David J. Earnest

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)

Role of Brain-Derived Neurotrophic Factor in the Circadian Regulation of the Suprachiasmatic Pacemaker by Light

The central pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus mediates the generation of mammalian circadian rhythms, including an oscillation in pacemaker sensitivity to photic signals conveyed by the retinohypothalamic tract. Because brain-derived neurotrophic factor (BDNF) has been implicated in the functional regulation of neural input to other targets of visual pathways, the present study examined whether changes in BDNF expression or blockade of its action in the SCN affect circadian pacemaker responses to light. In rats receiving infusion of exogenous BDNF into the SCN, the free-running rhythm of activity in constant darkness was characterized by large phase advances in response to light exposure during the midsubjective day, when the circadian pacemaker is normally insensitive to photic perturbation. In contrast, SCN infusion of BDNF did not potentiate either phase-delaying or phase-advancing effects of light on the rat activity rhythm during the subjective night. In heterozygous BDNF mutant mice, deficits and damped rhythmicity in SCN levels of this neurotrophin were accompanied by marked decreases in the amplitude of light-induced phase shifts during the subjective night. In agreement with the effects of decreased BDNF expression, SCN infusion of the tyrosine kinase inhibitor K252a blocked or strongly inhibited both the phase-delaying and -advancing effects of light during the subjective night. Collectively, these findings suggest that BDNF-mediated signaling may play an important role in the circadian regulation of SCN pacemaker sensitivity to light.

Circadian rhythm of brain-derived neurotrophic factor in the rat suprachiasmatic nucleus

This study was conducted to determine whether the rat suprachiasmatic nucleus (SCN) is characterized by circadian expression of brain-derived neurotrophic factor (BDNF). In constant darkness, SCN content of both BDNF mRNA and protein oscillated in a circadian fashion. BDNF mRNA and protein levels in the SCN reached peak values during the early subjective day and during the subjective night, respectively. In contrast, the hippocampus showed no sign of circadian rhythmicity in its expression of BDNF mRNA and protein. Since BDNF enhances synaptic transmission in other brain regions, the coincidence between peak expression of BDNF protein in the SCN and the known interval of circadian pacemaker sensitivity to the phase-shifting effects of light may have some implications for the role of BDNF in the circadian regulation of the SCN pacemaker by photic signals from the retinohypothalamic tract.

Expression and rhythmic modulation of circulating microRNAs targeting the clock gene Bmal1 in mice.

MicroRNAs (miRNAs) interact with 3′ untranslated region (UTR) elements of target genes to regulate mRNA stability or translation and thus play a role in regulating many different biological processes, including circadian rhythms. However, specific miRNAs mediating the regulation of essential clock genes remain largely unknown. Because vesicles containing membrane-bound miRNAs are present in the circulatory system, we examined miRNAs predicted to target the clock gene, Bmal1, for evidence of rhythmic fluctuations in circulating levels and modulatory effects on the 3′ UTR activity of Bmal1. A number of miRNAs with Bmal1 as a predicted target were expressed in the serum of mice exposed to LD 12∶12 and of these miRNAs, miR-152 and miR-494 but not miR-142-3p were marked by diurnal oscillations with bimodal peaks in expression occurring near the middle of the day and 8 or 12 hr later during the night. Co-transfection of pre-miR over-expression constructs for miR-494 and miR-142-3p in HEK293 cells had significant effects in repressing luciferase-reported Bmal1 3′ UTR activity by as much as 60%, suggesting that these miRNAs may function as post-transcriptional modulators of Bmal1. In conjunction with previous studies implicating miRNAs as extracellular regulatory signals, our results suggest that circulating miRNAs may play a role in the regulation of the molecular clockworks in peripheral circadian oscillators.

Circadian Regulation and Function of Voltage-Dependent Calcium Channels in the Suprachiasmatic Nucleus

Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC α1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC α1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns of rPer2 and rBmal1 expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.

Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes

The master circadian pacemaker located within the suprachiasmatic nucleus (SCN) of the mammalian brain controls system‐level rhythms in animal physiology. Specific SCN outputs synchronize circadian physiological rhythms in other brain regions. Within the SCN, communication among neural cells provides for the coordination of autonomous cellular oscillations into ensemble rhythms. ATP is a neural transmitter involved in local communication among astrocytes and between astrocytes and neurons. Using a luciferin–luciferase chemiluminescence assay, we have demonstrated that ATP levels fluctuate rhythmically within both SCN2.2 cell cultures and the rat SCN in vivo. SCN2.2 cells generated circadian oscillations in both the production and extracellular accumulation of ATP. Circadian fluctuations in ATP accumulation persisted with an average period (τ) of 23.7 h in untreated as well as vehicle‐treated and forskolin‐treated SCN2.2 cells, indicating that treatment with an inductive stimulus is not necessary to propagate these rhythms. ATP levels in the rat SCN in vivo were marked by rhythmic variation during exposure to 12 h of light and 12 h of dark or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night. Primary cultures of cortical astrocytes similarly expressed circadian oscillations in extracellular ATP accumulation that persisted for multiple cycles with periods of about 23 h. These results suggest that circadian oscillations in extracellular ATP levels represent a physiological output of the mammalian cellular clock, common to the SCN pacemaker and astrocytes from at least some brain regions, and thus may provide a mechanism for clock control of gliotransmission between astrocytes and to neurons.

Establishment and characterization of adenoviral E1A immortalized cell lines derived from the rat suprachiasmatic nucleus.

Primary cultured cells from the presumptive anlage of the rat suprachiasmatic nucleus (SCN) were immortalized by infection with a retroviral vector encoding the adenovirus 12S E1A gene. After drug selection, the resulting neural cell lines (SCN1.4 and SCN2.2) displayed (a) extended growth potential without evidence of transformed or tumorigenic properties, (b) expression of E1A protein within all cell nuclei, and (c) heterogeneous cell types in various stages of differentiation. A large proportion of the SCN1.4 and SCN2.2 cells were characterized by gliallike morphologies, but showed limited expression of corresponding cell type-specific antigens. In addition, both lines exhibited a stable population of cells with neuronlike characteristics. When treated so as to enhance differentiation, these cells were often distinguished by fine, long processes and immunocytochemical expression of neuronal markers and peptides found within SCN neurons in situ. Observations on SCN neuropeptide immunostaining, content, release, and mRNA expression followed a concordant pattern in which somatostatin and vasopressin cells were the most and least common peptidergic phenotypes in both lines, respectively. Since these results indicate that constituents of E1A-immortalized lines derived from the primordial SCN can differentiate into cells with phenotypes resembling parental peptidergic neurons, it will be critical to explore next whether these lines also retain the distinctive function of the SCN to generate circadian rhythms. Cloning of immortalized cell types could subsequently yield useful tools for studying the development of SCN glial and peptidergic cell types and delineating their distinct roles in mammalian circadian time-keeping.

Circadian clock and cell cycle gene expression in mouse mammary epithelial cells and in the developing mouse mammary gland

Mouse mammary epithelial cells (HC‐11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC‐11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation‐dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC‐11 cells and mammary glands, elevated Per2 expression was positively correlated with c‐Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with β‐casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation. Developmental Dynamics 235:263–271, 2006.

Mitochondrial Calcium Signaling Mediates Rhythmic Extracellular ATP Accumulation in Suprachiasmatic Nucleus Astrocytes

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN, and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of ATP. Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Because ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca2+ and ATP accumulation and have demonstrated that intracellular Ca2+ levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca2+ levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca2+. In contrast, our finding that peak mitochondrial Ca2+ occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca2+ uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium, and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Together, these results demonstrate that mitochondrial Ca2+ mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

Immortalized cells from the rat suprachiasmatic nucleus express functional melatonin receptors

Immortalized SCN2.2 cells retain most biochemical and biophysical characteristics of the native rat SCN including the expression of clock genes and circadian regulatory proteins, and its distinctive pacemaker function. This study assessed the expression and signaling of MT(1) and MT(2) melatonin receptors in SCN2.2 cells. SCN2.2 cells express MT(1) and MT(2) receptors mRNA as detected by RT-PCR. In situ hybridization with digoxigenin-labeled probes demonstrated that mRNA for MT(1) and MT(2) melatonin receptors is expressed mostly in cells with neuronal-like morphology, representing 10.8+/-2.2% and 9.8+/-0.2%, respectively, of the SCN2.2 cell population. MT(1) and MT(2) melatonin receptor proteins are expressed in both rat SCN2.2 cells and rat SCN tissue as demonstrated by Western blot analysis with specific receptor antiserum. Melatonin (0.1-100 nM) inhibited forskolin (20 microM)-stimulated cAMP formation in a dose-dependent manner and this effect was blocked by the competitive melatonin receptor antagonist luzindole (100-1000 nM). Furthermore, melatonin (1 nM) stimulated protein kinase C (PKC) activity by approximately 2-fold. The selective MT(2) receptor antagonist 4P-PDOT (100 nM) blocked this effect, indicating that the melatonin-mediated increase in PKC activity occurs through activation of MT(2) melatonin receptors. We conclude that SCN2.2 cells express functional melatonin receptors, providing an in vitro model to unveil the melatonin signaling pathway(s) involved in the regulation of circadian rhythms.