Slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus.

Authors:
Sergey A Savelyev
Sergey A Savelyev
Swedish Medical Nanoscience Center
Karin C Larsson
Karin C Larsson
Karolinska Institutet
Sweden
Anne-Sofie Johansson
Anne-Sofie Johansson
Karolinska Institutet
Sweden

J Vis Exp 2011 Feb 15(48). Epub 2011 Feb 15.

Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function. The SCN is a small, well-defined bilateral structure located right above the optic chiasm. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 μm (length, rostrocaudal axis) x 424 μm (width) x 390 μm (height). To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane, a technique developed for SCN tissue culture by Yamazaki et al. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi.

Download full-text PDF

Source
http://dx.doi.org/10.3791/2439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197397PMC
February 2011
10 Reads

Publication Analysis

Top Keywords

scn
9
clock gene
8
per2 protein
8
per2 expression
8
tissue culturing
8
suprachiasmatic nucleus
8
clock
7
tissue
6
per2
5
luciferase
5
procedure scn
4
slicing procedure
4
expression clock
4
scn mouse
4
rat brains
4
mouse rat
4
dissecting slicing
4
identified describe
4
brain scn
4
level brain
4

Similar Publications

The Tau mutation of casein kinase 1ε sets the period of the mammalian pacemaker via regulation of Period1 or Period2 clock proteins.

J Biol Rhythms 2014 Apr;29(2):110-8

MRC Laboratory of Molecular Biology, Cambridge, UK.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker in mammals, coordinating daily metabolic and physiological rhythms with the cycle of sleep and wakefulness. SCN neurons define circadian time via an auto-regulatory feedback loop in which the activation of Period (Per) and Cryptochrome genes is periodically suppressed by their own protein products. Casein kinase 1 (CK1) enzymes have a critical role in circadian pacemaking because they phosphorylate PER proteins and thereby direct their proteasomal degradation. Read More

View Article
April 2014

IA Channels Encoded by Kv1.4 and Kv4.2 Regulate Circadian Period of PER2 Expression in the Suprachiasmatic Nucleus.

J Biol Rhythms 2015 Oct 6;30(5):396-407. Epub 2015 Jul 6.

Department of Biology, Washington University, St. Louis, MO, USA.

Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 (Kv1. Read More

View Article
October 2015

Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation.

eNeuro 2017 Jul-Aug;4(4). Epub 2017 Aug 18.

Department of Psychiatry and Center for Circadian Biology, University of California, San Diego, La Jolla, CA 92093-0603.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca ([Ca]), and neuronal firing rate. [Ca] oscillates in SCN neurons even in the absence of neuronal firing. Read More

View Article
May 2018

Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms.

Eur J Neurosci 2015 Dec;42(12):3128-37

Department of Chronomedicine, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo, 060-8638, Japan.

The temporal order of physiology and behaviour in mammals is regulated by the coordination of the master circadian clock in the suprachiasmatic nucleus (SCN) and peripheral clocks in various tissues outside the SCN. Because the circadian oscillator(s) in the olfactory bulb (OB) is regarded as SCN independent, we examined the relationship between the SCN master clock and the circadian clock in the OB. We also examined the role of vasoactive intestinal peptide receptor 2 in the circadian organization of the OB. Read More

View Article
December 2015