Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice

Abstract

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein ( Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;Dbp KI/+ “liver reporter” mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Arginine-Vasopressin Expressing Neurons in the Murine Suprachiasmatic Nucleus Exhibit a Circadian Rhythm in Network Coherence In Vivo

“…AVP-specific Per2::luc rhythms in SCN slices illustrate the presence of functional molecular clocks[26]. We have observed similar results using a D site-binding protein (DBP) reporter in AVP-expressing SCN cells [42], however our selection of cell-based regions-of-interest used rhythmicity as a criterion and thus excluded non-rhythmic cells which may have been present. Imaging from SCN slices from AVPEluc/+ mice demonstrates the rhythmic transcription of avp in the SCN[63], and there is a circadian rhythm in the secretion of vasopressin[64, 65] and GABA[34] from AVP neurons in the SCN.…”

“…This fact limited our ability to demonstrate robust colocalization of AVP and gcAMP7 expression using immunofluorescence. However the model is well-documented to selectively target AVP neurons in the SCN and elsewhere [42,[44][45][46]. Additionally, there is the possibility that a minority of AVP-expressing neurons dorsal to, but not part of, the SCN that are marked by the cre-line may have been recorded [82].…”

Using bioluminescence to image gene expression and spontaneous behavior in freely moving mice

“…Luciferin was provided through drinking to avoid the stress induced by handling during luciferin injection, anesthesia, or recovery after receiving an implanted pump to deliver luciferin [17,19]. This simple yet elegant approach provided long-term imaging of luciferase bioluminescence throughout the Hr-Per1 mice, as was reported recently through freely moving BLI or other mice [35]. Furthermore, the proposed method of restricting imaging in darkness mostly to the…”

“…Circadian rhythms in Per2 expression have been recorded in freely moving hairless albino hr-/hr-mice (SKH1) using an EMCCD camera and a photomultiplier tube that collected an integrated luciferase signal while luciferin was supplied through an implanted pump [17]. A recent study described circadian rhythms in expression of the D-site albumin promoter binding protein gene measured in freely moving Cre-recombinase-dependent mice by BLI after oral luciferin delivery [35]. Another research group measured circadian rhythms in gene expression by repeatedly anesthetizing and imaging mice during a single day, although they were unable to correct for any stress effects on gene expression and the circadian clock in response to frequent handling and anesthesia [36].…”

Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster

“…Such terminal qRT-PCR outputs from explanted organs measure clock function only for that time point in the lifespan of the organism and can be time-, cost-, and labor-intensive. Given that circadian clocks appear to be linked to a wide range of physiologies, including metabolism as well as various behavioral disorders, there is a need to monitor distinct cell- and tissue-specific circadian clocks directly, in vivo, and in real time in Drosophila , similar to reporters developed in mouse models ( Sinturel et al, 2021 ; Smith et al, 2022 ).…”