Brains were removed from ICR mice (Samtako, Osan, Korea) on postnatal day (P) 1 and 1, 2, 4, and 8 weeks of age (i.e., P1d, P1w, P2w, P4w and P8w, n = 5 in each group), and then fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) at pH 7.4 for 2 hr. All animal experiments were approved by the Institutional Animal Care and Use Committee in Chungnam National University (2009-3-17). The tissue was cryoprotected by infiltration with a 10%-30% sucrose solution, embedded in an Optimal Cutting Temperature compound, and then rapidly frozen in 2-methyl butane precooled to its freezing point with liquid nitrogen. Tissue specimens were cut into 10 µm sagittal or horizontal sections on a cryostat and then mounted. Immunohistochemical staining of the tissue sections was done using the avidin-biotin peroxidase complex (ABC) method described previously (13). Sections were treated with blocking buffer (1% fetal bovine serum and 0.3% Triton X-100 in PBS) for 30 min after endogenous peroxidase blocking with 1% H₂O₂ in PBS and then rinsed thoroughly with PBS. Sections were incubated with the primary antibody for 24 hr at 4℃. The antibodies and their dilutions were ETA receptor (anti-rabbit, Sigma, St. Louis, MO, USA, E3651, 1:100), CD31 (anti-rat, BD Biosciences, Frankin Lakes, NJ, USA, 550274, 1:500), myelin basic protein (MBP, anti-rat, Millipore, Billerica, MA, USA, MAB386, 1:100), and adenomatous polyposis coli protein (APC, anti-mouse, Merck, Darmstadt, Germany, OP80, 1:500). Tissues were exposed to the biotinylated anti-mouse IgG and the streptavidin peroxidase complex (Vector labs, Burlingame, CA, USA) for 1 hr at room temperature after rinsing with PBS. Immunostains were visualized with diaminobenzidine (DAB, Sigma) for 3-5 min and then mounted using Polymount. Double immunofluorescent experiments were done using Cy™2-conjugated anti-rabbit IgG (1:400) and Cy™3-conjugated anti-mouse IgG (1:600, Amersham, Buckinghamshire, UK). All immunoreactions were examined under a fluorescent microscope (IX71, Olympus, Tokyo, Japan).

We prepared primary OPC cultures by mechanical dissociation according to the method by Chen et al. (14). Briefly, a P1 Sprague-Dawley rat pup (Samtako, Osan, Korea) was decapitated in an ice-chilled dish and the brain was removed. Mixed cultures were shaken overnight to detach OPCs from the astrocyte monolayer. Oligodendrocyte progenitors were plated on poly-D-lysine-coated culture dishes and grown in basal defined media (BDM) consisting of DMEM20S (DMEM, 4 mM L-glutamine, 1 mM sodium pyruvate, 20% FBS), 0.1% bovine serum albumin, 50 µg/mL human transferrin, 10 nM hydrocortisone, 10 nM biotin, 5 µg/mL insulin and 30 nM selenium. After stabilization of the oligodendrocyte progenitor cells for 3 days, 20 ng/mL each of PDGFAA and bFGF (Peprotech, Rocky Hill, CT, USA) were added to the media to differentiate the OPCs which were used after 3 days in vitro (DIV). The cultured cells were characterized immunocytochemically with cell-type-specific antibodies. More than 95% of the cells were positive for the monoclonal antibody A2B5 (clone 105, IgM, R&D systems, Minneapolis, MN, USA) and for the PDGFA receptor (CD140a, Millipore), a marker for OPCs in culture, while less than 5% of the cells were GalC-positive OLGs, GFAP-positive astrocytes or complement type-3-positive microglia (15, 16).

Total RNA was extracted from cultured OPCs using TRizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The amount of RNA and its purity was determined by measuring the optical density at 260 nm and the optical density ratio at 260 and 280 nm (ratio ~ 2), respectively. RNA integrity was checked by electrophoresis on agarose gel. Approximately 10 µg of total RNA from each sample was used to generate cDNA in a 40 µL reaction using 200 U of the SuperScript II (Invitrogen, Carlsbad, CA, USA) reverse transcriptase and oligo (dT). Subsequently, 2 µL of cDNA was subjected to PCR amplification in order to measure the expression of MBP gene. To amplify rat MBP, we used the following forward primer: 5'-CTGCCCTCGGCTTCTTAAT-3' (456-474) and reverse primer 5'-GTTTTAGCCAGTCAGGGTGC-3' (576-595). The nucleotide position reported in brackets corresponded to the published rat MBP cDNA sequence (GenBank accession number, M25889). Genes were amplified as follows: denaturation at 94℃ for 1 min, annealing at 60℃ for 1 min, extension at 72℃ for 1 min, and this was repeated for 28 cycles. PCR products were resolved by 1.2% agarose gel electrophoresis and visualized under UV light.

At P1d, P1w, P2w, P4w and P8w, the ICR mice (Samtako) were anesthetized. The entire brain was removed quickly from each mouse and homogenized in lysis buffer (50 mM TrisCl, 150 mM NaCl, 0.02% sodium azide, 100 µL/mL PMSF, 1 µg/mL aprotinin, 1% Triton X-100). Supernatant protein concentrations were determined with Micro BCA protein assay kits. Bovine serum albumin was used as a standard (Pierce Chemical, Rockford, IL, USA). Aliquots containing 20 µg of protein were boiled in 2 × sample buffer (0.8 M TrisCl, 10% glycerol, 20% β-mercaptoethanol, 10% SDS, and 0.02% bromophenol blue) for 5 min and loaded onto a polyacrylamide gel. Proteins were transferred onto nitrocellulose membranes after electrophoresis at 250 mA for 1 hr. Membranes were incubated in 5% skim milk in PBST (0.3% Triton X-100 in PBS) for 1 hr to block non-specific binding and then probed with a primary antibody: ETA receptor (1:500), MBP (1:500), and β-actin (1:5,000, Sigma). Membranes were washed 3 times for 10 min in PBST and incubated for 1 hr with a peroxidase-labeled secondary antibody (Vector Labs, Burlingame, CA, USA) diluted 1:2,000 in PBST. Immunolabeled proteins were detected by chemiluminescence after 3 additional washes using a Supersignal ECL kit (Pierce Chemical, Rockford, IL, USA) and Biomax Light-1 films. For blotting of OPCs, cells were treated with ET-1 and/or BQ123 and then harvested and cell extracts were obtained as above. Primary antibodies for ETA-receptor (1:500) and MBP (1:500) were used and detected as above.

OPCs grown on coverslips were loaded with the Ca²⁺ indicator Fura-2/AM according to the manufacturer's instructions (Molecular Probes, Carlsbad, CA, USA). Images were obtained by confocal microscopy (Zeiss LSM510 Meta; Carl Zeiss, Jena, Germany). For kinetic measurements, Ca²⁺-free HBSS buffer solution containing 10 mM EGTA was used to eliminate the extracellular Ca²⁺, and images were obtained every 5 sec for 15 min at 37℃ using a Cool-SNAP microscope (HQ, Photometrics, Tucson, AZ, USA).

During early development (P1d), ETA receptor immunoreactivity has been associated with blood vessels (Fig. 1A). However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the round oligodendrocyte-like cells of the corpus callosum and the white matter, apart from the vessels (Fig. 1B-D). MBP immunoreactivity was weakly found in the primitive corpus callosum region and then intensified in the corpus callosum during later development (Fig. 1E-H). ETA receptor immunoreactivity was observed on the exterior surface of blood vessels and appeared as small, oval cell bodies with processes that extended for some distance parallel to the vessel axes (Fig. 1I). To examine the localization of the ETA receptor in the perivascular elements of the vessels, double labeling with the ETA receptor and CD31, a marker of endothelial cells, was done. The ETA receptor was associated with blood vessels, but its perivascular elements were found to not co-localize with CD31, suggesting that the ETA receptor was expressed in the perivascular cells during early development (Fig. 2A-C). Since ETA receptor immunoreactivity was found in the primitive corpus callosum, double labeling with MBP, a marker of mature oligodendrocytes, was done. The ETA receptor co-localized with MBP positive cells at P1w (Fig. 2D-F) and the amount of ETA receptor-positive labeling in oligodendrocytes was higher at later stages of development. In addition, labeling with the other oligodendrocyte marker APC was also confirmed (Fig. 2G-I). Western blot was done to examine changes in the ETA receptor and MBP levels. Intense ETA receptor bands were detected at P1d and the intensity of these bands increased in ages approaching adulthood (P8w). On the other hand, MBP levels were found at P2w and they increased thereafter. Immunoblots for these ETA receptor proteins in the developing brain generally confirm the immunochemical findings (Fig. 3).

We first examined whether treatment of OPCs with an ETA receptor agonist, ET-1, would lead to increased oligodendrocyte myelination. OPCs treated with ET-1 exhibited an aggregation of cells and induction of MBP which is indicative of myelination (Fig. 4). To further examine the effect of ET-1 and ETA receptors on the myelination of oligodendrocytes, we measured MBP gene expression after 48 hr of treatment with ET-1. The RT-PCR analysis of MBP mRNA by a MBP-specific primer (17) amplified a band of 140 bp. As expected, the application of ET-1 dramatically increased MBP expression (Fig. 5A). The ET-1 response was totally abolished when cells were pretreated with BQ123, an ETA receptor antagonist. A relative estimate of PCR products was carried out by evaluating the ratio of MBP and the corresponding GAPDH bands for each template. Next, we used a pharmacological approach to show that ETA receptors are activated by ET-1 during oligodendrocyte myelination. MBP protein was elevated with ET-1 and down-regulated with BQ123 (Fig. 5B). The data suggest that ET-1 increases the myelination of oligodendrocytes by activating the endogenously expressed main ETA receptor subtype.

It has been shown that Ca²⁺ signaling is essential in the development and functioning of oligodendrocytes. For example, Ca²⁺ uptake is required for the initiation of myelination (18). Therefore, we examined the effect of ET-1 on calcium release kinetics with a Cool-SNAP microscope. A 4-fold increase in fluorescence intensity was detected within 2 min after ET-1 stimulation in normal bath solution (HBSS with normal [Ca²⁺]), which went down immediately and out of phase at 3 min (Fig. 6). This data suggests that ET-1 stimulation induces the myelination of oligodendrocytes through Ca²⁺ release.