European Journal of Pharmacology, 175 (1990) 63-69
63
Elsevier EJP 51109
Involvement of central/t and 8 opioid receptors in mediating the reinforcing effects of fl-endorphin in the rat R e g i n e B a l s - K u b i k , T o n i S. S h i p p e n b e r g a n d A l b e r t H e r z Department of Neuropharmacology, Max Planck Institute for Psychiatry, Am Klopferspitz 18a, D-8033 Planegg-Martinsried, F.R.G.
Received 29 June 1989, revised MS received 27 September 1989, accepted 17 October 1989
An unbiased place preference conditioning procedure was used to identify the central opioid receptor types through which the endogenous opioid peptide, fl-endorphin, acts to exert its reinforcing effects in rats in vivo. The intracerebroventricular administration of fl-endorphin, and selective /t (DAGO) or 8 (DPDPE) opioid receptor agonists produced marked preferences for the drug-associated place. Intracerebroventricular pretreatment with the selective tt antagonist, CTOP, eliminated the place preference produced by DAGO but not that produced by DPDPE. Pretreatment with the selective 8 antagonist, ICI 174,864, abolished the place preference induced by DPDPE. It did not modify the effect of DAGO. In contrast, pretreatment with either ICI 174,864 or CTOP abolished the effects of fl-endorphin. These data demonstrate that both /z and 8 receptors are involved in mediating the reinforcing effect of fl-endorphin and indicate that the activation of both receptor types is required for the expression of the motivational effects of fl-endorphin. Further they suggest that fl-endorphin produces its motivational effects via an interaction with an opioid receptor complex composed of both ~ and 8 receptors. Place conditioning; Opioid receptors; fl-Endorphin; CTOP (D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2); ICI 174,864; (Reinforcement)
1. Introduction The endogenous opioid peptide, fl-endorphin, exerts p r o f o u n d effects on motivational processes. Rats will self-administer fl-endorphin into the cerebral ventricles (Van Ree et al., 1979) and will exhibit a preference for an environment previously associated with its intracerebroventricular (i.c.v.) administration (Amalric et al., 1987; Bals-Kubik et al., 1988). In addition to its ability to function as a positive reinforcer, recent studies with rats have indicated that this peptide is also released in
Correspondence to: R. Bals-Kubik, Department of Neuropharmacology, Max Planck Institute for Psychiatry, Am Klopferspitz 18a, D-8033 Planegg-Martinsried, F.R.G.
response to ' n a t u r a l ' rewarding stimuli such as expecting a n d / o r receiving highly palatable food ( D u m and Herz, 1987). It is not clear through which receptor type(s) fl-endorphin acts to exert its effects in vivo. Although evidence for a distinct fl-endorphin binding site, the E receptor, in the periphery has been presented (Schulz et al., 1981; G a r z r n et al., 1985), its existence in the central nervous system (CNS) is less well d o c u m e n t e d (Johnson et al., 1982; G o o d m a n et al., 1983; H o u g h t e n et al., 1984). In addition, it has been reported that fl-endorphin binds with high affinity to/~ as well as 8 opioid receptors in the C N S (Akil et al., 1981). In a recent place conditioning study examining the motivational effects of opioids, it was shown that the opioid antagonist, naloxone, blocks the rein-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64 forcing effects of fl-endorphin (Amalric et al., 1987). Since, however, this antagonist binds with high affinity to both # and ~ receptors, the attenuation observed could have resulted from an antagonism at /~, 8 or both /~ and 6 opioid receptors. Thus in the present study a place preference conditioning procedure with rats was used to characterize the central opioid receptor type(s) upon which fl-endorphin acts to produce its motivational effects. Specifically, we examined the influence of the opioid receptor selective antagonists, ICI 174,864 (6: Cotton et al., 1984) and CTOP (if: Pelton et al., 1986), upon fl-endorphin-induced place conditioning. The use of these agents permits the identification of the functions associated with each receptor type and, in the case of the present study, the evaluation of the possible role of each in mediating the motivational effects of this endogenously occurring peptide.
2. Materials and methods
2.1. Subjects Male Sprague Dawley rats (Charles River Wiga, Sulzfeld, F R G ) weighing 250-300 g were anesthetized with sodium hexobarbital (60 m g / k g ; i.p.) and stereotaxically implanted with 23 gauge guide cannulas aimed at the lateral ventricle (AP = - 0.9 mm, L = + 1.5 mm, DV = 3.5 mm) (Paxinos and Watson, 1982). The rats were housed individually in wire cages in a climatically controlled colony room with a 12 h l i g h t / d a r k cycle (lights on at 6 a.m.). Food and water were available ad libitum.
2.2. Procedure Place conditioning commenced one week after cannula implantation and was conducted as described previously (Mucha and Herz, 1985; Shippenberg et al., 1987). The apparatus consisted of 30 x 60 x 30 cm wooden shuttleboxes with a clear plexiglas front. For conditioning sessions, each box was divided into two equal-sized compartments by means of a sliding wall. One compartment was white with a textured floor; the other
black with a smooth floor. For testing, the central wall was raised 12 cm above the floor and a 5 x 2 c m ' neutral' steel mesh platform was inserted along the line separating the two compartments. Conditioning sessions were conducted once a day for 6 days and consisted of injecting the conditioning drug or its vehicle on alternate days. The rats were immediately confined to one compartment of the shuttlebox following drug injection and to the other compartment following vehicle injection. The treatment c o m p a r t m e n t and the order of presentation of the drug and vehicle was counterbalanced for each drug dose. All conditioning sessions lasted 40 min. Test sessions were carried out 1 day after the last training session, in the drug-free state. The rats, which had not received any drugs, were placed on the 'neutral' platform of the testbox and allowed free access to both sides of the box for 15 min. An Olympus VX-351 videocamera with integrated stop-watch was used for data recording. The time spent in a particular place (drug, vehicle or neutral platform) was assessed by visual analysis of the recorded videotape. All sessions were conducted during the light phase of the light-dark cycle under conditions of dim illumination (14.5 lux) and with masking white noise. We (Shippenberg et al., 1987) have previously shown that rats exhibit no preference for either of the place cues under these conditions. The i.c.v, injections were given with a 30 gauge injection needle attached to a Hamilton microsyringe (10 /~l) via polyethylene tubing (I.D.: 0.7 mm). The drug solutions were administered over a 60 s period and the injection needles were left in place for an additional 30 s to ensure complete delivery of the solution. For antagonism tests, groups of rats were injected i.c.v, with ICI 174,864, C T O P or vehicle 10 min before the microinjection of the conditioning drug, or its vehicle. For these antagonism studies the lowest dose of each agonist producing significant conditioning was tested. At the end of the experiments, the animals were anesthetized and killed by decapitation. The brains were removed and sectioned in a cryostat to verify the location of the cannulas. Only data from those animals with histologically correct cannula placements were used for statistical analysis.
65
2.3. Statistical analysis Conditioning scores represent the time spent in the drug-paired place minus the time spent in the vehicle-paired place, and are expressed as means ___S.E. Dose-response curves were analysed with a one-way r a n d o m factorial analysis of variance. The Wilcoxon test, in which the time spent in the drug-associated place was c o m p a r e d to that spent in the vehicle-paired place, was used to determine whether individual doses produced significant conditioning. A one-way analysis of variance ( A N O V A ) followed by the Student N e w m a n Keul's test (SNK) was used to determine the statistical significance of effects of the antagonist pretreatments. The accepted level of significance for all tests was P < 0.05.
2.4. Drugs All drugs were dissolved in sterile water and were administered i.c.v, in a volume of 3.0/~1. The conditioning drugs were: h u m a n fl-endorphin (kindly d o n a t e d by C.H. Li, San Francisco, CA, USA); DAGO [D-Ala2,N-methyl-Phe4,Gly K ol]enkephalin (Bachem, Bubendorf, Switzerland); D P D P E [D-Pen2,D-PenS]enkephalin (Peninsula, Merseyside, U K ) ; C T O P ( D - P e n - C y s - T y r - D - T r p O r n - T h r - P e n - T h r - N H 2 ) (Peninsula, Merseyside, U K ) ; ICI 174,864 (Allyl2-Tyr-Aib-Aib-Phe-LeuO H ) (Cambridge Research Biochemicals, Cambridge, UK).
3. Results
3.1. Control tests of preference The rats that received injections of vehicle during each of the conditioning sessions did not exhibit a significant preference for either compartment of the testbox, thus confirming that the conditioning procedure used was unbiased. The mean time spent in the white and black compartments were 351 + 33 and 399 + 45 s (n = 10), respectively.
TABLE 1 Place conditioning produced by DAGO, DPDPE and fl-endorphin. Values represent the mean time+_S.E, spent in the drug- and vehicle-paired sides of the testbox. Drug
~tg (i.c.v.)
Place conditioning score (s)
DAGO
0.001 0.05 0.1 0.5 2.5 10.0 25.0 1.0 2.5 5.0
+ 16.5 + 69.3 + 105.2_+41.4 + 271.3 _+34.3 ~ + 264.1 _+38.7 b - 15.9 +_53.1 + 187.4_+41.4 + 174.0+_54.3 a + 72.0 _+93.5 + 90.8 _+42.7 + 234.9 +_49.9 b
DPDPE fl-Endorphin
Asterisks denote a significant preference for the drug-paired place (a p < 0.05; b p < 0.01).
3.2. Place conditioning produced by DAGO, DPDPE and B-EP The i.c.v, administration of D A G O as well as D P D P E resulted in significant preferences for the drug-associated place (table 1). The magnitude of these effects was linearly related to the dose of D A G O [F(1,33) = 19.1; P < 0.0001] and D P D P E [F(1,22) = 6.44; P < 0.02], respectively, fl-Endorphin p r o d u c e d significant effects at a dose of 5.0 /~g (Wilcoxon: P < 0.001). However, at the doses tested, no linear dose-response relationship was observed [F(1,31) = 3.48; P < 0.06]. F o r subsequent antagonism studies the lowest dose producing significant place conditionings was always tested.
3.3. Influence of CTOP pretreatment upon DAGOand DPDPE-induced place conditioning The i.c.v, administration of the selective /z antagonist, CTOP, at a dose of 1.0 /~g p r o d u c e d no preference for either the drug- or vehicle-paired place ( - 6 . 8 ± 90.4 s; n = 10) (Bals-Kubik et al., 1989). The effects of C T O P pretreatment on the place conditioning p r o d u c e d by D A G O and D P D P E are shown in fig. la,b, respectively. The administration of the bt agonist, D A G O (0.1 /zg), resulted in a significant preference for the drugpaired place in both the control (Wilcoxon: P
63
Elsevier EJP 51109
Involvement of central/t and 8 opioid receptors in mediating the reinforcing effects of fl-endorphin in the rat R e g i n e B a l s - K u b i k , T o n i S. S h i p p e n b e r g a n d A l b e r t H e r z Department of Neuropharmacology, Max Planck Institute for Psychiatry, Am Klopferspitz 18a, D-8033 Planegg-Martinsried, F.R.G.
Received 29 June 1989, revised MS received 27 September 1989, accepted 17 October 1989
An unbiased place preference conditioning procedure was used to identify the central opioid receptor types through which the endogenous opioid peptide, fl-endorphin, acts to exert its reinforcing effects in rats in vivo. The intracerebroventricular administration of fl-endorphin, and selective /t (DAGO) or 8 (DPDPE) opioid receptor agonists produced marked preferences for the drug-associated place. Intracerebroventricular pretreatment with the selective tt antagonist, CTOP, eliminated the place preference produced by DAGO but not that produced by DPDPE. Pretreatment with the selective 8 antagonist, ICI 174,864, abolished the place preference induced by DPDPE. It did not modify the effect of DAGO. In contrast, pretreatment with either ICI 174,864 or CTOP abolished the effects of fl-endorphin. These data demonstrate that both /z and 8 receptors are involved in mediating the reinforcing effect of fl-endorphin and indicate that the activation of both receptor types is required for the expression of the motivational effects of fl-endorphin. Further they suggest that fl-endorphin produces its motivational effects via an interaction with an opioid receptor complex composed of both ~ and 8 receptors. Place conditioning; Opioid receptors; fl-Endorphin; CTOP (D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2); ICI 174,864; (Reinforcement)
1. Introduction The endogenous opioid peptide, fl-endorphin, exerts p r o f o u n d effects on motivational processes. Rats will self-administer fl-endorphin into the cerebral ventricles (Van Ree et al., 1979) and will exhibit a preference for an environment previously associated with its intracerebroventricular (i.c.v.) administration (Amalric et al., 1987; Bals-Kubik et al., 1988). In addition to its ability to function as a positive reinforcer, recent studies with rats have indicated that this peptide is also released in
Correspondence to: R. Bals-Kubik, Department of Neuropharmacology, Max Planck Institute for Psychiatry, Am Klopferspitz 18a, D-8033 Planegg-Martinsried, F.R.G.
response to ' n a t u r a l ' rewarding stimuli such as expecting a n d / o r receiving highly palatable food ( D u m and Herz, 1987). It is not clear through which receptor type(s) fl-endorphin acts to exert its effects in vivo. Although evidence for a distinct fl-endorphin binding site, the E receptor, in the periphery has been presented (Schulz et al., 1981; G a r z r n et al., 1985), its existence in the central nervous system (CNS) is less well d o c u m e n t e d (Johnson et al., 1982; G o o d m a n et al., 1983; H o u g h t e n et al., 1984). In addition, it has been reported that fl-endorphin binds with high affinity to/~ as well as 8 opioid receptors in the C N S (Akil et al., 1981). In a recent place conditioning study examining the motivational effects of opioids, it was shown that the opioid antagonist, naloxone, blocks the rein-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64 forcing effects of fl-endorphin (Amalric et al., 1987). Since, however, this antagonist binds with high affinity to both # and ~ receptors, the attenuation observed could have resulted from an antagonism at /~, 8 or both /~ and 6 opioid receptors. Thus in the present study a place preference conditioning procedure with rats was used to characterize the central opioid receptor type(s) upon which fl-endorphin acts to produce its motivational effects. Specifically, we examined the influence of the opioid receptor selective antagonists, ICI 174,864 (6: Cotton et al., 1984) and CTOP (if: Pelton et al., 1986), upon fl-endorphin-induced place conditioning. The use of these agents permits the identification of the functions associated with each receptor type and, in the case of the present study, the evaluation of the possible role of each in mediating the motivational effects of this endogenously occurring peptide.
2. Materials and methods
2.1. Subjects Male Sprague Dawley rats (Charles River Wiga, Sulzfeld, F R G ) weighing 250-300 g were anesthetized with sodium hexobarbital (60 m g / k g ; i.p.) and stereotaxically implanted with 23 gauge guide cannulas aimed at the lateral ventricle (AP = - 0.9 mm, L = + 1.5 mm, DV = 3.5 mm) (Paxinos and Watson, 1982). The rats were housed individually in wire cages in a climatically controlled colony room with a 12 h l i g h t / d a r k cycle (lights on at 6 a.m.). Food and water were available ad libitum.
2.2. Procedure Place conditioning commenced one week after cannula implantation and was conducted as described previously (Mucha and Herz, 1985; Shippenberg et al., 1987). The apparatus consisted of 30 x 60 x 30 cm wooden shuttleboxes with a clear plexiglas front. For conditioning sessions, each box was divided into two equal-sized compartments by means of a sliding wall. One compartment was white with a textured floor; the other
black with a smooth floor. For testing, the central wall was raised 12 cm above the floor and a 5 x 2 c m ' neutral' steel mesh platform was inserted along the line separating the two compartments. Conditioning sessions were conducted once a day for 6 days and consisted of injecting the conditioning drug or its vehicle on alternate days. The rats were immediately confined to one compartment of the shuttlebox following drug injection and to the other compartment following vehicle injection. The treatment c o m p a r t m e n t and the order of presentation of the drug and vehicle was counterbalanced for each drug dose. All conditioning sessions lasted 40 min. Test sessions were carried out 1 day after the last training session, in the drug-free state. The rats, which had not received any drugs, were placed on the 'neutral' platform of the testbox and allowed free access to both sides of the box for 15 min. An Olympus VX-351 videocamera with integrated stop-watch was used for data recording. The time spent in a particular place (drug, vehicle or neutral platform) was assessed by visual analysis of the recorded videotape. All sessions were conducted during the light phase of the light-dark cycle under conditions of dim illumination (14.5 lux) and with masking white noise. We (Shippenberg et al., 1987) have previously shown that rats exhibit no preference for either of the place cues under these conditions. The i.c.v, injections were given with a 30 gauge injection needle attached to a Hamilton microsyringe (10 /~l) via polyethylene tubing (I.D.: 0.7 mm). The drug solutions were administered over a 60 s period and the injection needles were left in place for an additional 30 s to ensure complete delivery of the solution. For antagonism tests, groups of rats were injected i.c.v, with ICI 174,864, C T O P or vehicle 10 min before the microinjection of the conditioning drug, or its vehicle. For these antagonism studies the lowest dose of each agonist producing significant conditioning was tested. At the end of the experiments, the animals were anesthetized and killed by decapitation. The brains were removed and sectioned in a cryostat to verify the location of the cannulas. Only data from those animals with histologically correct cannula placements were used for statistical analysis.
65
2.3. Statistical analysis Conditioning scores represent the time spent in the drug-paired place minus the time spent in the vehicle-paired place, and are expressed as means ___S.E. Dose-response curves were analysed with a one-way r a n d o m factorial analysis of variance. The Wilcoxon test, in which the time spent in the drug-associated place was c o m p a r e d to that spent in the vehicle-paired place, was used to determine whether individual doses produced significant conditioning. A one-way analysis of variance ( A N O V A ) followed by the Student N e w m a n Keul's test (SNK) was used to determine the statistical significance of effects of the antagonist pretreatments. The accepted level of significance for all tests was P < 0.05.
2.4. Drugs All drugs were dissolved in sterile water and were administered i.c.v, in a volume of 3.0/~1. The conditioning drugs were: h u m a n fl-endorphin (kindly d o n a t e d by C.H. Li, San Francisco, CA, USA); DAGO [D-Ala2,N-methyl-Phe4,Gly K ol]enkephalin (Bachem, Bubendorf, Switzerland); D P D P E [D-Pen2,D-PenS]enkephalin (Peninsula, Merseyside, U K ) ; C T O P ( D - P e n - C y s - T y r - D - T r p O r n - T h r - P e n - T h r - N H 2 ) (Peninsula, Merseyside, U K ) ; ICI 174,864 (Allyl2-Tyr-Aib-Aib-Phe-LeuO H ) (Cambridge Research Biochemicals, Cambridge, UK).
3. Results
3.1. Control tests of preference The rats that received injections of vehicle during each of the conditioning sessions did not exhibit a significant preference for either compartment of the testbox, thus confirming that the conditioning procedure used was unbiased. The mean time spent in the white and black compartments were 351 + 33 and 399 + 45 s (n = 10), respectively.
TABLE 1 Place conditioning produced by DAGO, DPDPE and fl-endorphin. Values represent the mean time+_S.E, spent in the drug- and vehicle-paired sides of the testbox. Drug
~tg (i.c.v.)
Place conditioning score (s)
DAGO
0.001 0.05 0.1 0.5 2.5 10.0 25.0 1.0 2.5 5.0
+ 16.5 + 69.3 + 105.2_+41.4 + 271.3 _+34.3 ~ + 264.1 _+38.7 b - 15.9 +_53.1 + 187.4_+41.4 + 174.0+_54.3 a + 72.0 _+93.5 + 90.8 _+42.7 + 234.9 +_49.9 b
DPDPE fl-Endorphin
Asterisks denote a significant preference for the drug-paired place (a p < 0.05; b p < 0.01).
3.2. Place conditioning produced by DAGO, DPDPE and B-EP The i.c.v, administration of D A G O as well as D P D P E resulted in significant preferences for the drug-associated place (table 1). The magnitude of these effects was linearly related to the dose of D A G O [F(1,33) = 19.1; P < 0.0001] and D P D P E [F(1,22) = 6.44; P < 0.02], respectively, fl-Endorphin p r o d u c e d significant effects at a dose of 5.0 /~g (Wilcoxon: P < 0.001). However, at the doses tested, no linear dose-response relationship was observed [F(1,31) = 3.48; P < 0.06]. F o r subsequent antagonism studies the lowest dose producing significant place conditionings was always tested.
3.3. Influence of CTOP pretreatment upon DAGOand DPDPE-induced place conditioning The i.c.v, administration of the selective /z antagonist, CTOP, at a dose of 1.0 /~g p r o d u c e d no preference for either the drug- or vehicle-paired place ( - 6 . 8 ± 90.4 s; n = 10) (Bals-Kubik et al., 1989). The effects of C T O P pretreatment on the place conditioning p r o d u c e d by D A G O and D P D P E are shown in fig. la,b, respectively. The administration of the bt agonist, D A G O (0.1 /zg), resulted in a significant preference for the drugpaired place in both the control (Wilcoxon: P
