Moderate iron deficiency in infancy: Biology and behavior in young rats

Behavioural Brain Research 170 (2006) 224–232

Research report

Moderate iron deficiency in infancy: Biology and behavior in young rats夽 John L. Beard a,∗ , Barbara Felt b , Tim Schallert c , Maggie Burhans a , James R. Connor d , Michael K. Georgieff e a Department of Nutrition, Penn State University, PA 16802, United States Center for Human Growth and Development and Department of Pediatrics and Communicable Diseases, The University of Michigan, Ann Arbor, MI 48109, United States c Department of Psychology, University of Austin, Austin, TX 78712, United States d Department of Neurosurgery, Penn State College of Medicine, PA 17033, United States e Departments of Pediatrics and Child Psychology, University of Minnesota, Minneapolis, MN, United States b

Received 20 September 2005; received in revised form 13 January 2006; accepted 20 February 2006 Available online 29 March 2006

Abstract Iron deficiency anemia in early childhood is associated with developmental delays and perhaps, irreversible alterations in neurological functioning. The goals were to determine if dietary induced gestational and lactational iron deficiency alters brain monoamine metabolism and behaviors dependent on that neurotransmitter system. Young pregnant rats were provided iron deficient or control diets from early in gestation through to weaning of pups and brain iron concentration, regional monoamine variables and achievement of specific developmental milestones were determined throughout lactation. Despite anemia during lactation, most brain iron concentrations did not fall significantly until P25, and well after significant changes in monoamine levels, transporter levels, and D2 R density changed in terminal fields. The changes in D2 R density were far smaller than previously observed models that utilized severe dietary restriction during lactation or after weaning. Iron deficient pups had normal birth weight, but were delayed in the attainment of a number of milestones (bar holding, vibrissae-evoked forelimb placing). This approach of iron deficiency in utero and during lactation sufficient to cause moderate anemia but not stunt growth demonstrates that monaminergic metabolism changes occur prior to profound declines in brain iron concentration and is associated with developmental delays. Similar developmental delays in iron deficient human infants suggest to us that alterations in iron status during this developmental period likely affects developing brain monaminergic systems in these infants. © 2006 Elsevier B.V. All rights reserved. Keywords: Iron deficiency; Brain iron; Catecholamines; Development; Rat; Anemia; Dopamine; Serotonin; Developmental delays

1. Introduction Iron deficiency and anemia during infancy have been associated with poorer performance on mental and motor measures and altered social–emotional behavior [18,49]. Despite iron treatment, developmental alterations persisted in infancy [29,36,45] and may continue into adolescence [28]. The neu夽 Supported by PO1 HD39386 (Brain and Behavior in Early Iron Deficiency, Betsy Lozoff, Principal Investigator), and RO1 NS35088 (JB). ∗ Corresponding author at: Nutrition Department, 125 S Henderson Bldg., The Pennsylvania State University, University Park, PA 16802, United States. Tel.: +1 814 863 2917; fax: +1 814 863 6103. E-mail address: [email protected] (J.L. Beard).

0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.02.024

robiological alterations that account for these findings are not fully delineated or clearly identified in a causal model [5]. Investigators have explored central nervous system (CNS) effects of iron deficiency anemia (IDA) in rodent models for several decades [18,50] utilizing research designs that incorporate both the pre-weaning and post-weaning effects of iron deficiency [37,47,50]. Brain regions particularly iron rich in adulthood (striatum, substantia nigra and deep cerebellar nuclei) are not particularly rich in iron in early development, as the process of regional acquisition of iron appears to be developmentally bound [35]. Thus, dietary iron restriction during early periods of growth and development results in a very different profile of regional brain iron deficits than does iron

J.L. Beard et al. / Behavioural Brain Research 170 (2006) 224–232

deficiency during later periods of life. Indeed, our working hypothesis is that a significant portion of the consequences of iron restriction to the brain is likely to be related directly to the developmental biology occurring at the time of this nutrient deficiency. The direct and indirect effects of iron deficiency are not clear, but likely include effects on cell growth and differentiation [22], cellular bioenergetics [9] and biochemistry [5,37]. Previous studies identified manifestations of iron deficiency in neurotransmitter systems [8,11,12,30,50,51], myelin biology [6,31,52], and behavior [6,10,11,13,34]. Recently, new observations regarding metabolism and dendritic arborization in the hippocampus document lasting effects of pre- and postnatal iron deficiency on brain morphology [22,37]. Changes in monoamine metabolism can be used as a marker of developmental brain pathology or compensatory reorganization in the forebrain involving other neurotransmitters and cellular and molecular events [32,38,41]. Recently published studies indicate that relatively mild early iron deficiency in human infants result in delays in achievement of both short term and long-term developmental milestones [3]. The observations of a paucity of motor movements while iron deficient as infants and poorer executive functioning years later as adolescents may both be explained, in part, by alterations in the monoamergic systems. Most previous rat animal model studies that examined brain iron, DA, and generally utilized a post-natal model of iron deficiency [4,11,12,30,50,51]. Some studies commenced an iron deficient diet at P4 [11,12], others at P10 [34,35], others at P21 [4,6,50,51] and all used a severe dietary restriction to produce animals that generally had brain iron losses >50% in many brain regions. Other rodent studies utilized designs of pre-natal iron deficiency to examine the influences of intrauterine iron deficiency on brain development and subsequent functioning [9,13,22,25,42]. Many of the cited rodent studies utilized designs in which the brain iron deficiency was quite severe and concentrations were reduced by more than 50% in many brain regions. In some studies, the severity was chosen to match the degree of iron deficiency that occurs in human conditions such as intrauterine growth retardation and diabetes mellitus during pregnancy where fetal (but not post-natal) iron balance is compromised [17,32,33,41]. For instance, some infants of diabetic mothers may have a greater than 40% reduction in brain iron concentration and a 60% reduction in liver iron. Studying severe iron deficiency in rodent models has been very useful in defining changes in biology in the extreme. However, such situations fail to answer questions regarding less severe degrees of iron deficiency anemia like that commonly occurring throughout the period of human pregnancy and infancy that result in biologically meaningful alterations in CNS functioning. The objective of this study was to define the effects of a modest degree of brain iron deficiency during intrauterine life and lactation on regional iron concentrations, monoamine metabolism, and developmental milestones in a rat model. In order to avoid the teratology and profound growth failures

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of some earlier studies of iron deficiency during gestation in rodents, we utilized a dietary protocol designed to reduce brain iron by 10–20%. The hypothesis was that moderate iron deficiency that persisted through gestation and lactation would alter developmental progress and monoamine metabolism The model was chosen to be relevant to common conditions in the developing world where maternal ID is widespread, infants are likely born with proportionately reduced iron stores, and the iron deficiency continues for the maternal-infant dyadic through the period of lactation. To test this hypothesis, we used a developmental model of iron deficiency in rodents designed (by dietary means) to maintain a moderate level of anemia in the pups during lactation. 2. Methods 2.1. Design Young Sprague–Dawley (Harlan, Sprague–Dawley) females, approximately 125 g, were obtained and fed an iron sufficient diet (40 ppm iron, Harlan Teklad Nutritionals) for 2 weeks prior to mating. Pregnant dams were then randomly assigned to either a 4 ppm iron deficient diet, or continued the 40 ppm iron diet (the control group, CN) from gestation day (G5) to post-natal day (P7). All litters were culled to 10 pups per litter by P2 retaining equal numbers of males and females as able. At this time, the low iron dams were provided a second iron deficient diet of 10 ppm Fe until P20. This level of dietary iron for dams was to provide an adequate milk iron content to prevent growth faltering in iron deficient pups yet maintain a moderate level of body iron depletion. The control dams continued the CN diet until P20. At P20, all pups and moms received the CN diet through weaning at P23 and continued until pup brain assessments. To control for possible differences in maternal care of their pups and effects on pup development, male and female pups from each litter were used at each time point until a minimum sample size of n = 10 for each sex was obtained [27,46]. Development was assessed for each pup within each litter. Male and female pups were removed from each litter for brain assessments at P10 and P25. The number of litters per diet group was 16. Animals were housed in a temperature controlled animal facility with a reversed, 12:12 h light/dark cycle. The experimental protocol was approved by the University Committee for the Care of Animals at the University of Michigan.

2.2. Growth and physical development Animals were weighed on a top loading balance to the nearest 0.01 g during gestation and lactation: mothers—G1, G10, G20, P1, P10, P20, and P24. Pups were weighed at P1, P5, P10, P15, P20, and P25. Pup development was assessed for all pups between 09:00 and 14:00 h (during the dark cycle) on a five-point scale for fur, ear and eye development at 3-day intervals during lactation [1,3,26,38]. Individual pup scores were averaged within each litter to give a mean score for each sex.

2.3. Hematology and iron status Maternal blood was obtained by tail vein puncture, and hematocrit, hemoglobin, and serum iron were measured by standard methods. Tail vein punctures were done on G1, G10, G20, P1, P10, and P20. One male and one female pup were removed from the litters at P10 and P25 for sacrifice to measure brain iron or monoamines. The animals were anesthetized with pentobarbital and blood was obtained by intra-cardiac sampling. After perfusion with PBS via the left ventricle, the organs were rapidly removed. Blood samples for serum iron were centrifuged at 3000 × g at 4 ◦ C for 15 min and then sera were frozen at −80 ◦ C. The following brain regions were quickly dissected on ice: frontal cortex, caudate putamen, hippocampus, thalamus, ventral tegmentum-substantia nigra, pons, superficial cerebellum, and deep cerebellar nuclei. The regions were

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placed immediately in storage tubes and frozen at −80 ◦ C. A hemisphere of the brain reserved for autoradiography was frozen slowly in dry ice: isopentane slurry and then stored frozen at −80 ◦ C. Livers were rapidly removed, weighed, and immediately frozen. Liver non-heme iron was determined using published methods [10,35].

so separate experiments were conducted where we utilized multiple membranes isolated from caudate putamen for a Scatchard plot analysis of the affinity for RTI-55. The apparent affinity constant, Kd , for DAT was approximately 3.0 nM for controls and 3.3 nM in the iron deficient animals. These two Kd values were not significantly different from one another.

2.4. Regional brain iron and monoamines

2.7. Behavioral assessments

Frozen brain regions were digested by published and standard procedures and analyzed by atomic absorption spectrophotometry [35]. Catecholamine analysis was conducted by HPLC with coulometric detection. Briefly, brain regions were sonicated at 4 ◦ C in 0.1 M ultrapure perchloric acid (5:1, v/w) with DHBA added as an external standard. Samples were immediately centrifuged at 15,000 × g for 5 min at 4 ◦ C, filtered through a 0.2 ␮m filter, and 10 ␮l was injected into an ESA HPLC utilizing coulometric detection and reverse phase chromatography [30]. Efficiency of recovery was corrected for the DHBA external standard and all data expressed as pmoles/mg tissue.

Assessments of sensorimotor development [1,3,15] and activity were performed between 09:00 and 14:00 h (during the dark cycle) on all pups individually. Pups were placed on a warming-pad for the interval of separation from mother and testing and total separation interval did not exceed 10 min. Testers were blind to the diet group of litters. A developmental battery, based on basal ganglia, particularly monoaminergic neurotransmitter functionality, was administered at P6, P9, P12, P15, and P18 to assess the pattern of emergence for the following sensorimotor behaviors: auditory startle, surface righting, negative geotaxis, bar holding, bilateral forelimb head-on placing and unilateral vibrissae-evoked forelimb placing [3,17,26,27,33,38,39,42]. Auditory startle was measured as the presence or absence of body twitch in response to a handclap. A five-point scale was used for surface righting, negative geotaxis and bar holding, with “0” being absence of response and “5”signifying that the response was fully present immediately (surface righting and negative geotaxis) or was maintained for at least 10 s (bar holding). Surface righting and negative geotaxis involve vestibular systems and motor development [15]. Surface righting was assessed by placing pups supine on a flat surface and measuring the time to righting (pup returns to prone posture with all four paws on the surface). Negative geotaxis was measured by placing the pup, snout down, on a 30◦ wire-mesh inclined surface and measuring the time (within a 15 s trial) for the pup to turn 180◦ and begin crawling upward (away from gravity). For bar holding which involves development of forelimb grip and muscle development [15], pups were allowed to grasp a slender plastic bar (2 mm diameter) with both front paws and the time (within a 10 s trial) they could hang suspended, supporting body weight was measured. The final two measures of forelimb placing, sensitive to striatal dopamine somato-sensorimotor function [20,38,39,44,48] were used because these can be examined early in development [14,38,39,43] Placing assessments continued to P21 or P24. For bilateral forelimb placing, the ventral surface of the snout was placed in contact with a horizontal surface while holding the torso gently. When this skill is fully developed, both forepaws immediately and simultaneously place on the horizontal surface in response. The numbers of right and left front paw placements were counted for a total of 10 trials. Unilateral vibrissae-evoked forelimb placing was assessed by holding the pups securely at the torso and allowing one upper extremity to hang free. The vibrissae ipsilateral to the free extremity were then passed by a corner surface. When fully developed, stimulation of vibrissae results in immediate placement of the ipsilateral forelimb. Successful placements were counted for each forelimb separately, ten trials each. There was no asymmetry (right versus left) for bilateral or unilateral placing in either diet group. Therefore, the number of right and left placements was averaged for each pup. The performances of pups within litter were then averaged by sex and multiplied by 10 to give percent placing by litter and sex at each age. Pups from each diet group were also observed for general activity level (number of quadrants entered in 3 min) in a 1-m diameter open field at P15 and P25. The open field was sub-divided into sectors and the number of sectors entered with all four paws was counted. The open field was cleaned with alcohol between assessments. Individual pup scores were averaged within each litter to give a mean score for each sex.

2.5. Ligand binding protocol Procedures for dopamine transporter and serotonin transporter ligand binding were slightly modified from those previously described elsewhere [2], using [125 I]-RTI-55 (Perkin-Elmer, Boston, MA). Slides were incubated in a solution of [125 I]-RTI-55 (55.8 pM, 2200 Ci/mmol; approximately 150,000 cpm/ml) and protease inhibitor cocktail (PIC, pH 7.4, 25 ␮g/ml chymostatin, 25 ␮g/ml leupeptin, 100 ␮M EDTA and 100 ␮M EGTA) diluted in a phosphate buffer (50 mM NaH2 PO4 ; 50 mM Na2 HPO4 ) 1:10 for 90 min at 4 ◦ C. For the dopamine transporter (DAT), the solution also contained 10 ␮M fluoxetine hydrochloride (Eli Lilly and Company, Indianapolis, IN) to block serotonin transporter (SERT) binding; for SERT density analysis, the solution contained 1 ␮M GBR 12935 (Sigma) to block DAT binding. Nonspecific binding was determined by the addition of both GBR 12935 (1 ␮M) and fluoxetine hydrochloride (10 ␮M) to the solution. After the incubation period, the slides were washed three times in ice-cold fresh phosphate buffer (50 mM NaH2 PO4 ; 50 mM Na2 HPO4 ) for 5 min each. Immediately following the final wash, the slides were quickly dipped once in ice-cold ddH2 O to desalt the tissue and dried by a steady flow of air at room temperature overnight. DAT and SERT slides and an autoradiographic [125 I] Microscale (Amersham Biosciences, Piscataway, NJ) were exposed to Kodak BioMax MR-1 film (Amersham Biosciences). SERT slides were exposed for 7.5 h and DAT slides were exposed for 6.5 h.

2.6. Quantification of transporter densities Brain region densities were quantified using NIH Image (Bethesda, MD) computer software. A minimum of four consecutive slices was analyzed for each individual animal. The level of radioactivity of the Microscales on the day the film was developed was determined to provide the standard curve. Individual brain regions were outlined, and the average density of the bound radioligand was measured by NIH Image using the standard curve and the Rodbard prediction equation that assumes curvilinear relationships of optical density and radioligand binding. Densities were specified in units of nCi/mg polymer. The [125 I] Microscales were 46–48% efficient at predicting the radioactive signals from tissue, and the inefficiency was corrected for by dividing the initial density (nCi/mg polymer) by 0.47 to estimate the nCi/mg tissue. The efficiency of tritium ligand being transmitted through tissue to film was only 31–47% as efficient as the Microscales and was not a constant at all levels of radioactivity. We corrected for this inefficiency by dividing the initial density (nCi/mg polymer) by the corresponding inefficiency at each standard on the Microscales (i.e., 0.31, 0.33, 0.34, 0.36, 0.38, 0.41, 0.44, or 0.47) Further calculations involved conversion of radioactivity to moles of ligand bound by utilization of the specific activity of each isotope. Final densities are thus reported in fmoles of bound radiochemical/mg tissue. The coefficient of variation for within-animal replicates was <3% in all cases. The average of all replicates (ranged from 4–8 slices) was utilized as the density for that particular animal. These slice ligand-binding assays included only a single concentration of ligand,

2.8. Statistical analysis All biological and behavioral data were examined for normal distributions and log transformed when necessary prior to Chi-square or ANOVA. Behavioral measures were analyzed using the mean scores for males and females in each litter. The fundamental analysis was ANOVA with diet and sex and age as the main effect variables. Brain region was also considered as a main effect variable for analysis of brain iron. Interactions between main effects were examined with the level of significance for interactions set at P < 0.05. General linear modeling was used to assess the influence of sex as a covariate. Significance was set at P < 0.05.

26.33 ± 16.97† 4.07 ± 2.80 11.9 ± 5.1† 5.2 ± 2.0 11.1 ± 3.1† 6.0 ± 1.9

CN [38] (19M 19F) ID [41] (20M 21F)

CN [32] (16M16F) ID [25] (13M 12F)

P10

P25

There was no significant difference by sex at each age nor was there a sex × diet interaction. a Mean ± S.D.; values in parentheses are organ weight/g body weight. † P < 0.05 CN > ID. †† P < 0.05 CN < ID.

38.63 ± 9.01† 22.14 ± 6.69 1522 ± 112† (23.61 mg/g) 1379 ± 197†† (29.21 mg/g) 64.5 ± 7.5† 49.7 ± 14.2

2.843 ± 0.518† (43.83 mg/g) 2.470 ± 0.656†† (50.45 mg/g)

10.4 ± 6.8 ± 3.9 32.99 ± 19.03 ± 7.52 0.654 ± 0.186† (34.81 mg/g) 0.564 ± 0.095 (34.88 mg/g) 842 ± 103 (45.27 mg/g) 806 ± 80†† (50.16 mg/g)

Brain (mg) Body weight (g) Diet (n) Day

Table 1 Pup body, brain, liver weight, and hematology at P10 and P25a

3.3. Monoamine metabolism

19.4 ± 16.3 ± 2.7

Liver (g)

Hematocrit, hemoglobin, and serum iron were reduced by 40–50% in pups of ID dams during lactation. Non-heme liver iron was reduced by 70–80% at both P10 and P25 as compared to CN group pups (Table 1). Regional brain iron concentrations were lower in ID pups at P10 only in the pons and superficial cerebellum (Table 2). In contrast, at P25, five out of eight brain regions were > 20% lower in the ID rats (the cortex, nucleus accumbens, pons, superficial cerebellum, and thalamus).

At P10, there were already changes in brain catecholamine concentrations in the caudate putamen despite the lack of a significant change in caudate iron concentration (Fig. 1a). Regional dopamine was elevated >270% and norepinephrine (NE) >50% in ID caudate putamen compared to controls. Other monoamines or metabolites did not differ between treatment groups. At this same age, the DAT was doubled in the caudate putamen and was 75% greater in the nucleus accumbens of ID rats compared to that of CN rats (Fig. 2). The D2 R density was relatively unaffected by iron deficiency in terminal fields of the caudate putamen and nucleus accumbens (Fig. 3). In contrast, the substantia nigra had significantly greater D2 R (180% elevation) than controls. The other monoamine transporter studied in these animals, the serotonin transporter (SERT), was not significantly different in the caudate putamen at P10 in ID rats (Fig. 4). However, there was a significant and substantial elevation in SERT density in a number of projection areas in thalamus and elsewhere (45% increase in the vestibular nucleus, 110% increase in locus ceruleus, 100% in the lateral parabrachial nucleus, 25% increase in anteroventral thalamic nucleus, and 20% increase in the reticular thalamic nucleus).

12.3 ± 6.7 ± 1.6

Liver iron (␮g/g wet weight)

3.2. Pup iron status

3.9†

HCT (%)

7.12†

HGB (g/dl)

Serum iron (␮g/ml)

Maternal body weight did not differ significantly by diet group during gestation or lactation. The mothers consuming the ID diets had significantly lower hemoglobin concentrations than controls at G20 (13.6 ± 3.1 g/dl versus 15.9 ± 3.0 g/dl), P1 (12.5 ± 1.6 g/dl versus 15.2 ± 2.1 g/dl), P10 (12.3 ± 2.8 g/dl versus 15.6 ± 3.9 g/dl) and a trend at P20 (14.6 ± 3.2 g/dl versus 17.2 ± 3.9 g/dl). This indicates that pups from these dams experienced a modest iron deficiency in utero and during lactation. The ID group pup weight was not significantly different from control pups at delivery (5.7 ± 0.7 g versus 5.9 ± 0.6 g) but was significantly lower by P5 (9.8 ± 1.8 g versus 10.5 ± 1.1 g) and thereafter during lactation (Table 1). Whole brain weight was significantly lower for ID pups at P25 and liver weight was significantly lower for ID pups at P10 and P25 (Table 1). The only significant differences in physical development were later eye opening and ear development for ID pups. However, both measures were normal-for-age by P18.

0.6†

3.1. Growth and physical development

2.3†

3. Results

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40.89 ± 19.58† 11.24 ± 6.45

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Table 2 Pup brain iron concentrations at post-natal days 10 and 25 Cortex

Deep cerebellar nuclei

Nucleus accumbens

Superficial cerebellum

Pons

Hippocampus

Caudate putamen

Thalamus

P10

CN ID

0.64 ± 0.03 0.65 ± 0.03

0.59 ± 0.04 0.56 ± 0.04

0.56 ± 0.04 0.53 ± 0.04

0.69 ± 0.05 0.57 ± 0.03†

0.70 ± 0.04 0.55 ± 0.04†

0.68 ± 0.05 0.65 ± 0.05

0.84 ± 0.09 0.83 ± 0.04

0.59 ± 0.02 0.61 ± 0.04

P25

CN ID

0.91 ± 0.04 0.73 ± 0.03†

0.93 ± 0.05 0.82 ± 0.07

0.95 ± 0.04 0.75 ± 0.03†

0.66 ± 0.02 0.51 ± 0.02†

0.90 ± 0.02 0.75 ± 0.02†

0.80 ± 0.03 0.75 ± 0.03

0.98 ± 0.09 0.87 ± 0.05

0.62 ± 0.02 0.49 ± 0.04†

Units are nmoles Fe/mg tissue and data are expressed as the mean ± S.E.M., regions differed significantly from one another (P < 0.001). Sex was not a significant main effect variable nor was there a sex × dietary treatment interaction. † P < 0.05 by diet group.

Rats at P25 showed a different pattern of effect of iron deficiency in monoamines than what was observed at P10 (Fig. 1b). 5-HT, DA, and NE were no longer different from controls of the same age, though the metabolites, 5-HIAA and HVA, were significantly higher in these iron deficient rats. Despite more brain regions having lower brain iron concentrations at P25, there were no significant differences between ID and CN rats in DAT, D2 R, or SERT density at this age (Figs. 2–5). 3.4. Developmental measures The pattern of sensorimotor skill attainment was significantly altered in pups that experienced early iron deficiency. Audi-

Fig. 1. Brain monoamine concentration in caudate putamen at P10 (panel a) and P25 (panel b) in iron deficient (ID) and control (CN) rats. Figure of means ± S.E.M. NE = norepinephrine, EPI = epinephrine, DOPAC = dihyroxyphenyl acetic acid, DA = dopamine, 5-HIAA = 5 hydroxyindole acetic acid, HVA = homovanillic acid, 5 HT = serotonin (5-hydroxy tryptamine). “*” designates a highly significant difference between ID and CN values (P < 0.001).

tory startle, present for all control pups by P18, was present in only 52% of ID group pups by that age. Surface righting and negative geotaxis were significantly delayed for the ID pups at P12 and P15, but did not differ from controls by P18. Bar holding ability (Fig. 5a) was significantly lower for the ID pups at all ages assessed. Bilateral forelimb placing and vibrissae-evoked forelimb placing were also significantly reduced for the ID pups (Fig. 5b and c). Notably, all of these behaviors demonstrated abnormalities prior to significant loss of brain iron but during the time period of altered monoamine metabolism. There was evidence of “catch up” for bilateral forelimb placing at P21 (78% placing for ID pups versus 100% placing for CN pups) but not for vibrissae-evoked placing by the last assessment at P24. The number of sectors entered in the open field did not differ by diet group at P15 but was significantly less for ID pups at P25 (23.2 ± 14.4 g/dl versus 32.2 ± 16.6 g/dl).

Fig. 2. Dopamine transporter (DAT) density as measured by RTI-55 binding in four brain regions at P10 (panel a) and P25 (panel b). Figure of mean ± S.E.M. CPU = caudate putamen, NA = nucleus accumbens, OT = olfactory tubercle, SN = substantia nigra. “*” designates a highly significant difference between ID and CN values (P < 0.01).

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Fig. 3. Density of dopamine D2 R in iron deficient (ID) and control (CN) rat brains at P10 (panel a) and P25 (panel b). Figure of mean ± S.E.M. for each group and brain region. CPU = caudate putamen, NA = nucleus accumbens, OT = olfactory tubercle, SN = substantia nigra. “*” designates a significant differences exists between ID and CN rats (P < 0.01).

Fig. 5. Figure depicts (a) bar holding, (b) bilateral forelimb placing, (c) vibrasse forelimb placing of iron deficient and control rat pups at five different post-natal days. Figure of means ± S.E.M. “*” denotes a significant differences between ID and CN rats at that age (P < 0.02).

4. Discussion

Fig. 4. Density of the serotonin transporter (SERT) in brain regions at P10 (panel a) and P25 (panel b). CPU = caudate putamen; NA = nucleus accumbens; OT = olfactory tubercle; SN = substantia nigra; VN = vestibular nucleus; LC = locus ceruleus; LPB = lateral parabrachial; SUG = superficial gray layer; OPT = optic tract; LTN = laterodorsal thalamic nucleus; AV = anteroventral thalamic nucleus; RTN = reticular thalamic nucleus; ZI = zona incerta; CX = cortex. Figures of group means ± S.E.M. “*” designates a significant differences exists between ID and CN rats (P < 0.05).

The current study extends our knowledge regarding the consequences of iron deficiency during both pregnancy and lactation on the developing brain and on behavior in several ways. The rat model developed in this study demonstrates that iron deficiency anemia from gestation through mid-lactation at P10, does not necessarily lead to early deficits in regional brain iron concentration. But prolonging iron deficiency to the end of weaning does change the brain iron concentration demonstrated that whatever reserves of brain iron that existed at mid-lactation were exhausted by weaning. Nevertheless, monoamine metabolism was altered in some regions prior to detectable depletion of brain iron. Importantly, the attainment of sensorimotor skills associated with these monaminergic systems was similarly retarded prior to brain iron depletion. These modest delays in development of sensorimotor behaviors are consistent with delays observed in iron deficient human infants at analogous develop-

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mental stages [11,24]. The findings provide strong evidence for a causal brain–behavior relationship involving the monoamine system in early iron deficiency. Iron deficiency, induced early in lactation, results in severe reductions in brain iron at mid-lactation (P14) with continued worsening in brain iron content by weaning at P21 [6,12]. The period of rapid brain growth from P4- to mid-lactation and on to P21 was the chief component in the design of prior studies and indicated that commencing severe iron deficiency during this rapid period of growth resulted in dramatic reductions in brain iron [5,12]. In contrast, the current gestational model started at early gestation and continuing through lactation results in normal brain iron concentrations at mid-lactation despite substantial hematological deficits and severe reductions in liver iron content. Different patterns can be attributed to differences in the timing, dose (degree) and/or duration of iron deficiency [39]. Thus, despite depletion of liver iron and a moderate anemia, these developing rat pups did not deplete brain iron in many regions until the end of lactation. This finding suggests that rats avidly maintain brain iron uptake across the blood–brain barrier in the face of dwindling systemic supplies, and is consistent with up-regulation of the transferrin receptor and the iron transporter, DMT-1, in iron deficient rat pup brains [26]. Prioritization of iron among functional compounds as a result of diminution of iron delivery to cells takes place and is likely affected by the stage of organ growth [17,19,23,33,43]. The biological explanation for the ability of some brain regions to “hold on” to iron better than other regions, or perhaps, to acquire iron from other regions or the vasculature is not understood. It is likely imbedding in the ability of regions to up-regulate their iron acquisition proteins such as transferrin receptor, DMT-1 while also minimizing their obligatory losses of iron during a period of increased demands for regional growth and development. The iron deficient pups in the current protocol appear to be at the edge of the threshold in which brain iron is marginally preserved, despite dramatic reductions in liver and systemic iron status. As a result of this different model of inducing iron deficiency during lactation, the established relationship between lower brain iron and reduction in monoamine concentrations, DAT, and D2 R levels is not replicated [12]. Iron deficiency induced in gestation results in increased DA concentration, DA transporter level, and levels of metabolites at mid-lactation not the lower levels associated with ID commenced early in post-natal life. The substantial changes in the nigra-striatal and mesolimbic dopaminergic pathways prior to measurable changes in brain iron concentration may suggest yet another “sensor” of brain iron status. That is, an early “adaptive” stage of brain iron deficiency may have been missed in previous studies where protocols depleted the brain iron too rapidly for the detection of this stage. With prolonged depletion of body and brain iron to P25, we again observe the significant alterations in DA levels, DAT density that corresponds to lower brain iron concentrations [4,12,50]. The nature of this “compensation” for early ID at mid-lactation is not clear at the moment, but recent studies in the hippocampus demonstrate alterations in dendritic arborization with this dietary protocol [22,37]. It is possible the activity of the dopaminergic

neurons in terminal fields of P14 ID animal is increased in compensation for decreased cell numbers and synaptic plasticity. Cell culture experiments using iron chelators in neuroblastoma cells show periods of increased accumulation of dopamine in the intracellular space very soon after the chelator is added and precedes the subsequent loss in surface DA transporter and DA receptors (unpublished observations). The mechanism of effect of neuronal iron deficiency on the monoaminergic systems is unknown and may well involve multiple pathways. The present study documents that iron deficiency during gestation and lactation delays acquisition of sensorimotor behaviors. These delays were not universal, nor in all cases longlasting. For example, surface righting and negative geotaxis were again normal by P18 while auditory startle, bar hold, and forelimb placing remained below that of controls over the period of testing. Abnormal movement has been related to nigrostriatal and mesolimbic structures in iron deficiency during lactation and weaning [5,12,50,51] and both neurotransmitter and hypomyelination explanations were posited. We cannot, at this time, attribute any of the observed developmental delays directly to alterations in striatal or mesolimbic monoamine metabolism (e.g. this was not designed as an intervention study). However, alterations in striatal dopamine metabolism in early life are known to alter vibrissae forelimb placing [14,41]. The current experiment may have created a situation in which there was a transient “activation” of the dopaminergic system and DAlinked intracellular molecular events prior to the expected loss in expression of DAT, D2 R, DA, and serotonin [32]. The altered dopaminergic state at P10 may be related to the decreased vibrissae placing due to a transitory “activation–deactivation” to other aspects of brain biology [39,40]. Important neural structures such as the cerebellum that are fundamental to surface righting and negative geotaxis where the head position triggers a reaction to reposition the head and body relative to gravity and vertical position [8]. In addition, the delays in surface righting and negative geotaxis may indicate fundamental structural differences in the development of the vestibular system as noted in animal studies of micro and hypergravity conditions [8,16]. There have really been no concrete studies of structure and function relationships regarding iron deficiency and the vestibular system so only conjecture can be supplied at this time. Alterations in bioenergetics and myelination certainly exist in the iron deficient rodent brain and may also play a role in these delays [9,31,37]. None of these possibilities have been carefully examined yet relative to developmental iron deficiency. Future efforts may provide clues as to biologic causality for these behavioral developmental delays. The current report makes the new observation that perinatal iron deficiency does result in modest developmental of sensorimotor behaviors that are consistent with delays observed in human infants at analogous developmental stages [6,13]. Ongoing studies indicate that long-term behavioral effects of early mild iron deficiency emerge in adulthood. These include abnormalities in cognitive and sensorimotor function (Felt et al., Unpublished data). In a complex environment in which animals tend to compete for any advantage, developmental behavioral effects could either be exaggerated as they enter adulthood [14]

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or attenuated due to use-dependent neurobehavioral “therapy” [7,21,24]. Additionally, compensatory restoration of function could be fragile and vulnerable to later stress, drugs, or other events [14]. In conclusion, this protocol is designed to mimic the human condition where pre-conceptional maternal iron status is marginal, gestational iron intake is inadequate, and mothers are likely to remain mildly to moderately iron deficient while lactating. Our model suggests that this “moderate perinatal” iron deficiency has different consequences than previous studies that utilize a later, and more severe restriction in dietary iron, in their models. The differences exist with respect to brain iron depletion, alterations in neurochemistry, and behavioral sequelae. The iron deficient pups appear to engage compensatory mechanisms in the brain to preserve brain iron in mid-brain structures but this ultimately is overwhelmed by the lack of supply from the vasculature. Importantly, monoaminergic systems also seem to be sensitive to this looming deficit in iron in cell bodies and go through a period of elevation of expression of certain transporters and receptors prior to their loss of expression and the iron deficiency persists. But, the question remains: “is this a good model for the human condition”? Inter-species comparisons are complicated from the perspective that the rat fetus and pre-weanling pup have a rapid period of neurogenesis and iron demands relative to the developing human infant. The iron requirements far exceed that of the human infant during early development [37] but the timing of evaluation of brain biology at P10 and P25 were purposefully chosen to correspond to periods of very rapid neurogenesis relevant to human infant neurodevelopment [26]. The adaptive changes in dopamine biology that precede significant changes in brain iron content are, to our knowledge, a novel finding. Do such things happen in human infants? Some of the short term [3] and later effects [28] of early life iron deficiency in human infants certainly are consistent with this biology but do not exclude other possibilities as well. Follow-up reports will document the long term persistent behavioral and biological phenotypes of this model.

Acknowledgements The entire group of investigators participating in the Brain and Behavior in Early Iron Deficiency Program Project contributed to our thinking about the issues in this study. The work in this study and contributions of the authors are as follows. John Beard assisted in design, directed the analysis of all biologic variables, and was primary author of the manuscript. Barbara Felt designed the model, trained and supervised staff performing behavioral testing, conducted the behavioral data analysis, assisted in writing and interpretation. Tim Schallert assisted in the design of the behavioral testing, trained the staff at the University of Michigan for some behavioral tests, assisted in writing behavioral sections and interpretation. Maggie Burhans conducted all monoamine analysis and statistical analysis of the data. James Connor assisted in the design of the study. Michael Georgieff assisted in the design of the study, editing of the manuscript, and interpretation of the data.

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