The hippocampus, a structure critical for many forms of learning and memory, is particularly sensitive to the effects of prenatal and neonatal iron deficiency, and some of these effects persist long after brain iron levels have normalized (Jorgenson et al., 2003
; Jorgenson et al., 2005
). We hypothesized that prenatal/neonatal iron deficiency would affect rats’ subsequent ability to acquire trace fear conditioning, a form of learning that requires the dorsal hippocampus, while sparing delay fear conditioning, a form of learning that does not depend on the dorsal hippocampus. Unexpectedly, however, both delay and trace fear conditioning were impaired in preadolescent (P28 and P35) rats. These deficits did not extend into adulthood, by which time iron repletion was complete. In fact, at P56, the offspring of iron-deficient mothers actually showed enhanced trace fear conditioning compared to control rats. Thus, prenatal/neonatal iron deficiency produced a general impairment in fear conditioning in preadolescent rats, but a specific potentiation of dorsal hippocampus-dependent fear conditioning in early adulthood.
There are at least two plausible explanations for the attenuation of delay and trace fear conditioning seen at P28 and P35. The first is that the impairment was related to a disruption of amygdala function. At first blush this may appear unlikely, because amygdala metabolism is only mildly disturbed, at least at P10, by the same diet employed here (de Deungria et al., 2000
). However, it is worth noting that fear conditioning and memory consolidation are impaired by disruption of dopaminergic (Nader and LeDoux, 1999
) and noradrenergic neurotransmission (Hatfield and McGaugh, 1999
) respectively in the amygdala. Hence, even in the absence of direct metabolic disturbances in the amygdala, fear conditioning in this study may have been affected in this study via the well-documented effects of iron deficiency on the brains’ catecholamine systems (Burhans et al., 2005
The second possibility is that the impairment in both delay and trace conditioning was attributable to a loss of function in the ventral hippocampus. The rodent hippocampus can be divided into three rostrocaudal neuroanatomical segments that are segregated in terms of their inputs, outputs, and interconnectivity. Most investigations of the hippocampus’s role in fear conditioning, including trace fear conditioning, have focused on its septal (i.e., dorsal) portion, (Quinn et al., 2002
; Quinn et al., 2008
). However, it is the temporal (i.e., ventral) pole of the hippocampus that is most closely associated with the amygdala, and recent evidence indicates that damage to this area impacts fear conditioning in general (Bast et al., 2001
; Kjelstrup et al., 2002
; Richmond et al., 1999
; Trivedi and Coover, 2004
), including delay fear conditioning trained and tested using parameters similar to those of the current study (Burman et al., 2006
). Thus, fear conditioning is unlikely ever to occur entirely independently of the hippocampus, but rather independently of its septal pole. Because there is no reason to assume that iron deficiency differentially affects portions of the hippocampus along its rostrocaudal axis, it is quite plausible that delay fear conditioning is disrupted due to the effects of iron deficiency on ventral hippocampus function.
Although the involvement of the amygdala cannot be ruled out, the pattern of expression of PKC-gamma protein observed post-mortem in the P35 trace-conditioned animals is at least consistent with the possibility of hippocampal involvement in the observed fear conditioning deficits. Thus, expression of this plasticity-related protein was higher in trace-conditioned than in naïve, age-matched controls, but was reduced in previously iron deficient animals. While this study assessed PKC staining in the hippocampus because of the targeted nature of the behavior being tested, it is likely that PKC would be affected in other areas of the brain as well. To resolve the issue of the relative impact of iron deficiency on hippocampus-dependent versus amygdala-dependent memory, further research is required to establish whether these alterations in protein expression are specific to trace fear conditioning, and whether they occur in the amygdala, as well as in the hippocampus.
The recovery of fear conditioning seen in adult animals is congruent with the trajectory of brain iron and hematocrit content, which had reached normal levels by P56. Our behavioral data might suggest, therefore, that the impairments in learning seen at earlier time points were related to current iron status rather than to the long-term sequelae of metabolic dysfunction occurring during periods of gestation and infancy. In contrast, McEchron et al. (2005)
(McEchron et al., 2005
) found that late gestational and early-life iron deficiency impaired trace fear conditioning trained as late as P60, at which time point hematocrit levels were normal. Despite a number of differences between the two studies, their contrasting outcomes are most likely attributable to differences in iron deficiency regimens. Whereas the rats in the current study were returned to an iron-rich diet starting on P7, a low-iron diet was maintained in the earlier study until P31. This extension of iron deficiency throughout the juvenile preadolescent period may have been crucial in inducing long-term changes in proteins closely associated with differentiation and plasticity (Carlson et al., 2007
), and in dendritogenesis, which is maximal between P15 and P25 (Pokorny and Yamamoto, 1981
). In strikingly parallel findings, striatum-dependent learning is similarly spared in adult rats exposed to iron deficiency until P7 (Schmidt et al., 2007
), but not in adult rats exposed to iron deficiency throughout the period of late brain development. Taken together, the behavioral data derived from these disparate learning paradigms are consistent in suggesting that the cognitive deficits produced by prenatal iron deficiency may be recoverable through later iron supplementation, whereas the deficits induced through postnatal, extra-uterine iron deficiency are more liable to be irreversible.
An alternative explanation of the divergent effects of iron deficiency on trace fear in this and the McEchron et al (2005)
study is that a putative deficit in trace fear conditioning was mitigated by our adoption of more extensive fear conditioning procedures. Indeed, such a pattern has been reported in spatial learning (Schmidt et al., 2007
), where deficits in formerly ID rats were manifested only in the earliest phase of training. However, this interpretation could only account for an absence of differences in the relative strengths of delay and trace conditioning, but not for the frank enhancement of trace fear conditioning in previously ID adults that was observed in the current study.
The most striking aspect of the current findings was, perhaps, that trace fear conditioning was increased in formerly iron-deficient adult rats. Iron deficiency has a profound effect on brain, and specifically, hippocampal metabolism as evidenced by decreased cytochrome c oxidase (de Deungria et al., 2000
) and altered intracellular phosphocreatine:creatine ratio (Rao et al., 2003
) during the period of early iron deficiency. Others have postulated the induction of a relative hypothyroid state by iron deficiency (Beard et al., 1998
) that would also slow metabolic rate during development. This downregulation of metabolism during late fetal and early neonatal life may not only influence ongoing morphologic development and function of the brain while iron deficient but also induce long-term genomic and metabolic changes in a manner similar to how gestational protein-energy restriction that results in intrauterine growth restriction or choline administration during pregnancy modifies metabolism setpoints in the offspring for life (Gluckman and Hanson, 2004
; Meck and Williams, 2003
). These changes likely involve epigenetic mechanism of methylation and histone modification. While the effect of iron deficiency on methylation and acetylation of genes has not been specifically studied, there is circumstantial evidence that those processes are affected. For example, Carlson et al report in another article in this special issue that iron deficiency upregulates APP, Htatip, and appb1 expression in the hippocampus. The protein products of these genes combine to form a complex that translocates to the nucleus, inducing acetyl transferase activity (Cao and Sudhof, 2001
Iron is required to support highly metabolic activities such as dendritogenesis (Jorgenson et al., 2003
) and synaptic plasticity (Carlson et al., 2007
). The genes that regulate these processes are active in late gestation and early postnatal life and include those in the mTOR (mammalian target of rapamycin) pathway and vamp1 suggesting that they may be most malleable at that time (Carlson et al., 2007
). They are responsive to gestational iron status and their regulatory setpoints may be set during this time period, metabolically adapting to the low iron environment (Meck and Williams, 2003
). While there are other potential reasons for the enhanced performance of the formerly iron deficient animals at P56 in the current study, it is possible that iron treatment with standard amounts of iron actually represents a state of supplementation to a system trained in early life to function (albeit suboptimally) when far less iron is available. The result of provision of this critical metabolic substrate could be to enhance efficient energy production and thus synaptic plasticity to experience.
Interestingly, the unanticipated finding of stronger trace fear conditioning is actually consistent with an earlier literature showing an enhancement of inhibitory avoidance learning in previously iron deficient rats (Findlay et al., 1981
; Weinberg et al., 1979
; Weinberg et al., 1980
). Long-term memory of inhibitory avoidance learning, like trace fear conditioning, is dependent on both the dorsal and ventral hippocampus (Martinez et al., 2002
). Thus, collectively, these studies may suggest that early life iron deficiency produces a long-term enhancement in the expression of dorsal hippocampus-dependent fear.