EXPERIMENTAL
NEUROLOGY
117,216-218
(19%)
BRIEF COMMUNICATION Analgesic and Aversive Effects of Naloxone in BALB/c Mice ANTHONY L. VACCARINO,**~HI?L&NEPLAMONDON,~ANDRONALDMELZACK~ *Department
of Psychology,
University of New Orleans, Lakefront, New Orleans, McGill University, 1205 Dr. Penfield Avenue, Montreal,
Press,
Inc.
We have previously reported that systemic administration of either naloxone (20) or naltrexone (21) produces a dose-dependent analgesia in the formalin test in BALB/c mice. In addition, naloxone has been shown to have aversive properties as well (14). Although a number of investigators have reported naloxone-induced analgesia at low doses and hyperalgesia at high doses, naloxone produces analgesia in BALB/c mice at doses up to lOOO-fold greater than those previously reported to be analgesic (6,9,11,19,23). The finding that large doses of naloxone produce analgesia in BALB/c mice allows us to further examine the relationship between analgesia and affect, and to elucidate the neurochemistry underlying the actions of opiate antagonists. The formalin test was used to assess analgesia (7). The plantar surface of one hind paw was injected subcutaneously with 20 ~1 of 5% formalin. The amount of time the animal spent licking the injected paw was recorded for the period of lo-60 min following injection and provided an index of pain (8). Fifteen minutes prior to formalin, the mice were injected subcutaneously with saline or 0.1, 1, or 10 mg/kgnaloxone HCl (Endo Laboratories) in saline. Each group contained seven mice. The conditioned place preference test was used to assess the affective property of naloxone in BALB/c mice. The apparatus consisted of a start-box and two treat’
To whom
0014-48w92 Copyright All rights
correspondence
should
be addressed.
$5.00 0 1992 by Academic Press, Inc. of reproduction in any form reserved.
of Psychology,
ment compartments. One compartment was black with a wire grid floor, and the other was white with a wood chip floor. On the day before conditioning, the mice were placed in the start-box and allowed 15 min of free access to both compartments. The amount of time spent in the two compartments was recorded to detect any initial bias. No significant differences were found for the mean time spent in the black or white compartments (336.7 f. 35.0 and 356.7 + 37.1 s, respectively, t(17) = 0.29, n.s.) Three groups of six mice served as subjects. On conditioning days, the mice were injected subcutaneously with one of three doses of naloxone (0.1, 1.0, or 10 mg/ kg) and confined to one compartment for 45 min. On alternating days, the mice received saline and were confined for an equal amount of time to the other compartment. Half the mice received naloxone pairings in the black compartment and half in the white compartment. The mice received a total of six pairings (3-naloxone, 3-saline). On the test day (24 h after the last conditioning session), the mice were placed in the start-box in a drugfree state and allowed free access to the compartments for 20 min. The amount of time spent in the naloxoneand saline-paired compartments was recorded. Figure 1 shows the results for both the formalin test (expressed as percentage analgesia) and the conditioned place preference test (expressed as percentage aversion). Naloxone produced a dose-dependent analgesia in the formalin test, F(3,24) = 17.00, P < 0.0001. Significant analgesia, compared to saline controls, was obtained at 1 and 10 mg/kg (P < 0.05, Sheffe’s test). These results are in agreement with our previous report of naloxone analgesia in BALB/c mice (20). In the conditioned place preference test, naloxone produced a dose-dependent aversion, F(2,15) = 4.10, P < 0.05, see Fig. 1. The mice spent a significantly shorter amount of time in the naloxone-paired compartment than in the saline-paired compartment at both the 1 and 10 mg/kg doses of naloxone (P < 0.05, Sheffe’s test). Microinjection studies often demonstrate a common neurochemical substrate for morphine reward (22) and morphine analgesia (5, 16, 17, 24), suggesting that
Opioid antagonists have been shown to produce dosedependent analgesia in the formalin test in BALB/c mice. In light of this paradoxical finding, the motivational-affective property of naloxone was examined in BALB/c mice. Naloxone produced a conditioned place aversion at doses which were also found to produce analgesia in the formalin test (1 and 10 mglkg). In addition, the analgesia produced by 1 mg/kg naloxone was completely abolished in mice pretreated with nor-binaltorphimine, a highly selective K-opioid antagonist. Norbinaltorphimine on its own, however, had no effect. These results suggest that the analgesic actions of naloxone may be due to an interaction with K receptors. o ioo2 Academic
Louisiana 70148; and TDepartment Quebec, Canada H3A lB1
216
BRIEF
217
COMMUNICATION
opiate analgesia is accompanyed by positive affect. However, the present results fail to confirm this relationship. While naloxone produced a dose-dependent analgesia in the formalin test, the same doses were also found to produce aversion. Therefore, naloxone can reduce pain in animals in whom it also produces aversion. Exposure to stressful situations has been shown to have pain suppressing effects, a phenomenon known as stress-induced analgesia (2). It could be argued that since naloxone produces aversion it exerts its analgesic effect by a mechanism similar to that observed during stress. If the effect was indeed due to the stress produced by naloxone-induced aversion then naloxone analgesia should be commonly observed. However, with the exception of BALB/c mice, aversive doses of naloxone fail to produce analgesia in the formalin test in rats (10, 15) and other strains of mice (20). One possible site for naloxone’s analgesic action is at the K-opioid receptor. Levine et al. (12) reported that very low doses of naloxone potentiate analgesia produced by the kappa ligand, pentazocine. Furthermore, Abbott et al. (1) have demonstrated that morphine analgesia in the formalin test is partially K-mediated. These results are consistent with the properties of naloxone in BALB/c mice and may reflect an interaction of naloxone at the K receptor in the formalin test. BALB/c mice were therefore injected ip with 5 mg/kg of nor-binaltorphimine (Research Biochemicals Inc.), a highly selective K-opioid antagonist, 45 min prior to administration of an analgesic dose of naloxone (1.0 mg/ kg, see Fig. 1) or saline (n = 6 for both). The animals were then tested for analgesia in the formalin test as described above. The results, expressed as total time spent licking the injected paw, are shown in Fig. 2. Analysis of variance revealed a significant group effect,
Sal
Nal
NBin+Nal
NBin
FIG. 2. Effects of nor-binaltorphimine on naloxone analgesia. Data are expressed as mean time (+SEM) spent licking the formalininjected paw after injection of saline (Sal), naloxone (Nal), nor-binaltorphimine prior to naloxone (NBin + Nal), or nor-binaltorphimine alone (NBin). *Significant difference (P < 0.05, Sheffe’s test).
F(3,22) = 9.43, P < 0.001. More specifically, the analgesia produced by 1.0 mg/kg naloxone was completely abolished in rats pretreated with nor-binaltorphimine (P < 0.05, Sheffe’s test). However, nor-binaltorphimine had no effect when administered alone. These results suggest that the analgesic actions of naloxone in the formalin test in BALB/c mice may be due to an activation of K receptors, either directly, or indirectly via a nonopioid mechanism. The role of the K receptor in mediating the conditioned place aversion observed in this study is not known. While p agonists produce reward (4, 13), selective K agonists have been shown to produce aversion (3, 13, 18). It is possible, therefore, that the aversion produced by naloxone in this study is due to both the traditional antagonist properties of naloxone and the potential activation of Kreceptors by naloxone. However, further studies are needed to examine the role of the K-opioid receptor in mediating naloxone aversion in BALB/c mice. ACKNOWLEDGMENTS This NSERC
work was Fellow.
supported
by NSERC
Grant
A7891.
A.L.V
is an
REFERENCES
Naloxone
FIG.
1. Effects
1 Dose (mglkg)
of naloxone in the formalin and conditioned place preference tests. Data from the formalin test are expressed as percentage analgesia (+SEM). Data from the conditioned place preference test are expressed as percentage aversion (2SEM). Percentage analgesia = (S-N/S) X 100, where S is the mean time spent licking in the saline control group and N is the mean time spent licking after injection of naloxone. Percentage aversion = (S-N/S+N) X 100, where S is the mean time spent in the saline-paired compartment and N is the mean time spent in the naloxone-paired compartment. *Significant difference from saline (P < 0.05, Sheffe’s test).
ABBOTT, F. V., K. B. J. FRANKLIN, AND R. B. LIBMAN. 1986. A dose-ratio comparison of mu and kappa agonists in the formalin test and thermal pain. Life Sci. 39: 2017-2024. AKIL, H. M., J. MADDEN, R. L. PATRICK, AND 1976. Stress-induced increases in endogeneous Concurrent analgesia and it’s partial reversal Opiates and Endogenous Opiate Peptides (H. W. pp. 62-70. Elsevier Press, Amsterdam.
J. D. BURCHAT. opiate peptides: by naloxone. In Kosterlitz, Ed.),
BALS-KUBIK, R., A. HERZ, AND T. S. SHIPPENBERG. 1989. Evidence that the aversive effects of opioid antagonists and kappaagonists are centrally mediated. Psychopharmacology 98: 203206. BEACH, H. D. 1957. Morphine addiction in rats. 1957. Can. J. Psychol. 11: 104-112.
218
BRIEF
COMMUNICATION
CARR, K. D., AND S. UYSAL. 1985. Evidence of a supraspinal opioid analgesic mechanism engaged by lateral hypothalamic electrical stimulation. Bruin Res. 336: 55-62. 6. DICKENSON, A. H., D. LE BARS, AND J. M. BESSON. 1981. Endogeneous opiates and nociception: A possible functional role in both pain inhibition and detection as revealled by intrathecal naloxone. Neurosci. L&t. 24: 161-164. 7. DUBUISSON, D., AND S. G. DENNIS. 1977. The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brainstem stimulation in rats and cats. Pain 4: 161174. 8. HUNSKAAR, S., 0. B. FASMER, AND K. HOLE. 1985. Formalin pain in mice, a useful technique for evaluating mild analgesics. J. 5.
Neurosci.
Methods.
14:
69-76.
9. KAYSER, V., AND G. GUILBAUD. 1981. Dose-dependent analgesic and hyperalgesic effects of naloxone in arthritic rats. Brain Res. 226: 344-348. 10. KOCHER, L. 1988. Sytemic naloxone does not affect pain related behaviour in the formalin test in rat. Physiol. Behav. 43: 265268. 11. LEVINE, J. D., N. C. GORDON, AND H. L. FIELDS. 1979. Naloxone dose-dependently produces analgesia and hyperalgesia in postoperative pain. Nature 278: 740-741. 12. LEVINE, J. D., N. C. GORDON, Y. 0. TAIWO, AND T. J. CODERRE. 1988. Potentiation of pentazocine analgesia by low-dose naloxone. J. Clin. Invest. 82: 15’74-1576. 13. MUCHA, R. F., AND A. HERZ. 1985. Motivational properties of kappa and mu opioid agonists studied with place preference conditioning. Psychopharmacology 86: 274-280. 14. MUCHA, R. F., D. VAN DER KOOY, D. ~‘SHAUGHNESSY, AND P. BUCENIEKS. 1982. Drug reinforcement studied by the use place conditioning in rat. Bruin Res. 243: 91-105.
15. NORTH, M. A. 1977. Naloxone reversal of morphine analgesia but failure to alter reactivity to pain in the formalin test. Life Sci. 22:
295-302.
16. PERT, A., AND T. YAKSH. 1974. Sites of morphine analgesia in the primate brain: relation to pain pathways. Brain Res. 80: 135-140. 17. SHARPE, L. G., J. E. GARNE?T, AND T. J. CICERO. 1974. Analgesia and hyperreactivity produced by micro-injections of morphine into the periaqueductal grey matter of the rats. Behav. Biol. 11: 303-313. 18. SHIPPENBERG, T. S., AND A. HERZ. 1986. Differential effects of mu and kappa agonists on motivational processes. NZDA Res Mono.
75: 563-566.
19. UEDA, H., N. FUKUSHIMA, T. KITAO, M. GE, AND H. TAKAGI. 1986. Low doses of naloxone produce analgesia in the mouse brain by blocking presynaptic autoinhibition of enkephalin release. Neurosci. Lett. 65: 247-252. 20. VACCAFUNO, A. L., R. A. R. TASKER, AND R. MELZACK. 1988. Systemic administration of naloxone produces analgesia in BALBlc mice. Neurosci. Lett. 84: 103-107. 21. VACCARINO, A. L., R. A. R. TASKER, AND R. MELZACK. 1989. Analgesia produced by normal doses of opiate antagonists alone and in combination with morphine. Pain 36: 103-109. 22. VAN DER KOOY, D., R. F. MUCHA, M. ~‘SHAUGHNESSY, AND P. BUCENIEKS. 1982. Reinforcing effects of brain micro-injections of morphine revealled by conditioned place preference. Brain Res. 243: 107-117. 23. WOOLF, C. J. 1980. Analgesia and hyperalgesia produced in the rat by intrathecal naloxone. Bruin Res. 189: 593-597. 24. ZHOU, Z. F., Y. T. XUAN, AND J. S. HAN. 1974. Analgesic effect of morphine injected into the habenula, nucleus accumbens or amygdala of rabbits. Actu Pharm. Sinica. 5: 150-153.
NEUROLOGY
117,216-218
(19%)
BRIEF COMMUNICATION Analgesic and Aversive Effects of Naloxone in BALB/c Mice ANTHONY L. VACCARINO,**~HI?L&NEPLAMONDON,~ANDRONALDMELZACK~ *Department
of Psychology,
University of New Orleans, Lakefront, New Orleans, McGill University, 1205 Dr. Penfield Avenue, Montreal,
Press,
Inc.
We have previously reported that systemic administration of either naloxone (20) or naltrexone (21) produces a dose-dependent analgesia in the formalin test in BALB/c mice. In addition, naloxone has been shown to have aversive properties as well (14). Although a number of investigators have reported naloxone-induced analgesia at low doses and hyperalgesia at high doses, naloxone produces analgesia in BALB/c mice at doses up to lOOO-fold greater than those previously reported to be analgesic (6,9,11,19,23). The finding that large doses of naloxone produce analgesia in BALB/c mice allows us to further examine the relationship between analgesia and affect, and to elucidate the neurochemistry underlying the actions of opiate antagonists. The formalin test was used to assess analgesia (7). The plantar surface of one hind paw was injected subcutaneously with 20 ~1 of 5% formalin. The amount of time the animal spent licking the injected paw was recorded for the period of lo-60 min following injection and provided an index of pain (8). Fifteen minutes prior to formalin, the mice were injected subcutaneously with saline or 0.1, 1, or 10 mg/kgnaloxone HCl (Endo Laboratories) in saline. Each group contained seven mice. The conditioned place preference test was used to assess the affective property of naloxone in BALB/c mice. The apparatus consisted of a start-box and two treat’
To whom
0014-48w92 Copyright All rights
correspondence
should
be addressed.
$5.00 0 1992 by Academic Press, Inc. of reproduction in any form reserved.
of Psychology,
ment compartments. One compartment was black with a wire grid floor, and the other was white with a wood chip floor. On the day before conditioning, the mice were placed in the start-box and allowed 15 min of free access to both compartments. The amount of time spent in the two compartments was recorded to detect any initial bias. No significant differences were found for the mean time spent in the black or white compartments (336.7 f. 35.0 and 356.7 + 37.1 s, respectively, t(17) = 0.29, n.s.) Three groups of six mice served as subjects. On conditioning days, the mice were injected subcutaneously with one of three doses of naloxone (0.1, 1.0, or 10 mg/ kg) and confined to one compartment for 45 min. On alternating days, the mice received saline and were confined for an equal amount of time to the other compartment. Half the mice received naloxone pairings in the black compartment and half in the white compartment. The mice received a total of six pairings (3-naloxone, 3-saline). On the test day (24 h after the last conditioning session), the mice were placed in the start-box in a drugfree state and allowed free access to the compartments for 20 min. The amount of time spent in the naloxoneand saline-paired compartments was recorded. Figure 1 shows the results for both the formalin test (expressed as percentage analgesia) and the conditioned place preference test (expressed as percentage aversion). Naloxone produced a dose-dependent analgesia in the formalin test, F(3,24) = 17.00, P < 0.0001. Significant analgesia, compared to saline controls, was obtained at 1 and 10 mg/kg (P < 0.05, Sheffe’s test). These results are in agreement with our previous report of naloxone analgesia in BALB/c mice (20). In the conditioned place preference test, naloxone produced a dose-dependent aversion, F(2,15) = 4.10, P < 0.05, see Fig. 1. The mice spent a significantly shorter amount of time in the naloxone-paired compartment than in the saline-paired compartment at both the 1 and 10 mg/kg doses of naloxone (P < 0.05, Sheffe’s test). Microinjection studies often demonstrate a common neurochemical substrate for morphine reward (22) and morphine analgesia (5, 16, 17, 24), suggesting that
Opioid antagonists have been shown to produce dosedependent analgesia in the formalin test in BALB/c mice. In light of this paradoxical finding, the motivational-affective property of naloxone was examined in BALB/c mice. Naloxone produced a conditioned place aversion at doses which were also found to produce analgesia in the formalin test (1 and 10 mglkg). In addition, the analgesia produced by 1 mg/kg naloxone was completely abolished in mice pretreated with nor-binaltorphimine, a highly selective K-opioid antagonist. Norbinaltorphimine on its own, however, had no effect. These results suggest that the analgesic actions of naloxone may be due to an interaction with K receptors. o ioo2 Academic
Louisiana 70148; and TDepartment Quebec, Canada H3A lB1
216
BRIEF
217
COMMUNICATION
opiate analgesia is accompanyed by positive affect. However, the present results fail to confirm this relationship. While naloxone produced a dose-dependent analgesia in the formalin test, the same doses were also found to produce aversion. Therefore, naloxone can reduce pain in animals in whom it also produces aversion. Exposure to stressful situations has been shown to have pain suppressing effects, a phenomenon known as stress-induced analgesia (2). It could be argued that since naloxone produces aversion it exerts its analgesic effect by a mechanism similar to that observed during stress. If the effect was indeed due to the stress produced by naloxone-induced aversion then naloxone analgesia should be commonly observed. However, with the exception of BALB/c mice, aversive doses of naloxone fail to produce analgesia in the formalin test in rats (10, 15) and other strains of mice (20). One possible site for naloxone’s analgesic action is at the K-opioid receptor. Levine et al. (12) reported that very low doses of naloxone potentiate analgesia produced by the kappa ligand, pentazocine. Furthermore, Abbott et al. (1) have demonstrated that morphine analgesia in the formalin test is partially K-mediated. These results are consistent with the properties of naloxone in BALB/c mice and may reflect an interaction of naloxone at the K receptor in the formalin test. BALB/c mice were therefore injected ip with 5 mg/kg of nor-binaltorphimine (Research Biochemicals Inc.), a highly selective K-opioid antagonist, 45 min prior to administration of an analgesic dose of naloxone (1.0 mg/ kg, see Fig. 1) or saline (n = 6 for both). The animals were then tested for analgesia in the formalin test as described above. The results, expressed as total time spent licking the injected paw, are shown in Fig. 2. Analysis of variance revealed a significant group effect,
Sal
Nal
NBin+Nal
NBin
FIG. 2. Effects of nor-binaltorphimine on naloxone analgesia. Data are expressed as mean time (+SEM) spent licking the formalininjected paw after injection of saline (Sal), naloxone (Nal), nor-binaltorphimine prior to naloxone (NBin + Nal), or nor-binaltorphimine alone (NBin). *Significant difference (P < 0.05, Sheffe’s test).
F(3,22) = 9.43, P < 0.001. More specifically, the analgesia produced by 1.0 mg/kg naloxone was completely abolished in rats pretreated with nor-binaltorphimine (P < 0.05, Sheffe’s test). However, nor-binaltorphimine had no effect when administered alone. These results suggest that the analgesic actions of naloxone in the formalin test in BALB/c mice may be due to an activation of K receptors, either directly, or indirectly via a nonopioid mechanism. The role of the K receptor in mediating the conditioned place aversion observed in this study is not known. While p agonists produce reward (4, 13), selective K agonists have been shown to produce aversion (3, 13, 18). It is possible, therefore, that the aversion produced by naloxone in this study is due to both the traditional antagonist properties of naloxone and the potential activation of Kreceptors by naloxone. However, further studies are needed to examine the role of the K-opioid receptor in mediating naloxone aversion in BALB/c mice. ACKNOWLEDGMENTS This NSERC
work was Fellow.
supported
by NSERC
Grant
A7891.
A.L.V
is an
REFERENCES
Naloxone
FIG.
1. Effects
1 Dose (mglkg)
of naloxone in the formalin and conditioned place preference tests. Data from the formalin test are expressed as percentage analgesia (+SEM). Data from the conditioned place preference test are expressed as percentage aversion (2SEM). Percentage analgesia = (S-N/S) X 100, where S is the mean time spent licking in the saline control group and N is the mean time spent licking after injection of naloxone. Percentage aversion = (S-N/S+N) X 100, where S is the mean time spent in the saline-paired compartment and N is the mean time spent in the naloxone-paired compartment. *Significant difference from saline (P < 0.05, Sheffe’s test).
ABBOTT, F. V., K. B. J. FRANKLIN, AND R. B. LIBMAN. 1986. A dose-ratio comparison of mu and kappa agonists in the formalin test and thermal pain. Life Sci. 39: 2017-2024. AKIL, H. M., J. MADDEN, R. L. PATRICK, AND 1976. Stress-induced increases in endogeneous Concurrent analgesia and it’s partial reversal Opiates and Endogenous Opiate Peptides (H. W. pp. 62-70. Elsevier Press, Amsterdam.
J. D. BURCHAT. opiate peptides: by naloxone. In Kosterlitz, Ed.),
BALS-KUBIK, R., A. HERZ, AND T. S. SHIPPENBERG. 1989. Evidence that the aversive effects of opioid antagonists and kappaagonists are centrally mediated. Psychopharmacology 98: 203206. BEACH, H. D. 1957. Morphine addiction in rats. 1957. Can. J. Psychol. 11: 104-112.
218
BRIEF
COMMUNICATION
CARR, K. D., AND S. UYSAL. 1985. Evidence of a supraspinal opioid analgesic mechanism engaged by lateral hypothalamic electrical stimulation. Bruin Res. 336: 55-62. 6. DICKENSON, A. H., D. LE BARS, AND J. M. BESSON. 1981. Endogeneous opiates and nociception: A possible functional role in both pain inhibition and detection as revealled by intrathecal naloxone. Neurosci. L&t. 24: 161-164. 7. DUBUISSON, D., AND S. G. DENNIS. 1977. The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brainstem stimulation in rats and cats. Pain 4: 161174. 8. HUNSKAAR, S., 0. B. FASMER, AND K. HOLE. 1985. Formalin pain in mice, a useful technique for evaluating mild analgesics. J. 5.
Neurosci.
Methods.
14:
69-76.
9. KAYSER, V., AND G. GUILBAUD. 1981. Dose-dependent analgesic and hyperalgesic effects of naloxone in arthritic rats. Brain Res. 226: 344-348. 10. KOCHER, L. 1988. Sytemic naloxone does not affect pain related behaviour in the formalin test in rat. Physiol. Behav. 43: 265268. 11. LEVINE, J. D., N. C. GORDON, AND H. L. FIELDS. 1979. Naloxone dose-dependently produces analgesia and hyperalgesia in postoperative pain. Nature 278: 740-741. 12. LEVINE, J. D., N. C. GORDON, Y. 0. TAIWO, AND T. J. CODERRE. 1988. Potentiation of pentazocine analgesia by low-dose naloxone. J. Clin. Invest. 82: 15’74-1576. 13. MUCHA, R. F., AND A. HERZ. 1985. Motivational properties of kappa and mu opioid agonists studied with place preference conditioning. Psychopharmacology 86: 274-280. 14. MUCHA, R. F., D. VAN DER KOOY, D. ~‘SHAUGHNESSY, AND P. BUCENIEKS. 1982. Drug reinforcement studied by the use place conditioning in rat. Bruin Res. 243: 91-105.
15. NORTH, M. A. 1977. Naloxone reversal of morphine analgesia but failure to alter reactivity to pain in the formalin test. Life Sci. 22:
295-302.
16. PERT, A., AND T. YAKSH. 1974. Sites of morphine analgesia in the primate brain: relation to pain pathways. Brain Res. 80: 135-140. 17. SHARPE, L. G., J. E. GARNE?T, AND T. J. CICERO. 1974. Analgesia and hyperreactivity produced by micro-injections of morphine into the periaqueductal grey matter of the rats. Behav. Biol. 11: 303-313. 18. SHIPPENBERG, T. S., AND A. HERZ. 1986. Differential effects of mu and kappa agonists on motivational processes. NZDA Res Mono.
75: 563-566.
19. UEDA, H., N. FUKUSHIMA, T. KITAO, M. GE, AND H. TAKAGI. 1986. Low doses of naloxone produce analgesia in the mouse brain by blocking presynaptic autoinhibition of enkephalin release. Neurosci. Lett. 65: 247-252. 20. VACCAFUNO, A. L., R. A. R. TASKER, AND R. MELZACK. 1988. Systemic administration of naloxone produces analgesia in BALBlc mice. Neurosci. Lett. 84: 103-107. 21. VACCARINO, A. L., R. A. R. TASKER, AND R. MELZACK. 1989. Analgesia produced by normal doses of opiate antagonists alone and in combination with morphine. Pain 36: 103-109. 22. VAN DER KOOY, D., R. F. MUCHA, M. ~‘SHAUGHNESSY, AND P. BUCENIEKS. 1982. Reinforcing effects of brain micro-injections of morphine revealled by conditioned place preference. Brain Res. 243: 107-117. 23. WOOLF, C. J. 1980. Analgesia and hyperalgesia produced in the rat by intrathecal naloxone. Bruin Res. 189: 593-597. 24. ZHOU, Z. F., Y. T. XUAN, AND J. S. HAN. 1974. Analgesic effect of morphine injected into the habenula, nucleus accumbens or amygdala of rabbits. Actu Pharm. Sinica. 5: 150-153.
