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Loss of striatal mu1 opiate binding by substantia nigra lesions in the rat
Life Sciences, vol. 43, Printed in the U.S.A.
pp. 1697-1700
Pergamon Press
LOSS OF STRIATAL MU1 OPIATE BINDING BY SUBSTANTIA NIGRA LESIONS IN THE RAT
Richard J. Bodnar*yf, Janet A. Clark*, ptclline L. Cooper1 and Gavril W. Pasternak 9 *The George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettering Cancer Center **Departments of Neurology and Pharmacology Cornell U. Medical College New York, NY 10021 IDepartment of Psychology Queens College, CUNY Flushing, NY 11367 (Received in final form September 22, 1988) SUNMARY
Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5] enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. MUI binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and No change in mu2 binding assays supported these observations. delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.
Soon after the isolation of the endogenous opioid peptides, Kosterlitz and coworkers identified discrete receptors for morphine (mu) and the enkephalins Since then, we have suggested the division of mu receptors into (delta; 1). two discrete subpopulations: mu1 and mu2 (2; for review see 3). The mu2 receptor is highly morphine-selective. In contrast, mu1 receptors bind many enkephalins as potently as morphine. In addition to their differences in binding selectivity, they mediate different pharmacological actions (3). For example, mu1 receptors mediate morphine analgesia in the periaqueductal gray, nucleus raphe magnus and locus coeruleus (4), whereas mu2 receptors mediate respiratory depression and gastrointestinal transit (S-7). Previous workers have reported both a pre- and post-synaptic localization of opiate binding However, none of these earlier studies sites in the striatum (8-16). discriminated between the two mu receptor subtypes, both of which are present in the striatum (17). We now report the effects of substantia nigra lesions on striatal levels of mul, mu2 and delta binding.
0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1698
Striatal
Mu1
Opiate
Binding
Vol.
43, No.
21,
1988
METHODS (250-450 g) were administered haloperidol (1 Male Sprague-Dawley rats i.m.) prior to stereotaxically and ketamine (100 mglkg, mglkg, i.P.) the left substantia nigra (incisor bar implanting a bipolar electrode into -5mm,,coordinates 2.8-3.5 mm posterior to the bregma suture, 2.0 mm lateral to the top of the skull). Rats received a the sagittal suture, and 8.0 mm from lesion (lo-20 mA; Grass LM-4 15 second radiofrequency alternating current Lesion Maker). The electrode was then removed, the rats suture and permitted to permit degeneration of the nigro-striatal pathway to recover for 21 days and the striata immediately were sacrificed To assay binding, rats (10). dissected for binding studies and the mesencephalon preserved in 10% formalin. (40 pm) and stained The mesencephalin was subsequently sectioned coronally with cresyl violet to document lesion placement. individually homogenized in 2.5 ml For binding studies, the striata were EDTA (1.0 mM) and NaCl (100 of 50 mM TRIS buffer containing PMSF (0.1 mM), centrifuged at 49,000xg for 15 mM), incubated at 25'C for 15 minutes, and then immediately resuspended and assayed. minutes at O'C. The pellets were All binding assays were performed in Three separate assays were performed. 2.5 hr in TRIS buffer (pH 7.6) containing triplicate (250 pl) at 25'C for At the end of the incubation period, 2.5 ml of TRIS buffer were MgS04 (5 mM). Nonspecific added and the samples filtered using a Brandel Cell harvester. binding was determined with levallorphan (1 PM) and only specific binding was specific 3H-DADL binding (1 nM) was determined reported. In the first study, (mu1 and delta) and presence of morphine in triplicate samples in the absence sulfate (5 nM; delta) and specific 3H-DAG0 binding (1 nM) was determined in The inhibition of the absence (mu1 and mu2) and presence of DADL (5 nM; mu2). by DADL represented mu1 binding. The 3H-DADL binding by morphine and 3H-DAGO the inhibitors represented delta and mu2 binding remaining in the presence of examined mu1 binding directly with respectively. The second study binding, DPDPE (10 r&l) which selectively competes 3H-DADL (1 nM) in the presence of Mu2 binding was measured directly with 3Hdelta binding, leaving mu1 (16). DAGO (1 nM) in the presence of DSLET (5 r&l) which competes mu1 binding, leaving mu2 binding (16). Protein determinations were performed with each sample using the method of and binding normalized. Analysis of Lowry (18) binding was performed using a matched t-test. Only rats with histologically verified nigral lesions were included. RESULTS
AND
DISCUSSION
The substantia nigra lesions clearly lowered 3H-opioid binding in the striatum (Table 1). Total specific 3H-DADL and 3H-DAGO binding ipsilateral to the lesions were lowered 12 and 24%, compared to the respectively, contralateral controls. When we examined the effects of competitors to determine the effects on subtypes, we observed that delta binding was not significantly affected by the lesions. Rather, the entire decrease in binding could be attributed to the loss of approximately 30% of mu1 binding. In the 3H-DAG0 assay we also observed a highly significant inhibition of mu1 binding and a slightly lower loss of mu2 binding. We next (Table 2). assessed by measured in leaving only decrease in
examined mu1 binding directly using a mu1 selective binding assay In contrast to the previous studies in which mu1 binding was the inhibition of total binding, in this assay 3H-DADL binding is the presence of DPDPE (10 r&l), a highly selective delta ligand, mu1 binding remaining. Again, we observed a significant 25% striatal binding on the lesioned side. The lesions also lowered
Vol. 43, No. 21, 1988
1699
Striatal Mu1 Opiate Binding
mu2 binding in a second assay using DSLET as the competitor instead of DADL. These results illustrate several points. First, substantia nigral lesions lowered only mu binding. Delta binding, measured with 3H-DADL in the presence morphine, was not altered by the lesions. Both mu receptor subtypes were significantly lowered by the lesions. We used several approaches to measure muI binding. The first involved the competition of mu1 binding. Although useful, this approach suffers from the need to subtract two relatively large numbers to calculate a smaller one. We therefore also examined mu1 binding directly with 3H-DADL in the presence of DPDPE. Another advantage of this approach is the high selectivity of DPDPE for delta receptors. DPDPE labels delta receptors (KD N 3 nM) almost 20-fold more potently than mu1 receptors. The decreases in mu1 binding using all three assays were quite similar, with
Table lr Effects of nigral lesions on striatal %I-DADL and 3E-DAGD binding Binding (cpm/mg protein) Mul-- Mu2
Total 3H-DADL Control Lesion
3H-DAGO Control Lesion
3970 f 240 3501 f 330 -12 x p < 0.05
1260 f 760 898 f 490 -29 x p < 0.001
1600 f 140 1210 f 100 -24% p < 0.002
430 f 63 270 f 60 -36% p < 0.005
Delta 2710 2200 2603 k220 -4% n.s.
1170 f 96 940 f 65 -20% p < 0.007
Binding was performed with either 3H-DADL (0.7 nM; n=l2) or 3H-DAG0 (1 nM; n=9) in the absence or presence of morphine (5 nM) or DADL (5 nM), respectively. In the 3H-DADL assay, muI binding corresponded to the binding inhibited by morphine and delta binding as the remainder In the 3H-DAG0 assay, mu1 binding corresponded to the binding inhibited by DSLET and mu2 binding as the remainder. Results are the means f s.e.m. of individual rats. Table 2: Effect of nigral lesions on striatal mu1 and mu2 binding assays Binding (cpm/mg protein) Mu1
assay
Mu? assay
Control
1767 f
80
2521 2 160
Lesion
1337 f 140
1904 f 220
-25 X p < 0.013
-24% p < 0.001
The muI assay employed 3H-DADL (0.7 nM) in the presence of DPDPE (10 nM) to compete delta binding, leaving only mu1 binding. Results are Mu2 binding was determined using 3Hthe means f s.e.m. of 7 rats. DAGO (1 nM) in the presence of DSLET (5 nM) to compete mu1 binding. Results are the means f s.e.m. of 9 rats.
1700
Striatal Mu1 Opiate Binding
Vol. 43, No. 21, 1988
a mean loss of 30X. Mu1 binding sites were slightly more sensitive than mu2 sites, which were lowered 20-24X in two separate assays. However, the significance of these differences remains unclear. In conclusion, these results indicate that nigral lesions lower both mu1 and mu2 binding in the striatum with little effect on delta binding and suggest that the majority of striatal opioid binding is associated with intrinsic neurons. Previous reports showed similar binding losses (B-12), but none of them discriminated between the two mu receptor subtypes. The decreased striatal binding of radiolabeled enkephalins reflects their labeling of mu1 sites. The lost binding may be located presynaptically on the nigrostriatal neurons which are effectively destroyed by electrolytic lesions of the substantia nigra (lo), but other possibilities also must be considered, such as the loss of other striatal inputs and even transsynaptic degeneration. Acknowledgements We thank Drs. Shapiro and Posner for their support. This work was supported by a grant from the National Institute on Drug Abuse (DA02615) to GWP and a core grant from the National Cancer Institute to MSKCC (CA08748). RJB was supported, in part, by a sabbatical from Queens College.
REFERENCES 1. J.A.H. LORD, A.A. WATERFIELD, J. HUGHES and H.W. KOSTERLITZ, Nature 267:495-499 (1977). 2. B.L. WOLOZIN and G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA78:6181-6185 (1981). 3. G.W. PASTERNAK and P.L. WOOD, Life Sci. 38:1889-1898 (1986). 4. R.J. BODNAR, C.L. WILLIAMS, S.J. LEE, and G.W. PASTERNAK, B rain Res., in press. 5. G.S.F. LING and G.W. PASTERNAK, J. Pharmacol. Exp. Ther. (1985) 6. J.S. HEYMAN, C.L. WILLIAMS, T.F. BURKS, H.I. MOSBERG and F. PORRECA, J. Pharmacol. Exp. Ther., in press. 7. D. PAUL and G.W. PASTERNAK, Eur. J. Pharmacol., in press. 8. B. ABOU-KHALIL, A.B. YOUNG, and J.B. PENNEY, Brain Res. 323 :21-29 (1984). 9. L. ANTKIEWICZ-MICHALUK, u. HAVEMANN, J. VETULANI, A. WELLSTEIN and K. KUSCHINSKY, Life Sci. 35: 347-355 (1984). 10. L.C. MURRIN, J.T. COYLE and M.J. KUHAR, Life Sci. 27: 1175-1183, (1980). 11. W.D. BOWEN, C.B. PERT and A. PERT, Life Sci. 31:1679-1682 (1982). 12. H. PARENTI, F. TKIRONE, V.R. OLIGIATI and A. GROPPETTI, Brain Res. 280:317-332 (1983). 13. H. POLLARD, C. LLORENS-CORTES and J.C. SCHWARTZ, Nature 268:745-747 (1977). 14. II. POLLARD, C. LLORENS-CORTES, J.C. SCHWARTZ, C. GROS, and F. DRAY. Brain Res. 151:392-398 (1978). '.5.T.D. REISINE, J.I. NAGY, K. BEAUMONT, B.C. FIBIGER, and H.I. YAMAMURA, Brain Res. 177:241-252 (1979). 16. J.A. CLARK, R. HOUGHTEN and G.W. PASTERNAK, Mol. Pharmacol., submitted. 17. R.R. GOOiiMAN and G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA 82~6667-6671 (1985). 18. O.H. LOWERY, N.H. ROSEBROUGH, A.L. FARR and P.J. RANDALL, J. Biol. Chem. 193:447-450 (1951).
pp. 1697-1700
Pergamon Press
LOSS OF STRIATAL MU1 OPIATE BINDING BY SUBSTANTIA NIGRA LESIONS IN THE RAT
Richard J. Bodnar*yf, Janet A. Clark*, ptclline L. Cooper1 and Gavril W. Pasternak 9 *The George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettering Cancer Center **Departments of Neurology and Pharmacology Cornell U. Medical College New York, NY 10021 IDepartment of Psychology Queens College, CUNY Flushing, NY 11367 (Received in final form September 22, 1988) SUNMARY
Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5] enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. MUI binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and No change in mu2 binding assays supported these observations. delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.
Soon after the isolation of the endogenous opioid peptides, Kosterlitz and coworkers identified discrete receptors for morphine (mu) and the enkephalins Since then, we have suggested the division of mu receptors into (delta; 1). two discrete subpopulations: mu1 and mu2 (2; for review see 3). The mu2 receptor is highly morphine-selective. In contrast, mu1 receptors bind many enkephalins as potently as morphine. In addition to their differences in binding selectivity, they mediate different pharmacological actions (3). For example, mu1 receptors mediate morphine analgesia in the periaqueductal gray, nucleus raphe magnus and locus coeruleus (4), whereas mu2 receptors mediate respiratory depression and gastrointestinal transit (S-7). Previous workers have reported both a pre- and post-synaptic localization of opiate binding However, none of these earlier studies sites in the striatum (8-16). discriminated between the two mu receptor subtypes, both of which are present in the striatum (17). We now report the effects of substantia nigra lesions on striatal levels of mul, mu2 and delta binding.
0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1698
Striatal
Mu1
Opiate
Binding
Vol.
43, No.
21,
1988
METHODS (250-450 g) were administered haloperidol (1 Male Sprague-Dawley rats i.m.) prior to stereotaxically and ketamine (100 mglkg, mglkg, i.P.) the left substantia nigra (incisor bar implanting a bipolar electrode into -5mm,,coordinates 2.8-3.5 mm posterior to the bregma suture, 2.0 mm lateral to the top of the skull). Rats received a the sagittal suture, and 8.0 mm from lesion (lo-20 mA; Grass LM-4 15 second radiofrequency alternating current Lesion Maker). The electrode was then removed, the rats suture and permitted to permit degeneration of the nigro-striatal pathway to recover for 21 days and the striata immediately were sacrificed To assay binding, rats (10). dissected for binding studies and the mesencephalon preserved in 10% formalin. (40 pm) and stained The mesencephalin was subsequently sectioned coronally with cresyl violet to document lesion placement. individually homogenized in 2.5 ml For binding studies, the striata were EDTA (1.0 mM) and NaCl (100 of 50 mM TRIS buffer containing PMSF (0.1 mM), centrifuged at 49,000xg for 15 mM), incubated at 25'C for 15 minutes, and then immediately resuspended and assayed. minutes at O'C. The pellets were All binding assays were performed in Three separate assays were performed. 2.5 hr in TRIS buffer (pH 7.6) containing triplicate (250 pl) at 25'C for At the end of the incubation period, 2.5 ml of TRIS buffer were MgS04 (5 mM). Nonspecific added and the samples filtered using a Brandel Cell harvester. binding was determined with levallorphan (1 PM) and only specific binding was specific 3H-DADL binding (1 nM) was determined reported. In the first study, (mu1 and delta) and presence of morphine in triplicate samples in the absence sulfate (5 nM; delta) and specific 3H-DAG0 binding (1 nM) was determined in The inhibition of the absence (mu1 and mu2) and presence of DADL (5 nM; mu2). by DADL represented mu1 binding. The 3H-DADL binding by morphine and 3H-DAGO the inhibitors represented delta and mu2 binding remaining in the presence of examined mu1 binding directly with respectively. The second study binding, DPDPE (10 r&l) which selectively competes 3H-DADL (1 nM) in the presence of Mu2 binding was measured directly with 3Hdelta binding, leaving mu1 (16). DAGO (1 nM) in the presence of DSLET (5 r&l) which competes mu1 binding, leaving mu2 binding (16). Protein determinations were performed with each sample using the method of and binding normalized. Analysis of Lowry (18) binding was performed using a matched t-test. Only rats with histologically verified nigral lesions were included. RESULTS
AND
DISCUSSION
The substantia nigra lesions clearly lowered 3H-opioid binding in the striatum (Table 1). Total specific 3H-DADL and 3H-DAGO binding ipsilateral to the lesions were lowered 12 and 24%, compared to the respectively, contralateral controls. When we examined the effects of competitors to determine the effects on subtypes, we observed that delta binding was not significantly affected by the lesions. Rather, the entire decrease in binding could be attributed to the loss of approximately 30% of mu1 binding. In the 3H-DAG0 assay we also observed a highly significant inhibition of mu1 binding and a slightly lower loss of mu2 binding. We next (Table 2). assessed by measured in leaving only decrease in
examined mu1 binding directly using a mu1 selective binding assay In contrast to the previous studies in which mu1 binding was the inhibition of total binding, in this assay 3H-DADL binding is the presence of DPDPE (10 r&l), a highly selective delta ligand, mu1 binding remaining. Again, we observed a significant 25% striatal binding on the lesioned side. The lesions also lowered
Vol. 43, No. 21, 1988
1699
Striatal Mu1 Opiate Binding
mu2 binding in a second assay using DSLET as the competitor instead of DADL. These results illustrate several points. First, substantia nigral lesions lowered only mu binding. Delta binding, measured with 3H-DADL in the presence morphine, was not altered by the lesions. Both mu receptor subtypes were significantly lowered by the lesions. We used several approaches to measure muI binding. The first involved the competition of mu1 binding. Although useful, this approach suffers from the need to subtract two relatively large numbers to calculate a smaller one. We therefore also examined mu1 binding directly with 3H-DADL in the presence of DPDPE. Another advantage of this approach is the high selectivity of DPDPE for delta receptors. DPDPE labels delta receptors (KD N 3 nM) almost 20-fold more potently than mu1 receptors. The decreases in mu1 binding using all three assays were quite similar, with
Table lr Effects of nigral lesions on striatal %I-DADL and 3E-DAGD binding Binding (cpm/mg protein) Mul-- Mu2
Total 3H-DADL Control Lesion
3H-DAGO Control Lesion
3970 f 240 3501 f 330 -12 x p < 0.05
1260 f 760 898 f 490 -29 x p < 0.001
1600 f 140 1210 f 100 -24% p < 0.002
430 f 63 270 f 60 -36% p < 0.005
Delta 2710 2200 2603 k220 -4% n.s.
1170 f 96 940 f 65 -20% p < 0.007
Binding was performed with either 3H-DADL (0.7 nM; n=l2) or 3H-DAG0 (1 nM; n=9) in the absence or presence of morphine (5 nM) or DADL (5 nM), respectively. In the 3H-DADL assay, muI binding corresponded to the binding inhibited by morphine and delta binding as the remainder In the 3H-DAG0 assay, mu1 binding corresponded to the binding inhibited by DSLET and mu2 binding as the remainder. Results are the means f s.e.m. of individual rats. Table 2: Effect of nigral lesions on striatal mu1 and mu2 binding assays Binding (cpm/mg protein) Mu1
assay
Mu? assay
Control
1767 f
80
2521 2 160
Lesion
1337 f 140
1904 f 220
-25 X p < 0.013
-24% p < 0.001
The muI assay employed 3H-DADL (0.7 nM) in the presence of DPDPE (10 nM) to compete delta binding, leaving only mu1 binding. Results are Mu2 binding was determined using 3Hthe means f s.e.m. of 7 rats. DAGO (1 nM) in the presence of DSLET (5 nM) to compete mu1 binding. Results are the means f s.e.m. of 9 rats.
1700
Striatal Mu1 Opiate Binding
Vol. 43, No. 21, 1988
a mean loss of 30X. Mu1 binding sites were slightly more sensitive than mu2 sites, which were lowered 20-24X in two separate assays. However, the significance of these differences remains unclear. In conclusion, these results indicate that nigral lesions lower both mu1 and mu2 binding in the striatum with little effect on delta binding and suggest that the majority of striatal opioid binding is associated with intrinsic neurons. Previous reports showed similar binding losses (B-12), but none of them discriminated between the two mu receptor subtypes. The decreased striatal binding of radiolabeled enkephalins reflects their labeling of mu1 sites. The lost binding may be located presynaptically on the nigrostriatal neurons which are effectively destroyed by electrolytic lesions of the substantia nigra (lo), but other possibilities also must be considered, such as the loss of other striatal inputs and even transsynaptic degeneration. Acknowledgements We thank Drs. Shapiro and Posner for their support. This work was supported by a grant from the National Institute on Drug Abuse (DA02615) to GWP and a core grant from the National Cancer Institute to MSKCC (CA08748). RJB was supported, in part, by a sabbatical from Queens College.
REFERENCES 1. J.A.H. LORD, A.A. WATERFIELD, J. HUGHES and H.W. KOSTERLITZ, Nature 267:495-499 (1977). 2. B.L. WOLOZIN and G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA78:6181-6185 (1981). 3. G.W. PASTERNAK and P.L. WOOD, Life Sci. 38:1889-1898 (1986). 4. R.J. BODNAR, C.L. WILLIAMS, S.J. LEE, and G.W. PASTERNAK, B rain Res., in press. 5. G.S.F. LING and G.W. PASTERNAK, J. Pharmacol. Exp. Ther. (1985) 6. J.S. HEYMAN, C.L. WILLIAMS, T.F. BURKS, H.I. MOSBERG and F. PORRECA, J. Pharmacol. Exp. Ther., in press. 7. D. PAUL and G.W. PASTERNAK, Eur. J. Pharmacol., in press. 8. B. ABOU-KHALIL, A.B. YOUNG, and J.B. PENNEY, Brain Res. 323 :21-29 (1984). 9. L. ANTKIEWICZ-MICHALUK, u. HAVEMANN, J. VETULANI, A. WELLSTEIN and K. KUSCHINSKY, Life Sci. 35: 347-355 (1984). 10. L.C. MURRIN, J.T. COYLE and M.J. KUHAR, Life Sci. 27: 1175-1183, (1980). 11. W.D. BOWEN, C.B. PERT and A. PERT, Life Sci. 31:1679-1682 (1982). 12. H. PARENTI, F. TKIRONE, V.R. OLIGIATI and A. GROPPETTI, Brain Res. 280:317-332 (1983). 13. H. POLLARD, C. LLORENS-CORTES and J.C. SCHWARTZ, Nature 268:745-747 (1977). 14. II. POLLARD, C. LLORENS-CORTES, J.C. SCHWARTZ, C. GROS, and F. DRAY. Brain Res. 151:392-398 (1978). '.5.T.D. REISINE, J.I. NAGY, K. BEAUMONT, B.C. FIBIGER, and H.I. YAMAMURA, Brain Res. 177:241-252 (1979). 16. J.A. CLARK, R. HOUGHTEN and G.W. PASTERNAK, Mol. Pharmacol., submitted. 17. R.R. GOOiiMAN and G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA 82~6667-6671 (1985). 18. O.H. LOWERY, N.H. ROSEBROUGH, A.L. FARR and P.J. RANDALL, J. Biol. Chem. 193:447-450 (1951).