The current results indicate that hippocampal volume is reduced in healthy young E4 carriers relative to non-carriers with the right hippocampus being more susceptible to atrophy than the left hippocampus. Analysis of regional shape changes also highlighted specific regions of the right hippocampus where ApoE4 carriers experienced atrophy relative to non-carriers. Such regional shape changes in ApoE4 carriers were absent in the left hippocampus. Overall, these results suggest that in ApoE4 carriers, the right hippocampus is directly vulnerable to atrophy in healthy young subjects.
Despite the negative effects of ApoE4 genotype on hippocampal volume, ApoE4 carriers still maintained an equivalent cognitive performance relative to non-carriers in a range of tests that probed verbal learning and memory, visual memory, working memory and attention. This suggests that although early atrophy may be occurring in ApoE4 carriers in a structure that is known to be affected in the early stages of AD, actual memory performance is not yet undermined by this atrophy.
The ApoE4 allele is the most well studied risk gene for AD, and previous work has found that the E4 allele is associated with increased atrophy of the hippocampus in Alzheimer's disease 
. In healthy middle-aged and older non-demented E4 carriers, lower hippocampal volumes, decreased cortical thickness and increased rate of hippocampal atrophy relative to E4 non-carriers have been noted 
In healthy young subjects there are still relatively few studies which have examined the effect of ApoE genotype on brain structure and function. In the current study we hypothesised that hippocampal volume would be reduced in healthy young E4 carriers relative to non-carriers. The rationale for this hypothesis stems from earlier studies in younger populations, for example in young children and adolescents, E4 carriers were found to have thinner entorhinal cortices (EC) relative to E4 non-carriers 
. Shaw et al. also showed a stepwise increase in cortical thickness in the EC, with E4 carriers having the thinnest cortex, E2 carriers having the thickest, and E3 homozygotes having an intermediate position. Similarly, in healthy young subjects (age ~25 years) E3 homozygotes were found to have hippocampal volumes that were intermediate between E4 carriers who had the lowest hippocampal volume and E2 carriers who had the highest hippocampal volumes 
The results from the current study are in general agreement with these previous works. A generalised mixed-effect model for the right hemisphere indicated that genotype has an influence on right hippocampal volume and right amygdalar volume. However, only the right hippocampus was a significant fixed effect in this model. For the mixed-effect model of the left hemisphere, only the left hippocampus remained as a fixed effect following model simplification. Overall, the results from our mixed-effects models indicate that ApoE genotype has a significant effect on hippocampal volume. The volumes of no other structures were found to be significantly affected by genotype in the current study. Therefore our results extend the current literature by highlighting that the vulnerability of the E4 carriers to structural atrophy is localised to the right hippocampus while there is a general preservation of all other grey matter structures examined. One previous study has also noted that there were no differences in ventricular or hemisphere volumes between healthy young E4 carriers and non-carriers 
. However, the current results provide more detailed confirmation of a preservation of deep grey matter structures outside of the hippocampus in healthy young E4 carriers. Together, these results support the concept that E4 status does not have a global effect on the brain regions, but rather leads to a selective targeting of the hippocampal structure.
There are some earlier studies which failed to find differences in hippocampal volume between healthy young E4 carriers and non-carriers 
. These discrepancies may stem partly from low sample sizes and partly from differences in the genotypes being studied. One previous study 
examined differences between 10 E2/E3, 10 E3/E3 and 13 E3/E4 subjects and did not find hippocampal volume differences between these three groups, while the current study found hippocampal volume differences between a non-carrier group comprised of 22 E3/E3 subjects and a carrier group comprised of 21 E3/E3 subjects and one E4/E4 subject. The larger sample size of the current study, together with the automated algorithm for segmentation may enable more accurate detection of subtle volume changes between carriers and non-carriers. A second study which failed to find hippocampal volume differences between carrier and non-carrier groups 
also employed manual segmentation and included a very heterogeneous group of carriers (4 E4/E4 subjects, 12 E3/E4 subjects and 2 E2/E4 subjects) and non-carriers (100 E3/E3 subjects, 2 E2/E2 subjects, 15 E2/E3 subjects). Additionally, the E2 allele variant has been reported to have a protective effect against AD 
and cardiovascular diseases 
, and is also associated with increased longevity 
. Thus it is preferable to exclude the E2/E4 genotype from the E4 carrier group. Future studies with larger cohorts should consider stratifying ApoE groups into more homogenous subgroups. Considering that the differences in hippocampal volume between these groups are subtle, more consistent stratification might help to clear up some of the discrepancies in the literature.
Volume changes within E4 carriers may be related to changes in synaptic connections and myelination of the peripheral cortical neuropil in E4 carriers 
. Within young ApoE4 targeted replacement (TR) mice also show lower spine density in cortical layers II/III compared to ApoE2 TR mice 
. These differences may be related to increased oxidative insults resulting from changes in the pro-oxidant/antioxidant balance in E4 carriers 
. WM tract volume has also been shown to be reduced in healthy young E4 carriers 
. These findings suggest that E4 status has a negative effect on both GM and WM structures in healthy young people. However, the absence of differences in memory performance between carriers and non-carriers in the current study and in earlier studies 
, suggests that the brain retains enough reserve capacity at a young age to avoid decline in cognitive performance despite the structural deficits outlined above in E4 carriers. Deficits associated with ApoE4 are more apparent later in life when E4 carriers are more vulnerable to the cortical thinning observed in aging 
and AD 
, since less cortical thinning is necessary in key brain regions in E4 carriers before a critical anatomical threshold is passed, and neural dysfunctions become clinically evident.
Our finding of a more pronounced main effect of ApoE4 genotype on right hippocampal volume also extends the literature regarding laterality which has focused to date on older subjects where greater atrophy in the right hippocampus in E4 carriers has also been consistently reported 
as well as among AD patients 
. Interestingly, in healthy controls a “normal” asymmetry appears to exist with the right hippocampus generally being larger than the left hippocampus; a finding which has been confirmed in a meta-analysis of 82 studies 
. In older subjects, reversal of this typical asymmetry has been proposed as an indicator of early pathology 
In ApoE4 non-carriers, our results show that mean normalised volume of the right hippocampus was marginally, though non-significantly, larger than mean left hippocampal volume. This finding is consistent with the usual asymmetry reported by the meta-analysis noted above 
. Conversely, in ApoE4 carriers mean normalised volume of the right hippocampus was marginally, though non-significantly, smaller
than left hippocampal volume. Thus, the current results point to a trend towards a reduction in the “normal” asymmetry of the hippocampus which has been noted in earlier studies in healthy older and AD cohorts 
. Importantly, when comparing both the left and right hemisphere models, the right hemisphere model was also found to be a significantly better fit for the data, a finding which again emphasizes the selective vulnerability of the right hippocampus in ApoE4 carriers.
A greater predilection for damage in the right hemisphere has been noted in fMRI studies. Older E4 carriers have been found to exhibit more intense activation in parietal, frontal and right medial temporal lobe regions than non-carriers during the encoding of a picture learning task 
. E4 carriers have also been found to show reduced activation in left hippocampal regions compared to E3 carriers, which also supports the model of greater compensatory changes occurring in the right hemisphere 
. These studies are broadly compatible with the concept of greater right hemisphere involvement in normal aging as proposed by the Hemispheric Asymmetry Reduction in Older Adults (HAROLD) model of Cabeza 
Although not all fMRI studies have reported increased recruitment of right hemisphere activation in E4 carriers 
, differences between studies may be partly accounted for by the choice of functional tasks employed. A spatial context memory task which involves the right hemisphere in visuospatial processing was used in the study which found the greatest amount of compensatory right hemisphere activation 
. The lack of a right hemisphere effect in other studies 
may be related to tasks with an emphasis on language that would activate the left rather than the right hemisphere 
Findings of increased functional connectivity between medial temporal lobe (MTL) regions and other regions known to be affected by AD (e.g. posterior cingulate) in young E4 carriers also suggest that ApoE begins to be expressed in AD-associated brain regions long before cognitive decline 
. Filbey et al. reported that young E4 carriers showed more medial frontal, cingulate and MTL activity compared to non-carriers in a working memory task 
. In general agreement with this, other work has found that E4 carriers have more default mode network (DMN) connectivity and more hippocampal activation during a memory encoding task than non-carriers 
. However, a study by Mondadori et al. 
found that E4 carriers exhibited less neural activity in bilateral MTL and left frontal regions during the encoding and retrieval portions of an episodic memory task than performance-matched non-carriers. This was attributed to enhanced neural efficiency of memory networks in young adult E4 carriers which offers some support for a model of antagonistic pleiotrophy. Although hippocampal volume is reduced in the current cohort of healthy young E4 carriers, no cognitive differences were noted between carriers and non-carriers. Whether or not this equivalence of performance is achieved through extra compensation in the E4 carriers is not possible to say. It may be the case that cognitive deficits only become evident in E4 carriers when the risk allele is compounded by an additional risk factor such as AD history in the family 
In old age, the majority of studies note that E4 carriers have greater rates of cognitive decline compared with non-carriers 
. It could be hypothesized that structural changes occurring in healthy twenty year olds as a result of possession of the E4 allele, may not affect cognitive function at this early stage but may lay the ground work for faster cognitive decline in older age. Although there are exceptions, most studies have noted that E4 carriers performed worse in tasks of verbal and visual episodic memory compared with non-carriers. Also, studies have noted that those with two E4 alleles experienced more memory decline before those with only one E4 allele 
A limitation of the current study is that we do not know how the subjects progress over time. A longitudinal study which would follow healthy young carriers and non-carriers of the E4 allele over a period of ten or more years is warranted. Although there may be some limitations with regards to the FIRST algorithm, each subject's segmentations were carefully examined and found to be of good quality. The FIRST algorithm may offer some advantages over voxel-based morphometry (VBM) as VBM is prone to registration artefacts in deep GM structures 
. FIRST is also more objective than manual segmentation methods which may not be sufficiently sensitive to detect subtle regional changes and localised volume loss. The algorithm proceeds with segmentation based on the intensity values of voxels and avoids the biases that arise when a researcher must visually judge contrasts in order to delineate boundaries during manual segmentation.
Overall, our results suggest that in the E4 carrier group, even among healthy subjects as young as 25 years of age, there are subtle structural changes in the hippocampus leading to volume reduction which are significant in the right hemisphere. Our results lend support to a growing body of evidence that indicates that the right hemisphere may have a greater predilection for damage in the very early stages of neurodegeneration. Our results also suggest that E4 carriers that exhibit volume reduction in the right hippocampus may be at greater risk of neurodgeneration in later life and that the structural deficits found in young carriers may not be clinically manifest until much later time points. However, future studies with larger sample sizes, as well as longitudinal studies will be needed to confirm this.