The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.
A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called “clock genes” and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 μm (length, rostrocaudal axis) x 424 μm (width) x 390 μm (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.
1. Solution Preparation
2. Preparations Before Cutting and Culturing Slices
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.
4. Organotypic SCN Culture
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.
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 mm2 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.
Benefits and disadvantages with luciferase reporter technology
In contrast to ex vivo methods, such as RT-PCR, in situ hybridization and Western blot that require tissue sampling at many different time points (giving a low time resolution of normally 2-4 hrs depending on sampling frequency) in order to study diurnal variations in gene and protein expressions, the luciferase reporter technology allows high resolution (1-10 min) studies of circadian oscillations for many days in the same preparation. Thus, the number of used animals is minimized and detailed studies of effects on phase and period of the rhythm are feasible, which normally is not possible using conventional sampling techniques with low time resolution. However, although relative amplitude measurements of the rhythm are possible, it should be emphasized that reporter technology is not quantitative and can therefore not be used to measure amounts of transcribed genes or translated proteins.
The tissue recordings can be started and analyzed immediately after culturing, a great advantage for directly studying molecular effects of in vivo stress, in vivo light-induced phase shifts etc. The organotypic SCN culture allows long-term (weeks) pharmacological manipulation of adult brain tissue containing an intact mature synaptic network and the luciferase reporter technology allows stable recordings for many weeks. Thus, the tissue does not need to be neonatal or early postnatal in order to obtain healthy cultures, and in contrast to the acute slice chronic pharmacological treatments can be performed. In contrast to plasmid-reporter transfections in cell cultures, which do no lead to incorporation of DNA into the genome, the transgenic luciferase-animal models (as well as lenti-virus transfections) are favorable if epigenetic alterations are to be studied. It should in this context also be mentioned that luminescence imaging is nowadays possible with highly sensitive CCD cameras 11, allowing recordings even in single SCN neurons (~ 6-10 μm) and other cell types, utilized by major laboratories studying circadian rhythms. Finally, several circadian investigators commonly use monitoring of molecular expression using other reporters, such as the Green Fluorescent Protein, in order to study clock gene/protein oscillations 16-19.
Aspects of slice thickness
The here recommended thicknesses of the SCN slice (200-300 μm) could be reduced or increased if desired. However, although the tissue flattens out on the membrane after a few days in culture, it is not recommended to exceed 500 μm thickness of the initially cut slice in order to preserve viability of the tissue 7. A slice thinner than 100 μm is of mechanical and practical reasons difficult to handle. Because the SCN tissue is heterogeneous the thickness of the slice may affect the phase of the luciferase output signal, since different regions within the SCN (for instance the “core” versus the “shell”, and dorsal versus ventral regions) oscillate with different phases 20 and differentially re-synchronize after phase shifts 21. The thickness of the slice also affects the baseline of photon count since more tissue emits a larger number of photons. The amplitude of molecular oscillations may in this way indirectly be affected by the size of the explant. For these reasons, control and treated cultures should always be of the same size, thickness and containing the same region of SCN in order to oscillate in the same phase and with the same amplitude. The two unilateral nuclei, on the other hand, oscillate in phase with each other and with similar amplitudes as long as the vibratome cut is horizontal and not angled (which in turn is dependent on that the separation cut between cerebellum and the two hemispheres is perpendicular to the table surface). An investigator who performs SCN culturing might experience that SCN cultures do not oscillate in phase with each other. Practicing and standardization of the slicing technique, i.e. the slices are always cut at the same rostro-caudal level of the SCN, will improve the outcome.
Critical steps
Possible modifications
Significance
In transgenic mouse and rat strains (mPER2::LUC; mPer1-luc 8, 10, 27) luciferase enzyme activity reflects protein or gene expression rhythms and can be assessed by bioluminescence recording. The luciferase-generated bioluminescence gives a weak signal but the background luminescence is close to zero, making this method advantageous. Moreover, because the luciferase molecule is unstable and rapidly degraded there is no phototoxicity, which can appear during long-term excitatory illumination 28. Taken together, these properties allow long-lasting experiments making the luciferase reporter technology highly advantageous in circadian research.
The authors have nothing to disclose.
This work was funded by The Swedish Medical Research Council (K2009-75SX-21028-01-3, K2008-61X-20700-01-3); FONCICYT 000000000091984; the foundations of Jeansson, Söderström Königska sjukhemmet, Märtha Lundqvist and Sigurd och Elsa Goljes Minne; and Swedish Society of Medicine SLS-95151. Professor Gene D. Block, UCLA, is gratefully acknowledged for valuable comments on the manuscript. We thank Dr Michael Andäng and Dr Helena Johard for the HCN channel blocker, and Prof. Abdel El-Manira for providing video microscope.
Ethical considerations:
All experiments on animals were performed in accordance with the guidelines and regulations set forth by Karolinska Institutet and “Stockholm’s Norra Djurförsöksetiska Nämnd”. All animal experiments are performed with the intention to minimize any possible stress or discomfort to the animal.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
B27 supplement 50x | Invitrogen/Gibco | 17504-044 | ||
Cover glasses | Menzel-Gläser | 40#1 | ≈ 40 mm | |
Culture membranes | Millipore | PICMORG50 | 0.4 μm | |
DMEM low glucose w/o phenol red | Sigma | D2902 | Powder for 1L | |
Filter paper | Whatman | 1003 055 | ≈ 55 mm | |
Filtration set | Corning | 431097 | Pore size 0.22 μm Polyethersulfone | |
Forceps | Allgaier Instrumente | 0203-7-PS | Dumant #7 | |
HEPES | Invitrogen/Gibco | 15630-056 | 100 ml | |
HBSS 10x | Invitrogen/Gibco | 14065-049 | 500 ml | |
NaOCH3 7.5% | Invitrogen/Gibco | 25080-060 | 50 ml | |
Luciferin | Promega | E1602 | Beetle luciferin, potassium salt | |
Micro rongeur tool | Allgaier Instrumente | 332-097-140 | ||
PenStrep 10,000 U/ml | Invitrogen/Gibco | 15140-122 | 100 ml | |
Petri dishes | Corning | 430165 | ≈ 35 mm | |
PMT | Hamamatsu | H9319-11MOD | ||
Disposable scalpels | Paragon | P503 | Size 11 | |
Vacuum filter | Fisher | 09-761-5 | ||
Vacuum grease | Dow Corning | 50 g | ||
Vibratome | Campden Instruments |