Hello my name is Takako and I’m a researcher and an instructor of the BioClock Studio at
UC San Diego.
Today we are in the Gorman Lab, where I will show you how we study circadian rhythms in
neurons using a bioluminescence reporter.
Specifically, in this video I will show you the basics of how we can visually monitor
circadian rhythms in the SCN of a mouse using a luciferase reporter.
Circadian rhythms in mammals are controlled by the suprachiasmatic nucleus (referred to
as SCN).
The SCN is found in the hypothalamus right on top of the optic chiasm, where the two
optic nerves from the eyes cross.
The optic nerve delivers light input from the retina.
The SCN contains about 20,000 neurons and controls circadian patterns of behaviors,
such as: sleep and wake; hormone secretion, such as melatonin, and cortisol; and other
physiological functions, such as heart rate and body temperature.
In mammals, the SCN functions as a central pacemaker that adjusts the circadian rhythm
of the whole body, synchronizing the peripheral clocks in other organs.
To observe circadian rhythms in individual neurons in the SCN, we use a mouse line expressing
a fusion protein of a clock gene called PER2, and luciferase.
There are several techniques to insert a luciferase gene in mice.
Our mouse line is generated by the gene knock-in method.
In the mice that we are using today, the per2 DNA sequence in the genome is replaced by
a sequence encoding Per2 fused to luciferase creating a fusion protein.
This way light will be produced proportional to how much Per2 is present.
This PER2::LUCIFERASE knock-in mouse line was first generated in a laboratory at Northwestern
University and donated to our laboratory.
Per2 is an important core clock component that generates circadian rhythms at the molecular
level through transcription-translation feedback loops.
Per2 protein levels exhibit circadian rhythms in the SCN and in most tissues in the body.
Luciferase fused to PER2 reports the level of PER2 protein.
We use PER2 protein expression as a marker or “hand of the clock” that allow us to
tell time.
By looking at what point PER2 protein is in its daily cycle, we can infer the time of
day, because it is known when it peaks and troughs.
To prepare the samples for the experiment, we first humanely euthanize the mouse.
We extract the whole brain carefully to avoid damaging the hypothalamus.
Brains are stored in ice-cold saline.
Here you can see the extracted mouse brain.
We cut the hypothalamus out of the brain.
Once we have dissected the hypothalamus, we glue the hypothalamus (containing the SCN)
onto a specimen holder, and hold it in place with a piece of 2% agarose gel.
Then we will use a specialized instrument called the Vibratome to carefully prepare
very thin slices of the dissected hypothalamus.
The Vibratome water bath is filled with ice-cold saline, and brain slices are cut slowly with
a vibrating blade to minimize the damage to neurons.
For imaging purposes, we will slice into 200 um thickness.
In front of the SCN, the optic chiasm forms a thick triangular shape.
Right under the SCN, the optic chiasm is flat and forms a symmetric round dent.
The SCN can be identified as gray spots of about 1 mm width.
The optic chiasm and SCN are trimmed with knives.
The SCN slice is transferred on a culture insert in a sterile petri dish with medium
that contains luciferin.
We seal a plate with grease and Parafilm.
Finally we will load the sample dish to the imaging system with a very sensitive cooled
CCD camera.
The sample is kept at 37 degrees Celsius by a heater.
Inside this chamber it is completely dark.
Bioluminescence is a very faint light, so it is very important to eliminate any stray
light coming into the chamber.
At first, we check the focus of the microscope on the SCN with the lights on using bright-field
imaging.
Once that is set, we now keep the cells in total darkness except for the light produced
by the luciferin/luciferase reaction.
Every 30 minutes we take a picture with a 29-minute exposure time of that bioluminescence.
After several days of recording, we compile the images and create a video or a "stack
file" to observe how PER2 luciferase was expressed throughout the SCN over time.
Bioluminescence intensity of single neurons is analyzed using a program called MetaMorph.
We select a region of interest in the stack file.
The values within the region are exported to Excel.
Circadian rhythm parameters such as strength of rhythm, period, phase, and amplitude, are
analyzed in another specialized software.
This is a phase map of PER2 generated by Matlab (we use matlab scripts made by Dr. Leise at
Amherst College).
Here we see the phase-leading part in blue and lagging part in red.
Using Matlab, we can visualize spatio-temporal patterns of PER2 rhythms.
With these techniques, we can study synchrony within SCN cells, the effect of drugs or different
light conditions, and compare wild type SCN with a mutant strain.
Thank you for watching this video.
If you are interested in circadian biology and want more information, please visit BioClock
Studio’s website.