Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice

Abstract

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

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

“…Thus, the presumed rhythm of substrate intake, secondary to the rhythm of water intake, does not change the time of peak of the bioluminescence rhythm. This is consistent with recent results from Sinturel et al (2021) and Martin-Burgos et al (2022) using Per2 Luciferase mice. Subsequent studies used d- luciferin (2 mM) administered in the drinking water.…”

“…Additional technical development may allow in vivo detection of bioluminescence rhythms from neuronal populations in awake behaving mice. Approaches to optimize the signal detected from brain include use of highly efficient and cell-and brain-penetrant substrates (M. S. Evans et al, 2014;Iwano et al, 2018), cranial windows (Miller et al, 2014), and hairless or albino mice (Burgos et al, 2022;Iwano et al, 2018). (Note, the tyrosinase mutation leading to albinism in C57BL/6J mice is linked to Dbp on mouse Chromosome 7; we have nevertheless generated recombinants and produced albino reporters, including the kidney reporter mice in Suppl.…”

“…Bioluminescence was measured in freely moving Alb-Cre + ; Dbp KI/+ reporter mice with the "Lumicycle In Vivo" system (Actimetrics, Wilmette, IL) using methods as recently described (Martin-Burgos et al, 2022). Animals were checked daily at varied times using an infrared viewer (Carson OPMOD DNV 1.0), or goggles (Pulsar Edge Night Vision Goggles PL75095).…”

“…Other studies have localized the source of bioluminescence from widely expressed reporter genes in specific peripheral tissues based on photomultiplier tube placement on the body surface (Hamada et al, 2016; Sawai et al, 2019). Peripheral organ reporter gene activity has been assessed by in vivo imaging in anesthetized mice (Saini et al, 2013; Tahara et al, 2012) and more recently in ambulatory mice (Martin-Burgos et al, 2022; Saini et al, 2013; Sinturel et al, 2021). In some cases, viral vectors that afford anatomical specificity (through their site of injection, tropism, and/or by their design) have been used to direct reporter expression to specific tissues (Mei et al, 2018; Saini et al, 2013; Sinturel et al, 2021).…”

Targeting the Maladaptive Effects of Binge Drinking on Circadian Gene Expression

“…We observed a pronounced circadian peak (ZT 15) and trough (ZT 3) in the NAc expression of Per2 (with similar patterns for Per1 and Per3 ). Per2 is considered a reliable proxy for the overall circadian gene turnover [ 60 ], which supports its candidacy as a primary reporter gene in bioluminescent, luciferase-based reporter mice [ 61 , 62 ]. Considering our hypothesis that the CK1e / d inhibitor, PF-670462, and the PDE4 inhibitor, apremilast, would increase circadian gene expression following chronic harmful drinking, we decided to test the effects of these drugs at ZT 15, wherein the most pronounced ethanol-induced blunting of Per2 expression was seen.…”