Randall L. Hudson

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.—Gerdin, M. J., Masana, M. I., Rivera‐Bermúdez, M. A., Hudson, R. L., Earnest, D. J., Gillette, M. Ú., Dubocovich, M. L. 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. FASEB J. 18, 1646–1656 (2004)

Effect of MT1 melatonin receptor deletion on melatonin‐mediated phase shift of circadian rhythms in the C57BL/6 mouse

Abstract:  In the mouse suprachiasmatic nucleus (SCN), melatonin activates MT1 and MT2 G‐protein coupled receptors, which are involved primarily in inhibition of neuronal firing and phase shift of circadian rhythms. This study investigated the ability of melatonin to phase shift circadian rhythms in wild type (WT) and MT1 melatonin receptor knockout (KO) C57BL/6 mice. In WT mice, melatonin (90 μg/mouse, s.c.) administered at circadian time 10 (CT10; CT12 onset of activity) significantly phase advanced the onset of the circadian activity rhythm (0.60 ± 0.09 hr, n = 41) when compared with vehicle treated controls (−0.02 ± 0.07 hr, n = 28) (P < 0.001). In contrast, C57 MT1KO mice treated with melatonin did not phase shift circadian activity rhythms (−0.10 ± 0.12 hr, n = 42) when compared with vehicle treated mice (−0.12 ± 0.07 hr, n = 43). Similarly, in the C57 MT1KO mouse melatonin did not accelerate re‐entrainment to a new dark onset after an abrupt advance of the dark cycle. In contrast, melatonin (3 and 10 pm) significantly phase advanced circadian rhythm of neuronal firing in SCN brain slices independent of genotype with an identical maximal shift at 10 pm (C57 WT: 3.61 ± 0.38 hr, n = 3; C57 MT1KO: 3.45 ± 0.11 hr, n = 4). Taken together, these results suggest that melatonin‐mediated phase advances of circadian rhythms of neuronal firing in the SCN in vitro may involve activation of the MT2 receptor while in vivo activation of the MT1 and possibly the MT2 receptor may be necessary for the expression of melatonin‐mediated phase shifts of overt circadian activity rhythms.

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.

Cell Na+ activities and transcellular Na+ absorption by descending colon from normal and Na+-deprived rabbits

The relation between intracellular Na+ activities, (Na)c, determined employing Na+-selective microelectrodes, and the rates of active Na+ absorption,INa, by rabbit descending colon was examined whenINa was varied over a wide range by chronic dietary Na+ deprivation. (Na)c averaged 13 mM and was independent ofINa over a sixfold range. Further, the ratios of the slope resistance of the apical membrane (rm) to that of the basolateral membrane (rs) (i.e.rm/rs) in low-transporters (control diet) and high-transporters (Na+-deprived) did not differ significantly inspite of the fact that the Na+ conductance of the apical membranes of high-transporters was, on the average, three times greater than that of the low-transporters. These findings, together with the results reported by other laboratories, strongly suggest that the aldosterone-induced increase in the conductance of the apical membrane to Na+ and, in turn, the rate of entry of Na+ into the absorptive cells are followed by parallel increases in the ability of cells to extrude Na+ across the basolateral membrane in the absence of a sustained increase in (Na)c as well as the conductance of that barrier.

Current-voltage relations of sodium-coupled sugar transport across the apical membrane ofNecturus small intestine

SummaryThe current-voltage (I−V) relations of the rheogenic Na-sugar cotransport mechanism at the apical membrane ofNecturus small intestine were determined from the relations between the electrical potential difference across the apical membrane, Ψmc, and that across the entire epithelium, Ψms, when the latter was varied over the range ±200 mV, (i) under steady conditions in the presence of galactose and (ii) after the current across the apical membrane carried by the cotransporter,ISNam, is blocked by the addition of phloridzin to the mucosal solution.ISNam was found to be strongly dependent upon Ψmc over the range −50 mV < Ψmc <ESNam whereESNam is the “zero current” or “reversal” potential. Over the range of values of Ψmc encountered under physiological conditions the cotransporter may be modeled as a conductance in series with an electromotive force so thatISNam =gSNam (ESNam− Ψmc) wheregSNam is the contribution of this mechanism to the conductance of the apical membrane and is “near constant”. In several instancesISNam “saturated” at large hyperpolarizing or depolarizing values of Ψmc.The values ofESNam determined in the presence of 1, 5, and 15mm galactose strongly suggest that if the Na-galactose cotransporters are kinetically homogeneous, the stoichiometry of this coupled process is unity.Finally, the shapes of the observedI−V relations are consistent with the predictions of a simple kinetic model which conforms with current notions regarding the mechanico-kinetic properties of this cotransport process.

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine-induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract:  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 MT1 and/or MT2 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), MT1KO and MT2KO 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 MT1/MT2KO 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, MT1KO and MT2KO mice was statistically different from VEH‐treated controls. However, WT and MT2KO, but not MT1KO 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 MT1 melatonin receptor activation by endogenous melatonin. We suggest that MT1 and MT2 melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.

Genetic deletion of MT1 melatonin receptors alters spontaneous behavioral rhythms in male and female C57BL/6 mice

Behaviors vary over the 24h light/dark cycle and these temporal patterns reflect in part modulation by circadian neural circuits and hormones, such as melatonin. The goal of this study was to investigate the involvement of MT1 melatonin receptors in behavioral regulation by comparing male and female C57 wild type (WT) mice with C57 mice with genetic deletion of the MT1 receptor (MT1KO). A comprehensive array of fifteen distinct spontaneous behaviors was recorded continuously in the homecage over multiple days using the HomeCageScan system. Behaviors assessed were activity-like (i.e. come down, hang, jump, walk), exploration-like (i.e. dig, groom, rear up, sniff, stretch), resting-like (i.e. awake, remain low, rest, twitch) and ingestion-like (i.e. drink, eat). Phenotypic array and temporal distribution analysis revealed distinct behavioral rhythms that differed between WT and MT1KO mice. The rhythms were consistent from day to day in males and varied with the estrous cycle in females. We also studied the role of MT1 receptors on depressive and anxiety-like behaviors. Genetic deletion of MT1 receptors increased immobility time in the forced swim test and decreased the number of marbles buried in the marble burying test in both male and female C57 mice. We conclude that MT1 melatonin receptors are involved in neural pathways modulating diurnal rhythms of spontaneous behavior in the homecage as well as pathways regulating depressive and anxiolytic-like behaviors.

Automated video analysis system reveals distinct diurnal behaviors in C57BL/6 and C3H/HeN mice.

Advances in rodent behavior dissection using automated video recording and analysis allows detailed phenotyping. This study compared and contrasted 15 diurnal behaviors recorded continuously using an automated behavioral analysis system for a period of 14 days under a 14/10 light/dark cycle in single housed C3H/HeN (C3H) or C57BL/6 (C57) male mice. Diurnal behaviors, recorded with minimal experimental interference and analyzed using phenotypic array and temporal distribution analysis showed bimodal and unimodal profiles in the C57 and C3H mice, respectively. Phenotypic array analysis revealed distinct behavioral rhythms in Activity-Like Behaviors (i.e. walk, hang, jump, come down) (ALB), Exploration-Like Behaviors (i.e. dig, groom, rear up, sniff, stretch) (ELB), Ingestion-Like Behaviors (i.e. drink, eat) (ILB) and Resting-Like Behaviors (i.e. awake, remain low, rest, twitch) (RLB) of C3H and C57 mice. Temporal distribution analysis demonstrated that strain and time of day affects the magnitude and distribution of the spontaneous homecage behaviors. Wheel running activity, water and food measurements correlated with timing of homecage behaviors. Subcutaneous (3 mg/kg, sc) or oral (0.02 mg/ml, oral) melatonin treatments in C57 mice did not modify either the total 24 h magnitude or temporal distribution of homecage behaviors when compared with vehicle treatments. We conclude that C3H and C57 mice show different spontaneous activity and behavioral rhythms specifically during the night period which are not modulated by melatonin.

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

RationaleMelatonin 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.ObjectiveThe 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.MethodsMice 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.ResultsMethamphetamine 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.ConclusionsDeletion 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.

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.