Activation of μ opioid receptors in the medial preoptic area following copulation in male rats

Neuroscience 124 (2004) 11–21

ACTIVATION OF ␮ OPIOID RECEPTORS IN THE MEDIAL PREOPTIC AREA FOLLOWING COPULATION IN MALE RATS L. M. COOLEN,* M. E. FITZGERALD, L. YU AND M. N. LEHMAN

rodents, several reports have indicated that endogenous opioids administered systemically, affect different aspects of sexual behavior in. In general, opioids have inhibitory effects on consummatory aspects of mating. Systemic administration of the opioid agonist morphine decreases the proportion of males that copulate, increases mount and intromission latencies, and decreases the frequency of mounts and intromissions (Agmo and Paredes, 1988). In contrast, administration of the opioid receptor antagonists naloxone and naltrexone facilitate consummatory aspects of sexual behavior by increasing the percentage of males that copulate, by decreasing mount, intromission, and ejaculation latencies, as well as the numbers of mounts and intromissions achieved before an ejaculation (van Furth et al., 1994). In addition to the effects on consummatory aspects of behavior, systemic administration of opioids also affects appetitive elements of sexual behavior, since naloxone reduces anticipatory level changes in a bi-level mating chamber paradigm, which is used as a measure of sexual motivation (van Furth et al., 1994; van Furth and van Ree, 1996a). In addition, opioids are involved in the initiation of sexual behavior after ejaculation, since naloxone extends the post-ejaculatory interval (Szechtman et al., 1981; van Furth et al., 1994) and inhibits resumption of mating in sexually sated males, after the re-introduction of a female (Miller and Baum, 1987). Finally, opioids play a role in reward-related aspects of sexual behavior, since naloxone also blocks the expression of ejaculation-induced place preference (Agmo and Berenfeld, 1990; Mehrara and Baum, 1990). Opioids exert these different effects on male sexual behavior by acting in multiple brain sites, possibly via multiple receptor systems. For example, opioid ligands ␤-endorphin, met-enkephalin, and morphiceptin, microinjected into the medial preoptic area (MPOA), impaired aspects of copulation (Hughes et al., 1987, 1990; Matuszewich and Dornan, 1992; Matuszewich et al., 1995; van Furth et al., 1995) and ␤-endorphin infused into the medial amygdala inhibited initiation of copulation (McGregor and Herbert, 1992a,b). Opioids also affect sexual reward-related aspects of sexual behavior when administered into the MPOA; sexual reinforcement is produced by intraMPOA infusion of met-enkephalin (Agmo and Gomez, 1991) and blocked by intra-MPOA naloxone (Agmo and Gomez, 1993). In addition, opioids influence sexual motivation and reward when administered into the nucleus accumbens (Band and Hull, 1990) or ventral tegmental area (VTA; Mitchell and Stewart, 1990), while intra-VTA

Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267-0521, USA

Abstract—The current study tested the hypothesis that sexual behavior is a biological stimulus for release of endogenous opioid peptides. In particular, activation of ␮ opioid receptors (MOR) in the medial preoptic area (MPOA), a key area for regulation of male sexual behavior, was studied in male rats. MOR endocytosis or internalization was used as a marker for ligand-induced receptor activation, utilizing confocal, electron, and bright microscopic analysis. Indeed, mating including one ejaculation induced receptor activation in the MPOA, demonstrated by increased immunoreactivity for MOR, increased numbers of endosome-like particles immunoreactive for MOR inside the cytoplasm of neurons, and increased percentage of neurons with three or more endosome-like particles inside the cytosol. Moreover, it was demonstrated that MOR activation occurred within 30 min following mating and was still evident after 6 h. Mating-induced internalization was prevented by treatment with the opioid receptor antagonist naloxone before mating, suggesting that mating-induced receptor activation is a result of action of endogenous MOR ligands. I.c.v. injections of MOR ligand [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin resulted in internalization of the MOR in a similar manner observed following mating. Finally, mating induced Fos expression in MOR containing neurons in the MPOA. However, naloxone pretreatment did not prevent Fos activation of MOR neurons, suggesting that Fos induction was not the result of MOR activation. In summary, these results provide further evidence that endogenous opioid peptides are released in the MPOA during male sexual behavior. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: endocytosis, G protein-coupled receptor, sexual behavior, hypothalamus, Fos.

The effects of opiates and opioid peptides on male copulatory behavior in humans and non-human animals have been long recognized (Pfaus and Gorzalka, 1987). In humans, acute opioid administration results in an intense euphoria, often described in sexual terms (Mirin et al., 1980). In contrast, long term use of opioids is associated with decreased sexual drive and performance. In male *Corresponding author. Tel: ⫹1-513-558-1210; fax: ⫹1-513-5584454. E-mail address: [email protected] (L. M. Coolen). Abbreviations: DAB, 3,3⬘-diaminobenzidine tetrahydrochloride; DAMGO, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin; IR, immunoreactivity; MOR, ␮ opioid receptor; MPOA, medial preoptic area; PB, sodium phosphate buffer; PBS, phosphate-buffered saline; VTA, ventral tegmental area.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.10.045

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L. M. Coolen et al. / Neuroscience 124 (2004) 11–21

naloxone administration inhibits sexual motivation (van Furth and van Ree, 1996b). One common suggestion from these pharmacological studies is that copulation may be a biological stimulus for the release of endogenous opioid peptides in the brain. This hypothesis is further supported by the demonstration that sexual behavior activates the physiological mechanisms of analgesia and reward, both of which can be blocked by opioid receptor antagonists (Szechtman et al., 1981; Forsberg et al., 1987; Agmo and Berenfeld, 1990). The goal of the current study is to test the hypothesis that endogenous opioids are acting within the brain during male sexual behavior. In particular, we investigated action of opioids in the MPOA. The MPOA is a key brain region for the control of male sexual behavior (Hull et al., 2002) and has been shown to mediate effects of exogenous opioids on mating (see previous paragraph). The current study tests the hypothesis that endogenous opioids are acting in the MPOA during sexual behavior, by visualization of ligand-induced internalization of opioid receptors, as a specific index of receptor activation. Specifically, the mating-induced internalization of ␮ opioid receptors (MORs) in the MPOA were investigated. There is considerable evidence for the involvement of MORs on the regulation of male sexual behavior. First, pharmacological studies have indicated that MORs play a major role in the effects of opioids on sexual behavior, based on differential effects on sexual behavior of microinjections of MOR ligands and antagonists. Moreover, sexual behavior is drastically impaired in mice that lack the MOR gene, as evidenced by increased mount latencies, decreased numbers of mounts, and decreased percentage of animals displaying ejaculation (Tian et al., 1997). Furthermore, anatomical evidence supports a role for the MOR in the regulation of male sexual behavior, because the distribution of the MOR appears to closely correlate with brain regions involved in mediating copulation, including the MPOA (Delfs et al., 1994; Mansour et al., 1994a,b; Ding et al., 1996). Although these studies do not rule out additional roles for ⌬ and ␬ opioid receptors, MORs appear a prime candidate for opioid action in the MPOA and the current study was restricted to investigation of MOR activation during male sexual behavior. MORs have previously been demonstrated to undergo endocytosis in vitro (Keith et al., 1998) as well as in vivo (Eckersell et al., 1998; Trafton et al., 2000; Sinchak and Micevych, 2001, 2003) and visualization of receptor endocytosis can thus be used as a marker for ligand induced receptor activation.

EXPERIMENTAL PROCEDURES Subjects Young adult male (250 –260 g) and female (n⫽20; 210 –220 g) Sprague–Dawley rats obtained from Harlan laboratories (Indianapolis, IN, USA), were housed in same-sex pairs in artificially lighted rooms on a reversed 12-h light/dark cycle (lights off at 10:00 AM). Food and water were available at all times. Females were ovariectomized and implanted s.c. with 5% 17-␤-estradiol benzoate Silastic capsules. Progesterone, 500 ␮g in 0.1 ml of sesame oil, was injected s.c. 4 – 6 h prior to testing, to induce

sexual receptivity. All procedures were approved by the Animal Care and Use Committee of the University of Cincinnati and conformed to NIH guidelines involving vertebrate animals in research. All efforts were made to minimize the number of animals used and their suffering.

Behavioral testing All male rats were sexually experienced. Male rats were allowed to copulate during six pre-test mating sessions (30 min duration each) and were considered sexually experienced after they displayed multiple ejaculations during the last two mating sessions. All testing of experimental groups was performed 4 h after onset of the dark period, in a rectangular mating arena (60⫻45⫻50 cm) under dim red illumination. During the final test, males were allowed to mate until completion of one copulatory series, after which the female partners were removed, while males remained in the test cage until being killed. In experiments 1 and 3, males were killed 1 h following display of ejaculation; in experiment 2, males were killed 10, 20, 30, 60, or 360 min following ejaculation.

Drug injections DAMGO. In experiment 4, males received i.c.v. injections of the MOR-selective agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO; Sigma, St. Louis, MO, USA). One week prior to DAMGO injection, males were deeply anesthetized using sodium pentobarbital (50 mg/kg). The males’ heads were shaved and they were then placed into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA), with Lambda and Bregma at the same level. A single midline scalp incision was made. A unilateral guide cannula (length 7.4 mm; Plastics One, Roanoke, VA, USA) was implanted into the third ventricle, in close proximity to the MPOA, using the coordinates 0.4 mm caudal and 0 mm lateral to Bregma, and secured to the skull with dental cement and bone screws. After surgery, animals were allowed to recover for 7 days, during this time they were handled daily. The day of injection, an internal cannula, i.e. injector (projection 0.05 mm), attached via tubing to a 10 ␮l Hamilton syringe (Hamilton, Reno, NV, USA), was inserted into the guide cannula. After the injector was inserted into the guide cannula, the animal was returned to its home cage. DAMGO (0.1 or 1 ␮g in 5 ␮l volumes, dissolved in saline; n⫽3 each) or saline (5 ␮l; n⫽3) was infused over a period of 1 min using a microinjection pump. One hour following infusion, animals were perfused using 4% paraformaldehyde. Naloxone. In experiment 5, sexually experienced male rats were injected with naloxone (s.c.; 5 or 10 mg/kg body weight) or saline, 30 min before introduction of a receptive female. Sexual behavior was observed and measured until display of one ejaculation. Control males were injected with saline or naloxone hydrochloride (10 mg/kg; Sigma; n⫽4 each) and returned to their home cage, without engaging in sexual activity. Animals were killed 60 min following ejaculation, or the equivalent for control males (approximately 105 min).

Perfusion, tissue processing and immunocytochemistry Following the end of the behavioral test or drug infusion, animals were anesthetized using sodium pentobarbital (200 mg/kg; i.p.) and perfused transcardially with 100 ml 0.9% saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB; pH 7.3–7.4). Brains were removed and post fixed for 1 h at room temperature in the same fixative. Coronal sections were cut at 35 ␮m on a freezing microtome (Microm; Richard Allen, Kalamazoo, MI, USA) and were collected in four parallel series in cryoprotectant solution (30% sucrose, 30% ethylene glycol in 0.1 M PB; Watson et al., 1986). All incubations were performed at room temperature with gentle agitation.

L. M. Coolen et al. / Neuroscience 124 (2004) 11–21 Two series of sections from each brain in experiments 1, 2, 4, and 5 were processed for MOR-immunoreactivity (IR) using immunoperoxidase staining protocols (described below). One series of sections from experiments 1, 2, 4, and 5 was stained for MOR-IR using immunofluorescence. Finally, one series of sections from experiment 5 was dual stained for MOR and Fos-IR using a dual immunoperoxidase procedure. All sections were extensively rinsed in phosphate-buffered saline (PBS) between incubations. Free-floating sections were blocked with 1% H2O2 for 10 min at room temperature and then soaked for 1 h in incubation solution (PBS containing 0.1% bovine serum albumin and 0.4% Triton X-100). Next, sections were incubated overnight with a primary antiserum in incubation solution to recognize MOR (polyclonal anti-MOR antiserum raised in rabbit; raised against amino acids 384 –398 of MOR1; 1:10,000; Diasorin, Stillwater, MN, USA). Subsequently, sections were exposed for 60 min to biotinconjugated donkey anti-rabbit IgG (1:400 in incubation solution; Jackson Immunoresearch, Westgrove, PA, USA), and for 60 min to avidin– biotin– horseradish peroxidase (ABC-elite; 1:1000 in PBS; Vector Laboratories, Burlingame, CA, USA). The peroxidase complex was visualized by exposure for 10 min to a chromogen solution containing 0.02% 3,3⬘-diaminobenzidine tetrahydrochloride (DAB; Sigma) enhanced with 0.02% nickel sulfate in 0.1 M PB with hydrogen peroxide (0.015%) to produce a blue– black reaction product. Extensive washing in 0.1 M PBS terminated the reaction. Sections were mounted on Superfrost plus glass slides (Fisher, Pittsburgh, PA, USA) and coverslipped with DPX (Electron Microscopy Sciences, Fort Washington, PA, USA). For dual labeling of MOR and Fos, sections were processed as described above and then blocked with 1% H2O2 for 10 min at room temperature. Next, sections were incubated overnight with primary antiserum in incubation solution, to recognize Fos (polyclonal anti-Fos antiserum raised in rabbit; 1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and processed using the same avidin– biotin immunoperoxidase procedure described above with the second chromogen reaction performed without nickel sulfate, to produce a brown reaction product. Sections processed for immunofluorescence detection of MOR were processed as described above including incubation in primary antibody recognizing MOR (1:10,000), secondary antibody and ABC-complex. Next, sections were incubated for 10 min with biotinylated tyramide (1:250 in PBS with 0.003% H2O2 added; Tyramide Signal Amplification Kit; NEN Life Sciences, Boston, MA, USA) and for 30 min with CY-3-conjugated streptavidin (1: 200; Jackson ImmunoResearch Laboratories). Next, sections were washed, mounted on Superfrost plus glass slides, air-dried, and coverslipped with an aqueous mounting medium (Gelvatol) containing an anti fading agent (1,4-diazabicyclo (2,2)octane; Sigma). Immunocytochemical controls included omission of primary antibodies for Fos or MOR.

Pre-embedding EM immunocytochemistry For EM analysis (experiment 3), male rats were allowed to copulate to one ejaculation (n⫽3) and 1 h later were anesthetized using sodium pentobarbital (200 mg/kg; i.p.) and perfused transcardially with 5 ml 0.9% saline, followed by 50 ml of 2% paraformaldehyde containing 3.75% acrolein in 0.1 M PB and by 200 ml of 2% paraformaldehyde in 0.1 M PB. Control animals (n⫽3) were removed from the home cage and perfused using the same protocol. Brains were removed and post fixed for 1 h at room temperature in 2% paraformaldehyde in 0.1 M PB. Coronal sections (50 ␮m) were cut using a vibratome-series 1000 (Ted Pella, Redding, CA, USA) and were immunostained for MOR (polyclonal anti-MOR antiserum raised in rabbit; 1:1000; Diasorin) using the immunoperoxidase procedure described above, with the exception that 0.02% Triton X-100 was used as a detergent. The MOR-IR area in the MPOA was microdissected out of tissue sections with the aid of a dissecting microscope. Tissue bits were then post-fixed in 2%

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osmium tetroxide containing 1.5% potassium ferricyanide in 0.1 M sodium cacodylate, dehydrated in alcohols, transferred to propylene oxide, and infiltrated with a 50% propylene oxide/50% EM bed 812 (EM Sciences, Fort Washington, PA, USA) mixture, followed by EM bed 812 overnight at room temperature. The bits were flat-embedded in fresh EM bed-812 at the bottom of Beem capsules and cured at 60 °C overnight. Thick (1 ␮m) sections were cut on a Reichert Ultracut E ultramicrotome and examined under the light microscope. Once reaction product was seen, thin (70 nm) sections were taken and mounted on formvar-coated grids. Grids were stained with uranyl acetate and lead citrate and photographed using a JEOL 100CX TEM (Japan Electron Optics Limited, Peabody, MA, USA).

Data analysis: sexual behavior The number of mounts, intromissions and total duration of the mating tests, were recorded for all experiments. Statistical analysis was only performed in experiment 5 to compare numbers of mounts and intromissions, as well as duration of test, between mated animals treated with saline or different doses of naloxone. A one-way ANOVA and post hoc comparisons using the Fisher’s PLSD test were performed with an ␣ of 0.05 required for rejection of the null hypothesis.

Analysis of internalization of MORs Confocal analysis. Fluorescent stained sections were examined with a Zeiss laser-scanning confocal microscope system (Zeiss LSM510, Heidelberg, Germany). CY3-fluorescence was imaged with a 567 nm emission filter and a HeNe laser. To demonstrate the location of MOR-IR, images of MOR-IR MPN neurons were created by projecting (stacking) several optical sections obtained at 1 ␮m intervals through a section in the z axis. Of each stack of images through the neurons, two consecutive optical sections in the middle of a neuron were used for analysis. Internalized neurons had intracellular particle staining, presumably endosomes (Trafton et al., 2000). Numbers of MOR-IR intracellular particles were counted for 25–30 cells (in three to four sections containing the MPN) per animal, by an observer blind to experimental group, and were averaged per animal. In addition, the percentage of internalized MOR-IR cells was quantified: MOR-IR neurons containing three or more MOR-IR intracellular particles were considered internalized. Percentages of internalized neurons and average numbers of immunoreactive particles were averaged for each group, and compared using Student’s t-tests (experiment 1) or one-way ANOVAs with Fisher’s PLSD post hoc comparisons with 5% significance levels (experiment 4). Finally, in experiment 1, mating-induced internalization of MOR was analyzed in a region located dorsal to the MPOA, i.e. the triangular septal nucleus and septofimbrial nucleus. These areas are located in the same sections as the MPOA, contain abundant MOR-IR, but are not involved in regulation of male sexual behavior. For these areas, group averages of percentages of internalized neurons and average numbers of immunoreactive particles were compared using Student’s t-tests with 5% significance levels. Optical density measurements. The density of receptor IR was quantified to obtain an estimate of the relative level of internalization, on material processed for brightfield microscopy with nickel-enhanced DAB. For each animal, four images of the MPN were captured with a CCD camera attached to a Nikon microscope. Camera settings (gain and amplitude) were identical for all images of all control and mated animals. Optical density was analyzed using NIH image analysis software (Object-Image for Macintosh). An average threshold was calculated based on the average gray value of all the images, this threshold was then applied to all images. The area of staining above the threshold was measured (in ␮m) and reflected as a percentage of the total

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L. M. Coolen et al. / Neuroscience 124 (2004) 11–21

area of the entire image. Group averages were calculated and compared using Student’s t-tests (experiment 1), or a one-way ANOVA with Fisher’s PLSD post hoc comparisons (experiment 2, 4, 5), all using significance levels of 5%.

does not occur in adjacent brain regions not involved in regulation of sexual behavior.

Fos-MOR counts. Numbers of cells expressing Fos-IR, MOR-IR, or both were counted in a standard area (800⫻800 ␮m) in the MPN in two sections per animal using a drawing tube attached to a Leica microscope (Leica Microsystems, Wetzlar, Germany). Neurons single labeled for MOR-IR were determined by black staining of cytoplasm with a nucleus devoid of brown (Fos) reaction product. Neurons with only brown nuclei were considered single-labeled for Fos. Cells that contained both stained nucleus (brown) and cytoplasm (black) were considered dual labeled. Percentages of single and dual labeled cells were calculated and group averages of percentages and numbers of single and dual labeled cells were calculated for each group. To determine differences between groups a two-way ANOVA (factors: sex versus control and naloxone versus saline) and post hoc comparisons using the Fisher’s PLSD test were performed, both with a 0.05 level of significance. Digital images of immunostained sections were captured using a digital camera (Magnafire, Optronics, Goleta, CA, USA) attached to a Leica microscope. Images were imported into Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA) to compose the figures. Images were not adjusted or altered, except for occasional adjustment of brightness.

Experiment 2 tested the time course for internalization of MOR following mating behavior. Males were killed at different time intervals (10, 20, 30, 60, or 360 min following ejaculation; n⫽4 each). Control males were removed from the home cage without engaging in sexual activity (n⫽9). Mating induced a significant increase in optical density for MOR-IR (Fig. 1H; F(5,23)⫽4.72; P⫽0.0041), reflective of internalization, as early as 30 min following copulation (P⫽0.005– 0.03 compared with control), and this increase persisted at 60 and 360 min following copulation.

RESULTS Experiment 1: Internalization of MOR in the MPOA The first experiment tested the hypothesis that endogenous opioid peptides are released in the MPOA during male sexual behavior, resulting in internalization of MOR. MOR-IR was compared in males taken from their home cages without engaging in sexual activity (control; n⫽7), and males that were killed 1 h following mating that included one ejaculation (n⫽7). Fig. 1A and B illustrate that mating resulted in a dramatic increase in MOR staining in the MPOA compared with control males. Fig. 1C illustrates a quantitative analysis of this increase in MOR-IR as seen in immunoperoxidase stained sections. Indeed, the mean optical density of MOR-IR in the MPOA was significantly higher in mated males compared with control males (P⫽0.0053). Moreover, confocal analysis of alternate sections stained for MOR-IR using immunofluorescence indicated internalization of MOR (Fig. 1D, E). In particular, significantly increased numbers of endosome-like particles immunoreactive for MOR were observed within the cytoplasm of MPOA neurons in mated males compared with control males (Fig. 1F; P⫽0.002). In addition, the percentages of MOR-IR neurons that had three or more MOR-IR particles inside the cytoplasm also increased with mating (Fig. 1G; P⬍0.0001). In contrast, mating-induced internalization of MOR was not observed in neurons in a region located dorsal to the MPOA, including the triangular septal nucleus and septofimbrial nucleus (mean number of endosomes per neuron: 0.72⫾0.27 in control versus 0.97⫾0.27 in mated males). This region is located in the same coronal sections as the MPOA, but is not involved in the regulation of male sexual behavior. Hence, these data suggest that matinginduced MOR activation is restricted to the MPOA and

Experiment 2: time course

Experiment 3: EM localization In order to confirm the cellular localization of the MOR in MPOA neurons we examined sections stained for MOR using EM immunocytochemistry of sexually experienced male rats killed 60 min following one ejaculation (n⫽3), or after being removed from their home cage (control; n⫽3). Localization of MOR-IR was examined in six cells per animal. In MOR neurons examined in the MPOA of control males, MOR-IR was closely associated with the plasma membrane (Fig. 2) and not seen elsewhere within the cell. Conversely, in MOR neurons in MPOA of mated males, MOR-IR was localized within the cytoplasm, specifically associated with endosome-like vesicles and with the Golgi apparatus. Although some MOR-IR, in mated males was also associated with the plasma membrane, this was substantially less than that seen in neurons from control males. Experiment 4: DAMGO To further validate the use of receptor internalization as a marker for endogenous ligand-induced activation of the receptor, mating-induced internalization was compared with internalization induced by the MOR-selective agonist DAMGO. DAMGO (0.1 or 1 ␮g in 5 ␮l) or saline was infused into the third ventricle of male rats. Animals were killed 60 min following infusion. DAMGO resulted in internalization of the MOR in MPOA that appeared identical to that observed following mating (Fig. 3A). DAMGO increased the average number of endosome-like particles in MOR neurons in MPOA in a manner similar to that seen following mating (Fig. 3B; F(2,6)⫽27.976; P⫽0.001). Moreover, DAMGO resulted in increased percentage of MOR MPOA neurons that contained three or more endosome-like particles in the cytoplasm (Fig. 3C; F(2,6)⫽ 234.296; P⬍0.0001). In addition, DAMGO resulted in increased optical density of immunoperoxidase-stained MOR in the MPOA, similar to the effects of copulation (data not shown). Experiment 5: naloxone To test the assumption that the mating-induced internalization of the MOR and the associated changes in IR are indeed a result of activation of the MOR by endogenous opioids,

L. M. Coolen et al. / Neuroscience 124 (2004) 11–21

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Fig. 1. A, B) MOR-IR detected by immunoperoxidase staining in MPOA of control males (A) and of mated males, 1 h following mating including ejaculation (B). Scale bar⫽100 ␮m. 3V, third ventricle. C) Quantitative analysis of MOR internalization following mating, illustrating the sex-induced increase in MOR-IR optical density, represented by the mean (⫾S.E.M.) percentage of the total area of the image containing staining above threshold, compared with control males. Star indicates a statistically significant difference compared with control (P⫽0.0053). D, E) Confocal images (1 ␮m optical sections) illustrating MOR neurons in MPOA. D shows MOR-IR confined to the membrane of an MPOA neuron in a representative control male. E illustrates mating-induced internalization of MOR, indicated by the presence of endosome-like particles (marked with arrows) inside the cytoplasm. Scale bars⫽20 ␮m. F) Sex-induced increase of the average numbers of endosome-like particles inside the cytoplasm of MOR neurons in MPOA. Star indicates statistically significant difference compared with control (P⫽0.002). G) Mating-induced increase in mean (⫾S.E.M.) percentage of MPOA MOR neurons that contain three or more endosome-like particles inside the cytoplasm. Star indicates a statistically significant difference compared with control (P⬍0.0001). H) Analysis of MOR-IR optical density measurements (mean⫾S.E.M.) at different time points following display of ejaculation (10, 20, 30, 60, or 360 min; depicted on x axis). Sex induced an increase in optical density shown as the percentage of the total area of the image containing MOR-IR staining above threshold, at 30, 60, and 360 min following mating compared with control males (C). Stars indicate statistically significant differences compared with control or 10 min (P⫽0.005– 0.03).

experiment 5 tested the hypothesis that the MOR antagonist naloxone would prevent mating-induced internalization of MOR neurons in MPOA. Because naloxone generally facilitates sexual behavior (Pfaus and Gorzalka, 1987) only naloxone-treated males with similar ejaculation latencies and numbers of mounts and intromissions as saline-treated males were included in this study. Thus, there were no significant differences in behavior between the naloxone and saline-treated groups (Table 1; n⫽3 each). The results demonstrated that naloxone completely blocked mating-induced

increase of MOR-IR density in the MPOA (Fig. 4A). In particular, there was a significant interaction between the factors mating and drug (F(1,11)⫽5.250; P⫽0.0427), and post hoc analysis revealed a mating-induced increase in MOR-IR in saline-treated (P⫽0.016), but not in naloxone-treated males. No differences were detected between males treated with 5 or 10 mg/kg naloxone. Moreover, both doses of naloxone prevented the appearance of mating-induced MOR-IR endosome-like particles inside the cytoplasm of neurons in the MPOA (Fig. 4C).

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Fig. 2. EM photomicrographs showing the location of MOR-IR. In control males (A), MOR-IR was observed associated with the plasma membrane (indicated by arrows), whereas in mated males (B) MOR-IR was also within the cytoplasm in association with endosome-like structures and the Golgi apparatus (indicated by arrows) and less receptor staining was visible on the plasma membrane. Scale bars⫽500 nm.

Experiment 6: MOR and Fos colocalization Previous studies have demonstrated that mating induces Fos expression in the MPOA (Baum and Everitt, 1992; Coolen et al., 1997; Veening and Coolen, 1998). Therefore, the final experiment investigated if mating-induced Fos is expressed in MOR-IR neurons in the MPOA. Moreover, we tested the hypothesis that Fos induction is a result of MOR activation and can thus be prevented by naloxone administration. Brain sections from the male rats included in experiment 5 were processed for dual immunoperoxidase detection of MOR and Fos-IR and single and doublelabeled cells expressing MOR and/or Fos were counted. Indeed, 60.5% of MOR-IR neurons in the MPOA of saline injected males contained mating-induced Fos-IR, compared with 0% in non-mated control saline-injected males (Fig. 4D). However, not all MOR-IR neurons with internal-

ized MOR-IR endosome-like particles were also Fos-positive (Fig. 4E). Moreover, naloxone infusions did not prevent Fos expression in MOR-IR neurons, as there were no significant differences in percentages of MOR cells that were Fos positive following mating in saline-treated versus naloxone-treated males (Fig. 4D).

DISCUSSION The present study demonstrates activation of MOR in the MPOA following sexual behavior in male rats, evidenced by increased immunostaining for MOR and increased numbers of endosome-like particles inside the cytoplasm of MOR cells. The activation of MORs in the MPOA is likely caused by endogenous opioids, since pretreatment with the MOR antagonist naloxone prevented mating-induced activation of MORs. Hence, these results support the hy-

Fig. 3. A) Confocal image (1 ␮m optical section) demonstrating internalization of MOR-IR following DAMGO infusion. Arrows indicate endosome-like particles in the cytoplasm of MOR neurons. Scale bars⫽20 ␮m. B, C) Quantitative analysis of DAMGO-induced MOR internalization. B illustrates the DAMGO-induced increase in the average numbers (⫾S.E.M.) of endosome-like particles inside the cytoplasm of MOR neurons in MPOA. C demonstrates the DAMGO-induced increase in mean (⫾S.E.M.) percentage of MPOA MOR neurons that contain three or more endosome-like particles inside the cytoplasm. In both graphs white bars represent the control saline-treated males and gray bars represent males treated with 0.1 ␮g DAMGO (D 0.1) or 1 ␮g DAMGO (D 1). Stars indicate a statistically significant difference from control (P⬍0.001).

L. M. Coolen et al. / Neuroscience 124 (2004) 11–21

pothesis that endogenous opioid peptides are acting in the MPOA during mating. The present data illustrate the use of visualization of opioid receptors as a marker for endogenous ligand-induced activation during a naturally occurring behavior. The use of receptor internalization as a marker for receptor activation was first suggested by Mantyh and coworkers (Mantyh et al., 1995a,b; Allen et al., 1997), who used the phenomenon of receptor internalization of substance P receptor IR, another G-protein-coupled receptor, to determine activation of these receptors after specific somatosensory stimuli. After the initial agonist-receptor interaction that induces signal transduction, opioid receptors, like sub-

Table 1. Sexual behavior following saline or naloxone treatmenta

Saline (n⫽3) Naloxone 5 mg/kg (n⫽3) Naloxone 10 mg/kg (n⫽3)

Mounts

Intromissions

Ejaculation latency (seconds)

7.7⫾5.2 10.3⫾4.3

17.3⫾5.4 17.7⫾2.7

589.3⫾107.4 695.7⫾255.6

9.3⫾3.2

15.0⫾1.7

654.7⫾49.6

a

Parameters of sexual behavior, including numbers of mounts and intromissions, and ejaculation latency, are presented as group means⫾S.E.M. There were no significant differences between the groups.

Percentage above threshold

100 90 80 70 60 50 40 30 20 10 0

A

*

Percentage of MOR containing Fos

CONT SEX SALINE 100 90 80 70 60 50 40 30 20 10 0

10 5 10 CONT SEX SEX NALOXONE

17

B

C

SALINE

NALOXONE

E

F

D

*

CONT SEX SALINE

* *

10 5 10 CONT SEX SEX NALOXONE

Fig. 4. A) Optical density measurements (mean⫾S.E.M.) for MOR-IR in control males (CONT; white bars) or following sexual behavior (SEX) in males treated with saline (black bar) or naloxone (gray bars). Mating significantly increased the percentage of staining above threshold in saline-treated males. In contrast, naloxone prevented increased MOR-IR by mating. Star indicates a statistically significant difference compared with control. Numbers inside bars indicate dose of naloxone: 10 mg/kg or 5 mg/kg body weight. B, C) Confocal images (1 ␮m optical sections) illustrate mating-induced MOR internalization of MOR-IR in males treated with saline (B), while naloxone (5 mg/kg) completely prevented mating-induced internalization (C). Arrows indicate endosome-like particles in the cytoplasm of MOR neurons. Scale bars⫽20 ␮m. D) Quantitative analysis of mean (⫾S.E.M.) percentages of MOR neurons that also contain mating-induced Fos. Mating induced Fos in MOR neurons in both saline- (black bar) and naloxone (gray bars)-treated males. Stars indicate statistically significant differences compared with control (white bars). Numbers inside the bars indicate dose of naloxone: 10 mg/kg or 5 mg/kg body weight. E) Confocal image (1 ␮m optical section) illustrating two MOR cells with internalized endosomes (indicated by arrows) following mating in a saline-treated male. One of the two cells also contains mating-induced Fos (green). F) Image illustrating Fos expression in a MOR neuron following mating using DAB for Fos (brown) and nickel-enhanced DAB for MOR (black) in a representative naloxone (5 mg/kg)-treated male. Arrows indicate endosome-like particles in the cytoplasm of MOR neurons. Scale bars⫽20 ␮m.

18

L. M. Coolen et al. / Neuroscience 124 (2004) 11–21

stance P receptors, typically undergo phosphorylation, endocytosis, and dissociation from the ligand in the endosome, after which the receptors are recycled to the plasma membrane (Kobilka, 1992; Caron and Lefkowitz, 1993; Lefkowitz et al., 1993; Garland et al., 1996; Tsao and von Zastrow, 2000a,b, 2001; Tsao et al., 2001; von Zastrow, 2001). Internalization of MORs has been demonstrated in transfected cells and peripheral neurons (Arden et al., 1995; Keith et al., 1996; Sternini et al., 1996; Gaudriault et al., 1997; Burford et al., 1998) and rat brain neurons (Eckersell et al., 1998; Keith et al., 1998; Trafton et al., 2000). Thus, ligand-induced internalization of the opioid receptor can be used as a specific index of receptor activation. The current study is the first demonstration of internalization of MOR neurons by endogenous opioids evoked by a behavioral event. Previously, MOR internalization by endogenous opioids was demonstrated in the female rat MPOA following treatment with estrogen (Eckersell et al., 1998; Sinchak and Micevych, 2003) or opioid agonists (Trafton et al., 2000). Interestingly, MOR activation was not observed in spinal cord neurons following noxious stimuli sufficient to evoke endogenous opioid release, although internalization was evident following administration of opioid receptor agonists (Trafton et al., 2000). In contrast, the current study was able to demonstrate MOR activation following a natural event, i.e. sexual behavior including only one copulatory series leading to ejaculation. The present study demonstrates mating-induced internalization of MOR neurons in MPOA, evidenced by the increased presence of endosome-like particles inside the cytoplasm, as well as increased IR for MOR. Recently, Micevych and coworkers have demonstrated estrogen induced MOR internalization in MPOA and other brain sites in female rats (Eckersell et al., 1998; Sinchak and Micevych, 2001, 2003; Micevych et al., 2003). They reported an increase in MOR staining as well as translocation of the receptor from plasma membrane to the intracellular compartment. Similar to the present study, the internalization of the receptor produced a concentration of IR that allowed for the visualization of greater number of fibers, and thus resulted in an increased density of MORIR. It is important to note that the estrogen-induced increase in MOR-IR is not related to increased expression of the receptor, and instead reflects internalization of the receptor, since [3H]DAMGO binding levels did not change after estrogen treatment (Eckersell et al., 1998). Moreover, results from the current study demonstrate that increased MOR-IR within 30 min following mating, and new MOR protein synthesis is unlikely at this early time interval. These results are in agreement with the time course for internalization observed by in vitro studies (Keith et al., 1996, 1998), as well as with the time course for estrogeninduced changes in MOR-IR (Eckersell et al., 1998). The mating-induced increase of MOR-IR appears to be reflective of MOR internalization, although the cause of the increase in IR is unclear. Possible explanations include a more robust visualization of MOR-IR following the redistribution or clustering of MOR inside the dendrites and axons and a reduction in plasma membrane after internalization

(Mantyh et al., 1995b; Allen et al., 1997; Liu et al., 1997). Alternatively, the translocation of the receptor may provide a more easily accessible target for the antibody, which recognizes the c-terminus of MOR, resulting in a more robust reaction product. The current study also demonstrates that mating-induced increase in MOR-IR can be blocked with the MOR antagonist naloxone. Therefore, we conclude that matinginduced internalization of the receptor is reflective of receptor activation by endogenous opioids, released during or following sexual behavior. DAMGO infusions resulted in internalization of the MOR in the MPOA, which appeared identical to the internalization observed following mating; again supporting the view that mating-induced internalization is attributable to receptor binding by endogenous opioids, released during mating. Interestingly, DAMGO resulted in a similar internalization compared with those following mating, reflected by the number of endosomes per cell or percentage of internalized neurons. Moreover, the two doses of DAMGO did not differ in their ability to cause internalization. Together, these findings suggest that MOR neurons in the MPOA may have a maximum capacity for internalization, with remaining unoccupied receptors located on the membrane of the cell. Alternatively, endogenous opioids or DAMGO may be degraded by peptidases, which may prevent activation of all MORs, similar to the action of peptidases in the dorsal horn of the spinal cord (Song and Marvizon, 2003). However, this latter possibility is not supported by the results from the time course of mating-induced internalization, which indicate that MORs remain internalized for an extended period of time (as long as 360 min). Currently, it is unclear what underlies the extended receptor activation. It is possible that endogenous opioids may be released in the MPOA for an extended period. Alternatively, MORs may be slow to recycle back to the membrane, although recent evidence indicates that the MOR can recycle well within 60 min (Trafton et al., 2000). Identification of the stage within the recycling or degradative pathways after initial endocysosis of the endosomes at the different time intervals following mating, may shed some light on this issue (Tsao and von Zastrow, 2001). For instance, the presence of “early” endosomes at all time points may suggest recent activation of the receptor by endogenous peptides, thus an extended period during which opioids are present in the synaptic (or extrasynaptic) cleft. Alternatively, the presence of “late” endosomes at later time points following mating may suggest a slower degradation process of the MOR. There are major advantages to the use of internalization as a marker for receptor activation. However, like other markers for neural activation, this approach also has limitations. Most importantly, receptor internalization is a marker for receptor activation, and is therefore an indirect measure for the release of the endogenous ligand that caused activation of the receptors. Therefore, the current study does not provide information about the endogenous ligand that is activating MORs in the MPOA. Axon terminals containing ligands capable of activating MOR, including ␤-endorphin, enkephalins, and endomorphins, are lo-

L. M. Coolen et al. / Neuroscience 124 (2004) 11–21

cated in the MPOA (Simerly et al., 1986, 1988; MartinSchild et al., 1999). Recent work by Rodriguez-Manzo et al. (2002) demonstrated increased IR for enkephalins in the hypothalamus following sexual behavior in male rats, suggesting that enkephalins are endogenously released during or following sexual behavior. Interestingly, it was shown that enkephalins remain elevated for extended periods of time (24 – 48 h), possibly attributing to the long term internalization observed in the current study. In addition, enkephalinase inhibition in the MPOA facilitates male sexual behavior in the rat (Agmo et al., 1994). Some evidence also exists for a possible role of ␤-endorphin in male sexual behavior, since aged rats with impaired sexual activity have diminished levels of ␤-endorphin in the hypothalamus (Dorsa et al., 1984). A potential role of endomorphins in male sexual behavior has not yet been explored. Future studies utilizing microdialysis are needed to further address the nature of the opioid ligand(s) released in the MPOA during male sexual behavior. It is well established that sexual behavior induces neural activity in the MPOA, evidenced by increased expression of Fos (Baum and Everitt, 1992; Coolen et al., 1996; Veening and Coolen, 1998; Hull et al., 2002). However, the current results demonstrate that Fos induction in MPOA neurons during mating is not a result of MOR activation by endogenous opioids. Although a majority of MOR-IR neurons in the MPOA were Fos-positive, MOR blockade by naloxone did not prevent Fos induction while it did block MOR internalization. This finding is not entirely surprising since Fos is a marker for excitation of a neuron (Morgan and Curran, 1995; Kovacs, 1998; Hoffman and Lyo, 2002), while MOR activation results in inhibition of adenylyl cyclase and decreases Ca2⫹ conductance, thus generally inhibits the cell (Feldman et al., 1997). However, MOR activation has also been demonstrated to activate the MAP kinase signaling cascade (Kramer and Simon, 2000; Trapaidze et al., 2000), which in turn can result in production of Fos. Nonetheless, the current results indicate that MOR activation is not required for Fos activation in MPOA neurons; instead, Fos-induction is caused by signals other than endogenous opioids. Indeed, many neurotransmitter and neuropeptide systems have been implicated in the control of MPOA on male sexual behavior (Hull et al., 2002), but it is currently unknown which neurotransmitters contribute to neural activation of MPOA neurons. It is likely that activation of MOR and induction of Fos of the MOR containing neuron do not occur at the same time during sex behavior. Instead, one possible scenario is that activation of MPOA neurons occurs to stimulate expression of mating, while MOR activation subsequently inhibits the same neurons, contributing to inhibition of sex behavior. In agreement, opioid infusion into MPOA has been shown to inhibit sexual performance (Hughes et al., 1987, 1990; Matuszewich and Dornan, 1992; Matuszewich et al., 1995; van Furth et al., 1995) and naloxone blocked the increase in postictal behavioral depression following MPOA kindling (Paredes et al., 1992). Hence, MOR activation in MPOA may contribute to initiation of the postejaculatory interval or sexual satiety.

19

In conclusion, the current study demonstrated internalization of MORs in the MPOA following male sexual behavior, indicative of endogenous release and action of opioids. This is the first demonstration of MOR endocytosis induced by a behavioral event. Moreover, these results support the hypothesis that mating is a biological stimulus for endogenous opioid release. Acknowledgements—We would like to acknowledge the contributions of Adam Wells and Sidney Van Ess for technical assistance and Margaret Balfour for helpful comments during preparation of this manuscript. This research was supported by National Institutes of Health Grant DA14591 to L.M.C.

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(Accepted 20 October 2003)