We thank members in the Zhang lab for providing stimulating discussions and members in the Zhan lab for providing technical assistance. This research was supported by grants 31500860 (to C.Z.) of the NSFC, 2012CB837700 (to E.E.Z. and C.Z.) of the 973 Program from the M.O.S.T. of China, and by funding from the Beijing Municipal Government. E.E.Z. was supported by the Chinese &#34;Recruitment Program of Global Youth Experts&#34;.

All procedures in this protocol were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Biological Sciences, Beijing, in accordance with the governmental regulations of China.
1. Construction of the Cry1 Fluorescence Reporter
Note: Previous circadian studies using the bioluminescence system2,12,13 fuse ...

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In contrast to ex vivo methods, such as slice culture4,5, RT-PCR16, and in situ hybridization17, which require that animals be killed, the in vivo recording method allows investigators to study circadian gene expression in a living animal. As such, this technology provides the ability to evaluate the effect of different physical perturbations (e.g., sleep deprivation, stress, food...

In mammals, the suprachiasmatic nucleus (SCN) governs the whole body&#39;s circadian rhythm to coordinate an individual&#39;s response to exogenous environmental changes (e.g., light, temperature, stress, etc.)1. Core clock components consist of Per1-3, Cry1-2, Clock, and Bmal1, and play a central role in regulating the circadian clock of each cell. Each cell in the SCN contains the transcriptional activator, CLOCK/BMAL1, which acts as a heterodimer to induce the expression of PER and CRY. The PER/CRY complex then inhibits the function of CLOCK/BMAL1 to form a transcription-translation feedback loop that takes about 24 h to complete2,3.
Previous studies on the SCN have mainly employed the ex vivo SCN slice culture method4,5,6 and, while this approach has provided valuable information, its limitations have inhibited our ability to obtain data regarding the influence of other brain nuclei on the SCN, as well as the effect of exogenous stimuli (e.g., light) on cells residing in this critical region. In 2001, Hitoshi Okamura&#39;s group was the first to use the bioluminescence system to in vivo monitor circadian clock gene expression in the SCN in freely moving mice7. Ken-ichi Honma&#39;s group has spent the past few years further developing the bioluminescence in vivo recording system in the SCN8,9,10. Together, these studies have provided researchers with the ability to monitor the circadian clock in constant darkness or after a light pulse. However, because bioluminescence is too dim to allow for continuous monitoring during the light/dark cycle, coupled with the fact that light is the predominant signal required for the SCN-mediated entrainment of circadian clocks11, there is increasing demand for the development of experimental methods which overcome the limitations associated with bioluminescence recording. The current report describes a fluorescence-based system which was constructed to monitor the circadian clock of the SCN in vivo in freely moving mice. This easy-to-use method permits continuous monitoring during the light/dark cycle and allows for the long-term observation of the transcription of circadian clock genes in the SCN in real-time and at high temporal resolution.

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			<td>Sigma-Aldrich</td>
			<td>E1014</td>
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			<td>Sigma-Aldrich</td>
			<td>D5670</td>
			<td>Make fresh solution for each batch</td>
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			<td height="19" style="height:19px;">mouse stereotaxic apparatus</td>
			<td>B&#38;E TEKSYSTEMS LTD</td>
			<td>#SR-5M/6M</td>
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			<td>SigmaAldrich</td>
			<td>#1507002</td>
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			<td height="19" style="height:19px;">mouse stereotaxic apparatus</td>
			<td>B&#38;E TEKSYSTEMS LTD</td>
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			<td>SigmaAldrich</td>
			<td>#216763</td>
			<td>Reagent</td>
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			<td>Thorlabs</td>
			<td>FT200EMT</td>
			<td>0.39 NA, &#216;200 &#181;m</td>
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			<td height="19" style="height:19px;">microsyringe pump</td>
			<td>Nanoliter 2000 Injector, WPI</td>
			<td></td>
			<td>Equipment</td>
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			<td height="19" style="height:19px;">ceramic ferrule</td>
			<td>Shanghai Fiblaser</td>
			<td></td>
			<td>230&#8201;&#956;m I.D., 2.5 mm O.D.</td>
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			<td>BiolinkOptics</td>
			<td></td>
			<td>Equipment</td>
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in vivo monitoring of circadian clock gene expression in the mouse suprachiasmatic nucleus using fluorescence reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

Fluorescence reporter design of Cry1 was show in Figure 1A. Using the approach detailed above, 500 nL of rAAV-P(Cry1)-intron336-Venus-NLS-D2 was successfully injected into the SCN of an adult mouse, and exhibited robust Venus expression (Figure 1B, 1C). Fluorescence signals recorded under 12h/12h light/dark (LD) and dark/dark (DD) conditions (Figure 2) yielded robus...

Watch this Scientific Journal Video about In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters at JoVE.com

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

This method provides a way to monitor the circadian clock gene expression in real time in high temporal resolution in freely moving mice. The main advantage of this technique is that it works in both light-dark, and dark-dark conditions, which are critically important for circadian clock studies. Using an optical fiber knife, cut a fiber 17 millimeters in length.Making sure that each side of the optic fiber is smooth. Insert the optic fiber into the ceramic ferrule and fix the optic fiber to a ceramic ferrule with methyl ethyl ketone peroxide. Ensuring that the surfaces of the optic fiber and ceramic ferrule are flush.Clean the optic fiber with a cotton swab soaked with 75%ethnol. And keep it clean until ready for insertion. Begin the injection procedure by first loading five microliters of recombinant AAV into a microsyringe.Then after anesthetizing an adult mouse using an institutionally approved protocol, check for a sufficient depth of anesthesia by the lack of a toe pinch response. Next, use scissors to remove fur from the head of the mouse. And then mount the head of the mouse into a stereotaxic apparatus.After making an incision in the scalp, expose the skull. And adjust the stereotaxic apparatus so that bregma and lambda are in the same horizontal plane. Then use a cotton swab soaked in 4%hydrogen peroxide and water, to corrode the brain periosteum.Followed with a clean cotton swab to clear and dry the surface of the skull. Using the stereotaxic arm, identify the surgical site, 0.46 millimeters posterior and 0.25 millimeter lateral to bregma. Then using a micro drill, make a hole, about one millimeter in diameter at this point.Use a clear cotton swab with physiological saline solution to clear bone fragments from the exposed surface of the brain. Next, insert a microsyringe into the brain to a depth of 5.7 millimeters from the surface of the skull. And inject 500 nanoliters of recombinant AAV at a rate of 50 nanoliters per minute into the SCN.Leave the microsyringe in place for 10 minutes after the injection to ensure complete diffusion of recombinant AAV. After 10 minutes, slowly withdraw the microsyringe. Immediately after recombinant AAV injection, insert the ceramic ferrule containing the optic fiber, into the SCN.Then fix the ceramic ferrule and optical fiber onto the skull using dental resin and allow to dry. After drying, smear the surface of the dental resin with black nail polish. Place the mouse in a warmed recovery cage and monitor it, as it comes out of anesthesia.Once the animal has regained a sternal recumbency, it can be single housed and returned to the housing room with a 12 hour light-dark cycle. Eight days after surgery, measure the florescent signal in the SCN of sham operated control mice to establish the background signal. First, connect the fiber on the mouse head, with the fluorescent signal monitor.Set the software to measure the florescence for 15 seconds every 10 minutes at a frequency of 100 hertz. Record the signal from the sham injected mouse. Repeat the process to evaluate the fluorescent signal of mice injected with the recombinant AAV crypto chrome reporter.Exclude mice which have received the viral vector, but only display background signal as this likely indicates the the tip of the optic fiber has missed the SCN. After these initial measures, return the mice to their home cages for another three weeks, to allow the fluorescent signal to stabilize. At the conclusion of the experiment, check the placement of the optic fiber in the fixed brains.Exclude mice from analysis if the optic fiber tip is not correctly implanted in the SCN. Using the approach detailed above, 500 nanoliters of virus harboring the crypto chrome gene reporter was successfully injected into the SCN of an adult mouse. Robust venus expression was seen eight days after the virus injection.This magnified image demonstrates the high level of venus expression in the SCN. Background florescence in the SCN for a sham operated animal during light on and light off periods is seen here. This animal showed no detectable rhythm under both light-dark and dark-dark conditions.This image shows crypto chrome gene reporter expression over the course of seven days in a 12, 12 light-dark condition. And over seven days in a dark-dark condition. This image shows analysis of circadian rhythm in the light-dark condition.The red line indicates the converged fit in the sin curve. The result of which is shown here. Analysis of circadian rhythm in the dark-dark condition is shown here.Again, the red line indicates the converged fit in the sin curve. And the result of the fit is shown here.

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8.2K vistas.  Peking University. This method provides a way to monitor the circadian clock gene expression in real time in high temporal resolution in freely moving mice. The main advantage of this technique is that it works in both light-dark, and dark-dark conditions, which are critically important for circadian clock studies. Using an optical fiber knife, cut a fiber 17 millimeters in length.Making sure that each side of the optic fiber is smooth. Insert the optic fiber into the ceramic ferrule and fix the optic fi...