suboptimal tissue iron concentrations of the dam prior to conception that does not result in a stage of severe maternal IDA by the time the pups are born. While this dietary regimen led to neural impairment in the offspring, the development of IDA in the offspring, lack of weight gain, and lower core body temperature were all factors that could have contributed to the ABR latency defect. In order to begin to better define which of these factors might play a defining role to the development of neural impairment in the offspring, we changed the onset of the timing of the iron restriction along specific gestational windows. As outlined in Figure 4, we generated three additional groups of rats in which the iron-deficient diet (2–6 mg Fe/g) was introduced at the beginning of pregnancy (ID E0), and at the beginning of the 2nd (ID E7) or the 3rd (ID E14) trimester. All experimental groups remained on the iron- deficient diet during lactation and after weaning until the pups reached postnatal day 45, when the animals were subjected to hematological analysis along with weight and temperature measurements followed by an ABR measurement as outlined before. Control group (CTL) consisted of age-matched rats whose dams were maintained on a control diet which contains 240 mg Fe/g and is identical to the iron deficient diet in all other ingredients. As shown in Figure 5A, the hematological analysis revealed that the offspring from all the dietary groups had established severe anemia by the time of the ABR measurement at P45. All groups showed a significant reduction in hematocrit values, hemoglobin concentrations, red blood cell counts (RBC), mean corpuscular volumes (MCV) and serum iron concentrations relative to the CTL group. Other parameters that could impact neural function were also affected but to various degrees depending on the time when the ID diet was introduced to the pregnant dam. As shown in Figure 5B, all iron deficient groups showed a significant decrease in body weight, while only the ID- 2wks offspring presented with a significant decrease in core body temperature compared to controls. Core body temperatures were not affected when the iron restriction started at embryonic day 0 (ID E0), day 7 (ID E7) or day 14 (ID E14) and were comparable to control values (Figure 5C). Impact of Maternal Iron Restriction on Offspring The establishment of a neural impairment in the offspring is dependent on the onset of the iron restriction during pregnancy defining a window of vulnerability To monitor whether the delayed onset of the maternal iron restriction changes the functional neural impairments in the offspring we had detected in the ID-2wks dietary group, we performed ABR testing of the P45 offspring as outlined before. In all three groups DPOAE analysis revealed again that outer hair cell function was comparable to control animals (data not shown). ABR tests in the ID E0 and ID E7 offspring showed prolonged P2-P1 interpeak latencies ranging from 0.1760.06 ms up to 0.3960.05 ms compared to CTL values. ABR latency responses at the highest frequency (24 kHz) did not reach statistically significant levels in the ID E7 group (Figure 6A and 6B). There was also a decreased severity of the ABR defects with a later occurring insult. Surprisingly, when pregnant females were exposed to iron restriction at the beginning of the third trimester (ID E14), and the offspring was subjected to ABR testing, we did not find any statistically significant interpeak latency differences in the ID E14 group compared to the control group across all frequencies (Figure 6C). We also analyzed the P4-P1 interpeak latency differences to determine whether the impairment affects the entire brainstem auditory response in such a manner that changes observed at later peaks are even greater in magnitude than those detected through analysis of the P2-P1 interpeak latencies. As shown in Figures 6 and 7, ID animals showed a relatively larger increase than CTL when comparing the P4-P1 latencies (0.25–0.9 ms) to the P2-P1 latencies (0.5 ms - .1.5 ms). While all P4-P1 latencies, except one, were increased by over 1 ms in the ID E0 group (Figure 7A), the increase in latency was less severe in the ID E7 group. Nonetheless, even in this group, the alterations seen in the P4-P1 analysis were greater than in the P2-P1 analysis and also were significantly increased compared to control animals at all frequencies tested (Figure 7B). In contrast, and consistent with the measurements of P2-P1 changes, the ID E14 animals did not show a significant difference in P4-P1 interpeak latencies compared to the control group (Figure 7C). Taken together, our data show that dietary regimens that are not severe enough to cause a transition of maternal ID to severe IDA Figure 4. Experimental design of the dietary regimens. To determine the restricted developmental window in which maternal iron restriction impacts fetal development, we established one control (CTL) group and four experimental groups of iron deficient animals: CTL group: animals received control iron diet (240 mg Fe/g food) throughout the experiment; ID-2wks group: animals began the iron-deficient diet (2–6 mg Fe/g food) two weeks before conception; ID E0 group: the iron-deficient diet was introduced the day of plug detection (E0); ID E7 group: animals were placed on the iron-deficient diet on embryonic day 7 (E7); ID E14 group: animals were fed the iron-deficient diet starting on embryonic day 14 (E14); All pregnant Sprague–Dawley female rats in different diet regimens groups were maintained on the respective diet from the assigned start point throughout pregnancy and lactation. After weaning, the pups were maintained on iron-deficient diet until the day of auditory testing (postnatal day 45). doi:10.1371/journal.pone.0017483.g004 PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17483