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  • Bmal1 is an essential regulator for circadian cytosolic Ca²⁺ rhythms in suprachiasmatic nucleus neurons.

Bmal1 is an essential regulator for circadian cytosolic Ca²⁺ rhythms in suprachiasmatic nucleus neurons.

The Journal of neuroscience : the official journal of the Society for Neuroscience (2014-09-05)
Masayuki Ikeda, Masaaki Ikeda
ABSTRACT

The hypothalamic suprachiasmatic nucleus (SCN) plays a pivotal role in the mammalian circadian clock system. Bmal1 is a clock gene that drives transcriptional-translational feedback loops (TTFLs) for itself and other genes, and is expressed in nearly all SCN neurons. Despite strong evidence that Bmal1-null mutant mice display arrhythmic behavior under constant darkness, the function of Bmal1 in neuronal activity is unknown. Recently, periodic changes in the levels of intracellular signaling messengers, such as cytosolic Ca(2+) and cAMP, were suggested to regulate TTFLs. However, the opposite aspect of how clock gene TTFLs regulate cytosolic signaling remains unclear. To investigate intracellular Ca(2+) dynamics under Bmal1 perturbations, we cotransfected some SCN neurons with yellow cameleon together with wild-type or dominant-negative Bmal1 using a gene-gun applied for mouse organotypic cultures. Immunofluorescence staining for a tag protein linked to BMAL1 showed nuclear expression of wild-type BMAL1 and its degradation within 1 week after transfection in SCN neurons. However, dominant-negative BMAL1 did not translocate into the nucleus and the cytosolic signals persisted beyond 1 week. Consistently, circadian Ca(2+) rhythms in SCN neurons were inhibited for longer periods by dominant-negative Bmal1 overexpression. Furthermore, SCN neurons transfected with a Bmal1 shRNA lengthened, whereas those overexpressing wild-type Bmal1 shortened, the periods of Ca(2+) rhythms, with a significant reduction in their amplitude. BMAL1 expression was intact in the majority of neighboring neurons in organotypic cultures. Therefore, we conclude that proper intrinsic Bmal1 expression, but not passive signaling via cell-to-cell interactions, is the determinant of circadian Ca(2+) rhythms in SCN neurons.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Spermidine, ≥99% (GC)
Sigma-Aldrich
Spermidine, BioReagent, for molecular biology, suitable for cell culture, ≥98%
Sigma-Aldrich
Spermidine, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Pristane, synthetic, ≥98% (GC)
Supelco
Spermidine, analytical standard
SAFC
Pristane, synthetic, liquid, sterile-filtered, BioReagent