Direct Visualization and Mapping of the Spatial Course of Fiber Tracts at Microscopic Resolution in the Human Hippocampus - PubMed (original) (raw)
Direct Visualization and Mapping of the Spatial Course of Fiber Tracts at Microscopic Resolution in the Human Hippocampus
Michael M Zeineh et al. Cereb Cortex. 2017.
Abstract
While hippocampal connectivity is essential to normal memory function, our knowledge of human hippocampal circuitry is largely inferred from animal studies. Using polarized light microscopy at 1.3 µm resolution, we have directly visualized the 3D course of key medial temporal pathways in 3 ex vivo human hemispheres and 2 ex vivo vervet monkey hemispheres. The multiple components of the perforant path system were clearly identified: Superficial sheets of fibers emanating from the entorhinal cortex project to the presubiculum and parasubiculum, intermixed transverse and longitudinal angular bundle fibers perforate the subiculum and then project to the cornu ammonis (CA) fields and dentate molecular layer, and a significant alvear component runs from the angular bundle to the CA fields. From the hilus, mossy fibers localize to regions of high kainate receptor density, and the endfolial pathway, mostly investigated in humans, merges with the Schaffer collaterals. This work defines human hippocampal pathways underlying mnemonic function at an unprecedented resolution.
Keywords: entorhinal cortex; hippocampus; human circuitry; perforant pathway; polarized light microscopy.
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Figures
Figure 1.
Hippocampal circuitry and validation in the vervet monkey. Top: A–F indicate the components of the perforant path system largely identified from animal studies. Major pathways are represented by solid lines, whereas minor pathways are represented by dotted lines. A = “sublamina supratangentialis” (dotted red), a minor superficial entorhinal bundle projecting to the parasubiculum and presubiculum. B = entorhinal projection to the angular bundle (yellow). C = angular bundle fibers (yellow) perforating through the subiculum. D + E = these same fibers projecting superiorly (D) into the dentate gyrus or inferiorly (E) into the hippocampal CA fields (yellow). F = angular bundle fibers subjacent to the subiculum projecting into the CA fields (alvear path, light blue: note, for the human diagram, this pathway is larger in humans and is depicted as solid, whereas it is smaller in monkeys and depicted as dotted light blue on the bottom left). The remaining hippocampal circuitry includes: mossy fibers projecting from the dentate gyrus to CA3 and CA4 (dark blue), the endfolial pathway projecting from CA4 toward CA3 (light brown), CA3 projecting to the fornix (green) and to CA1 (Schaffer collaterals, orange), followed by CA1 and subiculum output to ERC (pink), and subicular output to the fornix (green). CA, cornu ammonis; DG, dentate gyrus; ERC, entorhinal cortex; PaS, parasubiculum; PHC, parahippocampal cortex; PRC, perirhinal cortex; PreS, presubiculum; ProS, prosubiculum; Sub, subiculum. Bottom: Polarized light microscopic images from the vervet monkey illustrate the above pathways.
Figure 2.
Sectioning and segmentation procedure from the left (A,B) and right (C,D) hemispheres of brain #1. (A) Example coronal blockface image from the left hemisphere from brain #1, section 548. (B) Sagittal reformat of coregistered serial coronal blockface sections with the numbers indicating the coronal slice number. (C) Coronal reformat of coregistered serial sagittal blockface sections from the right hemisphere from brain #1, with the numbers indicating the sagittal slice number. (D) Sagittal blockface image from the right hemisphere, slice 209. (E_–_G) Coronal transmittance images with overlaid medial temporal subregion demarcations in green. For the remainder of the figures, subregion demarcations will also be in green. The green asterisks highlight the darkening associated with myelin in the deep presubicular pyramidal cell layer that facilitates its segmentation. The blue scale bar is 1 cm in length.
Figure 3.
Entorhinal pathways and the angular bundle. (A) Coronal PLI at the level of the posterior hippocampal head of the left hemisphere of brain #1. The approximate localization of sagittal section S190 (shown in C) is indicated by peach vertical line. The green boundaries correspond to the subregion demarcations on the transmittance images from Figure 2_E_. The hue on the color wheel indicates the direction of the in-plane fiber orientation (see Supplementary Fig. 1), and the brightness/darkness of the color (e.g., more peripheral/central in the color wheel) indicates a primarily in-plane/through-plane orientation. Arrows with cross hairs denote a through-plane orientation. Best seen on the zoomed in coronal section from the box on the left are dual superficial entorhinal pathways (red arrows) running tangentially throughout the ERC to the parasubiculum and presubiculum. Both project longitudinally in the molecular layer of the presubiculum (red cross hair), confirmed on sagittal plane S190 of the right hemisphere in (C). In yellow are medial entorhinal projections to the angular bundle (lower 2 curvilinear yellow arrows) and from the angular bundle through the presubiculum into the molecular subiculum (top yellow sigmoidal arrows). Subicular lamination on the far right is indicated as mol, molecular; pyr, pyramidal; a–m, alvear/multiforme. (B) A slightly more anterior section (C500) with Nissl staining performed after PLI for visualization of cell bodies. Lamination of the ERC derived from the Nissl stain is superimposed on the transmittance and fiber orientation maps. Perforant pathway fibers originate in approximately layer Pre-α of the ERC and radially project to the angular bundle (yellow curved arrow). (C) Sagittal PLI slice S190 from the right hemisphere of brain #1 at the approximate location indicated by the vertical peach line in A. The approximate localization of the coronal section from the left hemisphere in A is indicated by the vertical black line on this right sagittal section. Red arrows highlight the presubicular longitudinal components of the superficial entorhinal pathways from A. The lower yellow arrow signifies perforant path projections from the medial ERC, through the angular bundle, and toward the presubiculum and subiculum. More superiorly is a longitudinal entorhinal pathway (thin yellow arrow) running in the pyramidal layer of the presubiculum, depicted as the yellow ellipses in Figure 4. The green arrow depicts fibers projecting to the fimbria; in blue is the alvear bundle to the posterior hippocampus; and in orange is the cingulum bundle within the parahippocampal gyrus (see Supplementary Fig. 3). (D) Diagrammatic representation of entorhinal pathways in the coronal and sagittal planes.
Figure 4.
Resolving crossing fibers in the parahippocampal gyrus. (A) Coronal sections anteriorly (C518) to posteriorly (C723). (B) Zoom in on the white box on C643. (C) Schematic of crossing fiber bundles. Yellow ellipse: the ERC most anteriorly (C518) projects to a longitudinal bundle that extends through the entire presubiculum within the pyramidal cell layer, deep to the molecular layer (see also Fig. 7 for the dark signal underneath the presubicular label). From and deep to this, obliquely oriented thick perforant pathway bundles can be seen extending to the subiculum proper (yellow arrows). Green arrows: a crossing curvilinear pathway projects from the subiculum to the parahippocampal gyrus and then turns to enter the alveus, representing subicular fibers projecting to the fornix (see also Fig. 8_B_). Thus, PLI can discern that the subicular output (dark purple fibers in C643) and perforant input fibers (yellow-green fibers in C643) cross one another subjacent to the subiculum, confirmed on the spotlight images of Supplementary Figure 4. Pink arrows: distally in the parasubiculum are CA1 and subiculum output fibers extending to the entorhinal region.
Figure 5.
Perforant path system longitudinally. (A) Medial sagittal section through the subiculum indicated by the medial gray dotted line in C (S210). The bottom left inset shows thick steeply longitudinal bundles of perforant fibers ascending through the subiculum (yellow arrow). The bottom right inset shows perforant path fibers going posteriorly to the hippocampal tail, targeting the outer one-third of the dentate superiorly and CA1 inferiorly (yellow arrows). Alvear fibers can be seen coursing into CA1 (light blue arrow). Superiorly, the alveus and fimbria contain extensive longitudinal fibers heading to the fornix (green arrow). (B) Lateral sagittal section indicated by the more lateral gray dotted line in C (S310). Extensive longitudinal fibers occupy CA1 lacunosum-moleculare (corresponding to the relatively dark portions on the coronal images from Fig. 8_B_,C, and yellow encircled regions in Supplementary Fig. 5). The alveus also contains extensive longitudinal fibers (green arrow). (C) Medial (S210) and lateral (S310) sagittal schematics on the right, with the corresponding coronal schematic cross-referenced by the gray dotted lines on the left.
Figure 6.
Dentate fibers, mossy fibers, endfolial pathway, and Schaffer collaterals. (A) Coronal PLI at the level of the hippocampal body showing a relatively diminished color orientation in fields CA4 and CA3 pyramidal cell layers, circumscribed in blue, representing the unmyelinated target of the mossy fibers. Endfolial fibers originate in CA4 (upper light brown curvilinear arrow) and project to stratum oriens. The Schaffer collaterals originate in CA3 (orange arrow) and project to the stratum radiatum. Fibers project from the endfolial pathway to the Schaffer collaterals (lower light brown sigmoidal arrow). The overlying alveus contains largely longitudinal forniceal fibers (see Figs 3_C_ and 5_A_,B). (B) Zoomed in section on the medial and inferior dentate gyrus indicated by the blue box in A at the bottom left. p, polymorphic layer, g, granular cell layer, and i, inner one-third of the dentate gyrus. This demonstrates tangential fibers in the polymorphic layer and inner one-third of the dentate gyrus, likely representing mossy cell associational fibers. Some subtle fibers can be seen traversing the granular layer to bridge these 2 layers (dark blue arrow).
Figure 7.
Coronal PLI of hemisphere #3. (A) Transmittance image of an anterior left coronal section from brain #2. (B) Corresponding color orientation map of section shown in A. alv, Alveus; CA, cornu ammonis; h, hilus; mol, molecular layer; lm, stratum lacunosum-moleculare; LV, temporal horn of lateral ventricle; PaS, parasubiculum; PreS, presubiculum; ProS, prosubiculum; Put, putamen; pyr, pyramidal layer; r, stratum radiatum.
Figure 8.
Perforant path system in-plane. (A) Angular bundle fibers projecting to the more posterior portion of the hippocampal head. (B) Perforant (yellow), alvear (light blue), and forniceal (green) pathways, shown at a higher magnification (see box in A). Yellow arrows: Sigmoidal perforant pathway fibers of varying obliquities can be seen extending from the angular bundle, through the subiculum, and into the deeper part of the molecular layer of the subiculum. Light blue arrows: Deeper angular bundle alvear pathway fibers extend subjacent to the prosubiculum and into the stratum oriens where they penetrate the pyramidal layer of CA1. Green arrows: output fibers emanating from the subiculum obliquely cross the fibers of the angular bundle en route to the parahippocampal gyrus and then alveus, ultimately to reach the fornix. Subicular lamination is indicated as mol, molecular; pyr, pyramidal; a–m, alvear/multiforme. CA1 lamination is indicated as oriens, stratum oriens; alv, alveus. (C) Higher magnification of the box shown in B depicting the dentate layers (polymorphic, granular, and inner/middle/outer thirds of the molecular layer) and CA1 lacunosum-moleculare (L-M), radiatum, and pyramidal layers. Perforant pathway bifurcation: Tangential perforant pathway fibers in the superficial molecular layer of the subiculum extend into the superficial L-M and project to the outer one-third and to a lesser degree to the middle one-third of the dentate molecular layer (yellow arrows, top). Fibers in the deeper molecular layer of the subiculum continue in a tangential manner in the radiatum and then turn inferiorly to penetrate deeper into the CA1 pyramidal cell layer (yellow curved arrow, bottom). (D) Graphical depiction of the perforant, alvear, and forniceal pathways.
Figure 9.
Kainate receptor autoradiography. (A) Nissl stain of one representative medial temporal section. (B) Kainate autoradiography. (C) Mean concentration of kainate receptors across hippocampal subfields and layers (n = 5). The highest concentration is in the pyramidal and lucidum layers of CA3 followed by the hilus. or, oriens; pyr, pyramidal; rad, radiatum; lac-mol, lacunosum-moleculare; luc, lucidum; mol, molecular; gr, granular; hilus, both the polymorphic layer of dentate gyrus and CA4.
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