Severe Postnatal Iron Deficiency Alters Emotional Behavior and Dopamine Levels in the Prefrontal Cortex of Young Male Rats

Abstract

Iron deficiency in early human life is associated with abnormal neurological development. The objective of this study was to evaluate the effect of postnatal iron deficiency on emotional behavior and dopaminergic metabolism in the prefrontal cortex in a young male rodent model. Weanling, male, Sprague-Dawley rats were fed standard nonpurified diet (220 mg/kg iron) or an iron-deficient diet (2–6 mg/kg iron). After 1 mo, hematocrits were 0.42 ± 0.0043 and 0.16 ± 0.0068 (mean ± SEM; P < 0.05; n = 8), liver nonheme iron concentrations were 2.3 ± 0.24 and 0.21 ± 0.010 μmol/g liver (P < 0.05; n = 8), and serum iron concentrations were 47 ± 5.4 and 23 ± 7.1 μmol/L (P < 0.05; n = 8), respectively. An elevated plus maze was used to study emotional behavior. Iron-deficient rats displayed anxious behavior with fewer entries and less time spent in open arms compared to control rats (0.25 ± 0.25 vs. 1.8 ± 0.62 entries; 0.88 ± 0.88 vs. 13 ± 4.6 s; P < 0.05; n = 8). Iron-deficient rats also traveled with a lower velocity in the elevated plus maze (1.2 ± 0.15 vs. 1.7 ± 0.12 cm/s; P < 0.05; n = 8), behavior that reflected reduced motor function as measured on a standard accelerating rotarod device. Both the time on the rotarod bar before falling and the peak speed attained on rotarod by iron-deficient rats were lower than control rats (156 ± 12 vs. 194 ± 12 s; 23 ± 1.5 vs. 28 ± 1.6 rpm; P < 0.05; n = 7–8). Microdialysis experiments showed that these behavioral effects were associated with reduced concentrations of extracellular dopamine in the prefrontal cortex of the iron-deficient rats (79 ± 7.0 vs. 110 ± 14 ng/L; P < 0.05; n = 4). Altered dopaminergic signaling in the prefrontal cortex most likely contributes to the anxious behavior observed in young male rats with severe iron deficiency.

Introduction

Iron deficiency is the most common single-nutrient deficiency disease in the world. The greatest prevalence of iron deficiency is found in infants between 6 and 24 mo of age, a stage when rapid brain growth occurs and cognitive and motor skills are developing. Among the numerous biological effects of iron, it is known that iron sufficiency is important for normal neurological function and iron deficiency during infancy, and early childhood is associated with diminished mental, motor, and behavioral functions (1). The effects of iron deficiency in infancy persist even after reversal of anemia with iron treatment (2–6).

Brain iron concentrations are highest in the substantia nigra, globus pallidus, nucleus caudate, red nucleus, and putamen, suggesting that the rapid accumulation of iron observed during the development of the nervous system may participate in behavioral organization (7). The biological basis for functional deficits of iron deficiency have been considered mainly in 3 areas: morphology, neurochemistry, and bioenergetics (8). Iron deficiency in early development affects brain myelination and consequently behavioral development (5, 9–13). Iron is required for the development and function of many neuronal circuits, especially the monoaminergic pathways (6, 14–22). Iron deficiency has also been linked to alterations in γ-amino butyric acid-mediated neurotransmission (20). Metabolism is affected by iron concentrations (23), e.g., the metabolic activity of cytochrome oxidase is reduced in prenatal iron deficiency, thus altering the generation and utilization of metabolic energy in the hippocampus (24).

Given the prevalence of iron deficiency in infants and the susceptibility of the developing brain to nutrient factors, understanding the neurochemical and behavioral effects of iron deficiency is critical to evaluate the metal’s role in mental health. Numerous animal studies have addressed the relationship between iron status and behavior. As early as 1972, decreased activity and disturbed diurnal rhythm were reported in iron-deficient rats (25). Many subsequent studies have consistently shown that iron-deficient animals are hypoactive (26–30). Iron deficiency also was reported to alter the response to environmental stimuli, stereotypic behavior, and exploratory behavior (22, 29, 31–34). Iron-deficient animals display deficits in fear conditioning (35, 36) and impaired spatial memory (6, 34). Other behavioral studies of emotion have led to inconclusive results; Beard et al. observed increased anxiety (37), whereas Eseh and Zimmerberg (38) reported no effect in adult rats with previous iron deficiency. In this investigation, we examined different aspects of behavioral function in the same cohort of young male rats with severe iron deficiency. Changes in neurotransmitter, receptor, and transporter concentrations were also explored with a focus on the dopaminergic pathway in the prefrontal cortex.

Materials and Methods

Animals and diets.

Animal protocols were approved by the Harvard Medical Area Animal Care and Use Committee. Male Sprague Dawley rats were purchased from Taconic (Taconic Farms) at 3 wk old. Upon receipt, rats were weighed and ranked in order of descending weight. Alternate rats were assigned to be fed either an iron-deficient diet containing 2–6 mg Fe/kg (TD99397, Harlan Teklad) or a control diet containing 220 mg Fe/kg (PicoLab 5053, LabDiet) (Supplemental Table 1). The latter was higher than typically considered adequate (35 mg Fe/kg). Other micronutrients known to influence behavior were present at adequate concentrations in the iron-deficient diet, e.g. zinc (36 mg/kg) and vitamin A (1.7 μg/g). Although higher concentrations of micronutrients were present in the control diet, their ratio to iron was still lower compared to the iron-deficient diet. Impaired behavior due to high iron or interactions with other micronutrients might be reflected in the control rats. In a previous neurobehavioral study of high dietary iron, Sobotka et al. (39) used the same reference level (35 mg Fe/kg) to compare behavior in rats fed higher iron diets of 350, 3500, and 20,000 mg/kg. Significant impairment was observed only at 20,000 mg Fe/kg, nearly 100 times greater than the concentration used in our experiments.

Rats were maintained on a 12-h-light/-dark cycle and consumed reverse osmosis deionized water ad libitum. Daily food consumption by the iron-deficient group was measured and the amount of food provided to the control group was adjusted to 120–125% to maintain the same body weight between 2 groups (pair-feeding). Behavioral tests were performed between postnatal d 45 and 52. At the end of the study, rats were killed by isoflurane overdose followed by exsanguination for collection of blood, liver, and brain tissues to analyze hematocrit, nonheme iron, and neurochemistry.

Elevated plus maze.

Emotional behavior was tested using an elevated plus maze (EPM, Harvard Apparatus). The apparatus was composed of 2 open arms (45 L × 10 W cm) and 2 closed arms (45 L × 10 W × 50 cm, height of wall). The EPM was 66 cm from the floor. At postnatal d 45, each rat was placed in the center of the maze, facing one of the open arms, and allowed to explore the maze for 5 min. The light intensity was dimmed and the test area was enclosed by black curtains. The latency of the first entry into an open arm, number of entries, and time spent in the center, open, and closed arms were recorded by a CCD camera (Sony Color Camera, 1.3 Lens, 0.7 Lux) and analyzed by Smart software v2.5 (Panlab, Harvard Apparatus). The EPM was cleaned with Quatricide TB (Phamacal Research Laboratories) between each animal test.

Rotarod.

Motor function was assessed with a standard accelerating rotarod device (Panlab, Harvard Apparatus), which consisted of a motor-driven knurled nylon cylinder 6 cm in diameter mounted horizontally 35.5 cm above a padded surface. Before the test, rats were trained on the rotarod with 3 sessions/d, 3 min/trial, with 5-min inter-trial intervals, at fixed speeds after habituation on the stationary rotarod for 1 min. Day 1 training speeds were 4, 7, and 10 rpm; d 2 training speeds were 7, 10, and 13 rpm; and d 3 training speeds were 10, 13, and 16 rpm. On d 4, rats of postnatal d 52 were tested on the rotarod with accelerating speed from 4 to 40 rpm over 5 min or until the rats fell off. Each rat was tested on the rotarod twice with a 10-min inter-trial interval. The rotarod device was cleaned using Quatricide TB between trials. Time on bar and speed attained on the rotarod before falling were recorded and the best score of each rat between 2 trials was used for analysis.

Nonheme iron analysis.

Nonheme iron in plasma and tissues was spectrophotometrically determined and expressed as μmol iron/g of wet tissue weight.

Neurotransmitter analysis.

Brains were micro-dissected to obtain prefrontal cortex and immediately frozen in liquid nitrogen and stored at –80° until analysis. Total tissue neurotransmitter concentrations were measured as described (40) using a Shisedo Capcell PAK MG C18 column (50 × 1.5 mm, 3-μm particles, ESA) for separation and ESA Coulochem III for detection.

Microdialysis.

Rats were anesthetized with urethane (1 g/kg, i.p.) and placed in a stereotaxic apparatus (Stoelting). A mid-sagittal incision was made over the skull and a small hole was drilled to expose the dura on the right side to allow for implantation of a guide cannula for the subsequent insertion of a precalibrated 3-mm concentric microdialysis probe (CMA-12 Elite, CMA/Microdialysis) into the right prefrontal cortex (Bregma: AP+3.2 mm, LR–0.8, DV–6.0). Following probe implantation, artificial cerebrospinal fluid (K⁺ 2.7 mmol/L) was continuously perfused at a rate of 1 μL/min with a Harvard Apparatus infusion pump for 2 h to allow for stabilization of injury-mediated release of neurotransmitters, after which four 20-min baseline samples were collected, followed by sample collection for an additional 60–80 min after s.c. injection of d-amphetamine (1 mg/kg) to stimulate dopamine release. Microdialysates were collected in a protective solution to prevent oxidative degradation (41). Samples were stored at –80° until analyzed as described above.

Western-blot analysis of dopamine receptors and transporters.

Prefrontal cortex was microdissected and homogenized in 150 mmol/L NaCl, 0.5% NP-40, 50 mmol/L Tris-HCl, pH 7.5) in the presence of protease inhibitors (Complete Mini Roche). Samples (50 μg protein) were electrophoresed on a 4–15% gradient SDS-polyacrylamide gel (Biorad) and transferred to a polyvinylidene difluoride membrane (Millipore) in SDS-free transfer buffer (2 mmol/L Tris base, 15 mmol/L glycine, 20% methanol). The membrane was blocked in 5 mmol/L Tris base, 150 mmol/L NaCl, pH 7.4 containing 5% nonfat dry milk, and incubated in goat anti-human dopamine transporter (DAT) antibody (Santa Cruz; 1:100; Supplemental Fig. 1) or mouse anti-human dopamine D2 receptor (D2R) antibody (Santa Cruz, 1:100) in the same buffer containing 0.1% Tween 20 and 2% nonfat dry milk. As a control for loading, the immunoblot was also incubated with mouse anti-actin (MP Biomedicals; 1:10,000). After washing, blots were incubated with IRDye800-conjugated secondary antibody (Li-COR, 1:10,000) and scanned using an Odyssey Infrared Imaging system (Li-COR). Relative intensities of protein bands normalized to actin were determined using Odyssey version 2.1 software.

Statistical analysis.

Data shown are the means ± SEM and based on power calculations, 6–8 rats/group were used for behavioral studies. Comparisons between groups were performed with either Student’s t test or Wilcoxon’s Rank Sum test as appropriate (NCSS 2004 and PASS 2005). Differences were considered significant at P < 0.05.

Results

Body weights, hematocrit, and nonheme iron concentrations.

Young male rats (postnatal d 52) were fed a control (220 mg Fe/kg) or iron-deficient (2–6 mg Fe/kg) diet for 1 mo. Body weights and brain weights did not significantly differ from pair-fed controls (Table 1). Hematocrit and nonheme iron concentrations in serum, liver, and brain were 51–92% lower in rats fed the low-iron diet than in controls (P < 0.05).

TABLE 1.

Characteristics of control and rats fed iron-deficient diet for 4 wk (postnatal d 52)¹

	Control	n	Iron-deficient	n
Body weight, g	221 ± 6.4	8	223 ± 12	8
Brain weight, g	1.9 ± 0.042	8	1.9 ± 0.061	8
Hematocrit, volume fraction	0.42 ± 0.0043	8	0.16 ± 0.0068†	8
Liver nonheme iron, μmol/g	2.3 ± 0.24	8	0.21 ± 0.010*	8
Serum nonheme iron, μmol/L	47 ± 5.4	8	23 ± 7.1*	8
Brain nonheme iron, μmol/g	0.19 ± 0.026	4	0.12 ± 0.012*	4

¹Values are means ± SEM. †Different from control, < 0.05 (Wilcoxon's Rank Sum test); *different from control, P < 0.05 (2-tailed Student's t test).

Elevated plus maze.

To compare the emotional behavior of iron-deficient and control rats, an elevated plus maze was used to test anxiety beginning at postnatal d 45. Rats fed the low-iron diet had fewer entries into open arms and spent less time in open arms (Fig. 1). Iron-deficient rats traveled with a lower velocity (P = 0.021) (Table 2).

FIGURE 1.

Elevated plus maze (A) and standard accelerating rotarod (B) tests of behavior and motor coordination of rats fed a control or iron-deficient diet for 4 wk on postnatal d 45 and 52, respectively. Data are mean ± SEM, n = 8. *Different from control, P < 0.05 (Wilcoxon’s Rank Sum test).

TABLE 2.

Behavior in elevated plus maze of rats fed control and iron-deficient diet (postnatal d 45)¹

	Control	Iron-deficient
Entries, n
Open arm	1.8 ± 0.62	0.25 ± 0.25†
Closed arm	9.5 ± 1.1	5.3 ± 1.0*
Central area	11 ± 1.1	4.4 ± 1.0*
Total	22 ± 2.3	9.9 ± 2.2*
Time, s
Open arm	13 ± 4.6	0.88 ± 0.88†
Closed arm	256 ± 10	265 ± 10
Central area	31 ± 5.6	34 ± 9.4
Latency, s	153 ± 56	264 ± 36

¹Values are mean ± SEM, = 8. †Different from control, P < 0.05 (Wilcoxon's Rank Sum test); *different from control, P < 0.05 (2-tailed Student's t test).

Rotarod.

To evaluate the effect of iron deficiency on motor function, rats were tested at postnatal d 52 on an accelerating rotarod. Iron-deficient rats fell off the rotarod sooner than control rats (P < 0.05) (Fig. 1B). The peak speed attained on the rotarod by iron-deficient rats was less than that attained by control rats (P < 0.05) (Fig. 1B).

Neurotransmitter concentrations in the prefrontal cortex.

Tissue serotonin in the prefrontal cortex did not differ between control (374 ± 18 ng/g) and iron-deficient rats (459 ± 50 ng/g, P = 0.19; n = 4/group). Tissue dopamine also did not differ between control (107 ± 46 ng/g) and iron-deficient rats (61 ± 15 ng/g, P = 0.40; n = 4/group). Microdialysis experiments demonstrated resting concentrations of extracellular dopamine were significantly lower in iron-deficient rats compared to controls (Fig. 2A). When stimulated by amphetamine, extracellular dopamine in the prefrontal cortex increased to the same extent for control (5.5-fold) and iron-deficient rats (5.3-fold); extracellular dopamine remained lower in iron-deficient rats since the basal resting concentration was reduced compared to controls. No significant changes in dopamine transporters or dopamine receptors were detected by Western-blot analysis of prefrontal cortex lysates (Fig. 2B).

FIGURE 2.

Microdialysis measurements of extracellular resting and stimulated dopamine (A) and Western-blot analysis of DAT and D2R (B) in prefrontal cortex of rats fed a control iron-deficient diet for 4 wk. Data are mean ± SEM, n = 4. *Different from control, P < 0.05 (Wilcoxon’s Rank Sum test). Western blot shows 3 representative samples with similar results observed for 7–8 different rats in each group. D2R, dopamine D2 receptor; DAT, dopamine transporter.

Discussion

The influence of postnatal iron deficiency on emotional behavior in young male rats was examined. Human studies suggest that anxiety-driven behavior is sensitive to poor iron status. Lozoff et al. (42) observed that anemic infants afflicted with iron deficiency display “increased fearfulness” even after treatment with iron therapy. Deinard et al. (43) showed that infants with even marginal iron deficiency display excessive fearfulness. Beard et al. (37) explored such behavior in a study of young iron-deficient rats at 6 wk of age and observed anxiety-related activities in light/dark box measures of distance traveled, repeated movements, center time, number of nose-pokes, and habituation to a novel environment. These investigators observed that iron-deficient rats moved more rapidly into dark compartments, although the time spent in dark compared to light did not differ from that of control (iron replete) rats. The iron-deficient rats also spent less time in the center of the box and such observations led to the proposal that the young rat is a model organism for behavioral studies to explore the influence of iron deficiency on brain function (37), particularly given the similarity of behaviors observed in iron-deficient children (42, 44). In this study, the elevated plus maze model was used to test for anxious behaviors based on avoidance of open and elevated spaces. Our results strongly support the idea that young male rats with severe iron deficiency display anxious behavior.

Eseh and Zimmerberg (38) also tested the behavioral influences of iron deficiency using ultrasonic vocalization to study rat pups born to iron-deficient or control dams. Although iron deficiency was associated with more distress calls, when later supplemented with iron, these rat pups did not have anxious responses. We have not tested whether anxious behavior could be reversed by iron supplementation and because our observations were made for postnatal iron deficiency, it is not appropriate to compare these results with observations reported for gestational and/or lactational iron deficiency. Many studies have shown that the period of development during which nutritional deficiency occurs later affects both behavioral and physiological endpoints (45). Interestingly, anxious responses in the elevated plus maze also have been observed in adult rats receiving daily i.p. injections of iron (46). Other behavioral impairments have been reported with dietary iron loading with carbonyl iron diet containing 20,000 mg Fe/kg (39). Clearly, imbalanced iron metabolism plays a critical role in anxiety and emotional behavior.

Another notable observation from our study is that iron-deficient rats also traveled in the elevated plus maze at a significantly lower speed, a result correlating with decreased physical activity (25, 28). Motor performance was directly tested in our study to confirm reduced activity due to iron deficiency. Testing on a standard accelerating rotarod device showed impaired motor function. Decreased physical activity and impaired skeletal motor activity along with reduced motivation caused by iron deficiency are observed in both human and animal studies (25, 27–29, 37, 44, 47–52). Iron deficiency may potentially affect many aspects related to motor function, including but not limited to neurochemistry, skeletal muscle strength, energy production, and oxygen supply. Behavioral alterations in motor performance could reflect the sum of these influences and contribute to the anxious behavior observed in the iron-deficient rats.

Many reports have demonstrated that iron deficiency affects monoaminergic pathways in the brain (53). The anxious behavior and motor dysfunction observed would reflect impaired dopaminergic functions. Iron deficiency has been associated with reduced dopamine receptor densities, but we are only aware of one study that reported findings in the prefrontal cortex (37) showing that D2R but not dopamine D1 receptor densities were reduced as measured by radioactive tracer binding. Western-blot analysis used in our study did not detect any difference in D2R between iron-deficient and control rats. Beard et al. (37) studied rats of similar age (~50 d) to our study cohort and a similar level of diet-induced postnatal iron deficiency was achieved, so the discordant data may simply reflect the different approaches used to detect D2R. Our results suggest that emotional behavior reflects changes in the neurotransmitter dopamine, which was reduced in iron-deficient rats. Although the fold-increase was similar to the amphetamine-evoked response observed in control rats, extracellular concentrations of the neurotransmitter were lower in iron-deficient rats.

To our knowledge, the present study is the first to examine the influence of iron deficiency on extracellular dopamine concentrations in the prefrontal cortex. Previous investigations using microdialysis determined extracellular dopamine increased in the caudate putamen of iron-deficient rats (17, 53). These observations correlated with decreased DAT (18), suggesting that dopamine uptake was affected by iron deficiency in this particular brain region. In contrast, we did not observe decreased DAT in the prefrontal cortex by Western-blot analysis. These combined observations may reflect differential regulation of the transporter by iron in different brain regions. Tissue serotonin in the prefrontal cortex of the iron-deficient rats was similar to controls. The fact that extracellular dopamine is significantly lower compared to control rats prompts the notion that neurotransmitter available for evoked release may be limiting in the prefrontal cortex of iron-deficient rats. Iron is a cofactor for tyrosine hydroxylase, a key enzyme in dopamine synthesis; whether its activity is specifically affected in the prefrontal cortex during iron deficiency must be better explored (54).

Male rats were used in this study, because estrogen effects on dopaminergic function in the context of iron deficiency have been reported (55). Establishing specific outcomes of the influence of iron deficiency in the prefrontal cortex will allow future study of gender effects. Whether iron repletion can reverse the behavioral influence of iron deficiency is another important question [reviewed in (56)]. We chose to induce postnatal iron deficiency in 21-d-old weanling rats, since newborn pups have immature development compared to humans with considerable and rapid brain growth until the weaning stage. Our study design was such that weanlings were fed iron-restricted diet to establish a cohort of young male rats (45–52 d old) with severe iron deficiency for behavioral and functional experiments after 4 wk. Future studies should explore supplementation to better understand how iron therapy influences the functional effects observed in the prefrontal cortex.