Ping Wu, Hai-Shui Shi, Yi-Xiao Luo, Ruo-Xi Zhang, Jia-Li Li, Jie Shi, Lin Lu

Tags: morphine withdrawal, J Pharmacol Exp Ther, Psychopharmacology, Ping Wu, Eur J Pharmacol, Hai-Shui Shi, Lu L, Koob GF, morphine dependence, Neuropeptides, withdrawal symptoms, withdrawal, Ma L, induced, Acta Pharmacol Sin, Eur J Neurosci, Lin Lu, mice, published article, Wei-Li Zhu, Jia-Li Li, Yi-Xiao Luo, morphine withdrawal symptoms, SpringerVerlag Berlin Heidelberg, Ruo-Xi Zhang, Jie Shi, transcription factor, Experimental procedure, drug treatment, personal copy, ACTH levels, naloxone, administration, learning and memory, glucocorticoid receptor, naloxone injection, Stinus L, locomotor activity, Maldonado R, opiate withdrawal, Body weight loss, Author, RU486, Alcohol Clin Exp Res
Content: Neuropeptide trefoil factor 3 attenuates naloxone-precipitated withdrawal in morphine-dependent mice Ping Wu, Hai-Shui Shi, Yi-Xiao Luo, Ruo-Xi Zhang, Jia-Li Li, Jie Shi, Lin Lu & Wei-Li Zhu Psychopharmacology ISSN 0033-3158 Volume 231 Number 24 Psychopharmacology (2014) 231:4659-4668 DOI 10.1007/s00213-014-3615-1 1 23
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Psychopharmacology (2014) 231:4659­4668 DOI 10.1007/s00213-014-3615-1 ORIGINAL INVESTIGATION
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Neuropeptide trefoil factor 3 attenuates naloxone-precipitated withdrawal in morphine-dependent mice Ping Wu & Hai-Shui Shi & Yi-Xiao Luo & Ruo-Xi Zhang & Jia-Li Li & Jie Shi & Lin Lu & Wei-Li Zhu
Received: 13 January 2014 / Accepted: 28 April 2014 / Published online: 14 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale The persistence of physical dependence and craving in addicts is considered to contribute to relapse. Increasing evidence indicates that neuropeptide systems are associated with several phases of drug addiction, but little is known about whether the neuropeptide trefoil factor affects withdrawal symptoms. Objectives This study aims to investigate the potential effects of the neuropeptide trefoil factor 3 (TFF3) on naloxoneprecipitated withdrawal symptoms in morphine-dependent mice. Results Mice received increasing doses of morphine over 3 days. On day 4, the mice were injected with TFF3 (1.0 mg/kg, i.p.) 30 min after the last dose of morphine. Thirty minutes after TFF3 treatment, naloxone (1 mg/kg, i.p.) was injected, and body weight, jumping behavior, wet-dog shakes, and locomotor activity were assessed 30 min later. Naloxone
caused significant weight loss and increased jumping behavior and wet-dog shakes in morphine-dependent mice. TFF3 (1.0 mg/kg) reversed these behavioral symptoms caused by morphine withdrawal, suggesting that TFF3 might ameliorate physical dependence associated with opiate addiction. Furthermore, TFF3 pretreatment significantly reduced morphine withdrawal-induced increases in plasma corticosterone and Adrenocorticotropic hormone levels. The glucocorticoid receptor agonist RU486 blocked the behavioral effects of TFF3 on morphine withdrawal symptoms. Finally, Fos expression in the medial prefrontal cortex which was decreased during morphine withdrawal was increased by TFF3 pretreatment. Conclusion These findings indicate that TFF3 might be a potential therapeutic candidate for opiate addiction by regulating glucocorticoid secretion and neuronal activation in the prefrontal cortex.
Ping Wu and Hai-Shui Shi equally contributed to this work. P. Wu : Y.National Institute on Drug dependence, Peking University, 38, Xue Yuan Road, Beijing 100191, China e-mail: [email protected] L. Lu Peking University Sixth Hospital/Institute of mental health, Peking University, Beijing 100191, China H.Biochemistry and Molecular Biology, College of Basic Medicine, Hebei Medical University, 361, Zhongshan East Road, Shijiazhuang 050017, China e-mail: [email protected] H.Keywords Addiction . Trefoil factor 3 . Morphine . Withdrawal . Corticosterone . Fos . c-fos Introduction Opiates are used clinically for their analgesic effects, but chronic exposure to opiates can lead to opiate misuse or addiction (Tang et al. 2006). Physical dependence and craving are considered the main causes of relapse in addicts after withdrawal. Physical dependence on morphine can be induced after acute withdrawal from repeated morphine administration (Heishman et al. 1989). Such physical dependence is clearly manifested by the presence of withdrawal symptoms induced by an injection the opioid receptor antagonist naloxone (Bickel et al. 1988). Naloxone-precipitated withdrawal in rodents is measured using a wide variety of assessments (e.g., jumping behavior, wet-dog shakes, diarrhea, tremors,
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and temperature changes). Significant increases in jumping behavior and wet-dog shakes and a decrease in body weight have been considered hallmarks of physical withdrawal after a naloxone injection in morphine-dependent mice (Lu et al. 2000c, 2005; Siegel et al. 1975). Neuropeptides are broadly distributed in the brain and considered the most common signaling molecules in the central nervous system (Merighi et al. 2011). Neuropeptides, such as neuropeptide Y, vasoactive intestinal polypeptide, neurotensin, galanin, opioid peptides, nociceptin, oxytocin, and angiotensin, have been shown to modulate various physiological processes, including drug addiction, learning and memory, stress reactivity, locomotor activity, anxiety, and depressive symptoms (Feany 1996; Huston and Hasenohrl 1995; Landgraf 2005; Merighi et al. 2011; Pan and Kastin 2008; Rotzinger et al. 2010). The neuropeptide trefoil factor 3 (TFF3), a major secretory product of mucin-producing cells, is produced in the gastro system but crosses the blood­brain barrier (Griepentrog et al. 2000). We previously found that acute systemic TFF3 administration (0.1 and 0.5 mg/kg, i.p.) promotes learning and enhances memory retention in a novel object recognition test in mice (Shi et al. 2012a). Considering the wide expression of TFF3 and the relatively few studies that have elucidated the functions of TFF3, the present study investigated the potential effect of TFF3 on opiate physical dependence using a model of naloxone-precipitated withdrawal in morphine-dependent mice. Many animal and human studies have shown that enhanced responsiveness of the hypothalamic­pituitary­ adrenal (HPA) axis, which induces an increase in corticosterone and adrenocorticotropic hormone (ACTH) secretion, has been associated with morphine withdrawal (Laorden et al. 2000, 2002). All drugs of abuse have the potential to induce the expression of immediate early genes (IEGs) in the brain, particularly c-fos, a marker of neural activation (Diaz et al. 2001; Kovacs 1998). Evidence indicates that morphine withdrawal increases Fos expression in several brain regions related to withdrawal symptoms, including the hippocampus, amygdala, and locus coeruleus (Gracy et al. 2001; Mannelli et al. 2004). Moreover, morphine-induced conditioned place preference (CPP) is associated with increased Fos expression in the prefrontal cortex and nucleus accumbens (Kaplan et al. 2003). Stress can induce drug seeking in animal models of relapse (Erb et al. 1996; Wang et al. 2001, 2006). Therefore, the purpose of the present study was to investigate whether TFF3 can reduce morphine withdrawal symptoms precipitated by naloxone in mice. Because stress and glucocorticoids play a critical role in the morphine withdrawal syndrome, another aim of the present study was to evaluate the possible involvement of glucocorticoid and Fos expression in the action of TFF3.
Materials and methods Animals Male ICR mice, weighing 18­22 g upon arrival, were obtained from the Peking University Experimental Animal Center. The mice were housed under a constant temperature (23± 2 °C) and humidity (50±5 %) and maintained on a 12-h/ 12-h light/dark cycle with free access to food and water. All of the procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the procedures were approved by the Local Animal Use Committee (LA2010-010). All of the behavioral tests were performed during the dark phase. Drugs administration and pretreatment time Morphine hydrochloride was obtained from Qinghai Pharmaceutical Co. Ltd (Xining, Qinghai, China). Naloxone hydrochloride and the glucocorticoid receptor agonist RU486 (mifepristone) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TFF3 was obtained from Yong Kang Jia Xin Biotechnology Co., Ltd (Beijing, China). Morphine, naloxone, and TFF3 were dissolved in sterile 0.9 % sodium chloride solution. RU486 was suspended in 0.9 % saline with Tween 80 (1 %v/v). The drug concentrations were adjusted to an appropriate injection volume of 10 ml/kg body weight. The dosage of RU486 was selected according to previous report (Mesripour et al. 2008), in which RU486 (50 mg/kg, s.c.) effectively inhibited glucocorticoid receptors and prevented memory deficit induced by morphine withdrawal in mice. Therefore, we used RU486 at the dosage of 50 mg/kg in the current study. The time interval of RU486 administration was decided according to the its pharmacokinetics data that the time to maximal serum concentration is 60 min and the half-life time is 34 h. The dosage of TFF3 was used based on our previous experiment that TFF3 at the dose of 1.0 mg/kg (i.p.) produced significant antidepressant-like effect (Shi et al. 2012b). The time interval of TFF3 administration was based on the pharmacokinetic parameter estimates of TFF3 in mice after s.c. administration with the time to maximal serum concentration of 30 min, the half-life time of 44 min, and the mean residence time of 76 min (Kjellev et al. 2007). To investigate the effects of TFF3 on morphine withdrawal, the mice were divided into eight groups. Four groups of mice were treated with morphine, and the other four groups were naive controls that were treated with an equal number of saline injections. In the "Results" section below, "saline" treatment refers to naive mice. TFF3 (1.0 mg/kg, i.p.) was administered 30 min before the naloxone injection in all eight groups. Changes in body weight, jumping behavior, wet-dog shakes, and locomotor activity were assessed 30 min after naloxone treatment.
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Induction of morphine withdrawal All of the mice were acclimated to the laboratory housing conditions for 7 days before the experiment and were randomly assigned to one of eight groups. Morphine dependence was induced according to previous reports (Farzin 1999; Zarrindast and Farzin 1996) with minor modifications. Briefly, the mice were injected (i.p.) with escalating morphine doses every 8 h (25, 25, 50, 50, 50, 75, 75, 75, and 100 mg/kg) during the first 3 days. The last morphine injection (100 mg/kg) was administered on day 4. The mice were then injected with naloxone (1 mg/kg, i.p.) or saline (i.p.), and withdrawal symptoms were monitored for 30 min immediately after the naloxone injections. Behavioral test Body weight was recorded immediately (BW1) and 30 min (BW2) after the naloxone injection and is expressed as body weight loss (BW2-BW1; (Gong et al. 2010). Jumping behavior was assessed by counting the number of jumps for 30 min immediately after the naloxone injection according to previous reports (Chen et al. 2011; Lu et al. 2000a, b). Each mouse was placed in a white opaque cylinder (32.0 cm heightЧ10.0 cm diameter) above a platform. The mouse remained in the cylinder for 30 min. One sensor under the platform monitored the vibration produced by jumping and transmitted the vibration signal to a computer to analyze the number of jumps. Additionally, wet-dog shakes were counted for 30 min. Wet dog shakes were defined as whole body shaking and were thought to be an adaptation of the body to an increase in set-point temperature in the central nervous system during withdrawal (Baumeister et al. 1992; Koob et al. 1992; Maldonado et al. 1992; Panchal et al. 2005). Moreover, to assess the possible effects of TFF3 on motor activity, the locomotor activity of another group of mice was detected immediately after naloxone injection (Chen et al. 2011). This experiment was performed using eight computerized animal locomotor monitoring cages. Each cage consisted of a Perspex cycloid box (12 cm heightЧ24 cm diameter) surrounded by three pairs of horizontal infrared beams arranged at 60° angles. Horizontal locomotion was recorded for 10 min as the number of light beam interruptions. Fos immunohistochemistry The immunohistochemical detection of Fos in brain tissue sections was performed using antiplasma (sc-52, Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to previous studies with minor modifications (Dong et al. 2005; Sundquist and Nisenbaum 2005). Thirty minutes after the naloxone injection, the mice, which were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) before perfusion, were perfused
with 4 % paraformaldehyde (PFA), and the brains were removed and postfixed for 24 h. The brains were then sectioned coronally with a microtome into 30 m thick sections. Every third serial section was collected on gelatin-coated microscope slides. All of the sections were placed in a freshly prepared methanol­H2O2 solution for 10 min to block endogenous peroxidase activity. After incubation with rabbit anti-Fos (Santa Cruz Biotechnology; 1:200 dilution in phosphatebuffered saline (PBS), 30 min, 37 °C), the tissue sections were washed three times in PBS followed by an additional 10 min incubation with biotin-conjugated second antibody and three washes with PBS. The sections were then incubated for 10 min in streptavidin­peroxidase and washed three times in PBS. The sections were then reacted with a 0.05 % solution of 3,3-diaminobenzidine (DAB; Beijing Zhongshanjinqiao Biological Technology Ltd, Beijing, China) and 0.01 % H2O2 in 0.1 M PBS. The incubation times varied from 3 to 10 min, depending on the expression levels of the DAB reaction product determined microscopically. The number of Fospositive cells in these brain regions was counted according to previous reports from our laboratory (Jiang et al. 2011, 2013), in which two or three sections from each brain region for each mouse were selected. The cell numbers on either side of the specific brain region were averaged and taken as the positive immunoreactive cell number for each mouse. The number of Fos-labeled cells was measured using a cast-grid microscope (MetaMorph/DP10/Bx41, UIC/Olympus, US/JP) with an Image Analysis program (MetaMorph, version 4.65). Two images were taken for each specimen under Ч 100 magnification. Serum corticosterone and ACTH measurement To determine serum corticosterone and ACTH levels in mice, 1 ml of blood was collected by decapitation bleeding. To avoid the acute stress of decapitation on the level of ACTH and corticosterone, we anesthetized mice with 50 mg/kg pentobarbital sodium (i.p.) before each decapitation. The blood samples were kept at room temperature for 1 h and then centrifuged at 3,000 rotations per minute for 10 min. The serum supernatant fraction was stored in another tube for the subsequent corticosterone and ACTH assays. Serum corticosterone and ACTH levels were measured using commercially available enzyme-linked immunosorbent assay kits (corticosterone ELISA, #2B870; ACTH ELISA, #2B350; Sun Biomedical Technology Co., Ltd, Beijing, China) according to the manufacturer's instructions. Because diurnal rhythms may induce fluctuations in hormone levels, blood samples were collected within the same time window (4:00­5:00 PM) for each mouse. The data are expressed as nanomole per liter for corticosterone and picogram per milliliter for ACTH.
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Statistical Analysis The data are expressed as mean ± SEM. The statistical analyses of the behavioral data, immunohistochemical data, and ELISA data were performed using two-way analysis of variance (ANOVA), followed by the Tukey post hoc test (for further details, see "Results" section). Values of p<0.05 were considered statistically significant. Results Acute TFF3 administration attenuated withdrawal symptoms in morphine-dependent mice The mice were treated with morphine for three consecutive days, and TFF3 or vehicle was administered 30 min after the last morphine injection on day 4. Thirty minutes later, saline or naloxone was injected in both the morphine and naive groups. Body weight, jumping behavior, and wet-dog shakes were detected immediately after the naloxone injection (Fig. 1a). The two-way ANOVA revealed that naloxone significantly decreased body weight (p<0.001; Fig. 1b) in morphine-dependent mice compared with saline treatment. In contrast, TFF3 pretreatment (1 mg/kg) reduced this body weight loss precipitated by naloxone (Fig. 1b). The two-way ANOVA of body weight loss revealed significant effects of naloxone (F1,32=70.05, p<0.001) and TFF3 (F1,32=35.08, p<0.001) and a significant naloxone Ч TFF3 interaction (F1,32=29.25, p<0.001). It would seem that the body weight loss was due to urination in 30 min. Although we did not measure the urination directly in the current study, previous evidence revealed that naltrexone challenge produced significant increases in urination and defecation in rats either chronically or acutely dependent on morphine (Kalinichev and Holtzman 2003). Therefore, we can explain that the body weight loss of mice in current investigation is due to the increase of urination and defecation. Naloxone-precipitated withdrawal also increased jumping behavior (p<0.01; Fig. 1c) and induced wet-dog shakes (p<0.01; Fig. 1d) in morphine-dependent mice compared with the saline control groups. TFF3 (1 mg/kg) reversed these withdrawal symptoms induced by naloxone. The two-way ANOVA revealed significant effects of naloxone (F1,32= 109.49, p<0.001) and TFF3 (F1,32=32.90, p<0.001) on jumping behavior and a significant naloxoneЧTFF3 interaction (F1,32=36.10, p<0.001). The two-way ANOVA also revealed significant effects of naloxone (F1,28=123.54, p<0.001) and TFF3 (F1,28=12.63, p<0.01) on wet-dog shakes and a significant naloxoneЧTFF3 interaction (F1,28=5.35, p<0.05). The dose of TFF3 used in the present study (1.0 mg/kg) was selected according to our pilot study, in which we used
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Fig. 1 Effects of TFF3 administration on naloxone-precipitated with- drawal in morphine-dependent mice. Naloxone (1 mg/kg, i.p.) was used to induce withdrawal after morphine treatment. TFF3 (1 mg/kg, i.p.) was injected 30 min before the naloxone injection. a Experimental procedure of Drug Treatment and behavioral measurement. b Body weight loss (n=9 per group); c jumping number (n=9 per group); and d wet-dog shakes (n=8 per group) during a 30-min period after the naloxone injection. The data are expressed as mean ± SEM. ***p<0.001, compared with salinetreated mice; ###p<0.001, compared with naloxone group. n=8­9 per group
several doses of TFF3. This dose of TFF3 was higher than the dose used in our previous study that investigated the effect of TFF3 on learning and memory. The blockade of naloxoneprecipitated withdrawal in morphine-dependent mice appears
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to require a higher TFF3 dose than the doses that affect learning and memory. Acute TFF3 administration had no effects on locomotor activity in the open field test A separate eight groups of mice were treated with morphine, TFF3, or naloxone as mentioned above and used to measure locomotor activity. No significant differences in locomotor activity were observed between morphine-dependent mice and morphine-naive mice. Additionally, pretreatment with TFF3 did not alter the distance traveled in the open field in morphine-dependent mice 30 min after the naloxone injection (p>0.05; Fig. 2). Acute TFF3 administration reduced morphine withdrawal-induced increases in plasma corticosterone and ACTH levels Morphine withdrawal can enhance the responsiveness of the HPA axis, reflected by increases in plasma corticosterone and ACTH secretion. To evaluate whether TFF3 reverses HPA axis hyperactivation during morphine withdrawal, we measured plasma corticosterone and ACTH levels in morphinedependent mice pretreated with TFF3 (1 mg/kg) 30 min before the naloxone injection (Fig. 3a). The levels of corticosterone (p<0.01) and ACTH (p<0.01) in plasma increased after naloxone treatment compared with saline-treated mice (Fig. 3b, c). TFF3 (1 mg/kg) significantly decreased both corticosterone and ACTH levels induced by naloxone (Fig. 3b, c). The two-way ANOVA revealed significant effects
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Fig. 2 Effect of TFF3 administration on locomotor activity in morphinedependent mice. TFF3 (1 mg/kg) was injected 30 min before naloxone (1 mg/kg, i.p.). Locomotion was recorded for 10 min in the open field test 30 min after the naloxone injection. The data are expressed as mean ± SEM. n=8 per group
of naloxone (F1,20=87.97, p<0.001) and TFF3 (F1,20=11.82, p<0.01) on corticosterone secretion and a significant naloxoneЧTFF3 interaction (F1,20=10.67, p<0.01). The statistical analysis also showed significant effects of naloxone (F1,20= 180.23, p<0.001) and TFF3 (F1,20=18.84, p<0.001) on plasma ACTH levels and a significant naloxoneЧTFF3 interaction (F1,20=24.56, p<0.001). Glucocorticoid receptor activation blocked the behavioral effects of TFF3 on withdrawal symptoms induced by naloxone in morphine-dependent mice TFF3 reduced naloxone-induced withdrawal symptoms and inhibited the production of corticosterone and ACTH, and we further investigated whether HPA function is involved in the behavioral effects of TFF3. In this experiment, the glucocorticoid receptor agonist RU486 (50 mg/kg, s.c.) was injected 30 min before naloxone treatment in morphine-dependent mice. Body weight, jumping behavior, and wet-dog shakes were then recorded (Fig. 4a). TFF3 significantly decreased body weight loss induced by naloxone in morphine-dependent mice (F1,28=7.26, p<0.05; Fig. 4b). Pretreatment with RU486 reversed the effects of TFF3 (F1,28=4.97, p<0.05). TFF3 also significantly reduced jumping behavior (F1,28 = 11.40, p<0.01) and wet-dog shakes (F1,28=8.67, p<0.01), and RU486 blocked the effects of TFF3 on both jumping behavior (F1,28=4.81, p<0.05; Fig. 4c) and wet-dog shakes (F1,28= 8.13, p<0.01; Fig. 4d). These results suggest that HPA hyperactivity might be associated with the onset of morphine withdrawal symptoms and effects of TFF3 in morphine-dependent mice. Acute TFF3 administration reduced the decrease in Fos expression in the mPFC induced by morphine withdrawal We compared Fos-positive cells in morphine-dependent mice. The statistical analysis included two factors (TFF/vehicleЧ naloxone/saline). Morphine withdrawal decreased the number of Fos-positive cells compared with the saline-treated group. Pretreatment with TFF3 (1 mg/kg) significantly increased the number of Fos-positive cells in the mPFC in morphinewithdrawn mice treated with vehicle (p<0.01; Fig. 5a, b). The two-way ANOVA revealed significant effects of naloxone (F1,20=158.81, p<0.001) and TFF3 (F1,20=14.77, p<0.01) on Fos-positive cells in the mPFC and a significant naloxoneЧTFF3 interaction (F1,20=17.30, p<0.001). Fos protein is a marker of neuronal activation. These results indicate that neurons in the mPFC were inactivated by naloxoneprecipitated morphine withdrawal and suggest that neuronal activity in the mPFC is involved in the inhibitory effect of TFF3 on the development of morphine withdrawal symptoms.
4664 Fig. 3 Effects of TFF3 administration on plasma corticosterone and ACTH levels evoked by naloxone in morphinedependent mice. a Experimental procedure of drug treatment and sample collection. Naloxone (1 mg/kg, i.p.) increased plasma b corticosterone and c ACTH concentrations. TFF3 (1.0 mg/kg) reduced the increase in blood corticosterone and ACTH levels. The data are expressed as mean ± SEM. ***p<0.001, compared with saline-treated mice; ##p<0.01, compared with naloxone group. n=6 per group
Corticosterone (nmol/L) ACTH (pg/mL)
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Discussion The present study demonstrated significant effects of TFF3 on naloxone-precipitated morphine withdrawal symptoms. The results showed that naloxone significantly decreased body weight and induced jumping behavior and wet-dog shakes in morphine-dependent mice. Systemic TFF3 administration significantly attenuated the abnormal changes in these behaviors caused by naloxone-precipitated morphine withdrawal, indicating an important effect of TFF3 on the morphine withdrawal syndrome. TFF3 administration did not elicit any alterations in spontaneous locomotor activity, thus excluding the possible sedative action of TFF3. Previous results from our laboratory showed that TFF3 can cross the blood­brain barrier and enter the brain 30 min after systemic treatment (Shi et al. 2012b), suggesting that the effects of TFF3 on the morphine withdrawal syndrome observed in the present study are attributable to central rather than peripheral actions. Increases in serum corticosterone levels induced by naloxone-precipitated withdrawal are a useful parameter of physical dependence on morphine in mice (Ueno et al. 2011). Consistent with previous reports, the present data showed that naloxone administration in morphine-dependent mice significantly elevated plasma corticosterone and ACTH levels, reflecting HPA axis activation. TFF3 pretreatment attenuated the secretion of corticosterone and ACTH induced by naloxone, suggesting that the reductions of withdrawal symptoms were associated with decreased responsiveness of the HPA axis after morphine withdrawal. Corticosterone and ACTH act mainly through glucocorticoid receptors that are highly localized in the mPFC, a critical region related to emotional, cognitive, and neuroendocrine processing. We evaluated whether glucocorticoid receptor blockade is involved in the
reversal effects of TFF3 on morphine withdrawal symptoms. The glucocorticoid receptor agonist RU486 blocked the behavioral effects of TFF3 on withdrawal symptoms induced by naloxone in morphine-dependent mice, suggesting that HPA hyperactivity may be associated with the onset of withdrawal symptoms and effects of TFF3 in morphine-dependent mice. Stress-exposed rodents exhibit alterations in the activity, transcriptional state, and morphological profile of neurons in the PFC, reflected by reduced expression of the IEG c-fos mRNA (Covington et al. 2010). Naloxone-precipitated withdrawal is a stressful event in morphine-dependent mice, with enhanced responsiveness of the HPA axis measured by increased plasma corticosterone and ACTH levels. Neuroadaptations in the PFC, reflected by IEG expression in rodents and neuroimaging studies in humans, have been linked to changes in the negative motivational component of opiate withdrawal and reward-based decision making (Li et al. 2009). The present results indicate that naloxone-precipitated morphine withdrawal decreased Fos expression in PFC neurons, which was reversed by acute TFF3 administration, suggesting that Fos expression in prefrontal cortical regions may be critically involved in coping with adverse effects induced by naloxone. Our results support the hypothesis that neuronal activity in the mPFC modulates morphine withdrawal. Although we did not detect the effects of RU486 on c-Fos expression increased by TFF3 in withdrawal groups directly, the previous evidence that RU486 altered supraoptic neuron Fos expression in late pregnancy (Antonijevic et al. 2000) and the data of present study support us to propose the possibility that RU486 could reverse the effects of TFF3 on withdrawalinduced changes in c-Fos expression in the mPFC. The frontal cortex consists of a heterogeneous collection of neurons, including excitatory pyramidal neurons and local -
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Fig. 4 Effects of glucocorticoid receptor agonist RU486 on withdrawal symptoms in TFF3treated morphine-dependent mice. a Experimental procedure of drug treatment and behavioral measurement. b Body weight loss; c jumping number; d and wet-dog shakes during a 30-min period after the naloxone injection. The data are expressed as mean ± SEM. *p<0.05, **p<0.01, compared with salinetreated mice; #p<0.05, ##p<0.01, compared with naloxone group. n=9 per group
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aminobutyric acid interneurons (Steketee 2005). The mPFC provides glutamatergic innervation to the ventral tegmental area (VTA) and nucleus accumbens. The decrease in the inhibitory modulation of excitatory transmission is thought to underlie the decreased inhibitory modulation of pyramidal projection neurons during early withdrawal from repeated drug exposure. Further studies are needed to directly assess the role of particular prefrontal cortical neurons and transmission from the mPFC to the VTA and nucleus accumbens in the reduction of morphine withdrawal induced by TFF3 administration. Neuropeptides, the most common signaling molecules in the central nervous system, act as neurotransmitters,
hormones, and neuromodulators and have been shown to participate in various physiological and pathologic processes, such as sleep, emotion, addiction, depression, anxiety, learning, and memory (Kirsz and Zieba 2011; Ogren et al. 2010; Portelli et al. 2012). Growing evidence suggests that neuropeptides regulate the withdrawal syndromes associated with several drugs, including morphine, alcohol, and cocaine (Leggio et al. 2011; Logrip et al. 2011; Picciotto 2010). Despite the absence of an identified cell surface receptor for TFF3, clear evidence indicates that TFF3 rapidly activates several signal transduction pathways (Baus-Loncar and Giraud 2005). TFF-triggered signaling cascades mainly include extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-
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naloxone + vehicle
naloxone + TFF3
level of phosphorylated-Akt (p-Akt) in the BLA and exerted antidepressant-like effects (Shi et al. 2012b). The previous finding of our lab revealed that TFF3 increased the expression level of phosphorylated-Akt (p-Akt) and that phosphatidylinositol 3-kinase (PI3K) inhibitor significantly blocked the antidepressant behavioral effects of TFF3, suggesting that PI3K/Akt pathway might be involved in the pharmacological effects of TFF3. Furthermore, activation of glucocorticoid receptor protects mice against ischemia/ reperfusion induced renal injury by suppressing PI3K/AKT signaling (Zhang et al. 2013). Therefore, it is possible that glucocorticoid receptor is the downstream target of TFF3 through PI3K/Akt pathway in morphine withdrawal. More research is necessary to determine exactly how TFF3 modulates naloxone-precipitated morphine withdrawal symptoms by regulating the HPA axis and neuronal activation and elucidate the direct receptors and downregulated signaling molecules that mediate the action of TFF3 in the central nervous system. Answers to these questions will provide a better understanding of how TFF3 modulates withdrawal syndromes and critical insights into the regulation of opiate addiction. In summary, we found that TFF3 significantly attenuated the withdrawal syndrome induced by naloxone in morphinedependent mice by downregulating HPA axis activity and increasing neuronal activation in the mPFC. These findings indicate that TFF3 exerts regulatory effects in drug abuse, providing a potential therapeutic candidate for opiate addiction.
Fig. 5 Effects of TFF3 administration on Fos expression in morphinedependent mice. a Naloxone (1 mg/kg, i.p.) decreased Fos expression in the mPFC. TFF3 (1.0 mg/kg) attenuated the reduction of Fos immunoreactivity in vehicle-treated mice. The data are expressed as mean ± SEM. ***p<0.001, compared with saline mice; ##p<0.01, compared with naloxone group. n=6 per group. b Representative images show Fospositive neuron expression in the mPFC. Scale bar=20 m Jun-N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K), and signal transducer and activator of transcription 3 (STAT3), which are associated with central nervous systemrelated behaviors (Chiang et al. 2010; Koo et al. 2010; Li et al. 2010; Ramin et al. 2010). Morphine withdrawal is a complicated process that requires the orchestration of numerous cellular events, including ERK, protein kinase A, and protein kinase C signaling. A previous report from our laboratory revealed that a systemic injection of TFF3 significantly increased Fos expression in the basolateral amygdale (BLA), mPFC, and hypothalamus in rats (Shi et al. 2012b). We also found that acute systemic TFF3 administration increased the
Acknowledgments This work was supported in part by the National Natural Science Foundation of China (no. 81201038, 31371140, 81201032, and 81371489), Beijing Higher Education Young Elite Teacher Project (YETP0068), Natural Science Foundation of Hebei Province (no. H2013206010), Youth Foundation of Science and Technology Studies of Hebei Province (no. Q2012078), Special Foundation from Ministry of Education of China Specialized Research Foundation for Doctoral Program of College from Ministry of National Education (no. 20121323120002), and National Science and Technology Major Project (no. 2012ZX09103-301-049, 2013ZX09103-003-012). Conflict of interest The authors declare that they do not have any conflicts of interest (financial or otherwise) related to the data presented in this manuscript. References Antonijevic IA, Russell JA, Bicknell RJ, Leng G, Douglas AJ (2000) Effect of progesterone on the activation of neurones of the supraoptic nucleus during parturition. J Reprod Fertil 120:367­376 Baumeister AA, Richard AL, Richmond-Landeche L, Hurry MJ, Waguespack AM (1992) Further studies of the role of opioid receptors in the nigra in the morphine withdrawal syndrome. Neuropharmacology 31:835­841 Baus-Loncar M, Giraud AS (2005) Multiple regulatory pathways for trefoil factor (TFF) genes. Cell Mol Life Sci 62:2921­2931
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