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

2021 ◽  
pp. 074873042110628
Author(s):  
Blanca Martin-Burgos ◽  
Wanqi Wang ◽  
Ivana William ◽  
Selma Tir ◽  
Innus Mohammad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Circadian Rhythms ◽  
Background Subtraction ◽  
Room Temperature ◽  
Small Difference ◽  
Peripheral Clocks ◽  
Freely Behaving ◽  
Freely Moving ◽  
Pharmacological Manipulations

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.

scholarly journals Methods for detecting PER2::LUCIFERASE bioluminescence rhythms in freely moving mice

2020 ◽  
Author(s):  
B. Martin-Burgos ◽  
W. Wang ◽  
I. William ◽  
S. Tir ◽  
I. Mohammad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drinking Water ◽  
Circadian Rhythms ◽  
Reporter Gene ◽  
Tissue Specific ◽  
Peripheral Clocks ◽  
Freely Behaving ◽  
Freely Moving ◽  
Pharmacological Manipulations

AbstractCircadian 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.

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

2022 ◽  
Author(s):  
Peter S Johnstone ◽  
Maite Ogueta ◽  
Inan Top ◽  
Sheyum Syed ◽  
Ralf Stanewsky ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Circadian Clocks ◽  
Specific Gene ◽  
Tissue Specific ◽  
In Vivo Measurement ◽  
Neuronal Tissues ◽  
Peripheral Clocks ◽  
The Brain

Circadian clocks are highly conserved transcriptional regulators that control 24-hour oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcription properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks; we found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. This result suggests that the presumed dominance of the brain clock in regulating peripheral clocks needs to be re-examined. This result further demonstrates that LABL allows rapid, affordable, and direct real-time monitoring of clocks in vivo.

scholarly journals GuPPy, a Python toolbox for the analysis of fiber photometry data

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Venus N. Sherathiya ◽  
Michael D. Schaid ◽  
Jillian L. Seiler ◽  
Gabriela C. Lopez ◽  
Talia N. Lerner
Keyword(s):  
Open Source ◽  
User Interfaces ◽  
Low Cost ◽  
Ease Of Use ◽  
Analysis Tool ◽  
Freely Behaving ◽  
Freely Moving ◽  
Computing Platforms ◽  
Graphic User Interfaces

AbstractFiber photometry (FP) is an adaptable method for recording in vivo neural activity in freely behaving animals. It has become a popular tool in neuroscience due to its ease of use, low cost, the ability to combine FP with freely moving behavior, among other advantages. However, analysis of FP data can be challenging for new users, especially those with a limited programming background. Here, we present Guided Photometry Analysis in Python (GuPPy), a free and open-source FP analysis tool. GuPPy is designed to operate across computing platforms and can accept data from a variety of FP data acquisition systems. The program presents users with a set of graphic user interfaces (GUIs) to load data and provide input parameters. Graphs are produced that can be easily exported for integration into scientific figures. As an open-source tool, GuPPy can be modified by users with knowledge of Python to fit their specific needs.

scholarly journals Regulation of Melanopsins andPer1byα-MSH and Melatonin in PhotosensitiveXenopus laevisMelanophores

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Maria Nathália de Carvalho Magalhães Moraes ◽  
Luciane Rogéria dos Santos ◽  
Nathana Mezzalira ◽  
Maristela Oliveira Poletini ◽  
Ana Maria de Lauro Castrucci
Keyword(s):  
Gene Expression ◽  
Circadian Rhythms ◽  
Intracellular Signaling ◽  
Cultured Cells ◽  
Clock Genes ◽  
Circadian System ◽  
Time Of Application ◽  
Intracellular Signaling Pathways ◽  
Peripheral Clocks ◽  
Internal Signal

α-MSH and light exert a dispersing effect on pigment granules ofXenopus laevismelanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely,Per1(one of the clock genes) and the melanopsins,Opn4xandOpn4m(photopigments).Per1showed temporal oscillations, regardless of the presence of melatonin orα-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreasedOpn4xandOpn4mexpressions, and abolished their temporal oscillations, opposite toα-MSH, which increased the melanopsins’ expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing theXenopusmelanophore population. This difference is probably due to the fact thatX. laevismelanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression.

scholarly journals Colocalized, Bidirectional Optogenetic Modulations in Freely Behaving Animals with a Wireless Dual-Color Optoelectronic Probe

2021 ◽  
Author(s):  
Xing Sheng
Keyword(s):  
Brain Research ◽  
Preference Test ◽  
Neural Activation ◽  
Light Emitting ◽  
Precise Control ◽  
Neural Activities ◽  
Dual Color ◽  
Freely Behaving ◽  
Freely Moving

The precise control of neural activities at both cellular and circuit levels reveals significant impacts on the fundamental neuroscience explorations and medical applications. Optogenetic methods provide efficient cell-specific modulations, but state-of-the-art technologies lack the ability of simultaneous neural activation and inhibition in a same brain region of freely moving animals. Here we report bidirectional neuronal activity manipulation accomplished by a wireless, dual-color optogenetic probe in synergy with the co-expression of two spectrally distinct opsins (ChrimsonR and stGtACR2) in a rodent model. Based on vertically assembled, thin-film microscale light-emitting diodes (micro-LEDs) on flexible substrates, the dual-color probe shows colocalized red and blue emissions and allows chronic in vivo operations with desirable biocompatibilities. In addition, we discover that neurons co-expressing the two opsins can be deterministically evoked or silenced under red or blue irradiations. Implanted in behaving mice, the wirelessly controlled dual-color probe interferes with dopaminergic neurons in the ventral tegmental area (VTA), increasing or decreasing dopamine levels with colocalized red and blue stimulations. Such bidirectional regulations further generate rewarding and aversive behaviors of freely moving animals in a place preference test. The technologies established here will create numerous opportunities and profound implications for the brain research.

scholarly journals Long-term in vivo recording of circadian rhythms in brains of freely moving mice

2018 ◽  
Vol 115 (16) ◽  
pp. 4276-4281 ◽  
Author(s):  
Long Mei ◽  
Yanyan Fan ◽  
Xiaohua Lv ◽  
David K. Welsh ◽  
Cheng Zhan ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Clock Gene ◽  
Constant Darkness ◽  
Behavioral Rhythms ◽  
Clock Gene Expression ◽  
Hippocampal Ca1 ◽  
Freely Moving ◽  
Term Monitoring

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1−/−, and Cry2−/− mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

scholarly journals GuPPy, a Python toolbox for the analysis of fiber photometry data

2021 ◽  
Author(s):  
Venus N Sherathiya ◽  
Michael D Schaid ◽  
Jillian L Seiler ◽  
Gabriela C Lopez ◽  
Talia Lerner
Keyword(s):  
Open Source ◽  
User Interfaces ◽  
Low Cost ◽  
Ease Of Use ◽  
Analysis Tool ◽  
Development Environment ◽  
Freely Behaving ◽  
Freely Moving ◽  
Graphic User Interfaces

Fiber photometry (FP) is an adaptable method for recording in vivo neural activity in freely behaving animals. It has become a popular tool in neuroscience due to its ease of use, low cost, the ability to combine FP with freely moving behavior, among other advantages. However, analysis of FP data can be a challenge for new users, especially those with a limited programming background. Here, we present Guided Photometry Analysis in Python (GuPPy), a free and open-source FP analysis tool. GuPPy is provided as a Jupyter notebook, a well-commented interactive development environment (IDE) designed to operate across platforms. GuPPy presents the user with a set of graphic user interfaces (GUIs) to load data and provide input parameters. Graphs produced by GuPPy can be exported into various image formats for integration into scientific figures. As an open-source tool, GuPPy can be modified by users with knowledge of Python to fit their specific needs.

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