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Functional alteration of opioid receptor subtypes in the mice exhibited conditioned suppression in motility
Neuroscience Letters, 53 (1985) 263-266
263
Elsevier Scientific Publishers Ireland Ltd.
NSL 03116
F U N C T I O N A L A L T E R A T I O N OF OPIOID R E C E P T O R S U B T Y P E S IN T H E MICE E X H I B I T E D C O N D I T I O N E D S U P P R E S S I O N IN MOTILITY
TSUTOMU KAMEYAMA, TOSHITAKA MATSUNO
NABESHIMA,
HIROYUKI
KAMEI and KIYOSHI
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468 (Japan) (Received June 1st, 1984; Revised version received November 5th, 1984; Accepted November 7th, 1984)
Key words." conditioned suppression - opiate binding - opioid receptor subtypes - mouse Mice exhibit a marked suppression of motility (conditioned suppression) when placed in the same environment in which they had previously received the electric footshock. The present study was designed to investigate the functional change of opioid receptor subtypes in the conditioned suppression group using an opioid binding assay technique. In the synaptic membrane of the conditioned suppression group, the binding capacities of [3H]naloxone at high and low affinity binding sites and of [3H]phencyclidine at high affinity binding site were significantly increased compared to those of the control group. On the other hand, the binding capacity of [3H]ethylketocyclazocine at both affinity binding sites in the conditioned suppression group was not changed. These results suggest that the binding function of different opioid receptor subtypes may be altered differently by stress.
Rats and mice exhibit a marked suppression in motility when they are placed in the same environment in which they had previously received an electric footshock. This suppression is considered to be a conditioned emotional response to the environment (conditioned suppression) [1, 4-8, 12, 13]. Furthermore, the conditioned suppression is attenuated by morphine and the effect of morphine is completely antagonized by pretreatment with naloxone. Thus, it is possible that the opiateinduced reduction of conditioned suppression is mediated by opioid receptor sites [4]. Pharmacologically, opioid receptors have been assigned to one of three major groups named after prototypic drugs: mu for morphine, kappa for ketocyclazocine and sigma for SKF-10047 [11]. The discovery of the enkephalins [3] has led to the fourth classification for peptides: delta [9]. These observations have led to numerous studies attempting to elucidate each physiological function of different opioid receptors. In addition, based on pharmacological results, it has been reported that the suppression of motility in the conditioned suppression group may be mediated through the opioid receptor subtypes, sigma and mu receptors [12]. Therefore, we attempted to investigate, using an opioid binding assay technique, whether the function o f opioid receptor subtypes in conditioned suppression group was different from that in control group. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
264
Male ddY mice, 8 weeks of age, were used. Experiments were carried out as previously described [12] using a transparent acrylic rectangular cage (23 x 28 x 12 cm) with a metal wire floor. The test cage was located in a sound attenuated room illuminated with a 20 W bulb. A mouse was left in the test cage for 6 rain and given the electric footshock (0.1 Hz, 200 ms, 300 V DC) through an isolated stimulator (Nihon Koden, Tokyo, Japan). The animal received electric shocks in the range of 1.2-3 mA since the resistance varied between 100 and 250 k~. The animal was placed again in the same cage 24 h after the shock treatment, but at the time no electric footshock was given (conditioned suppression; shocked group). The motility of the shocked group was measured for 6 rain in the test cage surrounded by an OptoVarimex (Columbus Instruments, OH, U.S.A.), a locomotor activity meter, 24 h after the shock treatment. The control group underwent the same procedure as the shocked group except for the electric footshock. The mice were sacrificed by decapitation 6 min after being returned to the shock treatment apparatus. The whole brains, excluding cerebellum, were rapidly removed and synaptic membrane was prepared by the method of Sivam et al. [15]. [3H]Opiate binding was initiated by the addition of 0.2 ml of the membrane preparation to a mixture containing 0.2-20.0 nM of [3H]naloxone ([3H]NLX: mu antagonist), [3H]phencyclidine ([3H]PCP: sigma agonist) and [3H]ethylketocyclazocine ([3H]EKC: kappa agonist) in a total volume of 1 ml. Incubations were carried out at 25°C for 30 rain. The binding reaction was stopped by rapid filtering through Whatman G F / B filters and filters were washed twice with 5 ml of ice-cold 25 mM Tris-HC1 buffer (pH 7.6 at 4°C). The filters were transferred to scintillation counting vials containing 10 ml of ACS 11 (Amersham Co., Arlington Heights, IL). The vials were shaken for 60 min and the radioactivity was measured using a Model 3255 Tri-Carb Liquid Scintillation Spectrometer System. Non-specific binding was defined by 5 /~M nonradioactive NLX, P C P and EKC in the cases of [3H]NLX, [3H]PCP and [3H]EKC, respectively. Filters presoaked in 0.1% poly-L-lysine at 4°C for 120 rain were used for the binding experiments of [3H]PCP to cut 'specific' ligand binding to the filters [18]. The protein concentration was determined by the method of Lowry et al. [10]. Analysis of biphasic Scatchard plots was performed by the method of Rosenthal [14] using a computer program to obtain the dissociation constant (Ka) and the maximum binding capacity (Bmax). When applicable, statistical significances were analyzed by Student's t-test for biochemical data or M a n n - W h i t n e y ' s U-test for behavioral data. When the mice were returned to the same apparatus in which they had been given an electric shock, they exhibited a marked suppression of motility (Control group: 2005.3 +68.5 counts (100%); Conditioned suppression group: 730.7 +_92.3 counts (36.4%), P < 0 . 0 1 ) , in agreement with our previous results [4-8, 12, 13]. On the other hand, in the synaptic membrane of the conditioned suppression group, the B .... but not Ka of [3H]NLX at high and low affinity binding sites and of [3H]PCP at high affinity binding sites increased significantly compared to those of the control
265
group (Table I). On the contrary, the Kd and Bmaxvalues of [3H]EKC at both affinity binding sites in the conditioned suppression group were not changed (Table I). We have reported that both cyclazocine (CLZ) and PCP, sigma agonists, diminish and morphine, a m u agonist, partially reduces the conditioned suppression of motility, while EKC, a kappa agonist, and methionine-enkephalin, a delta agonist, fail to affect it [12]. CLZ is classified as an opiate agonist-antagonist. However, since it has been reported that CLZ causes behavioral arousal and bizarre behavior, as does SKF-10047 [2], CLZ may be a relative sigma-like agonist. In addition, PCP binds to a sigma receptor although it does not belong to the opiates [16, 17]. Taking these findings into consideration, we have suggested that sigma and mu receptors may be important for opiate-induced attenuation of the conditioned suppression of motility [12]. The present results confirmed our hypothesis that in the synaptic membrane of mice showing conditioned suppression, the binding capacities of [3H]PCP and [3H]NLX binding sites increased significantly compared to the control group, but that of [3H]EKC binding sites did not alter. Taking the present and previous results together, we could consider that the binding capacities of the sigma and mu receptors might increase in compensation since the neuronal function mediated by these might decrease in the conditioned suppression group. On the other hand, though methionine-enkephalin does not affect the conditioned suppression of motility [12], the binding capacity of [3H]D-Ala-Met-enkephalinamide (a delta agonist) decreased in the conditioned suppression group (unpublished result). Therefore, a role of the delta receptor in the conditioned suppression remains to be investigated. TABLE 1 KINETIC P A R A M E T E R S OF O P I A T E BINDING TO BRAIN SYNAPTIC M E M B R A N E IN CONT R O L A N D C O N D I T I O N E D SUPPRESSION (CS) MICE The dissociation constant (Kd) and m a x i m u m binding capacity (Bmax) were determined by the method of Rosenthal [14] using a computer program. The numbers in parentheses show the number of independent experiments, each in triplicate. * P < 0 . 0 5 , * * P < 0 . 0 1 compared to control (Student's t-test). [3H]Ligand
Treatment
Affinity
Ka (nM)
Bmax ( f m o l / m g protein)
[3H]Naloxone
Control (4)
High Low High Low
0.44 _+0.08 32.52+3.79 0.59_+0.08 33.72 _+ 1.50
14.20_+ 2.62 1358.95+99.28 35.51 _+ 4.76** 1816.53 _+ 13.45"
High Low High Low
0.32 _+0.02 40.87 _+2.35 0.45_+ 0.05 42.21 _+4.80
22.92 _+ 1.18 1462.88 _+64.38 63.03 _+ 6.92** 1305.36+87.07
High Low High Low
0.13 -+ 0.02 19.93 -+ 4.01 0.12_+0.02 15.17_+0.56
CS (4)
[3H]Phencyclidine
ControL (3) CS (3)
[3H]Ethylketocyclazocine
Control (3) CS (3)
19.57 -+ 2.06 309.24 _+40.27 23.00_+ 1.77 286.34_+ 8.03
266
In conclusion, the binding function of different opioid receptor subtypes may be altered differently by stress. In addition, sigma and mu receptors may be important for the conditioned suppression of motility in mice. We thank Mr. Y. Noda and Mr. K. lshikawa for their excellent technical assistance, H. Furukawa, Ph.D., for synthesis of phencyclidine hydrochloride, and Sterling-Winthrop Research Institute (Rensselaer, NY) and Endo Laboratories (Wilmington, DE) for supplying ethylketocyclazocine methanesulfonate and naloxone hydrochloride, respectively. I Babbini, M., Gaiardi, M. and Bartoletti, M., Effect of morphine on a quickly learned conditioned suppression in rats, Psychopharmacologia, 33 (1973) 329-332. 2 Cowan, A., Simple in vivo tests that differentiate prototype agonists at opiate receptors, Life Sci., 28 (1981) 1559-1570. 3 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. and Morris, H.R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975) 577-579. 4 Kameyama, T. and Nagasaka, M., The effect of analgesics oil quickly learned conditioned suppression in mice, Neuropharmacology, 21 (1982) 1283-1289. 5 Kameyama, T. and Nagasaka, M., Effects of apomorphine and diazepam oil a quickly learned conditioned suppression in rats, Pharmacol. Biochem. Behav., 17 (1982) 59-63. 6 Kameyama, T. and Nagasaka, M., Effects of apomorphine and methamphetamine on a quickly learned conditioned-suppression response in rats, Neuropharmacology, 22 (1983) 813 817. 7 Kameyama, T. and Nagasaka, M., Effect of naloxone on a quickly learned conditioned suppression in rats following cessation of chronic haloperidol treatment, Brain Res., 275 (1983) 389-391. 8 Kameyama, T. and Nagasaka, M., A mode of aclion of morphine on a quickly learned conditioned suppression in mice, J. Pharm. Dyn., 7 (1984) 151 -155. 9 1,ord, J., Waterfield, D., Hughes, J. and Kosterlitz, H.W., Endogenous opioid peptides: multiple agonists and receptors, Nature tLond.), 267 (1977) 495-499. 10 Lowry, O.H,, Rosebrough, N..I., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.A. and Gilbert, P.E., The effect of morphine and nalorphine-like drugs in non-dependent and morphine dependent chronic spinal dog, J. Pharmacol. Exp. Ther., 197 (1976) 517-522. 12 Nabeshima, T., Yamada, K. and Kameyama, T., Effects of opiate agonists oil the conditioned suppression in motility of mice, Neurosci. Lett., 39 (1983) 301-306. 13 Nagasaka, M. and Kameyama, T., Effects of diazepam, meprobamate, chlorpromazine and apomorphine on a quickly learned conditioned suppression in mice, J. Pharm. Dyn., 6 (1983) 523-526. 14 Rosenthal, H.E., A graphic method for the determination and presentation of binding parameters in a complex system, Analyt. Biochem., 20 (1967) 525 532. 15 Sivam, S.P., Nabeshima, T. and Ho, I.K., Alterations of synaptic high and low affinity opiate binding sites after acute and chronic morphine administration in mice, Progr. Neuro-Psychopharmacol. Biol. Psychiat., 6 (1982) 119-127. 16 Zukin, R.S. and Zukin, S.R., Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites, Molec. Pharmacol., 20 (1981) 246-254. 17 Zukin, R.S, and Zukin, S.R., Multiple opiate receptors: emerging concepts, Life Sci., 29 (1981) 2681-2690. 18 Zukin, S.R., Fitz-Syage, M.L., Nichtenhausar, R. and Zukin, R.S., Specific binding of [3H]phencyclidine in rat central nervous tissue: Further characterization and technical consideration, Brain Res., 258 (1983) 277 284.
263
Elsevier Scientific Publishers Ireland Ltd.
NSL 03116
F U N C T I O N A L A L T E R A T I O N OF OPIOID R E C E P T O R S U B T Y P E S IN T H E MICE E X H I B I T E D C O N D I T I O N E D S U P P R E S S I O N IN MOTILITY
TSUTOMU KAMEYAMA, TOSHITAKA MATSUNO
NABESHIMA,
HIROYUKI
KAMEI and KIYOSHI
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468 (Japan) (Received June 1st, 1984; Revised version received November 5th, 1984; Accepted November 7th, 1984)
Key words." conditioned suppression - opiate binding - opioid receptor subtypes - mouse Mice exhibit a marked suppression of motility (conditioned suppression) when placed in the same environment in which they had previously received the electric footshock. The present study was designed to investigate the functional change of opioid receptor subtypes in the conditioned suppression group using an opioid binding assay technique. In the synaptic membrane of the conditioned suppression group, the binding capacities of [3H]naloxone at high and low affinity binding sites and of [3H]phencyclidine at high affinity binding site were significantly increased compared to those of the control group. On the other hand, the binding capacity of [3H]ethylketocyclazocine at both affinity binding sites in the conditioned suppression group was not changed. These results suggest that the binding function of different opioid receptor subtypes may be altered differently by stress.
Rats and mice exhibit a marked suppression in motility when they are placed in the same environment in which they had previously received an electric footshock. This suppression is considered to be a conditioned emotional response to the environment (conditioned suppression) [1, 4-8, 12, 13]. Furthermore, the conditioned suppression is attenuated by morphine and the effect of morphine is completely antagonized by pretreatment with naloxone. Thus, it is possible that the opiateinduced reduction of conditioned suppression is mediated by opioid receptor sites [4]. Pharmacologically, opioid receptors have been assigned to one of three major groups named after prototypic drugs: mu for morphine, kappa for ketocyclazocine and sigma for SKF-10047 [11]. The discovery of the enkephalins [3] has led to the fourth classification for peptides: delta [9]. These observations have led to numerous studies attempting to elucidate each physiological function of different opioid receptors. In addition, based on pharmacological results, it has been reported that the suppression of motility in the conditioned suppression group may be mediated through the opioid receptor subtypes, sigma and mu receptors [12]. Therefore, we attempted to investigate, using an opioid binding assay technique, whether the function o f opioid receptor subtypes in conditioned suppression group was different from that in control group. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
264
Male ddY mice, 8 weeks of age, were used. Experiments were carried out as previously described [12] using a transparent acrylic rectangular cage (23 x 28 x 12 cm) with a metal wire floor. The test cage was located in a sound attenuated room illuminated with a 20 W bulb. A mouse was left in the test cage for 6 rain and given the electric footshock (0.1 Hz, 200 ms, 300 V DC) through an isolated stimulator (Nihon Koden, Tokyo, Japan). The animal received electric shocks in the range of 1.2-3 mA since the resistance varied between 100 and 250 k~. The animal was placed again in the same cage 24 h after the shock treatment, but at the time no electric footshock was given (conditioned suppression; shocked group). The motility of the shocked group was measured for 6 rain in the test cage surrounded by an OptoVarimex (Columbus Instruments, OH, U.S.A.), a locomotor activity meter, 24 h after the shock treatment. The control group underwent the same procedure as the shocked group except for the electric footshock. The mice were sacrificed by decapitation 6 min after being returned to the shock treatment apparatus. The whole brains, excluding cerebellum, were rapidly removed and synaptic membrane was prepared by the method of Sivam et al. [15]. [3H]Opiate binding was initiated by the addition of 0.2 ml of the membrane preparation to a mixture containing 0.2-20.0 nM of [3H]naloxone ([3H]NLX: mu antagonist), [3H]phencyclidine ([3H]PCP: sigma agonist) and [3H]ethylketocyclazocine ([3H]EKC: kappa agonist) in a total volume of 1 ml. Incubations were carried out at 25°C for 30 rain. The binding reaction was stopped by rapid filtering through Whatman G F / B filters and filters were washed twice with 5 ml of ice-cold 25 mM Tris-HC1 buffer (pH 7.6 at 4°C). The filters were transferred to scintillation counting vials containing 10 ml of ACS 11 (Amersham Co., Arlington Heights, IL). The vials were shaken for 60 min and the radioactivity was measured using a Model 3255 Tri-Carb Liquid Scintillation Spectrometer System. Non-specific binding was defined by 5 /~M nonradioactive NLX, P C P and EKC in the cases of [3H]NLX, [3H]PCP and [3H]EKC, respectively. Filters presoaked in 0.1% poly-L-lysine at 4°C for 120 rain were used for the binding experiments of [3H]PCP to cut 'specific' ligand binding to the filters [18]. The protein concentration was determined by the method of Lowry et al. [10]. Analysis of biphasic Scatchard plots was performed by the method of Rosenthal [14] using a computer program to obtain the dissociation constant (Ka) and the maximum binding capacity (Bmax). When applicable, statistical significances were analyzed by Student's t-test for biochemical data or M a n n - W h i t n e y ' s U-test for behavioral data. When the mice were returned to the same apparatus in which they had been given an electric shock, they exhibited a marked suppression of motility (Control group: 2005.3 +68.5 counts (100%); Conditioned suppression group: 730.7 +_92.3 counts (36.4%), P < 0 . 0 1 ) , in agreement with our previous results [4-8, 12, 13]. On the other hand, in the synaptic membrane of the conditioned suppression group, the B .... but not Ka of [3H]NLX at high and low affinity binding sites and of [3H]PCP at high affinity binding sites increased significantly compared to those of the control
265
group (Table I). On the contrary, the Kd and Bmaxvalues of [3H]EKC at both affinity binding sites in the conditioned suppression group were not changed (Table I). We have reported that both cyclazocine (CLZ) and PCP, sigma agonists, diminish and morphine, a m u agonist, partially reduces the conditioned suppression of motility, while EKC, a kappa agonist, and methionine-enkephalin, a delta agonist, fail to affect it [12]. CLZ is classified as an opiate agonist-antagonist. However, since it has been reported that CLZ causes behavioral arousal and bizarre behavior, as does SKF-10047 [2], CLZ may be a relative sigma-like agonist. In addition, PCP binds to a sigma receptor although it does not belong to the opiates [16, 17]. Taking these findings into consideration, we have suggested that sigma and mu receptors may be important for opiate-induced attenuation of the conditioned suppression of motility [12]. The present results confirmed our hypothesis that in the synaptic membrane of mice showing conditioned suppression, the binding capacities of [3H]PCP and [3H]NLX binding sites increased significantly compared to the control group, but that of [3H]EKC binding sites did not alter. Taking the present and previous results together, we could consider that the binding capacities of the sigma and mu receptors might increase in compensation since the neuronal function mediated by these might decrease in the conditioned suppression group. On the other hand, though methionine-enkephalin does not affect the conditioned suppression of motility [12], the binding capacity of [3H]D-Ala-Met-enkephalinamide (a delta agonist) decreased in the conditioned suppression group (unpublished result). Therefore, a role of the delta receptor in the conditioned suppression remains to be investigated. TABLE 1 KINETIC P A R A M E T E R S OF O P I A T E BINDING TO BRAIN SYNAPTIC M E M B R A N E IN CONT R O L A N D C O N D I T I O N E D SUPPRESSION (CS) MICE The dissociation constant (Kd) and m a x i m u m binding capacity (Bmax) were determined by the method of Rosenthal [14] using a computer program. The numbers in parentheses show the number of independent experiments, each in triplicate. * P < 0 . 0 5 , * * P < 0 . 0 1 compared to control (Student's t-test). [3H]Ligand
Treatment
Affinity
Ka (nM)
Bmax ( f m o l / m g protein)
[3H]Naloxone
Control (4)
High Low High Low
0.44 _+0.08 32.52+3.79 0.59_+0.08 33.72 _+ 1.50
14.20_+ 2.62 1358.95+99.28 35.51 _+ 4.76** 1816.53 _+ 13.45"
High Low High Low
0.32 _+0.02 40.87 _+2.35 0.45_+ 0.05 42.21 _+4.80
22.92 _+ 1.18 1462.88 _+64.38 63.03 _+ 6.92** 1305.36+87.07
High Low High Low
0.13 -+ 0.02 19.93 -+ 4.01 0.12_+0.02 15.17_+0.56
CS (4)
[3H]Phencyclidine
ControL (3) CS (3)
[3H]Ethylketocyclazocine
Control (3) CS (3)
19.57 -+ 2.06 309.24 _+40.27 23.00_+ 1.77 286.34_+ 8.03
266
In conclusion, the binding function of different opioid receptor subtypes may be altered differently by stress. In addition, sigma and mu receptors may be important for the conditioned suppression of motility in mice. We thank Mr. Y. Noda and Mr. K. lshikawa for their excellent technical assistance, H. Furukawa, Ph.D., for synthesis of phencyclidine hydrochloride, and Sterling-Winthrop Research Institute (Rensselaer, NY) and Endo Laboratories (Wilmington, DE) for supplying ethylketocyclazocine methanesulfonate and naloxone hydrochloride, respectively. I Babbini, M., Gaiardi, M. and Bartoletti, M., Effect of morphine on a quickly learned conditioned suppression in rats, Psychopharmacologia, 33 (1973) 329-332. 2 Cowan, A., Simple in vivo tests that differentiate prototype agonists at opiate receptors, Life Sci., 28 (1981) 1559-1570. 3 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. and Morris, H.R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975) 577-579. 4 Kameyama, T. and Nagasaka, M., The effect of analgesics oil quickly learned conditioned suppression in mice, Neuropharmacology, 21 (1982) 1283-1289. 5 Kameyama, T. and Nagasaka, M., Effects of apomorphine and diazepam oil a quickly learned conditioned suppression in rats, Pharmacol. Biochem. Behav., 17 (1982) 59-63. 6 Kameyama, T. and Nagasaka, M., Effects of apomorphine and methamphetamine on a quickly learned conditioned-suppression response in rats, Neuropharmacology, 22 (1983) 813 817. 7 Kameyama, T. and Nagasaka, M., Effect of naloxone on a quickly learned conditioned suppression in rats following cessation of chronic haloperidol treatment, Brain Res., 275 (1983) 389-391. 8 Kameyama, T. and Nagasaka, M., A mode of aclion of morphine on a quickly learned conditioned suppression in mice, J. Pharm. Dyn., 7 (1984) 151 -155. 9 1,ord, J., Waterfield, D., Hughes, J. and Kosterlitz, H.W., Endogenous opioid peptides: multiple agonists and receptors, Nature tLond.), 267 (1977) 495-499. 10 Lowry, O.H,, Rosebrough, N..I., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.A. and Gilbert, P.E., The effect of morphine and nalorphine-like drugs in non-dependent and morphine dependent chronic spinal dog, J. Pharmacol. Exp. Ther., 197 (1976) 517-522. 12 Nabeshima, T., Yamada, K. and Kameyama, T., Effects of opiate agonists oil the conditioned suppression in motility of mice, Neurosci. Lett., 39 (1983) 301-306. 13 Nagasaka, M. and Kameyama, T., Effects of diazepam, meprobamate, chlorpromazine and apomorphine on a quickly learned conditioned suppression in mice, J. Pharm. Dyn., 6 (1983) 523-526. 14 Rosenthal, H.E., A graphic method for the determination and presentation of binding parameters in a complex system, Analyt. Biochem., 20 (1967) 525 532. 15 Sivam, S.P., Nabeshima, T. and Ho, I.K., Alterations of synaptic high and low affinity opiate binding sites after acute and chronic morphine administration in mice, Progr. Neuro-Psychopharmacol. Biol. Psychiat., 6 (1982) 119-127. 16 Zukin, R.S. and Zukin, S.R., Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites, Molec. Pharmacol., 20 (1981) 246-254. 17 Zukin, R.S, and Zukin, S.R., Multiple opiate receptors: emerging concepts, Life Sci., 29 (1981) 2681-2690. 18 Zukin, S.R., Fitz-Syage, M.L., Nichtenhausar, R. and Zukin, R.S., Specific binding of [3H]phencyclidine in rat central nervous tissue: Further characterization and technical consideration, Brain Res., 258 (1983) 277 284.