This is the first study, to our knowledge, to investigate the effects of repleting male adult rats deficient in Fe and n-3 FA with ALA and Fe, alone and in combination, on brain monoamine concentrations and cognitive performance. The experimental model was chosen to reflect the common scenario in low-income countries where children born to mothers with poor n-3 FA status consume a diet low in Fe and n-3 FA throughout childhood and only begin to consume a diet sufficient in one or both nutrients in early adulthood as dietary quality and variety improves.
The provision of ALA to ID + n-3 FAD rats for 5 weeks (PND 56–91) significantly increased the relative composition of DHA in total phospholipid FA of different brain regions to ~ 50 - 70% of control values. In contrast, the provision of Fe resulted in near complete recovery of brain Fe (~90–100% of controls). In the Hip, the synergistic Fe × ALA interaction for higher DHA levels may be explained by Fe being a co-factor of hepatic desaturases and elongases, which are responsible for the conversion of ALA to EPA and DHA [26, 27]. Thus, providing ALA in combination with Fe to double-deficient rats might enhance the conversion of ALA into its respective long-chain derivative DHA. Whether this finding can be translated to the human situation is not clear, as previous studies have shown that, in contrast to humans, the conversion of ALA to EPA and DHA is more efficient in rats [19, 20]. Nevertheless, Smuts et al. showed that providing ID school children with iron-fortified soup not only improved Fe but also n-3 FA status .
Rats that remained ID during the depletion period showed a ~15% reduction in food intake, which was also reflected by ~15% lower body weight compared to the groups repleted with Fe. This is consistent with several previous studies reporting decreased food intake associated with poor growth in rats suffering from ID anaemia [12, 28–30]. However, previous depletion studies showed that severe ID decreased relative weight gain (g weight gain per g food intake) [12, 28], while in the current study, relative weight gain was the same in rats repleted with Fe and remaining ID. Since neurotransmitters, including DA and 5-HT, are involved in the regulation of food intake , it is possible that the reduced food intake of ID rats is related to alterations in monoaminergic neurotransmission. Also, the reduced body weight in ID rats may explain why swimming speed in the MWM was lower in ID + ALA and ID + n-3 FAD rats.
We previously showed that the provision of an ID + n-3 FAD diet for 5 weeks post-weaning resulted in an additive 1- to 2-fold increase in DA concentrations in the OB and Str, and decreased 5-HT concentrations relative to controls in the OB at PND 56 . In the current study, at PND 91, rats that remained ID + n-3 FAD had significantly higher DA concentrations than controls (+189%) in the FC. In contrast, DA concentrations in rats that received ALA and Fe, alone or in combination, did not differ from controls, consistent with our previous study in which DHA/EPA was provided alone and in combination with Fe . However, in contrast to our previous study, where we found a significant Fe × DHA/EPA interaction on the DA metabolite dihydroxyphenylacetic acid (DOPAC) in the OB and Str , we found no Fe × ALA interactions on DOPAC in the current study. However, we did find significant antagonistic Fe × ALA interactions on 5-HT in FC and OB, which suggest that the provision of Fe in combination with ALA to double-deficient rats affects 5-HT concentrations differently from the provision of Fe and ALA alone. The FC is one of the last brain areas to become fully mature and previous studies have reported dramatic increases in DA- and 5-HT-mediated neurotransmission in the FC during adolescence [32–34]. This may explain why most effects of Fe and ALA repletion and of continued ID + n-3 FAD depletion at PND 91 were observed in the FC. Several rodent studies have previously investigated whether alterations in monoaminergic neurotransmission caused by ID can be reversed by Fe repletion [35–40]. These studies varied in timing and severity of ID, in timing and dose of iron repletion, as well as in the brain regions observed. Generally, the effects of ID induced during gestation and pre-weaning have been shown to be persistent [35, 36, 39, 40]. Rodent studies using a model of post-weaning ID are limited. To our knowledge, to date, only one study has shown that alterations in extracellular DA in the caudate putamen caused by ID introduced post-weaning were mostly reversible by iron repletion . Also, few studies have investigated whether the detrimental effects of n-3 FAD (induced over two generations) on monoaminergic neurotransmission can be reversed with ALA repletion [42, 43]. Kodas et al. investigated synaptic levels of DA and 5-HT in basal conditions and after pharmacological stimulation in rats that were n-3 FA depleted and in rats that were repleted with an ALA sufficient diet starting at different time points (birth, PND 7, PND 14 or PND 21) [42, 43]. N-3 FA deficiency altered DA and 5-HT release in basal conditions and under stimulation in the rat Hip. The provision of ALA during the first two weeks of postnatal life reversed these alterations, while they persisted in rats that received the repletion diet only from PND 21.
Nonetheless, our results cannot be directly compared with the results of these studies, as the experimental rats in our study were deficient in both Fe and n-3 FA before receiving the repletion diets. Thus, the effects of Fe or n-3 FA repletion could have been confounded by the untreated deficiency. It is well known that neurotransmitter systems can adapt to chronic stressors, such as drug and alcohol exposure . This could explain why certain monoamines that were altered in the ID + n-3 FAD rats after the depletion study (PND 56) were no longer altered in the rats that remained ID + n-3 FAD during the repletion study (at PND 92). We speculate that providing double-deficient rats with one nutrient only affects the adapted system differently than the provision of both nutrients, e.g. by re-activating different mechanisms, which may explain the different effects on brain 5-HT concentrations when Fe and ALA were provided alone and in combination.
Disturbances in DA and 5-HT neurotransmission can impair learning and memory . The results from the MWM testing indicate that the provision of ALA alone to double-deficient rats significantly impairs working memory performance compared to age-matched controls. This finding is consistent with our previous study, where the provision of DHA/EPA alone also produced significant deficits in working memory performance compared to age-matched controls . The mechanism of this effect is uncertain. Spatial working memory is mainly hippocampal-dependent but is sub-served in part by the prefrontal cortex [45, 46]. Therefore, the working memory results might be explained by the differential effects of ALA on 5-HT in the FC when provided alone and in combination with Fe. There is evidence suggesting that 5-HT is involved in working and reference memory processes [45, 47]. Unfortunately, data on monoamine concentrations in the Hip are not available in the current study.
We consistently demonstrated that rats repleted with ALA or Fe alone showed no learning effect in the reference memory task, indicated by a lack of improvement in distance swum across trials and days to find the hidden platform. On the other hand, rats that received ALA in combination with Fe exhibited a marked improvement. This finding could again be attributed to the differential effects of ALA and Fe on 5-HT concentrations in the FC when provided alone or in combination. Consistently, a recent study reported that ID rats fed with Fe and a mixture of essential FA, containing equal amounts of LA and ALA, exhibited improved learning and memory performance in the MWM compared with controls, while the rats fed with Fe alone performed worse . In contrast to our previous study that provided DHA/EPA, alone and in combination , we did find that the provision of ALA significantly shortened overall distance moved to find the hidden platform during the reference memory task. However, this beneficial effect of ALA was only apparent in the Fe + ALA group, which swam a significantly shorter distance to find the platform than the groups that remained double-deficient or received Fe or ALA only. Since spatial learning and memory tested in the MWM is mainly hippocampus-dependent [49, 50], the higher relative composition of DHA in total phospholipid FA in the Hip of rats that were fed ALA in combination with Fe might explain the improved reverence memory performance in this group. Nevertheless, it may be that ALA exerted beneficial effects on cognitive performance independent of its conversion to EPA and DHA.
Since spatial working and reference memory is mainly hippocampal-dependent, it is a limitation of this study that monoamine concentrations were not analyzed in the Hip. Also, the assessment of other neurotransmitters, such as glutamate and gamma-aminobutyric acid, or other factors involved in learning and behaviour, such as myelination, neuronal inflammation, and processes of morphogenesis and cell growth, would have been valuable.