Endomorphin1-like immunoreactivity in the limbic system of Syrian hamsters (Mesocricetus auratus)

Brain Research Bulletin, Vol. 56, No. 6, pp. 563–567, 2001 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter

PII S0361-9230(01)00644-X

Endomorphin1-like immunoreactivity in the limbic system of Syrian hamsters (Mesocricetus auratus) Richard D. Whitten,1 Sheryl Martin-Schild,2 James E. Zadina2 and H. Elliott Albers3* 1

Center for Behavioral Neuroscience, Department of Psychology, Georgia State University, Atlanta, GA, USA; 2 VA Medical Center and Tulane University School of Medicine, New Orleans, LA, USA; and 3Center for Behavioral Neuroscience, Departments of Biology and Psychology, Georgia State University, Atlanta, GA, USA [Received 12 June 2001; Accepted 25 July 2001] ABSTRACT: Recently, endomorphin-1 (Tyr-Pro-Trp-Phe-NH2; EM1), an endogenous peptide that has high affinity and selectivity for the ␮-opiate receptor, has been shown to modulate emotional behavior in mice and social behavior in Syrian hamsters. Endomorphin-1 (EM1) is present throughout the central nervous system in rats, mice, and guinea pigs; however, the distribution of EM1 in hamsters has not been described. The purpose of the present study was to investigate the distribution of EM1-like immunoreactivity (EM1L-IR) in the limbic system of Syrian hamsters using immunocytochemistry. Perikarya containing EM1L-IR were present in the anterior area, dorsomedial, ventromedial, periventricular, posterior, and arcuate nuclei of the hypothalamus. Fibers expressing EM1L-IR were present in the nucleus accumbens, caudate putamen, septum, bed nucleus of the stria terminalis, amygdaloid complex, and hypothalamus. The distribution of EM1 suggests a potential endogenous role for this peptide in major processes modulated by opiates, including affective states and social behavior. © 2002 Elsevier Science Inc.

anxiety. In hamsters, a single i.c.v. injection of EM1 prior to testing blocked the expression of conditioned defeat. Conditioned defeat is a phenomenon in which hamsters that have been defeated subsequently fail to exhibit normal territorial aggression and instead display submissive/defensive behaviors even when paired with a nonaggressive intruder. Furthermore, the presence of EM1-like immunoreactivity in Syrian hamster brain has not been described. In the present study, we report the presence of EM1-like immunoreactivity in limbic structures and describe its differential distribution in areas containing ␮-opiate receptors. MATERIALS AND METHODS Tissue Preparation and Immunocytochemistry All experimental protocols were approved by the Georgia State University Institutional Animal Care and Use Committee. Syrian hamsters (Mesocricetus auratus, Charles Rivers Laboratories, Wilmington, MA, USA; n ⫽ 6) weighing 110 –130 g were housed in Plexiglas cages (20 ⫻ 40 ⫻ 20 cm) with corn cob (Bed-O-Cob) bedding and free access to food and water at all times. The colony room was temperature-controlled (20°C ⫾ 2°) and maintained on a 14:10 light/dark cycle with lights off at 1100 h. Three hamsters were injected with 320-␮g colchicine in 2-␮l sterile normal saline into the lateral ventricle through indwelling cannulae 48 h before perfusion. Hamsters were anesthetized with ketamine (80 mg/kg)/xzylazine (8 mg/kg) administered intraperitoneally, and then animals were perfused transcardially under 70-mm Hg pressure with 50 ml of 2% NaNO3 in normal saline followed by 200 ml of 2.5% acrolein in 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde for 3 h.

KEY WORDS: Endomorphin-1, Opioids, Neuroanatomy, Limbic system, Hypothalamus, Amygdala.

INTRODUCTION Endogenous opioids are present throughout the limbic system [12] and are involved in modulating affect and social behavior, including anxiety, aggression, and response to social stress [4,15,22]. Endomorphins are endogenous ligands that bind selectively and with high affinity to the ␮-opiate receptor [26]. Endomorphin-1 (EM1) and endomorphin-2 (EM2) have been isolated from bovine [26] and human [10] brain, and EM1- and EM2-like immunoreactivity are found throughout the rat, mouse, and guinea pig central nervous system [13]. Endomorphin-1-like immunoreactivity, however, was more concentrated in forebrain areas, including limbic structures, of rodents as compared with EM2-like immunoreactivity. Recently, EM1 was shown to act as an anxiolytic in mice [4] and to modulate social behavior in Syrian hamsters [23]. In mice, a single intracerebroventricular (i.c.v.) injection of EM1 significantly decreased the normal preference for the closed arms of the elevated plus maze, indicating a reduction in

Immunocytochemistry The methods used for immunocytochemistry have been described elsewhere [13,14]. Brains were sectioned at 50 ␮m on a Lancer Series 1000 Vibratome TPI, O’Fallon, MO, USA) filled with ice-cold 0.05-M phosphate buffered saline (PBS; pH 7.4). The tissue was washed in NaBH4 (1 g/100 ml PBS) for three, 5-min intervals. After two, 10-min washes in PBS, the

* Address for correspondence: Dr. H. Elliott Albers, Department of Biology, Georgia State University, Atlanta, GA 30303, USA. Fax: (404) 651-3929; E-mail: [email protected]

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FIG. 1. Photomicrograph of endomorphin-1 (EM1)-like immunoreactive (EM1L-IR) perikarya in (A) dorsomedial hypothalamic nucleus (DMH) (magnification 10⫻, scale bar ⫽ 200 ␮m), (B) DMH (magnification 20⫻, scale bar ⫽ 100 ␮m), (C) ventromedial hypothalamic nucleus (VMH) (magnification 20⫻, scale bar ⫽ 100 ␮m), and (d) posterior hypothalamic (PH) nucleus (magnification 20⫻, scale bar ⫽ 100 ␮m).

tissue was incubated for 10 min in serum blocker containing 1% normal goat serum (NGS), 0.2% Triton-X 100 (TX), and 1% H2O2 in PBS. The tissue then was incubated for 15 min in 2% NGS and 0.4% TX in PBS. The tissue then was incubated for 40 h at 4°C on an orbital shaker in rabbit EM1 antiserum (1:5000) diluted in PBS, 2% NGS, 0.4% TX, and 0.1% BSA. Following three washes in cold PBS, the tissue was incubated for 2 h in 1:200 goat anti-rabbit biotinylated secondary antibody (VECTASTAIN Elite kit; Vector Laboratories, Burlingame, CA, USA), 1% NGS, and 0.2% TX in PBS. After three, 10-min washes in PBS, the tissue was incubated in

avidin-biotin complex (ABC), 2 drops A and 2 drops B per 10-ml PBS (ABC Elite Vector kit), solution for 1 h at room temperature. After four 15-min washes in PBS, the tissue was incubated in a 50-mg diaminobenzidine/100 ml 0.05-M Trizma base, pH 7.6 (Sigma Chemical Co., St. Louis, MO, USA) with 0.003% H2O2. Sections were washed twice for 5 min each in 0.01-M phosphate buffer, mounted on slides, and air-dried overnight. The prepared slides were cleared with xylene and coverslipped with a 60% Permount/40% xylene mixture. All controls were processed for the same duration and in identical solutions as described above. In addition, the EM1 antiserum at a working dilution of 1:5000 was preabsorbed with 10 ␮M of

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FIG. 2. (A) Photomicrograph of EM1L-IR cell bodies in the region between the DMH and VMH. (B) Photomicrograph of alternate section incubated in EM1 antiserum preabsorbed with 10-␮m EM1. Magnification 20⫻, scale bar ⫽ 100 ␮m.

EM1. After rotating for 2 h at room temperature, the antiserum/ peptide solution was agitated for 24 h at 4°C. RESULTS Distribution of EM1-like immunoreactive perikarya In colchicine-treated animals only, EM1L-IR cell bodies were present in several regions of the hypothalamus. Endomorphin-1like immunoreactive cell bodies were especially abundant in the medial zone, including the dorsomedial nucleus (Fig. 1 A,B), ventromedial nucleus (Fig. 1 C), and the region between the dorsomedial and ventomedial nuclei (Fig. 2A). EM1L-IR cell bodies were present also in moderate abundance in the posterior nucleus (Fig. 1D) and in low abundance in the anterior area, periventricular nucleus, and arcuate nucleus. Preabsorption of EM1 antiserum at its working concentration of 1:5000 with 10 ␮M of EM1 effectively blocked EM1-like immunoreactivity (Figs. 2A,B). The results are summarized in Fig. 3A–E. Distribution of EM1-like immunoreactive fibers The results are summarized in Table 1. The presence of EM1L-IR in the nucleus accumbens was concentrated in the anterior and medial portion of the nucleus. Immunoreactive fibers were located in both the core and the shell. EM1L-IR fibers in the caudate putamen were limited to the anterior portion of the nucleus. EM1L-IR varicose fibers were in high abundance in the lateral septum (Fig. 4A), and nonvaricose fibers were moderately abundant in the medial septum (Fig. 4B). The bed nucleus of the stria terminalis contained a moderate abundance of EM1L-IR fibers from the anterior portion of the nucleus through the posterior portion (Fig. 4C). EM1L-IR fibers in the amygdaloid complex were sparse. A low abundance of fibers were located in the anterior area, anterior cortical nucleus, central nucleus, medial nucleus, basomedial nucleus, and basolateral nucleus. In the hypothalamus, EM1L-IR fibers were located in the medial preoptic area, lateral preoptic nucleus, medial preoptic nucleus (Fig. 4D), median pre-

optic nucleus, anterior hypothalamic nucleus, lateral hypothalamic nucleus, ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, paraventricular nucleus (Figs. 4E,F), and periventricular nucleus. DISCUSSION Similar to rats [13], EM1L-IR is widespread in the limbic system of Syrian hamsters. EM1L-IR cell bodies are found in the hypothalamus, and EM1L-IR fibers are located throughout the limbic system. In the diencephalon, the distribution of EM1L-IR matches that reported for ␮-opiate receptors in the lateral and medial preoptic areas, the anterior hypothalamic area, lateral hypothalamic nucleus, dorsomedial and ventromedial hypothalamic nuclei, paraventricular nucleus, periventricular nucleus, suprachiasmatic nucleus, and the arcuate nucleus [7,8]. Telencephalic sites that contain EM1L-IR fibers in which ␮-opiate receptors also can be identified [7,8] include the nucleus accumbens, caudate putamen, lateral and medial septal nuclei, bed nucleus of the stria terminalis, and amygdaloid complex. The distribution of EM1 in these limbic structures indicates a potential endogenous role in major processes modulated by opiates, including affective states and social behavior. The presence of EM1 in the amygdala and hypothalamus, for example, places this peptide in an area of the brain that plays a pivotal role in fear, anxiety, flank marking, and agonistic behavior [1,2,6,9,11,20,24]. EM1L-IR also is present in the nucleus accumbens, which is part of an important mesolimbic pathway that is involved in reward [21,25] and social defeat stress [16,18]. Furthermore, EM1 is present in the septum, which is involved in anxiety-related responses [19] and defensive behavior [3], and the bed nucleus of the stria terminalis, which is involved in social communication [5]. The localization of EM1 is consistent with the possibility that this peptide plays a modulatory role in affective states and social behavior. ACKNOWLEDGEMENT

Supported by NS37232 to H.E.A.

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WHITTEN ET AL. TABLE 1 DISTRIBUTION OF ENDOMORPHIN-1–LIKE IMMUNOREACTIVITY IN SYRIAN HAMSTER LIMBIC STRUCTURES; ⫹, LOW ABUNDANCE; ⫹⫹, MODERATE ABUNDANCE; ⫹⫹⫹, HIGH ABUNDANCE Structure Abbreviation

AcbC AcbS AAA ACo AH Arc BLA BMA BST Ce Cpu DMH LH LPO LS MeA MnPO MPN MPO MS PVN Pe PH SCN VMH

FIG. 3. Camera lucida drawings of the hypothalamus illustrating the distribution of EM1L-IR perikarya in the colchicine-treated hamsters. AH ⫽ anterior hypothalamic area; Arc ⫽ arcuate nucleus (modified from Morin and Wood [17]).

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Structure

accumbens nucleus, core accumbens nucleus, shell anterior amygdaloid nucleus anterior cortical amygdaloid nucleus anterior hypothalamic area arcuate hypothalamic nucleus basolateral amygdaloid nucleus basomedial amygdaloid nucleus bed nucleus of the stria terminalis central amygdaloid nucleus caudate putamen dorsomedial hypothalamic nucleus lateral hypothalamic area lateral preoptic nucleus lateral septal nucleus medial amygdaloid nucleus median preoptic nucleus medial preoptic nucleus median proptic area medial septal nucleus paraventricular hypothalamic nucleus periventricular hypothalamic nucleus posterior hypothalamic nucleus suprachiasmatic nucleus ventromedial hypothalamic nucleus

EM1-IR

⫹⫹ ⫹ ⫹ ⫹ ⫹⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹

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FIG. 4. Photomicrograph of EM1L-IR fibers in (a) lateral septum (magnification 20⫻, scale bar ⫽ 100 ␮m), (B) medial septum (magnification 40⫻, scale bar ⫽ 50 ␮m), (C) bed nucleus of the stria terminalis (magnification 40⫻, scale bar ⫽ 50 ␮m), (D) medial preoptic nucleus (magnification 20⫻, scale bar ⫽ 100 ␮m), and (E) and (F) paraventricular hypothalamic nucleus (magnification 40⫻, scale bar ⫽ 50 ␮m).

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