Biomedical subjects

David J Earnest

Publications and source records attributed to David J Earnest.

14 recordsLinked to original sources

PubMed2005

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 alpha1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC alpha1 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.

ARNTL Transcription Factors↗

PubMed2005

Circadian profiling of the transcriptome in immortalized rat SCN cells.

Endogenous oscillations in gene expression are a prevalent feature of the circadian clock in the mammalian suprachiasmatic nucleus (SCN) and similar timekeeping systems in other organisms. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain these intrinsic rhythm-generating properties, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared with that found in the rat SCN in vivo using rat U34A Affymetrix GeneChips. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations with a 1.5-fold or greater difference in their mRNA abundance for two cycles. Many (35%) of these rhythmically regulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. Functional analyses and cartography indicate that a diverse set of cellular pathways are strategically regulated by the circadian clock in SCN2.2 cells and that the largest categories of rhythmic genes are those involved in cellular and systems-level communication or in metabolic processes like cellular respiration, fatty acid recycling, and steroid synthesis. Because many of the same genes or nodes within these functional categories were rhythmically expressed in both SCN2.2 cells and the rat SCN, the circadian regulation of these pathways may be important in modulating input to or output from the SCN clock mechanism. In summary, global expression and circadian regulation of the SCN2.2 transcriptome retain many SCN-like properties, suggesting that genes displaying rhythmic profiles in both experimental models may be integral to their function as both circadian oscillators and pacemakers.

Animals↗

PubMed2005

TrkB-deficient mice show diminished phase shifts of the circadian activity rhythm in response to light.

Brain-derived neurotrophic factor (BDNF) has been implicated in the mechanism underlying the circadian sensitivity of the clock in the hypothalamic suprachiasmatic nucleus (SCN) to the phase-shifting effects of light. In the present study, we examined the role of the cognate receptor for BDNF, the TrkB tyrosine kinase, in the photic regulation of the SCN clock by determining whether the phase-shifting action of light is impaired in mice with targeted mutation of the TrkB gene. In comparison with wild-type littermates, heterozygous TrkB mutant mice (trkB(+/-)) showed marked reductions in SCN and cortical levels of this neurotrophin receptor that were accompanied by decreases in the amplitude of light-induced phase shifts during the subjective night. These results provide further evidence indicating that BDNF-mediated signaling through the TrkB receptor is an important process in the gating of SCN responses to light and its phase-shifting effects on circadian rhythms.

Animals↗

PubMed2005

Long-term effects of neonatal alcohol exposure on photic reentrainment and phase-shifting responses of the activity rhythm in adult rats.

In rats, neonatal alcohol (EtOH) exposure coinciding with the period of rapid brain growth produces structural damage in some brain regions that often persists into adulthood and thus may have long-term consequences in the neural regulation of behavior. Because recent findings indicate that the circadian clock located in the rat suprachiasmatic nucleus is vulnerable to alcohol-induced insults during development, the present study examined the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior in adult rats. Rat pups were exposed to alcohol (3.0, 4.5, or 6.0 g x kg(-1) x day(-1)) or isocaloric milk formula on postnatal days 4-9 using artificial-rearing methods. At 2 months of age, animals were housed individually and circadian wheel-running behavior was continuously analyzed to determine the effects of neonatal alcohol treatment on the rate of reentrainment to a 6-h advance in the 12-h light:12-h dark photoperiod and the phase-shifting properties of free-running rhythms in response to discrete light pulses on a background of constant darkness. For all doses, neonatal alcohol exposure had a significant effect in reducing the time for reentrainment such that EtOH-treated rats required four to five fewer days than control animals for stable realignment of the activity rhythm to the shifted light-dark cycle. Coupled with the accelerated rate of reentrainment, the amplitude of light-evoked phase delays at circadian time 14 and advances at circadian time 22 in the 4.5 and 6.0 g x kg(-1) x day(-1) EtOH groups was almost twofold greater than that observed in control animals. The present observations indicate that the mechanisms by which photic signals regulate circadian behavior are permanently altered following alcohol exposure during the period of rapid brain development. These long-term alterations in the photic regulation of circadian rhythms may account, at least partially, for some neurobehavioral consequences of prenatal alcohol exposure in humans such as depression.

Animals↗

PubMed2005

Cell culture models for oscillator and pacemaker function: recipes for dishes with circadian clocks?

Primary cell cultures of avian pinealocytes and the mammalian suprachiasmatic nucleus (SCN), immortalized cell lines derived from the SCN (SCN2.2), and fibroblasts derived from mice and rats have been employed as in vitro models to study the cellular and molecular mechanisms underlying circadian biological clocks. This article compares and contrasts these model systems and describes methods for avian pinealocyte cultures, immortalized SCN2.2 cells, and mouse fibroblast culture. Each of these culture models has advantages and disadvantages. Avian pinealocytes are photoreceptive, contain a circadian pacemaker, and produce rhythms of an easily assayed endocrine output-melatonin. However, the molecular mechanisms underlying pinealocyte function are not understood. SCN2.2 cells express metabolic and molecular rhythms and can impose rhythmicity on cocultured cells as well as rat behavior when transplanted into the brain. Yet, the entrainment pathways are not experimentally established in these cells. Fibroblast cultures are simple to produce and express molecular clock gene rhythms, but they express neither physiological rhythmicity nor pacemaker properties. The relative merits of these culture systems, as well as their impact on understanding circadian organization in vivo, are also considered.

Animals↗

PubMed2005

Circadian rhythms from multiple oscillators: lessons from diverse organisms.

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.

Animals↗

PubMed2005

Neonatal alcohol exposure permanently disrupts the circadian properties and photic entrainment of the activity rhythm in adult rats.

BACKGROUND: Alcohol exposure during the period of rapid brain development produces structural damage in different brain regions, including the suprachiasmatic nucleus (SCN), that may have permanent neurobehavioral consequences. Thus, this study examined the long-term effects of neonatal alcohol exposure on circadian behavioral activity in adult rats. METHODS: Artificially reared Sprague-Dawley rat pups were exposed to alcohol (EtOH; 4.5 g/kg/day) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4-9. At 2 months of age, rats from the EtOH, GC, and suckle control (SC) groups were housed individually, and properties of the circadian rhythm in wheel-running behavior were continuously analyzed during exposure to a 12-hr light:12-hr dark photoperiod (LD 12:12) or constant darkness (DD). RESULTS: Neonatal alcohol exposure had distinctive effects on the rhythmic properties and quantitative parameters of adult wheel-running behavior. EtOH-treated animals were distinguished by unstable and altered entrainment to LD 12:12 such that their daily onsets of activity were highly variable and occurred at earlier times relative to control animals. In DD, circadian regulation of wheel-running behavior was altered by neonatal alcohol exposure such that the free-running period of the activity rhythm was shorter in EtOH-exposed rats than in control animals. Total amount of daily wheel-running activity in EtOH-treated rats was greater than that observed in the SC group. In addition, the circadian activity patterns of EtOH-exposed rats were fragmented such that the duration of the active phase and the number of activity bouts per day were increased. CONCLUSIONS: These data indicate that neonatal alcohol exposure produces permanent changes in the circadian regulation of the rat activity rhythm and its entrainment to LD cycles. These long-term alterations in circadian behavior, along with the developmental alcohol-induced changes in SCN endogenous rhythmicity, may have important implications in clinical sleep-wake disturbances observed in neonates, children, and adults exposed to alcohol in utero.

Animals↗

PubMed2004

Overlap in the distribution of TrkB immunoreactivity and retinohypothalamic tract innervation of the rat suprachiasmatic nucleus.

Brain-derived neurotrophic factor (BDNF) may regulate the circadian sensitivity of the clock in the hypothalamic suprachiasmatic nucleus (SCN) to light, possibly by modulating retinohypothalamic tract (RHT) input. In the present study, the anatomical distribution of the cognate receptor for BDNF, the TrkB tyrosine kinase, in RHT fibers and the SCN of rats was analyzed using combined immunohistochemical and anterograde tracing methods. Fluorescent immunostaining for the TrkB receptor was evident in fibers and cell bodies throughout the SCN. Dual labeling analysis revealed that there was substantial overlap in the distribution of TrkB immunostaining and cholera toxin subunit B (CTB)-labeling within RHT terminals and fibers projecting from the optic chiasm to the ventrolateral SCN. The present results suggest that RHT fibers may express TrkB receptors and thus provide a paracrine target for BDNF-mediated regulation of photic input to the SCN.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

Developmental alcohol exposure alters light-induced phase shifts of the circadian activity rhythm in rats.

BACKGROUND: Developmental alcohol (EtOH) exposure produces long-term changes in the photic regulation of rat circadian behavior. Because entrainment of circadian rhythms to 24-hr light/dark cycles is mediated by phase shifting or resetting the clock mechanism, we examined whether developmental EtOH exposure also alters the phase-shifting effects of light pulses on the rat activity rhythm. METHODS: Artificially reared Sprague-Dawley rat pups were exposed to EtOH (4.5 g/kg/day) or an isocaloric milk formula (gastrostomy control; GC) on postnatal days 4 to 9. At 2 months of age, rats from the EtOH, GC, and suckle control groups were housed individually, and wheel-running behavior was continuously recorded first in a 12-hr light/12-hr dark photoperiod for 10 to 14 days and thereafter in constant darkness (DD). Once the activity rhythm was observed to stably free-run in DD for at least 14 days, animals were exposed to a 15-min light pulse at either 2 or 10 hr after the onset of activity [i.e., circadian time (CT) 14 or 22, respectively], because light exposure at these times induces maximal phase delays or advances of the rat activity rhythm. RESULTS: EtOH-treated rats were distinguished by robust increases in their phase-shifting responses to light. In the suckle control and GC groups, light pulses shifted the activity rhythm as expected, inducing phase delays of approximately 2 hr at CT 14 and advances of similar amplitude at CT 22. In contrast, the same light stimulus produced phase delays at CT 14 and advances at CT 22 of longer than 3 hr in EtOH-treated rats. The mean phase delay at CT 14 and advance at CT 22 in EtOH rats were significantly greater (p < 0.05) than the light-induced shifts observed in control animals. CONCLUSIONS: The data indicate that developmental EtOH exposure alters the phase-shifting responses of the rat activity rhythm to light. This finding, coupled with changes in the circadian period and light/dark entrainment observed in EtOH-treated rats, suggests that developmental EtOH exposure may permanently alter the clock mechanism in the suprachiasmatic nucleus and its regulation of circadian behavior.

Animals↗

PubMed2003

Regulation of basal rhythmicity in protein kinase C activity by melatonin in immortalized rat suprachiasmatic nucleus cells.

Melatonin phase shifts circadian rhythms of the neuronal firing rate and stimulates PKC activity at dusk (CT 10) and dawn (CT 23) in the rat suprachiasmatic nucleus (SCN) slice via activation of the MT(2) melatonin receptor. We demonstrated that in the SCN2.2 cells basal PKC activity follows a rhythmic oscillation with an acrophase during the subjective dark phase (CT 14-CT 22) and nadirs during the subjective light phase at CT 2 and CT 10. Melatonin (0.01-10 nM, 10 min) significantly doubled basal PKC activity at CT 2 and CT 10, and decreased basal PKC activity at CT 6. We conclude that melatonin regulates the basal rhythm in PKC activity generated in SCN2.2 cells at the same periods of sensitivity observed in the native SCN.

Animals↗

PubMed2002

Real-time analysis of rhythmic gene expression in immortalized suprachiasmatic nucleus cells.

Immortalized cells derived from the suprachiasmatic nucleus (SCN) retain many properties of the SCN including the capacity to generate circadian rhythms. Stably transfected SCN2.2 cells expressing the human c- promoter linked to a luciferase reporter gene ( /luc) were examined for evidence of transgene responses to stimuli known to induce c- expression and of endogenous rhythmic variation. Bioluminescence-reported transgene expression was induced in SCN2.2 /luc cells following stimulation with fetal bovine serum or KCl. SCN2.2 /luc cells showed 24 h rhythms of bioluminescence with a 9- to 19-fold difference between peak and minimum levels. These results demonstrate that the regulation of /luc transgene expression in SCN2.2 cells is similar to that of the endogenous c- gene in the SCN.

Animals↗

PubMedDate not supplied

Developmental alcohol exposure disrupts circadian regulation of BDNF in the rat suprachiasmatic nucleus.

In rats, damage to neuronal populations in some brain regions occurs in response to neonatal alcohol exposure coinciding with the period of rapid brain growth. These alcohol-induced defects in brain development may persist into adulthood and thus have long-term implications for the functional characteristics of damaged neuronal populations. The present study examined the effects of neonatal alcohol exposure on endogenous rhythmicity of the circadian clock located in the rat suprachiasmatic nucleus (SCN). Specifically, experiments were conducted to determine whether neonatal alcohol exposure alters the circadian rhythm of brain-derived neurotrophic factor (BDNF) content in the rat SCN because this neurotrophin is an important rhythmic output from the SCN clock. Male rat pups were exposed to alcohol (4.5 g/kg/day) or isocaloric milk formula on postnatal days 4-9 using artificial rearing methods. At 5-6 months of age, SCN and hippocampal tissue was harvested and subsequently examined for content of BDNF protein. Time-dependent fluctuations in BDNF protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). In alcohol-treated rats, SCN levels of BDNF were significantly decreased and were characterized by a loss of circadian rhythmicity relative to those observed in control animals. In comparison, hippocampal levels of BDNF displayed no evidence of circadian regulation in all three treatment groups, but were slightly lower in alcohol-treated animals than in control groups. Importantly, these observations suggest that alcohol exposure during the period of rapid brain development may cause permanent changes in the SCN circadian clock.

Aging↗