Ethics Statement

All animal experiments were approved by the Institutional Animal Care and Use Committee, the Division of Health Sciences, Graduate School of Medicine, Osaka University (approval no. 31–03-3).

Animals

For 7-day-old mice study, pregnant embryonic day 12–13 female ICR mice were obtained from Japan SLC Inc. (Shizuoka, Japan). Experiments were performed when females gave birth to 10–15 pups to avoid variations in growth, because litter size affects the postnatal growth and development. For 3-week-old mice study, 3-week-old male ICR mice were obtained from Japan SLC Inc. (Shizuoka, Japan). All animals were housed under a 12-h light–dark cycle with free access to food and water. Pups were kept warm on a mat at 38 °C during the period when they were separated from their mother.

Chemicals

[2-14C]acetate (specific activity, 1.99 GBq/mmol) was purchased from PerkinElmer Life Science Inc. (Boston, MA, USA). 14C-microscale (RPA 511) was obtained from Amersham Biotech UK Ltd. (Buckinghamshire, UK). PTZ was obtained from Sigma-Aldrich (St. Louis, MO, USA). DL-fluorocitric acid barium salt was obtained from Sigma-Aldrich (St. Louis, MO, USA), and prepared as described by Paulsen et al. [15]. All other chemicals used were of the highest commercially available purity.

Surgery and Microinjection of Fluorocitrate

At 3-week-old, the mice were anesthetized using isoflurane (induction, 5%; maintenance, 2%), and placed in a stereotaxic apparatus. Fluorocitrate was injected into the striatum (0.5 nmol/µL, at a rate of 0.25 µL/min for 4 min) via a 30-gauge cannula using an automated syringe pump. The cannula was left in place for an additional 5 min after injection to reduce the reflux of the injected chemicals along the cannula track. Four hours following fluorocitrate injection, the animals were subjected to [2-14C]acetate administration.

[2-14C]Acetate Administration

Seven-day-old animals were maintained in the supine position and intranasally administered 5 µL [2-14C]acetate (92.5 kBq/animal) over a period of 1 min by a micropipette. For intranasal administration to 3-week-old mice, animals were placed in an anaesthesia box filled with isoflurane (5%) for 1 min, after which they were removed, held in the supine position, and immediately administrated 5 µL [2-14C]acetate (92.5 kBq/animal) intranasally. Following intranasal administration, the animals were kept in the supine position for an additional 1 min, returned to their cages, and allowed to move freely. For intravenous administration, the animals were administrated a bolus injection of [2-14C]acetate (92.5 kBq/animal), dissolved in 0.2 mL saline via the tail vein. For oral administration, 7-day-old animals were maintained in the supine position and orally administered 5 µL [2-14C]acetate (92.5 kBq/animal) by a micropipette.

[2-14C]Acetate Uptake

In the time course experiment using 7-day-old mice, the animals were sacrificed by decapitation under brief anaesthesia (isoflurane 5%) 5, 20, and 60 min after intranasal administration of [2-14C]acetate. In other experiments, 20 min after [2-14C]acetate administration, the animals were sacrificed by decapitation under brief anaesthesia (isoflurane 5%). For autoradiography, brains were quickly removed, surrounded with powdered dry ice and frozen. Next, coronal or sagittal slices (20-µm-thick) were prepared using a cryostat at –20 °C, arranged onto glass slides, and placed in contact with an imaging plate (Fuji Film Co., Tokyo, Japan) for several days. The photo-stimulated luminescence (PSL) values in each region of the prepared autoradiograms were subsequently determined using a multipurpose imaging scanner (FLA-7000; Fuji Film Co., Tokyo, Japan). The radioactivity concentrations in the regions of interest (ROIs) were obtained as (PSL-background)/area (mm2) [(PSL-BG)/A], calibrated in Bq/g tissue using 14C-microscale, and expressed as the distribution absorption ratios (DAR) to correct for differences in animal body weight and injected dose. DAR = radioactivity concentration in tissue (Bq/g) / total injected dose (Bq) × body weight (g). For the dissection study, tissues were quickly removed and weighed, and subsequently solubilised using the tissue solubilizer Soluene-350 (PerkinElmer Co., Ltd. MA, USA). Radioactivity was subsequently measured using a liquid scintillation counter. Radioactivity concentrations are expressed as the DAR values.

Radio Thin-Layer Chromatography (Radio TLC) Analysis

The animals were sacrificed by decapitation under brief anaesthesia (isoflurane 5%) 20 min after [2-14C]acetate intranasal administration. The brains were then quickly dissected, and the cerebral cortex was removed, homogenized with 0.5 N HClO4 solution (1:5 w/v), and centrifuged (1000 × g, 10 min). The supernatant was spotted onto thin-layer silica gel plates, and TLC was performed using 99% EtOH:28% NH3, 3:1 (v/v) as the eluent. Glutamate and glutamine were spotted on the same TLC plate and used as standard. The TLC plates were exposed to an imaging plate (Fuji Film Co., Tokyo, Japan) for several days. After the autoradiograms were obtained, the TLC plates were sprayed with ninhydrin to determine the Rf value for glutamine and glutamate. Quantitative analysis of the PSL values at each spot was performed using a multipurpose imaging scanner (FLA-7000; Fuji Film Co., Tokyo, Japan), as described above.

Behavioural Seizure Analysis

Seven-day-old animals were intraperitoneally injected with PTZ (80 mg/kg/25 mL saline) or an equivalent volume of saline. Following this injection, the animals were isolated in plastic cages, and their behaviour was observed for 2 h in 5-min intervals. The presence or absence of any type of behavioural seizure activity was scored as follows: stage 0, no abnormality; stage 1, exploring, and becoming immobilised; stage 2, head nodding, facial and forelimb clonus; stage 3, continuous myoclonic jerk, tail rigidity; stage 4, forelimb and hindlimb clonus, and kangaroo posture; stage 5, tonic–clonic convulsion. These scores align with Racine scale [16] and revised Racine scale applied to mice [17].

Statistical Analysis

All values are expressed as the mean ± SD (for each group). P-values < 0.05 on repeated measures ANOVA or Student's t-test were considered to indicate statistical significance.