Research Article
The Journal of Comparative Neurology
Research in Systems Neuroscience
DOI 10.1002/cne.23383
James C Dooley1, João G. Franca2, Adele M. H. Seelke1, Dylan F. Cooke1,3, Leah A.
Krubitzer1,3
1
Center for Neuroscience and 3Department of Psychology
University of California, Davis
Davis, California, 95618, USA
2
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, 219415902, Brazil
Running Head: Connections of somatosensory cortex in gray short5tailed opossums
Key Words: Marsupial, cortical evolution, multimodal cortex, 3a, 3b, posterior parietal
Corresponding Author:
Leah Krubitzer
Center for Neuroscience
1544 Newton Ct.
Davis, CA 95616
Phone:
530575758868
Email:
lakrubitzer@ucdavis.edu
Support: This project was supported by funds to Leah Krubitzer from NINDS (R21NS071225)
and NEI (R01 EY022987) to Joao Franca from CNPq – Brazil (proc. no. 200713/200856) and
James Dooley from NEI (T325EY015387505).
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/cne.23383
© 2013 Wiley Periodicals, Inc.
Received: Mar 06, 2013; Revised: May 29, 2013; Accepted: Jun 07, 2013
Journal of Comparative Neurology
The current experiment is one of a series of comparative studies in our laboratory designed to
determine the network of somatosensory areas that was present in the neocortex of the
mammalian common ancestor. Such knowledge is critical for appreciating the basic functional
circuitry that all mammals possess and how this circuitry was modified to generate species
specific, sensory mediated behavior. Our animal model, the gray short5tailed opossum
(
is a marsupial that is proposed to represent this ancestral state more
closely than most other marsupials and to some extent, even monotremes. We injected
neuroanatomical tracers into the primary somatosensory area (S1), rostral and caudal
somatosensory fields (SR and SC, respectively), and multimodal cortex (MM) and determined
their connections with other architectonically defined cortical fields. Our results show that S1
has dense intrinsic connections, dense projections from the frontal myelinated area (FM), and
moderate projections from S2 and SC. SR has strong projections from several areas, including
S1, SR, FM and piriform cortex. SC has dense projections from S1, moderate to strong
projections from other somatosensory areas, FM, along with connectivity from the primary (V1)
and second visual areas. Finally, MM had dense intrinsic connections, dense projections from
SC and V1, and moderate projections from S1. These data support the proposition that ancestral
mammals likely had at least four specifically interconnected somatosensory areas, along with at
least one multimodal area. We discuss the possibility that these additional somatosensory areas
(SC and SR) are homologous to somatosensory areas in eutherian mammals.
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The emergence of the neocortex and its expansion is the hallmark of mammalian
evolution (Krubitzer, 2007). While both comparative paleontology and modern genomic analysis
have provided exciting new insights into the chronology (Luo et al., 2003; Luo et al., 2011;
O'Leary et al., 2013), brain size and encephalization (Rowe et al., 2011), and interordinal
relationships (Hallstrom and Janke, 2008) of early mammals, our understanding of ancestral
brain organization remains limited; brain tissue does not fossilize, and cranial endocasts provide
information only on gross brain morphology and size. Yet brain organization is a window into
the behavior of our earliest ancestors, not only providing a view of how they perceived and
interacted with the world, but also what types of sensory5mediated behaviors were subject to
selection (Krubitzer and Seelke, 2012). The goal of the present investigation is to illuminate the
basic plan of cortical organization and connectivity of early mammals by examining the gray
short5tailed opossum, proposed to resemble this early ancestor more than other living species.
Although monotremes form the earliest mammalian radiation whose ancestors diverged
approximately 2075237 million years ago (MYA), they are highly derived, so studies of their
brain organization and connectivity provide useful, but somewhat limited information on the
brains of early, ancestral mammals (Woodburne et al., 2003; Pereira and Baker, 2006; van
Rheede et al., 2006; Hugall et al., 2007). Metatherians, including marsupials, diverged later than
monotremes (127.55190 MYA Meredith et al., 2011; O'Leary et al., 2013) and unlike
monotremes, many small brained, less derived members of this clade are thought to be
morphologically similar to ancestral mammals (Wong and Kaas, 2009; Rowe et al., 2011).
There are well over 300 species of marsupials in Australia and the Americas, and their external
morphology and behavior evolved in parallel to that of eutherian mammals to adapt to a
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multitude of terrestrial, arboreal and aquatic environments (Karlen and Krubitzer, 2007),
suggesting that a basic plan of body and brain organization transformed independently, but in a
similar manner, in these two major lineages. Despite their prolificacy and their importance for
understanding brain evolution, relatively little is known about the cortical sensorimotor circuitry
that drives their behavior.
The gray short5tailed opossum (
) is a small crepuscular marsupial
native to South America, and one of the few marsupial species currently bred in laboratory
conditions. We study
for several critical reasons. First, as noted above, because
marsupials’ ancestors arose very early in mammalian evolution, examining the cortical
organization and connectivity of modern marsupials could provide important insights into the
brains of the first mammals, allowing us to appreciate the basic cortical circuitry common to all
mammals. A second reason is that studies of marsupial motor cortex indicate that it is
completely embedded in or overlapping with somatosensory cortex (e.g. Lende, 1963a; Lende,
1963b; Lende, 1963c; Rees and Hore, 1970; Magalhaes5Castro and Saraiva, 1971; Beck et al.,
1996; Frost et al., 2000; Karlen and Krubitzer, 2007). This feature of the marsupial cortex is
reflected in patterns of connectivity of S1, which receives inputs associated with both somatic
and motor cortex from the thalamus (Killackey and Ebner, 1973; Joschko and Sanderson, 1987).
How this type of organization ultimately effects behavior is unclear since basic patterns of
behavior in marsupials are remarkably similar to their morphologically equivalent eutherian
counterparts (e.g. Australian striped possum vs. Malagsy aye aye; Erickson et al., 1998; Rawlins
and Handasyde, 2002). Third, marsupials are born extremely immature, at developmental time
points that correspond to embryonic events in eutherian mammals. These animals therefore make
excellent models of cortical development. Fourth, apart from this methodological advantage is
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the overlooked effect of this premature birth on the development of the somatosensory and motor
systems. Unlike most eutherian mammals, marsupials (particularly those that develop in a
pseudopouch like gray short5tailed opossums) have access to extremely early tactile inputs
before and during the development of central somatosensory pathways. How this impacts the
organization, connectivity and ultimate sensory mediated behavior of marsupials is an important
issue that has yet to be addressed.
Marsupials including
have been shown to have two complete
somatotopic representations within the cortex, the primary somatosensory area, S1, and the
second somatosensory area, S2 (Huffman et al., 1999; Catania et al., 2000; Frost et al., 2000;
Karlen and Krubitzer, 2007). Based on electrophysiological recordings, architectonic analysis
and patterns of connections, some studies indicate that marsupials have two to three additional
fields associated with somatosensory processing; a rostral field termed R or SR, a caudal field
termed C or SC, and a parietal ventral area, PV (Beck et al., 1996; Elston and Manger, 1999;
Huffman et al., 1999; Wong and Kaas, 2009; Anomal et al., 2011). Despite the important
phylogenetic position of marsupials, and the implications that cortical processing networks of
early mammals may be more complex than were previously thought, little is known about the
cortical connectivity of somatosensory areas in this group of mammals (see discussion).
The specific aim of the present investigation was to examine the cortical connections of
three somatosensory fields in
, S1, SR and SC. This was done by
injecting anatomical tracers into architectonically and/or electrophysiologically defined locations
within these fields, and relating patterns of connectivity to architectonically defined cortical
fields in the ipsilateral and contralateral hemisphere. These data from each hemisphere were
then quantified so that relative density of connections could be assessed. Comparisons of
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opossum cortical circuitry with other marsupials, monotremes and eutherian mammals indicate
that its cortical network likely reflects that of the common mammalian ancestor better than that
of the highly derived extant monotremes.
A total of 7 adult opossums (
), including 4 males and 3 females,
were used for these experiments. Animals ranged in age from 7 to 19 months old and weighed
between 73 and 128 g. The animals were housed in standard laboratory cages in which food and
water were available
and maintained on a 125hour light/dark cycle. All experiments
were performed under National Institutes of Health guidelines for the care of animals in research
and all protocols were approved by the Institutional Animal Care and Use Committee of the
University of California, Davis.
Animals were placed in an induction chamber and anesthetized with the inhalant
anesthetic isoflurane (153%). After induction, a specially fitted mask was placed over the
animal’s snout and the surgical plane of anesthesia was maintained with 152% isoflurane. Once
anesthetized, 2% lidocaine was injected subcutaneously at the scalp and around the ears, and the
animals were placed in a stereotaxic apparatus. Animals were given dexamethasone (0.452.0
mg/kg, intramuscularly, IM) and atropine (0.04 mg/kg, IM) at the start of surgery. Temperature
was maintained by placing the animal on a heating pad, and body temperature and respiration
were monitored throughout the experiment. An incision was made at the midline of the scalp and
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a small craniotomy was performed over cortical fields S1, SR, SC or MM. The exposed cortex
was photographed and this image was used to record the position of tracer injections relative to
blood vessels. A custom5beveled Hamilton syringe was lowered approximately 300 Lm into the
cortex, and 0.350.4 LL of 10% fluoro5emerald (FE), fluoro5ruby (FR), or biotinylated dextran
amine (BDA; Molecular Probes, Eugene, OR) was injected into the cortex. Several animals
received multiple tracer injections, resulting in a total of eleven injections in seven animals (see
Table 2). Two injections spread into surrounding areas, and were not included in quantitative
analysis, however were included as figures. The opening was then covered with an acrylic skull
cap or bone wax, and the temporal muscle and skin were sutured. Following the injection,
antibiotics (Baytril, 5 mg/kg, IM) and analgesics (buprenorphine, 0.03 mg/kg, IM) were
administered. The animal was allowed to recover for 557 days, enabling transport of the tracer.
Following this recovery period, animals were euthanized with an overdose of sodium
pentobarbital (Beuthanasia; 250 mg/kg, IP) and transcardially perfused with 0.9% saline,
followed by 4% paraformaldehyde in phosphate buffer (pH 7.4), and then 4% paraformaldehyde
in 10% phosphate buffered sucrose.
In three cases (08580, 09532, and 12518) electrophysiological recording experiments were
performed following the recovery period to confirm the placement of the injections. For these
terminal mapping procedures, subjects were anesthetized using either 152% isoflurane or 30%
urethane in propylene glycol (1.25 g/kg, IP). All other surgical procedures are like those
described above for neuroanatomical injection experiments, except a larger craniotomy was
made and the dura over S1 and the surrounding cortical regions was retracted. Digital images
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were taken so that electrophysiological recording sites could be directly related to cortical
vasculature.
Multiunit electrophysiological recordings of somatosensory cortex were performed using
tungsten microelectrodes (0.010 inches, 5 M ; A5M Systems, Inc., Sequim, WA). The electrode
was lowered into cortical layer 4, at a depth of approximately 2005400 Lm below the pial
surface. Multi5unit activity was amplified and filtered (100–5000 Hz; A5M Systems Model 1800
Microelectrode AC Amplifier; A5M Systems, Carlsborg, WA), captured and quantified (CED
Power1401 mk II hardware with Spike2 software), monitored through a loudspeaker, and
visualized on a computer monitor. At each recording site responses to somatosensory stimulation
were identified. Somatosensory stimulation consisted of light taps, displacement of hairs, light
brushing of skin with a paintbrush, hard taps, and manipulation of muscles and joints.
Descriptions of the receptive fields and the type of stimulus required to elicit a response were
documented and drawn on illustrations of the opossum body. Responses were recorded at
multiple recording sites (approximately 500 Lm apart). The location of each recording site was
marked on the digital image of the cortical surface relative to the vascular pattern, and was used
to aid in the process of tissue reconstruction and receptive field quantification. Following
electrophysiological recording, animals were euthanized with an overdose of sodium
pentobarbital (Beuthanasia; 250 mg/kg IP) and transcardially perfused with 0.9% saline,
followed by 4% paraformaldehyde in phosphate buffer, and then 4% paraformaldehyde in 10%
phosphate buffered sucrose.
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Following perfusion, the brain was extracted, weighed, and photographed, and the cortex was
removed from the subcortical structures and manually flattened between glass slides. The
flattened cortex was sectioned at 30 Lm using a freezing microtome. Alternating cortical
sections were stained for myelin or mounted for fluorescent microscopy. In cases in which BDA
was injected, tissue was divided into three series, one of which was reacted for BDA (Vectastain
Elite; Vector Laboratories, Burlingame, CA). In one case, the brain was cut in the coronal plane
at 40 Lm, and alternate sections were stained for cytochrome oxidase (CO) or mounted for
fluorescent microscopy. In this coronally sectioned case, block face images were taken of each
section to aid in 3D reconstruction (see Data analysis).
In each case in which the cortex was flattened, camera lucida reconstructions of
individual myelin sections were made on a stereomicroscope (Fig. 1A – B). As described
previously (Seelke et al., 2012), although individual sections can contain many partial
anatomical boundaries, the entire series of sections must be examined and combined into a single
comprehensive reconstruction to determine the full extent of cortical field boundaries. Each
reconstructed section contained the outline of the section, blood vessels, tissue artifacts, probes,
visible electrode tracks, and architectonic borders of cortical fields. Sections were aligned using
these landmarks and compiled into one composite image. When necessary, brightness and
contrast were adjusted of photomicrographs using Adobe Photoshop.
Injection sites (Fig. 1C) and retrogradely labeled cell bodies (Fig. 1D5E) were plotted
using an X/Y stage encoding system (MD Plot, Minnesota Datametrics, MN) that was mounted
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to a fluorescent microscope and connected to a computer. Blood vessels and tissue artifacts were
used to align connectional data from multiple sections and architectonic data from tissue stained
for myelin; all data were combined into one comprehensive reconstruction that contained
injection sites, retrogradely labeled cells and architectonic boundaries of cortical fields. These
methods have been described previously (Campi and Krubitzer, 2010; Cooke et al., 2012).
In the coronally sectioned case (12519) alternating sections were stained for CO and
fluorescence, and fluorescent sections containing labeled cells were reconstructed as described
above. However, rather than a compressed, flattened reconstruction, all sections were then
aligned to the section outlines from block face images, and imported to 3D reconstruction
software (Amira 3.1, Visage Imaging). Cortical field boundaries were reconstructed onto the
adjacent fluorescent sections, and the total number of labeled cells within a cortical area across
all sections was calculated.
Connectional data were quantified by calculating the percentage of labeled cells in each
cortical field. This was calculated separately for each hemisphere: in any given cortical field this
value was expressed as the percentage of total cells counted in that hemisphere. Labeled cells
inside the injection halo where extracellular label indicates the extent of passive tracer spread
were not included. This allowed us to normalize the data across cases and across different size
injections. Means were calculated from multiple injections in different animals using only
injections whose halos were entirely contained within the injected field. We used this data to
assign connection strength when describing our connections for each cortical field. The criteria
used are:
Strong: >10%
Moderate: 9% to 3%
Weak: 3% to 1%
Intermittent: >3% and inconsistent
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The goal of this study was to examine the corticocortical connections of S1 and
surrounding fields in
. Regardless of which hemisphere was injected (see
Table 2 for details), all cases were illustrated with injections shown in the left hemisphere for
ease of comparison across cases. Below we describe the architectonic boundaries of cortical
fields in
followed by descriptions of ipsilateral and contralateral connections of S1,
SR, SC and cortex immediately caudal to SC termed MM (see Table 1 for abbreviations).
!
"
#
Cortical field boundaries determined with myelin and other stains have been described
previously in
by our own and other laboratories (Huffman et al., 1999; Catania et
al., 2000; Frost et al., 2000; Kahn et al., 2000; Karlen and Krubitzer, 2006; Wong and Kaas,
2009). In several of these studies, cortical architecture was directly related to
electrophysiologically identified boundaries of S1, S2, SR, SC, V1 and V2. As in previous
studies, primary sensory areas including S1, V1, and A1 were densely myelinated and their
boundaries were readily determined in all animals (Fig. 1A). In Monodelphis, the extrastriate
region termed V2 consists of a lightly myelinated area immediately rostral to V1. While V2 has
not been extensively mapped, previous studies have shown that, as in other species, at the V1/V2
border there is a reversal in the progression of receptive field locations. Both functional and
architectonic evidence indicate it is approximately 0.5 mm wide (Kahn et al., 2000). The densely
myelinated S1 has been shown to be co5extensive with a complete representation of the
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contralateral body, and neurons in this field respond to cutaneous stimulation (Huffman et al.,
1999; Catania et al., 2000; Frost et al., 2000). The lateral portion of this field also contains a
motor map of the face (Frost et al., 2000). The darkly myelinated V1 has been shown to be co5
extensive with a retinotopic map of the contralateral hemifield (Kahn et al., 2000), as described
in all mammals studied. Although A1 was not defined eletrophysiologically in
, its
functional organization has been described in Australian quolls (Aitkin et al., 1986) and other
small brained eutherian mammals such as mice (Stiebler et al., 1997), and it is in the location and
has the appearance of the A1 we describe in
$ Further, in favorable preparations
neurons in this densely myelinated region in Monodelphis respond well to auditory stimulation
(Kahn and Krubitzer, 2002). For these reasons we term this field A1 in
$ Other areas
that were darkly myelinated include S2, the caudotemporal areas (CT), and the frontal
myelinated area (FM). Of these, S2 has been shown to be co5extensive with a functional map of
the body in a range of marsupials (Beck et al., 1996; Huffman et al., 1999). SC was a moderately
myelinated field bounded rostrally by S1 and laterally by S2. Caudally, SC borders a slightly
less myelinated field that previous studies have shown to be co5extensive with multimodal (MM)
cortex (Kahn and Krubitzer, 2002; Karlen and Krubitzer, 2006). MM is bounded caudally by V2
(Fig. 1). The moderately myelinated SR is bounded caudally by S1 and rostrolaterally by FM.
Without functional data, the rostromedial boundary of SR is difficult to determine because it
abuts cortex that is similar in appearance (i.e. moderately myelinated).
In three cases, electrophysiological recordings were made in and around the injections
sites (e.g. see Figs. 2, 4, and 7). Consistent with previous studies in
other marsupials, S1 contained neurons that were responsive to cutaneous stimulation of the
contralateral body. Although our mapping density was low, a topographic organization was
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found within S1 similar to other studies in which S1 was mapped in detail (Catania et al., 2000;
Frost et al., 2000). The goal of our mapping experiments was to define the body part
representation injected and the functional borders of S1, not to generate comprehensive maps.
This was possible in three cases in which anesthetic conditions were optimal.
!
% &
In 5 cases, injections were placed within S1 (Table 2, Figs 2 – 4; two cases not shown).
In 4 of these cases, injection sites and their halo were under 0.15 mm2 while the final case, the
injection site was 1.6 mm2 as measured in the flattened cortical sections (Fig. 4). The first 4
injections were entirely contained within S1, while the final injection slightly extended into
cortex at the rostral and caudal boundaries of S1 (in SR and SC). For this reason this final case
(08580) was not included in the statistical analysis for S1.
In two cases, injections were placed in lateral portions of S1, and electrophysiological
mapping indicated that these injections were within portions of the face representation (Figs. 2C
and 4C). In the other two cases, injections were placed in more medial portions of the field, in
the expected locations of the forelimb representation (Fig. 3; other case not shown). The overall
patterns of connections were similar in all cases. Most labeled cells were found within S1 (mean
= 72.6%, Fig. 5A, Table 3). Much of this intrinsic labeling was concentrated close to the
injection site, however in all cases labeled cells were seen throughout S1 (Figs. 254) and was not
exclusively localized to the representation of a particular region of the body. This result was
consistent for all injections, regardless of whether they were placed into the medially located
forepaw/body representation or the laterally located face representation. Moderate label was also
found in FM (mean 10.6%), and sparse labeling was observed in S2, SC and SR (mean 4.8%,
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3.9% and 1.6% respectively). In three cases (Figs. 2A; 4A; case 12519) sparse labeling was
observed in MM, A1, V1, and V2, but each of these regions contained less than 1% of labeled
cells (Fig. 5A). In two cases (Fig. 2A; case 12519), sparse labeling was observed on the medial
wall, just caudal to S1.
Within the contralateral hemisphere, in all cases the majority of labeled cells were found
in locations that were homotopic to both the injection site in the opposite hemisphere and to
areas that had dense ipsilateral label. Further, the overall patterns of interhemispheric
connectivity were similar across cases (Figs. 2B, 3B and 4B). As in the ipsilateral hemisphere,
the majority of label was in S1 (mean = 43.5%), and was not restricted to representation of a
particular region of the body. Other regions projecting contralaterally to S1 included dense label
in FM (mean = 22.2%), and moderate label in SC and SR (mean = 6.2% and 8.3%, respectively).
S2 contained sparse label (mean = 2.0%), and less than 1% of label was observed in other
sensory areas including A1, V1 and V2. Unsurprisingly, the most widespread and dense patterns
of ipsilateral and contralateral connections were observed in the case which had the largest
injection, which spread into adjacent fields (Fig. 4), however this injection also showed some
characteristics of connections found to SC and SR (i.e., Figs. 6, 7, and 8). Specifically, the
injected hemisphere showed higher than expected label in SC (9.6%), SR (9.9%), MM (4.5%)
and A1 (2.1%) compared to injections entirely contained within S1. Similarly, the contralateral
hemisphere showed increased label to SC (13.1%) and MM (5.6%, see Table 3).
!
%
Two cases had injections in SR; one injection was restricted to the boundaries of SR
(case 08580; Fig. 6) and one injection spread into S1 and FM (case 09532; Fig. 7). The
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calculation of cell percentage illustrated in Figure 5, cited below, and listed in Table 3 is taken
only from the case in which the injection was clearly restricted to SR (08580; Fig. 6), however
patterns of connections were largely similar between the two cases, and both clearly differed
from the patterns of S1 connections (09532; Fig. 7). Specifically, both cases show extensive
projections from frontal cortex, dense connections with the rostral portion of S1, piriform and
entorhinal cortex, and distributed connections with areas caudal to S1 including visual cortex.
The primary difference between these two cases is an increase in projections from entorhinal and
piriform cortex in the case not restricted to SR. For the case restricted to SR, intrinsic
connections to SR accounted for 22.5% of labeled cells, while area FM contained 15.1% of
labeled cells (Fig. 5B). Both S1 and piriform cortex also contained significant proportions of
labeled cells (11.1% and 15.0%, respectively). We found additional connections with S2 (3.6%),
SC (3.4%), V1 (4.0%) and A1 (2.4%). In general, the pattern of connectivity of SR was much
more distributed compared to connections of S1 and included projections from auditory, visual
and multimodal areas as well as piriform cortex.
Contralateral connectivity was also broadly distributed and appeared to be most dense in
the homotopic locations that had dense ipsilateral connections (Figs 5B, 6B, 7B). Thus, label was
most dense in areas SR (10.9%), FM (19.6%), and S1 (22.4%) and moderate in SC (4.4%).
However, there were two notable trends in which contralateral connectivity differed. In both
cases there were proportionately fewer contralateral projections from piriform cortex (0.2%)
compared to ipsilateral connections (15%, Fig. 6, 7). Additionally, one case (08580) showed
moderate projections from contralateral MM (4.3%), V1 (5.9%), and V2 (3.2%), increased
connectivity than was found in the ipsilateral hemisphere.
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!
% !
Four injection sites were located caudal to S1, two of these cases were primarily located
in SC, with the injection halos partially extending into S1 (Fig. 8A; Fig. 8B) and two cases were
placed caudal to this in MM (Fig. 9). While both injections into SC show similar patterns of
connections, the results from one of these cases were not included in quantitative analysis due to
damage to the cortical surface. For the SC injections, the majority of labeled cells were in S1
(60.4%). These labeled cells were not evenly distributed throughout S1, but were located in a
dense focus adjacent to the injection site, with moderate labeling scattered throughout S1 (Fig.
8A, 8B). Moderate to sparse labeling was also observed intrinsically within SC (3.9%), S2
(9.9%), MM (0.4%), V2 (0.6%) and V1 (1.2%; see Fig. 5C). In one case label was observed in
SR and FM (12.6 and 10.1, respectively) along with more extensive labeling throughout S1, but
this may be due to the slight spread of the injection site into S1 (Fig. 8B).
Contralateral label resulting from SC injections was most dense in mediolateral locations
in S1 (46.8%), SC (12.6%), and S2 (9.1%; Fig. 8E, 8F) that matched the mediolateral location of
the injection site in SC. In both cases, relatively dense label was also observed in FM (12.6%) in
the contralateral hemisphere (see Fig. 5C), although label in FM was only observed in the
ipsilateral hemisphere in one case.
In two cases, the injection sites were located more caudal compared to the previously
described injections. Previous studies in which electrophysiological recordings were made in
this region indicate that neurons are multimodal, responding to somatosensory, auditory or visual
stimuli, and this field was termed MM. These connections differed from connections of SC in
that the majority of labeled cells were observed in SC (29.4%), MM (32.9%) and V1 (20.4%;
Figs. 5D, 9A, 9C). Moderate to sparse label was observed in S1 (6.3%), S2 (2.9%) and A1
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(1.1%), and in one case, SR (1.4%). Contralateral projections were only determined in one case,
due to lack of transport in the other case (Fig. 9B). Label was most dense in MM (24.2%), SC
(14.1%), S1 (12.8%) and V1 (24.2%; Fig. 5D; 9B). Moderate to sparse label was observed in
other cortical fields including S2 (3.6%), FM (4.0%) and SR (0.2%).
The present investigation is part of a series of ongoing studies in our laboratory designed to
understand how the brains of our common ancestors were organized. Our study builds upon
recent developments exploring the issue of gross brain morphology in both mammaliaforms and
early crown mammals that have been discussed in current literature, particularly in genomic and
comparative paleontological studies (Luo et al., 2011; Rowe et al., 2011; O'Leary et al., 2013).
While the issue of brain organization of early mammals has been deliberated by our own and
other laboratories, the present study is the first comprehensive study of connectivity of multiple
cortical fields in any marsupial. Our study, interpreted in the context of studies in other
mammals, represents the only available avenue for understanding how the soft tissue of the brain
was organized and interconnected in early mammals. In turn, this prototypical organization
informs our understanding of how modifications to this organization ultimately generated the
remarkable diversity in behavior of extant species including humans.
!
!
%
!
'
While several studies have used electrophysiological recording methods to examine the
functional organization of somatosensory cortex in marsupials (Huffman et al., 1999; Catania et
al., 2000; Frost et al., 2000; for review see Karlen and Krubitzer, 2007), studies of corticocortical
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connections of somatosensory cortex are more limited. To date, the cortical connectivity of
primary somatosensory cortex has only been investigated in three marsupials (Fig. 10). These
include the North American Virginia opossum,
Australian brush5tailed possum,
American big5eared opossum,
(
(
(Beck et al., 1996), the
(Elston and Manger, 1999), and the South
(Anomal et al., 2011). All of these studies report
dense ipsilateral connections with other somatosensory areas, including S2, PV, SR, SC, and a
region of cortex immediately lateral to S1, in and around the rhinal sulcus (we term this
entorhinal cortex; EC). In the big5eared opossum and the brush5tailed possum, moderate to
sparse connections were also observed with cortex immediately caudal to SC (PS, peristriate
cortex in $
and PP, posterior parietal cortex in $ (
rostral to SR, and $
), with cortex immediately
, in cortex in the location of auditory cortex in other marsupials and
other mammals in. Interhemispheric connections via the anterior commissure where only
described for S1 in the brush5tailed possum and these were in a homotopic location with S1 and
with SR, PV and EC.
Our data in
are similar to those reported in these previous studies of
marsupials with a few important exceptions. First, unlike other marsupials, intrinsic connections
of S1 were not restricted to the representation of the body part injected, but instead extended
throughout S1 and this will be discussed below. Additionally, in contrast to most other
mammals, electrophysiological recording studies indicate that
does not have a
parietal ventral area (Catania et al., 2000; Frost et al., 2000), and connections did not appear to
differentiate S2 from other cortical fields in this region. While we did observe projections from
S2 (as in other marsupials), the degree of projections from S2 varied considerably between cases.
This is likely due to the location of injections in S1 and the nature of topographic interactions
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between S1 and S2. The laterally placed S1 injection was made into the vibrissae/naris
representation of the animal (see Fig. 2C), and the face representation occupies a large portion of
S2 (Catania et al., 2000; Frost et al., 2000). However, the medially placed injection was in the
expected representation of the forepaw or body; this representation occupies only a very small
lateral portion of S2 (Fig. 3A). Thus, these data suggest that unlike intrinsic connections of S1,
connections with S2 are more tightly topographically organized. Further, we only saw very
sparse connections with EC in a few animals, and moderate to dense connectivity with area FM,
a heavily myelinated area rostral to the lateral portion of S1 and lateral to SR. The function of
FM is not known. As with S2, the percent of labeled neurons in FM projecting to S1 differed
between cases, and the ambiguous function of this area in opossums make it difficult to speculate
why this difference was found. However, this observed difference may be due to the location of
the injection within S1, an idea supported by the observation that medial portions (representing
the body and forepaws) of S1 showed greater connectivity with FM than lateral (representing the
face) portions of S1. While previous marsupial studies demonstrate a similar darkly myelinated
region of cortex in the location of our FM (e.g. see Fig 13B of Beck et al., 1996; and Fig. 5A of
Elston and Manger, 1999; and Fig. 2A of Anomal et al., 2011), only the study in $
(Anomal et al., 2011) demonstrates dense projections with cortex in this region (termed the
frontral region rather than FM). Other studies show sparse connections with this field. As in the
brush5tailed possum, dense interhemispheric projections were from S1 and SR of the opposite
hemisphere. However, we also observed interhemispheric projections from S2 (rather than PV),
and from SC and FM (not reported in possums) to S1.
There are several factors that may contribute to the differences and similarities in
organization and connectivity observed between
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and other marsupials investigated.
Journal of Comparative Neurology
The first is phylogeny. Virginia opossums are relatively closely related to the gray short5tailed
opossum (both from the Didelphidae family) and both appear to have similar brain organization
and patterns of connectivity of S1, while the more distantly related brush5tailed possum differs
more significantly from these didelphids in both cortical organization, connections and cortical
sheet size. Another possibility is that of brain size. The gray short5tailed opossum has a very
small brain (~0.8 g) compared to the brush5tailed possum (11 g) and Virginia opossum (6.7 g).
Several studies have shown that smaller brains often have more widespread connections
compared to larger brains (Burkhalter and Charles, 1990), and that more local processing occurs
in larger brains (Manger et al., 1998; for review see Kaas, 2012). Thus, some differences in
connection patterns may be due to differences in processing strategies evolved in larger versus
small brains. Finally, it is possible that differences in connectivity are due to differences in
lifestyle and geographic dispersion. For example, the big5eared and gray short5tailed opossums
have remarkably similar patterns of connectivity. While phylogeny may explain some of this
similarity, it is important to consider that both species are omnivorous marsupials (primarily
carnivorous in the wild), have partially overlapping habitats in Brazil, Paraguay, and Argentina,
and are both predominantly terrestrial foragers and hunters that are also scansorial (have adapted
the ability to climb). Thus, adaptations associated with niche and lifestyle may also contribute to
similarities and differences in patterns of connectivity.
!
!
% &
In monotremes, an order of mammals whose ancestors branched off early in evolution,
connectional data are extremely limited. In both platypus and echidnas, several different
somatosensory areas have been identified, including S1, a second somatosensory area, which
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may correspond to S2 or PV or some combination of both fields, and a rostral area in which
neurons are responsive to stimulation of deep receptors (R or SR; Krubitzer et al., 1995).
Injections in S1 in the echidna result in a similar basic pattern of connectivity to that described
for marsupials, but connections are more limited in extent. S1 has dense intrinsic connections
and moderate connections with a field immediately rostral, (R/SR) and caudal to S1, although,
due to its organization, the caudal field is likely to be S2/PV rather than SC. S1 receives
moderate projections from cortex immediately rostral to SR, and light connections with V1 (See
Fig. 10).
In contrast to monotremes, there are numerous studies on connections of S1 in eutherian
mammals, and a complete review is beyond the scope of this discussion. However, comparisons
of eutherian mammals with small to moderate sized brains, as well as those that occupy a variety
of ecological niches indicate that a similar basic pattern of connectivity of S1 is present
(Manzoni et al., 1989; Krubitzer et al., 1993; Catania and Kaas, 2001; Kaas, 2011). In these
species S1 has dense intrinsic connections, and extrinsic connections with one or more lateral
fields corresponding to S2 and PV, with cortex immediately caudal to S1, connections with
cortex immediately rostral to S1, and often with cortex located far rostrally (Krubitzer et al.,
1986, squirrel; Chapin et al., 1987, rat; Weller et al., 1987, tree shrew; Schwark et al., 1992, cat;
Juliano et al., 1996, ferret).
Although the field immediately caudal to S1 is known by a variety of names in different
species, such as PPC (Reep et al., 1994) and PM (Slutsky et al., 2000), neurons here often
respond to deep somatic stimulation or are multimodal (Slutsky et al., 2000). Terminology for
cortex immediately rostral to S1 has also varied (DZ, Chapin and Lin, 1984; R, Slutsky et al.,
2000; 3a, Wong and Kaas, 2008; Cooke et al., 2012), but most studies indicate that like the
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Journal of Comparative Neurology
marsupials in which this region of cortex has been explored extensively (Huffman et al., 1999),
neurons in this region respond to stimulation of deep receptors of the skin, muscles and joints
(Chapin and Lin, 1984; Slutsky et al., 2000; Cooke et al., 2012; see Krubitzer et al., 2011 for
review).
One obvious difference in connectivity is the presence of an architectonically distinct M1
in eutherian mammals with extensive projections to S1. In some eutherians such as rats, S1 and
M1 partially overlap at the representation of the hindlimb and forelimb (Donoghue and Wise,
1982). In contrast, in other rodents such as squirrels (Cooke et al., 2012) and in other eutherians
such as primates (Powell and Mountcastle, 1959; Stepniewska et al., 1993; Huffman and
Krubitzer, 2001) the motor map of M1 is a distinct field, separated from S1 by area 3a (see
Krubitzer et al., 2011 for review). On the other hand, S1 and the presumptive M1 in marsupials
almost completely overlap (Beck et al., 1996; see Karlen and Krubitzer, 2007 for review) and in
gray short5tailed opossums there is not even evidence for a complete motor representation, since
microstimulation in S1 only elicits movements of the face (Frost et al., 2000). Further, there is
still some debate about whether marsupials even have an M1 that is homologous to M1 described
in eutherian mammals (Beck et al., 1996). Thus, direct comparisons of connectivity to S1, even
between different species of marsupials, can be difficult to interpret, because the presence of an
M1, its location, and its extent is still contentious in marsupials.
%
% &
One of the most novel findings in the gray short5tailed opossum was broadly distributed
intrinsic connections of S1. This was true both for laterally placed injections in the face
representation (Fig. 2A) as well as medially placed injections in the forepaw representation (Fig.
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3A) suggesting that information across the body is highly integrated within S1. This pattern of
intrinsic connectivity does not appear to be a general feature of marsupials. A single injection in
the forepaw representation of S1 in the big5eared opossum shows that, despite a large injection
site, intrinsic connections are mostly restricted to the medially located forepaw/body
representations within S1 (see Fig. 8A of Anomal et al., 2011). Additionally, injections placed
both medially and laterally in the Virginia opossum show similar topographic restriction between
body and face representations (see Fig. 16 and 17 of Beck et al., 1996). The more distantly
related Brush5tailed possum’s S1 also has topographically restricted intrinsic projections (Elston
and Manger, 1999).
A more extensive comparative analysis of non5marsupial mammals provides further
support that the pattern of intrinsic connectivity of S1 observed in the present investigation is not
a general mammalian feature.. Small5brained eutherian mammals such as rats, star5nosed moles
(Catania and Kaas, 2001), and naked mole rats (Henry and Catania, 2006) largely have restricted
intrinsic connectivity of S1 suggesting the pattern observed in the present study is not simply a
feature of a small brains. Other mammals, including squirrels (Krubitzer et al., 1986), tree
shrews (Weller et al., 1987), cats (Schwark et al., 1992), and a variety of primates (e.g. Jones et
al., 1978; Krubitzer and Kaas, 1990; Fang et al., 2002) show similar topographic restriction of
intrinsic connection of S1 (3b). Interestingly, injections of S1 in monotremes (echidna)
demonstrate scattered connections throughout S1 (Krubitzer, 1998). Thus, broadly distributed
intrinsic connections of S1 across topographically mismatched body part representations appear
to be observed in only two species examined (echidna and short5tailed opossum), both of which
may have retained aspects of organization from their shared common ancestor.
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Journal of Comparative Neurology
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While the limited data in monotremes indicates that broadly distributed intrinsic
connection are, in part, due to retention of a primitive form, there are several additional factors
which may contribute to this type of intrinsic processing within S1 in
. Of all the
marsupial species studied, the gray short5tailed opossum is the only one not reared in a pouch.
Thus, during development these pups naturally have access to more tactile stimulation than other
marsupials, and certainly experience more early tactile stimulation for all body parts than
eutharian mammals. This could lead to an enhanced integration of information across the body
compared to integration of information only for closely related body parts. Another possibility is
that these inputs serve to modulate response on the snout. Electrophysiological studies have
shown that the facial representation of the gray short5tailed opossum is expanded relative to the
representation of the rest of the body (Catania and Kaas, 2000; Frost et al., 2000), suggesting
functional importance of this area. Thus, it is possible that these inputs from different parts of S1
may be modulating the response of the snout. Finally, while the gray short5tailed opossum has
cortical somatic representation of its entire body, as mentioned previously motor responses have
only been elicited through microstimulation in lateral portions of S1, and have only resulted in
movements of the face (Frost et al., 2000). Thus, these widespread inputs across S1 may, to some
extent, represent connectivity with the gray short5tailed opossum “motor cortex”. This idea is
particularly intriguing, as it could explain a difference in connectivity with the Virginia opossum,
which has been shown to have a motor map which overlaps completely with somatosensory
cortex (Beck et al., 1996).
!
%
!
(
%
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)
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Journal of Comparative Neurology
Based on receptive field characteristics and stimulus preference, electrophysiological recording
studies have identified fields SC and SR in the Virginia opossum (Beck et al., 1996), the brush5
tailed possum (Elston and Manger, 1999), the northern quoll, the striped possum (Huffman et al.,
1999) and the big5eared opossum (Anomal et al., 2011). While other electrophysiological
recording studies in the gray short5tailed opossum have not differentiated SR and SC based on
neural response properties (Catania et al., 2000; Frost et al., 2000; present study), this is likely
due to anesthetic recording conditions. When anesthetic preparation was optimal and responses
could be studied in SR and SC in different marsupials, neurons in these areas responded to
stimulation of deep receptors in muscles and joints, had large receptive fields and contained
course topography of the contralateral body. A smaller percentage of recording sites in SR and
SC contained neurons that responded to cutaneous stimulation. Further, these previous studies
describe SC and SR as moderately to lightly myelinated fields compared to S1, and studies of
connections of S1 add additional evidence to support a role in somatosensory processing for
these two fields. However, to date there are no studies of connections of SR in any marsupial
and only one study has described the connections of SC by injecting neuroanatomical tracers into
this field (Anomal et al., 2011). In the big5eared opossum, connections of SC were similar to
those described in the present investigation for
(compare Fig. 6A of Anomal et al.,
2011 with Fig. 8A of the present study). They found strong intrinsic connectivity, as well as
connections to S2/PV and a region just caudal to SC termed peristriate cortex. While peristriate
cortex generally refers to cortex surrounding V1 (such as V2), the region of cortex labeled as
“peristriate” in the big5eared opossum was large, stretching from the rostral boundary of V1 to
the caudal boundary of SC, and likely contained V2 caudally as well as cortex that may be
homologous to MM in this study (see below). Anomal and colleagues (2011) also reported weak
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Journal of Comparative Neurology
projections to FM, SR, V1 and A1. The presence of visual and auditory projections to SC in
these marsupials indicates that SC is also involved in multimodal processing.
In the present study, injections in MM produced a pattern of connections that was distinct
from that of the more rostrally5located SC. Most noteworthy were the relatively strong
projections from non5somatosensory areas including V1 and V2. There is only one other study
of marsupials in which one injection was placed in a similarly located region (termed PP) in
brush5tailed possums (Elston and Manger, 1999). The patterns of connections observed in
brush5tailed possums were similar to those of gray short5tailed opossums in that dense
projections were observed from visual and auditory areas, and sparse projections were observed
from S1, S2, PV, and cortex rostral to S1 in what may be equivalent to SR and FM in the current
investigation. In terms of contralateral connectivity, in both gray short5tailed opossum’s MM and
brush5tailed opossum’s PP, projections were seen from V1 and V2, SC, along with sparse
projections from S1and S2. In small eutherians like rats, a field in a similar location (PPC) also
receives convergent sensory inputs from visual and somatosensory areas (Reep et al., 1994)
suggesting that this area may have a similar integrative role in sensory processing. Further,
although more data need to be collected from eutherian mammals other than mice and rats, it is
possible that this multimodal region is a primitive form of the greatly expanded posterior parietal
cortex of primates.
As mentioned previously, this is the first study investigating cortical connections of SR in
marsupials. In squirrels, the field immediately rostral to S1 (termed 3a) is densely connected to
motor cortex, S1 and PP, and sparsely connected to PV/S2, but does not have connections with F
(likely equivalent to marsupial FM; Cooke et al., 2012). Other studies in this rostral field in
rodents show this pattern of connectivity with S1, PP and motor cortex (Lee et al., 2011; for
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review see Krubitzer et al., 2011). While
SR receives projections from S1 as well
as SC and MM, unlike rodents, they also have additional projections from FM and piriform
cortex. In both
and other mammals, this rostral strip (SR and 3a, respectively) is
poorly myelinated and poorly laminated (Wong and Kaas, 2009). Thus, while marsupials and
rodents have areas rostral to S1 that contain neurons with similar somatosensory response
properties, architectonic appearance, and connections with S1, the marked differences in
connectivity between these areas suggest differences in function. Further, the issue of homology
of cortical areas rostral to S1 in marsupials and eutherian mammals is complicated by the current
uncertainty on the status of motor cortex in marsupials.
One of our SR injections included portions of FM and S1 (Fig. 7), the pattern of
connectivity of this injection was remarkably similar, with the exception of a larger percentage
of connectivity with EC and piriform cortex (15% in the restricted injection vs. 38.9% for the
injection containing FM). Since the primary difference between these injections is the inclusion
of FM, we would therefore hypothesize that FM is extensively connected with EC and piriform
cortex. While further investigation of this hypothesis is needed, it is supported by an injection
restricted to the heavily myelinated area F in squirrel, which also projects to a rhinal region of
cortex (termed the parietal rhinal area; Cooke et al., 2012).
Comparisons across major groups of mammals indicate that there is a common plan of
somatosensory cortex, composed of four interconnected fields, which to some degree is
represented in marsupials, monotremes, and eutherian mammals (Fig 10). Further, the present
study provides evidence suggesting additional fields such as FM and MM are components of this
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Journal of Comparative Neurology
plan as well. Our connectional data for multiple cortical fields associated with somatosensory
processing are supported by electrophysiological recording data from these different fields in
most marsupials examined. On the other hand, monotremes appear to have a relatively simple
network of somatosensory areas (S1, SR and S2 or S2/PV) compared to marsupials. While
monotremes radiated earlier in evolution than marsupials, due to specializations like the bill,
extant monotremes may have lost cortical areas (e.g. SC, MM, FM) and associated connections
to support the enormous expansion of the bill representation in S1 and the acquisition of
electrosensory reception. Because of these derivations, we propose that the organization and
connectivity of marsupial neocortex, particularly those that are terrestrial/scansorial with small
brains, may better reflect the ancestral state than that of monotremes, and certainly better
represents the common ancestor of marsupials and placentals. Finally, data from eutherian
mammals also provide support for the functionally interconnected somatosensory network of
cortical areas present in marsupials [S1, S2, PV, a rostral (R, 3a, or DZ), and a caudal (PM/PPC)
field], as well as for of multisensory cortex between caudal somatosensory areas and V1.
Based on these data, one can infer that the common mammalian ancestor had aspects of
this plan. Rather than a simple network with one or two fields, the cortical networks involved in
processing somatic inputs (both cutaneous and proprioceptive) were likely more complex than
previously thought and included at least four interconnected cortical fields, as well as a primitive
form of posterior parietal cortex involved in higher5order functions such as multisensory
integration. Further, the large proportion of cortex devoted to somatosensory processing
suggests that this sensory system was essential for survival in the Jurassic niche inhabited by
early mammals.
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Acknowledgments:
The authors thank Cindy Clayton, DVM and the rest of the animal care staff at the UC Davis
Psychology Department Vivarium. Additional thanks to Becky Grunewald for technical
assistance, Conor Weatherford for assistance in XY stage encoding, and Anika K Colopy for
additional data collection and analysis.
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Journal of Comparative Neurology
Conflict of Interest Statement:
The authors JCD, JGF, AMHS, DFC, and LAK, have no conflicts of interest.
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Role of Authors:
All authors had full access to all data in the study and take responsibility for the integrity of the
data and the accuracy of the data analysis. Study concept and design: JGF, DFC, and LAK.
Acquisition of data: JCD, JGF, DFC, and LAK. Analysis and interpretation of data: JCD, JGF,
AMHS, DFC, and LAK. Drafting of the manuscript: JCD and LAK. Critical revision of the
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manuscript for important intellectual content: JCD, JGF, AMHS, DFC, and LAK. Obtained
funding: LAK. Study supervision: LAK.
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Figure 1. (A) A light field digital image from case 09532 of a left hemisphere that has been
flattened, sectioned parallel to the cortical surface, and stained for myelin. Cortical field
boundaries have been drawn based on myeloarchitecture (solid black lines) or estimated (dashed
black lines). Where the medial wall has been unfolded, the junction with surface cortex has been
indicated with longer dashed lines. Although many cortical field boundaries can be determined
from a single section, the entire series of sections is used to make a comprehensive
reconstruction. (B) Light field image from case 12518 shows an injection site in S1 (arrow) in
relation to myeloarchitectonic boundaries. (C) The same injection site under fluorescent
illumination from the boxed region in B. The injection is small (~3005400 \m in diameter) and
confined to the boundaries of S1. Circles in B and C mark the same blood vessels used to align
sections. FR5labeled cell bodies from this case could be readily identified (D) within S1 and
other regions. Cell bodies and processes were also well labeled following injections of BDA into
John Wiley & Sons
Journal of Comparative Neurology
S1 in case 08580 (E). In all sections, rostral is to the left and medial is to the top. See Table 1 for
abbreviations.
Figure 2. Patterns of ipsilateral (A) and contralateral (B) cortical connections resulting from an
injection of FR in the vibrissae/snout representation of S1 for case 12518. Black dot and
surrounding black disc (A, C) are the FR injection site and halo, respectively. (Black dot
surrounded by gray disc in C is a separate injection – see Fig. 9C). In both A and B, red dots
indicate individual cells labeled by the neuroanatomical tracer. Most ipsilateral connections are
intrinsic to S1. Moderate label is also observed in ipsilateral S2 and FM, with weak label in SC
and SR. In the contralateral hemisphere, homotopic locations within S1 are most densely
labeled. Moderate contralateral connections are observed with SC, FM, and V2 and sparse
connections are observed in SR, MM, and V1. For quantification of labeled cells in each cortical
area, see Table 3 and Figure 5A. Details of neuroanatomical tracers used and injection
parameters are found in Table 2. In this case, electrophysiological recordings (A, C) were
performed to confirm the placement of the tracer injection. Grey dots indicate recording sites at
which neurons responded to somatosensory stimulation while Xs indicate sites at which no clear
response was elicited. (C) Expanded view of injection site and electrophysiological map from A,
showing different body part representations (separated by thin lines), and the receptive field
location (light red shaded region on face) of neurons at sites surrounding the injection site. Solid
lines mark cortical field boundaries determined from the entire series of myelin stained sections.
Medial (M) and rostral (R) directions are indicated by arrows. Other conventions as in the
previous figure.
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Journal of Comparative Neurology
Figure 3. Patterns of ipsilateral (A) and contralateral (B) cortical connections resulting from an
injection of FR centered in a medial portion of S1 in case 12513. As in the previous case (125
18), most ipsilateral connections are intrinsic to S1. Moderate to dense label is also observed in
SC and FM and sparse label is observed in SR and S2. In the contralateral hemisphere, dense
connections are observed in S1 and FM, and moderate connections are seen in SR, S2, and
piriform cortex. Other conventions as in previous figures.
Figure 4. Patterns of ipsilateral (A) and contralateral (B) cortical connections resulting from a
large injection of BDA in the vibrissae/face representation of S1 for case 08580. This injection
spread slightly into SR and SC. In both A and B, blue dots indicate individual labeled cells.
Most ipsilateral connections are intrinsic to S1. Strong label is observed in S2. Moderate label is
observed in FM, SC, SR, and MM. Homotopic locations within S1 in the contralateral
hemisphere are most densely labeled; dense label is also observed in SC and moderate label is in
SR, S2, MM and FM. In this case, electrophysiological recordings were performed to confirm
the placement of the tracer injection. (C) Expanded view of electrophysiological map from A.
Black dots surrounded by gray disk represent separate injections (see Fig. 6 and Fig. 9A, 9B).
Conventions as in previous figures.
Figure 5. Percentage of labeled cells in ipsilateral (black) and contralateral (gray) cortex resulting
from injections in S1 (A), SR (B), SC (C) and MM (D). The distribution of labeled cells across
different cortical areas is different for each cortical field injected. Results are mean + standard
deviation (when applicable) of injections from different animals. For results from individual
cases, see Table 3. For a list of abbreviations, see Table 1.
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Journal of Comparative Neurology
Figure 6. Patterns of ipsilateral (A) and contralateral (B) cortical connections resulting from an
injection of FR in SR for case 08580. Most ipsilateral connections are intrinsic to SR; dense label
is also observed in FM, S1, and EC/piriform cortex. Moderate to sparse label is widely
distributed and found is S2, SC, MM, A1, V2 and V1. In the contralateral hemisphere, dense
label is observed in SR, S1, FM; moderate label is observed in SC, MM, V2 and V1. In this case,
electrophysiological recordings were performed to confirm the placement of the tracer injection.
(C), Injection site (black circle surrounded by red halo) and adjacent electrophysiological map of
S1 as well as the injection sites in other cortical areas (BDA in S1, Fig. 4, and FE in SC, Fig. 9A,
9E, black circles surrounded by gray halos). Conventions as in previous figures.
Figure 7. Patterns of ipsilateral (A) and contralateral (B) cortical connections resulting from a
large injection of BDA that contained portions of FM, SR, and S1 for case 09532. Moderate label
is widely distributed across multiple cortical fields including FM, S1, S2, SR. One of the most
dense and widespread labeled areas is in EC and piriform cortex. Sparse label was also widely
distributed and found is SC, MM, A1, V2 and V1. The black box shows the region of cortex in
the inset BDA and myelin stained sections. The arrow points to the injection site. In the
contralateral hemisphere dense label was observed in FM and cortex just rostral to FM and
moderate label was observed in SR and S1, SC and piriform cortex. Sparse label was observed in
SC. In this case, electrophysiological recordings were performed to confirm the placement of the
tracer injection. (C) Injection site and adjacent electrophysiological map of S1 as well as the
location of a receptive field for neurons at a recording site located within the injection zone (light
blue shading on animal face). Conventions as in previous figures.
John Wiley & Sons
Page 40 of 57
Page 41 of 57
Journal of Comparative Neurology
Figure 8. Patterns of ipsilateral (A, B) and contralateral (D, E) cortical connections resulting
from an injection of FE into SC in cases 09518 and 08529. Green dots indicate individual cells
labeled by the neuroanatomical tracer. In A, grey areas are regions of cortex with superficial
cortical damage. In both (A) and (B), black boxes show areas from which pictures were taken in
(C) and (D). In the case without cortical damage (B), most of the labeled cells were observed in
S1, with strong label also in SR and FM and moderate label in S2 and SC. Weak label was
observed in V1. In the contralateral hemispheres (E, F), dense label was observed in S1, SC and
FM and moderate label was observed in S2 and MM, and weak label was observed in SR and
V2. Conventions as in previous figures.
Figure 9. Patterns of ipsilateral and contralateral cortical connections resulting from an injection
of FE in MM in case 08580 (A and B) and case 12518 (C). In both cases, the majority of labeled
cells in the hemisphere ipsilateral to the injection site (A and C) are in MM and SC with
additional dense labeling in V1. Moderate label was observed in S1, and weak label was
observed in S2, FM, A1 and V2. In the contralateral hemisphere of 08580 (B), strong label was
found in MM, SC, S1 and V1. Moderate label was seen in S2, FM and V2. Conventions as in
previous figures.
Figure 10. Stylized cladogram summarizing cortical connections of S1 in mammals. Despite
some differences, all marsupials share a basic pattern of connectivity that we propose represents
that of the common ancestor of all mammals. Monotremes diverged earlier than marsupials and
eutherians, and retain some basal traits like egg5laying, but have highly derived oral facial
John Wiley & Sons
Journal of Comparative Neurology
Page 42 of 57
specializations, skulls, brains and lifestyles. Fossils of early mammals (
) , bottom center), early marsupials (
eutherians (+
*
,
, center) and early
, upper right) share many characteristics with skulls of some extant
marsupials (left and top), particularly didelphids, while differing sharply from extant
monotremes (right). The orafacial configuration, body size, and brain size of present day
(top left) indicate that it may be the best extant model for early
marsupials and early mammals. The brain organization and connectivity of didelphids and
especially
may therefore reflect that of the common ancestor of all marsupials and
all mammals. Inherited connections are drawn in black arrows; derived (independently acquired)
connections are drawn in red arrows. The thickness of the arrow lines indicates the strength of
connections. Cortical fields are outlined in boxes positioned such that the spatial relationships of
fields have been preserved. The status of the presence of a separate S2 and PV (gray box) in the
common ancestor is difficult to deduce since some marsupials have separate fields and some
marsupials have a single field that could be either S2 or PV. mya, million years ago. Scale bars, 5
mm. Cladogram branch lengths are not to scale. Skull drawings adapted with permission from
Macrini (2001,
2007, $ (
$
; 2004,
), Luo and colleagues (2001, $ ) . 2003, $ *
Kermack and colleagues (1981;
colleagues (1996;
; 2005a, $ ,
(
Anomal and colleagues (2011;
; 2005b, -$ anatinus;
( . 2011, +$
), and
). Connectional data adapted from Beck and
), Elston and Manger (1999;
).
John Wiley & Sons
(
), and
Page 43 of 57
Journal of Comparative Neurology
3a
A1
BDA
CO
Contra
CT
DZ
EC
FE
fl
FM
fp
FR
IM
IP
Ipsi
ll
M1
MM
PM
PP/PPC
PV
Pir
R
S1
S2
SC
S.D.
SR
ul
V1
V2
vib
Somatosensory area (deep)
Primary auditory cortex
Biotinylated dextran amine
Cytochrome oxidase
Contralateral
Caudal temporal area
Dysgranular zone
Entorhinal cortex
Fluoro-emerald
forelimb
Frontal myelinated area
forepaw
Fluoro-ruby
Intramuscular
Intraperitoneal
Ipsilateral
lower lip
Primary motor cortex
Multimodal cortex
Parietal medial area
Posterior parietal cortex
Parietal Ventral area
Piriform cortex
Rostral somatosensory field
Primary somatosensory area
Secondary somatosensory area
Somatosensory caudal area
Standard Deviation
Somatosensory rostral area
upper lip
Primary visual area
Secondary visual area
vibrissae
John Wiley & Sons
Journal of Comparative Neurology
Case
12-18
12-13
09-18
12-19
08-80
08-80
09-32
09-18
08-29
12-18
08-80
Area
injected
+ halo
S1
S1
S1
S1
S1+SR, SC
SR
SR+FM,S1
SC
SC
MM
MM
Hemisphere
injected
Right
Right
Left
Right
Left
Left
Right
Left
Left
Right
Left
Amount Injection
Injection
Recording receptive
Injected area
sites
Tracer (µl)
(mm2)
field
FR
0.3
0.06
53 Vibrissae
FR
0.3
0.11
0
FR
0.3
0.07
0
FR
0.32
0.10
12
BDA
0.3
1.61
20 Vibrissae
FR
0.3
0.07
20 Lips
BDA
0.3
1.02
41 Vib/Lip/Naris
FE
0.3
0.14
0
FE
0.3
0.08
0
FE
0.4
0.04
53 unresponsive
FE
0.3
0.07
20 unresponsive
John Wiley & Sons
Page 44 of 57
Figure
Number
2
3
4
6
7
8
8
9
9
Journal of Comparative Neurology
Table 3:
Ipsilateral
connections
Pir/
EC
0
S1
S2
SC
SR
MM
FM
A1
V1
V2
CT
12-13
Area
Injected
S1
58.2
0.5
7.8
2.3
0.3
26.8
0
0
0
0
12-18
S1
83.8
3.3
1.8
1.3
0.2
3.2
0.6
0.1
0.1
0
0
5.5
12-19
S1
75.9
10.7
2.1
1.1
0.2
1.7
0.5
0
0.1
0
1.6
6.1
Avg.
72.6
4.8
3.9
1.6
0.2
10.6
0.4
0
0
0
0.5
5.3
S.D.
13.1
5.2
3.4
0.6
0
14
0.3
0.1
0
0
0.9
1
Case
Other
4.1
08-80
SR
11.1
3.6
3.4
22.5
3.2
15.1
2.4
4
1.1
0.3
15
18.3
08-29
SC
60.4
9.9
3.9
12.6
0.4
10.1
0.1
1.2
0.6
0
0
0.8
12-18
MM
4.4
3.1
33
0
38.1
3.1
1.8
9.9
0.2
0.4
0.9
5.1
08-80
MM
8.2
2.8
25.8
1.4
27.6
0.1
0.4
30.8
2.5
0
0.1
0.4
Avg.
6.3
2.9
29.4
0.7
32.9
1.6
1.1
20.4
1.3
0.2
0.5
2.7
S.D.
2.7
0.2
5.1
1
7.4
2.1
1
14.8
1.6
0.3
0.5
3.3
08-80
S1+SR, SC
46.7
10.9
9.6
9.9
4.5
4.8
2.1
0.9
0.5
0
1.5
8.4
09-32
SR+FM,S1
8.7
5.9
2.8
8.5
1.7
9.1
1.3
0.3
0.6
0
38.9
22.3
09-18
SC
65.5
6
6.4
0
4.6
0
0
10.3
5
0
0.4
1.8
V2
CT
Contralateral
connections
12-13
Area
Injected
S1
26
5.8
0
4.3
0
44.6
0
0
0
0
Pir/
EC
11.6
09-18
S1
35.7
0.2
2.9
21.4
0.7
30
0
0
0.5
0
0
12-18
S1
61.3
0
15.4
2.5
2.8
12.3
0
0.6
4.4
0
0
0.6
12-19
S1
50.9
1.9
6.6
5.1
2.3
1.9
0.2
0.2
0
0
1.1
29.8
Avg.
43.5
2
6.2
8.3
1.5
22.2
0
0.2
1.2
0
3.2
11.7
S.D.
15.7
2.7
6.7
8.8
1.3
18.9
0.1
0.3
2.1
0
5.6
12.6
Case
S1
S2
SC
SR
MM
FM
A1
V1
Other
7.8
8.6
08-80
SR
22.4
0.9
4.4
10.9
4.3
19.6
0.1
5.9
3.2
0
0.2
28
09-18
SC
53.8
1.4
10.6
3.6
6.3
14
0
0.9
2.1
0
0
7.5
08-29
SC
39.8
16.9
14.7
1.7
6.9
11.2
0
0.3
0.9
0
0
7.6
Avg.
46.8
9.1
12.6
2.7
6.6
12.6
0
0.6
1.5
0
0
7.5
S.D.
9.9
10.9
2.9
1.3
0.4
2
0
0.4
0.8
0
0
0.1
08-80
MM
12.8
3.6
14.1
0.2
24.2
4
0.2
24.2
9.1
0
0
7.6
08-80
09-32
S1+SR, SC
SR+FM,S1
61.4
8.7
5.4
0.2
13.1
1.2
3.8
7.3
5.6
0
5.9
39.2
0
0
0.1
0
0.7
0
0
0
0.2
8.4
3.8
35
John Wiley & Sons
Journal of Comparative Neurology
Page 46 of 57
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Using neuroanatomical experiments in which neuroanatomical tracers were injected, the
connections of different areas of somatosensory cortex were examined and both corticocortical
and interhemispheric connection patterns of connections were quantified. The pattern of
connectivity in somatosensory cortex suggests a network with at least four interconnected
cortical fields, and the presence of a primitive form of posterior parietal cortex.
John Wiley & Sons
Journal of Comparative Neurology
John Wiley & Sons