Opioid regulation of proopiomelanocortin (POMC) gene expression in the rat brain as studied by in situ hybridization

Opioid regulation of proopiomelanocortin (POMC) gene expression in the rat brain as studied by in situ hybridization

Neuropeptides (1993) Z&91-94 0 Longman Group UK Ltd 1993 Opioid Regulation of Proopiomelanocortin (POMC) Gene Expression in the Rat Brain as Studied ...

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Neuropeptides (1993) Z&91-94 0 Longman Group UK Ltd 1993

Opioid Regulation of Proopiomelanocortin (POMC) Gene Expression in the Rat Brain as Studied by in situ Hybridization E. GARCIA DE YEBENES and G. PELLETIER MRC Group in Molecular Endocrinology, CHUL Research Quebec, Canada (Reprint requests to GP)

Center and Lava/ University,

Abstract-Proopiomelanocortin (POMC) is the precursor of the potent opioid peptide P-endorphin as well as a number of other active peptides. On the basis of neuroanatomical data indicating the presence of contacts between POMC neurons in the rat arcuate nucleus,3.’ it has been proposed that POMC neurons could be autoregulated. In order to investigate the role of opiates in the regulation of POMC gene expression in the rat arcuate nucleus, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on POMC mRNA levels as measured by in situ hybridization. 4-day treatment with naloxone (4 mg/kg/day) produced a 60% increase in the number of silver grains overlying POMC neurons. Conversely, morphine (40 mg/kg/day) also administered during 4 days decreased the hybridization signal by 30%. The concomitant administration of morphine and naloxone completely prevented the effect of morphine on POMC gene expression indicating that the inhibitory influence of morphine is likely to be mediated by opioid receptors. The data obtained clearly indicate that activation of opioid receptors decreased the biosynthetic activity of POMC neurons and that conversely opiate receptor blockade caused an increase in the activity of these neurons. They are consistent with the hypothesis of an autoregulation of the POMC neuronal system by endogenous opiate peptide( Introduction

stimulating hormone (MSH). Anatomical evidence obtained from immunohistochemical studies has demonstrated that axions from several neurotransmitter systems, such as catechoiaminergic, serotoninergic and GABAergic systems1J4J8 are making synaptic contacts with POMC-containing neurons located in the arcuate nucleus. Moreover, lodal interconnections of POMC neurons have been described which may be indicative of autoregulatory processes.3*7Electrophysiological studies performed

Proopiomelanocortin (POMC) is the precursor of the potent opioid peptide j3-endorphin as well as a number of other bioactive peptides including adrenocorticotropin (ACTH) and melanocyteDate received 18 March 1993 Date accepted 22 March 1993 Correspondence to: Dr Georges Pelletier, CHUL Research Center, 2705 Laurier Blvd. Quebec GlV 4G2, Canada.

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on hypothalamic slices have also shown that opioid peptides including p-endorphin could decrease the spontaneous activity of arcuate neurons.6 This might also suggest that there is an autoregulation of POMC neurons via POMC-derived peptides. Recently, it has been shown that neuronal POMC gene expression could be modulated by dopamine as well as

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glucocorticoids and sex steroids.‘S1*J9 In order to determine the influence of opioids on POMC neurons in the rat arcuate nucleus, we have studied the effects of chronic administration of the opioid agonist morphine and naloxone, an opioid antagonist, on POMC mRNA levels using in situ hybridization at the cellular level.

Fig. 1 Light microscope autoradiographs showing typical POMC-labelled cells within the arcuate nucleus. X 800. (A) Vehicletreated male rats; (B) Naloxone-treated animals; (C) Morphine-treated animals; (D) Animals treated with both morphine and naloxone; Naloxone (B) increased and morphine (C) decreased the number of silver grains overlying POMC neurons. As shown in (D) the concomitant administration of morphine and naloxone did not modify the hybridization signal.

OPIOID REGULATION

OF POMC GENE EXPRESSION

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IN THE RAT BRAIN

Materials and methods Animals and treatments 32 adult male Sprague-Dawley rats (obtained from Charles River Canada Inc.), weighing 225-250 g were divided in four groups (8 animals per group). They were treated for 4 days with twice daily (8:00 and 20:00) intraperitoneal injections of morphine (40 mg/kg), naloxone (4.5 mg/kg) or a combination of both morphine and naloxone at the same doses. The control group received only the vehicle (0.9% NACl). On the morning of the fifth day, the animals were treated at 8:00 and were perfused 2 h later for histological procedures as described below.

for 30 min at room temperature before prehybridization. Also, sections from cerebellum, liver, and kidney were hybridized with the same probe. The number of silver grains overlying the labelled neurons was calculated using a micrometer disk in the eyepiece of the microscope. They were expressed as the number of grains per neurons. For each animal, 40 cells rostrocaudally distributed and randomly selected were analyzed. The statistical analysis according to the multiple range test of Duncan-Kramer9 was performed on the number of grains overlying 320 cells per experimental group.

Preparation of the POkEprobe

Results

The plasmid containing the rat POMC cDNA cloned in the EcoRI site of pBR 322 was generously provided by Dr J. Drouin (Montreal, Canada).4 The HindIII-XhoI restriction fragments corresponding to Exon III of the POMC gene (1.6 kb) were electrophoresed on a 1% agarose gel and electroeluted with an electroeluter (model 422: Bio-Rad, Richmond, CA, USA). The fragments were then labelled with [a-YS]deoxycytidine triphosphate (1000 Ci/mmol) (Amersham, IL, USA) by the random primer method5 to a specific activity ranging between 0.8 and 1.2 x log dpm/pg.‘5-17

Analysis of the autoradiograms indicated that a strong hybridization signal could be obtained in the arcuate nucleus and periarcuate regions of the hypothalamus after 7 days of exposure (Fig. 1A). In sections pretreated with RNase prior to hybridization, no autoradiographic reaction could be detected. Moreover, sections from cerebellum, liver and kidney did not show any hybridization (data not shown). As illustrated in Figures IB & 2, treatment with naloxone produced a 60% increase (p < 0.001) in the mean number of silver grains overlying labelled neurons. In contrast, morphine treatment decreased the hybridization signal by 30% (p < 0.001) (Fig. 1C). Administration of naloxone to morphine-

In situ hybridization Animals were fixed by intracardiac perfusion with 300 ml of 4% parafonnaldehyde, and then rinsed overnight in 0.05 M phosphate-buffered saline (PBS) containing 15% sucrose. 15-17 Thereafter, the brains were removed and frozen in isopentane chilled in liquid nitrogen. 10 pm coronal sections were cut throughout the complete length ofthe arcuate nucleus. One section out of two was collected. The sections were then mounted on gelatin- and polylysine-coated glass slides and kept at -70°C until used. In situ hybridization was performed as previously described.15-I7After hybridization, the sections were dehydrated and coated with liquid photographic emulsion (Kodak NTB-2). After 10 days of exposure, sections were processed and stained with hematoxylin and eosin. As control, sections from each group were treated with pancreatic RNase (20 pg/ml) (Boehringer Mannheim, Montreal, Canada)

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HYPOlHALAMUS-ARCUATE NUCLEUS ***

MA&ONE

Fig. 2

Effect of the administration of morphine and naloxone injected alone or in combination on POMC mRNA levels. ***p < 0.001, vehicle-treated animals (control) vs all the other experimental groups.

94 treated animals completely prevented the depressing effect of morphine (Fig. 1D). Discussion The present data clearly establish that POMC mRNA levels in neurons in the arcuate nucleus are modulated by opiates in the male rat. The prevention of the inhibitory effect of morphine by naloxone indicates that this morphine-induced effect is mediated by opiate receptors. The present finding showing that a short-term treatment with morphine decreased POMC mRNA levels in neurons of the arcuate nucleus is consistent with previous observations indicating a decrease in POMC mRNA levels in the rat hypothalamus during morphine tolerance11,12or following chronic administration of morphine.2 There are also recent studies which have shown that opiates can induce inhibition of POMCrelated peptide release. *,I3Moreover, electrophysiological studies performed on hypothalamus have also shown that p opioid agonists could decrease the spontaneous firing of arcuate neurons.6 Our results on the stimulating effect of naloxone on POMC gene expression are in agreement with recent results showing that chronic treatment with the opioid receptor antagonist naltrexone induced an increase in the concentration of mRNA levels in the hypothalamus of intact and castrated rats.‘O Opioid antagonism has also been demonstrated to increase P-endorphin release. In perfused hypothalami in vitro, both basal and potassium-stimulated j3-endorphin released increased in response to naloxone (1 0”M).13 All these results are consistent with the hypothesis that there is a feedback regulation of POMC gene expression and POMC-related peptide release by endogenous opioids. The present data as well as previous results from other groups do not allow us to identify the categories of endogenous opioid peptides involved in the regulation of POMC neurons. Since there are synaptic contacts between POMC neurons in the arcuate nucleus,3v7the opioid POMC-derived peptide P-endorphin appears a likely candidate to exert a direct tonic effect on POMC neuronal activity. Clearly, other studies especially the cellular localization of opioid receptors in the arcuate nucleus are required to firmly establish the site(s) of action of the endogenous opioid peptides.

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