Barbas1,2,3
Prefrontal Cortical Systems in the Rhesus Departments of Behavioral Neuroscience and 1Anatomy and
Neurobiology, Boston University School of Medicine, Boston,
Monkey MA, 2Department of Health Sciences, Boston University, 635
Commonwealth Ave., #431, Boston MA, 02218 and 3New
England Regional Primate Research Center, Harvard Medical
School, Boston, MA, USA
The prefrontal cortex encompasses a large and heterogeneous set of role of distinct prefrontal cortices in complex cognitive, mne-
areas, whose borders have been variously mapped in different monic and emotional processes [for reviews see (Goldman-Rakic,
architectonic studies. Differences in cortical maps present a 1988; Petrides, 1989; Fuster, 1989, 1993; Barbas, 1995)].
formidable problem in comparing data across studies and in Architectonic areas of the prefrontal cortex in macaque
constructing databanks on the connections and functional attributes monkeys, first mapped on the basis of cellular features, and the
of cortical areas. Here we used quantitative approaches to cortical distribution of myelin (Brodmann, 1905; Vogt and Vogt, 1919;
mapping to investigate (i) if architectonic areas of the prefrontal Walker, 1940; Von Bonin and Bailey, 1947; Sanides, 1970; Barbas
cortex in adult rhesus monkeys have unique profiles and (ii) if groups and Pandya, 1989; Preuss and Goldman-Rakic, 1991; Morecraft
of architectonic areas belonging to distinct cortical types, ranging et al., 1992) have been subdivided further with the aid of
from agranular to eulaminate, have similar features. In addition, we
modern cellular and molecular stains [e.g. (Hof and Nimchinsky,
used multidimensional analyses to see if, and how, prefrontal areas
form clusters when multiple features are considered simultaneously. 1992; Carmichael and Price, 1994; Nimchinsky et al., 1996)].
We used quantitative unbiased sampling procedures to estimate the However, architectonic studies rely on qualitative differences in
areal and laminar density of neurons, glia and neurons positive for a number of morphological cellular and neurochemical features
the calcium binding proteins parvalbumin (PV), calbindin (CB) and among areas, which may account for disagreements in areal
calretinin (CR) among 21 prefrontal areas or subdivisions of areas. subdivisions. Differences in maps and nomenclature present a
Neuronal density varied among the prefrontal cortices (range: 38 569 formidable problem in constructing central databanks on the
4078 to 58 708 2327 neurons/mm3); it was lowest in caudal connections and functional attributes of areas obtained from
orbitofrontal and medial areas (OPAll, OPro, 13, 24a, 32, M25) and different studies (Stephan et al., 2000).
highest in lateral prefrontal areas (subdivisions of areas 46 and 8). In this study we used an alternative approach to cortical
Neurons positive for PV were most prevalent in lateral prefrontal mapping by investigating whether architectonic areas of the
areas and least prevalent in caudal orbitofrontal and medial pre- prefrontal cortex can be characterized by a set of quantitative
frontal areas, whereas the opposite trend was noted for neurons
criteria. Architectonic areas vary in a number of laminar, cel-
that expressed CB. Neurons positive for CR did not show regional
differences, and the density of glia showed small variations among lular and molecular features. Here we focused on fundamental
prefrontal cortices. The differences among areas, along with differ- architectonic criteria that form the basis of classic architectonic
ences in the thickness of individual areas and layers, were used studies, namely, density of neurons and glia, as well as some
to establish a quantitative profile for each area. The results showed neurochemical markers for calcium binding proteins, which
that differences in the density of neurons, and the preponderance of label distinct classes of cortical neurons and have proved valu-
neurons positive for PV and CB, were related to different architec- able in architectonic studies [e.g. (Jones et al., 1995; Gabbott
tonic types of areas found within the prefrontal cortex. Conventional and Bacon, 1996b)]. We asked the following questions. Do
as well as multi-parameter statistical analyses distinguished at one architectonic areas of the prefrontal cortex have unique profiles
extreme the agranular and dysgranular (limbic) cortices, which were that can be described and illustrated quantitatively? Conversely,
characterized by prominent deep layers (VVI), the lowest neuronal do groups of architectonic areas have similar features that may
density, the highest ratio of glia/neurons, and the lowest density of suggest common functions? We addressed the latter question in
PV and the highest for CB. At the other extreme, lateral eulaminate
two ways. First, we tested whether areas belonging to distinct
cortices were characterized by the highest density of neurons, a
prominent granular layer IV, denser supragranular (IIIII) than cortical types have similar features, according to the structural
infragranular (VVI) layers, and a balanced distribution of neurons model of the prefrontal cortex (Barbas and Rempel-Clower,
positive for PV and CB. The results provide insights into potentially 1997). Unlike architectonic parcellation, which is based on
different rates of development or maturation of limbic and eu- the detailed cellular and molecular features of areas, cortical
laminate prefrontal areas, and their differential vulnerability in type relies on broad structural features shared by more than
neurological and psychiatric diseases. The quantitative methods one architectonic area, including the number and distinction
used provide an objective approach to construct maps, address of identifiable layers. In the prefrontal cortex we previously
differences in nomenclature across studies, establish homologies identified several types of cortices, ranging from agranular,
in different species and provide a baseline to identify changes in which have only three identifiable layers, to eulaminate, which
pathologic conditions. have six distinct layers. The significance of cortical type is based
on our previous findings indicating that the pattern of cortico-
Introduction cortical connections can be predicted on the basis of the broad
The prefrontal cortex in primates extends from the frontal pole laminar features of the interconnected areas [e.g. (Barbas, 1986;
to the premotor cortex, and is composed of several heterogene- Barbas and Rempel-Clower, 1997; Rempel-Clower and Barbas,
ous areas identified in classic architectonic studies (Brodmann, 2000)]. In a second approach, we used multidimensional
1905; Vogt and Vogt, 1919; Walker, 1940; Von Bonin and Bailey, analyses to see if, and how, prefrontal areas form clusters when
1947; Sanides, 1970). Structural differences may underlie the multiple features are considered simultaneously. The latter
Oxford University Press 2001. All rights reserved. Cerebral Cortex Oct 2001;11:975988; 10473211/01/$4.00
approach explored the structural similarities and differences of coated slides. A few sections were counterstained for Nissl to help
prefrontal areas independent of cortical type. The study of even delineate architectonic borders. Sections were dehydrated in graded
a few architectonic features within the context of the complex series of alcohols and xylenes and coverslipped with Permount (Fisher
areal and laminar features of the prefrontal cortex provided up Scientific). Control experiments were conducted in which the primary
antibody was omitted while all other steps were identical to the experi-
to 18 parameter dimensions. The quantitative approaches to
mental conditions.
cortical architecture provided key insights that may justify the
use of similar approaches to cortical mapping to begin to resolve
Prefrontal Areal and Laminar Boundaries
differences in nomenclature across studies, establish homologies
Twenty-one prefrontal areas or subdivisions of areas were investigated
in different species and provide a baseline to identify changes based on the architectonic map of Barbas and Pandya (Barbas and Pandya,
in pathologic conditions [e.g. (OKusky and Colonnier, 1982; 1989), modified from classic maps (Vogt and Vogt, 1919; Walker, 1940).
Selemon et al., 1995, 1998; Gabbott and Bacon, 1996b)]. Prefrontal cortices included medial areas 24a, 32, M25, M9, M10 and M14,
orbitofrontal areas OPAll, OPro, area 13, area 11 and O12, and lateral areas
46 and 8. Areas 46 and 8 were further subdivided into their dorsal (D) and
Materials and Methods
ventral (V) components. Areas 13 and 8 were subdivided into their gyral
(g) and sulcal (s) parts. Areas 24a and 46, which extend almost along
Tissue Processing
Data were obtained from seven adult rhesus monkeys (Macaca mulatta). the entire rostrocaudal span of the prefrontal region, were divided into
Seven animals were used to estimate the regional density of neurons and rostral, middle and caudal thirds. Only the rostral (r) and caudal (c) parts
glia, and five of these were used to study the distribution of the calcium of areas 24a and 46 were included for analysis.
binding proteins parvalbumin (PV), calbindin (CB) and calretinin (CR). Neurons and glia were distinguished in Nissl stained sections by
Experimental procedures were conducted according to the NIH Guide cell size and shape, and nuclear staining characteristics. Neurons were
for the Care and Use of Laboratory Animals (NIH publication #86-23, relatively large with a pale blue cytosol and clearly identified nucleolus,
revised 1987). while glia were usually smaller with intensely stained nucleus. All glia,
including astrocytes, oligodendrocytes and microglia, were placed in one
category.
Perfusion and Cryoprotection
Prefrontal areas identified in Nissl stained sections were matched to
Animals were given an overdose of anesthetic (sodium pentobarbital) corresponding sections stained for PV, CB and CR. The rostral and caudal
and perfused through the heart with saline, followed by 2 l of fixative limits of each area were identified through serial coronal sections. For
(4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The brain was each section architectonic boundaries were identified under a micro-
removed from the skull, photographed and cryoprotected in graded scope (Nikon, Optiphot). Drawings of architectonic borders were
solutions of sucrose (12, 16, 20 and 30%). The brain was then frozen in transferred from the slides onto paper by means of a digital plotter
70C isopentane according to Rosene and Rhodes (Rosene and Rhodes, (Hewlett Packard, 7475A) electronically coupled to the stage of the
1990), cut on a freezing microtome in the coronal plane at 40 m in 10 microscope and to a PC computer, as described previously [e.g. (Barbas et
series and collected in 0.1 M phosphate buffer. al., 1999)]. The boundary of each area was plotted and stored on disc as a
series of x and y coordinates.
Nissl Staining Each prefrontal area was subdivided into laminar subgroups to allow
One series of sections was stained for Nissl using either thionin or cresyl comparison in density across areas. Layer I is easily distinguishable in
violet to identify neurons and glia, and to delineate architectonic borders all areas and made up one laminar subgroup. Layers IIIII were included
(Ling and Leblond, 1973; Ling et al., 1973; Gabbott and Stewart, 1987; into one group as were layers VVI because there is no clear distinction
Barbas and Pandya, 1989). Nissl staining using thionin (five cases) and between each pair of layers in some prefrontal cortices (the agranular and
cresyl violet (two cases) yielded similar results. Sections mounted on dysgranular). For comparison, the same grouping was used for all pre-
chromealum coated slides were dried, defatted in a 1:1 solution of frontal cortices. In most prefrontal areas layer IV was distinct and formed
chloroform and 100% ethanol for 1 h, rehydrated through a series of an additional group. Agranular (areas OPAll and 24a) and dysgranular
graded alcohols and dH2O, stained with 0.05% thionin (pH 4.5) or 0.1% (areas OPro, 13, M25, and 32) areas were subdivided into three laminar
cresyl violet (pH 3.8) for 3 min, differentiated through graded alcohols subgroups, (i) layer I, (ii) layers IIIII and (iii) layers VVI, because they
and xylenes, and coverslipped with Permount (Fisher Scientific, either lack (agranular) or have only a rudimentary granular layer IV
Springfield, NJ). (dysgranular). In dysgranular areas an incipient and poorly defined layer
IV was included with layers V and VI.
Immunocytochemistry for PV, CB and CR In sections stained for PV, CB and CR, areas were subdivided into two
laminar subgroups, layers IIII and layers IVVI, because it was not
Matched series of sections were used for immunocytochemical staining of
possible to parcel the cortex into finer laminar subgroups in unstained
PV, CB, and CR. Antibodies for the calcium binding proteins were
sections, where neuropile positive for the calcium binding proteins
purchased from commercial sources (Swiss Antibodies, anti-PV #235,
further obscured the fine laminar boundaries seen in Nissl stained
monoclonal; anti-CB D-28k #300, monoclonal; anti-CR #7696, polyclonal).
sections.
Antibodies were specific for PV, CB and CR with no cross-reactivity (Celio
et al., 1988, 1990; Schwaller et al., 1993). Free-floating sections were
washed (3 10 min) in 0.1 M phosphate buffered saline (PBS, pH 7.4) and Counting Method
placed in 6% horse serum (PV and CB) or 6% goat serum (CR) for 1 h. Laminar subgroups for each area were subdivided from coronal sections
They were then rinsed in 0.1 M PBS (3 10 min), placed in a 0.1 M PBS into counting boxes (50 50 m, width height). This was accomplished
solution containing the antiserum for either PV, CB or CR (PV dilution using computer software to superimpose an electronic grid over an area
1:3000; CR and CB 1:2000) and incubated for 72 h at 4C with gentle and identify counting boxes by a series of x and y coordinates. The spe-
agitation. Sections were then rinsed (3 10 min) in 0.1 M PBS and cific counting boxes used to gather data were selected randomly by a
processed for immunocytochemistry according to the instructions in the computer program. The stage was then positioned to the precise x and
Vector ABC kit (Vector Labs, Burlingame, CA). Sections were placed in a y coordinates of each counting box. Counting was performed through
0.1 M PBS solution containing either anti-mouse IgG (PV and CB, Vector the optics of the microscope using a crosshair microgrid with known
Labs BA-2000) or anti-rabbit IgG (CR, Vector Labs BA-1000) and incubated dimensions as reference.
for 2 h at room temperature, then rinsed (3 10 min) in 0.1 M PBS and The density of neurons and glia was estimated using a counting
placed in the avidin-biotin-HRP complex for 90 min at room temperature. method adapted from previous studies (Sterio, 1984; Braendgaard and
Sections were rinsed twice (10 min each) in 0.1 M PBS and once in 0.1 M Gundersen, 1986; Williams and Rakic, 1988; Gundersen et al., 1988; West
PB (10 min), placed in a solution containing 0.05% 3,3-diaminobenzidine and Gundersen, 1990; West, 1993) [for a review see (Coggeshall and
(DAB) and 0.03% hydrogen peroxide in 0.1 M phosphate buffer for 3 min, Lekan, 1996)]. This method is based on the principle that each cell within
then rinsed in 0.1 M PB (3 10 min) and mounted on chromealum a region of interest has an equal chance of being counted (West, 1993).
Figure 1. Density of neurons in 21 prefrontal areas or subdivisions of areas. Each bar represents the average density and standard error in seven cases. Density values for each area
represent overall values measured across all cortical layers. A map of the prefrontal cortex above the bar graphs shows areas on the medial (left), orbital (center) and lateral (right)
surfaces, adapted from the map of Walker (Walker, 1940) by Barbas and Pandya (Barbas and Pandya, 1989). Letters in connection with architectonic areas (designated by numbers)
represent: C, caudal; D, dorsal; g, gyral; M, medial; O, orbital; R, rostral; s, sulcal; V, ventral.
Figure 4. Neuronal density profile in different types of prefrontal cortices. Three-dimensional graphs showing differences in neuronal density (y-axis), laminar thickness by layer
(x-axis) and number of neurons under 1 mm2 of cortical surface (z-axis) in: (A) agranular, (B) dysgranular, (C) eulaminate I and (D) eulaminate II cortices. Dotted lines for the x-axis
demarcate extent of cortical layers, and solid lines indicate 500 m intervals of cortical thickness.
Figure 8. DA plot showing the separation of prefrontal areas for the three most informative experimental measures, ND, THI and PV staining. Diagram fields contain normal curves
describing the distribution of individual data points for each area for single measures (e.g. ND ND) and bivariate confidence ellipses for pairwise combinations of measures (e.g. ND
THI). Areas are colored according to their memberships in regional prefrontal groups: green to blue spectrum indicates caudal orbital and caudal medial areas (OPAll, OPro, 13 gyral
and sulcal, and 24a rostral and caudal subdivisions, as well as areas 32 and 25); red or orange colors symbolize rostral orbital and medial areas (10, 11, O12, M9, M14); blue to green
spectrum indicates lateral areas (all parts of areas 46 and 8). The clear separation particularly of ND measurements belonging to different areas is also borne out by the results of the
DA described in Results.
accordance with the outcome of the DA, all subsequent statist- between at least the ventral subcomponents of area 46, and
ical analyses made use of the highly informative ND measure, underlined the dissimilarity of area 25 from all other areas.
combining it with information from other experimental vari- The initial impressions were confirmed when selected
ables, such as the THI and PV data. experimental measures, such as ND and THI, were combined for
laminar areal profiles. The results (not shown) also suggested a
similarity grouping of the gyral components of A8 and A9. The
Global Similarity of Areas Based on Different Experimental combined analysis of ND and PV variables for the laminar area
Measures profiles also validated the main prefrontal subdivisions, but
The NMDS plot in Figure 9A expresses the relative similarity of additionally indicated a more gradual change in the areal profiles
all prefrontal cortical areas in terms of their normalized laminar along these two measures. Such a gradient was borne out most
profiles for the combination of all experimental measures (ND, clearly in the analyses of the relative laminar ND profiles on
GD and THI for layers I, IIIII and VVI, and PV, CR and CB for their own. The NMDS (not shown) indicated a striking one-
layers IIII; layer IV information for ND, GD and THI as well as dimensional similarity gradient from medial and orbital to rostral
layer IVVI information for PV, CR and CB was omitted owing to lateral and caudal lateral prefrontal cortices, when the areas
the normalization process described in Materials and Methods). were compared in the absence of information about layer IV.
Clearly apparent was the existence of two main groups, com- In this gradual distribution of profiles, however, some areas still
prising granular areas on the one hand and agranular/dysgranular appeared closely related (e.g. areas A25 and A14, areas A32 and
areas on the other, indicating a larger dissimilarity between the A13, areas A46VC and A8V).
groups than within groups. Also apparent were some outliers,
which were dissimilar from most other areas (most notably A25),
Discussion
as well as a sequential similarity structure within both groups
of areas. The latter feature may hint on a regional organization
Profiles of Prefrontal Areas Established by Cellular
along structural gradients. The tree shown in Figure 9B con-
Density, PV and CB
firmed this global structure from the independent perspective of
Several findings emerged from this study. First, architectonic
a hierarchical cluster analysis. It shows again the separation of
areas have distinct profiles that can be described quantitatively.
all areas into two main groups and one very dissimilar outlier,
The conventional and multidimensional analyses employed
area 25. The composition of the two main clusters once more
emphasized that among several cellular, laminar and molecular
followed the assignment of areas to an agranular/dysgranular features, neuronal density was highly informative in establishing
group, on the one hand (at the top of the tree), and to a group architectonic profiles, followed by cortical thickness and PV.
of granular areas, on the other. The diagram also indicates a Glial density, and CB were less informative, and CR not at all, as
subdivision of the granular area group into gyral areas A8, A9, summarized in Figures 7, 8 and 10. Graphic representations from
A10 and A12, and the remaining granular areas. Further, the multidimensional analyses positioned areas along similarity
organization of area 46 seems to follow mainly a ventraldorsal dissimilarity axes and demonstrated that there are graded
divide, whereas area 8 appears to be split along sulcal and gyral architectonic differences in the prefrontal cortex, confirming
subcomponents. and extending previous findings (Barbas and Pandya, 1989).
More detailed relationships became apparent when the two Profiles thus established can be used to determine whether
main groups of areas, that is, the granular and agranular/dys- neighboring cortices are distinct or similar. These representa-
granular areas, were each analyzed on their own (data not tions highlighted, for example, area 25 as distinct from its
shown). These analyses confirmed the clear divide between the neighboring areas. In addition, area 46 appears to have several
gyral and sulcal portions of A8 and the relative similarity subdivisions, as noted in a recent study (Petrides and Pandya,