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.
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 × 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.
In mammals, the suprachiasmatic nucleus (SCN) governs the whole body's circadian rhythm to coordinate an individual'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'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'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.
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 a circadian promoter with a destabilized luciferase (dLuc) to induce rhythmic dLuc expression. To develop a fluorescence reporter, destabilized luciferase was replaced with destabilized fluorescence protein (dVenus) to induce rhythmic dVenus expression. The generation of the P(Cry1)-dVenus reporter is described below, the construct of which is currently being submitted to Addgene and will be available soon for academic use.
2. Production of Adeno-associated Virus15
3. rAAV Injection and Optic Fiber Insertion into the Adult Mouse SCN
4. In vivo Fluorescence Signal Monitoring and Data Collection
5. Data Analysis and Presentation
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 robust circadian rhythm in both conditions. Finally, fluorescence signal was recorded in long and short photoperiod conditions (Figure 3).
Figure 1. Fluorescence reporter design and it expression in the SCN
(A) The fluorescence reporter design of Cry1. (B) Photomicrograph of the mouse brain showing reporter expression 8 days after the virus injection. (C) Magnification of the area boxed in red on the photomicrograph shown in (B). Please click here to view a larger version of this figure.
Figure 2. Fluorescence in vivo recording in the SCN under LD and DD conditions
(A) Background fluorescence in the SCN showing no detectable rhythm under both LD and DD conditions. The white and gray areas indicate light-on and light-off periods, respectively. (B) P(Cry1)-intron336-Venus-NLS-D2 expression over the course of 7 days in a 12-12 LD condition and 7 days in a DD condition. (C) Analysis of circadian rhythm in the LD condition (~24.4 h); the red line indicates the converged fit in the Sine curve (the result of the fit is shown in the left panel). (D) Analysis of circadian rhythm in the DD condition (~23.25 h); the red line indicates the converged fit in the Sine curve (the result of the fit is shown in the left panel). Please click here to view a larger version of this figure.
Figure 3. In vivo fluorescence recordings in the SCN under 20-4 long and 4-20 short photoperiod conditions
Example of a fluorescence recording of P(Cry1)-intron336-Venus-NLS-D2 expression in the SCN for 7 days under a 12-12 LD condition, followed by 7 days under a 20-4 long photoperiod condition, 3 days under a DD condition, 3 days under a 12-12 LD condition, and then 7 days under a 4-20 short photoperiod condition. White and gray areas indicate the light-on and light-off periods, respectively. Please click here to view a larger version of this figure.
Table 1. Specific reagents required for protocol (See Table of Materials)
primer name | sequence | ||
primer F1 | cactaggggttcctgcggccgcACGCGTGTAAAGATGCACATGTG | ||
primer R1 | TTGTAACCTTGATACTTACCTACTTAGATCGCAGATCTCGTCCGG | ||
primer F2 | GTTATGACACAGTGTAGAAACTATGGCATAGGACAGATGACTGTG | ||
primer R2 | CATGGTCTTTGTAGTCCATGGTGGGTACCtCTTGACAGCTCTACC | ||
primer F3 | ctgtattttcagggcCCTGCAGGtGTGAGCAAGGGCGAGGAGCTG | ||
primer R3 | TACCTTTCTCTTCTTTTTTGGAGGCTTGTACAGCTCGTCCATGCC | ||
primer F4 | GCATGGACGAGCTGTACAAGCCTCCAAAAAAGAAGAGAAAGGTAG | ||
primer R4 | tgatatcgaattcGGATCCCTACACATTGATCCTAGCAGAAGCAC |
Table 2. Primer used in the protocol
Table 3. DNA sequence used in the protocol Please click here to download this file.
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 intake, etc.) on the neural circadian clock. In contrast to the in vivo bioluminescence7,8,10 method that can only work in constant dark conditions, the fluorescence in vivo recording method can be applied under light conditions-a key advantage of the technology as light signals re-entrainment of the SCN circadian clock18. Although relatively constant, robust circadian rhythms can be detected with this method, it should be mentioned that the reporter technology does not provide quantitative information and can therefore not be used to measure levels of gene transcription and translation.
There are many critical steps of this protocol. The concentrated rAAV titer should be as high as 2 – 8x 1012 viral particles per mL; lower viral titers would give off dim or even undetectable fluorescence signals. When fixing the head of the mouse into the stereotaxic apparatus, ensure that the skull is horizontal to secure accurate positioning for virus injection and optical fiber implantation. Injection of the virus should be done slowly (≤50 nL min-1) to ensure that the virus remains in the SCN. The ceramic ferrule and optical fiber should be securely fixed on the skull and dental resin should not contact the skin. If the ceramic ferrule and optical fiber are not adequately fixed, they will gradually detach from the mouse head.
This technique can be extended to include other gene reporters (e.g., Per2, Bmal1, c-Fos, Pomc, etc.) by changing the promoter and the corresponding intron19. By reversing the flag-inton336-Venus-NLS-D2 cassette with two reversed lox sequences and combining the reporter with a Cre mouse line, this technology can be used to record activity in specific neurons. For example, combining this approach with a Vip-cre mouse line would allow for the recording of Cry1 transcriptional rhythms in vasoactive intestinal polypeptide-expressing neurons in the SCN. This method can also employ GCaMP6 as a Ca2+ indicator to record the Ca2+ circadian rhythm in the brain.
The authors have nothing to disclose.
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 "Recruitment Program of Global Youth Experts".
KOD Plus Neo | TOYOBO | KOD-401 | Reagent |
pVENUS-N1 | addgene | #61854 | Plasmid |
pcDNA3.3_d2eGFP | addgene | #26821 | Plasmid |
pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA | addgene | #20297 | Plasmid |
MluI | Thermo Scientific | FD0564 | Reagent |
EcoRI | Thermo Scientific | FD0274 | Reagent |
Gibson Assembly Mix | NEB | E2611s | Reagent |
Lipofectamine 2000 | Thermo Scientific | 12566014 | Reagent |
Syringe Filter | EMD Millipore | SLHV033RS | 0.45 µm |
HiTrap heparin columns | gelifesciences | 17-0406-01 | 1 mL |
Amicon ultra-4 centrifugal filter | EMD Millipore | UFC810024 | 100,000 MWCO |
Benzonase nuclease | Sigma-Aldrich | E1014 | Reagent |
Sodium deoxycholate | Sigma-Aldrich | D5670 | Make fresh solution for each batch |
mouse stereotaxic apparatus | B&E TEKSYSTEMS LTD | #SR-5M/6M | Equipment |
pentobarbital | SigmaAldrich | #1507002 | Reagent |
mouse stereotaxic apparatus | B&E TEKSYSTEMS LTD | #SR-5M/6M | Equipment |
Hydrogen peroxide solution | SigmaAldrich | #216763 | Reagent |
Optical Fiber | Thorlabs | FT200EMT | 0.39 NA, Ø200 µm |
microsyringe pump | Nanoliter 2000 Injector, WPI | Equipment | |
ceramic ferrule | Shanghai Fiblaser | 230 μm I.D., 2.5 mm O.D. | |
Gene Observer | BiolinkOptics | Equipment |