Twenty-six pregnant ICR mice were purchased from Japan SLC Inc. (Shizuoka, Japan) and housed under controlled conditions with 12 hours light/12 hours dark cycle and ad libitum access to food and water. They were divided into two groups: diesel exhaust exposure group (n = 12) and control group (n = 14). The mice of the exposure group were exposed to diesel exhaust for 8 hours/day (9:00 - 17:00 h.), for 5 days per week (Monday-Friday) in an inhalation chamber at the Research Institute of Tuberculosis (Japan Anti-Tuberculosis Association, Tokyo, Japan) from gestational days (GD) 2 - 16. After the exposure period, mothers and pups were maintained in a clean room. Pregnant mice delivered their pups on GD 19. The number and the sex ratio of pups in the exposure group and the control group were 114 (male:female = 75:39) and 161 (75:86), respectively. On postnatal day (PND) 4, the number of pups per litter was adjusted randomly to ten. In each group, pups were weaned on PND 21, after which male mice were transported to Tokyo University of Science (Chiba, Japan). Mice were transported carefully to minimize stress factors by Sankyo Labo Service Co., Inc. (Tokyo, Japan) and Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan). All experimental animals were handled in accordance with institutional and national guidelines for the care and use of laboratory animals.
Exposure to diesel exhaust
A 2369 cc diesel engine (Isuzu Motors Ltd., Tokyo, Japan) was operated at a speed of 1050 rpm and at 80% load with a commercial oil. The exhaust was introduced into a stainless steel dilution tunnel (450 mm diameter × 6250 mm), where the exhaust was mixed with clean air, and average concentrations of exhaust constituents were maintained at 1.06 × 104 suspended particles/cm3 (171 μg/m3), 1.25 ppm for carbon monoxide (CO), 0.04 ppm for nitrogen dioxide (NO2), and less than 0.01 ppm for sulfur dioxide (SO2).
The SLA of each mouse was measured in a transparent acrylic cage (20 cm × 31 cm × 13 cm) with an activity monitor with an infrared ray sensor (NS-AS01; Neuroscience Inc., Tokyo, Japan). The analysis was done when the mice were 4 weeks old (n = 10/group). Movement was measured according to the release of temperature-associated infrared rays. SLA counts were collected at 10 min intervals for 3 days. Data were analyzed automatically with a computerized system (multidigital 32-port counter system; Neuroscience Inc.). The analysis was conducted in "a new environment" and in "a home cage environment", which refers to the first 60 min test period just after moving into a new cage and the subsequent test period each day, respectively. Statistical analysis was done with two-way, repeated-measures analysis of variance (ANOVA), in which the variables were diesel exhaust exposure and time, followed by post hoc Student's t-test. The level of statistical significance was set at P < 0.05.
Following the behavioural test, brain and trunk blood were obtained from the animals (5 weeks of age). The body weight of the animals was 28.56 - 37.51 g and there was no significant difference in body weight between the exposure group (32.7 ± 2.4 g) and the control group (32.9 ± 2.0 g). The brain was dissected into six regions, immediately frozen in liquid nitrogen, and stored at -80°C. Serum was separated in a gel barrier capillary blood collection tube (Capiject T-MG; Terumo Medical Corp., Elkton, MD) followed by centrifugation at 2200 g at 4°C for 15 min and stored at -80°C until analysis.
Brain dissection was done according to the modified method of Heffner et al.  and was based on the atlas described by Paxinos and Franklin . The following four regions were dissected from frozen forebrain and midbrain coronal sections on a silicon plate chilled with dry ice: prefrontal cortex (PFC; containing cingulated cortex and motor cortex areas 1 and 2); striatum (dorsal); hippocampus (caudal) and midbrain (containing ventral tegmental area and substantia nigra). Determination of monoamine levels was done in PFC, striatum, hippocampus, midbrain, cerebellum, and brainstem.
Preparation of homogenates
Frozen brain tissues was homogenized in ice-cold 0.2 M perchloric acid (Nacalai Tesque Inc., Kyoto, Japan) containing 100 μM Na2-EDTA (Dojinto Laboratories, Kumamoto, Japan) and 1 ng/mL isoproterenol as an internal standard (Sigma-Aldrich Co., St. Louis, MO). The homogenates were kept on ice for 30 min and centrifuged at 20,000 g at 0°C for 15 min. The supernatant was mixed with 1 M sodium acetate to adjust the pH to 3.0 (Kanto Chemical Co., Inc., Tokyo, Japan) and were frozen immediately in liquid nitrogen and stored at -80°C. The precipitate was used for the protein assay.
High-performance liquid chromatography (HPLC)
Each group contained samples from 10 mice. A 10 mL sample of the final supernatant was injected with a microsyringe (702SNR; Hamilton Co., Reno, NV) into an HPLC system equipped with an electrochemical detector (HTEC-500MAB; Eicom Co., Kyoto, Japan). The standard solution contained the monoamines dopamine and noradrenaline and their metabolites. The dopamine metabolites were 3-methoxytyramine hydrochloride (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The noradrenaline metabolites were normetanephrine hydrochloride (NM) and 4-hydroxy-3-methoxyphenylglycol hemipiperazinium (MHPG). Standards dopamine, HVA, 3-MT, NM and MHPG were obtained from Sigma-Aldrich. Standards noradrenaline and DOPAC were obtained from Nacalai Tesque and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. Separation of monoamines and their metabolites was done by passage through a C18 reverse-phase column (Eicompak SC-5ODS; 3.0 mm × 150 mm; Eicom), maintained at 25°C and connected to an electrochemical detector (EPC-500, Eicom). The mobile phase was 0.1 M acetic acid/citric acid buffer (pH 3.5) containing Na2-EDTA (5 mg/L), octanesulfonic acid (190 mg/L; Nacalai Tesque), and methanol (15% (v/v); Kanto Chemical Co., Inc.). The flow rate was maintained at 0.5 mL/min for 35 min. Data were collected and analysed with the PowerChrom 280 System (eDAQ Pty Ltd., New South Wales, Australia). To determine the protein concentration, pellets were dissolved in 100 mM Tris-HCl for protein determination by a high-sensitivity version of the Bradford method with a commercial reagent (ADV-01; Cytoskeleton Inc., Denver, CO), and measurements were done according to the manufacturer's protocol. The absorbance at 595 nm was measured with a 96-well microplate reader (model 550; Bio-Rad Laboratories Inc., Hercules, CA), and protein concentration was calculated from a standard curve generated with bovine γ-globulin (Pre-Diluted Protein Assay Standards: Bovine Gamma Globulin Set; Thermo Fisher Scientific Inc., Rockford, IL). Concentrations of monoamines and their metabolites are expressed as pg mg-1 of protein, and the catabolism rate is expressed as the ratio of metabolite to monoamine (e.g. HVA/dopamine). Indices were calculated from individual tissue samples. Statistical analysis was done with the Mann Whitney U-test. The level of statistical significance was set at P < 0.05.
Measurement of serum corticosterone
The concentration of corticosterone in serum was determined with a Correlate-EIA Corticosterone Enzyme Immunoassay Kit (Assay Designs Inc., Ann Arbor, MI).