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Circadian and ultradian rhythms of clock gene expression in the suprachiasmatic nucleus of freely moving mice.

Ono D, Honma K, Honma S - Sci Rep (2015)

Bottom Line: We found robust circadian rhythms in the clock gene expression, the phase-relation of which were the same as those observed ex vivo.Episodic bursts often accompanied activity bouts, but stoichiometric as well as temporal analyses revealed no causality between them.Clock gene expression in the SCN in vivo is regulated by the circadian pacemaker and ultradian rhythms of unknown origin.

View Article: PubMed Central - PubMed

Affiliation: Photonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.

ABSTRACT
In mammals, the temporal order of physiology and behavior is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Rhythms are generated in cells by an auto-regulatory transcription/translation feedback loop, composed of several clock genes and their protein products. Taking advantage of bioluminescence reporters, we have succeeded in continuously monitoring the expression of clock gene reporters Per1-luc, PER2::LUC and Bmal1-ELuc in the SCN of freely moving mice for up to 3 weeks in constant darkness. Bioluminescence emitted from the SCN was collected with an implanted plastic optical fiber which was connected to a cooled photomultiplier tube. We found robust circadian rhythms in the clock gene expression, the phase-relation of which were the same as those observed ex vivo. The circadian rhythms were superimposed by episodic bursts which had ultradian periods of approximately 3.0 h. Episodic bursts often accompanied activity bouts, but stoichiometric as well as temporal analyses revealed no causality between them. Clock gene expression in the SCN in vivo is regulated by the circadian pacemaker and ultradian rhythms of unknown origin.

No MeSH data available.


Circadian rhythms in clock gene bioluminescence from the SCN and in behavior in freely moving mice(a) Typical examples of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) rhythms in the SCN in vivo (upper) and ex vivo (lower). In vivo bioluminescence is plotted as raw counts at one min intervals (dots) and as a 4 h moving average (solid lines). Ex vivo bioluminescence is plotted at 10 min intervals as a 50 min moving average. Vertical lines in each panel indicate local times (solid line, 06:00 h; broken line, 18:00 h). (b) Circadian peak phases of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) in vivo (open circle) and ex vivo (closed circle) in the first circadian cycle (Day 1 in constant darkness (DD)) for the in vivo experiment and on Day 1 of culturing for the ex vivo experiment. Smaller circles indicate individual peaks and larger circles with a bar indicate the group means and SD. Grey and black bars at the bottom of each panel indicate the antecedent light-dark (LD) cycle (black: dark phase) before transfer to DD. Student’s t-test: *, <0.05; **, <0.01. (c) Representative examples of in vivo circadian Per1-luc (blue), PER2::LUC (red) or Bmal1-ELuc (green) rhythms in the SCN are illustrated with behavior (black) in actograms. Chi square periodograms for clock gene reporter and behavior activity are demonstrated under each actogram with the same colors as in the actogram. An oblique line in the periodogram indicates a significance level of p = 0.05. Each line shows the gene expression and activity distribution across a day, and sequential days are plotted from top to bottom. For better visualization, actograms are double plotted (48-h x axis). In vivo circadian rhythms are smoothed by a 4 h moving average method and detrended by a 24 h moving average subtraction method.
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f1: Circadian rhythms in clock gene bioluminescence from the SCN and in behavior in freely moving mice(a) Typical examples of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) rhythms in the SCN in vivo (upper) and ex vivo (lower). In vivo bioluminescence is plotted as raw counts at one min intervals (dots) and as a 4 h moving average (solid lines). Ex vivo bioluminescence is plotted at 10 min intervals as a 50 min moving average. Vertical lines in each panel indicate local times (solid line, 06:00 h; broken line, 18:00 h). (b) Circadian peak phases of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) in vivo (open circle) and ex vivo (closed circle) in the first circadian cycle (Day 1 in constant darkness (DD)) for the in vivo experiment and on Day 1 of culturing for the ex vivo experiment. Smaller circles indicate individual peaks and larger circles with a bar indicate the group means and SD. Grey and black bars at the bottom of each panel indicate the antecedent light-dark (LD) cycle (black: dark phase) before transfer to DD. Student’s t-test: *, <0.05; **, <0.01. (c) Representative examples of in vivo circadian Per1-luc (blue), PER2::LUC (red) or Bmal1-ELuc (green) rhythms in the SCN are illustrated with behavior (black) in actograms. Chi square periodograms for clock gene reporter and behavior activity are demonstrated under each actogram with the same colors as in the actogram. An oblique line in the periodogram indicates a significance level of p = 0.05. Each line shows the gene expression and activity distribution across a day, and sequential days are plotted from top to bottom. For better visualization, actograms are double plotted (48-h x axis). In vivo circadian rhythms are smoothed by a 4 h moving average method and detrended by a 24 h moving average subtraction method.

Mentions: In the present study, several technical improvements were made to enable bioluminescence recordings of the SCN in freely moving mice (Fig. 1, Supplementary Figure S1). First, cooling the photomultiplier tube (PMT) in the photon counting devise (In vivo Kronos, Atto) to 10 °C reduced dark counts and consequently increased the signal to noise ratio. Background noise was reduced from 834.2 ± 91.0 to 36.2 ± 6.5 counts/min (mean ± SD) and became stable (Supplementary Figure S1). In addition, a convex lens (Plano-Convex lens, #45-081, Edmond) between the optical fiber and PMT optimized the detection area and increased signal strength by ca 40%. Second, the substrate luciferin was delivered to the SCN via an osmotic pump implanted in the body, instead of perfusing it through the lateral ventricle7. Third, to ensure that the mice could move freely, a long (3 m) plastic optical fiber was employed in order to reduce fiber torque. These improvements all together enabled us to monitor bioluminescence in the SCN of freely moving mice up to 3 weeks (Supplementary Figure S1e).


Circadian and ultradian rhythms of clock gene expression in the suprachiasmatic nucleus of freely moving mice.

Ono D, Honma K, Honma S - Sci Rep (2015)

Circadian rhythms in clock gene bioluminescence from the SCN and in behavior in freely moving mice(a) Typical examples of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) rhythms in the SCN in vivo (upper) and ex vivo (lower). In vivo bioluminescence is plotted as raw counts at one min intervals (dots) and as a 4 h moving average (solid lines). Ex vivo bioluminescence is plotted at 10 min intervals as a 50 min moving average. Vertical lines in each panel indicate local times (solid line, 06:00 h; broken line, 18:00 h). (b) Circadian peak phases of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) in vivo (open circle) and ex vivo (closed circle) in the first circadian cycle (Day 1 in constant darkness (DD)) for the in vivo experiment and on Day 1 of culturing for the ex vivo experiment. Smaller circles indicate individual peaks and larger circles with a bar indicate the group means and SD. Grey and black bars at the bottom of each panel indicate the antecedent light-dark (LD) cycle (black: dark phase) before transfer to DD. Student’s t-test: *, <0.05; **, <0.01. (c) Representative examples of in vivo circadian Per1-luc (blue), PER2::LUC (red) or Bmal1-ELuc (green) rhythms in the SCN are illustrated with behavior (black) in actograms. Chi square periodograms for clock gene reporter and behavior activity are demonstrated under each actogram with the same colors as in the actogram. An oblique line in the periodogram indicates a significance level of p = 0.05. Each line shows the gene expression and activity distribution across a day, and sequential days are plotted from top to bottom. For better visualization, actograms are double plotted (48-h x axis). In vivo circadian rhythms are smoothed by a 4 h moving average method and detrended by a 24 h moving average subtraction method.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4508664&req=5

f1: Circadian rhythms in clock gene bioluminescence from the SCN and in behavior in freely moving mice(a) Typical examples of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) rhythms in the SCN in vivo (upper) and ex vivo (lower). In vivo bioluminescence is plotted as raw counts at one min intervals (dots) and as a 4 h moving average (solid lines). Ex vivo bioluminescence is plotted at 10 min intervals as a 50 min moving average. Vertical lines in each panel indicate local times (solid line, 06:00 h; broken line, 18:00 h). (b) Circadian peak phases of Per1-luc (blue), PER2::LUC (red), and Bmal1-ELuc (green) in vivo (open circle) and ex vivo (closed circle) in the first circadian cycle (Day 1 in constant darkness (DD)) for the in vivo experiment and on Day 1 of culturing for the ex vivo experiment. Smaller circles indicate individual peaks and larger circles with a bar indicate the group means and SD. Grey and black bars at the bottom of each panel indicate the antecedent light-dark (LD) cycle (black: dark phase) before transfer to DD. Student’s t-test: *, <0.05; **, <0.01. (c) Representative examples of in vivo circadian Per1-luc (blue), PER2::LUC (red) or Bmal1-ELuc (green) rhythms in the SCN are illustrated with behavior (black) in actograms. Chi square periodograms for clock gene reporter and behavior activity are demonstrated under each actogram with the same colors as in the actogram. An oblique line in the periodogram indicates a significance level of p = 0.05. Each line shows the gene expression and activity distribution across a day, and sequential days are plotted from top to bottom. For better visualization, actograms are double plotted (48-h x axis). In vivo circadian rhythms are smoothed by a 4 h moving average method and detrended by a 24 h moving average subtraction method.
Mentions: In the present study, several technical improvements were made to enable bioluminescence recordings of the SCN in freely moving mice (Fig. 1, Supplementary Figure S1). First, cooling the photomultiplier tube (PMT) in the photon counting devise (In vivo Kronos, Atto) to 10 °C reduced dark counts and consequently increased the signal to noise ratio. Background noise was reduced from 834.2 ± 91.0 to 36.2 ± 6.5 counts/min (mean ± SD) and became stable (Supplementary Figure S1). In addition, a convex lens (Plano-Convex lens, #45-081, Edmond) between the optical fiber and PMT optimized the detection area and increased signal strength by ca 40%. Second, the substrate luciferin was delivered to the SCN via an osmotic pump implanted in the body, instead of perfusing it through the lateral ventricle7. Third, to ensure that the mice could move freely, a long (3 m) plastic optical fiber was employed in order to reduce fiber torque. These improvements all together enabled us to monitor bioluminescence in the SCN of freely moving mice up to 3 weeks (Supplementary Figure S1e).

Bottom Line: We found robust circadian rhythms in the clock gene expression, the phase-relation of which were the same as those observed ex vivo.Episodic bursts often accompanied activity bouts, but stoichiometric as well as temporal analyses revealed no causality between them.Clock gene expression in the SCN in vivo is regulated by the circadian pacemaker and ultradian rhythms of unknown origin.

View Article: PubMed Central - PubMed

Affiliation: Photonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.

ABSTRACT
In mammals, the temporal order of physiology and behavior is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Rhythms are generated in cells by an auto-regulatory transcription/translation feedback loop, composed of several clock genes and their protein products. Taking advantage of bioluminescence reporters, we have succeeded in continuously monitoring the expression of clock gene reporters Per1-luc, PER2::LUC and Bmal1-ELuc in the SCN of freely moving mice for up to 3 weeks in constant darkness. Bioluminescence emitted from the SCN was collected with an implanted plastic optical fiber which was connected to a cooled photomultiplier tube. We found robust circadian rhythms in the clock gene expression, the phase-relation of which were the same as those observed ex vivo. The circadian rhythms were superimposed by episodic bursts which had ultradian periods of approximately 3.0 h. Episodic bursts often accompanied activity bouts, but stoichiometric as well as temporal analyses revealed no causality between them. Clock gene expression in the SCN in vivo is regulated by the circadian pacemaker and ultradian rhythms of unknown origin.

No MeSH data available.