ARTICLE IN PRESS

Neurodevelopmental
disorders of the prefrontal
cortex in an evolutionary
context
Branka Hrvoj-Mihica, Katerina Semendeferia,b,*
a
University of California San Diego, Department of Anthropology, La Jolla, CA, United States
b
University of California San Diego, Kavli Institute for Brain and Mind, La Jolla, CA, United States
*Corresponding author: Tel.: +1-858-8220750, e-mail address: ksemende@ucsd.edu

Abstract
The prefrontal cortex consists of several cytoarchitectonically defined areas that are involved
in higher-order cognitive and emotional processing. The areas are highly variable in terms of
organization of cortical layers and distribution of specific neuronal classes, and are affected in
neurodevelopmental and psychiatric disorders. Here the focus is on microstructural anatomical
characteristics of human prefrontal cortex in an evolutionary context with special emphasis on
Williams syndrome. We include a pilot analysis of distribution of neurons labeled with
an antibody to non-phosphorylated neurofilament protein (SMI-32) in the frontal pole of
Williams syndrome to further examine microstructural characteristics of the prefrontal cortex
in Williams syndrome and implications of the distribution of SMI-32 immunoreactive neurons
for connectivity between the frontal pole and other cortical areas in the disorder.

Keywords
Pyramidal neurons, SMI-32, Neurofilament, Williams syndrome, Neurodevelopmental disor-
ders, Frontal pole, Orbitofrontal cortex

1 Introduction
The prefrontal cortex (PFC) plays a fundamental role in information processing, and
subserving higher-order cognitive and emotional functions. It forms the anterior-
most portion of the primate cerebrum, encompassing a set of areas that are distinct
in their function, cellular organization, and connectivity (Barbas, 2000; Bobic
Rasonja et al., 2019). During primate evolution, selection acted on the behaviors
involving PFC areas, and hyperscaling of PFC relative to the rest of the neocortex
Progress in Brain Research, ISSN 0079-6123, https://doi.org/10.1016/bs.pbr.2019.05.003
© 2019 Elsevier B.V. All rights reserved.
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2 Disorders of the prefrontal cortex

represents a general evolutionary trend across primates (Bush and Allman, 2004).
In humans, PFC underwent a reorganization, with enlargement of certain cytoarch-
itectonically defined areas—notably Brodmann area 10 in the frontal pole (BA 10;
Semendeferi et al., 2001)—and relative reduction in parts of the orbitofrontal cortex
(BA 13; Semendeferi et al., 1998). Reorganization was also observed in subcortical
structures that are selectively interconnected with the PFC, including the amygdala
in humans (Barger et al., 2007, 2012), thalamus (Armstrong, 1986), and basal ganglia
(Raghanti et al., 2018), suggesting an important interplay in human evolution be-
tween emotional processing, and their evaluation and cognitive control of the
response. Our knowledge of PFC connectivity and its functional implications can
be expanded by including structures that were traditionally given less consideration
in the context of higher-order emotion and cognitive processing. One such example
is indusium griseum, a structure that was recently characterized as part of the
neocortex (Bobic Rasonja et al., 2019) and based on animal studies, plays an
important role in emotion regulation (Arroyo-Guijarro et al., 1988).
Various PFC areas are often sites that show altered organization in psychiatric
and neurodevelopmental disorders. Microstructural modifications, especially when
accompanied by distinct behavioral manifestations, offer a window into function of a
specific cortical area or, if coupled with information from the connecting subcortical
structures, into the functional aspects of specific circuitry (Mega and Cummings,
1994). Especially informative are the disorders with a clear genetic component, such
as Rett syndrome (RTT), Williams syndrome (WS), or selected cases of autism spec-
trum disorder (ASD), as they offer an opportunity to link the activity of genes with
their influence on specific aspects of brain structure.
The past several years have seen an increasing number of studies examining
various aspects of neuroanatomy in Williams syndrome (WS). WS is a neurodevelop-
mental disorder caused by a specific hemideletion on chromosome 7. It is character-
ized by an unusual increase in sociability, preservation of certain aspects of language
processing, and compromised spatial and general cognition (Bellugi et al., 1999,
2000). The syndrome offers an opportunity to link together different levels of brain
organization, and the specific role of PFC in processing complex behaviors compro-
mised in the disorder. Williams syndrome is well characterized genetically (Korenberg
et al., 2000), with a distinct behavioral and cognitive profile (Bellugi et al., 1999), and
the brain anatomy has been explored at macro- (e.g., Fan et al., 2017; Jernigan and
Bellugi, 1990; Reiss et al., 2004) and micro-structural (e.g., Hanson et al., 2018;
Lew et al., 2017, 2018; Wilder et al., 2018) levels. In addition, the organization of den-
drites on pyramidal neurons in the cortex of WS subjects has been described recently
(Hrvoj-Mihic et al., 2017) and functional implications of dendritic organization in WS
addressed using induced pluripotent stem cell techniques (Chailangkarn et al., 2016).
Here, we review phenotypical neural modifications of PFC in an evolutionary
context and in various disorders with a focus on WS. We report new observations
in WS lower layer III neurons in the frontal pole (BA 10), including a pilot analysis
of distribution of neurofilament proteins in neurons, and their implication for con-
nectivity between the frontal pole and other cortical areas.
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2 Prefrontal cortex anatomy in an evolutionary context 3

2 Prefrontal cortex anatomy in an evolutionary context

Diverse functions of PFC, grouped under the term “executive functions,” are mostly
processed in its dorsolateral divisions, including Brodmann’s areas (BA) 9, 10, and
46 (Baddeley, 1992; Fuster, 2000; Jurado and Rosselli, 2007). The orbital regions,
consisting of BA 10, 11, 12, and 13 are implicated in higher level emotional and so-
cial processing (Beer et al., 2003; Fellows, 2007a,b; Habib et al., 1996; Stone et al.,
1998). Both the executive functions, emotional responses, and processing of socially
relevant behaviors represent important adaptive complexes in primates navigating
changing environments and complex social systems, and thus are of evolutionary
importance (Lew and Semendeferi, 2017). Even though inferences about microstruc-
tural organization of the brain are not possible based on fossil remains, hominin en-
docasts suggest that some form of frontal lobe reorganization may have already taken
place in the late Pliocene. The endocranial casts of gracile australopithecine, includ-
ing Australopithecus afarensis and Australopithecus africanus, are characterized by
squared-off frontal lobes (Falk et al., 2000). A similar pattern is observed in A. sediba
(Carlson et al., 2011; Falk, 2014), a South African specimen dated to 1.9 mya (Berger
et al., 2010) coupled with possible transitional forms of the orbital sulcus, suggesting
a reorganization of PFC (Falk, 2014; but see also Carlson et al., 2011). The pattern
can be observed in comparative studies across primates, with human frontal lobes
already presenting distinct features at birth (Zollikofer and Ponce de León, 2013),
with the characteristic squared-off shape that sets them apart from other great apes.
Despite the distinct shape of the frontal lobe in extant humans, the frontal lobe as
a whole does not show expansion relative to great apes (Semendeferi et al., 2002), but
instead an increase in selected PFC areas, like BA 10 which forms the frontal pole
(Semendeferi et al., 2001). At the microstructural level, the same area is distinct in
humans by differing in neuronal density (Semendeferi et al., 2001), minicolumnar
spacing (Semendeferi et al., 2010), and morphology of dendrites of pyramidal neu-
rons, with longer, more branched dendrites with higher number of dendritic spines
relative to unimodal areas (Jacobs et al., 2001; see also Bianchi et al., 2012 for the
analysis of dendritic branching in chimpanzee). Layer III is of particular interest,
given the role of its neurons in corticocortical connections.
Within layer III of PFC, there are several differences between its upper and lower
parts that could carry important implications for the connectivity of a particular area.
For example, upper layer III is characterized by small and medium-sized cells, in
contrast to the larger neurons found in the lower layer III (Petrides and Pandya,
1999; Semendeferi et al., 2001). Across functionally distinct cortical areas, upper
layer III neurons vary in their dendritic morphology (Jacobs et al., 2001), whereas
the same differences are not present in the lower layer III (Zeba et al., 2008). Lower
layer III neurons in PFC also display a distinct pattern of dendritic development,
characterized by two rapid periods of growth, branching, and spinogenesis, separated
by a year-long period of stasis (Petanjek et al., 2008, 2011; Sedmak et al., 2018) that
has not been observed in upper layer III neurons. The maturation of lower layer III in
PFC coincides with important cognitive landmarks in early childhood—including
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4 Disorders of the prefrontal cortex

the establishment of initial components of the Theory of Mind (Casey et al., 2000)—
possibly reflecting maturation of cortical circuitry underlying higher-order integra-
tion of information from various sensory stimuli.
In further examining the observed morphological difference, immunohistological
staining can be particularly informative, since it is capable of distinguishing distinct
groups of neurons within the broad morphological class of pyramidal cells. One of
the stains, a monoclonal antibody SMI-32, labeling high and medium subunits of the
neurofilament protein triplet (Lee et al., 1988), stains a specific subset of neurons in
the cortex of primates (Campbell and Morrison, 1989). This class of neurons—SMI-
32 immunoreactive neurons (SMI-32ir)—was suggested to underlie long ipsilateral
cortico-cortical connections (Campbell et al., 1991) and displays differences in den-
sity across cortical areas and between layers within each area (Campbell and
Morrison, 1989; Hof et al., 1995a,b; Zeba et al., 2008). The distribution and density
of SMI-32 immunoreactive (SMI-32ir) neurons was also shown to vary across
species (Campbell and Morisson, 1989; Hof et al., 1995a) with higher number of
SMI-32ir cells observed in humans compared to macaques. Application of SMI-
32 staining has been used to subdivide various cortical areas in humans and non-
human primate (mostly macaque) cortex, resulting in further subdivision of macaque
orbitofrontal (Carmichael and Price, 1994) and motor (Preuss et al., 1997) areas.
Focusing specifically on the human cortex, the distribution of SMI-32ir neurons
was examined in various cortical areas (Hof et al., 1995b) and in the human orbito-
frontal cortex (Hof et al., 1995a) marked variations were observed within its cytoarch-
itectonically defined areas. In agranular PFC areas, for example, SMI-32ir are more
abundant in infragranular layers (layers V/VI) and show gradual increase in density in
layer III rostrally, toward dysgranular and granular areas (Hof et al., 1995a). Whereas
this general pattern is comparable to the distribution of SMI-32ir neurons in macaques
(Hof et al., 1995a), the number of SMI-32ir neurons and their increase especially in
lower layer III appears to distinguish human PFC areas from the same areas in
macaques. Therefore, existing studies suggest that SMI-32ir neurons display marked
variations in distribution across species, with differences not only across homologous
cortical areas, but limited in a specific layer within each area. Given that SMI-32ir
typically label neurons implicated into long cortico-cortical connections (Campbell
et al., 1991) and are especially prominent in deep layer III in humans (Campbell
and Morisson, 1989), the findings help narrow down the evolutionary studies on
one specific subset of neurons (SMI-32ir) within one unit (lower layer III) and one
functional role (long cortico-cortical connections) that may be emphasized in humans.

3 Psychiatric and neurodevelopmental disorders

Typical anatomy of different PFC areas is often compromised in neuropsychiatric
disorders. Several disorders—including schizophrenia, bipolar disorder, ASD,
RTT and WS—have been studied with a particular focus on the organization of
PFC relative to the non-affected controls (Table 1). Whereas PFC appears affected
Table 1 List of selected studies referenced in the text that utilize postmortem human tissue in the analysis of PFC areas.
Disorder PFC areas examined Layer Stain Findings References

Schizophrenia BA 9 IIIC Nissl Decreased number of large Pierri et al. (2001)

pyramidal cells
IIIC SMI-32a No difference in Pierri et al. (2003)
density/soma size
IIIC NF 200a Larger soma
No difference in density
BA 9, 46, 32 III V SMI 32a, N200a, FNP7a Trend for higher density Law and Harrison (2003)
(N200-ir)
No difference in soma size
BA 46 IIIC Golgi Decreased dendritic spine Glantz and Lewis (2000)
IIIC number
BA 10, 11, 45 rapid Golgi Lower dendritic spine density Garey et al. (1998)
Major depressive BA 9 NF 200a No difference in Miguel-Hidalgo et al. (2005)
disorder density/soma size

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BA 9, 46, 32 III SMI 32a, N200a, No difference in density Law and Harrison (2003)
V FNP7a
Bipolar disorder BA 9, 46, 32 III SMI 32a, N200a, No difference in density Law and Harrison (2003)
V FNP7a (layer III and V)
Smaller cell body (layer V)
BA 9 III Nissl Lower density (layer III) Rajkowska et al. (2001)
V Lower pyramidal neuron
density (layers III/Va)
Lower glial density (layer IIIc)
Down syndrome BA 9 IIIC rapid Golgi No difference prenatally/first
et al. (2002, 2011)
Vukšic
7 postnatal months
Rett syndrome BA 10 III Lucifer yellow Lack of areal specialization Belichenko et al. (1994)
V Decreased spine density
ASD dlPFC All layers Nissl Higher neuronal number Courchesne et al. (2011)
mPFC combined More prominent in dlPFC
BA 9 III V Golgi Kopsch Higher dendritic spine density (layer III) Hutsler and Zhang (2010)
III rapid Golgi Immature morphology Petanjek et al. (2019)
V SMI32a Decreased spine density
(layers II/IIIa and IIIb)
William syndrome BA 10 II/III V/VI Nissl Decreased neuronal density Lew et al. (2017)
BA 11
BA 10 II/III Golgi Kopsch Lack of areal specialization Hrvoj-Mihic et al. (2017)
BA 11 V/VI Compromised dendritic branching
BA 25 II/III V/VI NIssl Decreased neuronal density Wilder et al. (2018)
(layers II/III)
ASD, autism spectrum disorder; dlPFC, dorsolateral PFC; mPFC, mesial PFC; “-ir”, immunoreactive.
a
stain against neurofilament proteins.
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6 Disorders of the prefrontal cortex

in each of these disorders, closer analysis reveals interesting pattern of variations

across PFC, suggesting that the effects are often restricted to a specific area and,
within that area, may be limited to a specific cortical layer. Given that cortical layers
are characterized by a predictable set of connections (Barbas and Rempel-Clower,
1997; DeFelipe et al., 2002), linking each with a specific set of cortical areas and
subcortical structures (reviewed in Mega and Cummings, 1994), existing findings
suggest that specific circuits underlying connectivity between PFC and subcortical
structures may be more compromised than others in each disorder.
It has been proposed that the observed organization of PFC in schizophrenia, for
example, reflects compromises in thalamocortical and long-distance corticocortical
connectivity (Pierri et al., 2001). The hypothesis is based on the selective effects seen
on lower layer III neurons in PFC. Subjects with schizophrenia display a lower pro-
portion of pyramidal neurons with large cell bodies in deep layer III of BA 9 (Pierri
et al., 2001). The same population of neurons is characterized by a decrease in the
number of dendritic spines compared to controls, whereas the number of spines on
the dendrites of upper layer III neurons remains spared (Glantz and Lewis, 2000).
The subset of SMI-32ir lower layer III neurons did not show to be selectively affected
in schizophrenia (Pierri et al., 2003), displaying similarity in distribution and the
soma size with controls. However, the difference was found with labeling for a
different neurofilament epitope (NF 200), which labels both phosphorylated and
non-phosphorylated subunits of neurofilament proteins. NF 200 staining revealed
that schizophrenia is characterized by larger cell bodies in deep layer III (Miguel-
Hidalgo et al., 2005), suggesting that, within the deep layer III of BA 9, there
may be a selective effect on specific immuno-reactive subtype of neurons.
Analyses of cellular organization and dendritic morphology in schizophrenia
revealed another interesting pattern, potentially reflecting plasticity of PFC areas.
In the study examining number of dendritic spines on dendrites of pyramidal neurons
in lower layer III in BA 9, it has been reported that the use of antipsychotic agents
affected the number of spines, making the number of dendritic spines on dendrites of
patients who were undergoing therapy more similar to controls than to the individ-
uals with schizophrenia who did not take the medication (Glantz and Lewis, 2000).
A similar effect was revealed in a separate study that proposed the effects of
environmental factors—social deprivation associated with institutionalization,
and the role of age and alcohol abuse—on dendritic spines (Garey et al., 1998),
influencing the patterns observed between subjects with schizophrenia and
non-affected controls.
Similar patterns of selective involvement of specific cortical layers were reported
for the autism spectrum disorder (ASD). In a study examining a postmortem
sample of ASD, the number of neurons was increased in PFC relative to controls
in all cortical layers combined, although the effects were more prominent in dorso-
lateral PFC (dlPFC) compared to mesial PFC (mPFC; Courchesne et al., 2011). The
pathology in dendritic spines—in the case of ASD, manifested by increased density
of dendritic spines (Hutsler and Zhang, 2010)—was restricted to layer III of BA 9.
In contrast, higher density of spines in parietal cortex (BA 21) was distributed
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4 Prefrontal cortex in Williams syndrome 7

equally across layers II/III and layers V/VI (Hutsler and Zhang, 2010). It has been
previously shown that individuals with ASD do not perform well on tasks involving
dlPFC, even though there appear not to be structural differences detectable by MRI
(Griebling et al., 2010). The findings on neuronal density and dendritic spine
pathologies may thus suggest that subtle, microstructural abnormalities may affect
function of dlPFC in ASD, even in the absence of macrostructural pathologies.
In a separate report focusing on BA 9 in an adult subject with ASD (Petanjek
et al., 2019), the effects on the morphology of dendrites seem to be restricted to
the neurons in layer II and upper parts of layer III. Large pyramidal cells in the lower
layer III and neurons in infragranular layers did not display pathology, either in
dendritic morphology, density of neurons, or the distribution of a specific
subset—in this case, SMI-32ir—neurons. In the same subject, the neurons in upper
cortical layers were characterized by lower number of dendritic spines which also
displayed the morphology typical of immature spines (Petanjek et al., 2019). Given
the importance of upper layer III for short cortico-cortical connectivity (Hof et al.,
1995b), the observed pathology may result in decreased connectivity of BA 9 with
neighboring cortical areas.
In addition to the pathologies in the morphology and number of dendritic spines
in autism discussed above, deficiencies in RTT, a disorder previously classified un-
der ASD and characterized by a specific MECP2 mutation, displays distinct features
that set it apart from the cortex of controls. In analyzing dendritic morphology in
RTT, the most striking finding was that dendritic variations across functionally dif-
ferent cortical areas, as seen in controls, were absent in the cortex of RTT patients
(Belichenko et al., 1994). In the prefrontal cortex (BA 10), pathologies in layers II/III
were more prominent than in layers V/VI. In contrast, in the motor cortex (BA 4)
layers V/VI were more affected than layers II/III, whereas in the temporal cortex
(BA 22) both layers II/III and V/VI are affected equally (Belichenko et al., 1994).
The number of spines, however, was consistently reduced across all cortical layers
(Belichenko et al., 1994) suggesting, similarly to the pattern reported in Petanjek
et al. (2019), the role of early development for the appearance of disorder-specific
pathologies.

4 Prefrontal cortex in Williams syndrome

Among the disorders with a distinct social phenotype for which deficiencies in
some aspects of prefrontal circuitry have been suggested, Williams syndrome
(WS) deserves a particular mention. WS is underlined by a hemizygous deletion
of
25 genes on chromosome 7 (WS deleted region; Schubert, 2009) and charac-
terized by a unique set of preserved and compromised behavioral and cognitive fea-
tures: preservation of some aspects of language and face processing, compromised
spatial cognition and lower overall intelligence, and an increased desire to interact
socially and to approach strangers (Bellugi et al., 1999). Functional imaging has sug-
gested differences in brain activation during social tasks in WS individuals compared
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8 Disorders of the prefrontal cortex

to controls, especially in the frontal cortical areas. Individuals with WS display

lesser activation of dorsolateral PFC, dorsal anterior cingulate, and the striatum
in Go/NoGo tasks (Mobbs et al., 2007), and an overall higher activity of anterior
brain regions at the expense of primary and secondary visual areas in face and gaze
processing tasks (Mobbs et al., 2004). The behavioral phenotype in WS, therefore,
can at least in part be linked with the differences in higher-order processing of
socially relevant cues, which involve a distinct activity of PFC areas.
Despite the behavioral profile of WS, which would suggest abnormalities in the
organization of brain areas involved in processing of social behavior, frontal areas
underlying higher-level processing of socially relevant stimuli appear relatively
spared in WS. Based on MRI studies, the volume of the frontal lobes appears
comparable between WS and non-affected controls, despite the overall reduction
of the brain volume in WS (Reiss et al., 2004). Dorsal and medial subdivisions of
PFC similarly remain spared in WS (Fan et al., 2017). At the same time, certain
subdivisions of the frontal lobe show differences between WS and controls. The
differences are especially prominent in the orbitofrontal cortex and dorsal part of
the anterior cingulate cortex (Fan et al., 2017; Reiss et al., 2004) with WS individuals
displaying higher gray matter volumes than controls. These findings are paralleled
by the findings in two subcortical structures—the amygdala (Reiss et al., 2004) and
the putamen/nucleus accumbens region of the striatum (Fan et al., 2017), both of
which share reciprocal connections with PFC and are important parts of the circuits
underlying social behaviors.
Further analyses focusing on the cellular organization and morphology of
dendrites on pyramidal neurons revealed more specific patterns of organization of
cortex in WS, with a set of well-defined features the differ between WS and control
subjects (Fig. 1). In the prefrontal cortical areas, subjects with WS display decreased
neuronal density, with patterns that appear layer- and area-specific. Both in the fron-
tal pole (BA 10) and in the orbitofrontal cortex (BA 11), as well as in the ventral
portion of the anterior cingulate (BA 25), WS subjects have decreased density of neu-
rons relative to controls (Lew et al., 2017; Wilder et al., 2018). The decrease is
especially prominent in infragranular layers of BA 10, which contrasts with the
pattern observed in the somatosensory cortex (BA 3-1-2) and secondary visual area
(BA 18), where the density of neurons is higher in WS compared to control subjects
(Lew et al., 2017). Both BA 10 and BA 11 in WS display pathologies in the length,
branching complexity, and the number of spines on dendrites of pyramidal neurons,
lacking the typical increase in dendritic complexity relative to primary processing
areas, as seen in non-pathological individuals (Hrvoj-Mihic et al., 2017).
In subcortical structures, an increase in neuronal density was observed in the
lateral nucleus of the amygdala (Lew et al., 2018) and an increased density of glia
was reported in dorsal and medial caudate nucleus of the striatum (Hanson et al.,
2018). Both the amygdala and dorsal and medial caudate participate in processing
of social behavior, emotional responses, and cognitive control, and represent
important sources of subcortical inputs into medial and orbital regions of PFC
(Amaral and Price, 1984; Haber, 2003). In summary, existing findings suggest that
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4 Prefrontal cortex in Williams syndrome 9

FIG. 1
Summary of existing research conducted on microstructural aspects of prefrontal cortex in WS
syndrome and two sub-cortical structures with connections to prefrontal areas. WS subjects
display a decreased density of neurons in selected areas in the orbital cortex and the
frontal pole (A) and in the anterior cingulate (B), and an increased number of neurons in the
lateral nucleus of the amygdala (C). The medial and dorsal nuclei of the striatum in WS are
characterized by an increased number of glia compared to control subjects (D).
The image is based on Hanson, K.L., Lew, C.H., Hrvoj-Mihic, B., Groeniger, K.M., Halgren, E., Bellugi, U.,
Semendeferi, K., 2018. Increased glia density in the caudate nucleus in Williams syndrome: implications for
frontostriatal dysfunction in autism. Dev. Neurobiol. 78, 531–545. Lew, C.H., Brown, C., Bellugi, U.,
Semendeferi, K., 2017. Neuron density is decreased in the prefrontal cortex in Williams syndrome. Autism Res.
10, 99–112. Lew, C.H., Groeniger, K.M., Bellugi, U., Stefanacci, L., Schumann, C.M., Semendeferi, K., 2018.
A postmortem stereological study of the amygdala in Williams syndrome. Brain Struct. Funct., 223(4),
1897–1907. Wilder, L., Hanson, K., Lew, C., Bellugi, U., Semendeferi, K., 2018. Decreased neuron density and
increased glia density in the ventromedial prefrontal cortex (Brodmann area 25) in Williams syndrome. Brain Sci.
8, 209 referenced in the text.

the differences between WS and controls can be observed at the cellular level and
that they are more prominent in some cytoarchitectonically defined PFC areas—
and some layers within these areas—compared to others. Given that pyramidal
neurons encompass a variety of functionally distinct subclasses (DeFelipe et al.,
2002), existing findings suggest that, instead of assuming general pathologies af-
fecting all pyramidal neurons within a certain area or a cortical layer, it would be
meaningful to examine specific subsets of neurons that may be driving the ob-
served difference between WS and non-affected controls.
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10 Disorders of the prefrontal cortex

4.1 Distribution of neurofilament immunoreactive neurons in the

frontal pole of WS
Here, we report observations on SMI-32ir of pyramidal neurons in BA10 of WS.
Given the importance of supragranular layers for proper inter-cortical connectivity,
and since previous research suggests differences in supragranular layers between WS
and controls, we expect that the pathologies may be restricted to long-projecting,
magnopyramidal neurons in lower layer III, influencing the manifestation of behav-
ioral traits that suggest lack of adequate connectivity within the cortex. Given the
significance of identifying the distribution of SMI-32ir neurons, as discussed above,
we examined BA 10 of two subjects with WS and one non-affected control. One of
the two subjects, WS 1 (31-year-old male) was previously used in our study of den-
dritic morphology (Chailangkarn et al., 2016; Hrvoj-Mihic et al., 2017) and WS 9
(43-year-old female) was included in the study of density of neurons in the cortex
and in the amygdala of WS (Lew et al., 2017, 2018). Both subjects were also ana-
lyzed in the study of striatum (Hanson et al., 2018) and ventromedial PFC in WS
(Wilder et al., 2018). Details about the specimens are provided in the referenced
publications.
Tissue blocks corresponding to BA 10 were cryoprotected in successive concen-
trations of 10%, 20%, and 30% buffered sucrose solutions, and sectioned at 30 μm on
a Leica SM2010 freezing microtome. Immunohistochemical staining was performed
on free-floating sections, starting with an antigen retrieval step that consisted of
heating the sections for 30 min at 90 °C in citrate buffer (pH 2.5). Following the pre-
treatment in 3% hydrogen peroxide and 0.2 Triton-X, sections were blocked in 5%
bovine serum albumin with Triton-X, and placed into SMI-32 antibody (Biolegend,
San Diego, CA) in a dilution 1:1000. The sections were then processed using the sec-
ondary anti-mouse antibody in 1:200 concentration, followed by avidin-biotin
method with Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Staining
was visualized with the application of 3,30 -diaminobenzidine (DAB; Vector Labo-
ratories, Burlingame, CA).
Consistent with previous reports (Hof et al., 1995a), in the control subject SMI-32
staining gave the appearance of two distinctly stained bands, separating the cortex
into supra- and infra-granular layers divided by the unstained layer IV (Fig. 2A).
In supragranular layers, density of SMI-32ir neurons increased toward deeper parts
of layer III and the cell bodies of stained neurons became larger and more darkly
stained toward the border with layer IV (Fig. 2B, D). In infragranular layers,
SMI-32 again revealed two bands of stained neurons: densely packed neurons in
the layer V, immediately below the unstained layer IV and, toward the white matter,
another band of dense, but lightly stained neurons (Fig. 2C).
In the cortex of WS subjects, SMI-32 staining also revealed two distinctly stained
bands, corresponding to supra- and infra-granular layers, although the unstained
layer IV was less distinct than in controls. In WS subject, SMI-32ir neurons were
less densely packed and had more darkly stained and larger cell bodies (Fig. 2E).
In supragranular layers, there appeared to be less SMI-32ir neurons than in the
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4 Prefrontal cortex in Williams syndrome 11

FIG. 2
Photomicrographs of SMI-32 stained section of BA 10 in non-affected controls (subject 5552)
and WS (subjects WS 1 and WS 9). Image of both supra- and infra-granular layers taken at a
low (4 ) magnification in controls (A) and WS (E). Scale bar: 200 μm. A higher (10 )
magnification image illustrating distribution of SMI-32ir neurons in supra-granular layers
in controls (B) and WS (F), and in infragranular layers in controls (C) and WS (G). Scale bar:
100 μm. Example of SMI-32ir neurons in controls (D) and WS 9 (H) taken at 60
magnification. Scale bar: 50 μm.

controls. SMI-32ir also displayed a distinct organization, with areas containing only
a few, sparsely distributed neurons with very dark cell bodies and areas with densely
packed neurons (Fig. 2F, H). This pattern seems not to be related to the location of the
neurons relative to the top of the gyrus and bottom of the sulcus, since the areas with
sparse and the areas with dense SMI-32ir neurons were found adjacent to each other
(Fig. 2F). In the parts of supragranular layers where only a few neurons were positive
for SMI-32, those were found almost entirely in the
200 μm band above layer IV,
corresponding to the lower layer III (layer IIIC), although an occasional SMI-32ir
could be found in upper parts of supragranular layers (Fig. 2F). In infragranular
layers, staining revealed two bands of stained tissue, as seen in the control, but with
more neurophil staining in layer V than observed in the control subject (Fig. 2G).
Following these qualitative observations, we pursued quantification of the den-
sity of SMI-32ir neurons and their soma size. Number of SMI-32ir neurons and soma
size were obtained as described previously (Lew et al., 2017). Both WS subjects dis-
played lower density of SMI-32ir neurons/mm3 than the control (Fig. 3A). WS sub-
jects also displayed lower average soma volume compared to the control (Fig. 3B).
 ARTICLE IN PRESS

12 Disorders of the prefrontal cortex

FIG. 3
Graphs showing differences in density of SMI-32ir neurons (A) and volume of cell bodies
(B) between WS and the control subject. Dots in (B) represent values for individual neurons.

4.2 Implications of SMI-32ir neuron distribution in WS for

understanding variability in PFC organization
Previous research suggested that there is a decrease in the overall density of neurons
in BA 10 of WS subjects (Lew et al., 2017). When analyzed separately, layers II/III
also displayed a trend toward lower neuronal density in WS (Lew et al., 2017).
Here, we observed that same pattern by examining one subset of neurons—SMI-
32ir neurons located at the deep level III in BA 10 in two of the subjects used in
the study by Lew et al. (2017). The same population of neurons displayed lower av-
erage cell body size, with even the largest neurons in most cases falling below the
average for the control subject (Fig. 3B). It is, thus, possible that the differences seen
between WS and controls in neuronal density could be driven, at least in part, by
selective pathologies in SMI-32ir neurons in the lower layer III. If SMI-32ir neurons
indeed represent a neuronal population selectively affected in WS, we would expect
that the pathologies in their distribution are not limited to the BA 10, but that they
may be present in other prefrontal areas. Alternatively, additional sub-populations of
pyramidal cells could be affected specifically in the lower layer III, and SMI-32ir
neurons represent only one such population of affected neurons. In either case,
the present findings warrant use of additional cell-specific markers to examine pa-
thologies across layers in BA10 and other PFC areas of WS subjects.
Current findings on the differences in distribution of SMi-32ir neurons in deep
layer III carry implications for the connectivity between BA 10 and other cortical
area. SMI-32 typically stains layer III neurons with long ipsilateral projections
within the cortex (Hof et al., 1995a), and the decrease in SMI-32ir neurons in WS
suggests lesser connectivity between BA 10 and other cortical areas in the same lobe.
As BA 10 shares connections with the neighboring BA 9, BA 46 in the lateral PFC,
and BA 7 in the parietal lobe (reviewed by Mega and Cummings, 1994), weakening
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5 Future directions: Old questions with a new focus 13

the connectivity could explain compromised function of the “executive circuit”

within PFC. Behavioral manifestations seen in WS, including different responses
to social situations compared to age-matched controls (Jones et al., 2000) may at
least in part be explained by the effects lower density of long-projecting cortical neu-
rons has on corticocortical connectivity.
It is interesting that some social aspects of WS appear relatively early, and dif-
ferent responses to social situations are already present in late infancy/early child-
hood (Jones et al., 2000). In contrast, pathologies in other neurodevelopmental
disorders are not clearly present until much later—2 years in autism (Landa et al.,
2013) and late childhood in Down syndrome (DS; Fidler, 2005). The appearance
of symptoms in ASD and DS post-dates rapid development of cortical microcircuitry
and growth spurt in lower layer III pyramidal neurons. As already discussed, the neu-
rons in lower layer III of PFC are specific for displaying two growth spurts—from
birth until 2.5 months and from 18 months until 2.5 years (Petanjek et al., 2008;
Sedmak et al., 2018). In neurodevelopmental disorders in which pathologies in den-
dritic branching and length occur, they typically emerge after the second period of
rapid neuronal growth (Vukši
c et al., 2002, 2011), and behavioral symptoms do not
become prominent until much later. Early development of hypersocial behavior in
WS, indicative of compromised executive circuit within the brain (Mega and
Cummings, 1994), but predating maturation of layer IIIC neurons challenges the
hypothesis that the behavioral phenotype in WS in mostly driven by IIIc neurons,
suggesting instead that other, earlier maturing parts of cortical microcircuitry are
affected in WS.

5 Future directions: Old questions with a new focus

The present review highlighted variations within PFC and emphasized nuances in its
internal organization as an important topic in evolutionary studies. We have specif-
ically discussed one subtype of pyramidal neurons—SMI-32ir neurons—that dis-
plays marked variability across cortical areas and whose distribution may be
important for understanding organization of long ipsilateral cortico-cortical connec-
tions across species. Previous work has highlighted the significance of SMI-32ir neu-
rons in delineating functional boundaries within PFC areas resulting, for example, in
additional subdivisions of macaque orbitofrontal and medial prefrontal cortex
(Carmichael and Price, 1994). Further studies need to examine if these divisions
can be found in other species of primates, and whether or not the areas that show
evolutionary reorganization are characterized by an expansion of the existing basic
cytoarchitectonic plan of an area, or by additional subdivisions that cannot be
detected in Nissl-stained samples. As different layers within PFC can be distin-
guished based on the expression of different molecular markers (Arion et al.,
2007), further research warrants focus on specific layers and neuronal subtypes
within these layers.
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14 Disorders of the prefrontal cortex

By examining the distribution of SMI-32ir neurons in BA 10 of WS, we have

drawn attention to the importance of examining specific neuronal subclasses in neu-
rodevelopmental disorders to better understand the link between a structure’s orga-
nization and functional implications of the compromised neuronal organization.
Previously, SMi-32 staining has been used to study the pathology of Alzheimer’s
disease (Hof and Morrison, 1990), since SMI-32ir neurons appear especially vulner-
able to the decline in Alzheimer’s disease. Earlier work on WS has suggested de-
creased neuronal density in BA 10 in WS (Lew et al., 2017) and compromised
organization of dendrites on pyramidal neurons in BA 10 (Hrvoj-Mihic et al., 2017).
Here, we have expanded the analysis and presented pilot data that suggest that a specific
class of neurons staining positive for non-phosphorylated neurofilament epitopes
(SMI-32), located mostly in lower layer III of BA 10, may be in particularly compro-
mised in WS. Given the role of lower layer III in specifying cortico-cortical circuitry,
it is possible to hypothesize that the decrease in long distance connections between
PFC and other association areas in the cortex may underline the phenotype typical of
the disorder.
Further analyses focusing on neurodevelopmental disorders can yield important
insights into the function of SMI-32ir neurons. Given that SMI-32ir neurons are
sparse at birth and appear postnatally—making largest appearance 30–42 post-natal
days in the visual cortex of macaques (Kogan et al., 2000)—their development in
humans can yield important insights into the role of environment in specifying
the early establishment of cortical microcircuitry. It is worth nothing that lower layer
III neurons in human PFC are characterized by slow development that spans the first
2.5 years (Petanjek et al., 2008; Sedmak et al., 2018), and the maturation of spines
continues until the third decade of life (Petanjek et al., 2011). Given their long de-
velopment and the exposure to various factors influencing neuronal development,
examining lower layer III neurons in PFC can be informative for the patterns leading
to the development of pathologies.
It has been recently proposed (Petanjek et al., 2019) that slow maturation of layer
III neurons in PFC, with different parts of the dendrites maturing at different ages
over several years, may affect cortical network dysfunction at a scale larger than
a specific layer or one cortical area (Petanjek et al., 2019). In other words, if
developmental disturbance are experienced during the time when elongation of den-
drites takes place (in PFC lower layer III neurons, the second year of life), the effect
would be seen in reduced number of inputs into lower layer III neurons, changing
their functional properties and indirectly affecting their efferent targets, including
the pattern of processing within minicolumns (Petanjek et al., 2019). Given that
symptoms in neurodevelopmental disorders tend to appear in sequences, for example
with “precursors” to language problems noticed during pre-verbal infant vocaliza-
tions, but not having their verbal manifestation until much later (Fidler, 2005;
Karmiloff-Smith, 1997), analyses addressing the link between neuronal maturation
and early manifestations of later aberrant behaviors in various disorders can yield
important insights into the link between neuronal structure and functions.
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References 15

Acknowledgments
We would like to thank Natasa Jovanov Milosevic and Zdravko Petanjek for their advice and
for valuable discussions that helped shape some of the ideas presented here. We would also
like to acknowledge the help of Danica Budinscak, Bozica Popovic and Maja Horvat Bozic
from Croatian Institute for Brain Research in sharing their expertise on immunohistological
staining. A special thanks is due to Kari Hanson, Caroline Lew, Demi Greiner and Deion
Cuevas for their help with tissue processing and collection of the pilot data presented here,
and to Shyam Srinivansan for his (mostly) benevolent comments on some of the views
discussed in this review.

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