Estrogen receptors observed at extranuclear neuronal sites and in glia in the nucleus accumbens core and shell of the female rat: Evidence for localization to catecholaminergic and GABAergic neurons

Abstract

Estrogens affect dopamine-dependent diseases/behavior and have rapid effects on dopamine release and receptor availability in the nucleus accumbens (NAc). Low levels of nuclear estrogen receptor (ER) α and ERβ are seen in the NAc, which cannot account for the rapid effects of estrogens in this region. G-protein coupled ER 1 (GPER1) is observed at low levels in the NAc shell, which also likely does not account for the array of estrogens' effects in this region. Prior studies demonstrated membrane-associated ERs in the dorsal striatum; these experiments extend those findings to the NAc core and shell. Single- and dual-immunolabeling electron microscopy determined whether ERα, ERβ, and GPER1 are at extranuclear sites in the NAc core and shell and whether ERα and GPER1 were localized to catecholaminergic or γ-aminobutyric acid-ergic (GABAergic) neurons. All three ERs are observed, almost exclusively, at extranuclear sites in the NAc, and similarly distributed in the core and shell. ERα, ERβ, and GPER1 are primarily in axons and axon terminals suggesting that estrogens affect transmission in the NAc via presynaptic mechanisms. About 10% of these receptors are found on glia. A small proportion of ERα and GPER1 are localized to catecholaminergic terminals, suggesting that binding at these ERs alters release of catecholamines, including dopamine. A larger proportion of ERα and GPER1 are localized to GABAergic dendrites and terminals, suggesting that estrogens alter GABAergic transmission to indirectly affect dopamine transmission in the NAc. Thus, the localization of ERs could account for the rapid effects of estrogen in the NAc.

Keywords: G-protein coupled estrogen receptor 1; electron microscopy; estrogen receptor alpha; estrogen receptor beta; ventral striatum; γ-aminobutyric acid.

FIGURE 1

Light microscopic examination of estrogen receptors (ERs) in the nucleus accumbens (NAc). a) Depiction of the region analyzed in electron microscopy experiments; the blue rectangle was considered the NAc core while the yellow was the NAc shell. b and c) Moderate levels of nuclear, but no extranuclear, labeling were observed for ERα. d and e) Very sparse nuclear labeling for ERβ was observed. f and g) Dense extranuclear labeling, but no nuclear labeling for G-protein coupled ER 1 (GPER1) was observed in the NAc. Black arrows, immunoreactive cells/nuclei. Dashed line indicates approximate delineation of the NAc core and shell. Scale bar b,d,f = 200 μm; c,e,g = 100 μm

FIGURE 2

Electron micrographs show examples of estrogen receptor α (Erα)-containing profiles in the nucleus accumbens (NAc) core and shell. ERα-immunoreactivity (IR) is observed in: a) a two axon terminals (TER), one that forms a synapse with an unlabeled dendritic spine (uSP), in the NAc core; b) at the membrane of a glial process (GL) in the NAc core; c) a dendrite (DEN), where it is associated with the membrane of a mitochondrion (mit), the cell membrane, and microtubules in the NAc shell; d) a dendritic spine (SP) that forms a synapse with an unlabeled terminal (uTER), and an axon (AX) in the NAc shell. Black arrow, immunoperoxidase for ERα. Scale bar, 500 nm

FIGURE 3

Electron micrographs show examples of estrogen receptor β (Erβ)-containing profiles in the nucleus accumbens (NAc) core and shell. a) ERβ-IR is observed in a terminal (TER), where it is localized to a mitochondrion (mit), small synaptic vesicles, and the plasma membrane. ERβ-IR is also associated with the membrane of a glial cell (GL) in the NAc shell; b) the membrane and microtubules of a dendrite (DEN) in the NAc core; c) an axon (AX). Black arrow, immunoperoxidase for ER. Scale bar, 500 nm

FIGURE 4

Electron micrographs show examples of G-protein coupled ER 1 (GPER1)-containing profiles in the nucleus accumbens (NAc) core and shell. GPER1 immunoreactivity is observed in: a) an axon (AX) and associated with the membrane of a glial process (GL) in the NAc shell; b) a soma where it is associated with Golgi bodies. GPER1-immunoreactivity is also associated with the membrane of a glial cell (GL) in the NAc core; c) a dendrite (DEN), where it is associated with the membrane of a mitochondrion (mit), a dendritic spine (SP) and vesicles in an axon terminal (TER), in the NAc shell; d) in two axons in the NAc core. Black arrow, immunoperoxidase for ER. Scale bar, 500 nm

FIGURE 5

Electron micrographs show examples of profiles containing of estrogen receptor α (Erα) or G-protein coupled ER 1 (GPER1) immunoreactivity and tyrosine hydroxylase (TH) immunoreactivity in the nucleus accumbens (NAc) core and shell. a) Immunoreactivity for ERα is associated with small synaptic vesicles and a mitochondrion in a catacholaminergic terminal (TER) that is adjacent to an unlabeled dendrite (uDEN). b) GPER1 immunoreactivity associated with synaptic vesicles close to the synapse in two catecholaminergic terminals (TER), and one noncatecholaminergic terminal (uTER). c) ERα-IR TER in close proximity to an uDEN. d) GPER1 immunoreactivity associated with synaptic vesicles in a TER, and an axon (AX). Black arrow, immunoperoxidase for ER; white arrow, immunogold for TH. Scale bar, 500 nm

FIGURE 6

Electron micrographs show examples of profiles containing of estrogen receptor α (Erα) or G-protein coupled ER 1-immunoreactivity (GPER1-IR) and GABA-IR in the nucleus accumbens (NAc) core and shell. a) ERα-IR is associated with microtubules and the plasma membrane of a γ-aminobutyric acid-ergic (GABAergic) dendrite (DEN). b) ERα-IR associated with synaptic vesicles and the membrane near a synapse in a GABAergic terminal (TER). c) GPER1-IR associated with synaptic vesicles in a GABAergic TER; d) a GABAergic dendrite (DEN) with a spine (SP). Black arrow, immunoperoxidase for ER; white arrow, immunogold for GABA. Scale bar, 500 nm