Animal protocol and Pb2+ exposure
Rats were exposed to Pb2+ during development as previously described [9, 27]. Briefly, twenty-four adult Sprague-Dawley rats were individually housed in plastic cages with bedding at 22 ± 2°C under a 12-hour light: dark cycle (male-female ratio 2:1, weight 200~250 g). Eight female rats were randomly selected and placed on control or 0.2% lead acetate water (Sigma-Aldrich, St. Louis, MO) from 10 days prior to mating and until postnatal day 21, namely gestational and lactational lead exposure. The lead-exposed group (4 litters) and control group (4 litters) both received the same treatment throughout the study and food and water were provided ad libitum. One day after parturition, litters were culled to 8 pups (male-female ratio 1:1) and the pups were weaned at 21 days of age. After weaning, all pups were fed deionized drinking water. All procedures complied with institutional guidelines regarding the ethical care and use of animals.
Blood lead and hippocampal lead analysis
In each litter, four weaning pups including 2 male and 2 female rats were randomly selected to analyze the blood lead and hippocampal lead levels to evaluate the actual lead content at the end of the exposure. Blood samples (0.3–0.5 ml) were collected by cardiac puncture in tubes containing EDTA-disodium. Blood lead levels were determined via Thermo Elemental Solaar M6 Series (Thermo Elemental, Franklin, MA, USA) by Graphite Furnace Atomic Absorption Spectrometry and the quality control procedure for the assessment of lead exposure was performed. Hippocampus from both left and right sides were collected from each rat, rinsed softly with saline, sopped up water with filter paper, pooled together as one sample and weighed. After hippocampal tissues were digested by nitric acid and hydrogen peroxide, they were heated in the microwave digestion oven (CEM MARS5, USA). After that, hippocampal lead levels were measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500 CE, Agilent Technologies, USA). The operations are all performed in our ICP-MS lab, which meets the Chinese Standard (GBJ173-1984) and provides air to meet class 100 (class I) conditions.
Ultrastructural details of hippocampus were studied with electron microscopy as described [9, 28]. Briefly, after 32 weaning rats (8 litters, 4 pups/litter) were sampled by cardiac puncture as above, they were immediately decapitated and collected from both sides of hippocampi and immediately cut into tissue blocks (1 mm × 1 mm × 5 mm) and were processed for electron microscopy. Ultrathin sections (50 nm) were cut with an ultramicrotome (Ultracut, Reichert-Jung) and stained with 4% uranyl acetate for 20 minutes and with 1% Pb for 10 minutes prior to examination by electron microscopy (H-500, HITACHI, Japan). The slides were read by a designated and experienced pathologist who was blinded to the dose groups.
Morris water maze (MWM)
A test using the MWM was performed when young rats were 30 days old. In each litter, only one male and one female pups were randomly selected. The MWM was originally designed by English psychologist Morris in the 1980s , which consisted of a dark circular pool 150 cm in diameter and 50 cm in height. The pool was filled to a height of 35 cm with water at 22°C ± 0.5°C stained by black ink. A transparent Plexiglas® escape platform (12 cm in diameter) 5 cm below the water surface and invisible to the rats was located in the center of the southwest quadrant. The room had numerous extramaze cues that remained constant throughout the experiment and no intramaze cues to ensure that the rats had to rely on the location of extramaze cues to locate the platform. The procedure included a training portion and test portion. Each training day consisted of 4 trials per animal, with a quasi-randomly selected release location from each compass point (N, E, S, W). On trial 1 of day 1, the animal was released from the appropriate starting location and once the rat located the platform it was allowed to stay on it for 10 seconds. If the rat did not find the platform within 120 seconds, it was guided to reach it and allowed to remain on it for 10 seconds and then was returned to its heated cage following completion of the task. Twenty-four hours after last training trial (postnatal day (PND) 35), 7 days later (PND 42), and 1 month later (PND72), spatial memory was repeatedly examined. On each occasion experimental procedures and surroundings were kept constant. The time required to reach the platform (escape latency), distance swimming to the platform, and the swimming speed as well as the time and distance spent in each quadrant were recorded by a video tracking system. The measures were averaged per rat within each daily session.
The MWM originally was aimed to test short-term memory (STM), namely spatial reference memory. In previous studies, the retention tests including the inhibitory avoidance task , hippocampal dependent discrimination task , and conditioned taste aversion , were performed to examine long-term memory (LTM) of rats which were conducted at 5 days , 7 days  or 1 month  after training. However, there have not any studies to assess the MWM test for evaluation of LTM. In this study, we tried to modify the classic MWM procedure and add our self-designed retention test, which might be a new and practical way to apply the MWM to evaluate LTM.
Total RNA isolation
At 21 days of age, both sides of hippocampus of pups (8 litters, one male and one female pups/litter) were harvested and stored frozen at -80°C prepared for PCR. RNA was isolated using a Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Extracted RNA concentrations and purity were evaluated by measuring the A260 nm-to-A280 nm absorbance ratio with an ultraviolet spectrophotometer (Perkin Elmer, Wellesley, MA, USA). Integrity of RNA was assessed by agarose gel electrophoresis.
Real-time reverse transcription (RT)-PCR
Highly purified oligonucleotide primers were commercially generated (SBS Genetech, China). Primer design and optimization were performed with Oligo software (National Biosciences Inc., Plymouth, MN, USA) . The primers used were the following: mGluR3 [GenBank: M92076], sense 5'-GAC GTG GTC CTG GTG ATC CTA T-3', antisense 5'-CTA ACG GAG ATG CAC ATT G-3', 197 bp; mGluR7 [GenBank: D16817], sense 5'-CCA GAC AAC AAA CAC AAC CAACC-3', antisense 5'-GCG TTC CCT TCT GTG TCT TCT TC-3', 173 bp; β-actin, sense 5'-AGA CCT CTA TGC CAA CAC AGT GCT G-3', and antisense 5'-TCA TCG TAC TCC TGC TTG CTG A-3', 218 bp.
One-step, real-time quantitative RT-PCR was carried out with a LightCycler instrument (Roche, Mannheim, Germany) by using the LightCycler SYBR Green I RNA Master Kit (Roche, Mannheim, Germany). All reactions were conducted in duplicate. Negative control was performed with sterile purified deionized water. Each cycle of PCR included denaturation at 95°C for 5 seconds, primers annealing at 62°C for 5 seconds, and a final extension at 72°C for 12 seconds. The fluorescence of each sample was measured at 5°C below the melting temperatures (Tms) to eliminate background fluorescence due to primer-dimer . Results were analyzed with LightCycler Software version 3.5 by using the second derivative maximum method to set the CT. E was calculated using the equation E = 10(-1/slope) [36–38]. Agarose gel electrophoresis analyses were also performed to verify whether the amplified product corresponded to the size predicted for gene-specific product.
Relative quantification was carried out with the Relative Expression Software Tool (REST, Roche, Mannheim, Germany). Because the expression level of the β-actin gene was constant regardless of lead exposure , relative qualification was presented by means of normalization with the β-actin gene. Relative and normalized expression ratios (R) were calculated on the basis of the median of the performed duplicates and computed according to the following equation: R = Etargetexp(ΔCTtarget)/Erefexp(ΔCTref) [29, 36, 37].
Wilcoxon test was used in the analyses . The variations in mGluR3 and mGluR7 expression were compared using coefficients of variability and the Wilcoxon two group test. Blood lead levels and hippocampal lead levels were analyzed with one-way analysis of variance (ANOVA). In the MWM task, distance traveled (cm) and escape latency were the principal measures to evaluate the performance of the rats during acquisition training. The baselines of pretraining latency and swimming distance of two groups were analyzed with one-way ANOVA. Because the experimental design involves both a between-subjects factor (lead dose condition) and a within-subjects factor (days), repeated measures ANOVA was performed. Data are presented as mean ± SD and the level of significance is P < 0.05 (two tailed). All statistical evaluations were performed using standard statistical software (SAS Institute Inc., Cary, NC, USA).