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ORIGINAL ARTICLE
Cytoarchitectonic and chemoarchitectonic characterization
of the prefrontal cortical areas in the mouse
H. J. J. M. Van De Werd
•
G. Rajkowska
•
P. Evers
•
Harry B. M. Uylings
Received: 4 August 2009 / Accepted: 18 February 2010 / Published online: 12 March 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract This study describes cytoarchitectonic criteria
to define the prefrontal cortical areas in the mouse brain
(C57BL/6 strain). Currently, well-illustrated mouse brain
stereotaxic atlases are available, which, however, do not
provide a description of the distinctive cytoarchitectonic
characteristics of individual prefrontal areas. Such a
description is of importance for stereological, neuronal
tracing, and physiological, molecular and neuroimaging
studies in which a precise parcellation of the prefrontal
cortex (PFC) is required. The present study describes and
illustrates: the medial prefrontal areas, i.e., the infralimbic,
prelimbic, dorsal and ventral anterior cingulate and Fr2
area; areas of the lateral PFC, i.e., the dorsal agranular
insular cortical areas and areas of the ventral PFC, i.e., the
lateral, ventrolateral, ventral and medial orbital areas. Each
cytoarchitectonically defined boundary is corroborated by
one or more chemoarchitectonic stainings, i.e., acetylcho-
line esterase, SMI32, SMI311, dopamine, parvalbumin,
calbindin and myelin staining.
Keywords Cortical parcellation
Infralimbic, prelimbic, anterior cingulate, Fr2, agranular
insular and orbital cortical areas Nissl Myelin
Acetylcholinesterase Dopamine
Calcium binding proteins SMI-32 SMI-311
Abbreviations
ACd Dorsal agranular cingulate area
ACv Ventral agranular cingulate area, dorsal and
ventral part
AId
1
Dorsal agranular insular area, dorsal part
AId
2
Dorsal agranular insular area, ventral part
AIv Ventral agranular insular area
AIp Posterior agranular insular area
CB Calbindin
CL Claustrum
cc Corpus callosum
DA Dopamine
DI Dysgranular insular area
DLO Dorsolateral orbital area
FPl Lateral frontal polar area
FPm Medial frontal polar area
Fr1 Frontal area 1
Fr2 Frontal area 2
GI Granular insular area
IG Indusium griseum
IL Infralimbic area
LO Lateral orbital area
MO Medial orbital area
OB Olfactory bulb
H. J. J. M. Van De Werd H. B. M. Uylings (&)
Department of Anatomy and Neuroscience,
VU University Medical Center, P.O. Box 7057,
1007 MB Amsterdam, The Netherlands
e-mail: hbm.uylings@vumc.nl
H. J. J. M. Van De Werd H. B. M. Uylings
Graduate School Neuroscience Amsterdam,
Amsterdam, The Netherlands
G. Rajkowska
Department of Psychiatry and Human Behavior, University
of Mississippi Medical Center, Jackson, MS, USA
P. Evers
Netherlands Brain Bank, KNAW, Amsterdam, The Netherlands
H. B. M. Uylings
Department of Psychiatry and Neuropsychology,
School for Mental Health and Neuroscience,
Maastricht University, Maastricht, The Netherlands
123
Brain Struct Funct (2010) 214:339–353
DOI 10.1007/s00429-010-0247-z
PFC Prefrontal cortex
PL Prelimbic area
PV Parvalbumin
RSA Agranular retrosplenial cortex
RSG Granular retrosplenial cortex
VLO Ventrolateral orbital area
VLO
p
Posterior ventrolateral orbital area
VO Ventral orbital area
Introduction
Only a few publications, describing cytoarchitectonic fea-
tures of the mouse cerebral cortex, have been found in lit-
erature (Rose 1929; Caviness 1975; Wree et al. 1983). None
of these studies has focused on the prefrontal cortex (PFC), in
contrast to studies of the rat PFC (Krettek and Price 1977;
Van Eden and Uylings 1985; Uylings and Van Eden 1990;
Ray and Price 1992; Reep et al. 1996; Van De Werd and
Uylings 2008). The available mouse cytoarchitectonic/ste-
reotaxic atlases (Franklin and Paxinos 2008; Hof et al. 2000;
Slotnick and Leonard 1975) provide fine architectonic fig-
ures, but given their scope do not describe the cytoarchi-
tectonic criteria for the parcellation of the PFC.
The cytoarchitectonic definition of mouse prefrontal
areas is essential for stereological studies on the total
number of neurons and/or glia cells in the distinct cortical
areas using Nissl stainings (e.g., Rajkowska et al. 2004). In
addition, this is necessary in studies examining the question
whether a differential pattern of connectivity with cortical,
striatal and thalamic regions corresponds to different
cytoarchitectonically defined cortical areas (Uylings et al.
2003; Groenewegen and Witter 2004). In these and also in
physiological studies, different cytoarchitectonic prefrontal
areas need to be defined applying consistent and repro-
ducible criteria. Finally, such a cytoarchitectonic study is
needed due to the present intensive use of mice as animal
models for human brain disorders.
This study aims to provide cytoarchitectonic criteria to
characterize the boundaries between different cortical areas
in the medial, lateral and ventral/orbital regions of the
mouse PFC. As in other studies (e.g., Van de Werd and
Uylings 2008), the cytoarchitectonic boundaries are com-
pared with boundaries visible in chemoarchitectonic
stainings for myelin, acetylcholinesterase (AChE), dopa-
minergic fibers, SMI-32, SMI-311 and parvalbumin (PV)
and calbindin (CB)-positive neurons.
Materials and methods
The cytoarchitecture of the PFC was studied in ten
adult, male mice (strain C57BL/6) of similar weight
(approximately 20 g). These control mouse brains were
kindly donated and immersion fixed by Dr. H. Manji,
NIMH, USA. All animal procedures were in strict accor-
dance with the NIH animal care guidelines. The histolog-
ical processing of these brains was performed at the
laboratory of Dr. Rajkowska. The brains were embedded in
12% celloidin, cut into 40-lm serial sections using a
sliding microtome and Nissl (1% cresyl violet) stained.
Celloidin was chosen as an embedding medium to allow
for the preparation of ‘thick’ sections with clear morphol-
ogy and high contrast of Nissl-stained neurons and glial
cells. In these immersion-fixed brains, any spots showing
pycnotic reaction were not incorporated in this study.
In addition to these ten mice, four adult male mice
(C57BL/6 strain) were stained for dopamine and four adult
male mice for AChE, myelin, and immunohistochemically
for SMI, PV and CB. For each staining, a different set of
sections with several consecutive sections stained with
Nissl at HBMU’s laboratory was used. The antibodies
applied were the dopamine (DA) antibody (Geffard et al.
1984), SMI-32 antibody (Sternberger Monoclonals Inc.,
Baltimore, MD, USA: monoclonal antibody to one epitope
of non-phosphorylated tau neurofilaments, lot number 11),
SMI-311antibody (pan-neuronal neurofilament marker
cocktail of several monoclonal antibodies for several epi-
topes of non-phosphorylated tau protein, Sternberger
Monoclonals Inc., Baltimore, MD, USA: lot number 9)
(SMI antibodies are presently distributed through Covance
Research Products, USA), monoclonal anti-CB D-28K
antibody (Sigma, St. Louis, MO, USA: product number C-
9848, clone number CB-955, lot number 015K4826), and
monoclonal anti-PV antibody (Sigma, St. Louis, MO,
USA: product number P-3171, clone number PA-235, lot
number 026H4824). Mice to be stained for DA were
intracardially perfused under deep pentobarbital anesthesia
(1 ml/kg body weight, i.p.), with saline followed by fixa-
tive. For DA staining, the fixative was 5% glutaraldehyde
in 0.05 M acetate buffer at pH 4.0. After perfusion, the
brains were immersed in 0.05 Tris containing 1% sodium
disulfite (Na
2
S
2
O
5
) at pH 7.2 (De Brabander et al. 1992).
Mouse PFC was sectioned at 40 lm by a vibratome. These
sections were stained overnight in a cold room at 4°C using
the polyclonal primary antibody sensitive to DA that was
raised in the Netherlands Institute for Brain Research
(NIBR) (Geffard et al. 1984), the specificity of which had
been demonstrated previously (Kalsbeek et al. 1990). DA
antiserum was diluted 1:2,000 in 0.05 M Tris containing
1% Na
2
S
2
O
5
and 0.5% Triton X-100, pH 7.2. After over-
night incubation, the sections were washed three times with
Tris-buffered saline (TBS) and subsequently incubated in
the secondary antibody goat–antirabbit, also raised
in NIBR at 1:100 for 1 h. After having been rinsed 39 in
TBS, it was incubated in the tertiary antibody, peroxidase–
340 Brain Struct Funct (2010) 214:339–353
123
antiperoxidase, at 1:1,000 for 60 min. Both the secondary
and the tertiary antibodies were diluted in TBS with 0.5%
gelatine and 0.5% Triton X-100. For visualization, the
sections were transferred into 0.05% diaminobenzidine
(DAB; Sigma) with 0.5% nickel ammonium sulfate. The
reaction was stopped after a few minutes by transferring
the sections to TBS (3 9 10 min), then the sections were
mounted on slides, air dried, washed, dehydrated and
coverslipped.
Mice to be stained with anti-PV, anti-CB and SMI-32
and SMI-311 were fixed with 4% formaldehyde solution in
0.1 M phosphate buffer at pH 7.6. Mouse PFC was sec-
tioned at 40 lm by a vibratome. To prevent endogenous
peroxidase activity, free-floating sections were pretreated
for 30 min in a Tris-buffered saline (TBS) solution con-
taining 3% hydrogen peroxide and 0.2% Triton X-100. To
prevent non-specific antibody staining, these sections were
placed in a milk solution (TBS containing 5% nonfat dry
milk and 0.2% Triton X-100) for 1 h. Incubation of the
primary antibody, directly after the milk step was carried
out overnight in a cold room at 4°C. The primary anti-
bodies were diluted in the above-mentioned milk solution:
SMI-32 and SMI-311 at 1:1,000, PV antibody at 1:1,000,
and CB antibody at 1:250. For the monoclonal SMI-32,
SMI-311, PV and CB antibodies, raised in mice, we used
peroxidase-conjugated rabbit–antimouse (1:100 in 5% milk
solution with 0.2% Triton X-100) as a secondary antibody.
Visualization took place in 0.05% diaminobenzidine
enhanced with 0.2% nickel ammonium sulfate. The reac-
tion was stopped after a few minutes by transferring these
sections to TBS (3 9 10 min), after which the sections
were rinsed in distilled water, mounted on slides, air dried,
washed, dehydrated and coverslipped. Control sections that
were incubated according to the same procedure as
described above, omitting the primary antibody, were all
negative. All sections were cut coronally, because the
coronal plane offers in general the best view to differentiate
between the subareas of the rodent PFC (Uylings et al.
2003; Van de Werd and Uylings 2008).
Sections were processed for AChE staining according to
the protocol described by Cavada et al. (1995). The sec-
tions were incubated overnight in a solution of cupric
sulfate and acetate buffer at pH 5 to which acetylthiocho-
line iodide and ethopropazine were added just before the
start of incubation. After rinsing, the sections were devel-
oped in a sodium sulfide solution until a light brown color
appeared and subsequently intensified to a dark brown
color in a silver nitrate solution. Finally, the sections were
differentiated after rinsing in a thiosulfate solution, dehy-
drated and mounted. In all steps, the solutions and sections
were shaken constantly. The myelin was stained with silver
by physical development according to Gallyas (1979). The
sections were first placed in 100% ethanol and then
immersed in a 2:1 solution of pyridine and acetic acid for
30 min. After rinsing, they were placed in an ammonium
silver nitrate solution and after rinsing with 0.5% acetic
acid, the sections were immersed in the optimal physical
developer solution at room temperature (Gallyas 1979)
until they showed good stain intensity under the micro-
scope. Then the development of the staining was stopped in
0.5% acetic acid and the sections were dehydrated and
mounted with Histomount. The sections were studied at
intervals of 80–160 lm, and examined under a light
microscope at a 639 magnification.
Nomenclature
Our PFC nomenclature is mainly based on the one used
for the rat PFC by Krettek and Price (1977), Uylings
and Van Eden (1990), Ray and Price (1992), Reep et al.
(1996), Uylings et al. (2003) and Van De Werd and
Uylings (2008). Some names used for areas of the rat
PFC have been left out by us because they could not be
distinguished from other areas having different names
(see below).
Results
In mice, as in rats, the PFC is defined as the agranular part
of the frontal lobe. On the lateral side of the frontal lobe,
the dorsal part of the dorsal agranular insular area (AId1),
borders on the (dys)granular insular cortex (DI/GI), while
on the mediodorsal side, the second frontal area, Fr2,
borders on the (dys)granular cortex (Fr1). The position of
the subareas of the PFC are shown in Fig. 1, while in Fig. 2
in the rostrocaudal coronal sections, the PFC subareas are
shown.
Medial areas
Border between the prefrontal area Fr2
and the (dys)granular area Fr1
In the medial part of Fr1, layer V moves to a more
superficial position due to the gradual disappearance of
layer IV. We take the medial end of this shift as the
location where the granular layer IV has disappeared
completely, thus more medially than the visual appearance
of layer IV. At the border on Fr2, layer V has reached its
most superficial position. This is the main characteristic of
the Fr1–Fr2 boundary. In Fr2, the cells of layer II are, as a
layer, more clearly separated from layer I, while in Fr1
layer II shows clefts (Figs. 3, 4). In Fr2, the columns of the
layers V and VI are clearly situated closer to each other
than in Fr1 (e.g., Fig. 4a). In Fr2, the cells of the layer VI
Brain Struct Funct (2010) 214:339–353 341
123
are always in columns, while in Fr1, instead of columns a
horizontal arrangement of cells might be present. The cells
of the layer V of Fr2 are smaller than those of Fr1 (Figs. 3,
4, 5a).
Border between Fr2 and dorsal anterior cingulate areas
In Fr2, the outer surface of layer II is generally smooth, but
in dorsal agranular cingulate area (ACd) it is irregular and
has a darker, denser appearance (Figs. 3, 4, 5a). The col-
umns that can readily be seen in the layers V and VI of
both Fr2 and ACd are more densely packed in ACd than in
Fr2 (Figs. 3, 4, 5a). In Fr2, layer III tends to be broader and
lighter in appearance than in ACd (e.g., Fig. 4b).
Border between dorsal anterior cingulate and prelimbic
areas
The main characteristic of the border between ACd and
prelimbic (PL) is the columnar arrangement of layer V
cells in ACd, while in PL the cells of the layer V appear in
disorderly arrangement (Figs. 3, 4a, b, 6a). Layer VI in
ACd shows columns, while in PL the cells of the layer VI
are mainly arranged in horizontal rows (Figs. 3, 4a, b, 6a).
In ACd, layer II is narrow with its cells mostly concen-
trated at the boundary with layer I, while in PL, layer II is a
little broader with its cells more equally spread over the
layer. Layer III of ACd is lighter in appearance than layer
III of PL, due to the number of cells in ACd being less
dense than in PL (e.g., Fig. 4b).
Distinction in prelimbic between PLd and PLv
In PL, we discern a dorsal and a ventral part based on a
different aspect of layer II, which is narrow and compact in
the dorsal part of PL, but broad and less compact in the
ventral part. Layers III and V are more compact in the
ventral part of PL. The transition is rather sharp and
marked in our pictures by a small arrow (Figs. 3, 4, 6, 7).
Border between prelimbic and infralimbic areas
In PL, the layers II, III and V are clearly distinguishable,
i.e., the dark layer II is separated from layer V by the
lighter layer III, whereas in IL these layers are more or less
homogeneous. In IL, the size and distribution of the cells of
layers II, III and V are about the same. A typical feature of
IL is that cells of layer II in IL spread far into layer I, while
in PL only few cells of layer II are seen in layer I (Figs. 4 a,
b, 6). Therefore, layer II appears wider in IL than in PL. In
addition, the size of the somata in layer II in IL appears
smaller than in PL (e.g., Fig. 6).
Border between prelimbic and medial orbital areas
PL borders ventrally on medial orbital (MO) area only in
the anterior part of the frontal lobe (i.e., level a, b and c in
Figs. 1, 2). In MO, the cells are more homogeneously
distributed in layer II, while in PL they are unevenly spread
over layer II with the cells more densely packed on the
boundary with layer I (Fig. 3). In addition, the border
between layer II and III is less difficult visible in MO than
in ventral PL (Fig. 3).
Fig. 1 Schematic view of PFC areas. Top panel medial view; middle
panel lateral view; and lower panel orbital view. The lines above the
medial view mark the positions of the ten sections shown in Fig. 2.
For abbreviations, see ‘‘Abbreviations’’
342 Brain Struct Funct (2010) 214:339–353
123
Border between infralimbic and medial orbital areas
Caudal to panel c in Fig. 2, IL borders on MO. In IL the
layers II, III and V are homogeneous, while in MO those
layers can be distinguished from each other (e.g., Figs. 4b,
6b). This difference between IL and MO is most evident in
the transition of layer II into layer III (Figs. 4 a, b, 6).
Border between dorsal and ventral anterior cingulate
areas
At the level of genu (Figs. 1, 2), MO and IL have disap-
peared and PL changes into ventral agranular cingulate
area, dorsal and ventral part (ACv). Thus, caudal to genu,
ACd borders on ACv. In ACd, cells are arranged in col-
umns, while in ACv they are not. This is especially clear in
layer VI, which is columnar in ACd, while in ACv the cells
are mainly arranged in horizontal rows (Figs. 4c, 5a).
Layers II and III in areas ACd and ACv differ less from
each other than they do between ACd and PL. The somata
in layers III and V in ACd are generally larger than those in
ACv (Figs. 4c, 5a).
The difference between prelimbic and ventral anterior
cingulate areas
Generally, PL is more densely packed than ACv (Figs. 3,
4a, b, 6 for PL and Figs. 4c, 5a for ACv). In PL, the
layers are less distinguishable than in ACv. In ACv, the
horizontal laminar arrangement of cells in layer VI is
well distinguishable from the cells of layer V that are
larger and less densely packed than in PL (Fig. 4). Layer
III in ACv has a lighter appearance than layer III in PL,
due to the less dense packing of cells in ACv. As a
consequence, ACv and ACd differ less than ACd and
PL. The transition of PL into ACv, however, is a gradual
Fig. 2 Overview of PFC subareas in Nissl-stained sections at ten different levels indicated in Fig. 1. For abbreviations, see ‘‘Abbreviations’’.
Scale bar 1mm
Brain Struct Funct (2010) 214:339–353 343
123
process. Usually, PL changes into ACv at the genu of
the corpus callosum.
Distinction between the dorsal and ventral part in ACv
As in PL, we can discern a difference between the dorsal
and ventral part in ACv. In the dorsal part, layer II is
narrower with some clustering and with the layers III, V
and VI less compact than in the ventral part (Figs. 4c, 5a).
Layer II in the ventral part is broader with sometimes an
aspect of a spindle (e.g., Fig. 4c). Layer VI in the ventral
part of ACv is often darker than in the dorsal part of ACv.
In Figs. 4c and 5a, the border between the dorsal and
ventral part of ACv is marked by a small arrow.
Caudal border of the medial prefrontal areas
and the retrosplenial cortex
The first appearance of the fine, darkly stained granules in
layers II–III marks the transition of ACv into the granular
retrosplenial region (RSG). The agranular area dorsally to
RSG is the agranular retrosplenial area (RSA). For com-
parison of the cortex of the PFC with the retrosplenial
cortex see Fig. 5a, b.
Lateral areas
Border between the dysgranular insular cortex or granular
insular cortex and the dorsal agranular insular areas
In DI, layer IV is homogeneous with layers II and III, and
in the GI, layer IV is well distinguished from the layers III
and V by its small, densely and equally dispersed cells. In
AId
1
, layer IV is absent and the layers II, III, and V are
well distinguished from each other. Layer II is broad, dark
and compact in AId
1
, while homogeneous with layers III
and IV in DI and less compact in GI. In AId
1
, the cells of
the layersV and VI have been arranged in columns, but not
in DI or GI. In AId
1
, layer V lies more closely to the
surface than in DI or GI due to lack of layer IV (Figs. 3, 8,
9a).
Two subareas in the AId: dorsal AId
1
and ventral AId
2
The layer II of AId
1
has a dark and compact appearance,
while in dorsal agranular insular area, ventral part (AId
2
)
layer II is less compact and some of its cells spread into
layer I. In AId
2
, the columns of the layers V and VI are
more densely packed than in AId
1
(Figs. 3, 8).
Border between the dorsal part of the AId
1
and the posterior agranular insular areas
In AId
1
, the transition from one layer of the cortex to
another is gradual, while in posterior agranular insular area
(AIp) the layers appear to be clearly separated from each
other and are more compact than in AId
1
. This gives the
impression of eight or more layers, including the claus-
trum. In AId
1
, the cells are arranged in columns but not in
AIp (Fig. 9a).
More caudally, when AId becomes progressively smal-
ler and ultimately disappears, AIp borders directly on DI
(Fig. 9b).
Ventral or orbital areas
Border between the ventral part of the dorsal agranular
insular and the lateral orbital areas
In AId
2
, layer II is broad, the cells are not densely packed
and some cells extend into layer I; in LO, layer II is narrow
and its cells form clusters (Figs. 3, 8). These are the main
characteristics that distinguish AId
2
from LO. In AId
2
the
Fig. 3 Section through the frontal pole. In the medial part of granular
Fr1, layer V moves to a more superficial position due to the
diminishing layer IV. In Fr2, the cells of layer II are more
homogeneously separated from layer I than in Fr1. In ACd and in
dorsal PL, the cells of layer II are densely packed on the border with
layer I, but the cells of layer III are more densely packed in PL than in
ACd. Arrow indicates the border between dorsal PL and ventral PL.
In ventral PL, layer II is wider and less densely packed. In VLO, the
columns in layers II and III distinguish this area from VO and LO. See
also the clustering of layer II cells in LO, the wide loosely packed
cells of layer II in AId 2 and the densely packed layer II of AId1.
Scale bar 150 lm
344 Brain Struct Funct (2010) 214:339–353
123
cells of the layers III and V are arranged in columns, but
not in LO (Figs. 3, 8a). Layer I of LO is narrower than
layer I of AId
2
(Figs. 3, 8a).
Border between lateral orbital and ventrolateral areas
In LO, layer II shows clustering of cells and a sharp
boundary with layer III, while in VLO the cells of the
layers II and III show columns and the layers II and III are
homogeneous. In VLO, columns are sometimes seen in
layer V, but not in layer V in LO. On the whole, VLO is
more homogeneous than LO, so its layers are less distin-
guishable than in LO (Figs. 3, 8).
The homogeneity of the layers in VLO is nearly total in
the most posterior part of VLO, justifying the description
VLOp for this part of VLO (Fig. 8b).
Fig. 4 a Medial PFC at the fusion of the frontal lobe with the
retrobulbar region. Layer III cells in ACd are less densely packed than
in PL. Arrow indicates the border between dorsal and ventral PL.
Layer II in the ventral PL is wider than in the dorsal PL. The cells of
layer II are evenly dispersed in MO, but its boundary on layer I and III
is less sharp than in Fig. 3. Layer II of area IL extends into layer I.
The columns in layers II and III of VLO are typical. b Medial PFC at
the level of the forceps minor of corpus callosum. In Fr2 and ACd, the
layer VI shows columns, while a horizontal arrangement is visible in
PL, IL and MO parallel to pia. Arrow indicates the border between the
dorsal and ventral PL C Medial PFC at the supragenual level. In ACd
note that layers II and III are denser than in Fr2 and ACv. The
columns in ACd are denser in VI and V than in Fr2. Arrow indicates
the border between the dorsal ACv and ventral ACv. Layer II is wider
in the ventral ACv and layer VI cells are not in columns, but rather in
a horizontal arrangement. Scale bars 150 lm
Fig. 5 a Medial PFC just
anterior to i in Fig. 2. The
highest point of layer V and a
change in layer II are the
indications for the boundary
between Fr2 and Fr1. The
columns in layer VI and the
densely packed cells in layer II
in ACd differ from the
horizontal arrangement of layer
VI cells and the wide layer II in
ACv. Arrow indicates the border
between dorsal ACv and ventral
ACv. b Section through the
retrosplenial region at the level
of j in Fig. 2. Scale bars
150 lm
Brain Struct Funct (2010) 214:339–353 345
123
Border between ventrolateral and ventral orbital areas
The characteristic of VLO is the columnar arrangement of
the cells in layers II and III, whereas in VO layer II cells
show a more disorderly appearance (Figs. 3, 4a, 8). VO is
also the cortical area that provides the first contact between
the frontal lobe and the retrobulbar region (Fig. 4a).
Border between ventral and medial orbital areas
In VO, layer II has a disorderly appearance with a vague
border with layer III, while in MO the cells in layer II are
evenly dispersed and layer II has a clearer boundary with
layer III. The cortical layers are less distinct in VO than in
MO, but this difference is small (Figs. 3, 4a, 8).
Fig. 6 a Ventral part of the medial PFC for details of the areas IL and
MO; level: e in Fig. 2. In ACd, the layer II is characteristic, and the
columns in layers V and VI are distinguishable albeit with some
difficulty. Columns are absent in PL and layer VI of PL is denser.
Layers II and III of the dorsal and ventral PL differ in density.
Homogeneity of layers is characteristic in IL. In MO, the cells in layer
II are equally dispersed. VLO is characterized by columns in layers II
and III. b Medial PFC at a level between e and i in Fig. 2. In dorsal
PL, layer II is narrower than in ventral PL. Layer VI in PL and IL
have a more horizontal arrangement. In IL, homogeneity is mainly
seen in layers II and III. In MO, the evenly dispersed cells in the well
visible layer II is characteristic. VLO shows the characterizing
columns and homogeneity of layers II and III. Arrow indicates the
border between the dorsal and ventral PL. Scale bars 150 lm
Fig. 7 Acetylcholinesterase staining. a Section through the frontal
lobe. The strongest staining is in dorsal PL and in AId1. In ACd and
Fr2, staining is mainly seen in layer V. Staining in the ventral PL, IL
and MO is light and mainly in layer V. In VLOp, staining is mainly in
the lateral part. In LO, staining is seen in layer V and in the superficial
layers. Area AId2 shows less staining than its neighboring areas.
Arrow indicates the border between the dorsal and ventral PL.
b Medial PFC at the start of ACv. In ACd, staining is less than ACv
and mainly deep in layer V; in Fr2, staining is less than in ACd. Scale
bars 150 lm
346 Brain Struct Funct (2010) 214:339–353
123
Chemoarchitectonic features
We examined (immuno)cytochemical stainings to compare
the borders visible in these stainings with those in Nissl-
stained sections to determine to what extent cytochemical
borders coincide with cytoarchitectonically defined PFC
subareas. For comparison, we extrapolated the borders into
these cytochemical stainings from Nissl-stained sections in
the same mouse brain.
Acetylcholinesterase staining
In AChE-stained sections, the dorsal parts of PL and AId1
(Fig. 7a) are the most strongly stained areas in the prec-
allosal PFC. In this staining, LO is more heavily stained
than AId2, and the pattern of staining in LO differs from
that in VLO (Fig. 7 a). In the supracallosal part of the PFC,
the staining is strongest in ACv. It is less in ACd, and least
in Fr2 (Fig. 7b). Table 1 summarizes the visibility of
borders in the AChE staining: between the PFC areas only
the borders ACd/PL, AId1/AId2 and AId2/LO are well
detected with this staining.
Fig. 8 a Orbital and lateral areas of the PFC at the anterior fusion of
the frontal lobe with the retrobulbar region. Typical features are the
columns and the homogeneity in the layers II and III of VLO, the
clusters of cells in layer II of LO, the loose layer II of AId 2 with cells
spreading into layer I and densely packed columns, the wide and
rather homogeneous layer II of AId 1 and the less densely packed
columns in the lower layers. b The same areas at a more posterior
level: f in Fig. 2. Typical features are the homogeneity of IL, the
sharply outlined layer II with evenly dispersed cells in MO and the
homogeneity of VLOp. Scale bars 150 lm
Fig. 9 a Lateral PFC at the
transition to AIp. Typical are
the clearly distinguishable
(sub)layers of AIp and the
columns in AId1. b AIp
posterior to the PFC, CL
claustrum. In DI, layers II, III
and IV are homogeneous.
Scale bars 150 lm
Table 1 Comparison borders visible in different stainings
Borders Nissl AChE SMI-
32
SMI-
311
Myelin PV Cb DA
Fr1/Fr2 ?? ±
Fr2/ACd ?? ± ? ? ?
ACd/PL ?? ?? ? ? ? ?
ACd/ACv ?? ? ? ? ? ?
PL/IL ?? ± ±
PL/MO ?? ?? ??
IL/MO ?? ?
DI/AId1 ?? ?? ± ±
AId1/
AId2
?? ?? ± ± ? ± ??
AId1/AIp ??
DI/AIp ?? ??
AId2/LO ?? ?? ?? ?? ?? ?? ? ??
LO/VLO ?? ?? ?? ?? ?? ?? ?
VLO/VO ?? ? ? ? ±
VO/MO ?? ? ±
PFC/RS ??
?? Clearly definable, ? definable, ± difficult to define, no symbol
not definable
Brain Struct Funct (2010) 214:339–353 347
123
SMI-32 and SMI-311
The lack of SMI-32 staining in most areas of the mouse
PFC is striking (Fig. 10a). Only in LO, some layers display
clear immunopositive reaction. In VLO, less staining is
present.
In the corresponding SMI-311 section (Fig. 10b), the
strongest staining is also seen in LO. In VLO, the deeper
layers are well stained, but staining of layer III is much less
than in LO. Table 1 summarizes that in SMI-32 sections
only the borders of LO with AId2 and with VLO are well
defined and in SMI-311, in addition, the border PL/MO in
the anterior part of the frontal lobe.
Myelin staining
In the myelin-stained sections, VLO and VLOp are con-
spicuous by the paucity of staining. (Fig. 11a, b), while LO
is more heavily myelinated. The myelin staining in VO and
MO is also much stronger than in VLO (Fig. 11a, b).
Figure 11b shows that PL is not differentiated from IL;
both are lightly myelinated. The myelinization in ACd
appears stronger than in PL (e.g., Fig. 11b).
In the supracallosal region of the PFC, the length and
density of fibers are larger in ACd and Fr2 than in ACv
(Fig. 11c).
Table 1 summarizes that in myelin-stained sections only
the borders of LO with VLO and with AId2 are clearly
definable.
Parvalbumin
In the precallosal region of the PFC, the strongest and most
general PV staining is in LO (Fig. 12a). The staining in
VLOp is stronger and more general than in VLO (Fig. 12a, b).
The staining in AId2 tends to be stronger than in AId1
Fig. 10 a Section through the frontal lobe, stained by SMI-32.
Layers III and V are well stained in LO. In VLO, staining is seen
mainly in the lateral part; in AId, staining is limited to the deepest part
of layer V; and in the medial PFC subareas, very sparse staining is
present. b Section at the corresponding level, stained by SMI-311.
Staining is strongest in LO, and in VLO staining is more in its lateral
part. In Fr2, many large neurons are stained in layer V. PL shows less
staining than ACd and Fr2. AId1 and AId2 show only few stained
cells. Scale bars 150 lm
Fig. 11 Myelin-stained sections. a Section through the frontal lobe.
Nerve fibers are most strongly stained in LO. Sparsest staining is
present in VLO. b At the level of the forceps minor of the corpus
callosum. The nerve fibers are most strongly stained in LO.
c Supracallosal PFC. Longest and most densely packed fibers are
seen in Fr2 and ACd; in ACv, fibers are shorter and less densely
packed. Scale bars 150 lm
348 Brain Struct Funct (2010) 214:339–353
123
(Fig. 12b). In general, PV staining in the medial PFC is
poor, but in the more posterior sections Fr2 and ACd
show stronger staining (Fig. 12a, b). No border is visi-
ble between PL, IL and MO in PV-stained sections
(Fig. 12a, b).
In the supracallosal part of the PFC, the PV staining is
heaviest in Fr2, less in ACd and least in ACv, especially in
the ventral part of ACv (Fig. 12c). The highest density of
PV-positive cells is, however, visible in layer V of ACv,
especially in the dorsal part (Fig. 12c).
In AIp, the pattern of PV-stained layers shows a higher
PV-positive lamination than in AId1, which resembles
Nissl-stained sections (Fig. 12d).
Table 1 summarizes that only the borders of LO with
AId2 and with VLO are well detectable in PV stainings.
Calbindin
Differences of pattern and intensity of CB staining cor-
roborate the PFC areas extrapolated from Nissl sections,
but they are much less distinctive than in Nissl staining
(Fig. 13a–c). Table 1 summarizes that between PFC areas
in CB-stained sections, no border is consistently clearly
definable.
Dopamine
Dopamine staining is present in most areas of PFC except
for VLO, LO and the part of VO anterior to the fusion of
the frontal lobe with the retrobulbar region (Figs. 14a, 15).
At the fusion of the retrobulbar region with VO,
Fig. 12 Parvalbumin-stained sections. a Level of the frontal lobe.
Strongest staining in LO and lateral part of VLO. In AId1 and AId2,
sparse cells are seen in nearly all the layers. This is also seen in VO.
b Level after fusion of the frontal lobe with the retrobulbar region.
Strongest staining in LO and VLOp. Staining of cells in deep layers
more in AId2 than in AId1. c Supracallosal PFC. Parvalbumin-
positive cells in ACv are much more packed than in ACd or Fr2.
d Level of AIp. Most cells are seen in layer V of AIp. Scale bars
150 lm
Brain Struct Funct (2010) 214:339–353 349
123
dopaminergic fibers run through VO (Fig. 14b), as in the
rat (Van de Werd and Uylings 2008). In the supracallosal
region, the staining of ACv is stronger than in ACd and Fr2
(Fig. 14c). In the anterior part of PL, the dopaminergic
fibers run mainly in layers II and V (Fig. 14a), while in the
PL part caudal to the fusion of the frontal lobe with the
retrobulbar region, the dopaminergic fibers are mainly in
layer VI (Fig. 14b). This laminar pattern differs from the
one in the AId2. In fact, in AId2 the dopaminergic fibers
are in all layers with a slight preference for the deeper and
the upper part of the cortex (Fig. 15). As in the rat, less
dopaminergic fibers are present in the mouse claustrum in
comparison to the AIp layers (Fig. 15c).
Table 1 summarizes, that in dopamine-stained sections,
the borders of AId2 with AId1 and with LO are clearly
detectable.
Discussion
‘‘ Results’’ and Table 1 demonstrate that to define mouse
prefrontal areas, cytoarchitectonic staining is preferred to
cytochemical stainings as in rat (Van De Werd and Uylings
2008) and human (
}
Ongu
¨
r et al. 2003) studies. Cytochemical
staining did show two or more borders of mouse PFC areas
clearly, but cytochemical staining did not show all the bor-
ders definable in Nissl sections. Taking all the cytochemical
stainings together, 7 of the 14 borders are clearly detectable
(Table 1). The PFC areas clearly definable in cytochemical
stainings are LO, AId1 and AId2, and largely VLO and PL.
LO, as defined in Nissl staining, is visible in SMI 32 and SMI
311 because of its double-layered staining, as in AChE and in
PV staining. LO is also more strongly myelinated. AId1, as
defined in Nissl staining, is well definable in AChE staining,
and also as a very sparsely stained AId1 in the PV staining.
AId2, as defined in Nissl staining, is recognizable in dopa-
mine staining by the abundant DAergic fibers, which are
much less dense in the neighboring areas. In AChE staining,
however, AId2 is much less stained than the neighboring
AId1 and LO. VLO, as defined in Nissl-stained sections, is
visible in CB because of its columnar pattern, and in dopa-
mine staining due to a lack of dopaminergic fibers. PL, as
defined in Nissl staining, is detectable in dopamine staining
by its strong staining of layers II and V. Dorsal PL is cor-
roborated by the three strongly stained layers seen in dorsal
PL in AChE staining.
Our nomenclature of the subareas of the PFC in the
mouse corresponds, in general, to the one Ray and Price
Fig. 13 Calbindin-stained
sections. Boundaries have been
extrapolated from Nissl-stained
sections. Except for columns
and densely packed cells in
VLO and clustering of cells in
LO and MO, typical features for
identifying areas are not
abundant. a Level of frontal
pole. Small cells in AId2,
clusters in layer II in LO, and in
VLO densely packed cells and
columns. b Level after fusion of
the frontal lobe with the
retrobulbar region. Densely
packed cells in AId2, clusters of
cells in LO and homogeneity of
cells in VLOp. Arrow indicates
border between the dorsal and
ventral PL. c Supracallosal PFC.
Concentration of cells of layer II
at the boundary with layer I
characterizes ACd. No strong
staining in Fr2 and ACv. Arrow
indicates the border between the
dorsal and ventral ACv,
extrapolated from Nissl staining
in the adjacent section. Scale
bars 150 lm
350 Brain Struct Funct (2010) 214:339–353
123
(1992) used for the rat PFC, but differs in the following
aspects. We prefer using the neutral name of Fr2 as
introduced by Zilles (1985) instead of the medial precentral
area (PrCm), since the mouse and rat do not have a central
sulcus (Uylings and Van Eden 1990). The terms lateral and
medial frontal polar subareas (Ray and Price 1992), i.e.,
FP
l
and FP
m
, respectively, are not adopted by us, since we
can extrapolate the prefrontal cortical areas into this
frontopolar region. As we did in the rat PFC (Van de Werd
and Uylings 2008), we define two subareas in the mouse
AId, i.e., AId1 and AId2. In contrast to the rat PFC, we
could not distinguish a dorsolateral orbital area (DLO) in
the mouse among AId in a reproducible way, nor could we
distinguish a ventral agranular insular subarea (AIv) among
LO. In mouse, we prefer the term AId and LO on the basis
of its architectonic structure, which is more like the rat AId
and LO than the rat DLO and AIv, respectively (Van de
Werd and Uylings 2008). In the mouse, LO maintains its
features from the rostral to the caudal end, while in the rat,
LO is replaced caudally by an area AIv. This area is
characterized by a layer III, which is very cell-sparse
compared to layer III in LO. In general, such a change from
LO into AIv is not detected in the mouse brain. Therefore,
we prefer defining the whole area as LO and have not
specified an AIv area in the mouse.
An important macroscopic aspect of the mouse frontal
lobe is the very short frontal pole that is detached from the
retrobulbar region. This is different from the rat frontal
pole, which is (relatively) longer. In fact, the anterior–
posterior distance of the free part of the mouse frontal lobe
is hardly as large as the thickness of the six layers of the
cortex. Therefore, the characteristics of the superficial
layers have been used by us to distinguish the prefrontal
areas in the frontal pole in the coronal sections. Yet on the
basis of our experience with rat brains cut in coronal,
sagittal and horizontal planes, coronal sections are pre-
ferred by us for PFC area definition.
The cytoarchitectonic characteristics described in this
study are also visible in the ‘‘Atlas of the Mouse Brain’’ by
Franklin and Paxinos. This does not mean that all PFC
areas defined in this study are similar to the areas specified
in this atlas. We agree repeatedly for AId1, AId2, LO,
VLO, IL, PL, ACd and ACv, but differ for MO, Fr2 and
AIp. We also note that AId2 is called AIv in Franklin and
Paxinos (2008). We prefer the term AId2, since AIv is
located inside the rhinal sulcus in rat PFC studies (e.g.,
Uylings and Van Eden 1990; Ray and Price 1992; Reep
et al. 1996; Uylings et al. 2003; Van De Werd and Uylings
2008). In a following study (Van de Werd and Uylings, in
preparation), we will review in detail with maps the
Fig. 14 Dopamine-stained
sections. a Frontal lobe.
Dopamine-stained fibers present
in all areas of the medial PFC,
especially in PL. b Medial PFC
at the pregenual level.
Dopaminergic fibers in all areas
of the medial PFC, especially in
layer VI in PL. Arrow indicates
the border between the dorsal
and ventral PL, extrapolated
from Nissl staining in the
adjacent section. Fiber density
in layer V in the ventral PL was
higher than that in the dorsal
PL. c Supracallosal PFC.
Dopaminergic fibers were
mostly in ACv and less in ACd
and Fr2. Scale bars 150 lm
Fig. 15 Dopamine staining. a
The abundance of dopaminergic
fibers in AId2. b Dopaminergic
fibers are abundant in AId2 and
less in AId1 and LO. c AIp
shows dopaminergic fibers in all
layers, but no dopamine is seen
in the claustrum. Scale bars
150 lm
Brain Struct Funct (2010) 214:339–353 351
123
similarities and differences of the prefrontal areas defined
in this study with the mouse stereotaxic atlases (Hof et al.
2000; and Franklin and Paxinos 2008), the mouse cytoar-
chitectonic atlas by Rose (1929) and the mouse cytoar-
chitectonic studies by Caviness (1975) and Wree et al.
(1983).
In comparing mouse studies, it will be important to
consider whether size and defining features of the pre-
frontal areas differ between different genetic/inbred mouse
strains (Leinga
¨
rtner et al. 2007). Strain differences in the
visual cortex and the ‘barrel’ cortex, but not between the
entire size of the somatosensory cortex and the auditory
cortex, have been reported between C57BL/6J and DBA/2J
(Airey et al. 2005). On the basis of the figures of Hof et al.
(2000), we can expect differences in the ACv areas of the
129/Sv strain.
The prefrontal cortical areas can only be defined as such
on the basis of reciprocal connectivity patterns with the
dorsomedial nucleus of the thalamus, the intralaminar
thalamic nuclei, the neocortical areas, the basal ganglia, the
hypothalamus and the brain stem (Uylings et al. 2003). To
date, little is known about such connectivity patterns in the
mouse brain. Only one mouse study was published
29 years ago on the connections of the medial and lateral
PFC with the mediodorsal nucleus of the thalamus (Guldin
et al. 1981). This study demonstrates that the medial and
lateral ‘PFC’ areas receive mediodorsal connections. For
area AC
v
, however, such connections were not detected in
this study, but more refined tracing techniques have been
developed afterward. In the rat, the reciprocal projection
pattern of AC
v
with the mediodorsal thalamic nucleus was
also revealed in a later, more extensive study by Groe-
newegen (1988). By extrapolating the rat tracing studies it
is quite likely that the cytoarchitectonic cortical areas
described here are indeed prefrontal areas, with the possi-
ble exception of VLO (Reep et al. 1996; Uylings et al.
2003). It is still debatable whether the VLO area in the rat
can be considered to be part of PFC. On the basis of current
knowledge of connectivity patterns (Cechetto and Saper
1987; Ray and Price 1992; Uylings et al. 2003), in the rat
brain the DI is not considered to be part of the PFC.
Detailed tracing studies are necessary to further establish
whether or not the cytoarchitectonically defined frontal
mouse areas are all prefrontal areas indeed. Our study
provides an appropriate description of the characteristics of
the cytoarchitectonic delineation of these cortical areas that
can be applied to future detailed tracing studies, which can
be expected from a large US program on comparative
mouse neuroanatomy (Bohland et al. 2009).
Future tracing studies will demonstrate whether DLO
and AId, and LO and AIv, respectively, are ‘intermingled’
in the mouse, or whether a DLO and an AIv can be dis-
tinguished from AId and LO, respectively. In the rat, AId
projects onto the core of the accumbens nucleus, whereas
DLO projects more to the dorsolateral striatum (Fig. 2
in Groenewegen and Uylings 2010) and LO projects to
the lateral striatum, whereas AIv projects to the lateral
accumbens shell and ventral to this area (Fig. 2 in
Groenewegen and Uylings 2010).
In conclusion, the cytoarchitectonic definitions of mouse
prefrontal cortical areas described in this study will be of
use in stereological studies (e.g., Rajkowska et al. 2004) for
which borders of individual areas have to be determined to
estimate the total number of neurons and/or glial cells.
Moreover, these cytoarchitectonic criteria will be very
useful for a more precise localization of recording elec-
trodes (e.g., Herry and Garcia 2002), microdialysis probes
(e.g., Van Dort et al. 2009), receptor binding sites and
mRNAs expression (e.g., Amargo
´
s-Bosch et al. 2004;
Lidow et al. 2003), as well as for anatomical guidance of
neuroimaging studies (e.g., Barrett et al. 2003) and tracing
neural connections to and from mouse frontal cortical
areas.
Acknowledgments We thank Mrs. G. Clarke for the preparation of
histological Nissl-stained sections, Mr. H. Stoffels for his drawings in
Fig. 1 and Dr. L.J.A. Huisman for correcting the English. This study
was supported by Grants RO1 MH61578 (G.R.; H.B.M.U.),
MH60451 (GR) and RR17701 (GR).
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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