This study examined the consequences of combined gestational and lactational iron deficiency on the metabolome of the developing striatum and on striatum-dependent behaviors. The dietary model produced changes in the metabolome, most of which were resolved by P37 with iron replenishment. The concurrent metabolite changes correlated with and accounted for the delayed acquisition of striatum-dependent behaviors. Yet, despite the correction of the striatal metabolome with iron treatment and the resolution of anemia, behavioral abnormalities persisted.
The metabolomic analysis demonstrated that, although the overall developmental trajectories of metabolites were similar in the 2 dietary groups, multiple metabolites were altered in the striatum due to iron deficiency. Whereas some of the metabolite changes were similar to those demonstrated in the ID hippocampus reported from our laboratory previously (16
), changes in others, especially those on P22, were similar to those described for the ID hippocampus exposed to chronic hypoxia (17
). Taken together, this suggests that brain iron deficiency, combined with potential hypoxia due to anemia, is likely responsible for the metabolomic alterations in the striatum of the ID group.
The metabolite changes demonstrate that markers of energy metabolism (phosphocreatine and creatine), energy substrates (glucose and lactate), amino acids, neurotransmitters (glutamate, γ-aminobutyric acid, and taurine), and markers of neuronal and glial integrity and myelination (glutamine, myo-inositol, and NAA) are altered in the striatum due to gestational and lactational iron deficiency. Alterations in multiple metabolites suggest that the effect of iron deficiency is pervasive and likely involves multiple biochemical pathways in the developing striatum.
The ID rats in this study had a delayed acquisition of striatum dependent behaviors. Whether these behavioral impairments are permanent is not known, because the assessment did not extend beyond P37. Recent studies using the same dietary model showed similar delayed acquisition of these striatal tasks (12
). These striatum-based behavioral changes were largely attributed to alterations in dopaminergic homeostasis based on an extensive literature demonstrating changes in dopamine concentration, receptor concentrations, and reuptake mechanisms in this dopamine-rich structure (9
). Furthermore, the behavioral phenotypes of ID animals are thought to closely resemble animals with lesions to dopaminergic neurons (13
This study showed that ~80% of the variability in the 2 tested behaviors was accounted for by concurrent alterations in metabolite markers of energy metabolism, glutamatergic neurotransmission, glial integrity, and myelination. These processes were also implicated in prior whole brain and hippocampal iron deficiency models (14
) but were not previously assessed in the striatum.
The phosphocreatine concentrations were, overall, higher in the ID group. This was accompanied by lower creatine concentrations on P22, resulting in an increased phosphocreatine:creatine ratio during the period of peak anemia. We propose that hypoxia due to severe anemia is responsible for the lower creatine in the ID striatum. This is supported by a similar decrease in creatine demonstrated in the hippocampus subjected to chronic hypoxia (17
) and the normalization of creatine with correction of anemia on P37 in the present study. The increased phosphocreatine and decreased creatine suggests that energy production needed for cellular structure and function, including myelin production and synaptic transmission, may be impaired in the ID striatum (31
As in our previous study of the hippocampus (16
), iron deficiency altered glutamate and glutamine concentrations in the striatum so that the glutamate:glutamine ratio was increased. We postulate that increased glutamate:glutamine ratio represents suppressed glutamatergic neurotransmission in the ID striatum. Glutamate released from the neurons during glutamatergic neurotransmission is taken up by the adjacent astrocytes, converted to glutamine, and transferred back to the neurons, where it is reconverted to glutamate. Glutamate uptake and conversion to glutamine in the astrocytes are energy-demanding processes (35
) that are likely to be compromised in the energy-limited ID striatum. As further discussed below, we propose that suppressed glutamatergic neurotransmission may be responsible for the altered dopamine metabolism demonstrated in the ID striatum, insofar as both neurotransmitters modulate each other’s action in the striatum (38
). Finally, increased glutamate and decreased glutamine concentrations may also suggest the potential for increased vulnerability to oxidative injury in the ID striatum. Similar alterations in glutamate and glutamine were demonstrated in the striatum of mice lacking H-ferritin, a model of iron deficiency and oxidative stress (39
The logistic regression model identified 2 concurrent metabolites, NAA and myo
-inositol, related to the decreased behavioral performance during iron deficiency. In addition, multiple regression analysis demonstrated that the striatal concentrations of glutamine, NAA, myo
-inositol, and GPC + PCho on P22 predicted 77-83% of the variance in the behavioral performance. This leads to the speculation that the striatal process most affected by gestational and lactational iron deficiency is oligodendroglial cell integrity and myelination, because the affected metabolites are markers of glial integrity and membrane phospholipid biosynthesis (40
). Human and rat studies have shown that brain iron is vital for myelination (14
). In addition, oligodendrocytes require iron for energy production and myelin synthesis (49
Higher levels of striatal NAA in ID rats could represent a defect in deacetylation of NAA required during myelin production. During rapid postnatal brain growth, the peak uptake of brain iron coincides with the period of peak myelinogenesis. Human studies have found poorer motor control and altered auditory brainstem responses in formerly ID infants (8
), both ascribed to impaired myelination. The results of our study may provide metabolomic evidence for a delay in myelin maturation.
Previous literature, including work from our own group, demonstrated that impairments in striatum-dependent behaviors in iron deficiency are associated with alterations in dopamine metabolism (12
). In the present study, ~80% of the observed behavioral changes from iron deficiency were attributable to metabolite alterations that did not include dopamine, because the latter cannot be measured by NMR spectroscopy. Without simultaneous assessment of dopaminergic metabolites, it is not possible to determine the relative roles of dopamine and nondopamine metabolites in accounting for these specific behavioral impairments, which are strongly driven by the monoamine system (13
). As proposed earlier, it is possible that the glutamatergic changes we observed in the striatum and the hippocampus (16
) interact with the striatal dopaminergic system. Extensive evidence demonstrates a complex interaction between the dopaminergic and glutamatergic systems in the striatum, nucleus accumbens, prefrontal cortex, and hippocampus (53
). For example, the hippocampal glutamatergic system affects the tonic state of the dopamine neurons in the ventral tegmental area (55
). Adequate glutamatergic hippocampal input to the striatum is important for maintaining striatal dopaminergic neurons in a potentiated state. With reduced neuronal output from the hippocampus (59
), characterized by altered glutamatergic homeostasis (16
), it is possible that changes in the glutamatergic system affect the likelihood with which striatal dopaminergic neurons fire (55
). In this study and in the previous one (16
), the neurotransmitter abnormalities on NMR spectroscopy appear as glutamatergic in the hippocampus (16
) and glutamatergic input to the striatum; however, the resultant downstream behavioral effects in the striatum (12
) appear to be dopaminergic.
In conclusion, iron deficiency induced metabolite changes that accounted for a large proportion of striatum-dependent behavioral changes. The observed changes were the ones expected to be affected by iron deficiency and included alterations in markers of energy metabolism, neurotransmission, neuronal and glial integrity, and myelination. Additional studies are necessary to determine whether these metabolite alterations are selective to the striatum or also occur in other brain areas in gestational and lactational iron deficiency. Hierarchically, changes in metabolite markers of myelination were the ones most prominently affected; however, the interaction of the glutamatergic and dopaminergic changes, as well as metabolite alterations that are not detected by conventional NMR methods, or other, as yet to be determined factors induced by brain iron deficiency, could also account for the observed behavioral abnormalities. Of potential clinical significance, correction of iron deficiency anemia by P37 normalized the striatal metabolome but not the behaviors, suggesting that the striatum underwent fundamental and perhaps irreversible changes due to iron deficiency during development.