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Titre: Von Economo Neurons in the Anterior Insula of the Macaque Monkey
Auteur: Henry C. Evrard

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Neuron

Report
Von Economo Neurons in the Anterior Insula
of the Macaque Monkey
Henry C. Evrard,1,* Thomas Forro,1,3 and Nikos K. Logothetis1,2
1Max

Planck Institute for Biological Cybernetics, 72076 Tu¨bingen, Germany
Science and Biomedical Engineering, University of Manchester, Manchester M13 9PT, UK
3Present address: Department of Cognitive Neurobiology, Center for Brain Research, Medical University Vienna, 1090 Vienna, Austria
*Correspondence: henry.evrard@tuebingen.mpg.de
DOI 10.1016/j.neuron.2012.03.003
2Imaging

SUMMARY

The anterior insular cortex (AIC) and its unique
spindle-shaped von Economo neuron (VEN)
emerged within the last decade as having a potentially major role in self-awareness and social cognition in humans. Invasive examination of the VEN
has been precluded so far by the assumption that
this neuron occurs among primates exclusively in
humans and great apes. Here, we demonstrate the
presence of the VEN in the agranular anterior insula
of the macaque monkey. The morphology, size,
laminar distribution, and proportional distribution of
the monkey VEN suggest that it is at least a primal
anatomical homolog of the human VEN. This finding
sheds new light on the phylogeny of the VEN and
AIC. Most importantly, it offers new and muchneeded opportunities to investigate the primal
connections and physiology of a neuron that could
be crucial for human self-awareness, social cognition, and related neuropsychiatric disorders.
INTRODUCTION
The von Economo neuron (VEN) is an atypical projection neuron
that differs from the typical pyramidal neuron by its large spindleshaped perikaryon and unique and equally thick basal and apical
dendrites (von Economo, 1926; Seeley et al., 2012). Concentrations of VENs occur in the anterior insular cortex (AIC) and
anterior cingulate cortex (ACC) in humans and great apes (Nimchinsky et al., 1999; Allman et al., 2010) as well as in mammals
with large brains and complex social organization, such as cetaceans and elephants (Butti et al., 2009; Hakeem et al., 2009).
A wealth of imaging and lesion evidence indicates that AIC has
a central role in interoceptive, emotional, and social awareness
and cognition in humans (Critchley et al., 2004; Craig, 2009;
Lamm and Singer, 2010). Neuropathological evidence suggests
that VENs in the ventral portion of AIC could take part in that role.
Notably, the VEN is selectively depleted in the behavioral variant
of frontotemporal dementia (bvFTD) that is characterized by
a subtle loss of self-conscious emotion and empathy (Kim
et al., 2012). Alterations in the number of VENs, among other
symptoms, also suggest an implication in autism (Santos et al.,
482 Neuron 74, 482–489, May 10, 2012 ª2012 Elsevier Inc.

2011), suicidal psychosis (Bru¨ne et al., 2011), and agenesis of
the corpus callosum (Kaufman et al., 2008), all of which are
characterized in part by impaired interoception, emotion, and/or
empathy. These findings emphasize the need for an animal
model to help examine the fundamental organization, connections, and physiology of the VEN and its characteristic architectonic region. Comparative examinations in more than 20 primate
species concluded that concentrations of VENs occur exclusively in hominids among primates and that the VEN is
completely absent in lesser apes, monkeys, and nonanthropoid
primates (Nimchinsky et al., 1999; Allman et al., 2005, 2010). This
conclusion implies a late evolutionary emergence of the VEN
within the last 15 million years and a specific relationship to humans’ sophisticated awareness and cognitive abilities. This
conclusion also precludes invasive examination of the VEN in
the laboratory. Here, we demonstrate the presence of the VEN
in the agranular anterior insula (and ACC) in two species of
macaque monkeys commonly used in the laboratory (rhesus
and cynomolgus).
RESULTS
As in humans (Nimchinsky et al., 1999), the macaque VEN
stained with cresyl violet has a large spindle-shaped perikaryon
with a unique basal dendrite that is proximally as thick as its
apical dendrite (Figure 1A). The volume of the macaque VEN,
stereologically estimated with the optical planar vertical rotator
(Stark et al., 2007), is on average 50% and 70% larger than local
pyramidal and layer 6 fusiform neurons, respectively
(M. fascicularis: F2,4 = 53.457, p = 0.0013; M. mulatta: F2,4 =
23.438, p = 0.0062) (Figure 1B; see Table S1 available online
for details). Similar volume differences were observed in humans
(Figure 1B; Nimchinsky et al., 1999) and great apes (Nimchinsky
et al., 1999). The macaque VEN is significantly smaller than the
human VEN measured in the present (F2,7 = 26.041, p =
0.0006) and prior (Nimchinsky et al., 1999) studies and smaller
than chimpanzee and bonobo VENs, but it is within the range
of gorilla VENs (Nimchinsky et al., 1999). The human VEN is
slightly larger than local pyramidal neurons by comparison with
the monkey VEN (human VEN index [mean ± SD] = 3.0 ± 0.8;
macaque VEN index = 2.0 ± 0.4; F1,8 = 6.2, p = 0.0375).
VENs in both species of macaques are distributed in layer 5b in
the agranular insula (Figures 1C and 1D; Figure S1A), where they
are systematically commingled with sparsely distributed fork
cells (Figures 1D and 1E; Figure S1A). The fork cell is another

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Von Economo Neurons in the Macaque Insula

unique cell morphotype that is characterized by a bifid apical
dendrite and unique basal dendrite, is present among VENs in
human and great apes AIC (and ACC), and is selectively depleted,
along with the VEN, in bvFTD (Ngowyang, 1932; Kim et al., 2012).
The VENs and fork cells are concentrated within a small region in
the agranular anterior insula that is located, as in humans, just
anterior to the limen insula and medial to the superior limiting
sulcus (Figure 1F). (In order to avoid the impression that we infer
a clear anatomical and functional homology with the human AIC,
in the macaque we use the term ‘‘agranular anterior insula’’ [AAI]
to designate the agranular portion of the insula that lies anterior to
the limen. We ascribe the concentration of VENs to a small region
within the ventral AAI.) A preliminary examination of the cytoarchitecture of this region in Nissl preparations does not reveal major
interspecies differences and suggests that it constitutes a distinct
architectonic area. In this area, the small granule cells of layer 4
are absent; layer 2 is thin but darkly stained; layer 3 is distinctly
sublaminated; and layer 5 is wide with a clear sublamination
into a thin superficial layer 5a and a large layer 5b, which contains
larger neurons including VENs and fork cells (Figure 1C).
VENs and fork cells are also found in the ACC, particularly in
area 24b (Figures S1B and S1C), but these are scarcer and
more dispersed than those in ventral AAI. Isolated VENs are infrequently found in other regions of the prefrontal cortex including
areas 14 and 10. The present study focuses on the concentration
of VENs in the ventral AAI, consistent with the overall aim of our
research on primate insula. Other than frequency and number,
there are no obvious differences in morphology, size, or laminar
distribution between the insular and cingulate VENs. Details on
the VEN distribution in ACC will be reported separately.
A stereological estimate of the number of VENs in ventral AAI
with the optical fractionator (West et al., 1991) indicates an
average of 1,500 and 2,000 VENs per hemisphere in
M. fascicularis and M. mulatta, respectively (Figures 1G and
1H; see Table S1 for details). This number is, as expected,
much lower than the numbers reported previously in humans
and the average number in great apes, but it is within the lower
range of VEN numbers in some individual great apes (Allman
et al., 2010). Macaque VENs represent 2%–3% of the total
number of neurons in layer 5, which fits the trend for progressively higher VEN/pyramidal ratio from human to phylogenetically more distant primate species (Allman et al., 2010). Finally,
a comparison of the number of VENs in the left and right AAI
reveals a significantly higher number of VENs (F1,5 = 100.358;
p = 0.0002) and a nonsignificant trend for a higher VEN/
pyramidal ratio (F1,6 = 4.213; p = 0.0953) in the right AAI, also
in accordance with the asymmetry reported in hominids (Allman
et al., 2010). Although the rhesus brain is on average significantly
larger than the cynomolgus brain (F1,6 = 31.255; p = 0.0014),
there is not significant species difference in VEN number or
volume nor a significant correlation between VEN volumes or
numbers and absolute brain volume or encephalization quotient,
perhaps because of the small size of our sample.
Although the strongest evidence that the large spindle-shaped
neurons in the macaque insula correspond to human VENs
comes from their signature morphology, size, laminar distribution, and small percentage, it remains possible that these
neurons could in fact be unusually large local inhibitory interneu-

rons. Golgi staining, immunohistochemical labeling, and tract
tracing were used to verify the proposition that monkey VENs
are indeed projection and excitatory neurons. The Golgi preparation readily confirms the typical morphology of the VEN perikarya, and it shows that the apical dendrites of VENs typically
branch distally into several thinner spiny dendrites that spread
radially into layers I–III (Figure 2A, left), similar to typical layer 5
pyramidal projection neurons (Figure 2A, right). The basal
dendrite usually branches out into thinner spiny dendrites essentially in layer VI, again similar to human VENs (Watson et al.,
2006). In contrast to the VENs, the pyramidal neurons characteristically have highly branched spiny basal tufts that spread
proximally into layers V and VI.
Macaque VENs are immunoreactive for SMI-32 (Figure 2B),
an antibody that binds nonphosphorylated epitopes of the
neurofilament triplet protein expressed in pyramidal neurons,
particularly in those with long range projections, and it has
been reported to label human VENs (Hof et al., 1995; Nimchinsky
et al., 1995). Interestingly, the soma of the SMI-32-immunoreactive VENs in the macaque are conspicuously almost the only
labeled somata in layer 5b in AAI (Figure 2B), suggesting that
their unique morphology might correlate with a distinct function
and hodology. Macaque VENs are also immunoreactive for an
antipeptide antibody raised against the kidney-type glutaminase
(KGA) isoform of the phosphate-activated glutaminase (Figure 2D), a major enzyme isoform involved in the synthesis of
the excitatory neurotransmitter glutamate in cortical neurons of
the mammalian cerebral cortex (Akiyama et al., 1990).
Most brains examined here were collected from monkeys that
were used for tract-tracing experiments of various types. In
particular cases, we found retrogradely labeled VEN perikarya
dispersed among retrogradely labeled pyramidal neurons in
AAI (Figure 2G; Figures S1E0 and S1F0 ). Two such cases had
an injection of fluorescent dextran or cholera toxin b in contralateral AAI (Figures S1D and S1E), and two cases had a tracer injection in the ipsilateral portion of the insula (e.g., Figure S1F) that
receives gustatory afferent inputs from the thalamus (Pritchard
et al., 1986). The observation of retrogradely labeled VENs,
together with the Golgi and SMI-labeling evidence, confirms
that macaque VENs are not local inhibitory neurons, rather
they are projection neurons.
VENs in humans are immunopositive for a host of proteins
that may be variably related to the role of AIC in the control of
autonomic functions (e.g., serotonin receptor 2b [5ht2br])
(Allman et al., 2005), as well as in neuropsychiatric disorders
such as schizophrenia (e.g., disrupted-in-schizophrenia-1
[DISC-1]) (Allman et al., 2010), and also craving and
addiction (e.g., dopamine D3 receptor [D3]) (Allman et al.,
2005). Many macaque VENs are immunopositive for DISC-1
(Figures 2C and 2C0 ), 5ht2br (Figure 2E), and D3 (Figure 2F).
DISC-1 immunopositive VENs are clearly conspicuous, because
there are few immunopositive pyramidal neurons (Figure 2C). A
stereological estimate as to whether the DISC-1 population in
monkeys represents a large fraction of the total number of
VENs in the insula, as it does in humans ( 95%) (Allman et al.,
2010), would make an interesting future study. Although all of
the proteins examined here are also present in local pyramidal
neurons (and are thus not specific markers of the VENs), the
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484 Neuron 74, 482–489, May 10, 2012 ª2012 Elsevier Inc.

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Von Economo Neurons in the Macaque Insula

Figure 2. The Macaque VEN Shares
Numerous Characteristics with the Human
VEN
(A) A rapid Golgi stain reveals that the macaque
VEN possesses all morphological characteristics
of typical pyramidal projection neurons and of the
human VEN (Watson et al., 2006). S, soma; b,
dendritic branching; a, axon. Arrowheads point to
the distal extension of the apical dendrite. The
insert shows the distal arborization of the apical
dendrite into spiny branches. Scale bar represents
25 mm. (B and B0 ) Macaque VENs are distinctively
immunopositive for SMI-32 in layer 5b, whereas
pyramidal neurons are immunopositive predominantly in layer 5a. Scale bar represents 100 mm.
(C and C0 ) Macaque VENs are distinctively immunopositive for DISC-1 in layer 5b. Scale bar
represents 100 mm. (D–F) Macaque VENs are immunopositive for the KGA isoform of PAG (D), the
serotonin receptor 2a (E), and the dopamine
receptor D3 (F). Scale bar represents 15 mm in
(D)–(F). (G) Macaque VEN retrogradely labeled
in AAI with an injection of Alexa 594 dextran in
contralateral AAI. (See Figures S1D–S1F for an
illustration of the injection site and for more examples in two other cases.) Scale bar represents
25 mm in the main panel. The insert shows retrograde labeling of dendritic spines.

examinations of hominoids (Stimpson
et al., 2011). Thus, a dedicated stereological analysis of protein expression in the
VENs of humans and macaques could
help establish the much-needed primate
neurochemical model for disorders such
as schizophrenia and addiction.
DISCUSSION

similarity in the immunohistochemical characteristics of monkey
and human VENs suggests that subtle, rather than marked,
phylogenetic variation may reflect the hypothesized more
sophisticated role of the VENs in humans, as suggested in prior

Prior comparative studies concluded that
concentrations of VENs in primates occur
exclusively in humans and great apes (Nimchinsky et al., 1999;
Allman et al., 2010). The present report provides compelling
evidence that there is at least a primal anatomical homolog of
the human VEN in the monkey AAI (and ACC). There are at least

Figure 1. The Von Economo Neuron Is Present in Layer 5b in a Restricted Portion of the Agranular Anterior Insula in the Macaque Monkey
(A) High-magnification photomicrographs demonstrating the identical morphology of the macaque and human VENs. Scale bar represents 25 mm. (B) Histogram
demonstrating the larger volume of the macaque and human VENs compared to their respective pyramidal and fusiform neurons (p < 0.01). Volumes were
measured in three rhesus, three cynomolgus, and four human hemispheres. Ten VENs, 20 local pyramidal, and 10 layer 6 fusiform neurons were measured in each
hemisphere. (C) Low-magnification photomicrographs showing the similar location of the macaque and human VENs (red asterisks) and fork cells (green
asterisks) in layer 5b in coronal sections though the agranular insula. Scale bar represents 250 mm. (D) Intermediate magnification photomicrograph showing the
exact location of VENs (red arrowheads) and fork cells (green arrowheads) in layer 5 in the M. mulatta section illustrated in (C). The framed field corresponds to the
framed field in (C). High-magnification photomicrographs of each numbered cell are shown in Figure S1A. Scale bar represents 100 mm. (E) Photomicrographs of
typical fork cells in the agranular insula in macaques and human. (The photomicrograph from M. fascicularis also shows a VEN in the top left corner.) Scale bar
represents 25 mm. (F) Plots of VENs (red) and fork cells (green) in one individual section per species illustrating the similar distribution of the macaque and human
VENs in a restricted portion of the agranular insula, anterior to the limen (data not shown) and medial to the superior limiting sulcus (slsi). (See also Figures S1B and
S1C for plots and photomicrographs of VENs and fork cells in the anterior cingulate cortex.) (G) Table comparing the average number (±SD) of VENs and local
pyramidal neurons in layer 5b. (H) Bivariate scattergram showing individual counts of VENs in the left and right side of the agranular anterior insula in three
cynomolgus (open circles) and three rhesus (closed circles) monkeys.

Neuron 74, 482–489, May 10, 2012 ª2012 Elsevier Inc. 485

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three possible explanations for this discrepancy with earlier
observations. First, the large human VENs unambiguously stand
out at low microscope magnifications. Searching for relatively
smaller VENs among the densely packed cell population in layer
5 in the monkey required the highest microscope magnification,
which would be unusual for anyone accustomed to examining
the more obvious VENs in hominids. Second, the cytoskeletal
matrix of the small monkey VENs might be more fragile during
histological processing than that of the larger human VENs. In
the course of this work, we rejected many cases because
swelling of the perikarya prevented morphological differentiation. Third, in the major prior study, the number of VENs in humans and great apes was counted in consecutive sections that
were apparently spaced at 1 mm intervals (Nimchinsky et al.,
1999). Although the sampling used for nonhominid primates in
that study was not specified, such a sampling paradigm would
likely have been inadequate for the identification of VENs within
the small VEN-containing region of the ventral AAI that measures
2 3 2 3 1 mm3 in macaques.
The present results push back the emergence of the VEN in
primates from 15 to at least 25 million years ago (the cercopithecid/hominoid divergence node; Fabre et al., 2009) and
recommend a reexamination of the idea that VENs separately
evolved multiple times in phylogenetically distant species having
in common a large absolute brain size (>300 g) and a sophisticated social organization (Nimchinsky et al., 1999; Hof and Van
der Gucht, 2007; Butti et al., 2009; Hakeem et al., 2009; Allman
et al., 2010). The presence of VENs in the lighter brain of the
macaque ( 40–80 g) raises the question of whether VENs occur
in other primate species, perhaps less frequently, or even in nonprimates such as rodents, cats, and dogs. VENs have been
recently identified in the insula of the manatee (Butti and Hof,
2010).
Functional and neuroanatomical evidence indicates that the
AIC in humans provides the neural basis for awareness in the
form of an ultimate unified representation of all salient bodily
and emotional feelings, or a ‘‘sentient self,’’ at each moment in
time (Craig, 2009). The right AIC appears to be preferentially
associated with aversive, egocentric, and negative affects
relating to sympathetic activity and the left AIC with appetitive,
affiliative, and positive affects relating to parasympathetic
activity (Craig, 2005). Reports of selective alteration in the
number of VENs in mental disorders including bvFTD (Kim
et al., 2012), an asymmetric distribution (R > L; Allman et al.,
2010), and expressions of proteins related to homeostasis (Allman et al., 2005, 2010; Stimpson et al., 2011) have suggested
that VENs in humans, while preserving their basic physiological
role, evolved to be part of a ‘‘body loop’’ that monitors and incorporates physiological states and emotional salience relevant to
human awareness (Allman et al., 2010).
In the monkey, the general region of the ventral anterior insula
has been related to viscerosensory and -motor functions and is
interconnected with subcortical structures regulating autonomic
and behavioral responses to stressors, including vocalization
(Kaada et al., 1949; Price, 2007; Barbas et al., 2011). Microstimulation in the left anterior insula (Caruana et al., 2011) produced
facial motor responses associated with disgust as well as bradycardia and affiliative behaviors (reassuring ‘‘lip smacking’’),
486 Neuron 74, 482–489, May 10, 2012 ª2012 Elsevier Inc.

which could reflect the hypothesized association of parasympathetic activity (Craig, 2005). The presence of VENs in ventral AAI
in the macaque suggests that this region could share a common
evolutionary origin with the frontoinsular region (FI) that concentrates VENs in the ventral AIC in humans. The VENs and their
inclusive area in ventral AAI probably engender a much more
primitive function (perhaps essentially feeding related; Allman
et al., 2010) than do FI VENs in humans. Nevertheless, invasive
studies of their organization, hodology, and physiology could
provide significant insights into the evolutionary basis for selfawareness and empathy in humans. Regarding the latter, it
would be particularly interesting to examine whether the VENs
share functional similarities with the ‘‘mirror’’ neurons of the
ventral premotor cortex (Gallese et al., 2004).
The frontoinsular VENs in humans have been proposed to
project to ipsilateral ACC and contralateral AIC (Craig, 2009; Allman et al., 2010). Consistent with prior studies (Mesulam and
Mufson, 1982), our preliminary tract-tracing experiments indicate that ventral AAI of the macaque receives input from many
pyramidal neurons in contralateral AAI and ipsilateral ACC; yet,
the scarce retrograde labeling of VENs in those regions suggests
that the main projection target of VENs might lie somewhere else
in the brain. The relatively large size, small percentage, and
laminar distribution of the VENs are reminiscent of the specialized Betz cells in primary motor cortex (Butti et al., 2009). The
size of the VENs in humans (Nimchinsky et al., 1999) is within
the lowest range of the size of the Betz cells projecting to the
cervical segments of the spinal cord (Rivara et al., 2003). This
suggests the possibility of projections to distant brain regions
including the periaqueductal gray (PAG) and the parabrachial
nucleus (PBN) (Craig, 2002; Allman et al., 2005; Seeley, 2008;
Butti et al., 2009). PAG and PBN receive interoceptive afferents
from spinal lamina I (Craig, 1995), might receive inhibitory feedback from the insula (Craig, 2002), and have been identified as
subcortical nodes in a ‘‘salience network’’ anchored by FI and
ACC in humans (Zhou et al., 2010). PAG is also central in vocalization and speech (Ju¨rgens, 2009), which is in keeping with the
possible role of the left AIC in speech (Ackermann and Riecker,
2010) and with the presence of VENs in species with elaborate
vocalization repertoires (Hof and Van der Gucht, 2007). The
region concentrating VENs in the monkey shares architectonic
characteristics with the ‘‘lateral agranular insula (Ial),’’ defined
by Carmichael and Price (1994). Although the bulk of AAI projections to PAG arises from a directly adjacent ‘‘intermediate agranular insula (Iai),’’ retrograde tracing from PAG labeled few cells
in layer 5 in Ial (An et al., 1998).Tracing evidence in the rat (Saper,
1982) and intrinsic connectivity network functional magnetic
resonance imaging in humans (Zhou et al., 2010) suggests that
PBN might be interconnected with the anterior insula in primates;
and there is intriguing evidence that passive avoidance in the rat
requires lateralized contributions of the PBN and cerebral cortex
(Tassoni et al., 1992). It would be interesting to examine whether
VENs have preferential projections to PAG and PBN and, in light
of the model of forebrain asymmetry for emotions (Craig, 2005),
whether right and left VENs project differently to the lateral and
ventrolateral columns of PAG that respectively mediate active
(‘‘flight-fight’’) and passive (‘‘quiescent’’) autonomic and behavioral responses to stressors (Bandler and Shipley, 1994).

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The presence of VENs in the macaque does not discredit prior
evidence for a crucial role of the VENs and AIC in the emergence
of self-awareness and social cognition in humans (Craig, 2009; Allman et al., 2011). VENs in humans appear to be disproportionally
slightly larger than in macaques (see above); they may also have an
enhanced immunopositivity (and perhaps gene expression) for
proteins that are typically involved in homeostasis, which perhaps
favors higher interoceptive sensitivity (Stimpson et al., 2011).
Although there are reports of self-recognition in the mirror test (Macellini et al., 2010; Rajala et al., 2010) (a phenomenon considered
to be an indicator of underlying self-awareness) and self-agency
(Couchman et al., 2012) in the macaque monkey, evidence of
self-recognition in monkeys is certainly not as straightforward as
it is in great apes and humans (Anderson and Gallup, 2011).
Most importantly, the presence of VENs in the macaque monkey
clearly opens extraordinary opportunities to empirically examine
the fundamental organization, connections, and physiology of a
neuronal morphotype and a brain region, which appear to have
acquired a crucial role in self-awareness, social cognition, and
their related neuropsychiatric disorders in humans.
EXPERIMENTAL PROCEDURES
Histology
Entire brains or blocks containing the anterior insula were obtained from five
rhesus macaques (Macaca mulatta), four cynomolgus macaques (Macaca
fascicularis), and two humans. The macaque brains were obtained in the
context of separate tract-tracing experiments approved by the local authorities (Regierungspra¨sidium) and in full compliance with the European Parliament and Council Directive 2010/63/EU on the protection of animals used
for experimental and other scientific purposes. The human samples were
collected and processed in the context of an unrelated study (Koch et al.,
1985). A detailed description of all the procedures summarized below is
provided in the Supplemental Experimental Procedures.
Four rhesus and four cynomolgus brains were fixed with 4% formalin, sliced
in 50-mm-thick coronal sections, and processed for Nissl staining for related
or unrelated tract-tracing examination or for immunohistochemistry using
ABC and DAB. The primary antibodies were raised against SMI-32 (1:1,000;
Covance), the DISC-1 protein (1:1,000; Zymed Laboratories), serotonin
receptor 2b (1:1,000; Sigma), dopamine D3 receptor (1:1,000; Chemicon), or
the KGA isoform (1:1,000; Kenny et al., 2003).
Four cases with tracer injection are included here to demonstrate that VENs
are projection neurons. Nanoinjections of cholera toxin b (List) or Alexa 594
fluorescent dextran (Molecular Probes) were made in the AAI or middle dorsal
fundus of the insula. The processing of the slides was carried out as described
elsewhere (Evrard and Craig, 2008).
One unfixed rhesus brain was used for a rapid Golgi-Cox staining (Ramo´nMoliner, 1970) with the FD Rapid GolgiStain Kit (FD NeuroTechnologies).
Microscope Examination
Sections were independently examined by two observers (H.C.E. and T.F.).
A neuron was considered to be a large spindle-shaped neuron if it was located
in layer 5b among typical pyramidal neurons, had a unique basal dendrite that
was as thick as its apical dendrite, had an elongated perikaryon larger than or
as large as local pyramidal neurons, was symmetrical about its vertical and
longitudinal axes, and had a nucleus and nucleolus located in the middle of
the perikaryon (Nimchinsky et al., 1999). Spindle-shaped neurons that were
much smaller than the local pyramidal neurons or had very thin basal or apical
dendrite were excluded. These criteria prevented elongated pyramidal (or lanceolated) neurons, inverted pyramidal neurons, and small vertical fusiform
interneurons in layer 6 from being counted as large spindle-shaped neurons.
A fork cell was defined by a unique basal dendrite and a ‘‘forked’’ or ‘‘bifid’’
apical dendrite (Ngowyang, 1932).

Stereological Estimations
The total number of VENs was estimated at 633 with the optical fractionator
(West et al., 1991) using a set of 225 3 168 mm2 optical dissectors with
a 225 3 168 mm2 scan grid size for an exhaustive counting of the VENs (Butti
et al., 2009; Stimpson et al., 2011) (Figure S2A). The targeted number of
counted VENs was set at 300. Using this strategy, we obtained averages of
202.8 ± 85.9 (mean ± SD) and 177.2 ± 35.2 VENs in the rhesus and cynomolgus
macaques, respectively. The coefficients of error (CE) were all below the
threshold CE of 0.1 (Schmitz and Hof, 2005), except for one hemisphere
with a tolerated CE of 0.11 (Table S1). The thickness of the optical dissector
was set to 80% of the section thickness; the top and bottom guard zones
were each set to 10%. The thickness of the section was measured for each
counting frame and averaged for the calculation of the total number estimate
by using the Fractionator method (West et al., 1991). The total number of pyramidal neurons was estimated within the same region of interest (ROI) and in the
same sections. The dimensions of the sampling grid were set to 260 3 165 mm2
and the counting frame to 50 3 50 mm2 to count at least 300 pyramidal neurons
per hemisphere. The counts were made in a sequence of counting frames
selected by using a systematic random sampling.
Perikaryal volumes were estimated by using the optical vertical planar principle (Stark et al., 2007) in 10 VENs, 20 local pyramidal neurons, and 10 layer 6
fusiform neurons in 3 rhesus, 3 cynomolgus, and 4 human AICs (left or right)
(Figure S2B). The measured VEN and fusiform cells were selected randomly.
The measured pyramidal neurons were always randomly selected within the
direct vicinity of the measured VEN.
Comparisons of the number or volume of cells across species or sides of the
brain were made by using one-way or repeated-measure ANOVA and post hoc
t test. Possible correlation between count or volume estimates and brain
weight, body weight, and encephalization quotient (EQ) were analyzed by
using correlation test. EQ was obtained by using EQ = brain weight/0.12
(body weight)0.67 (Butti et al., 2009).
SUPPLEMENTAL INFORMATION
Supplemental Information includes two figures, Supplemental Experimental
Procedures, and one table and can be found with this article online at
doi:10.1016/j.neuron.2012.03.003.
ACKNOWLEDGMENTS
We thank A.D. (Bud) Craig for his relecture of our revision and insightful
suggestions. We thank J. Schramm, Z. Aschrafi, O. Groht, K. Piasecka,
K. Bogdanova, and S. Sa`nchez-Ancora for their excellent technical assistance.
We thank A. Bartels, M. Herdener, R. Diogo, and C. Kayser for their feedback
on a previous version of this manuscript.
Accepted: February 23, 2012
Published: May 9, 2012
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