Proopiomelanocortin (POMC) mRNA expression: distribution and region-specific down-regulation by chronic morphine in female guinea pig hypothalamus

Molecular Brain Research 55 Ž1998. 1–8

Research report

Proopiomelanocortin žPOMC / mRNA expression: distribution and region-specific down-regulation by chronic morphine in female guinea pig hypothalamus Yuan Fang a

a,b

, Martin J. Kelly a , Oline K. Rønnekleiv

a,b,)

Department of Physiology and Pharmacology, Oregon Health Sciences UniÕersity, Portland, OR 97201, USA b Neuroscience DiÕision, Oregon Regional Primate Research Center, BeaÕerton, OR 97006, USA Accepted 29 October 1997

Abstract There is compelling evidence that endogenous opioid peptides are regulated by exogenous opiates. Our previous studies have shown that the m-opioid receptor protein and mRNA are down-regulated in the mediobasal hypothalamus of the female guinea pig following chronic morphine treatment. In addition, electrophysiological studies have shown that hypothalamic b-endorphin Ž b-EP. neurons express m-opioid receptors that are uncoupled and down-regulated following chronic morphine treatment. Currently, we tested the hypothesis that chronic morphine, which produces down-regulation of m-opioid receptors, causes a down-regulation of pro-opiomelanocortin ŽPOMC, the precursor of b-EP. mRNA expression in female guinea pig hypothalamus. Female guinea pigs were ovariectomized and implanted subcutaneously Žs.c.. with 4 = 75 mg pellets for 2 days plus six more pellets of either morphine Ž n s 6. or placebo Ž n s 6. for another 5 days. Animals were sacrificed between 1000 and 1100 h on day 7. The expression of POMC mRNA were investigated using in situ hybridization histochemistry with a guinea pig specific 35S-labeled cRNA probe in hypothalamic tissue sections. POMC mRNA was localized to the arcuate nucleus ŽArc. and median eminence ŽME. of the medial basal hypothalamus. The distribution pattern was the same in both morphine and placebo control animals. However, the density of silver grains was less in morphine treated animals versus placebo control animals. Overall, the level of POMC mRNA was decreased by 22% in the Arc of morphine-treated guinea pigs as compared with the placebo controls Ž p - 0.05.. This decrease in POMC mRNA expression was even greater in the caudal Arc Ž28%, p - 0.01. in morphine-treated animals. These results suggested that the biosynthetic activity of POMC neurons is down-regulated with chronic exposure to morphine. q 1998 Elsevier Science B.V. Keywords: Arcuate nucleus; b-endorphin; Female guinea pig; In situ hybridization histochemistry; Median eminence; Morphine pellet

1. Introduction Proopiomelanocortin ŽPOMC., one of three endogenous opioid peptide precursors in the central nervous system, produces a biologically active opioid peptide b-endorphin Ž b-EP. w8,16,44,52,64x. The b-EP-containing POMC neurons are primarily located in the arcuate nucleus ŽArc. and the periarcuate area of the medial basal hypothalamus of rat and guinea pigs w7,22,36,65,68x. In addition, a small group of POMC neurons is also found in the nucleus of the solitary tract ŽNTS. in the lower brainstem w12,33x; how-

)

Corresponding author. Dept. of Physiology and Pharmacology, L334, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. Fax: q 1-503494-4352; E-mail: [email protected] 0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 4 8 - 3

ever, the physiological role of the NTS POMC neurons is not clear. b-EP neurons in the basal hypothalamus send projections to multiple brain regions and participate in a wide range of functions such as neuroendocrine control of pituitary hormone secretion, stress responses, antinociception, feeding behavior and reward w2,3,17,18,25,60,62, 63,66x. Chronic morphine treatment has complex actions on endogenous POMC-derived peptides, including their biosynthesis, release and physiological effects w11,23,26,45,48,49,67,69x. Several studies in male rats and one study in female rats have shown that chronic morphine treatment decreases POM C mRNA expression w11,23,48,49,67x, but hypothalamic immunoreactive b-EP levels remain unchanged or show little change after chronic treatment w11,48,49,67x. In addition, potassium-stimulated release of b-EP is not affected by chronic morphine

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treatment w11x, which may indicate that the releasable pool of b-EP does not change w11,48,67x. Previously, we found that m-opioid receptors and receptor mRNA in the mediobasal hypothalamus were downregulated in female guinea pig following chronic morphine treatment w57,69x. Based on the electrophysiological response to selective m-opioid agonists, the b-EP neurons appeared to be the most sensitive to these effects of chronic morphine w69x. Therefore in the present study, we tested the hypothesis that chronic morphine, which produces down-regulation of m-opioid receptors, causes a decrease in POMC mRNA expression in morphine-treated female guinea pigs. Some of these data have been reported in abstract form w21x.

2. Materials and methods 2.1. Animal treatment and tissue preparation All procedures with the animals were performed according to a protocol approved by our Institutional Animal Care Committee and in accordance to NIH guidelines. Adult female guinea pigs ŽTopeka strain. were ovariectomized and subcutaneously Žs.c.. implanted with 4 = 75 mg pellets of either morphine Ž n s 6. or placebo Ž n s 6. under ketamine Ž33 mgrkg, s.c..rxylazine Ž6 mgrkg. anesthesia. Two days later, each animal received six more pellets of morphine or placebo, respectively, for a total of 7 days. Animals were killed between 1000 and 1100 h on day 7, and the brains were quickly dissected and frozen in isopentane on dry ice at y55 to y658C. The fresh tissues were sectioned on a cryostat at 15 m m and thaw-mounted onto charged slides ŽFisher Scientific, Pittsburgh, PA, USA.. The slides were stored at y808C within an air-tight container with desiccant. 2.2. Preparation of the POMC probe A pGEM-3Z plasmid, containing a 516 bp insert corresponding to bases 383 to 899 of the guinea pig POMC cDNA sequence, was obtained from Dr. Peter J. Fuller w34x. This segment of the POMC cDNA sequence is complementary to the POMC mRNA sequence which codes the POMC peptides, adrenocorticotropic hormone ŽACTH., b-melanocyte-stimulating hormone Ž b-MSH., and b-EP. Radiolabeled antisense cRNA was transcribed with SP6 RNA polymerase and 35 S-rUTP from the POMC plasmid construct linearized with NcoI. The labeled product was purified through a G-50 Nick column ŽPharmacia Biotech, Alemeda, CA, USA.. 2.3. In situ hybridization histochemistry For the in situ hybridization histochemistry procedure, matched brain sections through the medial basal hypothala-

mus from placebo- and morphine-treated animals were always processed together. The tissue sections were fixed in 4% paraformaldehyde in 0.3 M Sorensen’s buffer, pH 7.4 ŽNa 2 HPO4rKH 2 PO4 , 67 mMr67 mM, 20r20,vrv. for 20 min, rinsed briefly in Sorensen’s buffer, treated in 0.1 M triethanolamine ŽTEA. for 3 min and for 10 min in 0.25% acetic anhydride and then hybridized as previously described w58,59x. Briefly, sections were covered with 30 m l of hybridization buffer Ž50% formamide, 10% dextran sulfate,1 = Denhardt’s solution, 2 = SSC, 100 m grml of yeast transfer RNA, 125 m grml sonicated salmon sperm DNA and 100 mM dithiothreitol. and allowed to prehybridize for 60 min at 588C. After prehybridization, the sections were quickly rinsed in 2 = SSC buffer. The 35 Slabeled antisense riboprobe was heat denatured in a 908C water bath, chilled on ice, diluted with hybridization buffer and used at a final saturating concentration of 2 = 10 4 dpmrm l. Subsequently, the sections were covered with glass coverslips, sealed with DPX ŽGallard and Scleisenger. and hybridized for at least 18 h at 58–608C. After hybridization, the slides were rinsed in 2 = SSC buffer, reacted with RNase Ž20 m grml. and washed in 2.0 = , 1.0 = , 0.5 = SSC at 55–608C, to a final stringency of 0.1 = SSC at 658C. The sections were dehydrated in increasing concentrations of ethanol, and together with autoradiographic w 14 Cxmicroscales ŽAmersham Life Science, Arlington Heights, IL, USA. were exposed to hyperfilmbmax X-ray film ŽNEN, Boston, MA, USA. for 3 to 6 days. The slides were then dipped in Kodak NTB-3 nuclear track emulsion and exposed for up to 14 days at 48C. Slides were developed in D19 developer, fixed in Kodak fixer, and counterstained with Gill’s 3 = Hematoxylin Ždiluted 1:12 with water.. Slides were then dehydrated in ethanol, cleared in xylene and cover slipped with Entellan. 2.4. Quantification Quantification of film images was performed using a Quadra Macintosh computer equipped with a flat bed scanner ŽHewlett Packard Scan Jet IIcx. and the NIH Image 1.54 software. The autoradiographic w 14 Cxmicroscale was used as a standard to calculate the POMC signal density in the X-ray film. The arcuate nucleus was divided into rostral and caudal parts of approximately equal length based on a guinea pig brain atlas w5x. Three sections, approximately 300 m m apart, from each subdivision were used for quantification and an average value of the three was used for further analysis. The films containing the autoradiographic signals were scanned and the optical densities of the signals were measured. The outline of the POMC mRNA images were traced in both groups of animals in order to perform the densitometry measurements. The optical density was converted to nCirg using the standard autoradiographic w 14 Cxmicroscale. The area encompassing the POMC mRNA signal was also measured. The emulsion coated slides were evaluated and

Y. Fang et al.r Molecular Brain Research 55 (1998) 1–8

photographed using a Leitz microscope with bright-field and dark-field condensers. The total number of cells with positive POMC mRNA signal Žat least 10 = background. were counted in the emulsion coated sections under bright-field microscopy. Using bright-field microscopy individual POMC mRNA-containing cells were identified by the higher concentration of grains immediately surrounding the cell nucleus. Thus the cell counting was possible, although the grains from these cells often overlapped which made grain quantification in individual cells difficult. The average number of positive cells in each section was used for further analysis. Statistical analysis was

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performed using a paired two-tailed Student’s t-test. A p-value less than 0.05 was considered a significant difference.

3. Results 3.1. Anatomical distribution Based on the analysis of the X-ray films and of microscopic examination of emulsion-coated brain sections, POMC mRNA was found almost exclusively in the arcuate

Fig. 1. Dark-field autoradiograms ŽA, C, E and G. taken from film images, and bright-field photomicrographs ŽB, D, F and H. taken from the emulsion-coated sections showing the distribution of POMC mRNA in the arcuate nucleus ŽArc. and median eminence ŽME. of the basal hypothalamus from a representative animal. A and B, C and D, E and F, G and H are matched anatomically and come from the same tissue sections. The scale bar in G s 1.0 mm for A, C, E and G. The scale bar in H s 0.5 mm for B, D, F and H. 3V: the third ventricle.

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Fig. 2. Bright-field ŽA, C. and dark-field ŽB, D. view of autoradiograms taken from film images which illustrate the densities of POMC mRNA signals in the caudal part of the arcuate nucleus ŽArc. of control ŽA, B. and morphine-treated ŽC, D. animals. Note that the density of POMC mRNA signal in the morphine-treated animal is less than the density in the control animal. 3V: the third ventricle. ME, median eminence. The scale bar s 0.8 mm for A, B, C and D.

nucleus ŽArc. and the median eminence ŽME. of the medial basal hypothalamus ŽFigs. 1 and 2.. Cells expressing POMC mRNA were distributed throughout the Arc from rostral to caudal regions ŽFigs. 1 and 2.. Moreover, POMC cells were also found in the ME ŽFig. 1.. Within the rostral ME, there were a few scattered POMC mRNAcontaining cells ŽFig. 1D.; however in the caudal ME, there was a higher density of positive cells in both the internal and external zones of the ME ŽFig. 1F,H.. A few scattered cells were also observed in the ventral hypothalamus lateral to the arcuate nucleus and in the medial part of the ventromedial nucleus of the hypothalamus. 3.2. Effects of morphine We have used a morphine treatment regimen similar to Goldstein and coworkers w13,28x to produce tolerance in female guinea pigs and have observed tolerance of hypothalamic neurons to m-opioid receptor activation w69x. With this morphine treatment regimen, we obtain serum morphine levels of 663.2 " 150.5 ngrml w57x, which is similar to the levels reported by the Goldstein lab w28x. The present study was aimed at elucidating the changes in hypothalamic POMC mRNA expression in this guinea pig model of morphine tolerance. Within the Arc, cells expressing POMC mRNA were highly concentrated with overlapping clusters of grains, which made grain counting from individual cells difficult ŽFigs. 1 and 3.. Therefore, all quantification was done through measurements of autoradiographic film density. The area encompassing the POMC mRNA signal was measured and was similar in

Fig. 3. Dark-field photomicrographs of emulsion-coated sections comparing the distribution of POMC mRNA in the rostral arcuate nucleus ŽArc. of the hypothalamus in a representative control ŽA. and morphine-treated ŽB. ovariectomized female guinea pig. The scale bar s 0.5 mm for both A and B. 3V: the third ventrical.

Y. Fang et al.r Molecular Brain Research 55 (1998) 1–8 Table 1 POMC mRNA levels ŽnCirg. in the arcuate nucleus ŽArc. of placebo and morphine-treated guinea pigs Treatment

Arc Whole

Rostral

Caudal

Placebo Morphine %, change

176.17"31.52 137.60"33.24 ) 21.9

162.10"27.55 135.33"31.28 16.5

190.37"39.73 136.93"39.30 ) ) 28.0

The effect of chronic morphine on the expression of POMC mRNA in the arcuate nucleus was quantified from film images. This analysis revealed that morphine treatment decreased POMC mRNA levels particularly in the caudal part of the Arc. ) : p- 0.05; ) ) : p- 0.01 vs. placebo groups, paired two-tailed Student’s t-test; shown as mean"S.E.M Ž ns6 per group..

both the control and the morphine-treated animals. The densities of X-ray film signal, however, were significantly less in morphine-treated animals versus placebo controls ŽFig. 2; Table 1.. The quantitative data derived from the films showed that the level of POMC mRNA in the Arc of morphine-treated guinea pigs Ž n s 6. was decreased overall by 22% as compared with the placebo controls Ž n s 6. Ž p - 0.05.. Based on regional differences in physiological responses of the Arc POMC neurons w15,41,69x, we subdivided the Arc into two equal parts along its rostral to caudal axis. Based on this division, we found that there was a significant decrease of 28% in POMC mRNA level in the caudal part of the Arc Ž p - 0.01., whereas there was not a significant decrease Žonly 16%. in POMC mRNA expression in the rostral part of the Arc in morphine-treated versus placebo-treated animals ŽTable 1.. In order to establish if the decrease in POMC mRNA level that was measured in the film images from morphine-treated animals was due to a decrease in POMC mRNA-containing cell numbers, we counted the total number of positive cells in each section. Overall, an average of 183.8 " 21.5 versus 157.1.5 " 12.1 POMC-containing cells were counted per section in control and morphine-treated guinea pigs, a difference of 14.5% which was not statistically significant. Within the rostral Arc, the number of cells expressing POMC mRNA was 172.8 " 13.5 and 164.7 " 17.5 in control and morphine-treated animals, respectively. Within the caudal Arc, 194.0 " 33.1 POMC mRNA-containing cells were found in controls versus 149.2 " 19.2 in morphine-treated guinea pigs, a difference of 23% which was not statistically significant.

4. Discussion In the present study, we have delineated the distribution of cells expressing POMC mRNA within the medial basal hypothalamus of the guinea pig using in situ hybridization histochemistry. This procedure revealed that POMC mRNA-containing cells are found in the arcuate nucleus and the median eminence of the medial basal hypothala-

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mus with a few cells in the ventromedial nucleus and in the area lateral to the arcuate nucleus. There were no cells that expressed POMC mRNA in any other hypothalamic area. The distribution pattern of POMC mRNA-containing cells reported here is consistent with the immunohistochemical localization of the POMC-derived peptides in guinea pig w65x, rat w7,30,68x, cat w56x, monkey w36x and human w6x hypothalamus. Similarly, the localization of POMC mRNA cells in the guinea pig arcuate nucleus is also consistent with previous studies of POMC mRNA distribution in the rat w4,24x. However in the rat, POMC mRNA neurons are also found in the posterior periventricular region w4x, but as currently demonstrated these cells are absent in the guinea pig hypothalamus. In addition, we have found that POMC mRNA-containing cell bodies are distributed in both the internal and external zones of the median eminence in the guinea pig. This finding has been confirmed using immunocytochemical staining of b-endorphin cells in the guinea pig median eminence ŽRønnekleiv, unpublished observations.. In contrast, cells expressing POMC in the rat median eminence are localized only in the internal zone w4,31x. The functional significance of the high abundance of POMC-containing cells throughout the median eminence of the guinea pig is currently unknown, but may reflect a greater role of POMC-derived peptides in the control of neurosecretion in the guinea pig. This, however, needs to be elucidated. In the present study, we found that chronic morphine decreased the level of POMC mRNA expression in the ArcrME of ovariectomized female guinea pigs. The greatest decrease in POMC mRNA levels were found in the caudal versus the rostral Arc in the morphine group as compared with the placebo group. This is the first report in female guinea pig showing a regional specific down-regulation of POMC mRNA expression induced by chronic morphine exposure. In addition, these results suggest that exposure to chronic morphine induces a down-regulation of POMC biosynthesis, which supports the hypothesis that chronic morphine inhibits the endogenous opioid system via a negative feedback mechanism w39x. It should be noted that the number of POMC mRNA-containing cells was decreased by 23% in the caudal Arc of morphine-treated guinea pigs in the present study, which was not statistically significant. However, the reduction in the number of cells that express detectable levels of POMC mRNA may have contributed to the overall reduction in POMC mRNA density in the morphine-treated animals versus placebotreated controls. The present study is consistent with previous reports which studied the same brain region with similar regimen of morphine treatment but in different species with different methods. For example in the rat hypothalamus, POMC mRNA expression is decreased by 50% with Northern blot analysis following a 5-day morphine treatment regimen w48,49x, by 30% as quantified by in situ hybridization histochemistry with a 4-day exposure to morphine w23x,

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and by 20–50% as analyzed by solution hybridization with a 7-day morphine treatment w9x. Recently, Wardlaw et al. w67x reported that a longer morphine treatment regimen Ž10 days. also induced a significant decrease in POMC mRNA expression in the hypothalamus of castrated male and female rats as analyzed with nuclease protection assay of POMC mRNA. Therefore, our results as well as the results from others suggest that the inhibitory effects of chronic morphine on POMC mRNA expression is evident as early as day 4 of treatment and lasts continually for 10 days and perhaps even longer with chronic morphine exposure. In addition, POMC and corticotropin-like intermediate lobe peptide content are also decreased in the hypothalamus w48,49x. This would indicate that the synthesis of POMC and related peptides Že.g., b-EP. is down-regulated by chronic morphine. Therefore, it is apparent that chronic morphine inhibits the endogenous POMC system. Furthermore, exogenous opiates may replace endogenous opioid peptides such as b-EP and participate in the adverse physiological responses during morphine withdrawal as proposed by Kosterlitz and Hughes w39x. The question arises as to what is the mechanism involved in the inhibitory effects of chronic morphine on POMC mRNA expression. Our previous studies have shown that b-EP neurons in the arcuate nucleus of the guinea pig hypothalamus are directly hyperpolarized by m-opioid receptor selective agonist such as wD-Ala2 , MePhe 4 , Gly-ol 5 xenkephalin ŽDAMGO. via the opening of inwardly rectifying Kq channels and these effects of DAMGO are totally blocked by the opioid antagonist naloxone. In contrast, the k-selective agonist U50, 488H and d-selective agonist cyclic wD-penicillamine 2 , D-penicillamine 5 xenkephalin ŽDPDPE. have no effect on b-EP neuronal excitability w35,46x. Therefore, the m-opioid is autoreceptor in b-EP neurons, which fits with the known physiological actions of m-opioid receptor activation on b-endorphin secretion w54x. In addition, we recently found that chronic morphine treatment decreases both the functional coupling of m-opioid receptors to Kq channels in b-EP neurons and the density of m-opioid receptors in the guinea pig medial basal hypothalamus after 6 to 8 days of morphine treatment w69x. This would suggest that down regulation of m-opioid receptors in b-EP neurons should increase neuronal excitability, but chronic morphine treatment caused a down-regulation of POMC-b-EP expression. The cause of this overall decrease in excitability may be related to an increase in the GABAergic input onto b-endorphin neurons as a result of uncoupling of postsynaptic m-opioid receptors in GABA neurons w35,42x. Therefore, the release of opioid inhibition of GABAergic neurons would have pronounced negative feedback effects on b-endorphin neurons. In fact, GABAergic synapses constitute half of all of the synaptic input onto Arc neurons w19x. Furthermore, results from studies using the opioid receptor antagonists naloxone and naltrexone would support this circuitry. A 4-day treatment regimen with nalox-

one produces a 60% increase in POMC mRNA level in the rat Arc, an effect which is opposite to the agonist morphine w23x. In addition, chronic treatment with naltrexone results in an up-regulation of b-EP biosynthesis and release in hypothalamus Arc neurons w10x. The release of POMC-related peptides is also increased by acute treatment with naltrexone in vitro and in vivo from the rat hypothalamus w32x. Therefore, Arc POMC-b-EP neurons appear to be under a tonic inhibitory control, and this negative feedback may involve both m-opioid receptors and GABA interneurons. Another possibility is that Arc POMC-b-EP may be down-regulated by chronic morphine through inhibition of corticotrophin-releasing factor ŽCRF.. CRF plays a major role in activating POMC mRNA gene expression and release of POMC-related peptides in the pituitary w1,43,61x and b-endorphin release from hypothalamic slices w53,55x. However, based on several studies in the rat, CRF mRNA levels are not affected by either chronic morphine or naloxone treatment w29,45x. Therefore, it appears that CRF neurons may not be involved in the down-regulation of POMC-b-EP system by chronic morphine in Arc. We observed a regional difference in the effects Ži.e., caudal versus rostral. of chronic morphine on POMC expression, which may be explained by differences in physiological function. Arcuate b-endorphin neurons synapse on a number of neurosecretory neurons with the rostral group projecting to the rostral neurosecretory cells Že.g., preoptic and supraoptic nucleus. and the caudal group synapsing on Arc neurosecretory neurons w14,27x. Arcuate neurosecretory neurons are inhibited via activation of a Kq channel which is coupled to m-opioid receptors w35,40,47x. In addition, a caudal group of these b-endorphin neurons project to the ventral tegmental area ŽVTA. of the midbrain w50,51x, and the activation of VTA dopamine neurons probably underlies the reward mechanism for endogenous opioids w20,37x. Moreover, these VTA projecting neurons are thought to be involved in the activation of the natural reward circuits such as feeding, drinking, and sexual and maternal behavior w20,37,38x. Indeed, it is this caudal group of POMC neurons that appear to be most susceptible to chronic opiate exposure based on the present findings and our previous electrophysiological results w69x. Interestingly, there is a regional difference in POMC expression following gonadal steroid treatment in the rat with the rostral Arc being more sensitive than the caudal Arc w15x. Therefore, based on the distinct physiological role of the caudal versus the rostral b-endorphin neurons, it is reasonable that we should see a regional difference in POMC expression following chronic morphine. In summary, the current study demonstrates that chronic morphine produces a down-regulation of POMC mRNA expression in the arcuate nucleus of the hypothalamus in the ovariectomized female guinea pig. The decrease in POMC mRNA expression appears to be most pronounced

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in the caudal part of the arcuate nucleus following chronic morphine exposure. Therefore, the present results taken together with the previous studies support the hypothesis that the endogenous POMC-b-EP system is under negative feedback control by m-opioid receptors. Acknowledgements This study was supported by NIH grants DA07165, DA05158, and DA00192 ŽRSDA to MJK.. We would like to thank Dr. Peter J. Fuller for his generous gift of POMC cDNA plasmid, and also thank Martha Bosch, Barry Naylor and Matthew J. Cunningham for their helpful assistance with experiments. References w1x D.J. Autelitano, M. Blum, M. Lopingco, R.G. Allen, J.L. Roberts, Corticotropin-releasing factor differentially regulates anterior and intermediate pituitary lobe proopiomelanocortin gene transcription, nuclear precursor RNA and mature mRNA in vivo, Neuroendo. 51 Ž1990. 123–130. w2x R.A. Baker, W.J. Shoemaker, Effect of prenatal ethanol and stress on levels of b-endorphin in different brain regions of the rat, Alcoholism: Clin. Exp. Res. 19 Ž1995. 727–734. w3x J.V. Bartolome, M.B. Bartolome, B.A. Lorber, S.J. Dileo, S.M. Schanberg, Effects of central administration of beta-endorphin on brain and liver DNA synthesis in preweanling rats, Neuroscience 40 Ž1991. 289–294. w4x V. Baubet, M. Fevre-Montange, N. Gay, G. Debilly, P. Bobillier, R. Cespuglio, Effects of an acute immobilization stress upon proopiomelanocortin ŽPOMC. mRNA levels in the mediobasal hypothalamus: a quantitative in situ hybridization study, Mol. Brain Res. 26 Ž1994. 163–168. w5x R. Bleier, The Hypothalamus of the Guinea Pig: A Cytoarchitectonic Atlas, University of Wisconsin Press, Madison, 1983. w6x B. Bloch, C. Bugnon, D. Fellman, D. Lenys, Immunocytochemical evidence that the same neuron in the human infundibular nucleus are stained with anti-endorphins and antisera of other related peptides, Neurosci. Lett. 10 Ž1978. 147–152. w7x F.E. Bloom, E. Battenberg, J. Rossier, N. Ling, R. Guillemin, Neurons containing b-endorphin in rat brain exist separately from those containing enkephalin: immunocytochemical studies, Proc. Natl. Acad. Sci. USA 75 Ž1978. 1591–1595. w8x A.F. Bradbury, D.G. Smyth, C.R. Snell, N.J.M. Birdsall, E.C. Hulme, C fragment of lipotropin has a high affinity for brain opiate receptors, Nature 260 Ž1976. 793–795. w9x D.M. Bronstein, H. Akil, In vitro release of hypothalamic b-endorphin Ž b E. by arginine vasopressin, corticotropin-releasing hormone and 5-hydroxytryptamine: evidence for release of opioid active and inactive b E forms, Neuropeptides 16 Ž1990. 33–40. w10x D.M. Bronstein, N.C. Day, H.B. Gutstein, K.A. Trujillo, H. Akil, Pre- and post-translational regulation of b-endorphin biosynthesis in the CNS: effects of chronic naltrexone treatment, J. Neurochem. 60 Ž1993. 40–49. w11x D.M. Bronstein, R. Przewlocki, H. Akil, Effects of morphine treatment on pro-opiomelanocortin systems in the rat brain, Brain Res. 519 Ž1990. 102–111. w12x D.M. Bronstein, M.K.-H. Schafer, S.J. Watson, H. Akil, Evidence that b-endorphin is synthesized in cells in the nucleus tractus solitarius: detection of POMC mRNA, Brain Res. 587 Ž1992. 269– 275.

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