Diverse Synaptic Distributions of G Protein-coupled Estrogen Receptor 1 in Monkey Prefrontal Cortex with Aging and Menopause

Abstract

Age- and menopause-related impairment in working memory mediated by the dorsolateral prefrontal cortex (dlPFC) occurs in humans and nonhuman primates. Long-term cyclic 17β-estradiol treatment rescues cognitive deficits in aged ovariectomized rhesus monkeys while restoring highly plastic synapses. Here we tested whether distributions of G protein-coupled estrogen receptor 1 (GPER1) within monkey layer III dlPFC synapses are sensitive to age and estradiol, and coupled to cognitive function. Ovariectomized young and aged monkeys administered vehicle or estradiol were first tested on a delayed response test of working memory. Then, quantitative serial section immunoelectron microscopy was used to determine the distributions of synaptic GPER1. GPER1-containing nonperforated axospinous synapse density was reduced with age, and partially restored with estrogen treatment. The majority of synapses expressed GPER1, which was predominately localized to presynaptic cytoplasm and mitochondria. GPER1 was also abundant at plasmalemmas, and within cytoplasmic and postsynaptic density (PSD) domains of dendritic spines. GPER1 levels did not differ with age or treatment, and none of the variables examined were tightly associated with cognitive function. However, greater representation of GPER1 subjacent to the PSD accompanied higher synapse density. These data suggest that GPER1 is positioned to support diverse functions key to synaptic plasticity in monkey dlPFC.

Keywords: Area 46; GPER1; delayed response; estradiol; synapse.

Figure 1.

DR task performance accuracy. V, vehicle; E, estradiol. Group results are expressed as the mean ± SEM. Young OXV + V, n = 7; young OVX + E, n = 6; aged OVX + V, n = 7; aged OVX + E, n = 6.

Figure 2.

Age effects on areal densities of synapses. (A), Areal density of the total population of synapses. (B), Areal density of synapses that contained GPER1 immunogold particles. (C), Areal density of GPER1-containing synapses that possessed nonperforated postsynaptic densities. (D), Areal density of GPER1-containing synapses that possessed perforated postsynaptic densities. V, vehicle; E, estradiol. Group results are expressed as the mean ± SEM. Significant age effects indicated by *P < 0.05 and **P < 0.01. Young OXV + V, n = 7; young OVX + E, n = 6; aged OVX + V, n = 7; aged OVX + E, n = 6.

Figure 3.

Densities of GPER1 immunogold particles in the presynaptic and postsynaptic compartments. V, vehicle; E, estradiol. Group results are expressed as the mean ± SEM. Young OXV + V, n = 7; young OVX + E, n = 6; aged OVX + V, n = 7; aged OVX + E, n = 6.

Figure 4.

Subcellular synaptic distribution of GPER1 immunogold particles. (A), Representative electron micrographs of 5 serial sections through a GPER1-containing perforated synapse spine. The postsynaptic density is readily apparent (black arrowheads). GPER1 immunogold particles are shown localized to mitochondrial (orange arrowheads), cytoplasmic (light beige arrowheads), synaptic (light green arrowheads), and subsynaptic (dark green arrowheads) domains. For each series, the third section (outlined with a black box) was used as a reference section, and all synapses that possessed a dendritic spine with a clear postsynaptic density in this section were marked and followed throughout the series for morphological and immunolabeling assessments. At, axon terminal; sp, dendritic spine; sa, spine apparatus. Scale bar, 250 nm. (B), Schematic diagram illustrating the 8 synaptic domains used to categorize the location of each GPER1 immunogold particle: active zone (1); mitochondrial (2); plasmalemmal, axon terminal (3); cytoplasmic, axon terminal (4); synaptic (5); subsynaptic (6); plasmalemmal, spine (7); and, cytoplasmic, spine (8). (C), Plots of the number of GPER1 immunogold particles within axon terminal (left), and dendritic spine (right) compartments averaged across all monkeys. Data are expressed as the mean ± SEM. n = 3141 synapses.

Figure 5.

Percentage of postsynaptic GPER1 immunogold particles localized to the subsynaptic domain, and its relationship to synapse areal density. (A), Bar graph of the percentage of postsynaptic GPER1 immunogold particles localized to the subsynaptic domain of GPER1-containing synapses. (B), Positive correlation between the percentage of postsynaptic GPER1 immunogold particles localized to the subsynaptic domain and the areal density of GPER1-containing synapses (left), and of GPER1-containing synapses with nonperforated PSDs (right) for each monkey. V, vehicle; E, estradiol. Group results are expressed as the mean ± SEM. Significant age effect indicated by **P < 0.01. Young OXV + V, n = 7; young OVX + E, n = 6; aged OVX + V, n = 7; aged OVX + E, n = 6.

Figure 6.

Schematic diagram illustrating proposed relationships of GPER1 distributions to aging and estradiol treatment. Nonperforated synapse spines containing GPER1 are lost with age, but are partially restored to levels of young monkeys with estrogen treatment (1). The percentage of GPER1 immunogold particles localized to the subsynaptic domain of nonperforated, but not of perforated, synapse spines is lower in aged than in young monkeys, and is associated with a low synapse density (2). Diagrams were drawn based on current results as well as data from previous work (Hao et al. 2006, 2007). Npf, nonperforated; pf, perforated.