Probabilistic maps of neocortical areas and subcortical fiber tracts, warped to a common reference brain, have been published using microscopic architectonic parcellations in ten human postmortem brains. The maps have been successfully applied as topographical references for the anatomical localization of activations observed in functional imaging studies. Here, for the first time, we present stereotaxic, probabilistic maps of the hippocampus, the amygdala and the entorhinal cortex and some of their subdivisions. Cytoarchitectonic mapping was performed in serial, cell-body stained histological sections. The positions and the extent of cytoarchitectonically defined structures were traced in digitized histological sections, 3-D reconstructed and warped to the reference space of the MNI single subject brain using both linear and non-linear elastic tools of alignment. The probability maps and volumes of all structures were calculated. The precise localization of the borders of the mapped regions cannot be predicted consistently by macroanatomical landmarks. Many borders, e.g. between the subiculum and entorhinal cortex, subiculum and Cornu ammonis, and amygdala and hippocampus, do not match sulcal landmarks such as the bottom of a sulcus. Only microscopic observation enables the precise localization of the borders of these brain regions. The superposition of the cytoarchitectonic maps in the common spatial reference system shows a considerably lower degree of intersubject variability in size and position of the allocortical structures and nuclei than the previously delineated neocortical areas. For the first time, the present observations provide cytoarchitectonically verified maps of the human amygdala, hippocampus and entorhinal cortex, which take into account the stereotaxic position of the brain structures as well as intersubject variability. We believe that these maps are efficient tools for the precise microstructural localization of fMRI, PET and anatomical MR data, both in healthy and pathologically altered brains.

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. Due to 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.

The human hippocampal formation is relevant for various aspects of memory and learning, and the different hippocampal regions are differentially affected by neuropsychiatric disorders. Therefore, the hippocampal formation has been subject of numerous cytoarchitectonic and other mapping studies, which resulted in divergent parcellation schemes. To understand the principles of hippocampal architecture, it is necessary to integrate different levels of hippocampal organisation, going beyond one modality. We here applied a multimodal mapping approach combining cyto-and multi-receptorarchitectonic analyses, and generated probabilistic maps in stereotaxic space of the identified regions. Cytoarchitecture in combination with the regional and laminar distribution of 15 neurotransmitter receptors visualized by in vitro receptor autoradiography were analysed in seven hemispheres from 6 unfixed shock frozen and serially sectioned brains. Cytoarchitectonic delineations for generation of probabilistic maps were carried out on histological sections from ten fixed, paraffin embedded and serially sectioned brains. Nine cyto-and receptorarchitectonically distinct regions were identified within the hippocampal formation (i.e., fascia dentata, cornu Ammonis (CA) regions 1-4, prosubiculum, subiculum proper, presubiculum and parasubiculum), as well as the hippocampal-amygdaloid transition area and the periallocortical transsubiculum. Subsequently generated probabilistic maps quantify intersubject variability in the size and extent of these cyto-and receptorarchitectonically distinct regions. The regions did not differ in their volume between the hemispheres and gender. Receptor mapping revealed additional subdivisions which could not be detected by cytoarchitectonic analysis alone. They correspond to parcellations previously found in immunohistochemical and connectivity studies. The multimodal approach enabled the definition of regions not consistently reported, e.g., CA4 region or prosubiculum. The ensuing detailed probabilistic maps of the hippocampal formation constitute the basis for future architectonically informed analyses of in vivo neuroimaging studies.

While the amygdala is considered to play a critical role in temporal lobe epilepsy, conclusions on underlying pathophysiological mechanisms have been derived largely from experimental animal studies. Therefore, the present study aimed to characterize synaptic network interactions, focusing on spontaneous interictal-like activity, and the expression profile of transmitter receptors in the human lateral amygdala in relation to temporal lobe epilepsy. Electrophysiological recordings, obtained intra-operatively in vivo in patients with medically intractable temporal lobe epilepsy, revealed the existence of interictal activity in amygdala and hippocampus. For in vitro analyses, slices were prepared from surgically resected specimens, and sections from individual specimens were used for electrophysiological recordings, receptor autoradiographic analyses and histological visualization of major amygdaloid nuclei for verification of recording sites. In the lateral amygdala, interictal-like activity appeared as spontaneous slow rhythmic field potentials at an average frequency of 0.39 Hz, which occurred at different sites with various degrees of synchronization in 33.3% of the tested slices. Pharmacological blockade of glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, but not N-methyl-D-aspartate receptors, abolished interictal-like activity, while the γ-aminobutyric acid A-type receptor antagonist bicuculline resulted in a dampening of activity, followed by highly synchronous patterns of slow rhythmic activity during washout. Receptor autoradiographic analysis revealed significantly higher α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, metabotropic glutamate type 2/3, muscarinic type 2 and adrenoceptor α(1) densities, whereas muscarinergic type 3 and serotonergic type 1A receptor densities were lower in the lateral amygdala from epileptic patients in comparison to autopsy controls. Concerning γ-aminobutyric acid A-type receptors, agonist binding was unaltered whereas antagonist binding sites were downregulated in the epileptic lateral amygdala, suggesting an altered high/low-affinity state ratio and concomitant reduced pool of total γ-aminobutyric acid A-type receptors. Together these data indicate an abnormal pattern of receptor densities and synaptic function in the lateral nucleus of the amygdala in epileptic patients, involving critical alterations in glutamate and γ-aminobutyric acid receptors, which may give rise to domains of spontaneous interictal discharges contributing to seizure activity in the amygdala.

Introduction Heterogeneity of segmentation protocols for medial temporal lobe regions and hippocampal subfields on in vivo magnetic resonance imaging hinders the ability to integrate findings across studies. We aim to develop a harmonized protocol based on expert consensus and histological evidence. Methods Our international working group, funded by the EU Joint Programme–Neurodegenerative Disease Research (JPND), is working toward the production of a reliable, validated, harmonized protocol for segmentation of medial temporal lobe regions. The working group uses a novel postmortem data set and online consensus procedures to ensure validity and facilitate adoption. Results This progress report describes the initial results and milestones that we have achieved to date, including the development of a draft protocol and results from the initial reliability tests and consensus procedures. Discussion A harmonized protocol will enable the standardization of segmentation methods across laboratories interested in medial temporal lobe research worldwide.

The human amygdala consists of subdivisions contributing to various functions. However, principles of structural organization at the cellular and molecular level are not well understood. Thus, we re-analyzed the cytoarchitecture of the amygdala and generated cytoarchitectonic probabilistic maps of ten subdivisions in stereotaxic space based on novel workflows and mapping tools. This parcellation was then used as a basis for analyzing the receptor expression for 15 receptor types. Receptor fingerprints, i.e., the characteristic balance between densities of all receptor types, were generated in each subdivision to comprehensively visualize differences and similarities in receptor architecture between the subdivisions. Fingerprints of the central and medial nuclei and the anterior amygdaloid area were highly similar. Fingerprints of the lateral, basolateral and basomedial nuclei were also similar to each other, while those of the remaining nuclei were distinct in shape. Similarities were further investigated by a hierarchical cluster analysis: a two-cluster solution subdivided the phylogenetically older part (central, medial nuclei, anterior amygdaloid area) from the remaining parts of the amygdala. A more fine-grained three-cluster solution replicated our previous parcellation including a laterobasal, superficial and centromedial group. Furthermore, it helped to better characterize the paralaminar nucleus with a molecular organization in-between the laterobasal and the superficial group. The multimodal cyto- and receptor-architectonic analysis of the human amygdala provides new insights into its microstructural organization, intersubject variability, localization in stereotaxic space and principles of receptor-based neurochemical differences.

The ‘BigBrain’ is a high-resolution data set of the human brain that enables three-dimensional (3D) analyses with a 20 µm spatial resolution at nearly cellular level. We use this data set to explore pre-α (cell) islands of layer 2 in the entorhinal cortex (EC), which are early affected in Alzheimer’s disease and have therefore been the focus of research for many years. They appear mostly in a round and elongated shape as shown in microscopic studies. Some studies suggested that islands may be interconnected based on analyses of their shape and size in two-dimensional (2D) space. Here, we characterized morphological features (shape, size, and distribution) of pre-α islands in the ‘BigBrain’, based on 3D-reconstructions of gapless series of cell-body-stained sections. The EC was annotated manually, and a machine-learning tool was trained to identify and segment islands with subsequent visualization using high-performance computing (HPC). Islands were visualized as 3D surfaces and their geometry was analyzed. Their morphology was complex: they appeared to be composed of interconnected islands of different types found in 2D histological sections of EC, with various shapes in 3D. Differences in the rostral-to-caudal part of EC were identified by specific distribution and size of islands, with implications for connectivity and function of the EC. 3D compactness analysis found more round and complex islands than elongated ones. The present study represents a use case for studying large microscopic data sets. It provides reference data for studies, e.g. investigating neurodegenerative diseases, where specific alterations in layer 2 were previously reported.

The hippocampus is an archicortical structure, consisting of subfields with unique circuits. Understanding its microstructure, as proxied by these subfields, can improve our mechanistic understanding of learning and memory and has clinical potential for several neurological disorders. One prominent issue is how to parcellate, register, or retrieve homologous points between two hippocampi with grossly different morphologies. Here, we present a surface-based registration method that solves this issue in a contrast-agnostic, topology-preserving manner. Specifically, the entire hippocampus is first analytically unfolded, and then samples are registered in 2D unfolded space based on thickness, curvature, and gyrification. We demonstrate this method in seven 3D histology samples and show superior alignment with respect to subfields using this method over more conventional registration approaches. Hippocampal subfields contain microcircuits that are critical for memory and vulnerable to neurological disease. Hippocampi have variable folding patterns between individuals, making them hard to register or parcellate. We present a surface-based hippocampal registration method that is analogous to neocortical inflation to a sphere and registration. Testing in seven detailed 3D histology samples revealed successful registration with respect to hippocampal subfields, and outperformed more conventional methods. This method provides groundwork for detailed multimodal hippocampal mapping across subjects and datasets in the future. The methodological advancements described here are made easily accessible in the latest version of open source software HippUnfold 1 . Code used in the development and testing of these methods, as well as preprocessed images, manual segmentations, and results, are openly available 2 .