Zingg Et Al (2014) Neural Networks of The Mouse Neocortex

Resource
Neural Networks of the Mouse Neocortex

Brian Zingg,1,4 Houri Hintiryan,2,4 Lin Gou,2 Monica Y. Song,2 Maxwell Bay,2 Michael S. Bienkowski,2 Nicholas N. Foster,2
Seita Yamashita,2 Ian Bowman,2 Arthur W. Toga,2,3 and Hong-Wei Dong2,3,*
1Zilkha Neurogenetic Institute
2Institutefor Neuroimaging and Informatics
3Department of Neurology
Keck School of Medicine of USC, University of Southern California, Los Angeles, CA 90032, USA
4These authors contributed equally to this work
*Correspondence: Hongwei.Dong@loni.usc.edu
http://dx.doi.org/10.1016/j.cell.2014.02.023
SUMMARY mammalian species have been assembled on substantially

smaller scales and for specific functional systems (Felleman
Numerous studies have examined the neuronal and Van Essen, 1991; Saleem et al., 2008). For the cerebral cor-
inputs and outputs of many areas within the mamma- tex, a brain structure involved in regulating cognition, motivation,
lian cerebral cortex, but how these areas are orga- and emotion, it remains largely unclear how different areas
nized into neural networks that communicate across across the entire structure communicate at the network level
the entire cortex is unclear. Over 600 labeled neuronal to guide its complex functions. Recently, significant progress
has been made in assembling structural and functional cortical
pathways acquired from tracer injections placed
networks in the human brain using functional MRI and diffusion
across the entire mouse neocortex enabled us to
tensor imaging (DTI) with graph theoretical analysis (Andrews-
generate a cortical connectivity atlas. A total of 240 Hanna et al., 2010; Behrens and Sporns, 2012; Toga et al.,
intracortical connections were manually recon- 2012). These efforts have advanced our understanding of how
structed within a common neuroanatomic frame- neural network disruptions may be associated with neurological
work, forming a cortico-cortical connectivity map and neuropsychiatric diseases. Nevertheless, it is necessary
that facilitates comparison of connections from to validate these networks using reliable neural tract tracing
different cortical targets. Connectivity matrices were methods in animal models at a higher resolution, which will facil-
generated to provide an overview of all intracortical itate exploration of the molecular and cellular etiologies of these
connections and subnetwork clusterings. The con- disorders.
nectivity matrices and cortical map revealed that the As part of the effort to chart long-range connectivity in the
mouse brain (Marx, 2012; Osten and Margrie, 2013; Pollock
entire cortex is organized into four somatic sensori-
et al., 2014), we launched the Mouse Connectome Project
motor, two medial, and two lateral subnetworks that
(MCP, www.MouseConnectome.org). We generated a cortical
display unique topologies and can interact through connectivity atlas, which accommodates over 600 labeled
select cortical areas. Together, these data provide a neural pathways from tracer injections applied across the entire
resource that can be used to further investigate neocortex. Two hundred and forty pathways were then manually
cortical networks and their corresponding functions. reconstructed onto a common neuroanatomic frame to create an
online interactive cortico-cortical connectivity map to ease com-
INTRODUCTION parison of connectivity patterns across injections. We report the
development of this resource and identify three major cortical
Decades of research have converged on the idea that cognition subnetworks: the somatic sensorimotor, medial, and lateral
and behavior are network-level phenomena (Bressler and subnetworks, each of which displays unique network topologies.
Menon, 2010; Sporns, 2010; Swanson and Bota, 2010). The We also provide evidence for how these relatively segregated
expression of complex behaviors requires the integration of networks may interact through highly associative regions like
various sensory inputs, the synchronization of multiple motor the prefrontal cortex, entorhinal cortex, and the claustrum.
outputs, and the coordination of activity within large-scale net-
works that link the two. Therefore, constructing a brain-wide RESULTS
connectivity diagram for all well-defined gray matter regions,
i.e., the macro- or meso-connectome (Sporns et al., 2005; Boh- Data Production and Collection
land et al., 2009; Bota et al., 2012) that captures the organiza- The MCP neuronal connectivity data were produced using dou-
tional principles of neural networks will help inform a multitude ble coinjection tract tracing (Thompson and Swanson, 2010),
of testable hypotheses regarding the neural underpinnings of which simultaneously reveals four types of information for a
cognitive function and motivated behavior. given region (i.e., A): its (1) inputs (A)B), (2) outputs (A/B),
Unlike the recently assembled connectome of the C. elegans (3) reciprocal or recurrent connections (A5 B), and (4) interme-
(White et al., 1986; Jarrell et al., 2012), wiring diagrams for diate stations, which bridge brain structures that are not directly
1096 Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc.
connected (A/C/B). In one animal, two confined, nonoverlap- that all nodes fall into a few relatively distinct cortico-cortical
ping coinjections are placed into different brain regions (Figures subnetwork modules (Figures 2C–2E). Each of the four somatic
1A and 1B and Figure S1A available online). Each coinjection sensorimotor modules (orofaciopharyngeal, upper limb, lower
consists of one anterograde (Phaseolus vulgaris leucoagglutinin limb/trunk, and whisker) formed distinct subnetworks. The
[PHAL] or biotinylated dextran amine [BDA]) and one retrograde medial, visual, and auditory modules formed one big network
(cholera toxin subunit b [CTb] or Fluorogold [FG]) tracer. Antero- that was also highly connected with the lower limb/trunk and
grade tracers label axons arising from coinjection sites and their whisker subnetworks. The insular and temporal areas along
terminals in targeted regions and retrograde tracers label up- the lateral aspect of the cortex formed two distinct clusters
stream neurons that innervate the coinjection sites, thus simulta- and the broad and unique intracortical connections of the claus-
neously revealing four pathways (Figures 1A–1C and S1A). trum and lateral entorhinal area suggested they may serve as
The size of coinjections are 250–500 mm and mostly confined hubs or regions of high network interaction (Sporns, 2010).
within individual cortical areas (Figure 1B), although when im-
ages are adjusted to reveal fine fibers, injection sites are over- Cortico-Cortical Connectivity Map
exposed, misrepresenting their actual size (Figure S1A). The The matrices provide a condensed view of cortical connec-
confinement of the injections can be verified from the cytoarch- tivity patterns, but exclude details like projection routes,
itectural background provided by a Nissl stain of the same sec- laminar specificity of projections, and topographical and topo-
tion (Figures 1B and S1B) and by observing their unique thalamic logical connectivity patterns, which are critical features of
labeling (Figures S1A and S1B). The specificity of injections are networks. Consequently, labeled pathways were manually
cross-validated by the application of retrograde tracers to re- reconstructed onto corresponding atlas levels to create a
gions targeted by anterogradely labeled axon terminals and comprehensive cortico-cortical connectivity map available
vice versa (Figure S1 for details on data validation). All images through the iConnectome map viewer (Figures 1D, S2B, and
were processed through informatics pipelines and presented S4A; http://www.MouseConnectome.org/CorticalMap/).
on the MCP website through an interactive visualization tool, This map includes 80 anterograde (PHAL) and 160 retrograde
the iConnectome (www.MouseConnectome.org; Figure S2A). pathways (CTb and FG). The coinjection sites are represented by
The fluorescent connectivity data are presented in four different circles, PHAL pathways by shaded regions, and retrograde la-
channels: PHAL (green), BDA (red), FG (yellow), and CTb (pink). beling by small dots that reflect regional and laminar distribution
Two additional channels aid in data analysis: the inverted fluo- patterns (Figures 1D and S4A). Each of these pathways was as-
rescent Nissl of the same section and the corresponding atlas signed a unique RGB value and rendered into an individually
level from a standard mouse atlas, the Allen Reference Atlas layered document such that multiple layers representing multiple
(ARA; Dong, 2007). Currently, a total of 600 pathways (304 injection sites could be viewed simultaneously within the same
efferent and 296 afferent) associated with 317 coinjections anatomic frame (ARA), thus revealing topographic trends and in-
are available (Figure S3 for injections; Table S1 for list of selected teractions between regions. Within the connectivity map, when
cases; Table S2 for abbreviations). Injections span the entire nodes within the same module of the connectivity matrix are
neocortex and selected regions of the entorhinal cortex, hippo- viewed together (e.g., anteromedial and anterolateral visual
campus, amygdala, and olfactory areas. Although this report areas), intermixed and overlapping cortical connectivity patterns
focuses on intracortical pathways, the subcortical connections are observed, suggesting a high degree of integration within the
of all injections also are available. same subnetwork (Figures S4B and S4C). Conversely, nodes of
different modules show divergent cortical connections.
Connectivity Matrices
To begin building cortical networks, data within all regions was The Somatic Sensorimotor Subnetworks
annotated by manually recording the distribution of anterograde To build the somatic sensorimotor subnetworks, four general pri-
and retrograde labeling. Using the annotation data, two label- mary somatosensory (SSp) domains were defined based on their
based weighted directional connectivity matrices were created subcortical and intracortical connections (Figures 3A and 3B):
(Figures 2A–2D). Iterating through each matrix entry, 89% of the mouth and nose (SSp-m/n), upper limb (SSp-ul), lower limb
connections exhibited mutual labeling by both anterograde and trunk (SSp-ll/tr), and barrel field (SSp-bfd). The specificities
and retrograde labeling methods, suggesting that the data sets of these domains were validated by examining their specific
were significantly similar. To reveal subnetworks, a composite somatotopic projections in sensory and motor related nuclei
matrix was constructed in which the nodes (ROIs) were reor- in the lower brainstem (Figures S5A and S5B). Each of these
dered via a clustering algorithm (Figure 2E; Supplemental Infor- SSp domains displays unique connectional patterns with other
mation for annotation and analysis details). The clustering orders somatic sensorimotor areas like the primary (MOp) and second-
nodes into modules that maximize the connectivity and arrange ary (MOs) somatomotor areas, and the secondary somato-
highly interconnected nodes such that they are clustered along sensory area (SSs) (Figures 3A, 3B, and S5C). These distinct
the matrix diagonal, facilitating visualization of grouped regions connections provided a structural basis for delineating related
that can be considered subnetworks. The number of connec- subdomains within each of these sensorimotor areas, which
tions within these groupings near the diagonal reflects the den- are largely unknown in the mouse. Parcellations were confirmed
sity of intraconnectivity within a subnetwork, while connections by the application of coinjections into the corresponding body
farther from the diagonal demonstrate a subnetwork’s intercon- subfield domains of the somatic sensorimotor cortical areas
nectivity with other subnetworks. The clustering demonstrated (i.e., SSp-ll/tr, MOp-ll/tr, MOs-ll/tr; Figure 3C).
Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. 1097
Figure 1. Strategy for Generating the Cortical Connectivity Atlas
(A) Schematic illustrating a PHAL/CTb and BDA/FG double coinjection in two different structures labeling both input to, and output from each injection site.
Reciprocal interactions between brain regions and circuit interactions between each injection site may also be revealed.
(B) A coronal section showing coinjections made into the MOs and ACAv viewed with Nissl background to reveal cytoarchitecture. Scale bar, 1 mm.
(C) Intermixed anterogradely labeled axons (green, PHAL) and retrogradely labeled neurons (pink, CTb) in the MOs following a coinjection in the contralateral
hemisphere (first two panels, arrows). Note: the PHAL/CTb coinjection is the same as pictured in B. Image histogram was adjusted differently for the two
hemispheres so that PHAL/CTb labeling on the left side can be viewed properly without over exposing the injection site on the right side. Last panel, comparison
of retrogradely labeled neurons from injection in ACAv (arrow, yellow, Fluorogold [FG]) and fibers and cells from injection in MOs (PHAL/CTb). Fluorescent Nissl in
blue; scale bar, 200 mm.
(D) Strategy for mapping fluorescent labeling from a raw image (left, scale bar, 1 mm) onto the corresponding level of the ARA (middle) to generate a
comprehensive map of projection pathways for all injection sites (right). Note: anterogradely labeled pathways were rendered as layer and regional-specific
shading, while retrogradely labeled neurons were represented by individual dots. The large circle on the right hemisphere represents an injection site (see
corresponding region on raw image).
See Figures S1, S2, S4, and Table S1 for more information.
These somatic sensorimotor areas formed four distinct sub- region of the MOp (MOp-orf; Yamada et al., 2005), (3) the rostro-
networks. The orofaciopharyngeal subnetwork is composed of dorsolateral MOs (MOs-rdl), (4) anterolateral SSp-bfd (SSp-
five major nodes (Figures 2 and 3B): (1) SSp-m/n, (2) the orofacial bfd.al), and (5) the rostral and caudoventral SSs (SSs-r and cv).
(legend on next page)
Coinjections into each of these orofaciopharyngeal nodes critical area that integrates inputs from the VIS, AUD, and SSp-ll/
showed they are all heavily reciprocally interconnected (Fig- tr (Figures 2, 3B, and S6A). The RSPd and RSPagl receive much
ure 3D). The gustatory (GU), visceral (VISC), and dorsal agranular stronger visual inputs, but only sparse AUD and SSp projections
(AId) areas also connect with this subnetwork (Figures 2 and 3D), (Figures 2 and S6A). In the ACA, axons from different VIS and
which could contribute relevant information (i.e., gustation and AUD areas intermix in layer 1 (Figure S6A), while neurons that
food safety; Carleton et al., 2010; Maffei et al., 2012). project to the VISp and VISam form different clusters in deeper
Following the same topological organization (i.e., reciprocity layers (Figure 4E).
among all nodes), the upper limb subnetwork is composed of Moreover, the ORBvl and all of these higher-order association
four somatic sensorimotor nodes: (1) the SSp-ul, (2) caudodorsal areas (PTLp, RSP, ACA) heavily interconnect with regional and
SSs caudodorsal (SSs-cd), (3) MOp-ul, and (4) rostrodorsal MOs laminar specificity (Figures 4A–4C, 4F, and S6B). For example,
(MOs-rd) (Figures 3D and S5B). The lower limb/trunk subnetwork in the PTLp, the densest labeling from ORB injections are distrib-
includes the SSp-ll/tr, MOp-ll/tr, and rostrodorsomedial MOs uted in layer 5 (Figures 4B and 4C); ACAd axons are distributed
(MOs-rdm) (Figures 3B–3D). Finally, the whisker subnetwork is primarily in PTLp layers 1 and 6 and retrogradely labeled neurons
composed of the caudomedial SSp-bfd (SSp-bfd.cm), MOp-w, across layers 2 to 6 (Figure S6B). Projections from these areas to
which corresponds to the vibrissal primary motor cortex (vM1) lower-order sensory areas also are laminar specific with ORBvl
(Mao et al., 2011; Gerdjikov et al., 2013), and the caudodorsal axons primarily distributing in layers 1 and 5 of visual areas, while
SSs (SSs-cd; Figures 3B and 3D). The upper limb, lower limb/ ACAd axons reside in layers 1 and 6 (Figures 4C, 4D, and S6B).
trunk, and whisker subnetworks also share connections with Interestingly, this medial subnetwork provides an interface
lateral subnetwork nodes like the temporal association (TEa), for direct interactions between different sensory modalities via
perirhinal (PERI), and ectorhinal (ECT) areas. Relevant informa- reciprocal connections among the visual and auditory areas
tion for the lower limb/trunk subnetwork also may be provided and the SSp-ll/tr and SSp-bfd.cm (Figures 2, 3B, S6C, and
by inputs from the visual (VIS), auditory (AUD), and several areas S6D). This supports the concept of crossmodal modulation,
within the medial networks (Figures 2 and 3D). which challenges the idea that mammalian primary sensory
Finally, a specific frontal eye field MOs domain (MOs-fef, Fig- cortices are strictly unisensory (Driver and Noesselt, 2008; Stein
ure S5D; Reep et al., 1990) was identified, which shares dense and Stanford, 2008). Finally, this medial network may be impor-
reciprocal connections with the visual, auditory, posterior parie- tant for translating sensory information into motor action since
tal, anterior cingulate, and restrosplenial areas of the medial the ORBvl, ACAd, and PTLp connect with the entire MOs
subnetworks. including the MOs-fef and the ORBvl and PTLp further connect
with the MOp-ll/tr (Figures 4B, 4C, 4F, and S5D).
The Medial Subnetworks The second medial subnetwork successively relays informa-
Cortical areas along the medial bank of the cortex cluster to form tion from the dorsal subiculum (SUBd) to the medial prefrontal
two parallel medial subnetworks (Figures 2 and 4A). The first cortex (Figure S6E). The RSPv is the only neocortical recipient
medial subnetwork is organized for transferring visual, auditory, of dense inputs from the SUBd, through which information pro-
and somatic sensory information to the ORBvl. ORBvl coinjec- cessed in the dorsal hippocampus can reach the neocortex
tions showed its direct reciprocal connections with the primary (Fanselow and Dong, 2010). The RSPv shares massive recip-
(VISp) and secondary visual (anteromedial, VISam; anterolateral, rocal connections with the ACAv. It in turn projects to medial pre-
VISal) and auditory areas (AUD), as well as the SSp-ll/tr and SSp- frontal cortical areas like the infralimbic (ILA), prelimbic (PL), and
bfd.cm (Figures 2 and 4A–4C). Coinjections into each of these medial orbitofrontal areas (ORBm) (Figure S6E), each of which
areas confirmed these connections (Figure S4B–S4C for VISam receive only sparse inputs from the RSPv (Figure 4F).
and VISal; Figure S6A for VISp and AUD; Figure 3B for SSp-ll/tr). Importantly, the two medial networks can interact (Figure 4F)
Further, multiple retrograde tracers injected into different visual since the RSPv and ACAv are connected with the ORBvl,
areas revealed dense ORBvl neuronal labeling that was inter- ACAd, RSPd, RSPagl, and PTLp. Finally, these medial subnet-
mixed but mostly not colocalized, suggesting multiple parallel work areas are all connected with the CLA (Figures 2, 4B, 4F,
ORBvl/VIS pathways (Figures 4E–4F). and S6B).
These VIS and AUD areas also reciprocally connect with areas
along the cortical medial bank, including the posterior parietal The Lateral Subnetworks
(PTLp), three subdivisions of the retrosplenial area (dorsal, Cortical areas in the lateral aspect of the neocortex form two
RSPd; agranular, RSPagl; and ventral, RSPv), and two subdivi- distinctive, highly interconnected networks (Figures 2 and 5A):
sions of the anterior cingulate area (dorsal, ACAd; ventral, the anterolateral insular and posterolateral temporal subnet-
ACAv) (Figures 2, 4A–4C, 4F, S6A, and S6B). The PTLp is another works. Distinguishing the lateral networks from the medial and
Figure 2. Weighted and Directed Cortico-Cortical Connectivity Matrices

(A–E) Connectivity matrices were constructed based on either anterograde (PHAL, A) or retrograde (FG/CTb, B) tract tracing data. In both matrices, connection
origin is listed along the row while targets are listed across the columns (sorted alphabetically). The weighting of each connection is indicated by red (strong),
orange (moderate), and yellow (light) coloring. In (C) and (D), the anatomical data in (A) and (B) has been reordered, illustrating a total of 12 distinct modules in
different cortical subnetworks. Combining retrograde and anterograde tracing methods formed the composite matrix (E), a consensus perspective of cortico-
cortical subnetwork connectivity. See Extended Experimental Procedures for details regarding construction of the matrices, Figure S3 for injection cases, and
Table S2 for list of abbreviations.
somatic sensorimotor subnetworks is the fact that the lateral Three areas located in the vicinity of the rhinal fissure, namely
subnetworks share connections with olfactory cortical areas the temporal association area (TEa), ECT, and PERI form the
(piriform cortex, endopiriform nucleus, dorsal taenia tecta), ba- highly interconnected posterolateral temporal subnetwork (Fig-
solateral amygdalar nucleus, and ventral hippocampus. ures 2, 5A, and 5C). A retrograde tracer injection in all three struc-
The anterolateral insular subnetwork is composed of the three tures back labeled neurons in layers 2 and 5a of the entire
agranular insular areas: the dorsal (AId), ventral (AIv), and poste- neocortex with the exception of the ACAv, RSP, and VISp (Fig-
rior (AIp). The neural connections of the rat AI have been investi- ures 5C and 5D), suggesting that this subnetwork receives input
gated (e.g., Saper, 1982; Allen et al., 1991; Jasmin et al., 2004), from nearly the entire neocortex. Complementarily, the TEa,
but their functional and structural differences remains controver- ECT, and PERI collectively project back to nearly the entire
sial. Our data show that these subdivisions are substantially in- neocortical mantle including the VISp (Figures 5C and 5E).
terconnected, but generate distinguishable cortical projections Injections in all other cortical areas revealed heterogeneous
(Figure 2). First, connections between these three areas and zones within the TEa (Figure S7B). The rostral TEa shares bidi-
the medial prefrontal cortex display a rough topography (Figures rectional connections with areas in the orofaciopharyngeal
5B and S1E): the AIp is heavily connected with the poorly defined network; the middle TEa and its adjacent PERI and ECT share
dorsal peduncular area (DP) (all layers) and its dorsally adjacent stronger connectivity with somatic sensorimotor areas of the
ILA (layers 1 and 6) but relatively sparsely connected with the PL. upper limb and lower limb/trunk subnetworks and visual and
The AIv has the densest reciprocal connections with the PL, ILA, auditory areas, as well as the PTLp; the caudal TEa more specif-
and DP. The AId is interconnected with the PL but sparsely con- ically connects with the medial prefrontal areas and ventral
nected with the ILA and not at all with the DP. An ILA coinjection hippocampus.
validated this preferential interaction between AIv and ILA (Fig- Finally, coinjections in the TEa, PERI, and ECT result in dense
ure S1E). Second, all three AI areas connect with the VISC, but labeling in layers 3-5 of the ENTl (Figure 5C).
only AId and AIp are significantly connected with the GU. The
VISC receives visceral inputs from the parvicellular part of the ENTl and CLA
ventral posterolateral thalamic nucleus (VPLpc), while the GU The ENTl and CLA are two reciprocally connected structures
shares massive reciprocal connections with the parvicellular that both share connections with the medial prefrontal (ILA,
part of the ventral posteromedial thalamic nucleus (VPMpc; Fig- PL, ORBm) and orbitofrontal areas (ORBvl, ORBl) (Figures 2,
ure S1B) (Jones, 2007). Third, the AId also is connected with the 6A, 6E, and 6G). The CLA further shares massive connections
orofaciopharyngeal (MOp-orf and MOs-rdl) and upper limb with cortical areas within the medial (ACAv, ACAd, PTLp,
(MOs-rd) subnetworks, while the AIp receives significant inputs RSPd, RSPagl) and lateral subnetworks (AId, AIv, AIp, TEa,
from the SSs and SSp-bfd (Figure S7A). PERI, ECT), as well as with the entire MOs and MOp (Figures
Caudally, these AI subdivisions preferentially connect with 6A–6D). Dense CLA axons travel through layers 5 and 6 of all
three components of the posterolateral temporal subnetwork sensory areas (SSp, AUD, VIS), although these cortical areas
(Figure 5B): the perirhinal (PERI) receives dense inputs from the contain relatively few neurons that project back to the CLA.
AId and AIv, while the ectorihinal (ECT) is more heavily innervated The intracortical connectivity of the CLA displays a unique asym-
by inputs from the AIp. Finally, all three generate dense inputs metric pattern: cortical inputs to the CLA are bilateral, but out-
specifically to layers 3–5 of the lateral entorhinal cortex (ENTl; puts from CLA are almost exclusively ipsilateral (Figures 6B
Figures 5B and S1B). and 6C).
Figure 3. The Somatic Sensorimotor Subnetworks

(A) Overview of the four major components of somatic sensorimotor areas (SSp, SSs, MOp, MOs). Each region is extensively interconnected with all others.
Parcellation of cortical areas in map based on ARA and drawn to scale. Diamond shape on midline indicates bregma.
(B) Projections from representative injection sites (colored dots) in each of four basic body representations in primary somatosensory cortex: orofaciopharyngeal
(orf, blue), upper limb (ul, green), lower limb and trunk (ll/tr, red), and whisker-related caudomedial barrel field (bfd.cm, yellow). A cartoon (inset) shows
approximate size and location of the four body areas defined here (inspired from Brecht et al., 2004). Top-down view (left) shows topographic organization of
projections from each area to corresponding primary and secondary motor areas (MOp, MOs). ARA defined boundary between MOs and MOp added for
reference. Projection data in top-down view were drawn to scale using coronal sections (right) and shaded regions represent the areal extent of the most dense
projections from each of the injected regions. Representative coronal sections also show projection trends to supplemental somatosensory area (SSs). Numbers
indicate position of sections relative to bregma (mm).
(C) Projections from each of the somatosensory subregions define presumably functionally related MOs and MOp subregions, which are tightly reciprocated, as
indicated by closely overlapped axonal fibers and retrogradely labeled cell bodies following coinjection. Here, coinjections of PHAL (green)/CTb (pink) in either
SSp-ll (top, right) or a corresponding MOp region (middle, middle) reveal intermixed labeling in other corresponding domains of the somatic sensorimotor region,
confirming their strong reciprocal connectivity. Both coinjections reveal intermixed labeling in the same MOs domain (left images on the top and middle).
Retrograde injection in the same MOs domain (left image on the bottom) confirms the specificity of this interaction, showing retrogradely labeled neurons in the
former two areas (middle and right images). Their anatomical locations and interactions are summarized in corresponding atlas levels in the bottom panel. Scale
bars, 500 mm and 100 mm (inset).
(D) Building on these observations, four network graphs were created using each of the defined somatosensory regions as starting points. Each subnetwork is
distinct and all components within it share a high degree of interconnection. Each are composed of several somatic sensorimotor ‘‘nodes’’ (color coded to match
anatomically defined functional domains in (B) that are reciprocally connected (as indicated with red arrows). Each of these subnetworks also includes other
nonsomatic ‘‘peripheral’’ nodes (gray circles) and their connections are shown with gray arrows.
See also Figure S5. For abbreviations of nomenclatures, please see Figure 2 and Table S2.
The ENTl shares much stronger reciprocal connections with rives at the medial prefrontal zone via two routes (Figure 7A).
all areas of the lateral subnetworks (Figures 5A, 6C and 6F), The dorsal route links the dorsomedial corner of the prefrontal
amygdala (basomedial and anterior basolateral nuclei), and the cortex (PL, ACAd) with the ventrolaterally located AId. The axons
ventral and intermediate CA1 and SUBd (data not shown). The through this route make a 45 cut that demarcates the border be-
ENTl receives direct inputs from the main olfactory bulb (Hin- tween the dorsolateral and ventromedial halves of the prefrontal
tiryan et al., 2012) and shares massive reciprocal connections cortex. The ventral route links the medial (ILA) and lateral (AIv)
with olfactory cortical areas like the piriform and taenia tecta areas across the orbitofrontal areas.
(Figure 6E). These data suggest that the ENTl is not only a Overall, most components of the medial subnetwork commu-
gateway for neocortical information to the hippocampus (de nicate with the orbitofrontal zone. The ORBvl and ORBl are
Curtis and Paré, 2004), but may also be a site of interaction for targets of projections from the VIS, AUD, and PTLp. The orbito-
various cortical areas and between these neocortical areas frontal zone also receives input from the TEa (lateral subnet-
and the amygdala, hippocampus, and olfactory cortical areas. work), suggesting that, like the medial prefrontal cortex, it may
Compared to the CLA, the ENTl receives very sparse or no also serve as a site of integration for the medial and lateral
direct inputs from regions within the medial subnetworks (ACA, subnetworks.
RSP, PTLp) and somatic sensorimotor subnetworks; however, Aside from the strong interaction between the medial prefron-
coinjections into layers 4/5 of the rostrodorsal ENTl revealed tal and insular zones, very little interaction is observed among
dense axons throughout layer 1 of almost the entire neocortex the other neighboring structures of the prefrontal cortex. For
(Figure 6E) with a few exceptions—the RSPv and areas within example, coinjections in the ORBvl show no projections to or
the orofaciopharyngeal subnetwork. Notably, the ENTl layer 1 from the PFCdl and medial prefrontal areas. The relative segre-
axons are denser in the contralateral visual, auditory, and SSp- gation of these prefrontal zones combined with their specific
bfd areas. cortical inputs and subcortical targets may help define their
unique processing role and contribution to behavior.
Interactions with the Prefrontal Cortex
Projections from each of the identified subnetworks topograph- DISCUSSION
ically converge onto discrete regions of the prefrontal cortex.
The somatic sensorimotor subnetworks primarily converge The iConnectome: An Open Resource of Multiformat
onto the dorsolateral, dorsal, and dorsomedial sectors of the Connectivity Data
rostral-most MOs (Figures 3B, 7A, and 7B). Together, these three Open resources providing access to neurohistological images
neighboring zones occupy the dorsolateral half of the prefrontal are revolutionizing neuroanatomy (Jones et al., 2011). The
cortex (PFCdl, Figures 7A and 7B). iConnectome is an online resource that presents high-resolution
The ventromedial half of the prefrontal cortex (PFCvm) is also whole-brain images of neural connectivity in several different
composed of three distinct zones: the medial prefrontal (ILA, PL, formats. Imaging data are presented in which labeled axonal
ACAd, ORBm), orbitofrontal (ORBvl, ORBl), and the anterior- pathways and neurons can be viewed with their own Nissl
most part of the agranular insular areas (AId, AIv). The medial background or their corresponding anatomic ARA map. Cor-
prefrontal zone reciprocally connects with the AI and caudal tico-cortical connectivity matrices (Figure 2) provide an overview
TEa of the lateral subnetworks potentially acting as a site for of intracortical connections. Cortical connectivity matrices that
medial and lateral subnetwork integration. This information ar- comprise virtually all of the neocortex have been generated in
Figure 4. The Medial Subnetworks

(A) Major components of the medial subnetworks, which mediate transduction of information between sensory areas (VIS, AUD, and caudal-most SSp) and
higher-order association areas along the medial bank of the neocortex, such as the retrosplenial (RSP), parietal (PTLp), anterior cingulate (ACA), and orbital (ORB)
areas.
(B) Connectivity pathways of the medial subnetwork revealed by coinjections in the ORB (note: these are aggregated pathways for three different cases, see three
coinjection sites in ORB, colored pink, light brown, and dark brown, medial to lateral).
(C) Representative raw images from an ORBvl coinjection. PHAL-labeled axons and CTb-labeled neurons are found in other medial network components such as
the ACAd and adjacent MOs-fef, PTLp, RSPd, and primary and secondary VIS areas (VISp, VISal, and VISam). Scale bars, 500 mm (first panel) and 200 mm.
(D) Laminar-specific differences in axonal projections to primary visual cortex (VISp) arising from either ORBvl (red, BDA labeling) or ACAd (green, PHAL). Injection
sites in the same brain, left panels, scale bars, 500 mm (left) and 1 mm. Projections to different layers of the same section of VISp (right). Underlying fluorescent
Nissl was inverted to aid visualization of layers (right-most panel). Scale bar, 200 mm.
(E) Four different retrograde tracers were injected into the VISp (two), VISam, and VISpm within the same brain, resulting in distinct, topographically arranged
clusters of neurons in the ACA and adjacent MOs-fef. In the ORBvl, these retrogradely labeled neurons are intermixed, but mostly not colocalized (bottom right,
5% colocalization for any combination of tracers in ORBvl, 436 cells counted, 21 had two or more tracers present). Scale bars, 1 mm (top left), 500 mm (bottom
left), 200 mm (top right), 100 mm (bottom right).
(F) Summary of interactions among the medial subnetworks. Left, interaction between sensory and association areas. Dashed lines indicate sparse connection.
Claustrum (CLA) is included due to high degree of interconnection with medial network. Middle, connections between the association areas. Thicker arrows
indicate dense projection patterns between regions. Dashed line separates a direct pathway to medial prefrontal region along the ventro-medial bank of the
cortex (second medial subnetwork). Asterisks indicate a unidirectional connection between CLA and RSP. Right panel, overview of medial network interactions
including TEa and parahippocampal structures (i.e., SUBd, ENTm), which project to RSP (red arrows). Reciprocal connections of visual (blue) and auditory (green)
areas with all major medial network components shown. Caudal-most somatosensory areas (SSp-ll/tr; SSp-bfd.cm) are included as well (gray arrows).
See also Figure S6.
different species using data available in the literature (Honey navigation (Feierstein et al., 2006; Bucci, 2009; Vann et al.,
et al., 2007; Sporns et al., 2007; Markov et al., 2011; for rat see 2009; Weible, 2013).
BAMS: http://brancusi.usc.edu/). In contrast, the networks The second medial subnetwork is topologically distinct from
reported here are based on data collected and analyzed in a the first in that it successively transmits information from the
homogenous fashion rather than gathered piecemeal from the SUBd to the RSPv to the ACAv and then to the ILA, PL, and
literature. The cortical connectivity map allows users to directly ORBm. This multisynaptic subnetwork may provide a structural
compare connectivity patterns of different cortical areas within basis for relaying information processed in the dorsal hippocam-
the same neuroanatomic framework. Taken together, these re- pus and SUBd, perhaps regarding spatial orientation, naviga-
sources allow researchers to conceptualize any cortical region tion, and episodic memory, to the medial prefrontal cortex
of interest in the context of larger network interactions. (Vann et al., 2009; Fanselow and Dong, 2010; Weible, 2013).
The lateral subnetworks represent a point of massive conver-
The Cortical Subnetworks gence in the cortex. Interactions are centered on two major com-
Examination of the full data set revealed that the neocortex is ponents: the AI in the anterolateral insula subnetwork, and the
organized into several subnetworks that display unique topolog- TEa/PERI/ECT complex in the posterolateral temporal subnet-
ical organization, perhaps reflecting different information pro- work. Each receives input from, and projects back to, an exten-
cessing strategies for each. Within the somatic sensorimotor sive number of cortical areas. For example, the anterolateral
network, all main somatic nodes within the four subnetworks insular subnetwork integrates gustatory, visceral, and olfactory
are heavily and reciprocally connected. This organization allows information, while the posterolateral temporal subnetwork pro-
direct interactions between sensory and motor areas in the cesses more visual, auditory, somatosensory, and motor infor-
absence of higher-order association areas. This pattern could mation. Both subnetworks then transfer this information rostrally
enable rapid integration of different sensory modalities for to the medial prefrontal cortex and caudally to the ENTl. These
dynamically regulating motor actions, such as the integration connectivity patterns may support the proposed role of the
of tactile information in the oral cavity and proprioception of AI in self-awareness of internal states (Craig, 2009) and the
the jaw for initiation, maintenance, or termination of rhythmic role of the TEa/PERI/ECT in perception, object recognition,
jaw movements throughout the masticatory period (Yamada and contextual memory associated with emotion (Winters
et al., 2005). et al., 2008; Aggleton et al., 2010).
Unlike direct sensorimotor interactions that occur within the
somatic sensorimotor subnetworks, the medial subnetworks pri- Interactions among the Subnetworks
marily mediate interactions between the sensory and higher-or- Importantly, several regions of the cortex potentially serve as
der association areas. The first medial subnetwork serves sites for subnetwork interaction. The PFCdl receives a conflu-
to transmit sensory information from the visual, auditory, and so- ence of information from all four somatic sensorimotor subnet-
matic sensory (SSp-ll /tr and SSp-bfd.cm) areas to the ORBvl works. The PFCvm receives convergent inputs from the medial
and is organized differently than the sensorimotor subnetworks. and lateral subnetworks and provides an interface for integrating
All of the sensory areas directly connect with the ORBvl through or communicating information regarding external stimuli (such as
multiple parallel pathways. Each of these areas is also con- visual, auditory, somatic sensory) and internal stimuli (such as
nected with higher-order association areas like the RSPd, visceral and gustatory information). The CLA provides another
RSPagl, RSPv, PTLp, ACAd, and ACAv, within which sensory means by which the medial, lateral, and even the somatic sen-
inputs can be integrated prior to reaching the ORBvl. Almost sory subnetworks may directly interact. Both the PFCvm and
all cortical areas in this network (ORBvl, ACA, RSP, PTLp) have CLA are directly interconnected with the ENTl, which further re-
been implicated in orientating and coordinating movements of ceives massive, highly integrated sensory information from the
the eyes, head, and body in object searching tasks and spatial two lateral subnetworks. Through the ENTl, this information
Figure 5. The Lateral Subnetworks

(A) Sagittal view of the major components of two lateral subnetworks: the anterolateral insular (including the AId, AIv, AIp, VISC, GU) and posterior temporal
(including TEa, ECT, PERI). These two interconnected subnetworks are also connected with olfactory (e.g., PIR) and medial prefrontal (mPFC) areas and with the
ENTl. TEa in particular forms extensive connections with much of the rest of the neocortex (gray arrows).
(B) Distinct projection patterns of the anterior agranular areas (PHAL injections involved both AId and AIv, left panel) and AIp (right panel) with the mPFC areas (PL,
ILA, DP), posterior temporal areas (TEa, ECT, PERI), and ENTl. The AIp targets more ventral structures in mPFC and more heavily innervates the central nucleus of
the amygdala (CEA). Scale bars, 500 mm.
(C) Map of neuronal inputs to (left) and output from (right) the TEa, which are arranged topographically along the rostrocaudal direction. Note: these pathways are
aggregated from six coinjections made into different parts of the TEa from rostral (red) to caudal (blue) direction (top most sagittal image, numbers relative to
bregma in mm). Retrogradely labeled neurons are indicated as colored dots and demonstrate the layer-specific origin of cortical projections to TEa. Axonal
pathways arising from TEa (outputs) are rendered as shaded areas of color.
(D) Raw image of retrograde labeling (FG, yellow) following injection in TEa. Cells are distributed extensively across numerous cortical regions following a single,
small injection, suggesting a high level of convergence. Bottom left panel shows close up of layer specificity in somatosensory barrel field, with most cell bodies
residing in layers 2/3, 5a, and some layer 6. Fluorescent Nissl inverted (right) to aid in discriminating layers. Layer 4 ‘‘barrels’’ indicated with arrow. Scale bars,
500 mm (top) and 200 mm (bottom).
(E) Raw image of coinjection in TEa. Fibers are predominately ipsilateral, but retrogradely labeled inputs are evenly distributed across both hemispheres. See right
panel, middle, comparing labeling in contralateral and ipsilateral MOs.
Scale bars, 1 mm (top right), 200 mm (middle), 500 mm (bottom). See also Figure S7.
may reach the hippocampus, amygdala, and olfactory cortical coverslipped, and scanned as high-resolution virtual slide image (VSI) files
areas, or be routed directly back to the medial prefrontal cortex using an Olympus VS110 high-throughput microscope. The VSI files were con-
verted to tiff format prior to being registered. Following registration and regis-
(Figure 7C). In addition, the ENTl is also the starting point of the
tration refinement pipelines, the NeuroTrace fluorescent Nissl was converted
classic trisynaptic circuit that transfers information to the hippo- to bright-field. Next, each of the five channels for every image was adjusted
campus, which may ultimately reach one of its main output tar- for brightness and contrast to maximize labeling visibility and quality in
gets, the SUBd (Witter, 2007), to re-enter the medial network iConnectome. Following final modifications (i.e., skewness, angles) and
through its projections to RSPv. Consequently, through the pre- JPEG2000 file format conversions, images were published to iConnectome.
frontal cortex, ENTl, and CLA, information has the potential to be For more details on experimental procedures, see Supplemental Information.
represented and communicated throughout the limbic loop sur-
Data Annotation and Construction of Cortical Connectivity Matrices
rounding the entire neocortex (Figure 7C).
and Connectivity Map
Currently, informatics tools that automatically and precisely identify fine
CONCLUSIONS AND PERSPECTIVE anatomic boundaries in histological brain sections are nonexistent. Conse-
quently, analysis of the data necessitates manual annotation to index anatomic
locations and semiquantitative strengths of labeled cortico-cortical pathways.
In conclusion, we and other groups have demonstrated the
Analysis is performed in two formats. The first is for the purpose of construct-
feasibility of producing and collecting large-scale connectivity ing a comprehensive connectivity database and is comprised of an excel sheet
data (Osten and Margrie, 2013; Pollock et al., 2014); however, that indexes anatomic locations and corresponding semiquantitative densities
interpretation of this wealth of anatomical data presents an of labeling (PHAL-labeled axons/terminal boutons; CTb- and FG-labeled
ongoing challenge. This resource provides a reference for deter- neurons). These data were used to generate connectivity matrices (see Sup-
mining the complete set of inputs and outputs for a given cortical plemental Information). The second method consists of manually rendering
region and for implicating it in a broader network context. Any of the observed labeling patterns using Adobe Photoshop. Each pathway is
rendered in a separate layer and all layers across all experiments are stacked
these long-range interactions may be validated at the synaptic
to allow for a composite view of labeling trends.
level using transsynaptic viral tracing and may be further
investigated to determine cell-type-specific connections using SUPPLEMENTAL INFORMATION
methods such as channelrhodopsin-assisted circuit mapping
(Luo et al., 2008; Osakada et al., 2011; Petreanu et al., 2009). Supplemental Information includes Extended Experimental Procedures, seven
Moreover, these projections may be assessed functionally using figures, and two tables and can be found with this article online at http://dx.doi.
available optogenetic techniques that allow one to measure the org/10.1016/j.cell.2014.02.023.
circuit level or behavioral consequences of manipulating a given
AUTHOR CONTRIBUTIONS
pathway within a neural network (Yizhar et al., 2011).
B.Z., H.H., L.G., M.Y.S, M.B, M.S.B., N.N.F., and H.-W.D. produced, pro-
EXPERIMENTAL PROCEDURES cessed, and analyzed the data and prepared the images for publication into
the iConnectome. B.Z. constructed the cortico-cortical connectivity map.
Data Generation, Collection, and Online Presentation S.Y. developed the interactive iConnectome map viewer. S.Y. also partici-
All experimental procedures have been described previously (Hintiryan et al., pated in the initial design of the iConnectome visualization tool and in the
2012). In brief, double coinjections of tracers were made into different areas development of the informatics pipeline for data processing. I.B. and M.S.B.
of the entire neocortex, hippocampus, olfactory cortical areas, and amygdala performed network analysis, constructed the connectivity matrices, and wrote
of 8-week-old male C57Bl/6J mice. PHAL (2.5%; Vector Laboratories) a description of corresponding employed methods in the manuscript. H.-W.D.,
and CTb (647 conjugate, 0.25%; Invitrogen) were coinjected, while BDA H.H., and B.Z. wrote the manuscript. All authors made constructive comments
(FluoroRuby, 5%; Invitrogen) was injected in combination with FG (1%; Fluoro- on the manuscript. A.W.T. served as project advisor and participated in the
chrome, LLC). One week was allowed for tracer transport after which animals planning and organizing of the project. H.-W.D. conceived and led the project.
were perfused and their brains extracted. All brains were sliced at 50 mm thick-
ness using a Compresstome (VF-700, Precisionary Instruments, Greenville, ACKNOWLEDGMENTS
NC). One series of sections was stained for PHAL using Alexa Fluor 488
(Invitrogen). All sections were counterstained with a fluorescent Nissl stain, The authors would like to thank Drs. Larry Swanson and Harvey Karten for
NeuroTrace 435/455 (NT; 1:1000; Invitrogen). The sections were then mounted, advising this project, Daren Lee, Anand A. Joshi, Nikhil G. Sane, and Queenie
Figure 6. The CLA and ENTl

(A) Axonal projections arising from the CLA are distributed throughout the entire neocortex and ENTl on the ipsilateral hemisphere. Note: these axons display
different regional and laminar distribution specificity (on right panel) Scale bars, 1 mm (left), 500 mm (top right), 200 mm (bottom).
(B) and (C) Asymmetric connections of the CLA with other cortical areas in the two hemispheres. Cortical inputs to CLA project to both sides with equal densities
(B, labels: dorsal and ventral claustrum (CLAd, CLAv), and endopiriform nucleus (EPd)), while outputs from CLA to other cortical areas indicated by retrograde
tracers are almost exclusively ipsilateral (C). Moreover, the CLA has a dorsal to ventral topography in its projections to the cortex, with cells in CLAv (yellow, C,
right) preferentially targeting ventral cingulate (C, left, injection site in ACAv). Very little colabeling was observed among cells labeled from a neighboring, more
dorsal injection (pink). Scale bars: 1 mm (left) and 200 mm (right).
(D) Neural inputs to the CLA from almost all cortical areas in medial, somatic, and lateral subnetworks. The somatomotor inputs preferentially target the dorsal-
most aspect of CLA.
(E) Representative images of PHAL-labeled axons in layer 1 of a wide range of neocortical areas arising from the rostrodorsal ENTl.
(F and G) Laminar specificity of PHAL-labeled axons and CTb-labeled neurons in the ENTl after coinjections made into the AI or ILA (F). Both cortical regions
provide strong, direct input to ENTl, further supported by retrograde data in (G). These data also confirm the CLA is a specific source of input to ENTl (G, middle).
Scale bars, 1 mm (F, top left and G, left), 500 mm (F, bottom and G, middle), 100 mm (F, top right).
See also Figures S1B–S1E.
Figure 7. Interactions with Prefrontal Cortex
(A and B) Cumulative projections from components of the somatic sensorimotor, lateral, and medial networks in two representative coronal sections of the
prefrontal cortex (PFC). Collectively these represent inputs from the entire neocortex to the PFC. Inputs were color coded based on the location of the injection
sites in different components of the network (A). For example all primary motor projections arising from multiple injections along the length of this structure were
(legend continued on next page)
Ng for the initial development of the iConnectome visualization tool and Betty Craig, A.D. (2009). How do you feel—now? The anterior insula and human
W. Lee, Carlos Mena, Vaughan Greer, and Robert De La Cruz for their contri- awareness. Nat. Rev. Neurosci. 10, 59–70.
butions to the website. We acknowledge Arleen Grewal, Annie Chen, Amy de Curtis, M., and Paré, D. (2004). The rhinal cortices: a wall of inhibition
Hwang, and Hyojin Ryu for their contributions in image processing. This between the neocortex and the hippocampus. Prog. Neurobiol. 74, 101–110.
work was supported by NIH/NIMH, MH094360-01A1 (HWD) and P41 Supple-
Dong, H.-W. (2007). The Allen Reference Atlas: A Digital Color Brain Atlas Of
ment (AWT 3P41RR013642-12S3). The Mouse Connectome Project (MCP)
The C57BL/6J Male Mouse (Hoboken: John Wiley and Sons, Inc.).
was initiated by the authors while still at the University of California, Los An-
geles (UCLA), but the entire program and project now fully reside at the Univer- Driver, J., and Noesselt, T. (2008). Multisensory interplay reveals crossmodal
sity of Southern California (USC). This manuscript is dedicated to Dr. Edward influences on ‘sensory-specific’ brain regions, neural responses, and judg-
(Ted) G. Jones, a devoted scientist, colleague, and beloved friend who will be ments. Neuron 57, 11–23.
greatly missed. Fanselow, M.S., and Dong, H.-W. (2010). Are the dorsal and ventral hippocam-
pus functionally distinct structures? Neuron 65, 7–19.
Received: July 23, 2013 Feierstein, C.E., Quirk, M.C., Uchida, N., Sosulski, D.L., and Mainen, Z.F.
Revised: January 25, 2014 (2006). Representation of spatial goals in rat orbitofrontal cortex. Neuron 51,
Accepted: February 10, 2014 495–507.
Published: February 27, 2014
Felleman, D.J., and Van Essen, D.C. (1991). Distributed hierarchical process-
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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Subjects
Data from 300 8-week-old male C57BL/6J mice (Jackson Laboratories) were used. They were housed in pairs in a room that was
temperature (21-22 C), humidity (51%), and light controlled (12 hr light:12 hr dark cycle with lights on at 6:00 am and off at 6:00
pm). The mice were allowed at least 1 week to adapt to their living conditions before surgery. Subjects had ad libitum access to
tap water and mouse chow throughout the experiments. The experiments were conducted according to the standards set by the
National Institutes of Health Guide for the Care and Use of Laboratory Animals and the institutional guidelines of the University of
California, Los Angeles (UCLA). The data presented in this report were collected during the time when the MCP was part of
UCLA; however, currently the project fully resides at the University of Southern California (USC).
Tracers
Double coinjections of tracers were made into different areas of the entire neocortex (Figure S1A) and into select regions of the
hippocampus, olfactory cortical areas, and amygdala (see Table S1 for list of injections; Figure S3 shows schematic summary of
selected injections). Each coinjection contained an anterograde (phaseolus vulgaris leucoagglutinin [PHAL] or dextran tetramethylr-
hodamine [BDA]) and a retrograde (cholera toxin subunit b [CTb] or Fluorogold [FG]) tracer. PHAL (2.5%; Vector Laboratories) and
CTb (647 conjugate, 0.25%; Invitrogen) were coinjected, while BDA (FluoroRuby, 5%; Invitrogen) was injected in combination
with FG (1%; Fluorochrome, LLC). In some cases, AAV1.hSyn.GFP or AAV1.CAG.RFP from Penn Vector Core was used. This virus
was originally created at the Allen Institute for Brain Sciences.
Validation of Connectivity Data

The specificity of injections were cross-validated by the application of retrograde tracers to regions targeted by anterogradely labeled
axon terminals and vice versa (Figures S1B and S1C). Generally, several injections were made into the same cortical areas of different
animals to validate connections. Injections made into the same brain region (e.g., the infralimbic area, ILA) of different mice showed
identical projection patterns (Figure S1D), while injections in neighboring regions displayed distinct connection patterns (Figure S1B),
indicating the robustness of the data. In some cases, injection sites encroached into smaller neighboring cortical areas, for example
an injection involved both the dorsal and ventral agranular insular area (AId and AIv) (Figure S1E); however, coinjections applied to
their targeted areas distinguished their connectivity patterns. For example, coinjections in the rostral AId and ILA showed that the AIv
(as well as CLA), but not AId, receives inputs from the ILA, while the AId, but not AIv, is connected with rostral AId (Figure S1E).
Injections
Efforts were made to make all of the injection sites approximately the same size (300–500 mm) by following the same injection
parameters for each (injection time, pipette tip size, infusion time), with a few injections being reduced in size to target smaller areas
(e.g., agranular insula). This facilitates drawing comparisons of strength across the data set, but we do recognize the limitations of our
subjective scoring system. It was not feasible for us to score projection strengths in a quantitative, automated fashion across the
whole data set due to the technical limitations of registering or aligning each tissue section with a corresponding atlas section,
and thus we relied on a manual approach.
Importantly, some injections are made into mixed areas; however, labeling from injections in multiple areas may be compared with
injections that are more clearly centered within one structure or the other since the same cortical region was targeted several times in
order to validate connections. Moreover, examining each of the distributed target areas of a given injection with subsequent retro-
grade tracer injections reveals the relative contribution and grouping of neuronal populations that gave rise to the original projection
pattern. Figure S1E further expands on this idea.
Connectivity-Based Parcellations of the Somatosensory Cortical Areas

To assemble the somatic sensorimotor subnetworks, we first defined the general anatomical boundaries of the body subregions for
the SSp, MOp, MOs, and SSs, on the basis of their topographic connectivity patterns. This approach is consistent with the connec-
tivity-based parcellation method (Beckmann et al., 2009; Sporns, 2010), which is based on the hypothesis that projection neurons
within a coherent brain region share extrinsic (interregional) projection sources and targets, while projection neurons in different
regions exhibit different connectivity trends.
Surgeries
The surgeries were performed under isoflurane anesthesia (Hospira). Mice were initially anesthetized in an induction chamber primed
with isoflurane and were subsequently mounted to the stereotaxic apparatus where they were maintained under anesthetic state via
a vaporizer (Datex-Ohmeda). The isoflurane was vaporized and mixed with oxygen (0.5 l/min) and nitrogen (1 l/min). The percent of
isoflurane in the gas mixture was maintained between 2 and 2.5 throughout the surgery. PHAL/CTb and BDA/FG infusions were deliv-
ered iontophoretically using glass micropipettes whose outside tip diameters measured approximately 15–20 mm. A positive 5 mAmp,
7 s alternating injection current was delivered for 10 min (Stoelting Co.). Pipettes were left in situ for an additional 10 min to avoid
Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. S1

diffusion of tracers along the needle track. Animals survived for 7 days prior to being sacrificed. AAV1.hSyn.GFP or AAV1.CAG.RFP
were also injected using the iontophoresis method as described above, except animals were sacrificed 2 weeks following surgeries.
Tissue Preparation
Following an overdose injection of sodium pentobarbital, each animal was transcardially perfused with approximately 50 ml of 0.9%
NaCl followed by 50 ml of 4% paraformaldehyde solution (PFA; pH 9.5). The brains were postfixed in 4% PFA for 24-48 hr at 4 C after
which they were embedded in 3% Type I-B agarose (Sigma-Aldrich) prior to sectioning. Four series of coronal sections were sliced at
50 mm thickness with a Compresstome and prepared for processing.
To ensure high quality of tissue, all brains were sliced using a Compresstome (VF-700, Precisionary Instruments, Greenville, NC).
The Compresstome slices nonfrozen tissue and circumvents issues related to low sectioning speed, tissue distortion, and slice sur-
face chatter marks. Tissue damage resulting from the freezing process also is obviated. The resulting superior tissue quality enables
maximal preservation of axon morphology and acquiesces to desirable Nissl staining. To maximize correspondence between our
sections and those from the ARA, all brains were consistently sliced at the same cutting angle as was used for the creation of the
reference atlas and the series containing sections with specific landmarks that most closely matched those in the Allen Reference
Atlas (ARA, Dong, 2007) was selected for processing (i.e., coronal section containing crossing of the anterior commissure).
Immunofluorescence Staining
One series of sections was stained for PHAL using the free-floating method for immunofluorescence. Briefly, sections were trans-
ferred to a blocking solution containing normal donkey serum (Vector Laboratories) and Triton X (VWR) for 1 hr. Following 3-5 min
rinses, sections were incubated in a KPBS solution comprised of donkey serum, Triton, and a 1:1000 concentration of rabbit anti-
PHAL antibody (Vector Laboratories) for 48-72 hr at 4 C. Sections were rinsed 3 times in KPBS and then soaked for 3 hr in the sec-
ondary antibody solution, which contained donkey serum, Triton and a 1:500 concentration of anti-rabbit IgG conjugated with Alexa
Fluor 488 (Invitrogen). Following 3 KBS rinses, the sections were counterstained with a fluorescent Nissl stain, NeuroTrace 435/455
(NT; 1:1000; Invitrogen). The sections were then mounted and coverslipped using 65% glycerol.
Imaging and Postacquisition Processing

The entire postacquisition processing procedure, which requires many time consuming steps, were standardized and pipelines were
created for speedier processing. Initially, all sections were scanned as high-resolution virtual slide image (VSI) files using an Olympus
VS110 high-throughput microscope fitted with a 10X objective lens. Images were captured tile by tile and then assembled together as
whole-brain images. Each image, containing 5 channels (PHAL, CTb, BDA, FG, and NT), was flipped to the correct left-right orien-
tation, matched to the nearest ARA atlas level, and converted to tiff format prior to being registered (detailed below). Following regis-
tration and registration refinement, the NeuroTrace fluorescent Nissl stain wass converted to a bright-field image. Next, each channel
for every image was adjusted for brightness and contrast to maximize labeling visibility and quality in iConnectome. Following final
modifications (i.e., skewness, angles) and JPEG2000 file format conversions, images were published to iConnectome (www.
MouseConnectome.org).
Semiautomated Image Registration

A combination of automatic and manual registration steps was used to resample, align, and coregister the acquired brain images with
the ARA atlas image. Each individual image was manually matched to its closest corresponding ARA atlas level and the information
was manually inserted into a registration table. Although manual, this reduces the 2D registration variability and improves the regis-
tration accuracy versus using a fully automated registration algorithm purely based on fiducial markers (like the Allen Brain Atlas,
www.Brain-Map.org). We used a 2D diffeomorphic demons algorithm for performing the image registration. Affine registration
was chosen to minimize distortion to preserve axonal morphology. The deformation matrix resulting from the registration process
was applied on the original resolution images to get the high-resolution warped images. The algorithm was implemented in Matlab
and standalone binaries were generated using Matlab compiler. The Laboratory of Neuro Imaging (LONI) pipeline was used for par-
allel execution of registration for multiple subjects.
Data Annotation
The data were analyzed manually since currently manual annotation provides the most accurate anatomic information. Analysis was
performed in two formats. The first was for the purpose of constructing a comprehensive connectivity database and is comprised of
an excel sheet that indexes anatomic locations and corresponding semiquantitative densities of labeling (PHAL-labeled axons/ter-
minal boutons and CTb- or FG-labeled neurons).
The second method consists of manually rendering the observed labeling patterns using Adobe Photoshop to create the cortico-
cortical connectivity map (Figure S2B; also see Figure 1F). Raw images are matched to corresponding atlas levels, and anatomical
landmarks and the underlying Nissl stain are used as guides in the accurate representation of observed axonal and cell body loca-
tions (Figure S4A). Each pathway is rendered in a separate layer and all layers across all experiments are stacked to allow for a com-
posite view of labeling trends. Labeling from different injections can be easily visualized and compared by selectively manipulating
layers (Figure S2B).
S2 Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc.

In the connectivity map, each coinjection site is represented by a circle. PHAL pathways were rendered as shaded regions with
regional and laminar specificity. Retrograde labeling (both FG and CTb) is represented by small dots that reflect regional and laminar
distribution patterns, but also relative densities (Figures 1F and S4A). Each of these pathways was color coded with a unique RGB
value. A general color scheme of red-orange-yellow-green-blue-indigo-violet, medial to lateral, was implemented for the online con-
nectivity map, although for analysis, colors were manipulated to reveal patterns more clearly.
Construction of Connectivity Matrix

To initialize building cortical networks, labeled data within all cortical regions were annotated by manually recording the distribution of
anterograde and retrograde labeling. If labeling was present in a given cortical region of interest (ROI), we subjectively weighted the
connection (from 1 to 3) based on the observed labeling density within the ROI, where 1 represented sparse, 2 moderate, and 3 dense
labeling. Both the anterograde and retrograde labeling was used to assess the strength assigned to each cortical area. This method
was used to decipher whether a labeled area was assigned a 1 or 0 (for no labeling). For example, if a PHAL injection in A resulted in
only a few labeled fibers in B, B was assigned a 1 if retrograde tracers in B back labeled a few neurons in A. Further, since each area
was targeted several times, the consistency of labeling across the cases was also used in our assessment of strength. For reliability,
the same cases were annotated by at least 2 raters. Only PHAL labeling for anterograde tracing was used due to its reliability and
robustness. Both FG and CTb were used for retrograde tracing data. Directionality was noted so that a region’s input and output
connections were distinguished. Importantly, since injection sites span all analyzed cortical ROIs, the output of one cortical region
is confirmed by input data of all other cortical areas (Figure S1) thereby validating connectivity and limiting false positives (Figures 2A
and 2B). Iterating through each matrix entry, 89% of connections exhibited mutual labeling by both anterograde and retrograde
labeling methods. Most of the inconsistent data came from anterograde labeled ENTl connections that were restricted to cortical
layer 1 on the outer edge of the brain, as deeper targeting of tracer injections tended to exclude. For a linear algebra-based compar-
ison, the Frobenius norm (Weisstein Eric W. http://mathworld.wolfram.com/FrobeniusNorm.html) of the difference of the two
matrices (i.e., the Frobenius distance) was calculated to be 16.6, while the mean Frobenius distance between two randomly con-
structed matrices with the same degree distributions was 28.9 (±0.29 SD, n = 1,000 comparisons), suggesting that the anatomical
data sets gathered by anterograde and retrograde tracers were significantly similar. Using the annotation data we constructed two
label-based weighted directional connectivity matrices (Figures 2C and 2D). For each connection, the origin of the connection is
listed along the row while the targets are listed across the columns. For visual purposes, the clustered ordering of both matrices
was made to be uniform with the composite matrix (discussed in the following paragraphs).
To ascertain an overview of all anatomical data, a composite matrix was built with the aid of the Brain Connectivity Toolbox (BCT)
(http://www.brain-connectivity-toolbox.net). We performed an element-wise logical ‘And’ operation between the anterograde and
retrograde matrices to form a third composite binary directed matrix in which the corresponding element was set to 1 if a connection
was present in both anterograde and retrograde, 0 otherwise. We then reordered the cortical ROI nodes of the composite matrix. Our
goal was to improve the effectiveness of the matrix for grouping and visualizing subnetwork modules (Rubinov and Sporns, 2011),
without altering the connectivity described. We first manually constructed an affiliation vector between the ROIs and their neuroana-
tomic modules (based on previous knowledge), including somatic sensorimotor (four subgroups, the orofaciopharyngeal, upper limb,
lower limb and trunk, as well as whisker), visual, auditory, medial (including all cortical areas along the medial bank of the neocortex,
such as the prelimbic, infralimbic, orbitofrontal, anterior cingulate, retrosplenial, and posterior parietal association areas), and insular
and temporal (three areas in the vicinity of rhinal fissure including the perirhinal, ectorhinal and temporal association areas) modules
(see list below for specific affiliations). Providing the input of the composite matrix and described affiliation vector to a module-based
reordering method (reorder_mod, Rubinov and Sporns, 2011) of the BCT rendered the ordering shown in (Figure 2E). As previously
mentioned, identical node ordering was applied to the anterograde and retrograde connectivity matrices to facilitate comparison.
Neuroanatomic Modules for Constructing Connectivity Matrices

ROI: module
ACAd: medial
ACAv: medial
Aid: insular
AIp: insular
AIv: insular
AON: olfactory
AUDd: auditory
AUDp: auditory
AUDv: auditory
CLA: CLA
DP: medial
ECT: rhinal
ENTl: ENTl
ENTm: spatial

EPd: olfactory
GU: insular
ILA: medial
MOp-ll/tr: SM-lltr
MOp-m/n: SM-orf
MOp-ul: SM-ul
MOp-w: SM-w
MOs-c: SM-lltr
MOs-fef: medial
MOs-rd: SM-ul
MOs-rdl: SM-orf
MOs-rdm: SM-lltr
ORBl: medial
ORBm: medial
ORBvl: medial
PERI: rhinal
PIR: olfactory
PL: medial
POST: spatial
PRE: spatial
PTLp: medial
RSPagl: medial
RSPd: medial
RSPv: medial
SUBd: spatial
SSp-bfd.al: SM-orf
SSp-bfd.cm: SM-w
SSp-ll/tr: SM-lltr
SSp-m/n: SM-orf
SSp-ul: SM-ul
SSs-c & cd: SM-ul
SSs-r & cv: SM-orf
Tea: rhinal
TTd: olfactory
TTv: olfactory
VISal: visual
VISam: visual
VISC: insular
VISl: visual
VISp: visual
VISpl: visual
VISpm: visual
SUPPLEMENTAL REFERENCES
Beckmann, M., Johansen-Berg, H., and Rushworth, M.F. (2009). Connectivity-based parcellation of human cingulate cortex and its relation to functional special-
ization. J. Neurosci. 29, 1175–1190.
Erzurumlu, R.S., Murakami, Y., and Rijli, F.M. (2010). Mapping the face in the somatosensory brainstem. Nat. Rev. Neurosci. 11, 252–263.
Killackey, H.P., Koralek, K.A., Chiaia, N.L., and Rhodes, R.W. (1989). Laminar and areal differences in the origin of the subcortical projection neurons of the rat
somatosensory cortex. J. Comp. Neurol. 282, 428–445.
Li, X.G., Florence, S.L., and Kaas, J.H. (1990). Areal distributions of cortical neurons projecting to different levels of the caudal brain stem and spinal cord in rats.
Somatosens. Mot. Res. 7, 315–335.
Nord, S.G. (1967). Somatotopic organization in the spinal trigeminal nucleus, the dorsal column nuclei and related structures in the rat. J. Comp. Neurol. 130,
343–356.
Rubinov, M., and Sporns, O. (2011) Brain Connectivity Toolbox [Computer program]. Available at http://www.brain-connectivity-toolbox.net.
Welker, E., Hoogland, P.V., and Van der Loos, H. (1988). Organization of feedback and feedforward projections of the barrel cortex: a PHA-L study in the mouse.
Exp. Brain Res. 73, 411–435.

Figure S1. Demonstration of Specificity and Accuracy of Injection Strategy, Related to Figure 1
(A) Representative images of coronal brain section with double coinjections in two neighboring domains of the primary somatosensory area (SSp): PHAL (green)/
CTb (pink) in the lower limb domain, and BDA(red)/FG(yellow) in the trunk domain. To demonstrate the specificity of these two coinjections, two adjacent clusters
of labeling (intermixed labeling of PHAL with CTb or BDA with FG) were observed in the posterior thalamic (PO) and ventroposteromedial (VPM) thalamic nuclei,
indicating their reciprocal connections with the SSp-ll or SSp-tr. Also, three distinct topographically arranged cortical columns associated with each injection
were observed in the auditory cortical area (AUD) (right panel, retrograde labeling shown), which provides further evidence that each injection site is discrete and
is not contaminating labeling from its neighboring injection site. Note, however, that to show proper labeling throughout the brain using the iConnectome viewer
(www.MouseConnectome.org), histogram levels were adjusted to reveal fine fibers, which causes overexposure at the coinjection sites such that they appear to
be larger (second panel, same section as left-most panel as it appears in iConnectome browser. Arrow indicates thalamic labeling that becomes visible when
contrast is adjusted). We estimate injection sites are typically 250-500 mm in diameter. Scale bars, 1 mm (left), 100 mm (middle), and 200 mm (right).
(B) Further demonstration of specificity of injection size and location. Coinjections (PHAL/CTb) were made into two small, adjacent cortical areas, the gustatory
(GU, top) and posterior agranular insular area (AIp, lower). Anatomical locations of these two injections can be validated by using underlying Nissl-stained cy-
toarchitecture within the same section (middle images, right panel is an inverted image of the fluorescent Nissl to aid viewing). Coinjection within the GU resulted
in a distinct cluster of intermixed labeling in the VPMpc, which relays taste information to the cortex (Jones, 2007), further validating specificity of the GU co-
injection. Coinjection in the AIp, but not in the GU, resulted in dense labeling in the deep layers of the ENTl (lower panel); a FG injection in the same region of the
ENTl resulted in dense retrograde labeling in the AIp (and adjacent CLA), but only sparse in the GU, providing further validation of the projection patterns. Scale
bars, 1 mm (left), 200 mm (middle), 1 mm (right).
(C) Connections between any two cortical areas in our project can be cross validated using both the anterograde and retrograde tracing method. Here, a PHAL
injection within the SSp (top left) results in dense axonal terminals in the MOp (top right). A FG injection in the same location of the MOp (bottom right) results in
retrogradely labeled neurons in the corresponding SSp location (bottom left). Importantly, using this strategy also reveals the regional and laminar specificity of
both the axons projecting to MOp and the layer-specific arrangement of cell bodies that give rise to this projection.
(D) Reproducibility and control of individual variability of labeling in different cases with injections in the same locations. PHAL injections made into identical
locations of the ILA in two different mice resulted in identical projection patterns in the cortex and midbrain. Several other injection cases were repeated as further
controls and resulted in identical labeling patterns. Scale bar, 1 mm.
(E) Resolving fine-scale organization of cortical structures smaller than the diameter of a single injection site. One coinjection involves two small, adjacent areas,
the dorsal (AId) and ventral (AIv) agranular insular areas (left) and resulted in labeling in the rostral AId and ILA, among other areas. To examine the specificity of
these connections, two coinjections were made into the ILA and rostral AId respectively in different animals. They show that only the AIv, but not AId, shares
reciprocal connections with the ILA, while the rostral AId interacts with only the more caudal AId, but not AIv. Cross examining all targets of the original, large
coinjection in AId/v (left panel) in this fashion revealed preferential interaction of each of the targets with either AId or AIv, thus allowing for parcellation of
connectivity patterns for these small cortical regions. Scale bar, 500 mm.

Figure S2. Online Resources: Screenshot of iConnectome Visualization Tool and iConnectome Map Viewer, Related to Figure 1
(A) Screenshot of the iConnectome image browser which allows viewing of all raw imaging data across the entire brain at 103 magnification for all injection cases.
In iConnectome, the multiple fluorescent connectivity data are presented in 4 different channels: PHAL in the green, BDA in the red, FG in the yellow, and CTb in
the pink. Two additional channels that aid in data analysis are a bright-field Nissl stain of the same section and the corresponding atlas level from a standard
mouse atlas the Allen Reference Atlas (ARA; Dong, 2007). For each section, the four labeled pathways related to each of two coinjections may be viewed
simultaneously by turning on and adjusting the transparency of the layers using the control panel indicated by arrow (1). Sections across the entire brain were
collected in order and may be selected for viewing from the thumbnail images in the panel indicated by arrow (2). The thumbnail images shown for this case
currently display the Nissl stain, but this can be changed to view any of the tracers using the controls indicated by arrow (3). This is especially useful for finding the
injection site location associated with a particular neural tracer.
(B) Screenshot of the iConnectome map viewer. Users may pan and zoom onto any of the 29 representative cortical sections and may select up to 10 projection
pathways simultaneously for viewing using the panel indicated by arrow (1). Cases are listed alphabetically by location of injection site, and coinjection labeling is
separated as input (retrograde) and output (anterograde) layers. Injection site locations are indicated by large colored circles, indicated by arrow (2), here for a
VISam injection (yellow) and VISal injection (green). In general, the color scheme used for the cortical connectivity map follows a rainbow-like pattern from medial
to lateral, with red used for deep structures along the midline, fading to orange for dorsal cingulate, yellow for secondary motor, and so on, with violet used for
lateral most structures like the insula.

Figure S3. Summary of Injection Site Locations for the Majority of Cases Used in the Study, Related to Figure 2
Collectively, all injection sites in our databases span throughout the entire neocortex. See Table S1 for a list of these injections. Diamond indicates PHAL/CTb
coinjection, circle indicates BDA/FG coinjection.

Figure S4. Generating the iConnectome Cortical Connectivity Map and Its Use in Establishing Interactive Networks between Cortical Re-
gions, Related to Figure 1
(A) General approach to mapping fluorescent labeling from a raw image (left column) onto the corresponding level of the ARA to generate the connectivity map
(middle column). Each coinjection site is represented by a circle. PHAL pathways were rendered as shaded regions with regional and laminar specificity.
Retrograde labeling (FG and CTb) is represented by small dots that reflect regional and laminar distribution patterns, but also relative densities (Figures 1F and
S4A). Each of these pathways was color coded with a unique RGB value and rendered into an individually layered document with transparency such that multiple
combinations of layers representing multiple injection sites could be viewed simultaneously within the same anatomic frame provided by the Allen Reference
Atlas (Dong, 2007), thus revealing topographic trends for large areas and interaction between regions. A general color scheme of red-orange-yellow-green-blue-
indigo-violet, medial to lateral, was implemented for the online map, although for analysis, colors were manipulated to reveal patterns more clearly. The PHAL
(green)/CTb (pink) coinjection site in the VISam is represented by a large circle; PHAL-labeled axonal pathways were rendered as layer and regional-specific
shading (see labeling in VISal); while retrogradely labeled neurons (CTb or FG) were represented as individual dots reflecting the density and layer-specific
distribution of observed cell bodies. The cortical parcellations in the reference atlas were, in part, created by using cytoarchitectural information revealed by Nissl
stain. Examination of the underlying Nissl stain in each injection case, along with major anatomical landmarks, allowed for accurate placement of labeling within
the reference atlas framework. PHAL and CTb labeling are intermixed in the VISal, as indicated by overlapping of shaded areas with individual dots, suggesting
reciprocal connectivity between the VISal and VISam regions (see right column for higher magnification raw images). Using this method, we have reconstructed a
total 240 cortico-cortical pathways (80 PHAL-labeled efferent pathways; 160 FG or CTb-labeled afferent pathways) across the entire neocortex. These
projection maps are available online in the iConnectome map viewer (www.MouseConnectome.org) (Figures 1F and S4B). Scale bars, 500 mm (left) and 200 mm
(right).
(B) Strategy for establishing connections between cortical regions. Coinjections reveal reciprocal connectivity between the injection site locations and other
target areas. For example, a VISam coinjection (top panel, yellow) labels both axonal fibers and cells in VISal and ORBvl, providing initial evidence for a reciprocal
interaction between these regions. A follow up coinjection in VISal (green) confirms this reciprocal connectivity and reveals a common target in ORBvl (Right
column shows raw images of the ORBvl with fluorescent labeling after coinjections in either VISam or VISal, scale bar, 200 mm). To validate these connections, a
PHAL/CTb coinjection was made into the ORBvl (bottom image on the right column), which reveals dense PHAL-labeled axons and CTb-labeled neurons in the
VISam and VISal (brown colored pathways in the connectivity map; see bottom left raw image for ORBvl labeling in the visual areas), confirming their strong
reciprocal connections. It also shows relatively sparse labeling in the VISp, suggesting its moderate connections with the ORBvl. Collectively, these data suggest
these structures interact as a network, summarized in bottom right panel. Following this strategy, larger interactive networks can be built using additional data
collected from across the entire cortex. For example, further examination of VISp interactions revealed shared connectivity with VISam and VISal areas as well
(not pictured), further broadening the scope of interaction for the network.
(C) Collecting all projection data for each case into a common atlas space allows for quick comparison of labeling trends and determination of common and
unique targets for different injection sites. For a single coinjection, projection data from across the entire cortex was rendered onto 29 corresponding atlas
sections, from the front of the cortex to the back, as viewed in the iConnectome map viewer (see Figure S2B). Five representative sections are shown here (left
panel, VISam coinjection, yellow) demonstrating anterograde and retrograde labeling in other cortical regions. Additional projection pathways may be overlayed
in the map for a convenient comparison of the interactions between each injection site (right panel, VISal injection in green). Based on this data, a summary of the
shared and unique targets of VISal and VISam is provided in the diagram on the right. The connectivity map may be useful for determining these trends and
interactions for any cortical region of interest.

Figure S5. Demonstration of Specificity of Different Functional Domains of Somatic Sensorimotor Areas, Related to Figure 3
(A) The specificity of SSp-m/n (orofacial domain) and SSp-bfd (whisker domain) were validated by their projections to either the dorsal or ventral part of the spinal
trigeminal nucleus (Nord, 1967; Welker et al., 1988; Killackey et al., 1989; Erzurumlu et al., 2010); while the specificity of the upper limb (SSp-ul) or lower-limb
(SSp-ll) domains were validated by their projections to the cuneate or gracile nucleus in the lower brainstem, which process sensory information related to upper
and lower limbs, respectively (Nord, 1967; Li et al., 1990). Scale bars, 1 mm (top), 500 mm (bottom).
(B) Direct comparison of projection patterns for different domains of SSp within the same brain. Top: Specificity of cortico-cortical connectivity of the SSp-ul and
SSp-ll domains. Two anterograde tracer injections were made into the SSp-ul (AAV1.hsyn.GFP, green) and SSp-ll (AAV1.CAG.RFP, red), respectively. Each
injection site location was validated by their topographic projections to the cuneate or gracile nuclei (top right). These two domains generate topographically
arranged projections in two domains of the MOs in the prefrontal cortex, the MOs-rd and MOs-rdm. The use of viral tracers in this example allowed visualization of
native fluorescence prior to cutting the brain (last panel, scale bar, 1 mm) which enabled localization of the injection sites and eased translation between the top-
down view of the cortex and the corresponding coronal sections. The very last panel of the figure superimposes the cortical connectivity map and the injection site
locations of both cases. Bottom: Two injections in the middle and caudal parts of the SSp-bfd (as validated by their axonal terminals in the ventral two third of the
spinal trigeminal nucleus) generate distinct projection patterns within the MOp and MOs.
(C) Topographic projections from different SSp functional domains (as well as the PTLp) to the MOp and MOs in the prefrontal cortex (anatomical locations and
coordinates of injection sites are shown in right column). These projections provide a structural basis to determine different functional domains of the MOp and
MOs, for example, the MOs-rdm, MOp-bfd, MOs-rd, MOs-rdl, and MOp-orf.
(D) The more caudal aspects of MOs and anterior cingulate are not heavily innervated by somatosensory projections, but rather receive extensive input from more
posterior cortical regions (e.g., visual, auditory, parietal) as seen in the schematic (left). Moreover, this region interacts with the posterior-most aspects of motor
cortex. For example, a retrograde injection in this motor region (FG, yellow, top left panel) results in extensive labeling of neurons in this MOs/ACA region (bottom
left). This interaction is further confirmed by anterograde injection in the MOs/ACAd region (PHAL, green), which projects to the corresponding caudal motor
region as well as visual, retrosplenial, and superior colliculus areas (middle). The brainstem projections to superior colliculus and other pretectal regions
implicated in controlling eye movements suggest this cortical area may correspond to the frontal eye field (MOs-fef in the text) identified in primates (also see
Reep et al., 1990 for rats). The layer-specific origin of primary visual cortex projections to this area are also shown (right panels), which presumably contribute
visual information for processing in the region.

Figure S6. Additional Information for Assembling the Medial Subnetworks, Related to Figure 4
(A) Parallel projection pathways from the VISp and AUDp to the association cortical areas within the medial subnetwork, including the ORBvl, ACAd, ACAv, and
PTLp. Please note that the VISp also has dense projections to the RSPd and RSPagl, which receives very sparse inputs from the AUDp. On the other hand, AUDp
projects heavily and unidirectionally to VIS areas (last panels).
(B) Projection map (left) of the ACAv (red injection site and pathways), ACAd (orange), and MOs-fef (yellow). All three areas are heavily connected with other
regions within the medial subnetworks, such as the ORB, PTLp, RSP, and CLA. These three areas also share connectivity with the visual, auditory, and somatic
areas with different densities. Raw images of one representative coinjection in the ACAd and its labeling in other areas (such as the ORB, CLA, PTLp, VISp, VISam,
and VISal) are found in the right panels.
(C) Summary of direct interactions between sensory areas in different modalities. A portion of caudomedial barrel field (SSp-bfd.cm) is reciprocally connected
with primary visual cortex, and the lower-limb and trunk domains of the SSp (SSp-ll/tr) interacts with auditory areas. The most striking example of sensory-
sensory interaction is a strong, unidirectional projection from all auditory areas to all visual areas (large arrow with asterisk, see also (A, last panel). A weaker
projection specifically from VISal appears to bridge AUD and VIS areas and is noted with a dashed line.
(D) Summary of additional interactions between the auditory and visual areas with the primary somatosensory area (SSp-ll/tr) (left), and direct interactions of the
SSp-ll/tr and MOp-ll/tr with other nodes within the medial subnetworks (such as the ORB, ACA, PTLp, and RSP).
(E) Connectivity map of the ACAv and RSPv, which form another medial subnetwork to relay information from the SUBd to the medial prefrontal cortex (ORBm,
PL, and ILA).
Figure S7. Additional Information for the Lateral Subnetworks, Related to Figure 5
(A) Raw images of direct projections from the SSp and SSs to the AIp. These projections are unique to specific portions of the somatosensory area and are
predominately contralateral. Scale bars, 1 mm (top, middle) and 100 mm (bottom).
(B) Topographic inputs from other cortical areas to the TEa, ECT, and PERI. These projections help define different subregions within these structures. The most
anterior aspect (1) mostly receives inputs from orofacial related somatosensory and motor areas, followed by all other somatomotor inputs (2). These inputs dip
below other cortical inputs in (3) and (4) and remain segregated in the perirhinal (PERI) region. The infralimbic area provides a very strong and specific input to
caudo-ventral regions of TEa (4) and (5) and many medial network structures (e.g., PTLp, VIS, AUD) interact with the adjacent dorso-caudal aspect of TEa (5).

Zingg Et Al (2014) Neural Networks of The Mouse Neocortex

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Neural Networks of the Mouse Neocortex

SUMMARY mammalian species have been assembled on substantially

Figure 2. Weighted and Directed Cortico-Cortical Connectivity Matrices

Figure 3. The Somatic Sensorimotor Subnetworks

Figure 4. The Medial Subnetworks

Figure 5. The Lateral Subnetworks

Figure 6. The CLA and ENTl

EXTENDED EXPERIMENTAL PROCEDURES

Validation of Connectivity Data

Connectivity-Based Parcellations of the Somatosensory Cortical Areas

Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. S1

Imaging and Postacquisition Processing

Semiautomated Image Registration

S2 Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc.

Construction of Connectivity Matrix

Neuroanatomic Modules for Constructing Connectivity Matrices

Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. S3

S4 Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc.

Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. S5

S6 Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc.

Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. S7

S8 Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc.

Cell 156, 1096–1111, February 27, 2014 ª2014 Elsevier Inc. S9

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