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Differential 5-HT-mediated regulation of stress-induced activation of proopiomelanocortin (POMC) gene expression in the anterior and intermediate lobe of the pituitary in male rats
Brain Research 772 Ž1997. 115–120
Research report
Differential 5-HT-mediated regulation of stress-induced activation of proopiomelanocortin Ž POMC. gene expression in the anterior and intermediate lobe of the pituitary in male rats Luis Garcıa-Garcıa, ´ ´ Jose´ A. Fuentes ) , Jorge Manzanares Departamento de Farmacologia, Facultad de Farmacia and Instituto Pluridisciplinar, UniÕersidad Complutense de Madrid, 28040 Madrid, Spain Accepted 1 July 1997
Abstract The purpose of the present study was to examine the role of 5-hydroxytryptamine Ž5-HT. neurons in mediating the effects of stress on proopiomelanocortin ŽPOMC. gene expression in the anterior and intermediate lobes of the pituitary gland. To this aim, the effects of 5-HT depletion induced by administration of the neurotoxin 5,7-dihydroxytryptamine Ž5,7-DHT; 200 m grrat; i.c.v.; 7 days. were investigated on POMC mRNA levels in the anterior and intermediate lobe of control and restraint-stressed rats. Three hours after brief exposure to diethyl ether Ž2 min. followed by 60 min of restraint stress increased POMC mRNA levels in the anterior and intermediate lobe of the pituitary. 5,7-DHT neurotoxic lesion, which resulted in a marked depletion of 5-HT Žbelow the level of sensitivity of the neurochemical assay, 6 pgrsample. but not of dopamine or norepinephrine concentrations in the periventricular nucleus of the hypothalamus, had no effect on basal POMC mRNA levels in the anterior or intermediate lobe of the pituitary. However, 5-HT depletion further increased POMC mRNA levels in the anterior pituitary and completely blocked POMC mRNA level enhancement induced in the intermediate lobe of stressed rats. These results suggest a possible inhibitory 5-HT tone on POMC gene expression in the anterior pituitary and a stimulatory 5-HT tone in the intermediate lobe of the pituitary under these experimental conditions of stress. It appears, therefore, that 5-HT exerts a differential regulation of stress-induced activation of POMC gene expression in the anterior and intermediate lobes of the pituitary in male rats. q 1997 Elsevier Science B.V. Keywords: Proopiomelanocortin; mRNA; Pituitary; 5-Hydroxytryptamine; 5,7-Dihydroxytryptamine
1. Introduction Proopiomelanocortin ŽPOMC. is the precursor molecule of the adrenocorticotropin hormone ŽACTH., the opioid peptide b-endorphin, b-lipotropin Ž b-LPH., and a-, band g-melanocyte-stimulating hormones ŽMSH.. POMC mRNA has been detected in the anterior and neurointermediate lobes of the pituitary gland, as well as in the arcuate nucleus of the hypothalamus and the amygdala w4,8x. Although POMC is synthesized in the pituitary gland, its post-translational proteolytic processing is different in the anterior and intermediate lobes. The major peptides produced in the anterior lobe are ACTH and b-LPH, while the predominant POMC related peptides in the intermediate lobe are a-MSH and b-endorphin w7x. POMC mRNA ) Corresponding author. Instituto Pluridisciplinar, Paseo Juan XXIII 1, Universidad Complutense, 28040 Madrid, Spain. Fax: q34 Ž1. 394-3264; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 6 3 - 9
levels in the intermediate lobe are under inhibitory dopaminergic control mediated by dopamine D 2 receptors w18x and under positive control by b-adrenergic agonists w5x. By contrast, in the anterior lobe POMC mRNA levels are regulated by glucocorticoid feedback inhibition w6x and corticotropin releasing factor ŽCRF. w2x. 5-Hydroxytryptamine Ž5-HT.-like immunoreactivity has been demonstrated in axons and axon terminals of all three pituitary lobes. A relatively dense network of 5-HT-labeled varicose fibers was present in the intermediate lobe. By contrast, only scattered fibers have been detected in the anterior and neural lobes w16x. The effects of acute physical and psychological stressful stimuli have been reported to increase POMC mRNA levels in the anterior and neurointermediate lobes of the pituitary gland w12,14,17x. In addition, it has been shown that 5-HT neurons mediate the increase in the secretion of a-MSH, the major cleavage product of POMC in the intermediate lobe, by regulating the inhibitory effects of
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restraint stress on periventricular hypophysial dopaminergic neurons w11x. However, little is known on the 5-HTmediated regulation of POMC mRNA levels in the anterior and intermediate lobe during stress. The purpose of the present study was to examine the role of 5-HT neurons in mediating the effects of stress on POMC gene expression in the anterior and intermediate lobes of the pituitary gland. To this aim, the acute effects of diethyl ether Ža stressor by itself. followed by restraint stress were examined after selective 5-HT neurochemical lesion induced by 5,7-dihydroxytryptamine Ž5,7-DHT. on POMC mRNA levels in the anterior and intermediate lobes of the pituitary gland. The results reveal that 5-HT neurons regulate distinctly stress-induced activation of POMC gene expression in both lobes of the pituitary.
2. Material and methods 2.1. Animals Adult male Sprague–Dawley rats, weighing 200–225 g, were obtained from Interfauna Iberica Laboratories ŽSan Feliu de Codines, Barcelona, Spain. and maintained under conditions of controlled temperature Ž23 " 18C. and lighting Žlights on 07.00–19.00 h., with food and water provided ad libitum. 2.2. Neurotoxin lesion 5,7-Dihydroxytryptamine creatinine sulfate Ž5,7-DHT; Sigma, St. Louis, MO, USA. was dissolved in a solution containing 0.3% saline and 0.1% ascorbic acid, stored over ice with minimal exposure to light. Animals were anesthetized with Equithesin Ž3 mlrkg, i.p.., and placed in a stereotaxic frame ŽDavid Kopf Instruments, Tujunga, CA, USA. with the incisor bar set 2.4 mm below the horizontal plane. A 10 m l Hamilton syringe was inserted into the lateral ventricle at coordinates A 0.0 mm, L 1.4 mm, and V y3.2 mm and 200 m g of 5,7-DHT or its vehicle Ž0.3% saline containing 0.1% ascorbic acid; 5 m l. were injected over a period of 2 min. The syringe remained in the brain for an additional 10 min to prevent fluid reflux. Following the operation the wound was sealed with surgical clips, and the animals allowed to recover for 7 days. The placement of the syringe was determined using a dissecting microscope and only those animals with syringe tracts in the lateral ventricle were included in the study. 2.3. Stress procedure On the day of the experiment, control rats were removed from their home cages and quickly decapitated. Stress-treated rats were removed from their home cages and placed in a glass desiccator containing cotton batting
saturated with diethyl ether. These animals lost their righting reflex within 30 s and remained anesthetized for 2–4 min after being removed from the desiccator. During this time, the rats were immobilized in the supine position by taping them to the laboratory bench top with adhesive tape, remained in this position for 60 min, then returned to their home cages and were decapitated 3 h later. In order to avoid POMC mRNA changes induced by circadian rhythms that could mask our results, experiments were performed between 08.00 and 13.00 h. 2.4. Tissue dissection and neurochemical analyses In order to confirm the extent of the 5-HT depletion in 5,7-DHT-treated rats versus vehicle-treated rats, frontal brain sections Ž600 m m. beginning at approximately A 9220 m m w15x were prepared in a cryostat Žy98C. and the periventricular nucleus of the hypothalamus was dissected from these sections according to a modification w19x of the method of Palkovits w22x. Tissue samples were placed in 60 m l of 0.1 M phosphate-citrate buffer ŽpH 2.5. containing 15% methanol and stored at y808C until assayed. On the day of the assay tissue samples were thawed, sonicated for 3 s ŽVibra Cell, model VC-501, Sonics and Materials, Danbury, CT, USA., and centrifuged for 60 s in a Microfuge ŽIEC, model Centra-MP4R, Needham, MA, USA.. 5-HT, dopamine ŽDA. and norepinephrine ŽNE. concentrations in tissue extracts were measured by high performance liquid chromatography ŽHPLC. with electrochemical detection. Twenty m l of the supernatant were injected onto a C18 reverse-phase analytical column Ž5 m m spheres; 250 = 4.6 mm; Nucleosil; Scharlau, Barcelona, Spain. which was protected by a precolumn cartridge filter Ž5 m m spheres; 30 = 4.6 mm.. The HPLC column was coupled to a single coulometric electrode conditioning cell in series with dual electrode analytical cells ŽCoulochem II, ESA, Bedford, MA, USA.. The conditioning electrode potential was set at 100 nA, and the analytical electrodes were set at q0.12 V and y0.31 V relative to internal Ag reference electrodes. The HPLC mobile phase consisted of 0.1 M phosphatercitrate buffer ŽpH 2.8. containing 0.1 mM ethylenediaminetetraacetic acid ŽEDTA., 0.045% sodium octylsulfate and 25% methanol. 5-HT, DA and NE contents of each sample were quantitated by comparing peak heights with those peaks of standards assayed the same day as determined by Shimadzu integrator ŽShimadzu, model C-R4AX-CHROMATOPAC, Kyoto, Japan.. The lower limit of sensitivity of this assay for these compounds was 2–6 pg per sample. Tissue pellets were dissolved in 1.0 N NaOH and assayed for protein w20x. 2.5. In situ hybridization histochemistry (ISHH) Twelve m m pituitary sections Ž24 sectionsranimal; six sectionsrslide, from seven to eight animalsrgroup., were
L. Garcıa-Garcıa ´ ´ et al.r Brain Research 772 (1997) 115–120
mounted onto gelatin-coated slides and stored at y808C until the day of the assay. ISHH was performed as described previously w26x using a synthetic 48-base oligonucleotide probe complementary to the 96–134 sequence of the rat POMC gene Žobtained from the Advanced Biotechnology Center, Charing Cross and Westminster Medical School, London, UK.. The oligonucleotide probe was labeled using terminal deoxytransferase ŽBoehringer, Madrid, Spain. to add a 35 S-labeled deoxyATP Ž1000 Cirmmol; Amersham, Madrid, Spain. tail to the 3X end of the probe. The probe Žin 45 m l of hybridization buffer. was applied to each section and left overnight at 378C for hybridization. Following hybridization, sections were washed 4 times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2 Ž1 = saline sodium citrate, SSC. at 558C, followed by two 30 min washes in 1 = SSC at room temperature, one brief water dip and blown dry with air. The dried slides were apposed to Hyperfilm b-max ŽAmersham, Madrid, Spain. 18 h for anterior lobe and 1 h for the intermediate lobe of the pituitary gland. Autoradiograms were analyzed with a Macintosh computer using the public domain NIH Image program Ždeveloped at the US National Institutes of H e a lth a n d a v a ila b le o n th e In te rn e t a t http:rrrsb.info.nih.govrnih-image.. The mean hybridization signal in each experimental group was calculated from the mean of the averages of all pituitary sections per animal. 2.6. Statistical analyses Statistical analyses were performed using two-way ANOVA followed by the Student Newman-Keul’s test.
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Table 1 Effects of 5,7-DHT administration on 5-HT, NE and DA concentrations in the periventricular nucleus of the rat hypothalamus
5-Hydroxytryptamine Norepinephrine Dopamine
Control
5,7-DHT
11.5"0.5 85.2"5.7 8.2"0.7
n.d. 85.5"9.6 8.9"0.8
Rats were injected with 5,7-DHT Ž200 m grrat, i.c.v.. or its saline vehicle Ž5 m lrrat. and decapitated 7 days later. Values represent means"S.E.M. Ž ns 7–8. of 5-HT, NE and DA concentrations Žngrmg protein.. n.d., values below the level of sensitivity of the neurochemical assay Ž2–6 pgrsample..
Differences were considered significant if the probability of error was less than 5%.
3. Results In order to ascertain whether central 5-HT neurons were involved in the stress-induced stimulation of POMC gene expression in anterior and intermediate lobes of the pituitary, rats were pretreated with the selective 5-HT neurotoxin 5,7-DHT w1x. One week after the intracerebroventricular injection of 5,7-DHT, 5-HT concentrations in the periventricular nucleus of the hypothalamus Žregion that contains DA cell bodies of the periventricular hypophysial neurons projecting to the intermediate lobe of the pituitary. were markedly reduced, but 5,7-DHT injection was without effect on dopamine or norepinephrine concentrations in the same region ŽTable 1.. As shown in Fig. 1, an increase Žapproximately 20%. in POMC mRNA levels in the anterior and intermediate lobes
Fig. 1. Effects of 5,7-DHT administration on stress-induced changes in POMC gene expression in the anterior ŽA. and intermediate lobes ŽB. of the pituitary gland. Rats were injected with 5,7-DHT Ž200 m grrat, i.c.v.. or its saline vehicle Ž5 m lrrat. and decapitated 7 days later. On the day of the experiment, animals were removed from their home cages and immediately decapitated Žcontrol. or briefly anesthetized with diethyl ether, restrained in the supine position for 60 min and decapitated 3 h later Žstress.. Optical densities were calculated from the uncalibrated mode expressed in grey scale values. Results are presented considering mean control values as 100%. Columns represent means and vertical lines S.E.M. Ž n s 7–8. of POMC mRNA levels. ) Values from stressed animals that are significantly different Ž P - 0.05. from controls. a Values from 5,7-DHT-stressed rats that are significantly different Ž P - 0.05. from vehicle-stressed rats.
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of the pituitary was found 3 h after brief exposure to diethyl ether followed by 60 min of restraint stress. In the 5,7-DHT-pretreated group, basal POMC mRNA levels in the anterior and intermediate lobes of the pituitary were unaltered. By contrast, prior administration of 5,7-DHT further enhanced POMC mRNA levels Žapproximately 40% when compared with control vehicle-treated group. in the
anterior lobe and blocked the effects of stress on POMC mRNA in the intermediate lobe of the pituitary gland. These results are clearly reflected by differences on the hybridization signal of the autoradiograms of Fig. 2. Basal POMC mRNA levels in the intermediate lobe are higher than in the anterior lobe of the pituitary. Therefore, in order to detect modifications on POMC mRNA levels, film
Fig. 2. Effects of 5,7-DHT and stress on POMC mRNA levels in the anterior and intermediate lobes of the pituitary gland. Autoradiograms of coronal pituitary sections of anterior ŽA–D. and intermediate ŽE–H. lobes. A,E: vehicle control; B,F: 5,7-DHT control; C,G: vehicle stress; D,H: 5,7-DHT stress. Film was exposed 18 h for the anterior lobe and 1 h for the intermediate lobe of the pituitary. Experimental design was as indicated in Fig. 1. Bar s 1 mm.
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was exposed 18 h for the anterior lobe and 1 h for the intermediate lobe of the pituitary. As a consequence of the differences in film exposure time, note that in Fig. 2A–D the intermediate lobe is overexposed whereas in Fig. 2E–H the anterior lobe is hardly visible. As shown in Fig. 2, the anterior lobe in panel D Ž5,7-DHT-stress-treated group. exhibits the highest hybridization signal when compared with the anterior lobes presented in panels A–C. By contrast, in panel G Žvehicle-stress group. the intermediate lobe shows the highest hybridization signal when compared with intermediate lobes presented in panels E, F and H.
4. Discussion The results of the present study demonstrated a differential 5-HT-mediated regulation of POMC gene expression in the anterior and intermediate lobes of the pituitary in stressed rats. This conclusion is based on the fact that, during restraint stress, lesion of 5-HT neurons by previous intracerebroventricular administration of the selective neurotoxin 5,7-DHT enhanced in the anterior lobe, while markedly attenuated POMC gene expression in the intermediate lobe of the pituitary. These results suggest a possible 5-HT inhibitory activity on POMC gene expression in the anterior lobe and a 5-HT stimulatory activity in the intermediate lobe of the pituitary. In the intermediate lobe, 5-HT lesions attenuated the stress-induced increase in POMC mRNA expression, which is consistent with previous results w11x suggesting that disruption of 5-HT neuronal activity may perhaps prevent stress-induced increase in the secretion of a-melanocyte stimulating hormone Ž a-MSH., one of the major posttranslational products of POMC gene in the intermediate lobe. Under basal conditions, the secretion of a-MSH is under dopaminergic inhibitory control from the intermediate lobe of the pituitary where terminals of the periventricular hypophysial DA neurons are located. It has been suggested that under stress experimental conditions, the lesion of 5-HT neurons blocks the secretion of a-MSH by disinhibiting dopaminergic neuronal activity in the intermediate lobe w11x. Similarly, POMC gene expression is under inhibitory dopaminergic control, mediated by D 2 receptors w18x and, in this respect, the involvement of DA neuronal activity in the intermediate lobe may not be ruled out to explain the blockade of POMC gene expression stress-induced increase in the present study. However, lesion of 5-HT neurons appears not to affect dopaminergic neuronal activity in the periventricular nucleus of the hypothalamus whose axons project to the intermediate lobe of the pituitary w10x. Furthermore, the chain of regulatory processes involved in the secretion of the hormone a-MSH and the stress experimental conditions are different than those implicated in the regulation of POMC gene expression in the intermediate lobe of the pituitary.
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On the other hand, a moderately dense network of serotonin fibers has been found in the intermediate lobe of the pituitary w16,23x, whose axons originate in both the raphe pontis and raphe magnus nuclei w25x. Synaptic-like contacts have been detected between these nerve fibers and proopiomelanocortin endocrine cells w16x. In addition, 5-HT has been shown to increase a-MSH secretion from melanotrophs in vitro w24x. Taken together, these facts imply that during stress 5-HT may be acting through a direct mechanism on POMC gene expression. The results of the present study suggest that, under stress conditions, 5-HT may be stimulating POMC gene expression in the intermediate lobe of the pituitary; therefore, depletion of 5-HT neurons induced by previous administration of 5,7-DHT would block the increase in POMC gene expression. Unlike the intermediate lobe, the anterior lobe of the pituitary is very scarce in serotonin fibers w16x suggesting that the additional increase found in POMC gene expression induced by stress may be mediated by an indirect mechanism. In the anterior lobe of the pituitary POMC mRNA levels are regulated by glucocorticoid feedback inhibition w6x and increased by CRF w2x. It has been reported that inhibition of 5-HT neuronal activity after administration of the selective 5-HT1A receptor agonist 8-OH-DPAT w13x resulted in a dose dependent increase in plasma ACTH w9x and corticosterone concentrations w21x. In addition, 8-OH-DPAT stimulates CRF in explanted rat hypothalami w3x. Taken together, these studies suggest that inhibition of 5-HT neuronal activity increases CRF activity. The enhancement of POMC gene expression found in 5,7-DHT stressed rats may be mediated, at least in part, by stimulation of CRF induced by depletion of 5-HT neuronal activity. Furthermore, the lack of effect of disruption of 5-HT neuronal activity after pretreatment with 5,7-DHT indicates that 5-HT neurons do not tonically regulate basal POMC gene expression in either lobe of the pituitary gland. In summary, the results of the present study suggest that 5-HT neurons are differentially involved in the regulation of POMC gene expression in the anterior and intermediate lobes of the pituitary. While 5-HT depletion did not affect POMC gene expression under basal conditions, pretreatment with 5,7-DHT during stress enhanced in the anterior lobe and blocked in the intermediate lobe POMC gene expression.
Acknowledgements This work was supported by Concerted Action from the European Union Grant BMH1-CT-94-1108 and Spanish Ministry of Education Grant DGICYT UE95-0017. L.G.-G. is a predoctoral fellow supported by the Spanish Ministry of Education. J.M. is a Senior Fellow supported by the Spanish Ministry of Education.
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pylamino.-tetralin Ž8-OH-DPAT. a potent and selective simplified ergot congener with central 5-HT-receptor stimulating activity, J. Neural Transm. 55 Ž1982. 169–188. V. Hollt, R. Przewlocki, I. Haarman, O.F.X. Almeida, N. Kley, M.J. Millan, A. Herz, Stress-induced alterations in the levels of messenger RNA coding for proopiomelanocortin and prolactin in rat pituitary, Neuroendocrinology 43 Ž1986. 277–282. J.F.R. Konig, R.A. Klippel, The Rat Brain: a Stereotaxic Atlas of ¨ Forebrain and Lower Parts of the Brainstem, Krieger, Huntington, 1963. C.s. Leranth, M. Palkovits, D.T. Krieger, Serotonin immunoreactive ´ nerve fibers and terminals in the rat pituitary-light and electron-microscopic studies, Neuroscience 9 Ž1983. 289–296. S.L. Lightman, W.S. Young, Corticotrophin-releasing factor, vasopressin and proopiomelanocortin mRNA responses to stress and opiates in the rat, J. Physiol. 403 Ž1988. 511–523. J.P. Loeffler, B.A. Demeneix, N.A. Kley, V. Hollt, Dopamine inhibition of proopiomelanocortin gene expression in the intermediate lobe of the pituitary, Neuroendocrinology 47 Ž1988. 95–101. K.J. Lookingland, K.E. Moore, Dopamine receptor mediated regulation of incerto-hypothalamic dopaminergic neurons in the male rat, Brain Res. 304 Ž1984. 324–338. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. M.J. Owens, E. Edwards, C.B. Nemeroff, Effects of 5HT1A receptor agonists on hypothalamo-pituitary-adrenal axis activity and corticotropin-releasing factor containing neurons in the rat brain, Eur. J. Pharmacol. 190 Ž1990. 113–122. M. Palkovits, Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Res. 59 Ž1973. 449–450. M. Palkovits, E. Mezey, C.C. Chiueh, D.T. Krieger, K. Gallatz, M.J. Browstein, Serotonin-containing elements of the rat pituitary intermediate lobe, Neuroendocrinology 42 Ž1986. 522–525. J.C.R. Randle, B.C. Moor, J. Kraicer, Differential control of the release of pro-opiomelanocortin-derived peptides from the pars intermedia of the rat pituitary: responses to serotonin, Neuroendocrinology 37 Ž1983. 131–140. N.J. Shannon, K.E. Moore, Determination of the source of 5-hydroxytryptaminergic neuronal projections to the neural and intermediate lobes of the rat pituitary gland through the use of electrical stimulation and lesioning experiments, Brain Res. 416 Ž1987. 322– 329. W.S. Young, E. Mezey, R.E. Siegel, Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats, Neurosci. Lett. 70 Ž1986. 198–203.
Research report
Differential 5-HT-mediated regulation of stress-induced activation of proopiomelanocortin Ž POMC. gene expression in the anterior and intermediate lobe of the pituitary in male rats Luis Garcıa-Garcıa, ´ ´ Jose´ A. Fuentes ) , Jorge Manzanares Departamento de Farmacologia, Facultad de Farmacia and Instituto Pluridisciplinar, UniÕersidad Complutense de Madrid, 28040 Madrid, Spain Accepted 1 July 1997
Abstract The purpose of the present study was to examine the role of 5-hydroxytryptamine Ž5-HT. neurons in mediating the effects of stress on proopiomelanocortin ŽPOMC. gene expression in the anterior and intermediate lobes of the pituitary gland. To this aim, the effects of 5-HT depletion induced by administration of the neurotoxin 5,7-dihydroxytryptamine Ž5,7-DHT; 200 m grrat; i.c.v.; 7 days. were investigated on POMC mRNA levels in the anterior and intermediate lobe of control and restraint-stressed rats. Three hours after brief exposure to diethyl ether Ž2 min. followed by 60 min of restraint stress increased POMC mRNA levels in the anterior and intermediate lobe of the pituitary. 5,7-DHT neurotoxic lesion, which resulted in a marked depletion of 5-HT Žbelow the level of sensitivity of the neurochemical assay, 6 pgrsample. but not of dopamine or norepinephrine concentrations in the periventricular nucleus of the hypothalamus, had no effect on basal POMC mRNA levels in the anterior or intermediate lobe of the pituitary. However, 5-HT depletion further increased POMC mRNA levels in the anterior pituitary and completely blocked POMC mRNA level enhancement induced in the intermediate lobe of stressed rats. These results suggest a possible inhibitory 5-HT tone on POMC gene expression in the anterior pituitary and a stimulatory 5-HT tone in the intermediate lobe of the pituitary under these experimental conditions of stress. It appears, therefore, that 5-HT exerts a differential regulation of stress-induced activation of POMC gene expression in the anterior and intermediate lobes of the pituitary in male rats. q 1997 Elsevier Science B.V. Keywords: Proopiomelanocortin; mRNA; Pituitary; 5-Hydroxytryptamine; 5,7-Dihydroxytryptamine
1. Introduction Proopiomelanocortin ŽPOMC. is the precursor molecule of the adrenocorticotropin hormone ŽACTH., the opioid peptide b-endorphin, b-lipotropin Ž b-LPH., and a-, band g-melanocyte-stimulating hormones ŽMSH.. POMC mRNA has been detected in the anterior and neurointermediate lobes of the pituitary gland, as well as in the arcuate nucleus of the hypothalamus and the amygdala w4,8x. Although POMC is synthesized in the pituitary gland, its post-translational proteolytic processing is different in the anterior and intermediate lobes. The major peptides produced in the anterior lobe are ACTH and b-LPH, while the predominant POMC related peptides in the intermediate lobe are a-MSH and b-endorphin w7x. POMC mRNA ) Corresponding author. Instituto Pluridisciplinar, Paseo Juan XXIII 1, Universidad Complutense, 28040 Madrid, Spain. Fax: q34 Ž1. 394-3264; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 6 3 - 9
levels in the intermediate lobe are under inhibitory dopaminergic control mediated by dopamine D 2 receptors w18x and under positive control by b-adrenergic agonists w5x. By contrast, in the anterior lobe POMC mRNA levels are regulated by glucocorticoid feedback inhibition w6x and corticotropin releasing factor ŽCRF. w2x. 5-Hydroxytryptamine Ž5-HT.-like immunoreactivity has been demonstrated in axons and axon terminals of all three pituitary lobes. A relatively dense network of 5-HT-labeled varicose fibers was present in the intermediate lobe. By contrast, only scattered fibers have been detected in the anterior and neural lobes w16x. The effects of acute physical and psychological stressful stimuli have been reported to increase POMC mRNA levels in the anterior and neurointermediate lobes of the pituitary gland w12,14,17x. In addition, it has been shown that 5-HT neurons mediate the increase in the secretion of a-MSH, the major cleavage product of POMC in the intermediate lobe, by regulating the inhibitory effects of
116
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restraint stress on periventricular hypophysial dopaminergic neurons w11x. However, little is known on the 5-HTmediated regulation of POMC mRNA levels in the anterior and intermediate lobe during stress. The purpose of the present study was to examine the role of 5-HT neurons in mediating the effects of stress on POMC gene expression in the anterior and intermediate lobes of the pituitary gland. To this aim, the acute effects of diethyl ether Ža stressor by itself. followed by restraint stress were examined after selective 5-HT neurochemical lesion induced by 5,7-dihydroxytryptamine Ž5,7-DHT. on POMC mRNA levels in the anterior and intermediate lobes of the pituitary gland. The results reveal that 5-HT neurons regulate distinctly stress-induced activation of POMC gene expression in both lobes of the pituitary.
2. Material and methods 2.1. Animals Adult male Sprague–Dawley rats, weighing 200–225 g, were obtained from Interfauna Iberica Laboratories ŽSan Feliu de Codines, Barcelona, Spain. and maintained under conditions of controlled temperature Ž23 " 18C. and lighting Žlights on 07.00–19.00 h., with food and water provided ad libitum. 2.2. Neurotoxin lesion 5,7-Dihydroxytryptamine creatinine sulfate Ž5,7-DHT; Sigma, St. Louis, MO, USA. was dissolved in a solution containing 0.3% saline and 0.1% ascorbic acid, stored over ice with minimal exposure to light. Animals were anesthetized with Equithesin Ž3 mlrkg, i.p.., and placed in a stereotaxic frame ŽDavid Kopf Instruments, Tujunga, CA, USA. with the incisor bar set 2.4 mm below the horizontal plane. A 10 m l Hamilton syringe was inserted into the lateral ventricle at coordinates A 0.0 mm, L 1.4 mm, and V y3.2 mm and 200 m g of 5,7-DHT or its vehicle Ž0.3% saline containing 0.1% ascorbic acid; 5 m l. were injected over a period of 2 min. The syringe remained in the brain for an additional 10 min to prevent fluid reflux. Following the operation the wound was sealed with surgical clips, and the animals allowed to recover for 7 days. The placement of the syringe was determined using a dissecting microscope and only those animals with syringe tracts in the lateral ventricle were included in the study. 2.3. Stress procedure On the day of the experiment, control rats were removed from their home cages and quickly decapitated. Stress-treated rats were removed from their home cages and placed in a glass desiccator containing cotton batting
saturated with diethyl ether. These animals lost their righting reflex within 30 s and remained anesthetized for 2–4 min after being removed from the desiccator. During this time, the rats were immobilized in the supine position by taping them to the laboratory bench top with adhesive tape, remained in this position for 60 min, then returned to their home cages and were decapitated 3 h later. In order to avoid POMC mRNA changes induced by circadian rhythms that could mask our results, experiments were performed between 08.00 and 13.00 h. 2.4. Tissue dissection and neurochemical analyses In order to confirm the extent of the 5-HT depletion in 5,7-DHT-treated rats versus vehicle-treated rats, frontal brain sections Ž600 m m. beginning at approximately A 9220 m m w15x were prepared in a cryostat Žy98C. and the periventricular nucleus of the hypothalamus was dissected from these sections according to a modification w19x of the method of Palkovits w22x. Tissue samples were placed in 60 m l of 0.1 M phosphate-citrate buffer ŽpH 2.5. containing 15% methanol and stored at y808C until assayed. On the day of the assay tissue samples were thawed, sonicated for 3 s ŽVibra Cell, model VC-501, Sonics and Materials, Danbury, CT, USA., and centrifuged for 60 s in a Microfuge ŽIEC, model Centra-MP4R, Needham, MA, USA.. 5-HT, dopamine ŽDA. and norepinephrine ŽNE. concentrations in tissue extracts were measured by high performance liquid chromatography ŽHPLC. with electrochemical detection. Twenty m l of the supernatant were injected onto a C18 reverse-phase analytical column Ž5 m m spheres; 250 = 4.6 mm; Nucleosil; Scharlau, Barcelona, Spain. which was protected by a precolumn cartridge filter Ž5 m m spheres; 30 = 4.6 mm.. The HPLC column was coupled to a single coulometric electrode conditioning cell in series with dual electrode analytical cells ŽCoulochem II, ESA, Bedford, MA, USA.. The conditioning electrode potential was set at 100 nA, and the analytical electrodes were set at q0.12 V and y0.31 V relative to internal Ag reference electrodes. The HPLC mobile phase consisted of 0.1 M phosphatercitrate buffer ŽpH 2.8. containing 0.1 mM ethylenediaminetetraacetic acid ŽEDTA., 0.045% sodium octylsulfate and 25% methanol. 5-HT, DA and NE contents of each sample were quantitated by comparing peak heights with those peaks of standards assayed the same day as determined by Shimadzu integrator ŽShimadzu, model C-R4AX-CHROMATOPAC, Kyoto, Japan.. The lower limit of sensitivity of this assay for these compounds was 2–6 pg per sample. Tissue pellets were dissolved in 1.0 N NaOH and assayed for protein w20x. 2.5. In situ hybridization histochemistry (ISHH) Twelve m m pituitary sections Ž24 sectionsranimal; six sectionsrslide, from seven to eight animalsrgroup., were
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mounted onto gelatin-coated slides and stored at y808C until the day of the assay. ISHH was performed as described previously w26x using a synthetic 48-base oligonucleotide probe complementary to the 96–134 sequence of the rat POMC gene Žobtained from the Advanced Biotechnology Center, Charing Cross and Westminster Medical School, London, UK.. The oligonucleotide probe was labeled using terminal deoxytransferase ŽBoehringer, Madrid, Spain. to add a 35 S-labeled deoxyATP Ž1000 Cirmmol; Amersham, Madrid, Spain. tail to the 3X end of the probe. The probe Žin 45 m l of hybridization buffer. was applied to each section and left overnight at 378C for hybridization. Following hybridization, sections were washed 4 times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2 Ž1 = saline sodium citrate, SSC. at 558C, followed by two 30 min washes in 1 = SSC at room temperature, one brief water dip and blown dry with air. The dried slides were apposed to Hyperfilm b-max ŽAmersham, Madrid, Spain. 18 h for anterior lobe and 1 h for the intermediate lobe of the pituitary gland. Autoradiograms were analyzed with a Macintosh computer using the public domain NIH Image program Ždeveloped at the US National Institutes of H e a lth a n d a v a ila b le o n th e In te rn e t a t http:rrrsb.info.nih.govrnih-image.. The mean hybridization signal in each experimental group was calculated from the mean of the averages of all pituitary sections per animal. 2.6. Statistical analyses Statistical analyses were performed using two-way ANOVA followed by the Student Newman-Keul’s test.
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Table 1 Effects of 5,7-DHT administration on 5-HT, NE and DA concentrations in the periventricular nucleus of the rat hypothalamus
5-Hydroxytryptamine Norepinephrine Dopamine
Control
5,7-DHT
11.5"0.5 85.2"5.7 8.2"0.7
n.d. 85.5"9.6 8.9"0.8
Rats were injected with 5,7-DHT Ž200 m grrat, i.c.v.. or its saline vehicle Ž5 m lrrat. and decapitated 7 days later. Values represent means"S.E.M. Ž ns 7–8. of 5-HT, NE and DA concentrations Žngrmg protein.. n.d., values below the level of sensitivity of the neurochemical assay Ž2–6 pgrsample..
Differences were considered significant if the probability of error was less than 5%.
3. Results In order to ascertain whether central 5-HT neurons were involved in the stress-induced stimulation of POMC gene expression in anterior and intermediate lobes of the pituitary, rats were pretreated with the selective 5-HT neurotoxin 5,7-DHT w1x. One week after the intracerebroventricular injection of 5,7-DHT, 5-HT concentrations in the periventricular nucleus of the hypothalamus Žregion that contains DA cell bodies of the periventricular hypophysial neurons projecting to the intermediate lobe of the pituitary. were markedly reduced, but 5,7-DHT injection was without effect on dopamine or norepinephrine concentrations in the same region ŽTable 1.. As shown in Fig. 1, an increase Žapproximately 20%. in POMC mRNA levels in the anterior and intermediate lobes
Fig. 1. Effects of 5,7-DHT administration on stress-induced changes in POMC gene expression in the anterior ŽA. and intermediate lobes ŽB. of the pituitary gland. Rats were injected with 5,7-DHT Ž200 m grrat, i.c.v.. or its saline vehicle Ž5 m lrrat. and decapitated 7 days later. On the day of the experiment, animals were removed from their home cages and immediately decapitated Žcontrol. or briefly anesthetized with diethyl ether, restrained in the supine position for 60 min and decapitated 3 h later Žstress.. Optical densities were calculated from the uncalibrated mode expressed in grey scale values. Results are presented considering mean control values as 100%. Columns represent means and vertical lines S.E.M. Ž n s 7–8. of POMC mRNA levels. ) Values from stressed animals that are significantly different Ž P - 0.05. from controls. a Values from 5,7-DHT-stressed rats that are significantly different Ž P - 0.05. from vehicle-stressed rats.
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of the pituitary was found 3 h after brief exposure to diethyl ether followed by 60 min of restraint stress. In the 5,7-DHT-pretreated group, basal POMC mRNA levels in the anterior and intermediate lobes of the pituitary were unaltered. By contrast, prior administration of 5,7-DHT further enhanced POMC mRNA levels Žapproximately 40% when compared with control vehicle-treated group. in the
anterior lobe and blocked the effects of stress on POMC mRNA in the intermediate lobe of the pituitary gland. These results are clearly reflected by differences on the hybridization signal of the autoradiograms of Fig. 2. Basal POMC mRNA levels in the intermediate lobe are higher than in the anterior lobe of the pituitary. Therefore, in order to detect modifications on POMC mRNA levels, film
Fig. 2. Effects of 5,7-DHT and stress on POMC mRNA levels in the anterior and intermediate lobes of the pituitary gland. Autoradiograms of coronal pituitary sections of anterior ŽA–D. and intermediate ŽE–H. lobes. A,E: vehicle control; B,F: 5,7-DHT control; C,G: vehicle stress; D,H: 5,7-DHT stress. Film was exposed 18 h for the anterior lobe and 1 h for the intermediate lobe of the pituitary. Experimental design was as indicated in Fig. 1. Bar s 1 mm.
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was exposed 18 h for the anterior lobe and 1 h for the intermediate lobe of the pituitary. As a consequence of the differences in film exposure time, note that in Fig. 2A–D the intermediate lobe is overexposed whereas in Fig. 2E–H the anterior lobe is hardly visible. As shown in Fig. 2, the anterior lobe in panel D Ž5,7-DHT-stress-treated group. exhibits the highest hybridization signal when compared with the anterior lobes presented in panels A–C. By contrast, in panel G Žvehicle-stress group. the intermediate lobe shows the highest hybridization signal when compared with intermediate lobes presented in panels E, F and H.
4. Discussion The results of the present study demonstrated a differential 5-HT-mediated regulation of POMC gene expression in the anterior and intermediate lobes of the pituitary in stressed rats. This conclusion is based on the fact that, during restraint stress, lesion of 5-HT neurons by previous intracerebroventricular administration of the selective neurotoxin 5,7-DHT enhanced in the anterior lobe, while markedly attenuated POMC gene expression in the intermediate lobe of the pituitary. These results suggest a possible 5-HT inhibitory activity on POMC gene expression in the anterior lobe and a 5-HT stimulatory activity in the intermediate lobe of the pituitary. In the intermediate lobe, 5-HT lesions attenuated the stress-induced increase in POMC mRNA expression, which is consistent with previous results w11x suggesting that disruption of 5-HT neuronal activity may perhaps prevent stress-induced increase in the secretion of a-melanocyte stimulating hormone Ž a-MSH., one of the major posttranslational products of POMC gene in the intermediate lobe. Under basal conditions, the secretion of a-MSH is under dopaminergic inhibitory control from the intermediate lobe of the pituitary where terminals of the periventricular hypophysial DA neurons are located. It has been suggested that under stress experimental conditions, the lesion of 5-HT neurons blocks the secretion of a-MSH by disinhibiting dopaminergic neuronal activity in the intermediate lobe w11x. Similarly, POMC gene expression is under inhibitory dopaminergic control, mediated by D 2 receptors w18x and, in this respect, the involvement of DA neuronal activity in the intermediate lobe may not be ruled out to explain the blockade of POMC gene expression stress-induced increase in the present study. However, lesion of 5-HT neurons appears not to affect dopaminergic neuronal activity in the periventricular nucleus of the hypothalamus whose axons project to the intermediate lobe of the pituitary w10x. Furthermore, the chain of regulatory processes involved in the secretion of the hormone a-MSH and the stress experimental conditions are different than those implicated in the regulation of POMC gene expression in the intermediate lobe of the pituitary.
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On the other hand, a moderately dense network of serotonin fibers has been found in the intermediate lobe of the pituitary w16,23x, whose axons originate in both the raphe pontis and raphe magnus nuclei w25x. Synaptic-like contacts have been detected between these nerve fibers and proopiomelanocortin endocrine cells w16x. In addition, 5-HT has been shown to increase a-MSH secretion from melanotrophs in vitro w24x. Taken together, these facts imply that during stress 5-HT may be acting through a direct mechanism on POMC gene expression. The results of the present study suggest that, under stress conditions, 5-HT may be stimulating POMC gene expression in the intermediate lobe of the pituitary; therefore, depletion of 5-HT neurons induced by previous administration of 5,7-DHT would block the increase in POMC gene expression. Unlike the intermediate lobe, the anterior lobe of the pituitary is very scarce in serotonin fibers w16x suggesting that the additional increase found in POMC gene expression induced by stress may be mediated by an indirect mechanism. In the anterior lobe of the pituitary POMC mRNA levels are regulated by glucocorticoid feedback inhibition w6x and increased by CRF w2x. It has been reported that inhibition of 5-HT neuronal activity after administration of the selective 5-HT1A receptor agonist 8-OH-DPAT w13x resulted in a dose dependent increase in plasma ACTH w9x and corticosterone concentrations w21x. In addition, 8-OH-DPAT stimulates CRF in explanted rat hypothalami w3x. Taken together, these studies suggest that inhibition of 5-HT neuronal activity increases CRF activity. The enhancement of POMC gene expression found in 5,7-DHT stressed rats may be mediated, at least in part, by stimulation of CRF induced by depletion of 5-HT neuronal activity. Furthermore, the lack of effect of disruption of 5-HT neuronal activity after pretreatment with 5,7-DHT indicates that 5-HT neurons do not tonically regulate basal POMC gene expression in either lobe of the pituitary gland. In summary, the results of the present study suggest that 5-HT neurons are differentially involved in the regulation of POMC gene expression in the anterior and intermediate lobes of the pituitary. While 5-HT depletion did not affect POMC gene expression under basal conditions, pretreatment with 5,7-DHT during stress enhanced in the anterior lobe and blocked in the intermediate lobe POMC gene expression.
Acknowledgements This work was supported by Concerted Action from the European Union Grant BMH1-CT-94-1108 and Spanish Ministry of Education Grant DGICYT UE95-0017. L.G.-G. is a predoctoral fellow supported by the Spanish Ministry of Education. J.M. is a Senior Fellow supported by the Spanish Ministry of Education.
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