A novel mechanism of dendritic spine plasticity involving estradiol induction of prostaglandin-E2

Abstract

The mechanisms establishing and maintaining dendritic spines in the mammalian CNS remain primarily unknown. We report a novel mechanism of neuronal spine plasticity in the developing preoptic area in which estradiol induces prostaglandin-E2 (PGE2) synthesis that in turn increases the density of spine-like processes. Estradiol requires PGE2 synthesis, in vivo and in vitro, and upregulates the dendritic spine protein spinophilin, an effect attenuated by antagonism of the AMPA-kainate receptor. This signaling pathway may involve cross talk between neighboring neurons and astrocytes and appears specific to the preoptic area in that hippocampal neurons responded with an increase in spinophilin to estradiol but not PGE2. Regionally specific mechanisms of estradiol-mediated synaptic plasticity allow for epigenetic control of complex sex-typic behaviors.

Fig. 1.

Microdissection of the preoptic area and the rostral hippocampal formation. A, Sagittal drawing of the developing rat brain demonstrating the areas collected (shaded regions) and the landmarks used to guide the microdissection, including the anterior commissure (ac) and the optic chiasm (ox) for the preoptic area (POA), and the corpus callosum and the dorsal portion of the third ventricle (d3v) for the hippocampal formation (hf). B,Coronal drawing of the neonatal brain at the level of the POA.Top left, Three-dimensional reconstruction of the neonatal brain at the level of the section (taken from Altman and Bayer, 1995). Top right, Sagittal view of the neonatal brain displaying the rostral to caudal coordinates used for collecting the POA. Shaded region represents the area collected for microdissections that were guided by the anterior commissure and the optic chiasm. cc, Corpus callosum;BST, bed nucleus of the stria terminalis;3v, third ventricle. Grid scale, 0.5 mm.

Fig. 2.

E₂ or PGE₂ increase spine-like processes of preoptic area neurons in vitro. A, Representative photomicrograph of cultured neurons treated with 0.5 nm PGE₂ on DIV1 and DIV2 and visualized on DIV3 after immunocytochemical detection of MAP-2. Numerous aspects of neuronal morphology were assessed, but the only changes observed were in the number and density of spine-like processes, defined as any protrusion <5 μm in length. Scale bar, 25 μm. B, Representative photomicrographs of the neurites of cultured neurons treated with vehicle, 5 nmE₂, or 5 nm E₂coadministered with 1 μm of the cyclooxygenase inhibitor indomethacin, and visualized after MAP-2 immunocytochemistry. Scale bar, 5 μm. C, Treatment with E₂ (5 nm) or PGE₂ (0.5 nm) for 48 hr resulted in a significant increase in the number of spine-like processes on neurites. PGF_2α (0.5 nm) had no effect compared with vehicle-treated controls (10 neurons from each coverslip, n = 6 per group, ANOVA; *p < 0.001 compared with PGF_2α and vehicle). D, Similar effects of E₂ and PGE₂ were seen on mean spine density in a separate experiment. The combination of E₂ and PGE₂ was not different from either one alone. The induction of spine-like processes by E₂ was blocked by simultaneous administration of indomethacin (1 μm) but had no effect on the actions of exogenous PGE₂ (10 neurons from each coverslip,n = 6 per group, ANOVA; *p < 0.001 compared with E₂ + Indo and vehicle).

Fig. 3.

E₂ or PGE₂ increase expression of spinophilin protein in the preoptic area in vitro. A, Representative photomicrograph of the neurites of cultured neurons treated with 5 nmE₂ and visualized after spinophilin immunocytochemistry. Scale bar, 25 μm. B, Enlargement of process encapsulated in A. Each punctum corresponds to one locus of concentrated protein. Scale bar, 5 μm. C,Representative Western immunoblot from treated cultures loaded onto each lane 3 d after plating. Cultures were exposed to the following either individually or in combination for 48 hr: (1) 0.5 nm PGE₂, (2) 5 nmE₂, (3) 1 μm indomethacin, or (4) vehicle. The blot was probed with an anti-spinophilin/neurabin II rabbit polyclonal antibody that recognized an appropriate band at 120 kDa. D, Analysis of Western immunoblots of cultured newborn preoptic area neurons treated for 48 hr in vitro. Data represent a ratio of iad with the iad of the vehicle controls of the same gel serving as the comparison group (n = 6 per group). This standardization allowed for the analysis of seven groups across three gels. Treatment with E₂ or PGE₂ resulted in a significant increase in expression of spinophilin. Coadministration of both did not augment effects seen when applied individually. Indomethacin blocked the estradiol-induced effect on spinophilin expression and had no effect alone (ANOVA; *p < 0.001 compared with Indo, E₂ + Indo, or vehicle).

Fig. 4.

Antagonism of the AMPA–kainate receptor attenuates E₂- or PGE₂-induced spinophilin expression. A, Representative Western immunoblot from treated POA cultures 3 d after plating. Cultures were exposed to the following either individually or in combination for 48 hr: (1) 0.5 nm PGE₂, (2) 5 nmE₂, (3) 10 μm glutamate, (4) 100 μm AP-5, (5) 40 μm MK801, (6) 40 μm DNQX, or (7) vehicle. The blot was probed with an anti-spinophilin/neurabin II rabbit polyclonal antibody that recognized an appropriate band at 120 kDa. B, C, Analysis of Western immunoblots of cultured newborn preoptic area neurons treated for 48 hr in vitro. Data represent a ratio of iad with the iad of the vehicle controls of the same gel serving as the comparison group (n = 6 per group). Treatment with glutamate resulted in an increase in the expression of spinophilin comparable with that induced by either E₂ or PGE₂. Coadministration of the AMPA–kainate antagonist DNQX significantly reduced both the E₂ and PGE₂-induced increase in expression, whereas coapplication of NMDA receptor antagonists had no effect on expression (ANOVA; *p < 0.001 compared with vehicle; **p < 0.001 compared with E₂ + DNQX, PGE₂ + DNQX, and vehicle; ***p < 0.001 compared with E₂ + AP-5, E₂ + MK801, and vehicle).

Fig. 5.

The in vitro effects of E₂ and PGE₂ on spinophilin expression are maintained in vivo. A, The mRNA for three of the four PGE₂ receptor (EP1–4) subtypes are detectable within the POA as early as 4 hr post-utero. Tissue from rat kidney was used as a positive control for EP4. PN0, Postnatal day 0.B, Representative Western immunoblot of tissue from the POA collected from animals treated for 48 hr beginning on P0. Animals were exposed to the following either individually or in combination: (1) 0.1 cc sesame oil (subcutaneously), (2) 100 μg of E₂in 0.1 cc sesame oil (subcutaneously), (3) 25 μg of indomethacin in 0.1 cc sesame oil (subcutaneously), (4) 2 μg of PGE₂ in 0.1 cc in 0.9% saline (intracerebroventricularly), or (5) 0.1 cc of 0.9% saline (intracerebroventricularly). The blot was probed with an anti-spinophilin/neurabin II rabbit polyclonal antibody that recognized an appropriate band at 120 kDa. C, D, Quantification of Western immunoblots of preoptic area tissue treated for 48 hr in vivo. Each analysis represents one immunoblot quantified as a ratio of iad to that of controls of the same gel (n = 4 per group). Treatment with E₂ or PGE₂ resulted in a significant increase in levels of spinophilin. Indomethacin blocked the estradiol-induced effect on spinophilin, whereas its coapplication with PGE₂ had no effect (ANOVAs; *p < 0.001 compared with E₂ + Indo and vehicle; **p < 0.001 compared with vehicle).

Fig. 6.

E₂ increases PGE₂ levels in the developing preoptic area. Quantitative GC–MS analysis was used to measure the levels of various prostanoids and their metabolites in the preoptic area of newborn female rats treated with E₂ or vehicle for 36 hr. A, PGE₂ levels were increased threefold in rats treated with 100 μg of E₂ in 0.1 cc sesame oil (subcutaneously) (t test, *p < 0.002). B, There was no change in PGD₂, PGF_2α, or 6-keto-PGF_1α. TXB₂ was undetectable above background. C, The pathway of arachidonic acid conversion to the prostanoids via cyclooxygenase suggests the effects of E₂ may be acting directly at PGE synthase or indirectly via COX-2.

Fig. 7.

Different mechanisms appear involved in the regulation of spinophilin protein in hippocampal tissue.A, Representative Western immunoblot of tissue from the POA collected from animals treated for 48 hr beginning on P0. Animals were exposed to the following either individually or in combination: (1) 0.1 cc sesame oil (subcutaneously), (2) 100 μg of E₂in 0.1 cc sesame oil (subcutaneously), (3) 25 μg of indomethacin in 0.1 cc sesame oil (subcutaneously), (4) 2 μg of PGE₂ in 0.1 cc in 0.9% saline (intracerebroventricularly), or (5) 0.1 cc of 0.9% saline (intracerebroventricularly). The blot was probed with an anti-spinophilin/neurabin II rabbit polyclonal antibody that recognized an appropriate band at 120 kDa. B, Analysis of Western immunoblots from cultured newborn rostral hippocampal tissue treated for 48 hr in vitro. The analysis represents one immunoblot quantified as a ratio of iad to that of controls of the same gel (n = 4 per group). Treatment with E₂ (5 nm) but not PGE₂ (0.5 nm) resulted in a significant increase in expression of spinophilin (ANOVA; *p < 0.001 compared with PGE₂ or vehicle). C, D, Quantification of Western immunoblots from animals treated 48 hr in vivoas described in A. Each analysis represents one immunoblot quantified as a ratio of iad to that of controls of the same gel (n = 4 per group). Treatment with E₂alone or in combination with indomethacin resulted in a significant increase in expression of spinophilin. PGE₂ alone or in combination with indomethacin had no effect on spinophilin expression (ANOVA; **p < 0.001 compared with vehicle).

Fig. 8.

Sexually dimorphic expression of spinophilin on the day of birth in the POA. A, Western immunoblot of preoptic area tissue collected from males and females on the day of birth. The blot was probed with an anti-spinophilin/neurabin II rabbit polyclonal antibody that recognized an appropriate band at 120 kDa.B, Quantification of the above Western immunoblot of male and female preoptic area tissue. We determined iad for each band in arbitrary units, and males were compared to females on the same gel (n = 5/group). Males displayed two and a half times the level of spinophilin protein in the POA as compared with that of females (t test, ∗p < 0.001). The magnitude of difference is consistent with the effects on spinophilin levels induced by estradiol or PGE₂ treatment bothin vivo and in vitro.