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Department of Pharmacology, University School of Medicine, 30100 Murcia, Spain
Address all correspondence and requests for reprints to: Professor M. Victoria Milanés, Department of Pharmacology, University School of Medicine, Campus de Espinardo, 30100 Murcia, Spain. E-mail: milanes{at}um.es.
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Introduction |
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It has been shown that stressors can influence different facets of drug addictions, including the rewarding properties of drugs of abuse and relapse vulnerability after cessation of drug use (4, 5). An important component of the stress-responsive system is the hypothalamic-pituitary-adrenal (HPA) axis. The parvocellular division of the hypothalamic paraventricular nucleus (PVN) is the primary location of corticotropin-releasing factor (CRF) neurons and constitutes a key site for adaptive responses to stress. CRF-containing neurons in the PVN are innervated by numerous afferents from other brain regions. Major afferent innervations of the PVN originate in brain stem noradrenergic neurons located in the medulla oblongata. Direct noradrenergic inputs from the brain stem nucleus tractus solitarius (NTS)-A2 to the parvocellular PVN have been described (6). At the ultrastructural level, several studies have confirmed the noradrenergic innervation of CRF neurons in the PVN, and evidence suggests that these inputs primarily have a facilitatory effects on HPA axis regulation (7, 8).
A variety of physiological stressors stimulate catecholamine synthesis through regulatory effects on TH. Short-term regulation of catecholamine biosynthesis occurs through phosphorylation of TH, which enhances enzymatic activity, whereas long-term regulation is achieved through changes in the level of TH protein (9, 10). TH activity is directly regulated by phosphorylation at Serine (Ser)-31 and Ser40 (9, 11). Changes in the state of phosphorylation of TH are critically involved in the regulation of catecholamine synthesis. In particular, increases in the phosphorylation of Ser31 and Ser40 accelerate TH activity, thereby stimulating production of neurotransmitter (11).
We have previously shown that morphine withdrawal but not chronic morphine treatment stimulates NA turnover in the PVN (12) as well as the activity of NTS-A2 TH-positive neurons (as reflected by c-Fos expression) (13). This response was accompanied by a rise in TH enzymatic activity in the PVN (14). The present study was designed to assess the changes in TH phosphorylation after chronic morphine administration and after morphine withdrawal in the PVN and brain stem catecholaminergic cell groups innervating the PVN. To this end, we first evaluated the changes in TH phosphorylation at Ser31 and Ser40 in addition to total levels of TH and TH enzymatic activity. It is known that phosphorylation of TH at each site is associated with distinct signaling pathways. Because ERK phosphorylates Ser31 in vitro and in situ (9, 11), we then tested whether activation of the ERK kinase (MEK)-ERK1/2 pathway contributes to morphine withdrawal-induced TH phosphorylation at Ser31. This was accomplished by investigating the effects of SL327, a selective inhibitor of ERK activation (15), on levels of TH phosphorylated at Ser31. In addition, double-label immunostaining was used to investigate c-Fos expression during morphine withdrawal in NTS neurons positive for TH phosphorylated at Ser31. In the final part of the study, we evaluated whether activation of ERK1/2 signaling pathway by MEK participates somehow in the regulation of HPA axis during morphine withdrawal, using plasma corticosterone and ACTH levels as markers of the axis activity.
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Materials and Methods |
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On the basis of previous studies (16), rats were rendered dependent on morphine by sc implantation of morphine base pellets (75 mg), one on d 1, two on d 3, and three on d 5, under light ether anesthesia. This procedure has repeatedly been shown to induce dependence as measured behaviorally and biochemically (17). Control animals were implanted with placebo pellets containing lactose instead morphine on the same time schedule. On d 8, the animals pretreated with morphine or placebo pellets were injected with saline sc or naloxone (2 mg/kg sc). The weight gain of the rats was checked during treatment to ensure that the morphine was liberated correctly from the pellets because it is known that chronic morphine treatment induces a decrease in body weight gain due to lower caloric intake (18). In addition, body weight loss was determined as the difference between the weight determined immediately before saline or naloxone injection and a second determination made 60 and 90 min later.
To determine the effect of inhibiting ERK phosphorylation on the morphine withdrawal-induced changes in TH phosphorylation at the PVN and brain stem catecholaminergic areas as well as in HPA axis activity, TH phosphorylated (p) at Ser31 (pSer31-TH) levels, and plasma corticosterone and ACTH concentrations were determined in morphine-dependent and control rats treated with SL327 (a selective inhibitor of MEK) (15) 1 h before the administration of naloxone or saline. This inhibitor was dissolved in dimethylsulfoxide (DMSO) 100% and injected ip at an injection volume of 1 ml/kg and at doses of 50 and 100 mg/kg. On the basis of our initial experiments of SL327-induced inhibition of ERK phosphorylation in PVN, the 100-mg/kg dose was chosen for our experiments.
Tissue preparation for Western blot analysis
Sixty and 90 min [for total TH(tTH) determination] and 90 min (for TH phosphorylation analysis and for pERK evaluation) after administration of naloxone or saline, rats were killed by decapitation, the brains were removed rapidly, fresh frozen, and stored immediately at –80 C until use. The hypothalamic tissue containing the PVN was dissected according to the technique of Palkovits (19), and the PVN corresponds to those in plates 25 and 26 in the atlas of Palkovits and Brownstein (20). Because noradrenergic neuronal groups in the NTS extend from the level of the area postrema (AP), rostrally, to upper cervical segment of spinal cord caudally (6), brain stem tissue corresponding to NTS-A2 cell group was dissected between the AP, rostrally, to the pyramidal decussation, caudally (plane of sections relative to bregma: –13.68 to –14.60) (21).
Western blotting
PVN and brain stem samples were placed in homogenization buffer (PBS, 2% sodium dodecyl sulfate, protease inhibitors plus a phosphatase inhibitor cocktail set), homogenized, and sonicated for 30 sec before centrifugation at 12,000 x g for 10 min at 4 C. The total protein concentrations were determined spectrophotometrically using the bicinchoninic assay method (22). The optimal amount of proteins to be loaded was determined in preliminary experiments by loading gels with increasing protein contents from samples of each experimental group. Samples containing 40 µg protein were loaded on a 10% sodium dodecyl sulfate/polyacrylamide gel, electrophoresed, and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) using a minitransblot electrophoresis transfer cell (Bio-Rad, Hercules, CA). Parallel gels were stained with Comassie Blue to verify loading, sample integrity, and protein separation. Similar transfer was ascertained by cutting the lower portion of the blots and staining for total protein with Amido Black. Nonspecific binding of antibodies was prevented by incubating membranes in 1% BSA in TBST [10 mM Tris HCl (pH 7.6), 150 mM NaCl, 0.15% Tween 20]. The blots were incubated overnight at room temperature (for tTH and pTH) or 4 C (for pERKs) with the following primary antibodies: polyclonal anti-tTH (against phosphorylated and nonphosphorylated TH; 1:1000 dilution; AB152; Chemicon International, Temecula, CA); polyclonal anti-pSer31-TH (1:500; AB5423; Chemicon International); polyclonal anti-pSer40 TH (1:500; AB5935; Chemicon International); or monoclonal anti-pERK1/2 (1:1000; sc-7383; Santa Cruz Biotechnology, Santa Cruz, CA) in TBST with BSA. After extensive washing with TBST, the membranes were incubated for 1 h at room temperature with peroxidase-labeled secondary antibodies (antirabbit sc-2004 for tTH and pTH; Santa Cruz; antimouse sc-2005 for p-ERK1/2; Santa Cruz) at 1:5000 dilution. After washing, immunoreactivity was detected with an enhanced chemiluminescence Western blot detection system (ECL; Amersham Ibérica, Madrid, Spain) and visualized by Amersham Hyperfilm-ECL. Quantification of immunoreactivity corresponding to tTH and TH phosphorylated at Ser31 and Ser40 (60 kDa) and pERK (42 and 44 kDa) bands was carried out by densitometry (Gel Doc; Bio-Rad). The integrated OD of the bands was normalized to the background values. Relative variations between bands of the experimental samples and control samples were calculated in the same image.
Tissue preparation for immunohistochemistry
Sixty and 90 min after administration of naloxone or saline, rats were deeply anesthetized with an overdose of pentobarbital (100 mg/kg ip) and perfused transcardially with 300 ml PBS [PBS (pH 7.4); 1 mM NaF, which was included in all buffers and incubation solutions] followed by 500 ml of fixative containing 4% paraformaldehyde in PBS. After removal of the perfused brains, they were postfixed in the same fixative and stored at 4 C overnight. Free-floating coronal brain stem sections (30 µm thickness) were obtained on a Vibratome (Leica, Nussloch, Germany) and stored in cryoprotectant at –20 C until histochemical processing. Analysis of TH phosphorylated at Ser31 and Ser 40 as well as double staining c-Fos/pSer31-TH was made at various levels, ranging from the AP, rostrally, to the pyramidal decussation, caudally (plane of sections relative to bregma: –13.68 to –14.60; 21). Sections used for histological analysis were regularly spaced at intervals of 60 µm.
Immunohistochemistry
Before all immunocytochemical steps, sections were rinsed several times in PBS. Expression of TH phosphorylated at Ser31 and Ser40 was examined in free-floating sections and processed for immunohistochemistry. Briefly, the sections were preincubated for 20 min in absolute methanol containing 10% H2O2, rinsed twice in PBS (15 min each rinse), and treated with NSS-PBS [PBS containing 2% goat serum and 0.5% Triton X-100] for 30 min. Sections were then incubated in the same anti-pTH antibodies at a dilution of 1:400 in NSS-PBS overnight at room temperature. This was followed by application of a biotinylated antirabbit IgG (diluted 1:200 in NSS-PBS; Vector, Burlingame, CA) and then with the avidin-biotin complex at room temperature for 1 h each. Visualization of the antigen-antibody reaction sites was performed using 0.033% 3, 3'-diaminobenzidine (DAB; Sigma, St. Louis, MO) and 0.014% H2O2 in 0.05 M Tris-HCl buffer for 7 min. The reaction was stopped in PBS.
For c-Fos/pSer31-TH double-label immunohistochemistry, brain stem tissue sections from each rat in each treatment group were processed as follows: c-Fos was revealed with DAB intensified with nickel in the first position, and the enzyme was revealed with DAB in the second position. c-Fos immunohistochemistry was performed as described previously, using a polyclonal anti-(c-Fos) antibody (1:3000 dilution; non-cross-reactive with Fos-B, Fra-1, or Fra-2; sc-52; Santa Cruz) and a secondary antirabbit IgG (diluted 1:200 in NSS-PBS; Vector), and c-Fos antibody-peroxidase complex was visualized by using a mixture of NiSO4.6H2O (33.2 mg/ml), DAB (0.033%), and 0.014% H2O2 in 0.175 M sodium acetate solution (pH 7.5). When the level of staining was appropriate, tissue sections were transferred into distilled water (Milli-Q water; Millipore Corp.) to stop the color reaction. After the c-Fos staining, sections were rinsed twice in PBS (15 min each rinse), treated with NSS-PBS for 30 min, and then incubated overnight with the rabbit polyclonal anti-pSer31-TH antibody (diluted 1:400 in NSS-PBS). The same immunohistochemistry procedures described above were followed. The pSer31-TH antibody-peroxidase complex was stained in 0.033% DAB and 0.014% H2O2 in 0.05 M Tris-HCl buffer. The reaction was stopped in PBS. With this staining procedure, the c-Fos nuclear protein stains blue-black, and the pSer31-TH cytoplasmatic enzyme stains yellow-brown. The sections were mounted onto glass slides coated with gelatin, air dried, dehydrated through graded alcohols, cleared in xylene, and coverslipped with dibutylphtalate.
Quantification of phosphorylated TH immunoreactivity
Evidence of TH phosphorylated at Ser31 and Ser40 immunoreactivity was examined under a light microscope. The number of pTH-immunopositive cells was determined using a computer-assisted image analysis system (Q500MC; Leica, Madrid, Spain). This system consists of a light microscope (DMLB; Leica) connected to a videocamera (Sony 151-AP; Sony, Madrid, Spain) and the image analysis computer. A square field (129 µm side) was superimposed on the captured image (x40 magnification) for use as a reference area. The sections showing a discernible level of pTH immunoreactivity were selected for quantitative image analysis. For the NTS-A2 analysis, the right and left sites of five to six sections were analyzed and averaged per rat. The number of pTH-positive neurons of both the right and left sides of eight to 12 correlative sections was averaged per animal. Measures were also averaged in each experimental group.
Quantification of c-Fos-positive pSer31-TH-positive neurones
Positive nuclei for c-Fos immunoreactivity were detected using the same conventional light microscopy described above, and counted at x40 magnification. c-Fos-positive pSer31-TH-positive neurons were identified as cells with brown cytoplasmic deposits for pSer31-TH-positive staining and blue/dark nuclear staining for c-Fos. A square field (129 µm) was superimposed on captured image for use as reference area. The number of single- and double-labeled c-Fos neurons observed bilaterally was counted in four to five sections from each animal in the NTS-A2. Because pSer31-TH-positive neurones could be counted as c-Fos positive only if the nucleus was visible, the pSer31-TH-positive cells without a visible nucleus were excluded from the analysis. Counts comprised: 1) activated cells (c-Fos positive nucleus), 2) specific c-Fos-containing cells with no pSer31-TH (c-Fos positive nuclei and pSer31-TH-negative cytoplasm), and 3) double-labeled neurones (positive for both c-Fos and pSer31-TH) in each section.
Measurement of TH activity
Ninety minutes after saline or naloxone injection to placebo- or morphine-pelleted animals, rats were killed by decapitation, the brain removed, and the hypothalamic PVN dissected according to the method described above. TH catalyzes the hydroxylation of tyrosine to generate L-DOPA and water using tetrahydropterine as a cofactor. TH activity is measured by quantifying tritiated water production from 3,5-[3H]L-tyrosine. PVN samples were placed in homogenization buffer (PBS, protease inhibitors plus a phosphatase inhibitor cocktail set), and homogenized before centrifugation at 10,000 rpm for 10 min at 4 C. Twenty-five microliters of the supernatants were incubated at 37 C in a final volume of 50 µl of a reaction mixture containing the following components: 0.2 M Tris HCl (pH 7), 1 mM tetrahydrobiopterin, 10 mM ß-mercaptoethanol, 0.02% catalase, 50 µM isotopically diluted L-[3,5-3H]tyrosine (radioactive concentration 10 µCi/ml, specific activity 0.2 mCi/µmol; Amersham). For the blank reaction, samples were replaced by sodium orthovanadate. After 4 h of incubation, the reaction was stopped by the addition of 1% trichloroacetic acid, and the radioactive organic compounds were separated from tritiated water by absorption onto activated charcoal. After centrifugation (12,000 rpm, 4 C, 5 min), tritiated water was quantified in the supernatant by scintillation counting in a Wallac 1409 liquid scintillation counter (PerkinElmer, Waltham, MA). The assays were performed in duplicate.
Corticosterone and ACTH assays
At the end of the treatment, rats were killed by decapitation between 1000 and 1100 h to avoid circadian variations in plasma levels of corticosterone and ACTH. Trunk blood was collected into ice-cooled tubes containing 5% EDTA and then was centrifuged (2500 rpm; 4 C; 15 min). Plasma was separated, divided into two aliquots and stored at –80 C until assayed for corticosterone or ACTH. Plasma levels of corticosterone and ACTH were estimated, as sensitive markers of the HPA axis activity, with commercially available kits for rats (125I-corticosterone and 125I-hACTH RIA; MP Biomedicals, Orangeburg, NY). The sensitivity of the assay was 7.7 ng/ml for corticosterone and 5.7 pg/ml for ACTH. The inter- and intraassay coefficients of variation were 10.3 and 7.1%, respectively, for corticosterone, and the intraassay coefficient of variation for ACTH was 3.9%. For corticosterone, the antibody cross-reacted 100% with corticosterone and less than 0.01% with other steroids. For ACTH, the antibody cross-reacted 100% with ACTH and 0.1 or less with other pituitary hormones.
Drugs and reagents
Pellets of morphine base (Alcaliber Laboratories, Madrid, Spain) or lactose were prepared by the Department of Pharmacy and Pharmaceutic Technology (School of Pharmacy, Granada, Spain); naloxone HCl was purchased from Sigma, dissolved in sterile 0.9% NaCl (saline), and administered in volumes of 0.1 ml per 100 g body weight. SL327, kindly provided by Dr. R. Santos (Bristol-Myers Squibb Co., Princeton, NY) was dissolved in 100% DMSO and administered in volumes of 0.1 ml per 100 g body weight. DMSO was purchased from Sigma. Drugs were prepared fresh every day. Reagents included: protease inhibitors (Boehringer Mannheim, Mannheim, Germany); phosphatase inhibitor cocktail set (Calbiochem, Darmstadt, Germany); goat serum (Sigma); avidin-biotin complex (ABC kits; Vector); and nickel sulfate (Sigma). TH activity reagents were purchased from Sigma.
Statistical analysis
Data are presented as mean ± SEM and were analyzed using two-way or one-way ANOVA followed by the Newman-Keuls post hoc test. Student’s t test was used when comparisons were restricted to two experimental groups. Differences with a P < 0.05 were considered significant.
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Results |
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The body weight loss after saline or naloxone injection to placebo-pelleted and morphine-dependent rats was also recorded. In agreement with our previous results (23), two-way ANOVA revealed that morphine pretreatment, naloxone injection, and the interaction between pretreatment and acute treatment have a significant effect on body weight loss at 60 (pretreatment: F(1, 21) = 23.73, P < 0.0001; treatment: F(1, 21) = 10.72, P = 0.0036; interaction: F(1, 21) = 9.67, P = 0.0053) and 90 min (pretreatment: F(1, 22) = 14.36, P = 0.0010; treatment: F(1, 22) = 14.36, P = 0.0010, interaction: F(1, 22) = 9.15, P = 0.0062). Post hoc analysis showed that naloxone injection to morphine-dependent animals significantly increased (P < 0.001) the body weight loss when measured 60 (21.50 ± 2.10 g; n = 4) and 90 min (12.57 ± 1.49 g; n = 7) after naloxone injection when compared with the placebo-pelleted group also receiving naloxone (60 min: 4.00 ± 1.23 g, n = 4; 90 min: 2.17 ± 1.78 g, n = 6) and with morphine-treated rats given saline (60 min: 6.33 ± 1.45 g, n = 3; 90 min: 2.17 ± 0.31 g, n = 6). However, administration of naloxone to control rats resulted in no significant changes in body weight when measured 60 min after drug injection, compared with control rats receiving saline (3.50 ± 1.32 g at 60 min, n = 4;1.29 ± 1.92 g at 90 min; n = 7).
Effects of morphine dependence and withdrawal on total TH levels in PVN and NTS
The influence of morphine dependence and withdrawal on the immunoreactivity of tTH was examined by Western blot analysis in the PVN and NTS 60 and 90 min after sc injection of saline or naloxone (2 mg/kg) to control rats and animals considered dependent on morphine. In the PVN, the ANOVA for tTH at 60 min revealed no significant effects of acute treatment [F(1, 12) = 0.43; P = 0.5223], pretreatment [F(1, 12) = 1.04; P = 0.3285], or an interaction between pretreatment and acute treatment [F(1, 12) = 0.05; P = 0.8272]. The ANOVA for tTH at 90 min showed a significant effect of pretreatment [F(1, 11) = 9.44; P = 0.0116], with no main effect of acute treatment [F(1, 11) = 1.45; P = 0.2546] or an interaction between pretreatment and acute treatment [F(1, 11) = 0.00; P = 0.9919]. As shown in Fig. 1
, A and B, tTH in the PVN from rats treated with morphine did not significantly differ from that in the placebo control group 60 or 90 min after saline or naloxone administration, indicating that the amount of total protein was not changed during morphine dependence or withdrawal. Two-way ANOVA for tTH in the NTS at 60 min revealed no significant effects of acute treatment [F(1, 11) = 0.06; P = 0.8075], pretreatment [F(1, 12) = 0.85; P = 0.1993], or an interaction between pretreatment and acute treatment [F(1, 12) = 0.34; P = 0.5225]. Total TH levels in the NTS were unchanged 60 min after saline or naloxone administration to morphine-dependent rats (Fig. 2A). Ninety minutes after saline or naloxone injection to morphine-dependent rats, there was a significant enhancement in total protein levels, compared with placebo-pelleted animals also receiving saline or naloxone (Fig. 2B). The ANOVA for tTH at 90 min showed a significant effect of pretreatment [F(1, 11) = 24.38; P = 0.0004], with no main effect of acute treatment [F(1, 11) = 0.15; P = 0.7088] or an interaction between pretreatment and acute treatment [F(1, 11) = 0.90; P = 0.3619].
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) 90 min after the opioid antagonist injection, compared with the corresponding control group receiving naloxone and the morphine-dependent animals receiving saline. No changes in pSer40-TH levels were found in this nucleus in morphine withdrawn rats (Fig. 1D). In the NTS, the ANOVA for pSer31-TH showed significant effect of morphine administration [F(1, 12) = 7.09; P = 0.0207] and main effect of acute treatment [F(1, 12) = 6.04; P = 0.0301] with no main acute treatment-pretreatment interaction [F(1, 12) = 2.74; P = 0.1237]. The ANOVA for pSer40-TH revealed no significant main effects of morphine pretreatment, naloxone injection or an interaction between pretreatment and acute treatment [F(1, 12) = 0.15; P = 0.7060; F(1, 12) = 0.03; P = 0.8740; F(1, 12) = 0.39; P = 0.5464, respectively]. As shown in Fig. 2C, rats dependent on morphine and given naloxone also showed a significant enhancement in pSer31-TH levels in the NTS compared with the corresponding control group receiving naloxone and with the morphine-dependent animals receiving saline. By contrast, either injection of naloxone to control rats or saline administration to morphine-dependent animals did not alter TH phosphorylation at Ser31. Nonetheless, no changes in TH phosphorylation at Ser40 were found after naloxone-induced morphine withdrawal in this brain stem region (Fig. 2D).
These results were confirmed by immunohistochemical procedures using the same pSer31-TH and pSer40-TH antibodies (see Materials and Methods) in the NTS. Rats were treated with placebo or morphine under conditions known to elicit levels of dependence, as described in Materials and Methods, and were killed at 60- and 90-min time points after saline or naloxone administration. Two-way ANOVA for pSer31-TH at 60 min showed an interaction between pretreatment and acute treatment [F(1, 17) = 52.66; P < 0.0001], with main effect of acute treatment [F(1, 17) = 33.51; P < 0.0001] and pretreatment [F(1, 17) = 14.50; P = 0.0014]. The ANOVA for pSer31-TH at 90 min revealed significant effect of morphine administration [F(1, 14) = 20.28; P = 0.0005], main effect of acute treatment [F(1, 14) = 62.59; P < 0.0001], and an acute treatment-pretreatment interaction [F(1, 14) = 30.76; P < 0.0001]. As shown in Fig. 3, there were few cells exhibiting pSer31-TH staining in the NTS from control rats receiving saline or naloxone and from morphine-treated rats given saline. By contrast, high levels of pSer31-TH immunoreactivity were observed in the NTS-A2 cell group 60 and 90 min after naloxone injection to morphine-dependent rats (Fig. 3A, d and h). Quantitative analysis revealed that morphine withdrawal induced a significant elevation in the number of pSer31-TH-positive neurons (Fig. 3B). These immunohistochemical results are consistent with Western blot analysis in the present study.
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As shown in Fig. 4A, there are no important changes in the number of cells exhibiting pSer40 labeling in the NTS-A2 cell group from morphine-withdrawn rats, compared with the corresponding control group. Quantitative analysis at 60- and 90-min time points showed no significant differences in the number of pSer40-TH-positive neurons in the NTS from morphine-dependent rats treated with naloxone with regard to the placebo control groups receiving naloxone. Thus, two-way ANOVA for pSer40-TH at 60 min showed no significant main effects of morphine pretreatment, naloxone injection, or an interaction between pretreatment and acute treatment [F(1, 16) = 0.01; P = 0.9415; F(1, 16) = 0.00; P = 0.9494; F(1, 16) = 0.3581; P = 0.3581, respectively]. The ANOVA for pSer40-TH at 90 min revealed no significant effect of morphine administration [F(1, 13) = 0.76; P = 0.3997], a main effect of acute treatment [F(1, 13) = 6.49; P = 0.0243] with an acute treatment-pretreatment interaction [F(1, 13) = 7.55; P = 0.0166]. NTS from morphine-treated rats receiving saline shows lower pSer40-TH-positive neurons at 90 min, compared with morphine withdrawn rats (Fig. 4B).
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, morphine withdrawal-induced c-Fos expression was partly localized in neurons expressing phosphorylated TH at Ser31 in the NTS. Two-way ANOVA revealed a main effect of morphine treatment [F(1, 12) = 13.27; P = 0.0034], naloxone injection [F(1, 12) = 14.64; P = 0.0024], and an interaction between pretreatment and acute treatment [F(1, 12) = 16.26; P = 0.0017]. Post hoc analysis revealed that morphine withdrawal induced an increase of the number of pSer31-TH-containing neurons expressing c-Fos in NTS-A2, compared with placebo controls receiving naloxone and morphine-treated rats given saline instead of naloxone (Fig. 5E), whereas administration of the opioid antagonist to control rats did not induce any modification, compared with control groups receiving saline. The number of single-labeled neurons for c-Fos and pSer31-TH as well as the number of c-Fos-positive/non-TH phosphorylated cells in NTS for each treatment is shown in Table 1. Two-way ANOVA for TH-positive and for c-Fos-positive neurons showed significant effects of acute treatment [pSer31-TH-positive neurons: F(1, 12) = 15.08, P = 0.0022; c-Fos-positive neurons: F(1, 13) = 13.47, P = 0.0028] and two-way interaction between pretreatment/acute treatment [pSer31-TH-positive neurons: F(1, 12) = 16.73, P = 0.0015; c-Fos-positive neurons: F(1, 13) = 16.64, P = 0.0013]. A significant effect of morphine treatment was found for pSer31-TH-positive neurons [F(1, 12) = 13.10, P = 0.0035] but not for c-Fos-positive neurons [F(1, 13) = 4.30, P = 0.0585]. ANOVA for c-Fos-positive/pSer31-TH-negative neurons showed no significant effect of pretreatment [F(1, 13) = 0.25, P = 0.6280], acute treatment F(1, 13) = 0.33, P = 0.5775], or an interaction between pretreatment and acute treatment [F(1, 13) = 0.15, P = 0.7079]. As Table 1 exhibits, all the pSer31-TH-positive neurons were activated, as revealed by c-Fos expression.
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First, we determined the basal levels of phosphorylated (activated) ERK1/2 (pERK1/2) in the PVN and NTS from control and from morphine-withdrawn rats pretreated with SL327. As shown in Fig. 6, A–D, Student’s t test showed a significant decrease in phosphorylation of ERK1/2 in the presence of 100 mg/kg SL327 (a dose that selectively blocks MEK1/2) (26) in both controls (PVN: t6 = 6.146; ***, P < 0.001 for pERK1 and t6 = 5.176; **, P < 0.01 for pERK2; NTS: t6 = 4.592; **, P < 0.01 for pERK1 and t6 = 4.010; **, P < 0.01 for pERK2) and morphine-withdrawn animals (PVN: t6 = 3.301; *, P < 0.05 for pERK1 and t6 = 3.040; *, P < 0.05 for pERK2; NTS: t6 = 5.949; **, P < 0.01 for pERK1 and t6 = 8.304; ***, P < 0.001 for pERK2). Importantly, SL327 effectively reduced basal levels of phospho-ERK1/2 immunoreactivity. Next, we injected SL327 at the dose of 100 mg/kg ip to control rats and to animals made dependent on morphine 1 h before saline or naloxone administration and determined pSer31-TH levels by Western blot in the PVN and NTS. As shown in Fig. 6, pSer31-TH levels strongly decreased in the PVN (Fig. 6E) and returned to basal levels in the NTS (Fig. 6F). Two-way ANOVA (Fig. 6, E and F) in rats pretreated with the ERK phosphorylation inhibitor, SL327, revealed a significant effect of pretreatment on TH phosphorylation at Ser31 in the PVN [F(1, 10) = 27.67, P = 0.0004] and NTS [F(1, 10) = 8.00, P = 0.0179]. No significant effect of either acute treatment [PVN: F(1, 10) = 3.42, P = 0.0943; NTS: F(1, 10) = 4.00, P = 0.0734], or interaction for pretreatment-acute treatment was observed [PVN: F(1, 10) = 2.49, P = 0.1453; NTS: F(1, 10) = 2.13, P = 0.1751]. As mentioned above, SL327 effectively reduced basal levels of phospho-ERK1/2 immunoreactivity, thereby suggesting that the decrease in pSer31-TH levels after treatment with SL327 is not caused by a nonspecific action of the compound on MEK. Thus, these results suggest that TH phosphorylation at Ser31 after morphine withdrawal occurs downstream of ERK (Fig. 7).
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To evaluate whether a causal link exists between ERK phosphorylation by MEK and HPA axis hyperactivation during morphine withdrawal, we measured plasma corticosterone and ACTH concentrations in animals made dependent on morphine and pretreated with SL327 (100 mg/kg ip) or vehicle (DMSO) 1 h before naloxone administration. Student’s t test showed that pretreatment with SL327 did not significantly revert the increase in either corticosterone (490.50 ± 41.18 ng/ml, n = 4, t(6) = 1.970, P = 03569 vs. morphine+DMSO+naloxone: 634.80 ± 60.66 ng/ml, n = 4) or ACTH (297.80 ± 51.19 pg/ml, n = 5, t(6) = 0.9405, P = 0.3833 vs. morphine+DMSO+naloxone: 237.80 ± 38.07 pg/ml, n = 4) release during morphine withdrawal. Pretreatment with DMSO (vehicle) did not significantly modify the increase in corticosterone (t(6) = 0.9978, P = 0.3569 vs. morphine+naloxone) or ACTH (t(6) = 2.226, P = 0.0676 vs. morphine+naloxone) secretion after naloxone injection to morphine-dependent rats.
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Discussion |
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The parvocellular division of the PVN is the primary location of CRF neurons and constitutes a key site for adaptive responses to stress. The involvement of the HPA axis in development of addiction has been a subject of recent investigation (4), but the specific pattern of its molecular activation in the context of addiction has not been thoroughly investigated. CRF and noradrenergic activity have been shown to be activated during withdrawal from most drug of abuse (32, 33), and it has been hypothesized that the interaction of CRF/NA may be a stress mechanism contributing to the development of addiction. Dysregulation of the HPA response to stress also occurs with acute and chronic opioid administration (17, 24, 34, 35) and has been implicated as a contributing factor to continued substance abuse (29, 34).
Recent data provide evidence that activation of HPA axis during naloxone-precipitated morphine withdrawal involves transcriptional up-regulation of hypophyseotropic CRF expression in the PVN of the hypothalamus (33). Previous results from this laboratory have shown that activation of noradrenergic terminals innervating the PVN modulates the HPA axis activity in response to morphine withdrawal. Thus, morphine withdrawal but not chronic morphine treatment increases NA turnover and c-Fos expression in the PVN concomitantly with an increase in corticosterone secretion (a marker of HPA axis activity) as well as the activity of NTS-A2 TH-positive neurons (as reflected by c-Fos expression) (13, 17, 23). These effects were inhibited by adrenoceptor antagonist, which indicates that the hyperactivity of the axis during morphine withdrawal is mediated via a stimulatory noradrenergic pathway (12). Additionally, we reported that morphine withdrawal is associated with an increase in TH enzymatic activity in the PVN, a projection area of noradrenergic neurons arising from NTS-A2 (14). However, the identities of the TH serine residues that are phosphorylated during morphine withdrawal are not well characterized.
Changes in the state of phosphorylation of TH, the rate-limiting enzyme in the synthesis of catecholamine, are critically involved in the regulation of catecholamine synthesis and function. In particular, increases in the phosphorylation of Ser31 and Ser40 accelerate TH activity, thereby stimulating production of neurotransmitter in catecholamine terminals (for review, see Refs. 10 and 11). The first aim of the present study was to assess the changes in TH phosphorylation after morphine withdrawal in the PVN and brain stem catecholaminergic cell groups innervating the PVN. The results of this work provided evidence for TH phosphorylation during naloxone-induced morphine withdrawal in both catecholaminergic cell bodies in NTS and terminals innervating the PVN. Using phosphorylation state-specific antibodies directed toward Ser31 or Ser40, in the present study, we have shown that naloxone-induced morphine withdrawal greatly increased the level of TH phosphorylation at Ser31 in rat PVN, concomitantly with enhanced TH activity. We also found that morphine withdrawal increased levels of TH phosphorylated at Ser31 in the NTS (as assessed by Western blot and immunohistochemistry). Together, these data suggest that Ser31 phosphorylation of TH may be an important modulator of TH activity during naloxone-induced morphine withdrawal and might be directly involved with regulating NA turnover in morphine withdrawn rats. Our results show that phosphorylation of TH at Ser31 in the NTS 90 min after naloxone administration to morphine-dependent rats was above that observed at 60 min. Because phosphorylation of TH is closely associated with the activation of the enzyme, present data are in agreement with our previous findings showing that the increase in TH activity was maximal 90 min after naloxone-induced morphine withdrawal.
It is well known that phosphorylation of Ser40 results in a considerable increase in TH activity (11). Association of Ser40 phosphorylation with TH activity and catecholamine synthesis in vivo has been shown. Striatal Ser40 phosphorylation was increased concomitantly with enhanced TH activity and DOPA biosynthesis (36). However, TH activation has been shown to occur in absence of Ser40 phosphorylation (37). In the present work, similar number of TH Ser40-positive cells was seen in the NTS from control and morphine-dependent rats at the 60-min time point. Similar results were found at 90 min, although the number of cell bodies was more numerous. Additionally, no changes in pSer40 TH levels were found in the PVN from morphine-withdrawn rats. From these results one is tempted to suggest that Ser40 phosphorylation is unlikely to be of major importance in the activation of TH by morphine withdrawal, although it might play a role in maintaining activity under basal conditions. Present data are in agreement with recent findings suggesting that sustained phosphorylation of TH at Ser40 could be the mechanism for maintenance of catecholamine synthesis to replace those released in response to nerve stimulation (38). Caution should be taken, however, before reaching this conclusion. Thus, whereas it can be reasonably concluded that TH phosphorylation at Ser31 is required for the activation of TH during morphine withdrawal, the precise role of TH phosphorylation at Ser40 remains somewhat equivocal. In addition, from present results it remains to be determined what functional role might be associated with the higher number of TH Ser40 cell bodies at the 90-min time point than observed at 60 min. On the other hand, the immunohistochemical study show that morphine-treated rats have a lower number of pTH Ser40-positive neurons in the NTS 90 min after saline administration, compared with both control rats receiving saline and dependent rats receiving naloxone. Because the Western blot analysis showed no changes in pSer40 TH levels in this nucleus in morphine-saline-treated rats, it is possible that it reflects only a lower number of neurons but not a lower level of TH phosphorylated at Ser40 in this group of animals.
As previously mentioned, morphine withdrawal results in c-Fos expression in the NTS-A2 catecholaminergic cell group (13, 14, 23). The present study extended these findings by demonstrating that morphine withdrawal gives rise to an increase in c-Fos in the NTS-A2. Using dual immunolabeling for c-Fos and TH phosphorylated at Ser31, present data show that all the TH-positive neurons phosphorylated at Ser31 coexpress c-Fos. These results support the hypothesis that catecholaminergic pathways innervating the PVN are activated during morphine withdrawal.
Chronic opiate exposure induces numerous neurochemical adaptations in the noradrenergic system, including increased expression of TH (1, 2, 3, 39). Recent findings from our group have also shown the involvement of an up-regulated cAMP-dependent protein kinase A in activating TH levels and c-Fos expression in the NTS during morphine withdrawal (40). In addition, we have also shown that activated PKC may be critical for the activation of the catecholaminergic brain stem cell groups in response to opioid withdrawal (41). In the present study, it should be noted that TH protein expression was increased in NTS in morphine dependent rats, suggesting that the effect of morphine is mediated through both the activation (via phosphorylation) of TH and the up-regulation of its expression, which is in agreement with previous results from this laboratory (14). It is well known that the regulation of TH enzyme through number and activity represents the central means for controlling the synthesis of catecholamine. There is now evidence that TH protein and activity levels can be regulated by two different categories: short-term regulation of enzyme activity (e.g. enzymatic phosphorylation) and medium- to long-term regulation of gene expression (transcriptional regulation) (10, 11). One of the most consistent biochemical changes seen in response to chronic morphine exposure is up-regulation of TH (1, 39). In the locus coeruleus, chronic morphine administration has been shown to increase levels of TH immunoreactivity, which could be expected to increase the capacity of these neurons to synthesize NA (3). In addition, it has been proposed that drugs that perturb catecholaminergic function can induce changes in the TH mRNA and protein expression. Thus, morphine and cocaine increases TH immunoreactivity in dopaminergic brain reward regions.
An activator protein-1 sequence (the Fos/Jun binding site) has been identified in the TH gene (42). Because both cocaine and morphine induced the expression of Fos, it has been hypothesized that this transcription factor augment TH transcription by binding to the activator protein-1 site (10). Additionally, it has been proposed that glucocorticoids represent one of several different classes of hormones that regulate levels of TH mRNA. Thus, TH protein and mRNA were shown to be increased by glucocorticoids (43, 44). Moreover, a glucocorticoid regulatory element is postulated to exist in the TH gene (10). According to the present results showing an increase in ACTH and corticosterone release, previous findings from our laboratory have demonstrated that morphine withdrawal produces a marked increase in plasma corticosterone levels (17, 45).
Together, all these data might suggest that morphine withdrawal could increase TH immunoreactivity levels in the NTS through Fos expression and the increased corticosterone release, which agree with recent findings from this laboratory showing an increase in TH protein levels in the NTS-A2 (14). Similar results have also been shown in the locus ceruleus and ventral tegmental area, in which chronic morphine up-regulates TH immunoreactivity by a transcriptional mechanism (1, 39). Our results also show increased TH immunoreactivity in the NTS from morphine-dependent animals given saline instead naloxone. Because this treatment induced neither c-Fos expression nor corticosterone release, our findings might suggest that posttranscriptional mechanisms could be responsible for the enhanced TH levels, as has been proposed for other brain areas (39).
As previously mentioned, it is well established that acute activity of TH is regulated by protein phosphorylation. Four serine residues, namely Ser8, 19, 31, and 40, located near the N-terminal end of the protein, can be phosphorylated by a number of protein kinases. The only protein kinases reported to phosphorylate TH at Ser31 in vitro were ERK1/2 (25). Phosphorylation of TH at only Ser31 in situ increases TH activity and catecholamine synthesis (11). It is known that morphine dependence and withdrawal alters the levels and/or activity of various signaling elements. These chronic adaptive molecular mechanisms involve gene expression and/or some protein kinases, which are relevant for signaling processes involving protein phosphorylation (5, 34, 46, 47). ERKs are a family of serine/threonine protein kinases that have been functionally linked to addiction (46). ERK phosphorylates various substrates, including many enzymes, transcription factors, and proteins. Increases in ERK activation directly translate into downstream phosphorylation of cytosolic effector proteins that govern specific neuronal function. Given that TH is phosphorylated on a specific serine residue (Ser31) by the ERK, it is possible that activation of ERK1/2 provides a way in which TH is regulated under morphine dependence.
Recently we observed, by Western blot and immunohistochemistry, that naloxone-induced morphine withdrawal increases phosphorylated ERK1/2 in rat PVN and NTS neurons, indicating that this treatment increased ERKs activity (our unpublished results). In the present study, we therefore used the MEK inhibitor SL327 to check the involvement of ERK1/2 in the TH phosphorylation at Ser31 during morphine withdrawal. We found that treatment with 100 mg/kg (a dose that selectively blocks MEK) (26) decreased the morphine withdrawal stimulation of Ser31 phosphorylation in the PVN and abolished this stimulation in the NTS. These data suggest that morphine withdrawal induced an activation of ERKs, which results in enhanced Ser31 phosphorylation. Whereas pSer31 immunoreactivity returned to basal levels in the NTS, the MEK inhibitor did not reduce pSer31 immunoreactivity to baseline in the PVN. ERK activation, and then TH phosphorylation, is well documented to involve both a raf-MEK-dependent and a raf-MEK-independent pathway (25, 48). Thus, phorbol esters, which are activators of protein kinase C (PKC), elicit phosphorylation of TH at Ser31, suggesting that ERK can be activated in a protein kinase C-dependent manner (48, 49). Additionally, it has been shown that, although PKC does not directly phosphorylate Ser31, it does trigger modification of the site by other kinases (10). Given that Ser31 phosphorylation of TH in the PVN was not totally reversed by the MEK inhibitor, it is tempting to suggest that morphine withdrawal achieves this phosphorylation, at least in part, via a PKC-dependent activation of ERK1/2.
Consistent with previous results (12, 33), present data show that morphine withdrawal produced corticosterone and ACTH hypersecretion. Whereas data presented here clearly indicate that morphine withdrawal increases Ser31 phosphorylation and that this is probably mediated in part by a MEK activation of ERK1/2, MEK activation does not appear to be necessary for the withdrawal-induced increase in the secretor activity of the HPA axis. Thus, the increase in corticosterone and ACTH release in response to morphine withdrawal was not blocked by the MEK inhibitor SL327, leaving us to conclude that the secretor activity of the HPA axis after morphine withdrawal is most likely because of other signaling pathways. Previous reports from our laboratory indicate that inhibition of PKC significantly blocked the corticosterone secretion during morphine withdrawal (50). These findings are consistent with the observation that PKC is up-regulated in the PVN during morphine dependence and may suggest that PKC activity is necessary for the increased HPA axis activity during morphine withdrawal (41).
In conclusion, the present study suggests that morphine withdrawal might stimulate TH activity and accelerate NA turnover in the PVN via a mechanism involving activation of ERKs and phosphorylation of TH at Ser31 (Fig. 7), which agree with other findings showing that morphine increases ERK phosphorylation in the ventral tegmental area, which, in turn, contributes to drug-induced increases in TH expression (51). In addition, morphine withdrawal appears to stimulate the secretor activity of the HPA axis in a MEK-ERK-independent manner.
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Disclosure Statement: The authors have nothing to disclose.
First Published Online September 6, 2007
Abbreviations: AP, Area postrema; CRF, corticotropin-releasing factor; DAB, 3, 3'-diaminobenzidine; DMSO, dimethylsulfoxide; HPA, hypothalamic-pituitary-adrenocortical; MEK, ERK kinase; NA, noradrenaline; NSS-PBS, PBS containing goat serum and Triton X-100; NTS, nucleus tractus solitarius; p, phosphorylated; PKC, protein kinase C; PVN, paraventricular nucleus; Ser, serine; TBST, Tris HCl, NaCl, and Tween 20; TH, tyrosine hydroxylase; tTH, total TH.
Received April 20, 2007.
Accepted for publication August 28, 2007.
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