THE JOURNAL OF COMPARATIVE NEUROLOGY 492:145177 (2005)

Prefrontal Cortex in the Rat: Projections

to Subcortical Autonomic, Motor, and
Limbic Centers
PAUL L.A. GABBOTT,1,2* TRACY A. WARNER,1 PAUL R.L. JAYS,1
PHILLIP SALWAY,1 AND SARAH J. BUSBY1
1
University Department of Pharmacology, Oxford, OX1 3QT United Kingdom
2
Department of Biological Sciences, The Open University, Milton Keynes MK7 6AA,
United Kingdom

ABSTRACT
This paper describes the quantitative areal and laminar distribution of identied neuron
populations projecting from areas of prefrontal cortex (PFC) to subcortical autonomic, motor, and
limbic sites in the rat. Injections of the retrograde pathway tracer wheat germ agglutinin
conjugated with horseradish peroxidase (WGA-HRP) were made into dorsal/ventral striatum
(DS/VS), basolateral amygdala (BLA), mediodorsal thalamus (MD), lateral hypothalamus (LH),
mediolateral septum, dorsolateral periaqueductal gray, dorsal raphe, ventral tegmental area,
parabrachial nucleus, nucleus tractus solitarius, rostral/caudal ventrolateral medulla, or thoracic
spinal cord (SC). High-resolution at-map density distributions of retrogradely labelled neurons
indicated that specic PFC regions were differentially involved in the projections studied, with
medial (m)PFC divided into dorsal and ventral sectors. The percentages that WGA-HRP retro-
gradely labelled neurons composed of the projection neurons in individual layers of infralimbic
(IL; area 25) prelimbic (PL; area 32), and dorsal anterior cingulate (ACd; area 24b) cortices were
calculated. Among layer 5 pyramidal cells, approximately 27.4% in IL/PL/ACd cortices projected
to LH, 22.9% in IL/ventral PL to VS, 18.3% in ACd/dorsal PL to DS, and 8.1% in areas IL/PL to
BLA; and 37% of layer 6 pyramidal cells in IL/PL/ACd projected to MD. Data for other projection
pathways are given. Multiple dual retrograde uorescent tracing studies indicated that moderate
populations (9%) of layer 5 mPFC neurons projected to LH/VS, LH/SC, or VS/BLA. The data
provide new quantitative information concerning the density and distribution of neurons in-
volved in identied projection pathways from dened areas of the rat PFC to specic subcortical
targets involved in dynamic goal-directed behavior. J. Comp. Neurol. 492:145177, 2005.
2005 Wiley-Liss, Inc.

Indexing terms: cognition; visceral information; pyramidal cells; descending pathways; subcortical
targets

The rat prefrontal cortex (PFC) consists of cytoarchitec-
turally and functionally distinct areas located over the
medial, orbital, and insular surfaces of the rostral cerebral
hemispheres (Cechetto and Saper, 1990; Neafsey, 1990;
Neafsey et al., 1993; Thierry et al., 1994; Paxinos et al.,
1999; Uylings et al., 2000, 2003; Westerhaus and Loewy,
2001; Bohn et al., 2003). The medial (m)PFC is strategi-
cally involved in both cognitive and autonomic visceromo-
tor functions (Neafsey et al., 1986, 1993; Loewy, 1991;
Verberne and Owens, 1998; Groenewegen and Uylings,
2000; Uylings et al., 2003). In contrast, the ventrolaterally
located orbital cortices mediate aspects of reward associ-
ations that underlie discriminatory behavior (Schoen-
baum et al., 2003). By comparison, the insular cortex is
primarily a viscerosensory region directly involved in the
processing of afferent cardiovascular, cardiopulmonary,
gastrointestinal, gustatory, and related sensory informa-
tion (Cechetto and Chen, 1990; Allen et al., 1991; Zhang
and Oppenheimer, 1997; Jasmin et al., 2004).
Grant sponsor: The Wellcome Trust; Grant number: 047314/96/Z/WRE/MB
(to P.L.A.G.); Grant sponsor: the Beit Memorial Fund (to P.L.A.G.); Grant
sponsor: the Medical Research Council (U.K.); Grant number: G9312936N (to
P.L.A.G.); Grant sponsor: The Royal Society (to P.L.A.G.); Grant sponsor: The
Open University, Milton Keynes (to P.L.A.G.).
*Correspondence to: Paul L.A. Gabbott, Department of Biological Sci-
ences, The Open University, Milton Keynes MK7 6AA, United Kingdom.
E-mail: p.l.gabbott@open.ac.uk
Received 29 April 2004; Revised 30 November 2004; Accepted 26 May
2005
DOI 10.1002/cne.20738
Published online in Wiley InterScience (www.interscience.wiley.com).

2005 WILEY-LISS, INC.

146 P.L.A. GABBOTT ET AL.

Neural information processed by the PFC is relayed to axons innervating multiple subcortical targets (Pinto and
subcortical sites through populations of projection pyra- Sesack, 2000).
midal neurons situated mainly in deep layers 5 and 6 of The importance of this study is that it presents high-
the cortex (DeFelipe and Farinas, 1992). The terminal resolution at-map density distributions and quantitative
arbors of the descending axonal projections monosynapti- areal and laminar data concerning the main projection
cally innervate distinct subcortical circuits, thereby inte- pathways out of rat PFC innervating subcortical struc-
grating the results of information processing in the PFC tures strategically involved in autonomic, motor, and lim-
with the activity of specic neural networks in subcortical bic functions. Such data provide a rich anatomical frame-
target regions (Bacon and Smith, 1993; Carr and Sesack, work for construction of realistic quantitative
2000). Although the identities of subcortical target regions computational models of how neural activity in specic
receiving input from dened areas of the rat PFC have regions of the rat mPFC might inuence (through parallel
been well described in anterograde tract-tracing studies multiple-distribution output pathways) dened subcorti-
(Sesack et al., 1989; Heidbreder and Groenewegen, 2003; cal regions subserving autonomic, limbic, and other be-
Vertes, 2004), there is little information regarding the havioral functions (Heidbreder and Groenewegen, 2003;
overall quantitative areal and laminar density distribu- Uylings et al., 2003). Preliminary results from this study
tions of the specic PFC neuron populations participating have been reported previously (Gabbott and Busby, 2000).
in descending projection pathways to autonomic, motor,
and limbic centers in the rat.
The principal aims of this study were to provide coronal MATERIALS AND METHODS
and high-resolution at-map laminar density distribu-
The experimental procedures are described in three
tions of neurons in the rat PFC projecting to a wide selec-
parts. All surgical and animal procedures were conducted
tion of strategically important subcortical target sites in-
in accordance with the Society for Neuroscience Policy on
volved in a range of behaviors (see Table 1). By using the
the use of animals in neuroscience research and were
data from such maps in combination with previous esti-
licensed under the U.K. Animals (Scientic Procedures)
mates of the areal and laminar volume numerical densi-
Act, 1986.
ties (Nv; number of cells per unit volume) of pyramidal
cells and -aminobutyric acid (GABA)-ergic local circuit Retrograde tract-tracing using WGA-HRP:
inhibitory neurons (Gabbott et al., 1997), it has been pos- tracer injections
sible to dene the proportions of pyramidal cells in the
various layers of the infralimbic (IL; Brodmann area 25; Eighty-three male Sprague-Dawley rats (225280 g)
see Brodmann, 1909), prelimbic (PL; area 32), and dorsal were used in these experiments. Animals were deeply
anterior cingulate (ACd; area 24b) elds of mPFC project- anesthetized with equithesin (0.3 ml/100 g body weight
ing to each subcortical region investigated. An ancillary i.p.; Millenbruck and Wallinga, 1946). Unilateral pressure
aim was to use two uorescent retrograde tracers in indi- injections of the conventional retrograde tracer wheat
vidual animals, to derive initial percentage estimates of germ agglutinin conjugated with horseradish peroxidase
projection mPFC neurons with divergent corticoefferent (WGA-HRP; 510%) in 0.01 M phosphate-buffered saline
(PBS; pH 7.4) were made via glass microelectrodes (tip
diameters 20 30 m) into a selection of identied regions
of the rat brain and spinal cord (Table 1). WGA-HRP was
chosen because of its known transport properties, ease of
Abbreviations detection, and permanent labelling properties (Bolam,
1992). Details of the stereotaxic locations of the injections
ACd dorsal anterior cingulate cortex (area 24b)
AId dorsal anterior insular cortex
and the number of animals used for each brain region are
AIv ventral anterior insular cortex also given in Table 1. The locations of representative in-
BLA basolateral amygdala jection sites are shown in Figures 3, 5 8. Volume of WGA-
DP dorsal peduncular cortex HRP tracer injected varied according to injection site;
DR dorsal raphe
DS dorsal striatum
volumes ranged between 50 and 200 nl, with the VTA,
GU gustatory insular area NTS, rVLM, and cVLM having smaller volumes (50 100
IG indusium gresium nl) of tracer injected than DS, VS, and SC (150 200 nl).
IL infralimbic cortex (area 25) Injections were made continually over a period of 10 min-
LH lateral (posterolateral) hypothalamus
MD mediodorsal thalamus
utes (Bolam, 1992). After tracer delivery, the microelec-
MOP primary motor area trodes remained in place for a further 5-minute period
MOS secondary motor area before being slowly withdrawn.
OBl lateral orbital cortex After a survival period of 510 days, animals were re-
OBm medial orbital cortex
OBvl ventrolateral orbital cortex
anesthetized with equithesin and perfused transcardially,
PAG periaqueductal gray initially with 50 ml of 0.9% physiological saline and then
PB parabrachial nucleus with 500 ml of a mixture of aldehyde xatives 0.52%
PL prelimbic cortex (area 32) glutaraldehyde (G)/3 4% paraformaldehyde (P) in 0.1 M
PrCm precentral motor area
SC thoracic spinal cord
phosphate buffer (PB; pH 7.4) at room temperature. After
Sep lateral septum perfusion, the brains were carefully removed from the
SSp primary somatosensory cortex skulls and postxed for 1 hour in fresh xative. The brains
cVLM caudal ventrolateral medulla were cut into two coronal blocks. The front block extended
rVLM rostral ventrolateral medulla
VP ventral peduncular cortex
from the tip of the cerebral hemispheres to a short way
VS ventral striatum (nucleus accumbens) behind the start of the corpus callosum (see Fig. 1AE).
VTA ventral tegmental area The second block of brain tissue or segment of spinal cord
RAT PFC PROJECTIONS
TABLE 1. Summary Data Detailing the Total Number (n) of Animals Used per Injection Site and the Range of Stereotaxic Coordinates of the WGA-HRP
Injection Sites (Paxinos and Watson, 1986, 1998)1
Region
DS Dorsal striatum
VS Core/shell ventral striatum Heimer et al. 1991
BLA Basolateral amygdala
LH Lateral hypothalamus; including part of the
dorsomedial and posterolateral segment
Sep Mediolateral septum (incl. rostroventral part)
dlPAG Dorsolateral periaqueductal grey
DR Dorsal raphe
VTA Ventral tegmental area
PB Parabrachial nucleus
NTS Nucleus tractus solitarius
rVLM Rostral ventrolateral medulla
cVLM Caudal ventrolateral medulla
SC Thoracic spinal cord (segments T5T12)
MD Mediodorsal nucleus of the thalamus
Total
1
Stereotaxic coordinate range given as lateral, vertical, anterior/posterior from Bregma.
was taken from around the site of the tracer injection
(Table 1).
Serial coronal Vibratome sections (80 m thick) were
cut from both blocks of tissue, and longitudinal sections
were similarly prepared from the thoracic spinal cord.
Sections were collected in 0.1 M PB. After sectioning, the
tissue sections were thoroughly washed in 50 mM Tris
buffer (pH 7.4) and reacted for peroxidase activity by
incubating the sections in 0.01% H2O2 and 0.05% 33-
diaminobenzidine (DAB) for 310 minutes (Bolam, 1992).
The sections were then washed, dehydrated in an ascend-
ing series of alcohols, passed through xylene, and nally
embedded under glass coverslips in DPX mountant.
Nissl cytoarchitectonics of PFC and
construction of cytoarchitectural at-maps
Four additional male rats (250 g) were anesthetized
with equithesin and perfused with aldehyde xatives
0.52% G/3% P in 0.1 M PB (pH 7.4). Serial coronal Vi-
bratome sections (50 m thick) were then cut from the
anterior half of the brains [from 5.0 to 1.0 mm anterior
to Bregma (B); Fig. 1AD,af]. These tissue sections were
mounted in order onto chrome gelatin-coated slides, air
dried, and subsequently stained with cresyl violet (Gab-
bott et al., 1997). The sections were nally dehydrated in
an ascending series of alcohols, taken through xylene, and
embedded in DPX moutant.
By consulting previously published cytoarchitectonic
maps of rat mPFC (Ray and Price, 1992; Swanson, 1998),
areal borders and the boundaries between cytological lay-
ers within each area were dened for each set of Nissl-
stained sections (see Fig. 1A,B,af). Drawings of the areal
borders and laminar boundaries were made via a drawing
tube attached to a light microscope. The drawings were
scanned digitally and electronic les subsequently im-
ported into graphics software (Adobe Illustrator/
Photoshop) and measurement programs (NIH Image,
MacStereology). All measuring programs incorporated an
assumed linear isotropic tissue shrinkage of 15% (Gabbott
et al., 1997).
Section alignment. To ensure spatial congruence,
sections were rotated and their medial walls aligned op-
timally along the dorsoventral midline axis (Fig. 1F). For
each section, a top left hand corner tangential reference
point (TRP) was then dened (see Figs. 1G, 5C,C). The
TRP represented the intersection point of orthogonal tan-
147
n Range of injection coordinates (mm) Code No.
5 1.2, 7.4, 1.0 to 1.0, 7.2, 1.7 31
6 2.0, 4.8, 1.2 to 2.0, 4.8, 1.7 56
6 4.8, 8.4, 2.9 to 4.9, 8.2, 2.2 4
6 1.9, 8.8, 2.6 to 1.9, 8.2, 1.9 71
5 0.0, 7.2, 0.2 to 0.0, 7.4, 0.7 62
8 0.6, 5.2, 8.0 to 0.6, 5.4, 7.6 25
7 0.0, 6.3, 8.0 to 0.0, 6.3, 7.5 16
4 0.7, 8.3, 5.1 to 0.4, 8.4, 5.6 51
5 1.9, 6.7, 9.5 to 1.9, 6.6, 9.2 67
6 0.5, 8.0, 13.8 to 0.6, 8.1, 13.6 42
7 2.2, 10.2, 12.0 to 2.0, 10.1, 12.6 22
6 2.0, 9.8, 14.1 to 2.0, 9.9, 13.9 39
7 Thoracic spinal cord T5-12 11
5 0.5, 5.5, 3.3 to 0.6, 5.4, 2.8 46
83
gents to the vertical dorsoventral (midline) axis and a
horizontal tangent to the dorsal border of the tissue (Figs.
1af, 5C,C,D). Section alignment was performed sepa-
rately for both hemispheres.
Upper layer 5 root point. For each section, an oblique
45 line was drawn from the TRP into the tissue and its
intersection with the upper layer 5 boundary dened
(Figs. 1af,G, 5C,C,D). This transection represented the
upper layer 5 root point and served as a common ducial
mark in each section for the subsequent reconstructions
(Figs. 1e,G, 5D). Clockwise (positive) and anticlockwise
(negative) distances of all the areal borders along the
upper layer 5 boundary, with respect to the upper layer 5
root point, were measured in the four sets of serial Nissl-
stained sections.
The upper layer 5 boundary is a continuous line in
sections anterior to approximately 3.5 mmB (Fig. 1a c).
To produce a at-map reconstruction of anterior frontal
areas, this boundary line was necessarily interrupted at
the lateral edge of the lateral orbital cortex. However, in
posterior sections, continuity of the boundary line is nat-
urally interrupted between the infralimbic/dorsal pedun-
cular cortices and the ventrolateral/lateral orbital cortex
as the rostral pole of the nucleus accumbens becomes
established (3.0 mmB). Because the lateral orbital cor-
tex (OBl) is the main area affected by this discontinuity, it
was necessary to arbitrarily divide the OBl into a medial
anterior part (shown at top left of map in Fig. 1H) and a
posterior lateral part (shown at right of map in Fig. 1H).
This arrangement ensured minimal distortion of the or-
bital cortex.
Areal borders. Linear distributions of areal borders
around the upper layer 5 boundary line were then con-
structed for each section. Distribution maps of all the
areal borders along this laminar boundary were subse-
quently produced by aligning the individual section distri-
butions rostrocaudally with respect to the upper layer 5
root point; each distribution was spaced apart by the
thickness (50 m) of each section. Corresponding areal
boundaries were then joined between sections to produce a
at-map of areal borders.
The areal border at-maps derived from the four ani-
mals were then superimposed, and a composite map was
constructed. Areal borders in this nal, overall at-map
were smoothed by using a nearest-neighbor least-
difference algorithm to accommodate differences among
148 P.L.A. GABBOTT ET AL.

Figure 1
RAT PFC PROJECTIONS
the four individual reconstructions (Fry, 1992). The nal
generic at-map of rostral cortical areal borders shown in
Figure 1H served as a template for mapping the quanti-
tative distributions of projection neuron populations in
rostral cortices.
Construction of projection neuron at-maps
Animal selection. For each projection pathway stud-
ied, the histological sections from individual animals were
inspected to ascertain not only the accuracy and size of the
WGA-HRP injection site, and possible encroachment into
surrounding areas, but also the degree of retrograde
transport and the subsequent quality and extent of retro-
grade cellular labelling in the areas of cortex under inves-
tigation. According to these criteria, the histological sec-
tions from specic animals were selected for
reconstruction and quantitative analysis. At least two
complete sets of coronal sections in which labelling was
considered (by several investigators) to be optimal were
used per projection pathway. Projection at-maps were
constructed only for the hemisphere with the greater
number of labelled cells (ipsilateral/contralateral to the
side of tracer injection).
Digitization of coronal sections. The perimeter of
each coronal section and the positions of individual retro-
gradely labelled cells were dened and recorded with a
light microscope (Leitz Dialux 22) tted with a high-
resolution three-axis motorized stage interfaced to a com-
puter system (Neurolucida; MicroBrightField, Colchester,
VT). The tissue sections were observed at several optical
magnications (15, 25, and 40 objectives) to verify
positively labelled cells. Several researchers indepen-
dently checked a selection of different sections to assess
interobserver variability and standardize the detection of
labelled cells. To avoid double counting of cells bisected by
the plane of section, only the cut labelled cellular proles
lying at the upper surface of each section were included for
Fig. 1. A: Medial surface of the right hemisphere of rat brain.
Scale (in mm) anterior to Bregma (B, dot). Rostrocaudal (r c) extent
of reconstructed medial cortical areas lie between 4.3B and 2.2B.
B: Transparent surface of rat brain showing rostrocaudal (r c) posi-
tion of coronal section at 3.2 mm B. C: Drawings showing rostro-
caudal location of tissue block containing left prefrontal cortex. The
position of a rostral section (dark) located at 3.2 mm anterior to
Bregma reference is also given. D: Corresponding reconstructed areas
of lateral (i), dorsal (ii), and orbital (iii) prefrontal cortex are shown.
E: ACd, PL, and IL areas on the medial wall of the right hemisphere;
Bregma (B). Scales in mm. F: Superposition of rostral coronal sections
in their correct relative positions perpendicular to the axis of section-
ing. G: Transformed alignment of coronal sections so that the top left
tangential reference points (TRPs; see Fig. 5) are superimposed. The
central axis of all TRPs is indicated in the at-map distribution shown
in F. af: Six sets of coronal sections (Nissl section and drawing of
areal boundaries) from 2.3B to 4.2B adapted from Swanson (1998).
Cortical areas are indicated (PL cortex in gray). Note location of TRP
in each section (cf. Fig. 1E). PrCm is a medial subdivision of the
secondary motor area MOs (Ray and Price, 1992). H: Flat-map pro-
jection showing layer 5 borders between the areas of rostral cortex
examined in this study. This at-map of areal boundaries provided a
template for mapping the laminar densities and distributions of the
retrogradely labelled cell populations. Common tangential reference
point (TRP; vertical line). Note discontinuity (horizontal dotted line)
where lateral orbital cortex (arrows) is divided and separated from
anterior ventromedial areas of orbital cortex (sections df). Scale
bars 0.5 cm in B,D; 2 mm in F,G; 1 mm in c,e (applies to a,b,d,e,f).
149
analysis, whereas those at the lower surface were ex-
cluded (Howard and Reed, 1998). By reference to coronal
Nissl-stained sections, areal borders and laminar bound-
aries were drawn onto the sections containing the retro-
gradely labelled cell populations.
Segmentation of rostral cortex into sampling strips.
In each coronal section, the upper layer 5 root point was
determined. This point dened the center of a segmenta-
tion procedure applied to the upper layer 5 boundary line.
Taking the root point as the center of sampling strip 0, the
upper layer 5 laminar boundary was then segmented into
uniform sampling intervals 100 m wide, extending clock-
wise (positive numbers) and anticlockwise (negative num-
bers) around the tissue section. As shown in Figure 5D,
the complete segmentation of the upper layer 5 boundary
line allowed cortical areas in a tissue section to be divided
into a continuous set of nonoverlapping trapezoid sam-
pling strips with the long axes extending perpendicularly
from the pial surface to the white matter. This segmenta-
tion procedure was carried out for each section analyzed.
Quantication of retrogradely labelled cell popula-
tions. In each section, the absolute numbers of retro-
gradely labelled cells occurring in each layer 1 6 of each
cortical sampling strip were determined. Labelled cells
that crossed the left side of each strip and the upper
laminar boundary were not included (Fry, 1992). Simi-
larly, within each strip, only cut labelled cellular proles
lying at the upper surface of the section were included for
analysis, those at the lower surface being excluded. This
procedure was carried out on the histological sections from
at least two animals for each pathway investigated.
Density distribution at-maps. The results from in-
dividual animals were subsequently imported into data
spreadsheets (Excel Microsoft Ofce XP and SPSS 12),
with each cortical layer treated separately. The spread-
sheet data for each layer were arranged relative to count-
ing strip 0, the results for each section being ordered
serially along the rostrocaudal axis of the brain. The sizes
of data cells on all spreadsheets were adjusted to repre-
sent the ratio between the sampling interval used to seg-
ment the upper layer 5 boundary line (100 m) and the
absolute thickness of the corresponding histological sec-
tions (80 m). Marker points for areal borders in each
layer of each section were also included. The nal data
spreadsheets therefore represent high-resolution unfolded
density-distribution at-maps of the absolute numbers of
retrogradely labelled cells in trapezoid sampling volumes
(oriented perpendicular to the axis of viewing) through
dened layers of the cortical areas sampled (see Fig. 5D).
To aid visualization, the data spreadsheets were subse-
quently transformed into gray-scale density-distribution
maps (see Fig. 5E,F). Comparisons between different lay-
ers of a given projection pathway or between different
pathways were facilitated by using the same range of
gray-scale density coding for each map (see Figs. 9 11;
maximal range 0 70 labelled cells per data cell in spread-
sheet divided equally into 10 gray levels from white to
black, respectively). Gray-scale density-distribution at-
maps for each layer in the areas of PFC studied were
generated for all pathways investigated. One advantage of
using at-maps is that the rostrocaudal and dorsoventral
density distributions of projection neurons are simulta-
neously charted, allowing local regional variations to be
readily detected.
150 P.L.A. GABBOTT ET AL.

TABLE 2. Summary Laminar and Areal Data for Rat Cortical Areas 25 (IL), 32 (PL), and 24b (ACd)1
Total Nv N %GABA %Pyram Nv Pyram L (m) Abs.n.pyramids
Area 25 IL
1 10,138 15.6 84.4 8,557 178 1,522
2 74,721 11.2 88.8 66,352 101 6,702
3 54,906 19.7 80.3 44,089 186 8,201
5 51,349 19.1 80.9 41,541 264 10,966
6a 54,846 10.1 89.9 49,307 232 11,439
6b 79,999 13.1 86.9 69,519 39 2,711
Mean 54,327 14.8 85.2 Sum 41,541
Area 32 PL
1 6,832 17.4 82.6 5,643 207.4 1,168
2 75,696 14.9 85.1 64,417 88.5 5,701
3 59,690 18.8 81.2 48,468 236.2 11,448
5 42,121 21.2 78.8 33,191 340.5 11,302
6a 56,601 12.7 87.3 49,412 264.4 13,064
6b 80,416 14 86 69,157 56.9 3,935
Mean 53,559 16.5 83.5 Sum 46,618
Area 24b ACd
1 5,879 8.1 91.9 5,403 201.4 1,085
2 72,453 25.7 74.3 53,832 77.3 4,161
3 45,740 20.9 79.1 36,180 345.2 12,489
5 41,283 21.6 78.4 32,365 444.2 14,377
6a 52,398 10.1 89.9 47,105 332 15,639
6b 63,795 17 83 52,949 137.4 7,275
Mean 46,924 17.2 82.8 Sum 55,026
1
Total Nv N, mean numerical density per 1 mm3 of all neurons; %GABA and %Pyram, percentages that GABA immunoreactive and pyramidal cells, respectively, constitute of all
neurons; Nv Pyram, overall mean numerical density of pyramidal neurons; L, laminar thickness; Abs.n.pyramids, absolute number of pyramidal cells in each layer under 1 mm2
of pial surface area. Total number for a column of cortex under 1 mm2 of pial surface area also given. Data derived from Tables 2,3,5,6 and 7 in Gabbott et al. (1997).

Standardization of at-maps. Individual gray-scale
density distributions were imported into graphics pro-
grams (Adobe Illustrator 10 and Photoshop 7) and then
transformed electronically so that the areal boundary
markers were maximally aligned with borders on the
standardized areal at-map (Fig. 1H; Fry, 1992). This
standardization procedure allowed comparisons to be
made between the laminar density-distribution maps for
each of the pathways investigated. Because layer 5 was
demonstrated to be the principal layer from which de-
scending projections originated (see Fig. 13), an overall
density-distribution at-map composed of all the projec-
tion cell populations, derived from the different pathways
studied, was constructed to identify specic regions of
layer 5 with high total numbers of labelled layer 5 projec-
tion cells.
Quantitative parameters for retrogradely
labelled cell populations in medial PFC
Quantitative data were extracted from a previously
published report dening specic aspects of the neuronal
composition of the rat mPFC (Gabbott et al., 1997). For
each layer in IL, PL, ACd cortices, the following mean
values were obtained (Table 2): 1) volume numerical den-
sity of all neurons (Nv Total Neurons); 2) percentage of
GABA-immunoreactive neurons among the total number
of neurons (%GABA); and 3) Nv of pyramidal neurons
(dened as GABA-immunonegative cells): [Nv Pyram
(Nv Total Neurons %GABA)/100]. The latter parameter
assumed that all non-GABA-containing neurons were py-
ramidal cells projecting out of the cortex (White, 1989;
DeFelipe and Farinas, 1992; Gabbott et al., 1997).
By considering the depth of each layer (thickness L
m) the absolute number of pyramidal cells in a given
lamina under 1 mm2 of pial surface was subsequently
calculated (Abs.n.pyramids). Mean values for these pa-
rameters are presented in Table 2.
Quantitative parameters for retrogradely labelled
cell populations in mPFC. Figure 2A,B shows the cyto-
logical and morphometric procedures schematically. By
using the histological material containing identied pro-
jection cell populations in mPFC, separately labelled from
14 subcortical sites with the retrograde pathway tracer
WGA-HRP, in conjunction with data presented in Table 2,
the following quantitative parameters were derived for
each layer in IL, PL, and ACd cortices: 1) volume numer-
ical density of retrogradely labelled projection cells for
each pathway (Nv Project.); 2) percentage oft retrogradely
labelled neurons among the total number of pyramidal
neurons in a layer or area of mPFC (%Project.); and 3)
combined percentage of individual retrogradely labelled
neuron populations among the total number of pyramidal
projection neurons (Total %Project.). [Note: In the projec-
tion from mPFC to dorsal striatum, the ventral (v) and
dorsal (d) PL cortex were investigated separately; see
Figs. 6, 13.]
Sets of alternate coronal sections (1:2 series) through
the rostrocaudal extents of IL, PL, and ACd cortices from
three animals in which the sites of WGA-HRP injection,
retrograde labelling, and cellular transport were consid-
ered to be optimal were selected for study. By using a
computer-assisted light microscope system (Neurolucida;
MicroBrightField), 30 50 square nonoverlapping sam-
pling quadrats (either 100 100 m or 250 250 m,
depending on laminar thickness; Table 2) were randomly
superimposed over each layer of the mPFC regions inves-
tigated. While focussing through the tissue section, the
absolute numbers of retrogradely labelled somata con-
tained within each sampling quadrat were recorded (Gab-
bott and Bacon, 1996a,b; Howard and Reed, 1998). The
left and lower quadrat boundaries, as well as the lower
surface of the tissue section, were dened as exclusion
boundaries (Gabbott et al., 1997; Howard and Reed, 1998).
From the density of retrogradely labelled cells occurring
per quadrat and the thickness of each section, the mean
volume numerical densities of retrogradely labelled pro-
jection cells (Nv Project. per mm3; Fig. 2B) in each layer of
IL, PL (vPL and dPL calculated separately for the dorsal
striatal pathway), and ACd cortices were subsequently
RAT PFC PROJECTIONS
Fig. 2. Quantitative methodology. A: The numerical densities of
all neurons (Nv Total Neurons), pyramidal projection cells, and local
circuit GABAergic neurons (LCNs) in each of layers 1 6 of areas 25,
32, and 24b have been dened in a previous quantitative GABA
immunocytochemical and Nissl cytoarchitectonic investigation (Gab-
bott et al., 1997). The latter study also calculated the percentage of
GABA-immunoreactive LCNs in each layer of areas 25, 32, and 24b
(%GABA, asterisk). The numerical density of pyramidal projection
cells (Nv Pyram.) can therefore be calculated, assuming that all LCNs
are GABA immunoreactive and that all pyramidal neurons are
GABA-immunonegative projection cells. B: From the histological ma-
terial prepared in this study, the numerical density of WGA-HRP
retrogradely labelled neurons projecting to specic subcortical target
sites (Nv Project.) can be dened stereologically for each layer of areas
25, 32, and 24b. C: The percentage that neurons projecting to a
specic subcortical nucleus constitute of the overall pyramidal cell
population in each layer of each area (%Project.) can be subsequently
derived. Scale bars 250 m.
obtained (Gabbott and Bacon, 1996b; Howard and Reed,
1998).
The mean percentages that identied neuron popula-
tions in IL, PL, and ACd cortices (known to project to
specic subcortical autonomic and limbic sites) consti-
tuted of the total population of pyramidal cells were de-
rived for each layer of each area (or subarea) of rat mPFC
151
[%Project. (Nv Project/Nv Pyram) 100; Fig. 2C]. Fi-
nally, the total percentages that all 14 retrogradely la-
belled neuron populations composed of the total number of
pyramidal projection neurons (Total %Project.
%Project. for each pathway) were calculated for each
layer in each area. Mean group values SEM (n 3) were
subsequently derived (Fry, 1992; Howard and Reed,
1998).
Dual-uorescence retrograde tract-tracing
studies
Part three was a series of retrograde pathway-tracing
studies using two uorescent tracers in the same animal.
The aim was to dene the identity, location, and numeri-
cal size of projection neuron populations in mPFC with
divergent efferent axons that simultaneously innervated
two subcortical target structures (Fig. 3A,Biiii; Catsman-
Berrevoets and Kuypers, 1981; Heimer and Zaborsky,
1989; Meredith and Arbuthnott, 1993; Pinto and Sesack,
2000). Separate injections of compatible retrograde uo-
rescent tracers were made into specic pairwise combina-
tions of the 14 subcortical brain regions being investigated
(Fig. 3A, Table 5; Bolam, 1992; Meredith and Arbuthnott,
1993). Control experiments were also undertaken (see be-
low).
This part of the investigation used 128 Sprague-Dawley
rats (240 285 g) of both sexes. Animals were anesthetized
with equithesin (Millenbruck and Wallinga, 1946; 0.3 ml/
100 g body weight,. i.p.) and placed in a stereotaxic frame
(Kopf Instruments). After appropriate surgery, separate
pressure injections of 50 200 nl of 25% fast blue (FB;
Sigma, St. Louis, MO) and 100 200 nl of 20 25% rhoda-
mine red-labelled latex microspheres (RLM; Lumauor) in
Tris-buffered saline (TBS; pH 7.4) were injected in se-
quence via glass microelectrodes (tip diameters 20 30
m) into specic paired combinations of subcortical sites
(see Table 5). The ranges of stereotaxic coordinates for the
injection sites (Paxinos and Watson, 1986, 1998) are given
in Table 1.
After a survival period of 5 8 days, the animals were
given an overdose of anesthetic and transcardially per-
fused, initially with 0.9% physiological saline at room
temperature (RT) and then with 500 ml of 4% P xative in
0.1 M PB, pH 7.4, at RT. The brains and spinal cords were
carefully removed and postxed in fresh xative in 0.1 M
PB, pH 7.4, for 1 hour at 4C.
By using the histological methods described above, se-
rial coronal sections were collected throughout the rostro-
caudal extent of the mPFC and also from the correspond-
ing subcortical injection sites in the brain and spinal cord.
Tissue blocks were cut at a thickness of 80 m with a
vibratome. Tissue sections were washed thoroughly (3
10 minutes) in 0.1 M PBS (pH 7.4), mounted in 100%
glycerol, coverslipped, and viewed under uorescent optics
by using a custom lter block (Chroma Technologies) that
permitted the simultaneous visualization and discrimina-
tion of both FB and RLM uorescent tracers (Fig. 3DF).
Fluorescence visualization procedures. Care was
taken not to quench the uorescent signal when examin-
ing the sections under uorescent optics. In animals with
optimally located injection sites, areas of mPFC contain-
ing retrogradely labelled uorescent somata were care-
fully examined at high magnication to determine the
presence of neurons containing both uorescent tracers
152 P.L.A. GABBOTT ET AL.

(FB/RLM). When double-labelled neurons were encoun-

tered in the mPFC, the region of maximal dual labelling
was analyzed quantitatively (Howard and Reed, 1998).
Sampling quadrats (100 100 m) were used to count the
number of cells throughout the thickness of the section
containing either FB or RLM or both FB/RLM tracers (Fig.
3E,F). Because of the paucity of labelled neurons in layers
3, 2, and 1, data were collected only for layers 5 and 6 in
each area. The percentages of double retrogradely labelled
neurons (FB/RLM) within the total number of retro-
gradely labelled layer 5 (6) cells present [FB RLM
(FB/RLM)] were calculated. [In total, 150 300 retro-
gradely labelled neurons were sampled per layer 5 (6), per
area per animal.] However, because of the methodological
difculties (see below), the results were classied into 5%
data bins (see Table 6); quantitative data were obtained
from at least two animals for each paired combination of
injection sites. It was necessary to transform the ob-
served uorescence data (assuming that the two parent
populations were present in the same proportion as in
the single-pathway tracing studies; Table 3AC), to de-
rive the percentage of pyramidal neurons in layer 5 (6)
of a given area projecting to a known pair of subcortical
target structures (Tables 5, 6; Fig. 3; see also Pinto and
Sesack, 2000).
Control retrograde tracing experiments. Pilot injec-
tions of WGA-HRP into immediately adjacent nonproject-
ing structures did not produce retrogradely labelled neu-

Fig. 3. A: Dual retrograde labelling. Two different uorescent

retrograde tract tracers (rhodamine-labelled latex microspheres,
RLM; fast blue, FB) are injected into two subcortical targets (X and Y,
respectively) containing terminal axonal arbors of mPFC projection
neurons. Two distinct cell populations, projecting to either X or Y, are
retrogradely labelled in the cortex with either RLM or FB. A third cell
population projecting to both X and Y will be retrogradely labelled
with both RLM and FB tracers (X/Y). Unlabelled neurons project to
other subcortical targets. Bi: Two separate populations of cortical
projection neurons, which project to either subcortical target site X or
target site Y. The two population may be composed of different num-
bers of cells. Bii: Overlapping populations of cortical projection neu-
rons. Distinct neuron populations project solely to either target sites
X or Y or to both X and Y. Biii: Population of neurons projecting to
target site X contains a subpopulation of projection cells that also
project to target site Y. No cells projecting to target site Y alone.
C: Injection of RLM into the upper segments of the thoracic spinal
cord. The central canal is indicated (thick arrow). The injection is
bilateral and includes the central autonomic area and the interme-
diolateral cell column of the thoracic spinal cord (single-headed thin
arrow). Labelled cells are visible in the longitudinally aligned lateral
ber tracts (double-headed thin arrow). r, Rostral; c, caudal. D: Layer
5 dPL cells retrogradely labelled after separate injections of FB into
dorsal striatum (DS) and RLM into the basolateral nucleus of the
amygdala (BLA). Neurons only projecting to DS are indicated (single-
headed arrows). A population of cells, out of the plane of focus, con-
taining both tracers is present (double-headed arrows). E: Layer 5
vPL cells retrogradely labelled after injections of FB into ventral
striatum/nucleus accumbens (VS) and RLM into the basolateral nu-
cleus of the amygdala (BLA). One retrogradely labelled neuron
projects to VS (single-headed arrow), whereas another projects to
BLA. Two neighboring retrogradely labelled neurons contain both
RLM and FB and therefore project to both VS and BLA (double-
headed arrows). F: Layer 5 ACd cells retrogradely labelled after
injections of FB into lateral hypothalamus (LH) and RLM into the
thoracic spinal cord (SC). Two corticospinal cells are indicated (single-
headed arrows). Another corticospinal neuron contains both RLM and
FB (double-headed arrow), indicating that it also projected to the
lateral hypothalamus. Scale bars 500 m in C; 25 m in DF.
RAT PFC PROJECTIONS
ron populations in the mPFC. For example, injections of
tracer directly into 1) the posterior, centrolateral, or para-
central nuclei of the thalamus (for MD injections), 2) area
postrema of the medulla (for NTS injections), or 3) zona
incerta, ventromedial thalamus (for LH injections) did not
produce retrogradely labelled PFC neurons. In different
animals, injections (of similar sizes and locations) into the
target nuclei studied here consistently retrogradely la-
belled neurons in the same regions of the PFC and other
brain nuclei.
In the dual-uorescence tracer study, control tract-
tracing experiments were performed to test for possible
pathway specicity of the individual tracers. For specic
combinations of subcortical targets (LH/SC, NTS/SC, VS/
BLA), the sites of tracer injection were interchanged. In
such experiments, there was no signicant qualitative or
quantitative difference in the distributions of retrogradely
labelled neurons in mPFC. Overall, the resulting patterns
of retrograde tracing were in accordance with published
reports.
The images presented in this paper were digitized and
stored as Adobe Photoshop 7 PSD image les. Contrast,
brightness, and gray-level modications were performed
in Photoshop to optimize image quality. Final output les
were in TIFF format.
RESULTS
The approximate surface location of the transverse seg-
ment of rostral cortex (including PFC) examined in this
study is shown with respect to the lateral, dorsal, and
orbital surfaces of both hemispheres of the rat brain in
Figure 1Diiii. This segment lay bilaterally between ap-
proximately 4.3B and 2.2B (Fig. 1A,D). Figure 1AD appeared as diffuse DAB labelling throughout the neu-
shows the anteroposterior extent of this segment on the
rostral medial wall. The territories of the principal medial
regions contained within this segment are shown in Fig-
ure 1E. This region of cortex was studied because it in-
cluded signicant proportions of the main elds of rostral
cortex: orbital, peduncular, infralimbic, prelimbic, ante-
rior cingulate, medial precentral, motor, somatosensory,
and insular areas and their respective subdivisions (Fig.
1af). In addition, it represented the region with the
greatest degree of overlap between all the retrogradely
labelled cell populations.
Cytoarchitectural at-maps
A generic at-map of the cytoarchitectonic areal borders
present at the level of the upper layer 5 boundary was
derived and is shown in Figure 1H. The at-map is pre-
sented for the right cerebral hemisphere (Fig. 1H; lateral
to right, medial and orbital to left). The midvertical line in
Figure 1H connects the TRP dened through all sections
(cf. Fig. 1H and G; see also Fig.1af). It is important to
note that the at-map is obtained by unfolding the upper
layer 5 boundary orthogonally with respect to the TRP
line (Fig. 1G). A similar reconstruction for the left cerebral
hemisphere displayed no signicant differences in the dis-
tribution of the cortical elds.
All the major cytoarchitectonic boundaries together
with those of the principal subdivisions of the orbital,
insular, motor, and peduncular cortices are displayed in
Figure 1H. In constructing the generic at-map, OBl cor-
tex contains a lateral strip abutting the agranular insular
153
cortices (AIv and AId). In reality, this strip is continuous
with OBl and OBvl, which border areas of mPFC. Note
also that the medial portion of the secondary motor cortex
(MOS) is classied as the precentral motor area (PrCm) as
given by Ray and Price (1992). The cytoarchitectural
at-map shown in Figure 1H has been used as the tem-
plate for generating the at-map density distributions of
retrogradely labelled cell populations
Retrograde tract-tracing studies
Tracer injections. In total, 83 animals were used in
the tract-tracing experiments described in this study.
Although several animals per injection site had accu-
rate tracer injection, successful retrograde axonal
transport and cellular labelling, only the two optimally
labelled animals were used in the reconstructions for
each pathway. Tracer injections were considered opti-
mal when the region of peroxidase labelling of both the
intensely stained central core and the surrounding halo
(of very much weaker labelling) were conned to as
large a volume fraction of the targeted subcortical site
as possible. An additional requirement was that the
injection site did not encroach into neighboring nuclei
and ber tracts known to project to rostral cortical
areas. Table 1 provides a summary of the range of
injection site coordinates. The intense, darkly stained,
central area of each injection site represents the core
region from which the vast majority (if not all) of the
neurons were retrogradely labelled by HRP. Where
present, and depending on the precise location of the
injection site, the lightly stained halo surrounding the
central dark cores represents regions producing few
retrogradely labelled neurons in PFC. The halo regions
ropil that contrasted with the strongly labelled cores,
where both groups and individual neurons could be
found intensely labelled. The qualitative descriptions
are derived from all animals in which injections were
accurate and discrete, and subsequent cortical labelling
was representative of the pathways under investiga-
tion.
Distribution and morphology of retrogradely la-
belled cells. After the subcortical injections of WGA-
HRP, retrogradely labelled somata could be readily found
throughout areas of rostral cortex (Fig. 4AD,F). At low
magnication, populations of darkly stained cellular pro-
les could be readily discerned in the tissue and were
located in specic areas and laminae depending on the site
of the tracer injection (Fig. 4AC). Retrogradely labelled
cells were present throughout the full thickness of the
histological sections. Bisected labelled cellular proles
were present at the upper and lower surfaces of the sec-
tions. At higher magnication, the retrogradely labelled
somata contained the characteristic brown end-product of
the peroxidase reaction. The vast majority of labelled cells
were clearly visible against a relatively unstained back-
ground (Fig. 4A,F). For all the pathways examined, most
retrogradely labelled cortical cells were pyramidal in mor-
phology (Fig. 4F). These labelled cells had darkly stained,
triangular somata (containing numerous dark puncta),
frequently giving rise to the stout proximal segments of
ascending apical dendrites and the initial parts of basal
dendritic arbors (Fig. 4F). No retrogradely labelled corti-
cal cells were found that clearly displayed the morpholog-
154
Fig. 4. Projection to spinal cord (SC). A: WGA-HRP retrogradely
labelled layer 5 corticospinal neurons (arrow) in PL cortex. (capillary,
c). B: Labelled layer 5 corticospinal neurons (arrow) in vPL and IL
cortices. v, Ventral; d, dorsal; m, medial. C: Enlargement of the region
in B showing labelled layer 5 vPL corticospinal neurons (arrows).
D: Drawing of a coronal histological section taken 3.3 mm in front of
Bregma (animal 15). The boxed region represents the extent of the
histological section shown in E. E: Location and distribution of retro-
ical characteristics of local circuit (nonprojection) inter-
neurons (Gabbott et al., 1997).
Coronal, at-map, and quantitative
distributions of retrogradely labelled cells
The salient features of the coronal and at-map distri-
butions of retrogradely labelled neuron populations asso-
ciated with the pathways studied are described below and
shown in Figures 4 12. Quantitative data dening the
percentage that identied projection neurons composed of
the pyramidal cell populations in IL, PL, and ACd cortical
areas are also given in Figures 13 and Table 3AC.
Construction of maps. Volume distortions occur
when curved segments of the cerebral cortex are unfolded
to give at representations (see Fig. 1 in Gabbott et al.,
1987). The attening procedure used in this study un-
folded the cortex with respect to the upper layer 5 bound-
ary line (Fig. 5B,C). Because the cortical strips used to
obtain labelled cell density information were trapezoid-
like wedges (with long axis perpendicular to pial surface),
density-distribution at-maps produced for supercial
layers would be correspondingly larger than for maps
constructed in deep layers (Fig. 5C). That is, for nonover-
lapping sampling strips, the segmentation interval along
the layer 5 boundary line would not have been the same
interval as the lower layer 6 or upper layer 3 boundary
line (Fig. 5C). To overcome this problem, density distribu-
P.L.A. GABBOTT ET AL.
gradely labelled neurons (arrows) in rostral cortical areas following
tracer injection into thoracic spinal cord. F: Two WGA-HRP retro-
gradely labelled corticospinal PL pyramidal cells (P). Apical (single-
headed arrows) and basal dendritic processes (double-headed arrows)
are indicated. Note characteristic punctate reaction end-product in
labelled somata and processes. Scale bar 500 m in A,B; 200 m in
C; 2 mm in D; 1 mm in E; 25 m in F.
tion maps constructed for each layer were, in effect, col-
lapsed onto the same segmentation interval used to par-
tition the upper layer 5 boundary line (see Figs. 8E, 9E).
This procedure produced density-distribution at-maps
that facilitated interlaminar comparisons both within and
between projection pathways.
Thoracic spinal cord (SC; Figs. 4, 10H, 13). Retro-
gradely labelled cell populations in rostral cortex after SC
injections (Fig. 3C) were found in both hemispheres lo-
cated principally in the secondary motor area (MOs, in-
cluding PrCm; Fig. 4AC,E) and with greatly reduced
frequencies in PL and IL cortices (Fig. 4C,E). Corticospi-
nal neurons were found mainly in layer 5 (Figs. 4A,B,E,
13).
The at-map density distribution of cortical neurons in
layer 5 projecting to the thoracic spinal cord (Fig. 10H)
also indicated a peak density occurring in medial MOs
(area PrCm). Corticospinal neurons were present, albeit
in much reduced numbers, throughout large rostrocaudal
extents of PL and IL cortex (Fig. 10H). Cortical neurons
projecting to SC were absent from almost all areas of
orbital cortex and from MOp and GU, AId, and AIv corti-
ces (Fig. 10H).
Quantitative estimates indicated that neurons project-
ing to the thoracic spinal cord constituted 1% of projection
neurons in layer 5 of IL cortex, 9% of layer 5 projection
neurons in PL, and 13% of projection neurons in ACd layer
RAT PFC PROJECTIONS
Fig. 5. Projection to dorsal raphe (DR). A: Coronal section showing
the position and extent of the central foci (umbra and penumbra) of a
WGA-HRP injection into DR. Bregma level and animal identity code
(16) are indicated. B: Cytoarchitectonic division of the prefrontal
cortex at Bregma 3.2 mm. C: Location and distribution of retro-
gradely labelled neurons (dark punctate dots) in rostral cortex after
an injection similar to A. Drawing composed from six serial sections
(2328). Boundary between upper layer 5 and lower layer 3 is indi-
cated; layer 5 contains vast majority of corticoraphe neurons. Vertical
(V) and horizontal (H) axes (divided into mm) have been applied
tangentially to the tissue sections to dene the upper left hand tan-
gential reference point (TRP; open circle). An oblique line at 45 to
the V-H axes is indicated. d, Dorsal; l, lateral; a, anterior; p, posterior.
C: Nissl-stained section at the level of the sections shown in B and C.
An oblique line to the V-H axes is shown. D: Redrawing of C showing
distribution of retrogradely labelled corticoraphe neurons (punctate
dots) and the boundary between lower layer 3 and upper layer 5. The
point where the 45 line from TRP crosses the layer 5/layer 3 bound-
ary is dened as the center of sampling bin 0. The layer 3/5 boundary
has been segmented into sampling bins (100 m wide) extending
155
clockwise (positive bin numbers) and anticlockwise (negative bin
numbers) around the tissue section. The segmented layer 3/5 bound-
ary allows the whole cortex to be divided up into a continuous set of
nonoverlapping vertical trapezoid sampling strips extending from pia
to white matter. E: Corticoraphe cell distribution. The absolute num-
bers of cells in each layer of each sampling strip were recorded for
each section (n 6; sections 2328). Shown here is the number of
corticoraphe cells in layer 5 collapsed onto the segmented layer 3/5
boundary line. By using areal boundary map in B, the position of
borders between cortical areas have been dened. a-p, Anterior-
posterior axis. The distributions for each section are spaced apart by
the section thickness (t). F: Transformation of E into a at-map
gray-scale representation. The absolute number of retrogradely la-
belled corticoraphe neurons in each sampling bin is represented by a
dened gray-scale value. The position of the central sampling bin
(dened by the 45 line from the TRP) common to all sections is
indicated (asterisk). Note the distribution of the corticoraphe neuron
population throughout areas of mPFC and also in the dorsal and
ventral regions of AId. Scale bars 5 mm in A; 500 m in F.
156 P.L.A. GABBOTT ET AL.

TABLE 3A. Calculated Percentages That WGA-HRP-Labelled Neuron Populations (Retrogradely Labelled from Dened Subcortical Sites) Constituted of
the Total Population of Pyramidal Projection Neurons in Given Layers of Area 25 (Mean SEM; n 3)1
Layer BLA DS VS PB LH NTS PAG Sep VTA DR MD rVLM cVLM SC Total
1 0.27 0.3
0.20
2 4.59 0.14 6.79 0.71 12.2
0.35 0.05 0.39 0.22
3 3.74 0.27 5.56 2.45 0.13 12.2
0.48 0.06 0.72 0.31 0.09
5 7.92 1.66 18.59 8.91 26.02 6.79 3.02 5.30 3.81 1.16 0.41 3.70 2.10 0.87 90.3
0.66 0.34 0.51 1.04 1.61 0.46 0.42 0.45 0.56 0.20 0.16 0.46 0.47 0.16
6 3.93 1.30 2.94 1.37 10.68 34.68 0.05 56.3
0.47 0.25 0.34 0.46 0.99 0.85 0.01
1
The combined percentage (total) that the individual labelled neuron populations constituted of the total number of pyramidal projection neurons in a layer is also given.

TABLE 3B. Calculated Percentages That WGA-HRP-Labelled Neuron Populations (Retrogradely Labelled from Dened Subcortical Sites) Constituted of
the Total Population of Pyramidal Projection Neurons in Given Layers of Area 32 (Mean SEM; n 3)1
DS v DS d
Layer BLA A32 A32 VS PB LH NTS PAG Sep VTA DR MD rVLM cVLM SC Total
1 0.15 0.15
0.06
2 7.61 0.47 0.76 7.51 1.95 vA32dA32 17.517.8
0.34 0.07 0.15 0.44 0.51
3 3.30 0.71 1.28 6.20 0.59 2.45 0.11 13.313.8
0.43 0.12 0.23 0.53 0.23 0.31 0.08
5 8.29 7.23 18.85 27.20 2.47 26.17 1.08 2.86 2.76 5.68 4.68 3.61 1.20 1.50 8.88 103.6112.5
0.37 0.65 0.96 0.74 0.33 1.59 0.18 0.28 0.42 0.52 0.39 1.19 0.18 0.18 0.83
6 1.04 3.68 6.10 1.26 10.65 41.33 0.43 58.061.9
0.21 0.22 0.27 0.17 0.95 0.99 0.09
1
Because the projection to the dorsal striatum (DS) was markedly different between ventral (v) and dorsal (d) parts of PL cortex (see Figs. 5A, 6A), data are presented separately
for these territories in area 32. The combined percentage (total) that the individual labelled neuron populations constituted of the total number of pyramidal projection neurons
in a layer is given (a range is given for layers 2 6 because of the differences in the projection to DS between ventral and dorsal area 32).

TABLE 3C. Calculated Percentages That WGA-HRP-Labelled Neuron Populations (Retrogradely Labelled from Dened Subcortical Sites) Constituted of
the Total Population of Pyramidal Projection Neurons in Given Layers of Area 24b (Mean SEM; n 3)1
Layer BLA DS VS PB LH NTS PAG Sep VTA DR MD rVLM cVLM SC Total
1 0.0
2 5.35 0.28 1.96 5.62 13.2
0.41 0.08 0.30 0.98
3 1.58 0.56 0.21 4.99 0.11 7.6
0.27 0.14 0.08 0.49 0.08
5 1.07 17.57 5.04 0.65 29.86 1.11 2.69 1.65 4.43 4.34 3.61 0.30 0.30 13.34 85.9
0.16 0.76 0.61 0.10 1.68 0.14 0.35 0.26 0.42 0.34 0.62 0.14 0.14 1.11
6 0.33 7.99 0.05 0.15 12.77 36.36 1.07 58.7
0.06 0.62 0.03 0.06 0.66 0.92 0.23
1
The combined percentage (total) that the individual labelled neuron populations constituted of the total number of pyramidal projection neurons in a layer is also given.

5 (Fig. 13, Table 3AC). About 0.7% of layer 6 projection
neurons in PL and ACd projected to the thoracic spinal
cord (Fig. 13, Table 3AC).
Dorsal raphe (DR; Figs. 5, 10B, 13). Injections into
DR (Fig. 5A) retrogradely labelled layer 5 cells in the
PrCm subregion of MOs and in ACd, PL, and IL cortices as
well as in areas of AI cortex (Fig. 5CE). The at-map
density distribution (Fig. 10B) indicated that the eld of
neurons projecting to DR arose primarily from PL, ACd,
and IL cortices. However, regions of the orbital and AId
cortices (and anterior sectors of MOs and MOp) provided
additional but more diffuse projections to DR.
The projection to DR arose from layer 5 cells (Figs.
5C,D, 10B, 13, Table 3AC). Approximately 5% of projec-
tion neurons in PL innervated DR; corresponding values
of 4% and 1% were derived for ACd and IL, respectively
(Fig. 13).
Dorsal striatum (DS; Figs. 6AC, 9A, 13). WGA-
HRP tracer injections into dorsomedial DS (Fig. 6A) la-
belled populations of neurons located mainly ipsilaterally
in layers 2, 5, and 6 of dorsal PL, ACd, and the medial part
of MOs (PrCm; Fig. 6B,C). Retrogradely labelled neurons
were also found in layer 5 of ventrolateral and lateral OB
cortex (Fig. 6B). There was moderate labelling over the
same regions of contralateral cortex.
The density-distribution at-map of layer 5 neurons
projecting to DS across rostral areas of frontal cortex
(Fig. 9A) indicated high projection neuron densities oc-
curring in dorsal PL and ACd cortices and a very
marked reduction in the density of labelled cells in the
ventral part of PL and in IL cortices. There were, in
addition, prominent projection neuron densities in both
sectors of Obl (Fig. 9A).
In IL, neurons innervating DS accounted for less than
2% of projection cells in layers 5 and 6 (Fig. 13, Table 3A).
In ventral PL, about 7% of layer 5 and 3.5% of layer 6
projection neurons innervated DS. By comparison, in dor-
sal PL, about 19% of layer 5 and 6% of layer 6 output cells
projected to DS (Fig. 13, Table 3B). In ACd, 18% of layer 5
projection neurons and 8% of layer 6 projecting cells in-
nervated DS (Fig. 13, Table 3C).
Ventral striatum/nucleus accumbens (VS; Figs.
6DF, 9B, 13). WGA-HRP tracer injections into both the
core and the shell of the VS (Heimer et al., 1991; Fig. 6D)
RAT PFC PROJECTIONS
labelled predominantly populations of neurons located ip-
silaterally in layers 2,5,6 of PL and IL cortices (Fig. 6E,F).
Retrogradely labelled cells were also present in areas DP
and VP as well as in layers 5 and 6 of ventrolateral OB,
AId, and AIv cortices (Fig. 6E). In PL cortex, retrogradely
labelled cells were located ventrally in two prominent
tangential tiers, layer 2 and layer 5 (Fig. 6F). There was
limited contralateral labelling conned mainly to layer 5
in PL, dorsal IL, and AI cortices (Fig. 6E).
The density-distribution at-map of neurons in rostral
cortex projecting to VS clearly demonstrated a peak den-
sity occurring within IL and the ventral part of PL cortex
over the full rostrocaudal extent of the cortical territory
charted (Fig. 9B). There was a much reduced projection
originating from OBm, medial OBvl, and AId cortices.
Noteworthy is that the at-map density distribution of
cortical labelling after DS and VS injections were comple-
mentary with regard to the patterns of projection neurons
across areas of mPFC (Fig. 9A vs. B).
Neurons projecting to VS in layer 5 of IL and PL con-
stituted signicant proportions (19% and 27%, respec-
tively) of the projection cells in these regions (Fig. 13,
Table 3A,B). Additional projections from layers 2 and 3 in
IL and PL composed 6 8% of the pyramidal cell popula-
tions. Neurons in layer 5 of ACd projecting to VS ac-
counted for approximately 5% of projection cells in this
area (Fig. 13, Table 3C).
Basolateral nucleus of the amygdala (BLA; Figs.
6GM, 9C, 10).
Discrete injections of tracer into BLA, for example, as
shown in Figure 6GJ, produced consistent labelling in
medial MOs, ACd, PL, IL, and DP/VP cortices and in
anteromedial OB, AId, and ventral GU cortices (Fig.
6L). Retrogradely labelled neurons were located pre-
dominantly in two tiers situated in layers 2 and upper 5
in dorsal IL, vPL, dPL, ACd, and medial MOs (Fig.
6K,M). Of note is a signicant projection from both layer
2 and layer 5 of contralateral medial border cortices
(Fig. 6K).
The at-maps of this pathway show a comparatively
uniform density of labelled neurons in layers 2 and 5
projecting to BLA (Fig. 9C). There is a very sparse projec-
tion to BLA from lateral orbital cortex, with a virtual lack
of labelled cells in SSp and MOp (Fig. 9C). A specic
feature of these distributions is the prominent nonalign-
ment of peak densities in layer 2 and layer 5 of PL cortex
(Fig. 9C); that is, a density peak in layer 2 of anterodorsal
PL cortex did not align with a similar density peak occur-
ring ventroposteriorly in layer 5 (Fig. 9C). This nding
was also observed in the at-map produced from the sec-
ond animal and was corroborated in a reconstruction,
solely of PL cortex, from a third animal. Of interest is that
the distribution of cells in layer 3 of PL cortex, much
reduced compared with that in layers 2 and 5, was rela-
tively uniform. An additional observation is the relatively
high density of corticoamygdala neurons in layer 5 of AId
cortex (Fig. 9C).
Quantitative data indicated that, in PL, corticoamyg-
dala neurons composed about 8% of projection neurons in
layers 2 and 5, 3% of cells in layer 3, and 1% of projection
neurons in layers 6 (Fig. 13, Table 3B). In IL, the corre-
sponding values were 5% for layer 2, 4% in layer 3, 8% in
layer 5, and 4% in layer 6 (Fig. 13, Table 3B). In ACd, 5%
of projection neurons in layer 2 projected to BLA, with
157
lower values of 2% for layer 3, 1% for layer 5, and 0.3% for
layer 6 (Fig. 13, Table 3C).
Lateral hypothalamus (LH; Figs. 7AC, 9D, 13).
WGA-HRP injections into mainly lateral (also medial and
posterior) hypothalamic nuclei (Fig. 7A) produced strong
bilateral labelling in layers 2, 3, 5, and 6 of PrCm, ACd,
PL, IL, DP/VP, and medial orbital cortices (Fig. 7B,C).
Some retrogradely labelled neurons were also present ip-
silaterally in AId and AIv (Fig. 7B).
Quantitative data indicated that the prominent projec-
tions to LH arose from layers 5 and 6 (Fig. 7C, Table
3AC). For IL, PL, and ACd together, approximately 27%
of layer 5 and 12% of layer 6 projection neurons projected
to LH (Fig. 13, Table 3AC).
The at-map density distributions of projection neurons
in layers 2 6 indicated that neurons projecting to the
hypothalamus were distributed across medial MOs
(PrCm), in all areas of mPFC, and also in orbital and
insular areas (Fig. 9D). There was a coalignment of pro-
jections from all layers, especially along the shoulder of
the medial border (which included dorsal ACd and PrCm).
Of specic note is that the injection site slightly en-
croached into mediodorsal and posterior areas of the hy-
pothalamus (Fig. 7A).
Parabrachial nucleus (PB) and dorsolateral periaq-
ueductal gray (dlPAG; Figs. 7EJ, 9E, 10A, 13).
Tracer injections into these subcortical regions (Fig. 7E,H)
retrogradely labelled layer 5 neurons primarily in IL and
PL cortices (Fig. 7F,G,I,J). Labelled cells were also
present in AI cortex, principally layer 5 (Fig. 7F,I). Retro-
gradely labelled cells projecting to the dlPAG were also
found in layer 5 of ventral ACd (Fig. 7I,J). Although both
projections arose bilaterally, the ipsilateral projections
were numerically more extensive.
The at-map density distribution of cells projecting to
PB demonstrated that they were principally located in IL,
DP, and VP cortices (Fig. 10A). Other projections, al-
though comparatively sparse, came from throughout PL
cortex and from OBvl/OBl and AId/GU cortices (Fig. 10A).
Projections to dlPAG from rostral cortex originated in
IL, PL, and ACd cortices. Of note is the reduced projection
from the anterior parts of PL and ACd cortex (Fig. 9E).
Other areas of rostral cortex, including the orbital and
insular cortices, were virtually devoid of projections to
dlPAG (Fig. 9E).
The quantitative data indicated that neurons projecting
to PB were located mainly in layer 5, where they composed
9% of projection neurons in IL, 3% in PL, and about 0.5%
in ACd (Fig. 13, Table 3AC). Neurons with axons that
principally innervated dlPAG accounted for approxi-
mately 3% of layer 5 projection neurons in each of IL, PL,
and ACd (Fig. 13, Table 3AC).
Septum (Sep) and ventral tegmental area (VTA; Fig.
7K,L,NP). Injections of WGA-HRP into the mediolat-
eral septum, which included ventral sectors of this region
(Fig. 7K), labelled mainly neurons in layer 5 of ventral
mPFC, including ventral PL, IL, DP/VP, and medial or-
bital cortices (Fig. 7L,M). Labelled corticoseptal neurons
were not found in AI cortex (Fig. 7L). The projection to
mediolateral septum was bilateral with retrogradely la-
belled cells distributed mainly on the ipsilateral side. In-
jections into the VTA (Fig. 7N) retrogradely labelled layer
5 neurons in IL and ventral PL cortices (with additional
labelling in dorsal PL and ACd cortices) and also AId
cortex (Fig. 7O,P).
 Fig. 6. Projections to dorsal striatum (DS), ventral striatum (VS),
and basolateral amygdala (BLA). A: Drawing showing a WGA-HRP
injection into DS. Bregma level and animal identity code are indi-
cated. B: Bilateral areal distributions of retrogradely labelled DS
somata (dots) in a coronal section from 30 at approximately 3.2/3.3B
(see Fig. 1). The numbers of retrogradely labelled cells (n) in each
ipsilateral and contralateral section are given. C: Boxed region in B
showing laminar distribution of DS cells in dPL cortex. D: WGA-HRP
injection into VS (nucleus accumbens). Bregma level and animal
identity code are indicated. E: Bilateral areal distributions of retro-
gradely labelled VS somata (dots) in a coronal section from 59 at
approximately 3.2/3.3B. F: Boxed region in E showing laminar dis-
tribution of labelled VS cells. G: Part of a Nissl-stained coronal section
(from approximately 2.53.0 mm posterior to Bregma) illustrating the
cytoarchitecture of the amygdala and surrounding structures. H: Cy-
toarchitectural denition of section shown in G: Anterior (ant) and
posterior (post) parts of the basolateral amygdala (BLA), basomedial
amygdala (BMA), lateral amygdala (LA), central nucleus of the amyg-
dala (CEA), intercalated nuclei of the amygdala (IA), dorsal part of the
endopyriform nucleus (EPd), perirhinal cortex (Peri), piriform cortex
(PIR, with layer 2 indicated), amygdalar capsule (amc), optic tract
(OT). I: WGA-HRP injection (dark region; arrowed) into BLA (cf. G,H).
J: WGA-HRP injection into BLA (4). K: Bilateral areal distributions of
retrogradely labelled corticoamygdala somata (dots) in a coronal sec-
tion from 3 at approximately 3.2/3.3B. L: Line diagram showing the
boundaries between cortical areas. M: Boxed region in K showing the
laminar distribution of labelled cells projecting to BLA. Note predom-
inant bilaminar distribution in layers 2 and 5 (arrows). Scale bars
5 mm in A,D; 2 mm in B,E; 1 mm in H (applies to GI); 2.5 mm in J;
2 mm in M (applies to K,M).
RAT PFC PROJECTIONS
The at-map reconstruction indicated that neurons pro-
jecting to the septal regions injected originated principally
from IL, DP/VP areas, a posterior sector of PL cortex, and
to a lesser extent ACd and anterior PL cortices (Fig. 10C).
Additional diffuse cortical projections arose from orbital
cortex (Fig. 10C). No projections to the mediolateral
septum were observed from MOs, MOp, Ssp, or regions
of insular cortex (Fig. 10C). Projections to VTA origi-
nated over a widespread territory of PL, IL, and ACd
cortices (Fig. 10D). Additional, more diffuse projections
arose from anterior PrCm, orbital, and insular areas
(Fig. 10D).
Approximately 5% of layer 5 projection neurons in IL
were labelled after injections into the mediolateral sep-
tum (Fig. 13, Table 3A). Corresponding values of 3% and
2% were found for PL and ACd cortices, respectively
(Fig. 13, Table 3B,C). Between 4% and 6% of layer 5
output neurons in IL, PL, and ACd projected to VTA,
with the highest number occurring in PL cortex (Fig. 13,
Table 3AC).
Medullary nuclei: nucleus tractus solitarius (NTS)
and rostral and caudal ventrolateral medulla (rVLM,
cVLM; Figs. 8A,B, 10FH, 13). In contrast to all the
other pathways, injections of WGA-HRP into the con-
tralateral NTS (Fig. 8A) produced retrograde labelling of
layer 5 neurons in all of the dorsorostral areas of cortex
(Fig. 8B). The highest densities of labelled cells were,
however, found in IL and AId cortices (Fig. 8B). There was
an absence of projection neurons throughout the ventrally
situated orbital cortex (Fig. 8B). Contralateral tracer in-
jections into the rVLM and cVLM (Fig. 8A) primarily
labelled numerically small populations of layer 5 cells in
IL and AI cortices (Figs. 8B, 13).
The at-map density distributions indicated that the
cortico-NTS projection was sparsely distributed over the
majority of the rostral territory examined (Fig. 10E). Two
regions of relatively increased density occured throughout
the IL cortex and in AId and the gustatory part of the
insular cortex (Fig. 8B). Cortical projections to the rVLM
and cVLM were sparsely distributed over PL and IL cor-
tices and in AId (Fig. 10F,G).
In IL, NTS-projecting neurons constituted 7% of layer 5
projection cells; this value decreased to about 1% in PL
and ACd (Fig. 13, Table 3AC). Neurons projecting to
rVLM represented 4%, 1%, and 0.3%, respectively, of layer
5 projection neurons in IL, PL, and ACd (Fig. 13; Table
3AC). Similar percentages were found in layer 5 of the
respective areas for neurons projecting to cVLM (Fig. 13,
Table 3AC).
Mediodorsal thalamus (MD; Fig. 8DF, 11, 13). In-
jections of WGA-HRP into MD (Fig. 8D) produced the
greatest number of retrogradely labelled corticotha-
lamic cells compared with the other pathways; there
was a strong bilateral projection (Fig. 8E). Labelled
cells were found almost exclusively in layer 6 of the
following cortical areas: MOS (PrCm), ACd, PL, IL, DP,
OBvl, OBl, AIv, and AId. In more anterior sections,
labelled cells were present in the medial areas of the
orbital cortex (see Fig. 1a,b).
The at-map distribution of corticothalamic neurons in
layer 6 of rostral cortex displayed a uniformly intense
distribution in most areas, apart from a complete lack of
labelled cells in anterolateral MOs and in both MOp and
SSp areas (Fig. 11). The corticothalamic projection was
159
the most widespread and intensely labelled of the path-
ways investigated.
The quantitative results indicated that neurons in ACd,
PL, and IL projecting to MD thalamus constituted on
average about 37% of projection neurons in layer 6 of these
areas (Fig. 13, Table 3AC). Of note is that approximately
4% of layer 5 projection neurons in PL and ACd also
projected to MD thalamus (Fig. 13, Table 3AC).
Composite projection at-map and quantitative
data overview. The composite at-map density distri-
bution shown in Figure 12A,B clearly indicates the wide-
spread involvement of layer 5 neurons in the mPFC, pe-
duncular, insular, and orbital cortices projecting to the
subcortical regions investigated. In particular, the great-
est density of projection neurons for the individual path-
ways occurred across the shoulder of the mPFC in PrCm;
this density contour gradually declined through ACd and
remained high in posterior PL and IL cortices (Fig. 12B).
The potential dual projections of these distributions were
assessed quantitatively (see below).
Table 3AC provides combined data dening the total
percentage that labelled projection neurons belonging to
all the pathways studied (Total %Project) constituted of
the total number of projection neurons in specic layers of
IL, PL, and ACd cortices. These results indicate that the
combined populations of layer 5 projection cells accounted
for approximately 90%, 109%, and 86%, respectively, of
the projection neurons in layer 5 of IL, PL, and ACd
cortices. (Note: A value greater than 100% indicates the
existence of separate neuron populations projecting to
more than one subcortical target; see Discussion.) Simi-
larly, the combined labelled cell populations in IL, PL, and
ACd accounted for 56%, 60%, and 59%, respectively, of
projection neurons in layer 6. Figure 14 shows the relative
contribution of each projection pathway to the combined
population of projection neurons (given as 100%) in layers
5 and 6 of IL, dorsal and ventral PL, and ACd cortices.
Noteworthy are the projections to dorsal and ventral stri-
atum and lateral hypothalamus arising from layer 5 and
to the mediodorsal thalamus and lateral hypothalamus
originating in layer 6. Table 3AC also indicates the com-
paratively high value (1218%) that the combined projec-
tion cell populations constituted in layer 2 of the three
areas investigated.
Combined percentage that individual
retrogradely labelled neuron populations
composed of the total number of pyramidal
neurons in each area (Total %Project. per
area)
Table 3 presents data indicating that, among the path-
ways investigated here, the major projections emerging
from across all layers of IL, PL, and ACd cortices were to
LH (9%), to MD (8%), to VS (7.7% in ventral PL and
IL), to DS (5.6% in dorsal PL and ACd), and to BLA
(4% in IL and PL). Other projections came from between
0.1% and 2.1% of projection pyramidal cells in IL, PL, and
ACd.
Dual-uorescence retrograde tract-tracing
studies
Representative line drawings of the retrograde tracer
injection sites are shown in Figures 5 8. Figure 3C illus-
trates the injection site of rhodamine-labelled micro-
160 P.L.A. GABBOTT ET AL.

Figure 7
RAT PFC PROJECTIONS
spheres (RLM) into the thoracic spinal cord. Figure 3DF
shows populations of neurons retrogradely labelled by ei-
ther FB or RLM. Cells containing FB were frequently
pyramidal in shape and displayed distinct diffuse nuclear
and cytoplasmic labelling (Fig. 3D). Cells containing RLM
were also mainly pyramidal in outline, with the uores-
cent label appearing as clusters of distinct puncta in the
cytoplasmata (Fig. 3E,F). Cells containing both tracers
(FB/RLM) displayed typical FB uorescent nuclei and cy-
toplasmata lled with red-uorescing RLM particles (Fig.
3E,F). Double retrogradely labelled neurons were pyrami-
dal in shape and found principally in layer 5 of all mPFC
areas, except for numerically very small populations of
layer 6 neurons projecting to both SC and MD and layer 2
neurons projecting to both VS and BLA. There was no
signicant masking of one tracer by the other, but FB
enhanced the uorescence of RLM (Fig. 3E,F).
Table 5 details the pairwise combinations of injection
sites investigated and the relative frequency of double-
labelled neurons (cells containing FB/RLM uoresence)
across IL, PL and ACd cortices (Fry, 1992). Deep-layer
neurons in the mPFC projecting to BLA/VS, LH/VS, BLA/
DS, and LH/SC were most frequently observed in these
experiments. In each of these pathways there were neu-
rons that projected to both injection sites as well as neu-
rons that projected to each site alone (see Fig. 3Bii). (Only
single retrogradely labelled cells were found in orbital and
insular cortices).
A quantitative summary of the multiple divergent path-
ways from specic areas of mPFC is given in Table 6 and
shown graphically in Figure 15 (Fry, 1992). These data
provide initial indications of the percentage that dual-
labelled neurons constituted of the projection cell popula-
tions in layer 5 (or 6 for the MD projection). First esti-
Fig. 7. Projections to lateral hypothalamus (LH), parabrachial
nucleus (PB), periaqueductal gray (dlPAG), septum (Sep), and ventral
tegmental area (VTA). A: WGA-HRP injection into LH (71). Cytoar-
chitectural boundaries and distance relative to Bregma are indicated.
The inset shows the mediodorsal and posterior hypothalamic nuclei
included in the injection site. B: Bilateral areal distributions of la-
belled somata projecting to LH (dots) in a coronal section from 70
taken at approximately 3.2/3.3B (see Fig. 1). Note the extensive
labelling in the deep layers of orbitomedial areas and in the ventral/
dorsal AI. Numbers of retrogradely labelled cells (n) in each ipsilat-
eral and contralateral section are given. C: Boxed region in B showing
the laminar distribution of labelled cells projecting to LH. D: Line
diagram showing the boundaries between cortical areas. E: WGA-
HRP injection into PB (67). Scale same as A. F: Ipsilateral areal
distribution of retrogradely labelled somata projecting to PB (68) at
3.2/3.3B. Scale same as B. G: Boxed region in F showing the
laminar distribution of labelled cells projecting to PB. H: WGA-HRP
injection into PAG (25). I: Ipsilateral areal distribution of retrogradely
labelled somata projecting to dlPAG (24) at approximately 3.2/
3.3B.Scale same as B. J: Enlarged boxed region in I showing the
laminar distribution of labelled cells in IL cortex projecting to PB.
K: WGA-HRP injection into mediolateral septum (62). L: Ipsilateral
areal distribution of retrogradely labelled somata projecting to Sep
(66) at approximately 3.2/3.3B. Note labelled cells in orbital cortex.
Same scales as A and B, respectively. M: Boxed region in L showing
the laminar distribution of labelled cells projecting to Sep. N: WGA-
HRP injection into VTA (51). O: Ipsilateral areal distribution of ret-
rogradely labelled somata projecting to VTA (55) at 3.2/3.3B. Same
scale as B. P: Boxed region in I showing the laminar distribution of
labelled cells projecting to VTA. Note labelled cells in AI cortex. Scale
bars 5 mm in A,H,N; 2 mm in D (applies to B,D); 2 mm in I (applies
to F,I,L,O).
161
mates indicate that, of the layer 5 projection neuron
population, 9% in ventral PL cortex projected to both
LH/VS, 6% in PL projected to VS/BLA, 7% in ACd pro-
jected to LH/SC, and 2.5% in dorsal PL innervated DS/
BLA (Fig. 15).
DISCUSSION
This study presents important structural data about the
architecture of the rodent PFC: rst, a generic at-map of
PFC areas and, second, a set of representative coronal
sections and high-resolution at-map density distribu-
tions of identied neuron populations in PFC projecting to
dened subcortical centers involved in autonomic and lim-
bic functions. In addition, the mean percentages that iden-
tied projection neuron populations constituted of the
overall projection cell populations in IL, PL, and ACd
areas of the rat mPFC have been derived (Cechetto and
Saper, 1990; Loewy, 1991; Saper, 1996; Ongur and Price,
2000; Uylings et al., 2000). Quantitative information is
also presented dening the percentages that double-
labelled cells (retrogradely labelled with two uorescent
tracers from combinations of two subcortical sites; Table
5) composed of the layer 5(6) projection neuron popula-
tions in areas of mPFC.
Cytoarchitectonic at-maps of PFC
Previous cytoarchitectural studies have produced at-
maps of cortical areas in several mammalian species:
mouse (Paxinos and Franklin, 2001), rat (Ray and Price,
1992), monkey (Carmichael and Price, 1995; Gabbott and
Bacon, 1996a), and human (Vogt et al., 1995; Ongur et al.,
2003). The areal at-map presented here resembles the
rodent map of Ray and Price (1992) and the mouse map of
Paxinos and Franklin (2001). Ray and Price unfolded the
whole rat cortex with respect to the ventromedial edge of
IL cortex, the ventral edge of the corpus callosum, and the
dorsomedial edge of the presubiculum (Fig. 4. in Ray and
Price, 1992). This contrasts with the anteroposterior dor-
somedial TRP reference line dened for PFC in this study
(see Fig. 1G,H). The upper layer 5 boundary line was used
in the unfolding procedure, because the majority of pro-
jection neuron populations were located in layer 5 and
minimal volume distortions would occur in other layers
(especially layers 1 and 6) as a result of the attening
process (see Fig. 1; Gabbott et al., 1987).
Efferent projections of PFC
Injections of anterograde tracers (PHA-L and BDA) into
specic regions of PFC have been used extensively to trace
labelled efferent axons from sites in PFC to their terminal
axonal arbors in subcortical target nuclei. The most ex-
tensive anterograde study of projections from mPFC has
been by Sessack et al. (1989; see also Room et al., 1985;
Reep et al., 1987; McGeorge and Faull, 1989; Hurley et al.,
1991; Takagishi and Chiba, 1991; van Eden et al., 1992).
In a more recent anterograde tracing study, Vertes (2004)
has reassessed the projections of ventral and dorsal mPFC
and concludes that the two cortical regions innervate par-
tially overlapping sets of subcortical nuclei (Fig. 12 in
Vertes, 2004; cf. Fig. 16 in Hurley et al., 1991).
Anterograde tracing techniques label projection neu-
rons whose processes pass through the injection site,
162
Fig. 8. Projections to medullary nuclei and to mediodorsal thala-
mus (MD). A: Drawings of WGA-HRP injections into the nucleus of
the solitary tract (NTS; 42), rostral ventrolateral medulla (rVLM; 22),
and caudal ventrolateral medulla (cVLM; 39). Cytoarchitectural re-
gions and Bregma distances are indicated. B: Contralateral areal
distributions of retrogradely labelled somata projecting to NTS (44),
rVLM (20), and cVLM (40) at approximately 3.2/3.3B. The numbers
of retrogradely labelled cells (n) in each section are given. Layer 5
boundaries are indicated in NTS distribution. Projection neurons in
thereby dening the region of cortex from which projec-
tions originate. In contrast, retrograde tract tracers (HRP,
uorescent tracers) allow the laminar and areal distribu-
tion of parent neuronal somata involved in a specic pro-
jection from PFC to be identied.
Striatum. DS and VS at-map projections (Fig. 9A,B)
show a broad topographic division of the medial wall into
ventral and dorsal territories. The highest densities of
P.L.A. GABBOTT ET AL.
each pathway were located in layer 5. C: Diagram showing the bound-
aries between cortical areas. D: Drawing of a WGA-HRP injections
into MD (46). Bregma distance is given. E: Bilateral areal distribu-
tions of retrogradely labelled somata projecting to MD (48) taken at
approximately 3.2/3.3B. The numbers of retrogradely labelled cells
(n) in each section are given. F: Boxed region in E showing the
laminar distribution of labelled cells projecting to ipsilateral MD.
Scale bars 5 mm in A; 2 mm in B,E.
Fig. 9. AE: Flat-map areal density distributions of retrogradely
labelled neuronal somata in rostral cortical areas of the rat brain
following injections of WGA-HRP into DS (31), VS (56), BLA (5), LH
(71). Distributions are for ipsilateral layer 5 (DS, VS, PAG), layers 2
and 5 (BLA), and layers 2 6 (LH). Gray scale gives the number of
labelled cells per sampling bin (0 70). Inset shows at-map denition
of rostral cortical areas. Note neurons in lateral orbital cortex project-
ing to DS (double-headed arrow), complementary areas of mPFC
projecting heavily to either DS or VS (dotted lines and arrows), and
neurons in AI cortex projecting to BLA (arrow). Scale bars 1 mm.
RAT PFC PROJECTIONS 163

Figure 9
 Fig. 10. AH: Flat-map areal density distributions of retrogradely layer 5. Gray scale gives the number of labelled cells per sampling bin
labelled neuronal somata in rostral cortical areas following injections (0 70). Inset shows at-map denition of rostral cortical areas. Scale
of WGA-HRP into PB (67), DR (16), Sep (62), VTA (51), NTS (42), bars 1 mm.
rVLM (22), cVLM (39), and CS (11). Distributions are for ipsilateral
RAT PFC PROJECTIONS 165

Fig. 11. Flat-map areal density distributions of retrogradely labelled neuronal somata in rostral
cortical areas following injections of WGA-HRP into MD (46). Note: distribution is for ipsilateral layer 6.
Grayscale gives number of labelled cells per sampling bin (0 70). Inset shows at-map denition of
rostral cortical areas. Scale bars 1 mm.

neurons projecting to the caudate putamen were promi-
nently located in PrCm, ACd, and dPL cortices and ven-
trolateral orbital areas (Berendse et al., 1992; Gerfen and
Wilson, 1996), whereas the highest densities of neurons
projecting to the nucleus accumbens were situated in vPL
and IL cortices and to a lesser extent in AI cortex (Ding et
al., 2001).
Basolateral amygdala. Bilateral corticoamygdala
projections arising from layers 2 and 5 of different PFC
areas have been described (Sesack et al., 1989; Hurley et
al., 1991; McDonald et al., 1996; Shi and Cassell, 1998;
Heidbreder and Groenewegen, 2003; Vertes, 2004). Evi-
dence suggests that dPL/ACd and vPL/IL innervate sev-
eral amygdaloid nuclei (medial, basomedial, cortical, an-
terior, central and medial capsular, and dened
intercalated regions), whereas BLA is heavily innervated
by ACd, PL, and IL cortices (Fig. 6GL). In this study, the
projection to BLA originates from layers 2 and 5 across
mPFC, with additional projections arising from orbital
and AI cortices (Figs. 6L, 8C). Jasmin et al. (2004) also
report a strong projection from rostral AI to basal amyg-
daloid nuclei.
Lateral (medial/posterior) hypothalamus. Projec-
tions from PFC to the lateral hypothalamic area as well as
portions of the medial and posterior hypothalamic nuclei
are well described (Sesack et al., 1989; Thompson and
Swanson, 1998; Floyd et al., 2001; Pajolla et al., 2001;
Vertes, 2004). Of special note is that the distribution
shown in Figure 9D shows markedly increased numbers of
retrogradely labelled neurons in layers 2 6 of dorsal PL
and ACd along the rostrocaudal axis and in posterior
PL/IL cortices (Pajolla et al., 2001). The anterograde study
of Floyd et al. (2001) in the rat also reports differential
projections to hypothalamus from rostrocaudal areas of
PFC. In the latter study, rostral PL/IL and MO/VO corti-
ces principally innervated the rostrocaudal extents of the

Fig. 12. Summary at-map density distribution. A: Composite
diagram showing the combined densities of retrogradely labelled cell
distributions in layer 5 for the projection cell populations investigated
in this study (excluding layer 6 MD). B: Distribution in A subjected to
a nonlinear contrast threshold lter to identify specic density con-
tours. Readily apparent is the high total density of the labelled cell
populations near to the TRP axis (dorsal shoulder of the medial
prefrontal cortex; thick arrow) as well as a peak labelling contour that
passes through caudal PL into IL (thin arrow). C: Flat-map denition
of rostral cortical areas. Scale bars 1 mm.
Figure 13 (Continued)
RAT PFC PROJECTIONS 167

Fig. 13. Graphic representations showing the percentages that WGA-HRP retrogradely labelled
neurons constituted of the overall number of pyramidal cells in layers 1 6 of areas 25, 32, and 24b for the
subcortical projection pathways investigated in this study. Mean results SEM; n 3.

lateral and posterior hypothalamic areas, whereas projec-
tions from caudal PL/IL also terminated in dorsomedial
and perifornical regions (Thompson and Swanson, 1998).
In contrast, ACd targeted dorsal and posterior hypotha-
lamic regions, with dorsolateral orbital and AI cortices
projecting mainly to lateral hypothalamus (Heidbreder
and Groenewegen, 2003; Vertes, 2004).
Periaqueductal gray. Floyd et al. (2000) report that
ACd, PL, and IL cortices project differentially to the lon-
gitudinal cell columns in the dlPAG and vlPAG of the rat
(Hardy and Leichnetz, 1981; Bandler et al., 1991; Ongur
and Price, 2000; Jasmin et al., 2004). In addition, orbital
and AI cortices project to vlPAG (Floyd et al., 2000; Jas-
min et al., 2004). In this study, a WGA-HRP injection
centered on dlPAG (which also impinged on vlPAG) la-
belled projection neurons predominantly situated cau-
dally in mPFC and including parts of ACd, dorsal and vPL
and IL cortices (Fig. 6D), a nding similar to that of Floyd
et al. (2000).
Parabrachial nucleus. The location of neurons pro-
jecting to lateral and medial PB subnuclei in the ventro-
medial (principally IL) and orbital aspects of the PFC,
including AI cortex (Fig. 7F), agree with locations in pre-
vious reports (Saper, 1982a; Hurley et al., 1991). The
anterograde study of Vertes (2004) also decribes a projec-
tion from ventral mPFC regions to medial/lateral PB (see
Sesack et al., 1989, p 235).
Septum. Injections of retrograde tracer into medio-
lateral septum labelled cell populations located cau-
doventrally in ipsilateral mPFC, prominently in IL/
DP/VP cortices and posteromedial orbital areas. Specic
medial and lateral septal nuclei, and both the horizontal
part and the diagonal band of Broca, are known to
receive topographically organized inputs from mPFC
(Sesack et al., 1989; Hurley et al., 1991; Takagishi and
Chiba, 1991; Vertes, 2004). Gaykema et al. (1991) pro-
vide light microscopic evidence that anterogradely la-
belled axons from several PFC areas (including those
dened here) come into close contact with the somata
and processes of cholinergic septal neurons. The precise
synaptic interactions of PFC afferents with septohip-
pocampal projection neurons remain to be established
(Tomimoto et al., 1987; Brito and Brito, 1990; Gaykema
et al., 1990, 1991).
Ventral tegmental area. Carr and Sesack (2000)
have described the reciprocity and ultrastructural char-
acteristics of connections between PFC areas and neu-
rons in the VTA. The present study extends the light
microscopic observations by demonstrating that PFC3
VTA neurons are distributed over a large territory that
168
includes all of IL and PL cortices and regions of PrCm and
AI cortex.
Dorsal raphe. The prefrontal cortex projects to raphe
nuclei, in particular, the midline dorsal nucleus and to a
much lesser extent the magnus, obscurus, and pallidal
nuclei (Sesack et al., 1989; Peyron et al., 1998; Varga et
al., 2001; Vertes, 2004; Jankowski and Sesack, 2004).
Projection neurons innervating DR were found in this
study to be distributed mainly over the full rostrocaudal
extents of IL and PL cortices and anterior regions of ACd
and PrCm.
Medullary nuclei. PFC projections to autonomic and
cardiovascular centers in the NTS, rVLM, and cVLM have
been previously described (Terreberry and Neafsey, 1983,
1987; Sesack et al., 1989; Sun, 1992; Bacon and Smith,
1993; Torrealba and Muller, 1999). The present study
demonstrates sparse projections to NTS from the full ros-
trocaudal extents of most rostral cortical areas, especially
from IL cortex (Fig. 10E). By contrast, patchy projections
to rVLM and cVLM are conned mainly to caudoventral
regions of mPFC, principally caudoventral PL and IL cor-
tices, with supplementary projections from AI cortex
(Figs. 10F,G; Terreberry and Neafsey, 1987; Jasmin et al.,
2004).
Spinal cord. Cortical neurons projecting to the tho-
racic spinal cord were predominantly situated in PrCm
with decreasing frequency throughout ACd, PL, and IL
cortices. A nding that correlates with the studies of Nudo
and Masterton (1988, 1990) and Miller (1987; see Fig.
1A,B).
Mediodorsal thalamus. The topographic reciprocity
of connections between specic thalamic, intralaminar,
and midline nuclei with PFC have been described for the
rat (Conde et al., 1995; Vertes, 2004). The MD nucleus has
prominent bilateral reciprocal connections with many re-
gions of frontal cortex, especially mPFC and orbital and AI
cortices (Fig. 8E: Negyessy et al., 1998; Uylings et al.,
2003). The corticothalamic projection neurons are located
predominantly throughout layer 6 (Fig. 11; DeFelipe and
Farinas, 1992).
Composite layer 5 projection map (Fig. 12). A com-
bined at-map of all the layer 5 projection neuron popu-
lations investigated in this study, revealed the high den-
sity of retrogradely labelled cells in PrCM, ACd, dorsal PL,
and caudoventral PL and IL cortices as well as in caudal
aspects of AId and GU areas. One important feature is
that the populations of labelled projection neurons are
distributed across all areas of PFC, with much reduced
representations in primary somatosensory and motor cor-
tices (Fig. 12). This indicates a signicant and distributed
inuence of PFC operations in the function of subcortical
regions.
Quantitative data
The quantitative data presented in Table 3AC conrm
and extend the at-map density distributions. Mean areal
values across all layers of IL, PL, and ACd cortices (Table
4) indicated that the major projections were to LH (9.0%
of all projection neurons), to MD (8.0%), to VS (7.7% in
ventral PL and IL), to DS (5.6% in dorsal PL and ACd),
and to BLA (4.0% in IL and PL). Projections to other
subcortical targets accounted for between 0.1% and 2.1%
of the projection cells in IL, PL, and ACd. Of special note
is that, for mPFC as a whole, 37% of projection neurons
P.L.A. GABBOTT ET AL.
throughout layers 2 6 of the cortex were involved in the
subcortical pathways investigated; the vast majority of
these neurons were resident in layers 5 and 6 (Table
3AC).
mPFC: dorsal vs. ventral divisions. The quantita-
tive data presented here (Table 3AC) clearly emphasize
the partitioning of the mPFC into a dorsal (d) and a
ventral (v) part, as exemplied by the differential projec-
tions from IL/vPL and dPL/ACd to DS (Fig. 6E; Sesack et
al., 1989; Saper, 1996; Granon and Pouchet, 2000). The
projections from mPFC to Sep, NTS, cVLM, rVLM, PB,
and VS are greater from IL/PL than from ACd (Figs. 7,
10 12). Other projection systems to PAG, VTA, and MD
arise uniformly across IL, PL, and ACd, whereas projec-
tions to SC, DR, and LH show increasing gradients toward
ACd.
Laminar projections. In this study, although most
subcortical projection pathways originated predominantly
from layers 5 and 6 (Table 3AC, Fig. 4), several origi-
nated from more than one lamina. Notably, projections to
LH, DS, and VS came from across layers 2 6, and princi-
pally from layer 5. In contrast, the projection to BLA was
bimodal, with distinct peaks in layers 2 and 5 (Fig. 3A;
Pinto and Sesack, 2000; Ding et al., 2001), layers that
receive prominent amygdalocortical input (Bacon et al.,
1996). The bilaminar projection to BLA is particularly
strong from dIL and PL cortices (Fig. 8B,D). Cassell et al.
(1989) have demonstrated that a signicant proportion of
ipsilaterally projecting corticoamygdala neurons is
present in layers 2 and 5 of IL and PL; in addition, a
population of layer 5 neurons projects solely to the con-
tralateral BLA.
The quantitative data dene the very prominent de-
scending projections from IL, PL, and ACd cortices to
mediodorsal thalamus (Conde et al., 1995; Vertes,
2004); in PL, over 41% of layer 6 pyramidal neurons
project to MD. In addition, a small population of layer 5
neurons (4%) contributes to the corticothalamic path-
way. This information provides clear quantitative ana-
tomical evidence for the strong involvement of the
mPFC as an important cortical hub in the distribution
of cognitive and autonomic information to subcortical
limbic structures (MD, VS, BLA) and motor (DS) and
neuroendocrine effector (LH) regions (Sesack et al.,
1989; Hurley et al., 1991; Coutureau and Killross, 2003;
Vertes, 2004).
Multiple projection pathways from mPFC
The data presented in Table 3AC indicate that most
Total %Project values are less than 100%, indicating the
existence of other populations of projection neurons. How-
ever, for layer 5, in area 32 the Total %Project is approx-
imately 109%, suggesting either inherent quantitative er-
rors or that some neurons project to more than one
subcortical site (McGeorge and Faull, 1987; Perino et al.,
1987; Levesque et al., 1996; Levesque and Parent, 1998;
Negyessy et al., 1998). Insofar as reasonable estimates
were obtained for other areas/layers (with labelled neu-
rons accounting for 90% and 86% of layer 5 projection cells
in IL and ACd, respectively, and 56%, 60%, and 59% of
layer 6 projection neurons in IL, PL, and ACd), the data
appear to be consistent. Furthermore, because major
pathways were investigated in this study, it is unlikely
 Fig. 14. Pie charts for layers 5 and 6 in IL, PL, and ACd cortices
showing the mean cumulative percentages that the individual la-
belled cell populations constituted of the overall pyramidal cell pop-
ulations. Note that the projections from PL cortex have been divided
into a ventral (v) and a dorsal (d) component. This relates to the
widely differing number of projection neurons projecting to DS in
ventral vs. dorsal PL cortex. Note also that the individual percentages
for ventral and dorsal PL cortex exceed 100%, indicating that some
layer 5 neurons in both vPL and dPL project to more than one
subcortical target site. The remaining distributions represent 100% of
the total number of pyramidal cells in each layer.
170
Fig. 15. Graphs showing the mean percentages that double-
labelled neurons constituted of the total projection neuron population
in layers 5 and 6 of PL, IL, and ACd cortices that innervated identied
combinations of common subcortical targets. Note that neurons with
that other projection pathways would alter the data sub-
stantially.
Although large numbers of potential dual projection
combinations were examined (44; Table 5), only 13 gave
rise to dual retrogradely labelled neurons in mPFC (Table
6, Fig. 16). The data indicated that, among the layer 5
projection neuron populations, 9% in vPL cortex projected
to LH/VS, 6% in PL projected to VS/BLA, 7% in ACd
projected to LH/SC, and 2.5% in dPL innervated DS/BLA
(Fig. 15, Table 6). Thus there are limited numbers of
neurons in mPFC regions with axons that innervate com-
binations of the subcortical structures investigated in this
study. Similar ndings have been previous reported for
descending projections from the cortex (Akintunde and
Buxton, 1992; Levesque et al., 1996; Levesque and Parent,
1998; Pinto and Sesack, 2000).
In a technically challenging quadruple retrograde uo-
rescent tracer study in the rat, Akintunde and Buxton
(1992) found a very low mean percentage (2%) of dually
labelled neurons projecting from the cortex to combina-
tions of red nucleus, pons, striatum, or spinal cord. La-
belled corticorubral, corticopontine, and cortico(dorso)s-
triatal neurons were distributed throughout layers 3, 5,
and 6 of agranular PrCm and MOs cortices (Gerfen and
Wilson, 1996). Previous studies have also reported that
P.L.A. GABBOTT ET AL.
axonal projections both to the spinal cord (SC) and to the mediodorsal
nucleus of the thalamus (MD) were located in upper layer 6 of ACd
(asterisk).
individual descending corticoefferent projections rarely
provide collateral input to three or more subcortical tar-
gets (Ferino et al., 1987; Levesque and Parent, 1998); this
is also true for the corticoaccumbens pathway (Pinto and
Sesack, 2000; Rosell and Gimenez-Amaya, 2001; cf. Thi-
erry et al., 1983; Perino et al., 1987). Pinto and Sesack
(2000) also report that, of mPFC neurons in the rat pro-
jecting to the nucleus accumbens (VS), 13% sent a
branched projection to the contralateral prefrontal cortex,
7% sent a collateral projection to the BLA, and 3% sent a
collateral to the VTA (see Ding et al., 2001). Taking into
account the relative proportions and identities of the neu-
ron populations against which such percentages were de-
rived, the data presented here indicating that 5.9% of all
layer 5 projection neurons in PL cortex sent collaterals to
both BLA and VS (Table 6) are in general agreement with
the result of Pinto and Sesack (2000; Table 1, p 638).
It was possible to establish that the pathways from PL
to NTS/dlPAG and NTS/SC (Fig. 15) are likely to repre-
sent two separate dual pathways (and not a single path-
way with multiple branches), in that no double-labelled
neurons were found in PL after injections into dlPAG and
SC (Table 6). An additional point of interest, given the role
of ascending serotonergic and dopaminergic innervation of
mPFC (Dalley et al., 2004), is that both IL and PL cortices
RAT PFC PROJECTIONS
Fig. 16. A: Composite diagram showing individual populations of
labelled neuronal somata in the DP, IL, vPL, dPL ACd, PrCm, and
medial MOs cortices of the rat mPFC projecting to VS (blue), BLA
(green), dorsal striatum DS (red), and MD thalamus (purple) mapped
onto a single representative coronal section (at 3.2 mmB). (Each
projection population distribution was mapped in 80-m-thick sec-
tions.) Note the relative laminar distributions and the intermixing of
the projection cell populations (particularly DS and VS) between the
various regions of the mPFC, especially vPL and dPL. Note also that
the specic projection neuron populations displayed represent a frac-
tion of the projection neurons present in the mPFC. B: Enlargement
of the vPL region shown in A. In general, high numbers of VS com-
pared with DS projecting neurons are present in vPL. The situation is
contain populations of layer 5 projection neurons (1% and
2%, respectively) that innervate both DR and VTA nuclei
(Carr and Sesack, 2000; Varga et al., 2001; Jankowski and
Sesack, 2004).
Functional considerations
The present paper indicates a topographic segregation
of the rodent PFC (medial, orbital, and insular areas) into
rostrocaudal and mediodorsal territories associated with
descending projection pathways to common subcortical
nuclei (Sesack et al., 1989; Hurley et al., 1991). The most
pronounced territorial division occurs in mPFC, where a
clear partition into a dorsal group (mPFCd) comprising
PrCm, ACd, and dPL cortices and a ventral group of vPL,
IL, DP/VP, and OBm cortices is evident (see Fig. 16).
Neafsey and colleagues have presented a similar division
of rodent mPFC (see Fig. 6.2 in Neafsey et al., 1993; see
also Morgan and LeDoux, 1995; Heidbreder and Groe-
171
reversed in dPL, where there is a greater absolute number of DS-
compared with VS-projecting cells. Note that neurons projecting to
BLA reside in upper layer 5 and the massive layer 6 projection to MD
thalamus. Other projection systems could be similarly mapped.
C: Model diagram showing two distributions of projection neurons
across cortical layers in two related cortical regions. Neurons project-
ing to four subcortical targets (VS, BLA, DS, and MD) have been
identied for regions a and b. Quantitative analyses dene the
number/proportion of projection cells per pathway for each layer.
Characteristic laminar distribution proles are subsequently con-
structed. Such proles provide primary indices of differences (aster-
isks) in the four projection systems that govern how the two regions
affect the activity of the target structures. Scale bars 1 mm.
newegen, 2003; Morgan et al., 2003; Christakou et al.,
2004; Vertes, 2004; Dalley et al., 2004).
Physiological and anatomical evidence indicates that
the rat PFC may be compartmentalized into specic func-
tional domains (Seamans et al., 1995; Ragozzino et al.,
1998, 1999a,b; Delatour and Gisquet-Verrier, 2000; Groe-
newegen and Uylings, 2000; Coutureau and Killross,
2003; Heidbreder and Groenewegen, 2003; Kesner and
Ragozzino, 2003; Morgan et al., 2003; Vertes, 2004; Dalley
et al., 2004). The insular cortex is a viscerosensory region
processing afferent cardiovascular, cardiopulmonary, gas-
trointestinal, odour, gustatory, and related sensory and
pain information (Saper, 1982b; Ruggiero et al., 1987;
Yasui et al., 1991; Jasmin et al., 2004). This information is
subsequently relayed to a variety of subcortical autonomic
regions (VS, BLA, MD, LH, PB, dlPAG, VTA, DR, and
medullary regions) and to orbital and mPFC regions (Gab-
bott et al., 2003; Jasmin et al., 2004). Ventral and lateral
172
Fig. 17. Schematic illustration of projection neurons and struc-
tural minicolumns in PL cortex (area 32) of the rat. A: Dendritic
bundles have been described from rat PL cortex (Gabbott and Bacon,
1996c). Mean center-to-center distance between bundles is 45 m.
The cortical territory associated with an individual bundle extends
perpendicular to the pia through all cortical layers (depth 1,250
m). Dendritic bundles are derived from the apical dendrites of layer
6, 5, 3, and 2 pyramidal neurons and processes and dendrites of other
neurons (Gabbott and Bacon, 1996c; Gabbott, 2003). B: In cross-
section, minicolumns can be ideally packed into a clustered hexago-
nal honeycomb lattice parallel with the pia (Gabbott and Bacon,
1996c; Gabbott, 2003). In rat PL cortex, the mean surface area of each
minicolumn (SA) is 1,360 m2, and there are 735 minicolumns per 1
mm2 of pial surface (Gabbott and Bacon, 1996c). C: By using the data
provided in Table 1, the number of pyramidal neurons (non-GABA
containing cells) in each layer under 1 mm2 of pia (Gabbott et al.,
1997) can be calculated (see Table 6). Assuming these cells to be
projection pyramidal neurons, then the absolute numbers of projec-
tion cells in each layer of a minicolumn (volume 1,360 m2 1,250
areas of orbital cortex are implicated in lower order dis-
criminations, reversal learning, and choice-delayed rein-
forcement (Schoenbaum et al., 2003; Uylings et al., 2003;
Dalley et al., 2004).
The mPFCd is centrally involved in cognitive functions
such as decision making, memory for motor responses,
response selection and implementation, spatiotemporal
action sequencing, and spatial navigation learning and in
working memory (Stuesse and Newman, 1990; Harrison
and Mair, 1996; Kesner and Ragozzino, 2003; Dalley et al.,
2004). In contrast, the mPFCv is involved in the coordi-
nation of autonomic activities (such as cardiovascular and
pulmonary functions, hemodynamic shifts, and gastroin-
testinal and electrodermal actvitities) and in the media-
P.L.A. GABBOTT ET AL.
m) can be calculated. D: Schematic diagram using data provided in
Table 2 (also Fig. 3A,B) and Table 6 to estimate, to a rst approxi-
mation, number of pyramidal cells in a particular layer of a minicol-
umn projecting to a known target. Two markedly different example
pathways have been chosen: PL3 MD, where projection neurons con-
stitute 41.33% of layer 6 PL pyramids; and PL3 NTS, where projec-
tion neurons constitute 1.08% of layer 5 PL pyramids. Calculations
indicate that about 9.6 neurons in layer 6 of each minicolumn project
to MD and that 0.2 neurons in layer 5 of each minicolumn project to
NTS. The latter result suggests that, given uniform distribution, a
single PL3 NTS projection neuron would occur every ve minicol-
umns. This neuron could receive afferent pyramidal and local cir-
cuitry inhibitory input from neurons in the parent and surrounding
four minicolumns (intracortical connections highly schematized; as-
terisk). By comparison, in such an arrangement, 48 pyramidal neu-
rons distributed in layer 6 of the ve minicolumns would project to
MD; there may be distinct microcircuits associated with the MD
projection cells.
tion of these responses during conditional learning (Burns
and Wyss, 1985; Hurley-Guis and Neafsey, 1986; Hardy
and Holmes, 1988; Sun, 1992; Frysztak and Neafsey,
1994; Saper, 1996; Fisk and Wyss, 1997, 2000; Owens et
al., 1999; Owens and Verberne, 2001; Morgan et al., 2003).
The mPFCv is also involved in attentional supervisory
functions, stimulus saliency, action outcome rules (task
contingencies), and set-shifting underlying the dynamics
of behavioral exibility (Ragozzino et al., 1998, 1999a,b;
Groenewegen and Uylings, 2000; Granon and Pouchet,
2000; Delatour and Gisquet-Verrier, 2000; Morgan et al.,
2003; Dalley et al., 2004). The roles of both mPFCv (IL in
particular) and LO/AI cortices may center on providing
details of internal autonomic context (emotional saliency)
RAT PFC PROJECTIONS 173

about object/place relationships, with temporally related Projection systems and the superposition of func-
information being processed simultaneously by mPFCd tional domains in rat mPFC. Specic subregions of
(Kesner and Ragozzino, 2003); the idea of neural activity mPFC are involved in different aspects of cognitive and
related to autonomic context is analogous to a somatic sensory information processing required to construct real-
marker (Damasio et al., 1991). time strategies for exible goal-directed behavior
Because of its connections with central autonomic struc- (Goldman-Rakic, 1994, 1995; Ragozzino et al., 1999b;
tures in the medulla and spinal cord, mPFCv in rodents is Floresco et al., 1999; Romanides et al., 1999). The rat
considered to represent a visceromotor region at the cor- mPFC may therefore be organized dorsoventrally as a
tical head of the emotional motor system (Holstege et al., graded set of partially overlapping functional domains,
1996). An essential anterior component of this system is each domain with a characteristic complement of pyra-
LH, which receives a massive input from mPFC areas (see midal neurons that differentially project, via parallel
Figs. 7B and 9D; Pajolla et al., 2001) and projects directly distributive pathways, to specic sets of subcortical tar-
to autonomic medullary regions. Recently, Thompson and get nuclei (see Fig. 16A). The partial superposition of
Swanson (2003; Fig. 16) have described a hypothalamic the complementary projection systems (for example, su-
visceromotor pattern generator network in which excita- perposition of VS, DS, BLA, and MD projection neuron
tory inputs from mPFC and orbital cortices (Fig. 9D) are distributions, as shown in Fig. 16AC) could underlie a
strategically involved. Hardy (1994) provides anatomical morphological partition of rat mPFC responsible for the
evidence that the anterior insular PFC indirectly inu- functional operations described above. Indeed, Chu-
ences central autonomic functions via a hypothalamomed- dasama and Robbins (2003) have recently reported dis-
ullary relay (Saper et al., 1976; Hardy and Holmes, 1988; sociable functional contributions of the orbital and IL
Hardy and Mack, 1990; Hardy, 1994). Indeed, the rat cortices to Pavlovian autoshaping and discrimination
mPFC is centrally involved in cognitive and autonomic reversal learning, which provides further evidence for
processes underlying psychological stress (McDougall et the functional and areal heterogeneity of the frontal
al., 2004). cortex in rats (see also Dalley et al., 2004). The overall
functional architecture of the rodent mPFC would in-
clude not only targeted corticoafferent and intrinsic con-
nections (Audinat et al., 1988; Conde et al., 1995; Gab-
TABLE 4. Mean Percentage Across All Layers That the Total Number of bott et al., 1997, 2003), but also, as shown
Neurons Projecting to Specic Subcortical Targets Constituted of the Total hypothetically in Figure 16C, subregional laminar den-
Number of Pyramidal Cells under Unit Surface Area of Cortex (Mean sity variations in specic overlapping projection path-
Values SEM; n 3)1
ways that are possibly related to the functional inter-
PL cortex connectivity of structural minicolumns (see below).
(area 32) (%) Structural minicolumns. Dendritic bundles repre-
IL cortex ACd cortex
Region (area 25) (%) Ventral Dorsal (area 24b) (%) sent the central components of the vertically organized
cortical minicolumns and are derived from the ascending
BLA 4.0 1.0 4.0 0.8 1.7 0.6
DS 0.7 0.3 2.4 1.2 5.4 3.5 5.3 2.4 apical dendrites of layer 6, 5, 3, and 2 pyramidal neurons
VS 6.8 1.5 8.5 3.9 1.5 0.6 and the processes of other neurons (Fig. 17; Gabbott and
PB 2.1 0.8 0.6 0.3 0.2 0.1
LH 8.0 1.9 8.1 2.2 11.0 2.7 Bacon, 1996c; Gabbott, 2003). Bundles of apical dendritic
NTS 1.4 0.5 0.2 0.1 0.2 0.1 processes have been described in rat PL cortex, the mean
0.6 0.3 0.6 0.4 0.5 0.2
PAG
Sep 1.1 0.4 0.6 0.3 0.3 0.1
center-to-center distance between bundles being 45 m
VTA 0.8 0.2 1.1 0.3 0.9 0.3 (Gabbott and Busby, 1996c). The cortical territory associ-
DR 0.2 0.1 0.9 0.2 0.9 0.2 ated with an individual bundle extends perpendicularly to
MD 7.0 1.6 9.0 2.8 8.0 1.9
rVLM 0.7 0.4 0.2 0.1 0.1 0.05 the pia through all layers of the cortex (depth 1,250 m).
cVLM 0.4 0.1 0.3 0.2 0.1 0.05 In cross-section, the packing of minicolumns can be mod-
SC 0.2 0.1 1.9 0.7 2.9 1.0
Total (%) 34.0 38.4 41.4 33.6 elled ideally as a clustered hexagonal honeycomb lattice
1
parallel to the pial surface (Fig. 17). In rat PL cortex, the
The projection from PL cortex to DS has been divided into ventral and dorsal compo-
nents. The overall percentage that projection neurons involved in the pathways studied
mean surface area of each minicolumn (SA) is 1,360 m2,
composed of the total number of pyramidal neurons in a specic area is also given. and there are approximately 735 minicolumns per square

TABLE 5. Combinations of Two Subcortical Regions That Were Injected with Separate Fluorescent Retrograde Tract Tracers in Individual Animals1
DS
VS VS
BLA BLA
LH LH
Sep Sep
PAG 0 0 0 PAG
DR 0 0 DR
VTA 0 0 0 VTA
PB 0 0 PB
NTS 0 0 0 0 0 0 0 NTS
rVLM 0 0 rVLM
cVLM 0 0 cVLM
SC 0 0 0 0 SC
MD 0 0 0 0 0 0
1
For each animal, within each combination, tracers were alternated between injection sites. Dual retrograde labelling of single neurons in the mPFC (ACd, PL, IL) is indicated
(, , , low to high frequency of double-labelled neurons in layer 5 mPFC; 0, absence of dual labelling). For region abbreviations, see text. Very low dual SC/MD labelling
() occurred in upper layer 6. (Pathways not examined are indicated by dashes). Total number of combinations studied 44. Number of dual projection pathways from mPFC
13. See Table 6 and Fig. 16 for quantitative data on dual-projection neuron populations in specic areas of mPFC.
174 P.L.A. GABBOTT ET AL.

millimeter of pial surface (Fig. 17, Table 7A; see also targets. These calculations indicate that about 9.6 pyra-
Gabbott and Busby, 1996c). The data presented in Table midal projection neurons in layer 6 of each minicolumn in
7B and illustrated in Figure 17 give the total numbers of PL cortex project to MD and that 0.2 neurons in layer 5 of
corticoefferent neurons per PL minicolumn and the abso- each minicolumn project to NTS (Table 6B). Given uni-
lute number of cells projecting to identied subcortical form distribution, a single PL3 NTS projection neuron
would occur every ve minicolumns (Fig. 17). This neuron
could be associated with specic corticoafferent (Conde et
TABLE 6. Multiple Divergent Subcortical Pathways: Retrograde Tract al., 1995; Gabbott and Bacon, 1997), interareal (Audinat
Tracing with Two Separate Fluorescent Tracers1 et al., 1988; Gabbott et al., 2003), and intermodular (So-
P mPFC Q
mogyi et al., 1998) synaptic networks. In such an arrange-
Regions (%) area (%) ment, 48 pyramidal neurons distributed in layer 6 of the
LH VS 20 PL 8.9
same ve PL minicolumns would project to MD, where
LH SC 15 PL 4.5 they might also be embedded in distinct microcircuits
20 ACd 7.2
DS BLA 10 dPL 2.5
associated with the corticothalamic projection. There are
BLA PAG 20 IL 1.8 thus more output neurons per PL minicolumn in layers 5
15 PL 1.5
VS BLA 20 PL 5.9
and 6 given to limbic projections (LH, MD, VS, BLA, DS)
Sep BLA 5 IL 0.6 than to the more distant central autonomic centers in the
10 PL 1.0
NTS SC 10 IL 0.7
midbrain, medulla, and spinal cord (Table 6B). This dif-
20 PL 1.6 ference may relate directly to the content and spatiotem-
cVLM rVLM 10 IL 0.5
PAG NTS 20 PL 0.7
poral processing of information entering the two descend-
5 ACd 0.2 ing functional streams (Room et al., 1985; Groenewegen
PB NTS 5 IL 0.7
DR VTA 5 IL 0.2
and Berendse, 1994; Seamans et al., 1995; Floresco et al.,
20 PL 1.7 1999; Delatour and Gisquet-Verrier, 2000; Heidebreder
MD SC 1 ACd2 0.4
DS SC 5 dPL 1.3
and Groenewegen, 2003).
5 ACd 1.0 In conclusion, this paper has produced a comprehensive
1
P, quantitative data dening the percentage that double retrogradely labelled neurons
set of at-map density distributions and quantitative cy-
(AB, Fig. 3A), constituted of the total number of neurons retrogradely labelled with toarchitectural information of identied neuron popula-
uorescent tracers in layer 5 of IL, PL, and ACd areas of rat mPFC (see also Fig. 3A,
B, DF). Data were divided into 5% bins from 0 to 20%); Q, calculated percentage that
tions in specic areas and layers of rat PFC projecting (via
the dual retrogradely labelled populations of layer 5 (6) neurons constituted of the total a cascade of parallel pathways) to a major set of subcorti-
number of pyramidal projection neurons in the corresponding area of mPFC. See Figure
3A. From the data obtained in the pathway tracing studies using two uorescent
cal target nuclei involved in cognitive, limbic, motor, and
tracers injected separately into regions m and n, let the percentage that dual retro- autonomic functions. Such a biologically real quantitative
gradely labelled cells (mn) composed of the total labelled neuron populations (X Y
XY) equal P%. If the percentage of layer 5 neurons projecting separately to region A
framework, combined with details of the specic synaptic
equals x% and separately to region B equals y%, then, from the dual tracing studies, the circuits in which the various projections neurons are em-
percentage that dual retrogradely labelled cells (ab) compose of the overall layer 5 cell
population (Q%) can be calculated as [{(x y)/[1 (P/100)]} (P/100)] (Howard and
bedded (White, 1989; Somogyi et al., 1998), could provide
Reed, 1998). The corresponding values for X and Y can be found in Table 3AC. 2Very the basis for understanding the interactions between in-
low number of dual-labelled MD neurons in layer 6 only.
tegrated projection systems at the nal stage of cortical
processing. Insights could also be gained into the spatio-
TABLE 7A. Pyramidal Cell Composition of Structural Minicolumns in Rat temporal dynamics and resolution of information transfer
PL Cortex (Area 32) and Number of Neurons Projecting to NTS and MD: from the rat PFC to subcortical target nuclei involved in
Number of Pyramidal Cells in Each Layer of PL Cortex under 1 mm2 of specic goal-directed behaviors (Fuster, 1989; Damasio et
Pial Surface and in a Minicolumn1
al., 1991; Cummings, 1995; Dalley et al., 2004).
Abs.n.pyramids in Abs.n.pyramids
each layer under per hexagonal
Layer 1 mm2 of pial surface minicolumn*
1 1,168 1.6
2 5,701 7.8 ACKNOWLEDGMENTS
3 11,448 15.6
5 11,302 15.4 The authors thank Dr. Daniel Henderson, Statistics
6a 6b 16,999 23.1
Sum 46,618 63.5
Department, The Open University, Milton Keynes, for
1
help with the statistical analyses and quantitative proce-
Hexagonal minicolumn parameters: center to center spacing 45 m; surface area
1,360 m2; number per pial mm2 735; and extend from pia to white matter (approx.
dures. The assistance of Tina Wardhaugh, The Open Uni-
total length 1,250 m; see also Fig. 8). Data taken from Gabbott and Bacon (1996). versity, is greatly appreciated.

TABLE 7B. Absolute Numbers of Identied Deep Layer Subcortical Projection Neurons per Minicolumn1 in Rat PL Cortex2
DS

Layer BLA vPL dPL VS PB LH dlPAG Sep VTA DR NTS rVLM cVLM SC MD
5 1.3 1.1 2.9 4.2 0.4 4.0 0.4 0.4 0.9 0.7 0.22 0.2 0.2 1.4 0.6
6 1.4 2.5 9.64
1
Hexagonal minicolumn parameters: center to center spacing 45 m; surface area 1,360 m2; number per pial mm2 735; and extend from pia to white matter (approx. total
length 1,250 m; see also Fig. 8). Data taken from Gabbott and Bacon (1996).
2
DS projection to DS divided into vPL and dPL regions. Note that there are approximately 64 pyramidal neurons in a minicolumn in rat PL cortex.
3
Minimum value (e.g., 0.2 PL3 NTS projection neurons per PL minicolumn).
4
Maximum value [e.g., 9.6 PL3 MD (corticothalamic) projection neurons per PL minicolumn].
RAT PFC PROJECTIONS
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