C .-K. Won, Y.S. Oh Brain Research 887 2000 250 –258
251
induce prostaglandin release from medullary and cerebellar treated circular glass coverslips. The cells were then
astrocytes but not from cortical and hypothalamic as- maintained for an additional 3–5 days before experiments.
trocytes [42]. Furthermore, astrocytes cultured from differ- At this stage, we found that GFAP-positive cells were
ent regions of the brain display different degrees of gap- greater than 90 in astrocyte cultures derived from
junction coupling [20], different levels of muscarinic different regions of the brain. The culture medium was
1
acetylcholine receptors [4], differential modulation of Na changed every 3 days. Immunocytochemistry was per-
channel mRNA expression [29], and different rate of formed as previously reported [28] using monoclonal anti-
uptake in serotonin and glutamate [3]. These highly GFAP antibody Roche Mol. Biochem..
heterogeneous characteristics of different regional as- trocytes may suggest a specialized role of astrocytes in
2.2. Evaluation of astrocyte stellation different regions of the CNS during information processing
and brain development. Cells were fixed in 4 paraformaldehyde and stained
In the present study, we have examined the regional with coomassie blue 0.25 Coomassie brilliant blue,
heterogeneity of astrocyte morphology changes in response R-250, 50 methanol, and 10 acetic acid to determine
to cAMP treatment, which is a well known factor to affect the percentage of stellate cells in culture. In order to
astrocyte morphology changes [26,34,40], and blockage of eliminate any serum effect on different regional astrocytes,
cAMP- and isoproterenol-induced astrocyte stellation by astrocytes cultured on 24 well plates were maintained in
glutamate in six different regional astrocytes cultured from the absence of serum for 24 h, and the astrocyte medium
cerebral cortex, hippocampus, brain stem, mid brain, was changed to fresh DMEM containing various con-
cerebellum, and hypothalamus. The results suggest that centrations of 8-CPT-cAMP Biolog Life Science Institute
there is a regional heterogeneity of astrocyte morphology or isoproterenol Sigma, respectively, in the absence or
changes in response to cAMP treatment and glutamate the presence of
L
-glutamate Sigma. cAMP- or iso- effect on astrocyte stellation.
proterenol-induced astrocyte stellation was determined after incubating the cells for 2 h in a 378C CO incubator,
2
and the levels of stellation among different regional
2. Materials and methods astrocytes were compared. We have chosen 2 h cAMP or
isoproterenol stimulation since our preliminary study 2.1. Primary astrocyte cultures
showed the maximum astrocyte stellation could be achieved after 2 h of stimulation in all regional astrocytes.
Astrocytes were cultured as previously described [28] As a control, DMEM changes without 8-CPT-cAMP or
from postnatal day 0–1 Sprague–Dawley rats with a minor isoproterenol did not induce any astrocyte morphological
modification to enrich flat, polygonal-shape astrocytes. changes in all regional astrocytes data not shown.
Briefly, six different regions of the rat brain, including Cells with processes longer than their perinuclear diame-
cerebral cortex, hippocampus, cerebellum, mid brain su- ters were defined as stellate cells according to the criteria
perior and inferior colliculi, brain stem pons and medul- used by Kimelberg et al. [19] and Shao et al. [40]. Stained
la, and hypothalamus were isolated, minced, and incu- cells were mounted on slide glass and viewed in a
bated in a solution containing 30 units papain ml Roche, transmitted light with a 103 objective lens using an
Earle’s salts, 0.5 mM EDTA, and 1.65 mM
L
-cysteine at inverted TMS-F microscope Nikon that was connected
378C for 15 min followed by trituration in a complete with a CCD camera Pixera. For each coverslip, three
culture medium Dulbecco’s Modified Eagle’s Medium, randomly chosen fields were counted about 40–60 cells in
DMEM, containing 10 fetal bovine serum obtained from each field, and the percentage of stellate cells was
Hyclone and penicillin streptomycin, 100 units ml each. determined thus, a minimum of 150 cells per each
2
Resuspended cells were plated onto 75 cm culture flasks coverslip was counted. Usually two coverslips, sometimes
7
Corning at high density .2310 cells flask, and
three coverslips, were included for each experimental maintained overnight at 378C in vitro in a 5 CO 95
condition thus, a minimum of 300 cells was counted per
2
air atmosphere. On the following day, the culture medium experimental condition, and each experimental condition
was replaced with a fresh complete culture medium, and was repeated from 3 to 4 independent culture preparations
maintained for 7–10 days in vitro. After the cells became thus, a total of 900–1,200 cells were counted in each data
confluent, the culture flask was replaced with 10 ml of points in Figs. 1, 2, 4 and 5. The percentage of stellate
fresh complete medium and was shaken overnight on an cells in each experimental condition was expressed as
orbital shaker Lab-line Instruments at a speed of 250 rpm average6standard deviation. Student’s t-test was used for
in a 378C CO incubator. On the next day, the culture flask statistical analysis. The experiments were repeated from at
2
was washed five times with phosphate buffered saline least three independent astrocyte culture preparations
PBS, and then the attached astrocytes were dissociated corresponding to each brain region. Sister cultures of
4 2
with trypsin–EDTA and re-plated 5310 cells cm onto astrocytes were routinely stained with anti-GFAP antibody
24 well culture plates Corning containing 12 mm un- to determine the quality of astrocyte cultures. In this study,
252 C
Fig. 1. Dose–response curve of astrocyte morphology changes in response to 8-CPT-cAMP, a membrane permeable cAMP analog. Astrocytes were maintained in the absence of serum for 24 h, and then the cells were treated with various concentrations of 8-CPT-cAMP for 2 h. Note that cerebellar
astrocytes were the most sensitive astrocytes to 8-CPT-cAMP treatment while hypothalamic astrocytes were the least sensitive to 8-CPT-cAMP treatment. Each datum point represents the mean6S.D. from 3–4 independent culture preparations. , P,0.05; , P,0.01 compared to all other regional astrocytes.
CB, cerebellum; MB, mid brain; HP, hippocampus; BS, brain stem; CC, cerebral cortex; HT, hypothalamus.
we have not observed any dramatic changes in the able cAMP analog, induced astrocyte stellation with a
percentage of GFAP-positive cells among different as- dose-dependent manner in all regional astrocytes Fig. 1,
trocyte preparations. and there was a regional heterogeneity in astrocyte mor-
phology changes in response to 8-CPT-cAMP. Cerebellar astrocytes were the most sensitive cells to 8-CPT-cAMP
3. Results treatment, whereas hypothalamic astrocytes were the least
