Neuroscience

Modeling and Simulation of

Neocortical Micro- and
Reviewed Preprint
Mesocircuitry. Part I: Anatomy
v2 • November 26, 2024
Revised by authors Michael W Reimann , Sirio Bolaños-Puchet, Jean-Denis Courcol, Daniela Egas Santander,
Alexis Arnaudon, Benoît Coste, Fabien Delalondre, Thomas Delemontex, Adrien Devresse, Hugo Dictus,
Alexander Dietz, András Ecker, Cyrille Favreau, Gianluca Ficarelli, Mike Gevaert, Joni Herttuainen,
Reviewed Preprint
James B Isbister, Lida Kanari, Daniel Keller, James King, Pramod Kumbhar, Samuel Lapere, Jᾱnis Lazovskis,
v1 • August 13, 2024
Huanxiang Lu, Nicolas Ninin, Fernando Pereira, Judit Planas, Christoph Pokorny, Juan Luis Riquelme,
Armando Romani, Ying Shi, Jason P Smith, Vishal Sood, Mohit Srivastava, Werner Van Geit,
Liesbeth Vanherpe, Matthias Wolf, Ran Levi, Kathryn Hess, Felix Schürmann, Eilif B Muller,
Henry Markram , Srikanth Ramaswamy

Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland • Riga Business School,
Riga Technical University, Riga, Latvia • Nottingham Trent University, Nottingham, UK • ELKH-University of
Debrecen, Neuroscience Research Group, Debrecen, Hungary • University of Aberdeen, Aberdeen, UK • Laboratory
for Topology and Neuroscience (UPHESS), Brain Mind Institute, School of Life Sciences, École polytechnique
fédérale de Lausanne (EPFL), Lausanne, Switzerland • Neural Circuits Laboratory, Newcastle University, Newcastle,
UK

https://en.wikipedia.org/wiki/Open_access
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eLife Assessment

This manuscript reports a detailed model of juvenile rat somatosensory cortex,

consisting of 4.2 million morphologically and biophysically detailed neuron models,
arranged in space and connected according to diverse experimental data - a
valuable tool for the field. The construction of the model is based on a methodology
with solid supporting evidence. It should be noted that, by necessity, such a large-
scale model development involves many assumptions, interpolations, and decisions
that could have compounding downstream effects on further analyses that may be
difficult to disambiguate.

https://doi.org/10.7554/eLife.99688.2.sa4

Abstract
The function of the neocortex is fundamentally determined by its repeating microcircuit
motif, but also by its rich, interregional connectivity. We present a data-driven computational
model of the anatomy of non-barrel primary somatosensory cortex of juvenile rat, integrating
whole-brain scale data while providing cellular and subcellular specificity. The model
consists of 4.2 million morphologically detailed neurons, placed in a digital brain atlas. They
are connected by 14.2 billion synapses, comprising local, mid-range and extrinsic
connectivity. We delineated the limits of determining connectivity from neuron morphology
and placement, finding that it reproduces targeting by Sst+ neurons, but requires additional

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 specificity to reproduce targeting by PV+ and VIP+ interneurons. Globally, connectivity was
characterized by local clusters tied together through hub neurons in layer 5, demonstrating
how local and interegional connectivity are complicit, inseparable networks. The model is
suitable for simulation-based studies, and a 211,712 neuron subvolume is made openly
available to the community.

1 Introduction
Cortical dynamics underlie many cognitive processes and emerge from complex multi-scale
interactions. These emerging dynamics can be explored in large-scale, data-driven, biophysically-
detailed models (Markram et al., 2015 ; Billeh et al., 2020 ), which integrate different levels of
organization. The strict biological and spatial context enables the integration of knowledge and
theories, the testing and generation of precise hypotheses, and the opportunity to recreate and
extend diverse laboratory experiments based on a single model. This approach differs from more
abstract models in that it emphasizes anatomical completeness of a chosen brain volume rather
than implementing a specific hypothesis. Using a “bottom-up” modelling approach, many detailed
constituent models are combined to produce a larger, multi-scale model. To the best possible
approximation, such models should explicitly include different cell and synapse types with the
same quantities, geometric configuration and connectivity patterns as the biological tissue it
represents.

Investigating the multi-scale interactions that shape perception requires a model of multiple
cortical subregions with inter-region connectivity, and for certain aspects, the subcellular
resolution provided by a morphologically detailed model is also required. In particular, Barabási
et al., 2023 argued that the function of the healthy or diseased brain can only be understood
when the true physical nature of neurons is taken into account and no longer simplified into
point-neuron networks. Also, Einevoll et al. (2019) pointed out that simulations of large-scale
models are essential for bridging the scales between the neuron and system levels in the brain. In
that regard, modern electron-microscopic datasets have reached a scale that allows the
reconstruction of a ground truth wiring diagram of local connectivity between several thousand
neurons (MICrONS-Consortium et al., 2021). However, this only covers a small fraction of the
inputs a cortical neuron receives. While afferents from outside the reconstructed volume are
detected, one can only speculate about the identity of their source neurons and connections
between them. The scale required to understand inter-regional interactions is only available at
lower resolutions in the form of region-to-region or voxel-to-voxel connectivity data.

To help better understand cortical structure and function, we present a general approach to create
morphologically detailed models of multiple interconnected cortical regions based on the
geometry of a digitized volumetric brain atlas, with synaptic connectivity predicted from anatomy
and biological constraints (Figure 9 ). We used it to build a model of the juvenile rat non-barrel
somatosensory (nbS1) regions (Figure 9 , center). These regions were selected for the wealth of
available experimental data from various labs, and to build upon our previous modeling work
(Table 1 ). The workflow is based on the work described in Markram et al. (2015) , with several
additions, refinements and new data sources that have been independently described and
validated in separate publications (Table 1 ). The model captures the morphological diversity of
neurons and their placement in the actual geometry of the modeled regions through the use of
voxelized atlas information. We calculated at each point represented in the atlas the distance to
and direction towards the cortical surface (Figure 9 ; step 1). We used that information to select
from a pool of morphological reconstructions anatomically fitting ones and orient their dendrites
and axons appropriately (Figure 9 ; step 2). As a result, the model was anatomically complete in
terms of the volume occupied by dendrites in individual layers. We then combined established
algorithms for the prediction of local (Reimann et al., 2015 ) and mid-range (Reimann et al.,

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 2019 ) connectivity (Figure 9 ; step 3) as well as extrinsic connectivity from thalamic sources
(Markram et al., 2015 , (Figure 9 ; step 4) to generate a connectome at subcellular resolution
that combines those scales.

We characterized several emerging aspects of connectivity (Figure 9 ; step 5). First, we found
that brain geometry, i.e., differences in cortical thickness and curvature have surprisingly large
effects on how much individual layers contribute to the connections a neuron partakes in. Second
we characterized the predicted structure of connectivity at an unprecedented scale and determine
its implications for neuronal function. In particular, we analyzed how the widths of thalamo-
cortical axons constrains the types of cortical maps emerging. Furthermore, we characterized the
global topology of interacting local and mid-range connectivity, finding highly complex topology of
local and mid-range connectivity that specifically requires neuronal morphologies. Finally, we
systematically analyzed the higher-order structure of connectivity beyond the level of pairwise
statistics, such as connection probabilities. Doing so, we characterized highly connected clusters of
neurons, distributed throughout the volume that are tied together by mid-range synaptic paths
mediated by neurons in layer 5, which act as “highway hubs” interconnecting spatially distant
neurons in the model. The highly non-random structure of higher-order interactions in the
model’s connectivity was further validated in a range of follow-up publications using the model
(Ecker et al., 2024 , 2023 ; Egas Santander et al., 2024 ; Reimann et al., 2023 ).

Finally, we present an accompanying manuscript that details neuronal and synaptic physiology
modeled on top of these results, describes the emergence of an in vivo-like state of simulated
activity, and delivers a number of in silico experiments generating insights about the neuronal
mechanisms underlying published in vivo and in vitro experiments (Isbister et al. (2023) ; Figure
9 ; step 6).

The anatomically detailed modeling approach provides a one-to-one correspondence between

most types of experimental data and the model, allowing the data to be readily integrated.
However, this also leads to the difficult challenge to curate the data and decide which anatomical
trend should be integrated next. Due to the incredible speed of discovery in the field of
neuroscience an integrative model will always be lagging behind the latest results, and due to its
immense breadth, there is no clear answer to which feature is most important. We believe the
solution to this is to provide a validated model with clearly characterized strengths and
weaknesses, along with the computational tools to customize it to fit individual projects. We have
therefore made not only the model, but also most of our tool chain openly available to the public
(Figure 9 ; step 7).

Already the process of adding a new data source to drive a refinement of the model serves to
provide important insights. We demonstrate this by comparing the model to connectivity
characterized through electron microscopy (MICrONS-Consortium et al., 2021), finding
mismatches, and describing the changes required to fix them. This allowed us to determine which
rules are required to predict connectivity from the locations and densities of neuronal processes.
Previously, simple overlap of distributions of axonal and dendritic segments has been proposed
(Peters’ rule; Peters and Feldman, 1976 ; Garey, 1999 ), and contrasted with findings of
preference for specific cell types or subcellular domains (White and Keller, 1987 ; Mishchenko et
al., 2010 ). Our approach to local connectivity combines overlap with the principle of cooperative
synapse formation (Fares and Stepanyants, 2009 ; Reimann et al., 2015 ), additionally
Schneider-Mizell et al. (2023) proposed a combination of overlap and targeting preferences. Our
comparison to electron microscopy let us uncover the strength and nature of the targeting
preferences shaping connectivity beyond neuronal and regional anatomy. We found that
cooperative synapse formation explains some forms of apparent targeting. Additionally, we found
that the distribution of postsynaptic compartments targeted by connections from somatostatin
(Sst)-positive neurons is readily predicted from overlap only, while for parvalbumin (PV)-positive

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 Table 1

References to publications of input data and methods employed for individual modeling steps. An asterisk next to a reference
indicates that substantial adaptations or refinements of the data or methods have been performed that will be explained in
this manuscript. In the other cases, a basic summary will be provided and an exhaustive description in the Methods.

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 and vasoactive intestinal peptide (VIP)-positive neurons additional specificity plays a role. We
found no indication of additional specificity for excitatory neurons. The model is available both
with and without the characterized inhibitory specificity.

2 Results

2.1 Atlas-based neuron placement

The workflow for modeling the anatomy of juvenile rat nbS1 is based on the work described in
Markram et al. (2015) , with several additions and refinements. Most of the individual steps and
data sources have already been independently described and validated in separate publications
(Table 1 ). The basis of the work was a digital atlas of juvenile rat somatosensory cortex, based
on the classic work of Paxinos and Watson (2007) (Figure9, step 1.). The atlas provides region
annotations, i.e., each voxel is labeled by the somatosensory subregion it belongs to. It also
provided a spatial context for the model, with the non-modeled barrel region being surrounded on
three sides by modeled regions (specifically: S1DZ and S1ULp). The atlas was enhanced with
spatial data on cortical depth and local orientation towards the cortical surface (Bolaños-Puchet et
al., 2024 ; Bolaños-Puchet et al., 2023 ). We further added annotations for cortical layers,
placing layer boundaries at fixed normalized depths (Table S3 ). Using laminar profiles of cell
densities (Keller et al., 2019 ), we generated additional voxelized datasets with cell densities for
each morphological type (see Methods).

Next, we filled the modeled volume of the atlas with neurons (Figure 9 , step 2.). Neuronal
morphologies were reconstructed in slices, and repaired algorithmically (Anwar et al., 2009 ;
Markram et al., 2015 ). Out of 1017 morphologies, 58 were new in vivo stained reconstructions
used for the first time in this work (see Methods). The morphologies were classified into 60
morphological types (m-types, Table S1 ; 18 excitatory Figure 2A , 42 inhibitory), based on
expert knowledge and objectively confirmed by topological classification (Kanari et al., 2019 ).

As only 58 morphologies were in vivo stained reconstructions, the rest potentially suffered from
slicing artifacts (see Figure S5 for examples), despite applying a repair algorithm (Markram et
al., 2015 ). A topological comparison (Kanari et al., 2018 , 2019 ) between axons and dendrites
of neurons in all layers (Figure S4 ) revealed that in vitro reconstructions from slices could not
capture detailed axonal properties beyond 1000 µm, but could faithfully reproduce dendritic
arborization. Cell bodies for all m-types were placed in atlas space according to their prescribed
cell densities.

At each soma location, a reconstruction of the corresponding m-type was chosen based on the size
and shape of its dendritic and axonal trees (Figure S7 ). Additionally, it was rotated according to
the orientation towards the cortical surface at that point. These steps ensured that manually
identified features of the morphologies (Table S4 ) landed in the correct layers (Figure 2C ;
S8 ). Additionally, a random rotation around an axis orthogonal to layer boundaries was applied.

2.2 Biological dendrite volume fractions emerge in the model

The distribution over 60 m-types in the model captured the great morphological diversity of
cortical neurons and matched the reference data used (Figure 3A , see Methods). The placement
of morphological reconstructions matched expectation, showing an appropriately layered
structure with only small parts of neurites leaving the modeled volume (Figure 2C ). For a more
quantitative validation, we calculated the fraction of the volume occupied by neurites in 100 depth
bins of a cylindrical volume spanning all layers and with a radius of 100 µm (Figure 3B ). This
included an estimated volume for axons forming the mid-range connectivity between modeled
regions, based on its synapse count (see below and Methods).

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 Figure 1

Overview of the model building and analysis workflow.

Step 1: Building was based on a volumetric atlas of the modeled regions: (1) S1J; (2) S1FL; (3) S1Tr; (4) S1HL; (5) S1Sh; (6)
S1DZ; (7) S1DZO; (8) S1ULp. Additional atlases of biological cell densities and local orientation towards the surface were built.
Step 2: Neuron morphologies were reconstructed and classified into 60 morphological types (m-types). They were placed in
the volume according to the densities and orientations from step 1. Step 3: The anatomy of intrinsic synaptic connectivity was
derived as the union of one algorithm for local connectivity and one for mid-range connectivity. Step 4: Extrinsic inputs from
two thalamic sources were placed on modeled dendrites according to published methods. Step 5: Taken together, these
steps allowed us to predict the topology of connectivity at scale with (sub-)cellular resolution. Step 6: The anatomical model
served as the basis of a physiological model, ready to be simulated. This is presented in an accompanying manuscript. Step 7:
The model, simulation and analysis tools have been made publicly available. Left: During modeling, three types of
generalization had to be made to fulfill input data requirements: from mouse to rat, from adult to juvenile, and from one
cortical region to another. Generalizations used are indicated in each step.

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 Figure 2

Anatomy of the model

A: Exemplar 3D reconstructions of the 18 excitatory m-types. Rendering and visualization was done in NeuroMorphoVis
(Abdellah et al., 2018 ). Dendritic diameters are scaled (x3) for better resolution. B: Modeled cortical layers, with exemplars
of excitatory morphological types placed in the model. C: The placement of each morphological type recreates the biological
laminar anatomy of dendrites and axons, which then serves as the basis of local connectivity. Inset shows modeled brain
regions (solid colors) in the context of the non-modeled regions (transparent).

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 Figure 3

Volumetric anatomy of the model

A: M-type composition per layer: For each layer, stacked histograms of the relative fractions of each m-type, comparing the
model (right bar for each layer) to the input data (left bar). For simplicity, the layer designation is stripped from each m-type.
Left: For excitatory types; right: Inhibitory types. B: Stacked histograms of the fraction of space filled by neurites at various
depths in the model. The y-axis indicates the distance in µm from the bottom of layer 6. B1: For neurites of neurons in
different layers indicated in different colors. Grey: estimated lower bound for the volume of axons supporting the mid-range
connectivity. B2: For neurites of different m-types. Colors as in A, but inhibitory types are grouped together. B3: For different
types of neurites. C: Comparing fractions for axons and dendrites to the literature (Santuy et al., 2018 ). X-marks indicate
overall means, other marks means in individual layers; teal: reference, black: model.

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 We found a clearly layered structure also for the neurite volumes, with apical dendrites
contributing substantially to the volume in layers above their somas. The volume was clearly
dominated by dendrites, filling between 23% and 47% of the space, compared to 2% to 11% for
axons (Figure 3B3 ). The range of values for dendrites matched literature closely (between 26%
and 46%, Santuy et al., 2018 ). Conversely, values for axons were lower than literature (between
17% and 24%); however, this is explained by the vast amount of external input axons from
nonsomatosensory and extracortical sources that are not part of the model.

2.3 Local, mid-range and extrinsic connectivity modeled separately

Synaptic connectivity in comparable models is often modeled by imposing experimentally
characterized connection probabilities, potentially distance-dependent, onto the various neuronal
pathways (Potjans and Diesmann, 2014 ; Pronold et al., 2024 ). As this approach directly
parameterizes structural connections strengths at the population level it allows easy recreation of
meso-scale architectures, as characterized in, e.g., Jiang et al. (2015) . On the other hand, it has
been shown that top-down imposed connection probabilities, even distance-dependent ones,
cannot fully capture the non-random higher order structure of connectivity at cellular resolution
(Gal et al., 2021 ). Such non-random connectivity has been repeatedly demonstrated in cortical
circuitry (Song et al., 2005 ; Perin et al., 2011 ; Reimann et al., 2023 ; Egas Santander et al.,
2024 ) and has functional impact (Reimann et al., 2017b ; Egas Santander et al., 2024 ; Nolte
et al., 2020 ). Instead, we used a previously published approach (Reimann et al., 2015 ) that
selects synapses as a subset of axodendritic, axo-somatic and axo-axonic appositions, based on
biologically motivated rules (Figure 9 , step 3; 4A). Note that this algorithm is not equivalent to
the so-called “Peters’ rule” (Peters and Feldman, 1976 ; Garey, 1999 ; Rees et al., 2017 ), as the
subset is not selected completely randomly. Crucially, the concept of cooperative synapse
formation (Fares and Stepanyants, 2009 ) is used, which introduces statistical dependence for
potential synapses between the same pair of neurons: If one is selected the probability that others
are selected increases. The algorithm has been demonstrated to result in a nonrandom higher-
order structure of connectivity matching biological references (Reimann et al., 2015 ; Gal et al.,
2017 ; Reimann et al., 2017a ,b ). Conversely, connection probabilities are not directly
controlled by the modeler. We measured the emerging connection probabilities by emulating a
multi-patch procedure that is often used for that purpose in vitro (Figure 4B , S9A , see
Methods). We compared them to connection probabilities reported in 124 literature sources
gathered by Zhang et al. (2019) (Figure S9B-D ). We note that reported connection
probabilities vary drastically, for example, ranging from zero to 88% for L2/3 EXC to L2/3 PV.
Significant variability persisted when only data sources for rat or the age of the model (P14) were
considered, with ranges of values stretching up to 51% and 54% respectively. While values for the
model were sometimes outside the reported range, this was mostly restricted to pathways with
only one or two sources.

To create biologically realistic overall neuron out-degrees, our algorithm requires axon
reconstructions to be complete. While we try to ensure the completeness of axonal arborizations
through the use of in vivo stained reconstructions and repair algorithms, this becomes more
challenging, the more an axon stretches away from its soma, due to lack of dye penetration and
potentially slicing artifacts. According to our analysis of the morphologies used, most axons were
only accurately reconstructed up to 1000µm from the soma (see above). This contrast between
local axon around the soma and more mid-range collaterals stretching into neighboring regions
can also be seen in in vivo stained reconstructions (Figure 4A ). Therefore, to model connections
at larger distances, we used a second, previously published algorithm (Reimann et al., 2019 ). It
places mid-range synapses according to three biological principles that all need to be separately
parameterized based on experimental data (as detailed in the subsequent paragraphs): First,
connection strength: ensuring that the total number of synapses in a region-to-region pathway
matches biological estimates. Second, layer profiles: ensuring that the relative number of synapses
in different layers matches biological estimates. Third, topographical mapping: Ensuring that the
specific locations within a region targeted by mid-range connections of neurons describe a

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 Figure 4

Intrinsic connectivity as union of local and mid-range connectivity.

A: Local connectivity was derived as in (Markram et al., 2015 ; Reimann et al., 2015 ), i.e. based on neuron placement and
morphologies. Right: Axo-dendritic appositions were considered as potential synapses. They were then filtered to yield
among other biological constraints biologically realistic bouton densities. B: Range of resulting connection probabilities
within 100µm. Indicated are the minimum (inner circle) and maximum (outer circle) found over 20 repetitions of sampling
1000 pairs in a simulated multi-patch procedure (Fig S9A ). See Methods for mapping from morphological types to
inhibitory subclasses (PV/SST/INH). C: An exemplary layer 5 PC axon reconstruction from Janelia MouseLight
(mouselight.janelia.org, Gerfen et al. (2018) ) that includes long-range collaterals, shown in the context of mouse
somatosensory regions. The blue circle highlights local branches all around the soma. Red highlights depict more targeted
collaterals into neighboring regions. D1: Predicted pathway strengths as indicated in Figure S2C3 . D2: Pathway strength
emerging from the application of the apposition-based connectivity algorithm described in Reimann et al. (2015) . E: Values
of the diagonal of (D1) compared to the diagonal of (D2). F: Schematic of the strategy for connectivity derivation: Within a
region only apposition-based connectivity is used; across regions the union of apposition-based and mid-range connectivity.
G: Connection strength constraints for the mid-range connectivity derived by subtracting D2 from D1 and setting elements <
0 to 0 (colors as in D). H: Resulting total density from both types of modeled synaptic connections in individual regions
compared to the data in (D1).

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 biologically parameterized, topographical mapping. We note that in the literature on macro-scale
connectomics, the pathways between somatosensory regions would be considered “short-range,
inter-regional connections”. We will still refer to connections from the two algorithms as the local
and mid-range connectomes respectively, for simplicity and because of the intuitive contrast it
invokes.

To parameterize connection strengths, we used data from axon tracing experiments provided by
the Allen Institute (Harris et al., 2019), adapted from mouse to rat, yielding expected densities of
projection synapses between pairs of regions (Figure 4D1 ). First, we asked to what degree the
local connectome algorithm suffices to model the connectivity within a region. To that end, we
compared the mean excitatory synapse densities of local connectivity to the target values adapted
from (Reimann et al. (2019) , diagonal Figure 4D1 vs. D2; E). These target values were derived
from relative region-to-region connectivity strengths, estimated by axon tracing, scaled to match
overall volumetric synapse densities estimated from electron microscopy. We found that the
overall average matches the data fairly well, however the variability across regions was lower in
the model (0.123 ± 0.017 µm−1; mean ± std in the model vs. 0.097 ± 0.06 µm−1). Based on these
results, we decided that the local connectome sufficed to model connectivity within a region. It
also created a number of connections across region borders (Figure 4D2 ). Consequently, we
parameterized the strengths of additional mid-range connections to be placed as the difference
between the total strength from the data and the strengths resulting from local connectivity, with
connection strengths within a region set to zero (Figure 4G ). As a result synaptic connections
between neighboring regions will be placed by both algorithms (Figure 4F ), with a split ranging
from 20% local to 70% local. The lower spread of apposition-based synapse density within a region
(see above) will also reduce the variability of combined synapse density from both algorithms
(Figure 4H ). While this will halve the coefficient of variation of density across regions from 0.34
to 0.17, the overall mean density over all regions is largely preserved (0.23 data vs. 0.24 for the
combined algorithms; Figure 4H , red dot).

Finally, the dendritic locations of synaptic inputs from thalamic sources were modeled as in
(Markram et al., 2015 ), using experimental data on layer profiles of bouton densities of
thalamocortical axons, and morphological reconstructions of these types of axons (Figure 9 ,
step 4; S12A; Methods). Based on these data, each thalamic input fiber was assigned an innervated
domain in the model, recreating the layer profile along an axis orthogonal to layer boundaries and
spreading equally in the other dimensions (Figure S12B-E ). We modeled two types of thalamic
inputs, based on the inputs into barrel cortex from the ventral posteromedial nucleus (VPM-based)
and from the posterior medial nucleus (POm-based) respectively (Harris et al., 2019; Shepherd and
Yamawaki, 2021 ). While barrel cortex was not a part of the model, we used these projections as
examples of a core-type projection, providing feed-forward sensory input (VPM-based) and a
matrix -type projection, providing higher-order information (POm-based).

The numbers of thalamic input fibers innervating the model were estimated as follows. Laminar
synapse density profiles were summed over the volume to estimate the total number of
thalamocortical synapses, and the number of synapses per neuron was estimated from the lengths
of thalamo-cortical axons. The ratio of these numbers resulted in 72,950 fibers for the POm-based
matrix-type projection, and 100,000 fibers for the VPM-based core-type projection. These numbers
are consistent with the volume ratio of the two thalamic nuclei (1.25mm3 for POm to 1.64mm3 for
VPM; ratio: 0.76).

2.4 Specificity of axonal targeting

For the axons of various inhibitory neuron types, certain aspects of connection specificity have
been characterized (Tremblay et al., 2016 ), such as a preference for peri-somatic innervation for
PV+ basket cells. The local connectivity in the model is based axo-dendritic overlap, combined with
a pruning rule that prefers multi-synaptic connections, but does not take the post-synaptic
compartment type into account (Reimann et al., 2015 ). As such, it captures targeting preferences

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 resulting from specific axonal morphologies, but not potential effects due to potential molecular
mechanisms during synapse formation or pruning. This is based on the idea that the
developmental mechanisms underlying connectivity are manifold and complicated, but their
cumulative effect is at least partially visible in the characteristic shapes of the neurites. It has been
demonstrated by us and other groups that anatomy-based approaches suffice to create highly
nonrandom network topologies that match many biological trends (Gal et al., 2017 ; Reimann et
al., 2017a ; Udvary et al., 2022 ), but the analyses have so far been focused mainly on excitatory
sub-networks. To investigate to what degree the approach suffices to explain observed targeting
trends, we compared the model to characterizations of targeting specificity from the literature,
recreating the electron microscopy studies of Motta et al., 2019 and Schneider-Mizell et al., 2023
in silico.

Motta et al. (2019) analyzed an approximately 500,000 µm3 volume of tissue in layer 4,
considering the postsynaptic targets of the contained axons. As axons in the volume were
fragments, specificity was assessed by comparing the data to a binomial control fit against
observations of axons forming at least one synapse onto a postsynaptic compartment type (see
Methods). Axons with unexpectedly high fractions onto a compartment type were then considered
to target that type. We calculated targeting fractions of axons forming at least 10 synapses in a
comparable layer 4 volume of our model (Figure 5A ). Compared to Motta et al. (2019) ,
fractions of synapses onto apical dendrites were elevated, and more inhibitory synapses were
considered (Figure 5B ). As in the original study, for almost all compartment types, observed
distributions were more long tailed than expected with a number of axons showing a significantly
high targeting fraction (Figure 5C ). The fractions of axons with such specificities also matched
the reference, except for an even higher fraction being specific for apical dendrites in the model
(Figure 5D ). The match resulted from local connectivity in our model using the principle of
cooperative synapse formation (Fares and Stepanyants, 2009 ), where all synapses forming a
connection are kept or pruned together. This creates a statistical dependence between the
synapses that results in a significant deviation from the binomial control models. We demonstrate
this with a simple stochastic model of cooperative synapse formation, fitted to the data of Motta et
al. (2019) . For each axon in the data, we generated a random list of compartments types for
potential synapses matching the overall frequencies observed in the data. For each, the number
generated was 2.5 times the number of synapses of the corresponding axon. Next, we pruned
synapses to the original counts in two ways: First, we grouped potential synapses onto the same
type into groups of three that are kept or discarded together, thereby introducing statistical
dependence. The results provide the same amount of apparent targeting as the data (Figure S11 A-
C ). Second, we pruned without the grouping where no targeting was found (Figure S11 D-F ).
We do not claim that the stochastic model and its parameters provide an accurate description of
the underlying biological processes, only that it demonstrates the effect of cooperative synapse
formation. We conclude that the nonrandom trends observed by Motta et al. (2019) are an
indication of cooperative synapse formation captured by our model.

Recently, the MICrONS dataset (MICrONS-Consortium et al., 2021) has been analyzed with respect
to the axonal targeting of inhibitory subtypes in a subvolume of 100 × 100 µm2 surface area
spanning all layers of the cortex (Schneider-Mizell et al., 2023 ). Similar to Motta et al. (2019)
they considered their distributions of types of postsynaptic compartments. But due to the larger
reconstructed volume, they were able to analyze complete or almost complete axons, allowing for
a quantitative comparison rather than relying on a fitted binomial control model. Additionally,
they calculated the fraction of synapses that are part of a multisynaptic connections and the
fraction thereof that is within 15 µm of another synapse of the same connection.

A comparable volume of the model (see Methods) contained 173 interneurons vs. 163 in the
original study. Their distribution into four connectivity classes according to morphological and
molecular determinants hypothesized by Schneider-Mizell et al. (2023) approximately matched
as well (Figure 5E ; perisomatic targeting: Basket Cells; distal targeting: Sst+; sparsely targeting:

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 Figure 5

Recreating an electron-microscopic specificity analysis

In the top part we analyzed synapses, axons and dendrites in an approximately cubic volume in L4, emulating the techniques
of Motta et al., 2019 . In the bottom part we analyzed a 100 × 100 µm volume of the MICrONS dataset defined by Schneider-
Mizell et al., 2023 . A: Fraction of synapses placed on somata (SOM), proximal dendrites (PD), smooth dendrites (SD), apical
dendrites (AD) and axon initial segments (AIS) for axon fragments in the sampled volume. Black bars indicate mean values
over axons, arrows indicate binomial probabilities fit to observations of axons forming at least a single synapse; pink: for
excitatory axons, black: inhibitory. Fractions missing from 100% are onto other compartment types. B: Overall count on
synapses on different postsynaptic compartment classes inside the studies volumes, comparing the data of Motta et al.,
2019 to the model. Colors as indicated in the legend. C: Distributions of the fractions of synapses onto different
compartment types over excitatory (left) and inhibitory (right) axons. Expected from the binomial model in A (grey) against
the observations in our anatomical model (black outlines). D: Fraction of axons with significantly increased synapse counts
onto different compartment types compared to the binomial control. Indicated for two values of the false detection rate
criterion (q=0.05, 0.3, Storey and Tibshirani, 2003 ). Comparing the reference data to our anatomical model. E: Top numbers
of neurons in four connectivityderived classes in the 100 × 100 µm volume of the MICrONS dataset, defined by Schneider-
Mizell et al., 2023 . Bottom: Numbers in a comparable volume of the model. For assignment of m-types into the four classes
see the main text. F: Derivation of alternative connectivity with targeting specificity, using the example of the “PeriTC” class.
The axo-dendritic appositions of an axon (top) are classified as matching the targeting (green box; here: appositions with
somata or proximal dendrites) or not. Non-matching appositions are removed with probability pnt (middle). This is followed
by a non-specific removal of connections (formed by single or multiple synapses) until the biological density of synapses on
the axon is met (bottom).
G: Bouton densities resulting from the process in F. Left to right: For PeriTC neurons, targeting somata and proximal
dendrites; for InhTC neurons, targeting inhibitory neurons; for SparTC neurons, targeting the first synapse of a connections;
for SparTC neurons, without targeting preference. Respective optimized pnt values reported in the main text. H: Resulting
targeting of postsynaptic compartments, fraction of synapses in multisynaptic connections and clumped synapses (see
Schneider-Mizell et al., 2023 ). Red: Default model; blue: optimized alternative model with specific targeting; grey:
optimized alternative model without specific targeting; orange: data of Schneider-Mizell et al., 2023 Boxes outline the
characteristic feature of each class. I: Inhibitory targeting specificities combining the best performing models.

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 Neurogliaform Cells and L1; inhibitory targeting: bipolar and VIP+). The postsynaptically targeted
compartments matched for the distal targeting group (Figure 5H , top right; 5I), indicating that
their preference can be explained by their axonal morphology, specifically its trend to ascend to
and then branch in superficial layers. For the other three classes, their respective eponymous
trends were not recreated (Figure 5H , red vs. orange).

We then explored the strengths of targeting mechanisms required to explain postsynaptic targets
in a modified version of our connectivity algorithm: Beginning with all axo-dendritic appositions
as potential synapses, we first remove appositions that are not placed on the preferred
postsynaptic compartment with a probability pnt(Figure 5F ; top to middle). This replaces a non-
specific, but otherwise identical first pruning steps in the regular version of our algorithm. This is
followed by removing connections non-specifically, until the biological density of synapses on the
axon is matched (Figure 5F ; middle to bottom). As reference for biological densities of synapses
on axons, we use the sources listed in Reimann et al. (2015)

For the perisomatic targeting class, probability pnt to remove non-proximal, non-soma synapses
was optimized against the data of Schneider-Mizell et al. (2023) to 97%. The remaining synapses
had a density on the axons of perisomatic targeting cells that was three times higher than biology,
which could be reduced in the second, non-specific pruning step removing two thirds of the
connections (Figure 5G , left). This indicates substantial room for rewiring through structural
plasticity while preserving the targeting specificity of perisomatic targeting cells. The resulting
specificity of postsynaptic compartments and multi-synaptic connections then match the reference
data (Figure 5H , top left, blue vs orange; 5I).

For the inhibitory targeting class, probability pnt to remove synapses on non-inhibitory neurons
was optimized to a similar value of 96.5%. Curiously, this first step already reduced the resulting
axonal density of synapses to the biological value, indicating that this class of interneurons cannot
perform substantial rewiring without losing its targeting specificity (Figure 5G , second from
left).

For sparsely targeting cells, we evaluated two hypotheses: First, we note that this targeting class is
associated with Neurogliaform Cells, which are known to have volumetrically transmitting
synapses. It is possible that the sparseness of their targeting can be explained by very few of their
synapes having an anatomical postsynaptic partner, rather than by a targeting mechanism. Indeed
a non-specific removal of 96% of all synapses recreated the sparsity of these connections found in
Schneider-Mizell et al. (2023) (Figure 5H , bottom left, grey vs orange; 5I). This reduced the
axonal density of synapses to 30% of the biological value, implying that the remaining 70% may be
volumetrically transmitting (Figure 5G , right). Second, we randomly picked a “first” synapse
from each connection formed by this class that we considered to be targeted. Of the remaining
synapses, we removed pnt= 95%, which recreated the characteristic sparsity of the connections
equally well (Figure 5H , bottom left, blue vs orange; 5I). In this case, the second, non-specific
pruning step was required, indicating substantial room for rewiring (Figure 5G , second from
right).

Using a recently developed computational tool (“Connectome-Manipulator”; Pokorny et al.

(2024) ), we created a rewired connectome instance that combines the existing excitatory
connectivity with the additional rules for the inhibitory connectivity in a single connectome. This
rewired connectome is openly available in SONATA format (Dai et al., 2020 ), see key resources
table in the Methods.

2.5 Structure of thalamic inputs

Though we have found that the anatomy-based prediction of connectivity underestimates the
specificity of some inhibitory connection types, it remains a powerful tool that has been
demonstrated to recreate non-random trends of excitatory connections that make up the majority

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 of synapses (Reimann et al., 2015 , 2017b ; Gal et al., 2017 ). We therefore set out to
characterize the anatomy and topology of connections at all scales considered in the model. We
began with the thalamic input connections, whose axons were generated based on the axon
density profiles and the statistics of their horizontal spread (Figure S12 A ). Synapses were
established based on their volumetric overlap with cortical dendrites to match the laminar
synapse density profiles of the presynaptic thalamic axons (see Methods and (Figure S12 D, E )).
The individual postsynaptic dendritic morphologies, cell placement, and orientation were
considered in this process (Figure S12 B, C ). The process did not prefer the dendrites of one
neuron type over another. In contrast, (Cruikshank et al., 2007 , 2010 ; Sermet et al., 2019 )
found physiologically stronger thalamo-cortical inputs onto excitatory and PV-positive neurons
than onto SST-positive neurons. While the authors of the studies make compelling arguments that
this at least partially reflects the anatomical strength of the pathways, others have cautioned
against mixing of the anatomy and physiology of connectivity (Sporns, 2013 ). We therefore
believe that further validation of the thalamo-cortical pathways is best performed in the
accompanying manuscript (Isbister et al., 2023 ), where the experimental methods of the
references are closely recreated.

Regarding the laminar structure, we found for both projections that the peaks of the mean
number of thalamic inputs per neuron occur at lower depths than the peaks of the synaptic
density profiles (Figure 6A ). This is consistent with synapses on apical dendrites of PCs being
higher than their somas, but the fact that most peaks occur at places where the synapse density is
close to zero gives a clear indication that synapse density profiles alone can be misleading about
the location of innervated neurons. At the level of individual neurons, the number of thalamic
inputs varied greatly, even within the same layer (Figure 6B ). Overall, the matrix-type
projection innervated neurons in layers 1 and 2 more strongly than the core-type projection, while
in layers 3, 4, and 6, the roles were reversed. Neurons in layer 5 were innervated on average
equally strongly by both projections, although layer 5a preferred the matrix-type and layer 5b the
core-type projection.

To characterize the horizontal structure, we introduced the common thalamic innervation (CTI,
see Methods) as a measure of the overlap in the thalamic inputs of pairs of neurons. As pairs with
many common inputs are likely to have similar stimulus preferences, the magnitude and range of
this effect has consequences for the emergence of functional assemblies of neurons. As expected
from the horizontal extent of individual fibers, the CTI was distance-dependent (Figure 6C, D ),
and showed strong variability. Even directly neighboring pairs might not share a single thalamic
afferent, leading to a sparse spatial distribution of pairs with strong overlap. A Gaussian fit of the
distance dependence of CTI revealed roughly equally strong overlapping innervation for both
coreand matrix-type projections (Figure 6E1 ). The strength of the overlap increased for lower
layers in the case of core-type and decreased for matrix-type projections, while being equally
strong in layer 5. The horizontal range of common innervation was larger for matrix-type
projections in all layers (Figure 6E2 ). In summary, while the anatomy of thalamo-cortical
projections introduces a spatial bias into the emergence of cortical maps, it is relatively weak on
its own and supports different stimulus preferences even for neighboring pairs of neurons.

2.6 Local brain geometry affects connectivity

Cortex is often seen as a homogeneous structure with parallel layers, but at the larger spatial
scales considered in this model, significant variability in its height and curvature can be observed.
Our approach to connectivity allowed us to predict the impact of local brain geometry on
connectivity (see also Ronan et al., 2011 ). We partitioned the model into columnar subvolumes
with radii of approximately 230 µm (Figure 7A ) that we then analyzed separately. We do not
claim that these columns have a biological meaning on their own, they only serve to discretize the
model into individual data points each representing localized circuitry affected by local geometry.

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 Figure 6

Anatomy of thalamic innervation.

A: Depth profiles of synapse densities (dashed lines) and mean number of thalamic inputs per neuron (solid lines) for core-
(red) and matrix-type (blue) thalamocortical projections. Shaded area indicates the standard error of mean. For a region-
specific validation of synapse densities (dashed lines) against experimental data, see Figure S13 . B: Mean and standard
deviation of the number of thalamic inputs for neurons in individual layers or all neurons. Colors as in A. C: Common thalamic
innervation (CTI) of an exemplary neuron (black dot) and neurons surrounding it, calculated as the intersection over union of
the sets of thalamic fibers innervating each of them. Scale bar: 200 µm. D: CTI of pairs of neurons at various horizontal
distances. Dots indicate values for 125 randomly picked pairs; lines indicate a sliding average with a window size of 40 µm. We
perform a Gaussian fit to the data, extracting the amplitude at 0 µm (A) and the standard deviation (σ). E: Values of A
(unitless) and σ (in flatmap coordinates) for pairs in the individual layers or all pairs. Colors as in A.

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 Figure 7

A1, A2: Parcellation of the modeled volume into 230 µm radius columns. Exemplary slice of columns highlighted in green. B:
Geometrical metrics of column subvolumes in the flat view. Peripheral columns masked out (grey); green outline: highlighted
columns in A. Left: Conicality, defined as the slope of a linear fit of depth against column radius. Negative values indicate
narrowing towards L6. Right: Column height, i.e. cortical thickness at the location of the column. C1: Modularity of the
networks of connections within each column. C2: Column conicality and height colored by modularity; D1: Conicality of
columns against their laminar neuronal composition, normalized against the overall composition of the model. Colored lines
indicate linear fits. D2: Conicality against the density of connections in subnetworks given by the intersections of columns
with individual layers. E: Counts of afferents formed onto neurons in individual columns from neurons in the entire model.
E1: Normalized in-degrees originating from neurons in individual layers, plotted against conicality. E2: In-degree of neurons
in individual layers normalized by the overall in-degree in the model into each layer plotted against column height. F: r-values
of linear fits against generalized, n-dimensional in-degree as in E2. F1: Of generalized in-degree against height; F2: Of
generalized in-degree against conicality.

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 We quantified the two main variable factors of geometry, measuring the height and conicality (a
feature that measures the convexity of a column, see Methods) of each column (Figure 7B ). We
found that differences in these factors lead to differences in relevant topological parameters, such
as the modularity of the local network (Figure 7C ). This effect was mostly mediated by
differences in the neuronal composition, both in terms of total neuron count (not shown) and
relative counts for individual layers (Figure 7D1 ). However, conicality also affected the density
of connections in local, layer-specific subnetworks (Figure 7D2 ), i.e., a measure that is
normalized against neuron counts.

Modularity in particular, is strongly correlated with column volume and neuronal count (Figure
S15AB ), which are in turn driven by the column’s height and conicality. This effect is shaped by
the network structure and not just by its size, since it is not present for controls where the number
of neurons and their connections are maintained but their pairing is assigned at random (i.e., ER-
controls). In fact, for ER-controls, modularity is anti-correlated with volume and neuron count
(Figure S15B ). One way in which the results could be artificial is as follows: Modularity is first
defined for a partition of neurons into modules; next the partition maximizing the measure is
considered. This can only be done heuristically, and it is possible that optimal partitions are more
readily found in certain connectomes than others (see also Methods). To rule this out, we focused
on the most modular columns (i.e, those with modularity higher than 0.4). For these, we showed
that on average random subnetworks on 50% of the neurons maintained the modularity values of
the full columns and that this is not an artifact of the algorithm computing the modularity values
(Figure S15C ).

Going beyond the local, purely internal networks, we considered how much each column was
innervated by the individual layers of the entire model. We predict a surprisingly strong impact of
geometry, e.g., the ratio of inputs from layer 3 to inputs from layer 6 shifts from almost 2-1 in
convex regions to 1-2 in concave regions (Figure 7E1 ). On the other hand, we consider the total
amount of inputs received, in each layer of a column. In layers 4, 5 and 6, neurons had a higher in-
degree if they were members of tall columns (i.e. placed at locations of large cortical thickness).
For layers 1, 2 and 3 the trend was weakened or nonexistent (Figure 7E2 ). The notion of in-
degree can be generalized to the n-dimensional in-degree measuring participation in specific,
directed motifs of n+1 neurons (see Methods). While trends differed between individual layers,
overall the dependence of generalized in-degree on geometrical measures increased with
dimension (Figure 7F1, F2 “full”). This was particularly driven by neurons in layer 6. Curiously,
in that layer the sign of the r-values, with respect to conicality, inverted from dimensions 1 and 2
to dimensions above 2, indicating the overall innervation of layer 6 is stronger in convex regions,
but the participation in higher-order motifs is stronger in concave regions.

The effect of conicality can be explained by convex / concave regions allocating more or less
relative space to individual layers, thereby weighing the contributions of the corresponding
subnetworks differently. The effect of column height is twofold: First, taller columns will contain
more neurons. Second, a different selection of neuron morphologies will be placed, depending on
the column height. Briefly, taller morphologies will be selected for taller columns (see also Figure
S7 ). Indeed, the selected morphologies in layers 2 to 5 were significantly different from the
overall composition in columns taller than 2 mm or shorter than 1.4 mm; in layer 6 only the short
columns led to significant deviance (Figure S14A , 7B ). Due to the finite number of
morphology reconstructions used there is the risk of an artificial effect: It is possible that in
geometrically outlying columns only a small number of non-representative morphologies were
placed. Testing this, we found that the morphological diversity overall increased from superficial
to deeper layers, as more morphological reconstructions were available for the thicker, deeper
layers (Figure S14B ). Over columns, diversity was relatively uniform. The largest decrease in
diversity was observed for short columns and layer 5. In that layer, the least diverse column used
around a third of the available reconstructed morphologies (Figure S14C ). As this still

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 amounted to 909 different morphological reconstruction with significant spread between them,
we do not believe that dominance by non-representative morphologies explains the qualitative
differences in connectivity.

2.7 The complexity of local and mid-range

connectivity requires neuronal morphologies
So far, we have analyzed single (thalamo-cortical) pathways making up less than 5% of the
synapses in the model, and connectivity at scales that are already readily achievable in electron-
microscopic reconstructions. The large size of the model also allowed us to characterize predicted
connectivity at cellular resolution, but scales that are experimentally only accessible with regional
or voxelized resolution (Oh et al., 2014 ; Bota et al., 2015 ; Scannell et al., 1995 ; Scholtens et
al., 2014 ), thereby bridging the scales as outlined in the introduction. Note that the topological
methods in the following sections represent and analyze the connectome as a graph, with neurons
as nodes and connections between neurons as directed edges, irrespective of the number of
synapses in the connection.

We began by analyzing the global structure of neuron-to-neuron connectivity in the entire model,
considering local and mid-range connectivity separately. The topology of synaptic connectivity at
single neuron resolution has previously been described in terms of the over-expression of directed
simplices (Reimann et al., 2017b ). A directed simplex of dimension n (or n-simplex, plural n-
simplices) is a neuron motif of n + 1 neurons that are connected in a purely feed-forward fashion,
with a single source neuron sending connections to all others, a single sink neuron receiving
connections from all others, and the connections between non-sink neurons forming an n − 1-
simplex (Figure 8A1 inset; Figure S17A ). In particular, 0-simplices correspond to single
nodes, and 1-simplices to directed edges. Simplex counts of different dimensions in a network
provide a metric of network complexity and can be used to discern their underlying structure
(Kahle, 2009 ; Curto et al., 2013 ; Giusti et al., 2015 ). Regarding function, high-dimensional
simplices have been demonstrated to shape the structure of spiking correlation between neurons
and membership in functional cell assemblies (Reimann et al., 2017b ; Ecker et al., 2024 ).

In line with previous results (Reimann et al., 2017b ), we found simplices up to dimension seven
in the local connectivity (Figure 8A1 , green). The maximal dimension did not increase
compared to Markram et al. (2015) even with the larger scale of the present model, in
accordance with using the same algorithm for local connectivity. However, the addition of mid-
range connectivity did produce a major change. In mid-range connectivity alone, simplices of
dimension up to 15 were observed (Figure 8A2 , orange). This holds true even though local and
mid-range connectivity have roughly the same number of edges (2.1 billion local, 2.5 billion mid-
range), indicating that the higher simplex counts are not simply due to a larger number of
connections. The simplex counts in the combined network of local and mid-range connections are
not simply the sum of the local and mid-range simplex counts (Figure 8A2 ). They are
consistently higher and also attain a higher dimensionality generating motifs of up to dimension
18, indicating a strong structural link between the two systems.

We compared the simplex counts of the model to a range of relevant controls that capture simple
anatomical properties, such as the density of connections or cortical layers, but ignore the impact
of neuronal morphologies (Figure 8A1 , A2). This allowed us to assess the degree to which the
neuronal geometry generates the complexity of the network. The control models and the
parameters on which they capture were the following: the Erdős–Rényi (ER) model used the
overall connection density, the stochastic block model (SBM) used density in m-type-specific
pathways, the configuration model (CM) used sequences of inand out-degrees, and finally the
distance block model (DBM) used distance-dependence and neuron locations for individual m-type-
specific pathways. See Methods for details.

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 Figure 8

Global connectivity structure local vs. mid-range.

A1: Simplex counts of the local connectivity network and several types of random controls (see text). Examples of d-simplices
for d = 1, 2, and 3. Intuitively, an n-simplex is formed by taking an n − 1-simplex and adding a new node to which all neurons
connect (sink). A2: In orange shades, simplex counts of the mid-range connectivity network and several types of random
controls. In gray, simplex counts of the combined network and in blue the sum of the local and the mid-range simplex
counts. Inset: local, mid-range and the sum of simplex counts on a linear scale. B1: Node participation per layer, normalized
to add up to one in each dimension. Top/bottom row: Local/mid-range connectivity network. Left to right: Participation as
source, in any position, and as a sink of a simplex. B2: From left to right: Spatial location of the cells in the simplicial n-cores in
flat coordinates for all n. Depth coordinates for each connected component in the simplicial cores (cells participating in
simplices of maximal dimension) each dot marks the depth of a neuron in that component. Adjacency matrix of the simplicial
cores with respect to local (green dots) and mid-range (orange dots) connections. B3: Number of unique neurons in the
simplicial cores present in each position of a simplex (i.e., position along the order from source to target. numbers: total
counts, bars: counts relative to the total). C1: Right: Distribution of path distances between pairs of neurons in the local
simplicial core; green: along only local edges; grey: along all edges. Left: Euclidean distances between pairs at a given path
distance. Arrows: See C2. C2: Total Number of neurons in all paths of length 3 between neurons of the local core which are at
distance 3 in the combined circuit (black arrow in C1) split by location. Red/purple: at positions 2 and 3 respectively; grey:
expected from randomly assigned m-types while maintaining the global distribution. Cross-/stripe-patterned: For pairs at
path distances greater/less-or-equal than 3 along local edges only (green arrows in C1).

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 Figure 9

Overview of the building workflow and tools used. Names of individual software tools are bold in the description (see also
Key resources table); descriptions of required inputs in italics. Where not listed, the inputs of a step are outputs of the
previous step. For completeness, also physiological modeling steps not described in this work are also depicted, but indicated
semi-transparent. Note that the Key resources table only lists tools related to anatomical modeling.

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 We found that the structure of connectivity is not only determined by the parameters captured by
the controls. The CM control was the closest control for mid-range connectivity, suggesting that the
effect of degree is important, as expected from the very long-tailed degree distributions of the mid-
range connectome (Figure S16A ). Nonetheless, this control model still largely underestimates
the complexity of the mid-range network. At the same time, the DBM control was the closest
control for local connectivity, showing that much of its structure is indeed determined by spatial
distance under certain morphological constraints.

2.8 Simplicial cores define central subnetworks,

tied together by mid-range connections
Next, we investigated in which layers the neurons forming these high-dimensional structures
resided by measuring node participation, i.e. the number of simplices to which a node belongs, and
a measure of the node’s centrality (Sizemore et al., 2018 ; see Methods for details). We found that
in local connectivity, most simplices of dimensions 2, 3, and 4 have their source in layer 3 and their
sink in layer 5 Figure 8B1 top. Yet, for dimensions 5 and above, we found a shift towards layer
6, with both sources and sinks found mostly in that layer. In mid-range connectivity the structures
are much more concentrated on layer 5, which contains both source and sink Figure 8B1
bottom. Only for simplices of dimension greater than 8 does layer 4 provide more sources. Taken
together, this indicates a robust local flow of information from superficial to deeper layers and
within deep layers, with layer 5 forming a backbone of structurally strong mid-range connectivity.
Neurons in layer 6 form numerous simplices among themselves with no apparent output outside
of layer 6, but they are known to be the source of many cortico-thalamic connections (Shepherd
and Yamawaki, 2021 ) that are not a part of this model.

The model made an explicit distinction between m-types forming outgoing mid-range connections
(i.e., projecting m-types) and those that do not (Figure S16C , “proper sinks”) leading to bimodal
distributions of out-and total degrees in the corresponding DBM and SBM controls (Figure
S16A ). In contrast, the actual mid-range network has a unimodal, long-tailed degree
distribution, similar to biological neuronal networks (Giacopelli et al., 2021 ). This further
demonstrates that the m-types alone can not capture the specific targeting between neuron groups
without their actual morphologies.

One type of connectivity specificity that has been found in biological neuronal networks (Towlson
et al., 2013 ) is the formation of a rich club (Zhou and Mondragon, 2004 ; van den Heuvel and
Sporns, 2011 ). This is characterized by a rich-club curve, which measures whether high degree
nodes are more likely to connect together, compared to the CM control (see Methods). We observe
that the local network has a rich-club effect, but it is not stronger than expected in a spatially finite
model with distance-dependent connectivity (Figure S17B1/2 ). On the other hand, the mid-
range network does not exhibit a rich-club effect, see Figure S17B2 bottom, showing that high
degree nodes are not central to the network.

When we generalized the rich club analysis to higher dimensions by considering node
participation instead of degree (Opsahl et al. (2008) ; see Methods for details) a different picture
emerged (see Figure S17B3 ). We found that the rich club coefficients in the mid-range network
increased with dimension, while the opposite happens for the local network. We speculate that
this effect persists after normalization with respect to relevant controls. Unfortunately, this cannot
be currently verified since generating appropriate random controls for these curves is an open
problem currently investigated in the field of random topology (Unger and Krebs, 2024 ).
Nonetheless, this indicates that central nodes strongly connect to each other forming a structural
backbone of the network that is not determined by degree alone.

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 We studied these structural backbones, by focusing on the simplicial cores of the networks, which
is a higher dimensional generalization of the notion of network core, which is determined by
degree alone (see Methods). The general trend observed is that the local network is distributed,
while the mid-range network is highly localized. First in terms of the locations of neurons
participating in the cores (Figure 8B2 left; green vs. orange), and their laminar locations (Figure
8B2 right). Second, even though the numbers of neurons in the local and mid-range cores are in
the same order of magnitude, the local core has 26 disconnected component while the mid-range
core is fully connected (Figure 8B3 bottom). Finally, the mid-range core was successively more
strongly localized towards the source position, where only seven unique neurons were the sources
of all of the 15, 108 highest-dimensional mid-range simplices (Figure 8B3 top).

Finally, we studied the interactions between the local and mid-range networks, specifically, the
ability of mid-range connnectivity to form short-cuts between neurons of the local core. Even
though the sub-network on the nodes in the local core has multiple connected components (Figure
8B2 ), when paths through nodes outside the core are allowed, it is fully connected. When only
local connectivity was considered, the path distances between core neurons were widely
distributed between one and seven with a median of four and a strong dependence on their
Euclidean distance (Figure 8C1 , green). On the other hand, when mid-range connections were
added the maximum path distance drops to five, with a negligible number of pairs at that path
distance. The median path distance drops to three, and the dependence of the path distance on the
Euclidean distance of the pairs nearly disappears (Figure 8C1 , grey). Crucially, even though the
number of edges almost doubled with the addition of mid-range connections, the number of direct
connections remained negligible; instead paths of length three seem to be dominant for
information exchange between neurons.

We therefore studied these paths of length three in the combined circuit in more detail, labeling
the neurons along them from position 1 (start-node) to position 4 (end-node). We found that layer
5 (the layer with the highest outgoing edge probability, see Figure S16B ) is over-represented in
nodes in positions 2 and 3 compared to a random assignment of m-types (Figure 8C2 ).
Moreover, the over-representation depends on the path distance between the start-node and end-
node within the local network: when the value is larger than three (bars hatched by crosses), the
effect is stronger than for pairs at a distance three or less (bars hatched by horizontal bars). This
demonstrates and quantifies how neurons in layer 5 act as “highway hubs” providing shortcuts
between neurons that are far away from each other in the local circuit.

3 Discussion
We have presented a model of the non-barrel primary somatosensory cortex of the juvenile rat
that represents its neuronal and - particularly - synaptic anatomy in high detail. The model
comprises a spatial scale that allows for the study of cortical circuits not only as isolated functional
units, but also their interactions along inter-regional connections. It also demonstrates how novel
insights that are not readily apparent in disparate data and individual models can be gained when
they are combined in a way that creates a coherent whole. Specifically, we were able to make
multiple predictions about the structure of cortical connectivity that required integration of all
anatomical aspects potentially affecting connectivity. At the scale of this model, anatomical aspects
affecting connectivity went beyond individual neuronal morphologies and their placement, and
included intrinsic cortical curvature and other anatomical variability. This was taken into account
during the modeling of the anatomical composition, e.g. by using three-dimensional, layer-specific
neuron density profiles that match biological measurements, and by ensuring the biologically
correct orientation of model neurons with respect to the orientation towards the cortical surface.
As local connectivity was derived from axo-dendritic appositions in the anatomical model, it was
strongly affected by these aspects.

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 One type of literature resource our approach to connectivity did not use is experimental
measurements of local connection probabilities. While these resources are valuable data for our
understanding of cortical processing, they are difficult to use in a biophysically detailed model
covering large spatial scales. First, connection probabilities are often identified through a PSP
response at the soma (Song et al., 2005 ; Perin et al., 2011 ; Thomson et al., 2002 ). However,
for connections formed by low numbers of synapses on distal dendrites the PSP may be attenuated
too much to be measurable at the soma (Neher, 1992 ). The connection may still be relevant, e.g.,
by collaboratively triggering non-linear dendritic events or affecting plasticity of nearby synapses
(Farinella et al., 2014 ; Iacaruso et al., 2017 ; Tazerart et al., 2020 ). These effects are not
considered in simplified models, leading to a focus on somatically-visible connections only.
However, in our case, we want to be able to study these effects with our model. Second, some
studies report connection probabilities, but only incidentally, being primarily interested in the
physiology of a synaptic pathway (Thomson et al., 2002 ; Markram et al., 1997 ; Mason et al.,
1991 ). Hence, they employ sampling strategies that aim to maximize the number of connected
pairs encountered. Third, even studies aiming to characterize the anatomy of circuitry can provide
contradictory estimates: Jiang et al. (2015) report 0 connections for 150 sampled pairs of PCs in
layer 5 of adult mouse V1, while in the proofread portion of the data of MICrONS-Consortium et al.
(2021) 13 out of 154 pairs (8.4%) within 100 µm are connected. Fourth, connection probabilities are
often measured by sampling pairs of neurons at inter-soma distances below 100 µm or even only
50 µm (see Table 4.1 of Zhang et al. (2019) for an overview). While connection probabilities drop
with distance, in a model covering large spatial dimensions, the number of potentially connected
pairs grows with the square of distance. Consequently, a connection probability of, e.g., 0.15% at
500 µm would represent as many connections as a probability of 15% at 50 µm, and it cannot be
rounded to zero. Recently, unbiased connection sampling techniques have been developed that
provide estimates at larger distances (Chou et al., 2023 ), that are likely to solve some of these
issues in the future.

Connectivity derived from axo-dendritic appositions alone was insufficient at the large spatial
scale of the model, as it was limited to connections at distances below 1000 µm. While we found
that it generated the right amount of connectivity within a somatosensory subregion, we
combined it with a second algorithm for inter-regional connectivity. The algorithm is
parameterized by a combination of overall pathway strength, topographical mapping and layer
profiles, which together describe a probability distribution for the segments of mid-range axons,
i.e., an average axonal morphology, using established concepts. On the dendritic side, the
algorithm takes individual neuronal morphologies and their placement into account. We have
demonstrated that the sum of local and non-local synapse densities matches the reference. It is
important to consider whether such a mixture of two independent algorithms captures potentially
important statistical interactions between the local and mid-range connectivity of individual
neurons. We note that local connectivity is fully constrained by morphology, and the non-local
connectivity is parameterized independently for individual morphological types. The combined
connectome therefore captures important correlations at that level, such as stronger and weaker
non-local cortico-cortical connections from slender-tufted and thick-tufted layer 5 PCs
respectively.When the model was created, interactions beyond this level had not yet been
described because local axon reconstructions are reconstructed from slices, which prevents the
possibility of obtaining long range information. Even in vivo staining still only reconstructs axons
within a region (Buzás et al., 2006 ). On the other hand, long-range axon reconstructions are
obtained through whole brain staining, which has low accuracy for local connectivity (Winnubst et
al., 2019 ). Analysis of new EM datasets, such as a characterization of distance-dependent
targeting of excitatory/inhibitory neurons by the axons of L5 thick-tufted pyramidal cells (Bodor et
al., 2023 ) using the data of MICrONS-Consortium et al. (2021) could be incorporated in future
models. Finding non-random correlations between local and non-local connections would require
a strong null model to compare measurements to and our model can serve as that.

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 Recreating electron-microscopic analyses of connectivity in silico, we could then predict
limitations of predicting connectivity from neuron placement and morphology and characterize
the additional mechanisms shaping connectivity. Conceptually, a number of mechanisms
determine the structure of synaptic connectivity, which we will list from general and large-scale to
specific and micro-scale: First, large-scale anatomical trends over hundreds of µm, given by non-
homogeneous (e.g. layered) soma placement and broad morphological trends (e.g. ascending
axons). These are trends that can be captured by simple distance or offset-dependent connectivity
models (Gal et al., 2020 ). Second, small-scale morphological trends captured by axonal and
dendritic morphometrics such as branching angles and tortuosity. These are trends that require
the consideration of individual morphologies and their variability instead of average ones. Third,
the principle of cooperative synapse formation (Fares and Stepanyants, 2009 ) formalizing an
avoidance of structurally weak connections. Fourth, any type-specific trends not captured by
neuronal morphologies, such as local molecular mechanisms and type-specific synaptic pruning.
Fifth, non-type specific synaptic rewiring, e.g. through structural plasticity. We will refer to these
aspects as (L)arge-scale, (S)mallscale, (C)ooperativity, (T)ype-specificity and (P)lasticity respectively,
and we argue that for the explanation of the connectome, the more general explanations should
be exhausted first, before moving on to more specific ones. Also note that this classification is not
considering the underlying developmental causes, but instead associates anatomical and non-
anatomical predictors with the structure of the connectome. (L) has been shown to accurately
predict large-scale connectivity trends, giving rise to Peters’ rule (Peters and Feldman, 1976 ;
Garey, 1999 , although the exact meaning of Peters’ rule is debated, see Rees et al., 2017 ). It
has been demonstrated that (L) alone does not suffice to explain non-random higher order trends
in excitatory connectivity, while the combination of (L,S,C) does (Gal et al., 2017 ; Reimann et al.,
2017a ; Gal et al., 2020 ; Udvary et al., 2022 ). More specifically, Reimann et al., 2017b found
that (L,C) explains overexpression of reciprocal connectivity in cortical circuits, but (L,S,C) is
required to match the biological trend for clustered connectivity, and Udvary et al., 2022 found
(L,S) recreates non-random triplet motifs. Billeh et al., 2020 combined (L) with highly refined (P)-
type rules, demonstrating great functional match of the resulting model.

Our comparison to the results of Motta et al. (2019) cannot capture the role of (L), as the scale of
the considered volume is too small. But it demonstrates that some non-random targeting trends
can be explained by (S,C) and highlights the importance of (C). Further, the overall lower
specificity of excitatory axon fragments indicates that (T) may not play a role for them. Similarly,
the comparison to Schneider-Mizell et al. (2023) shows that (T) is not required to explain the
connectivity of “distal-targeting” (i.e., Sst+) neuron types. Conversely, for “perisomatic-targeting”
types (i.e., PV+ basket cells) (T) was required to match the distribution of postsynaptic
compartment types. Additionally, the number of potential synapses remaining after applying an
optimized (T)-type mechanism was too large to be sustained by the axons, implying a crucial role
of (P) in reducing it further. This is in contrast to “inhibitory-targeting” neurons, where an
optimized (T)-type pruning lowered the synapse count so much that no space for (P) remained. For
the “sparsely-targeting” neurons of Schneider-Mizell et al., 2023 we developed two competing
hypotheses, one predicting no role for (T) and 70% of their synapses volumetrically transmitting,
i.e., without clear postsynaptic partner, and the other predicting a role for (T) similar to the
perisomatic-targeting neurons. We summarize our predictions in Table 2 .

In addition to targeting specificity, we predict the following: First, we were able to predict the
effect of cortical anatomical variability on neuronal composition by increasing or decreasing the
space available for individual layers. If we assume that each layer has a given computational
purpose (Felleman and Van Essen, 1991 ), then this may have functional consequences. It is
possible that cortical circuits compensate for this effect, either anatomically (e.g. using different
axon or dendrite morphologies), or non-anatomically. Either case would imply the existence of an
active mechanism with the possibility of malfunction. Alternatively, function of cortical circuits is
robust against the differences in wiring we characterized. This can be studied either in vivo, or in
silico based on this model.

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 Table 2

Anatomical, morphological and other aspects affecting connectivity and our predictions for their relevance for efferents of
different neuron types. See main text for an explanation of the individual aspects. The signs in the table indicate whether we
predict a certain aspect to be not relevant (-), relevant (+), or highly relevant (++) for a given neuron type.

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 Second, we predicted constraints on the emergence of cortical maps from the anatomy of thalamo-
cortical innervation, i.e., from the combination of: shape and placement of cortical dendrites, the
specific layer pattern formed by thalamo-cortical axons, and their horizontal reach. We
demonstrated how differences in these parameters may affect the distances between neuron pairs
with similar stimulus preferences. At the lower end, very different preferences are supported even
between neighboring neurons. At the higher end, neurons further than ∼350 µm apart are likely
to sample from non-overlapping sets of thalamic inputs. Other mechanisms will ultimately affect
this - chief among them structural synaptic plasticity - this can be thought of as the ground state
that plasticity is operating on.

Third, we predicted the topology of synaptic connectivity with neuronal resolution at an

unprecedented scale, i.e., combining local and mid-range connectivity. We found that the structure
of neither can be explained by connection probabilities, degree-distributions, or distance-
dependence alone, not even when individual pathways formed between morphological types are
taken into account. We expect the mid-range network to have a small-world topology, but
computing smallworld coefficients for a network of this size is infeasible (see Methods).

Fourth, we predict that the mid-range connectivity forms a strong structural backbone distributing
information between a small number of highly connected clusters. The paths within the mid-range
network strongly rely on neurons in layer 5 and form short-cut paths for neurons further away
than ∼2 mm; for smaller distances, local connectivity provides equivalent or shorter paths.

All these insights required the construction of an anatomically detailed model in a

threedimensional brain atlas; additionally, most of them required the large spatial scale we used.
Their functional and computational implications are difficult to predict without also considering
data on neuron activity. To that end, an accompanying manuscript describes our modeling of the
physiology of neurons and synapses, along with a number of in silico simulation campaigns and
their results. Additionally, more insights were gained from using the model in several
publications: In Ecker et al. (2023 , 2024 ); Egas Santander et al. (2024) it has been
demonstrated that non-random higher order structure affects functional plasticity, assembly
formation and the reliability and efficiency of coding of subpopulations. Moreover, two of these
insights were validated against electron microscopy data. Furthermore, in Reimann et al. (2023)
the model served as an important null model to compare an electron microscopic connectome to,
enabling the discovery of specific targeting of inhibition at the cellular level.

This iteration of the model is incomplete in several ways. First, several compromises and
generalizations were made to be able to parameterize the process with biological data, most
importantly, generalizing from mouse to rat. We note that such mixing is the accepted stateof-the-
art in the field, and also neuroscience in general. All 19 data-driven models of rodent
microcircuitry listed in Figure 2 of the recent review of Ramaswamy (2024) conduct some
sort of mixing, including the very advanced mouse V1 model of the Allen Institute (Billeh et al.,
2020 ). In this iteration we focused on investigating the general features of the (multi-region)
mammalian cortex, e.g., high-order motifs, connected by L5 neurons across subregions or the
effect of curvature on connectivity. In the future, more specific aspects of different cortical regions
could be investigated using more specific data sources. This would lead to different versions of the
model for different regions that can be compared and contrasted using the various structural
metrics we developed in this work. Further, we made a number of assumptions about the
biological systems that we explicitly list in Supplementary Section 5.2 . Should improved and
more specific data sources, such as EM-based connectomes or whole-brain neuron
reconstructions, become available in the future, our modeling pipepline is designed to readily
utilize them for refinement. There are also known limitations to the actual model building steps.
While we enforce cell density profiles along the depth-dimension, densities are assumed to be
otherwise uniform within a given subregion. For additional structure, the algorithms will need to
be updated; this will be required for example, for the modeling of the barrel field. Similarly,

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 during volume filling, orientation and placement of neuron morphologies is considered only along
the depth-axis. Modeling of the barrel system will also require the consideration of orientation
and location with respect to nearby barrel centers. Furthermore, we assumed uniform relative
layer thickness throughout the modeled region, due to sparsity of the required data. However,
given that Yusufŏgulları et al. (2015) has demonstrated there are substantial differences in layer
thickness between rat hindlimb and barrel field regions and Narayanan et al. (2017) and
Wagstyl et al. (2020) found lower but still significant differences within developed rat barrel cortex
and human somatosensory cortex respectively, the model pipeline should be updated once
sufficient data becomes available. This demonstrates that detailed modeling requires constant
iteration, as a model can never be proven to be right, only to be wrong. Thus, we made the tools to
improve our model also openly available (see Data and Code availability section). Figure 9
provides a technical overview of individual modeling steps, the software tools involved and the
duration required to run them, to serve as a guide to interested contributors.

4 Methods

Key resources table

Resource availability

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 Lead contact
Further information and requests for data and code should be directed to and will be fulfilled by
the lead contact: Michael W. Reimann ([email protected])

Materials availability

No materials were used in this computational work

Data and code availability

Data on cell densities, bouton densities and mean numbers of synapses per connection
were reported in original publications. Their DOIs are listed in the Key Resources Table.
Data on synapse densities of thalamo-cortical inputs and their projection axon widths are
reported in figures and text in this publication.
Volumetric atlases, neuron reconstructions, the parameterization of mid-range
connectivity in JSON format, and the description of the model in SONATA format (Dai et al.,
2020 ) have been deposited at Zenodo and are publicly available as of the data of the
publication. The DOIs are listed in the key resources table.
Original code has been deposited at Zenodo and is publicly available as of the date of
publication. DOIs are listed in the key resources table.
Any additional information required to reanalyze the data reported in this paper is
available from the lead contact upon request.

4.1 Method details

4.1.1 Preparation of voxelized atlases

First and foremost, the departure from a simplified hexagonal geometry (as in the NMC model
Markram et al. (2015) ), required a digital brain atlas defining the anatomy of the region. To that
end, we took as starting point the Paxinos and Watson (2007) adult rat brain atlas, processed
individual digitized slices, aligned them and labeled them to assemble a smooth three-dimensional
volume with region annotations. The resulting atlas was then scaled down from the dimensions of
adult rat to juvenile (P14) rat brain by reducing the size of individual voxels from 40µm to
38.7348µm. The scaling factor was based on the ratio of S1HL thicknesses at those ages (2082µm
for juvenile vs. 2150µm for adult). Finally, supplementary atlas datasets were generated as
described in Bolaños-Puchet et al. (2024) ; Bolaños-Puchet et al. (2023) . These datasets provide
additional spatial information required to ensure biologically accurate placement and orientation
of dendritic trees (see below). First, the normalized cortical depth (between 0 and 1) at each point;
second, the local orientation as a vector pointing towards the cortical surface at each point; third,
the total cortical thickness at each point, i.e., the length of the shortest path from the cortical
surface to the bottom of layer 6 passing through that point.

Additionally, we followed Bolaños-Puchet et al. (2024) ; Bolaños-Puchet et al. (2023) to produce

a flat map of somatosensory regions, that is, a coordinate transformation associating each voxel
with a two-dimensional projection into the plane. This can be used to create a flat view of region
annotations that is crucial for the description of the topographical mapping of mid-range synaptic
connectivity (see below). In short, to produce the flat map, a projection surface was defined by
reconstructing a mesh from all points at a relative cortical depth of 0.5. Next, the local orientation
field in the supplementary atlas was numerically integrated, yielding streamlines that were used
to project each voxel center onto the projection surface. Finally, the projection surface was

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 flattened with an authalic (area-preserving) algorithm. The main property of the resulting flat map
is that in any flat view derived from it, each pixel represents a subvolume of cortex that spans all
layers, akin to a cortical column.

4.1.2 Reconstruction of neuron morphologies

Neuronal reconstructions were collected with two techniques: neurons were either filled with
biocytin in a brain slice and reconstructed (Markram et al., 2015 ; Muralidhar et al., 2014 ), or
were filled with a fluorescent dye in vivo and reconstructed (Buzás et al. (1998) ; Karube and
Kisvárday (2011) ). In total 1017 unique reconstructions were used, 896 of which were previously
used in Markram et al. (2015) , 63 were new in vitro reconstructions, and 58 were new in vivo
stained reconstructions.

Animal surgery

Normal C57BL/6 mice (P52-60) were used which were bred and maintained in the animal house
facility of Department of Anatomy, Physiology and Embryology under appropriately controlled
conditions (approval of Local Ethics Committee for Animal Research Studies at the University of
Debrecen in line with European Union guidelines for the care of laboratory animals, Directive
2010/63/EU).

For initial anesthesia, animals were injected with pentobarbital (0.15ml (6mg/ml), i.p.). Prolonged
anesthesia was achieved by injecting 0.05 ml every 60-90 minutes depending on the reaction to the
toe-test. Head restraining was used and craniotomy performed in both hemispheres at coordinates
ML 1.5 - 2 mm ; AP 0.5 - 1 mm (Paxinos and Watson, 2007 ) in order to expose the primary
somatosensory cortex at the representation of the (hind-limb and fore-limb). We applied the
topical anesthetic Lidocaine (Xylocaine gel, Egis Gyógyszergyár ZRT, Hungary) to all surgical
wounds and pressure points. A custom-made plastic chamber (prepared by cutting a ring from a 1
ml plastic syringe) was mounted on the skull using super glue and was surrounded by 4% agar
(VWR International Kft, Hungary). Then, the cortex was exposed by making a slit on the dura
mater with the bent tip of a 14 gauge hypodermic needle.

Single neuron labelling

For this purpose, borosilicate glass pipettes (GB150F-8P, Science Products GmbH, Germany) were
pulled (Model P-97, Sutter Instrument Co., USA) with a resistance in the range of 60-80MΩ
(bevelled on a BV-10M beveler, Sutter Instrument Co., USA) and filled with 1M KCl containing 2%
biocytin (Sigma-Aldrich Chemie GmbH, Germany). The microelectrode was attached to a hydraulic
microdrive (MHW-4 Narishige, Japan ) and the tip guided in the cortex under the guidance of a
surgical microscope (OP-MI, Zeiss). Then the chamber was then filled with 4% agar (VWR
International Kft, Hungary) for better stability of the micropipette. Neuronal activity was recorded
and amplified with AxoClamp-2A (Axon Instruments Inc., USA). After filtering, the signal was
displayed on an oscilloscope and an audio monitor to aid and control intracellular penetration.
Successful penetration of the cell membrane was indicated by a sudden drop of the resting
membrane potential (below -40mV) while applying 0.05 nA in the step-current mode (put here
stimulus configuration from Master-8). Then, biocytin was injected with +2 nA using a duty cycle
of 400 msec on and 200 msec off typically for 20 min. In each hemisphere 1-3 penetrations were
made with approximately 0.3 mm spacing from each other. Neuronal activity was searched
blindly across the entire cortical depth.

Histology

The animals received a lethal dose of anesthetics and were perfused transcardially first with the
washing medium (oxygenated Tyrode’s solution) for 2 min or until the blood showed clearing and
then with a fixative (approx. 100 ml) containing 2% paraformaldehyde (VWR International Kft,

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 Hungary) and 1% glutaraldehyde (Sigma-Aldrich Chemie Gmbh, Germany) in 0.1 M phosphate
buffer (PB, pH 7.4) for 50 minutes. Next, the brain was removed from the skull and tissue blocks
containing the region of interest were dissected. A series of 60-80 um thick vibratome (Leica
VT1000S, Leica Biosystem) sections were cut in the coronal plane and collected in 5×10 lots in glass
vials. The sections were washed in 0.05M Tris-buffer saline (TBS, 10 min) and 0.05M TBS
containing 0.1% Triton X-100 (2 × 10 min) and incubated at 4°C in avidin-biotin complexed-HRP
(ABC-Elite kit, Vector Laboratories, Inc., USA), diluted 1:200 in 0.05M TBS containing 0.1% Triton X-
100 (Sigma-Aldrich Chemie Gmbh, Germany) for overnight. Then the sections were washed in TBS
for 2 × 10 min and in TB for 10 min. They were treated with 0.05% DAB (3.3’ diaminobenzidine-
4HCl, Sigma-Aldrich Chemie Gmbh, Germany) diluted in 0.05 M TB containing 0.0025% CoCl2 for
30 min while agitating on an electric shaker. Finally the labelling was visualized in the presence of
0.1% H2O2 (5 min to 10 mins). After washing the sections in 0.05M TBS (3 × 10 min) and 10 min in
0.1 M PB the quality of the DAB reaction and the presence of intracellularly labelled cells were
inspected while wet under a light microscope (x10 objective). All sections of blocks containing
strongly labelled neurons underwent osmification, dehydration and resin embedding in order to
retain the 3D structure of their axons and dendrites. Accordingly, sections were treated with 1%
OsO4 (osmium tetroxide, PI Chem Supplies, USA) diluted in 0.1% PB for 15 minutes. After rinsing in
0.1M PB for 3×10 min, they were dehydrated in ascending series of ethanol (50, 70, 90, 95 per cent
and abs ethanol), propylene oxide, each step for 2×10 min, and submerged in resin (Durcupan,
Sigma-Aldrich Chemie GmbH, Germany) overnight at room temperature. Finally, the sections were
mounted on glass slides, coversliped and cured at 56oC for 24 hours (Somogyi and Freund,
1989 ).

Morphological neuron reconstruction

Labelled neurons were reconstructed in 3D using the Neurolucida neuron reconstruction system
running on Windows XP platform (Neurolucida v.8.23, MicroBrightField Inc., Williston, USA). For
this purpose a Leica DMRB microscope (x100 objective) was coupled to a motorized XY-stage and a
z-motor via a stage controller (Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany). Each
neuron was reconstructed from 20-32 adjoining sections. Neighboring sections were aligned using
the 3-point alignment and the least-squares algorithm for the cut ends of labelled processes and
fiducial landmarks such as the contour of cut blood vessels. The cell body, dendrites, axons and
axon terminals were reconstructed together with their thickness value.

4.1.3 Morphology curation and classification

All morphological neuron reconstructions (see Section 4.1.2 ) were curated and repaired to
correct reconstruction errors and slicing artifacts, as described in Markram et al. (2015) . They
were then classified based on the following strategy. Both interneurons and pyramidal cells were
first classified by expert reconstructors according to their observed shapes by inspection through
the microscope. The expert classification of pyramidal cells, which is based on the shape of the
apical dendrites, was then used as input for the training of the algorithms for the objective
classification as presented in Kanari et al. (2019) . The objective classification was performed
based on the topological morphology descriptor (TMD) (Kanari et al., 2018 ), which encodes the
branching structure of neuronal trees. The TMD of apical trees was extracted from all
morphological reconstructions and was used to train different classifiers for the objective
classification of cells into distinct groups. See Fig. S6 for exemplary excitatory morphologies
and their features.

The expert-proposed scheme comprised 60 morphological types (m-types) (18 excitatory and 42
inhibitory). The m-types of pyramidal cells are distinguished first by layer and further by shape of
their tuft, such as untufted (UPC) and tufted (TPC) cells; and finally into subclasses (A:large tufted,
B:late bifurcating, C:small tufted). The results of the objective classification of pyramidal cells were
then used to validate this classification scheme. A classification scheme was deemed valid if the
TMD was significantly different between classes. This was the case for classes in all layers except

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 for a distinction between two subgroups in layer 3 (L3 TPC:A vs. L3 TPC:B), which was
consequently discarded. Instead we performed an unsupervised clustering of all tufted pyramidal
cells in layer 3 based on their TMD and found a different split into two classes, best described as
large tufted (L3 TPC:A) and small tufted (L3 TPC:C), which we then used in the model. The resulting
list of m-types is found in Table S1 .

For the remaining layers, the results of the objective classification were published in a paper ?
describing a variety of methodologies, using TMD Kanari et al. (2018) in combination with
advanced machine learning tools, such as Convolutional Neural Networks and Graph Neural
Networks. The expert classification could be supported by a variety of methods, ranging from 60%
in interneurons to 80 − 90% in pyramidal cells. Consequently, we considered the expert
classification to be sufficiently accurate to build the model. To increase the morphological
variability in the model we combined axon reconstructions with soma and dendrite
reconstructions from other neurons of the same m-type (mix & match; Markram et al. (2015) ).

4.1.4 Preparation of cell density data

Inhibitory cell densities were constrained following Keller et al. (2019) . In brief, several datasets
were combined to provide depth profiles of densities for successively more granular neuron
classes (Fig. S1 ). The first dataset consists of neuronal soma density estimations, using antibody
stains of neuronal nuclear protein (NeuN) and γ-aminobutyric acid (GABA) from rat neocortex (n =
6, Keller et al. (2019) ). Cell counts provided mean densities and a measure of inter-individual
variability (Markram et al., 2015 ). Cell counts were obtained from a single rat in this dataset, and
their positions were annotated and divided into 100 equal-width bins extending from the top of
layer 1 (L1) to the bottom of layer 6 (L6) (Keller et al., 2019 ). This provided depth profiles of both
total neuron (from NeuN) and inhibitory neuron densities (from GABA). A similar profile was also
estimated from immunostaining with calbindin (CB), calretinin (CR), neuropeptide Y (NPY),
parvalbumin (PV), somatostatin (SOM) and vasoactive intestinal peptide (VIP) (at least three slices
from at least two rats) (Keller et al., 2019 ). All stains were corrected for shrinkage (Ghobril,
2015 ). A single-neuron reverse transcription polymerase chain reaction (RT-PCR) dataset
(Toledo-Rodriguez et al., 2005 ) allowed mapping of biochemical markers to morphological types,
by finding spatial distributions of morphological cell types that would reproduce the biochemical
marker distribution (Keller et al., 2019 ). Whenever the classification could not be resolved down
to morphological types (i.e., excitatory cells, neurogliaform cells, L1 cells, etc.), an estimation of the
fractions of subtypes was used (Muralidhar et al., 2014 ). The depth profile of excitatory neuron
densities was further subdivided into subtypes, maintaining the same layerwise proportions of
these types as in previous work (Markram et al., 2015 ). The final output of this process was a
dataset of neuron density as a function of cortical depth for each of the 60 morphological types
(Table S1 ).

4.1.5 Voxelized neuronal composition

To prepare the modeled volume for cell placement, we first associated each voxel of the atlas with
a cortical layer. We assumed layer boundaries to be at the same normalized depth at each point of
the region. The depths, derived from Markram et al. (2015) , are listed in Table S3 . As a voxel
atlas of normalized depths was provided as an input, layer identities could be readily looked up
from that table. Similarly, neuron densities for each voxel of the atlas were produced from the
vertical density profiles that served as inputs (see Sec. 4.1.4) by looking up the corresponding
values using the normalized depth.

4.1.6 Neuron placement

Neurons were placed into the volume by first generating soma positions and annotating them with
a morphological type according to the voxelized densities generated. Next, for each location we
selected a reconstructed morphology from the annotated morphological type. As previously

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 (Markram et al., 2015 ), we tried to select an appropriate reconstruction taking also the
variability within a morphological type into account. For instance, the largest exemplars of layer 5
pyramidal cells cannot be placed close to the top of the layer, as otherwise their tuft would stick
out of the top of layer 1. We selected morphologies by scoring them according to how well
manually identified features, such as dendritic tufts or horizontal axonal branching, would land in
the biologically appropriate layers, when placed at any given location. The placement rules that
were used were the same as in Markram et al. (2015) and are listed in Table S4 .

Previously, this process was aided by the use of a simplified geometry where layer boundaries
were formed by parallel planes. To execute the algorithm in a realistic brain volume, we used
auxiliary voxel atlasses (see Sec. 4.1.1 ). The first atlas contained for each voxel the normalized
depth of its center point, i.e., a value between 0 and 1 where 0 would indicate its placement at the
top of layer 1 and 1 the bottom of layer 6. The second atlas contained for each voxel the total
thickness of the cortex at that location. As layer boundaries in the model were always placed at
fixed normalized depths (Table S3 ), we could calculate the absolute distance of the voxel to any
layer boundary by subtracting their normalized depths and multiplying the result with the local
cortical thickness. Based on this, we calculated the overlap of the dendritic and axonal features of
a candidate morphology with the target layer interval (Fig. S7 ). Morphology selection was then
performed as previously (Markram et al., 2015 ), that is, a morphology was selected randomly
from the top 10% scorers for a given position.

4.1.7 Modeling synaptic connectivity

Local connectivity

To determine the structure of synaptic connectivity between neurons, data on numbers of

synapses and their locations were required. Previous data for mean bouton densities on axons of
various neuron types, and number of synapses per connection (Reimann et al., 2015 ), were used
and generalized to all nbS1 subregions. The data was then used as described in (Reimann et al.,
2015 ). Similar to neuronal morphologies (see above), there is no evidence for anatomical
differences in connectivity between nbS1 subregions, with the exception of barrel cortex (not
included in the model).

Mid-range connectivity

Due to the larger spatial scale of the present model compared to Markram et al. (2015) ,
additional data was required to further constrain synaptic connectivity at a global scale. To that
end, we referred to data on relative strengths of synaptic connections from the Allen Mouse Brain
Connectivity Atlas (Harris et al., 2019), and generalized it for use with a rat model. We began by
scaling the relative connection densities of Harris et al. (2019) to absolute densities in units of
synapses per µm3. That is, the entire voxel-to-voxel connection matrix of intra-cortical connectivity
was scaled to match the average total density of synapses measured in electron microscopy (Schüz
and Palm, 1989 ).

Next, we summed the densities over voxels belonging to pairs of nbS1 subregions, resulting in a 6
x 6 connection matrix of synapse densities in pathways between and within regions. We mapped
this matrix to rat nbS1 by finding corresponding regions in the mouse and rat atlases (Fig. S2A ).
Here, we assumed that synapse densities in these pathways are comparable between mouse and
rat, although the larger dimensions of the rat brain resulted in larger absolute synapse counts (Fig.
S2C ).

We used the same mapping to generalize the spatial structure of the targeting of connections
between regions from mouse to rat. This refers to the question of which specific parts of a region
are innervated by individual axons in a different region. Reimann et al. (2019) modeled this

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 innervation as a topographical mapping between pairs of regions in a flat view of neocortex. We
used a flat map (see Sec. 4.1.1 ) to create a flat view of rat nbS1 subregions and recreated a
matching topographical mapping between them as follows (see Fig. S2A ).

First, three points were identified inside the flat view of a mouse region such that the area of the
enclosed triangle is maximized. Color labels (“red”, “green” and “blue”) are arbitrarily assigned to
the three points. Next, another three points defining a maximal area triangle were placed in the
flat map of the corresponding rat region(s). The same color labels were then manually assigned to
these points to best recreate their spatial context in the mouse triangle, e.g., if the point in SSp-n
closest to the SSp-bfd border was labeled “red”, then the point in S1ULp closest to the S1BF border
will also be labeled “red”. We then assume that any point in a rat region corresponds to the
equally labeled point in the corresponding mouse region, with linear interpolation and
extrapolation between points (Fig. S2B ). This assumption transplants the existing prediction of
the topographical mapping from mouse to rat, resulting in the mapping depicted in Fig. S2D . We
scaled the mapping variance by a factor of 2 to account for the larger size of S1 in our rat flatmap
(160 flat units) than in the mouse flatmap ( 80 flat units).

Finally, we derived predictions for the relative distributions of synapse locations across cortical
layers for connections between subregions. Reimann et al. (2019) provided predictions for all
pairs of source and target regions in mouse, which we applied to the corresponding pairs of
subregions of rat nbS1. That is, we assumed these layer profiles generalize to rat, albeit using the
thicker cortical layers of rats (Fig S2E1 vs. E2).

These constraints were then used to build mid-range connections as described in (Reimann et al.,
2019 ).

4.1.8 Preparation of thalamic input data

As previously described in Markram et al. (2015) , we aimed to add synaptic connections from
thalamic regions to the circuit model. While they will ultimately serve as one of the controllable
inputs for the simulation of in vivo-like experiments, they can also be used to predict innervation
strengths in our anatomical model. Specifically, we modeled two such inputs: one ”bottom-up”
input with a core-type laminar profile, and one ”top-down” input with a matrix -type laminar
profile (Harris et al., 2019; Guo et al., 2020 ; Shepherd and Yamawaki, 2021 ). To create
biologically realistic thalamo-cortical projections with a meaningful distinction between coreand
matrix-type inputs, we needed data on the strengths of these pathways and the laminar profiles of
their synapses.

As in Markram et al. (2015) , we used data on cortical innervation by thalamic sources from
Meyer et al. (2010) , which yielded information on the depth profiles of thalamo-cortical
projections in barrel cortex. To extrapolate to non-barrel somatosensory cortex, we considered
data for the VPM-S1BF pathway to be representative of core-type inputs, and generalized it to all
nbS1 regions (Fig. S3A1 , left). Similarly, we considered data for the POm-S1BF pathway as
representative of matrix-type inputs (S3A2, left), and generalized it to all nbS1 regions. Since the
data is reported as absolute volumetric bouton density of thalamo-cortical axons, we were able to
derive the total number of synapses to place by assuming one synapse per bouton and summing
over the entire innervated volume. The depth profiles for both pathways featured two clearly
separated peaks. We digitized the depth profiles and split them into 10 bins per peak.
Furthermore, we applied a threshold of 0.01/µm3 below which values were set to 0 (Fig S3A ).

To constrain the innervation strength and targeting of individual thalamic axons, we used
morphological reconstructions of thalamo-cortical projection neurons from the Janelia
MouseLight project (mouselight.janelia.org ; Economo et al. (2016) ). This is a generalization of
mouse data to a rat model, made necessary by the lack of a comparable resource for rat. To

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 parameterize core-type projections, we calculated the total axonal length in somatosensory areas
of n = 11 reconstructions with somata in VPM and axons reaching the somatosensory areas. We
found lengths between 5 and 60 mm (Fig. S3 B ) with a median of 27 mm that we combined with
an assumed synapse density of 0.2/µmReimann et al. (2015) to get an average number of 5400
synapses per projection fiber. To estimate the lateral spread of the area innervated by individual
axons (the vertical component is covered by the layer profiles), we considered the locations of
reconstructed axon segments contained within the somatosensory regions. We then fit a Gaussian
to the lateral distance of the segments from their center (Fig. S3C ), resulting in a median value
of 120µm.

Unfortunately, the MouseLight database contained only a single neuron with its soma in the POm
region and its axon reaching somatosensory areas (labeled as AA604). Visual inspection revealed
that its axon mostly avoided these areas to target more medial motor-related regions. As such, to
parameterize the matrix-type projections, we instead calculated the lateral spread of the single
axon in the motor areas (300µm in MOp; 172µm in MOs; mean: 236µm). Its total length in cortical
regions was 28 mm.

4.1.9 Modeling the structure of thalamo-cortical innervation

We determined the dendritic locations of thalamo-cortical input synapses as previously described

in Markram et al. (2015) , but adapted for the more complex geometry of this model. Briefly,
binned depth profiles of densities of thalamo-cortical synapses were used as input (see Sec.
4.1.8 , Fig. S12A1, A2 ). Next, we used the region atlas (see Sec. 4.1.1 ) to find the
corresponding depth bins in the model. This allowed the identification of all dendritic sections in
the model whose center point fell within a depth bin. We then performed a random selection of
those sections and placed synapses at random locations on each selected section, until the
prescribed number of synapses for a given depth bin was reached (Fig. S12A2 ). Sections were
selected with probabilities proportional to their lengths and with replacement.

After all synapses for a thalamo-cortical projection had been placed, we mapped each of them to a
presynaptic thalamic neuron. These neurons were not fully modeled, i.e., they were not assigned a
soma position or morphology, and the mapping simply allowed us to determine which synapses
would be activated together. To parameterize the process, we used an estimate of the number of
synapses formed by a single fiber and its horizontal spread (see Sec. 4.1.8 ). We divided the total
number of synapses placed (590 · 106 core-type; 380 · 106 matrix-type) by the number per fiber to
estimate the number of innervating fibers (approximately 100’000 for the ”core”-type projection;
73’000 ”matrix”-type). These numbers were split between the eight subregions according to their
relative volumes (Table S5 ), with the following steps being executed separately for each of
them.

We then abstractly modeled thalamo-cortical afferent axons as lines entering their respective
subregion at the bottom of layer 6 with a certain horizontal reach for the formation of synapses.
This was done by first randomly distributing locations (one per fiber) across the boundary of
layers 4 and 5 (Fig. S12 B ) and then moving them 1500µm along the negative voxel orientation
(towards layer 6; Fig. S12 B , black dots). The resulting positions and orientation vectors were
used as the starting position of the fibers and their directions, respectively (Fig. S12 B , black
arrows). The presynaptic fiber of a synapse was then determined by a stochastic process based on
the horizontal reach of individual fibers around their respective location (Fig. S12 B , red areas).
This was parameterized as a Gaussian with a σ = 120µm for core-type, and σ = 235µm for
matrixtype projections (see Sec. 4.1.8 ). For each placed synapse, its distance to neighboring
fibers was calculated and used as inputs into the Gaussian (Fig. S12D ). This distance was
calculated as the distance between the location of the synapse and the line defined by the fiber’s
starting point and direction. The probability that any fiber was chosen as innervating fiber of the
synapse was then proportional to the values (Fig. S12E ).

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 4.1.10 Estimated volume of intrinsic mid-range axons

As the model did not contain mid-range axons, we estimated their volume based on mid-range
synapse counts instead. We first counted the number of mid-range synapes on dendrites inside the
volume of interest. We then converted the count into a volume by assuming that an axonal
segment with a length of 5.4 µm and a diameter of 0.21 µm supported each synapse. The length
was based on the inverse of the mean bouton density of excitatory axons in cortex (Reimann et al.,
2015 ); the diameter was the mean diameter of axons in the model. As this excludes parts of the
axon not forming boutons, this is a lower bound estimate.

4.1.11 Measuring local connection probabilities

Local connection probabilities of the model were measured by emulating a multi-patch clamp
sampling procedure as is often employed to measure connectivity in vitro. First, a pathway to
sample, i.e. pre- and post-synaptic neuron types, was selected and all neurons of non-participating
types were discarded. Of the remaining neurons, one was randomly selected as an initial seed.
Next, eleven additional neurons were selected by repeating the following procedure: Let S = s0, s1,
…, sn be the set of already sampled neurons and T the remaining neurons. We calculate for all
neurons ti ∈ T :

where ɸ refers to the standard normal distribution and D refers to the distance between two
neuron somata in two dimensions. If the sampled pathway was intralaminar, the first dimension
was orthogonal to the layer boundaries at the location of the first sampled neuron, and the second
dimension was orthogonal to the first at a randomly chosen angle. If the sampled pathway was
interlaminar, both dimensions were parallel to layer boundaries at the location of the first
sampled neuron. The value of σ was set to 38.82. Then, the next sampled neuron would be
randomly picked from T with probabilities proportional to their respective Wi.

This roughly approximates the mainly two-dimensional sampling in slices of brain tissue while
remaining comparable between intra- and interlaminar pathways. The value of σ was determined
to yield pairs at distances below 100µm with high probability. Specifically, this puts D = 100µm at
around 2.5 standard deviations or the 95th percentile of the normal distribution used.

4.1.12 Literature sources for connection probabilities

We used the data collected in “Figure 4 -1 , XLSX file” of Zhang et al. (2019) . Reported
connection probability values were classified into a pathway as follows. An initial classification of
source and target into excitatory or inhibitory was extracted from the column “Connection type”.
Next, source and target layers were determined based on the columns “Presynaptic Type” and
“Postsynaptic Type”. This was straightforward, as the values were consistently formatted to list the
layer first, followed by additional specification, e.g. “L4 FS IN”. Inhibitory types were further
broken down: If “Presynaptic Type” or “Postsynaptic Type” specified one of “PV” or “BC” it was
classified as “PV”; if it specified one of “SOM”, “MC” or “DBC” it was classified as “SST”; otherwise it
was classified as ”INH”.

For Figure S9B we used all sources where the value of “Species” was either “rat” or “mouse”.
For Figure S9C we additionally ensured that the value of “Age” was prescribed in postnatal days
and the reported interval contained P14. For Figure S9D only sources where the value of
“Species” was “rat” were used.

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 4.1.13 Connection probability of L5 PCs in the MICrONS data
We analyzed the “minnie65 public v117” release of the MICrONS data (https://www.microns-explorer
.org/cortical-mm3 ). We restricted the analysis to neurons where the proofreading status was
listed as “clean” or “extended”. We then considered neurons where the type of the “allen soma
coarse cell class model” was listed as ”5P IT”, ”5P PT” or ”5P NP”, resulting in 37 neurons. In that
group, 154 pairs were within 100µm. Of those, 13 were connected according to the “synapses pni
2” table of the data release.

4.1.14 Validation of synaptic connectivity

The part of the connectome derived with a apposition-based approach (local connectivity) was
constrained by anatomical data on bouton densities and mean numbers of synapses per
connection for different morphological types (Reimann et al., 2015 ). As such, we validated that
the results match these data (Fig. S10A, B ). As previously found, the only mismatch lied in the
emerging bouton densities of Chandelier Cells. These neurons form synapses only onto the axon
initial segment of other neurons, which we model by disregarding appositions on the dendrites.
For two Chandelier types, this resulted in an insufficient number of appositions to fulfill bouton
density constraints (Fig. S10A , black arrow).

Arguably the most important constraint is the number or density of excitatory synapses in
pathways between individual subregions, as it determines the overall excitability of the model and
the velocity of the spread of activity. Here, we compared the total number of excitatory synapses,
i.e., the union of the output of both algorithms to the data (Fig. S10C ), finding a robust
qualitative match. Due to the stochastic nature of the connectivity algorithms, an exact match
cannot be expected.

Finally, the mid-range connectivity algorithm was further constrained by predicted topographical
mapping between regions (Fig. S10D ) and synapse layer profiles (Sec. 4.1.7 E), which we
validated against the data.

4.1.15 Validation of thalamo-cortical innervation

In silico synapse density profiles of VPM and POm projections were validated against the in vitro
layer profiles from Meyer et al. (2010) that were used as the input recipes (Fig. S13A, B ). To
account for non-uniform thicknesses across regions, the depth values of the density profiles were
normalized to the maximum thickness of each of the eight subregions. There is a decent match
between the recipe and the actual layer profiles, but with a 15% overshoot at peak densities. This
can be explained by the fact that the numbers of synapses to be placed were computed based on
region bounding boxes which were larger than the actual volumes. Therefore, the actual densities
are slightly higher when computing them on a voxel basis as it was done in this validation.

4.1.16 Calculation of common thalamic innervation

For a given thalamo-cortical pathway we calculated the common thalamic innervation (CTI) as
follows for all neurons: First, for each neuron the set of thalamic fibers innervating it with at least
one synapse was identified. Next, we iterated over all pairs of neurons in the model, comparing
their sets of innervating fibers. Specifically, we calculated the size of the intersection of the sets
and divided it by the size of their union. The resulting CTI was a measure between 0 (no overlap)
and 1 (complete overlap).

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 4.1.17 Generation of columnar subvolumes
We partitioned the modeled volume into subvolumes with shapes approximating hexagonal
prisms. To that end, we represented each voxel by the flattened location of its center. In the
resulting two-dimensional coordinate system, we distributed seed points in a hexagonal grid with
a distance of 460µm (based on the 230µm column radius of Markram et al. (2015) ) between
neighboring points. Next, for each voxel we determined to which seed point it was closest. All
voxels that were closest to the same seed point were then grouped together as parts of the same
subvolume. As subvolumes near the periphery of the model could be incomplete, i.e., not form
complete hexagonal prisms, we excluded them from further analysis if either of the following two
conditions was met: 1. The subvolume had fewer than six neighbors in the hexagonal grid; 2. Its
volume was less than 0.166mm3. That threshold is based on the volume of a circular prism with a
radius of 230µm and a height of 1000µm.

4.1.18 Columnar volumes and their conicality

Columnar volumes were defined in a flat view of the model, generated as in (Bolaños-Puchet et al.,
2024 ; Bolaños-Puchet et al., 2023 ). In the two-dimensional coordinate system, we defined a
hexagonal grid with a radius (large diagonal of a hexagon) as indicated in the text. Then, each
voxel (38.7348µm resolution) was associated with the hexagon that contained the flat coordinates
of its central point. Thus, each hexagon defined a continuous columnar volume that sampled all
cortical layers. To calculate its conicality, we first found its central vertical axis (orthogonal to
layer boundaries). Then we conducted a linear fit of cortical depth against distance from the
vertical axis of all voxels contained in a column. The slope of the linear fit was defined as the
conicality measure.

4.1.19 Generation of random connectivity control models

This section contains definitions of the random connectivity control models and explains how
these were generated, presented in weakly increasing order of complexity.

The Erdős–Rényi controls (ER) are random directed networks where the edges are added with a
fixed probability independently at random. The controls were constructed by taking each ordered
pair of nodes (i, j) and adding an edge from i to j at random with probability , where

E (respectively N ) is the number of edges (respectively nodes) in the original network.

The stochastic block model controls (SBM) are random networks where the edges are added
independently at random with a fixed probability dependent on the m-type pathway of the pre-
and post-synaptic neurons. These controls were built as for the ER-controls, but with a different
probability for each pathway. More precisely, if (i, j) are two neurons of m-types A and B, then
their probability of connection is

where NABis the number of neurons of m-type A and B and EAB is the number of edges from
neurons of type A to neurons of type B in the original network.

The directed configuration model controls (CM) are random networks that closely approximate a
given in/out-degree sequence, i.e., a vector of length the number of nodes, whose entries are their
in/out-degrees. To build these controls, we encode the edges of the original matrix by two vectors,
sources and targets, such that (sources[i], targets[i]) is a directed edge of the matrix (this
corresponds to a binary matrix in coordinate format, useful when working with sparse matrices).

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 Then, we shuffle the entries of both vectors sources and targets independently, which gives a new
directed network, with the same degree sequence as the original. This new network might contain
loops or parallel edges, so we remove them, thus this construction only approximates the original
degree sequence. The density of loops and parallel edges tends to decrease as the number of nodes
increases (Newman, 2003 ), so the approximation is good. Indeed, in our controls for local
connectivity, 235, 574.0±376.0 (mean ± std) connections out of 2, 050, 028, 490 were missing, which
is less than 0.012% of all connections. For mid-range connectivity the number was 629, 493.4 ± 2,
228.0 out of 2, 482, 296, 102, which is less that 0.026% of all connections.

Finally, the distance block model controls (DBM) are random networks where the edges are added
independently at random with a probability , that is, exponentially
decreasing with distance d, where αA is the probability at distance zero and βA the decay constant,
both depending only on the pre-synaptic cell m-type A. For local connectivity, the distance between
a pair of neurons used was the Euclidean distance of their position in space (in µm). For mid-range
connectivity, the specific method used to originally construct the connectome (Reimann et al.,
2019 ) was taken into account, preferentially connecting neurons at one location within a
subregion A to neurons in another subregion B by first parameterizing linear transformations of
the flattened coordinates of the two regions. The resulting virtual coordinate system VA,B is then
used to connect pairs of neurons from A and B in a distance-dependent way. Therefore, the
distance considered in this control between pairs of neurons was the Euclidean distance between
these virtual coordinates.

For computational reasons, the model coefficients αA and βA were estimated by randomly sub-
sampling (up to) 100 000 pre-synaptic neurons of m-type A and 100 000 post-synaptic neurons of
any type and computing their pairwise distances. For local connectivity, distances from 0 to
1000µm were then divided into 20 evenly spaced 50µm bins. For mid-range connectivity, distances
from 0 to 400 (a.u.) were divided into 20 evenly spaced 20 (a.u.) bins. Connection probabilities
were estimated in each bin by dividing the number of existing connections by the number of
possible connections between all pairs of neurons within that bin (excluding connections at
distance zero, that is, between one neuron with itself). Model coefficients were then determined
by fitting the exponential probability function p(d) to these data points. The whole procedure was
repeated ten times with different random sub-samples of neurons, and the averaged model
coefficients over these ten estimates were used to build the controls. Overall, the relative standard
errors of the mean over the ten estimates were on average across all m-types less than 1%. For
local connectivity, model coefficients of all 60 pre-synaptic m-types were found to be on average
= 0.138 ± 0.102 (SD) and = 0.0096 ± 0.002 (SD) respectively. For mid-range connectivity, the
model coefficients of all 18 m-types that had any outgoing mid-range connections were on average
= 0.0006 ± 0.001 (SD) and = 0.012 ± 0.001 (SD) respectively.

4.1.20 Modularity
The modularity of a network is a metric that determines to which extent it is structured by
modules or communities. Intuitively, a network has high modularity if nodes within the modules
are densely interconnected, while nodes of different modules are sparsely interconnected.

Formally, consider a directed network with no loops or double edges. It can be represented by a
binary matrix A with zeros in the diagonal where the entry Aij is 1 if there is an edge from i to j
and 0 otherwise. The modularity of a partition C of the nodes of the network into modules is given
by:

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 where m is the number of edges i.e., the number of non-zero entries in A, Pij is the probability of
having an edge from i to j in a directed configuration model of A, and

Thus, the function QC contrasts the existence of an edge between two nodes in the same module
with the probability that it would appear by chance. This probability is given by

where are the in- and out-degrees of the nodes i and j respectively. The modularity of

the network QA is the maximal value attained by QC across all possible partitions of the nodes of A.
See Nicosia et al. (2009) for further details.

It has been shown that computing the modularity of a network is NP-complete (Brandes et al.,
2007 ), thus only heuristic approximations are possible. To approximate the modularity of each
of the columns in the parcellation described in Figure 7 , we used the Louvain algorithm for
directed networks (Dugué and Perez, 2015 ) implemented in Scikit-network (Bonald et al.,
2020 ). To control for the effect of size and density of the columns on their modularity we also
computed the modularity of corresponding ER-controls.

Note that the output of the Louvain algorithm is not deterministic. To further asses the its effect on
the modularity approximation we focused on the columns with high modularity values i.e., those
with modularity values higher than 0.4. For each of these columns, we recomputed the modularity
of a subnetwork obtained by subsampling 50% of the nodes at random. We computed the
modularity of the subnetwork with respect to two partitions: First, where the modules are
inherited from the modules determined by the Louvain algorithm in the full column, we called
these the original labels. Second, by re-running the Louvain algorithm from scratch on the
subcolumn and thus determining new modules of the subnetwork, we called these the new labels.
We repeated this procedure for 50 different random samples for each column and contrasted the
distribution of the modularity values obtained in both modalities as well as those obtained for the
full column.

4.1.21 Node participation and simplicial core

For any given node in a directed network, its node participation is the number of simplices that it
is part of. This value can be further split by dimension, giving rise to the notion of n-node
participation. Given that any directed simplex has a single source and a single target, node
participation can be further refined to participation as a source or participation as a sink. For n =
1, source node participation is equivalent to out-degree, sink node participation is equivalent to in-
degree and node participation is equivalent to the total degree (i.e., the sum of in- and outdegrees).
Thus, node participation can be thought of as a higher dimensional version of degree.

The n-simplicial core of a network is the sub-network on the nodes that participate in simplices of
dimension n or higher, or equivalently on the nodes whose n-node participation is greater than 0.
The simplicial core is the sub-network on the nodes that participate in simplices of maximal
dimension. These concepts are a generalization of the notion of core of a network, which is a
notion that is solely based on degree, by taking into account higher order interactions. We refer to
the (n-) simplicial core of the local/mid-range network as the (n-)local/mid-range core, and we refer
to the union of both as the core.

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 4.1.22 Finding and counting simplices in connectivity networks

Directed simplices were computed using the custom-made C++ package flagser-count. This code is
a variation of the flagser package (Lütgehetmann et al., 2020 ). The code takes as input the
adjacency matrix of a directed network in compressed sparse row (or column) format, outputs the
number of directed cliques in the network, and includes the option to print all simplices. There is
also the option to output node participation, so for every node v and every dimension d a value is
given for the number of d-simplices that contain v. The algorithm considers each node
independently as a source node and then performs a depth-first search on that node to find all
simplices, where at each step it creates a new simplex by adding any node that is an out-neighbor
of all nodes in the current simplex.

Since each source node is considered independently, the computations can be easily parallelized.
The simplex count computations were conducted on the Blue Brain high performance computing
system. Each computation was run on two Intel Xeon Gold 6248 CPUs using a total of 40 physical
cores. To compute the simplex counts for the local network took 3 hours and 15 minutes using
8.2GB of memory, the mid-range network took 14 hours and 44 minutes using 9.8GB of memory
and the combined network took 115 days and 20 hours using 69.5GB of memory. The transpose
adjacency matrices of the mid-range and combined networks were used, which have the same
simplex counts, because these were significantly faster to compute due to the degree distributions.
The computation for the combined network was attempted on the non-transposed matrix and ran
for 121 days, and computed partial counts, but encountered 898 nodes for which the counts could
not be computed within 24 hours, such nodes were deemed too computational expensive and
skipped.

4.1.23 Rich-club and generalized rich-club

The k-rich-club coefficient of an undirected network is the density of the subnetwork on the nodes
of degree greater than k, and is given by the formula

where N>kis the number of nodes of degree greater than k, and E>k is the number of edges
between the nodes in N>k. We call the function ɸ the rich-club curve. For a directed network we
obtain three variations of this curve depending on whether we consider in-degree, out-degree, or
total-degree (the sum of in- and out-degree), and we remove the coefficient 2 due to the fact that
the maximum number of edges is now N (N − 1) instead of N (N − 1)/2. This gives us three formulas

where (resp. ) is equivalent to N>k (resp. E>k) but restricted to the total

degree, and similarly for in and out.

The rich-club coefficient can be naturally generalized by replacing the degree with any network
metric (Opsahl et al., 2008 ). In particular, we define the simplicial rich-club coefficient by

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 where is the number of nodes with d-node participation at least k, and is the

number of edges between them. Note that ɸ1 = ɸtotal since a 1-simplex is simply an edge of the
network.

A large rich-club coefficient value is said to indicate a preference of high degree nodes to connect
together. However, it is shown in Colizza et al. (2006) that the rich-club coefficient is increasing
even in Erdős–Rényi networks, and is actually a consequence of higher degree nodes naturally
being more likely to connect. Therefore, it is important to normalize the rich-club coefficient by
dividing by the rich-club of an appropriate control, that is, a control with the same degree
distribution such as a configuration model, see Section 4.1.19 . Hence we define the normalized
rich-club coefficient as

where is the rich-club coefficient of a configuration model control of the original

network, and we similarly define ρin(k) and ρout(k). A network is said to have a rich-club effect if
ρtotal(k) > 1, as this indicates the high degree nodes connect together more than is “expected”. The
importance of this normalization can be seen in Figure S16 , where B1 seems to indicate the
presence of a rich-club effect for the mid-range network, which disappears when normalized in
B2.

Normalizing the simplicial rich-club is difficult as it requires a control model on directed flag
complexes that fixes node participation. Randomly sampling simplicial complexes with fixed
simplex counts or other high dimensional network properties is an active area of research (Kahle
et al., 2014 ; Young et al., 2017 ; Unger and Krebs, 2024 ), but as of yet there is no known
appropriate control model for the simplicial rich-club, and it is a topic that warrants further
investigation.

4.1.24 Paths and path distances

Let G be a directed network on n nodes, with adjacency matrix A. A path of length k (or k-path)
from node u to node v is a sequence of nodes u = x1, . . . , xk+1 = v such that (xi, xi+1) is an edge of G
for all i = 1, . . . , k. The path distance between u and v is the length of the shortest path from u to v.
A path from u to v is geodesic if its length is the path distance from u to v.

The number of k-paths between all nodes in G can be easily computed using matrix multiplication,
since the number of k-paths between u and v is given by , where A is the adjacency matrix

of G (Diestel, 2005 ). Consequently, the number of k-paths from the nodes i1, . . . , it to all other
nodes in G is given by

where A[i1 ,…,it] is the t × n matrix obtained from A by taking only the rows i1, . . . , it. Note that the
smallest k for which entry i, j is nonzero is the path distance from i to j.

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 Using this approach we were able to compute the path distances in the combined network
between pairs of all 396 nodes in the local and mid-range cores. We ran the computation in
parallel by partitioning the nodes into 13 sets C1, . . . , C13 of approximately 23 nodes each and
computing

Note that the order of the brackets ensures that at each step a 23 × 4234929 matrix is computed,
rather than a 4234929 × 4234929 matrix.

Once we had the path distances between the nodes in the local and mid-range cores, we computed
the geodesic paths between them. This was done using the custom C++ package pathfinder. This
code functions in a similar way to flagser-count, except that at each step it creates a new path by
considering any node that is an out-neighbor of the final node in the current path. At input the
start-nodes, end-nodes, and path distances can be specified. To compute all geodesic paths of
length at most three between nodes within the local and mid-range cores took 8 hours and 47
minutes, using 51.9GB of memory. Between these 396 nodes there are 6, 383 geodesic 1-paths, 1,
039, 814 geodesic 2-paths, and 21, 701, 345 geodesic 3-paths. To compute all geodesic 4-paths was
attempted, but after running for 24 hours failed to finish, and is likely to be computationally
infeasible due to the exponential growth of the number of paths.

4.1.25 Generation of EM-like volumes

We emulated the specificities of the electron microscopic studies of Motta et al., 2019 and
Schneider-Mizell et al., 2023 as follows. First, in Motta et al., 2019 the fragments of axons and
dendrites inside a volume are reconstructed as well as their synaptic connections. Then, axon and
dendrite fragments with at least 10 synapses inside the volume are considered for analysis. In the
model, we began by loading the three-dimensional locations of all synapses between neurons in a
100µm columnar volume. Next, we determined which synapses are contained in a box-shaped
subvolume at the top of layer 4 of the model. The size of the box was determined to contain the
same number of synapses (approximately 145’000) as the volume of Motta et al., 2019 . This was
achieved at 110x110x85µm, about twice as large as the reference volume, as it does not contain
synapses extrinsic to the model (also we do not know to what degree the dimensions of the
reference volume were corrected for shrinkage). We then determined the types of postsynaptic
compartments. Synapses onto somata and axon initial segments were labeled as such; of the rest,
if the soma of the targeted neuron was inside the box volume, it was tagged as proximal dendrite;
of the rest, if the targeted neuron was inhibitory, it was tagged as smooth dendrite; remaining
synapses onto apical dendrites were tagged as such; all others were tagged ”other”. We applied the
same filtering as the reference, removing axons, dendrites (and their synapses) with less than 10
synaptic contacts.

On the other hand, Schneider-Mizell et al., 2023 determined which neurons had their somata
inside a volume, and then reconstructed their entire dendritic and axonal trees, even parts outside
the volume. The volume sampled all layers and was approximately 100x100µm wide, but was not
exactly box-shaped, as it followed the main direction of contained apical dendrites. We emulated
this by defining a subvolume in flat mapped coordinates (Bolaños-Puchet et al., 2024 ; Bolaños-
Puchet et al., 2023 ): We considered all neurons whose flattened coordinates were inside a
100x100µm square. As the flat map flattens away the vertical dimension along which apical
dendrites are oriented in the model, this corresponded to the the sampling of Schneider-Mizell et
al., 2023 . Then, all synaptic connections between the sampled neurons were analyzed as in the
reference.

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 4.1.26 Binomial ”first hit” model

Control models to compare the distributions of postsynaptic compartments against were

generated as described in the reference (Motta et al., 2019 ). The binomial model assumes that
each synapse of an axon is formed with a fixed probability ptype onto a given postsynaptic
compartment type, hence the expected distribution of the number of synapses onto that type is a
binomial one. A binomial model is fitted for each combination of axon type (excitatory vs.
inhibitory) and postsynaptic compartment type as follows. For all axon fragments of the given
type, we consider the total number of synapses formed and whether it forms at least one synapse
onto the given compartment type. For a given axon fragment, the likelihood of the observation is
(1 − ptype)naxon if no synapse onto the type is observed and 1 − (1 − ptype)naxon otherwise, where
naxon is the number of synapses of the axon fragment in the volume. Then, ptype is estimated by
maximizing the joint likelihood of all observations, i.e. over all axons of the given type.

Acknowledgements
The authors would like to thank Giuseppe Chindemi, Javier DeFelipe and Rajnish Ranjan for help
with the scientific development of the model; Tristan Carel, James Dynes, Stefan Eilemann, Bruno
Magalhães, Juan Hernando Vieites and Arseny Povolotsky for contributions and support to the
engineering challenges; the BBP Core Services team for responding to IT requests and services
surrounding the research; Marwan Abdellah, Elvis Boci and Nadir Roman Guerrero for help with
visualizations of the model; Zoltán Kisvárday for supervision of morphology reconstruction
efforts; Eva Kenny, Silvia Scarabelli and Riccardo Sinsi for help with project management; and
Karin Holm, Akiko Sato and Georges Khazen for support of manuscript development and helpful
discussions.

Additional information

Author contributions

Conceptualization: H.M., E.B.M., S.R., M.W.R.

Data curation: Y.S., W.V.G., S.B.P., H.L., D.K., L.K.
Formal analysis: L.K., S.B.P., A.E., D.E.S., M.W.R.
Funding acquisition: H.M.
Investigation: D.E.S., M.W.R., J.P.S., M.S.
Methodology: E.B.M, S.R., L.K., W.V.G, S.B.P., A.R., D.E.S., N.N., M.W.R., K.H., J.L., R.L., J.P.S.,
D.K.
Project administration: H.M., S.R., F.S., E.B.M, A.R., M.W.R., K.H., R.L., S.L., J.P.
Resources: F.S., J.K., P.K., F.P., J.-D.C.
Software: A.A., H.D., V.S., S.B.P, A.E., M.W.R., H.L., J.L., J.P.S., F.D., A.De., J.K., P.K., F.P., M.W.,
B.C., J.-D.C., T.D., G.F., M.G., J.B.H., L.V., J.H., J.L.R.
Supervision: S.R., H.M., F.S., E.B.M, A.R., M.W.R., K.H., R.L., J.K., J.-D.C.
Validation: L.K., W.V.G, H.D., V.S., S.B.P, A.E., C.P., M.W.R., A.Di., M.G.
Visualization: L.K., S.B.P, A.E., D.E.S., J.L., C.P., M.W.R., C.F.
Writing - original draft: S.B.P, D.E.S., S.R., L.K., C.P., J.P.S., M.W.R., D.K., M.S.
Writing - review & editing: A.A., S.B.P., S.R., A.E., J.I., M.W.R., K.H., R.L.

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 Funding
This study was supported by funding to the Blue Brain Project, a research center of the École
polytechnique fédérale de Lausanne (EPFL), from the Swiss government’s ETH Board of the Swiss
Federal Institutes of Technology. RL, JPS and JL were supported by EPSRC under grant number
EP/P025072/1. RL was supported by a collaboration grant from EPFL. S.R. is supported by a Marie
Skl-odowska-Curie Global Fellowship Agreement 842492; Newcastle University Academic Track
(NUAcT) Fellowship; Fulbright Research Scholarship; Lister Institute Prize Fellowship; Academy of
Medical Sciences Springboard Award; Research grant from the Air Force Office of Scientific
Research (FA9550-23-1-0533); International Brain Research Organization (IBRO) Earlycareer
Award; Theoretical Sciences Visiting Program (TSVP) at the Okinawa Institute of Science and
Technology (OIST).

Declaration of interests
The authors declare no competing interests.

5 Supplementary Material

5.1 Results of individual validations

We began by validating that the cortical layers placed in the atlas formed a stack of continuous
volumes. To that end, we calculated the fraction of voxels in each layer that were directly adjacent
to another layer. As expected, adjacent voxels were found only between adjacent layers. Fractions
of adjacent voxels decreased in lower layers as they are generally thicker and due to the curved
geometry of the volume. Additionally, we tested that layers are spatially continuous by confirming
that for each voxel there was at least one neighboring voxel in the same layer. This was the case
for over 99.99% of the voxels with the handful of violating voxels limited to the periphery of the
modeled volume (not shown).

To validate the neuronal composition of the model, we then compared the densities of excitatory
and inhibitory neurons placed in the model against the input constraints. The vertical density
profiles matched the input robustly, with minor numerical differences resulting from the need to
round the number of neurons to place in a voxel to the nearest integer.

The biologically correct placement of neuronal morphologies was first validated by visual
inspection of renderings of neurons in the context of the atlas (Figure S8 ). More quantitatively,
we considered for each neuron its placement score that quantifies to what degree manually
identified morphological features reach the correct layers and neurites remain within the model
volume (see Figure S7 ; STAR*Methods). Specifically, we ensured that the fraction of neurons
with poor placement score remained below 1%.

Connectivity was validated in terms of the following aspects: We ensured that the mean bouton
density on axons matched biological reference data (Figure S10A ). A mismatch was only
observed for Chandelier Cells in layers 2/3 and 4. This is a consequence of the highly specific
connectivity of those m-types targeting only axon initial segments of postsynaptic neurons, which
is challenging to model based on axonal appositions. Further, we ensured that the mean number
of synapses per connection in m-type-specific pathways matches the biological reference (Figure
S10B ). For midrange connectivity, we ensured that the topographical mapping between
subregions and laminar profiles of synapse locations matches the specified references (Figure
S10D, E ). For the union of local and mid-range connectivity, we validated the overall density of

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 synaptic connections between subregions (Figure S10C ). Finally, for thalamic inputs we
validated their overall strengths and layer specificities by comparing the laminar profiles of their
synapse locations to reference data (Figure S13 ).

5.2 List of assumptions made in model building

Inherited assumptions

We assume that the published data sources and modelling steps (Table 1 ) are correct and
consequently inherit their assumptions. Below, we will only list the assumptions made on top of
that.

Data assumptions

We make a number assumptions related to generalizing input data

We assume the juvenile rat SSCx volume is a uniformly scaled-down version of the adult
SSCx.
We assume no significant variability between nbS1 regions in terms of: Cell density layer
profiles, relative layer thickness, neuronal morphologies, bouton densities, number of
synapses per connection in pathways.
We assume the relative strengths of connectivity and structure axonal targeting is
comparable in SSCx regions between mouse and rat.
We assume that the VPM to barrel cortex projection in adult rat is representative for a
general core-type, feedforward thalamic input in juvenile rat. Similarly, POm to barrel
cortex for matrix-type, feedback input.
We assume mouse thalamo-cortical projection axons are informative for the structure of
the rat thalamo-cortical projection system.

Structuring assumptions

We assume that grouping of cortical neurons into 60 morphological classes is also useful
for the description of the neuronal composition (cell density profiles) and the synaptic
connectivity between them.
We assume that the eight established subregions provide a parcellation that is useful for
the description of mid-range connectivity in the model.

Modeling assumption

We assume that purely vertical cell density profiles capture all relevant details of neuronal
composition.
We assume that the union of synaptic connections from two separate algorithms accurately
describes the connectivity at all scales that are relevant in the model. Specifically, we
assume there is no ”midrange gap” remaining between the two algorithms.
We assume that thalamo-cortical axons do not target specific classes of neurons, beyond
what is given by their layer profiles.
We assume the horizontal spread of thalamo-cortical axons can be captured by a Gaussian
profile for the synapses formed.

5.3 Supplementary Tables and Figures

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 Table S1

Morphological types (m-types) used in the model

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 Table S2

Predictions

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 Table S2 (continued)

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 Table S2 (continued)

Figure S1

Derivation of neuron density depth profiles. Left: A vertical profile of neuron densities, calculated from antibody stains of
neuronal nuclear protein (NeuN). From left to right: Neuron densities in each bin are split into individual morphological types
through antibody stains for various markers (blue boxes) and an established mapping of markers to types (green), or by
applying established neuronal compositions (red). Information in this flow diagram is illustrative, not quantitative.

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 Figure S2

Data sources for connectivity modeling.

A: Mapping from mouse to equivalent rat subregions. Left: flat view of mouse somatosensory subregions, Right: equivalent
flat view for rat. Corresponding subregions are indicated with matching colors. Triangles are drawn on top of each subregion
(shown for SSp-ul and S1FL) using manually annotated points that are assumed to correspond to each other. Inset: These
triangles define affine transformations between mouse and rat subregions. B: Transformed rat subregions (colored outlines)
are shown overlaid to their corresponding mouse subregions (colored areas). The topographical mapping of connections
between subregions predicted in Reimann et al. (2019) (black arrows) can thus be generalized to rat connectivity. C: Mean
connection densities between rat somatosensory subregions were derived from the Allen Mouse Brain Connectivity Atlas by
summing over corresponding mouse somatosensory subregions. Labels on the left side of the connection density matrix
describe the mapping applied from mouse to rat subregions. D: The topographical mapping of long-range connectivity was
parameterized as in (Reimann et al., 2019 ): For each pair of subregions, e.g. SS1FL and S1HL, affine transformations TS1FL
and TS1HL were defined that transformed the top-down views of the regions into a common coordinate system. Connectivity
was then distance-dependent in the common coordinate system. Transformations that were optimized for mouse data in
(Reimann et al., 2019 ) were adapted through the process in A and B for use in rat. This described connectivity where parts
of one subregion were predominately connecting to specific parts of another subregion. The plot indicates parts that are
preferentially connecting to each other in the same color. Sharp transitions at subregion boundaries are the result of
modeling connectivity separately for each pair of subregions in this iteration of the model. E: For the vertical dimension,
predicted layer profiles of connections between subregions of (Reimann et al., 2019 ) are generalized to rat. One example is
illustrated. E1, left: Exemplary slice through the SSp-ll region of the Allen Mouse Brain Atlas. Right: Layer profile of synapses
for a feedforward connection targeting layer 4. E2: Generalized version we used for rat. Right: Slice through the S1HL region
of the atlas. Left: Generalized layer profile, still targeting layer 4.

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 Figure S3

Input data for the derivation of the locations of thalamic inputs.

A1: Layer profile of bouton densities of VPM axons in the rat barrel field. Light red: Digitized from Meyer et al. (2010) . Dark
red: Binned into 20 bins per peak with a lower cutoff of 1/mm3 applied. A2: Same for POm axons. B: Total length of 11
reconstructed axons of Janelia MouseLight neurons with somas in VPM in various somatosensory subregions. Dashed line:
Median C: Standard deviation of a Gaussian fit of the reconstruction points of the same axons around their centroid in
somatosensory areas. Dashed line: Median

Table S3

Normalized depths used for layer boundaries

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 Figure S4

Topological comparison as in (Kanari et al., 2018 , 2019 ) of in vivo stained and in-vitro (i.e., from slices) axonal (A) and
dendritic (B) reconstructions of rat somatosensory cortex of pyramidal cells from layers 2-6. Top row presents the topological
profiles of in vivo stained reconstructions, second row presents the topological profiles of in slices stained reconstructions and
bottom row the difference between them (red: in vivo stained, blue: in slices stained). Number of cells per layer are reported
in individual pannels.

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 Figure S5

Comparison of representative reconstructructed morphologies that have been stained in slice (top) or in vivo (bottom). One
pyramidal cell morphology per layer is depicted.

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 Figure S6

Classification and morphometrics of excitatory morphologies.

A: Same as Figure 2A . B: Morphometrics of all morphologies belonging to the 18 excitatory m-types.

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 Table S4

Placement features used for modeling. The right column denotes the
vertical target interval by specifying a layer and a relative offset within
the layer, with 0.0 indication the bottom and 1.0 the top of the layer.

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 Figure S7

Scoring the placement of a neuron morphology for a voxel.

A: Neurite features, here the apical tuft, were manually given a vertical annotation interval (grey) and assigned a target
interval, expressed as a layer interval (red). (Note: parts of the apical dendrite visually shortened.) B: Then the placement of a
neuron morphology in a given voxel (blue) is scored as follows: Normalized depth values of the target interval are calculated
(red); the normalized depth of the voxel is looked up in an atlas (blue); it is used to calculate the normalized depth of the
annotation interval when the morphology is placed in the voxel (grey); a score is calculated as in (Markram et al., 2015 )
based on the overlap of the intervals.

Table S5

Number of thalamic fibers providing inputs to the model and each of its subregions.

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 Figure S8

Exemplary neurons in the model, one per m-type, rendered in the context of a slice spanning all cortical layers (grey borders).

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 Figure S9

Comparing local connection probabilities to the literature.

A: Local connection probabilities were measured by emulating a multi-patch clamp sampling procedure (see Methods).
Resulting relative locations of 1000 sampled pairs of excitatory neurons in layer 5 (L5EXC) are indicated. Black triangle
location of the potential presynaptic partner. Grey: Locations of unconnected neurons. Orange: Of connected neurons. Top:
In the x-y plane that is roughly parallel to layer boundaries. Bottom: In the x-z plane; the z-axis is roughly orthogonal to layer
boundaries. B: Violinplots of distributions of connection probabilities reported in 124 literature sources, gathered by Zhang et
al. (2019) . Colors of the violins indicate means of the distributions. Numerals indicate the number of literature sources
used. Arrowheads indicate the mean values measured in the model. They are black, if the value falls inside the distribution of
literature values; purple if they are outside, but within 25% of the variability; red if they outside that. Where only a single
literature source was available, 10% variability was assumed. Literature sources for mouse and rat were used. C: As B, but
only literature sources where the reported animal age range contained P14, the target age of the model. D: As B, but only
literature sources for rat were used.

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 Figure S10

Validation of modeled connectivity.

A, B: Touch connectivity is validated by comparing emerging bouton densities (A) and mean numbers of synapses per
connection (B) to the target values from the data. Each data point depicts a single morphological type (A) or type-specific
pathway (B). Black arrows: Bouton density for Chandelier Cells (ChC) in layers 2, 3 and 4. C: Mean densities of synapses from
the union of touch connectivity and mid-range connectivity in pathways within and between regions (C2) compared to that
target values from the data (C1). D: Structure of the topographical mapping of connections; each part of the model
predominately connects to neurons at equally colored locations. (D1) Target mapping from the data. (D2) Analyzed in the
union of touch connectivity and mid-range connectivity. E: Layer profiles of synapses placed in mid-range connections
between regions (blue bars) compared to the predicted target profiles from (Reimann et al. (2019) , pink lines). Depicted is
the density of synapses in a depth bin relative to the overall mean density over the entire depth.

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 Figure S11

Caption

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 Figure S12

Modeling thalamo-cortical synaptic inputs.

A: Input vertical profiles of thalamocortical synapses from core-type projections (A1) and matrix-type projections (A3). A2:
Exemplary model neuron with projection synapses placed on dendrites according to the prescribed densities. B: Modeling of
afferent thalamic fiber locations: Depicted is an exemplary slice of the model. Shaded vertical areas indicate the upper edges
of layers, with colors consistent with the rest of the manuscript (L1: yellow, L2: orange, L3: red, L4: pink, L5: blue, L6: green).
For each fiber a location at the border between layers 4 and 5 is randomly chosen (10 examples shown; white spheres). A
point 1500µm towards the bottom of layer 6 is chosen as the starting point of each fiber (black dots). A domain of influence is
then assigned around a line starting at that location with the indicated direction (black arrows and red areas). Influence
weakens with distance from the line with a Gaussian profile; for details, see D, E. C: Locations of fibers in the flat view. 10% of
the full density shown. Inset: Locations of approximately 1mm2 shown in full density. The green circle indicates a single
standard deviation of the Gaussian of influence strength used for the core style projections (i.e., 120µm). D: For an exemplary
synapse its distances to surrounding fibers is measured and the value of the Gaussian for those distances calculated. It is cut
off at two standard deviations. E: The probability that a given fiber is chosen as innervator of a synaptic location (blue bar) is
proportional to its value in D (shown only for fibers with nonzero probability).

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 Figure S13

Validation of thalamo-cortical density profiles.

Synapse density profiles of thalamocortical synapses from core-type (VPM) projections (A) and matrix-type (POm) projections
(B), compared with the target profiles from Meyer et al. (2010) (pink lines). Depicted are the mean densities (black lines: ±
SD) over voxels within depth bins relative to the respective total cortical thickness in each of the eight sub-regions.

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 Figure S14

Deviance and diversity of the morphological composition.

The morphological composition of a subvolume is a vector that specifies for reconstructed morphology how often it was
placed into the subvolume. A: For each layer and columnar subvolume the deviance of its morphological composition, a
measure of its difference to the global composition of the entire layer. It is measured as the negative logarithm of the p-value
of a chi-square test of the hypothesis that the composition of the layer/column intersection matches the composition of the
layer. B: For each layer and columnar subvolume the diversity of its morphological composition, a measure of the degree to
which many different morphologies were placed. It is measured as the binary entropy of the morphological vector. C: For all
morphologies of layer 5 neurons their cumulative frequencies. Grey: in layer 5 globally; orange: in the column with the
highest layer 5 diversity; blue: in the column with the lowest layer 5 diversity. Cumulative frequencies calculated after sorting
by frequency.

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 Figure S15

Modularity across sub-columns

A: Left: Heat map of modularity values of the networks of connections within each column as parcellated in Figure 7 .
Right: The same but for ER-controls of each column, thus fixing the number of nodes and connections but modelling random
connectivity. B: Modularity is strongly correlated to the volume of the column and the number of neurons in it. The
modularity values are much higher than those obtained in columns of the same size with random connectivity. Moreover, for
the controls modularity is in fact anti-correlated with volume and number of neurons. C: Modularity values of random
subnetworks of the columns with highest modularity (higher than 0.4). For each column, 50% of the neurons were sampled at
random 50 times, providing 50 subnetworks for each column. The modularity of these was computed in two ways. Blue
restricting the modules of the full column to the sample. Pink: Re-optimizing to find the modules that maximize the
modularity value (see Methods). The modularity distributions are virtually the same between the two methods and coincide
in their mean with the modularity value of the full column (purple cross).

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 Figure S16

Basic properties of network connectivity.

A:Top/bottom degree distributions of the local/mid-range networks and their corresponding control models. On the second
row additional inserts are provided showing the bi-modality of the out and total degree distributions of the SBM and DBM
controls. B: Matrices showing the probability of connection between given pre/post-synpatic m-types. Vertically at the right of
each matrix the probability of connection for a fixed pre-synaptic m-type. Horizontally on top of each matrix, the probability
of connection for a fixed post-synaptic m-type. C: Left and center, percentage of special nodes in both networks, sources
(nodes of in-degree 0) and proper sinks (nodes of out-degree 0 that are not isolated nodes). Right: layer distribution of the
special nodes of the mid-range network.

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 Figure S17

Rich club analysis

A: Depiction of simplices and non-simplices in various dimensions with distinction between the source and target nodes.
B:Rich-club analysis, top/bottom rows for the local/mid-range networks. Left column, rich-club curves for the original
networks as well as their CM controls. Top left additionally shows the rich-club curves of the DBM controls of the local-
networks as well as their corresponding CM controls. Middle column shows normalized rich club curves obtained by dividing
the rich-club curves for the original networks by the mean of the rich-club curves of their corresponding controls. Right
column shows the simplicial rich club curves for color-coded dimensions ≥ 2.

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Author information

Michael W Reimann*
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-3455-2367

For correspondence: [email protected]

*Co-lead authors

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 Sirio Bolaños-Puchet*
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-4049-6488

*Co-lead authors

Jean-Denis Courcol*
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-9351-1461

*Co-lead authors

Daniela Egas Santander*

Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0001-9838-7992

*
Co-lead authors

Alexis Arnaudon
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0001-9486-1458

Benoît Coste
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Fabien Delalondre
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Thomas Delemontex
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-3218-5137

Adrien Devresse
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-0071-3265

Hugo Dictus
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Alexander Dietz
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

András Ecker
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0001-9635-4169

Cyrille Favreau
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-1381-1585

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 Gianluca Ficarelli
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-2366-4008

Mike Gevaert
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-7547-3297

Joni Herttuainen
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0001-7301-2350

James B Isbister
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-1013-3013

Lida Kanari
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-9539-5070

Daniel Keller
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

James King
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-0906-8389

Pramod Kumbhar
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-1756-801X

Samuel Lapere
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-4207-2622

Jᾱnis Lazovskis
Riga Business School, Riga Technical University, Riga, Latvia
ORCID iD: 0000-0002-3285-7522

Huanxiang Lu
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-3377-9359

Nicolas Ninin
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Fernando Pereira
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Judit Planas
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Michael W Reimann et al., 2024 eLife. https://doi.org/10.7554/eLife.99688.2 76 of 95

 ORCID iD: 0000-0002-8221-7988

Christoph Pokorny
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-9771-2180

Juan Luis Riquelme

Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-4604-7405

Armando Romani
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0001-6388-4286

Ying Shi
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-6442-9880

Jason P Smith
Nottingham Trent University, Nottingham, UK
ORCID iD: 0000-0002-4209-1604

Vishal Sood
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

Mohit Srivastava
ELKH-University of Debrecen, Neuroscience Research Group, Debrecen, Hungary

Werner Van Geit

Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-2915-720X

Liesbeth Vanherpe
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-1226-0526

Matthias Wolf
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0002-6997-6330

Ran Levi‡
University of Aberdeen, Aberdeen, UK
ORCID iD: 0000-0001-5297-8295

‡
Co-senior authors

Kathryn Hess‡
Laboratory for Topology and Neuroscience (UPHESS), Brain Mind Institute, School of Life
Sciences, École polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland
ORCID iD: 0000-0003-2788-9754

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 ‡Co-senior authors

Felix Schürmann‡
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

‡Co-senior authors

Eilif B Muller‡
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0003-4309-8266

‡Co-senior authors

Henry Markram‡
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland
ORCID iD: 0000-0001-6164-2590

For correspondence: [email protected]

‡Co-senior authors

Srikanth Ramaswamy‡
Blue Brain Project, École polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland,
Neural Circuits Laboratory, Newcastle University, Newcastle, UK
ORCID iD: 0000-0001-6642-7136

For correspondence: [email protected]

‡Co-senior authors

Editors
Reviewing Editor
Richard Naud
University of Ottawa, Ottawa, Canada

Senior Editor
Panayiota Poirazi
FORTH Institute of Molecular Biology and Biotechnology, Heraklion, Greece

Reviewer #1 (Public review):

Summary:

In this study, the authors describe the construction of an extremely large-scale anatomical
model of juvenile rat somatosensory cortex (excluding the barrel region), which extends
earlier iterations of these models by expanding across multiple interconnected cortical areas.
The models are constructed in a way to maintain biological detail from a granular scale - for
example, individual cell morphologies are maintained, and synaptic connectivity is founded
on anatomical contacts. The authors use this model to investigate a variety of properties,
from cell-type specific targeting (where the model results are compared to findings from

Michael W Reimann et al., 2024 eLife. https://doi.org/10.7554/eLife.99688.2 78 of 95

 recent large-scale electron microscopy studies) to network metrics. The model is also
intended to serve as a platform and resource for the community by being a foundation for
simulations of neuronal circuit activity and for additional anatomical studies that rely on the
detailed knowledge of cellular identity and connectivity.

Strengths:

As the authors point out, the combination of scale and granularity of their model are what
make this study valuable and unique. The comparisons with recent electron microscopy
findings are some of the most compelling results presented in the study, showing that certain
connectivity patterns can arise directly from the anatomical configuration, while other
discrepancies highlight where more selective targeting rules (perhaps based on molecular
cues) are likely employed. They also describe intriguing effects of cortical thickness and
curvature on circuit connectivity and characterize the magnitude of those effects on different
cortical layers.

The detailed construction of the model is drawn on wide range of data sources (cellular and
synaptic density measures, neuronal morphologies, cellular composition measures, brain
geometry, etc.) that are integrated together; other data sources are used for comparison and
validation. This consolidation and comparison also represents a valuable contribution to the
overall understanding of the modeled system.

Weaknesses:

The scale of the model, which is a primary strength, also can carry some drawbacks. In order
to integrate all the diverse data sources together, many specific decisions must be made
about, for example, translating findings from different species or regions to the modeled
system, or deciding which aspects of the system can be assumed to be same and which should
vary. All these decisions will have effects on the predicted results from the model, which
could limit the types of conclusions that can be made (both by the others and by others in the
community who may wish to use the model for their own work). However, the public release
of the models and most of the associated tools does provide others a somewhat easier path to
modify and evaluate this iteration of the model for their own studies.

Overall, the model presented in this study represents an enormous amount of work and
stands as the basis for other work by the same group as well as a unique resource for the
community, even while acknowledging that it may be somewhat unwieldy for the community
to employ due to the weight of its manifold specific construction decisions, size, and
complexity.

https://doi.org/10.7554/eLife.99688.2.sa3

Reviewer #2 (Public review):

Summary:

The authors build a colossal anatomical model of juvenile rat non-barrel primary
somatosensory cortex, including inputs from the thalamus. This enhances past models by
incorporating information on the shape of the cortex and estimated densities of various types
of excitatory and inhibitory neuron across layers. This is intended to enable analysis of the
micro- and mesoscopic organisation of cortical connectivity and to be a base anatomical
model for large-scale simulations of physiology.

Strengths:

Michael W Reimann et al., 2024 eLife. https://doi.org/10.7554/eLife.99688.2 79 of 95

 • The authors incorporate many diverse data sources on morphology and connectivity.
• This paper takes on the challenging task of linking micro- and meso-scale connectivity
• By building in the shape of the cortex, the authors were able to link cortical geometry to
connectivity. In particular they make an unexpected prediction that cortical conicality affects
the modularity of local connectivity, which should be testable.
• The author's analysis of the model led to the interesting prediction that layer 5 neurons'
connect local modules, which may be testable in the future, and provide a basis to link from
detailed anatomy to functional computations.
• The visualisation of the anatomy in various forms is excellent
• The model is openly shared

Weaknesses:

• There is no effort to determine how specific or generalisable the findings here are to other
parts of cortex.
• Although there is a link to physiological modelling in another paper, there is no clear
pathway to go from this type of model to understanding how the specific function of the
modelled areas may emerge here (and not in other cortical areas).
• Some of the decisions seem a little ad-hoc, and the means to assess those decisions is not
always easily available to the reader
• The shape of the juvenile cortex - a key novelty of this work - was based on merely a scalar
reduction of the adult cortex. This is very surprising, and surely an oversimplification. Huge
efforts have gone into modelling the complex nonlinear development of cortex, by teams
including the developing Human Connectome Project. For such a fundamental aspect of this
work, why isn't it possible to reconstruct the shape of this relatively small part of juvenile rat
cortex?
• The same relative laminar depths are used for all subregions. This will have a large impact
on the model. However, relative laminar depths can change drastically across the cortex (see
e.g. many papers by Palomero-Gallagher, Zilles and colleagues). The authors should
incorporate the real laminar depths, or, failing that, show evidence to show that the laminar
depth differences across the subregions included in the model are negligible.
• The authors perform an affine mapping between mouse and rat cortex. This is again
surprising. In human imaging, affine mappings are insufficient to map between two
individual brains of the same species, and nonlinear transformations are instead used. That
an affine transformation should be considered sufficient to map between two different
species is then very surprising. For some models, this may be fine, but there is a supposed
emphasis here on biological precision in terms of anatomical location.
o Live nature of the model. This is such a colossal model, and effort, that I worry that it may
be quite difficult to update in light of new data. For example, how much person and compute
time would it take to update the model to account for different layer sizes across subregions?
Or to more precisely account for the shape of juvenile rat cortex?

https://doi.org/10.7554/eLife.99688.2.sa2

Reviewer #3 (Public review):

This manuscript reports a detailed model of the rat non-barrel somatosensory cortex,
consisting of 4.2 million morphologically and biophysically detailed neuron models, arranged
in space and connected according to highly sophisticated rules informed by diverse
experimental data. Due to its breadth and sophistication the model will undoubtedly be of
interest to the community, and the reporting of anatomical details of modeling in this paper is
important for understanding all the assumptions and procedures involved in constructing
the model. While a useful contribution to this field, the model and the manuscript could be

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 improved by employing data more directly and comparing simple features of the model's
connectivity - in particular, connection probabilities - with relevant experimental data.

The manuscript is overall well-written, but contains a substantial number of confusing or

unclear statements, and some important information is not provided.

Comments on revisions:

The authors mostly addressed all my points and improved the paper substantially. I do not
have further extensive comments except one general point below.

Regarding section 2.3 and metrics of connectivity like pairwise connection probabilities, it is
great that the authors rewrote that section and added comparisons with experimental data in
Figs. 4 and S9. Unfortunately, what one finds when direct comparisons are made is that the
modeled pairwise connectivity is quite different from the data. Fig. S9 shows that the model's
results do not agree with data in about half of the cases (purple and red arrows). Similarly
large discrepancies can be seen for some other metrics, like in Fig. S10B and S10C1,C2. (And
similar concerns apply to thalamocortical connections in section 2.5, where it looks like little
to no data are available to verify the pairwise connectivity between the thalamic and cortical
neurons via a direct comparison.)

This is concerning since this model forms the basis for multiple other studies of cortical
dynamics and function by the same group and potentially others in the community, with
multiple papers relying on it, whereas basic properties of connectivity are apparently not
captured well.

On the other hand, this is also a "glass half full" situation, showing that the sophisticated
algorithms for establishing connections, developed by the authors, are working well in at
least half of the connection types explored. It is therefore imperative that the authors
continue refining these algorithms to capture the remaining half in future iterations and
producing improved models that the community can better rely on.

Please also note that Fig. S11 does not have a caption.

https://doi.org/10.7554/eLife.99688.2.sa1

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this study, the authors describe the construction of an extremely large-scale

anatomical model of juvenile rat somatosensory cortex (excluding the barrel region),
which extends earlier iterations of these models by expanding across multiple
interconnected cortical areas. The models are constructed in such a way as to maintain
biological detail from a granular scale - for example, individual cell morphologies are
maintained, and synaptic connectivity is founded on anatomical contacts. The authors
use this model to investigate a variety of properties, from cell-type specific targeting
(where the model results are compared to findings from recent large-scale electron
microscopy studies) to network metrics. The model is also intended to serve as a platform
and resource for the community by being a foundation for simulations of neuronal

Michael W Reimann et al., 2024 eLife. https://doi.org/10.7554/eLife.99688.2 81 of 95

 circuit activity and for additional anatomical studies that rely on the detailed knowledge
of cellular identity and connectivity.

Strengths:

As the authors point out, the combination of scale and granularity of their model is what
makes this study valuable and unique. The comparisons with recent electron microscopy
findings are some of the most compelling results presented in the study, showing that
certain connectivity patterns can arise directly from the anatomical configuration, while
other discrepancies highlight where more selective targeting rules (perhaps based on
molecular cues) are likely employed. They also describe intriguing effects of cortical
thickness and curvature on circuit connectivity and characterize the magnitude of those
effects on different cortical layers.

The detailed construction of the model is drawn on a wide range of data sources (cellular
and synaptic density measures, neuronal morphologies, cellular composition measures,
brain geometry, etc.) that are integrated together; other data sources are used for
comparison and validation. This consolidation and comparison also represent a valuable
contribution to the overall understanding of the modeled system.

We thank the reviewer for the kind comments.

Weaknesses:

The scale of the model, which is a primary strength, also can carry some drawbacks. In
order to integrate all the diverse data sources together, many specific decisions must be
made about, for example, translating findings from different species or regions to the
modeled system, or deciding which aspects of the system can be assumed to be the same
and which should vary. All these decisions will have effects on the predicted results from
the model, which could limit the types of conclusions that can be made (both by the
others and by others in the community who may wish to use the model for their own
work).

We agree that this is a downside of the principle of biophysically detailed modeling that is
best addressed by continuous refinement in collaboration with the community. We would like
to once again invite any interested party to participate in this process.

As an example, while it is interesting that broad brain geometry has effects on network
structure (Figure 7), it is not clear how those effects are actually manifested. I am not
sure if some of the effects could be due to the way the model is constructed - perhaps
there may be limited sets of morphologies that fit into columns of particular thicknesses,
and those morphologies may have certain idiosyncrasies that could produce different
statistics of connectivities where they are heavily used. That may be true to biology, but it
may also be somewhat artifactual if, for example, the only neurons in the library that fit
into that particular part of the cortex differ from the typical neurons that are actually
found in that region (but may not have been part of the morphological sampling).

We agree that the limited pool of morphological reconstructions can lead to artifactual results
in the way the reviewer pointed out. To investigate that hypothesis, we added a
supplementary figure (S14) where we characterize (1): to what degree the morphological
composition of a columnar subvolume reflects the overall composition of the model; and (2):
The level of morphological diversity in each columnar subvolume. We discuss the results at
the end of section 2.6. Briefly, while we cannot fully rule out the possibility of an artificial
result, we found a high and virtually uniform level of morphological diversity in all columns
and layers. This makes it unlikely that individual idiosyncratic morphologies strongly affect

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 the local connectivity. However, we acknowledge that the minimum level of morphological
diversity required is unknown. We believe that at this stage all we can do is characterize this
and leave final interpretation to the reader.

I also wonder how much the assumption that the layers have the same relative
thicknesses everywhere in the cortex affects these findings, since layer thicknesses do in
fact vary across the cortex.

We agree that layer thickness variation would affect circuit properties. Variability of layer
thickness can be split into two components: variability stemming from differences in total
thickness, which our model covers, and variability of relative, i.e., normalized layer
thickness, which we miss. In this region of cortex, though, data on the relative thickness of
cortical layers is sparse. The Waxholm Atlas does not distinguish somatosensory cortical
layers in its labels [Kleven et al, 2023]. Yusufoğulları (2015) compares layer thicknesses of rat
hindlimb and barrel field regions. After normalization against total thickness, the relative
difference increased towards the superficial layers from 0 in L6 to 33% in L1. Variability of
normalized thicknesses within developed rat barrel cortex, based on layer boundaries
reported in Narayanan et al. (2017) vary by 2% to 5% over approximately 2 mm. One major
effect of such variability would be to scale the number of neurons in a given layer locally by
the corresponding factors. For comparison, the resulting variability in neuron counts due to
differences in conicality (Fig. 7D1) was around +-25%. A further effect of variable relative
layer thickness would be its impact on the selection of suitable morphologies to be placed in
the volume.

In summary, adjustment of layer thickness is a refinement which should be done in future

versions of the model, once more data is available. The discussion section has been updated
to acknowledge this limitation. However, as outlined at the beginning of this point-by-point
reply, we will not conduct such updates to the model in the context of this manuscript, as it
describes the version of the model used for a number of follow-up studies.

In addition, the complexity of the model means that some complicated analyses and
decisions are only presented in this manuscript with perhaps a single panel and not
much textual explanation. I find, for example, that the panels of Figure S2 seem to
abstract or simplify many details to the point where I am not clear about what they are
actually illustrating - how does Figure S2D represent the results of "the process
illustrated in B"? Why are there abrupt changes in connectivity at region borders (shown
as discontinuous colors), when dendrites and axons span those borders and so would
imply interconnectivity across the borders? What do the histograms in E1 and E2 portray,
and how are they related to each other?

We apologize for the confusion. We have updated the figure caption of Figure S2 to better
explain its contents.

Overall, the model presented in this study represents an enormous amount of work and
stands as a unique resource for the community, but also is made somewhat unwieldy for
the community to employ due to the weight of its manifold specific construction
decisions, size, and complexity.

Reviewer #2 (Public Review):

Summary:

The authors build a colossal anatomical model of juvenile rat non-barrel primary
somatosensory cortex, including inputs from the thalamus. This enhances past models by
incorporating information on the shape of the cortex and estimated densities of various

Michael W Reimann et al., 2024 eLife. https://doi.org/10.7554/eLife.99688.2 83 of 95

 types of excitatory and inhibitory neurons across layers. This is intended to enable an
analysis of the micro- and mesoscopic organisation of cortical connectivity and to be a
base anatomical model for large-scale simulations of physiology.

Strengths:

• The authors incorporate many diverse data sources on morphology and connectivity.

• This paper takes on the challenging task of linking micro- and mesoscale connectivity.

• By building in the shape of the cortex, the authors were able to link cortical geometry to
connectivity. In particular, they make an unexpected prediction that cortical conicality
affects the modularity of local connectivity, which should be testable.

• The author's analysis of the model led to the interesting prediction that layer 5 neurons
connect local modules, which may be testable in the future, and provide a basis to link
from detailed anatomy to functional computations.

• The visualisation of the anatomy in various forms is excellent.

• A subnetwork of the model is openly shared (but see question below).

We thank the reviewer for their kind comments.

Weaknesses:

• Why was non-barrel S1 of the juvenile rat cortex selected as the target for this huge
modelling effort? This is not explained.

We have added an explanation of this decision to the third paragraph of the introduction.

• There is no effort to determine how specific or generalisable the findings here are to
other parts of the cortex. Although there is a link to physiological modelling in another
paper, there is no clear pathway to go from this type of model to understand how the
specific function of the modelled areas may emerge here (and not in other cortical
areas).

With respect to generality against specific findings, our philosophy is as follows: Despite the
fact that most of our source data comes from juvenile rat somatosensory cortex, we also had
to generalize many data sources across organisms, ages or regions. Hence, in this iteration we
focused on investigating the general features of the (multi-region) mammalian cortex, e.g.,
high-order motifs, connected by L5 neurons across subregions or the effect of curvature on
the connectivity. In the future, more specific data sources can be used to build diverging
versions of the model, e.g. one for adult vs. juvenile rat. They can then be used to contrast the
ages and focus on more specific findings. We already defined a number of structural metrics
that can be used to contrast more specific versions of the model quantitatively.

We now clarify this pathway to understanding more specific function in the last paragraph of
the discussion.

• In a few places the manuscript could be improved by being more specific in the
language, for example:

- "our anatomy-based approach has been shown to be powerful", I would prefer instead
to read about specific contributions of past papers to the field, and how this builds on
them.

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 - similarly: "ensuring that the total number of synapses in a region-to-region pathway
matches biology." Biology here is a loose term and implies too much confidence in the
matching to some ground truth. Please instead describe the source of the data, including
the type of experiment.

We have removed or rewritten the mentioned parts. We now clarify that we work based on
biological estimates from experiments and cite the experiment sources. We also provide brief
descriptions of the types of data and how they were derived.

• Some of the decisions seem a little ad-hoc, and the means to assess those decisions are
not always available to the reader e.g.

- pg. 10. "Based on these results, we decided that the local connectome sufficed to model
connectivity within a region.". What is the basis for this decision? Can it be formalised?

- "In the remaining layers the results of the objective classification were used to validate
the class assignments of individual pyramidal cells. We found the objective classification
to match the expert classification closely (i.e., for 80-90% of the morphologies).
Consequently, we considered the expert classification to be sufficiently accurate to build
the model." The description of the validation is a little informal. How many experts were
there? What are their initials? Was inter-rater or intra-rater reliability assessed? What are
these numbers? The match with Kanari's classification accuracy should be reported
exactly. There are clearly experts among the author list, but we are all fallible without
good controls in place, and they should be more explicit about those controls here, in my
opinion.

- "Morphology selection was then performed as previously (Markram et al., 2015), that is,
a morphology was selected randomly from the top 10% scorers for a given position." A
lot of the decisions seem a little ad-hoc, without justification other than this group had
previously done the same thing. For example, why 10% here? Shouldn't this be based on
selecting from all of the reasonable morphologies?

We have clarified that the density of local connectivity is verified against the validation
datasets by comparing the diagonals in Figure 4B, in addition to the quantification of Figure
4C.

For the classification, we have now published a detailed preprint describing the objective
confirmation of expert classification by a variety of methods (see Kanari et al. 2024 https://
www.biorxiv.org/content/10.1101/2024.09.13.612635v1). We cannot include the full
methodology in the current paper, due to its large extent. For the benefit of the reader, we
have included the appropriate citation and extended the short description of the
methodology. As described in this paper, the classification accuracy varies per layer, cell type,
etc. We have now described in more details these results, that can be accessed in details in
out preprint.

• I would like to know if one of the key results relating to modularity and cortical
geometry can be further explored. In particular, there seem to be sharp changes in the
data at the end of the modelled cortical regions, which need to be explored or explained
further.

We now explore these results further in supplementary figure S15, which we discuss in the
results Section 2.6.

• The shape of the juvenile cortex - a key novelty of this work - was based on merely a
scalar reduction of the adult cortex. This is very surprising, and surely an

Michael W Reimann et al., 2024 eLife. https://doi.org/10.7554/eLife.99688.2 85 of 95

 oversimplification. Huge efforts have gone into modelling the complex nonlinear
development of the cortex, by teams including the developing Human Connectome
Project. For such a fundamental aspect of this work, why isn't it possible to reconstruct
the shape of this relatively small part of the juvenile rat cortex?

We agree that a more complex approach should be used in the future. However, as outlined
at the beginning of this point-by-point reply, we will not conduct such updates to the model in
the context of this manuscript, as it describes the version of the model used for a number of
follow-up studies.

• The same relative laminar depths are used for all subregions. This will have a large
impact on the model. However, relative laminar depths can change drastically across the
cortex (see e.g. many papers by Palomero-Gallagher, Zilles, and colleagues). The authors
should incorporate the real laminar depths, or, failing that, show evidence to show that
the laminar depth differences across the subregions included in the model are negligible.

This point has also been raised by reviewer #1 above. For convenience, we repeat our reply
below.

We agree that layer thickness variation would affect circuit properties. Variability of layer
thickness can be split into two components: variability stemming from differences in total
thickness, which our model covers, and variability of relative, i.e., normalized layer
thickness, which we miss. In this region of cortex, though, data on the relative thickness of
cortical layers is sparse. The Waxholm Atlas does not distinguish somatosensory cortical
layers in its labels [Kleven et al, 2023]. Yusufoğulları (2015) compares layer thicknesses of rat
hindlimb and barrel field regions. After normalization against total thickness, the relative
difference increased towards the superficial layers from 0 in L6 to 33% in L1. Variability of
normalized thicknesses within developed rat barrel cortex, based on layer boundaries
reported in Narayanan et al. (2017) vary by 2% to 5% over approximately 2 mm. One major
effect of such variability would be to scale the number of neurons in a given layer locally by
the corresponding factors. For comparison, the resulting variability in neuron counts due to
differences in conicality (Fig. 7D1) was around +-25%. A further effect of variable relative
layer thickness would be its impact on the selection of suitable morphologies to be placed in
the volume.

In summary, adjustment of layer thickness is a refinement which should be done in future

versions of the model, once more data is available. The discussion section has been updated
to acknowledge this limitation. However, as outlined at the beginning of this point-by-point
reply, we will not conduct such updates to the model in the context of this manuscript, as it
describes the version of the model used for a number of follow-up studies.

• The authors perform an affine mapping between mouse and rat cortex. This is again
surprising. In human imaging, affine mappings are insufficient to map between two
individual brains of the same species and nonlinear transformations are instead used.
That an affine transformation should be considered sufficient to map between two
different species is then very surprising. For some models, this may be fine, but there is a
supposed emphasis here on biological precision in terms of anatomical location.

We agree that this is a weakness that we will address in future revisions of the model.

• One of the most interesting conclusions, that the connectivity pattern observed is in
part due to cooperative synapse formation, is based on analyses that are unfortunately
not shown.

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 We originally decided not to show this part as we underestimated the interest in this
particular result. We have now included the result in supplementary figure S10 and discuss
the figure in the results.

• Open code:

- Why is only a subvolume available to the community?

We have now made the entire model available under doi.org/10.7910/DVN/HISHXN. The Data
and Code availability section has been updated to clarify this.

- Live nature of the model. This is such a colossal model, and effort, that I worry that it
may be quite difficult to update in light of new data. For example, how much person and
computer time would it take to update the model to account for different layer sizes
across subregions? Or to more precisely account for the shape of the juvenile rat cortex?

To provide more information to people interested in participating in model refinements, we

have added a new Figure 9. We discuss potential opportunities for refinement at the end of
the discussion section.

Reviewer #3 (Public Review):

This manuscript reports a detailed model of the rat non-barrel somatosensory cortex,
consisting of 4.2 million morphologically and biophysically detailed neuron models,
arranged in space and connected according to highly sophisticated rules informed by
diverse experimental data. Due to its breadth and sophistication, the model will
undoubtedly be of interest to the community, and the reporting of anatomical details of
modeling in this paper is important for understanding all the assumptions and
procedures involved in constructing the model. While a useful contribution to this field,
the model and the manuscript could be improved by employing data more directly and
comparing simple features of the model's connectivity - in particular, connection
probabilities - with relevant experimental data.

The manuscript is well-written overall but contains a substantial number of confusing or

unclear statements, and some important information is not provided.

Below, major concerns are listed, followed by more specific but still important issues.

Major issues

(1) Cortical connectivity.

Section 2.3, "Local, mid-range and extrinsic connectivity modeled separately", and Figure
4: I am confused about what is done here and why. The authors have target data for
connectivity (Figure 4B1). But then they use an apposition-based algorithm that results in
connectivity that is quite different from the data (Figure 4B2, C). They then use a
correction based on the data (Figure 4E) to arrive at a more realistic connectivity. Why
not set the connectivity based on the data right away then? That would seem like a more
straightforward approach.

We have completely re-written our description and discussion of connectivity in the model.
We now more explicitly motivate our connectivity modeling choices in the first paragraph of
section 2.3 of the results and in the second paragraph of the discussion.

The same comment applies to Section 2.4., "Specificity of axonal targeting": the
distributions of synapses on different types of target cell compartments were not well

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 captured by the original model based on axon-dendrite overlap and pruning, so the
authors introduced further pruning to match data specificity. While details of this
process and what worked and what didn't may be interesting to some, overall it is not
surprising, as it has been well known that cell types exhibit connectivity that is much
more specific than "Peters rule" or its simple variations. The question is, since one has the
data, why not use the data in the first place to set up the connectivity, instead of using
the convoluted process of employing axon-dendrite overlap followed by multiple
corrections?

We would like to point out that we are not employing “Peters rule”, we now make this explicit
in the revision in the first paragraph of section 2.3 of the results. Furthermore, we would
argue that the match to the Motta et al. data indicates that our approach is more than just a
“simple variation”. Finally, we believe that there is important insight in: 1. The specific ways
in which the algorithm had to be changed to match the Schneider-Mizell data, e.g. that the
connectivity of SST positive neurons did not have to be adapted at all. 2. That the specificity of
the other two types could still be matched by a selection of a subset of axonal appositions (i.e.,
of potential synapses).

Most importantly, what is missing from the whole paper is the characterization of
connection probabilities, at least for the local circuit within one area. Such connection
probabilities can be obtained from the data that the authors already use here, such as
the MICRONS dataset. Another good source of such data is Campagnola et al., Science,
2022. Both datasets are for mouse V1, but they provide a comprehensive
characterization across all cortical layers, thus offering a good benchmark for
comparison of the model with the data. It would be important for the authors to show
how connection probabilities realized in their model for different cell types compared to
these data.

We now report connection probabilities in the reworked figure 4 and compare them to
reported connection probabilities from many different sources and labs in supplementary
figure S8. We prefer a comparison to a wide range of sources to relying on a single report.

(2) Section 2.5, "Structure of thalamic inputs" and Figure 6.

The text in section 2.5 should provide more details on what was done - namely, that the
thalamic axons were generated based on the axon density profiles and then synapses
were established based on their overall with cortical dendrites. Figure S10 where the
target axon densities from data and the model axon densities are compared is not even
mentioned here. Now, Figure S10 only shows that the axon densities were generated in a
way that matches the data reasonably well. However, how can we know that it results in
connectivity that agrees with data? Are there data sources that can be used for that
purpose? For example, the authors show that in their model "the peaks of the mean
number of thalamic inputs per neuron occur at lower depths than the peaks of the
synaptic density". Is this prediction of the model consistent with any available data?

Most importantly, the authors should show how the different cell types in their model are
targeted by the thalamic inputs in each layer. Experimental studies have been done
suggesting specificity in targeting of interneuron types by thalamic axons, such as PV
cells being targeted strongly whereas SST and VIP cells being targeted less.

We have updated the Results section to provide context for the thalamic axon placement, and
referred the reader to the methods for more detail. A reference to Figure S10 has now been
added to this section as well.

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 As for validations of the structure of the thalamo-cortical inputs: We found that the existing
literature on the topic, such as Cruikshank et al., 2007, 2010 and more recently Sermet et al.,
2019, is predominately on the physiological strengths of the pathways. We acknowledge that
the authors provide compelling arguments that their findings are likely partially due to
differences in the anatomical innervation strengths. On the other hand, Sporns, 2013
cautioned against mixing up structural and functional connectivity. Overall, we believe that it
is simply cleaner to perform this validation in the accompanying manuscript (“Part II:
Physiology and Experimentation”), using the full physiological model. Note that we have
actually performed that validation in the manuscript (see preprint under the following doi:
10.1101/2023.05.17.541168, Figure 3H1).

Note that a higher physiological strength onto PV+ neurons is observed.

(3) "We have therefore made not only the model but also most of our tool chain openly
available to the public (Figure 1; step 7)."

In fact it is not the whole model that is made publicly available, but only about 5% of it
(211,000 out of 4,200,000 neurons). Also, why is "most" of the tool chain made openly
available, and not the whole tool chain?

We have now made the entire model available under doi.org/10.7910/DVN/HISHXN. This has
also been added to the Key resource table.

With regard to the tool chain, everything is on our public github (https://github.com
/BlueBrain/) except for the algorithm for detecting axonal appositions. For that tool there are
currently unresolved potential copyright issues with former collaboration partners. We are
working to resolve them.

Other issues

"At each soma location, a reconstruction of the corresponding m-type was chosen based
on the size and shape of its dendritic and axonal trees (Figure S6). Additionally, it was
rotated to according to the orientation towards the cortical surface at that point."

After this procedure, were cells additionally rotated around the white matter-pia axis? If
yes, then how much and randomly or not? If not, then why not? Such rotations would
seem important because otherwise additional order potentially not present in the real
cortex is introduced in the model affecting connectivity and possibly also in vivo
physiology (such as the dynamics of the extracellular electric field).

They are indeed additionally randomly rotated. We have clarified this in the revision.

The term "new in vivo reconstructions" for the 58 neurons used in this paper in addition
to "in vitro reconstructions" is a misnomer. It is not straightforward to see where the
procedure is described, but then one finds that the part of Methods that describes
experimental manipulations is mostly about that (so, a clearer pointer to that part of
Methods could be useful). However, the description in Methods makes it clear that it is
only labeling that is done in vivo; the microscopy and reconstruction are done
subsequently in vitro. I would recommend changing the terminology here, as it is
confusing. Also, can the authors show reconstructions of these neurons in the
supplementary figures? Is the reconstruction shown in Figure 4A representative?

The term is used because the staining is done in vivo. To the best of our knowledge, the
reconstruction process cannot be performed in vivo. However, to avoid any confusion we
modified the text to clarify this distinction to in-vivo stained.

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 With respect to the reconstruction in Figure 4: The intent of the panel is to demonstrate the
concept of targeted long-range axons that our morphologies are missing, necessitating the use
of a second algorithm for longer-range connectivity. As such, it is not one of the
reconstructions we used, but one of Janelia MouseLight. While we mentioned MouseLight in
the figure caption, we formulated it in a way that could be misunderstood to mean that we
merely used the MouseLight browser to render one of our morphologies. We apologize for
the confusion, and we have fixed the figure caption.

In this revision we have added exemplars of representative morphology reconstructions (in

slice stained and in vivo stained) in a new supplementary figure, as requested (Figure S5). It
is referenced in the last paragraph of section 2.1.

In the Discussion, "This was taken into account during the modeling of the anatomical
composition, e.g. by using three-dimensional, layer-specific neuron density profiles that
match biological measurements, and by ensuring the biologically correct orientation of
model neurons with respect to the orientation towards the cortical surface. As local
connectivity was derived from axo-dendritic appositions in the anatomical model, it was
strongly affected by these aspects.

However, this approach alone was insufficient at the large spatial scale of the model, as
it was limited to connections at distances below 1000μm."

As mentioned above, it is not clear that this approach was sufficient for local connectivity
either. It would be great if the authors showed a systematic comparison of local
connection probabilities between different cell types in their model with experimental
data and commented here in the Discussion about how well the model agrees with the
data.

As mentioned in the reply to a previous comment, we now report connection probabilities.

In the Discussion: "The combined connectome therefore captures important correlations

at that level, such as slender-tufted layer 5 PCs sending strong non-local cortico-cortical
connections, but thick-tufted layer 5 PCs not." (Also the corresponding findings in
Results.)

If I understand this statement correctly, it may not agree with biological data. See
analysis from MICRONS dataset in Bodor et al., https://www.biorxiv.org/content/10.1101
/2023.10.18.562531v1.

Our statement was indeed misleading and formulated too strongly. While thick-tufted
pyramidal cells do form long-range intra-cortical connections, the structural strength of these
pathways is weaker than for slender-tufted PCs, which are associated with the IT (intra-
telencephalic) projection type. We have made this clear in the revision.

Table 2 is confusing. What do pluses and minuses mean? What does it mean that some
entries have two pluses? This table is not mentioned anywhere else in the text. If pluses
mean some meaningful predictions of the model, then their distribution in the table
seems quite liberal and arbitrary. It is not clear to me that the model makes that many
predictions, especially for type-specificity and plasticity. Also, why is the hippocampus
mentioned in this table? I don't see anything about the hippocampus anywhere else in
the paper.

We have clarified the description of the table in its caption and removed references to
hippocampus, which were left from an earlier draft of the paper.

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 In the Discussion, "Thus, we made the tools to improve our model also openly available
(see Data and Code availability section)."

As mentioned before, the authors themselves write that they made "most of our tool
chain openly available to the public", but not all of it.

With regard to the tool chain, everything is on our public github (https://github.com
/BlueBrain/) except for the algorithm for detecting axonal appositions. For that tool there are
currently unresolved potential copyright issues with former collaboration partners. We are
working to resolve them.

Table S2 has multiple question marks. It is not clear whether the "predictions" listed in
that table are truly well-thought-out and/or whether experimental confirmations are
real.

Some of the citations in that table were broken due to technical difficulties with the citation
manager used. We apologize and have fixed this in the revision.

Introduction: It would be quite appropriate to cite here Einevoll et al., Neuron, 2019 ("The
Scientific Case for Brain Simulations").

We now reference this important work.

Recommendations for the authors:

Reviewing Editor's note:

Consultation with the reviewers highlighted three main issues: the integration of
connection probability profiles, non-uniform cortical thickness, and the overall
organization of the manuscript.

Reviewer #1 (Recommendations For The Authors):

Apart from the points discussed in the public review, my main concern is that the
manuscript itself is not as tightly constructed as it should be, to the detriment of the
reader's ability to understand the model itself and the conclusions from the presented
analyses.

There are places where the text references seemingly incorrect figure panels or refers to
panels that don't exist:

- Section 2.2, first paragraph - refers to Figure 2D, E but those panels do not exist in
Figure 2.

- Section 2.2, second paragraph - refers to Figure 3D3 - perhaps it should be 3B3?

- Section 2.8, first paragraph - has no figure references but seems like it should be
referring to parts of Figure 8 (perhaps Figure 8B1 specifically?)

- Is the reference to Figure S11A on page 16 supposed to be to S12A?

In other places, figure labels and descriptions are not clear, and terminology is not
always well-defined or explained.

- Figure 8 and the associated section 2.8 are very difficult to draw conclusions from as
presented - several of the terms used are opaque and not clearly defined in the text or
legends. I could not easily infer how the normalization works for the "normalized node

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 participation per layer", or what "position in simplex" means for "unique neurons in
core", and what their "relative counts" are relative to.

- Are "targets" in Figure S12A the same as "sinks"? If so, it would be better to use a single
term consistently throughout.

- Figure S12 - figures in part B do not have enough labels to interpret - what is the y-axis
of the "rich-club analysis" graph? Also, the figures in part B bottom are labeled "long-
range" rather than "mid-range" connections.

In general, I found the use of both letters and numbers for figure panels (e.g. Figure 7E1)
more confusing than helpful - it didn't seem like panels with the same letter were visually
grouped consistently, and it sometimes made it more difficult to follow the flow of a
figure. I would recommend using only letters in nearly every case here.

We thank the reviewer for directing our attention to these issues. We have fixed them in the
revision. However, we have decided to keep our original panel numbering scheme. Panels
with the same letter are meant to be conceptually grouped as they address related or similar
measures.

Other minor points:

- Section 2.4 - paragraph 2 - sentence 5 "inhbititory" -> "inhibitory".

- Figure 5B figure legend - references Schneider-Mizell et al. 2023 but probably should be
Motta et al. 2019?

- Figure 5C - figure key "expcected" -> "expected".

- The lower part of Figure 7C looks like it belongs to panel D2 instead of panel C due to
relative spacing.

We once again thank the reviewer, and we have fixed the listed issues.

Reviewer #2 (Recommendations For The Authors):

(1) Abstract:

- Is it really 'integrating whole brain-scale data'? This seems a bit misleading.

- "We delineated the limits of determining connectivity from anatomy" - here I think you
mean determining connectivity from morphology, or dendrite/axon appositions. Electron
microscopy is still anatomy and presumably would be much closer to function.

We originally used the term “anatomy” as connectivity depends on the correct placement of
neurons in addition to their morphology. However, as the reviewer points out, this term is
misleading as it would encompass electron microscopy, which can go beyond what we do
with the model. We have updated the text to read “morphology and placement”.

(2) Introduction:

"Investigating the multi-scale interactions that shape perception requires a model of

multiple cortical subregions with inter-region connectivity, but it also requires the
subcellular resolution provided by a morphologically detailed model." - This statement,
as written, is not true in my opinion. You can argue for the value of morphologically-
detailed neuron models to the study of perception, but they are not required for the
investigation of perception.

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 We have updated the text to be clearer: subcellular resolution is only required for certain
aspects that are related to perception.

(3) Results:

- Pg. 9/10. There are three sentences in a row that are of the style: "ensuring that the
total number of synapses in a region-to-region pathway matches biology." Biology here
is a loose term and implies too much confidence in the matching to some ground truth.
Please instead describe the source of the data, including the type of experiment here
already. o Pg. 10. On the first read, I found it quite hard to follow what exactly was done
in Figure 4.

What are the target values adapted from Reimann et al., 2019, for example?

- Pg. 10. "Based on these results, we decided that the local connectome sufficed to model
connectivity within a region.". What is the basis for this decision? Can it be formalised? o
Pg. 16, Figure 7 B-C. The apparent effect of geometry on modularity is potentially very
interesting. However, are the sharp drop-offs in values for modularity (but also conicality
and height) true, or are some artefacts due to columns at the edges of the sampled area?

We have discussed these points above in the general comments and strengths and
weaknesses.

- Pg. 18. Simplicial cores define central subnetworks, tied together by mid-range
connections. This work, in particular leading to the conclusion of the layer 5 highway
hubs, stands out as being a successful attempt to simplify the highly detailed model to a
degree that it generates useable new understanding.

We thank the reviewer for the kind comment.

(4) Figures:

Figure 2: The caption doesn't seem to match the Figure (e.g. there are no brain regions
depicted in A). o Figure 4f. This is a key panel, but is squished into a small corner of
Figure 4, and therefore hard-to-read.

We have fixed this in the revision.

Reviewer #3 (Recommendations For The Authors):

In Major comments, point (1) discusses the issue of connectivity known from data. For all
the aspects of connectivity mentioned there, I would recommend the authors re-build
their model using the connectivity data directly. It would be interesting to test whether a
model constructed in such a way would have any difference in simulated neural activity
relative to the model they have constructed.

This is indeed a very interesting avenue of research. However, we believe that it is best
conducted in separate manuscripts. First, in Pokorny et al., 2024 (https://doi.org/10.1101/2024
.05.24.593860) we conduct this investigation, comparing the emerging activity in the model to
the one for simpler connectivity models. Additionally, in Egas-Santander et al., 2024 (https://
www.biorxiv.org/content/10.1101/2024.03.15.585196v3) we found that simpler connectomes
lead to less reliable spiking activity globally. Finally, in the accompanying manuscript (https://
www.biorxiv.org/content/10.1101/2023.05.17.541168v5) we compare activity with and
without the targeting specificity of Schneider-Mizell et al.

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 In Major comments, point (2) discusses thalamic inputs. I would recommend the authors
to address the issues mentioned there.

We have replied to those comments above.

In addition, panels F and G of Figure 6 are mentioned in the caption but are not shown in
the figure. In panel B, the choice of visualization is strange. It would make sense to show
box plots for all the data instead of bars for mean values and points for randomly
selected 50 cells. Panels E1 and E2 lack units.

We have removed mentions of panels F and G and changed the style of plot. Units for E1 and
E2 are now explained in the figure caption.

In Major comments, point (3) touches upon model and tool sharing. I would recommend
making such statements more accurate and reflecting what exactly is provided to the
community since not everything is shared.

We have now made the entire model available under doi.org/10.7910/DVN/HISHXN.

With regard to the tool chain, everything is on our public github (https://github.com
/BlueBrain/) except for the algorithm for detecting axonal appositions. For that tool there are
currently unresolved potential copyright issues with former collaboration partners. We are
working to resolve them.

I would recommend the authors address all the other points mentioned in the public
review as well. In addition, below are some smaller issues that should be fixed.

Figure 2: the caption appears to be partially wrong and partially misassigned to the
figure panels.

We fixed the issue.

Also, note that in L6 the types L6_TPC:A and L6_TPC:C are listed in the figure, but L6_TPC:B
is not mentioned.

There is indeed no TPC:B type in layer 6. The distinction between TPC:A and TPC:B is based on
early or late bifurcations of the apical dendrite and is only observed in layer 5.

Figure 3, panel B2: the caption refers to colors in panel (C), but the authors probably
meant to refer to panel (A).

We fixed the issue.

"The placement of morphological reconstructions matched expectation, showing an

appropriately layered structure with only small parts of neurites leaving the modeled
volume (Figure 2D, E)."

Figure 2 does not have panels D and E.

"The volume was clearly dominated by dendrites, filling between 23% and 47% of the
space, compared to 2% to 11% for axons (Figure 3D3)." There is no panel D or D3 in
Figure 3.

"Recently, the MICrONS dataset (MICrONS-Consortium et al., 2021) has been analyzed
with respect to the axonal targeting of inhibitory subtypes in a 100 x 100 μm subvolume

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 spanning all layers (Schneider-Mizell et al., 2023)."

100 x 100 μm is an area (and should be 100 x 100 μm^2), not a volume.

Figure S11B requires a legend for the color map.

We fixed the issues.

Table S1: What is the difference between L6_BP and L6_BPC? They both are referred to as
L6 bipolar cells.

We have changed the description of L6_BPC to “Layer 6 bitufted pyramidal cell”.

https://doi.org/10.7554/eLife.99688.2.sa0

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