Preparation of the MED probes
1, The MED probe is placed in a sterilized disposable Petri dish (100 mm diameter).
2, Rinse with sterilized distilled water (SDW) three times, and then with 70% EtOH one time about 15-30min.
3, Rinse the MED probe again with SDW 3 times, and dry up and expose to UV light for 15-30 min.
4, On the day before culture (or the same day of culture), treat the surface of the MED probe with brief (within one second) and repeated exposures to blue flame to increase its hydrophilicity. Exposure was repeated 8-12 times and each exposure followed by cooling for ~10 sec.
5, Put the MED probe in to 4 ºC for about 15min.
6, Put collagen Type 1-C 0.8ml (Nitta gelatin ; Cellmatrix), x10 DMEM 0.1 ml, and buffer (Table 1) 0.1 ml into the 1.5ml tube on ice, and gently mix.
7, The MED probe is place on a cold pack, and apply the mixed collagen solution into the probe. After evenly wet the whole surface of MED probe, rest of the collagen solution must be removed as much as possible.
8, Apply SDW in the Petri dish outside of the MED probe to keep moisture. Incubate the MED probe in 37 ºC for 1-2 hours to firm the collagen gel. Be sure that the collagen gel is formed by microscopic observation, since collagen some lot of Collagen Type I-C does not form collagen gel.
9, Rinse with SDW one time, and the MED probe is pre-incubated with culturing medium contained 5% FBS until it is used.
SCN slice preparation for long term culturing on the MED probes
10, Hypothalamic slices (200µm thick) containing the SCN are obtained from the hypothalamic block (≈ 2 x 1 x 5 mm) of mice of 2-5 days of age using a tissue chopper.
11, After trimming, hypothalamic slice containing the SCN is placed on the collagen-coated MED probe using a transfer pipette. Set the slice so that the SCN covers the electrodes.
12, Removed medium on the MED probe, and then the SCN slice is incubated in a CO2 incubator with 100% humidity for 1-2 h until the slice attaches to the collagen gel.
13, After 1-2 h incubation, add 250μl culturing medium contained 5% FBS into the MED probe, and the SCN is incubated in a CO2 incubator. Half volume of medium (add 250μl and remove 250μl) is exchanged every day for first 10 culture days, thereafter medium exchange is performed every other day. For first three days, medium should contain 5% FBS, but thereafter 5% of supplement instead of serum. Within a few days, neurites start to extend from the periphery of the explants as shown in Figure 1b.
Measurement of clock gene and neuronal activity rhythms from the SCN on the MED probe
14, To measure clock gene expression from the SCN neuron, transgenic mice carrying a luciferase reporter are used. Transfection of luciferase reporter may also be used. We used transgenic mice carrying a luciferase reporter listed below.
・ Per1-luc (Per1 promoter driven luciferase reporter)14
・ PER2::LUC (Luciferase reporter fused to Per2 protein)15
15, One to three weeks after preparation, recording is started by setting the MED probe with cultured SCN slice in an MED connector. The connector is then placed in the stage-top incubator of a microscope. To keep humidity of and oxygen level for cultured SCN slice stable, the MED probe is sealed with O2 permeable film by attaching the film to the edge of the probe wall with silicone grease.
16, Stage-top incubator (280mm width x 200mm depth x 70mm height) is made of an acrylic glass of 8 mm thick. Temperature in the incubator is controlled by circulated warmed air (Figure 1a).
17, Bioluminescence is captured by an EM-CCD camera, and microscope should be covered dark box to prevent unnecessary light. Since intensity of bioluminescence is very weak, expose time could vary 20-60 min depending on the reporter signals. Bioluminescence imaging of the SCN on the MED probe is shown in Figure 1b.
CCD camera is set at the top or the bottom port of the microscope. For the upright microscope, a CCD camera is set at the top port, and the stage-top incubator needs a whole for the objective lens. The hole should be sealed with insulation foam, and the lens may be warmed with e.g. heating band. For the inverted microscope, a CCD camera is set to the bottom port of the microscope. The stage-top incubator does not need a hole on the top, but the objective lens beneath the MED probe needs to be warmed. Electrodes interrupt the bioluminescence signals when images are taken from the bottom port of the microscope.
18, Spontaneous firing frequency is counted if an amplitude of an action potential is exceeded a threshold level. In the SCN slice, we set the threshold level to signal to noise ratio ≈ 2.0.