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Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase
Life Sciences 70 (2002) 3069 – 3075
Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase Tolga Uz*, Javaid I. Javaid, Hari Manev The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA Received 12 September 2001; accepted 11 January 2002
Abstract Circadian rhythms might be involved in addictive behaviors. The pineal secretory product melatonin decreases cocaine sensitization in rats; mice mutant for the critical melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase (AANAT), exhibit altered behaviors. We hypothesized that AANAT/melatonin system, which is up-regulated at night, affects cocaine sensitization in mice. Intraperitoneal cocaine treatment (10 and 20 mg/kg) dose-dependently increased locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice; this effect was similar during the day and at night. Injections of cocaine during the day for three days resulted in behavioral sensitization in normal and AANAT mutant mice whereas treatment at night triggered sensitization in AANATdeficient mice only. AANAT expression and synthesis of N-acetylserotonin/melatonin could play a role in addictive properties of cocaine. D 2002 Elsevier Science Inc. All rights reserved. Keywords: AANAT mutant; N-acetylserotonin; Melatonin; Addiction; Mice
Introduction Chronopharmacology has identified numerous drugs whose effects depend upon circadian rhythms. In humans, use of cocaine appears to be diurnally affected; most abuse occurs during the afternoon and cocaine-caused health problems peak soon thereafter [1]. In
* Corresponding author. Tel.: +1-312-413-4580; fax: +1-312-413-4569. E-mail address: [email protected] (T. Uz). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 5 9 - X
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experimental animals, psychostimulants, such as cocaine and amphetamine present circadian rhythmicity in their behavioral and neurophysiological effects such as feeding and locomotor activity [2–4]. Moreover, circadian changes were observed in cocaine self-administration in rats; these changes were not accompanied by a corresponding variance in the pharmacokinetic profiles of cocaine [5]. Thus, it is possible that circadian rhythm-driven mechanisms could play a pivotal role in development of cocaine addiction. The pineal gland is a key organ that influences mammalian circadian rhythms. Melatonin is synthesized from serotonin by an action of arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase; AANAT) which produces rate-limiting intermediate N-acetylserotonin (NAS). AANAT mRNA content [6] and NAS and melatonin levels (for review, see [7]) exhibit marked circadian rhythms with high levels at night and low or even undetectable levels during the day. Recently, Sircar [8] found in experiments with rats that the administration of melatonin prior to repeated cocaine injections prevented the development of cocaine-induced behavioral sensitization. These data point to a possible role for the AANAT/melatonin system in cocaine-induced behaviors and possibly in the development of drug addictions. Our recent findings suggest that AANAT mutant mice can be used to investigate the contribution of the AANAT/melatonin system to mouse behavior [9]. Moreover, the natural AANAT mutant (‘‘NAS/melatonin deficient’’) C57BL/6J strain of mice [9,10] shows less sensitivity to the locomotor-stimulating [11] and seizure-inducing [12] effects of cocaine than AANAT normal mice, such as the C3H/HeJ strain. Therefore, we hypothesized that the AANAT/melatonin system might be important for the development of locomotor sensitization to cocaine and that its role could be investigated with the help of the AANAT mutation. We tested our hypothesis using the natural AANAT mutant (C57BL/6J) and AANAT normal mice (C3H/HeJ), and we performed the experiments during the day (low AANAT expression) and at night (high AANAT expression).
Methods Animals and drugs Male C57BL/6J and C3H/HeJ mice, 8-weeks old and weighing 25-30 g, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Animals were housed in groups of three and had free access to laboratory chow and water except during behavioral experiments. They were kept in a temperature-controlled room on a 12-h light:dark cycle (lights off at 7 pm). Experiments were carried out during the day (8-11 am) and at night (9-12 pm). The experimental protocol was approved by the institutional Animal Care Committee. Cocaine hydrochloride (Sigma Chemical St. Louis, MO, USA) was dissolved in sterile physiological saline (0.9% NaCl) and administered i.p. in an injection volume of 0.05 ml/ 25g body weight (n = 6). Cocaine was injected within the last hour of the above-noted three-hour time windows. Vehicle treatments were administered in the same volume and manner (n = 6).
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Locomotor activity measurements, behavioral sensitization Mice that had not been exposed to the testing monitors previously were weighed and placed individually in 21 22 12 cm (length width height) activity cages for a 30-min adaptation period. After this period, locomotor activity was measured for 30 min using the Cage Rack Photobeam Activity Measurement System (San Diego Instruments, San Diego, CA, USA) equipped with computer-monitored photobeam frames. Immediately after the second 30-min interval, animals were injected with saline or 10 or 20 mg/kg free-base cocaine and returned to the activity cages; the recording procedure continued for additional 30 min. This schedule was selected based on the previous characterization of melatonin production in the two strains of mice we used [13]. These authors reported circadian melatonin production in C3H/HeJ but no detectable melatonin in C57BL/6J mice. In the C3H/HeJ mice they found very low melatonin during the day lasting up to two hours after lights off. [13]. The movement of each mouse was recorded as the number of beam interruptions and reported as locomotor activity (ambulation). The experiment room was equipped with a light timer; the nighttime drug administrations were done under dim red light. After each experiment session, mice were returned to their home cages. To measure locomotor sensitization, the same procedure was repeated for three consecutive days and nights. Statistical analysis Locomotor activity scores (mean ± s.e.m.) were analyzed by a two-way ANOVA with the factors of chronic treatment (cocaine vs. saline) and time of the treatment (day vs. night) for
Fig. 1. Stimulatory dose-dependent effects of acute cocaine administration on locomotor activity of AANAT normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice: absence of diurnal alterations. Results (mean ± s.e.m.) are expressed as a percentage of locomotor activity (number of beam breaks during a 30-min test session: 6 per each group) of the corresponding control, saline-injected mice (dashed line). Mice were injected during the day (open bars) or at night under dim red light (closed bars). *p < 0.05 and **p < 0.01 vs. corresponding salineinjected control group (Duncan’s test). Note a dose-dependent locomotor activation by cocaine. No differences were observed between day vs. night responses.
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both strains of mice (C57BL/6J and C3H/HeJ) separately, followed by Duncan’s multiplerange post-hoc test with a p < 0.05 accepted as statistically significant.
Results Intraperitoneal cocaine treatment (10 and 20 mg/kg) induced an acute dose-dependent increase in the locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice, and this effect was similar during the day and at night (in the dark) (Fig. 1). However, the actual extent of the stimulatory effect of cocaine was lower in AANAT mutant than in normal mice (Fig. 1: p < 0.05, Duncan’s test). Repeated (three consecutive days) injections of cocaine applied during the day only (low AANAT/melatonin in normal mice) resulted in behavioral sensitization both in normal and AANAT mutant/deficient mice (Fig. 2). Repeated injections of cocaine at night (high AANAT/melatonin in normal mice) failed to produce behavioral sensitization in normal mice but were effective in triggering sensitization in AANAT mutant mice (Fig. 2).
Fig. 2. Circadian alterations in the development of cocaine sensitization in AANAT normal (C3H/HeJ) mice and the absence of circadian influence in AANAT mutant (C57BL/6J) mice. Results (mean ± s.e.m.) are expressed as locomotor activity; i.e., the number of beam breaks during a 30-min test session measured individually. Mice (6 per group) were injected for three consecutive days i.p. with saline (4) or 10 (6) and 20 (5) mg/kg cocaine, either during the day or at night under dim red light. Note the development of sensitization to cocaine (increased locomotor activity after second and third injection) in all groups except C3H/HeJ mice injected at night; *p < 0.05 and **p < 0.01 vs. day 1; #p < 0.05 vs. day 2 (Duncan’s test).
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Discussion Behavioral sensitization to repeated administration of cocaine in inbred mice is well documented [14,15] and represents a behavioral model indicative of the addictive properties of this drug. Here we show evidence for a marked circadian alteration in the development of cocaine sensitization in normal mice: whereas repeated cocaine injections during the day produced robust behavioral sensitization, repeated administration at night failed to trigger sensitization. This circadian effect was completely absent in AANAT mutant mice that do not synthesize melatonin; in these mice, cocaine was effective in triggering behavioral sensitization both during the day and at night. Our findings suggest that endogenously-produced melatonin (at night) might be responsible for the nocturnal suppression of cocaine sensitization in normal mice. Supporting this possibility is a recent finding by Sircar [8]; this author reported that repeated daily melatonin injections prior to administration of cocaine for five days prevented the development of cocaine-triggered behavioral sensitization in rats. Thus, our data point to a putative role for AANAT in circadian alterations of behavioral sensitization to cocaine and also to the possibility of its addictive liability. Several mechanisms could participate in the link between AANAT– cocaine. Since AANAT is predominantly expressed in the pineal gland [6], it is likely that when synthesized and released in to the blood, which normally occurs at night, pineal serotonin metabolites produced by AANAT (i.e., NAS and its metabolite melatonin) affect their primary targets and subsequently modify the action of cocaine. One such target could be nitric oxide synthase (NOS); inhibition of NOS by melatonin [16] or NAS could reduce behavioral sensitization [8]. Alternatively, NAS and/or melatonin could bind specific receptors [17]; it is not clear how these receptors would modify the action of cocaine but one could envision their putative effect on the release of neurotransmitters such as dopamine or serotonin, as suggested by others [18]. Sircar [8] reported that pharmacological (30 mg/kg) melatonin increases acute cocaineinduced locomotor activity, whereas we did not observe different acute effect of cocaine during the day or at night (Fig. 1). Direct injections of melatonin into the nucleus accumbens either stimulate (100 mM) or decrease (3 mM) locomotor activity [19]. Thus, it is possible that endogenous melatonin affects the acute action of cocaine differently from pharmacological concentrations. Primarily pineal enzyme, AANAT is also expressed in the brain [9,20,21] although its role in the central nervous system (CNS) is not clear. It has been suggested that expression of AANAT in the CNS might be responsible for behaviors unrelated to circadian rhythms [9]; if relevant for behavioral actions of cocaine, the CNS AANAT might influence the sensitivity of mice to the acute stimulatory action of cocaine that was not affected by circadian rhythms. Specifically, we observed that AANAT-mutant mice are more resistant to cocaine-triggered acute locomotor stimulation, as previously reported [11]. It has to be stressed that our experimental model included circadian rhythms and a natural AANAT mutated inbred mice strain, C57BL/6J [10]. As a control, we used one of the few inbred mice strains with functional circadian levels of serotonin metabolites N-acetylserotonin and melatonin, C3H/HeJ [13,22]. Genetic differences other than AANAT mutations
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may differentiate these two strains of mice. For example, the C3H/HeJ mice carry a mutation for retinal degeneration; however, these mice express regular circadian melatonin synthesis and light-induced suppression of pineal melatonin [23]. To explore and address a possible interference of different genetic backgrounds, a transgenic AANAT knockout would be needed. Nevertheless, our results conclusively demonstrated that behavioral sensitization to cocaine in mice is strongly influenced by a circadian mechanism that appears to be absent in the C57BL/6J strain. For the first time, the present study demonstrates a marked difference in the development of cocaine-induced behavioral sensitization during the day vs. at night in inbred mice, and provides new insight into possible mechanisms involved in addiction. Since AANAT mutant; i.e., NAS/melatonin-deficient mice, develop cocaine-induced behavioral sensitization regardless of the time of the day, the nocturnal induction of the serotonin-metabolizing enzyme AANAT and the subsequent elevation of blood levels of the serotonin metabolites NAS and melatonin might be responsible for nocturnal suppression of cocaine-induced behavioral sensitization. Further studies are needed to elucidate the molecular mechanisms that link the AANAT system and the behavioral effects of drugs of abuse, such as cocaine. Considering that the mechanisms of drug sensitization (reverse tolerance) might be operative in the development of drug abuse [24], a better understanding of the nocturnal suppression of cocaine sensitization observed in our study could help us identify a novel target for prevention and/or treatment of addictions.
Acknowledgments Supported in part by NIH grant RO1 MH61572 (H.M.).
References 1. Erickson TB, Lee J, Zautcke JL, Morris R. Analysis of cocaine chronotoxicology in an urban ED. American Journal of Emergency Medicine 1998;16(6):568 – 71. 2. Giorgetti M, Zhdanova IV. Chronic cocaine treatment induces dysregulation in the circadian pattern of rats’ feeding behavior. Brain Research 2000;877(2):170 – 5. 3. Iijima Y, Shinoda M, Kuribara H, Asami T, Uchihashi Y. Evaluation of acute and sub-acute effects of cocaine by means of circadian variation in wheel-running and drinking in mice. Nihon Shinkei Seishin Yakurigaku Zasshi 1995;15(4):315 – 21. 4. Kita T, Takahashi M, Wagner GC, Kubo K, Nakashima T. Methamphetamine-induced changes in activity and water intake during light and dark cycles in rats. Progress in Neuro-Psychopharmacology and Biological Psychiatry 1998;22:1185 – 96. 5. Baird TJ, Gauvin D. Characterization of cocaine self-administration and pharmacokinetics as a function of time of day in the rat. Pharmacology Biochemistry and Behavior 2000;65(2):289 – 99. 6. Roseboom PH, Coon SL, Baler R, McCune SK, Weller JL, Klein DC. Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland. Endocrinology 1996;137:3033 – 44. 7. Vanecek J. Cellular mechanisms of melatonin action. Physiological Reviews 1998;78(3):687 – 721.
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8. Sircar R. Effect of melatonin on cocaine-induced behavioral sensitization. Brain Research 2000;857(1 – 2): 295 – 9. 9. Uz T, Manev H. Prolonged swim-test immobility of serotonin N-acetyltransferase (AANAT)-mutant mice. Journal of Pineal Research 2001;30(3):166 – 71. 10. Roseboom PH, Namboodiri MA, Zimonjic DB, Popescu NC, Rodriguez IR, Gastel JA, Klein DC. Natural melatonin ‘knockdown’ in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase. Brain Research Molecular Brain Research 1998;63:189 – 97. 11. Marley RJ, Arros DM, Henricks KK, Marley ME, Miner LL. Sensitivity to cocaine and amphetamine among mice selectively bred for differential cocaine sensitivity. Psychopharmacology (Berlin) 1998;140(1):42 – 51. 12. Golden GT, Ferraro TN, Smith GG, Snyder RL, Jones NL, Berrettini WH. Acute cocaine-induced seizures: differential sensitivity of six inbred mouse strains. Neuropsychopharmacology 2001;24(3):291 – 9. 13. Goto M, Oshima I, Tomita T, Ebihara S. Melatonin content of the pineal gland in different mouse strains. Journal of Pineal Research 1989;7(2):195 – 204. 14. Itzhak Y, Martin JL, Black MD, Ali SF. Effect of the dopaminergic neurotoxin MPTP on cocaine-induced locomotor sensitization. Pharmacology Biochemistry and Behavior 1999;63(1):101 – 7. 15. Henricks KJ, Miner LL, Marley RJ. Differential cocaine sensitivity between two closely related substrains of C57BL mice. Psychopharmacology 1997;132:161 – 8. 16. Bettahi I, Pozo D, Osuna C, Reiter RJ, Acuna-Castroviejo D, Guerrero JM. Melatonin reduces nitric oxide synthase activity in rat hypothalamus. Journal of Pineal Research 1996;20(4):205 – 10. 17. Dubocovich ML, Masana MI, Benloucif S. Molecular pharmacology and function of melatonin receptor subtypes. Advances in Experimental Medicine and Biology 1999;460:181 – 90. 18. Iuvone PM, Gan J. Functional interaction of melatonin receptors and D1 dopamine receptors in cultured chick retinal neurons. Journal of Neuroscience 1995;15:2179 – 85. 19. Paredes D, Rada P, Bonilla E, Gonzalez LE, Parada M, Hernandez L. Melatonin acts on the nucleus accumbens to increase acetylcholine release and modify the motor activity pattern of rats. Brain Research 1999;850 (1 – 2):14 – 20. 20. Uz T, Manev H. Chronic fluoxetine administration increases the serotonin N-acetyltransferase messenger RNA content in rat hippocampus. Biological Psychiatry 1999;45:175 – 9. 21. Stefulj J, Hortner M, Ghosh M, Schauenstein K, Rinner I, Wolfler A, Semmler J, Liebmann PM. Gene expression of the key enzymes of melatonin synthesis in extrapineal tissues of the rat. Journal of Pineal Research 2001;30(4):243 – 7. 22. von Gall C, Lewy A, Schomerus C, Vivien-Roels B, Pevet P, Korf HW, Stehle JH. Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonindeficient C57BL mice and melatonin-proficient C3H mice. Eur J Neurosci 2000;12(3):964 – 72. 23. Lucas RJ, Freedman MS, Lupi D, Munoz M, David-Gray ZK, Foster RG. Identifying the photoreceptive inputs to the mammalian circadian system using transgenic and retinally degenerate mice. Behav Brain Res 2001;125(1 – 2):97 – 102. 24. Phillips TJ. Behavior genetics of drug sensitization. Critical Reviews in Neurobiology 1997;11(1):21 – 33.
Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase Tolga Uz*, Javaid I. Javaid, Hari Manev The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA Received 12 September 2001; accepted 11 January 2002
Abstract Circadian rhythms might be involved in addictive behaviors. The pineal secretory product melatonin decreases cocaine sensitization in rats; mice mutant for the critical melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase (AANAT), exhibit altered behaviors. We hypothesized that AANAT/melatonin system, which is up-regulated at night, affects cocaine sensitization in mice. Intraperitoneal cocaine treatment (10 and 20 mg/kg) dose-dependently increased locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice; this effect was similar during the day and at night. Injections of cocaine during the day for three days resulted in behavioral sensitization in normal and AANAT mutant mice whereas treatment at night triggered sensitization in AANATdeficient mice only. AANAT expression and synthesis of N-acetylserotonin/melatonin could play a role in addictive properties of cocaine. D 2002 Elsevier Science Inc. All rights reserved. Keywords: AANAT mutant; N-acetylserotonin; Melatonin; Addiction; Mice
Introduction Chronopharmacology has identified numerous drugs whose effects depend upon circadian rhythms. In humans, use of cocaine appears to be diurnally affected; most abuse occurs during the afternoon and cocaine-caused health problems peak soon thereafter [1]. In
* Corresponding author. Tel.: +1-312-413-4580; fax: +1-312-413-4569. E-mail address: [email protected] (T. Uz). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 5 9 - X
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experimental animals, psychostimulants, such as cocaine and amphetamine present circadian rhythmicity in their behavioral and neurophysiological effects such as feeding and locomotor activity [2–4]. Moreover, circadian changes were observed in cocaine self-administration in rats; these changes were not accompanied by a corresponding variance in the pharmacokinetic profiles of cocaine [5]. Thus, it is possible that circadian rhythm-driven mechanisms could play a pivotal role in development of cocaine addiction. The pineal gland is a key organ that influences mammalian circadian rhythms. Melatonin is synthesized from serotonin by an action of arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase; AANAT) which produces rate-limiting intermediate N-acetylserotonin (NAS). AANAT mRNA content [6] and NAS and melatonin levels (for review, see [7]) exhibit marked circadian rhythms with high levels at night and low or even undetectable levels during the day. Recently, Sircar [8] found in experiments with rats that the administration of melatonin prior to repeated cocaine injections prevented the development of cocaine-induced behavioral sensitization. These data point to a possible role for the AANAT/melatonin system in cocaine-induced behaviors and possibly in the development of drug addictions. Our recent findings suggest that AANAT mutant mice can be used to investigate the contribution of the AANAT/melatonin system to mouse behavior [9]. Moreover, the natural AANAT mutant (‘‘NAS/melatonin deficient’’) C57BL/6J strain of mice [9,10] shows less sensitivity to the locomotor-stimulating [11] and seizure-inducing [12] effects of cocaine than AANAT normal mice, such as the C3H/HeJ strain. Therefore, we hypothesized that the AANAT/melatonin system might be important for the development of locomotor sensitization to cocaine and that its role could be investigated with the help of the AANAT mutation. We tested our hypothesis using the natural AANAT mutant (C57BL/6J) and AANAT normal mice (C3H/HeJ), and we performed the experiments during the day (low AANAT expression) and at night (high AANAT expression).
Methods Animals and drugs Male C57BL/6J and C3H/HeJ mice, 8-weeks old and weighing 25-30 g, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Animals were housed in groups of three and had free access to laboratory chow and water except during behavioral experiments. They were kept in a temperature-controlled room on a 12-h light:dark cycle (lights off at 7 pm). Experiments were carried out during the day (8-11 am) and at night (9-12 pm). The experimental protocol was approved by the institutional Animal Care Committee. Cocaine hydrochloride (Sigma Chemical St. Louis, MO, USA) was dissolved in sterile physiological saline (0.9% NaCl) and administered i.p. in an injection volume of 0.05 ml/ 25g body weight (n = 6). Cocaine was injected within the last hour of the above-noted three-hour time windows. Vehicle treatments were administered in the same volume and manner (n = 6).
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Locomotor activity measurements, behavioral sensitization Mice that had not been exposed to the testing monitors previously were weighed and placed individually in 21 22 12 cm (length width height) activity cages for a 30-min adaptation period. After this period, locomotor activity was measured for 30 min using the Cage Rack Photobeam Activity Measurement System (San Diego Instruments, San Diego, CA, USA) equipped with computer-monitored photobeam frames. Immediately after the second 30-min interval, animals were injected with saline or 10 or 20 mg/kg free-base cocaine and returned to the activity cages; the recording procedure continued for additional 30 min. This schedule was selected based on the previous characterization of melatonin production in the two strains of mice we used [13]. These authors reported circadian melatonin production in C3H/HeJ but no detectable melatonin in C57BL/6J mice. In the C3H/HeJ mice they found very low melatonin during the day lasting up to two hours after lights off. [13]. The movement of each mouse was recorded as the number of beam interruptions and reported as locomotor activity (ambulation). The experiment room was equipped with a light timer; the nighttime drug administrations were done under dim red light. After each experiment session, mice were returned to their home cages. To measure locomotor sensitization, the same procedure was repeated for three consecutive days and nights. Statistical analysis Locomotor activity scores (mean ± s.e.m.) were analyzed by a two-way ANOVA with the factors of chronic treatment (cocaine vs. saline) and time of the treatment (day vs. night) for
Fig. 1. Stimulatory dose-dependent effects of acute cocaine administration on locomotor activity of AANAT normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice: absence of diurnal alterations. Results (mean ± s.e.m.) are expressed as a percentage of locomotor activity (number of beam breaks during a 30-min test session: 6 per each group) of the corresponding control, saline-injected mice (dashed line). Mice were injected during the day (open bars) or at night under dim red light (closed bars). *p < 0.05 and **p < 0.01 vs. corresponding salineinjected control group (Duncan’s test). Note a dose-dependent locomotor activation by cocaine. No differences were observed between day vs. night responses.
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both strains of mice (C57BL/6J and C3H/HeJ) separately, followed by Duncan’s multiplerange post-hoc test with a p < 0.05 accepted as statistically significant.
Results Intraperitoneal cocaine treatment (10 and 20 mg/kg) induced an acute dose-dependent increase in the locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice, and this effect was similar during the day and at night (in the dark) (Fig. 1). However, the actual extent of the stimulatory effect of cocaine was lower in AANAT mutant than in normal mice (Fig. 1: p < 0.05, Duncan’s test). Repeated (three consecutive days) injections of cocaine applied during the day only (low AANAT/melatonin in normal mice) resulted in behavioral sensitization both in normal and AANAT mutant/deficient mice (Fig. 2). Repeated injections of cocaine at night (high AANAT/melatonin in normal mice) failed to produce behavioral sensitization in normal mice but were effective in triggering sensitization in AANAT mutant mice (Fig. 2).
Fig. 2. Circadian alterations in the development of cocaine sensitization in AANAT normal (C3H/HeJ) mice and the absence of circadian influence in AANAT mutant (C57BL/6J) mice. Results (mean ± s.e.m.) are expressed as locomotor activity; i.e., the number of beam breaks during a 30-min test session measured individually. Mice (6 per group) were injected for three consecutive days i.p. with saline (4) or 10 (6) and 20 (5) mg/kg cocaine, either during the day or at night under dim red light. Note the development of sensitization to cocaine (increased locomotor activity after second and third injection) in all groups except C3H/HeJ mice injected at night; *p < 0.05 and **p < 0.01 vs. day 1; #p < 0.05 vs. day 2 (Duncan’s test).
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Discussion Behavioral sensitization to repeated administration of cocaine in inbred mice is well documented [14,15] and represents a behavioral model indicative of the addictive properties of this drug. Here we show evidence for a marked circadian alteration in the development of cocaine sensitization in normal mice: whereas repeated cocaine injections during the day produced robust behavioral sensitization, repeated administration at night failed to trigger sensitization. This circadian effect was completely absent in AANAT mutant mice that do not synthesize melatonin; in these mice, cocaine was effective in triggering behavioral sensitization both during the day and at night. Our findings suggest that endogenously-produced melatonin (at night) might be responsible for the nocturnal suppression of cocaine sensitization in normal mice. Supporting this possibility is a recent finding by Sircar [8]; this author reported that repeated daily melatonin injections prior to administration of cocaine for five days prevented the development of cocaine-triggered behavioral sensitization in rats. Thus, our data point to a putative role for AANAT in circadian alterations of behavioral sensitization to cocaine and also to the possibility of its addictive liability. Several mechanisms could participate in the link between AANAT– cocaine. Since AANAT is predominantly expressed in the pineal gland [6], it is likely that when synthesized and released in to the blood, which normally occurs at night, pineal serotonin metabolites produced by AANAT (i.e., NAS and its metabolite melatonin) affect their primary targets and subsequently modify the action of cocaine. One such target could be nitric oxide synthase (NOS); inhibition of NOS by melatonin [16] or NAS could reduce behavioral sensitization [8]. Alternatively, NAS and/or melatonin could bind specific receptors [17]; it is not clear how these receptors would modify the action of cocaine but one could envision their putative effect on the release of neurotransmitters such as dopamine or serotonin, as suggested by others [18]. Sircar [8] reported that pharmacological (30 mg/kg) melatonin increases acute cocaineinduced locomotor activity, whereas we did not observe different acute effect of cocaine during the day or at night (Fig. 1). Direct injections of melatonin into the nucleus accumbens either stimulate (100 mM) or decrease (3 mM) locomotor activity [19]. Thus, it is possible that endogenous melatonin affects the acute action of cocaine differently from pharmacological concentrations. Primarily pineal enzyme, AANAT is also expressed in the brain [9,20,21] although its role in the central nervous system (CNS) is not clear. It has been suggested that expression of AANAT in the CNS might be responsible for behaviors unrelated to circadian rhythms [9]; if relevant for behavioral actions of cocaine, the CNS AANAT might influence the sensitivity of mice to the acute stimulatory action of cocaine that was not affected by circadian rhythms. Specifically, we observed that AANAT-mutant mice are more resistant to cocaine-triggered acute locomotor stimulation, as previously reported [11]. It has to be stressed that our experimental model included circadian rhythms and a natural AANAT mutated inbred mice strain, C57BL/6J [10]. As a control, we used one of the few inbred mice strains with functional circadian levels of serotonin metabolites N-acetylserotonin and melatonin, C3H/HeJ [13,22]. Genetic differences other than AANAT mutations
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may differentiate these two strains of mice. For example, the C3H/HeJ mice carry a mutation for retinal degeneration; however, these mice express regular circadian melatonin synthesis and light-induced suppression of pineal melatonin [23]. To explore and address a possible interference of different genetic backgrounds, a transgenic AANAT knockout would be needed. Nevertheless, our results conclusively demonstrated that behavioral sensitization to cocaine in mice is strongly influenced by a circadian mechanism that appears to be absent in the C57BL/6J strain. For the first time, the present study demonstrates a marked difference in the development of cocaine-induced behavioral sensitization during the day vs. at night in inbred mice, and provides new insight into possible mechanisms involved in addiction. Since AANAT mutant; i.e., NAS/melatonin-deficient mice, develop cocaine-induced behavioral sensitization regardless of the time of the day, the nocturnal induction of the serotonin-metabolizing enzyme AANAT and the subsequent elevation of blood levels of the serotonin metabolites NAS and melatonin might be responsible for nocturnal suppression of cocaine-induced behavioral sensitization. Further studies are needed to elucidate the molecular mechanisms that link the AANAT system and the behavioral effects of drugs of abuse, such as cocaine. Considering that the mechanisms of drug sensitization (reverse tolerance) might be operative in the development of drug abuse [24], a better understanding of the nocturnal suppression of cocaine sensitization observed in our study could help us identify a novel target for prevention and/or treatment of addictions.
Acknowledgments Supported in part by NIH grant RO1 MH61572 (H.M.).
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