Brain Research 887 2000 250–258 www.elsevier.com locate bres
Research report
cAMP-induced stellation in primary astrocyte cultures with regional heterogeneity
Chung-Kil Won, Young S. Oh
Departments of Medicine and Neurobiology , Sparks Center 865, University of Alabama at Birmingham, 1530 3rd Ave. South, Birmingham,
AL 35294, USA
Received 27 June 2000; accepted 29 August 2000
Abstract
It is well known that increased cAMP levels in cultured astrocytes can convert flat polygonal shaped astrocytes into process-bearing, stellate astrocytes. In this study, we have examined the possible existence of astrocyte regional heterogeneity in morphological changes in
response to cAMP stimulation. Primary astrocyte cultures were prepared from six different regions of neonatal rat brains, including cerebral cortex, hippocampus, brain stem, mid brain, cerebellum, and hypothalamus. After about 2 weeks in culture, the astrocyte culture
medium was changed to DMEM containing various concentrations of 8-CPT-cAMP, a membrane permeable cAMP analog, for 2 h. We found that 250 mM 8-CPT-cAMP produced a maximum effect causing .95 stellation in all regional astrocytes except hypothalamic
astrocytes 56 stellation. At lower cAMP concentrations, cell stellation most effectively occurred in cerebellar astrocytes. To examine further the regional heterogeneity of astrocyte morphological changes, glutamate was added together with 8-CPT-cAMP to block
cAMP-induced astrocyte stellation. Interestingly, glutamate blockage on cAMP-induced astrocyte stellation was brain region-specific in that cerebral and hippocampal astrocytes were effectively blocked by glutamate when compared to other regional astrocytes. Furthermore,
glutamate inhibited isoproterenol-induced astrocyte stellation in a region-specific manner similarly as in cAMP-induced stellation. The present study demonstrates that astrocytes derived from different regions of the neonatal rat brain maintain different levels of
morphological plasticity in culture.
2000 Elsevier Science B.V. All rights reserved.
Theme : Development and regeneration
Topic : Glia and other non-neuronal cells
Keywords : Cerebrum; Hippocampus; Cerebellum; Mid brain; Brain stem; Hypothalamus
1. Introduction thicker and longer processes and increased cellular content
of glial fibrillary acidic protein GFAP has been observed Primary cultures of astrocyte are diverse in their mor-
in the CNS after various types of injury caused by phology [8,24], and many factors can influence the mor-
physical, chemical, and pathological trauma [15,23]. It is phology of cultured astrocytes, such as the presence or the
suggested that astrocyte morphology changes in vivo may absence of neurons in culture [14,22], reagents which can
affect neuronal excitability and or synaptic activity by increase intracellular cAMP levels [13,26,34,40], and the
changing the volume of the extracellular space [16]. At presence of b-amyloid peptide [33] or various hormones
present, the level of regional heterogeneity in astrocyte [31,43]. Astrocyte morphology in vivo can also change in
morphology changes in response to various stimuli is not response to various stimuli. For example, during lactation
well understood. [38] or dehydration state [16], astrocyte morphology can
There is increasing evidence suggesting regional hetero- change responding to these normal physiological chal-
geneity of astrocyte functions. For example, dopamine lenges. The appearance of reactive astrocytes in vivo with
receptors were expressed in astrocytes cultured from the striatum but not from other brain regions [17]. A phos-
phoinositide-linked peptide response was different among
Corresponding author. Tel.: 11-205-934-3806; fax: 11-205-934-
cortical, cerebellar, and spinal cord astrocytes [10]. An-
1147. E-mail address
: yohuab.edu Y.S. Oh.
giotensin II could activate phospholipase C PLC and
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 2 2 - X
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
