Methods to Assess Melatonin Receptor-Mediated Phase-Shift and Re-entrainment of Rhythmic Behaviors in Mouse Models.

The neurohormone melatonin facilitates entrainment of biological rhythms to environmental light-dark conditions as well as phase-shifts of circadian rhythms in constant conditions via activation of the MT1 and/or MT2 receptors expressed within the suprachiasmatic nucleus of the hypothalamus. The efficacy of melatonin and related agonists to modulate biological rhythms can be assessed using two well-validated mouse models of rhythmic behaviors. These models serve as predictive measures of therapeutic efficacy for treatment of circadian phase disorders caused by internal (e.g., clock gene mutations, blindness, depression, seasonal affective disorder) or external (e.g., shift work, travel across time zones) causes in humans. Here we provide background and detailed protocols for quantitative assessment of the magnitude and efficacy of melatonin receptor ligands in mouse circadian phase-shift and re-entrainment paradigms. The utility of these models in the discovery of novel therapeutics acting on melatonin receptors will also be discussed.

Impact of endogenous melatonin on rhythmic behaviors, reproduction, and survival revealed in melatonin-proficient C57BL/6J congenic mice.

The hormone melatonin is synthesized from serotonin by two enzymatic reactions (AANAT and ASMT/HIOMT) in the pineal gland following a circadian rhythm with low levels during the day and high levels at night. The robust nightly peak of melatonin secretion is an output signal of the circadian clock to the whole organism. However, so far the regulatory roles of endogenous melatonin in mammalian biological rhythms and physiology processes are poorly understood. Here, we establish congenic mouse lines (>N10 generations) that are proficient or deficient in melatonin synthesis (AH+/+ or AH-/- mice, respectively) on the C57BL/6J genetic background by crossing melatonin-proficient MSM/Ms with C57BL/6J. AH+/+ mice displayed robust nightly peak of melatonin secretion and had significantly higher levels of pineal and plasma melatonin vs AH-/- mice. Using this mice model, we investigated the role of endogenous melatonin in regulating multiple biological rhythms, physiological processes, and rhythmic behaviors. In the melatonin-proficient (AH+/+) mice, the rate of re-entrainment of wheel-running activity was accelerated following a 6-hour phase advance of dark onset when comparted with AH-/- mice, suggesting a role of endogenous melatonin in facilitating clock adjustment. Further in the AH+/+ mice, there was a significant decrease in body weight, gonadal weight and reproductive performance, and a significant increase in daily torpor (a hypothermic and hypometabolic state lasting only hours during adverse conditions). Endogenous melatonin, however, had no effect in the modulation of the diurnal rhythm of 2-[125 I]-iodomelatonin receptor expression in the SCN, free-running wheel behavior in constant darkness, life span, spontaneous homecage behaviors, and various types of social-emotional behaviors. The findings also shed light on the role of endogenous melatonin in mice domestication and provide new insights into melatonin's action in reducing energy expenditure during a food shortage. In summary, the congenic mice model generated in this study offers a significant advantage toward understanding of the role of endogenous melatonin in regulating melatonin receptor-mediated rhythm behaviors and physiological functions.

Food-induced reinforcement is abrogated by the genetic deletion of the MT1 or MT2 melatonin receptor in C3H/HeN mice.

Palatable food is known for its ability to enhance reinforcing responses. Studies have suggested a circadian variation in both drug and natural reinforcement, with each following its own time course. The goal of this study was to determine the role of the MT1 and MT2 melatonin receptors in palatable snack food-induced reinforcement, as measured by the conditioned place preference (CPP) paradigm during the light and dark phases. C3H/HeN wild-type mice were trained for snack food-induced CPP at either ZT 6 - 8 (ZT: Zeitgeber time; ZT 0 = lights on), when endogenous melatonin levels are low, or ZT 19 - 21, when melatonin levels are high. These time points also correspond to the high and low points for expression of the circadian gene Period1, respectively. The amount of snack food (chow, Cheetos®, Froot Loops® and Oreos®) consumed was of similar magnitude at both times, however only C3H/HeN mice conditioned to snack food at ZT 6 - 8 developed a place preference. C3H/HeN mice with a genetic deletion of either the MT1 (MT1KO) or MT2 (MT2KO) receptor tested at ZT 6 - 8 did not develop a place preference for snack food. Although the MT2KO mice showed a similar amount of snack food consumed when compared to wild-type mice, the MT1KO mice consumed significantly less than either genotype. We conclude that in our mouse model snack food-induced CPP is dependent on time of day and the presence of the MT1 or MT2 receptors, suggesting a role for melatonin and its receptors in snack food-induced reinforcement.

Learned motivation drives circadian physiology in the absence of the master circadian clock.

The suprachiasmatic nucleus (SCN)-often referred to as the master circadian clock-is essential in generating physiologic rhythms and orchestrating synchrony among circadian clocks. This study tested the hypothesis that periodic motivation induced by rhythmically pairing 2 reinforcing stimuli [methamphetamine (Meth) and running wheel (RW)] restores autonomous circadian activity in arrhythmic SCN-lesioned (SCNX) C3H/HeN mice. Sham-surgery and SCNX mice were treated with either Meth (1.2 mg/kg, i.p.) or vehicle in association, dissociation, or absence of an RW. Only the association of Meth treatment and restricted RW access successfully reestablished entrained circadian rhythms in mice with SCNX. RW-likely acting as a link between the circadian and reward systems-promotes circadian entrainment of activity. We conclude that a conditioned drug response is a powerful tool to entrain, drive, and restore circadian physiology. Furthermore, an RW should be recognized as a potent input signal in addition to the conventional use as an output signal.-Rawashdeh, O., Clough, S. J., Hudson, R. L., Dubocovich, M. L. Learned motivation drives circadian physiology in the absence of the master circadian clock.

Melatonin receptor activation increases glutamatergic synaptic transmission in the rat medial lateral habenula.

Melatonin (MLT) is secreted from the pineal gland and mediates its physiological effects through activation of two G protein-coupled receptors, MT1 and MT2 . These receptors are expressed in several brain areas, including the habenular complex, a pair of nuclei that relay information from forebrain to midbrain and modulate a plethora of behaviors, including sleep, mood, and pain. However, so far, the precise mechanisms by which MLT control the function of habenula neurons remain unknown. Using whole cell recordings from male rat brain slices, we examined the effects of MLT on the excitability of medial lateral habenula (MLHb) neurons. We found that MLT had no significant effects on the intrinsic excitability of MLHb neurons, but profoundly increased the amplitude of glutamate-mediated evoked excitatory post-synaptic currents (EPSC). The increase in strength of glutamate synapses onto MLHb neurons was mediated by an increase in glutamate release. The MLT-induced increase in glutamatergic synaptic transmission was blocked by the competitive MT1 /MT2 receptor antagonist luzindole (LUZ). These results unravel a potential cellular mechanism by which MLT receptor activation enhances the excitability of MLHb neurons. The MLT-mediated control of glutamatergic inputs to the MLHb may play a key role in the modulation of various behaviors controlled by the habenular complex.

Genetic deletion of the MT1 or MT2 melatonin receptors abrogates methamphetamine-induced reward in C3H/HeN mice.

The drug of abuse methamphetamine (METH) is known for its ability to enhance reward responses. The rewarding properties of psychostimulants have been shown to vary across time of day in mice. The goal of this study was to determine the role of the MT1 and MT2 melatonin receptors in METH-induced reward, as measured by the conditioned place preference (CPP) paradigm during the light and dark phases. C3H/HeN wild-type mice were trained for METH-induced CPP at either ZT 6-8 (ZT: Zeitgeber time; ZT 0=lights on), when endogenous melatonin levels are low, or ZT 19-21, when melatonin levels are high. These time points also correspond to the high and low points for expression of the circadian gene Period1, respectively. The locomotor response to METH (1.2mg/kg, ip) treatment was of similar magnitude at both times; however only C3H/HeN mice conditioned to METH at ZT 6-8 developed a place preference. C3H/HeN mice with a genetic deletion of either the MT1 (MT1KO) or MT2 (MT2KO) receptor tested at ZT 6-8 or ZT 19-21 did not develop a place preference for METH, though both showed a similar increase in locomotor activity following METH treatment when compared to wild-type mice. We conclude that in our mouse model METH-induced CPP is dependent on time of day and the presence of the MT1 or MT2 receptors, suggesting a role for melatonin in METH-induced reward.

Role of MT1 melatonin receptors in methamphetamine-induced locomotor sensitization in C57BL/6 mice.

RATIONALE: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Melatonin potentiates running wheel-induced neurogenesis in the dentate gyrus of adult C3H/HeN mice hippocampus.

This study assessed the role of melatonin in modulating running wheel(RW)-induced hippocampal neurogenesis in adult C3H/HeN mice. Chronic melatonin (0.02 mg/mL, oral for 12 days) treatment did not affect cell proliferation or cell survival determined by the number of BrdU-positive cells in dentate gyrus of mice with access to fixed wheel (FW). RW activity significantly increased cell proliferation [RW (n = 8) versus FW (n = 6): dorsal, 199 ± 18 versus 125 ± 12, P < 0.01; ventral, 211 ± 15 versus 123 ± 13, P < 0.01] and newborn cell survival [RW (n = 7) versus FW (n = 8): dorsal, 45 ± 8.5 versus 15 ± 1.8, P < 0.01; ventral, 48 ± 8.1 versus 15 ± 1.4)] in the dorsal and ventral dentate gyrus. Oral melatonin treatment further potentiated RW activity-induced cell survival in both areas of the dentate gyrus [melatonin (n = 10) versus vehicle (n = 7): dorsal, 63 ± 5.4 versus 45 ± 8.5 P < 0.05; ventral, 75 ± 7.9 versus 48 ± 8.1, P < 0.01] and neurogenesis [melatonin (n = 8) versus vehicle (n = 8): dorsal, 46 ± 3.4, versus 34 ± 4.5, P < 0.05; ventral, 41 ± 3.4 versus 25 ± 2.4, P < 0.01]. We conclude that melatonin potentiates RW-induced hippocampal neurogenesis by enhancing neuronal survival suggesting that the combination of physical exercise and melatonin may be an effective treatment for diseases affecting the hippocampus neurogenesis.

Genetic deletion of MT(1) and MT(2) melatonin receptors differentially abrogates the development and expression of methamphetamine-induced locomotor sensitization during the day and the night in C3H/HeN mice.

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.

Circadian periods of sensitivity for ramelteon on the onset of running-wheel activity and the peak of suprachiasmatic nucleus neuronal firing rhythms in C3H/HeN mice.

Ramelteon, an MT(1)/MT(2) melatonin receptor agonist, is used for the treatment of sleep-onset insomnia and circadian sleep disorders. Ramelteon phase shifts circadian rhythms in rodents and humans when given at the end of the subjective day; however, its efficacy at other circadian times is not known. Here, the authors determined in C3H/HeN mice the maximal circadian sensitivity for ramelteon in vivo on the onset of circadian running-wheel activity rhythms, and in vitro on the peak of circadian rhythm of neuronal firing in suprachiasmatic nucleus (SCN) brain slices. The phase response curve (PRC) for ramelteon (90 µg/mouse, subcutaneous [sc]) on circadian wheel-activity rhythms shows maximal sensitivity during the late mid to end of the subjective day, between CT8 and CT12 (phase advance), and late subjective night and early subjective day, between CT20 and CT2 (phase delay), using a 3-day-pulse treatment regimen in C3H/HeN mice. The PRC for ramelteon resembles that for melatonin in C3H/HeN mice, showing the same magnitude of maximal shifts at CT10 and CT2, except that the range of sensitivity for ramelteon (CT8-CT12) during the subjective day is broader. Furthermore, in SCN brain slices in vitro, ramelteon (10 pM) administered at CT10 phase advances (5.6 ± 0.29 h, n = 3) and at CT2 phase delays (-3.2 ± 0.12 h, n = 6) the peak of circadian rhythm of neuronal firing, with the shifts being significantly larger than those induced by melatonin (10 pM) at the same circadian times (CT10: 2.7 ± 0.15 h, n = 4, p < .05; CT2: -1.13 ± 0.08 h, n = 6, p < .001, respectively). The phase shifts induced by both melatonin and ramelteon in the SCN brain slice at either CT10 or CT2 corresponded with the period of sensitivity observed in vivo. In conclusion, melatonin and ramelteon showed identical periods of circadian sensitivity at CT10 (advance) and CT2 (delay) to shift the onset of circadian activity rhythms in vivo and the peak of SCN neuronal firing rhythms in vitro.

Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin.

The hormone melatonin phase shifts circadian rhythms generated by the mammalian biological clock, the suprachiasmatic nucleus (SCN) of the hypothalamus, through activation of G protein-coupled MT2 melatonin receptors. This study demonstrated that pretreatment with physiological concentrations of melatonin (30-300 pM or 7-70 pg/mL) decreased the number of hMT2 melatonin receptors heterologously expressed in mammalian cells in a time and concentration-dependent manner. Furthermore, hMT2-GFP melatonin receptors heterologously expressed in immortalized SCN2.2 cells or in non-neuronal mammalian cells were internalized upon pretreatment with both physiological (300 pM or 70 pg/mL) and supraphysiological (10 nM or 2.3 ng/mL) concentrations of melatonin. The decrease in MT2 melatonin receptor number induced by melatonin (300 pM for 1 h) was reversible and reached almost full recovery after 8 h; however, after treatment with 10 nM melatonin full recovery was not attained even after 24 h. This recovery process was partially protein synthesis dependent. Furthermore, exposure to physiological concentrations of melatonin (300 pM) for a time mimicking the nocturnal surge (8 h) desensitized functional responses mediated through melatonin activation of endogenous MT2 receptors, i.e., stimulation of protein kinase C (PKC) in immortalized SCN2.2 cells and phase shifts of circadian rhythms of neuronal firing in the rat SCN brain slice. We conclude that in vivo the nightly secretion of melatonin desensitizes endogenous MT2 melatonin receptors in the mammalian SCN thereby providing a temporally integrated profile of sensitivity of the mammalian biological clock to a melatonin signal.