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Architectonic Mapping
of the Human Brain beyond Brodmann
Katrin Amunts1,2,3,* and Karl Zilles1,3,4,*
1Instituteof Neuroscience and Medicine (INM-1), Research Centre Jülich, 52425 Jülich, Germany
2C. and O. Vogt Institute for Brain Research, Heinrich-Heine-University Düsseldorf, 40001 Düsseldorf, Germany
3JARA-BRAIN, Jülich-Aachen Research Alliance, 52425 Jülich, Germany
4Department of Psychiatry, Psychotherapy, and Psychosomatics, RWTH Aachen University, 52072 Aachen, Germany

*Correspondence: (K.A.), (K.Z.)

Brodmann has pioneered structural brain mapping. He considered functional and pathological criteria for
defining cortical areas in addition to cytoarchitecture. Starting from this idea of structural-functional relation-
ships at the level of cortical areas, we will argue that the cortical architecture is more heterogeneous than
Brodmann’s map suggests. A triple-scale concept is proposed that includes repetitive modular-like struc-
tures and micro- and meso-maps. Criteria for defining a cortical area will be discussed, considering novel
preparations, imaging and optical methods, 2D and 3D quantitative architectonics, as well as high-perfor-
mance computing including analyses of big data. These new approaches contribute to an understanding
of the brain on multiple levels and challenge the traditional, mosaic-like segregation of the cerebral cortex.

Introduction well as potential future strategies of multimodal and multiscale

Korbinian Brodmann subdivided the cerebral cortex into approaches from areas to cells and molecules. Starting with
numerous areas based on regional differences in the distribution, Brodmann’s idea about structural-functional relationships at
density, shape, and size of cell bodies, i.e., the cytoarchitecture the level of cortical areas and its relationship to myeloarchitec-
(Brodmann, 1909). Although not proven at that time, he was ture, we will argue that intra-areal organization is more heteroge-
convinced that each cortical area subserves a certain function neous than classical cortical maps suggest. Consequently, it
within a larger network. In present neuroimaging studies, Brod- requires a new definition of the concept of a ‘‘cortical area’’ as
mann’s schematic drawings of cortical maps are still frequently central element of cortical segregation. We will highlight and
used references to register functional activations to anatomical discuss the impact of recent developments in quantitative cy-
structures, although his map does not match more recent toarchitectonics and probabilistic mapping of cortical areas, as
anatomical and functional data in many brain regions (Zilles well as novel methods to specifically label cellular and molecular
and Amunts, 2010), and new approaches are mandatory components of cortical architecture including ‘‘whole-brain
(Amunts et al., 2014b). approaches.’’ Finally, we will discuss the potential of and the
Brain mapping based on the regional distribution of cortical challenges on modern optic and computational methods for a
areas is a valuable concept beyond the generation of an atlas. deeper understanding of cortical organization, including its com-
It is a way to understand cortical organization by integrating plex fiber architecture and structural connectivity.
in vivo structural and functional imaging data and post mortem
high-resolution cytoarchitectonic observations in a common Cytoarchitectonic, Myeloarchitectonic, and Myelin
reference space (Amunts et al., 2007, 2014b; Mazziotta et al., Density Maps
2001; Roland et al., 1997; Roland and Zilles, 1994). This attempt The spatial distribution pattern of neuronal cell bodies is called
to compare architectonic and functional data has a long history cytoarchitecture, and that of myelinated nerve fibers represents
and goes back to Cecile and Oskar Vogt (Vogt and Vogt, 1919) the myeloarchitecture (Brodmann, 1909; Vogt and Vogt, 1919;
who collaborated with the neurosurgeon Otfried Förster (Foer- Von Economo and Koskinas, 1925; Zilles et al., 2015a, 2015b).
ster, 1931). They performed electrophysiological mapping in pa- Distinct layers of cell bodies (parallel to the cortical surface)
tients and monkeys and compared the independently achieved and myelinated fibers (vertically, horizontally, and obliquely ori-
architectonic and functional results in both species to under- ented) can be identified in the cerebral cortex. Cell bodies are
stand the functional role of architectonically defined areas also vertically arranged, thus forming (mini)columns (Buxhoeve-
(Vogt and Vogt, 1926) (Figure 1). Their approach conceptually den et al., 2000; Schlaug et al., 1995; Schleicher and Zilles,
foresees the development of brain mapping during the last de- 2005).
cades (Table 1). Most regions of the human cerebral cortex have a six-layered
In the present review, we will focus on recent developments in architecture (isocortex), with the notable exception of the motor
mapping the microscopical organization of the human cerebral cortex, which does not show a clearly recognizable layer IV in
cortex, which not only is based on new methods and tools for adult brains (Brodmann, 1909). Non-isocortical (i.e., allocortical)
observer-independent parcellations, but also includes the novel regions have more (e.g., entorhinal cortex) or less (e.g.,
concept of probabilistic mapping. It will also review available as hippocampus) layers than the isocortex. Regionally specific

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Figure 1. Early Maps of Cortical
Hand-drawn, myeloarchitectonic map by Oskar
Vogt (provided by the C. and O. Vogt Archive,
Heinrich Heine University Düsseldorf) and elec-
trophysiological stimulation in patients under
neurosurgery provided by Otfried Förster (from
Vogt and Vogt, 1926). One of the research aims of
the Vogts was to understand the physiology of
brain areas, identified in myelo- and cytoarchi-
tectonic studies, and to relate anatomical aspects
of brain organization to their function in terms
of neurobiological processes, but also mental

inferior parietal lobule (Caspers et al.,

2006), and various other areas (e.g.,
Choi et al., 2006; Eickhoff et al., 2006b;
Grefkes et al., 2001; Kurth et al., 2010;
Malikovic et al., 2007; Malikovic et al.,
2015). Areas of the fusiform gyrus, some
visual areas, and the entorhinal cortex
seem to be more closely related to sulci
(Fischl et al., 2009; Hinds et al., 2009; Lor-
enz et al., 2015; Weiner et al., 2014). Thus,
the inference from macroscopical land-
marks to cytoarchitectonic borders may
be useful for an approximate anatomical
orientation, but the relationships of such
landmark-based maps to the architecture
must be proven for each brain region.
Can ‘‘tedious anatomy’’ required for ar-
chitectonic studies at the level of layers
and sublayers (Devlin and Poldrack,
2007) be overcome by using high-resolu-
tion structural MRI in the living brain? This
would require a spatial resolution of less
than 40 mm because some cortical
layers are only 30–40 mm wide (Von Econ-
omo and Koskinas, 1925). High-resolu-
differences in cyto- and/or myeloarchitecture enable the parcel- tion post mortem (T2 weighted images using a 4.7 T scanner;
lation of the cerebral cortex into microscopically definable areas. in-plane resolution 59 3 68 mm2; slice thickness 0.6 mm) and
To take advantage of in vivo neuroimaging methods, maps in vivo (T1 weighted images using a 1.5 T scanner; in-plane res-
were proposed that rely on macroscopical landmarks, and olution 0.28 3 0.28 mm2; slice thickness 0.25/0.35 mm; gray-
take gyral and sulcal patterns as criteria for parcellating the scale normalized surface coil images) MRI (Eickhoff et al.,
cortex (Lancaster et al., 2000; Tzourio-Mazoyer et al., 2002). 2005b) compared with microscopic cyto- and myeloarchitec-
Brodmann (1914) and Vogt and Vogt (1919) denied any precise tonic observations in histological sections from the same tissue
correlation between macroscopically visible landmarks and bor- block demonstrate that the MRI signal mainly reflects the varia-
ders of architectonic areas. Only the border between the primary tion of the myelin density throughout the different cortical layers.
motor and primary somatosensory areas is regularly found in the Up to 84% of the signal variation is caused by the heterogeneous
fundus of the central sulcus. The border between motor and pre- distributed myelin, while only 9%–16% is explained by the
motor cortex, however, is not defined by a macroscopic land- laminar variation of the packing density of cell bodies.
mark like the precentral or any other sulcus (Geyer and Zilles, The Vogts identified 185 areas based on differences in the
2005). For other areas, some borders seem to be associated pattern of myelinated axons between cortical areas; for recent
with sulci, whereas others are not. A lack of co-localization of reviews and new maps based on the data of the Vogts and their
architectonic borders with macroscopical landmarks is found collaborators, see (Nieuwenhuys, 2013; Nieuwenhuys et al.,
in the Broca region (Amunts et al., 1999; Amunts et al., 2004), ex- 2015). The Vogts stated that Brodmann, who described 43
trastriate visual areas (Rottschy et al., 2007), areas within the su- areas, had probably missed numerous borders. By comparing
perior (Scheperjans et al., 2005; Scheperjans et al., 2008b) and cyto- and myeloarchitectonic maps, they were convinced that

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Table 1. Development of Cytoarchitectonic Maps and Concepts

Brodmann’s Cytoarchitecture Beyond Brodmann
43 cytoarchitectonic areas Estimated 150–200 cortical areas (Blumensath et al., 2013; Vogt and Vogt, 1919; von
Economo and Koskinas, 1925)
Definition of border between areas based Definition of borders between areas based on quantitative criteria and statistical measures
on visual inspection
Nissl staining labels neuronal cell bodies as Stainings specific for neuronal cell types and myelin using immunohistology, enzymes, and
well as glial nuclei and endothelial cells genetic labeling
Based on light microscopy Novel optical techniques, e.g., clearing techniques, light sheet imaging, optical
tomography, PLI
Mosaic-like pattern More complex pattern including within-area heterogeneities, such as at the borders of visual
cortical areas and gradations (Sanides, 1964; Vogt, 1910; Zilles and Amunts, 2012b)
Single-brain (hemisphere) study Studies on larger samples to reveal intersubject variability and inter-hemispheric
Maps of areas as schematic 2D drawing of 3D maps and cytoarchitectonic probabilistic maps as a tool to study intersubject variability
the surface of a ‘‘typical’’ brain

the borders detected by both modalities coincide (Vogt and licly available. They provide a new type of a human brain atlas,
Vogt, 1919). Myelin-stained sections show the distribution of indicating the brain’s regional distribution pattern in myelin den-
myelinated axons, while the unstained neuropil in cytoarchitec- sity and its segregation into areas. They supplement recent web-
tonic sections represents the space occupied by synapses, based atlas tools and microstructural maps (Table 2).
axons, dendrites, and blood vessels. In contrast to myelin den- Myelin density maps show approximately 180 different areas.
sity maps (see below), myeloarchitecture reflects the course This is in the same range as the estimated number of human
and composition of single axons; they form layers and sublayers, neocortical areas of 150–200 areas in surface-based atlases
run in parallel or orthogonal to the cortical surface, but also cross (Van Essen et al., 2012a) and the number of areas identified by
other axons in an oblique way (Vogt and Vogt, 1919). It can be the Vogt school (185 areas). Correspondences in extent and po-
concluded that both cytoarchitecture and myeloarchitecture sition between cyto- and myeloarchitectonic areas have been
reflect different aspects of connectivity (Figure 2). confirmed for several areas, but not for all from different regions,
Although less popular than Brodmann’s cytoarchitectonic including striate and extrastriate cortex, areas 4a and 4p of the
map, myeloarchitectonic parcellations recently received more primary motor cortex, areas 1, 2, and 3a/3b of the somatosen-
attention because high-resolution MRI enables the visualization sory cortex, and areas 44 and 45 of Broca’s region (Amunts
of regional differences in intracortical myelin density similar to, et al., 2000; Van Essen et al., 2012a).
but at much lower spatial resolution than, the classical mye- The comparison of the total number of areas, however, must
loarchitectonic studies of the Vogts. The delineation of a few be interpreted only as a first step toward a systematic and
cortical regions has been described using MRI (e.g., Barazany comprehensive comparison since similarities and differences
and Assaf, 2012; Dinse et al., 2015; Geyer et al., 2011; Walters vary between brain regions and types of areas. Higher associa-
et al., 2003). Single nerve fibers or fiber bundles have not been tive areas, e.g., of the frontal or temporal lobes, show a much
visualized in these studies, and their spatial orientation cannot finer-grained segregation in high-resolution analyses of post
be determined. Such features, however, were crucial criteria mortem brains as compared to myelin maps. An example is Bro-
for parcellation in the classical myeloarchitectonic studies ca’s region, where more than a dozen areas have been identified
(Vogt and Vogt, 1919). High angular resolution diffusion imaging based on its receptor architecture of different neurotransmitter
showed myeloarchitectonic features, e.g., inner and outer systems (Amunts et al., 2010); the same region appears to be
Baillarger stripes, Cajal-Retzius stripe, which enables a clear quite homogenous with only a few ‘‘hotspots’’ and ‘‘patches’’
distinction between a prefrontal area and primary visual, so- in recent multimodal maps based on functional connectivity,
matosensory and motor areas at an isotropic spatial resolution myelin, and fMRI of a large cohort (Van Essen, 2013). If it is
of 92 mm (Aggarwal et al., 2015). true that myelin maps underestimate the number of areas in
Whole-brain maps based on MRI-based myelin measure- some regions, such as Broca’s region and other prefrontal areas,
ments have also been provided (Augustinack et al., 2014; Fischl it can be expected that the real number of areas exceeds 180.
et al., 2004; Glasser et al., 2014; Glasser and Van Essen, 2011; The microscopic analysis of histological sections in post mor-
Moreno-Dominguez et al., 2014; Van Essen and Glasser, tem brains remains the ‘‘gold standard’’ to verify structural par-
2014), and retinotopic maps of the visual cortex correspond well cellations in the human brain. The higher spatial resolution not
with those myelin maps (Abdollahi et al., 2014). The T1w/T2w ra- only allows the study of laminar and sub-laminar patterns of
tio is a reasonable estimate of relative myelin content across the cell distributions, but also enables mapping with nearly similar
cortical surface, resulting in a myelin density map (Glasser and quality in all brain regions throughout the cortex. The gain in
Van Essen, 2011) with a spatial resolution above the laminar spatial resolution and level of detail opens new perspectives to
level. The maps of the Human Connectome Project (HCP) study the intrinsic structure of the cerebral cortex but is also
including myelin-based representations have been made pub- accompanied by new challenges. One concerns the number

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Figure 2. Principles of Cyto- and Myeloarchitecture of Human Neocortex and Their Relationship to Connectivity
Cytoarchitecture refers to the distribution of cells in cortical layers and sublayers, their density and morphology. The space surrounding cell bodies is called
neuropil—it contains space for synapses, dendrites, and axons. Myeloarchitecture refers to the area- and layer-specific distribution of axons with their myelin
sheaths. The drawings on the left and on the right panels were adapted from a drawing by the Vogts (Vogt and Vogt, 1919). Cells in particular layers have a specific
connectivity profile. For example, pyramidal cells in layer III project to other cortical areas, including those in the other hemisphere, while layer V neurons send
their axons to deeper structures of the brain stem or the spinal chord. The precise layer-specific connectivity for cortical areas of the human brain is largely

and localization of microstructural areas and corresponding organization of the visual cortex as proposed by Hubel and Wie-
maps, which differ in the literature even within one modality sel was described as an ‘‘ice-cube’’ model using electrophysio-
(for a review, see Zilles and Amunts, 2010; Zilles et al., 2015c). logical recordings and axonal tracing. The model emphasized
Examples of cyto- and myeloarchitectonic maps reach from 4 the existence of repeated units of similar function uniformly
major types of areas (Bailey and von Bonin, 1951) through 43 distributed throughout the primary visual cortex (Hubel and Wie-
of Brodmann (Brodmann, 1909) to about 150 by von Economo sel, 1972). Ocular dominance columns and hypercolumns were
and Koskinas (Von Economo and Koskinas, 1925) and 185 mye- identified as information processing units (for a review, see
loarchitectonic maps (Vogt and Vogt, 1919). Consequently, do Ts’o et al., 2009). The architecture of the barrel cortex reflecting
the maps differ only in their ‘‘granularity,’’ i.e., is one map just the topographic representation of the whiskers represents
more detailed than the other? If so, can the different parcellation another example of the congruence between an electrophysio-
schemes be mapped onto each other? Or, is one map ‘‘better’’ logically defined map and its underlying microarchitecture
than the other with respect to its methodical approach or func- (Woolsey and Van der Loos, 1970). In generalizing these findings
tional relevance? Do the maps perhaps reflect, to a different de- from primary and higher sensory cortices, architectonic similar-
gree, heterogeneity within areas? How fine-grained should a ities within an isocortical area were linked to the hypothesis of
map be? Can new multimodal mapping methods, e.g., of the repeated, canonical local circuits in the neocortex (Buxhoeveden
molecular or genetic organization contribute to solving these and Casanova, 2002; Defelipe et al., 2012; Douglas and Martin,
questions? 2004; Markram et al., 2015; Mountcastle, 1957; Szentágothai,
Multimodality of brain mapping was a perspective starting with 1975).
early architectonic work. Brodmann (1909) stated in the last chap- Others critically discussed the concept of ‘‘columns’’ and
ter of his pioneering monography: ‘‘The specific histological differ- ‘‘modules’’ and questioned the view of traditional columns form-
entiation of cortical areas provides irrefutable proof of their specific ing canonical units as a general principle of cortical organization,
functional differentiation.The large number of distinct structural beyond sensory areas (Catania, 2002; Ts’o et al., 2009). It has
zones suggests a spatial specialization of various individual func- been argued that they are not obligatory elements of the
tions, and finally the all-round sharp demarcation of many areas in- neocortex, they can also occur in subcortical structures, and
dicates inexorably a strictly circumscribed localisation of their they are very dynamic and overlapping (Rockland, 2010). More-
corresponding physiological function’’ (Brodmann, 1994). over, the types of columns are different and can be defined by
Early electrophysiological findings of Mountcastle in the so- cellular composition, pattern of connectivity, myelin content,
matosensory cortex are consistent with this modular organiza- enzyme activity, magnitude of gene expression, or functional
tion of the isocortex (Mountcastle, 1957, 1978). The functional properties (Rakic, 2008).

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Table 2. Examples of Human Brain Atlases Containing Microstructural Data, Cortical Segregation, and Tools
Modalities Comments Link Selected References
Allen Brain Atlas In situ hybridization, 3D-Viewer, search and
Nissl, microarray analysis tools; developmental display/humanbrain/
and mouse atlases Documentation
BigBrain Cytoarchitecture 3D reconstruction of an Amunts et al., 2013
individual human brain
at 20 mm; visualization and
download tools; labeling in
Caret atlases: MRI Surface-based atlas, myelin Van Essen et al., 2012a,
Conte69, maps, cortical parcellations, 2012b
PALS-B12 PC-CC and areal boundaries
FSL atlas fMRI, MRI, and DTI Parcellation and morphometry of Jenkinson et al., 2012
cortical and subcortical regions;
link to maps of the JuBrain atlas
Human Connectome Diffusion-based In vivo multimodal atlas, myelin http://www.humanconnectome. Van Essen et al., 2013
Project (HCP) connectivity, maps, surface and volume data org/
Jubrain Cytoarchitecture Probabilistic maps based on https://www.jubrain.fz-juelich. Zilles and Amunts, 2010;
mapping in ten post mortem de/apps/cytoviewer/cytoviewer- Schleicher et al., 2005
brains main.php
The Human Brain MRI, cell bodies, In vivo MRI from a different Tool developed by:
Atlas at Michigan myelin brain as compared to brains/human/index.html Sudheimer, Winn, Kerndt,
State University post mortem Shoaps, Davis, Fobbs Jr.,
Johnson, Michigan State
University; National Museum
of Health and Medicine,
Armed Forces Institute of
THE HUMAN Cell bodies, myelin, Different tools, book, and DVD Mai et al., 2007
BRAIN. INFO chemoarchitecture
MNI templates MRI In vivo atlas, link between Collins et al., 1994;
and atlas MNI-space to the BigBrain ServicesAtlases/HomePage Evans et al., 2012
The whole-brain MRI, PET Navigable brain volume, three Vidoni, 2012
atlas, Harvard planes, vasculature, aging aanlib/
Medical School (structure and function)
SPM anatomy Probabilistic Same maps as in JuBrain atlas; Eickhoff et al., 2005a,
toolbox cytoarchitectonic tools for superimposition of inm-1/EN/Forschung/_docs/ 2006c
maps cyto-maps with fMRI data SPMAnatomyToolbox/
Talairach daemon MRI Inference of Brodmann’s Lancaster et al., 1997,
map to the atlas of Talairach 2000
and Tournoux
The zoomable human Nissl and fiber Ultra-high-resolution sections Tool developed by
brain atlas staining Georg Striedter
Scalable brain atlas MRI, cell bodies, 3D interactive atlas, different Bakker et al., 2015
myelin tools, nomenclature, and main/index.php
delineation data. Link to
JuBrain atlas
IIT Human Brain Atlas DTI, HARDI Probabilistic maps of gray Zhang and Arfanakis, 2013;
and white matter Varentsova et al., 2014

From Individual to Population-Based Maps mann. The cytoarchitectonic maps of the JuBrain atlas, for
The integration of intersubject variability of cortical areas into example, rely on statistically testable definition of borders be-
brain maps and their comparison with functional imaging data tween cortical areas in histological, cell-body-stained sections
represents a major issue of modern brain mapping beyond Brod- in a sample of ten post mortem brains, which were acquired

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Figure 3. Analysis and Visualization of Cytoarchitectonic Borders

Cytoarchitecture of the secondary visual cortex V2 (area 18 by Brodmann) and the neighboring extrastriate area hOc3d (not defined by Brodmann; roughly
corresponding to part of Brodmann 17 area 19).
(A) The border has been defined based on measurements of the GLI (Amunts et al., 2000; Schleicher et al., 2009).
(B) GLI profiles, which quantify laminar changes in cortical depths from the layerI/II border to the cortex/white matter border, i.e., the cytoarchitecture, were then
extracted. The green and blue profiles characterize the cytoarchitecture in areas V2 and hOc3d, respectively.
(C) A sliding window approach was used to compare neighboring blocks of profile to each other. The Mahalanobis distance (y axis) was used as a measure for the
degree of difference in the shape of the profiles between neighboring blocks of profiles, i.e., of differences in cytoarchitecture, depending on the position of the
profile (x axis). A significant maximum at the Mahalanobis distances (at profile index 137) as a function of the profile position is indicative of a cytoarchitectonic
border at that position (Hotellings T2-test for significance). This analytical/statistical approach leads to ‘‘sharp’’ borders.
(D) The profiles were then stacked to each other to form a ‘‘mountain plot,’’ indicating the GLI values (z-axis) depending on both the cortical depths (layer, x axis)
and the position of the profile (y axis). Distance between profiles is 17 microns. Note the different profile positions, at which changes in the GLI appear in the
different layers and sublayers at the V2/hOc3d border. Moreover, this kind of plot also shows that cytoarchitectonic homogeneity is a relative feature, rather than
an absolute one. It demonstrates intra-areal variations in layer-wise GLI.

through the body donor program of the Anatomical Institute of certain area is found at each position in the reference brain.
the University of Düsseldorf (cf. Table 1) following the require- Our group has published probabilistic maps during the past
ments of the Ethics Committee of the University of Düsseldorf two decades for numerous brain regions including cortical areas
(Table 2, Figure 3). Maps of individual brains were superimposed and subcortical structures (Table 3 and Figure 4). Maps are
to the T1-weighted, single-subject template of the MNI as a com- available via the JuBrain atlas viewer, a web interface-based
mon reference space (Collins et al., 1994; Evans et al., 2012). The on WebGL (
variability in the topography of areas is captured by continuous cytoviewer-main.php). The cytoarchitectonic areas were in addi-
probabilistic maps, which show the probability with which a tion projected on a surface model of the ‘‘Colin27 brain,’’ which

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Table 3. Overview of Presently Available Cytoarchitectonic, Probabilistic Maps

Relationship to
Brain Region Name of Area Brodmann Areas Reference
Occipital cortex 17, 18 17, 18 Amunts et al., 2000
hOc3d, hOc4d 19 Kujovic et al., 2013
hOc3v, hOc4v 19 Rottschy et al., 2007
hOc5 19 Malikovic et al., 2007
hOc4la, hOc4lp 19 Malikovic et al., 2015
Fusiform gyrus FG1, FG2 37 Caspers et al., 2013
FG3, FG4 37 Lorenz et al., 2015
Frontal pole Fp1, Fp2 10 Bludau et al., 2014
Subgenual anterior cingulate s24a, s24b, 25a, 25p, s32, 24, 25, 32 Palomero-Gallagher et al., 2015
Orbitofrontal cortex Fo1, Fo2, Fo3 11 Henssen et al., 2015
Broca’s region 44, 45 44, 45 Amunts et al., 1999, 2004
Motor cortex 4a, 4p 4 Geyer et al., 1996
Posterior insula Ig1, Ig2 Regio insularis Kurth et al., 2010
Temporal cortex, superior Te1 41 Morosan et al., 2001
Te3 22 (post.) Morosan et al., 2005
Hippocampus, entorhinal CA1-4, FD, subicular complex, HATA, 28, 34, 35 Amunts et al., 2005
entorhinal cortex
Parietal cortex, anterior 1, 3a, 3b 1, 3 Geyer et al., 1999, 2000
2 2 Grefkes et al., 2001
Parietal cortex, posterior, inferior PF, PFcm, PFop, PFt, PGa, PGp 39, 40 Caspers et al., 2006, 2008
Parietal cortex, posterior, superior 5L, 5M, 5Ci, 7PC, 7A, 7P, 7M 5, 7 Scheperjans et al., 2008a, 2008b
Parietal cortex, intraparietal hIP1, hIP2 5, 7, 39, 40 Choi et al., 2006
hIP3 7 Scheperjans et al., 2008a, 2008b
Parietal cortex, operculum OP1-4 40, 43 Eickhoff et al., 2006a, 2006b
Amygdala superficial, centro-medial, laterobasal n.d. Amunts et al., 2005
Basal forebrain Ch1-2, Ch3, Ch4 n.d. Zaborszky et al., 2008
Cerebellar nuclei Dorsal and ventral dentate, emboliform, n.d. Tellmann et al., 2015
globose, fastigial
The relationship to Brodmann areas is only approximate, since Brodmann’s publication very often does not allow to an unambiguous definition, and the
ontologies may overlap. For a comparison of different nomenclatures (Bailey and von Bonin, 1951; Campbell, 1905; Smith, 1907; Rose, 1927, 1928;
Vogt and Vogt, 1919; Vogt, 1910; von Economo and Koskinas, 1925), see Zilles and Amunts (2012a).

was generated with FreeSurfer ( These proaches have been applied and modified during the following
maps supplement existing microstructural atlases (Table 2) decades (Adamson et al., 2005; Mackey and Petrides, 2009;
and constitute a tool to analyze other neuroimaging data. The Schmitt and Böhme, 2002).
SPM toolbox has been developed to facilitate the analysis of The measurement of regional distribution patterns of the
structure-functional relationships at the level of cortical areas gray level index (GLI), i.e., a reliable estimate of the volume
(Eickhoff et al., 2005a, 2006c). Such studies demonstrate the fraction of cell bodies (Wree et al., 1982), and of GLI profiles
close relationship of the segregation of the cerebral cortex into vertical to the cortical ribbon using a computer-controlled
areas and its functional segregation (Eickhoff et al., 2007). scanning procedure were crucial steps for the development
of observer-independent tools to define the position of areal
Quantitative Architectonics borders based on statistically testable criteria. The shapes of
The introduction of automated image analysis can be seen as a GLI profiles quantify the changes in the volume fraction of
major move in cytoarchitectonic mapping beyond the traditional cell bodies from the surface to the white matter boundary,
pure visual inspection of Nissl-stained sections. ‘‘Profiles’’ i.e., cytoarchitecture. Starting from the idea that the cellular ar-
capturing the changes of a morphometric parameter (e.g., the chitecture changes at the border between two areas, a method
volume fraction of cell bodies) from the surface of the brain to was established that compared immediately adjacent groups
the cortex/white matter border were extracted as measures of (‘‘blocks’’) of GLI profiles to each other by moving the blocks
the cortical architecture (Haug, 1956; Istomin and Amunts, like a sliding window along the cortical ribbon (Schleicher
1992; Istomin and Shkliarov, 1984; Schleicher et al., 1986, et al., 1998, 1999, 2005, 2009). Positions where cytoarchitec-
1998; Wree et al., 1982; Zilles, 1978). This and similar ap- ture changed were identified by significant differences in profile

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Figure 4. Probabilistic, Cytoarchitectonic JuBrain Atlas

The maps are based on observer-independent definitions of areal borders and quantitative cytoarchitectonics in serial histological sections of ten human post
mortem brains. Upper left, screenshot from the website showing the probabilistic map of frontal pole area Fp1 (Bludau et al., 2014). Upper right and lower row
present the maximum probability map (MPM), where each position of the reference space is associated to that area, which shows the highest probability (Eickhoff
et al., 2005a, 2006c). Different areas are labeled by different colors. Published maps are available for download at

shapes defined by the Mahalanobis distance between adjacent cell bodies, etc. (Schleicher et al., 2000). In contrast to clas-
blocks of GLI profiles (Figure 3). The shapes of profiles are sical cytoarchitectonic studies, the present approach takes
described by a set of ten features based on central moments into account a whole set of features, reflecting the cytoarchi-
and their derivatives. They are extracted from the profiles if in- tecture over the whole cortical cross section in contrast to
terpreted as frequency distributions. Although the central mo- single features such as the presence of a particular cell type,
ments are relatively abstract measures, they correspond well of the density of a layer. This is particularly important since sin-
with cytoarchitectonic features. For example, the first moment gle features are not powerful enough to parcellate the human
is similar to the mean GLI, averaged throughout the cortex. The cerebral cortex, an observation that was already made by
third moment, the center of gravity in x direction (direction from Von Bonin and Bailey (1961). In addition, these authors were
the surface to the white matter), corresponds to the relation- the most extreme advocates of gradual, in contrast to sharp,
ship of supra- to infragranular layers in the volume fraction of borders.

Neuron 88, December 16, 2015 ª2015 Elsevier Inc. 1093



How ‘‘Sharp’’ Are Architectonic Borders between The fringe area and the border tuft at the border between the
Cortical Areas, and How Uniform Is the Cytoarchitecture primary and secondary visual areas represent further examples
within an Area? of transitional phenomena (Amunts et al., 2000; Sanides and
Measurements of laminar changes of cell densities as obtained Vitzthum, 1965; Von Economo and Koskinas, 1925). GLI profiles
in observer-independent definitions of borders are a prerequisite here provide reproducible and reliable data for further analyses
to answer the question of how ‘‘sharp’’ or ‘‘gradual’’ borders be- of structural heterogeneity within areas. An example is given in
tween two cortical areas are, and to identify positions at the Figure 3, which shows two extrastriate areas and measures of in-
cortical ribbon, where cytoarchitecture changes abruptly. This ter-areal heterogeneity. It demonstrates that the transition of
relates also to the question of how ‘‘homogeneous’’ a cytoarchi- area V2 (BA 18) to the dorsally adjacent area hOc3d (Kujovic
tectonic area is or how much ‘‘heterogeneity’’ the definition of an et al., 2013) has indeed a complex nature. In addition, it reveals
area as a structural entity can tolerate. Various multimodal and repetitive and non-repetitive elements, which occur in a distance
multiscale heterogeneities within cortical areas have been of several millimeters from each site of the border. Fluctuations
described using different histochemical, electrophysiological, occur at different cortical depths, i.e., specific layers and sub-
and connectivity studies, e.g., in primary and secondary visual layers, at different distances from the border. Whether such fluc-
cortex (Baizer et al., 1991; Falchier et al., 2002). tuations in GLI correspond to the columnar organization of the
Cortical columns were described not only in sensory, but also visual cortex remains a project of future research. Measure-
in multimodal cortical areas (Bugbee and Goldman-Rakic, 1983; ments of the GLI nevertheless allow identifying a position, where
Buxhoeveden, 2012; Innocenti and Vercelli, 2010; Mountcastle, cytoarchitecture changes abruptly.
1997; Peters and Sethares, 1996; Raghanti et al., 2010; Rock- Notably, other borders of the visual cortex show abrupt
land and Ichinohe, 2004; Schlaug et al., 1995). These vertically changes in cytoarchitecture, i.e., an abrupt and significant in-
oriented structures, however, do not change the horizontal lami- crease of the Mahalanobis distance (e.g., borders of fusiform
nation pattern with its distinct differences in cell densities and areas FG1 and FG2; Caspers et al., 2013). In such cases, the
cell types between layers (Brodmann, 1909; Vogt and Vogt, border is easily visible by pure visual inspection.
1919; Zilles and Amunts, 2010). Columns and even more mini- Thus, the phenomenology of areal borders varies from abrupt
columns are too small for a concept of a cortical area at the to more gradual changes. The cytoarchitectonic heterogeneities
meso-scale. do not change the general characteristic features of an area to
Other structural or functional entities below the level of a such a degree as that found at the borders between areas, but
cortical area consist of barrels, ocular dominance columns, contribute to a more complex picture of the neurobiological con-
orientation columns, blobs, inter-blobs, but also local modifica- ditions underlying cortical cytoarchitecture. Quantitative mea-
tions of the architecture introduced by the geometry of the cor- surements and statistical testing of multivariate feature vectors
tex, i.e., cortical folding. Cortical zones, which contain callosal enable a localization of cytoarchitectonic borders despite this
projection neurons, are larger regions of specific cyto- and complexity. Additionally, multimodal imaging techniques may
myeloarchitectonic organization within or between cortical help to distinguish areas from each other.
areas and contain many columns. Well-described examples
are found between areas of the visual cortex, most of which Alternative Mapping Strategies of Cortical Structure
contain very large pyramidal cells, mainly located in deeper Cyto- and myeloarchitectonic analyses are now complemented
layer III (Clarke and Miklossy, 1990; Von Economo and Koski- by new strategies including long-range structural connectivity
nas, 1925; Zilles and Clarke, 1997). The magnopyramidal area analyses, mapping of intracortical circuits, cell types, synapses,
OBg of Von Economo and Koskinas (1925) is such an example matrix proteins, transmitters, receptors, and genetic markers at
but was clearly identified as part of area OB (BA 18 or V2) by the micro and molecular scale. All data come inevitably from
these authors and in following studies (Amunts et al., 2000) different (human) brains and should be registered in a common
without questioning the concept of OB or BA 18 as a cytoarch- reference model at their topographically correct position to
itectonic entity. BA 18 includes, therefore, the callosal termina- make a comparison feasible (Amunts et al., 2014a). Although
tion region OBg without changing its principal cytoarchitecture. the methods for collecting such multimodal data are principally
Patchy distributions of afferents from different sources have available and widely used in neurophysiology, cell biology, phar-
been frequently described within a cytoarchitectonic area, and macology, or genetics, it was and is presently difficult to apply
it has been argued that the prefrontal cortex shows a modular them to the mapping of whole human brains for producing
organization (Goldman and Nauta, 1977; Goldman-Rakic, such integrative maps.
1984; Goldman-Rakic and Schwartz, 1982). Another example Receptor autoradiography, imaging of trace elements and lip-
for within-area heterogeneity is given by the dendritic structure ofuscin, enzyme-immunohistochemistry, immuno-histochemis-
of neurons, which varies as a function of eccentricity in pri- try, and some methods based on genetic markers already
mary visual cortex, as demonstrated by a study focusing on proved to be useful to whole human brain mapping. Receptors
morphometric variability of NADPH-diaphorase neurons in the are key molecules of signal transmission and thus elucidate
rat cortex (da Rocha et al., 2012). Also, somatotopy is a well- important aspects of the molecular and functional organization
known aspect of cortical organization, which is found not only of the human brain (Zilles and Amunts, 2009; Zilles et al.,
in primary sensory and motor areas, but also in higher uni- 2002). Receptorarchitectonic analysis using quantitative in vitro
modal areas (e.g., SII cortex) and subcortical structures (e.g., receptor autoradiography offers an effective way to cortical par-
thalamus). cellation at the mesoscale (Zilles and Amunts, 2009; Zilles et al.,

1094 Neuron 88, December 16, 2015 ª2015 Elsevier Inc.



2002). The analysis of the regional and laminar distribution of immuno-histochemical mapping with antibodies against trans-
numerous receptor types of classical transmitter systems com- mitter synthesizing enzymes with a ChAT antibody represents
bined with cytoarchitectonic studies in the same brains has an early example of mapping the functional subdivisions of the
shown that borders between cortical areas identified with both human cerebral cortex (Mesulam et al., 1992). Since then
methods precisely coincide in many cases (e.g., Zilles et al., numerous further studies were performed using antibodies
2004). Whereas some receptor types do not detect all cytoarchi- against ChAT, GAD, neurotransmitters, and neuropeptides, but
tectonic borders and integrate several areas in a larger receptor- a complete architectonic mapping of the whole human cerebral
architectonic ‘‘family,’’ others reveal a finer-grained parcellation cortex was not intended in any of these studies. To our knowl-
than cytoarchitecture (Amunts et al., 2010; Zilles and Amunts, edge, the first study of areal segregation in the human cerebral
2009). Thus, multimodal receptor mapping not only represents cortex using antibodies against a cytoskeletal protein (non-
an evaluation of cytoarchitectonic parcellations, but also pro- phosphorylated site of neurofilament H; SMI-32) demonstrated
vides novel parcellations not detected by a single modality inter-areal differences of subpopulations of pyramidal neurons
(Amunts et al., 2010; Geyer et al., 1996; Palomero-Gallagher in the parainsular and parahippocampal cortex, superior tempo-
et al., 2009; Scheperjans et al., 2005; Zilles et al., 1995). Multi- ral gyrus including auditory areas, posterior inferior temporal gy-
receptor analyses of cortical areas can be visualized and rus (probably BA37), and primary visual cortex (Campbell and
analyzed as ‘‘receptor fingerprints’’ (Zilles et al., 2002). If the fin- Morrison, 1989) as well as human premotor, primary motor,
gerprints of numerous areas are used in principal component and somatosensory areas (Baleydier et al., 1997). Antibodies
and hierarchical cluster analyses, receptorarchitecture reveals against the calcium-binding proteins parvalbumin and calbindin,
the participation of cortical areas in functional networks (Zilles SMI-32, and the neuron-specific DNA-binding protein NeuN in
et al., 2015a). the human polar temporal and perirhinal cortex enabled a
Minerals and trace elements are distributed differentially with detailed parcellation of these regions (Ding and Van Hoesen,
respect to cortical layers and areas, as shown, for example, in 2010; Ding et al., 2009).
experiments based on focal injections of the tracer sodium sele- Since the laminar pattern of cortical areas is the most impor-
nite to identify zinc-positive projection neurons in the visual cor- tant criterion for architectonic brain mapping, the labeling of
tex (Ichinohe et al., 2010) and in laser ablation inductively distinct cortical layers by the demonstration of proteins of the
coupled plasma mass spectrometry, LA-ICP-MS, of brain sec- transcription factors Rfx3 as marker of layer II, Cart as marker
tions (Becker et al., 2005). Molecular mapping employing MALDI of layer III, Rspo1 as marker of layer IV, and Etv1 as marker of
imaging is a method that links quantitative data on transmitters, layer V (Boyle et al., 2011) as well as FoxP2 (Ferland et al.,
proteins, and lipids together with their localization in microtome 2003) and Tbr1 as markers of layer VI (Hevner et al., 2001) pro-
sections. The application of LA-ICP-MS and MALDI (Benavides- vided promising tools for brain mapping in rodent brains, which
Piccione and DeFelipe, 2007; Blazquez-Llorca et al., 2010; await their future application in human brain mapping.
DeFelipe et al., 2006, 2013) is methodically highly challenging Understanding the segregation of the cerebral cortex requires
for whole human brain analyses and limited until now to regions also considering the formation of areas during development. The
of interest. analysis of the origin of pyramidal cells in the ventricular zone re-
For decades, neurochemical aspects of brain organization vealed a protomap, which is conserved during the radial migra-
were visualized using enzyme histochemistry. For example, tion of the cells and their final arrival in the cortical plate (Lui et al.,
COX has been employed to identify regional long-term activity 2011; O’Leary et al., 2013; Rakic et al., 2009). This development
changes in animal models of brain diseases, e.g., depression is the biological basis of a multilevel organization of the cerebral
(Harro et al., 2014). AChE was used to map the basal forebrain cortex, with the radial placement of the cells generating its
and other brain regions (Mesulam and Geula, 1991, 1994). columnar structure, and with the projection of the protomap to
NADPH diaphorase staining was also successfully used for map- the mature cortex representing its segregation in cortical areas.
ping purposes (da Rocha et al., 2012; Franca et al., 1997; Oer- The migration of the later interneurons from their subcortical
mann et al., 1999). Although the enzyme histochemistry can be sources (Rubenstein and Campbell, 2013) leads to an enormous
replaced by the highly specific immuno-histochemical staining diversity of cell types in each cortical area and layer.
of proteins, it is still a valuable approach for whole human brain Recently, the usage of array-based gene analysis for human
mapping. brain mapping considerably broadened the horizon. Most
Efforts were made to classify different neuronal types in Golgi advanced efforts are provided by the Human Brain Atlas of the
de-impregnated and Nissl counterstained preparations (Werner Allen Institute, which also includes developmental data (https://
et al., 1989). Reliable mapping of neuronal cell types, however, It provides comprehensive in situ hy-
was first possible with the introduction of immuno-histochemical bridization datasets together with Nissl stained sections from
methods, which allowed separating pyramidal versus non-pyra- post mortem human brains (Hawrylycz et al., 2012; Shen et al.,
midal neurons and a further differentiation of numerous neuronal 2012; Sunkin et al., 2013) (Figure 5). The further development
types. Using an antibody against Wisteria floribunda agglutinin of the in situ hybridization approach and a complete and more
(WFA), a lectin that selectively labels N-acetylgalactosamines fine-grained coverage of the human brain would provide an ulti-
beta 1 residues of glycoproteins of the neuronal extracellular ma- mate database for mRNA mapping, allow a direct comparison
trix, the mapping of the anterior and posterior subdivisions of the with cyto-, multireceptor-, and and myeloarchitectonic maps of
primary motor cortex BA4, and the somatosensory areas 3a and the brain, and thus represent a major step toward a multimodal
3b was demonstrated in the human brain (Hilbig et al., 2001). The human brain map.

Neuron 88, December 16, 2015 ª2015 Elsevier Inc. 1095



The study of gene expression with microarrays in different

cortical regions of human and chimpanzee brains revealed
several overall conserved gene co-expression modules between
species, with a greater interspecies module conservation in the
primary sensory cortex than in association cortex and in subcor-
tical structures as compared to cortical (Oldham et al., 2006).
This fits well with the structural differentiation (Smaers et al.,
2010; Zilles, 2005) and cytoarchitectonic gradation streams (Sa-
nides, 1962) of association areas versus hierarchically lower, un-
imodal sensory and primary sensory areas during primate brain
evolution. Chimpanzees hardly show a gene co-expression
module, which is strongly represented in human cerebral cortex
and contains genes involved in energy metabolism. These genes
have similar patterns of connection strengths to other genes of
the electron transport chain in neurons of the human cerebral
cortex (Oldham et al., 2006). They control mitochondrial struc-
tural properties, synapse formation, and vesicle docking as
well as cytoskeleton regulation. Probably, the expansion of the
human neocortex during primate brain evolution, which led to
an enormous increase in processing capacity, was accompa-
nied by an increased energy metabolism required for structural
plasticity of neurons and their synapses (Oldham et al., 2006).
Genetic labeling of cortical cell types with bacterial artificial
chromosome (BAC), transgenic (Heintz, 2001), and knock-in as
well as intersectional strategies enable cell typing in mice
( It is also possible to study connectivity and
function of specific cell types. The response to genetic alter-
ations can be studied in any genetically identified cell type using
BAC array translational profiling (Heiman et al., 2008). The de-
gree of molecular similarities and dissimilarities between cell
types could be described with a hierarchical cluster analysis,
and groups of genes were identified that encode cell-type-spe-
cific functions. Furthermore, comparisons can be performed to
study the development of specific cell types (Doyle et al.,
2008). A further progress in genetic labeling to characterize cell
types using high-throughput methods is recently provided by
the MultiColor FlpOut (MCFO) approach, which revealed shape
and position of cells in Drosophila (Nern et al., 2015). A transcrip-
tional driver and stochastic, recombinase-mediated excision of
transcription-terminating cassettes controls the expression of
multiple membrane-targeted and distinct epitope-tagged pro-
teins. Gene targeting by knockin methods considerably
improved the reproducibility and strategic design of cell-type
targeting and identification of novel cell subtypes even within
the family of pyramidal cells (Sorensen et al., 2015). Genetic
dissection of cortical circuits already started in the mouse brain
by systematic targeting of cell types and fate mapping of neural
progenitors (for recent review, see Huang, 2014). This approach
can provide a cortical cell atlas of unprecedented detail and
Figure 5. Correspondence of Areal Borders across Different
(A) Section from the Allen Brain Atlas showing parvalbumin gene expression in (F) Benzodiazepine binding sites of the GABAA receptor labeled with [3H] flu-
neurons of the human primary visual area V1 and secondary visual area V2 mazenil. (G) Binding sites of the GABAB receptor labeled with [3H] CGP 54626.
(, specimen RP_070313_01_C07). Delineation Parvalbumin-positive cortical neurons have their termination field in the sur-
between V1 and V2, and laminar labeling by the authors of the present review. rounding of their cell bodies, where they release the inhibitory transmitter
(B–G) Cyto-, myelo-, and receptorarchitecture of human areas V1 and V2 from GABA. Notably, the laminar density distribution of parvalbumin-positive neu-
a different brain (Institute of Neuroscience and Medicine, INM-1, Research rons and GABA receptor binding sites are similar in both V1 and V2, with the
Centre Jülich). (B) Cell-body-stained section. (C) Myelin-stained section. (D) exception of GABAB receptors, which are also present at intermediate den-
Agonistic binding sites of the GABAA receptor labeled with [3H] muscimol. (E) sities in layers V and VI of V1. Roman numerals indicate cortical layers. The
Antagonistic binding sites of the GABAA receptor labeled with [3H] SR95531. scale bars code receptor densities in fmol/mg protein.

1096 Neuron 88, December 16, 2015 ª2015 Elsevier Inc.



develop into a tool to bridge various scales and modalities. Serial two-photon microscopy combines fluorescence imag-
Although BAC transgenics and gene targeting enable a tremen- ing of whole mouse brains with two-photon microscopy. Since
dous progress for the definition of specific cell types and sub- the imaging is performed on blockfaces during stepwise
types in the rodent brain, its application to brain mapping is sectioning using a vibratome, distortion-free and high-resolution
currently restricted to a few accessible species and not appli- datasets can be achieved and easily aligned. This fully auto-
cable to human brain mapping. mated procedure results in 3D reconstructions of the mouse
Mapping of single morphological cell types was not systemat- brain in which the distribution of immuno-stained neurons or
ically applied to whole cortex parcellation in the human brain, but traced fiber tracts can be visualized (Ragan et al., 2012).
it was used in cases in which highly differentiated cells occur only In the rodent brain, the combination of new optical with im-
in well-circumscribed areas. For example, the giant pyramidal muno-histological methods generated the methodical basis for
cells of Betz are mainly restricted to the primary motor cortex whole-brain analyses, even at a synaptic level. For example,
(Brodmann area 4; Brodmann, 1909). Since they are loosely combined focused ion beam milling and scanning electron mi-
distributed at the border to the premotor cortex, their role as croscopy (FIB/SEM) enabled us to obtain 3D samples from the
decisive structures for a precise delineation of BA4 is, however, six layers of the rat somatosensory cortex and identifying synap-
somewhat limited. Meynert cells represent another example, tic junctions. A total volume of tissue of approximately 4,500
found in layers V–VI of the primary visual cortex (Palay, 1978; micron3 and around 4,000 synapses from three different animals
Winfield et al., 1981). Economo cells are restricted to the fron- were analyzed (Anton-Sanchez et al., 2014).
toinsular and anterior cingulate cortex in the human brain (Allman Experimental tracing techniques, which are the ‘‘golden stan-
et al., 2010; Seeley et al., 2012). Notably, analyses of connectiv- dard’’ in animal research (Lanciego and Wouterlood, 2006; Nassi
ity at the macroscale showed that some cortical regions pre- et al., 2015; Wouterlood et al., 2014, to name only a few of them),
dominantly have local short-range connections, whereas other cannot be employed to study the human connectome, with a few
areas are preferentially connected via long-distance connec- exceptions (Galuske et al., 2000). 3D Polarized Light Imaging
tions with numerous other regions (Gong et al., 2009; Tomasi (3D-PLI) represents new tool to study fiber architecture across
and Volkow, 2010; Van den Heuvel and Sporns, 2011). There- scales reaching from the micrometer to the centimeter scale
fore, a systematic multiscale analysis of the regional distribution (Axer et al., 2011a, 2011b). It is based on the detection of light
of various pyramidal or other cell types, cytoarchitectonic par- signals caused by the birefringence of myelin sheaths surround-
cellation, and connectivity data at the macroscale represents a ing axons in unstained histological sections (Axer et al., 2001,
typical model for future multiscale studies in human brain 2011a, 2011b). Using the Jones calculus, these signals are trans-
mapping. ferred to vector orientation maps, where each vector indicates
the spatial orientation of axons in small volume elements. In
Optical Methods and Whole-Brain Approaches dependence on the methodology and equipment, a spatial res-
Methods without Clearing olution of up to 1.2 mm (in-plane) can be achieved. In contrast
Micro-optical sectioning tomography (MOST) is a light micro- to other novel optical methods, whole-brain imaging is feasible,
scopy-based optical tomography with simultaneous physical but single axons with a diameter at the range of the spatial res-
sectioning of whole mouse brains, which enables the visualiza- olution may not be dissolvable. The resulting images disclose a
tion of cell bodies and blood vessels at a spatial resolution of fiber architecture, which is comparable with classical mye-
1 mm without tissue clearing using a modified Nissl staining (Li loarchitectonic observations, but also findings based on diffu-
et al., 2010; Wu et al., 2014). Cell and vascular numbers, packing sion tensor imaging (Caspers et al., 2015; Reckfort et al.,
densities, distances from the cells to the nearest microvessel, 2015). They differ in at least two important aspects: they provide
microvascular length, and volume can be measured in such true 3D information and contain quantitative information. The re-
specimens. The method is potentially also applicable to tissue quirements for processing images of whole-brain sections with a
blocks of the human brain. The fluorescence micro-optical size of 1.5 TByte (and nearly 4 PBytes for a whole human brain)
sectioning tomography (fMOST) method (Gong et al., 2013) is a are immense, and high-performance computing and large stor-
further development of the MOST technique, which allows the age facilities are mandatory.
application of immunofluorescence and thus opens the door The dissection of human brains into small tissue blocks is in-
for more specific characterization of cell types including genetic evitable in electron microscopy (Denk and Horstmann, 2004;
cell targeting. Helmstaedter, 2013; Lichtman and Denk, 2011) and optical
Optical coherence tomography (OCT) is a method already coherence tomography (Magnain et al., 2015; Wang et al.,
used to analyze the microstructure of human brain tissue. It 2011). Besides an incredible amount of time and effort for prep-
avoids artifacts introduced by traditional histological processing aration and measurement, the methods require demanding, and
and enables imaging of individual neurons in tissue blocks with a in some applications presently not available, computing re-
size of several square millimeters and over 50 mm in depth. OCT sources to achieve the mapping of large brain regions or even
images can be compared with those obtained from cell body a whole human brain (Helmstaedter, 2013). Automated image
staining (Magnain et al., 2015). It is therefore an option to assess analysis systems cannot fully replace the human labor presently.
the cytoarchitecture of post mortem human brains with minimal Algorithm-based systems are being developed, which can be
distortion. Further development of OCT may allow imaging of un- easily used by hundreds of ‘‘amateurs’’ with minimum training
distorted volumetric data of centimeter cube tissue blocks or to enable a quality-controlled segmentation and reconstruction.
entire hemispheres (Magnain et al., 2015). Such a massive online crowd-source strategy may overcome

Neuron 88, December 16, 2015 ª2015 Elsevier Inc. 1097



current time restrictions (Helmstaedter et al., 2011). The main ping, but fiber tracts are also visible in 3D after immuno-stain-
challenge for mapping complete circuits is the imaging, recon- ing of myelin. When interpreting CLARITY results, it must be
struction, and analysis of the enormous amount of neuronal pro- kept in mind that refractive index mismatches and optical dis-
cesses, since a standard voxel in MRI (1 mm isotropic) contains tortions occur (Chung et al., 2013; Yang et al., 2014). CLARITY
several kilometers of such structures. In the case of the human has been further developed by improving the workflow, partic-
brain, multi-beam electron microscopy must be available to ularly with respect to the lipid removal without electrophoresis,
reduce the data acquisition time to a tolerable level for small tis- and the integration of light sheet optics (CLARITY-optimized
sue blocks, not to speak of the whole brain. In each of the hith- light sheet microscopy, COLM; Tomer et al., 2014), which im-
erto published datasets of the Drosophila brain, mouse retina, proves its applicability to larger tissue blocks. Other clearing
and primary visual cortex, the volume images required between techniques were also published. SCALE (Hama et al., 2011)
several hundred gigabytes and a dozen terabytes per dataset. In and CUBIC (Susaki et al., 2014) enable whole mouse brain
the near future, these storage space requirements will increase clearing with preserved fluorescence signal. However, the
to several petabytes per dataset (Helmstaedter, 2013). SCALE protocol requires months for the clearing process of
The ultimate goal of architectonic mapping at the nanometer somewhat larger tissue blocks, and optical distortions seem
scale is the visualization of neuronal circuits by the detection of to be inevitable (Hama et al., 2011).
dendrites, axons, synapses, and cellular components while pre- A promising advance toward a connectivity analysis with
serving their topography with respect to cortical layers and specification of the neural connections is provided by clearing
cortical areas. Presently, nanometer analysis is possible with with pH-adjusted 1-propanol or tert-butanol and its combina-
electron microscopical techniques: transmission electron micro- tion with Rabies virus labeling, which reveals long-range axonal
scopy of serial sections (Mishchenko et al., 2010), scanning elec- projections, and light sheet microscopy (for review, see Keller
tron microscopy after automated tape collection of serial and Dodt, 2012) in whole mouse brains (Schwarz et al.,
sections (Kuwajima et al., 2013), serial block-face scanning elec- 2015). With tissue clearing methods and confocal light sheet
tron microscopy (SBSEM; Denk and Horstmann, 2004), and microscopy, where out-of-focus and scattered light is filtered
focused ion beam scanning electron microscopy (Knott et al., out, mapping of Purkinje cells and tracing of fiber tracts was
2008). SBSEM is the method most intensively used in brains or performed (Silvestri et al., 2012), or the distribution of b-amyloid
tissue blocks of the cerebral cortex of various species. However, plaques can be visualized, counted, and measured (Jährling
only small blocks can be analyzed by the combination of a scan- et al., 2015). Light sheet microscopy can also be applied to
ning electron microscope with a device to cut off sections of blocks of human brain tissue (Costantini et al., 2015). Whereas
approximately 25 nm thickness from the surface of a block two-photon microscopy provides a higher sensitivity than light
(Denk and Horstmann, 2004). The surface of the block with the sheet microscopy, the major advantage of the newest genera-
embedded nervous tissue is imaged after each section, and tion of latter technique for human brain mapping purposes is its
thus a 3D reconstruction of the complete tissue block with per- higher speed (107 mm3/s versus 105 mm3/s) and its ability to
fect alignment is feasible. Since classical electron microscopy analyze larger tissue blocks (cm3 versus mm3) (Costantini
(EM) is necessary to measure single synaptic contacts, but et al., 2015).
cannot define the molecular composition of synapses, array to-
mography has recently proposed to bridge the distance between Computational Challenges for Super-High-Resolution,
immunofluorescence of proteins visible in light microscopy and Whole-Brain Models
the superior spatial resolution of electron microscopical imaging In addition to considerable storage requirements, there are pure
modalities (Collman et al., 2015; Micheva and Smith, 2007). Array time restrictions to analyze cellular and subcellular morphology
tomography has a better spatial resolution compared to confocal at super-high-resolution specimen and whole-brain level. For
microscopy, and the detectability of objects by immunofluores- example, to map the course of each single axon and to analyze
cent staining is depth independent. This method allows, for each of the 1.5 3 1014 synapses in a human brain (Pakkenberg
example, a volumetric mapping of immunohistochemically iden- et al., 2003) would simply be impossible during a researcher’s
tified synapses of mouse cortex at the nanometer scale in a lifespan. The situation is even worse when the dynamic nature
voxel-conjugate fashion (Collman et al., 2015). of synapses would be taken into account.
Methods with Tissue Clearing Considerable demands on data handling, storage, visualiza-
The last years also provided powerful optical and tissue tion, and analysis, i.e., big data analytics, therefore represent
clearing methods, allowing to specifically label and image neu- an increasing challenge in human brain anatomy and physiology
rons, microcircuits, as well as their distant connections in whole (Petersen and Sporns, 2015). The combination of large amount
rodent brains without any sectioning. CLARITY (clear, lipid- of data (TByte to PByte range per brain) and a complex recon-
exchanged, acrylamide-hybridized rigid, imaging/immunostain- struction pipeline for the human brain demands massively paral-
ing compatible, tissue hydrogel) allows the clearing of whole lel supercomputer resources. The BigBrain project resulted in a
rodent brains or tissue blocks of human brains, immunostaining 3D model of a human brain based on 7,404 cell-body-stained
of structures for which antibodies are available, and 3D visual- sections; the volume of the original data was approximately 1
ization without the time-consuming sectioning of the tissue and TByte for a resolution of 20 microns isotropic (Amunts et al.,
3D reconstruction (Chung et al., 2013; Yang et al., 2014). Not 2013). This dataset provides a reference space for integrating
only can immuno-histochemically labeled cell types and their other aspects of the microstructural brain organization. Consid-
regional and laminar distribution be visualized for brain map- ering that novel optical methods are often performed in tissue

1098 Neuron 88, December 16, 2015 ª2015 Elsevier Inc.



blocks and volumes of interest (VOI) in contrast to whole-brain The presence and expression of micro-maps is different in
approaches, new tools and methods are necessary to integrate different brain regions, and some forms seem to be specific for
VOI-based approaches into whole-organ datasets (Lippolis a certain area.
et al., 2013) such as the BigBrain dataset and is foreseen in the Meso-maps are non-repetitive entities of cortical areas, which
Human Brain Project (HBP, https://www.humanbrainproject. change in their cyto-, myeloarchitecture, connectivity, and func-
eu/). Importantly, not only does the spatial resolution differs be- tion at the border between areas. Brodmann’s atlas shows
tween these datasets, but also data modality, format, quality, meso-maps, subdividing the cortex into a set of cortical areas.
and other factors vary, and the documentation of where the tis- The JuBrain atlas also represents an atlas at the meso-level.
sue blocks come from should be improved to enable a registra- There is an ongoing debate on a hierarchical organization at
tion to the atlas considering its precise topography. the meso-level (Beul et al., 2015). Von Economo and Koskinas
Supercomputing resources are increasingly used in brain (1925) were among the first to propose a hierarchical classifica-
mapping. This is true in particular for mapping projects with res- tion of areas. This is supported by more recent architectonic
olutions of 1 micron or less, which are necessary to appreciate studies (e.g., for the cingulate cortex; Palomero-Gallagher
single-cell morphologies. For example, the total amount of a et al., 2008). Meso-maps reveal a pattern that is more complex
Golgi-stained mouse brain with a spatial resolution of 1 micron than Brodmann’s mosaic-like map.
is 8 TByte (Li et al., 2010), while the human brain is larger by a The advance of mapping with anatomical (including modern
factor of roughly 7,500. Handling such an amount of data means optical methods), neurophysiological, and neuroimaging tech-
to take care about efficient data processing performance on a niques produced a variety of maps beyond this parcellation.
large number of compute nodes. This requires optimized distrib- These different maps should be integrated into a common
uted processing pipelines and parallel programming. Detailed multi-level, i.e., multi-scale and multi-modal brain model and
documentation of the processing steps and the complex depen- atlas. Therefore, the scientific content and limits of each map
dencies of the datasets at each level of the multi-step recon- are to be defined, and tools for integration have to be developed.
struction pipeline represent additional challenges (Amunts Integration requires cross-validation of the different modalities
et al., 2014a). against each other. For example, the registration of maps pro-
The combination of the different approaches and new prepara- duced by functional neuroimaging, which operates mainly on
tions in combination with advanced computing and data handling the macro-level, to meso-maps based on microscopic analysis
are prerequisites to consider the multi-level organization of the is more than the definition of anatomical localization of activation
human brain and not only to integrate different modalities into blobs and patterns. It also enables an inference of function to
an atlas, but also to cover different spatial scales of brain organi- columnar organization, micro-maps, and the meso-map of a
zation, bridging from the micrometer to the centimeter scale. cortical area, which together define the structural basis of the
function, which is relevant for modeling of the neocortex (Grillner,
2014; Markram et al., 2015; Ritter et al., 2013; Petersen and
From Brodmann Areas to a Triple-Scale Concept of Sporns, 2015).
Cortical Organization We propose to consider the following categories of brain orga-
In contrast to Brodmann’s maps, we propose to distinguish three nization and methodical prerequisites for defining a cortical area
levels of cortical organization (Figure 6): at the meso-level.
d repetitive modular-like structures
d micro-maps d Specific function in terms of cognitive functions and mental
d meso-maps processes. This can be tested empirically in electrophysi-
ological experiments and/or functional neuroimaging, but
The horizontal organization into cortical layers and sublayers also based on mathematical models (e.g., Shipp et al.,
runs across the three levels of organization. 2013). For example, is a certain brain area uniquely
Modular-like structures include columns, which represent involved in a mental process as measured by fMRI? Is a
different types of repetitive invariant structural and/or functional certain brain area part of one or more neural networks
entities such as mini-columns, as well as other repetitive with specific functions? Does the distribution of certain
invariant entities like hypercolumns, blobs, inter-blobs (visual neuronal types and their molecular characteristics corre-
cortex, primate brains), barrels (somatosensory cortex, e.g., ro- late with a specific function of the cortical area? The
dent brains), etc. This category also comprises patchy inputs to criterion ‘‘specific function’’ requires, however, a clear
layer 1, overlapping with patchy expression of M2 Acetylcholin ontological foundation, which is not given in many cases,
receptors as shown in the mouse V1 (Ji et al., 2015). particularly in higher associative areas and their role in
Micro-maps are non-repetitive elements, formed by many re- cognition. Functions are often defined on a mixed ontolog-
petitive modular-like structures, and represent local specializa- ical basis, e.g., psychological criteria are used together
tions within cortical areas (e.g., retinotopy, somatotopy). Further with neurophysiological parameters or linguistic concepts
examples are the fringe area and the border tuft at the transition when ‘‘functions’’ of language-related areas are described.
between V1/V2 (Sanides and Vitzthum, 1965), callosal input and d Connectivity. Does the proposed areal segregation corre-
output domains at the border between two areas (Clarke and spond to a specific connectivity pattern (e.g., Rockland,
Miklossy, 1990; Goldman-Rakic, 1984). Some of these micro- 2002) including both short-range and long-range connec-
maps show gradients in cyto- or myeloarchitecture (Figure 6). tions? Does the cytoarchitectonic cartography match

Neuron 88, December 16, 2015 ª2015 Elsevier Inc. 1099


Figure 6. Triple-Scale Concept of Cortical
It includes (I) modular-like structures, (II) micro-
maps, and (III) meso-maps. Cytoarchitecture of
two areas in the superior temporal gyrus (TE4/5).
Layers are labeled with Roman numbers (A). The
border between both areas is defined using an
observer-independent method (Schleicher et al.,
2009). The graph represents the Mahalanobis
distance, which quantifies the cytoarchitectonic
heterogeneity in dependence on the position in the
cortical ribbon (B). This distance function shows a
clear peak at the border between the two areas,
where cytoarchitecture significantly changes, as
proven by a Hotelling’s T2 test; areas constitute
meso-maps. The distance function increases
when moving toward the border, where it reaches
its maximum, and then decreases over the extent
of many modular-like structures. These increases
and decreases are gradual and occur in the border
region; they are an example of micro-maps (B and
C). Modular-like structures (C) can be seen as
vertically oriented columns of cell bodies; their
visibility depends on the orientation of the histo-
logical section relative to the orientation of the
column and varies between brain regions. In the
present example, columns are best visible in layer
VI of Te4.

d Evolutionary coherence. Can ho-

mologies be identified between hu-
man brain areas and those of
nearly related primates? This
approach is presently difficult to
prove beyond its attractive hypo-
thetical basis. In the strict sense,
homology requires a common
ancestor and the proof of the
development of a cortical area in
recent species from this ancestor.
However, brains of such ancestors
are not available. The term homol-
that of the myeloarchitecture and/or connectivity (for a re- ogy is also used to indicate similarities in microstructure,
view, see Zilles et al., 2015b)? The distribution of myelin topography, and/or connectivity, which requires multi-
density seems to be an indirect, but promising, aspect of modal brain mapping strategies including genetic ana-
connectivity-based mapping as outlined before. lyses. To identify the emergence of putatively new cortical
d Reproducibility, convergence, and multimodality. Reliable areas during human brain evolution from hypothetical
strategies and criteria based on measurements and statis- ancestral states, or ‘‘evolutionary recycling’’ of already
tics should be applied for the definition of a cortical area. available areas, is a matter of ongoing debate (Dehaene
The quality and reproducibility of maps should be testable and Cohen, 2007). There are strong arguments that differ-
and not depend on the frequently described agreement of ences in the cortical organization between human brains
‘‘experienced observers.’’ Reproducibility and conver- and those of animals are not just quantitative (Hermansen
gence also refer to multimodality, i.e., different modalities et al., 2007; Rakic, 2008).
should produce the same or non-conflicting segregation d In addition, inter-subject variability of cortical areas repre-
patterns. ‘‘Non-conflicting’’ may also mean that several sents a further dimension in defining a cortical area. It is
areas can form a ‘‘family’’ of areas; this is the case, for relevant with respect to the above-mentioned criteria: vari-
example in multimodal receptor architecture of different ability in brain function/behavior and connectivity is
neurotransmitter systems, where certain receptors ‘‘lump considerable as well as dynamic and interacts with the
together’’ areas, identified as separate units in other recep- possibility to generalize parcellations from one brain to
tor types (Zilles and Amunts, 2009). It may also be the case another. Different degrees of variability in cytoarchitecture,
when several areas do not differ in their myelin density and size, and relationship to sulcal landmarks have been
thus do not indicate finer parcellations. shown in different brain regions—from highly variable

1100 Neuron 88, December 16, 2015 ª2015 Elsevier Inc.



areas like Broca’s region to low variable subcortical nuclei Aggarwal, M., Nauen, D.W., Troncoso, J.C., and Mori, S. (2015). Probing re-
gion-specific microstructure of human cortical areas using high angular and
such as the amygdala (Amunts et al., 2005; Amunts et al., spatial resolution diffusion MRI. Neuroimage 105, 198–207.
2004). Inter-subject variability is not simply ‘‘noise,’’ which
has to be eliminated by statistical procedures, but repre- Allman, J.M., Tetreault, N.A., Hakeem, A.Y., Manaye, K.F., Semendeferi, K.,
Erwin, J.M., Park, S., Goubert, V., and Hof, P.R. (2010). The von Economo neu-
sents a topic of research (Zilles and Amunts, 2013). rons in frontoinsular and anterior cingulate cortex in great apes and humans.
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In a strict sense, Brodmann’s cortical areas do not fulfill any of
Amunts, K., Schleicher, A., Bürgel, U., Mohlberg, H., Uylings, H.B., and Zilles,
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(e.g., Filimonoff, 1932; Kononova, 1935; Von Economo and Kos- variable? Neuroimage 11, 66–84.
kinas, 1925). From today’s perspective, many areas that Brod-
Amunts, K., Weiss, P.H., Mohlberg, H., Pieperhoff, P., Eickhoff, S., Gurd,
mann has described do not match recent data, e.g., in the J.M., Marshall, J.C., Shah, N.J., Fink, G.R., and Zilles, K. (2004). Analysis
extrastriate visual cortex, frontal operculum, and fusiform gyrus. of neural mechanisms underlying verbal fluency in cytoarchitectonically
Physiological, anatomical, and imaging studies have provided defined stereotaxic space–the roles of Brodmann areas 44 and 45. Neuro-
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Taken together, the last years have shown an immense advance
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the hypothesis that cortical organization is composed of at least
three different spatial scales—columns, micro-, and meso Amunts, K., Axer, H., and Bücker, O. (2014a). Towards a multi-scale, high-res-
maps—thus emphasizing the multi-level nature of brain organiza- olution model of the human brain. In Brain-Inspired Computing, L. Grandinetti,
N. Petkov, and T. Lippert, eds. (Switzerland: Springer International Publishing
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that human brain models and simulation are conceptualized.
The new mapping approaches can bridge the gaps between Amunts, K., Hawrylycz, M.J., Van Essen, D.C., Van Horn, J.D., Harel, N., Po-
line, J.B., De Martino, F., Bjaalie, J.G., Dehaene-Lambertz, G., Dehaene, S.,
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The authors thank Dr. Sebastian Bludau, Daniel Zachlod, Dr. Axel Schleicher, 93, 252–259.
and Hartmut Mohlberg for their contribution to cytoarchitectonic mapping and
the development of the JuBrain atlas and Professor Jochen Staiger, University Axer, H., Axer, M., Krings, T., and Keyserlingk, D.G. (2001). Quantitative esti-
Göttingen, for valuable suggestions regarding new imaging techniques. The mation of 3-D fiber course in gross histological sections of the human brain us-
research leading to this study has been supported by the portfolio theme ing polarized light. J. Neurosci. Methods 105, 121–131.
‘‘Supercomputing and Modeling for the Human Brain’’ by the Helmholtz Asso-
ciation and has received funding from the European Union Seventh Frame- Axer, M., Amunts, K., Grässel, D., Palm, C., Dammers, J., Axer, H., Pietrzyk, U.,
work Programme (FP7/2007- 2013) under grant agreement no. 604102 and Zilles, K. (2011a). A novel approach to the human connectome: ultra-high
(Human Brain Project). resolution mapping of fiber tracts in the brain. Neuroimage 54, 1091–1101.

Axer, M., Grässel, D., Kleiner, M., Dammers, J., Dickscheid, T., Reckfort, J.,
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