receptor agonists. Moreover, the effects of estradiol treatment on PSD-95 expression are consistent with a growing body of evidence that this postsynaptic protein is a key marker of estrogen action related to spine synapse formation.

Figure 1

Examination of synaptic protein expression in the hippocampal formation. A: Schematic representation of hippocampal formation with boxes to demonstrate the areas of the CA1, CA3 and dentate gyrus (DG) analyzed [59]. B: Distribution of synaptic proteins in the CA1 region of the hippocampus. Representative light photomicrographs of synaptophysin (Syn), spinophilin (Spn), PSD-95, GluR1, GluR2, GluR3, VGaT, and VGlut1 peroxidase labeling. Dotted lines demonstrate the medial portion of the stratum radiatum (SR) relative to the strata pyramidale (SP) and lacunosum-moleculare (SLM). Scale bar = 20 µm.

Figure 2

Effects of selective ER agonist administration on synaptophysin, spinophilin, and PSD-95 immunoreactivity in the CA1 stratum radiatum of the dorsal hippocampus. A: Synaptophysin expression was not altered by any of the steroid regimens when compared to vehicle (Veh). B: Labeling for spinophilin was not altered in any treatment group compared to vehicle. In comparison to EB treatment, DPN plus PPT significanly reduced synatophysin levels (post hoc test * p < 0.05). C: PSD-95 immunoreactivity was significantly higher in EB, PPT alone and DPN + PPT treated hippocampi compared to vehicle (post hoc test * p < 0.05). In the DPN alone group there was a trend for increased PSD-95 expression (post hoc test # p = 0.061). For this and all subsequent figures error bars represent SEM.

Figure 3

Expression of vesicular transporters in stratum radiatum of CA1 did not change with any of the steroid regimens. EB, PPT alone, DPN alone and PPT + DPN did not significantly alter VGluT1 or VGaT expression levels in comparison to vehicle.

Figure 4

Estradiol and ER selective agonists differentially regulated AMPA receptor subunits GluR1-3 in CA1 stratum radiatum. A: The level of GluR1 immunoreactivity was significantly increased relative to vehicle by PPT alone and DPN alone (post hoc test * p < 0.05). There was a trend for EB to increase the levels of GluR1 immunoreactivity (post hoc test # p = 054). B: GluR2 labeling was significantly increased by DPN alone (post hoc test * p < 0.05) but not by EB, PPT alone or DPN + PPT when compared to vehicle. C: A significant decrease in the levels of GluR3 immunoreactivity was observed with DPN alone (post-hoc test * p < 0.05) but not administration of EB or PPT alone.

Figure 5

Steroid replacement alters GluR2 and PSD-95 labeling selectively in other CA1 layers and in the dentate gyrus. A: DPN administration significantly increased GluR2 immunoreactivity is in the CA1 stratum pyramidale (SP) and stratum oriens (SO) (post hoc test * p < 0.05). There was no effect of DPN in the stratum lacunosum-moleculare (SLM) and the other steroid regimens had no effect in any of the layers. B: In the dentate gyrus, compared to vehicle, EB increased PSD-95 levels in the inner molecular layer (IML) and in the middle molecular layer (MML) (post hoc test * p < 0.05). There was no effect of any treatment on the outer molecular layer (OML), granule cell layer (GCL) or central hilus (CH).