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© 2008 American Society for Nutrition J. Nutr. 138:931-937, May 2008

Nutrient Requirements and Optimal Nutrition

Perinatal Iron Deficiency Affects Locomotor Behavior and Water Maze Performance in Adult Male and Female Rats1,2

Stephane L. Bourque, Umar Iqbal, James N. Reynolds, Michael A. Adams and Kanji Nakatsu*

Department of Pharmacology and Toxicology, Queen's University, Kingston, Canada K7L 3N6

* To whom correspondence should be addressed. E-mail: nakatsuk{at}queensu.ca.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Methods
 Results
 Discussion
 LITERATURE CITED
 
Iron deficiency during early growth and development adversely affects multiple facets of cognition and behavior in adult rats. The purpose of this study was to assess the nature of the learning and locomotor behavioral deficits observed in male and female rats in the absence of depressed brain iron levels at the time of testing. Adult female Wistar rats were fed either an iron-enriched diet (>225 mg/kg Fe) or an iron-restricted diet (3 mg/kg Fe) for 2 wk prior to and throughout gestation, and a nonpurified diet (270 mg/kg Fe) thereafter. Open-field (OF) and Morris water maze (MWM) testing began when the offspring reached early adulthood (12 wk). At birth, perinatal iron-deficient (PID) offspring had reduced (P < 0.001) hematocrits (–33%), liver iron stores (–83%), and brain iron concentrations (–38%) compared with controls. Although there were no differences in iron status in adults, the PID males and females exhibited reduced OF exploratory behavior, albeit only PID males had an aversion to the center of the apparatus (2.5 vs. 6.9% in controls, P < 0.001). Additionally, PID males required greater path lengths to reach the hidden platform in the MWM, had reduced spatial bias for the target quadrant, and had a tendency for greater thigmotactic behavior in the probe trials (16.5 vs. 13.0% in controls; P = 0.06). PID females had slower swim speeds in all testing phases (–6.2%; P < 0.001). These results suggest that PID has detrimental programming effects in both male and female rats, although the behaviors suggest different mechanisms may be involved in each sex.


   Introduction
TOP
 ABSTRACT
 Introduction
Methods
 Results
 Discussion
 LITERATURE CITED
 
Iron deficiency (ID)3 is the most widespread single nutritional disorder in the world. It is the only global nutritional health risk that is highly prevalent in populations of both developing and industrialized nations (1,2). Although ID is common among the general population, a preponderance of pregnant women are affected by this condition, due in part to an expansion in blood volume and increased erythropoeisis, as well as increased iron demands from the fetal-placental unit (3). According to conservative estimates, more than half of pregnant women in developing countries (4) and between 20 and 40% of women in western industrialized countries are anemic, due mainly to ID (4).

ID during and shortly after pregnancy can affect multiple facets of cognition and behavior in the offspring, including learning, conduct, motivation, and attentiveness. These deficits remain after the insult has been corrected (i.e., despite subsequent iron replenishment). This "developmental programming" of cognition and behavior has been reported in intervention and nonintervention studies involving newborn infants and young school children throughout a number of geographical regions [for review, see (5)]. Most of these studies were epidemiological, and therefore of limited use in elucidating the specific mechanisms involved in this model of developmental plasticity. Consequently, the use of rodent models to study perinatal iron deficiency (PID) has been quintessential in characterizing the biochemical and neurophysiologic changes that underlie these cognitive and behavioral deficits.

Independent from the programming effects described above, ID can induce cognitive and behavioral deficits in adult rats, as well as humans (6). Depletion of brain iron appears to affect numerous neurotransmitter systems and may be linked to reduced learning capacity and locomotor activity (7,8). Interestingly, in certain studies, these effects persist even when the ID is corrected (7). Thus, in some cases, it may be difficult to distinguish between the programming effects of developmental ID and the effects of acute brain iron deprivation. Studies in which iron is restricted during gestation and through the subsequent postnatal period may induce, in addition to the fetal-programmed behavioral effects, persistent decreases in brain iron that also influence cognition and behavior. In studies by Felt et al. (9) and Kwik-Uribe et al. (10), ID was induced in the mother during gestation and continued throughout the postnatal phase, resulting in persistent decreases in brain iron at the time of behavioral testing. In a more recent study, Felt et al. (11) demonstrated that maternal ID throughout pregnancy and lactation resulted in persistent neurophysiological changes in the offspring at 12 wk of age, despite brain iron levels being normalized at that time. Behavioral deficits were also observed in the offspring of iron-deficient mothers at 5 wk of age, although brain iron levels at that time were not reported (11).

To further investigate the nature of the behavioral deficits previously reported in adult PID rats, we conducted spontaneous locomotor and Morris water maze (MWM) testing in the offspring of dams that were iron-restricted during the gestational period. Our overall objective was to determine whether behavioral and cognitive differences persisted in adulthood in the absence of changes in brain iron content. Furthermore, previous work done with PID and learning in the MWM has largely focused on males. Thus, the purpose of this study was to determine the affect of PID on exploratory behavior and MWM performance in both adult male and female rats.


   Methods
TOP
 ABSTRACT
 Introduction
 Methods
Results
 Discussion
 LITERATURE CITED
 
Animals and diets

The experimental protocols described herein were approved by the Queen's University Animal Care Committee. Sixteen 8-wk-old female Wistar rats were purchased from Charles River and housed separately in plastic cages in the Queen's University Animal Care Facility, which maintained a 12-h light/12-h dark cycle and an ambient temperature of 23°C. All rats were given 1 wk to acclimatize to the novel surroundings before beginning treatment.

All purified diets (Research Diets Inc.) were based on the AIN-93G diet, which has been described elsewhere (12). The purified diets were identical in composition, with the exception of added ferric citrate, which was adjusted to obtain the following iron concentrations: 225 mg/kg in the control diet (D03072504) and 3 mg/kg in the low-iron diet (D03072501). The nonpurified diet (Lab Diets; 5001), which has been described elsewhere (13), contained 270 mg/kg iron. All rats consumed food and water ad libitum.

Following the acclimatization period, 8 female rats were randomly chosen and assigned to the low-iron diet, and 4 different females were randomly selected and assigned to the purified control diet. The remaining 4 dams were fed the nonpurified diet. After 2 wk on their respective diets, all dams were mated to 10-wk-old male Wistar rats that were fed the nonpurified diet. All rats were fed their respective diets throughout pregnancy. Within 8 h after giving birth, all dams were given the nonpurified control diet. Food consumption and body weights (BW) of offspring were monitored every 2 d until postnatal day (PD) 35 and weekly thereafter. At 24-h postpartum, litters were culled to 8 rats (4 males and 4 females) to standardize postnatal conditions. At weaning (PD21), the remaining pups were separated from their mothers and given the nonpurified diet. Behavioral testing began when rats reached 12 wk of age. Adult offspring were killed at 24 wk of age by pentobarbitol overdose and exsanguination.

Behavioral testing

All behavioral testing was done between 1200 h and 1600 h.

    Spontaneous locomotor activity. At ~12 wk, adult offspring were tested for spontaneous locomotor activity as previously described (14). Rats were left in the open-field (OF) apparatus for a total of 20 min. Outcome measures included distance traveled, distance traveled in the center of the apparatus, time spent at rest, and time spent hyperactive (defined as movement faster than 15 cm/s).

    MWM. Swim path, speed, and distance to reach platform were obtained using the Videomot computer program (TSE Systems Inc.) and recorded for off-line analysis. Escape latency was also measured and analyzed; however, distance to platform (path length) was used instead for statistical comparisons and is presented in this study because it is considered to be a more reliable indicator of overall performance (15).

Day 1: visible platform phase A black, circular escape platform (12-cm diameter) was placed in the center of a quadrant of a circular pool (1.8-m diameter), filled with water (maintained at 26 ± 1°C) and made opaque with nontoxic white paint. The platform protruded 1–2 cm above the water's surface. The top of the visible platform (as well as the hidden platform, see below) was covered with a fine rubber mesh to facilitate the rats' climbing onto it.

Rats were trained in blocks, each block consisting of 4 consecutive trials. Each trial lasted a maximum of 60 s. If the rat could not locate the platform in 60 s, the experimenter showed the rat the location by placing it on the platform. All rats remained on the platform for 15 s between each trial. A different release point (from the intersection of each of the 4 quadrants near the wall) was used for each trial. Rats underwent 2 blocks of 4 consecutive trials, separated by a resting period of 5 min. After each block, rats were towel-dried and returned to their cages, which were warmed by a heat lamp.

Day 2 to 5: hidden platform and probe trial phase A white circular escape platform (12-cm diameter) was submerged 1–2 cm below the surface of the water in the same pool. The location of the hidden platform was the middle of the quadrant adjacent to the quadrant where the visible platform had been previously located. The hidden platform did not change locations from day 2 to day 5. The same testing pattern was used for the hidden platform phase as was used for the visible platform phase with the following exception: following the 2 blocks of testing, the hidden platform was removed, and after a 5 min rest period, rats were placed in the center of pool and released; this probe trial lasted 60 s.

Tissue collection and analysis

Pups that were culled at 24 h were killed by decapitation, and blood samples were collected in heparinized microcapillary tubes. The tubes were then centrifuged (11,500 x g; 15 min) and packed cell volume was determined as a measure of hematocrit (Hct). Organs were excised into ice-cold saline, cleaned of extraneous connective tissue, blotted dry, weighed, snap frozen in liquid nitrogen, and stored at –80°C until processing. Adult rats (24 wk) were first anesthetized with sodium pentobarbital (90 mg · kg BW–1, intraperitoneally). Blood samples were subsequently obtained from the inferior vena cava, and Hct were determined as described above. Adult rats were then perfused through the aorta with ice-cold saline using a peristaltic pump to remove blood, and tissues were collected as described above.

For tissue iron analysis, frozen tissues were thawed and dried at 65°C for a minimum of 72 h. Dried tissues were reduced to ash in a muffle furnace at 200°C for 2 h and at 500°C for 18 h. The ash was then dissolved in hot, concentrated nitric acid and then diluted with distilled and deionized water. Iron concentrations were determined using a SpectrAA-20 flame atomic absorption spectrophotometer (Varian Canada). Bovine liver standards (National Institute of Standards, 1577b) and blanks were included as quality controls.

Statistical analysis

All offspring data from the 3 diet groups were initially compared by 2-way ANOVA. There were no behavioral or biochemical differences between offspring born to dams fed the control purified diet and control nonpurified diets, so these groups were combined for all subsequent statistical analyses. BW, brain weights, Hct, and tissue iron levels were analyzed by 2-way ANOVA (by treatment and sex) with Student's t test as post hoc analysis. Spontaneous locomotor activity, as well as MWM swim speeds and thigmotactic behavior, were analyzed by repeated measures 2-way ANOVA (by treatment and time), with Student's t test (for between treatment group comparison), or 1-way ANOVA with Neuman-Keul's (for between time comparisons). MWM path lengths and spatial bias data were analyzed by repeated measures ANOVA with Dunnett's post hoc test (for comparisons of the first block and subsequent blocks within a particular testing phase; e.g., blocks H2-H8 were compared with block H1). This comparison was done because a 2-way ANOVA using all the data from a testing phase would mask treatment effects that occur only during particular intervals within that testing phase (i.e., delayed acquisition). No comparisons were made between males and females to identify treatment effects in behavior, because of the extensive body of work showing that males perform substantially better than females in the water maze task [for review see (16)]. Data were analyzed using GraphPad Prism software (Version 5). Offspring data from each litter were pooled, and each n value refers to number of litters. Data were analyzed for homogeneity of variance using Bartlett's test for samples of unequal size; all data sets compared were found to be homogeneous. Results are presented as means ± SEM. Differences with P < 0.05 were considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Methods
 Results
Discussion
 LITERATURE CITED
 
    Parental and offspring outcomes. The quantities of food ingested in the period before and throughout pregnancy did not differ among treatment groups (data not shown). Maternal ID did not affect the pregnancy success rate (87.5% in both groups), or the number of pups per litter, which was 14.3 ± 0.9 in the ID group and 15.4 ± 0.5 in controls. However, the low-iron treatment markedly affected iron status of the offspring at birth (Table 1). Hct were 32% lower (P < 0.001), liver iron was 83% lower (P < 0.001), and brain iron was 38% lower (P < 0.001) in the PID offspring than in controls. There were no sex differences or treatment-by-sex interactions for any of these variables. The treatments did not affect the Hct, brain, or liver iron concentrations in 24-wk-old male or female adult offspring. However, the liver iron concentration was greater in females (P < 0.001) (Table 1).


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  		TABLE 1 Iron status of control and PID offspring at 24 h and 24 wk of age1

 
PID male BW were lower than controls between PD5 and PD15 (P < 0.05) (Fig. 1A); these data are also expressed as a fraction of control male BW (Fig. 1A, inset). PID females had reduced BW compared with controls between PD1 and PD9 (P < 0.05) (Fig. 1B); these data are also expressed as a fraction of control BW (Fig. 1B, inset). Sex-based differences in BW were present from PD29 (P < 0.05) onward. No treatment-by-sex interactions were observed on any postnatal day. There were no treatment effects or sex differences in brain weights at birth (data not shown). Adult brain weights did not differ between PID and control rats at the end of the study, but males had greater brain weights than females, as expected (P < 0.001).


Figure 1
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  		FIGURE 1  Effect of maternal dietary iron treatment prior to and throughout gestation on BW of male (A) and female (B) offspring of Wistar rats between PD1 and PD35. Diets administered to dams of control and PID offspring contained > 225 mg/kg Fe and 3 mg/kg Fe, respectively. Data are also expressed as percentage of control BW (insets). Data are means ± SEM, n = 6. *Different from controls on that day, P < 0.05. Males differed from females beginning on PD29, P < 0.05.

 
    Locomotor behavioral outcomes. Adult males and females from the PID group had markedly decreased spontaneous locomotor activity and exploratory behavior in the OF compared with adult controls (Fig. 2). PID males, compared with control males, traveled less total distance (P < 0.01) (Fig. 2A) and had fewer rearings (P < 0.001) (Fig. 2B). They also spent more time at rest (P < 0.01) and spent less time hyperactive (P < 0.05) (data not shown). Additionally, PID males spent less time in the center of the apparatus than control offspring (P < 0.01) (Fig. 2C). PID females, in turn, traveled less total distance (P < 0.05) (Fig. 2A), had fewer rearings (P < 0.01) (Fig. 2B), spent less time hyperactive (P < 0.05), and tended to spend more time at rest (P < 0.07). In contrast, low-iron treatment did not affect the total amount of time females spent in the center of the apparatus (Fig. 2C). Total distances traveled, percent time hyperactive, and number of rearings in all males and females decreased as time progressed (P < 0.001 for all variables). There were no treatment-by-time interactions for any of the parameters analyzed in either male or female offspring.


Figure 2
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  		FIGURE 2  Effect of maternal dietary iron treatment prior to and throughout gestation on distance traveled (A), number of rearings (B), and total time spent in center of an OF apparatus of adult male and female offspring of Wistar rats. Diets administered to dams of control and PID offspring contained >225 mg/kg Fe and 3 mg/kg Fe, respectively. Data are means ± SEM, n = 5–6. *Different from controls at that time interval, P < 0.05. Distance traveled and number of rearings were affected by time and treatment in both sexes, P < 0.001. Percent time spent in the center of the OF apparatus differed between treatment groups only in the males, P < 0.001.

 
    MWM outcomes. PID treatment had no affect on path length to reach the escape platform during the pretraining/visible platform phase of testing (day 1) in either the males or females (data not shown). In the hidden-platform phase, control males showed immediate improvement in path length to reach the platform compared with block H1 (P < 0.001) (Fig. 3A). In contrast, PID males did not show improvement until block H4 (day 2 of the hidden-platform phase) (P < 0.01) (Fig. 3B). Whereas PID females showed improvement in performance as early as block H2 (P < 0.05) (Fig. 3D), performance in the control females did not improve until block H3 (P < 0.05) (Fig. 3D), although performance tended (P < 0.07) to improve in block H2.


Figure 3
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  		FIGURE 3  Effect of maternal dietary iron treatment prior to and throughout gestation on path lengths to reach the hidden platform in a MWM of adult control male (A), PID male (B), control female (C), and PID female (D) offspring of Wistar rats. Diets administered to dams of control and PID offspring contained >225 mg/kg Fe and 3 mg/kg Fe, respectively. Shaded areas in the background represent different days of testing. Data are means ± SEM, n = 6. *Different from block H1 in the same group, P < 0.05.

 
Control males demonstrated improvement in spatial bias for the target quadrant (defined as the propensity to remain in the quadrant wherein the platform is located) on block H2 (P < 0.05), and remained significantly elevated thereafter (Fig. 4A). In contrast, PID males did not improve until block H5 (day 3 of the hidden platform phase) (P < 0.05) (Fig. 4B). Control females showed improvement in spatial bias performance in block H5 (P < 0.05) (Fig. 4C), whereas PID females showed signs of improved performance in block H6 (P < 0.01) (Fig. 4D). Spatial bias between treatment groups during the visible platform phase or probe trials did not differ (data not shown).


Figure 4
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  		FIGURE 4  Effect of maternal dietary iron treatment prior to and throughout gestation on spatial bias for the target quadrant (expressed as a percentage of total distance traveled) in the hidden platform phase of a MWM of adult control male (A), PID male (B), control female (C), and PID female (D), offspring of Wistar rats. Diets administered to dams of control and PID offspring contained >225 mg/kg Fe and 3 mg/kg Fe, respectively. Shaded areas in the background represent different days of testing. Data are means ± SEM, n = 6. *Different from block H1 in the same group, P < 0.05.

 
Males in the PID group tended (P < 0.06) to travel an overall greater proportional distance in the thigmotaxis zone, defined as the outer tenth of the perimeter of the pool (16.5 ± 2.5%), compared with control rats (13.0 ± 2.1%) during the probe trials. There was also a treatment-by-time interaction in the males (P < 0.05), but there were no time effects. Conversely, thigmotactic behavior in the probe trials did not differ between PID females (21.6 ± 3.8%) and female controls (21.5 ± 4.3%). There was a significant effect of time (P < 0.001) in the females, although there was no treatment-by-time interaction. Thigmotactic behavior was not assessed in the visible or hidden platform phases of the MWM task, because there are inherent limitations of this component of the analysis (the release point is within the thigmotactic boundary, and therefore inappropriately affects the proportion of distance traveled in that region).

Overall, swim speeds did not differ between the PID (29.6 ± 0.3 cm/s) and control (29.9 ± 0.7 cm/s) males throughout MWM testing. In contrast, PID females swam slower (31.0 ± 0.5 cm/s) than control females (33.0 ± 0.5 cm/s) (P < 0.001). There were also treatment-by-time interactions in the swim speeds of both males (P < 0.05) and females (P < 0.01).


   Discussion
TOP
 ABSTRACT
 Introduction
 Methods
 Results
 Discussion
LITERATURE CITED
 
The major differences in the offspring after maternal iron-restriction during pregnancy included marked reductions in hematocrits and tissue iron levels at birth and overall reduced exploratory behavior in an OF apparatus in adulthood. Additionally, PID males, but not females, tended to avoid the center of the OF apparatus and had overall poorer performance in the MWM than control males. PID females in turn, had markedly slower swim speeds than controls. Taken together, these results suggest that PID induces long-term changes in behavior despite subsequent iron replenishment after birth and that male and female rats may have different susceptibilities to these adverse programming effects.

Our treatment paradigm was intended to restrict iron in the gestational and immediate postnatal period. Dams were given a high-iron diet immediately after giving birth, which replenished maternal Hct and hemoglobin levels within 7 d (17). However, whether this was sufficient time to replenish the iron in maternal milk is not known (18). Consequently, the supply of iron to the pups may have remained inadequate throughout the nursing period, despite recovered hematological indices in the mothers. Indeed, in this study, PID rats' weights continued to drop until postnatal week 2, indicating that nutrition during lactation was inadequate for optimal growth (Fig. 1). Moreover, Beard et al. (19) recently showed that exploratory deficits in an OF apparatus are reversed when ID pups are out-fostered to control fed dams within days of birth, suggesting that iron restriction during the postnatal period is important for certain behavioral deficits in this model of developmental programming. Nevertheless, our treatment regimen produced marked reductions in tissue iron concentrations and hematocrits at birth that did not persist in adulthood, a stage of maturity at which behavioral abnormalities were observed. That is, behavioral differences were evident in the PID adult male and female rats despite the lack of PID affect on adult tissue levels of iron. Thus, these data reveal the programming effects of PID, and not changes associated with reduced brain iron per se, an alteration in adults which is known to cause impaired task acquisition in behavioral studies (7).

In the OF apparatus, both PID males and females exhibited reduced exploratory behavior. However, only PID males spent less time in the center of the OF apparatus than their respective controls, a behavior that has been interpreted as a sign of anxiety and fear (20,21). The finding that the PID females did not display the same aversion to the center of the OF apparatus suggests that their reduced exploratory behavior is not a consequence of an elevated response to stress, but may be due to motor deficits instead. This hypothesis is supported by the observation that PID females had slower swim speeds than controls in all phases of the MWM.

The MWM has been important to the study of behavioral neuroscience, particularly as an indicator of altered function of the hippocampus, among other brain regions (22). Improvement in the MWM requires numerous integrated processes, including task acquisition, spatial navigation, and implementation of search strategies, in addition to memory consolidation (16). Thus, disturbances in any of these processes could potentially affect performance. Consequently, analysis of the behavioral patterns in the MWM can provide valuable information about the underlying functional deficits. In this study, we used a pretraining phase, involving a visible platform task, to habituate the rats to the novel water environment. Lack of this pretraining has been shown to mask subtle differences in treatment groups (23). That is, habituation or pretraining to the water maze is important in controlling for other factors that can be misinterpreted as learning or memory deficits (e.g., learning to swim away from the wall to escape the pool). Furthermore, having a visible platform phase allows for identification of sensorimotor deficits that could affect performance in the MWM (16). This concept is important given that the present data suggest that there are motor deficits in the PID females, and both Felt et al. (11) and Beard et al. (24) reported that PID rats show signs of sensorimotor deficits using the forelimb placement test.

PID males displayed less improvement in reaching the hidden platform than control males in the first 2 d of the invisible platform phase of testing: a finding that indicates delayed task acquisition. This is further supported by the finding that PID males displayed no significant improvement in spatial bias toward the target quadrant until day 3 of the invisible platform phase compared with the control males; this analysis controls for the element of chance encounters with the platform. Moreover, we showed previously that the pretraining/visible platform phase can mask the degree of impairment in a different model of fetal insult (25), despite being critically important in MWM testing as stated above. Therefore, the true extent of impairment might be revealed by a more complex MWM protocol.

The delayed task acquisition observed in the male PID offspring, much like the differences observed in the OF, may be related to increased anxiety and fearfulness. Stress has been shown to contribute to learning and memory impairment in the MWM (23,26,27). This notion is further supported by the finding that PID males had increased thigmotaxis (wall-seeking behavior) during the probe trials of the MWM. Increased thigmotactic behavior has also been interpreted as a sign of anxiety and fear, which would likely be manifested when the platform is removed (i.e., probe trials). Indeed, altered hypothalamic-pituitary-adrenal (HPA) axis responsiveness has been reported in a PID model of developmental plasticity (28).

The underlying mechanisms that are responsible for the locomotor deficits in the female, and the proposed elevated state of anxiety in the PID male offspring are beyond the scope of this study. Nevertheless, numerous alterations in central neurotransmitter signaling have been implicated (24,29–33), in addition to altered growth and development of brain regions involved in influencing behavior (32–34). Interestingly, despite evidence showing an apparent altered responsiveness of the HPA axis in PID males (i.e., increased anxiety and fear), these same studies indicate that there are no long-term differences in circulating levels of corticotropin or corticosterone (28). Enhanced HPA axis responses may result from an increased density of receptors without a change in hormone levels. Indeed, we have previously shown that increased glucocorticoid receptor densities in the hippocampus produce enhanced glutamate release in response to a standard dose of dexamethasone in a guinea pig model of chronic prenatal ethanol exposed (35).

In summary, we have characterized the behavioral outcomes in a model of developmental plasticity due to PID to provide a foundation for ongoing mechanistic studies. An important concept emphasized herein is that changes in maternal iron intake during pregnancy can affect long-term behavior in the adult offspring, even without persistent differences in brain iron levels in adulthood. Adverse programming effects have been associated with a number of macro- and micronutrient deficiencies; however, the worldwide prevalence of ID in humans, especially in pregnant women, makes this an especially relevant form of developmental insult to study.


   FOOTNOTES
 
1 Supported by the Canadian Institutes of Health Research (MOP-68993, MOP-74521) and the Bickell Foundation of Canada. S.L.B was supported by the Canadian Hypertension Society/Pfizer/Canadian Institutes of Health Research Research and Development Doctoral Fellowship. Back

2 Author disclosures: S. L. Bourque, U. Iqbal, J. N. Reynolds, M. A. Adams, and K. Nakatsu, no conflicts of interest. Back

3 Abbreviations used: BW, body weight; Hct, hematocrit; HPA, hypothalamic pituitary adrenal; ID, iron deficiency; MWM, Morris water maze; OF, open field; PID, perinatal iron deficiency; PD, postnatal day. Back

Manuscript received 22 December 2007. Initial review completed 31 December 2007. Revision accepted 25 February 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Methods
 Results
 Discussion
 LITERATURE CITED
 

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