Pentad: A reproducible cytoarchitectonic protocol and its application to parcellation of the human hippocampus

Abstract

Introduction: The hippocampus is integral for learning and memory and is targeted by multiple diseases. Neuroimaging approaches frequently use hippocampal subfield volumes as a standard measure of neurodegeneration, thus making them an essential biomarker to study. Collectively, histologic parcellation studies contain various disagreements, discrepancies, and omissions. The present study aimed to advance the hippocampal subfield segmentation field by establishing the first histology based parcellation protocol, applied to n = 22 human hippocampal samples.

Methods: The protocol focuses on five cellular traits observed in the pyramidal layer of the human hippocampus. We coin this approach the pentad protocol. The traits were: chromophilia, neuron size, packing density, clustering, and collinearity. Subfields included were CA1, CA2, CA3, CA4, prosubiculum, subiculum, presubiculum, parasubiculum, as well as the medial (uncal) subfields Subu, CA1u, CA2u, CA3u, and CA4u. We also establish nine distinct anterior-posterior levels of the hippocampus in the coronal plane to document rostrocaudal differences.

Results: Applying the pentad protocol, we parcellated 13 subfields at nine levels in 22 samples. We found that CA1 had the smallest neurons, CA2 showed high neuronal clustering, and CA3 displayed the most collinear neurons of the CA fields. The border between presubiculum and subiculum was staircase shaped, and parasubiculum had larger neurons than presubiculum. We also demonstrate cytoarchitectural evidence that CA4 and prosubiculum exist as individual subfields.

Discussion: This protocol is comprehensive, regimented and supplies a high number of samples, hippocampal subfields, and anterior-posterior coronal levels. The pentad protocol utilizes the gold standard approach for the human hippocampus subfield parcellation.

Keywords: CA1; CA2; CA3; hippocampal subfields; histology; pyramidal neurons; segmentation; subiculum.

FIGURE 1

Nine rostrocaudal hippocampal levels represented by line drawing schemata in the coronal plane. Each level shows different landmarks that makes it unique. Accordingly, each anterior-posterior level has been given a unique name: (A) Genu, (B) genu-pes, (C) pes, (D) pes-DG, (E) full DG, (F) separated DG, (G) x-region, (H) uncus-body, and (I) anterior body. The solid black lines represent the pial tissue surface and the dentate gyrus. The gray lines denote the gray-white matter boundaries, while the dashed lines indicate gray-white matter boundaries in the entorhinal cortex. The lightest gray lines depict the pyramidal layer, and dotted lines denote the presubicular clouds, LPE, and ParaS.

FIGURE 2

Microscopic images depict the neuronal characteristics, as outlined in the pentad protocol, of the hippocampal subfields. Images have been positioned so that the superior portion of the pyramidal layer is on the top of the image. Note that CA2 (F), CA3 (G), and CA4 (H) display intense chromophilia in their pyramidal neurons as well as medial counterparts CA2u (J) and CA3u (K). Subfields CA1 (E), Sub (C), and ProS (D) show a moderate amount of chromophilia, as do their medial counter parts CA1u (I) and Subu (L). ParaS (A), PreS (B), CA2, and CA3 present a dense packing density, while CA1, Sub, and CA4 display sparser neuronal populations. ParaS and PreS contain extremely small neurons in the superficial layer (layer II) while CA3 and CA4 in pyramidal layer exhibit the largest neurons. The CA2 and CA3 pyramidal neurons reveal the highest degree of collinearity.

FIGURE 3

CA fields (CA1–CA3) from three different cases (Cases 12, 21, and 13, respectively) stained for Nissl substance. All slides at the level of the hippocampal body. (A–C) Macro scale photographs of three cases, and a zoomed in mesoscale image (D–F) showing CA1, CA2, and CA3 from the same Nissl stained section. Arrows point to the characteristic jaggedness found in CA1’s pyramidal layer. Note the change in staining intensity from CA1 and CA2, with CA1 showing lighter staining. CA1 contains smaller neurons than both CA3 and CA2, but CA2 and CA3 have similar neuron sizes and staining intensity. CA3’s neurons exhibit more collinearity than CA2. Arrowheads in panels (D–F) denote the clustering of neurons at the inferior edge of the pyramidal layer. Magnification bars, 1 mm.

FIGURE 4

Subfields CA1, ProS, and Sub from two different cases (Cases 18 and 6, respectively). Histologic slides illustrated at the full DG level with photomicrographs of two respective Nissl stained sections (A,B), and zoomed mesoscale images (C,D). Black dashed rectangles correspond to the close-up view of panels (C,D), showing Sub, ProS, and CA1. Arrowheads point to the superficial neuronal clustering in the ProS pyramidal layer. Note the smaller and more densely packed neurons in ProS. Magnification bar for panels (A,B): 2 mm, for panels (C,D): 1 mm.

FIGURE 5

Macro (A) and mesoscopic views (B,C) of the subicular cortices from Case 3. Panel (B) shows ProS and Sub, while panel (C) shows ParaS, PreS, and Sub. Note the presubicular clouds overlay the distal part of Sub (C), resulting in the staircase, or oblique boundary. Arrowheads point to the presubicular clouds. Medial to ParaS is the entorhinal cortex. Magnification bar for panel (A): 2 mm, for panels (B,C): 1 mm.

FIGURE 6

The medial CA subfields versus the lateral CA subfields. Macro view of fully parcellated Nissl stained section at the full DG (A) and x-region (B) levels from Case 12. Panels (A,B) show the macro views of the meso views of Sub (C), Subu (D), CA1 (E), CA1u (F), CA2 (G), CA2u (H), CA3 (I), and CA3u (J). Note the clustering of neurons in the middle of the pyramidal layer in Subu, and the more homogenously distributed pyramidal layer of Sub. CA1 and CA1u contain smaller and more lightly stained neurons, as well as less densely packed, than those in CA2, CA2u, CA3, and CA3u. Both CA2 and CA2u contain neurons that exhibit less collinearity than the neurons observed in CA3 and in CA3u. Finally, note how all medial subfields (denoted with a “u” at the end of the subfield name; far right panels) have smaller neurons than their lateral counterpart. Magnification bar for panels (A,B): 2 mm, for panels (C–J): 500 μm.

FIGURE 7

Parcellated human hippocampus from rostral to caudal levels from one case (Case 17). Panels (A–I) shows Nissl stained sections from each of the nine established levels (see Figure 1): genu (A), genu-pes (B), pes (C), pes-DG (D), full DG (E), separated DG (F), x-region (G), uncus-body (H), and body (I), respectively. (A) The start of the hippocampus, typically only Sub, CA1, and rarely PreS is present. (B) ProS, ParaS emerge by this rostrocaudal point, as well as Subu. (C) As pes show a definite shape, similar subfield parcellation to panel (B). (D) CA2, CA4, and CA1u appear, as well as the DG. (E) CA2 and CA4 both become larger, while Subu reduces in territory. (F) CA3 present, as well as CA2u. (G) Uncinate gyrus (and uncus) separates from the hippocampal body, and Subu and CA1u continue to get smaller. (H) ParaS and PreS shift more medially, Subu has disappeared. (I) ParaS and all medial subfields have ended. Note how subfields fluctuate in size (i.e., medial-lateral width) as hippocampus moves anterior to posterior. Magnification bar, 2 mm.