Bmal1Is an Essential Regulator for Circadian Cytosolic Ca²⁺Rhythms in Suprachiasmatic Nucleus Neurons

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 2ϩ 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 2ϩ 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 2ϩ 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 2ϩ 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 2ϩ rhythms in SCN neurons.

Robust gene expression changes in the ganglia following subclinical reactivation in rhesus macaques infected with simian varicella virus

“…Examples of genes that mapped to these two GO processes include those involved with double-stranded DNA damage repair ( BAZ1A , FC 3) (Lan et al , 2010); transcriptional regulation ( MIER1 , FC 3) (Blackmore et al , 2008); mRNA degradation ( CNOT7 , FC 2) (Aslam et al , 2009); and regulation of circadian calcium rhythms in neurons ( ARNT , FC 2) (Ikeda and Ikeda, 2014). An additional 17 genes that mapped to “multicellular organismal reproductive process” played a role in microtubule stabilization (Emanuele and Stukenberg, 2007) ( CEP57 , FC3, confirmed by RT-PCR, Fig.…”

BK Channels in the Central Nervous System

“…The state-dependent effects of BK channels on neural excitability have also been demonstrated in the suprachiasmatic nucleus (SCN) of the hypothalamus, a GABAergic circuit controlling circadian rhythms where [Ca 2+ ] i , cAMP-dependent second messengers, fast-activating K + currents, and BK currents display circadian oscillations (Doi et al, 2011; Granados-Fuentes, Norris, Carrasquillo, Nerbonne, & Herzog, 2012; Hong, Jeong, Min, & Lee, 2012; Ikeda & Ikeda, 2014; Ikeda et al, 2003; Kudo, Loh, Kuljis, Constance, & Colwell, 2011; Meredith et al, 2006; O’Neill, Maywood, Chesham, Takahashi, & Hastings, 2008; Panda et al, 2002; Pitts et al, 2006). Paradoxically, total BK currents are inversely correlated with neuronal Ca 2+ levels, which positively regulate BK currents (Meredith et al, 2006); however, they are also inversely correlated with fast-activating K + currents that mediate fast-firing rates (Granados-Fuentes et al, 2012; Kudo et al, 2011).…”

Neuronal oscillations on an ultra‐slow timescale: daily rhythms in electrical activity and gene expression in the mammalian master circadian clockwork

“…Interestingly, pharmacological disruption of RyR function abolishes circadian rhythms in [Ca 2+ ] i level, electrical activity and behaviour (Ikeda et al., ; Mercado et al., ), suggesting that this is a key link between the molecular and electrical oscillations in SCN neurons. Indeed, members of the molecular clock, Bmal1 and Cry1 , interact to modulate the activity of the RyR2 transcription (Pfeffer et al., ; Ikeda & Ikeda, ), while pharmacological activation of the RyRs causes excitation in SCN neurons (Aguilar‐Roblero et al., , ). Together, this suggests that clock‐operated intracellular calcium store release contributes to the up‐state of SCN neurons during the day.…”

“…Elsewhere in the nervous system, oscillations in intracellular calcium signalling underlie most of the fast rhythms in neuronal excitability (Berridge, 1998(Berridge, , 2014. In SCN neurons, steady-state intracellular calcium [Ca 2+ ] i concentration/level oscillates in a circadian manner, peaking during the day and entering a nadir at night [ (Colwell, 2000;Ikeda et al, 2003a;Irwin & Allen, 2010;Enoki et al, 2012;Hong et al, 2012;Brancaccio et al, 2013;Belle et al, 2014;Ikeda & Ikeda, 2014;Noguchi et al, 2017); but see (Ikeda et al, 2003b)]. This peak in global SCN [Ca 2+ ] i anticipates the peak in electrical activity (Ikeda et al, 2003a;Enoki et al, 2017b), raising the possibility that the initial source of [Ca 2+ ] i in SCN neurons is largely through clock-operated intracellular calcium store release (COi-CaSR), and not through depolarised RMP-and action potentialevoked membrane calcium entry via voltage-gated calcium channels (VGCCs).…”