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Departments of
*
Nutrition and
Internal Medicine, University of California, Davis, CA 95616
2To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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KEY WORDS: • iron • iron deficiency • development • brain iron • behavior • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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). According to data
from the third National Health and Nutrition Examination Survey (NHANES III, 1988–1994) (Alaimo et al. 1994
), the prevalence of
IDA in children 1–2 y old in the United States was 3% and the
prevalence of iron deficiency without anemia was 9%. Nationwide, this
prevalence represents ~240,000 and 700,000 toddlers, respectively.
Furthermore, the prevalence of iron deficiency among women of
child-bearing age (20–49 y) has been reported to range from 8 to
20% (Looker et al. 1997
), demonstrating that infants
may be at risk for marginal-to-low iron status not only as a result of
postnatal factors, but also as a result of limited prenatal iron
sources.
Iron deficiency is reported to have an effect on cognition [reviewed
in Pollitt (1993)
]. Several epidemiologic studies
suggest that for anemic children, iron supplementation is correlated
with improved performance outcomes, including attention and learning
(Idjradinata and Pollitt 1993
, Pollitt et al. 1986
, Seshadri and Gopaldas 1989
,
Soewondo et al. 1989
), as well as motor development
(Idjradinata and Pollitt 1993
). A study of nonanemic
adolescent girls also demonstrated a positive correlation between iron
supplementation and improved verbal learning and memory (Bruner et al. 1996
). That study suggested that not only can improved
iron status affect cognitive ability well past the first few years of
life, but also that improvements in cognitive skills can be seen
without significant changes in hematologic parameters. The importance
of adequate iron status during early development is underscored further
by a recent study that demonstrated that early childhood anemia was an
independent risk factor associated with mild-to-moderate mental
retardation later in life (Hurtado et al. 1999
). Given
the number of women and children in the United States affected by iron
deficiency and IDA, it is important to recognize how both prenatal and
postnatal iron nutrition affect early infant health and development.
Numerous animal studies have been done to study the behavioral effect
of iron deficiency. Similar to what has been seen in human
epidemiologic work, animal models demonstrate that iron deficiency
anemia can cause altered activity patterns (Edgerton 1972
) and responsiveness (Weinberg et al. 1979
and 1980
), as well as altered learning (Massaro and Widmayer 1981
). The developing brain can be particularly sensitive to
changes in iron status not only because of its rapid growth and
development, but also because certain developmental events occur in a
small window of time during which the timing and duration of a nutrient
insult can have a significant, long-term effect on brain maturation
and function [reviewed in Morgane et al. (1993)
]. This
concept is supported in a study by Felt and Lozoff (1996)
. Despite several weeks of iron supplementation, which
resulted in the normalization of plasma and liver iron concentrations,
rats that were made anemic during select periods of gestation and early
lactation experienced persistent behavioral abnormalities and low brain
iron concentrations. Given the prevalence of iron deficiency in infants
and the susceptibility of the brain to nutrient insults during early
development, understanding the biochemical and behavioral effect of
iron deficiency on these processes is critical not only in improving
our understanding of iron’s role in these events, but also in
ameliorating and treating the consequences of the deficiency.
Most animal models have examined the behavioral effect of a
severe iron deficiency; therefore, limited information is available
concerning the physiologic and behavioral consequences of chronic
marginal iron deficiency during early development. We recently
developed a murine model for marginal iron deficiency during early
development. Brain development in mice follows a chronological pattern
similar to that seen in humans (Bayer 1989
,
Morgane et al. 1993
); therefore, this model allows us to
examine the biochemical and behavioral effects of chronic marginal iron
status. Our initial studies demonstrated that mice fed marginal iron
diets through pre- and early postnatal development experienced
persistent changes in grip strength, which were independent of lower
body weights (Kwik-Uribe et al. 1999
). The current
experiment extends the observations of this first study as follows:
1) by incorporating a period of postnatal iron repletion;
2) by reexamining the effects of iron status on grip
strength and auditory startle; and 3) by including a Morris
maze test of learning and memory.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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The animal protocol was in accordance with the Guide for the
Care and Use of Laboratory Animals (NRC 1996
) and was
approved by the University of California, Davis Animal Use and Care
Committee. Virgin female Swiss-Webster mice were purchased from a
commercial supplier (Charles River, Wilmington, MA) at 3 wk of age.
Mice were housed in groups of 2–3 in suspended stainless steel cages
and maintained on a normal 12-h light:dark cycle. All mice were adapted
to a control purified diet containing 75 µg Fe/g diet
(Table 1
) for 1 wk before the onset of the study. After this acclimation period,
the mice were randomly assigned to a diet group and fed this diet
throughout the duration of the study. After 8 wk of consuming the diet,
the females were mated and a successful pregnancy was identified by the
presence of a vaginal plug. On gestation day 17, pregnant mice were
transferred from the stainless steel cages to plastic hanging maternity
cages. Cages were filled with a shallow layer of sawdust shaving and a
small ball of cotton was provided for use as nesting material. When
necessary, litter size was reduced to 8 pups (1:1 male-to-female ratio
when possible) within 1 wk of birth. At weaning [postnatal (PND) d
21], 1 male and 1 female pup from each litter and their corresponding
dam were killed for assessment of iron status. The remaining pups (3
males and 3 females/litter) were separated and same-sex littermates
were housed in suspended stainless steel cages (3 pups/cage). The mice
were earmarked for identification purposes and were assigned to a
treatment group. Housing constraints made individual food intake
measurements for offspring impossible, but food intake was measured
daily on a per cage basis (intake over a 24-h period minus spillage).
Individual weight gain was measured weekly from weaning (PND 21)
through PND 75.
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The purified experimental diets used in this study were based on
egg white as the protein source and contained either 14
µg Fe/g diet (marginal iron diet) or 75
µg Fe/g diet (control iron diet) (Table 1)
. All other
minerals were consistent with current recommendations (NRC 1996
), and mineral concentrations were verified by
inductively coupled plasma spectroscopy (trace scan ICP; ThermoJarrel
Ash, Franklin, MA) before the onset of the study. The marginal iron
diet (14 µg Fe/g diet) used in this study was shown
previously to result in lower iron status without causing significant
changes in hematologic iron markers in adult offspring
(Kwik-Uribe et al. 1999
). All mice consumed deionized
water ad libitum throughout the duration of the study.
Design
Dams. After 1 wk of consuming the purified control diet, weight-matched females were fed either a control (n = 21) or marginal iron (n = 45) diet. To further deplete iron stores, each mouse in the study was subjected to a tail bleed every 3–5 d starting at wk 5 of the study. Blood (100 µL) was collected into heparinized collection tubes at each draw. Depending on the weight of the mouse, 6–9 blood collections were done over the 3-wk draw period; thus the total volume of blood collected represented 45–55% of the animal’s total blood volume. One week after the last tail bleed, mice were mated with males fed a commercial diet (Harlan-Teklad Rodent Chow, Madison, WI). All mice were pregnant within 3 wk of the start of breeding. All females were fed their respective diets from the start of the study through PND 21 of their offspring. Weights of each female were recorded weekly from the start of the study and every 3 d during gestation. To ensure an adequate number of offspring for the study, only dams with 8 or more live pups at PND 21 were considered eligible for the study.
Offspring. Offspring were weaned on PND 21 and assigned to one of three experimental groups. Offspring born to marginal iron females were fed either the marginal iron diet (14 µg Fe/g diet; marginal iron group; n = 16 litters/sex) or were switched to the control diet (75 µg Fe/g diet; replete group; n = 13 litters/sex). Offspring of control dams were fed the control diet (75 µg Fe/g diet; control group; n = 13 litters/sex). Diets were fed from PND 21 through 75.
Neurobehavioral testing
Grip strength and auditory startle.
On the basis of our previous findings, we focused the
neurobehavioral testing in this study to include grip strength and
auditory startle measurements (Kwik-Uribe et al. 1999
).
One male and one female mouse per litter were tested for either grip
strength or auditory startle responsiveness. Once assigned to either
grip or auditory testing, the same pup was tested on PND 30, 45 and 60.
Morris maze.
Beginning on PND 50, a group of males from each experimental group
(n = 10/group) was assigned to Morris maze testing
(Lamberty and Gower 1991
). Because one test may affect
an animal’s ability to perform another test, mice selected for the
Morris maze did not undergo grip or auditory testing. The testing
procedure used was modified to minimize body temperature losses and is
described briefly below.
Testing was done daily beginning at 0800 h, during the mouse’s
light cycle. Before testing, each mouse was removed from his stainless
steel cage ("home cage") and transferred to a plastic cage with a
shallow layer of sawdust ("transport cage"). Mice were housed
individually in the transport cage. Once in the transport cage, the
mice were brought into the testing room in groups of 3–4. To ensure
consistency, testing for each mouse occurred in the same order and
within 1–2 h of the original testing time on d 1 throughout the
duration of maze testing. After the mouse’s initial body temperature
was taken and the black arc (visual cue) and platform were placed in
the appropriate locations in the pool, the mouse was lowered and
released by hand into one of the four quadrants of the water maze. The
mouse was allowed 90 s to find and climb onto the submerged
platform. If the mouse did not find the platform, the mouse was removed
from the water through the same opening from which he was released and
placed onto the submerged platform. After finding the platform or being
placed on the platform by the tester, the mouse remained there for
30 s. The mouse was then removed from the pool, gently dried by
hand with a towel and placed into a warming cage. Because the mouse’s
body temperature can drop 4–9°C during the course of the trial
(unpublished data), each mouse was allowed to warm up between trials
according to a modification of the procedure described by
Middaugh et al. (1996)
. Each mouse in the group of 3–4
mice brought into the room for testing was allowed to complete one
trial through a given quadrant before the next trial was begun; thus
the intertrial interval was 5–10 min for each mouse. Each mouse
completed 4–5 trials daily, with the mouse entering through a
different quadrant for each trial. The quadrant entry order used was
randomized for each testing session.
For the first four consecutive days of testing, the submerged platform and black arc were positioned in the pool and maintained in these positions for each trial. The frequency and time spent by the mouse in each quadrant was visually monitored and recorded with the assistance of a computer-based timer program until the 90-s trial was completed or the mouse had climbed onto the platform. In addition to completing the four trials on d 4 of testing, an additional trial, a probe trial, was conducted to assess spatial learning. During the probe trial, the black arc remained in its position in the pool, but the submerged platform was removed. For the 60-s trial interval, the frequency and time spent swimming in each quadrant were measured. After testing on d 4, there was no additional testing for 72 h. After this break period (d 5 of testing), the arc and platform were positioned in the pool as they were for the first 4 d of testing. The performance of each mouse was monitored for four trials and an additional probe trial. On d 6 of testing, the platform and arc were rotated 180° and the same five trials (4 trials in each quadrant and 1 probe trial) were performed. On d 7, the same set-up used for the previous day was repeated; however, the probe trial was conducted without the black arc or submerged platform in the pool. On the final day of testing (d 8), a prominent visual cue (a flag) was attached to the platform and escape latencies were recorded for four trials.
Hemoglobin and tissue mineral analysis.
Offspring were killed by cervical dislocation on PND 75. Blood was
collected from the heart into heparinized syringes after dislocation
and the mice were then perfused through the heart with ice-cold 8.8
g/L sodium chloride. After perfusion, whole brain, liver and
gastrocnemius muscle were removed from each mouse. Once removed and
weighed, tissues used for mineral analysis were frozen in liquid
nitrogen and stored at -20°C until analyzed. For tissue iron, zinc,
copper and manganese concentrations, mineral analysis was done using
inductively coupled plasma spectroscopy (trace scan ICP; ThermoJarrel
Ash) after wet-ashing with nitric acid as previously described
(Clegg et al. 1981
). Hemoglobin (Hb) concentrations in
the dams and their offspring were determined using a standard
colorimetric assay for cyanmethemoglobin (Sigma Chemical, St. Louis,
MO).
Statistical analysis.
Statistical analysis of maternal variables was done using one-way
ANOVA (StatView, version 5.4, Abacus Concepts, Berkeley CA). Data for
offspring were analyzed using one- (treatment) and two-way
(treatment and sex or treatment and time) ANOVA. When data were
collected for more than one offspring from a litter (e.g., body weight,
Hb), the results were averaged to give a litter mean for each sex. In
addition, analysis of covariance (ANCOVA), using body weight as the
covariate, was done for selected behavioral parameters. For each sex,
auditory startle data were analyzed by repeated-measures ANOVA
using five-trial blocks with diet as the independent variable in
the analysis. These data were analyzed using Statistical Analysis
System (SAS, Cary, NC). Fisher’s Protected Least Significant
Difference was used to determine significant differences among groups
and an 
of P < 0.05 was defined as significant
for all tests. Data throughout the text, tables and figures are
expressed as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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Dietary treatment had no effect on weight gain during the initial 9-wk period of the study or during gestation (data not shown). The number of mice in each group that became pregnant and successfully maintained their litters through gestation and lactation did not differ significantly as a result of dietary treatment (61 and 63% for control and marginal iron females, respectively). The mean litter size at birth was similar between groups (8.0 ± 0.2 pups/litter in the control group and 8.2 ± 0.3 pups/litter in the marginal iron group), with a total of 13 control litters and 29 marginal iron litters meeting the criteria for eligibility in the study at PND 21.
Feeding a marginal iron diet for the initial 6 wk had no effect on Hb status because there was no significant difference in Hb concentrations between control and marginal iron females (data not shown). By the time of the last tail-bleed, there was a significant difference in Hb values between control and marginal iron female mice (P < 0.0001), with Hb values of 163.5 ± 3.7 and 134.0 ± 4.2 g/L for control and marginal iron females, respectively. Significantly lower Hb concentrations in marginal iron dams (P < 0.0001) were also measured at the time of weaning of their offspring (PND 21), with Hb values of 171.9 ± 3.8 and 154.0 ± 8.0 g/L for control and marginal iron dams, respectively.
Pup growth and food intake.
Weights of male and female offspring were not affected by dietary
treatment at weaning; thus no group differences were apparent when the
mice were assigned to their treatment groups. Within 1 wk of being
weaned, both marginal iron and replete mice weighed significantly less
than their male (P < 0.05) and female (P
< 0.05) counterparts in the other treatment groups. Body weights
remained significantly lower until PND 49 for marginal iron and replete
males and until PND 70 for marginal iron and replete females
(Fig. 1
). When the mice were killed on PND 75, body weights did not differ
among dietary groups for either sex (data not shown).
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Hb status in male and female offspring.
On PND 21, Hb concentrations were significantly lower in marginal Fe
mice than in controls (P < 0.0001); they were nearly
50% lower in both male and female offspring born to dams fed marginal
iron diets (Table 2
). By PND 75, Hb concentrations did not differ among groups.
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Neurobehavioral testing |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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For males, there was an effect of dietary treatment on startle
performance for select blocks during PND 30 and 45 (Fig. 2
). On PND 30, both marginal and replete males demonstrated a
significantly lower startle response than control males (P
< 0.05) during block 5. In addition, marginal iron males had a
lower startle responsiveness than control males during the first block
on PND 45 (P < 0.05). There was also a trend for lower
startle responsiveness in marginal iron males compared with control
males during block 3 (P = 0.0831) and block 4
(P = 0.0787) on PND 45. There were no differences among
dietary groups for the remaining blocks at PND 30, 45 and 60. Further
examination of the startle data revealed what appeared to be a lag in
the development of peak startle responsiveness from PND 30 to 60 in the
marginal iron males. When mean response amplitudes across ages were
analyzed for block 1 (block in which maximum startle responsiveness is
achieved) by repeated-measures ANOVA, a significant increase in
startle response with age occurred only in control (P = 0.0094) and replete males (P = 0.0112); marginal iron
males demonstrated no significant increase in mean response amplitude
with age (P = 0.1821). Dietary treatment had no effect
on auditory startle responsiveness in females for any of the testing
periods (data not shown).
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There was an overall increase in grip strength across testing sessions
for all males. Repeated-measures ANOVA of male forelimb grip
strength data demonstrated a significant effect of diet on this
variable. Marginal iron males had lower forelimb grip strengths on PND
30, 45 and 60 compared with control and replete males (Fig. 3
). Because body weight and grip strength can be correlated, the data
were reanalyzed using body weight as the covariate. The
F-values obtained for the analysis are presented in
Table 3
. Even with adjustment of the data for differences in body weight, the
lower forelimb grip strength in marginal iron males on PND 30, 45 and
60 remained significant (P < 0.05; post-hoc
differences of unadjusted means are depicted in Fig. 3
).
Repeated-measures ANOVA of male hindlimb data demonstrated that
marginal iron males had lower grip strength; however, when these data
were reanalyzed to adjust for body weight, the differences in hindlimb
grip strength were no longer significant (data not shown). Replete
males had forelimb and hindlimb grip strength measurements that did not
differ from those recorded for control males.
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|
. On PND 30, 45 and 60, forelimb grip
was 17–37% lower in the marginal females relative to control females.
After adjustment for body weight differences (Table 3)
, group
differences in forelimb grip strength remained significant
(P < 0.05). Hindlimb grip strength was significantly
lower only on PND 30 in the marginal iron (48.5 ± 1.9 g) and
replete (50.1 ± 1.5 g) females compared with control females
(65.5 ± 1.2 g); ANCOVA using body weight as the covariate
demonstrated that the lower hindlimb grip strength at PND 30 in these
females was independent of body weight (Table 3)
. At PND 45 and 60,
hindlimb grip strength in the females did not differ among dietary
groups (data not shown). Morris maze.
Dietary treatment influenced performance in the Morris maze (Fig. 4
).
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When the data were analyzed within groups for differences across testing days, only control and replete males demonstrated a significant reduction in latency across all 7 testing days [control: F(6,63) = 5.903, P < 0.0001; replete: F(6,63) = 3.113, P = 0.0098]; the marginal iron males had no improvement in escape latencies across testing days [F(6,63) = 0.637, P = 0.7004]. Furthermore, when the linear regression coefficients for latency across d 1–3 were obtained for each mouse and analyzed by ANOVA, control males demonstrated a significantly greater rate of change in escape latencies across days (-14.929 ± 2.093) than either marginal iron (-4.236 ± 1.883; P = 0.0048) or replete males (-7.589 ± 2.729; P = 0.0028); marginal iron and replete males did not differ in their rate of change across these days.
When the orientation of the maze was switched by 180° on d 6 of testing, there was not a dramatic increase in time required to find the platform in any of the treatment groups, suggesting that all males were able to use relevant visual cues to orient themselves spatially within the maze. In addition, probe trials conducted on d 4, 5 and 6 revealed that all of the mice spent the greatest amount of time during the 60-s trial in the quadrant that had once contained the submerged platform (data not shown). There were no group differences for the probe trials, demonstrating that all of the mice had learned the spatial orientation of the maze. In addition, the latency to locate the platform during the cued-performance task on d 8 of testing revealed no differences among groups (data not shown).
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Tissue mineral concentrations |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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On PND 21, brain iron concentrations in both marginal iron male and
female offspring were lower than in controls (Table 4
; P < 0.05). The only other brain mineral affected by
dietary treatment at this age was copper. Marginal iron males
demonstrated a slight, yet significant increase in brain copper
concentrations (control: 0.036 ± 0.001 µmol/g; marginal: 0.041
± 0.002 µmol/g; P < 0.05); for
females, brain copper concentrations did not differ among groups
(data not shown). At PND 21, brain manganese (Table 4)
and zinc
concentrations (data not shown) did not differ among dietary groups.
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. At
this age, there was a significant effect of diet (P = 0.0005) and sex (P = 0.0141) on brain iron
concentrations. In control mice, brain iron concentrations increased 66
and 53% in males and females, respectively. Marginal iron males and
females doubled their brain iron concentrations over this same time
period; however, brain iron concentrations remained lower in both
sexes. Brain iron concentrations in replete males did not differ
significantly from control mice, demonstrating a normalization of brain
iron concentration by this age. Brain iron in replete females, however,
did not match control, remaining 10% lower in these mice at PND 75.
Brain manganese concentrations were ~18% higher in marginal iron
males and females compared with controls at PND 75 (Table 4)
. Both
replete males and females had brain manganese concentrations that were
similar to the concentrations measured in control mice at this age.
Brain copper and zinc concentrations at PND 75 did not differ among
diet groups in both sexes (data not shown).
Liver.
Marginal iron diets resulted in significant changes in liver mineral
concentrations on PND 21 and 75 (Table 4)
.
The marginal iron male and female offspring had liver iron concentrations that were ~16% higher than that measured in control offspring at PND 21. In addition to elevated iron status, liver manganese concentrations in the marginal iron males and females were nearly twice that measured in control offspring. Copper concentrations in the liver of marginal iron males (0.383 ± 0.033 µmol/g) and females (0.374 ± 0.051 µmol/g) were significantly higher (P < 0.001) than those measured in male (0.215 ± 0.035 µmol/g) or female (0.230 ± 0.036 µmol/g) controls. Liver zinc concentrations were unaffected by dietary treatment (data not shown).
By PND 75, liver iron concentrations reflected the dietary treatments. Despite having higher liver iron concentrations on PND 21, marginal iron males had 62% lower and females 80% lower liver iron concentrations than control mice by PND 75. Liver iron concentrations in replete males and females increased significantly by PND 75; they were 2–5 times higher than the level in marginal offspring and 30–50% higher than the amount in control mice. In addition to changes in liver iron, liver manganese concentrations were significantly elevated in both marginal iron males and females relative to control offspring. Manganese concentrations in replete offspring were not significantly different from the levels measured in control mice. Marginal iron males and females experienced a small, but significant decrease in total liver weight (data not shown); however, correction of liver iron and manganese concentrations for the smaller tissue size did not eliminate the statistical differences among groups for these minerals (data not shown).
At PND 75, liver copper concentrations were higher only in marginal iron females as a result of dietary treatment; however, adjustment of these data for their smaller liver weights eliminated any group differences (data not shown). For males and females, liver zinc concentrations did not differ among treatment groups at PND 75 (data not shown).
Muscle.
In both male and female marginal iron mice, muscle iron and manganese concentrations at PND 75 were significantly affected by dietary treatment. In males and females, control (males: 0.209 ± 0.010 µmol/g; females: 0.231 ± 0.014 µmol/g) and replete mice (males: 0.194 ± 0.021 µmol/g; females: 0.209 ± 0.007 µmol/g) had significantly higher muscle iron concentrations than marginal iron mice (males: 0.155 ± 0.009 µmol/g; females: 0.156 ± 0.009 µmol/g; P < 0.05). In addition, marginal iron females had significantly greater muscle manganese concentrations (2.024 ± 0.078 nmol/g) than control (1.715 ± 0.121 nmol/g) and replete females (1.551 ± 0.056 nmol/g) (P < 0.05). In males, marginal mice had significantly greater (P < 0.05) muscle manganese concentrations (1.923 ± 0.121 nmol/g) only compared with replete male offspring (1.490 ± 0.070 nmol/g). In males and females, muscle iron and manganese concentrations did not differ between control and replete offspring. Muscle copper and zinc concentrations were not affected by dietary treatment (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSNeurobehavioral testingTissue mineral concentrationsDISCUSSIONREFERENCES |
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In marginal iron males and females, grip strength was the developmental
variable consistently affected. Forelimb grip strength in marginal
offspring was 20–40% lower than that measured in control mice and
independent of differences in body weight. Hindlimb grip was also lower
in marginal iron mice for several testing sessions; however, the lower
grip strength was related to differences in body weight. These data
show that there are multiple mechanisms by which iron deficiency can
affect motor development. Whether it is by inducing changes in body
weight, possibly by altering food efficiency, or by altering metabolism
within the central nervous system, as suggested in previous work with
this model demonstrating increased oxidative stress in the cerebellum
(Kwik-Uribe et al. 1999
), the mechanism does not lessen
the effect of the outcome. The marginal iron mice had lower grip
strength. This alteration in motor function, in addition to affecting
absolute strength, may have affected the mouse’s ability to interact
with and explore its environment (Idjradinata and Pollitt 1993
). Understanding that different mechanisms may be working
to produce similar outcomes demonstrates the complexity of the role of
iron in this developmental process.
In addition to disruptions in motor function, marginal iron males
demonstrated altered performance in the Morris water maze. During d 1
of testing, the marginal iron males located the submerged platform in
less time than control males for each of the four trials. Because this
was the first time that the mice had been in the water maze, their
performance on this day is unlikely to reflect learning, but can
perhaps be attributed to increased activity levels. A similar
performance outcome was observed by others in iron-deficient rats.
Williamson and Ng (1980)
reported that increased
exploratory behavior during the initial stages of testing resulted in
improved performance by iron-deficient rats in a T-maze;
however, as the deficiency progressed, normal maze learning occurred.
Similar to this enhanced T-maze learning, improved performance of
marginal iron males in the Morris maze may be a reflection of
heightened activity levels contributing to shorter escape latencies.
Although increased activity levels may have assisted the marginal iron males in navigating the water maze during the early stages of testing, additional observations indicate that these mice exhibited alterations in learning that affected maze performance. Disruptions in learning by marginal iron males during the first days of testing (acquisition phase) was demonstrated by the following observations concerning marginal iron males: 1) they were not different in performance times across test days; 2) they demonstrated a diminished rate of change in escape latencies across days; 3) they found the platform less often on d 4 of testing; and 4) they had a longer latency to locate the platform for two of the four trials on d 3 and 4 of testing. These differences in performance during the initial period of Morris maze testing suggest that chronic marginal iron deficiency had a significant effect on how the mice performed and learned in the maze.
Other behavioral studies of iron deficiency have shown that low iron
animals demonstrate behavioral abnormalities in various testing
paradigms. A common theme that has emerged from several of these
studies is that low iron animals have altered responsiveness to novel
or environmental stimuli. A study in rats (Massaro and Widmayer 1981
) found that during the first phase of testing, low iron
animals had impaired performance in an association learning task but,
with time, performed at a level indistinguishable from that observed in
control animals. One hypothesis that is put forth in the study by
Massaro and Widmayer (1981)
suggests that the nutrient
deficiency limits the animal’s ability to interact with its
environment; thus the animal "acquire(s) information about those
stimuli ... which are more biologically meaningful and serve to
reinforce physiological needs ...." Work by Weinberg et al. (1980)
also suggests that low iron animals have an
attenuated responsiveness to novel stimuli, but a heightened
responsiveness to a more aversive stimulus. These studies suggest that
marginal iron males in this study did not have an impairment in
absolute learning, but rather in how they learned. On d 1 of testing,
the marginal iron males were perhaps less affected by the novelty of
the water maze, and thus could focus on obvious environmental cues,
which in the maze, helped to guide the discovery of the submerged
platform. When information was recorded during a trial, it was obvious
that each mouse adopted a strategy to navigate the water maze.
Instrumentation available during this study did not permit an analysis
of additional aspects of maze performance such as swim path, swim
distance and swim velocity, thus we do not know if any of these
endpoints were affected by marginal iron deficiency. In future
experiments, this information may provide new insights into how
alterations in arousal, attention or motor activity may be affecting
performance.
Alterations in motivation may also have affected how the marginal iron
males performed during water maze testing. Iron deficiency has been
reported in both human (Beard et al. 1990
,
Lukaski, Hall et al. 1990
, Martinez-Torres et al. 1984
) and animal (Beard et al. 1984
,
Dillman et al. 1980
, Smith and Beard 1989
) studies to result in attenuated thermoregulation. With
the animal’s exposure to cold, iron deficiency results in a failure to
maintain normal body temperature, resulting in core body temperatures
that are 2–5°C below that measured in iron-sufficient controls.
Environmental temperature has previously been shown to be a factor that
affects learning and memory (Sandi et al. 1997
) in
control (nutrient-sufficient) animals, with colder temperatures
resulting in enhanced maze performance (19 vs. 21°C). Although water
temperature during testing was maintained at 20–22°C, it is possible
that alterations in thermoregulation within the mice may have
contributed to the observed changes in maze performance. Once in the
water, marginal iron mice could have demonstrated impaired
thermoregulation, resulting in lower core body temperatures. Increased
activity in an attempt to increase body temperature or simply to escape
from the water may have been motivating factors that enhanced the
performance of marginal iron males during early phases of maze testing.
Previous work has suggested that iron deficiency during early
development may have a lasting effect on behavioral performance
(Felt and Lozoff 1996
), thus suggesting that there may
be a window of time during brain development in which adequate iron
status is critical. This is supported by the current study, which found
that despite iron supplementation of marginal iron offspring at
weaning, disturbances in maze performance persisted. Escape latencies
of control and replete males were similar at the start of testing, but
on d 2–4, replete males required more time to find the submerged
platform. Because performance of control and replete males did not
differ during the last few days of testing, the increased latency of
replete males during the acquisition phase suggests that these mice
experienced learning delays. In addition to altered maze performance
compared with control mice, replete males also demonstrated a
difference in their performance pattern compared with marginal iron
males. If the observed effect on maze performance was due solely to
chronic marginal iron intakes during gestation and lactation, the
marginal iron and replete males would be expected to perform in a
similar manner. The variation in performance of these mice suggests
that adequate iron status is important not only during early brain
formation, but also later in development. During gestation and
lactation, the various cell types in the brain proliferate and
differentiate, resulting in the formation of brain structures
[reviewed in Morgane et al. (1993)
]. As the animal
approaches puberty and adolescence, the brain continues to develop
[reviewed by Golub (2000)
]. The brain
maturation that occurs during this phase largely reflects a decline in
synaptic density and an increase in myelination of the cortex. Because
these steps are integral events for the development of higher learning
and cognitive function, the altered performance of marginal mice
relative to their replete counterparts may represent distinct effects
of marginal iron status during the adolescent period of brain
development. Although the early formation of brain structures is
typically considered the critical window of vulnerability, the altered
maze performance of marginal iron mice likely reflects the cumulative
effect of marginal iron intakes during brain formation and the later
maturation of these structures.
The finding that chronic marginal iron intakes resulted in persistent
changes in brain iron concentrations is consistent with previous
reports. On PND 21, brain iron was 31% lower in marginal iron
offspring and, despite increasing at PND 75, brain iron remained
16–19% lower in the marginal iron animals. This lowering of brain
iron is within the range reported in studies of more severe iron
deficiency during gestation and early lactation (Dallman et al. 1975
, Felt and Lozoff 1996
) and within the range
previously reported with this model (Kwik-Uribe et al. 1999
). These data support the concept that the duration of a
low iron diet can be as physiologically relevant to the animal as the
severity of the iron deficiency. In addition, this study demonstrates
that feeding a control diet from weaning can increase brain iron
concentrations in offspring born to marginal iron dams; however, only
in males does the repletion increase whole-brain iron to a
concentration similar to that measured in control males. Replete
females had whole-brain iron concentrations that remained 11%
lower than brain iron in control females. It is important to consider
that whole-brain iron concentrations were measured in this study.
Previous work has shown that iron is not distributed uniformly in the
brain, with regions such as the basal ganglia reported to have the
highest brain iron concentrations in the adult animal [reviewed in
Beard et al. (1993)
]. Despite an increase in
whole-brain iron in both males and females, it is unclear from the
work presented here whether all brain regions would have benefited
equally from improved iron intakes or whether regional differences may
have persisted. One study demonstrated persistent changes in the
concentration of iron regulatory proteins (i.e., transferrin, ferritin)
despite the normalization of regional brain iron concentrations
(Erikson et al. 1997
), demonstrating that despite this
normalization, disturbances in the proteins that regulate iron status
can still exist.
In summary, the results of this study demonstrate that chronic marginal iron intakes can affect not only tissue iron concentrations, but also can have a functional effect on behavioral measures. Importantly, the postweaning period of iron repletion used in this study reversed some, but not all of the biochemical and behavioral effects of marginal iron diets during gestation and lactation. The various factors that may have influenced the behavioral outcomes in this study are currently under investigation and will be reported in future work.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: Hb, hemoglobin; IDA, iron deficiency anemia; PND, postnatal day.
Manuscript received December 13, 1999. Initial review completed February 3, 2000. Revision accepted April 3, 2000.
|
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