Materials and methods those described previously [2,27]. Twenty-four hours after
H . Katsuki et al. Brain Research 885 2000 240 –250
241
enzymatic activity [3,23]. Moreover, phosphatidylinositol in the case of calbindin immunocytochemistry was added
PI 3-kinase, which catalyzes phosphoinositide phos- for 1 h, then avidin–biotin peroxidase complex method
phorylation at the D-3 position of the myo-inositol ring, is was performed with Vectastain Elite ABC kit Vector
activated by growth factor receptor stimulation via interac- according to the manufacturer’s instructions.
tions of SH2 domains of the kinase regulatory subunit with Cell counting was performed as described previously
2
the receptor [24]. These signal transduction mechanisms [1]. Four areas of 1 mm , the total of which corresponds to
2
may be differentially involved in diverse biological conse- 4 of the whole area 1 cm , were chosen at random
quences of receptor tyrosine kinase activation in various from each well and the number of neurons in the areas
cell types [12,18,43]. 400–1300 cells in each well was counted under a
In the present study we set out for the experiments to microscope. Number of surviving neurons in each well
2
discriminate which signaling pathways play an important was expressed as cells cm
and data from four culture role in the bFGF-induced biological responses in fetal rat
wells from the same sister culture were presented in figures hippocampal neurons. We have previously demonstrated
as mean6S.E.M. Reproducibility of results was confirmed that increase in the amount of L-type voltage-dependent
by using different sister cultures. In the present cultures,
21 21
Ca channels VDCCs and consequent increases in Ca
more than 90 of the cells were labeled by antibodies to influx may play a critical role in the acceleration of
neurofilament and microtubule-associated protein-2 and neuritic branch formation by bFGF [27]. Accordingly, the
identified as neurons by their morphology. Therefore, potential involvement of VDCCs in promotion of neuronal
possible contaminating non-neuronal cells including as- survival and enhancement of calbindin expression by
troglia and fibroblast cells were estimated to be less than bFGF was also investigated.
10. Cultures on 35-mm dishes were used for the evaluation
of neurite branching, the methods essentially according to
2. Materials and methods those described previously [2,27]. Twenty-four hours after
plating, the medium was changed to serum-free modified Cultures of dissociated hippocampal neurons were pre-
Eagle’s medium containing 2 B-27 supplement Gibco. pared as described previously [1,27]. Briefly, hippocampal
As reported in a previous study [27], we found a difficulty tissues were isolated from the brains of embryonic day 18
in examining the effect of nicardipine on low-density Wistar rats. Dissociated cells were obtained by enzymatic
culture maintained
in N2-supplemented
serum-free digestion of minced tissues by 0.25 trypsin and 0.01
medium, because nicardipine showed potent cytotoxicity deoxyribonuclease I, and subsequent mechanical dissocia-
under these conditions by unidentified mechanisms. This tion. The cell suspension was diluted in modified Eagle’s
problem was resolved by usage of B-27 supplement. medium containing 10 fetal bovine serum and plated on
Therefore, we used B-27-supplemented medium in all 48-well plastic plates Costar coated with poly-
L
-lysine to experiments on low-density culture in the present study.
4 2
obtain a final density of 5.0310 cells cm , or on 35-mm The effect of bFGF on neurite morphogenesis was similar-
culture dishes Iwaki Glass coated with poly-
L
-lysine to ly observed in the presence of either N2 or B-27 supple-
3 2
obtain a final density of 2.0310 cells cm . They were ments. We selected neurons that have neurites longer than
cultured at 378C in a humidified 5 CO 95 air their soma diameters and were free from contact with other
2
atmosphere. cells at 2 days in vitro DIV, and recorded their locations
In the case of cultures on 48-well plates, the medium in the culture dish using an ACAS 470 work station
was changed to drug-containing, serum-free DMEM F-12 Meridian, Okemos, MI. These cells were photographed
medium supplemented with N2 hormones at 24 h after and then drugs were added to the cultures. The same cells
plating. After a certain period of culture usually 3 days were again photographed 48 h later at 4 DIV. In several
after the change of the medium, cells were fixed with 4 experiments, immunocytochemical staining was performed
paraformaldehyde for 30 min at 48C. Cultures were thereafter, the methods according to those described above.
subsequently processed for immunocytochemical examina- The number of branch points along the longest neurite was
tion. They were initially treated with 0.3 Triton X-100 counted on the photographs. If the selected neurons died
for 30 min, then treated with 0.3 H O in methanol for 1 within 48 h of drug treatment, data from these cells were
2 2
h after wash with phosphate-buffered saline PBS. Fol- discarded.
lowing wash with PBS, 10 normal goat serum 10 Data are expressed as mean6S.E.M. Statistical signifi-
normal horse serum in the case of calbindin immuno- cance of difference between groups was evaluated by
cytochemistry in PBS was added. After 1 h, dishes were two-way analysis of variance followed by Dunnett’s test. P
treated with either anti-g-aminobutyric acid GABA anti- values less than 0.05 were considered significant. bFGF
body 1:10 000, rabbit anti-GABA, Sigma or anti-calbin- used in this study is CS23, a modified human bFGF in
din antibody 1:200, mouse anti-calbindin D28k, Sigma in which serine is substituted for cysteine at amino acid
PBS for overnight at 48C. After wash with PBS, residues 70–88 to prevent conformational changes and
biotinylated anti-rabbit IgG biotinylated anti-mouse IgG increase stability [1,26]. Calphostin C was a generous gift
242 H
from Kyowa Hakko Kogyo Ltd. N-Acetyl-S-farnesyl-
L
- cysteine AFC was from Calbiochem, U73122 was from
Biomol Research Lab., and wortmannin and forskolin were from Sigma. Other drugs were purchased from Wako
Chemicals.