Homologous laminar organization of the mouse and human subiculum

Abstract

The subiculum is the major output component of the hippocampal formation and one of the major brain structures most affected by Alzheimer's disease. Our previous work revealed a hidden laminar architecture within the mouse subiculum. However, the rotation of the hippocampal longitudinal axis across species makes it unclear how the laminar organization is represented in human subiculum. Using in situ hybridization data from the Allen Human Brain Atlas, we demonstrate that the human subiculum also contains complementary laminar gene expression patterns similar to the mouse. In addition, we provide evidence that the molecular domain boundaries in human subiculum correspond to microstructural differences observed in high resolution MRI and fiber density imaging. Finally, we show both similarities and differences in the gene expression profile of subiculum pyramidal cells within homologous lamina. Overall, we present a new 3D model of the anatomical organization of human subiculum and its evolution from the mouse.

Figure 1

Structural comparison of the mouse and human hippocampus. (a) Three-dimensional representations of the mouse and human hippocampus showing the relative locations of the DG/CA3 (yellow), CA1 (red), and SUB (green); see also Supplementary Movie 1 or use Schol-AR app). The longitudinal hippocampal axis (red in axes chart) in mouse is oriented dorsoventrally, whereas the human longitudinal axis is rotated into the anterior–posterior axis. In addition the anterior/posterior (septo-temporal; blue color) axis in mice is oriented in the superior-inferior direction in humans. (b) Sagittal view of human brain volume representing the spatial location of all in situ hybridization datasets from the Allen Human Brian Atlas (top). Two tissue blocks containing posterior (middle) and anterior (bottom) parts of the hippocampus (white arrows) are shown overlaid on sagittal 3 T structural MRI images. All images in (b) are from www.brain-map.org. Data viewable with Schol-AR augmented reality app, for details visit https://www.ini.usc.edu/scholar/download.html.

Figure 2

Complementary gene expression patterns in the posterior subiculum. (left) In situ hybridization staining for Htr2a, Nts, and Chrm2 in adjacent tissue sections alongside Nissl stained cytoarchitecture and corresponding atlas drawing (based on Nissl section). Htr2a is strongly expressed in the CA1 and superficial ProSUB area (outlined in orange), Nts is strongly expressed in superficial SUB cells located near the molecular layer (m; expression area outlined in red), and Chrm2 is expressed in a deep layer of cells dorsal to the alveus (alv) within the ProSUB and SUB (outlined in yellow). Tissue Index (TI) number references section number within the overall tissue series. Red and blue boxes represent zoomed in image areas of the SUB and ProSUB, respectively, shown in middle and right columns. Together, the three gene expression patterns represent disparate molecular domains as shown by the red, orange, and yellow colored regions in the atlas drawings in top row. All Nissl and in situ hybridization images downloaded from www.brain-map.org.

Figure 3

Gene expression domain boundaries shift across the longitudinal axis, but maintain their complementary relationship. In situ hybridization images of distribution patterns for Pcp4, Chrm2, Th, Htr2a, and Nts gene expression (outlined in yellow, red, or orange colors) at three different rostrocaudal levels spanning 8 mm of the longitudinal axis (posterior to anterior from left to right). Tissue Index (TI) numbers on each image reference section number in tissue series. Pcp4 and Th expression patterns closely mirror the distribution patterns of Chrm2 and Nts, respectively. On the top are corresponding atlas drawings demarcating each of the three laminar molecular domains (drawings based on the boundaries drawn in the Th-labeled sections). Note, at most posterior levels (TI: 99–136), the area of the ProSUB is small compared to the more anterior levels. All in situ hybridization images downloaded from www.brain-map.org.

Figure 4

Complementary gene expression patterns in the anterior SUB (TI: 901–930). (middle row) In situ hybridization staining for Chrm2 , Th, and Htr2a (colored outlined) in adjacent tissue sections alongside Nissl stained cytoarchitecture and corresponding atlas drawing (based on Nissl section). In anterior coronal sections, the SUB appears separated by the CA1 into a dorsal and ventral region. The ventral region of the SUB contains complementary Chrm2, Th, and Htr2a expression patterns in the SUB and ProSUB as observed in posterior hippocampal levels (zoomed in images of red boxed regions are shown in bottom row). In contrast, the dorsal region of the SUB at this anterior level contains Chrm2 and Htr2a expression, but very little Th expression (zoomed in images in blue boxed regions are shown in top row). Closer examination of the Nissl staining suggests a tri-laminar cytoarchitecture containing a distinct intermediate layer (arrow in top row Nissl image, orange domain in atlas drawing) with cells that are more darkly-stained and densely packed than the cells located deeper (yellow in atlas drawing) and more superficially (blue in atlas drawing). Based on this trilaminar organization, Chrm2-expressing cells are primarily distributed in the deep layer adjacent to the alveus (alv) whereas Htr2a-expressing cells are located in the intermediate and superficial layers near the molecular layer (m). All Nissl and in situ hybridization images downloaded from www.brain-map.org.

Figure 5

Gene expression patterns at the SUB anterior tip. (top row) 4 mm anterior to the section shown in Fig. 4, the CA1 is no longer present and the ventral and dorsal regions of the SUB become continuous along with their laminar gene expression patterns. In situ hybridization images of Chrm2, Th, and Htr2a expression (colored outlines) are presented with adjacent Nissl and corresponding atlas drawings. Chrm2-expressing cells form a continuous external layer adjacent to the alveus (alv), whereas Th and Htr2a expression is distributed within cells located more internally near the molecular layer (m). Unlike the tissue level shown in Fig. 4, there is abundant Th expression in the more dorsal part of the SUB. In the most anterior coronal section (bottom row, 2 mm anterior to sections shown in top row), the molecular layer is no longer present. Chrm2-expressing cells form a continuous ring layer that surrounds a core of Th expressing cells (few Htr2a expressing cells remain at this level). Together this data shows that Chrm2, Th, and Htr2a expressing cells form three distinct layers that wrap continuously around the anterior/medial tip of the hippocampus. In relation to the section in Fig. 4, the layer of Th expression appears to end dorsally approximately ~ 6 mm from the anterior pole of the SUB, whereas Chrm2 and Htr2a expression continues posteriorly within the dorsal part of the anterior SUB. ). All Nissl and in situ hybridization images downloaded from www.brain-map.org.

Figure 6

Tissue microstructure imaging resolution compared to gene expression segmentation. (a) 2D turbo spin echo T2w whole brain 7 T in vivo MRI image acquired at 200 µm in-plane resolution with 2 mm thickness (arrow points to hippocampus). (b) Zoomed in 7 T MRI image of the hippocampus at a different rostrocaudal level showing the major hippocampal strata. The strata radiatum and stratum lacunosum moleculare in hippocampus proper as well as the SUB molecular layer appear as a dark band (arrow) that can help distinguish major hippocampal subregions. (c) Ex vivo T1w 16.4 T MRI image ((50 μm) resolution) of a dissected post-mortem tissue sample from the temporal lobe. At this resolution, additional anatomical features are apparent including the granule cell layer of the dentate gyrus (arrow). (d) Fiber track density of the postmortem tissue sample obtained from high resolution angular diffusion imaging at 16.4 T (150 μm 3D isotropic resolution). (e) Zoomed in view of the fiber track density in the hippocampus of the post-mortem sample with comparison to the gene expression-based atlas segmentation (f). Arrowheads in (e) and (f) mark the corresponding gene expression domain boundary positions. The ProSUB is distinguished from the adjacent SUB by the presence of several thicker dorsoventrally-oriented fiber bundles (orange/red boundary in atlas). In addition, a mediolateral fiber track dorsal to the alveus (asterisk) corresponds to the deep layer of cells located in both the ProSUB and SUB (yellow atlas area). (g) Average fiber track density within each region’s voxel (colors correspond to atlas regions in (f); mean ± S.D.). Abbreviations: agranular bundle (agb), dentate gyrus (DG), fimbria (fi), molecular layer of the subiculum (m), presubiculum (PRE), prosubiculum (ProSUB), stratum pyramidale of the subiculum (sp), subiculum (SUB).

Figure 7

Homologous SUB laminar organization with similarities and differences in gene expression. (a) Comparison of Nts and Chrm2 expression in the mouse dorsal subiculum (specifically SUBdd in the HGEA nomenclature, top row) and human posterior SUB (bottom row). Despite the difference in cell packing density, the laminar organization of the SUB pyramidal cells appears conserved across species. In the mouse HGEA, the Nts-expressing cells are located in SUB layer 1 (SUB_1), whereas Chrm2 expression is located in SUB layer 4 (SUB_4). In both mouse and human, the Nts-expressing cells are located superficially near the molecular layer (m) whereas the Chrm2-expressing cells are located deep near the alveus (alv). The position of the alveus and molecular layer (as well as the laminar organization of the pyramidal layer) is dorsoventrally inverted due to the rotation of the hippocampal axis between the two species. (b) In contrast to the highly similar Nts and Chrm2 expression patterns, Htr2a and Pcp4 are differentially expressed between the mouse (top, sagittal-cut images, HGEA atlas to left)) and human SUB (bottom, coronal sections). Htr2a expression is not strongly expressed in the mouse SUB (some minor expression in ventral CA1), but strongly expressed in human CA1 and SUB (bottom left). Pcp4′s combinatorial expression pattern in SUB is bilaminar in the mouse (SUB_2 and SUB_4, top right) but only expressed in the deep layer of human SUB cells (corresponding to SUB_4 only, bottom right).

Figure 8

A 3D translational model of mouse and human SUB laminar organization. (a) Using the 3D HGEA model of the mouse SUB (left), we developed a 3D model of the human SUB based on our observations of the gene expression-based SUB lamina and the relative position of corresponding SUB subregions. Our model suggests two changes to the SUB have occurred across evolution between the mouse and human: 1) rotation of the longitudinal axis (middle), and 2) the folding back of the anterior SUB against the long axis (right). Images on the right show the view of the human SUB model from the medial (top) or lateral perspective (bottom; see also Supplementary Movie 2 or use Schol-AR app). (b) Coronal atlas section series from the mouse HGEA with the 4 colored gene expression layers and subregions (left) with similarly corresponding coronal human atlas drawings with similarly colored gene expression layers and subregions (right). Data viewable with Schol-AR augmented reality app, for details visit https://www.ini.usc.edu/scholar/download.html.