Repeated exposure to substances of abuse results in an increase in some behavioral responses. This phenomenon, called behavioral sensitization (BS), is well described in preclinical models. However, its existence in humans is still a matter of debate. After a review of preclinical evidence of BS and its mechanisms in animal models, we reviewed the evidence supporting the existence of BS in humans, despite the limited research available in this regard. We focused our review on opioids and psychostimulants, since they share the ability to promote addictive behaviors. Further, they induce BS despite their distinct sedative and stimulant properties. Moreover, we proposed future research perspectives in this review to address the remaining unsolved questions, especially regarding BS in humans using a harm reduction approach.

Time-of-day is a crucial, yet often overlooked, biological variable in biomedical research. We examined the top 25 most cited papers in several domains of behavioral neuroscience to determine whether time-of-day information was reported. The majority of studies report behavioral testing conducted during the day, which does not coincide with the optimal time to perform the testing from an functional perspective of the animals being tested. The majority of animal models used in biomedical research are nocturnal rodents; thus, testing during the light phase (i.e. animals' rest period) may alter the results and introduce variability across studies. Time-of-day is rarely considered in analyses or reported in publications; the majority of publications fail to include temporal details when describing their experimental methods, and those few that report testing during the dark rarely report whether measures are in place to protect from exposure to extraneous light. We propose that failing to account for time-of-day may compromise replication of findings across behavioral studies and reduce their value when extrapolating results to diurnal humans.

Investigations have shown that the circadian rhythm can affect the mechanisms associated with drug dependence. In this regard, we sought to assess the negative consequence of morphine withdrawal syndrome on conditioned place aversion (CPA) and lateral paragigantocellularis (LPGi) neuronal activity in morphine-dependent rats during light (8:00-12:00) and dark (20:00-24:00) cycles. Male Wistar rats (250-300 g) were received 10 mg/kg morphine or its vehicle (Saline, 2 mL/kg/12 h, s.c.) in 13 consecutive days for behavioral assessment tests. Then, naloxone-induced conditioned place aversion and physical signs of withdrawal syndrome were evaluated during light and dark cycles. In contrast to the behavioral part, we performed in vivo extracellular single-unit recording for investigating the neural response of LPGi to naloxone in morphine-dependent rats on day 10 of morphine/saline exposure. Results showed that naloxone induced conditioned place aversion in both light and dark cycles, but the CPA score during the light cycle was larger. Moreover, the intensity of physical signs of morphine withdrawal syndrome was more severe during the light cycle (rest phase) compare to the dark one. In electrophysiological experiments, results indicated that naloxone evoked both excitatory and inhibitory responses in LPGi neurons and the incremental effect of naloxone on LPGi activity was stronger in the light cycle. Also, the neurons with the excitatory response exhibited higher baseline activity in the dark cycle, but the neurons with the inhibitory response showed higher baseline activity in the light cycle. Interestingly, the baseline firing rate of neurons recorded in the light cycle was significantly different in response (excitatory/inhibitory) -dependent manner. We concluded that naloxone-induced changes in LPGi cellular activity and behaviors of morphine-dependent rats can be affected by circadian rhythm and the internal clock.

Genome-wide association meta-analysis (GWAMA) is an effective approach to enlarge sample sizes and empower the discovery of novel associations between genotype and phenotype. Independent replication has been used as a gold-standard for validating genetic associations. However, as current GWAMA often seeks to aggregate all available datasets, it becomes impossible to find a large enough independent dataset to replicate new discoveries. Here we introduce a method, MAMBA (Meta-Analysis Model-based Assessment of replicability), for assessing the "posterior-probability-of-replicability" for identified associations by leveraging the strength and consistency of association signals between contributing studies. We demonstrate using simulations that MAMBA is more powerful and robust than existing methods, and produces more accurate genetic effects estimates. We apply MAMBA to a large-scale meta-analysis of addiction phenotypes with 1.2 million individuals. In addition to accurately identifying replicable common variant associations, MAMBA also pinpoints novel replicable rare variant associations from imputation-based GWAMA and hence greatly expands the set of analyzable variants.

The circadian system organizes circadian rhythms (biological cycles that occur around 24 h) that couple environmental cues (zeitgebers) with internal functions of the organism. The misalignment between circadian rhythms and external cues is known as chronodisruption and contributes to the development of mental, metabolic and other disorders, including cancer, cardiovascular diseases and addictive disorders. Drug addiction represents a global public health concern and affects the health and well-being of individuals, families and communities. In this manuscript, we reviewed evidence indicating a bidirectional relationship between the circadian system and the development of addictive disorders. We provide information on the interaction between the circadian system and drug addiction for each drug or drug class (alcohol, cannabis, hallucinogens, psychostimulants and opioids). We also describe evidence showing that drug use follows a circadian pattern, which changes with the progression of addiction. Furthermore, clock gene expression is also altered during the development of drug addiction in many brain areas related to drug reward, drug seeking and relapse. The regulation of the glutamatergic and dopaminergic neurocircuitry by clock genes is postulated to be the main circadian mechanism underlying the escalation of drug addiction. The bidirectional interaction between the circadian system and drug addiction seems to be mediated by the effects caused by each drug or class of drugs of abuse. These studies provide new insights on the development of successful strategies aimed at restoring/stabilizing circadian rhythms to reduce the risk for addiction development and relapse.

Anhydroecgonine methyl ester (AEME), also called methylecgonidine, is a pyrolysis product of crack cocaine that is neurotoxic and potentiates cocaine-induced sensitization. The sensitization induced by drugs of abuse can be influenced by melatonin, a neuroprotective pineal hormone. In the same way, drugs of abuse like alcohol and methamphetamine can modify melatonin synthesis. The aim of the present work was to investigate the AEME effects on melatonin synthesis in the rat pineal gland. Neurotransmitter systems involved in its effects, antioxidant enzyme activities and the melatonin protective role in AEME-induced toxicity were also evaluated. The animals were injected with AEME i.p. (1.12 mg per kg of body weight per day) or vehicle for 10 consecutive days and the nocturnal pineal melatonin synthesis profile and SOD, GPx and GR activities in the cerebral cortex and hippocampus were assessed. Cultured pineal glands were incubated with AEME for 30 min or 48 h before norepinephrine stimulation and melatonin synthesis, arylalkylamine N-acetyltransferase activity, cAMP and [Ca2+]i were determined. The involvement of cholinergic and glutamatergic systems was analyzed using different antagonists. The protective role of melatonin in AEME toxicity on hippocampal neurons was evaluated by a viability assay. AEME impaired melatonin synthesis both in vivo and in vitro and this effect seems to be mediated by muscarinic receptors and [Ca2+]i elevation. AEME reduced neuronal viability and melatonin was able to protected hippocampal neurons against AEME toxicity. The melatonin synthesis impairment observed could lead to the worsening of the direct AEME neurotoxicity and to the exacerbation of the crack cocaine addiction and sensitization.

Melatonin, or 5-methoxy-N-acetyltryptamine, is synthesized and released by the pineal gland and locally in the retina following a circadian rhythm, with low levels during the day and elevated levels at night. Melatonin activates two high-affinity G protein-coupled receptors, termed MT1 and MT2, to exert beneficial actions in sleep and circadian abnormality, mood disorders, learning and memory, neuroprotection, drug abuse, and cancer. Progress in understanding the role of melatonin receptors in the modulation of sleep and circadian rhythms has led to the discovery of a novel class of melatonin agonists for treating insomnia, circadian rhythms, mood disorders, and cancer. This review describes the pharmacological properties of a slow-release melatonin preparation (i.e., Circadin®) and synthetic ligands (i.e., agomelatine, ramelteon, tasimelteon), with emphasis on identifying specific therapeutic effects mediated through MT1 and MT2 receptor activation. Discovery of selective ligands targeting the MT1 or the MT2 melatonin receptors may promote the development of novel and more efficacious therapeutic agents.

While it is known that tobacco use varies across the 24-h day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however, the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments.

The objective of this study is examine the role of melatonin and melatonin receptors in nicotine free-choice consumption

A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin-deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin-proficient mice lacking both or one of the high-affinity melatonin receptors (MT1 and MT2; double-null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild-type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns.

Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin-proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However, single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference.

This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and potential mechanistic explanations.

Here, we review work over the past two decades that has indicated drug reward is modulated by the circadian system that generates daily (i.e., 24h) rhythms in physiology and behavior. Specifically, drug-self administration, psychomotor stimulant-induced conditioned place preference, and locomotor sensitization vary widely across the day in various species. These drug-related behavioral rhythms are associated with rhythmic neural activity and dopaminergic signaling in the mesocorticolimbic pathways, with a tendency toward increased activity during the species typical wake period. While the mechanisms responsible for such cellular rhythmicity remain to be fully identified, circadian clock genes are expressed in these brain areas and can function locally to modulate both dopaminergic neurotransmission and drug-associated behavior. In addition, neural and endocrine inputs to these brain areas contribute to cellular and reward-related behavioral rhythms, with the medial prefrontal cortex playing a pivotal role. Acute or chronic administration of drugs of abuse can also alter clock gene expression in reward-related brain regions. Emerging evidence suggests that drug craving in humans is under a diurnal regulation and that drug reward may be influenced by clock gene polymorphisms. These latter findings, in particular, indicate that the development of therapeutic strategies to modulate the circadian influence on drug reward may prove beneficial in the treatment of substance abuse disorders.

Drug addiction is a brain disease involving alterations in anatomy and functional neural communication. Drug intake and toxicity show daily rhythms in both humans and rodents. Evidence concerning the role of clock genes in drug intake has been previously reported. However, the implication of a timekeeping brain locus is much less known. The epithalamic lateral habenula (LHb) is now emerging as a key nucleus in drug intake and addiction. This brain structure modulates the activity of dopaminergic neurons from the ventral tegmental area, a central part of the reward system. Moreover, the LHb has circadian properties: LHb cellular activity (i.e., firing rate and clock genes expression) oscillates in a 24-h range, and the nucleus is affected by photic stimulation and has anatomical connections with the main circadian pacemaker, the suprachiasmatic nucleus. Here, we describe the current insights on the role of the LHb as a circadian oscillator and its possible implications on the rhythmic regulation of the dopaminergic activity and drug intake. These data could inspire new strategies to treat drug addiction, considering circadian timing as a principal factor.

We have demonstrated that mitochondrial oxidative damage and PKCδ overexpression contribute to methamphetamine-induced dopaminergic degeneration. Although it is recognized that antioxidant melatonin is effective in preventing neurotoxicity induced by methamphetamine, its precise mechanism remains elusive. C57BL/6J wild-type mice exhibited a similar degree of dopaminergic deficit when methamphetamine was administered during light and dark phases. Furthermore, dopaminergic neuroprotection by genetic inhibition of PKCδ during the light phase was comparable to that during the dark phase. Thus, we have focused on the light phase to examine whether melatonin modulates PKCδ-mediated neurotoxic signaling after multiple high doses of methamphetamine. To enhance the bioavailability of melatonin, we applied liposomal melatonin. Treatment with methamphetamine resulted in hyperthermia, mitochondrial translocation of PKCδ, oxidative damage (mitochondria > cytosol), mitochondrial dysfunction, pro-apoptotic changes, ultrastructural mitochondrial degeneration, dopaminergic degeneration, and behavioral impairment in wild-type mice. Treatment with liposomal melatonin resulted in a dose-dependent attenuation against degenerative changes induced by methamphetamine in wild-type mice. Attenuation by liposomal melatonin might be comparable to that by genetic inhibition (using PKCδ((-/-)) mice or PKCδ antisense oligonucleotide). However, liposomal melatonin did not show any additional protective effects on the attenuation by genetic inhibition of PKCδ. Our results suggest that the circadian cycle cannot be a key factor in modulating methamphetamine toxicity under the current experimental condition and that PKCδ is one of the critical target genes for melatonin-mediated protective effects against mitochondrial burdens (dysfunction), oxidative stress, pro-apoptosis, and dopaminergic degeneration induced by methamphetamine.

Cocaine sensitization and reward are reported to be under the influence of diurnal rhythm. However, no previous studies have reported brain areas that play a role as modulators and underlie the mechanism of diurnal variations in cocaine reward. We examined (1) the diurnal rhythm of glycogen synthase kinase-3β (GSK-3β) activity in the suprachiasmatic nucleus (SCN) and reward-related brain areas in naive rats; (2) the effect of day and night on the acquisition of cocaine-induced conditioned place preference (CPP); (3) the influence of cocaine-induced CPP on GSK-3β activity in the SCN and reward-related brain areas; and (4) the effect of the GSK-3β inhibitor SB216763 microinjected bilaterally into the ventral tegmental area (VTA) on cocaine-induced CPP. A significant diurnal rhythm of GSK-3β activity was found in the SCN and reward-related brain areas, with diurnal variations in cocaine-induced CPP. GSK-3β activity in the SCN and reward-related brain areas exhibited marked diurnal variations in rats treated with saline. GSK-3β activity in rats treated with cocaine exhibited distinct diurnal variations only in the prefrontal cortex and VTA. Cocaine decreased the expression of phosphorylated GSK-3β (i.e. increased GSK-3β activity) only in the VTA in rats trained and tested at ZT4 and ZT16. SB216763 microinjected into the VTA bilaterally eliminated the diurnal variations in cocaine-induced CPP, but did not affect the acquisition of cocaine-induced CPP. These findings suggest that the VTA may be a critical area involved in the diurnal variations in cocaine-induced CPP, and GSK-3β may be a regulator of diurnal variations in cocaine-induced CPP.

The disruptive effects of cocaine on physiological, behavioral and genetic processes are well established. However, few studies have focused on the actions of cocaine on the adult circadian timekeeping system, and none have explored the circadian implications of long-term (weeks to months) cocaine exposure. The present study was undertaken to explore the actions of such long-term cocaine administration on core circadian parameters in mice, including rhythm period, length of the nocturnal activity period and photic entrainment. For cocaine dosing over extended periods, cocaine was provided in drinking water using continuous and scheduled regimens. The impact of chronic cocaine on circadian regulation was evidenced by disruptions of the period of circadian entrainment and intrinsic free-running circadian period. Specifically, mice under a skeleton photoperiod (1-min pulse of dim light delivered daily) receiving continuous ad libitum cocaine entrained rapidly to the light pulse at activity onset. Conversely, water controls entrained more slowly at activity offset through a process of phase-delays, which resulted in their activity rhythms being entrained 147° out of phase with the cocaine group. This pattern persisted after cocaine withdrawal. Next, mice exposed to scheduled daily cocaine presentations exhibited free-running periods under constant darkness that were significantly longer than water controls and which also persisted after cocaine withdrawal. These cocaine-induced perturbations of clock timing could produce chronic psychological and physiological stress, contributing to increased cocaine use and dependence.

Melatonin modifies physiological and behavioral responses to psychostimulants, with the MT₁ and MT₂ melatonin receptors specifically implicated in facilitating methamphetamine (METH)-induced sensitization in melatonin-proficient mice.

The objective of the study is to assess differences in locomotor sensitization after a single dose of methamphetamine in low-melatonin-expressing C57BL/6 wild-type and MT₁ receptor knockout (MT₁KO) mice, comparing with melatonin-expressing C3H/HeN mice.

Mice received a vehicle or methamphetamine (1.2 mg/kg, i.p.) pretreatment (day 1) during the light (ZT5-9) or dark (ZT 19-21) periods in novel test arenas. Locomotor sensitization was assessed by methamphetamine challenge after an eight-day abstinence (day 9). TH protein expression was evaluated by immunofluorescence and Western blot analysis.

Methamphetamine pretreatment induced statistically significant locomotor sensitization upon challenge after eight-day abstinence in C3H and C57 wild-type mice during the light period. The magnitude of sensitization in C57 mice was diminished in the dark period and completely abrogated in MT₁KO mice. No differences were observed in tyrosine hydroxylase immunoreactivity in the mesolimbic dopamine system. Additional exposures to the test arenas after methamphetamine pretreatment (nights 2-6) enhanced sensitization.

Deletion of the MT₁ melatonin receptor abolishes sensitization induced by a single METH pretreatment. The magnitude of sensitization is also altered by time of day and contextual cues. We conclude that the MT₁ melatonin receptor is emerging as a novel target of therapeutic intervention for drug abuse disorders.

Perinatal asphyxia (PA) increases the likelihood of suffering from dopamine-related disorders, such as ADHD and schizophrenia. Since dopaminergic transmission plays a major role in cocaine sensitization, the purpose of this study was to determine whether PA could be associated with altered behavioral sensitization to cocaine. To this end, adult rats born vaginally (CTL), by caesarean section (C+), or by C+ with 15 min (PA15, moderate PA) or 19 min (PA19, severe PA) of global anoxia were repeatedly administered with cocaine (i.p., 15 mg/kg) and then challenged with cocaine (i.p., 15 mg/kg) after a 5-day withdrawal period. In addition, c-Fos, FosB/ΔFosB, DAT, and TH expression were assessed in dorsal (CPu) and ventral (NAcc) striatum. Results indicated that PA15 rats exhibited an increased locomotor sensitization to cocaine, while PA19 rats displayed an abnormal acquisition of locomotor sensitization and did not express a sensitized response to cocaine. c-Fos expression in NAcc, but not in CPu, was associated with these alterations in cocaine sensitization. FosB/ΔFosB expression was increased in all groups and regions after repeated cocaine administration, although it reached lower expression levels in PA19 rats. In CTL, C+, and PA15, but not in PA19 rats, the expression of TH in NAcc was reduced in groups repeatedly treated with cocaine, independently of the challenge test. Furthermore, this reduction was more pronounced in PA15 rats. DAT expression remained unaltered in all groups and regions studied. These results suggest that moderate PA may increase the vulnerability to drug abuse and in particular to cocaine addiction.

This study explored the role of the melatonin receptors in methamphetamine (METH)-induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT(1) and/or MT(2) melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild-type (WT), MT(1)KO and MT(2)KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH-pretreated but not in vehicle (VEH)-pretreated mice. In MT(1)/MT(2)KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH-pretreated WT, MT(1)KO and MT(2)KO mice was statistically different from VEH-treated controls. However, WT and MT(2)KO, but not MT(1)KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT(1) melatonin receptor activation by endogenous melatonin. We suggest that MT(1) and MT(2) melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.

Despite the evidence that there is a daily rhythm in smoking behavior and that the effects of drugs of abuse exhibit diurnal variations, very few studies have explored the extent to which sensitivity to the effects of nicotine vary over the course of the day. In the studies described in this report, the melatonin proficient mouse strain C3H/Ibg and the melatonin deficient mouse strains C57BL/6J and DBA/2J were assessed for diurnal variations in sensitivity to the effects of nicotine. Results indicated that there is significant variation in sensitivity to both activity and body temperature depressant effects of nicotine in the melatonin proficient C3H/Ibg strain with maximal sensitivity occurring during the latter third of the light period of the light cycle and minimal sensitivity taking place during the last third of the dark phase of the light cycle. The melatonin deficient strains did not exhibit diurnal differences in sensitivity to the effects of nicotine suggesting a potential role for melatonin in modulating the effects of nicotine. Experiments with knockout mice lacking both the Mtnr1a and Mtnr1b melatonin receptors confirmed that the reduced sensitivity observed during the dark phase is melatonin dependent. Diurnal variation in nicotinic receptor expression also was measured in cortex, hippocampus, hypothalamus and striatum using [(125)I]-α-bungarotoxin and [(125)I]-epibatidine. [(125)I]-α-bungarotoxin binding in hypothalamus of C3H mice exhibited a diurnal pattern with maximal binding observed in the latter third of the light portion of the light cycle. No other significant differences in binding were detected.

Circadian pattern of activity regulates many aspects of mammalian physiology and behavior to particular times of the day by entraining the circadian clocks to external environmental signals. Since circadian rhythms are sensitive to many pharmacological agents, it is important to understand if the repetitive use of psychostimulants such as amphetamine will alter the circadian rhythm behavioral activity pattern. The present study uses male Sprague-Dawley rats to study the long-term effects of amphetamine on the locomotor circadian rhythm activity pattern. Rats were randomly assigned to a testing cage that recorded their locomotor activity nonstop for eleven days using the open field assay, as follows: one day of baseline activity was recorded and then the experimental group was injected with amphetamine (0.6mg/kg) for 6days, no treatment for 3days (i.e., washout days) and then re-challenged with amphetamine for one more day while the control group was treated similarly with saline. The Cosine Curve Statistical Analysis (CCSA) test was used to fit a 24-hour curve to activity pattern. Results indicate that repetitive daily amphetamine injections cause behavioral sensitization and a significant change of circadian rhythm of locomotor activity pattern, and elicit behavioral expectation to receive the drug or expression of withdrawal during the washout days. The results suggest that either changes in circadian rhythm caused sensitization and withdrawal or sensitization and withdrawal caused the change in circadian rhythm activity.

The rewarding influence of drugs of abuse varies with time of day and appears to involve interactions between the circadian and the mesocorticolimbic dopamine systems. The circadian system is also intimately involved in measuring daylength. Thus, the present study examined the impact of changing daylength (photoperiod) on cocaine-seeking behaviors. Male Sprague-Dawley rats were trained and tested on a 12L:12D light:dark schedule for cocaine-induced reinstatement of conditioned place preference (CPP) at three times of day (Zeitgeber time (ZT): 4, 12, and 20) to determine a preference score. Rats were then shifted to either shorter (6L:18D) or longer (18L:6D) photoperiods and then to constant conditions, re-tested for cocaine-induced reinstatement under each different condition, and then returned to their original photoperiod (12L:12D) and tested once more. Rats exhibited a circadian profile of preference score in constant darkness with a peak at 12 h after lights-off. At both ZT4 and ZT20, but not at ZT12, shorter photoperiods profoundly suppressed cocaine reinstatement, which did not recover even after switching back to 12L:12D. In contrast, longer photoperiods did not alter reinstatement. Separate studies showed that the suppression of cocaine reinstatement was not due to repeated testing. In an additional experiment, we examined the photoperiodic regulation of tyrosine hydroxylase (TH) and dopamine transporter (DAT) proteins in drug-naive rats. These results revealed photoperiodic modulation of proteins in the prefrontal cortex and dorsal striatum, but not in the nucleus accumbens or ventral tegmental area. Together, these findings add further support to the circadian genesis of cocaine-seeking behaviors and demonstrate that drug-induced reinstatement is modulated by photoperiod. Furthermore, the results suggest that photoperiod partly contributes to the seasonal expression of certain drug-related behaviors in humans living at different latitudes and thus our findings may have implications for novel targeting of circadian rhythms in the treatment of addiction.

Many features of the suprachiasmatic nucleus (SCN) are the same in diurnal and nocturnal animals, suggesting that differences in phase preference are determined by mechanisms downstream from the SCN. Here, we examined this hypothesis by characterizing rhythmic expression of Period 1 (PER1) and Period 2 (PER2) in several extra-SCN areas in the brains of a diurnal murid rodent, Arvicanthis niloticus (grass rats). In the shell of the nucleus accumbens, dorsal striatum, piriform cortex, and CA1 of the hippocampus, both PER1 and PER2 were rhythmic, with peak expression occurring at ZT10. PER1 in the dentate gyrus also peaked at ZT10, but PER2 was arrhythmic in this region. In general, these patterns are 180 degrees out of phase with those reported for nocturnal species. In a second study, we examined inter-individual differences in the multioscillator system of grass rats. Here, we housed grass rats in cages with running wheels, under which conditions some individuals spontaneously adopt a day active (DA) and others a night active (NA) phase preference. In the majority of the extra-SCN regions sampled, the patterns of PER1 and PER2 expression of NA grass rats resembled those of nocturnal species, while those of DA grass rats were similar to the ones seen in grass without access to running wheels. In contrast, the rhythmic expression of both PER proteins was identical in the SCN and ventral subparaventricular zone (vSPZ) of DA and NA animals. Differences in the phase of oscillators downstream from the SCN, and perhaps the vSPZ, appear to determine the phase preference of particular species, as well as that of members of a diurnal species that show voluntary phase reversals. The latter observation has important implications for the understanding of health problems associated with human shift work.

The impact of the circadian timing system upon behavior and physiology is pervasive, and previous evidence suggests a circadian modulation of drug-seeking behavior and responsiveness to drugs of abuse. To further characterize daily rhythms in reward and to extend these observations to natural reinforcers, diurnal variation in the rewarding value of sex and systemic amphetamine was assessed via the conditioned place preference paradigm. To identify potential mechanisms for rhythmicity in reward, levels of tyrosine hydroxylase (TH) and core clock proteins (Period1 and Bmal1) were examined across the day in the ventral tegmental area (VTA) and the nucleus accumbens (NAcc). During an initial training period, male rat sexual performance varied diurnally with a nadir near the light-to-dark transition. Diurnal rhythms also were evident for both mating and amphetamine-related reward. However, the rhythms for these particular stimuli exhibited differences in their pattern of timing, with sex reward showing a peak during the middark period and amphetamine reward exhibiting high points during the late night and midday with a nadir prior to the light-to-dark transition. A diurnal variation also was seen for the locomotor-activating effect of acute amphetamine administration with a peak during the late night. Western blot analyses revealed that Period1 and Bmal1 protein levels were rhythmic in the NAcc but not in the VTA. By contrast, TH protein levels were rhythmic in both the NAcc and VTA, but the peaks differed with that in the NAcc coinciding with the peak of sex reward and that in the VTA associated with the peak in amphetamine reward. Thus, it appears that both natural and drug-related reward vary in a diurnal fashion but differ in the timing of their peak and nadir levels. The phase relationships between reward rhythms and mesolimbic TH protein levels suggest that an increased capacity for the release of dopamine in the NAcc may underlie the rhythms in sex-related reward, while amphetamine-related reward occurs at a time when the likelihood of evoked NAcc DA release is relatively low.

The concept of the dosing time-dependent (DTD) actions of drugs has been used to describe the effects of diurnal rhythms on pharmacological responsiveness. Notwithstanding the importance of diurnal variability in drug pharmacokinetics and bioavailability, it appears that in the central nervous system (CNS), the DTD actions of psychotropic drugs involve diurnal changes in the CNS-specific expression of genes encoding for psychotropic drug targets and transcription factors known as clock genes. In this review, we focused our discussion on the DTD effects of the psychostimulants cocaine and amphetamines. Both cocaine and amphetamines produce differential lasting behavioral alterations, that is, locomotor sensitization, depending on the time of the day they are administered. This exemplifies a DTD action of these drugs. The DTD effects of these psychostimulants correlate with diurnal changes in the system of transcription factors termed clock genes, for example, Period 1, and with changes in the availability of certain subtypes of dopamine receptors, for example, D2 and D3. Diurnal synthesis and release of the pineal hormone melatonin influence the DTD behavioral actions of cocaine and amphetamines. The molecular mechanism of melatonin's effects on the responsiveness of CNS to psychostimulants appears to involve melatonin receptors and clock genes. It is proposed that the DTD characteristics of psychostimulant action and the contributions of the melatonergic system may have clinical implications that include treatments for the attention deficit hyperactivity disorder and possibly neurotoxicity/neuroprotection.

In the last decade, the fruit fly Drosophila melanogaster, highly accessible to genetic, behavioral and molecular analyses, has been introduced as a novel model organism to help decipher the complex genetic, neurochemical, and neuroanatomical underpinnings of behaviors induced by drugs of abuse. Here we review these data, focusing specifically on cocaine-related behaviors. Several of cocaine's most characteristic properties have been recapitulated in Drosophila. First, cocaine induces motor behaviors in flies that are remarkably similar to those observed in mammals. Second, repeated cocaine administration induces behavioral sensitization a form of behavioral plasticity believed to underlie certain aspects of addiction. Third, a key role for dopaminergic systems in mediating cocaine's effects has been demonstrated through both pharmacological and genetic methods. Finally, and most importantly, unbiased genetic screens, feasible because of the simplicity and scale with which flies can be manipulated in the laboratory, have identified several novel genes and pathways whose role in cocaine behaviors had not been anticipated. Many of these genes and pathways have been validated in mammalian models of drug addiction. We focus in this review on the role of LIM-only proteins in cocaine-induced behaviors.

Ovarian hormones play a role in the use of drugs of abuse in women. In female rats estradiol has been shown to enhance acquisition of cocaine self-administration and behavioral sensitization induced by repeated cocaine treatment. Experiments were conducted to determine the effects of estradiol and/or progesterone on cocaine self-administration and behavioral sensitization to cocaine (10mg/kg; in animals with unilateral 6-hydroxydopamine lesions). Five groups of ovariectomized females were tested: (1) oil vehicle; (2) estradiol (E); (3) progesterone (P); (4) estradiol and progesterone given concurrently (EPC); (5) estradiol and progesterone given sequentially (EPS: 3 days of estradiol, 1 day progesterone, 1 day oil). All animals were tested during the dark phase of the light:dark cycle at ZT1600 and ZT2000-2100. Behavioral sensitization results: there was substantial conditioned turning throughout the habituation periods, and all animals exhibited behavioral sensitization with repeated cocaine treatment. Multivariate analysis indicated a significant effect of hormone treatment, time of day and day of testing. When individual groups were compared, however, only at ZT1600 did the E-treated and the EPS-treated animals show a trend (p<0.06) for greater behavioral sensitization to cocaine relative to the oil-treated animals. Self-administration results: all groups showed rapid acquisition of cocaine self-administration at 0.3 mg/kg/infusion, so we did not see an effect of ovarian hormones on acquisition, or a difference between groups tested at ZT1600 versus ZT2100 (p<0.05). There was, however, enhanced total intake of cocaine at 0.75 mg/kg/infusion in the E and the EPS groups. Concurrent administration of progesterone with estradiol counteracted the effect of estradiol on cocaine intake at 0.75 mg/kg/infusion, while progesterone alone did not enhance cocaine self-administration.

Recent work has shown that time-of-day influences drug-seeking behavior. The present experiments tested the hypothesis that the master circadian pacemaker located in the suprachiasmatic nucleus of the hypothalamus (SCN) is required for generating day:night differences in drug-seeking behavior, specifically the acquisition, extinction, and reinstatement of cocaine-induced conditioned place preference (CPP). Sham and SCN-lesioned (SCNx) rats were trained for cocaine-induced CPP behavior at either ZT4 (Zeitgeber time 4, 4 h after lights-on) or ZT12 (lights-off). After being tested for side preference, rats were allowed to extinguish CPP. This was followed by cocaine-induced reinstatement with 5 mg/kg and 10 mg/kg of cocaine. SCNx animals exhibited no 24-h locomotor activity rhythm. Acquisition of CPP behavior did not vary with time-of-day, but was greater in SCNx animals. Sham rats tested at ZT12 took significantly longer to extinguish CPP behavior compared to ZT4, an effect completely abolished by SCN lesions. Cocaine-induced reinstatement of CPP did not vary with time of day in sham rats. However, SCNx animals tested at ZT4 trended towards greater reinstatement to the low dose of cocaine, and displayed significantly less reinstatement to the higher dose of cocaine than sham rats. Additionally, SCNx rats tested for reinstatement to the lower dose of cocaine displayed greater reinstatement at ZT4 than at ZT12. We conclude that: 1) acquisition of CPP behavior does not vary between the two times of day tested but is influenced tonically by the SCN, 2) extinction of cocaine CPP varies with time-of-day and this variation depends critically on the SCN, and 3) reinstatement of cocaine CPP does not vary between the two times of day tested. However, day:night differences in reinstatement are unmasked in animals lacking an SCN, suggesting the possibility that an extra-SCN oscillator is responsible for generating variation in this cocaine-seeking behavior.

Time of day can influence cocaine-seeking behavior in rats, and this influence may depend on the suprachiasmatic nucleus (SCN). We used western blots to measure expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins in the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and caudate in sham and SCN-lesioned (SCNx) rats at ZT4 (Zeitgeber time 4, 4 h after lights-on) or ZT20 (8 h after lights-off). In the mPFC, DAT levels were lower at ZT20 than at ZT4 in sham but not SCNx rats. In the NAc, DAT expression was higher at ZT20 than at ZT4, and this effect was blunted in SCNx rats. Caudate DAT levels were unaffected by time of day or SCNx. Levels of TH did not change in mPFC with time of day or SCN lesion; TH levels were higher at ZT20 in the NAc, with a trend towards higher levels at this time in SCNx rats. Caudate TH expression was slightly elevated at ZT20 in sham but not in SCNx rats. We conclude that DAT and TH within these brain regions exhibit diurnal variation and dependence on the SCN. The results may have implications for new strategies to maximize pharmacological treatments for drug addiction.

Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals.

To test the hypothesis that time of day influences the locomotor effects of cocaine in rats, we measured short- and long-term sensitization to cocaine at five different Zeitgeber times (ZT). Short-term sensitization was expressed equally at all ZTs tested. Long-term sensitization was expressed only at dark onset, suggesting a possible influence of melatonin. Exogenous melatonin given immediately before cocaine challenge slightly enhanced long-term sensitization. Taken together, the present results support a time-of-day influence on locomotor sensitization to cocaine.

This study examined prevalence and patterns of co-use of opioids and cocaine in regular users of illicit opioids (N = 729) recruited from 5 Canadian cities. Fifty-seven percent (n = 417) reported having used both opioids and cocaine in the month and week preceding the interview; of these, 73% (n = 304) were able to identify a typical pattern of daily co-use. In a typical day, injectors of opioids and cocaine (n = 119) and injectors of opioids who inhaled cocaine (n = 111) showed stable opioid use but variable cocaine use, which peaked at 21 hr. Overall, 30% of the individuals used both drugs exclusively in a sequential fashion, 35% reported taking opioids and cocaine within the same hour, and 35% reported taking them together at the same time or mixing them. These findings indicate that different individuals display different patterns of opioids and cocaine co-use.

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.

The Porsolt forced swim test (FST) is one of the most widely used behavioral tests in the evaluation of the antidepressant effects of drugs. It is based on the fact that these drugs reduce the depression-related behaviors of learned helplessness. The model has been modified for use in mice. In contrast to rats, mice are exposed to forced swimming only once and their immobility behavior is measured and considered a "depression-like" phenotype. Like many other behavioral tests, FST can be affected by observer-related artifacts. In recent years, automated testing systems have been developed to decrease artifacts that may greatly influence the interpretation of results. In this work, we used two strains of mice, i.e., C3H/HeJ and C57BL/6J, which differ in their FST immobility times. We employed a new commercially available automated FST device and a blinded observer-based FST, and we examined their ability to measure behavioral differences between these two mouse strains. Our results suggest that the tested automated FST system generates reliable data comparable to results obtained by trained observers.

Contribution of circadian mechanisms to the psychostimulant-induced behaviors has been suggested. The pineal gland is important component of circadian mechanisms. Using pinealectomized mice and sham-operated controls, we tested the contribution of pineal gland to the rewarding effects of cocaine in conditioned place preference test. Experiments were performed both during the day and at night. Controls with intact pineal glands demonstrated significant decrease in cocaine-induced conditioned place preference at night compared to daytime, whereas pinealectomized mice did not show any diurnal differences. Circadian mechanisms regulated by the pineal gland thus appear critically involved in cocaine-induced reward.

Dopaminergic drugs, including the D2/D3 agonist quinpirole, produce lasting changes in the brain that lead to altered behavioral responses. The action of these drugs is dosing time-dependent; in fruit flies, behavioral response to quinpirole shows a marked circadian variability. Here we demonstrate diurnal rhythm-dependent variations both in quinpirole-induced locomotor behaviors and in striatal D2 and D3 protein levels in mice. We found opposing diurnal rhythms in striatal D2 and D3 protein levels, resulting in a high D2/D3 ratio during the day and a low D2/D3 ratio at night. Protracted quinpirole treatment differentially altered striatal D2/D3 rhythms depending on the time of injection (i.e., day or night). When quinpirole-induced locomotor activity was analyzed for 90 min, we found hypomotility after the first day or nighttime drug injection. By the seventh injection, daytime quinpirole treatment produced clear hyperactivity while nighttime quinpirole treatment continued to induce a significant initial hypoactivity followed by a hyperactivity period. Our data indicate that quinpirole-induced long-term alterations in the brain include dosing time-dependent changes in dopamine receptor rhythms. Therefore, we propose that diurnal mechanisms, which participate in drug-induced long-term changes in the dopamine system, are important for the development of dopaminergic behaviors.

Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO) gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LN(v)s), is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LN(v)function,Lmomutants also show defects in circadian rhythms of behavior. However, the role for LN(v)s in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LN(v)s reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LN(v)s to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug.

Fluoxetine produces initial paradoxical anxiogenic effect in some patients. In an elevated plus-maze (EPM), fluoxetine triggers an anxiogenic-like effect in rodents. Behavioral responses to psychoactive drugs can be modified by the pineal gland. We assessed the actions of fluoxetine in the EPM in melatonin-proficient C3H mice, melatonin-deficient C57BL6 mice, and in sham-operated and pinealectomized mice. Mice were assayed 30 min after the first injection and on day 14. Protracted fluoxetine treatment reduced the time on the anxiogenic open arms and increased the entries into the safe closed arms in sham-operated C3H mice. Fluoxetine was ineffective in pinealectomized C3H or C57BL6 mice. It is possible that the pineal system contributes to the previously observed anxiogenic action of fluoxetine in humans.

Sensitization to psychostimulants can be influenced by circadian rhythms. The pineal gland, the main source of circadian melatonin synthesis, may influence behavioral sensitization to cocaine; mice with normal melatonin rhythms do not get sensitized at night. Clock genes such as Period1 (Per1) show rhythmic region- and strain-dependent expression in the mouse brain, and mice mutant for the Per1 gene lack cocaine sensitization. Here, for the first time we show circadian changes of PER1 protein levels in the mouse striatum, a brain region crucial for the development of locomotor sensitization to cocaine. In male C3H/HeJ mice, we found peak striatal PER1 protein levels during the day; this was preceded by a Per1 mRNA peak 16 h earlier. Pinealectomized mice did not show this circadian pattern. We analyzed circadian cocaine sensitization at times when striatal PER1 protein levels in control mice (naive and sham-pinealectomized) were high and low, respectively. Only mice with circadian changes in striatal Per1 expression showed the night-time absence of cocaine sensitization, whereas pinealectomized mice were without circadian changes in striatal Per1 and were sensitized to cocaine regardless of diurnal rhythm. Our results indicate that both the striatal circadian Per1 expression and diurnal locomotor cocaine sensitization are strongly influenced by pineal products. Since we found evidence for the expression of melatonin receptor mRNA in the striatum, we suggest that further studies on pineal-driven mechanisms will help us better understand the mechanisms of drug abuse and identify novel targets for the prevention and/or treatment of addictions.

An unlikely animal model is gaining popularity in neuropharmacological research: the 2-mm fruit fly (Drosophila melanogaster). Drugs have been administered to adult flies in their food and, more recently, via gasses and injections. Pharmacological tools have introduced behavioral alterations in Drosophila reminiscent of human behavior, rescued flies from gene-alteration-triggered neuropathologies, and triggered gene silencing. Combined, these methods hold promise for significant neuropharmacological advancement.

This study describes the effects of melatonin on cocaine-induced anxiety-like behavior and nucleus accumbens (NAc) cAMP levels in rats. Animals drinking a solution of melatonin (200 ng/ml) at night, either during repeated cocaine administration (15 mg/kg i.p., twice a day for 9 days) or during its withdrawal, showed less anxiety-like behavior in a defensive withdrawal paradigm 48 h after the last injection of cocaine. Melatonin did not alter behavior in control rats treated with saline. Animals exposed for 1 week to unrestricted free-choice oral melatonin self-administration (200 ng/ml) did not show preference for the drinking solution containing melatonin. Pretreatment with melatonin (200 ng/kg i.p. or 200 ng/ml orally) significantly attenuated the augmentation of cAMP levels in NAc following acute cocaine administration (15 mg/kg i.p.). Taken together, these results suggest that a low-dose night-time melatonin treatment results in anxiolytic-like effects in rats withdrawn from repeated cocaine administration, can antagonize cocaine-induced activation of NAc cAMP levels and has low dependence liability.