Impact of Maternal Iron Restriction on Offspring function that would also affect the general neural output [64,65,66,67]. To determine the impact of the early maternal ID on the CNS development of the offspring, we conducted an ABR analysis of the ID and normal offspring at P40–45 days, a time point at which the auditory system is fully developed. As shown in Figure 3A, ABR latency analysis revealed significantly increased P2-P1 interpeak latencies in ID offspring compared to CTLs at all frequencies examined, ranging from 0.2560.18 ms to 0.496 0.034 ms (* p,0.0001). When we compared the interpeak latency values of the individual animals in the ID group to control animals, it was apparent that iron deficient animals showed a more pronounced variability across all animals compared to controls (Figure 3B). We also analyzed the interpeak latency of P4-P1 to determine whether the impairment affects other aspects of auditory information processes detectable by ABR analysis. As shown in Figure 3C, interpeak latencies P4-P1 were also significantly increased in the ID group. In order to exclude possible defects of inner and outer hair cells that would influence the ABR measurements, we also conducted a DPOAE assess- ment. As shown in Figure 3D, DPOAE amplitude values of ID group were indistinguishable from the control group at all frequencies which strongly suggested normal hair cell function. Wave morphology was not different compared to CTL and hearing thresholds were not elevated in ID animals (data not shown). Taken together, our data show that animals exposed to ID during gestation develop an IDA that is associated with a significant defect in neural function as determined by ABR latency analysis. Delayed onset of gestational iron restriction does not prevent the development of IDA in the offspring The dietary regimen used so far in our study (ID -2wks) was selected to model a pre-pregnancy state that is defined by Figure 3. Auditory Brainstem Responses (ABR) measurement revealed decreased conduction velocities in the offspring born to iron deficient dams. Female Sprague-Dawley rats were provided with the iron-deficient diet two weeks before conception and remained on this diet throughout pregnancy and lactation. Rat pups born to these dams were maintained on iron-deficient diets until testing at postnatal day 45 (P45). Cochlear function was also assessed for all animals (CTL and ID -2wks groups) at ,P45. A. The ABR (P2-P1) interpeak latencies recorded from ID -2wks (red bars) animals showed a highly significant increase compared to CTL (black bars) at all frequencies tested. Auditory nerve function was analyzed by measuring the Auditory Brainstem Responses (ABR) to tone pips at 5 log-spaced frequencies from 5.6 to 24.4 kHz (at 70 dB sound pressure level- SPL). ABR peaks and troughs were identified using a semi-automated tool and verified by a trained observer. Latencies were calculated from onset of stimulus and the (P2-P1) interpeak latency was computed; Mean 6 SEM, p,0.0001). B. The ABR interpeak latencies displayed increased variability in the ID -2wks animals (red circles) compared to CTL (open circles) and the mean value was increased in iron deficient animals. C. The ABR (P4-P1) interpeak latencies recorded from ID -2wks (red hatched bars) animals were significantly prolonged compared to CTL (black bars) at all frequencies tested (Mean 6 SEM, p,0.0001). D. The ID dietary regimen was not associated with outer hair cell damage or cochlear dysfunction as revealed by Distortion-Product of Otoacoustic Emissions (DPOAE) analysis. There was no significant difference between control (black bars) and iron-deficient rats (red bars) in DPOAE amplitudes. (For A–D: n = 35 CTL rats from 5 separate litters and n = 15 ID -2wks rats from 3 separate litters; Mean 6 SEM). doi:10.1371/journal.pone.0017483.g003 PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17483