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 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 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. We tested delivery of D-luciferin via a subcutaneous minipump and in the drinking water. 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. 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 inDbpreporter mice

“…To overcome these difficulties with assessing the origin of bioluminescence in “whole-body” reporter mice, and to refine recently developed methods for long-term monitoring of peripheral rhythms in ambulatory mice 55 , we generated Albumin-Cre ; Dbp KI /+ (“liver reporter”) mice. First, we examined the potential impact of route of substrate administration on rhythms using a Lumicycle In Vivo system 55 (Actimetrics, Wilmette IL). Mice were entrained to 12L:12D followed by a skeleton photoperiod consisting of 4 1-h pulses of light every 24 hr (1L:1D:1L:6D:1L:1D:1L:12D) with the 12-h dark phase coinciding with 12-h dark phase of the preceding lighting cycle.…”

“…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 73, 74 , cranial windows 75 and hairless or albino mice 55, 74 . These approaches may allow interrogation of the SCN circuit in vivo , extending the elegant studies being performed with SCN slices in vitro.…”

“…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 55 . Each unit contained two PMTs (Hamamatsu H8259-01), and programmable LED lights.…”

“…Liver reporter mice were euthanized and dissected to confirm that bioluminescence originated exclusively from the liver. In Per2 LucSV mice, major sources of bioluminescence in the abdomen were liver, kidney and intestines (see also Martin-Burgos et al, 2020).…”

“…These studies complement work done by assessing population rhythms in gene expression in tissue samples 36–42 indicating altered rhythm amplitude, phase, and phase relationships in and between SCN and peripheral oscillators following resetting 43 . More recent advances include development of methods for monitoring bioluminescence rhythms from the SCN in vivo 44–50 , and for assessing peripheral rhythms in anesthetized 51–53 and ambulatory 54, 55 mice.…”