A critical challenge in neuroimaging is improving our detection of task-related, neural activity changes in subcortical structures and sub-structures in human brain. The hippocampus, a structure central to learning and memory, has proven difficult to image. Our prior work improved the resolution of functional and structural images of the hippocampus and surrounding cortex (Zeineh et al., 2001
; Zeineh et al., 2003
) and enhanced our ability to visualize activations in the hippocampus in groups of subjects. We present here modifications to these existing hippocampal anatomical imaging sequences that offer greater in-plane resolution of the hippocampus (Zeineh et al., 2000
) compared to other methods using MP-RAGE sequences (Miller et al., 2005
). This in-plane resolution confers a benefit of improved localization of cognitive task-dependent changes in the BOLD signal to specific spatial domains of the extended hippocampal formation. The modified sequences we present improve resolution in the longitudinal plane from 3mm to 1mm. While subfields can be most accurately identified in the coronal plane (medial-lateral plane), increased resolution in the anterior-posterior plane is also of importance to hippocampal function because of differences in inputs and processing in anterior compared to posterior hippocampus (Moser and Moser, 1998
). Greater slice thickness is also advantageous for improving accuracy in cortical thickness measurements, which can reveal subfield-specific atrophy that may underlie neurocognitive diseases such as Alzheimer’s disease (Burggren et al., 2008
; Han et al., 2006
) and medial temporal lobe epilepsy (Mathern et al., 1996
Following imaging with high-resolution anatomical and functional sequences, we next show that two-dimensional flat maps of the hippocampus can then be obtained from these imaging sequences. Flat maps are advantageous for several reasons. The structure of hippocampal subregions, as well activations along the long-axis of the hippocampus, can be visualized in a single image of the hippocampus (see also Ekstrom et al., 2008
). This permits visualization of patterns across the complex as a whole that are not easily discernable in slices. The organization of subregions in the HC is such that most extend along the long axis from the anterior to the posterior extent, but each area may comprise only a few voxels in-plane. In a study by Zeineh et al. (Zeineh et al., 2003
) for instance, the anterior/posterior dissociation between encoding and retrieval was easily discerned on the flat map. Similarly, the restriction of HC area activation to extrahippocampal structures (parahippocampal gyrus and fusiform gyrus) during the response of the hippocampus to novel stimuli was easily apparent on flat maps while this organization was difficult to discern in single slices (Zeineh et al., 2000
). In our case, activations during virtual navigation that span part of the posterior parahippocampal cortex and negative activations in the medial hippocampus are clearly visible on a single image (), consistent with previous literature (Aguirre et al., 1996
; Shipman and Astur, 2008
Flat maps also allow greater resolution than can be obtained with our original anatomical images. Specifically, flat maps are interpolated down to a final isotropic voxel resolution of 0.43
. These high-resolution maps can then be used to clearly visualize (interpolated) functional activations (Zeineh et al., 2003
), localize electrodes in subfields of the hippocampus (Ekstrom et al., 2008
), and provide enhanced information about the structural integrity of the hippocampus using measures such as cortical thickness (Burggren et al., 2008
Finally, and perhaps most importantly, our present use of flat maps allows the warping of individual hippocampi into a common flat image, allowing group analyses within the hippocampus proper. This type of analysis has several advantages over the more common approach of registering individual hippocampi to a templates based on different brains such as the MNI or Talaraich template. The group anatomical maps obtained with our methods represent the actual anatomy of the group, which is particularly important with patient populations where hippocampal subfields may be degraded and thus be greatly out of register with a template based on normals (Thompson et al., 2000
). Second, matching to templates is based on alignment of sulci and gyri of the entire brain, which in many cases will leave the hippocampi of individual subjects poorly aligned (Miller et al., 2005
). Thus, because flat maps allow visualization of the hippocampus in an entire image, are of higher resolution, and can be used to produce group maps, flat mapping is advantageous to conventional imaging.
Some possible limitations to working with hippocampal flat maps warrant discussion. One issue, discussed previously in Hyde et al. (2001), is that scans resulting in isotropic voxels (e.g., the same size in three dimensions) may have advantages over non-isotropic scans, an issue of particular importance in BOLD imaging. While our interpolated flat maps are isotropic (0.4 × 0.4 × 0.4), our hippocampal anatomical and BOLD sequences are of high-resolution in-plane but are non-isotropic (one to three millimeters in the longitudinal plane). We see no reason to believe, though, that significant amounts of information are lost when using non-isotropic voxels while much information is to be gained in a structure such as the hippocampus. In particular, our use of non-isotropic voxels in our scans permits higher in-plane resolution than is used in MR-RAGE sequences (0.4 vs. 0.75 mm), which is critical for accurately viewing and demarcating hippocampal subregions. Because variation in the HC architecture is minimal through plane and maximal within plane, it makes sense to maximize the in-plane resolution even at the cost of through plane resolution. Currently, SNR limitations limit our ability to improve from one millimeter to 0.4 mm in the longitudinal plane. We note that because of the linear relationship between SNR and magnet strength, higher Tesla magnets (.e.g, 7T) may offer one means of improving our overall voxel resolution and will be a focus of our future work.
A second concern may be related to the method or criteria we employed for demarcation of hippocampal subregions. Because MR technology cannot currently render in vivo
brains at a comparable resolution to that at which post-mortem brains may be viewed at, we cannot definitively localize different cell layers and cell types and therefore cannot say with confidence exactly where one subregion begins and another ends. To attempt to deal with this particular issue, we employ the Duvernoy and Amaral and Insausti atlases, which are based on histological images of the hippocampus, so as to identify subregional boundaries. We look for changes in hippocampal shape and signal intensity on the MR image and then match to the same areas on histological images in the atlases. We have shown significant intra-rater reliability in the locations of subregions unfolded (Burggren et al., 2008
). Future efforts based on probabilistic atlases and on patient population of post mortem hippocampi will ultimately be necessary to determine how reliable the boundary demarcations are with respect to the underlying cellular anatomy.
A final concern regarding functional activations is how clusters of activations map for 3-D to 2-D. One potential concern here might be that clusters spreading through the medial-lateral axis of the hippocampus (e.g., from CA3 to subiculum) appear as multiple clusters due to the flat mapping. We deal with this issue by producing beta-weight values in FSL for all voxels, mapping to 2D, and then producing the thresholded and cluster-smoothed 2-D hippocampal map for the group. Because thresholding is done at the group level, clusters on individual subjects that cross hippocampal boundaries will not be maintained. Another potential issue with viewing activations in flat space is that not all voxels present in 3-D space will be present in 2-D space. As we note in the Methods, activations in flat space were obtained by averaging voxels that project from 3-D (in cases where more than one voxel projects from 3-D space to 2-D space). Other investigators have attempted warping (“inflating”) to alternative spaces such as spherical spaces (Fischl et al., 1999
), which in principle would deal with the issue of voxel reduction in 2-D space. However, given the interdigitations of the CA fields and the dentate gyrus, this would potentially result in further distortions than flat space. Instead, future methodologic work may include 3-D spaces that involve less distortion. In particular, refolding our hippocampi (Zeineh et al., 2003
) could produce a 3-D template to which we can ultimately directly warp our hippocampi.
We present here a methodology for imaging the hippocampus at high-resolution. Our method is an improvement on earlier methods presented by our lab, demonstrating that the hippocampus can be imaged at high-resolution (0.4 × 0.4 × 1–3mm), computationally unfolded, and then combined with other hippocampal flat maps to produce group activations maps. These maps provide information about hippocampal subfields, information that cannot currently be obtained with conventional imaging techniques. Furthermore, the improvements to our methods that we present expand the resolution of our original high-resolution anatomical sequences from three millimeters to one millimeters, thus allowing clearer visualization of subregions such as CA3 and DG and CA1 from CA3-DG. Our sequences also provide clearer visualization of changes in hippocampal shape in the longitudinal plane. We also present an automated computational interpolation method that improves our ability to detect changes in the shape of the hippocampus along the longitudinal axis. Finally, we present improvements in our ability to detect functional changes in subjects’ hippocampi at the individual and group level by introducing software that is compatible with functional activation software such as FSL, SPM, and AFNI. The group maps produced using this method allow clear visualization of clusters of activation in subfields of the hippocampus across a group of subjects. Together, these methods enhance our ability to detect subregion specific changes in BOLD activity in the hippocampus during behavior.