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The effect of methadone addiction on cyclic nucleotide levels in regions of rat brain
Life Sciences, Vol. 39, pp. 477-481 Printed in the U.S.A.
Pergamon Journals
THE EFFECT OF METHADONE ADDICTION ON CYCLIC NUCLEOTIDE LEVELS IN REGIONS OF RAT BRAIN
D. Sadava and B. Mack Joint Science Department The Claremont Colleges Claremont, California, 91711
(Received in final form May 15, 1986) Summary Studies of tissue culture cells and tissue slices have implicated the nucleotides, cyclic AMP and cyclic GMP, in the mechanism of action of opiates. However, there are little in vivo data to corroborate this hypothesis. We addicted rats to the synthetic opiate, methadone, by providing the drug in their drinking water (dosage 2.1 mg./kg./day). The two cyclic nucleotides were measured in four brain areas which contain a high concentration of opiate receptors: amygdala, neostriatum, periventricular grey, and thalamus. Data were obtained after acute exposure of the rats to the drug (i day), tolerance (35 days), withdrawal (35 days on drug then l day off drug), and readjustment (35 days on drug then 21 days off drug). Cyclic GMP levels were low (0.03 pmol./mg, tissue) in the four regions and did not differ significantly during the experiment. Cyclic AMP levels were higher (1-3 pmol./mg.) and fluctuated consistently in the four regions. After acute methadone treatment, there was a reduction in cyclic AMP, which continued at lower levels after tolerance. One day of withdrawal led to increased cAMP, which rose to near control levels. After readjustment, the levels were reduced. These data indicate an involvement of cyclic AMP in the addiction and withdrawal processes in the intact animal. The nucleotides cyclic-3',5'-adenosine monophosphate (cAMP) and cyclic-3',5'-guanosine monophosphate (cGMP) have been implicated in the mode of action on the brain of endogenous and exogenous opiates (1,2). A model has been proposed in which neuronal cAMP is reduced during acute exposure to an opiate, slowly returns to normal during tolerance, increases during withdrawal and returns to baseline value after a period of readjustment in the absence of the opiate (3,4). Evidence in support of this model has come from studies on rat brain homogenates (5), guinea pig ileum (6), and mouse neuroblastoma-glioma hybrid cultured cells (7). In addition, increases in plasma cAMP (8) and platelet adenylate cyclase (9) have been noted during withdrawal from heroin in humans. Since cGMP often acts in opposition to cAMP (i0), and since cGMP is present in nervous tissue where it regulates depolarization (ii), opiates might be expected to affect neuronal cGMP levels in a manner opposite to their affect on cAMP. Indeed, increased cGMP has been noted after acute opiate incubation of rat brain slices (12) and hybrid cultured cells (13). There have been few in vivo studies of the effects of exogenous opiates on brain cyclic nucleotide levels. When rats were made tolerant to morphine, rapid withdrawal led to increased cAMP in the striatum (14). Implantation of morphine 0024-3206/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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pellets in rats led to decreased cAMP, and initially, increased cGMP, in caudate, hypothalamus, substantia nigra, and thalamus (15). While these data are consistent with an opiate-cyclic nucleotide relationship, there has not been a systematic study in which the nucleotides were measured following in vivo addiction and withdrawal. We report here such a study, in which rats were addicted to the synthetic narcotic, methadone. In addition, opiate receptors are well characterized, and appear to be concentrated in the limbic system and paleospinothalamic pathways (16-18). Therefore, we measured cAMP and cGMP in amygdala, neostriatum, periventricular grey, and thalamus.
Methods Male albino rats, initial weight 200-220 g., were obtained from Simonson Laboratories, Gilroy, California. They were housed individually at 20°C, with a a 12 hr. photoperiod, and fed standard laboratory chow. Methadone was presented in the drinking water as previously described (19). This is a model for addiction in which the animals show a compulsion, tolerance, and physical dependency for the drug. Tolerance develops after two weeks of exposure to methadone, and withdrawal symptoms are observed after 1 day of removal of the drug from the drinking water of addicted animals (19). Controls did not receive the drug in their water. The average dosages of methadone were: Day I: 0.8 mg/kg/day; day 6 : 1 . 2 mg/kg/day; day 1 0 : 1 . 4 mg/kg/day; day 1 8 : 1 . 8 mg/kg/day; day 2 5 : 2 . 0 mg/kg/day; day 3 5 : 2 . 1 mg/kg/day. Groups of four animals were sacrificed between 1-5 PM according to the following treatments: I. Control. 2. Acute: drug presented for 1 day. 3. Tolerance: drug presented for 35 days. 4. Withdrawal: drug presented for 35 days, then water without drug for 1 day. 5. Readjustment: drug presented for 35 days, then water without drug for 21 days. Animals were sacrificed by cervical dislocation and the heads frozen immediately in liquid nitrogen. The brain was removed, and, following location via a stereotaxic atlas (20), neostriatum, periventricular grey, thalamus, and amygdala were removed and weighed (mean 15.9 mg.). Tissues were homogenized immediately with i0 strokes of a teflon-glass homogenizer in 350 ul. of ice-cold 0.4 mM EDTA. Homogenates were heated in a boiling water bath for 3 min., and the samples centrifuged at 29,000 x g. for 15 min. to remove coagulated molecules. The supernatants were kept frozen at -20°C for up to 3 weeks prior to cyclic nucleotide assay, cAMP was measured by protein binding assay and cGMP by radioimmunoassay (kits from Amersham, Arlington Heights Illinois). All assays were done in duplicate. Statistical analyses were done by the t-test for paired data.
Results Levels of cAMP, expressed on a tissue weight basis (Table I), were similar to those previously reported for rat brain (14,15). This indicates that the methods of sacrifice and analysis did not lead to artificially altered cAMP levels. In the four regions which contain relatively high concentrations of receptors for methadone (17), similar changes in cAMP were noted over the course of addiction and withdrawal. After one day of exposure of the animals to the drug (acute treatment), there was a reduction cAMP. After five weeks of drug treatment (addiction), there was less tissue cAMP than the acute situation. In all tissues examined, withdrawal of methadone
Vol. 39, No. 5, 1986
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led to significantly increased cAMP after 1 day. Three weeks after initial withdrawal (readjustment), cAMP levels in all regions were reduced from the immediate withdrawal period to nearly tolerance levels.
TABLE I Cyclic AMP Levels in Brain Regions
Condition
PG
I. 2. 3. 4. 5.
2.25 1.36 1.28 1.85 1.37
Control Acute Tolerance Withdrawal Readjust
TH
• ± ± ± *
.18 .23 .21 .14 .17
NS
3.05" 2.39 ± 1.64 ± 2.51 * 1.89 ±
.26 .31 .17 .19 .12
1.45' 0.85 ± 0.69 ~ 1.59 ± 0.84 *
AM
.12 .27 .16 .19 .25
2.56 2.38 1.95 2.23 2.17
± ± ± ± ±
.21 .27 .08 .17 .19
Data are expressed as mean concentrations ± S.D. of each region from eight animals in pmol./mg, tissue weight. Abbreviations: PG: periventricular grey; TH: thalamus; NS: neostriatum; AM: amygdala. For each tissue, means were significantly different for each treatment ( p < 0.01) with the exceptions of the following: PG: 2,3, and 5; TH: 2 and 4; NS: 2,3, and 5; 1 and 4; AM: 1,2 and 5; 4 and 5.
As was found in previous studies (i1,15), cGMP was present in brain in much smaller amounts than cAMP (Table II). The low concentrations of cGMP, coupled with the small amounts of tissues examined, led to homogenate concentrations that were near the lower limits of the assay system used. Within these limits, the levels of cGMP were similar throughout the experimental period.
TABLE II Cyclic GMP Levels in Brain Regions
Condition
PG
i. 2. 3. 4. 5.
0.031' 0.032± 0.028± 0.030± 0.037 ±
Control Acute Tolerance Withdrawal Readjust
TH
.017 .019 .008 .Oll .012
0.025* 0.032± 0.029~ 0.034± 0.018 ±
NS
.007 .017 .021 .012 .008
0.037± 0.033* 0.048± 0.029± 0.027 ±
AM
.016 .011 .012 .019 .008
Expression of data and abbreviations are as in Table I. tissue set were not significantly different (p > 0.01).
0.024± 0.029 ± 0.027 ± 0.029± 0.035±
.009 .006 .016 .011 .026
Means within each
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Discussion Data from the four brain regions (Table I) are consistent in many respects with the model for fluctuations of cAMP during opiate addiction and withdrawal (3) and argue against an involvement of fluctuations of cGMP (Table II). Following acute methadone treatment, cAMP levels were reduced, as the model predicts and in accordance with previous in vitro studies (5-7). However, during tolerance, cAMP levels did not return to control levels as predicted but remained low. It is possible that sufficient time was not allowed for tolerance to develop (5 weeks); however, in the addiction model used in these experiments this is usually sufficient for tolerance (19) and drug consumption data indicate that tolerance did develop. Withdrawal led to an immediate increase in cAMP, as predicted, and during readjustment the levels lowered toward baseline. That control levels were not reached during readjustment (21 days) may indicate that the period given was not sufficient for complete biochemical normalization. In humans, withdrawal symptoms are detectable for up to one month after the cessation of methadone administration (21). The means by which cAMP acts as an intermediary between opiate binding and neuronal activity are not clear. The binding of an opiate to its neuronal receptor leads to an inhibition of membrane-bound adenylate cyclase (22,23). Cellular cAMP is important in ionic changes involved in neuronal firing (4), and via protein phosphorylation regulates the formation and release of neurotransmitters (24). Studies of these parameters in the four regions examined in this report should clarify the role of cAMP in opiate action.
Acknowledgements We thank Mallinckrodt Chemical Works, St. Louis, Missouri, for a gift of the methadone used in these studies. Dr. B. Baker provided assistance in scaling down the radioimmunoassay for cGMP. This research was partially supported by a grants from Pitzer College and the W.M. Keck Foundation.
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. II. 12. 13. 14. 15. 16. 17. 18.
M. WOLLMAN, Prog. Neurobiol. 16 145-154 (1980). J. KEBABIAN, Adv. Cyclic Nucl. Res. 8 421-508 (1977). S. SNYDER, Sci. Am. 236 44-56 (1977). R . E . WEST and R. J. MILLER, Brit. Med. Bull. 39 53-58 (1983). Z. MERALI, B. TSANG, R.H. SINGHEL and P.A. HRDINA, Res. Comm. Chem. Path. Pharm. 14 29-37 (1976). A. REZVASNI, J. HUIDOBORA-TORO, J. PABLO, J. HU, and E. L. WAY, J. Pharmacol. Exp. Ther. 225 251-255 (1983). P. LAW, D. HOM, and H. H, LOH, Mol. Pharmacol. 23 26-35 (1983). H . L . WEN, W. K. HO, H. K. WONG, Z. D. MEHAL, Y. NG, and L. MA, Comp. Med. East West. 6 241-245 (1979). G . N . PANDEr, F. DELEON-JONES, E. INWANG and J. M. DAVIS, Clin. Pharmacol. Ther. 27 607-611 (1980). N. D. GOLDBERG and M. HADDOX, Ann. Rev. Biochem. 46 823-896 (1977). U. WALTER and P. GREENGARD, Curr. Top. Cell. Regul. 19 219-256 (1981). R. GULLIS, J. TRABER, and B. HAMPRECHT, Nature. 256 57-59 (1975). G. GWYNN and E. COSTA, Proc. Natl. Acad. Sci. 79 690-694 (1982). C. MEHTA and W. E. JOHNSON, Life Sci. 16 1883-1888 (1975). K. BONNET, Life Sci. 16 1877-1882 (1975). G. PASTERNAK, S. CHILDERS and S. SNYDER, Science. 208 514-516 (1980). S. ATWEH and M. J. KUHAR, Brit. Med. Bull. 39 47-52 (1983). C. A. CUELLO, Brit. Med. Bull. 39 11-16 (1983).
Vol. 39, No. 5, 1986
19. 20. 21. 22. 23. 24.
Methadone and Cyclic Nucleotides
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M. FLAHERTY and D. SADAVA, Arch. Int. Pharmacodyn. Ther. 212 103-108 (1974). L. J. PELLIGRINO, A. S. PELLIGRINO, and A.J. CUSHMAN, A Stereotaxic Atlas of the Rat Brain edn.2, Plenum, New York (1979). M. GOSSOP, B. BRADLEY, J. STRANG, and P. CONNELL, Brit. J. Psychiat. 144 203-208 (1984). G. KOSKI and W. A. KLEE, Proc. Natl. Acad. Sci. 784185-4189 (1981). C. BARCHFIELD and F. MEDZIHRADSKY, Biochem. Biophys. Res. Commun. 121 641-648 (1984). M. KENNEDY, Ann. Rev. Neurosci. 6 493-518 (1983).
Pergamon Journals
THE EFFECT OF METHADONE ADDICTION ON CYCLIC NUCLEOTIDE LEVELS IN REGIONS OF RAT BRAIN
D. Sadava and B. Mack Joint Science Department The Claremont Colleges Claremont, California, 91711
(Received in final form May 15, 1986) Summary Studies of tissue culture cells and tissue slices have implicated the nucleotides, cyclic AMP and cyclic GMP, in the mechanism of action of opiates. However, there are little in vivo data to corroborate this hypothesis. We addicted rats to the synthetic opiate, methadone, by providing the drug in their drinking water (dosage 2.1 mg./kg./day). The two cyclic nucleotides were measured in four brain areas which contain a high concentration of opiate receptors: amygdala, neostriatum, periventricular grey, and thalamus. Data were obtained after acute exposure of the rats to the drug (i day), tolerance (35 days), withdrawal (35 days on drug then l day off drug), and readjustment (35 days on drug then 21 days off drug). Cyclic GMP levels were low (0.03 pmol./mg, tissue) in the four regions and did not differ significantly during the experiment. Cyclic AMP levels were higher (1-3 pmol./mg.) and fluctuated consistently in the four regions. After acute methadone treatment, there was a reduction in cyclic AMP, which continued at lower levels after tolerance. One day of withdrawal led to increased cAMP, which rose to near control levels. After readjustment, the levels were reduced. These data indicate an involvement of cyclic AMP in the addiction and withdrawal processes in the intact animal. The nucleotides cyclic-3',5'-adenosine monophosphate (cAMP) and cyclic-3',5'-guanosine monophosphate (cGMP) have been implicated in the mode of action on the brain of endogenous and exogenous opiates (1,2). A model has been proposed in which neuronal cAMP is reduced during acute exposure to an opiate, slowly returns to normal during tolerance, increases during withdrawal and returns to baseline value after a period of readjustment in the absence of the opiate (3,4). Evidence in support of this model has come from studies on rat brain homogenates (5), guinea pig ileum (6), and mouse neuroblastoma-glioma hybrid cultured cells (7). In addition, increases in plasma cAMP (8) and platelet adenylate cyclase (9) have been noted during withdrawal from heroin in humans. Since cGMP often acts in opposition to cAMP (i0), and since cGMP is present in nervous tissue where it regulates depolarization (ii), opiates might be expected to affect neuronal cGMP levels in a manner opposite to their affect on cAMP. Indeed, increased cGMP has been noted after acute opiate incubation of rat brain slices (12) and hybrid cultured cells (13). There have been few in vivo studies of the effects of exogenous opiates on brain cyclic nucleotide levels. When rats were made tolerant to morphine, rapid withdrawal led to increased cAMP in the striatum (14). Implantation of morphine 0024-3206/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
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pellets in rats led to decreased cAMP, and initially, increased cGMP, in caudate, hypothalamus, substantia nigra, and thalamus (15). While these data are consistent with an opiate-cyclic nucleotide relationship, there has not been a systematic study in which the nucleotides were measured following in vivo addiction and withdrawal. We report here such a study, in which rats were addicted to the synthetic narcotic, methadone. In addition, opiate receptors are well characterized, and appear to be concentrated in the limbic system and paleospinothalamic pathways (16-18). Therefore, we measured cAMP and cGMP in amygdala, neostriatum, periventricular grey, and thalamus.
Methods Male albino rats, initial weight 200-220 g., were obtained from Simonson Laboratories, Gilroy, California. They were housed individually at 20°C, with a a 12 hr. photoperiod, and fed standard laboratory chow. Methadone was presented in the drinking water as previously described (19). This is a model for addiction in which the animals show a compulsion, tolerance, and physical dependency for the drug. Tolerance develops after two weeks of exposure to methadone, and withdrawal symptoms are observed after 1 day of removal of the drug from the drinking water of addicted animals (19). Controls did not receive the drug in their water. The average dosages of methadone were: Day I: 0.8 mg/kg/day; day 6 : 1 . 2 mg/kg/day; day 1 0 : 1 . 4 mg/kg/day; day 1 8 : 1 . 8 mg/kg/day; day 2 5 : 2 . 0 mg/kg/day; day 3 5 : 2 . 1 mg/kg/day. Groups of four animals were sacrificed between 1-5 PM according to the following treatments: I. Control. 2. Acute: drug presented for 1 day. 3. Tolerance: drug presented for 35 days. 4. Withdrawal: drug presented for 35 days, then water without drug for 1 day. 5. Readjustment: drug presented for 35 days, then water without drug for 21 days. Animals were sacrificed by cervical dislocation and the heads frozen immediately in liquid nitrogen. The brain was removed, and, following location via a stereotaxic atlas (20), neostriatum, periventricular grey, thalamus, and amygdala were removed and weighed (mean 15.9 mg.). Tissues were homogenized immediately with i0 strokes of a teflon-glass homogenizer in 350 ul. of ice-cold 0.4 mM EDTA. Homogenates were heated in a boiling water bath for 3 min., and the samples centrifuged at 29,000 x g. for 15 min. to remove coagulated molecules. The supernatants were kept frozen at -20°C for up to 3 weeks prior to cyclic nucleotide assay, cAMP was measured by protein binding assay and cGMP by radioimmunoassay (kits from Amersham, Arlington Heights Illinois). All assays were done in duplicate. Statistical analyses were done by the t-test for paired data.
Results Levels of cAMP, expressed on a tissue weight basis (Table I), were similar to those previously reported for rat brain (14,15). This indicates that the methods of sacrifice and analysis did not lead to artificially altered cAMP levels. In the four regions which contain relatively high concentrations of receptors for methadone (17), similar changes in cAMP were noted over the course of addiction and withdrawal. After one day of exposure of the animals to the drug (acute treatment), there was a reduction cAMP. After five weeks of drug treatment (addiction), there was less tissue cAMP than the acute situation. In all tissues examined, withdrawal of methadone
Vol. 39, No. 5, 1986
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led to significantly increased cAMP after 1 day. Three weeks after initial withdrawal (readjustment), cAMP levels in all regions were reduced from the immediate withdrawal period to nearly tolerance levels.
TABLE I Cyclic AMP Levels in Brain Regions
Condition
PG
I. 2. 3. 4. 5.
2.25 1.36 1.28 1.85 1.37
Control Acute Tolerance Withdrawal Readjust
TH
• ± ± ± *
.18 .23 .21 .14 .17
NS
3.05" 2.39 ± 1.64 ± 2.51 * 1.89 ±
.26 .31 .17 .19 .12
1.45' 0.85 ± 0.69 ~ 1.59 ± 0.84 *
AM
.12 .27 .16 .19 .25
2.56 2.38 1.95 2.23 2.17
± ± ± ± ±
.21 .27 .08 .17 .19
Data are expressed as mean concentrations ± S.D. of each region from eight animals in pmol./mg, tissue weight. Abbreviations: PG: periventricular grey; TH: thalamus; NS: neostriatum; AM: amygdala. For each tissue, means were significantly different for each treatment ( p < 0.01) with the exceptions of the following: PG: 2,3, and 5; TH: 2 and 4; NS: 2,3, and 5; 1 and 4; AM: 1,2 and 5; 4 and 5.
As was found in previous studies (i1,15), cGMP was present in brain in much smaller amounts than cAMP (Table II). The low concentrations of cGMP, coupled with the small amounts of tissues examined, led to homogenate concentrations that were near the lower limits of the assay system used. Within these limits, the levels of cGMP were similar throughout the experimental period.
TABLE II Cyclic GMP Levels in Brain Regions
Condition
PG
i. 2. 3. 4. 5.
0.031' 0.032± 0.028± 0.030± 0.037 ±
Control Acute Tolerance Withdrawal Readjust
TH
.017 .019 .008 .Oll .012
0.025* 0.032± 0.029~ 0.034± 0.018 ±
NS
.007 .017 .021 .012 .008
0.037± 0.033* 0.048± 0.029± 0.027 ±
AM
.016 .011 .012 .019 .008
Expression of data and abbreviations are as in Table I. tissue set were not significantly different (p > 0.01).
0.024± 0.029 ± 0.027 ± 0.029± 0.035±
.009 .006 .016 .011 .026
Means within each
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Vol. 39, No. 5, 198~
Discussion Data from the four brain regions (Table I) are consistent in many respects with the model for fluctuations of cAMP during opiate addiction and withdrawal (3) and argue against an involvement of fluctuations of cGMP (Table II). Following acute methadone treatment, cAMP levels were reduced, as the model predicts and in accordance with previous in vitro studies (5-7). However, during tolerance, cAMP levels did not return to control levels as predicted but remained low. It is possible that sufficient time was not allowed for tolerance to develop (5 weeks); however, in the addiction model used in these experiments this is usually sufficient for tolerance (19) and drug consumption data indicate that tolerance did develop. Withdrawal led to an immediate increase in cAMP, as predicted, and during readjustment the levels lowered toward baseline. That control levels were not reached during readjustment (21 days) may indicate that the period given was not sufficient for complete biochemical normalization. In humans, withdrawal symptoms are detectable for up to one month after the cessation of methadone administration (21). The means by which cAMP acts as an intermediary between opiate binding and neuronal activity are not clear. The binding of an opiate to its neuronal receptor leads to an inhibition of membrane-bound adenylate cyclase (22,23). Cellular cAMP is important in ionic changes involved in neuronal firing (4), and via protein phosphorylation regulates the formation and release of neurotransmitters (24). Studies of these parameters in the four regions examined in this report should clarify the role of cAMP in opiate action.
Acknowledgements We thank Mallinckrodt Chemical Works, St. Louis, Missouri, for a gift of the methadone used in these studies. Dr. B. Baker provided assistance in scaling down the radioimmunoassay for cGMP. This research was partially supported by a grants from Pitzer College and the W.M. Keck Foundation.
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. II. 12. 13. 14. 15. 16. 17. 18.
M. WOLLMAN, Prog. Neurobiol. 16 145-154 (1980). J. KEBABIAN, Adv. Cyclic Nucl. Res. 8 421-508 (1977). S. SNYDER, Sci. Am. 236 44-56 (1977). R . E . WEST and R. J. MILLER, Brit. Med. Bull. 39 53-58 (1983). Z. MERALI, B. TSANG, R.H. SINGHEL and P.A. HRDINA, Res. Comm. Chem. Path. Pharm. 14 29-37 (1976). A. REZVASNI, J. HUIDOBORA-TORO, J. PABLO, J. HU, and E. L. WAY, J. Pharmacol. Exp. Ther. 225 251-255 (1983). P. LAW, D. HOM, and H. H, LOH, Mol. Pharmacol. 23 26-35 (1983). H . L . WEN, W. K. HO, H. K. WONG, Z. D. MEHAL, Y. NG, and L. MA, Comp. Med. East West. 6 241-245 (1979). G . N . PANDEr, F. DELEON-JONES, E. INWANG and J. M. DAVIS, Clin. Pharmacol. Ther. 27 607-611 (1980). N. D. GOLDBERG and M. HADDOX, Ann. Rev. Biochem. 46 823-896 (1977). U. WALTER and P. GREENGARD, Curr. Top. Cell. Regul. 19 219-256 (1981). R. GULLIS, J. TRABER, and B. HAMPRECHT, Nature. 256 57-59 (1975). G. GWYNN and E. COSTA, Proc. Natl. Acad. Sci. 79 690-694 (1982). C. MEHTA and W. E. JOHNSON, Life Sci. 16 1883-1888 (1975). K. BONNET, Life Sci. 16 1877-1882 (1975). G. PASTERNAK, S. CHILDERS and S. SNYDER, Science. 208 514-516 (1980). S. ATWEH and M. J. KUHAR, Brit. Med. Bull. 39 47-52 (1983). C. A. CUELLO, Brit. Med. Bull. 39 11-16 (1983).
Vol. 39, No. 5, 1986
19. 20. 21. 22. 23. 24.
Methadone and Cyclic Nucleotides
481
M. FLAHERTY and D. SADAVA, Arch. Int. Pharmacodyn. Ther. 212 103-108 (1974). L. J. PELLIGRINO, A. S. PELLIGRINO, and A.J. CUSHMAN, A Stereotaxic Atlas of the Rat Brain edn.2, Plenum, New York (1979). M. GOSSOP, B. BRADLEY, J. STRANG, and P. CONNELL, Brit. J. Psychiat. 144 203-208 (1984). G. KOSKI and W. A. KLEE, Proc. Natl. Acad. Sci. 784185-4189 (1981). C. BARCHFIELD and F. MEDZIHRADSKY, Biochem. Biophys. Res. Commun. 121 641-648 (1984). M. KENNEDY, Ann. Rev. Neurosci. 6 493-518 (1983).