1616. |
Four Distinct Phases of Basket/Stellate Cell Migration After Entering Their Final Destination (the Molecular Layer) in the Developign cerebellum
D. Bryant Cameron a, Kazue Kasai b, Yulan Jiang a, Taofang Hu a, Yoshinaga Saeki b, Hitoshi Komuro,
The Clevaland Clinic Foundation,
The Ohio State University,
Developmental Biology,
332(1616),
FILL,
(2009)
Link To Paper
Abstract:
In the adult cerebellum, basket/stellate cells are scattered throughout the ML, but little is known about theprocess underlying the cell dispersion. To determine the allocation of stellate/basket cells within the ML, weexamined their migration in the Keywords:
Stellate cell, Basket Cell, inhibitory interneurons, neuronal cell migration, cerebellum, early postnatal mice, time lapse imaging, brain slices Materials & Methods:
Monitoring cell migration in cerebellar slice preparations
Cerebella of postnatal day (P) 5–14 mice (CD-1, both sexes) were
sectioned transversely or sagittally into 180 μm-thick slices on a
vibrating blade microtome (VT1000S, Leica Instruments) (Komuro
and Rakic, 1992, 1993, 1995, 1998a; Komuro et al., 2001). Cerebellar
slices were placed on 24-mm diameter polyester membrane inserts
(0.4 μm pore size, Corning Inc.) in 6 well plates (Corning Inc.). The
bottom of each plate was filled with 2.5 ml of culture medium, which
consisted of DMEM/F12 (Invitrogen) with N2 supplement, penicillin
(90 U/ml) and streptomycin (90 μg/ml). Fifteen μl of the culture
medium with 0.7 μl of 2×108 TU/ml of HGY amplicon vector was
added to the center of the top surface of each slice. The slices were
subsequently put in a CO2 incubator (37 °C, 95% air, 5% CO2). Twentyfour
hours after sectioning, slices were transferred and placed on
35 mm-glass bottom microwell dishes (MatTek Co.) with 2.0 ml of the
culture medium. The dishes were placed into the chamber of a microincubator
(PDMI-2, Harvard Apparatus) attached to the stage of a
confocal microscope (Leica). The chamber temperature was kept at
37.0±0.5 °C, and the slices were provided with a constant gas flow
(95% air, 5% CO2). To prevent movement of the slices during
observation, a nylon net glued to a small silver wire ring was placed
over the preparations.
A laser scanning confocal microscope (TCS SP, Leica) was used to
visualize EGFP-expressing cells in the slices (Komuro and Rakic,
1998a; Komuro et al., 2001; Komuro and Kumada, 2005; Kumada et
al., 2006; Cameron et al., 2007). The use of this microscope permitted
high-resolution imaging of EGFP-expressing cells up to 100 μm deep
within the tissue slices. The tissue was illuminated with a 488-nm
wavelength light from an argon laser through an epifluorescence
inverted microscope equipped with a 40×oil-immersion objective,
and fluorescence emission was detected at 530±15 nm. Image data
were collected at an additional electronic zoom factor of 1.0–2.0. To
determine the location of EGFP-expressing cells within the ML, the
EGL–ML border and the ML–PCL border, at the beginning and the end
of each recording session, fluorescence images and transmitted
images were simultaneously recorded with 40×magnification. To
avoid the injured cells located near the sectioning surfaces, we
examined the migration of EGFP-expressing cells located 15–50 μm
below the surface of each slice. To monitor migration and morphological
changes, images of EGFP-expressing cells in up to 40 different
focal planes along the z-axis were collected with laser scans every
30 min for up to 70 h.
The long-term observation of cell movement allowed us to
examine the behavior of EGFP-expressing cells (stellate/basket
cells) from the entrance into the ML to the completion of the
migration within the ML. Therefore, in this study, the average speed of
migration and the average transit time in the four different phases
were obtained from the same EGFP-expressing cells (stellate/basket
cells). Microscopic Technique
Confocal Microscopy Cell Type(s)
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35 mm-glass bottom microwell dishes (MatTek Co.) 