Low brain iron effects and reversibility on striatal dopamine dynamics.

Iron deficiency (ID) in rodents leads to decreased ventral midbrain (VMB) iron concentrations and to changes in the dopamine (DA) system that mimic many of the dopaminergic changes seen in RLS patient where low substantia nigra iron is a known pathology of the disease. The ID-rodent model, therefore, has been used to explore the effects that low VMB iron can have on striatal DA dynamics with the hopes of better understanding the nature of iron-dopamine interaction in Restless Legs Syndrome (RLS). Using a post-weaning, diet-induced, ID condition in rats, the No-Net-Flux microdialysis technique was used to examine the effect of ID on striatal DA dynamics and it reversibility with acute infusion of physiological concentrations of iron into the VMB. This study replicated prior findings by showing that the ID condition is associated with increased extracellular striatal DA, reduced striatal DA uptake, and blunted DA-2-receptor-agonist feedback enhancement of striatal DA uptake. Despite the increase in extracellular striatal DA, intracellular striatal DA, as determined in tissue homogenates, was decrease in the ID rat. The study's key finding was that an infusion of physiological concentrations of iron into the VMB reversed the ID-induced increase in extracellular striatal DA and the ID-induced decrease in intracellular striatal DA but had no effect on the ID-induced changes in DA uptake or on the blunted DA-uptake response to quinpirole. In summary, the ID-rodent model provides highly reproducible changes in striatal DA dynamics that remarkably parallel dopaminergic changes seen in RLS patients. Some but not all of these ID-induced changes in striatal DA dynamics were reversible with physiological increases in VMB iron. The small changes in VMB iron induced by iron infusion likely represent biologically relevant changes in the non-transferrin-bound labile iron pool and may mimic circadian-dependent changes that have been found in VBM extracellular iron.

In male rats with concurrent iron and (n-3) fatty acid deficiency, provision of either iron or (n-3) fatty acids alone alters monoamine metabolism and exacerbates the cognitive deficits associated with combined deficiency.

Concurrent deficiencies of iron (Fe) (ID) and (n-3) fatty acids [(n-3)FAD)] in rats can alter brain monoamine pathways and impair learning and memory. We examined whether repletion with Fe and DHA/EPA, alone and in combination, corrects the deficits in brain monoamine activity (by measuring monoamines and related gene expression) and spatial working and reference memory [by Morris water maze (MWM) testing] associated with deficiency. Using a 2 × 2 design, male rats with concurrent ID and (n-3)FAD [ID+(n-3)FAD] were fed an Fe+DHA/EPA, Fe+(n-3)FAD, ID+DHA/EPA, or ID+(n-3)FAD diet for 5 wk [postnatal d 56-91]. Biochemical measures and MWM performance after repletion were compared to age-matched control rats. The provision of Fe in combination with DHA/EPA synergistically increased Fe concentrations in the olfactory bulb (OB) (Fe x DHA/EPA interaction). Similarly, provision of DHA/EPA in combination with Fe resulted in higher brain DHA concentrations than provision of DHA alone in the frontal cortex (FC) and OB (P < 0.05). Dopamine (DA) receptor D1 was upregulated in the hippocampus of Fe+DHA/EPA rats (fold-change = 1.25; P < 0.05) and there were significant Fe x DHA/EPA interactions on serotonin (5-HT) in the OB and on the DA metabolite dihydroxyphenylacetic acid in the FC and striatum. Working memory performance was impaired in ID+DHA/EPA rats compared with controls (P < 0.05). In the reference memory task, Fe+DHA/EPA improved learning behavior, but Fe or DHA/EPA alone did not. These findings suggest that feeding either Fe or DHA/EPA alone to adult rats with both ID and (n-3)FAD affects the DA and 5-HT pathways differently than combined repletion and exacerbates the cognitive deficits associated with combined deficiency.

Combined deficiency of iron and (n-3) fatty acids in male rats disrupts brain monoamine metabolism and produces greater memory deficits than iron deficiency or (n-3) fatty acid deficiency alone.

Deficiencies of iron (Fe) (ID) and (n-3) fatty acids (FA) [(n-3)FAD] may impair brain development and function through shared mechanisms. However, little is known about the potential interactions between these 2 common deficiencies. We studied the effects of ID and (n-3)FAD, alone and in combination, on brain monoamine pathways (by measuring monoamines and related gene expression) and spatial working and reference memory (by Morris water maze testing). Using a 2 × 2 design, male rats were fed an ID, (n-3)FAD, ID+(n-3)FAD, or control diet for 5 wk postweaning (postnatal d 21-56) after (n-3)FAD had been induced over 2 generations. The (n-3)FAD and ID diets decreased brain (n-3) FA by 70-76% and Fe by 20-32%, respectively. ID and (n-3)FAD significantly increased dopamine (DA) concentrations in the olfactory bulb (OB) and striatum, with an additive 1- to 2-fold increase in ID+(n-3)FAD rats compared with controls (P < 0.05). ID decreased serotonin (5-HT) levels in OB, with a significant decrease in ID+(n-3)FAD rats. Furthermore, norepinephrine concentrations were increased 2-fold in the frontal cortex (FC) of (n-3)FAD rats (P < 0.05). Dopa decarboxylase was downregulated in the hippocampus of ID and ID+(n-3)FAD rats (fold-change = -1.33; P < 0.05). ID and (n-3)FAD significantly impaired working memory performance and the impairment positively correlated with DA concentrations in FC (r = 0.39; P = 0.026). Reference memory was impaired in the ID+(n-3)FAD rats (P < 0.05) and was negatively associated with 5-HT in FC (r = -0.42; P = 0.018). These results suggest that the combined deficiencies of Fe and (n-3) FA disrupt brain monoamine metabolism and produce greater deficits in reference memory than ID or (n-3)FAD alone.

Iron deficiency alters the day-night variation in monoamine levels in mice.

Monoamine metabolism in the central nervous system is altered by dietary iron deficiency, with a stronger effect seen during the active than rest span of the circadian cycle. In this report, we examined changes in intracellular and extracellular monoamine levels, synthetic enzymes, transporter and receptor densities, and responses to amphetamine-induced dopamine (DA) efflux in iron-deficient and iron-sufficient mice. Extracellular striatal DA levels were 15-20% higher in all groups during the active dark phase compared to the inactive light phase, with correspondingly lower dopamine transporter (DAT) and higher tyrosine hydroxylase levels. Iron deficiency decreased DAT density by 20% and 28% in the light and dark phases, respectively, and elevated the DOPAC/DA ratio only in the dark, indicating that iron deficiency does interact with the normal diurnal cues for cyclicity. Enhanced DA efflux after amphetamine stimulation indicates no limitation on monoamine synthesis and release and is consistent with altered synaptic efficacy and perhaps recycling of DA in iron deficiency. These experimental findings provide new evidence that brain iron insufficiency does have a differential effect on the DA system at different biological times of the day and night and may be causally related to the phasic motor symptoms observed in Restless Legs Syndrome.

Iron deficiency alters dopamine uptake and response to L-DOPA injection in Sprague-Dawley rats.

Iron deficiency (ID) disrupts brain dopamine (DA) and norepinephrine (NE) metabolism including functioning of monoamine transporters and receptors. We employed caudate microdialysis and no net flux (NNF) in post-weaning rats to determine if ID decreased the extraction fraction (E(d)). Five micromolar quinpirole, a dopamine D(2) receptor agonist, resulted in 80% decrease in extracellular DA and 45% higher E(d) in control animals. The D(2) agonist had no effect on E(d) in ID animals despite a reduction in basal DA. DAT mRNA levels were reduced by 58% with ID, while DAT protein in ventral midbrain and caudate and membrane associated DAT were also reduced by ID. Carbidopa/l-DOPA was administered to determine if elevated extracellular DA in ID was due to increased release. The DA response to l-DOPA in ID rats was 50% smaller and delayed, whereas the NE response was threefold higher. The caudate concentration of NE was also elevated in ID. Elevated dopamine-beta-hydroxylase activity in ID provides a tentative explanation for the increased NE response to l-DOPA. These experiments provide new evidence that ID results in altered synthesis and functioning of DAT and perhaps suggests some compensatory changes in NE metabolism.

Early postnatal iron repletion overcomes lasting effects of gestational iron deficiency in rats.

Iron deficiency anemia in early childhood causes developmental delays and, very likely, irreversible alterations in neurological functioning. One primary goal for the present study was to determine whether the effects of late gestational iron deficiency on brain monoamine metabolism, iron content, and behavioral phenotypes could be repaired with iron intervention in early lactation. Young pregnant rats were provided iron-deficient or control diets from mid-gestation (G15). At postnatal d 4 (P4), pups from iron-deficient dams were out-fostered either to other ID dams or control dams while pups of control dams were similarly fostered to other control dams. Dietary treatments continued to adulthood (P65) when brain iron and regional monoamines were evaluated. P4 iron repletion normalized body iron status, brain iron concentrations, monoamine concentrations, and monoamine transporter and receptor densities in most brain regions. Dopamine transporter densities in caudate and substantia nigra were lower in ID rats but were normalized with iron repletion. Serotonin transporter levels in most brain regions and open-field exploration were also normalized with iron repletion. The success of this approach of early postnatal iron intervention following iron deficiency in utero contrasts to a relative lack of success when the intervention is performed at weaning. These data suggest that a window of opportunity exists for reversing the detrimental effects of iron deficiency in utero in rats and provides strong support of intervention approaches in humans with iron deficiency during pregnancy.

Acoustic startle response is disrupted in iron-deficient rats.

Diurnal effects on motor control are evident in the human disease of Restless Leg Syndrome (RLS), which is purported to be linked to brain iron deficiency as well as alterations in dopaminergic systems. Thus, we explored the relationship between daily rhythms, the onset of motor dysregulation and brain iron deficiency in an animal model of iron deficiency. Male and female weanling Sprague-Dawley rats consuming control (CN) or iron-deficient (ID) diets were examined weekly for acoustic startle response (ASR) and prepulse inhibition (PPI) for a 5-week period. Iron deficiency reduced the magnitude, but not timing, of the ASR at specific time points. ASR was elevated 60% at the onset of the dark cycle relative to the median of the light cycle in male CN and ID rats. The respective elevation was 400% and 150% in female CN and ID rats during the first 2 weeks of testing. The diurnal cycle of ASR response was attenuated by 3 weeks of testing in both dietary treatment groups. PPI was not affected by iron deficiency, sex, diurnal cycle or the interaction between these factors. These results thus demonstrate that iron deficiency moderately alters ASR signaling although the inhibitory pathways of ASR do not appear to be affected.

Filtration disrupts synaptosomes during radiochemical analysis of serotonin uptake: comparison with chronoamperometry in SERT knockout mice.

Radiochemical methods have failed to reveal decreases in synaptosomal serotonin uptake in mice lacking one functional copy of the serotonin transporter (SERT) gene. By contrast, uptake rates determined by chronoamperometry in synaptosomes from SERT+/- mice show gene-related reductions. We revisited [(3)H]5-HT uptake in SERT knockout mice to determine the effects of inclusion of O(2) in the incubation buffer on the kinetic parameters obtained by this method. In oxygenated synaptosomes prepared from frontal cortex and striatum, modest 25 and 35% reductions in radiolabeled 5-HT uptake were detected in SERT+/- versus SERT+/+ mice. However, even in the presence of O(2), no differences in [(3)H]5-HT uptake were detected between SERT+/- and SERT+/+ mice in brain stem in contrast to 60% reductions determined by chronoamperometry. Moreover, while inclusion of O(2) modestly increased the rates of [(3)H]5-HT uptake, rates determined by chronoamperometry in the presence of O(2) were 40-fold greater than those determined radiochemically. We present evidence that the filtration process used in the radiochemical method leads to substantial loss of transported 5-HT resulting in lower apparent uptake rates. These findings explain the relative insensitivity of radiochemical methods for determining biologically important alterations in uptake such as those occurring between SERT+/- and SERT+/+ mice and in response to O(2).