Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

1. Solution Preparation

1. Culture medium with air-buffering capacity
   1. Fill a 1 liter bottle with approximately 800 mL sterile H₂O (autoclaved milliQ H₂O).
   2. While stirring, add and mix following substances: 1 container low glucose, serum-free DMEM 2902 powder without sodium bicarbonate and phenol red (phenol red interferes with the bioluminescence signal), 20 mL B27 supplement 50x, 4.7 mL of a 7.5% NaHCO₃ solution (or 0.35 g NaHCO₃), 10 mL HEPES 1M, 2.5 mL PenStrep 10,000 U/ mL and 3.5 g D-glucose. Let the medium stir until the ingredients are completely dissolved.
      The final medium (1 liter) will contain:
      1x DMEM, 1x B27 supplement, 4.2 mM NaHCO₃, 10 mM HEPES, 25 U/ mL penicillin, 25 U/ mL streptomycin and 19 mM D-glucose.
   3. Adjust pH to 7.2, using NaOH to decrease or HCl to increase the pH, and bring volume up to 1 liter with sterile H₂O.
   4. Adjust osmolality with H₂O (decrease osmolality) or D-glucose (increase osmolality). The osmolality must be between 285-315 mOsm/Kg but the optimal range is 300-310 mOsm/Kg. Do not to dilute the medium more than 5 %.
   5. Filter the culture medium using Corning sterile vaccum filtration sets (e.g. 2 x 500 mL with pore size 0.22 μm) in a sterile hood. Keep the medium in 4°C and protect it from light using aluminum foil. We recommend that the medium is used within 3 months.
2. Hank’s balanced salt solution (HBSS) buffer with supplements (for cutting slices)
   1. Fill a 1 liter glass bottle with ~600 mL sterile (autoclaved milliQ) H₂O and add the following substances: 100 mL HBSS 10x stock, 10 mL Penicillin-Streptomycin 10,000 U/ mL, 5 mL of a 7.5% NaHCO₃ solution and 10 mL HEPES 1M.
      The final cutting solution will contain:
      1x HBSS, 10 mM HEPES, 4.5 mM NaHCO₃, 100 U/ mL penicillin and 100 U/ mL streptomycin.
   2. Check pH and, if necessary, adjust pH to 7.2 and bring the volume up to 1 liter with sterile H₂O.
   3. Check osmolality, which must be between 285-315 mOsm/Kg.
   4. Cool the HBSS to 4°C. The HBSS buffer needs to be very cold (4°C) during the slicing/cutting procedure in order to bring down the metabolism and preserve tissue viability.

2. Preparations Before Cutting and Culturing Slices

1. The tissues are cultured in a warm dry chamber without CO₂. To avoid drying out the cultures need to be sealed with cover glasses attached to the dishes by grease. For this purpose, fill 5 mL syringes with silicone-based vacuum grease. Cover the syringe tips with small pieces of aluminum foil and autoclave them. Also, autoclave filter papers (used during slicing procedure).
2. Right before slicing procedure, apply autoclaved vacuum grease on the top ring surface of 35 mm petri dishes. Prepare a separate petri dish for each slice culture.
3. Before starting slicing procedure all non-sterile instruments, razor blades, vibratome blades, cover glasses and other material used for the slice and culture procedure have to be sterilized. Spray all non-sterile equipment with 70% ethanol. Expose the instruments and the greased petri dishes with UV in a sterile hood for at least 30 minutes UV exposure before culturing.
4. At the same time, fill a Falcon tube with air-buffering culture medium (count 1.2 mL for each culture; for 8 cultures prepare for instance 10 mL medium) and add freshly thawed luciferin (0.1 M stock solution; Promega, Madison, WI) (10 μL luciferin to 10 mL medium; final concentration 0.1 mM).
5. The luciferin is light sensitive and both stock solutions and the luciferin-containing medium need to be protected from light. Place the Falcon tube with luciferin-medium in a dark 36-37°C heating chamber. If appropriate, the luciferin can also be added directly into the culture dishes at time of cutting. If luciferin is not added, there will be no light reaction between luciferase and luciferin and therefore no signal from the tissue.
6. After UV exposure, attach a sterile blade to the vibratome.

3. SCN Slicing Procedure

The following procedure describes slicing of adult, typically 2-4 months old, C57/BL6 mice. Please note: the cutting procedure, as well as light exposure of the animal during the night, can differentially reset the phase of the SCN. To avoid this, the cutting and culturing should be performed during the light hours, preferably between ZT 6-12, when no substantial phase shifts due to the procedure occur ¹². If SCN has to be sampled in darkness, 3.1 and 3.2 have to be performed in red light or with night goggles to avoid light-induced phase shifts.

1. Anesthetize the mouse preferably by isofluorane (Baxter) in a glass chamber. When the animal has lost its pain reflexes (check by pinching with the nails in the paw) but has not yet stopped breathing (to maintain oxygen supply as long as possible), rapidly decapitate the head with a pair of scissors or similar.
   Please note: in some countries CO₂ exposure (hypercapnia) is still permitted as anesthetic method, although it has been reported to promote animal anxiety. In addition, cervical dislocation may be required before decapitation. Please follow local legislations when euthanizing animals.
2. Remove the eyes from the head with scissors in order to prevent strain and further excitation of the optic nerves, which can damage the SCN.
3. If still attached, remove the last cervical vertebrate with scissors, remove the skin (Fig 1a) and make two cuts with a fine pair of scissors (for instance iris scissors), one cut at each side of the skull along the sides, thus making a removable “lid”.
4. Open the skull with a micro rongeur tool (fine dissecting scissors can also be used on a mouse) and remove all bone until the olfactory bulbs can be seen. Work upwards, never pressing down the brain with the tool, in order to prevent damage of the ventral SCN (Fig 1b).
5. Carefully cut the optic nerve between the olfactory bulbs and the hemispheres using fine micro dissecting spring scissors. Make sure that the nerve is completely cut since stretching of the optic nerve can lead to damage in the SCN and ruptured slices.
6. Turn the head upside down and let the intact brain fall out (if still attached to the brain, other cranial nerves may need to be cut caudal to the optic nerve) into a container (for instance a glass petri dish ≈ 10 cm) filled with 50-100 mL cold HBSS allowing rapid cooling of the brain. Ideally, the two optic nerves should remain intact (Fig 1c). Keep the brain in the HBSS 30-60 seconds to make sure the brain is cooled.
7. Use a spoon or similar and place the chilled brain, dorsal surface up, on a sterile cutting surface (for example a glass petri dish lid turned upside down). In order to prepare a coronal cut, make a perpendicular cut with sterile razor blades or scalpels between the cerebral hemispheres and the cerebellum, thus removing the cerebellum.
8. Apply superglue on the dry platform belonging to the vibroslicer/vibratome.
9. Pick up the brain (cerebral hemispheres without cerebellum and without olfactory bulbs) by inserting a sharp, curved forceps in the rostral part and carefully dry off the HBSS on a sterile filter paper, still holding the brain with the forceps. (Instead of inserting a forceps, a small piece of sterile filter paper can be used to attach and transfer the brain).
10. Fix the hemispheres onto the glued platform with the rostral tip upwards and ventral surface closest to the cutting blade. Attach the platform in the holder belonging to the vibratome (for instance from Campden Instruments, UK) and immediately fill it up with cold HBSS. If the perpendicular cut is made properly, the hemispheres should stand straight up thus providing a good angle necessary for making a coronal cut containing the bilateral SCN.
11. In order to reach the target SCN area, start cutting off thicker sections (500-800 μm) of the hemispheres at high or maximum speed of the vibratome. Moving the blade can be fairly quick in the beginning before reaching the hypothalamus but should be slowed down when the optic chiasm becomes visible (high frequency of the vibrating blade and slow movement horizontally decreases cell damage during slicing, thus increasing slice viability). Reduce the sections to 100 μm when the optic chiasm becomes larger (wider) and the anterior commissure becomes smaller. In order to acquire a mid-SCN section, work yourself “down” (caudal direction) until the two SCN nuclei start to appear. A magnifying glass may be necessary for visualizing the nuclei. Please note: the SCN in the mouse brain is located more caudal of the optic chiasm as compared to the rat brain.
12. When the desired level of SCN has been reached (the SCN will at this point appear as more defined, round or almond shaped structures; Fig 1d; approximately Bregma -0.46- -0.70 mm for the center region of SCN in mouse ¹³, Bregma -0.92- -1.40 mm for rat ¹⁴), cut the SCN section (Fig 1e). Suitable thickness of the slice is for mouse 250 ± 50 μm; for rat 350 ± 50 μm.
13. Using a soft brush, lift and transfer the SCN slice to a lid from a medium sized petri dish filled with cold HBSS, placed under a dissection microscope or stereoscope. Check under magnification if the bilateral SCN is clearly visible. If the mid-region of the SCN will be chosen (which may not necessarily be the only “optimal” slice level but that we consider is the easiest way to standardize the slicing procedure and reduce variation), it should be clearly seen at least on one side of the slice section. If the cut was too rostral, make another section and check for the SCN under magnification.

4. Organotypic SCN Culture

1. In the petri dish lid filled with HBSS, dissect out the bilateral SCN as a square tissue (~ 1.5 mm each side) with a pair of sterile scalpels under a dissecting microscope. A small piece of the optic chiasm will remain attached to the explant, but no other nuclei should be included. Cut as close as possible without removing SCN tissue.
2. If appropriate for the planned experiment, the bilateral SCN can then be cut in half to obtain two unilateral SCN. One unilateral SCN can then be used as control (Fig 2a).
3. Fill 1200 μL of the luciferin-medium into a ≈ 35 mm Petri dish and place a culture membrane (Milli-CM 0.4 μm, Millipore, Bedford, MA) on top of the liquid surface (2b). The culture medium volume is crucial, as the culture membrane should sit securely on the base of the culture dish, not float or rock in the medium ¹¹. Make sure there are no air bubbles under the membrane. Avoid unnecessary light exposure when working with luciferin.
4. The explants are small and difficult to pick up. Therefore use a 1000 μL pipette + tip to suck the SCN explant into the tip and press it out on the membrane. In case the explant is too big to easily fit into a 1000 μL pipette tip (e.g. rat SCN; and mouse bilateral SCN) the tip may be cut with a sterile tool to create a wider opening. Discard excessive HBSS on the membrane with the pipette. For luciferase recording, place only one SCN/dish (Fig 2b) as the recording tubes detect all photons emitted from the dish and do not distinguish between signals from different tissues.
5. Seal the dish with a cover glass (≈ 40 mm, Menzel-Gläser, Germany) and vacuum grease (Dow Corning Corp, USA)¹¹. Make sure the seal is tight (Fig 2c). If not, seal with more grease.
6. Transfer the dishes to a 36-37°C light-tight chamber and start PMT-recordings immediately.

5. Measurement of Luciferase Activity by Recording Bioluminescence

Luciferase-induced bioluminescence signals from the small SCN tissue are detected and amplified with photonmultiplier-tube (PMT) detector assemblies mounted inside a light tight chamber. The PMTs are normally positioned ~ 1-2 cm above the culture dishes ⁸. PMT recording setups can be custom made or are commercially available ¹¹.

1. Place the dish under a PMT (the heat produced from the PMT will remove condensation on the cover glass) and close the chamber. Make sure the chamber is 100% light-tight as the dark count of PMTs used for SCN tissues is very low (less than 20 photons/min). The PMTs detect even the weakest light leakage.
2. Start the data acquisition, which is performed with software (for instance LumiCycle; Actimetrics Inc., Wilmette, IL, USA). Photon counts are integrated over 1-10 min intervals to get high resolution of the gene/protein expression.
3. After the recording is finished, the obtained circadian expression of the gene/protein can be analyzed with appropriate software (Origin, OriginLab, Northampton, MA, USA; ClockLab, Actimetrics; LumiCycle, Actimetrics Inc; Chrono, Till Roenneberg, University of Munich, Munich, Germany) to determine phase, period (time for one cycle) and amplitude of the rhythm. The peak expression of gene or protein is mostly used as reference point and is defined as the highest photon count during one cycle. The data can be smoothed before analyses of phase, period and amplitude, especially if the signal/noise ratio is low. The baseline sometimes changes and need to be subtracted before the analyses are performed.

6. Representative Results:

We here present the oscillatory PER2::LUC expression as a read out for viability and condition of the cultured tissue. Under optimal conditions, and if the tissue is alive, the PER2::LUC expression oscillates with a circadian rhythm as shown in figure 3. PER2 in the SCN is maximally expressed typically around Zeitgeber Time 12-13 (where ZT 12 represents lights off in a 12:12 hr light:dark cycle). The larger in size the live tissue is, the higher the photon count becomes. However, the size and thickness of organotypically cultured tissue should be kept small in order to keep the tissue viable, preferably not larger than 15 mm^{2 11} and not thicker than 500 μm ⁷. The SCN, if dissected as described here, typically shows photon counts between 10.000-40.000/min if the tissue is sampled from a homozygous PER2::LUC animal. The amplitude of the oscillation in organotypic SCN cultures is typically very high during the first cycle as compared with the following cycles. It is not entirely clear why the first cycle has very high amplitude. One possible explanation is that a substantial portion of cells in the tissue may die shortly after initial cutting and culturing, thus not utilizing luciferin after the first cycle. The cutting procedure may also cause excessive excitation, which could amplify the luciferase signal during the first cycle.

Figure 3 shows luminescence traces from SCN slices and contains one trace (red) obtained from a slice that was not initially healthy. Dead or unhealthy tissues have low photon count baselines. (In addition, dead tissues often dissociate in the dish and cannot be removed from the membrane in one piece.)

The technique described in this report can beneficially be used in pharmacological experiments. Figure 4 shows a trace from a culture that we treated between day 1 and 2 with a HCN channel blocker (ZD7288, 10 μM). As can be seen in the figure and as published previously ¹⁵, the blocker significantly reduced the amplitude of the circadian oscillation of PER2; however, after washing out with normal culture medium the oscillation came back, demonstrating that the blocker affected the molecular clock but the tissue was viable and healthy.

Figure 1. Slicing procedure
A) The head of a euthanized decapitated mouse with eyes and skin removed. B) The skull is removed with a micro rongeur tool. When using the tool, one must work upwards and never press down the brain with the tool. C) The brain shown upside down (ventral side up) without olfactory bulbs. The white optic chiasm with the two intact optic nerves can be seen. The suprachiasmatic nucleus (SCN, the borders marked with red) is located close to the optic chiasm. D) A coronally cut brain attached to the platform in the vibroslicer, at the level of SCN. E) Coronal brain section (250 μm thick) containing the optic chiasm (OC), third ventricle (3V) and the bilateral suprachiasmatic nuclei (SCN).

Figure 2. Organotypic tissue culturing.
A) The two unilateral SCN nuclei (insert) dissected from the slice shown. B) Culture dish (35 mm Petri dish) with culture membrane, medium and explant, but without vacuum grease and cover glass. The medium (1.2 mL) can be seen as liquid between the dish and the membrane. One unilateral SCN nucleus is placed on the culture membrane. The whitest part of the small tissue is a piece of the optic chiasm (insert). C) The culture dish with its membrane and its SCN tissue, sealed with vacuum grease and a round cover glass.

Figure 3. Bioluminescence recordings from healthy and non-healthy SCN tissue cultures.
Examples of bioluminescence recordings of PERIOD2::LUCIFERASE (PER2::LUC) expression in suprachiasmatic nucleus (SCN) slices obtained from mice held in a 12h:12h light:dark cycle. The PER2::LUC protein oscillates with a circadian (~24 hr) variation in which the maximum expression of the protein occurs at Zeitgeber time 12-13. Thus, the phase of the gene rhythm is dependent on the light dark schedule in which the animal was kept before sacrificed. Typically, bioluminescence from unilateral SCN tissues dissected as described in the protocol shows photon counts between ~10.000-40.000/minute. The figure shows traces from one healthy (black) SCN culture, one non-healthy SCN culture (red) and one SCN culture that dried out (blue) after opening the sealed culture dish at day 4 and not re-sealing the dish properly (indicated by arrow).

Figure 4. Bioluminescence recording during and after drug exposure.
PER2::LUC expression in a culture before, during and after action of a HCN channel blocker (ZD7288, 10 μM). The first arrow indicates the time when the blocker was added. The second arrow indicates washout, which was made by replacing the drug containing medium with conditioned control medium. Note the reduced amplitude after 2 days of drug exposure, lack of oscillation at day 4 and the quick recovery of the protein rhythm after washout.