Sprague–Dawley rats were purchased with dams from SLC Ltd. (Shizuoka, Japan), and maintained under a 12-h light/dark cycle with free access to food and water. All of the experiments described were performed in accordance with the local and international guidelines on the ethical use of laboratory animals. Efforts were made to minimize the number of animals and their suffering.
The neocortices of day-19 rat embryos were dissected. The tissues were digested with papain (1 mg/ml), and mechanically dissociated with the aid of a plastic pipette. The dissociated cells were plated onto poly-d
-lysine-coated dishes at relatively low cell densities (100–200 cells/mm2
). Neocortical neurons were grown in serum-free Dulbecco’s modified Eagle medium containing nutrient mixture N2 (Narisawa-Saito et al., 1999a
). This procedure reduced astroglial contamination to less than 5% of the total cell population until 7 days in vitro
(DIV) (Narisawa-Saito et al., 1999a
). For biochemical assay, cultures were supplemented daily with purified recombinant human EGF (20 pg/ml) or amphiregulin (200 pg/ml) for 6 days. An ErbB1 receptor inhibitor, PD153035 (100 nM, Tocris Cookson Inc., St. Louis, MO, USA) was added 2 h before each EGF treatment. Alternatively, EGF treatment was extended until DIV12 for immunocytochemistry (see below).
Tissues or cells were lysed in SDS lysis buffer containing 1% SDS, 20 mM Tris–HCl (pH 7.4), 5 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride. The lysates were boiled for 3 min, and then clarified by centrifugation. The protein concentrations of the supernatants were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were then subjected to electrophoresis on 7.5% or 10% SDS–polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA) in 0.1 M Tris base, 0.192 M glycine and 20% methanol using a semi-dry electrophoretic transfer system. The membranes were blocked overnight at 4 °C with 0.1% Tween 20 in Tris-buffered saline (T-TBS) containing 5% bovine serum albumin. Membranes were then probed with the following primary antibodies: anti-SAP97 (1:800 dilution, StressGen, Victoria, BC, Canada), anti-GRIP1 (1:1000, BD Transduction Laboratory, San Diego, CA, USA), anti-pan-PDZ (1:1000, Upstate Biotechnology, Waltham, MA, USA), anti-PSD-95 (1:2000, Upstate Biotechnology), anti-β-actin (1:1000, Chemicon Int., Temecula, CA, USA), anti-neuron specific enolase (NSE; 1:1000, Polysciences, Inc., Warrington, PA, USA), anti-glial fibrillary acidic protein (GFAP; 1:4000, DakoCytomation, Glostrup, Denmark), anti-phospho-ErbB1 receptor (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-ErbB1 receptor (1:500, Santa Cruz Biotechnology) antibodies. The membranes were incubated with these antibodies diluted in T-TBS containing 5% bovine serum albumin at room temperature for 1 h. After several washes with T-TBS, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-mouse IgG or goat anti-rabbit IgG secondary antibodies (1:10,000, DakoCytomation) at room temperature for 1 h. The membranes were then washed at least four times with T-TBS and target proteins were visualized using an ECL chemiluminescence system (Promega, Madison, WI, USA). To quantify the amount of protein, we determined liner ranges of individual immunoblots with serial dilution of control samples; 1.0–40 μg/ lane for GFAP, β-actin; 2.5–20 μg/ lane for GRIP1; 5–20 μg protein/lane for SAP97, PSD95, NSE. Using immunoblots carrying an appropriate amount of protein, we measured the signal density from immunoblots using NIH Image analysis software. The changes in protein levels were expressed as a percentage of their respective controls. In parallel, data were confirmed by the immunoblot analysis with serial dilution of samples.
Reverse transcriptase–polymerase chain reaction (RT-PCR)
Using an RT-PCR High kit (Toyobo, Osaka, Japan), cDNA fragments for SAP97, GRIP1 and β-actin were directly synthesized from total RNA and amplified within the linear range of amplification using a Light Cycler (Roche Diagnostics, Indianapolis, IN, USA). The oligonucleotide primers were designed as follows: 5′-TCCAGCAGTGTGAAGACCTG-3′ and 5′-CAGCTTCTTGGGACTTGAGG-3′ generating a 371-bp fragment for GRIP1, 5′-AAATGCCATCAAGAGGTTGC-3′ and 5′-CAGGGCAGAGAGATGAGACC-3′ generating a 328-bp fragment for SAP97, and 5′-GGCATCCTGACCCTGAAGTA-3′ and 5′-GGGGTGTTGAAGGTCTCAAA-3′ generating a 203-bp product for β-actin. PCR primers were designed from DNA sequences of neighboring exons for SAP97 and GRIP1 not to allow genomic amplification. The relative abundance of the mRNAs was estimated with individual Cot values and logarithmic amplification curves by the Fit Point program (Roche Diagnostics). Final PCR products were electrophoresed in a 2% agarose gel, stained with ethidium bromide, and imaged with a CCD camera (Cosmicar; Pentax, Tokyo, Japan).
Cultured cells were washed with phosphate-buffered saline and fixed for 20 min with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). Fixed cells were immunostained with either anti-MAP2 antibodies (1:100, Chemicon Int.) or anti-GFAP antibodies (1:1000, DakoCytomation). Alternatively, the culture period was extended until DIV12 to allow synaptic formation (Jourdi et al., 2003
). Cultures were immunostained with anti-PSD-95 family/pan-PDZ (mAb K28/86.2, 1:500, Upstate Biotechnology) antibodies. Their immunoreactivity was revealed using the ABC-diaminobenzidine method (Vector Laboratories Inc., Burlingame, CA, USA) and visualized with the aid of a microscope (Axioskop; Carl Zeiss Oberkochen, Germany) fitted with an LCD color camera (DP50-CU; Olympus, Tokyo, Japan). All pictures were taken with a 20× or 60× objective, at 1/300 s shutter speed using Studio Lite software (Pixera Corporation Osaka, Japan).
EGF administration to neonatal rats
Recombinant human EGF or cytochrome c
(Sigma Chemical Co., St. Louis, MO, USA or Higeta-Syoyu Co., Chiba, Japan) was dissolved in physiologic saline and administered s.c. at the nape of the neck of newborn rats daily from postnatal days 2–10 at a dose of 875 ng/g of body weight (injection volume 0.0125 ml/g). Peripherally administered EGF penetrates the blood–brain barrier of neonatal rats and affects neurochemical markers in the brain (Futamura et al., 2003
). Cytochrome c
was used for control injections because it has similar chemical characteristics as EGF including its molecular weight and isoelectric point, but lacks the biological activity of EGF (Calamandrei and Alleva, 1989
). Brains were removed from EGF- or cytochrome c
-injected rats and used in the following experiments.
On postnatal day 11, rats were anesthetized by inhalation of diethyl ether (Wako Pure Chemical, Osaka, Japan), and killed by transcardial perfusion with 4% paraformaldehyde and 0.1% glutaraldehyde in a 0.1 M phosphate-buffered solution (pH 7.4). The brains were removed and immersed in the same fixative for 24 h. Tissues were dehydrated in a graded ethanol series and embedded in paraffin wax. Serial thin slices (4 μm thick) were cut from the blocked samples, deparaffinized with xylene and ethanol, and stained with anti-PSD-95 family/pan-PDZ antibodies (1:100, Upstate Biotechnology). Immunoreactivity was visualized with goat anti-mouse IgG antibodies conjugated to Alexa Fluor 546 (Invitrogen, Carlsbad, CA, USA). Brain slices were optically sectioned further with a laser-scanning confocal microscope (FV500 with 0.6-μm optical depth, Olympus).