- Email: [email protected]
Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex
Life Sciences, Vol. 48, pp. PL-25-PL-29 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGY LETTERS A c c e l e r a t e d Communication CIRCADIAN RHYTHM IN ADENOSINE A1 RECEPTOR OF MOUSE CEREBRAL CORTEX Chiara Elorio, Institute
Anna M a r i a R o s a t i ,
Ugo T r a v e r s a
and R o d o l f o V e r t u a
o f P h a r m a c o l o g y and P h a r m a c o g n o s y , U n i v e r s i t y v i a A. V a l e r i o 32 - 34100 T r i e s t e - I t a l y
( S u b m i t t e d A u g u s t 14, 1990; a c c e p t e d S e p t e m b e r 7, r e c e i v e d i n f i n a l f o r m December 5, 1990)
of T r i e s t e 1990;
Abstract. In order to investigate diurnal variation in adenosine AI receptors binding parameters, Bmax and Kd values of specifically bound N6 - cyclohexy1[3H]adenosine were determinated in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks (light on from 7.00 to 19.00 h). Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.
Introduction The existence of circadian rhythms regulating physiological and biochemical events in living beings has been recognized for many years. Circadian rhythms are generated by an endogenous pacemaker located within the suprachiasmatic nuclei (1) which influences, in a not yet clarified way, also the levels of several hormones, neurotransmitters and enzymes (2, 3, 4). Part of the pharmacological interest in circadian rhythms arises from studies revealing that the therapeutic efficacy of a drug may vary according to the time administration schedule (5, 6, 7) and that, in turn, some drugs like the antidepressants and short-acting benzodiazepines are able to alter circadian rhythms (8, 9, 10, ll). The existence of a diurnal variation has also been investigated for several neurotransmitter receptors, both in nervous and non-nervous tissues. Circadian rhythms have been demonstrated for serotonin (12, 13) alpha- and beta-adrenergic ( 1 4 ) opiate (15) muscarinic and GABA binding sites (16). Moreover, modifications in the timing of peak of adrenergic and dopamine receptors have been reported following chronic administration of imipramine (i7, 1 8 ) . It is now widely accepted that adenosine may play a functional role in modulating the neuronal firing, generally acting as an inhibitory agent, by means of specific extracellular receptors, widely distributed in the central nervous system (19). The aim of this study was to ascertain whether the adenosine receptors in the cerebral cortex of mice are subjected to a time-dependent fluctuation. In order to avoid any disturbance in the original rhythm, mice were housed in a separate semisound-proof room under controlled light-dark cycles for one month 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
PL-26
Circadian Rhythm in Adenosine A1 Receptor
Vol. 48, No. 6, 1991
before testing. Material and Method Male Swiss-Nos mice (Nossan, Corezzana, Italy) weighing approximately 20 gr at the begin of the study (the end of September), were housed in group of 5 per cage in a semisound-proof room. Water and food were continuously available and room cleaning was done every morning at random times. The room temperature was kept at 23±i °C and the humidity at 60±2 %. Mice were exposed to a 12 hour light/dark cycle with artificial light on at 7.00 h (off at 19.00 h). After 4 weeks under these conditions, mice were killed at 3-hr intervals by decapitation. Brains were rapidly dissected on ice and cerebral cortices were pooled, weighed and frozen in liquid nitrogen. Crude membrane preparations were obtained homogenizing the cortices in lO volumes ice-cold 50 mM Tris-BCl buffer, pH 7.4, with Ultra-Turrax (Ika-Werk, FR6). The homogenate was centrifuged at 40.O00xg for 20 min at 4 °C, the pellet resuspended in I0 volumes Tris-HCl buffer and again centrifuged as above. The final pellet was suspended in lO volumes Tris-HCl, incubated with adenosine deaminase (2 UI/ml) at 24°C for 30 min and used in the receptor assay. N6-Cyclohexyl-[3H]adenosine (3H-CHA) (New England Nuclear, specific activity 1779,7 GBq/mmol) saturation experiments were performed in 50 mM Tris-HCl buffer (i ml total volume) containing i0 mg wet tissue (approximately 500 ~g protein) and 3H-CHA ranging from 0.I to i0 nM, each dose in triplicate. After 120 min at 25 °C, the samples were filtered under vacuum through Whatman GF-B filters, previously soaked in 50 mM Tris-HCl buffer; the filters were washed four times with 2.5 ml aliquots of ice-cold buffer, placed dried in 5 ml scintillation vials containing Filter Count cocktail and counted by liquid scintillation spectrometry (Tri-Carb 300 CD, Packard Instrument Co., USA). The specific binding of 3H-CHA was defined as the difference between the total binding and that remaining in the presence of i0 ~M R-phenyl isopropyl adenosine (R-PIA) and represented 90-95% of the total binding. Analysis of the binding curves was performed by Ligand computerized program (20). One way analysis of variance (ANOVA) was used to compare Bmax and Kd values. Results The non-hydrolyzable adenosine analog 3H-CHA bound to mouse cerebral cortex in a saturable and reversible manner. The saturation data obtained at 3hr interval indicated that the maximal number of binding sites (Bmax) (3H-CHA ranging from 0.i to 10.0 nM) decreased during the light period, reaching the lowest value at 18.00 hr. Thereafter, the Bmax value began to increase, showing a peak between 24.00 and 03.00 hr (fig. i). The amplitude (defined as peak:nadir per cent) between 03.00 and 18.00 hr was 33%. One way analysis of variance (ANOVA) indicated that the changes were significant (F=46, p<0.005). In Fig.2, two representative Scatchard plots are shown, which correspond at times of greatest and least binding. No significant difference was found among the Kd values which ranged from 0.41 to 0.62 nM, mean ± SEM of the eight Kd values 0.52 ± 0.07 nM.
Discussion Computer-assisted analysis of saturation isotherms obtained over a 24-h period indicates that the density of A1 adenosine receptors in the cerebral cortex of mouse is subjected, like most if not all neurotransmitter receptors
Vol. 48, No. 6, 1991
Circadian Rhythm in Adenosine A1 Receptor
PL-27
o.32T "~
0.3
o.2,t
F °2'I
o
~ °.2',l
T FIG.I Circadian rhythm of Bmax for 3H-CHA specifically bound to cortical receptors; cortices of 5 animals were pooled and 2 saturation experiments were performed. The means ± SEM of the Bmax values are shown. The light period is 07.00-19.00 and the dark period 19.00-07.00. Statistical analysis by one-way ANOVA: F (03.00, 18.00)=46, p
B/F"
0.5 o.+i~
o0J
,
,
:
0,].00 hr
c3
18.00 hr
-'~d
~ ".
0.08 O.16 0.24 0.32 3H-CHA (pmol bound/1Omg wet weight)
FIG. 2 Scatchard analysis of the specific binding of 3H-CHA ranging from 0.i to i0.0 nM to cortical receptors at two different times of day. The Bmax value was 0.306 + 0.01 and 0.230 + 0.005 pmol bound/lO mg wet weight and the Kd 0.50 (0.46-0.59) and 0.41 (0.37-0.46) nM at 03.00 and 18.00 hr respectively.
PL-28
Circadian Rhythm in Adenosine A1 Receptor
Vol. 48, No. 6, 1991
in the central nervous system (CNS), to circadian fluctuations. The diurnal pattern is characterized by a decrease of the Bmax values during the light cycle (07.00 to 19.00), followed by an increase during the dark cycle. Since Scatchard analysis of the agonist binding showed no changes in the Kd values, it can be assumed that the loss in binding to A1 receptors was due to an actual loss of receptor sites (down-regulation) and not to an uncoupling of A1 receptors from the G protein. Since no data are available relative to time-dependent fluctuation of adenosine levels in the CNS, a cause and effect relationship between the circadian variations of the density of cortical A1 receptors and adenosine levels cannot be established. However, indirect evidence suggests that A1 receptor density can be modulated by adenosine in vivo (21). Endogenous adenosine acts as a natural anticonvulsant (22), apparently interacting at the A1 receptor level (23) and a reduced sensitivity to convulsants has been demonstrated when cerebral A1 receptors were chronically exposed to the adenosine receptor antagonist, theophylline (24). The reported increase of cortical binding sites in this latter study (29%) is comparable to the increase (33%) in Bmax in the current study observed between 03.00 and 18.00 hr. It is thus tempting to speculate that the increase in the density of A1 receptors occurring during the dark cycle might be related to a local fall in adenosine at the receptor level. In conclusion, the present results indicate that cortical A1 receptor density exhibits circadian variation, the knowledge of which may contribute to defining the physiological role of the purine agonist in the central nervous activity. ACKNOWLEDGMENTS This research was partially supported by a grant of the Ministero della Pubblica Istruzione (MPI 60%). A preliminary account of these results has been presented at the International Symposium on Pharmacology of the Purinergic Receptors, 1990 (Noordwijk, The Netherlands).
References i. F.W. TUREK, Trends Pharmacol. Sci. 8:212-217 (1987) 2. R.J. FEUERS and L.E. SCHEVING, Annu. Rev. Chronopharmacol. 4:209-256 (1988) 3. I.M. MAZURKIEWICZ-KWILECKI and G.D. PRELL, Pharmacol. Biochem. Behav. 12: 549-553 (1980) 4. S.M. REPPERT, W.J. SCHWARTZ and G.R. UHL, Trends Neurosci. 10:76-80 (1987) 5. P.H. REDFERN and P.C. MOSER, Annu. Rev. Chronopharmacol. 4:107-136 (1988) 6. M. OLLAGNIER, H. DECOUSUS and Y. CHERRAH, Clin. Pharmacokin. 12: 367-377 (1987) 7. M.T. MARTIN-IVERSON, S.M. STAHL and S.D. IVERSEN, Psychopharmacol. 95: 534539 (1988) 8. W.C. DUNCAN Jr. and T.A. WEHR, Annu. Rev. Chronopharmacol. 4: 137-170 (1988) 9. B.E.F. WEE and F.W. TUREK, Pharmacol. Biochem. Behav. 32:901-906 (1989) I0. O. VAN REETH, J.J. VANDERHAEGHEN and F.W. TUREK, Drain Res. 444: 333-339 (1988) ii. F.W. TUREK and S.H. LOSEE-OLSON, Life Sci. 4_O0:1033-i038 (1987) 12. J. AKIYOSHI, H. KURANAGA, K. TSUCHIYAMA and H. NAGAYAMA, Pharmacol. Biochem. Behav. 3_~2:491-493 (1989) 13. P. ROCCA, A.M. GALZIN and S.Z. LANGER, Naunyn-Schmiedeberg's Arch. Pharmacol. 340:41-44 (1989) 14. M.S. KAFKA, A. WIRZ-JUSTICE and D. NABER, Brain Res. 207:409-419 (1981)
Vol. 48, No. 6, 1991
Circadian Rhythm inAdenosineAl Receptor
PL-29
15. D. NABER, A. WIRZ-JUSTICE and M.S. KAFKA, Neurosci. Lett. 2 1 : 4 5 - 5 0 (1981) 16. M.S. KAFKA, M.A. BENEDITO, J.A. BLENDY and N.S. TOKOLA, Chronobiol. Int. 3: 91-100 (1986) 17. A. WIRZ-JUSTICE, M.S. KAFKA, D. NABER and T.A. WEHR, Life Sci. 27: 341-347 (1980) 18. D. NABER, A. WIRZ-JUSTICE, M.S. KAFKA and T.A. WEHR, Psychopharmacol. 68: 1-5 (1980) 19. M. WILLIAMS, Ann. Rev. Pharmacol. Toxicol. 2 7 : 3 1 5 - 3 4 5 (1987) 20. P.J. MUNSON and D. RODBARD, Anal. Biochem. 107:220-239 (1980) 21. R.C. SANDERS and T.F. MURRAY, Neuropharmacology 2 7 : 7 5 7 - 7 6 0 (1988) 22. M. DRAGUNOW, Trends Pharmacol. Sci. 7 : 1 2 8 - 1 3 0 (1986) 23. P.H. FRANKLIN, G.E. ZHANG, E.D. TRIPP and T.F. MURRAY, J. Pharmacol. Exp. Ther. 251:1229-1236 (1989) 24. P. SZOT, R.C. SANDERS and T.F. MURRAY, Neuropharmacology 26: 1173-1180 (1987)
Pergamon Press
PHARMACOLOGY LETTERS A c c e l e r a t e d Communication CIRCADIAN RHYTHM IN ADENOSINE A1 RECEPTOR OF MOUSE CEREBRAL CORTEX Chiara Elorio, Institute
Anna M a r i a R o s a t i ,
Ugo T r a v e r s a
and R o d o l f o V e r t u a
o f P h a r m a c o l o g y and P h a r m a c o g n o s y , U n i v e r s i t y v i a A. V a l e r i o 32 - 34100 T r i e s t e - I t a l y
( S u b m i t t e d A u g u s t 14, 1990; a c c e p t e d S e p t e m b e r 7, r e c e i v e d i n f i n a l f o r m December 5, 1990)
of T r i e s t e 1990;
Abstract. In order to investigate diurnal variation in adenosine AI receptors binding parameters, Bmax and Kd values of specifically bound N6 - cyclohexy1[3H]adenosine were determinated in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks (light on from 7.00 to 19.00 h). Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.
Introduction The existence of circadian rhythms regulating physiological and biochemical events in living beings has been recognized for many years. Circadian rhythms are generated by an endogenous pacemaker located within the suprachiasmatic nuclei (1) which influences, in a not yet clarified way, also the levels of several hormones, neurotransmitters and enzymes (2, 3, 4). Part of the pharmacological interest in circadian rhythms arises from studies revealing that the therapeutic efficacy of a drug may vary according to the time administration schedule (5, 6, 7) and that, in turn, some drugs like the antidepressants and short-acting benzodiazepines are able to alter circadian rhythms (8, 9, 10, ll). The existence of a diurnal variation has also been investigated for several neurotransmitter receptors, both in nervous and non-nervous tissues. Circadian rhythms have been demonstrated for serotonin (12, 13) alpha- and beta-adrenergic ( 1 4 ) opiate (15) muscarinic and GABA binding sites (16). Moreover, modifications in the timing of peak of adrenergic and dopamine receptors have been reported following chronic administration of imipramine (i7, 1 8 ) . It is now widely accepted that adenosine may play a functional role in modulating the neuronal firing, generally acting as an inhibitory agent, by means of specific extracellular receptors, widely distributed in the central nervous system (19). The aim of this study was to ascertain whether the adenosine receptors in the cerebral cortex of mice are subjected to a time-dependent fluctuation. In order to avoid any disturbance in the original rhythm, mice were housed in a separate semisound-proof room under controlled light-dark cycles for one month 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
PL-26
Circadian Rhythm in Adenosine A1 Receptor
Vol. 48, No. 6, 1991
before testing. Material and Method Male Swiss-Nos mice (Nossan, Corezzana, Italy) weighing approximately 20 gr at the begin of the study (the end of September), were housed in group of 5 per cage in a semisound-proof room. Water and food were continuously available and room cleaning was done every morning at random times. The room temperature was kept at 23±i °C and the humidity at 60±2 %. Mice were exposed to a 12 hour light/dark cycle with artificial light on at 7.00 h (off at 19.00 h). After 4 weeks under these conditions, mice were killed at 3-hr intervals by decapitation. Brains were rapidly dissected on ice and cerebral cortices were pooled, weighed and frozen in liquid nitrogen. Crude membrane preparations were obtained homogenizing the cortices in lO volumes ice-cold 50 mM Tris-BCl buffer, pH 7.4, with Ultra-Turrax (Ika-Werk, FR6). The homogenate was centrifuged at 40.O00xg for 20 min at 4 °C, the pellet resuspended in I0 volumes Tris-HCl buffer and again centrifuged as above. The final pellet was suspended in lO volumes Tris-HCl, incubated with adenosine deaminase (2 UI/ml) at 24°C for 30 min and used in the receptor assay. N6-Cyclohexyl-[3H]adenosine (3H-CHA) (New England Nuclear, specific activity 1779,7 GBq/mmol) saturation experiments were performed in 50 mM Tris-HCl buffer (i ml total volume) containing i0 mg wet tissue (approximately 500 ~g protein) and 3H-CHA ranging from 0.I to i0 nM, each dose in triplicate. After 120 min at 25 °C, the samples were filtered under vacuum through Whatman GF-B filters, previously soaked in 50 mM Tris-HCl buffer; the filters were washed four times with 2.5 ml aliquots of ice-cold buffer, placed dried in 5 ml scintillation vials containing Filter Count cocktail and counted by liquid scintillation spectrometry (Tri-Carb 300 CD, Packard Instrument Co., USA). The specific binding of 3H-CHA was defined as the difference between the total binding and that remaining in the presence of i0 ~M R-phenyl isopropyl adenosine (R-PIA) and represented 90-95% of the total binding. Analysis of the binding curves was performed by Ligand computerized program (20). One way analysis of variance (ANOVA) was used to compare Bmax and Kd values. Results The non-hydrolyzable adenosine analog 3H-CHA bound to mouse cerebral cortex in a saturable and reversible manner. The saturation data obtained at 3hr interval indicated that the maximal number of binding sites (Bmax) (3H-CHA ranging from 0.i to 10.0 nM) decreased during the light period, reaching the lowest value at 18.00 hr. Thereafter, the Bmax value began to increase, showing a peak between 24.00 and 03.00 hr (fig. i). The amplitude (defined as peak:nadir per cent) between 03.00 and 18.00 hr was 33%. One way analysis of variance (ANOVA) indicated that the changes were significant (F=46, p<0.005). In Fig.2, two representative Scatchard plots are shown, which correspond at times of greatest and least binding. No significant difference was found among the Kd values which ranged from 0.41 to 0.62 nM, mean ± SEM of the eight Kd values 0.52 ± 0.07 nM.
Discussion Computer-assisted analysis of saturation isotherms obtained over a 24-h period indicates that the density of A1 adenosine receptors in the cerebral cortex of mouse is subjected, like most if not all neurotransmitter receptors
Vol. 48, No. 6, 1991
Circadian Rhythm in Adenosine A1 Receptor
PL-27
o.32T "~
0.3
o.2,t
F °2'I
o
~ °.2',l
T FIG.I Circadian rhythm of Bmax for 3H-CHA specifically bound to cortical receptors; cortices of 5 animals were pooled and 2 saturation experiments were performed. The means ± SEM of the Bmax values are shown. The light period is 07.00-19.00 and the dark period 19.00-07.00. Statistical analysis by one-way ANOVA: F (03.00, 18.00)=46, p
B/F"
0.5 o.+i~
o0J
,
,
:
0,].00 hr
c3
18.00 hr
-'~d
~ ".
0.08 O.16 0.24 0.32 3H-CHA (pmol bound/1Omg wet weight)
FIG. 2 Scatchard analysis of the specific binding of 3H-CHA ranging from 0.i to i0.0 nM to cortical receptors at two different times of day. The Bmax value was 0.306 + 0.01 and 0.230 + 0.005 pmol bound/lO mg wet weight and the Kd 0.50 (0.46-0.59) and 0.41 (0.37-0.46) nM at 03.00 and 18.00 hr respectively.
PL-28
Circadian Rhythm in Adenosine A1 Receptor
Vol. 48, No. 6, 1991
in the central nervous system (CNS), to circadian fluctuations. The diurnal pattern is characterized by a decrease of the Bmax values during the light cycle (07.00 to 19.00), followed by an increase during the dark cycle. Since Scatchard analysis of the agonist binding showed no changes in the Kd values, it can be assumed that the loss in binding to A1 receptors was due to an actual loss of receptor sites (down-regulation) and not to an uncoupling of A1 receptors from the G protein. Since no data are available relative to time-dependent fluctuation of adenosine levels in the CNS, a cause and effect relationship between the circadian variations of the density of cortical A1 receptors and adenosine levels cannot be established. However, indirect evidence suggests that A1 receptor density can be modulated by adenosine in vivo (21). Endogenous adenosine acts as a natural anticonvulsant (22), apparently interacting at the A1 receptor level (23) and a reduced sensitivity to convulsants has been demonstrated when cerebral A1 receptors were chronically exposed to the adenosine receptor antagonist, theophylline (24). The reported increase of cortical binding sites in this latter study (29%) is comparable to the increase (33%) in Bmax in the current study observed between 03.00 and 18.00 hr. It is thus tempting to speculate that the increase in the density of A1 receptors occurring during the dark cycle might be related to a local fall in adenosine at the receptor level. In conclusion, the present results indicate that cortical A1 receptor density exhibits circadian variation, the knowledge of which may contribute to defining the physiological role of the purine agonist in the central nervous activity. ACKNOWLEDGMENTS This research was partially supported by a grant of the Ministero della Pubblica Istruzione (MPI 60%). A preliminary account of these results has been presented at the International Symposium on Pharmacology of the Purinergic Receptors, 1990 (Noordwijk, The Netherlands).
References i. F.W. TUREK, Trends Pharmacol. Sci. 8:212-217 (1987) 2. R.J. FEUERS and L.E. SCHEVING, Annu. Rev. Chronopharmacol. 4:209-256 (1988) 3. I.M. MAZURKIEWICZ-KWILECKI and G.D. PRELL, Pharmacol. Biochem. Behav. 12: 549-553 (1980) 4. S.M. REPPERT, W.J. SCHWARTZ and G.R. UHL, Trends Neurosci. 10:76-80 (1987) 5. P.H. REDFERN and P.C. MOSER, Annu. Rev. Chronopharmacol. 4:107-136 (1988) 6. M. OLLAGNIER, H. DECOUSUS and Y. CHERRAH, Clin. Pharmacokin. 12: 367-377 (1987) 7. M.T. MARTIN-IVERSON, S.M. STAHL and S.D. IVERSEN, Psychopharmacol. 95: 534539 (1988) 8. W.C. DUNCAN Jr. and T.A. WEHR, Annu. Rev. Chronopharmacol. 4: 137-170 (1988) 9. B.E.F. WEE and F.W. TUREK, Pharmacol. Biochem. Behav. 32:901-906 (1989) I0. O. VAN REETH, J.J. VANDERHAEGHEN and F.W. TUREK, Drain Res. 444: 333-339 (1988) ii. F.W. TUREK and S.H. LOSEE-OLSON, Life Sci. 4_O0:1033-i038 (1987) 12. J. AKIYOSHI, H. KURANAGA, K. TSUCHIYAMA and H. NAGAYAMA, Pharmacol. Biochem. Behav. 3_~2:491-493 (1989) 13. P. ROCCA, A.M. GALZIN and S.Z. LANGER, Naunyn-Schmiedeberg's Arch. Pharmacol. 340:41-44 (1989) 14. M.S. KAFKA, A. WIRZ-JUSTICE and D. NABER, Brain Res. 207:409-419 (1981)
Vol. 48, No. 6, 1991
Circadian Rhythm inAdenosineAl Receptor
PL-29
15. D. NABER, A. WIRZ-JUSTICE and M.S. KAFKA, Neurosci. Lett. 2 1 : 4 5 - 5 0 (1981) 16. M.S. KAFKA, M.A. BENEDITO, J.A. BLENDY and N.S. TOKOLA, Chronobiol. Int. 3: 91-100 (1986) 17. A. WIRZ-JUSTICE, M.S. KAFKA, D. NABER and T.A. WEHR, Life Sci. 27: 341-347 (1980) 18. D. NABER, A. WIRZ-JUSTICE, M.S. KAFKA and T.A. WEHR, Psychopharmacol. 68: 1-5 (1980) 19. M. WILLIAMS, Ann. Rev. Pharmacol. Toxicol. 2 7 : 3 1 5 - 3 4 5 (1987) 20. P.J. MUNSON and D. RODBARD, Anal. Biochem. 107:220-239 (1980) 21. R.C. SANDERS and T.F. MURRAY, Neuropharmacology 2 7 : 7 5 7 - 7 6 0 (1988) 22. M. DRAGUNOW, Trends Pharmacol. Sci. 7 : 1 2 8 - 1 3 0 (1986) 23. P.H. FRANKLIN, G.E. ZHANG, E.D. TRIPP and T.F. MURRAY, J. Pharmacol. Exp. Ther. 251:1229-1236 (1989) 24. P. SZOT, R.C. SANDERS and T.F. MURRAY, Neuropharmacology 26: 1173-1180 (1987)