All of the animal experiments described were approved by the Animal Use and Care Committee guidelines of Niigata University (Approval No18 on April 26, 2010) and performed in accordance with the guidelines of NIH-USA. Every effort was made to minimize the discomfort of the animals in addition to the number of animals used in the experiments.
Male newborn Sprague-Dawley rats (SLC Ltd., Hamamatsu, Japan) were housed with a dam under a 12-h light/dark cycle (lights on 8:00 a.m.) in a plastic cage (276×445×205 mm). The rats were allowed free access to food and water. After weaning [postnatal day (PND) 20–30], rats were separated and housed with 2–3 rats per cage. Each adult animal (PND 56–94) was used in each experiment. Naïve Sprague-Dawley rats (all male, PND 56–70; SLC) were also used in control experiments. Recombinant human EGF (Higeta Shouyu Co., Chiba Japan) was dissolved in saline. EGF (0.875 µg/g) was administered subcutaneously (s.c.) each day to half of the littermates during PND 2–10 
. Control littermates received an injection of the same dose of cytochrome c (control protein) on the same schedule. The dose of EGF used in this study did not produce any apparent growth retardation in rats and mice 
. Some of adult rats daily received risperidone (1 mg/kg, i.p.; Janssen Pharmaceuticals Inc) or saline for 14 days.
Enzyme-linked immunosorbent assay (ELISA)
Rats were anesthetized with halothane, and brains were removed and cut into 1-mm thick slices. Using published boundaries 
, we identified and punched out each brain region of interest. TH levels were measured using ELISA 
. In brief, brain tissues were homogenized in 10 volumes of homogenization buffer [0.1 mM phenylmethanesulfonyl fluoride, 0.1 mM benzethonium chloride, 1 mM benzamidine (Sigma Chemical Co., St. Louis, MO), and 10 µg/ml aprotinin]. Brain homogenates were centrifuged at 14000× g for 20 min at 4°C, and the supernatants were stored at −80°C until use. Protein concentrations were determined using a Micro BCA kit (Pierce, Rockland, IL) with bovine serum albumin (BSA) as a standard.
Tissue homogenates or striatal lysates (standards) were loaded into ELISA plate wells coated with mouse monoclonal anti-TH antibody (a gift from Dr. Hatanaka and Dr. Takei). Plates were incubated with rabbit polyclonal anti-TH antibody (Chemicon, Temecula, CA) followed by incubation with anti-rabbit IgG β-galactosidase (1
1000, American Qualex, San Clemente, CA). The fluorescence of the enzyme products from a reaction with 4-methylumbelliferyl-β-D-galactoside (MUG, Sigma) was measured using a microplate reader (COLONA electric Co., Ltd., Ibaraki, Japan).
Each brain tissue was dissected as described above and homogenized in 200 µl lysis buffer [2% sodium dodecylsulfate (SDS), 10 mM Tris-HCl buffer (pH 7.4), 5 mM ethylenediamine-N,N,N′,N′
-tetraacetic acid (EDTA), 10 mM NaF, 2 mM Na3
, 0.5 mM phenylarsine oxide], and boiled at 95°C for 5 min. After centrifugation at 12000 rpm for 20 min, the supernatant was harvested. The supernatant was mixed with 5× sample buffer [0.31 M Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 25% 2-mercaptoethanol] and boiled at 95°C for 5 min. Denatured protein samples were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher and Schull, Dassel, Germany) by electrophoresis. The membrane was probed with anti-TH (1
2000, Chemicon), anti-vesicular monoamine transporter 2 (VMAT2) (1
1000, Chemicon), anti-dopamine-beta-hydroxylase (DβH) (1
500, Chemicon), anti-dopamine D1 receptor (1
1000, Sigma) or anti-D2 receptor (1
1000, Chemicon) antibodies. After washing, membrane immunoreactivity was detected using anti-rat, anti-rabbit, or anti-mouse immunoglobulin antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratory, West Grove, PA) followed by a chemiluminescence reaction (ECL kit, GE Health Science Inc., Tokyo, Japan) and exposure to X-ray films. Film images carrying a liner range of darkness of bands were subjected to film scanning and converted to the 8-bit digital data. Densitometric quantification of band intensity was performed with the free software Image J (National Institutes of Health, USA).
Rats were anesthetized with halothane, perfused transcardially for 7 min with phosphate-buffered saline (150 mM NaCl, 0.1 M sodium phosphate; pH 7.5) followed by 4% paraformaldehyde in phosphate-buffered saline. Brains were removed and post-fixed in the same fixation solution for 24 h at 4°C. Fixed brains were immersed in 30% sucrose solution for 3–5 days, and frozen in resin (Tissue-Tek, Sakura Finetek U.S.A. Inc. Torrance, CA). Sections (40 µm) were cut with a cryostat (CM1510, Leica, Nussloch, Germany). After rinsing in Tris-buffered saline [TBS; 0.1 M Tris-HCl (pH 7.4), 150 mM NaCl] containing 0.2% Triton X-100, sections were pretreated with 6% BSA and 0.2% Triton X-100 in TBS and then incubated with anti-TH antibody (1
1000, Chemicon) in TBS containing 3% BSA and 0.2% Triton X-100 for 48–72 h at 4°C. After rinsing in TBS/0.2% Triton X-100 three times, sections were incubated with biotinylated anti-rabbit immunoglobulin antibody (1
200, Jackson Immunoresearch Laboratory). Immunoreactivity was visualized using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) using diaminobenzidine as a substrate.
Determination of monoamine contents
Each brain region was homogenized in 0.1 M perchloric acid containing 0.1 mM EDTA, and 100 nM isoproterenol. After centrifugation at 12000 rpm for 20 min, the supernatants and pellets were harvested. Concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in supernatants were analyzed by HPLC-electrochemistry 
. The mobile phase containing 50 mM trisodium citrate (pH 3.2), 25 mM NaH2
, 10 mM diethylamine, 0.03 mM EDTA, 2.5 mM 1-octane sulfonic acid sodium salt, 6% methanol, 1% dimethylacetamide was delivered at 0.5 mL/min. Monoamines were separated on an analytical HPLC column (CA-50DS, 4.6×150 mm, Eicom, Kyoto, Japan) and detected with a graphite electrode (WE-3G, Eicom) to which +700 mV was applied. Data analysis was performed with a data acquisition computer (Powerchrom, Eicom). Tissue pellets were homogenized in 0.5 N NaOH and subjected to protein determination with a Micro BCA kit (see above). Tissue monoamine contents were normalized with protein concentrations.
Local drug administration to the globus pallidus
Control and EGF-treated rats (PND 56–70) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.). After confirming deep anesthesia, a rat was mounted on a stereotaxic apparatus with an incisor bar set at 3.3 mm below the interaural line. The skull was exposed and two holes were drilled for bilateral implantation of guide cannulae (23 G stainless-steel pipes) into the GP. The stereotaxic coordinates were 0.9 mm anterior, ±3.0 mm lateral from the bregma, and 4.5 mm below the dura mater. After allowing the rat at least 10 days of recovery from surgery, a 30-G needle connected to Teflon tubing and a Hamilton syringe was placed 2 mm below the tip of the guide cannula. The drug (0.5 µl) was injected over a period of 30 sec, and the needle left in place for an additional 30 sec. Rats were placed in their home cage for 5–15 min, to allow for local diffusion of the drug; the rats were then subjected to behavioral tests (see below). When rats received reserpine, rats were placed in their home cage for 120 min to deplete the local dopamine pool. The cannula position was confirmed after the completion of the behavioral tests (see below).
Reserpine (Daiichi Sankyo Pharmaceutical Inc., Tokyo, Japan) was dissolved in phosphate-buffered solution (pH 4.0) containing 3 mg/mL DL-methionine and 70 mg/mL propylene glycol. Conventional dopamine receptor ligands, SCH23390, raclopride and quinpirole, were all obtained from Sigma and dissolved in 10% dimethyl sulfoxide (DMSO) in saline (vehicle).
Rats were anesthetized with sodium pentobarbital (65 mg/kg i.p.) and mounted in a stereotaxic apparatus. The skull was exposed and a hole was drilled for unilateral implantation of a guide cannula (AG-8, Eicom) into the GP. The stereotaxic coordinates were 0.9 mm anterior; 3.0 mm lateral from the bregma, and 4.8 mm below the dura mater. After allowing the rat at least 10 days of recovery from surgery microdialysis experiments were performed.
The microdialysis probe (2 mm active area, A-I-8-02, Eicom) was connected to Teflon tubing (0.65 mm o.d., 0.12 mm i.d.; Bioanalytical Systems Inc., West Lafayette, IN). The rat was perfused with artificial cerebrospinal fluid (pH 7.0) containing 147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, and 0.5 mM MgCl2 at a flow rate of 0.7 µL/min. Dialysate was discarded to obtain a steady state for at least 18 h after implantation of the probe, and then dialysate samples were collected every 30 min. The first five fractions were collected to determine basal levels of dopamine. The perfusion medium was switched to the medium containing a high concentration of potassium (80 mM KCl, 69.7 mM NaCl, 1.2 mM CaCl2, 0.5 mM MgCl2; pH 7) for 60 min (for two fractions). The perfusion medium was then switched back to the original medium and five fractions were additionally collected. In all, total 12 fractions were collected.
Dopamine in the dialysates was determined by HPLC with electrochemical detection. The mobile phase containing 48 mM citric acid, 24 mM sodium acetate, 10 mM NaCl, 0.5 mM EDTA, 2.5 mM SDS, and 16% acetonitrile (pH 4.8) was delivered at 50 µL/min. Dopamine was separated on an analytical column (BDS Hypersil C18 1×100 mm, Thermo Fisher Scientific, Yokohama, Japan) and detected with a 3 mm glassy carbon electrode detector (Unijet flow cell; Bioanalytical Systems Inc.) to which +550 mV was applied. Data analysis was performed with the analysis software (Epsilon LC; Bioanalytical Systems Inc.). Data were not compensated with the recovery rate.
Confirmation of cannula positioning
After local drug injection or microdialysis, rats were deeply anesthetized with halothane and decapitated. Brains were quickly removed and fixed in 4% paraformaldehyde for 3 days. Fixed brains were cut into 50-µm sections using a vibratome (Dosaka EM Ltd., Kyoto, Japan). Each section was stained with 0.5% cresyl violet solution. The location of a microdialysis probe or injection needle was determined under a microscope according to a stereotaxic atlas 
. Animals that exhibited incorrect cannula placement were removed from the data analysis.
Measurement of acoustic startle and prepulse inhibition of startle response
Acoustic startle amplitude was measured in a startle chamber (SR-Lab Systems, San Diego Instruments, San Diego, CA) 
. Rats were placed into a startle chamber with 70-dB background noise. Five minutes later, the startle amplitude was recorded in a session that included multiple trial types: (i) a 120-dB 40-ms noise burst presented alone (pulse); (ii–iv) 40-ms 120-dB noise burst following prepulses by 100 ms (20-ms noise burst) that were 5, 10, and 15 dB above background noise (i.e., 75-, 80-, 85-dB prepulse, respectively); and (v) no stimulus (background noise alone). The percentage PPI of startle responses was calculated as: 100−[(startle response on prepulse-pulse stimulus trials – no stimulus trials)/(pulse-alone trials – no stimulus trials)]×100. To match the magnitude of pulse alone startles between groups, a 120-dB noise burst was replaced with a 110-dB noise burst in some experiments.
Analysis of locomotor activity
Side effects of reserpine were estimated by monitoring spontaneous locomotor activity under novel conditions. Reserpine-infused rats were placed in an open field box (45 cm length×45 cm width×30 cm height, MED Associates, St. Albans, VA, USA) under a moderate light level (200 Lx). Line crossings and rearing counts were measured by photo-beam sensors (25 mm intervals for horizontal axis and 150 mm for vertical axis).
Social interaction test
The index for social interaction of rats was measured according to Futamura et al. (2003) 
. Following the above locomotor test, reserpine- or vehicle-infused rats were exposed to an unfamiliar male rat that was housed in another cage, and was age, body-weight, and gender-matched. All tests were videotaped and scored in blind. Scoring of social interaction times and duration was based on sniffing behaviors, defined as active chasing of the partner, shaking the nose near the partner, and contacting the partner with the nose.
Measurement of immobilization levels in the bar-hang test
Immobilization levels and the cataleptic effects of drugs were measured using a bar-hang test method 
. In the bar-hang test, the front paws of the rat were gently placed on a horizontal metal bar (5 mm diameter) and placed 10 cm above ground level. The test was terminated when the paw of animal was released from the bar or 300 sec had passed, and the total time until the animals removed the paw from the bar was recorded. Rats were subjected to three blocks (separated by 20-min intervals) of three trials. Scores at the three different time blocks (after 1-h acclimation, 20 and 40 min after drug infusion) were monitored for comparison purposes.
All data are expressed as means ± SEM. Statistical differences in the behavioral data were determined by analysis of variance (ANOVA). When univariate data were obtained from two groups, two-tailed t-test was used for comparison. Behavioral scores were initially subjected to factorial ANOVA using neonatal EGF treatment (two levels) and local drug infusion (two or three levels) as between-subjects factors, and prepulse magnitude (three levels) or test session (two levels) as a within-subjects factor. As the initial analyses yielded a significant factorial interaction, data were subjected to a Fisher's LSD post-hoc test with or without repeated measure. The interaction of a within-subjects factor with a between-subjects factor was estimated by multivariate analysis of variance (MANOVA). Individual statistical differences between data points are shown in the figures. Correlations between dopamine release and PPI were examined by Pearson's correlation analysis. A p value less than 0.05 was regarded as statistically significant. Statistical analyses were performed using StatView software (SAS Institute Inc., Cary, NC, USA). N values in parentheses represent the number of animals used in each group.