Notes

1. The thickness of slices can vary from 200 to 700 ^m. Thicker slices are easier to handle, but thinner slices get better diffusion of oxygen and nutrients. Considering these points, 400 ^m of thickness seems a good compromise in most cases.

2. Collagen gel can be used as an alternative to attach slices to cover slips. The cover slips are cleaned and sterilized as described above, then they are first coated with poly-D-lysine and finally with collagen.

Fig. 3. Suprachiasmatic nucleus (SCN) slice retaining its original topology in culture as confirmed by immunohistochemistry and in situ hybridization. (A) Antibody staining shows that the neurons containing vasoactive intestinal peptide are localized in the ventrolateral part of the SCN slice in culture, as they are in vivo. (B) Moreover, the neurons expressing vasopressin mRNA are localized in the dorsomedial part of the slice, as in the SCN in vivo. Scale bar is 100 ^m. Figure is adapted from ref. 3.

Fig. 3. Suprachiasmatic nucleus (SCN) slice retaining its original topology in culture as confirmed by immunohistochemistry and in situ hybridization. (A) Antibody staining shows that the neurons containing vasoactive intestinal peptide are localized in the ventrolateral part of the SCN slice in culture, as they are in vivo. (B) Moreover, the neurons expressing vasopressin mRNA are localized in the dorsomedial part of the slice, as in the SCN in vivo. Scale bar is 100 ^m. Figure is adapted from ref. 3.

a. Apply poly-D-lysine solution (25 ^g/mL in water; Sigma, cat. no. P-7280, sterilized with a 0.45-^m filter) to the surface of cover slips for 1 h.

b. Rinse the cover slips twice in distilled water and dry.

c. Apply a small amount of collagen solution (Cellmatrix Type I-A kit, Nitta-gelatin Inc., Japan) to the cover slips and spread over. Keep in a humidified incubator for 30 min at 37°C until the collagen solidifies.

d. Place each slice on a cover slip and add a small amount of collagen solution to cover the slice. Keep in a humidified incubator at 34°C for 1 to 4 h until the collagen solidifies.

3. Coating cover slips with 0.05 to 0.1% polyethylene imine (Sigma) prevents damage to the plasma clot during culturing. Apply the polyethylene imine solution to the surface of cover slips and keep them in a humidified incubator for 3 to 12 h. After the solution is removed, cover slips are washed in sterilized distilled water three times.

Fig. 4. Real-time recording of bioluminescence from suprachiasmatic nucleus (SCN) cultures of mPerl-luc transgenic mice. (A) High-power image of the bioluminescence of an SCN culture after addition of 1 mM luciferin to the medium. Note that the SCN occupies the majority of the slice, and the surrounding anterior hypothalamic area is thin. Scale bar = 500 pm. (B) Rhythmic change of bioluminescence in a SCN culture. The upper panels show the SCN culture imaged every 60 min for 5 d. In the lower graph, each circle represents the total intensity of luminescence counted for 20 min. Arrow indicates the change of the culture medium. (C) Waveform of the N-methyl-D-aspartate (NMDA)-treated cycle and the preceding cycle. Application of NMDA at 6 h after the peak induced a phase-delay (a), and at 12 h after the peak induced a phase-advance (b). Figures are adapted from ref. 14.

Fig. 4. Real-time recording of bioluminescence from suprachiasmatic nucleus (SCN) cultures of mPerl-luc transgenic mice. (A) High-power image of the bioluminescence of an SCN culture after addition of 1 mM luciferin to the medium. Note that the SCN occupies the majority of the slice, and the surrounding anterior hypothalamic area is thin. Scale bar = 500 pm. (B) Rhythmic change of bioluminescence in a SCN culture. The upper panels show the SCN culture imaged every 60 min for 5 d. In the lower graph, each circle represents the total intensity of luminescence counted for 20 min. Arrow indicates the change of the culture medium. (C) Waveform of the N-methyl-D-aspartate (NMDA)-treated cycle and the preceding cycle. Application of NMDA at 6 h after the peak induced a phase-delay (a), and at 12 h after the peak induced a phase-advance (b). Figures are adapted from ref. 14.

4. Mix plasma and thrombin solutions under neutral pH to obtain a firm plasma clot. Thrombin should be dissolved with Gey's BSS containing 10 mM HEPES.

5. There are two types of culture tubes: flat-bottom and round-bottom. We strongly recommend using flat-bottom tubes, as it is not necessary to take the cover slip outside the tube to observe the slice under a phase contrast microscope.

6. The volume of culture medium is a critical factor. In roller tube cultures, when replacing medium, aspirate the old medium and add 650 pL of fresh medium into each flat-bottom tube. Make sure that the slices are covered by medium for only half of the time during rotation. This allows optimal recovery of nutrients from the medium and oxygen from the air.

Fig. 5. (A) Suprachiasmatic nucleus (SCN) culture of mPerl-luc transgenic mice examined by light microscopy (a), and by cooled charge-coupled device (b). Scale bar = 100 ^m. (B) Temporal changes in bioluminescence signals from 100 SCN cells randomly chosen, were plotted and then superimposed. Figures are adapted from ref. 21.

Fig. 5. (A) Suprachiasmatic nucleus (SCN) culture of mPerl-luc transgenic mice examined by light microscopy (a), and by cooled charge-coupled device (b). Scale bar = 100 ^m. (B) Temporal changes in bioluminescence signals from 100 SCN cells randomly chosen, were plotted and then superimposed. Figures are adapted from ref. 21.

For porous membrane cultures, at the beginning of culturing add 1 mL of fresh medium into each well of a 6-well plate. When replacing medium, aspirate the old medium and add 700 to 800 ^L of fresh medium into each well. Alternatively, move the well inserts to a new culture plate with 1 mL of fresh medium for each well. Make sure that the slices are not immersed in the medium; the surface of the medium must just touch the membrane.

7. It is reported that slices cultured by the roller tube method show more gliosis than those cultured on porous membranes (2,22). To prevent growth of macrophages, glia, and fibroblasts, it is recommended that a low concentration (10-6 to 10-5 M working solution) mixture of antimitotic drugs (mixture of uridine, cytosine-p-D-arabino-furanoside, and 5-fluorodeoxyuridine) is administered for no more than 24 h at days 2 to 4 in vitro.

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Microscopy Analysis

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