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Endocrinology Vol. 141, No. 4 1366-1376
Copyright © 2000 by The Endocrine Society

ARTICLES

Activation of c-fos Expression in Hypothalamic Nuclei by µ- and {kappa}-Receptor Agonists: Correlation with Catecholaminergic Activity in the Hypothalamic Paraventricular Nucleus1

M. Luisa Laorden, M. Teresa Castells, M. Delia MartÍnez, Pedro J. MartÍnez2 and M. Victoria Milanés

Department of Physiology and Pharmacology, Unit of Pharmacology (M.L.L., M.D.M., P.J.M., M.V.M.), and Department of Cell Biology (M.T.C.), University School of Medicine, 30100 Murcia, Spain

Address all correspondence and requests for reprints to: Dr. M. V. Milanés, Unit of Pharmacology, School of Medici School of Medicine, 30100 Murcia, Spain. E-mail: milanes{at}fcu.um.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Administration of the preferential µ-opioid receptor agonist, morphine, and selective {kappa}-opioid receptor agonists elicits activation of the hypothalamus-pituitary-adrenocortical axis, although the site or the molecular mechanisms for these effects have not been determined. The expression of Fos, the protein product of the c-fos protooncogene, has been widely used as an anatomical marker of monitoring neuronal activity. In the present study we evaluated 1) the effects of the µ-opioid receptor agonist, morphine, and those of the selective {kappa}-opioid receptor agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl-]benzeneacetamide methane sulfonate (U-50,488H), administration on the expression of Fos in hypothalamic nuclei; and 2) the possible modification of the activity of noradrenergic neurons known to send afferent projections to the paraventricular nucleus (PVN), the site of CRF neurons involved in initiating ACTH secretion. Using immunohistochemical staining of Fos, the present results indicate that acute treatment with either morphine or U-50,488H induces marked Fos immunoreactivity within the hypothalamus, including the medial parvicellular PVN and supraoptic and suprachiasmatic nuclei. Pretreatment with naloxone attenuated the effect of morphine, whereas nor-binaltorphimine, a selective {kappa}-opioid receptor antagonist, abolished the effect of U-50,488H on Fos induction. Correspondingly, morphine and U-50,488H injection increased the production of the cerebral noradrenaline metabolite 3-methoxy-4-hydroxyphenylethylene glycol as well as noradrenaline turnover in the PVN. These effects were antagonized by naloxone and nor-binaltorphimine, respectively. All of these findings are discussed in terms of specific events that couple opioid-induced activation of the hypothalamus-pituitary-adrenocortical axis and noradrenergic activity with changes in gene expression in selective hypothalamic nuclei.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INDUCTION of the immediate-early gene (IEG), c-fos, and the expression of its protein product, Fos, in neuronal nuclei have been proposed to reflect second messengers activation and hence serve as sensitive indicators of cellular responses induced by various stimuli (1, 2) including opioids (3). The nuclear Fos protein probably translates transient synaptic events into long lasting changes in neuronal excitability and has been used as an anatomical marker for monitoring neuronal activity (2, 3).

Acutely administering the prototype of the µ-opioid receptor (MOR), morphine, and {kappa}-opioid receptor (KOR) agonists potently stimulates the hypothalamus-pituitary-adrenal (HPA) axis through a centrally mediated mechanism (4, 5). Activation of MOR and KOR by morphine and U-50,488H, respectively, stimulates the secretion of ACTH and corticosterone (6, 7, 8, 9). Previous experiments have shown that this stimulatory effect of opioids is mediated through the secretion of hypothalamic CRF (10, 11, 12). Several lines of evidence indicate that the ability of opioids to increase HPA axis activity is secondary to the actions of these drugs to stimulate the activity of noradrenergic neurons innervating the hypothalamic paraventricular nucleus (PVN). Thus, enhancement of 3-methoxy-4-hydroxyphenylethylen glycol (MHPG; the cerebral metabolite of noradrenaline (NA)] production and NA turnover was found in the hypothalamus after morphine or U-50,488H administration to rats, which was correlated with an augmented secretion of corticosterone (13, 14). Furthermore, the effect of morphine on the HPA axis activity was antagonized by {alpha}- and ß-adrenoceptor blockers (13).

Although one of the prominent actions of opioids is to depress electrical activity of neurons, it has been demonstrated that acute morphine administration activates expression of the IEG c-fos in different regions of the rat brain, such as striatum and nucleus accumbens (15, 16). However, other studies have reported the opposite effects or the absence of changes in different areas of the rat brain (17, 18). Fos is involved in the transcription genes encoding for neuropeptides, and it seems to function in the coupling of extracellular signals to long term alterations in cellular activity. This makes Fos reactivity a useful parameter to study the sensitivity of neuronal circuits involved in neuroendocrine reactivity to external stimuli.

In this report we extended the study of opioid receptor activation on hypothalamic nuclei by comparing the effects of µ and {kappa} agonists. The aim of the present study was to determine whether morphine and/or U-50,488H stimulates neuronal activity in the PVN (which has been implicated in the effects of opioids on HPA axis activity) by interacting selectively with µ- and {kappa}-opioid receptors, respectively. To test this possibility, we examined morphine- and U-50,488H-induced expression of Fos in the cells of the parvocellular PVN, the primary location of tuberoinfundibular CRF cells (19). We also studied the expression of Fos in hypothalamic areas involved in neuroendocrine control, such as the hypothalamic supraoptic (SON) and suprachiasmatic (SCN) nuclei. In addition, we studied the changes in NA turnover in the hypothalamic PVN after morphine and U-50,488H administration to examine a possible relation between the effects of opioids on the Fos expression and those on noradrenergic activity under identical conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats (220–250 g) were housed four per cage under a 12-h light, 12-h dark cycle (lights on, 0800 h; lights off, 2000 h) in a temperature-controlled room (22–24 C), with food and water available ad libitum. The animals were cared for in accordance with local committee and ethical NIH guidelines. As stress can affect the expression of immediate-early genes (20) and the activity of the HPA axis, the experimental design included efforts to reduce the potential effect of stress. For that, animals were handled daily for about 5 min (between 0900–1000 h) for at least 3 days before the experimental day in the experimental room to adapt them to manipulations and minimize nonspecific stress responses. As the expression of immediate-early genes may vary diurnally (21), the experiments were also designed such as perfusion-fixation or brain removing for catecholamine analysis would be performed between 1030 and 1200 h.

Experimental procedure
For immunohistochemistry studies saline was administered sc 20 min before and 20 min after morphine (30 mg/kg, ip) injection. For controls, saline was injected sc 20 min before and 20 min after ip saline administration. The ability of naloxone to reverse the effects of morphine was also examined. Naloxone was injected twice: 20 min before (5 mg/kg, sc) and 20 min after (3 mg/kg, sc) morphine (30 mg/kg, ip). To examine the effects of naloxone alone, naloxone was given to the rats in the same manner, and animals received ip saline instead of morphine. Other groups of rats were injected ip with Milli-Q water (vehicle) 2 h before receiving U-50,488H (15 mg/kg, ip). Controls were given ip vehicle 2 h before ip saline injection. The ability of the selective KOR antagonist nor-binaltorphimine (BNI) to reverse the effects of U-50,488H was examined. For that, BNI (5 mg/kg, ip) was administered 2 h before U-50,488H (15 mg/kg, ip) injection. For controls, BNI was injected at the same dose 2 h before ip saline administration.

For estimation of catecholamines in the PVN, morphine (30 mg/kg, ip) was injected to animals pretreated 20 min before with saline sc or naloxone (5 mg/kg, sc). Controls received saline ip 20 min after saline sc or naloxone (5 mg/kg, sc). Rats were killed by decapitation 15 and 30 min after morphine or ip saline administration. Other groups of animals received ip vehicle or BNI (5 mg/kg, ip) 2 h before the administration of U-50,488H (15 mg/kg, ip). Control rats were injected with ip vehicle or BNI 2 h before ip saline administration. Rats were killed 15 and 30 min after ip saline or U-50,488H injection.

Tissue preparation and Fos immunohistochemistry
Rats were killed with an overdose of pentobarbital (100 mg/kg, ip) 90 min after morphine, U-50,488H, or ip saline administration. The delay of 90 min after opioids or control injections was chosen, because it was previously demonstrated that the peak effect of stimulated Fos in brain is 90 min (2). After anesthesia, rats were perfused transcardially with 300 ml PBS (pH 7.4), followed by 500 ml cold 4% paraformaldehyde in PBS (pH 7.4). After perfusion, brains were removed, postfixed in the same fixative, and stored at 4 C overnight. Free floating coronal brain sections (50 µm in thickness) throughout the rostrocaudal extend of the hypothalamus were obtained on a Vibratome (Vibratome 1000, TPI, St. Louis, MO). A total of 20 hypothalamic sections were taken for each animal, corresponding to plates 22–26 in the atlas of Palkovits and Brownstein (22), which contain the hypothalamic SCN, SON, and PVN (plane of sections relative to bregma: -0.95 mm for SCN, -1.3 mm for SON, and -1.8 mm for PVN (23).

Expression of Fos protein was examined in free floating sections, which were collected serially in adjacent sets and shaken in PBS for at least 30 min to remove the fixative. The sections were preincubated for 20 min in absolute methanol plus 30% H2O2 to block endogenous peroxidase activity. They were rinsed in PBS twice (15 min each) and treated with NSS-PBS (PBS containing 1% normal swine serum; DAKO Corp., Glostrup, Denmark; and 0.5% Triton X-100) for 30 min. All sections were reacted with the primary polyclonal Fos antibody (1:3000 dilution in NSS-PBS; sc253, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 36 h at 4 C. The antibody was raised in rabbits against a peptide corresponding to amino acids 128–152 mapping within a highly conserved domain of c-fos p62 of human origin, which is identical to the corresponding rat sequence. The bound primary antibody was then localized by biotinylated antirabbit IgG (1:200 dilution in NSS-PBS; Vector Laboratories, Inc., Burlingame, CA) and subsequently with the avidin-biotin complex (ABC kits, Vector Laboratories, Inc.) at room temperature for 1 h each. Visualization of the antigen-antibody reaction sites used 0.033% 3,3'-diaminobenzidine (Sigma, St. Louis, MO) and 0.015% H2O2 in 0.05 M Tris-HCl buffer for 7 min. The reaction was stopped in PBS. Noncounterstained was performed. The sections were mounted onto glass slides coated with gelatin, air-dried, dehydrated through graded alcohols, cleared in xylene, and cover-slipped with DPX (BDM, Poole, UK).

Quantification of Fos-like immunoreactivity (Fos-IR)
Evidence of Fos-IR was examined under a light microscope. The density of Fos-like immunopositive nuclei was determined with a computer-assisted image analysis system. This system consists of an Axioskop microscope (Carl Zeiss, Oberkochen, Germany) connected to a videocamera and an Imco 10 computer (Kontron Instruments Ltd., Bildanalyse, Germany) with Microm Image Processing software (Microm, Barcelona, Spain). The three or four sections of each nuclei showing the highest level of Fos-IR were selected for quantitative image analysis. A standard square field (93.5-µm side) was superimposed upon the captured image to be used as reference area. The area of Fos immunolabeling included in this square was used for estimating the immunoreactivity, and the percentage of Fos-IR was evaluated. Based on orientation criteria, the medial parvocellular neurosecretory portion of the PVN was defined once the adjacent boundaries of the posterior magnocellular and periventricular parts were identified (24). For this nuclei, the density of Fos-like immunopositive nuclei at three or four rostrocaudal levels encompassing the parvocellular zone, the primary location of tuberoinfundibular CRF cells, was used for estimating Fos-IR. The percentage of Fos-IR of both the right and left sides of three or four correlative sections for each nuclei was averaged per animal. Measures were also averaged in each experimental group for the PVN, SON, and SCN.

Estimation of NA and its metabolite MHPG in the PVN
After decapitation, the brains were removed rapidly, fresh-frozen, and stored immediately at -80 C until use. The hypothalamic tissue containing the PVN was dissected from a coronal brain slice according the technique of Palkovits (25), and the PVN corresponds to those in plates 25–26, 1800–2100 µm caudally to the bregma (22). NA and its metabolite in the central nervous system (CNS), MHPG, were determined by HPLC with electrochemical detection. Bilateral tissue samples were weighed, placed in 600 µl cold perchloric acid (0.1 M), and homogenized with a Polytron-type homogenizer (setting 4 for 30 s). The homogenates were then centrifuged (15,000 rpm, 4 C, 15 min), and the supernatants were taken for analysis and filtered through 0.22-µm pore size GV filters (Millipore Corp., Bedford, MA). Two aliquots of the supernatant from the same tissue sample were used, the first for analysis of NA and the second for analysis of MHPG. Ten microliters of the first aliquot of each sample were injected into a 5-µm C18 reverse phase column (Waters Corp., Milford, MA) through a Rheodyne (Rheodyne Inc., Cotati, CA) syringe-loading injector 200-µl loop. Electrochemical detection was accomplished with a glassy carbon electrode set at a potential of +0.65 V vs. the Ag/AgCl reference electrode (Waters Corp.). The mobile phase consisted of a 95:5 (vol/vol) mixture of water and methanol with sodium acetate (50 mM), citric acid (20 mM), 1-octyl-sodium sulfonate (3.75 mM), di-n-butylamine (1 mM), and EDTA (0.135 mM), adjusted to pH 4.3. The flow rate was 0.9 ml/min, and chromatographic data were analyzed with Millennium 2010 Chromatography Manager (Millipore Corp.) equipment. NA was detected by the described HPLC method at an elution time of 4.35 min. Under these conditions, MHPG was not observed. As in the rat CNS most of MHPG is present in a sulfate conjugate form, the method for determination of total MHPG in the PVN is based on the acid-catalyzed hydrolysis of MHPG sulfate (26, 27). The aliquots for MHPG analysis were kept in polypropylene, screw-capped tubes for 5 min in a water bath at 100 C. The tubes were then cooled on ice and centrifuged (4000 rpm, 4 C, 10 min). The supernatant of the hydrolyzed samples was injected (20–40 µl) into the HPLC equipment. The eluent for MHPG determination was as described above, but without 1-octyl-sodium sulfonate. Under these conditions, MHPG eluted at 4.80–5 min. NA and MHPG were quantified by reference to calibration curves run at the beginning and the end of each series of assays. Linear relationships were observed between the amount of standard injected and the peak heights measured. The content of NA and MHPG in the PVN was expressed as nanograms per g wet wt of tissue.

Drugs and chemicals
Morphine hydrochloride (Alcaliber, Madrid, Spain) was dissolved in sterile 0.9% NaCl (saline) and administered in a volume of 0.15 ml/100 g. Naloxone hydrochloride (Sigma) was dissolved in saline and given in a volume of 0.1 ml/100 g. Trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane sulfonate (U-50,488H; a gift from Upjohn, Kalamazoo, MI) was dissolved in saline and given in a volume of 0.1 ml/100 g. Nor-binaltorphimine (Sigma) was dissolved in Milli-Q (Millipore Corp.) sterile water (vehicle) and given in a volume of 0.1 ml/100 g. NA bitartrate and MHPG hemipiperazinium salt (used as HPLC standards) were purchased from Sigma. Drugs were prepared fresh every day. Other reagents were of analytical grade.

Statistical analysis
All values are expressed as the mean ± SEM. Data were analyzed by two-way ANOVA, followed by the Newman-Keuls post-hoc test. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphine treatment induces Fos expression in hypothalamic nuclei
In untreated (naive) rats, neurons of the PVN, SON, and SCN showed detectable basal Fos expression. The signal was obviously due to the minor stress of handling. Compared with untreated animals, rats given injections of saline sc plus saline ip plus saline sc (controls) had similar numbers of Fos-IR in the PVN (1.38 ± 0.1 vs. 1.71 ± 0.2) and in the SON (1.97 ± 0.3 vs. 2.02 ± 0.1). However, saline-treated rats showed higher Fos-IR (P < 0.01) in the SCN compared with untreated animals (4.14 ± 0.1 vs. 2.54 ± 0.03). We found that nuclear Fos-IR was particularly induced in the SCN. Thus, the SCN of control rats showed higher (P < 0.001) basal Fos-IR than either the PVN or SON.

Compared with saline-injected rats, those treated with morphine showed a large number of Fos-IR cells within all of the hypothalamic regions studied. The signal was observed in the PVN (Fig. 1Go, A and B), SON (Fig. 2Go, A and B), and SCN (Fig. 3Go, A and B). Quantitative analysis showed that the Fos-IR after morphine administration was significantly higher in the PVN (P < 0.001; Fig. 4AGo), SON (P < 0.01; Fig. 5AGo), and SCN (P < 0.001; Fig. 6AGo) compared with that in control animals receiving saline instead morphine.



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  		Figure 1. Photomicrographs of Fos immunoreactivity in the PVN 90 min after saline sc plus saline ip (A), saline sc plus morphine ip (B), naloxone plus saline ip (C), and naloxone plus morphine (D). Note the induction of Fos-IR in the parvocellular subdivision (arrow) after morphine injection and its attenuation by pretreatment with naloxone. 3V, Third ventricle. Scale bar, 111 µm.

 


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  		Figure 2. Photomicrographs of Fos immunoreactivity in the SON 90 min after saline sc plus saline ip (A), saline sc plus morphine ip (B), naloxone plus saline ip (C), and naloxone plus morphine (D). Note the induction of Fos-IR after morphine injection and its attenuation by pretreatment with naloxone. Scale bar, 87 µm.

 


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  		Figure 3. Photomicrographs of Fos immunoreactivity in the SCN 90 min after saline sc plus saline ip (A), saline sc plus morphine ip (B), naloxone plus saline ip (C), and naloxone plus morphine (D). Note the induction of Fos-IR after morphine injection and its attenuation by pretreatment with naloxone. OC, Optic chiasm. Scale bar, 87 µm.

 


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  		Figure 4. Quantitative analysis of Fos-IR in the PVN 90 min after the administration of morphine (A) and U-50,488H (B) to control rats and to animals pretreated with naloxone or BNI. Both MOR and KOR agonists significantly increased the number of Fos-positive cells, and these effects could be attenuated by the administration of naloxone and BNI, respectively. ***, P < 0.001 (compared with control). +, P < 0.05; +++, P < 0.001 (compared with animals treated with morphine and U-50,488H, respectively). n = 4–6/group.

 


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  		Figure 5. Quantitative analysis of Fos-IR in the SON 90 min after the administration of morphine (A) and U-50,488H (B) to control rats and to animals pretreated with naloxone or BNI. Both MOR and KOR agonists significantly increased the number of Fos-positive cells, and these effects could be attenuated by the administration of naloxone and BNI, respectively. **, P < 0.01; ***, P < 0.001 (compared with control). +, P < 0.05; +++, P < 0.001 (compared animal treated with morphine and U-50,488H, respectively). n = 4–6/group.

 


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  		Figure 6. Quantitative analysis of Fos-IR in the SCN 90 min after the administration of morphine (A) and U-50,488H (B) to control rats and to animals pretreated with naloxone or BNI. Both MOR and KOR agonists significantly increased the number of Fos-positive cells, and these effects could be attenuated by the administration of naloxone and BNI, respectively. ***, P < 0.001 (compared with control). +++, P < 0.001 (compared with animals treated with morphine and U-50,488H, respectively). n = 4–6/group.

 
Inhibition by naloxone of morphine-induced Fos-IR
In rats injected with naloxone sc plus saline ip, there were no significant modifications in Fos-IR in any of the hypothalamic nuclei compared with control animals receiving injections of saline sc plus saline ip. However, treatment with naloxone 20 min before and 20 min after morphine administration attenuated the increase in Fos-IR produced by the opioid agonist in the PVN (Fig. 1Go, C and D), SON (Fig. 2Go, C and D), and SCN (Fig. 3Go, C and D). Quantitative analysis demonstrated that naloxone significantly inhibited the increase in Fos-IR induced by morphine in the PVN (P < 0.05; Fig. 4AGo), SON (P < 0.05; Fig. 5AGo), and SCN (P < 0.001; Fig. 6AGo).

U-50,488H induces Fos expression in hypothalamic nuclei
Compared with untreated animals, rats given injection of saline ip 2 h after ip vehicle administration showed similar expression of the nuclear Fos protein (PVN, 1.38 ± 0.1 vs. 1.46 ± 0.1; SON, 1.97 ± 0.3 vs. 1.86 ± 0.2; SCN, 2.54 ± 0.03 vs. 3.32 ± 0.6). Administration of U-50,488H 2 h after vehicle injection yielded marked Fos-IR within the PVN (Fig. 7Go, A and B), SON (Fig. 8Go, A and B), and SCN (Fig. 9Go, A and B) compared with that in vehicle- plus saline-treated animals. Quantitative analysis showed that there was significant increase in Fos-IR in the PVN (P < 0.001; Fig. 4BGo), SON (P < 0.001; Fig. 5BGo), and SCN (P < 0.001; Fig. 6BGo) compared with that in animals injected with vehicle plus saline.



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  		Figure 7. Photomicrographs of Fos-IR in the PVN 90 min after vehicle ip plus saline ip (A), vehicle plus U-50,488H (B), BNI plus saline (C), and BNI plus U-50,488H (D). Note the induction of Fos-IR in the parvocellular subdivision (arrow) after U-50,488H injection and its attenuation by pretreatment with BNI. Scale bar, 111 µm.

 


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  		Figure 8. Photomicrographs of Fos-IR in the SON 90 min after vehicle ip plus saline ip (A), vehicle plus U-50,488H (B), BNI plus saline (C), and BNI plus U-50,488H (D). Note the induction of Fos-IR after U-50,488H injection and its attenuation by pretreatment with BNI. Scale bar, 87 µm.

 


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  		Figure 9. Photomicrographs of Fos-IR in the SCN 90 min after vehicle ip plus saline ip (A), vehicle plus U-50,488H (B), BNI plus saline (C), and BNI plus U-50,488H (D). Note the induction of Fos-IR after U-50,488H injection and its attenuation by pretreatment with BNI. Scale bar, 87 µm.

 
Inhibition of U-50,488H-induced Fos expression by pretreatment with BNI
Animals given the selective KOR antagonist BNI plus saline showed no significant modification in Fos-IR in any of the hypothalamic nuclei explored compared with that in control rats receiving vehicle plus saline. However, pretreatment with BNI 2 h before U-50,488H injection antagonized the increase in Fos-IR produced by the KOR agonist in the PVN (Fig. 7Go, C and D), SON (Fig. 8Go, C and D), and SCN (Fig. 9Go, C and D). Quantitative analysis indicated that BNI significantly blocked the U-50,488H-induced increase in Fos expression in the PVN (P < 0.001; Fig. 4BGo), SON (P < 0.001; Fig. 5BGo), and SCN (P < 0.001; Fig. 6BGo).

MHPG production and NA turnover in the hypothalamic PVN after morphine administration
Fifteen minutes after morphine injection, a significant increase was observed in both the production of the cerebral NA metabolite MHPG (312 ± 88 vs. 35 ± 9 ng/g; P < 0.01) and NA turnover (P < 0.01; Fig. 10AGo). These effects were significantly (P < 0.01) antagonized by naloxone (MHPG, 100 ± 16 ng/g). Administration of naloxone to control rats produced no changes in the MHPG production (68 ± 9 ng/g) or in NA turnover (Fig. 10AGo). Similar results were obtained when MHPG and NA turnover were measured 30 min after morphine administration (data not shown).



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  		Figure 10. Turnover of NA (as estimated by the MHPG/NA ratio) in the PVN of rats 15 min after the injection of A) saline sc plus saline ip, saline sc plus morphine (30 mg/kg, ip), naloxone (5 mg/kg, sc) plus saline ip, and naloxone (5 mg/kg, sc) plus morphine (30 mg/kg, ip); B) vehicle ip plus saline ip, vehicle plus U-50,488H (15 mg/kg, ip), BNI (5 mg/kg, ip) plus saline ip, and BNI (5 mg/kg, ip) plus U-50,488H (15 mg/kg, ip). Rats were decapitated 15 min after saline ip, morphine, and U-50,488H injections. Each value represents the mean of six or seven rats ± SE. **, P < 0.01; ***, P < 0.001 (compared with control groups). ++, P < 0.01; +++, P < 0.001 (compared with rats receiving saline plus morphine and vehicle plus U-50,488H, respectively).

 
MHPG production and NA turnover in the hypothalamic PVN after U-50,488H administration
Rats injected with U-50,488H showed a significant (P < 0.001) increase in MHPG production (150 ± 16 vs. 65 ± 7 ng/g) and NA turnover (P < 0.001; Fig. 10BGo) 15 min after KOR agonist injection. These changes in MHPG production and NA turnover were significantly inhibited (P < 0.05; P < 0.001, respectively) in rats pretreated with the KOR-selective antagonist BNI (MHPG, 103 ± 11; Fig. 10BGo). However, pretreatment with BNI did not modify the MHPG content (64 ± 8 ng/g) or NA turnover in control rats. When NA turnover was measured 30 min after U-50,488H injection, no changes were observed in MHPG production or NA turnover (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Regulatory control of the HPA axis originates principally from the hypothalamic PVN. The parvocellular neurosecretory neurons of this nucleus serve as the origin of a cascade of events by delivering the two main CRF, CRF and arginine vasopressin (AVP), resulting in the ACTH-mediated release of corticosterone (28). A primary component of the rat response to opioids is known to involve activation of the HPA axis (6, 8, 14; for review, see Ref. 5). The present immunohistochemical study shows that the preferential MOR agonist, morphine, induces the neuronal expression of Fos protein within neurons that anatomically correspond to the parvocellular PVN, the primary location of tuberoinfundibular CRF cells that constitute the apex of the HPA axis. This transcription factor may regulate a subsequent wave pattern of gene expression, thereby mediating long term consequences of trans-synaptic stimulation. This study further shows that the selective KOR agonist U-50,488H also promotes the expression of Fos in the same hypothalamic nuclei, particularly in the neurosecretory nerve cells that constitute the parvocellular division of the PVN. Although the Fos-positive nuclei in the PVN were largely confined to the parvocellular region, the precise identification of the cell groups that respond to opioids awaits further study using double labeling techniques. However, as in other studies (29, 30), Fos expression within the medial PVN can be taken as an index of HPA axis activity and as an indicator of the degree of activation of the medial parvocellular cell populations (31). The fact that these nerve cells govern the HPA axis activity is indicative that a hyperactive functional state of PVN neurons may contribute to the opioid-induced activation of the HPA axis. Pretreatment with naloxone inhibited Fos induction produced by morphine; pretreatment with the selective KOR antagonist BNI completely blocked U-50,488H-induced Fos expression, indicating that the effects of morphine and U-50,488H were mediated by MOR and KOR, respectively.

The present investigation is in agreement with previous studies which demonstrated that administration of morphine induced expression of c-fos messenger RNA and its protein product, Fos, in different regions of the rat CNS, including the hypothalamic PVN and SON (32), striatum, and nucleus accumbens (15, 33). However, the results contrast with those from another studies where application of morphine to naive animals elicited only weak c-fos response in the hypothalamus (17, 34). From the present results we cannot identify cell types in the parvocellular part of the PVN that are activated after morphine and U-50,488H treatment. However, as intracerebroventricular or systemic administration of opioids resulted in raised corticosterone levels (6, 7, 8, 9, 14), Fos expression in the PVN can be correlated with ACTH release from the anterior pituitary.

The present results also show that activation of MOR or KOR induces the neuronal expression of Fos protein in hypothalamic nuclei adjacent to the PVN, such as SON and SCN, which are also implicated in neuroendocrine functions. SON receives projections from the PVN and may mediate adaptive neuroendocrine and autonomic responses after exposure to opioid agonists (35, 36). On the other hand, the SCN is an endogenous circadian pacemaker that governs daily behavioral, physiological, and hormonal rhythms (37). Recently, it has been proposed that the SCN is responsible for a circadian variation in plasma ACTH and corticosterone levels, in part through a direct innervation of CRF-producing neurons in the PVN, resulting in stimulation of CRF release (38). The effects of morphine on Fos-IR were similar qualitatively to those of U-50,488H, but the KOR agonist produced higher Fos-IR than did morphine in the three nuclei analyzed and may reflect the reported higher level of KOR binding and messenger RNA in the rat hypothalamus compared with those of MOR (39). On the other hand, our results show that the basal levels of Fos-IR in the SCN were higher than those in the PVN and SON, which agree with previous findings showing spontaneous Fos in the SCN early in the day (40, 41). Recent studies have shown that opioids play an important role in the control of oxytocin and AVP release by magnocellular neurons in the SON and SCN and, thereby, hormone release in the neural lobe of pituitary and the response of circadian clock to light (42, 43). As Fos expression is an indicator of neuronal activity, the present results indicate that opioids activate magnocellular neurons in the SON and SCN, although the functional significance of this activation requires more detailed study. On the other hand, our study showed that Fos-IR in the PVN were largely confined to the parvocellular region. Inspection of the magnocellular part showed it to be mostly unresponsive. Thus, there was expression of c-fos in magnocellular neurons of the SON and SCN, but not in magnocellular neurons of the PVN. Although c-fos activation after opioid administration was dependent on the activation of selective opioid receptors, we cannot conclude that there are not opioid receptors on magnocellular neurons of the PVN.

Studies on the mechanism of action of opioids have centered on the inhibition of adenylate cyclase activity, resulting in decreases in both intracellular cAMP levels (for review see Ref. 44) and neuronal activity, although it has been shown that opioids can actually inhibit inhibitory interneurons [such as {gamma}-aminobutyric acid (GABA)-ergic neurons] and lead to activation of specific circuits (45). Our results show activation of Fos protein, the product of the IEG c-fos, by acute administration of morphine and U-50,488H, suggesting a stimulatory effect of opioids on IEG expression in the hypothalamus. It has been demonstrated that opioids activate extracellular signal-related kinase, which has been shown to induce transcription of c-fos (46, 47). It has been proposed that this action may be involved in the dependence-inducing properties of these drugs (48). Despite robust increases in Fos-IR by opioids in the rat PVN, it is uncertain how Fos may signal the transcription of CRF gene, as this gene does not contain an activating protein-1-binding site to which the Fos-Jun transcription factor complex binds (49), although previous studies have suggested CRF to be a potential target of activating protein-1 transcription factor (50).

The most extensive characterized effect of morphine and U-50,488H on the endocrine system involves stimulation of the HPA axis (5). Both opioids increase ACTH and corticosterone release (6, 8, 51), and it has been postulated that this effect is produced through the secretion of CRF (11). However, the mechanism by which opioids produce activation of the HPA axis is still a matter of speculation. Previous studies have demonstrated that morphine facilitates the secretion of NA, which, in turn, releases CRF via stimulation of {alpha}-adrenoceptors, suggesting that the action of the opiate on the response of the HPA axis is probably mediated through stimulation of noradrenergic neurotransmission (52). This hypothesis is supported by the observation that noradrenergic projections from the nucleus of the solitary tract, distributed primarily throughout the parvocellular CRF-containing neurons of the PVN, are clearly involved in the regulation of HPA axis activity (53, 54, 55). Catecholaminergic involvement in the secretion of corticosterone in response to morphine, primarily via secretion of NA, has been well documented. Previous studies have proposed that an increase in hypothalamic noradrenergic activity could occur after morphine treatment, which was correlated with a simultaneous increase in corticosterone secretion (56). These effects of morphine were blocked in rats pretreated with reserpine or {alpha}- and ß-adrenoceptor antagonists, suggesting that the action of the opiate on the HPA axis may be dependent on stimulatory catecholaminergic systems (13). Considerable less is known about the role of KOR activation in modulating noradrenergic neurotransmission in the hypothalamus.

The present study confirms previous results showing that acute administration of morphine leads to an overproduction of the brain NA metabolite MHPG and to an elevation of NA turnover in the hypothalamic PVN. Furthermore, we show that stimulation of KOR produces an increase in noradrenergic pathways innervating the PVN. Noradrenergic hyperactivity after opioid administration seems to be dependent on activation of selective MOR and KOR, as it was partially or completely blocked by pretreatment with naloxone and BNI. Possible mechanisms underlying the hypothalamic Fos response to morphine and U-50,488H are not known. However, the fact that hypothalamic nuclei expressing Fos-IR, particularly the PVN, receive dense NA innervation (53) and the hypothesis that the effects of morphine on the HPA axis are mediated through noradrenergic pathways impinging on the PVN may suggest that NA could trigger the induction of the transcription factor gene product Fos. The hypothalamus is densely innervated by GABA-containing nerve terminals (for review, see Ref. 57). GABA is a major inhibitory neurotransmitter in the CNS and is presumed to negatively regulate the CRF neurons in the PVN (58). Therefore, the possibility that opioids may lead to increased noradrenergic activity by inhibiting inhibitory GABAergic projections cannot be dismissed.

In summary, our experiments show that activation of MOR and KOR trigger the expression of Fos in various hypothalamic nuclei, particularly in the PVN. As Fos expression is an indicator of neuronal activity, these results confirm that opioids activate neurosecretory neurons implicated in the activity of the HPA axis. As Fos protein functions as a transcription factor, its induction may extend to the level of gene regulation of the critical hypothalamic hormones precursor, such as CRF and AVP, which trigger HPA activation. Although the element that mediates c-fos induction after the stimulation of neurons has not been yet determined, there are a number of signals that induce c-fos expression, such as neurotransmitters (e.g. NA) and increased calcium influx (3, 59). As the present work also showed an enhanced noradrenergic activity in the PVN after opioid administration, these results suggest possible interaction between Fos induction and noradrenergic systems, which may lead to regulation of specific target genes, such as CRF and AVP, although a direct link between those elements remains to be demonstrated. Finally, the present study may indicate that morphine and U-50,488H activate the HPA axis through similar molecular mechanisms.


   Acknowledgments
 
We thank Profs. L. Puelles (University of Murcia), R. Coveñas (University of Salamanca), and Dr. E. Puelles (University of Murcia) for important advice and criticism.


   Footnotes
 
1 This work was supported by grants from the Ministry of Culture and Education, Spain (DGICYT PM 96–0095 and CICYT SAF 99–0047). Back

2 Fundación Séneca Fellow. Back

Received September 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dragunow M, Faull R 1989 The use of c-fos as a metabolic marker in neuronal pathways tracing. J Neurosci Methods 29:261–265[CrossRef][Medline]
  2. Morgan JI, Curran T 1991 Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 14:421–451[CrossRef][Medline]
  3. Hughes P, Dragunow M 1995 Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol Rev 47:133–178[Medline]
  4. Buckingham JC, Cooper TA 1984 Differences in hypothalamic-pituitary-adrenocortical activity in the rat after acute and prolonged treatment with morphine. Neuroendocrinology 38:411–417[Medline]
  5. Pechnick RN 1993 Effects of opioids on the hypothalamo-pituitary-adrenal axis. Annu Rev Pharmacol Toxicol 32:353–382
  6. Ignar DM, Kuhn CM 1990 Effects of specific µ and {kappa} opiate tolerance and abstinence on hypothalamo-pituitary-adrenal axis secretion in the rat. J Pharmacol Exp Ther 255:1287–1295[Abstract/Free Full Text]
  7. Gonzálvez ML, Milanés MV, Vargas ML 1991 Effects of acute and chronic administration of µ- and {delta}-opioid agonists on the hypothalamic-pituitary-adrenocortical (HPA) axis in the rat. Eur J Pharmacol 200:155–158[CrossRef][Medline]
  8. Martínez JA, Vargas ML, Fuente T, del Río-García J, Milanés MV 1990 Plasma ß-endorphin and cortisol levels in morphine-tolerant rats and in naloxone-induced withdrawal. Eur J Pharmacol 182:117–123[CrossRef][Medline]
  9. Milanés MV, Gonzálvez ML, Fuente T, Vargas ML 1991 Pituitary-adrenocortical response to acute and chronic administration of U-50,488H in the rat. Neuropeptides 20:95–102[CrossRef][Medline]
  10. Wang XQ, Imaki T, Shibasaki T, Yamauchi N, Demura H 1996 Intracerebroventricular administration of ß-endorphin increases the expression of c-fos and of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus of the rat. Brain Res 707:189–195[CrossRef][Medline]
  11. Buckingham JC, Cooper TA 1986 Pharmacological characterization of opioid receptor influencing the secretion of corticotrophin-releasing factor in the rat. Neuroendocrinology 44:35–40
  12. Nikolarakis K, Pfeiffer A, Stalla GK, Herz A 1987 The role of CRF in the release of ACTH by opiate agonists and antagonists in rats. Brain Res 421:373–376[CrossRef][Medline]
  13. Martínez-Piñero MG, Milanés MV, Alcaraz C, Vargas ML 1994 Catecholaminergic mediation of morphine-induced activation of pituitary-adrenocortical axis in the rat: implication of {alpha}- and ß-adrenoceptors. Brain Res 668:122–128[CrossRef][Medline]
  14. Milanés MV, Laorden ML, Chapleur-Chateau M, Burlet A 1997 Differential regulation of corticotropin-releasing factor and vasopressin in discrete brain regions after morphine administration: correlations with hypothalamic noradrenergic activity and pituitary-adrenal response. Naunyn Schmiedeberg Arch Pharmacol 356:603–610[CrossRef][Medline]
  15. Chang SL, Squinto SP, Harlan RE 1988 Morphine activation of c-Fos expression in rat brain. Biochem Biophys Res Commun 157:698–704[CrossRef][Medline]
  16. Liu L, Nickolenko J, Sharp FR 1994 Morphine induces c-fos and jun B in striatum and nucleus accumbens via D1 and N-methyl-D-aspartate receptors. Proc Natl Acad Sci USA 91:8537–8541[Abstract/Free Full Text]
  17. Erdtmann-Vourliotis M, Mayer P, Riechert UGG, Höllt V 1998 Identification of brain regions that are markedly activated by morphine in tolerant but not in naive rats. Brain Res Mol Brain Res 61:51–61[Medline]
  18. Carr KD, Kutchukhidze N, Park TH 1999 Differential effects of µ and {kappa} opioid antagonists on Fos-like immunoreactivity in extended amygdala. Brain Res 822:34–42[CrossRef][Medline]
  19. Sawchenko PE, Swanson LW 1983 The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J Comp Neurol 218:124–144
  20. Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ 1995 Pattern and time course of immediate-early gene expression in rat brain following acute stress. Neuroscience 64:477–505[CrossRef][Medline]
  21. Grassi Zuconi G, Menegazzi M, Carcereri De Prati A, Vescia S, Ranucci G, Bentivoglio M 1994 Different programs of gene expression are associated with different phases of the 24 hr and sleep-wake cycles. Chronobiologia 21:93–97[Medline]
  22. Palkovits M, Brownstein M J 1988 Maps and Guide to Microdissection of the Rat Brain. Elsevier, New York
  23. Paxinos G, Watson C 1982 The Rat Brain in Stereotaxic Coordinates. Academic Press, Sidney
  24. Swanson LW, Simmons DM 1989 Differential steroid hormone and neural influences on peptide mRNA levels in CRH cells of the paraventricular nucleus: a hybridization histochemical study in the rat. J Comp Neurol 285:413–435[CrossRef][Medline]
  25. Palkovits M 1973 Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res 59:449–450[CrossRef][Medline]
  26. Lookingland KJ, Ireland LM, Gunnet JW, Manzanares J, Tian Y, Moore KE 1991 3-Methoxy-4-hydroxyphenylethyleneglycol concentrations in discrete hypothalamic nuclei reflect the activity of noradrenergic neurons. Brain Res 559:82–88[CrossRef][Medline]
  27. Artigas F, Sarrias MJ, Adell A, Gelpí E 1986 Quantitation of total MHPG in the rat brain using a non enzymatic hydrolysis procedure. Effects of drugs. Life Sci 39:1571–1578[CrossRef][Medline]
  28. Antoni FA 1986 Hypothalamic regulation of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocr Rev 7:351–378[Medline]
  29. Dayas CC, Buller KM, Day TA 1999 Neuroendocrine responses to an emotional stressor: evidence for involvement of the medial but not the central amygdala. Eur J Neurosci 11:2312–2322[CrossRef][Medline]
  30. Li HY, Sawchenko PE 1998 Hypothalamic effector neurons and extended circuitries activated in "neurogenic" stress: a comparison of footshock effects exerted acutely, chronically, and in animals with controlled glucocorticoids levels. J Comp Neurol 393:244–266[CrossRef][Medline]
  31. Swanson LW, Sawchenko PE, Rivier J, Vale WW 1983 Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunocitochemical study. Neuroendocrinology 36:165–186[Medline]
  32. Gutstein HB, Thome JL, Fine JL, Watson SJ, Akil H 1998 Pattern of c-fos mRNA induction in rat brain by acute morphine. Can J Physiol Pharmacol 76:294–303[CrossRef][Medline]
  33. Chang SL, Patel NA, Romero AA, Thompson J, Zadina JE 1996 FOS expression induced by interleukin-1 or acute morphine treatment in the rat hypothalamus is attenuated by chronic exposure to morphine. Brain Res 736:227–236[CrossRef][Medline]
  34. Garcia MM, Brown HE, Harlan RE 1995 Alterations in immediate-early gene proteins in the rat forebrain induced by acute morphine injection. Brain Res 692:23–40[CrossRef][Medline]
  35. Blake AD, Bot G, Reisine T 1996 Structure-function of the cloned opiate receptors: peptide and small molecule interactions. Chem Biol 3:967–972[CrossRef][Medline]
  36. Bot G, Chahl LA 1996 Induction of Fos-like immunoreactivity by opioids in guinea-pig brain. Brain Res 731:45–56[CrossRef][Medline]
  37. Klein DC, Moore RY, Reppert SM 1991 Suprachiasmatic Nucleus: The Mind’s Clock. Oxford University Press, New York
  38. Buijs RM, Wortel J, Van Heerikhuize JJ, Kalsbeek A 1997 Novel environment induced inhibition of corticosterone secretion: physiological evidence for a suprachiasmatic nucleus mediated neuronal hypothalamo-adrenal cortex pathway. Brain Res 758:229–236[CrossRef][Medline]
  39. Mansour A, Fox CA, Akil H, Watson SJ 1995 Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications. Trends Neurosci 18:22–29[CrossRef][Medline]
  40. Abe H, Rusak B 1994 Physiological mechanisms regulating photic induction of Fos-like protein in hamster suprachiasmatic nucleus. Neurosci Biobehav Rev 18:531–536[CrossRef][Medline]
  41. Sumová A, Trávnícková Z, Mikkelsen JDIH 1998 Spontaneous rhythm in c-Fos immunoreactivity in the dorsomedial part of the rat suprachiasmatic nucleus. Brain Res 801:254–258[CrossRef][Medline]
  42. Allen CN, Jiang ZG, Teshima K, Darland T, Ikeda M, Nelson CS, Quigley DI, Yoshioka T, Allen RG, Rea MA, Grandy DK 1999 Orphanin-FQ/nociceptin (OFQ/N) modulates the activity of suprachiasmatic nucleus neurons. J Neurosci 19:2152–2160[Abstract/Free Full Text]
  43. Muller W, Hallermann S, Swandulla D 1999 Opioidergic modulation of voltage-activated K+ currents in magnocellular neurons of the supraoptic nucleus in rat. J Neurophysiol 81:1617–1625[Abstract/Free Full Text]
  44. Childers SR, Fleming L, Konkoy C, Marckel D, Pacheco M, Sexton T, Ward S 1992 Opioid and cannabinoid receptor inhibition of adenyl cyclase in brain. Ann NY Acad Sci 654:33–51[Abstract]
  45. Johnson SW, North RA 1992 Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483–488[Abstract]
  46. Hill C, Treisman R 1995 Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80:199–211[CrossRef][Medline]
  47. Gutstein HB, Rubie EA, Mansour A, Akil H 1997 Opioid effects on mitogen-activated protein kinase signaling cascades. Anesthesiology 87:1118–1126[CrossRef][Medline]
  48. Nestler EJ, Aghajanian GK 1997 Molecular and cellular basis of addiction. Science 278:58–63[Abstract/Free Full Text]
  49. Chan RKW, Brown ER, Ericsson A, Kovács K, Sawchenko PE 1993 A comparison of two immediate-early genes, c-fos and NGFI-B, as markers for functional activation in stress-related neuroendocrine circuitry. J Neurosci 13:5126–5138[Abstract]
  50. Imaki T, Shibasaki T, Hotta M, Demura H 1992 Early induction of c-fos precedes increased expression of corticotropin-releasing factor mRNA in the paraventricular nucleus after immobilization stress. Endocrinology 131:240–246[Abstract]
  51. Alcaraz C, Milanés MV, Vargas ML 1993 Chronic kappa opioid receptor antagonism produces supersensitivity to U-50,488H at the hypothalamo-pituitary-adrenocortical (HPA) axis level. J Pharmacol Exp Ther 266:1385–1389[Abstract/Free Full Text]
  52. Suemaru S, Dallman MF, Darlington DN, Cascio CS, Shisako J 1989 Role of alpha adrenergic mechanisms in effects of morphine on the hypothalamo-pituitary-adrenocortical and cardiovascular systems in the rat. Neuroendocrinology 49:181–190[Medline]
  53. Cunningham ET, Sawchenko PE 1988 Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol 274:60–76[CrossRef][Medline]
  54. Widmaier EP, Lim A, Vale W 1989 Secretion of corticotropin-releasing factor from cultured rat hypothalamic cells: effects of catecholamines. Endocrinology 124:583–590[Abstract]
  55. Itoi K, Suda T, Tozawa F, Dobashi I, Ohmori N, Sakai Y, Abe K, Demura H 1994 Microinjection of norepinephrine into the paraventricular nucleus of the hypothalamus stimulates corticotropin-releasing factor gene expression in conscious rats. Endocrinology 135:2177–2182[Abstract]
  56. Martínez-Piñero MG, Vargas ML, Milanés MV 1993 L-type Ca2+ channel ligands modulate morphine effects on the hypothalamus-pituitary-adrenocortical axis in rat. Eur J Pharmacol 232:191–198[CrossRef][Medline]
  57. Itoi K, Seasholtz AF, Watson SJ 1998 Cellular and extracellular regulatory mechanisms of hypothalamic corticotropin-releasing hormone neurons. Endocr J 45:13–33[Medline]
  58. Petrusz P, Merchenthaler I 1992 The corticotropin-releasing factor system. In: Nemeroff CB (ed) Neuroendocrinology. CRC Press, Boca Raton, pp 129–183
  59. Kovács K 1998 c-Fos as a transcription factor: a stressful (re)view from a functional map. Neurochem Int 33:287–297[CrossRef][Medline]



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