Psychopharmacology DOI 10.1007/s00213-014-3785-x
ORIGINAL INVESTIGATION
Striatal dopamine D1 and D2 receptors are differentially regulated following buprenorphine or methadone treatment Stéphane Allouche & Thierry Le Marec & Antoine Coquerel & Florence Noble & Nicolas Marie
Received: 23 June 2014 / Accepted: 20 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale Chronic administration of morphine induces adaptations in neurotransmission system such as the dopamine pathway, and these modifications could be influenced by the drug administration pattern. Methadone and buprenorphine are the two main opioid substitution therapies, and despite their protracted use in humans, no study has investigated their ability to regulate dopamine system after chronic exposure/ withdrawal.
Electronic supplementary material The online version of this article (doi:10.1007/s00213-014-3785-x) contains supplementary material, which is available to authorized users. S. Allouche : T. Le Marec : F. Noble : N. Marie Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8206, Paris, France S. Allouche : T. Le Marec : F. Noble : N. Marie Institut national de la santé et de la recherche médicale, U 705, Paris, France S. Allouche : T. Le Marec : F. Noble : N. Marie Université Paris Descartes, Laboratoire de Neuropsychopharmacologie des Addictions, Institut Médicament-Toxicologie-Chimie Environnement, Paris, France A. Coquerel Service de pharmacologie, CHU de Caen, France A. Coquerel UMR « COMETE », Université de Caen Basse-Normandie, Institut national de la santé et de la recherche médicale U 1075, 14032 Caen, France F. Noble (*) Laboratoire de neuropsychopharmacologie des addictions, CNRS UMR 8206 - INSERM U 705, Faculté de Pharmacie, 4, avenue de l’Observatoire, 75006 Paris, France e-mail: [email protected]
Objectives We evaluated the consequences of two administration patterns of methadone and buprenorphine on striatal dopamine D1 (D1R) and D2 (D2R) receptor levels. Methods Mice were treated with escalating doses of methadone or buprenorphine for 5 days either once daily (binge) or three times a day (TTD). D1R and D2R density in striatum was measured by autoradiography using [3H]-SCH23390 and [3H]-raclopride, respectively, at 1 (WD1), 14 (WD14), and 35 (WD35)days after the last opioid injection. Results A downregulation of D1R was observed upon TTD administration of buprenorphine and binge methadone treatment while an increase of those receptor levels was detected both with binge buprenorphine and TTD methadone treatments. Concerning the D2R, we rather measured an early or late downregulation with both agonists and administration patterns. Conclusions Our results demonstrated that methadone and buprenorphine were able to differentially regulate dopamine receptor density depending on the withdrawal period and the administration pattern. Keywords Methadone . Buprenorphine . Dopamine receptors . Striatum . Drug exposure pattern
Introduction Opioid abuse, largely due to heroin injection or misuse of opioid analgesics, is a substantial health problem. The most effective pharmacotherapies for treating opioid addiction are based on buprenorphine or methadone administration (Gerra et al. 2009). These compounds share some similarities with morphine as they are high-affinity mu opioid receptor (MOR) agonists (Huang et al. 2001; Raynor et al. 1994) with a partial agonist activity for buprenorphine (Huang et al. 2001; McPherson et al. 2010; Selley et al. 1997). Consistently, the
Psychopharmacology
long-term use of methadone and buprenorphine could also promote behavioral adaptations and neuroplastic changes in brain regions involved in drug addiction. We recently showed that chronic treatment with buprenorphine and methadone could promote long-lasting behavioral sensitization (until 35 days after withdrawal) depending on many factors including the pattern of administration. Thus, the one daily injection treatment was more effective than the three times daily injection regimen to promote locomotor sensitization (Allouche et al. 2013). Among the neurotransmission systems altered by chronic opioid administration, the dopaminergic system is of particular importance. Indeed dopamine projections from the mesencephalon (ventral tegmental area/substantia nigra) to striatum (ventral and dorsal) play a major role in the behavioral effects of drug abuse (Di Chiara 1999; Ting and van der Kooy 2012; Wise 1980). In striatum, dopamine is essentially acting through dopamine D1 (D1R) and dopamine D2 (D2R) receptors (Beaulieu and Gainetdinov 2011) which are strongly involved in the behavioral adaptations usually observed following chronic administration of drugs of abuse (Baik 2013), including relapse to drug seeking (Bossert et al. 2007; Shaham and Stewart 1996) or locomotor sensitization (Jeziorski and White 1995; Serrano et al. 2002; Yoo et al. 2006). Few studies report dopamine receptor regulation following long-lasting opioid treatments (Le Marec et al. 2011; May et al. 1998), and this is all the more true when considering opioid substitution therapies. Since these medications could be prescribed for months and even for years, it seems crucial to evaluate a putative differential impact of such treatments on the dopaminergic system after prolonged exposure and withdrawal. So, in the present study, we investigated the effect of methadone or buprenorphine administrations on D1 and D2 receptor expression level measured by autoradiography in the striatum after 1, 14, or 35 days of withdrawal. Mice received escalating doses of opioids either once (binge) or three times (TTD) daily for 5 days.
the faculty. Every effort was made to reduce the number of animals used and their discomfort. Drug treatments Methadone hydrochloride and buprenorphine hydrochloride (Francopia, France) were dissolved in saline (0.9 % (w:v) NaCl) and injected via intraperitoneal (i.p.) route. All animals received 0.1 ml/10 g of bodyweight. Administration patterns and rational choice of doses were described in a previous study (Allouche et al. 2013). The three times a day (TTD) treatment consisted of three daily injections of escalating doses of methadone (1.25, 2.5, 5, 7.5, and 10 mg/kg) or buprenorphine (0.05, 0.1, 0.2, 0.3, and 0.4 mg/kg) for 5 days at 09:00, 13:00, and 17:00 hours. The binge treatment consisted of one daily injection of escalating doses of methadone (3.75, 7.5, 15, 22.5, and 30 mg/kg) or buprenorphine (0.15, 0.3, 0.6, 0.9, and 1.2 mg/kg) for 5 days at 09:00 hours (Allouche et al. 2013). Control groups were treated with saline under the same conditions. Immediately after injection, mice were returned to their home cages. Mice were maintained in drug-free conditions during 1, 14, or 35 days after the last opioid agonist injection. Receptor autoradiography The procedure of dopamine receptor study was the same as described previously (Le Marec et al. 2011). Preparation of brain sections After 1, 14, or 35 days of withdrawal, mice were sacrificed by CO2 inhalation then brains were quickly removed, frozen in isopentane at −50 °C, and immediately stored at −20 °C until processing. Coronal sections of 20 μm thickness were cut in a cryostat (Jung frigocut 2800N, Leica, Germany) at −20 °C, mounted on Superfrost Plus slides (Menzel Gläser, Germany), and stored at −20 °C until used. Receptor binding
Materials and methods Animals Male OF1 mice (Charles River, France) weighing 23–25 g at the beginning of the experiments were housed eight per cage, on a 12 h light/dark cycle in a temperature-controlled room (21±2 °C) with food and water available ad libitum. Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) as well as French law, with the standard ethical guidelines and under the control of the ethical committee of
For D1 receptors, brain sections were preincubated in 50 mM Tris–HCl buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 100 nM ketanserin (for blocking 5-HT2 receptors) (Sigma-Aldrich, France) for 30 min at 25 °C and then incubated in the same buffer containing 1 nM [3H]-SCH23390 (Perkin Elmer, France) in the presence (non-specific binding) or in the absence (total binding) of 10 μM SKF-38393 (Sigma-Aldrich, France) for 60 min at 25 °C (Tien et al. 2003). For D2 receptors, sections were preincubated in 50 mM Tris–HCl buffer (pH 7.4) supplemented with 120 mM NaCl and 5 mM KCl for 30 min at 25 °C and then incubated in the
Psychopharmacology
Striatum was identified using the mouse brain atlas of Franklin and Paxinos (Franklin and Paxinos 1997). Sections were placed in a Beta imager (Biospace Lab, France) which allows an access to the absolute activity of the samples, with sensitive, rapid, and wide dose–response linearity (Charpak et al. 1989) (Supplemental figure 1). Radioactivity was quantified with the Beta imager software M3 Vision (Biospace Lab, France). Specific binding was determined by subtracting non-specific binding from total binding in corresponding brain areas. Data are expressed as the mean±SEM of specific binding (in cpm/mm2).
D1R 8
[3H]-SCH-23390 specific binding (cpm/mm2)
Radioactivity quantification
a
* 6 4 2 0
WD14
WD35
Withdrawal days
D2R
2.5 2.0 1.5
*** ***
1.0 0.5 0.0
Statistical analysis
WD1
b [3H]-Raclopride specific binding (cpm/mm2)
same buffer containing 2 nM [3H]-raclopride (Perkin Elmer, France) in the absence (total binding) or in the presence (nonspecific binding) of 10 μM 2-bromo-alpha-ergocryptine (Sigma-Aldrich, France) for 60 min at 25 °C (Tien et al. 2003). Following incubation with radioligands, sections were quickly washed (2×1 min) in preincubation buffers at 4 °C and rinsed in distilled water at 4 °C for 2 s.
WD1
WD14
WD35
Withdrawal days
Data were analyzed using a two-way analysis of variance (ANOVA) (treatment and withdrawal days, WD) followed by a Bonferroni post hoc test for multiple comparisons (GraphPad Prism 5.04, GraphPad software).
Results Effects of methadone treatments on striatal dopamine receptor density In a first set of experiments, we quantified dopamine receptors in striatum of mice treated with binge or TTD methadone treatment. Following the binge methadone treatment, D1R density was downregulated regardless of withdrawal time (Fig. 1a: Ftreatment (1,22) =6.613, p=0.017; Fwithdrawal days (2,22) =2.664, p=0.092; Fwithdrawal days × treatment (2,22) =0.413, p=0.666) whereas changes in D2R density was observed (Fig. 1b: Ftreatment (1,22) =59.11, p
ORIGINAL INVESTIGATION
Striatal dopamine D1 and D2 receptors are differentially regulated following buprenorphine or methadone treatment Stéphane Allouche & Thierry Le Marec & Antoine Coquerel & Florence Noble & Nicolas Marie
Received: 23 June 2014 / Accepted: 20 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale Chronic administration of morphine induces adaptations in neurotransmission system such as the dopamine pathway, and these modifications could be influenced by the drug administration pattern. Methadone and buprenorphine are the two main opioid substitution therapies, and despite their protracted use in humans, no study has investigated their ability to regulate dopamine system after chronic exposure/ withdrawal.
Electronic supplementary material The online version of this article (doi:10.1007/s00213-014-3785-x) contains supplementary material, which is available to authorized users. S. Allouche : T. Le Marec : F. Noble : N. Marie Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8206, Paris, France S. Allouche : T. Le Marec : F. Noble : N. Marie Institut national de la santé et de la recherche médicale, U 705, Paris, France S. Allouche : T. Le Marec : F. Noble : N. Marie Université Paris Descartes, Laboratoire de Neuropsychopharmacologie des Addictions, Institut Médicament-Toxicologie-Chimie Environnement, Paris, France A. Coquerel Service de pharmacologie, CHU de Caen, France A. Coquerel UMR « COMETE », Université de Caen Basse-Normandie, Institut national de la santé et de la recherche médicale U 1075, 14032 Caen, France F. Noble (*) Laboratoire de neuropsychopharmacologie des addictions, CNRS UMR 8206 - INSERM U 705, Faculté de Pharmacie, 4, avenue de l’Observatoire, 75006 Paris, France e-mail: [email protected]
Objectives We evaluated the consequences of two administration patterns of methadone and buprenorphine on striatal dopamine D1 (D1R) and D2 (D2R) receptor levels. Methods Mice were treated with escalating doses of methadone or buprenorphine for 5 days either once daily (binge) or three times a day (TTD). D1R and D2R density in striatum was measured by autoradiography using [3H]-SCH23390 and [3H]-raclopride, respectively, at 1 (WD1), 14 (WD14), and 35 (WD35)days after the last opioid injection. Results A downregulation of D1R was observed upon TTD administration of buprenorphine and binge methadone treatment while an increase of those receptor levels was detected both with binge buprenorphine and TTD methadone treatments. Concerning the D2R, we rather measured an early or late downregulation with both agonists and administration patterns. Conclusions Our results demonstrated that methadone and buprenorphine were able to differentially regulate dopamine receptor density depending on the withdrawal period and the administration pattern. Keywords Methadone . Buprenorphine . Dopamine receptors . Striatum . Drug exposure pattern
Introduction Opioid abuse, largely due to heroin injection or misuse of opioid analgesics, is a substantial health problem. The most effective pharmacotherapies for treating opioid addiction are based on buprenorphine or methadone administration (Gerra et al. 2009). These compounds share some similarities with morphine as they are high-affinity mu opioid receptor (MOR) agonists (Huang et al. 2001; Raynor et al. 1994) with a partial agonist activity for buprenorphine (Huang et al. 2001; McPherson et al. 2010; Selley et al. 1997). Consistently, the
Psychopharmacology
long-term use of methadone and buprenorphine could also promote behavioral adaptations and neuroplastic changes in brain regions involved in drug addiction. We recently showed that chronic treatment with buprenorphine and methadone could promote long-lasting behavioral sensitization (until 35 days after withdrawal) depending on many factors including the pattern of administration. Thus, the one daily injection treatment was more effective than the three times daily injection regimen to promote locomotor sensitization (Allouche et al. 2013). Among the neurotransmission systems altered by chronic opioid administration, the dopaminergic system is of particular importance. Indeed dopamine projections from the mesencephalon (ventral tegmental area/substantia nigra) to striatum (ventral and dorsal) play a major role in the behavioral effects of drug abuse (Di Chiara 1999; Ting and van der Kooy 2012; Wise 1980). In striatum, dopamine is essentially acting through dopamine D1 (D1R) and dopamine D2 (D2R) receptors (Beaulieu and Gainetdinov 2011) which are strongly involved in the behavioral adaptations usually observed following chronic administration of drugs of abuse (Baik 2013), including relapse to drug seeking (Bossert et al. 2007; Shaham and Stewart 1996) or locomotor sensitization (Jeziorski and White 1995; Serrano et al. 2002; Yoo et al. 2006). Few studies report dopamine receptor regulation following long-lasting opioid treatments (Le Marec et al. 2011; May et al. 1998), and this is all the more true when considering opioid substitution therapies. Since these medications could be prescribed for months and even for years, it seems crucial to evaluate a putative differential impact of such treatments on the dopaminergic system after prolonged exposure and withdrawal. So, in the present study, we investigated the effect of methadone or buprenorphine administrations on D1 and D2 receptor expression level measured by autoradiography in the striatum after 1, 14, or 35 days of withdrawal. Mice received escalating doses of opioids either once (binge) or three times (TTD) daily for 5 days.
the faculty. Every effort was made to reduce the number of animals used and their discomfort. Drug treatments Methadone hydrochloride and buprenorphine hydrochloride (Francopia, France) were dissolved in saline (0.9 % (w:v) NaCl) and injected via intraperitoneal (i.p.) route. All animals received 0.1 ml/10 g of bodyweight. Administration patterns and rational choice of doses were described in a previous study (Allouche et al. 2013). The three times a day (TTD) treatment consisted of three daily injections of escalating doses of methadone (1.25, 2.5, 5, 7.5, and 10 mg/kg) or buprenorphine (0.05, 0.1, 0.2, 0.3, and 0.4 mg/kg) for 5 days at 09:00, 13:00, and 17:00 hours. The binge treatment consisted of one daily injection of escalating doses of methadone (3.75, 7.5, 15, 22.5, and 30 mg/kg) or buprenorphine (0.15, 0.3, 0.6, 0.9, and 1.2 mg/kg) for 5 days at 09:00 hours (Allouche et al. 2013). Control groups were treated with saline under the same conditions. Immediately after injection, mice were returned to their home cages. Mice were maintained in drug-free conditions during 1, 14, or 35 days after the last opioid agonist injection. Receptor autoradiography The procedure of dopamine receptor study was the same as described previously (Le Marec et al. 2011). Preparation of brain sections After 1, 14, or 35 days of withdrawal, mice were sacrificed by CO2 inhalation then brains were quickly removed, frozen in isopentane at −50 °C, and immediately stored at −20 °C until processing. Coronal sections of 20 μm thickness were cut in a cryostat (Jung frigocut 2800N, Leica, Germany) at −20 °C, mounted on Superfrost Plus slides (Menzel Gläser, Germany), and stored at −20 °C until used. Receptor binding
Materials and methods Animals Male OF1 mice (Charles River, France) weighing 23–25 g at the beginning of the experiments were housed eight per cage, on a 12 h light/dark cycle in a temperature-controlled room (21±2 °C) with food and water available ad libitum. Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) as well as French law, with the standard ethical guidelines and under the control of the ethical committee of
For D1 receptors, brain sections were preincubated in 50 mM Tris–HCl buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 100 nM ketanserin (for blocking 5-HT2 receptors) (Sigma-Aldrich, France) for 30 min at 25 °C and then incubated in the same buffer containing 1 nM [3H]-SCH23390 (Perkin Elmer, France) in the presence (non-specific binding) or in the absence (total binding) of 10 μM SKF-38393 (Sigma-Aldrich, France) for 60 min at 25 °C (Tien et al. 2003). For D2 receptors, sections were preincubated in 50 mM Tris–HCl buffer (pH 7.4) supplemented with 120 mM NaCl and 5 mM KCl for 30 min at 25 °C and then incubated in the
Psychopharmacology
Striatum was identified using the mouse brain atlas of Franklin and Paxinos (Franklin and Paxinos 1997). Sections were placed in a Beta imager (Biospace Lab, France) which allows an access to the absolute activity of the samples, with sensitive, rapid, and wide dose–response linearity (Charpak et al. 1989) (Supplemental figure 1). Radioactivity was quantified with the Beta imager software M3 Vision (Biospace Lab, France). Specific binding was determined by subtracting non-specific binding from total binding in corresponding brain areas. Data are expressed as the mean±SEM of specific binding (in cpm/mm2).
D1R 8
[3H]-SCH-23390 specific binding (cpm/mm2)
Radioactivity quantification
a
* 6 4 2 0
WD14
WD35
Withdrawal days
D2R
2.5 2.0 1.5
*** ***
1.0 0.5 0.0
Statistical analysis
WD1
b [3H]-Raclopride specific binding (cpm/mm2)
same buffer containing 2 nM [3H]-raclopride (Perkin Elmer, France) in the absence (total binding) or in the presence (nonspecific binding) of 10 μM 2-bromo-alpha-ergocryptine (Sigma-Aldrich, France) for 60 min at 25 °C (Tien et al. 2003). Following incubation with radioligands, sections were quickly washed (2×1 min) in preincubation buffers at 4 °C and rinsed in distilled water at 4 °C for 2 s.
WD1
WD14
WD35
Withdrawal days
Data were analyzed using a two-way analysis of variance (ANOVA) (treatment and withdrawal days, WD) followed by a Bonferroni post hoc test for multiple comparisons (GraphPad Prism 5.04, GraphPad software).
Results Effects of methadone treatments on striatal dopamine receptor density In a first set of experiments, we quantified dopamine receptors in striatum of mice treated with binge or TTD methadone treatment. Following the binge methadone treatment, D1R density was downregulated regardless of withdrawal time (Fig. 1a: Ftreatment (1,22) =6.613, p=0.017; Fwithdrawal days (2,22) =2.664, p=0.092; Fwithdrawal days × treatment (2,22) =0.413, p=0.666) whereas changes in D2R density was observed (Fig. 1b: Ftreatment (1,22) =59.11, p
