Early-life iron deficiency persistently disrupts affective behaviour in mice

Abstract

Background/objective: Iron deficiency (ID) is the most common nutrient deficiency, affecting two billion people worldwide, including about 30% of pregnant women. During gestation, the brain is particularly vulnerable to environmental insults, which can irrevocably impair critical developmental processes. Consequently, detrimental consequences of early-life ID for offspring brain structure and function have been described. Although early life ID has been associated with an increased long-term risk for several neuropsychiatric disorders, the effect on depressive disorders has remained unresolved.

Materials and methods: A mouse model of moderate foetal and neonatal ID was established by keeping pregnant dams on an iron-deficient diet throughout gestation until postnatal day 10. The ensuing significant decrease of iron content in the offspring brain, as well as the impact on maternal behaviour and offspring vocalization was determined in the first postnatal week. The consequences of early-life ID for depression- and anxiety-like behaviour in adulthood were revealed employing dedicated behavioural assays. miRNA sequencing of hippocampal tissue of offspring revealed specific miRNAs signatures accompanying the behavioural deficits of foetal and neonatal ID in the adult brain.

Results: Mothers receiving iron-deficient food during pregnancy and lactation exhibited significantly less licking and grooming behaviour, while active pup retrieval and pup ultrasonic vocalizations were unaltered. Adult offspring with a history of foetal and neonatal ID showed an increase in depression- and anxiety-like behaviour, paralleled by a deranged miRNA expression profile in the hippocampus, specifically levels of miR200a and miR200b.

Conclusion: ID during the foetal and neonatal periods has life-long consequences for affective behaviour in mice and leaves a specific and persistent mark on the expression of miRNAs in the brain. Foetal and neonatal ID needs to be further considered as risk factor for the development of depression and anxiety disorders later in life.Key MessagesMarginal reduction of gestational alimentary iron intake decreases brain iron content of the juvenile offspring.Early-life ID is associated with increased depression- and anxiety-like behaviour in adulthood.Reduction of maternal alimentary iron intake during pregnancy is reflected in an alteration of miRNA signatures in the adult offspring brain.

Keywords: Iron deficiency; anxiety disorder; depression; hippocampus; miRNA profile; mouse behaviour.

Figure 1.

Paradigm for dietary iron restriction of pregnant mice. (A) Schematic depiction of the experimental timeline. No difference was detected in the (B) weight gain of mothers with an iron-deficient diet (IDD) (48 ppm) and iron-sufficient diet (ISD) (96 ppm) during pregnancy. (C) Litter size, (D) percentage of pups alive, and (E) average weight per pup did not differ between IDD and ISD treated animals on the day of delivery, as well as (F) average weight per pup on postnatal day 7 (P7) (n=38–43/group). All data are presented as mean ± S.E.M. Data were analysed using two-way ANOVA with repeated measures.

Figure 2.

Maternal alimentary iron restriction during pregnancy and lactation reduces the iron content in the pup brain. (A) Representative images of Prussian Blue staining of hippocampal sections of postnatal day 11 (PD11) offspring from mothers provided with iron-deficient diet (IDD) (48 ppm) or iron-sufficient diet (ISD) (96 ppm) during pregnancy and lactation. Scale bars: 200 µm, 10x magnification. (B) Quantification of iron levels in the hippocampus revealed a significant difference between IDD and ISD PD11 offspring (n=6/group). (C) Comparison of NeuN-positive cells per mm² indicated no difference in the amount of neuronal cells in the hippocampus of ISD and IDD offspring on PD11 (n=6/group). (D) Representative images of hippocampal sections with Prussian Blue staining in adult IDD and ISD offspring. Scale bars: 200 µm, 10x magnification. (E) Quantification showed no difference in hippocampal iron levels between IDD and ISD adult offspring (n=5/group). All data are presented as mean ± S.E.M. Data were analysed using two-way ANOVA with repeated measures; **p < 0.01.

Figure 3.

Reduced postnatal spontaneous maternal care behavior upon dietary ID during pregnancy in mice. Percentage of different (A) pup-directed (nursing, licking and grooming, and nest building) and (B) non pup-directed (self-grooming, sleeping, eating, and drinking) behaviours from postnatal day 1 (PD1) to PD6 of mothers provided with iron-deficient diet (IDD) (48 ppm) or iron-sufficient diet (ISD) (96 ppm) (n=8/group). IDD mothers showed significantly reduced licking and grooming behaviour but no other differences between groups were observed. (C) Exemplary depiction of a trace of recorded ultrasonic vocalizations (USVs) recorded from pups on PD4. (D) Mean number and (E) amplitude of USV calls of IDD and ISD pups on PD4 revealed no difference between groups (n=19/group). All data are presented as mean ± S.E.M. Data were analysed using mixed model ANOVA with repeated measures and Student’s t-test where appropriate; *p < 0.05.

Figure 4.

Increased depression-like behaviour in adult mice after early-life ID. Significant differences in behavioural despair in (A) the forced swim test in male and female and (B) the tail suspension test in male offspring from mothers provided with iron-deficient diet (IDD) (48 ppm) compared to iron-sufficient diet (ISD) (96 ppm) during pregnancy and lactation. (C) A significant decrease of time spent in the light compartment of the light/dark box in IDD male mice demonstrates increased anxiety-like behaviour. No differences between groups was found in anhedonic behaviour in the (D) sucrose preference test (percentage of sucrose preference), (E) exploratory activity in the open field test (total distance travelled) and (F) motor coordination in the rotarod (latency to fall off) (n=20–28/group). All data are presented as mean ± S.E.M. Data were analysed using two-way ANOVA; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

Figure 5.

Adult hippocampal neurogenesis is not altered upon after early-life ID. (A) Schematic depiction of the protocol timeline for the administration of BrdU ((+)-5’ Bromo-2’-deoxyuridine). (B) Representative images of BrdU staining in offspring of mothers provided with an iron-deficient diet (IDD) (48 ppm) or iron-sufficient diet (ISD) (96 ppm) during pregnancy and lactation (until postnatal day 10). Scale bars: 200 µm, 20x magnification. (C) Quantification of BrdU⁺ cells in the hippocampus (n=5–9/group), as well as (D) comparison of NeuN-positive cells per mm² in hippocampal areas indicated no difference between ISD and IDD animals (n=8/group). All data are presented as mean ± S.E.M. Data were analysed using two-way ANOVA and Student’s t-test where appropriate.

Figure 6.

The miRNA expression pattern in the adult hippocampus is modulated by early-life ID. (A) Results of miRNA sequencing in hippocampal tissue of offspring of mothers with an iron-deficient diet (IDD) (48 ppm) and iron-sufficient diet (ISD) (96 ppm) revealed 530 differentially expressed miRNAs (blue dots: q < 0.05). Only two miRNAs, miR 200a and miR 200b, had a log2-fold changes of ≥ + 1.5 or ≤ − 1.5. (B) TargetScanMouse (Version 8.0) revealed 2627 target genes of miR 200a and 1511 target genes of miR 200b. Of these, 253 genes were targeted by both miR 200a and miR 200b, depicted by a Venn diagram (Venny 2.1.0). (C) Results of an enrichment analysis of genes targeted by both miR 200a and miR 200b revealed the involvement in a variety of different biological processes (white bars; Enrichr, GO: Biological processes).