Early-life iron deficiency anemia alters the development and long-term expression of parvalbumin and perineuronal nets in the rat hippocampus.

Early-life iron deficiency anemia (IDA) alters the expression of critical genes involved in neuronal dendritic structural plasticity of the hippocampus, thus contributing to delayed maturation of electrophysiology, and learning and memory behavior in rats. Structural maturity in multiple cortical regions is characterized by the appearance of parvalbumin-positive (PV(+)) GABAergic interneurons and perineuronal nets (PNNs). Appearance of PV(+) interneurons and PNNs can serve as cellular markers for the beginning and end of a critical developmental period, respectively. During this period, the system progresses from an immature yet highly plastic condition, to a more mature and efficient state that is however less flexible and may exhibit poorer potential for recovery from injury. To test if fetal-neonatal IDA alters parvalbumin (PV) mRNA expression, protein levels, and the number of PV(+) interneurons and PNNs in the male rat hippocampus, pregnant dams were given an iron-deficient (ID) diet (3 mg iron/kg chow) from gestational day 2 to postnatal day (P) 7 and then placed on an iron-sufficient (IS) diet (198 mg/kg) for the remainder of the experiment. On this regimen, formerly ID animals become fully iron-replete by P56. Minimal levels of PV (mRNA and protein), PV(+) interneurons, and PNNs were found in IS and ID P7 rats. By P15, and continuing through P30 and P65, ID rats had reduced PV mRNA expression and protein levels compared to IS controls. While there were no differences in the number of PV(+) neurons at either P30 or P65, the percentage of PV(+) cells surrounded by PNNs was slightly greater in ID rats as compared to IS controls. The lower levels of these acknowledged critical period biomarkers in the ID group are consistent with studies that demonstrate later maturation of the acutely ID hippocampus and lower plasticity in the adult formerly ID hippocampus. The findings provide additional potential cellular bases for previously described electrophysiologic and behavioral abnormalities found during and following early-life IDA.

Gestational and neonatal iron deficiency alters apical dendrite structure of CA1 pyramidal neurons in adult rat hippocampus.

The hippocampus develops rapidly during the late fetal and early postnatal periods. Fetal/neonatal iron deficiency anemia (IDA) alters the genomic expression, neurometabolism and electrophysiology of the hippocampus during the period of IDA and, strikingly, in adulthood despite neonatal iron treatment. To determine how early IDA affects the structural development of the apical dendrite arbor in hippocampal area CA1 in the offspring, pregnant rat dams were given an iron-deficient (ID) diet between gestational day 2 and postnatal day (P) 7 followed by rescue with an iron-sufficient (IS) diet. Apical dendrite morphology in hippocampus area CA1 was assessed at P15, P30 and P70 by Scholl analysis of Golgi-Cox-stained neurons. Messenger RNA levels of nine cytoplasmic and transmembrane proteins that are critical for dendrite growth were analyzed at P7, P15, P30 and P65 by quantitative real-time polymerase chain reaction. The ID group had reduced transcript levels of proteins that modify actin and tubulin dynamics [e.g. cofilin-1 (Cfl-1), profilin-1 (Pfn-1), and profilin-2 (Pfn-2)] at P7, followed at P15 by a proximal shift in peak branching, thinner third-generation dendritic branches and smaller-diameter spine heads. At P30, iron treatment since P7 resulted in recovery of all transcripts and structural components except for a continued proximal shift in peak branching. Nevertheless, at P65-P70, the formerly ID group showed a 32% reduction in 9 mRNA transcripts, including Cfl-1 and Pfn-1 and Pfn-2, accompanied by 25% fewer branches, that were also proximally shifted. These alterations may be due to early-life programming of genes important for structural plasticity during adulthood and may contribute to the abnormal long-term electrophysiology and recognition memory behavior that follows early iron deficiency.

Delayed alternation performance in rats following recovery from early iron deficiency.

Early iron deficiency (ID) is one of the most common nutrient deficiencies in both developed and developing countries. This condition has been linked to perturbations in myelin formation, alterations of monoamine neurotransmitter systems particularly in the striatum, and deficits in energy metabolism particularly in the hippocampus (HP) and prefrontal cortex (PFC) in rats. Early ID has also been traced to long-term behavioral consequences in children in domains linked to these neuropathologies. The current experiment assesses formerly iron deficient (FID) adult rats on a delayed alternation (DA) task - a procedure thought to be sensitive to PFC dysfunction. Rat dams were started on an iron deficient chow at gestational day (G) 2 and maintained on this diet until postnatal day (P) 7; behavioral training began at P 65 when animals were iron replete. FID animals exhibited accelerated acquisition (p=0.002) and fewer errors (p=0.003) on the DA task compared to controls. These findings may reflect an imbalance between hippocampal and prefrontal modulation of this behavior most likely emanating from long-term hippocampal disinhibition by early ID that persists in spite of early iron treatment from P 7.

Effects of gestational iron deficiency on fear conditioning in juvenile and adult rats.

The hippocampus is especially sensitive to the effects of gestational and neonatal iron deficiency, even after iron repletion. This study compared the effects of iron deficiency, maintained from gestational day 2 to postnatal day (P)7, on "delay" and "trace" fear conditioning. Only the latter paradigm is critically dependent on the dorsal hippocampus. In different groups of rats, fear conditioning commenced either prior to puberty (P28 or P35) or after puberty (P56). Fear conditioning was measured using fear-potentiated startle. Both delay and trace fear conditioning were diminished by iron deficiency at P28 and P35. Hippocampal expression of the plasticity-related protein PKC-gamma was increased through trace fear conditioning, but reduced at P35 in the iron-deficient group. Trace fear conditioning was enhanced by prior iron deficiency in the P56 group. This unanticipated finding in iron-repleted adults is consistent with the effects of developmental iron deficiency on inhibitory avoidance learning, but contrasts with the persistent deleterious long-term effects of a more severe iron-deficiency protocol, suggesting that degree and duration of iron deficiency affects the possibility of recovery from its deleterious effects.

Influence of gestational age and fetal iron status on IRP activity and iron transporter protein expression in third-trimester human placenta.

Placental iron transport during the last trimester of pregnancy determines the iron endowment of the neonate. Iron transport is a function of the major iron transport proteins: transferrin receptor-1 (TfR-1) and ferroportin-1 (FPN-1). The mRNAs for TfR-1 and, potentially, FPN-1 are posttranscriptionally regulated by iron regulatory protein (IRP)-1 and IRP-2. We assessed the effect of gestational age and fetal iron status on IRP-1- and IRP-2-binding activity and on the localization and protein expression of TfR-1 and FPN-1 protein at 24-40 wk of gestation in 21 placentas obtained from iron-sufficient nonanemic mothers. Gestational age had no effect on cord serum ferritin concentration, IRP-2 RNA-binding activity, transporter protein location, and TfR-1 or FPN-1 protein expression. IRP-1 activity remained constant until full term, when it decreased (P = 0.01). Placental ferritin (r = 0.76, P < 0.001) and FPN-1 (r = 0.44, P < 0.05) expression increased with gestational age. Fetal iron status, as indexed by cord serum ferritin concentration, was inversely related to placental IRP-1 (r = -0.66, P < 0.001) and IRP-2 (r = -0.42, P = 0.05) activities. Placental ferritin protein expression correlated better with IRP-1 (r = -0.45, P = 0.04) than with IRP-2 (r = -0.35, P = 0.10) activity. Placental TfR-1 and FPN-1 protein expression was independent of fetal or placental iron status and IRP activities. Iron status had no effect on transport protein localization. We conclude that, toward the end of the third trimester of iron-sufficient human pregnancy, the placenta accumulates ferritin and potentially increases placental-fetal iron delivery through increased FPN-1 expression. IRP-1 may have a more dominant role than IRP-2 activity in regulating ferritin expression.

Iron deficiency alters iron regulatory protein and iron transport protein expression in the perinatal rat brain.

Iron plays an important role in numerous vital enzyme systems in the perinatal brain. The membrane proteins that mediate iron transport [transferrin receptor (TfR) and divalent metal transporter 1 (DMT-1)] and the iron regulatory proteins (IRP-1 and IRP-2) that stabilize their mRNAs undergo regional developmental changes in the iron-sufficient rat brain between postnatal day (P) 5 and 15. Perinatal iron deficiency (ID) affects developing brain regions nonhomogeneously, suggesting potential differences in regional iron transporter and regulatory protein expression. The objective of the study was to determine the effect of perinatal ID on regional expression of IRP-1, IRP-2, TfR, and DMT-1 in the developing rat brain. Gestationally iron-deficient Sprague Dawley rat pups were compared with iron-sufficient control pups at P10. Serial 12-mu coronal sections of fixed frozen brain from pups on P10 were assessed by light microscopy for IRP-1, IRP-2, DMT-1, and TfR localization. ID did not change the percentage of cells with positive staining for the four proteins in the choroid epithelium, ependyma, vascular endothelium, or neurons of the striatum. ID increased the percentage of neurons expressing the four proteins in the hippocampus and the cerebral cortex. Increased numbers of TfR- and DMT-1-positive cells were always associated with increased IRP-positive cells. The P10 rat responds to perinatal ID by selectively increasing the number of neurons expressing IRP-regulated transporters in brain regions that are rapidly developing, without any change at transport surfaces or in regions that are quiescent. Brain iron distribution during ID seems to be locally rather than globally regulated.

Perinatal iron deficiency alters apical dendritic growth in hippocampal CA1 pyramidal neurons.

Iron deficiency early in life is associated with cognitive disturbances that persist beyond the period of iron deficiency. Within cognitive processing circuitry, the hippocampus is particularly susceptible to insults during the perinatal period. During the hippocampal growth spurt, which is predominantly postnatal in rodents, iron transport proteins and their messenger RNA stabilizing proteins are upregulated, suggesting an increased demand for iron import during this developmental period. Rat pups deprived of iron during the perinatal period show a 30-40% decrease in hippocampal metabolic activity during postnatal hippocampal development. We hypothesized that this reduced hippocampal neuronal metabolism impedes developmental processes such as neurite outgrowth. The goals of the current study were to investigate the effects of perinatal iron deficiency on apical dendritic segment growth in the postnatal day (P) 15 hippocampus and to determine if structural abnormalities persist into adulthood (P65) following iron treatment. Qualitative and quantitative immunohistochemical analyses of dendritic structure and growth using microtubule-associated protein-2 as an index showed that iron-deficient P15 pups have truncated apical dendritic morphology in CA1 and a persistence of an immature apical dendritic pattern at P65. These results demonstrate that perinatal iron deficiency disrupts developmental processes in the hippocampal subarea CA1 and that these changes persist despite iron repletion. These structural abnormalities may contribute to the learning and memory deficits that occur during and following early iron deficiency.

Developmental changes in the expression of iron regulatory proteins and iron transport proteins in the perinatal rat brain.

The perinatal brain requires a tightly regulated iron transport system. Iron regulatory proteins (IRPs) 1 and 2 are cytosolic proteins that regulate the stability of mRNA for the two major cellular iron transporters, transferrin receptor (TfR) and divalent metal transporter-1 (DMT-1). We studied the localization of IRPs, their change in expression during perinatal development, and their relationship to TfR and DMT-1 in rat brain between postnatal days (PND) 5 and 15. Twelve-micron frozen coronal sections of fixed brain tissue were obtained from iron-sufficient Sprague-Dawley rat pups on PND 5, 10, and 15, and were visualized at 20 to 1,000x light microscopy for diaminobenzidine activity after incubation with specific primary IRP-1, IRP-2, DMT-1, and TfR antibodies and a universal biotinylated secondary and tertiary antibody system. IRP and transport protein expression increased in parallel over time. IRP1, IRP2, and DMT-1 were partially expressed in the choroid plexus epithelial cells at PND 5 and 10, and fully expressed at PND 15. The cerebral blood vessels and ependymal cells strongly expressed IRP1, IRP2, and DMT-1 as early as PND 5. Substantive TfR staining was not seen in the choroid plexus or ependyma until PND 15. Glial and neuronal expression of IRP1, IRP2, DMT-1, and TfR in cortex, hippocampal subareas and striatum increased over time, but showed variability in cell number and intensity of expression based on brain region, cell type, and age. These developmental changes in IRP and transporter expression suggest potentially different time periods of brain structure vulnerability to iron deficiency or iron overload.