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

Summary

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.

Abstract

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.

Introduction

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 complete^2,3.

Previous studies on the SCN have mainly employed the ex vivo SCN slice culture method^4,5,6 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 SCN^8,9,10. 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.

Protocol

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 system^2,12,13 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.

1. Amplify the functional mouse Cry1 promoter (+328 to -1208 bp region of the Cry1 gene, transcription start site designated as ‘‘-1’’). Use the PCR enzyme (KOD Plus Neo), primers F1 and R1 (see Table 2), mouse genome as a template and the manufacturer’s PCR protocol to amplify the Cry1 promoter DNA fragment.
2. Amplify intron 336 of the Cry1 gene, which is important for the circadian clock function of Cry1¹⁴. Use the PCR enzyme, primers F2 and R2, mouse genome as a template and the manufacturer’s PCR protocol to amplify the intron 336 DNA fragment.
3. Amplify Venus. Use the PCR enzyme, primers F3 and R3, pVENUS-N1 plasmid as a template and the manufacturer’s PCR protocol to amplify the Venus DNA fragment.
4. Amplify the NLS-D2 DNA fragment. Use the PCR enzyme, primers F4 and R4, pcDNA3.3-d2eGFP plasmid as a template and the manufacturer’s PCR protocol to amplify the NLS-D2 DNA fragment.
5. Synthesize DNA fragment 1 as exon 1.
6. Synthesize DNA fragment 2 as the linker between intron 336 and the Venus DNA fragment.
7. Cut the pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA vector plasmid with the MluI and EcoRI enzymes. Use the Gel Extraction Kit to purify the larger DNA fragment. Use the manufacturer’s protocol.
8. Using a Gibson assembly mix and its standard protocol, assemble all of the DNA fragments together to produce the pAAV-P(Cry1)-intron336-Venus-NLS-D2-WPRE-HGHpA reporter. Use the manufacturer’s protocol.

2. Production of Adeno-associated Virus¹⁵

1. Prepare the following plasmid DNA stocks: 31.25 µg of pRV1, 31.25 µg of pH21, 125 µg of pFdelta6, and 62.5 µg of pAAV-P(Cry1)-intron336-Venus-NLS- D2-WPRE-HGHpA.
2. Prepare five 15 cm dishes of 293T cells and wait until the cells become 90% confluent.
3. Transfect the plasmids into the 297T cells following the transfection reagent protocol. For each dish, use 6.25 µg of pRV1, 6.25 µg of pH21, 25 µg of pFdelta6, 12.5 µg of pAAV-P(Cry1)-intron336-Venus-NLS-D2-WPRE-HGHpA, and 120 µL of transfection reagent.
4. 72 h after transfection, detach the cells from each plate using a pipette gun; harvest the cells into two 50 mL tubes.
5. Pellet the cells at 800 x g for 10 min and discard the supernatant. Wash the cells by placing 20 mL of phosphate-buffered saline (PBS) into each tube; pellet the cells at 800 x g for 10 min, and discard the supernatant.
6. Resuspend pellets in 150 mM NaCl and 20 mM Tris solution, pH 8.0; use 25 mL of solution/tube. Add 1.25 mL of freshly prepared 10% sodium deoxycholate (in dH₂O) to each tube. Add benzonase nuclease to a final concentration of 50 units per mL, and mix thoroughly. Incubate at 37 °C for 1 h.
7. Remove cellular debris by centrifuging at 3000 x g for 15 min. Further ensure that all debris has been removed by pushing the mixture through a 0.45 μm syringe filter; this step will help prevent blocking of heparin columns.
8. Set up the heparin columns using a peristaltic pump; set the flow rate to 1 mL/min, ensuring that no air bubbles are introduced into the columns.
9. Equilibrate the columns with 10 mL of a 150 mM NaCl, 20 mM Tris solution, pH 8.0. Add the solution containing the virus to each column. Wash the columns with 20 mL of a 100 mM NaCl, 20 mM Tris solution, pH 8.0. Then, using a 5 mL syringe, wash the columns with 1 mL of a 200 mM NaCl and 20 mM Tris solution pH 8.0, followed by 1 mL of a 300 mM NaCl and 20 mM Tris solution, pH 8.0.
10. Use a 5 mL syringe to elute the virus with 3 mL of a 400 mM NaCl, 20 mM Tris solution, pH 8.0, followed by 4 mL of a 450 mM NaCl, 20 mM Tris solution, pH 8.0, and finally by a 3 mL of a 500 mM NaCl, 20 mM Tris solution, pH 8.0. Collect the extracted materials.
11. For recombinant adeno-associated virus (rAAV) concentration, load the extracted materials, 4 mL at a time, into centrifugal filter units with a 100,000 molecular weight cutoff. Centrifuge each 4-mL sample at 2000 x g for 5 min at room temperature, discarding the flow-through.
12. When all extracted materials have been run through the centrifugal filter units, add 4 mL of PBS and centrifuge the mixture into approximately 250 μL. Remove rAAVs (viral titer should be about 2 - 8 x 10¹² viral particles per mL) from the centrifugal filter units, aliquot and store at -80 °C until use. At this stage, the virus can be stored for several months.

3. rAAV Injection and Optic Fiber Insertion into the Adult Mouse SCN

1. Injection of circadian fluorescence reporter-carrying rAAVs into the SCN
   1. Load 5 µL of rAAV (the virus titer is about 2 - 8 x 10¹² virus particles per mL) into a micro-syringe.
   2. Anesthetize an adult mouse (2- to 5-month-old, C57BL/6 background) with Nembutal (80 mg/kg) via intraperitoneal injection. Check for sufficient depth of anesthesia by the lack of a toe-pinch response.
   3. Use scissors to remove fur from the head of the mouse and then mount the mouse into a stereotaxic apparatus. Provide a heat source to anesthetized animals via a circulating hot water blanket or another heating device.
      NOTE: Alternatively, use depilatory cream or electric hair clippers to remove the fur.
   4. Clean the surgical site with the appropriate disinfecting agents. For example, this could include sterile water or alcohol and iodine, betadine, or dilute chlorhexidine in alternating scrubs. Repeat at least 3 times. Drape the surgical site.
   5.  Use the scissors to make an incision in the scalp and expose the skull.
   6. Adjust the stereotaxic apparatus so that bregma and lambda are in the same horizontal plane; make two symmetrical points of bregma in the same horizontal plane.
   7. Use a sterile cotton swab soaked in 4% H₂O₂/H₂O to corrode the brain periosteum. Then use a clean, sterile cotton swab to clear and dry the surface of the skull.
   8. Using a micro drill, make a hole about 1 mm in diameter 0.46 mm posterior and 0.25 mm lateral to bregma.
   9. _fiUse a clean cotton sterile swab with physiological saline solution to clear bone fragments from the exposed surface of the brain.
   10. Insert a micro-syringe into the brain, ensuring that the tip of the micro-syringe is 0.46 mm posterior and 0.25 mm lateral to bregma, and 5.7 mm deep from the surface of the skull.
   11. Inject 500 nL of rAAV (50 nL min^-1) into the SCN, leaving the micro-syringe in place for 10 min after the injection to ensure complete diffusion of rAAV.
   12. Slowly withdraw the micro-syringe.
2. Insert the optical fiber into the SCN area.
   1. Using an optical fiber knife, cut a fiber 17-mm in length, making sure that each side of the optic fiber is smooth.
   2. Insert the optic fiber into the ceramic ferrule and fix the optic fiber to a ceramic ferrule with methyl ethyl ketone peroxide (AB glue), ensuring that the surfaces of the optical fiber and ceramic ferrule are flush.
   3. Clean the fiber with a cold sterilant solution or ethylene oxide.
   4. After rAAV injection (step 3.1.11), insert the ceramic ferrule-containing optic fiber into the SCN.
   5. Fix the ceramic ferrule and optical fiber onto the skull using dental resin and allow to dry.
   6. Smear the surface of the dental resin with back nail polish. Repeat the procedures outlined in 3.1.2 to 3.2.5, omitting the actual injection of rAAV for control.
3. Post-surgical care
   1. Provide mice with post-operative analgesics, such as buprenorphine 2 mg/kg body weight, subcutaneously, twice a day.
   2. Place mice in a recovery cage while they come out of anesthesia.
   3. House mice individually under a 12 h light/dark condition (light intensity ~100 lux at the bottom of the cage) with food/water available ad libitum. Eight days should be allotted before further experimentation to allow for sufficient recovery time and to ensure optimal fluorescence reporter expression.

4. In vivo Fluorescence Signal Monitoring and Data Collection

1. Eight days after surgery, connect the fiber on the mouse head with the fluorescence signal monitor (e.g., Gene Observer). Check the fluorescence signal in the SCN of control mice to establish the background signal. Conduct the observation and measurement of this signal using fluorescence signal monitor.
2. Evaluate the fluorescence signal of experimental mouse with the same operation of the control mouse. Exclude mice which have received the viral vector but which only display the background signal from analysis, as this likely indicates that the tip of the optic fiber has missed the SCN. After these initial measures, return mice to their home cages for another 3 weeks to allow the fluorescence signal to stabilize.
3. After 3 weeks, connect the mice with the fluorescence signal monitor. Measure the fluorescence for 15 s every 10 min, at a frequency of 100 Hz. During these measurements, the mice are freely moving in their cages with food/water available ad libitum. Use a light intensity at the bottom of the cage of ~100 lux, and a the laser power at the tip of the optic fiber between 15 - 20 µW.
4. After the completion of all recordings, perfuse mice with 4% paraformaldehyde, and remove and slice the brains to check placement of the optic fiber. Exclude mice from analysis if the optic fiber tip is not correctly implanted in the SCN.

5. Data Analysis and Presentation

1. Measure fluorescence signal every 10 min for 15 s, at a frequency of 100 Hz, to yield 1500 data points. The average of these 1500 points translates to the fluorescence signal at any given time point. Plotting multiple averages over time produces a signal-to-time curve which corresponds to the transcriptional rhythm of Cry1 under a control light condition in the SCN of freely moving mice.

Results

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...

Discussion

In contrast to ex vivo methods, such as slice culture^4,5, 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...

Materials

Name	Company	Catalog Number	Comments
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