Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala

Abstract

The mu-opioid receptor (mu-OR), like most G-protein-coupled receptors, is rapidly internalized after agonist binding. Although opioid peptides induce internalization in vivo, there are no studies that demonstrate mu-OR internalization in response to natural stimuli. In this study, we used laser-scanning microscopy to demonstrate that estrogen treatment induces the translocation of mu-OR immunoreactivity (mu-ORi) from the membrane to an internal location in steroid-sensitive cell groups of the limbic system and hypothalamus. Estrogen-induced internalization was prevented by the opioid antagonist naltrexone, suggesting that translocation was largely dependent on release of endogenous agonists. Estrogen treatment also altered the pattern of mu-ORi at the bright-field light microscopic level. In the absence of stimulation, the majority of immunoreactivity is diffuse, with few definable mu-OR+ cell bodies or processes. After stimulation, the density of distinct processes filled with mu-ORi was significantly increased. We interpreted the increase in the number of mu-OR+ processes as indicating increased levels of internalization. Using this increase in the density of mu-OR+ fibers, we showed that treatment of ovariectomized rats with estradiol benzoate induced a rapid and reversible increase in the number of fibers. Significant internalization was noted within 30 min and lasted for >24 hr after estrogen treatment in the medial preoptic nucleus, the principal part of the bed nucleus, and the posterodorsal medial amygdala. Naltrexone prevented the increase of mu-OR+ processes. These data imply that estrogen treatment stimulates the release of endogenous opioids that activate mu-OR in the limbic system and hypothalamus providing a "neurochemical signature" of steroid activation of these circuits.

Fig. 1.

Confocal image of cells in the medial preoptic nucleus stained with antibodies against μ-OR. A, From an OVX rat, the μ-ORi is associated with the plasma membrane.B, From an estrogen-treated rat, the μ-ORi is translocated to the interior of the cell. Magnification, 800×.

Fig. 2.

Confocal photomicrographs of μ-ORi processes in the medial part of the medial preoptic nucleus from an OVX (A), OVX plus EB (B), and OVX plus EB rat pretreated with naltrexone (C). Immunoreactive fibers were optically sectioned in thez-axis along the line indicated at thebottom of each photograph and rotated 90°. The resulting image (top of each panel) illustrates that the μ-ORi is associated with the plasma membrane (arrowheads) in ovariectomized rats (A). Treatment with estrogen induced a translocation of the μ-ORi into the interior of the neural process (B). The neural process had also become more varicose (B, arrowheads). Neural processes from rats treated with naltrexone and then estrogen had μ-ORi associated with the plasma membrane (C, arrowheads). The immunolabeling in naltrexone-treated rats was consistently less intense than in tissue from OVX and estrogen-treated rats. Magnification, 2400×.

Fig. 3.

Schematic representation of the quantification method of μ-OR+ fibers in the medial part of the medial preoptic nucleus (MPNm). μ-OR+ fibers that touched or intersected the perpendicular lines superimposed on the μ-ORi plexus within MPN were counted. The density of immunoreactive fibers was calculated by dividing the number of processes by the total length of the lines within μ-ORi plexus. The same type of analysis was applied to the MeApd. Results are presented in Figure 7. ac, Anterior commissure; BST, bed nucleus of the stria terminalis; MPNl, lateral portion of the MPN;LPO, lateral preoptic area; PS, parastrial nucleus.

Fig. 4.

Photomicrographs of the medial preoptic area stained with antibodies against the μ-OR. A, μ-OR-immunoreactive fibers are concentrated in the medial part of the medial preoptic nucleus, surrounding the central part (MPNc), which is devoid of labeled fibers. μ-ORi fibers form a dense plexus dorsolateral to the MPNc. B, Other concentrations of μ-ORi fibers at the level of the MPNc, including the striohypothalamic nucleus (StHy) and the principal portion of the bed nucleus of the stria terminalis (BST). f, Fornix;LV, lateral ventricle.

Fig. 5.

Bright-field photomicrographs of μ-OR immunocytochemical localization in the medial MPN, dorsal to the central part of the MPN in the area quantified (Fig. 3). OVX animals (A) had a low density of distinct μ-OR+ fibers (A, arrows). Although a number of distinct μ-OR+ fibers were detected (arrows), the majority of μ-OR immunoreactivity was diffuse (A, C, D, arrowheads). Treatment with estrogen produced a dramatic increase in the density of varicose μ-OR+ fibers at 4 hr (B), but by 72 hr (C) the distribution resembled tissue from ovariectomized rats. The estrogen-induced increase was blocked by pretreatment with naltrexone (D). Scale bar:D, 25 μm.

Fig. 6.

Bright-field photomicrographs of μ-OR immunocytochemical localization in the posterodorsal medial amygdala in OVX (A) and estrogen-treated rats (B, C, D). Estrogen induces an increase in the number of μ-OR+ fibers 4 hr after estrogen (B); by 72 hr (C) the density is similar to OVX levels. Naltrexone blocks the estrogen effect (D) and induces a significant decrease in the density of fibers. Scale bar:D, 50 μm.

Fig. 7.

Graphic representation of the density of μ-ORi fibers in the medial part of the MPN (MPNm) (solid line) and the posterodorsal medial amygdaloid nucleus (MeApd) (dashed line) after estrogen treatment. The moment of EB injections is time 0. The density of distinct μ-OR+ fibers was significantly elevated by 30 min and peaks at 4–6 hr. The density of μ-OR+ fibers is basal by 48 hr. The bars at the 4 hr time point illustrate the density of μ-OR+ fibers in the MPNm (solid bar) and MeApd (open bar) in OVX plus EB rats pretreated with naltrexone 24 and 4 hr before perfusion. The density of μ-OR+ fibers in the naltrexone-treated rats is significantly lower than in ovariectomized rats in the MPN and MeApd. Each point is the mean ± SEM (n = 4 per time point).

Fig. 8.

Histogram showing [³H]DAMGO binding in the MeA (solid bar) and MPO (open bar) at 0, 6, 12, and 24 hr after subcutaneous injection of 50 μg of EB. No significant change in [³H]DAMGO binding was observed in either the MeA or MPO after estrogen injection. Each point is the mean ± SEM (n = 4 per time point).