Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories.

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the entorhinal and parahippocampal cortices as well as Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 µm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized slices spaced 5 mm apart (pixel size 0.4 µm at 20× magnification). Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while the definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed less saliently. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed neuroimaging research on the human MTL cortex.

Entorhinal vessel density correlates with phosphorylated tau and TDP-43 pathology.

INTRODUCTION: The entorhinal cortex (EC) and perirhinal cortex (PC) are vulnerable to Alzheimer's disease. A triggering factor may be the interaction of vascular dysfunction and tau pathology. METHODS: We imaged post mortem human tissue at 100 µm3 with 7 T magnetic resonance imaging and manually labeled individual blood vessels (mean = 270 slices/case). Vessel density was quantified and compared per EC subfield, between EC and PC, and in relation to tau and TAR DNA-binding protein 43 (TDP-43) semiquantitative scores. RESULTS: PC was more vascularized than EC and vessel densities were higher in posterior EC subfields. Tau and TDP-43 strongly correlated with vasculature density and subregions with severe tau at the preclinical stage had significantly greater vessel density than those with low tau burden. DISCUSSION: These data impact cerebrovascular maps, quantification of subfield vasculature, and correlation of vasculature and pathology at early stages. The ordered association of vessel density, and tau or TDP-43 pathology, may be exploited in a predictive context. HIGHLIGHTS: Vessel density correlates with phosphorylated tau (p-tau) burden in entorhinal and perirhinal cortices. Perirhinal area 35 and posterior entorhinal cortex showed greatest p-tau burden but also the highest vessel density in the preclinical phase of Alzheimer's disease. We combined an ex vivo magnetic resonance imaging model and histopathology to demonstrate the 3D reconstruction of intracortical vessels and its spatial relationship to the pathology.

FreeSurfer-based segmentation of hippocampal subfields: A review of methods and applications, with a novel quality control procedure for ENIGMA studies and other collaborative efforts.

Structural hippocampal abnormalities are common in many neurological and psychiatric disorders, and variation in hippocampal measures is related to cognitive performance and other complex phenotypes such as stress sensitivity. Hippocampal subregions are increasingly studied, as automated algorithms have become available for mapping and volume quantification. In the context of the Enhancing Neuro Imaging Genetics through Meta Analysis Consortium, several Disease Working Groups are using the FreeSurfer software to analyze hippocampal subregion (subfield) volumes in patients with neurological and psychiatric conditions along with data from matched controls. In this overview, we explain the algorithm's principles, summarize measurement reliability studies, and demonstrate two additional aspects (subfield autocorrelation and volume/reliability correlation) with illustrative data. We then explain the rationale for a standardized hippocampal subfield segmentation quality control (QC) procedure for improved pipeline harmonization. To guide researchers to make optimal use of the algorithm, we discuss how global size and age effects can be modeled, how QC steps can be incorporated and how subfields may be aggregated into composite volumes. This discussion is based on a synopsis of 162 published neuroimaging studies (01/2013-12/2019) that applied the FreeSurfer hippocampal subfield segmentation in a broad range of domains including cognition and healthy aging, brain development and neurodegeneration, affective disorders, psychosis, stress regulation, neurotoxicity, epilepsy, inflammatory disease, childhood adversity and posttraumatic stress disorder, and candidate and whole genome (epi-)genetics. Finally, we highlight points where FreeSurfer-based hippocampal subfield studies may be optimized.

A deep learning toolbox for automatic segmentation of subcortical limbic structures from MRI images.

A tool was developed to automatically segment several subcortical limbic structures (nucleus accumbens, basal forebrain, septal nuclei, hypothalamus without mammillary bodies, the mammillary bodies, and fornix) using only a T1-weighted MRI as input. This tool fills an unmet need as there are few, if any, publicly available tools to segment these clinically relevant structures. A U-Net with spatial, intensity, contrast, and noise augmentation was trained using 39 manually labeled MRI data sets. In general, the Dice scores, true positive rates, false discovery rates, and manual-automatic volume correlation were very good relative to comparable tools for other structures. A diverse data set of 698 subjects were segmented using the tool; evaluation of the resulting labelings showed that the tool failed in less than 1% of cases. Test-retest reliability of the tool was excellent. The automatically segmented volume of all structures except mammillary bodies showed effectiveness at detecting either clinical AD effects, age effects, or both. This tool will be publicly released with FreeSurfer (surfer.nmr.mgh.harvard.edu/fswiki/ScLimbic). Together with the other cortical and subcortical limbic segmentations, this tool will allow FreeSurfer to provide a comprehensive view of the limbic system in an automated way.

as-PSOCT: Volumetric microscopic imaging of human brain architecture and connectivity.

Polarization sensitive optical coherence tomography (PSOCT) with serial sectioning has enabled the investigation of 3D structures in mouse and human brain tissue samples. By using intrinsic optical properties of back-scattering and birefringence, PSOCT reliably images cytoarchitecture, myeloarchitecture and fiber orientations. In this study, we developed a fully automatic serial sectioning polarization sensitive optical coherence tomography (as-PSOCT) system to enable volumetric reconstruction of human brain samples with unprecedented sample size and resolution. The 3.5 µm in-plane resolution and 50 µm through-plane voxel size allow inspection of cortical layers that are a single-cell in width, as well as small crossing fibers. We show the abilities of as-PSOCT in quantifying layer thicknesses of the cerebellar cortex and creating microscopic tractography of intricate fiber networks in the subcortical nuclei and internal capsule regions, all based on volumetric reconstructions. as-PSOCT provides a viable tool for studying quantitative cytoarchitecture and myeloarchitecture and mapping connectivity with microscopic resolution in the human brain.

A harmonized segmentation protocol for hippocampal and parahippocampal subregions: Why do we need one and what are the key goals?

The advent of high-resolution magnetic resonance imaging (MRI) has enabled in vivo research in a variety of populations and diseases on the structure and function of hippocampal subfields and subdivisions of the parahippocampal gyrus. Because of the many extant and highly discrepant segmentation protocols, comparing results across studies is difficult. To overcome this barrier, the Hippocampal Subfields Group was formed as an international collaboration with the aim of developing a harmonized protocol for manual segmentation of hippocampal and parahippocampal subregions on high-resolution MRI. In this commentary we discuss the goals for this protocol and the associated key challenges involved in its development. These include differences among existing anatomical reference materials, striking the right balance between reliability of measurements and anatomical validity, and the development of a versatile protocol that can be adopted for the study of populations varying in age and health. The commentary outlines these key challenges, as well as the proposed solution of each, with concrete examples from our working plan. Finally, with two examples, we illustrate how the harmonized protocol, once completed, is expected to impact the field by producing measurements that are quantitatively comparable across labs and by facilitating the synthesis of findings across different studies. © 2016 Wiley Periodicals, Inc.

A computational atlas of the hippocampal formation using ex vivo, ultra-high resolution MRI: Application to adaptive segmentation of in vivo MRI.

Automated analysis of MRI data of the subregions of the hippocampus requires computational atlases built at a higher resolution than those that are typically used in current neuroimaging studies. Here we describe the construction of a statistical atlas of the hippocampal formation at the subregion level using ultra-high resolution, ex vivo MRI. Fifteen autopsy samples were scanned at 0.13 mm isotropic resolution (on average) using customized hardware. The images were manually segmented into 13 different hippocampal substructures using a protocol specifically designed for this study; precise delineations were made possible by the extraordinary resolution of the scans. In addition to the subregions, manual annotations for neighboring structures (e.g., amygdala, cortex) were obtained from a separate dataset of in vivo, T1-weighted MRI scans of the whole brain (1mm resolution). The manual labels from the in vivo and ex vivo data were combined into a single computational atlas of the hippocampal formation with a novel atlas building algorithm based on Bayesian inference. The resulting atlas can be used to automatically segment the hippocampal subregions in structural MRI images, using an algorithm that can analyze multimodal data and adapt to variations in MRI contrast due to differences in acquisition hardware or pulse sequences. The applicability of the atlas, which we are releasing as part of FreeSurfer (version 6.0), is demonstrated with experiments on three different publicly available datasets with different types of MRI contrast. The results show that the atlas and companion segmentation method: 1) can segment T1 and T2 images, as well as their combination, 2) replicate findings on mild cognitive impairment based on high-resolution T2 data, and 3) can discriminate between Alzheimer's disease subjects and elderly controls with 88% accuracy in standard resolution (1mm) T1 data, significantly outperforming the atlas in FreeSurfer version 5.3 (86% accuracy) and classification based on whole hippocampal volume (82% accuracy).

Quantitative comparison of 21 protocols for labeling hippocampal subfields and parahippocampal subregions in in vivo MRI: towards a harmonized segmentation protocol.

OBJECTIVE: An increasing number of human in vivo magnetic resonance imaging (MRI) studies have focused on examining the structure and function of the subfields of the hippocampal formation (the dentate gyrus, CA fields 1-3, and the subiculum) and subregions of the parahippocampal gyrus (entorhinal, perirhinal, and parahippocampal cortices). The ability to interpret the results of such studies and to relate them to each other would be improved if a common standard existed for labeling hippocampal subfields and parahippocampal subregions. Currently, research groups label different subsets of structures and use different rules, landmarks, and cues to define their anatomical extents. This paper characterizes, both qualitatively and quantitatively, the variability in the existing manual segmentation protocols for labeling hippocampal and parahippocampal substructures in MRI, with the goal of guiding subsequent work on developing a harmonized substructure segmentation protocol. METHOD: MRI scans of a single healthy adult human subject were acquired both at 3 T and 7 T. Representatives from 21 research groups applied their respective manual segmentation protocols to the MRI modalities of their choice. The resulting set of 21 segmentations was analyzed in a common anatomical space to quantify similarity and identify areas of agreement. RESULTS: The differences between the 21 protocols include the region within which segmentation is performed, the set of anatomical labels used, and the extents of specific anatomical labels. The greatest overall disagreement among the protocols is at the CA1/subiculum boundary, and disagreement across all structures is greatest in the anterior portion of the hippocampal formation relative to the body and tail. CONCLUSIONS: The combined examination of the 21 protocols in the same dataset suggests possible strategies towards developing a harmonized subfield segmentation protocol and facilitates comparison between published studies.

MRI parcellation of ex vivo medial temporal lobe.

Recent advancements in radio frequency coils, field strength and sophisticated pulse sequences have propelled modern brain mapping and have made validation to biological standards - histology and pathology - possible. The medial temporal lobe has long been established as a pivotal brain region for connectivity, function and unique structure in the human brain, and reveals disconnection in mild Alzheimer's disease. Specific brain mapping of mesocortical areas affected with neurofibrillary tangle pathology early in disease progression provides not only an accurate description for location of these areas but also supplies spherical coordinates that allow comparison between other ex vivo cases and larger in vivo datasets. We have identified several cytoarchitectonic features in the medial temporal lobe with high resolution ex vivo MRI, including gray matter structures such as the entorhinal layer II 'islands', perirhinal layer II-III columns, presubicular 'clouds', granule cell layer of the dentate gyrus as well as lamina of the hippocampus. Localization of Brodmann areas 28 and 35 (entorhinal and perirhinal, respectively) demonstrates MRI based area boundaries validated with multiple methods and histological stains. Based on our findings, both myelin and Nissl staining relate to contrast in ex vivo MRI. Precise brain mapping serves to create modern atlases for cortical areas, allowing accurate localization with important applications to detecting early disease processes.

Blockface histology with optical coherence tomography: a comparison with Nissl staining.

Spectral domain optical coherence tomography (SD-OCT) is a high resolution imaging technique that generates excellent contrast based on intrinsic optical properties of the tissue, such as neurons and fibers. The SD-OCT data acquisition is performed directly on the tissue block, diminishing the need for cutting, mounting and staining. We utilized SD-OCT to visualize the laminar structure of the isocortex and compared cortical cytoarchitecture with the gold standard Nissl staining, both qualitatively and quantitatively. In histological processing, distortions routinely affect registration to the blockface image and prevent accurate 3D reconstruction of regions of tissue. We compared blockface registration to SD-OCT and Nissl, respectively, and found that SD-OCT-blockface registration was significantly more accurate than Nissl-blockface registration. Two independent observers manually labeled cortical laminae (e.g. III, IV and V) in SD-OCT images and Nissl stained sections. Our results show that OCT images exhibit sufficient contrast in the cortex to reliably differentiate the cortical layers. Furthermore, the modalities were compared with regard to cortical laminar organization and showed good agreement. Taken together, these SD-OCT results suggest that SD-OCT contains information comparable to standard histological stains such as Nissl in terms of distinguishing cortical layers and architectonic areas. Given these data, we propose that SD-OCT can be used to reliably generate 3D reconstructions of multiple cubic centimeters of cortex that can be used to accurately and semi-automatically perform standard histological analyses.

H.M.'s contributions to neuroscience: a review and autopsy studies.

H.M., Henry Molaison, was one of the world's most famous amnesic patients. His amnesia was caused by an experimental brain operation, bilateral medial temporal lobe resection, carried out in 1953 to relieve intractable epilepsy. He died on December 2, 2008, and that night we conducted a wide variety of in situ MRI scans in a 3 T scanner at the Massachusetts General Hospital (Mass General) Athinoula A. Martinos Center for Biomedical Imaging. For the in situ experiments, we acquired a full set of standard clinical scans, 1 mm isotropic anatomical scans, and multiple averages of 440 µm isotropic anatomical scans. The next morning, H.M.'s body was transported to the Mass General Morgue for autopsy. The photographs taken at that time provided the first documentation of H.M.'s lesions in his physical brain. After tissue fixation, we obtained ex vivo structural data at ultra-high resolution using 3 T and 7 T magnets. For the ex vivo acquisitions, the highest resolution images were 210 µm isotropic. Based on the MRI data, the anatomical areas removed during H.M.'s experimental operation were the medial temporopolar cortex, piriform cortex, virtually all of the entorhinal cortex, most of the perirhinal cortex and subiculum, the amygdala (except parts of the dorsal-most nuclei-central and medial), anterior half of the hippocampus, and the dentate gyrus (posterior head and body). The posterior parahippocampal gyrus and medial temporal stem were partially damaged. Spared medial temporal lobe tissue included the dorsal-most amygdala, the hippocampal-amygdalo-transition-area, ∼2 cm of the tail of the hippocampus, a small part of perirhinal cortex, a small portion of medial hippocampal tissue, and ∼2 cm of posterior parahippocampal gyrus. H.M.'s impact on the field of memory has been remarkable, and his contributions to neuroscience continue with a unique dataset that includes in vivo, in situ, and ex vivo high-resolution MRI.

Automatic Geometry-based Estimation of the Locus Coeruleus Region on T1-Weighted Magnetic Resonance Images.

The locus coeruleus (LC) is a key brain structure implicated in cognitive function and neurodegenerative disease. Automatic segmentation of the LC is a crucial step in quantitative non-invasive analysis of the LC in large MRI cohorts. Most publicly available imaging databases for training automatic LC segmentation models take advantage of specialized contrast-enhancing (e.g., neuromelanin-sensitive) MRI. Segmentation models developed with such image contrasts, however, are not readily applicable to existing datasets with conventional MRI sequences. In this work, we evaluate the feasibility of using non-contrast neuroanatomical information to geometrically approximate the LC region from standard 3-Tesla T1-weighted images of 20 subjects from the Human Connectome Project (HCP). We employ this dataset to train and internally/externally evaluate two automatic localization methods, the Expected Label Value and the U-Net. We also test the hypothesis that using the phase image as input can improve the robustness of out-of-sample segmentation. We then apply our trained models to a larger subset of HCP, while exploratorily correlating LC imaging variables and structural connectivity with demographic and clinical data. This report contributes and provides an evaluation of two computational methods estimating neural structure.

Automatic geometry-based estimation of the locus coeruleus region on T1-weighted magnetic resonance images.

The locus coeruleus (LC) is a key brain structure implicated in cognitive function and neurodegenerative disease. Automatic segmentation of the LC is a crucial step in quantitative non-invasive analysis of the LC in large MRI cohorts. Most publicly available imaging databases for training automatic LC segmentation models take advantage of specialized contrast-enhancing (e.g., neuromelanin-sensitive) MRI. Segmentation models developed with such image contrasts, however, are not readily applicable to existing datasets with conventional MRI sequences. In this work, we evaluate the feasibility of using non-contrast neuroanatomical information to geometrically approximate the LC region from standard 3-Tesla T1-weighted images of 20 subjects from the Human Connectome Project (HCP). We employ this dataset to train and internally/externally evaluate two automatic localization methods, the Expected Label Value and the U-Net. For out-of-sample segmentation, we compare the results with atlas-based segmentation, as well as test the hypothesis that using the phase image as input can improve the robustness. We then apply our trained models to a larger subset of HCP, while exploratorily correlating LC imaging variables and structural connectivity with demographic and clinical data. This report provides an evaluation of computational methods estimating neural structure.

The prosubiculum in the human hippocampus: A rostrocaudal, feature-driven, and systematic approach.

The hippocampal subfield prosubiculum (ProS), is a conserved neuroanatomic region in mouse, monkey, and human. This area lies between CA1 and subiculum (Sub) and particularly lacks consensus on its boundaries; reports have varied on the description of its features and location. In this report, we review, refine, and evaluate four cytoarchitectural features that differentiate ProS from its neighboring subfields: (1) small neurons, (2) lightly stained neurons, (3) superficial clustered neurons, and (4) a cell sparse zone. ProS was delineated in all cases (n = 10). ProS was examined for its cytoarchitectonic features and location rostrocaudally, from the anterior head through the body in the hippocampus. The most common feature was small pyramidal neurons, which were intermingled with larger pyramidal neurons in ProS. We quantitatively measured ProS pyramidal neurons, which showed (average, width at pyramidal base = 14.31 µm, n = 400 per subfield). CA1 neurons averaged 15.57 µm and Sub neurons averaged 15.63 µm, both were significantly different than ProS (Kruskal-Wallis test, p < .0001). The other three features observed were lightly stained neurons, clustered neurons, and a cell sparse zone. Taken together, these findings suggest that ProS is an independent subfield, likely with distinct functional contributions to the broader interconnected hippocampal network. Our results suggest that ProS is a cytoarchitecturally varied subfield, both for features and among individuals. This diverse architecture in features and individuals for ProS could explain the long-standing complexity regarding the identification of this subfield.

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories.

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the cortices that make up the parahippocampal gyrus (entorhinal and parahippocampal cortices) and the adjacent Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 µm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized (20X resolution) slices with 5 mm spacing. Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed more gradually. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed human neuroimaging research on the MTL cortex.

Predicting the location of human perirhinal cortex, Brodmann's area 35, from MRI.

The perirhinal cortex (Brodmann's area 35) is a multimodal area that is important for normal memory function. Specifically, perirhinal cortex is involved in the detection of novel objects and manifests neurofibrillary tangles in Alzheimer's disease very early in disease progression. We scanned ex vivo brain hemispheres at standard resolution (1 mm × 1 mm × 1 mm) to construct pial/white matter surfaces in FreeSurfer and scanned again at high resolution (120 µm × 120 µm × 120 µm) to determine cortical architectural boundaries. After labeling perirhinal area 35 in the high resolution images, we mapped the high resolution labels to the surface models to localize area 35 in fourteen cases. We validated the area boundaries determined using histological Nissl staining. To test the accuracy of the probabilistic mapping, we measured the Hausdorff distance between the predicted and true labels and found that the median Hausdorff distance was 4.0mm for the left hemispheres (n=7) and 3.2mm for the right hemispheres (n=7) across subjects. To show the utility of perirhinal localization, we mapped our labels to a subset of the Alzheimer's Disease Neuroimaging Initiative dataset and found decreased cortical thickness measures in mild cognitive impairment and Alzheimer's disease compared to controls in the predicted perirhinal area 35. Our ex vivo probabilistic mapping of the perirhinal cortex provides histologically validated, automated and accurate labeling of architectonic regions in the medial temporal lobe, and facilitates the analysis of atrophic changes in a large dataset for earlier detection and diagnosis.

Surface based analysis of diffusion orientation for identifying architectonic domains in the in vivo human cortex.

Diffusion tensor MRI is sensitive to the coherent structure of brain tissue and is commonly used to study large-scale white matter structure. Diffusion in gray matter is more isotropic, however, several groups have observed coherent patterns of diffusion anisotropy within the cerebral cortical gray matter. We extend the study of cortical diffusion anisotropy by relating it to the local coordinate system of the folded cerebral cortex. We use 1mm and sub-millimeter isotropic resolution diffusion imaging to perform a laminar analysis of the principal diffusion orientation, fractional anisotropy, mean diffusivity and partial volume effects. Data from 6 in vivo human subjects, a fixed human brain specimen and an anesthetized macaque were examined. Large regions of cortex show a radial diffusion orientation. In vivo human and macaque data displayed a sharp transition from radial to tangential diffusion orientation at the border between primary motor and somatosensory cortex, and some evidence of tangential diffusion in secondary somatosensory cortex and primary auditory cortex. Ex vivo diffusion imaging in a human tissue sample showed some tangential diffusion orientation in S1 but mostly radial diffusion orientations in both M1 and S1.

Quantitative optical coherence microscopy of neuron morphology in human entorhinal cortex.

Introduction: The size and shape of neurons are important features indicating aging and the pathology of neurodegenerative diseases. Despite the significant advances of optical microscopy, quantitative analysis of the neuronal features in the human brain remains largely incomplete. Traditional histology on thin slices bears tremendous distortions in three-dimensional reconstruction, the magnitude of which are often greater than the structure of interest. Recently development of tissue clearing techniques enable the whole brain to be analyzed in small animals; however, the application in the human remains challenging. Methods: In this study, we present a label-free quantitative optical coherence microscopy (OCM) technique to obtain the morphological parameters of neurons in human entorhinal cortex (EC). OCM uses the intrinsic back-scattering property of tissue to identify individual neurons in 3D. The area, length, width, and orientation of individual neurons are quantified and compared between layer II and III in EC. Results: The high-resolution mapping of neuron size, shape, and orientation shows significant differences between layer II and III neurons in EC. The results are validated by standard Nissl staining of the same samples. Discussion: The quantitative OCM technique in our study offers a new solution to analyze variety of neurons and their organizations in the human brain, which opens new insights in advancing our understanding of neurodegenerative diseases.

TDP-43 and tau concurrence in the entorhinal subfields in primary age-related tauopathy and preclinical Alzheimer's disease.

Phosphorylated tau (p-tau) pathology correlates strongly with cognitive decline and is a pathological hallmark of Alzheimer's Disease (AD). In recent years, phosphorylated transactive response DNA-binding protein (pTDP-43) has emerged as a common comorbidity, found in up to 70% of all AD cases (Josephs et al., Acta Neuropathol, 131(4), 571-585; Josephs, Whitwell, et al., Acta Neuropathol, 127(6), 811-824). Current staging schemes for pTDP-43 in AD and primary age-related tauopathy (PART) track its progression throughout the brain, but the distribution of pTDP-43 within the entorhinal cortex (EC) at the earliest stages has not been studied. Moreover, the exact nature of p-tau and pTDP-43 co-localization is debated. We investigated the selective vulnerability of the entorhinal subfields to phosphorylated pTDP-43 pathology in preclinical AD and PART postmortem tissue. Within the EC, posterior-lateral subfields showed the highest semi-quantitative pTDP-43 density scores, while the anterior-medial subfields had the lowest. On the rostrocaudal axis, pTDP-43 scores were higher posteriorly than anteriorly (p < 0.010), peaking at the posterior-most level (p < 0.050). Further, we showed the relationship between pTDP-43 and p-tau in these regions at pathology-positive but clinically silent stages. P-tau and pTDP-43 presented a similar pattern of affected subregions (p < 0.0001) but differed in density magnitude (p < 0.0001). P-tau burden was consistently higher than pTDP-43 at every anterior-posterior level and in most EC subfields. These findings highlight pTDP-43 burden heterogeneity within the EC and the posterior-lateral subfields as the most vulnerable regions within stage II of the current pTDP-43 staging schemes for AD and PART. The EC is a point of convergence for p-tau and pTDP-43 and identifying its most vulnerable neuronal populations will prove key for early diagnosis and disease intervention.

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

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.