Methods

Dissociate Neuron & Slice Culture

Preparation of a dispersed neuron and slice culture of rat suprachiasmatic nucleus (SCN)
Sato Honma, M.D., Ph.D.
Associate Professor
Department of Physiology
Hokkaido University Graduate School of Medicine
Sapporo, Japan
E-mail: sathonma@med.hokudai.ac.jp

1) Preparation of the MED probes
2) Dispersed cell culture of the rat SCN neurons
3) Dispersed cell culture of the mouse SCN neurons
4) SCN slice preparation for multi-channel analysis using MED probe
5) Tips for long term recording

1) Preparation of the MED probes
For initial preparation, careful cleaning and sterilization is necessary. Thereafter, the probe can be kept under clean conditions in a sterilized disposable Petri dish (100 mm diarneter). After rinsing the probe surface 3 times with sterilized distilled water (SDW), we wash the MED probes, both inside and outside of it, with mild detergent solution for laboratory ware (e g 7X) at 37 C for >-30 min while rotating slowly. Rinse with SDW -5 times and then incubate with 0.1 N HCI for 1-6 h which may reduce the impedance of the electrodes. After rinsing with SDW 3 times, we sterilize the probes with 70% Ethanol for 15 min. The probe is, then, rinsed again with SDW 3 times, dry up and expose to UV light for 15 to 30 min. The MED probe can be kept in dry and clean conditions afterwards.
On the day before the culture, the probe's dish surface is treated with brief repeated exposures to blue flame to increase its hydrophilicity.

(Blue Frame)
	Download Movies 1. Brief repeated exposures to blue frame DOWNLOAD (3.9MB, 1.2 min, wmv file)
(Hydrophilic surface after expose)
	Download Movies 1. Hydrophobic surface DOWNLOAD (0.7 MB, 15 sec, wmv file) 2. Hydrophilic surface after expose DOWNLOAD (0.7 MB, 15 sec, wmv file)

For the dispersed cell culture, the dish surface is then precoated with 0.02% poly-L-ornithine overnight in 4 C to facilitate cell attachment. After rinsing with SDW, the probe is covered with culture medium supplemented with 2~5% serum for at least 2 h in a CO 2 incubator. For slice culture, the dish surface should be covered with collagen gel sheet using either Tyoe 1A or Type 1C collagen ( Nitta gelatin ; Cellmatrix ). Prepare the solution according to the manufacture's instruction for Type 1A while the dish is kept cool. After applying the collagen solution, rest of the solution must be removed as much as possible. The MED probes were then incubated in 37'C for at least I h to firm the collagen gel. After rinsing with SDW once, we preincubate the probe with culture media supplemented with serum at least for a few hours.

2) Dispersed cell culture of the rat SCN neurons
Rat pups were anesthetized by hypothermia and decapitated. Coronal sections of the hypothalamus (600-700 um thick) are obtained using a tissue chopper (Mcllwain). Bilateral SCNs are dissected in ice-cold Preparation Buffer (Table 1a) under a dissecting microscope using scalpel blades. Depending on experimental design. SCN blocks of roughly 10 to 30 pups are pooled together. The SCNs are incubated in 0.03% trypsin in Preparation Buffer at 37 C for 15 min. After rinsing the SCNs with Preparation Buffer containing 0.022% trypsin inhibitor and 0.01% DNase. SCN cells are dissociated by flushing with a fire-polished Pasteur pipette. Cell dissociation is a critical step affecting
the cell viability. Cells are usually dissociated by pipetting for 3-5 times. Strong flush and intense pipetting (more than 10 times) reduce the cell viability. Single cells are obtained by filtering through a # 200 stainless-steel mesh. After centrifuging the cell suspension for 5 min at 1500 r.p.m., SCN cells are resuspended at lO" vial cells/ml in Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 15 mM NaHCO 3 , 10 mM HEPES and 20 mg/l Kanamycin (Gibco). High cell viability (-95%) is indispensable for the successful long term recording from dispersed SCN neurons.
Cell suspension is seeded in the central area of the probe at the relatively high density of 5,000-7,500 vial cells/mm 2 and incubated in a humidified atmosphere at 37 C with 5% C0 2 , 95% air. We use cloning rings with an inner diameter of 5 mm in order to seed the dispersed cells primarily in the central area of the MED probe where 64 electrodes locate. Six to 8 hours after plating the cells, we remove the cloning ring and overlay I ml of culture medium supplemented with 2% fetal bovine serum and 5% of 20 X Supplement Solutions (Table lb). Culture medium is exchanged every second day until the recording is started. A multi-spontaneous neuronal activity of the cultured SCN neurons is started to
monitor from 4 to 10 days after cell culturing and recorded continuously for 2-6 weeks. Figure la shows the phase contrast photomicrgraph of dispersed cell culture of the rat SCN neurons.

Table 1: Composition of Preparation Buffer and supplements for culture medium.
a. Preparation buffer (pH 7.3 / 0 C)
NaCl	8.6 (g/L ultrapure water)
KCl	0.3
HEPES	4.7
NaHCO 3	3.0
Kanamycin	0.02
b. 20x Supplement solution
Apotransferrin	2 mg/ml
Insulin (water soluble)	100 ug/ml
Putresecine	2 mM
Progesterone (water soluble)	0.4 mM
Sodium selenite	0.6 uM

Fig.1 Photomicrographs of the rat SCN on the MED probe. a. Dispersed cell culture at day 7. Black squares are the electrodes (50pm X 50/Im, inter polar distance between the two electrodes is 150 pm). Arrows indicate some of the neurons. b. Organotypic slice culture at day 14. OC: optic chiasm. VIII : the 3*d ventricle. A broken oval indicates the border of the unilateral SCN.

 

3) Dispersed cell culture of the mouse SCN neurons
Methods for the dispersed cell culture of the mouse SCN neurons are basically the same as those for the rat SCN. Only difference is the thickness of the hypothalamic slice, 350 um thick. If the number of pooled SCN is as small as 3-5, we transfer SCN blocks to a 1.5 ml tube containing -0.8 ml culture medium, where we dissociate cells by pipetting.

4) SCN slice preparation for multi-channel analysis using the MED probe
Hypothalamic slices containing the SCN are obtained from mice or rats of 4-6 days of age using a tissue chopper. We usually obtain coronal hypothalamic slices of 200-250 um and 250-350 pm thick for mice and rats, respectively. For the older animals, slicing with a Vibratome and bubbling of buffer with oxygen during the slice preparation maybe necessary to keep the viability of the SCN slice. After the trimming, hypothalamic slice containing the SCN is transferred to the collagen-coated MED probe using a transfer pipette with a large orifice. Mechanical stress to the slice, such as flushing with medium or touching, should be minimized while setting the slice on the electrodes of the MED probe. After removing the excess medium from the probe, the SCN slice is incubated in a CO 2 incubator with 100% humidity for 1-2 h until the slice attaches to the collagen gel Then the slices are incubated with ~250 ul DMEM/F12 medium supplemented with 5% fetal bovine serum and 5% of 20 X Supplement Solution for the first 2 days, and thereafter with the same medium but containing no serum. Of critical importance, slices are kept wet but not submerged in medium, so we exchange a half of the medium every day and adjust the medium volume. Within a few days, neurites start to extend from the periphery of the explants as shown in Figure 1b.

5) Tips for the long term recording
In order to keep the MED probe in saturated humidity while a connector and cables in dry condition, we cover the probes with 35 mm petri dish lined with wet sterile cotton pad (Fig. 2). Covering with a 100 mm diameter Petri dish lined with wet sterile cotton will increase the humidity furthermore. In order to keep the culture dish in 100% air, concentration of NaHC0 3 in the medium must be reduced so that the pH is kept ~7.4.
A problem in dispersed cell culture is the migration of neurons on the dish surface. Relatively large sized electrode of 50 by 50 pm for MED probes is advantageous. Furthermore, by supplementing the medium with fetal bovme serum at O 5-2.5%, we can reduce the neuronal migration. However, serurn-induced grial overgrowth must be prevented by treating with antimitotics (1 uM each of cytosine arabinoside, uridine, and fluorodeoxyuridine) for 24 h near the beginning of the culture (~3-7 days).

Fig.2 Simple methods to keep humidity. a. The MED probe is covered with 35 mm petri dish lined with sterilized cotton pad which is wet with culture medium. Slits and hole of the cotton pad are for the air bent and micorscopic observation. b. The MED probe is covered with 100 mm petri dish lined with wet cotton pad which further humidifies the culture dish. A hole of the cotton pad is for the microscopic observation.

References
Honma, S., Nakamura, W., Shirakawa T. and Honma, K.
Application of a Multielectrode Array Dish to Chronobiology: Monitoring the Circadian Firing Rhythm of Single SCN Neurons.
In: "Biological Rhythms" Proceedings of the Tenth Sapporo Symposium on Biological Rhythm, ed. by Honma K., and Honma S., Hokkaido University Press, Sapporo, 2005.

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