Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.
BACKGROUND
Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown.


RESULTS
In this study we isolated primary hepatocytes from transgenic Per2(Luc) mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/-) Per2(Luc) cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes.


CONCLUSIONS
Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.
Neuroscience Program, Smith College, Northampton, Massachusetts, United States of America.
Casey J
CJ
Guenthner
Neuroscience Program, Smith College, Northampton, Massachusetts, United States of America.
Martha E
ME
Luitje
Neuroscience Program, Smith College, Northampton, Massachusetts, United States of America.
Lorna A
LA
Pyle
Neuroscience Program, Smith College, Northampton, Massachusetts, United States of America.
Penny C
PC
Molyneux
Department of Mathematics, Amherst College, Amherst, Massachusetts, United States of America.
Jimmy K
JK
Yu
Department of Mathematics, Amherst College, Amherst, Massachusetts, United States of America.
Alexander S
AS
Li
Department of Mathematics, Amherst College, Amherst, Massachusetts, United States of America.
Tanya L
TL
Leise
Neuroscience Program, Smith College, Northampton, Massachusetts, United States of America.
Mary E
ME
Harrington
D000465:Algorithms; D000818:Animals; D002940:Circadian Rhythm; D018920:Coculture Techniques; D003198:Computer Simulation; D056931:Cryptochromes / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008858:Microscopy, Phase-Contrast; D008954:Models, Biological; D009154:Mutation; D009991:Oscillometry / Q000379:methods; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism*; D061251:Primary Cell Culture; D013997:Time Factors
C538967:Cry2 protein, mouse; D056931:Cryptochromes; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D008156:Luciferases