THE JOURNAL O F COMPARATIVE NEUROLOGY 364567-608 ( 1996)

Local Circuit Neurons in the Medial

Prefrontal Cortex (Areas 24a,b,c, 25 and 32)
in the Monkey: I. Cell Morphology
and Morphometrics
PAUL L.A. GABBOTT AND SARAH .J. HACON
University Department of Pharmacology, Oxford, England

ABSTRACT
This paper provides a comprehensive morphological description of local circuit neurons in
the medial prefrontal cortex (mPFC: areas 24a, 24b, 24c, 25 and 32) of the monkey.
Cortical interneurons were identified immunocytochemically by the expression of the
calcium binding proteins calretinin (CR), parvalbumin IPV) and calbindin D-28k (CB).
Interneurons were also identified using GABA immunocytochemistry. The areal and laminar
distributions of CR, PV, and CB cells were consistent across mPFC; their morphological
characteristics identified them as local circuit neurons. Throughout layers 2-6: CR immunore-
activity labelled double bouquet and bipolar neurons, PV was localised in large and small basket
neurons and in chandelier (axoaxonic)cells, while CB immunoreactivity was present in double
bouquet, Martinotti, and neurogliaform neurons. In addition, some cells in layer 1 (including
Cajal-Retzius neurons) were CR immunoreactive. Calbindin immunoreactivity also labelled a
population of large nonpyramidal neurons deep in the cortex. Other types of CR, PV and CB
cells were also immunolabelled. A small population of layer 3 pyramidal cells was weakly CB
immunoreactive. Peak cell densities occurred in layer 2iupper layer 3 for CR+ neurons and in
upper to midlayer 3 for CB+ cells. PV+ neuron density peaked in midcortex. These
observations support and extend a similar study of monkey prefrontal cortex (Conde et al.
119941J. Comp. Neurol. 341:95-116). The morphologies and combined cortical depth distribu-
tions of CR+, PV+, and CB+ neurons were similar to GABA-immunolabelled cells.
Local circuit neurons in mPFC displaying NADPH diaphorase activity composed less than
0.25% of the total neuron population, and were distributed in two horizontal strata, in mid- to
lower layer 3 and in lower layer 5iupper layer 6. CR, PV and CB immunoreactivity was
colocalised in NADPH diaphorase-reactive neurons.
The interrelationships between CR+, PV+ and CB+ neurons were investigated using dual
immunocytochemistry. CR+ puncta were found to be closely associated with the cell bodies and
proximal processes of PV+ neurons, whereas CR+ puncta were located more distally over
processes from CB+ cells. Additionally, PV+ puncta were found closely apposed to PV+ somata
and processes and C R + puncta abutted against CR+ cell bodies.
The companion paper (Gabbott and Bacon [ 19961J. Comp. Neurol.) presents quantitative
data regarding the areal and laminar distributions of the identified cell classes in mPFC. Such
data provide a realistic structural framework with which to investigate neuronal operations in
monkey mPFC. ' IXK Wile!.-Liss. Ine.

Indexing terms: limbic system, cingulate cortex, GABA, calcium binding proteins, NADPH diaphorase

The prefrontal cortex of primates is involved with com-
plex behaviours that require the integration of higher brain
functions. These associative functions include motivation,
attention, cognition, learning and memory and the spatio-
temporal organisation of goal-directed behaviours (Fuster,
1989; Goldman-Rakic, 1990).
Parts of the prefrontal cortex, located Over the
orbital and insular surfaces of the brain are not only
involved in these higher brain functions (Vogt and Gabriel,
19931, but also act together with the limbic system to
provide part of the complex physiologcal background to a
wide range of emotional reactions and expressions (Smith
Accepted J u n e 19, 1995.
Address reprint rcqucsts t.o Paul L.A. Gahhott, University Departmcnt of
Pharmacology, Mansfield Road, Oxford. OX1 3QT. United Kingdom.

( 1996 WILEY-LISS, INC.

568
and DeVito, 1984). The medial prefrontal cortex (mPFC)
subserves a variety of autonomic (somatovisceral) activities
that underlie emotional behaviour, for example, respiration
and cardiovascular functions (breathing, heart rate and
blood pressure), gastrointestinal mobility and secretion,
and pupillary dilation (Neafsey et al., 1993; Buchanan and
Powell, 1993; Powell et al. 1994).
On the basis of cytoarchitecture, mPFC is composed of
Brodmann areas 24a, 24b, and 24c (anterior cingulate cortex
or anterior limbic cortex), area 32 (prelimbic cortex) and
area 25 (infralimbic cortex). This structural definition of
mPFC is concordant across a number of mammalian spe-
cies, including humans (rat, Van Eden et al., 1991; rabbit,
Buchanan et al., 1994; cat, Room et al., 1985; Musil and
Olson, 1993; monkey, Carmichael and Price, 1994; see also
Figs. 1A,B of this study; and humans, Uylings and Van
Eden, 1990; Vogt et al. 1995).
The functional activity of each cortical area is governed
by the dynamic interplay between cortical afferents, intrin-
sic circuitry, and their relationship with the corticoefferent
pathways. Although the afferent and efferent connectivities
of the cortical areas composing the mPFC are beginning to
be unravelled in detail (Van Hoesen et al., 1993; Carmichael
and Price, 19941, comparatively little is known about the
internal architecture and synaptic connectivities of these
areas in the monkey. However, detailed wiring diagrams of
their internal construction would identify the routes via
which afferent information (derived from other cortical
areas and subcortical sources) is integrated and processed
within defined neural networks and subsequently relayed
to other cortical fields and brain regions.
Neural networks in the cortex are composed of intercon-
nected populations of pyramidal cells and local circuit or
nonpyramidal neurons (Douglas and Martin, 1990).Pyrami-
dal neurons are excitatory in function, and, in addition to
having local intracortical axon arbors, they project to other
cortical areas and/or subcortically. Golgi and immunocyto-
chemical studies show that local circuit neurons in the
monkey PFC are heterogeneous in axon and dendritic
morphologies and laminar location (Lewis and Lund, 1990;
Lund and Lewis, 1993). Moreover, the vast majority of
cortical interneurons use GABA to mediate their inhibitory
intracortical operations (see Mize et al., 1992; Jones et al.,
1994). Specific types of inhibitory cortical local circuit
neurons (e.g., axoaxonic, basket, and double bouquet neu-
rons; see review by Jones et al., 1994) have defined patterns
of synaptic connectivity with specific parts of pyramidal
cells (e.g., axon initial segments, somata, and dendrites).
Abbreviations
ABP avidin-biotin-peroxidase
CB calbindin D-28k
CBPs calcium binding proteins
CR calretinin
DAB 3,3’-diaminobenzidine
FITC Auorcscein isothiocyanate
GABA gamma aminobutyric acid
GAD glutamic acid decarboxylase
mPFC medial prefrontal cortex
PNADPH reduced form of nictotinamide adenine dinucleotide phos-
phate
NBT nitroblue tetrazolium
NO nitric oxide
NOS
PB
nitric oxide synthase
phosphate buffer
PBS phosphate-buffered saline
PV parvalbumin
RT room temperature
TRIS Tris (hydroxymethy1)-methylamine
P.L.A. GABBOTT AND SJ. BACON
Furthermore, extensive connections also exist between
cortical interneurons (Jones et al., 1994). In concert, local
circuit neurons play crucial and specific functional roles in
shaping the physiological response properties of pyramidal
cells, thereby strongly influencing neural activity in the
cortex (Douglas and Martin, 1992; Wilson et al., 1994).
Recent studies have shown that the separate types of
GABAergic local circuit neuron in monkey cerebral cortex
can be readily identified using immunocytochemistry for
the calcium binding proteins, calretinin, parvalbumin and
calbindin D-28k (DeFelipe et al., 1989a,b; Demeulemeester
et al., 1989, 1991; Blumcke et al., 1990; Van Brederode et
al., 1990; Lewis and Lund, 1990; Huntley and Jones, 1990;
DeFelipe and Jones, 1991, 1992; Morino-Wannier et al.,
1992; Hof et al., 1993; Conde et al., 1994). The current
investigation was undertaken with the specific aim of
expanding on the studies of Hof et al. (1993) and Conde et
al. (1994) by investigating both the morphology and quanti-
tative distribution of the structural components involved in
the local neuronal circuitry of the monkey mPFC.
The present study was performed at two related levels.
Firstly, a detailed and comprehensive morphological ac-
count of the areal and laminar distribution of local circuit
neurons in mPFC that show immunoreactivities for the
calcium binding proteins, calretinin, parvalbumin and cal-
bindin D-28k. In addition, the structural relationships
between neurons containing different calcium binding pro-
teins was investigated using double immunocytochemistry.
A further aim was a thorough documentation of interneu-
rons in monkey mPFC displaying NADPH diaphorase
activity (Sandell, 1986). Since diaphorase activity is coloca-
lised with nitric oxide synthase (the biosynthetic enzyme
for nitric oxide; Hope et al., 1991),these interneurons have
the potential to affect cortical operations, not only through
GABA-mediated mechanisms, but also via the actions of the
neuromodulator nitric oxide (NO; see reviews by Vincent,
1994, 1995). Indeed, NADPH diaphorase activity has been
colocalised with specific calcium-binding proteins within
interneurons (Vincent, 1995).
The second part of the study was a quantitative analysis
of the areal and laminar distributions of the immunocyto-
chemically and histochemically identified classes of local
circuit neuron in the separate areas of the monkey mPFC.
These data are presented in the companion paper (Gabbott
and Bacon, 1996).
The overall rationale behind this light microscopic inves-
tigation is that detailed qualitative and quantitative infor-
mation concerning the morphology and distribution of local
circuit neurons in the adult monkey mPFC is of prime
importance, firstly, towards understanding the involvement
of interneurons in cortical circuitry during normal and
abnormal development, and secondly, by providing a frame-
work with which to interpret alterations in the functional
architecture of local circuit neurons in the human mPFC
that are considered to underlie psychiatric disorders such
as schizophrenia, Pick’s disease, Wernicke-Korsakoff s syn-
drome, and Alzheimer’s disease (Benes et al., 1991; and
reviews by Benes, 1993a,b, 1995; Braak and Braak, 1993).
MATERIALS AND METHODS
Animals and tissue preparation
Material used in this study was obtained from five adult
female cynomologus monkeys (Macaca fasicularis; 6.0-
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 569

10.0 kg). The animals had been previously used in experi- immunocytochemical studies has been given previously
ments studying the primate visual system. At the end of (Table 1).All sera were made up in TRIS (pH 7.4) buffer
these experiments the animals had been given a lethal containing 0.01% sodium azide.
overdose (180mg) of sodium pentobarbitone (Baker and Immuolabelling was visualised using one of the following
May. UK). When sufficiently anaesthetised the animals methods:
were perfused transcardially with either 4% paraformalde- (i) The standard immunoperoxidase procedures using
hyde (animal M5-AC 302), or a mixture of aldehyde fixa- appropriate species matched Vectastain ABC kits (Vector
tives containing 3% paraformaldehyde with either 0.3% Laboratories) and developed with 3,3’-diaminobenzidine
(animal M2-KACM 4/94), 0.5% (animals M1-KACM 1/94 (DAB) as the chromagen (incubating sections in TRIS (pH
and M4-KACM) or 0.74 (animal M3-KACM 13/93) glutar- 7.4) with 0.05% DAB and 0.01% HzOzfor 3-10 minutes at
aldehyde in 0.1 M phosphate (PB) buffer (pH 7.4). The room temperature).
brains had been removed, postfixed in fixative and stored in (ii) Immunolabelling procedures using appropriate spe-
PB buffer (pH 7.4) at 4°C. cies-specific secondary antibodies tagged with alkaline phos-
For the studies presented here, blocks of tissue containing phatase. Prior to labelling with secondary antibody, tissue
the anterior medial wall of the cortex that included cortical sections were incubated in TRIS (pH 7.4) buffer containing
areas 24a, 24b, 24c, 25 and 32 were carefully excised from 0.015% levamisol for 3 x 10 minutes (to abolish endog-
the brains (Fig. 1A,B). In three of the animals tissue was enous phosphatase activity). Fast red TRinapthol AS-MX
taken from both hemispheres. These tissue blocks were (Kit F4523, Sigma Chemicals Ltd.) was then used as the
sectioned serially using a Vibratome in either the coronal chromagen to reveal immunospecific alkaline phosphatase
plane (animals M1-3 and M5; Fig. 1C) or parasagitally (ani- activity. The colouration of fast redinapthol-alkaline phos-
mal M4). Sections were collected as sets; each set contained phatase labelling is bright red.
5-8 serial sections cut at one of several thicknesses (50, 80 (iii) An indirect immunofluorescence method. After the
or 100 pm). After sectioning, the tissue was rinsed thor- primary antisera incubations, selected sections were thor-
oughly in 50 mM TRIS-HCl pH 7.4 buffer (TRIS). oughly washed in phosphate-buffered saline (PBS)and then
incubated in the appropriate species-specific secondary IgG
NADPH diaphorase histochemistry serum conjugated with fluorescein isothiocyanate (FITC,
and immunocytochemistry Sigma Chemicals Ltd).
In the above immunocytochemical procedures, sections
Overview. In each set of sections, individual sections were washed thoroughly in TRIS buffer pH 7.4 (3 x 15
were reacted for either: (i) diaphorase enzyme activity
minutes) between each of the incubation steps.
alone; ( i i ) for diaphorase activity in combination with
Control incubations. In control sections incubated with-
GABA immunocytochemistry or immunocytochemistry us-
out ,NADPH, no specific diaphorase enzyme reactivity was
ing an antiserum directed against one of the calcium
present. Specific immunolabelling was absent in control
binding proteins (CBPs):calretinin (CR),parvalbumin (PV),
sections that had been incubated without primary antise-
or calbindin (CB); or (iii) treated with osmium tetroxide,
rum. However, in these latter sections a uniform and weak
dehydrated, and embedded in resin (see below). The last
colouration was present throughout the tissue due to
group of sections was used in morphometric studies (Gab-
bott and Bacon, 1996). In addition, selected sets of sections nonspecific labelling.
were Nissl-counterstained and some sections were pro-
Double preembedding immunocytochemical labelling.
cessed solely for NOS immunocytochemistry. Following diaphorase enzyme histochemistry, certain sec-
The technical procedure used to demonstrate the immu- tions were reacted using a double preembedding immunocy-
nocytochemical localisation of a marker substance in mate- tochemical labelling procedure described recently by Bevan
rial previously reacted to reveal NADPH diaphorase activ- et al. (1994). Essentially, sections were incubated either
ity has been described in detail previously (Gabbott and simultaneously or sequentially with two primary antisera.
Bacon, 1994).The methodological procedures are therefore (Combinations of CRIPV, CRICB, CBIPV antisera were
described in brief. used during simultaneous incubations. During sequential
N A D P H diaphorase histochemistry. After several incubations the same antisera combinations were used but
washes in TRIS, tissue sections were reacted histochemi- the order of the antisera was varied to achieve optimal
cally for NADPH diaphorase activity in a solution contain- results.) Antisera dilutions and incubation times were the
ing 0.25 mgiml of Nitro Blue Tetrazolium (NBT) and 1.0 same as those for single antiserum incubations (Table 1).
mgiml of reduced nitotinamide adenine phosphate The primary antisera were sequentially tagged with avidin-
(,NADPH). Tissue sections were incubated at 37°C for biotin-peroxidase ( ABP) complex using appropriate Vec-
45-120 minutes and the reaction terminated by thorough tastain ABC kits (Vector Laboratories). The first ABP
rinses in cold (4°C) TRIS buffer. (NBT and ,,NADPH were complex was revealed using DAB as the chromagen and the
purchased from Sigma Chemicals Ltd). second ABP complex visualised with Vector SG (Vector
Preembedding immunocytochemistry. Selected sec- Laboratories). Excess peroxidase activity was quenched
tions were then rinsed for an extended period in TRIS after the first visualisation reaction by washing sections in
buffer containing 0.5% Triton X-100 for 1-2 hours or were 0.05%,H 2 0 2in TRIS buffer (pH 7.4) x 10 minutes. Sections
freeze-thawed as described previously (Bolam, 1992). Sec- were then washed thoroughly in TRIS buffer (pH7.4). In
tions were then washed in either 20% normal goat serum or addition, an avidinibiotin blocking step was included be-
20% normal swine serum (30-60 minutes) and then incu- tween first and second labelling sequences. Control incuba-
bated in a primary antiserum against either GABA, CR, PV, tions were undertaken to ensure method specificity (Bevan
CB or nitric oxide synthase (NOS, brain and endothelial et al., 1994).
forms; Vincent, 1994,1995).The source of the antisera, the Tissue processing. After the histochemical reactions,
range of working dilutions, incubation times, and sera tissue sections were processed in one of two ways. The
specificity are given in Table 1. The use of these antisera in majority of sections were mounted in order onto gelatin-
 570
A
c+R
V
Fig. 1. A: Photograph of the anterior medial wall of the left cerebral
hemisphere of monkey M3. Calibration bar = 0.5 cm. B: Diagram
showing the positions of the cytoarchitectonic areas 24a,b,c, 25 and 32
over the surface of the medial wall. Coronal sections (1-6) are shown in
C. (Note the proximity of sections 3 and 4 to accommodate the highly
curved aspect, the genu, of the corpus callosum.) C: Coronal sections
coated slides and air-dried. Of these, specific sets of sections
were lightly counterstained with cresyl violet. The slides
were finally passed through an ascending series of alcohols,
P.L.A. GABBOTT AND SJ. BACON
cc
taken at the rostrocaudal sampling levels in C. The rostrocaudal
extents of cortical areas 24a, 24b, 24c, 25 and 32 are indicated.
Abbreviations: CS, cingulate sulcus; LOS, lateral orbital sulcus; MOS,
medial orbital sulcus; PS, principal sulcus: RS, rostral sulcus: and CC,
corpus callosum. Orientation markers: R, rostral; C, caudal; M, medial;
L, lateral; V, ventral; and D, dorsal.
passed swiftly through xylene and the sections embedded in
DPX mountant. Other sections were treated with 0.5%
Os04 in 0.1 M PB for 0.5-1.0 hour, dehydrated through a n
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
TABLE 1. Primary Antisera: Suppliers, Incubation Dilutions and Times
Serum Abbreviation Source/ Ref'.
ascending series of alcohols (including 1%uranyl acetate in
70% alcohol for 1 hour), flat-embedded in resin (ACM
Durcupan) and cured for two days at 56°C.
Tissue sections reacted using immunofluorescence were
thoroughly washed ( 3 x 10 minutes) in PBS and then
mounted in 100%glycerol on glass slides.
Tissue examination. Sections were observed in a light
microscope and objects of interest recorded with photomi-
crographs or drawings. Immunofluorescence was detected
using a Leitz Lz filter system attached to a photomicroscope.
Postembedding GABA immunocytochemistry. Semi-
thin (0.5-2.0 pm) resin-embedded sections were obtained
from selected cortical areas and lamina and reacted using
the postembedding GABA immunocytochemistry proce-
dure described by Somogyi and Hodgson (1985).
Cell morphometry, statistical analysis
and spine density counts
At high magnification (using a 1 0 0 ~oil immersion
objective lens and 10x eyepieces) the cytoplasmic and
nuclear profiles of both strongly and weakly reactive diapho-
rase-positive cells as well as for CR+, PV+, CB+, and
GABA+ immunoreactive neurons were traced onto paper
using a drawing tube attached to a photomicroscope. The
samples of somatic and nuclear profiles were derived from
neurons situated in all layers of the cortex and from the
different cortical areas investigated in this study. Only cells
in which the nuclear profiles could be discerned were
included in the analysis. The cell populations were taken
from material reacted using the DABiimmunoperoxidase
procedure (see above).
Somatic profile areas were measured and then calculated
using a computerised planimeter (Apple QuadraiKurta
digtising tablet system operated using MacStereology).
Measuring programs incorporated a linear and isotropic
tissue shrinkage factor of 15% (see Gabbott and Bacon,
1995). The diameters of area equivalent circles (D.Circle)
were subsequently calculated for the individual somatic
profile populations (Weibel, 1980). Somatic profile data
were then analysed using a statistical software package
(InStat. GraphPad Software, CAI. Data were initially sub-
jected to Bartlett homogeneity tests before the population
means were compared using multiple t-tests (Fry, 1993).
Significant differences between means were considered to
occur when a P value of <0.05 was obtained (Fry, 1993).
Five animals were used to gather somatic profile data for
CR, PV, CB, and GABA-immunopositive cell populations,
while four animals were analysed to provide data concern-
ing the diaphorase-reactive neuron populations.
571
Working Incubation
Code Host animal dilutions times
I ' 1.000-5.000
I 200-.500
1 500-2.500
1~20(1-500
l.200-1.000
1.200-250
I . 100-200
1:500-1.500
1. 1.000-2.000
Spine density estimates were determined for selected
dendritic segments from NADPH diaphorase-reactive neu-
rons and immunoreactive cells using the procedure of
Feldman and Peters (1979).
Terminology and symbols. The terms diaphorase-
reactive ( D + 1 and immunoreactive or immunopositive (ei-
ther CR+, PV+, CB+, GABA+ or NOS+) refer to specific
stainingiimmunolabelling in cells and cellular processes.
The terms diaphorase-nonreactive (D- ) and immunonega-
tive (either CR-, PV-, CB-, GABA- or NOS-)refer to
the absence of specific stainingiimmunolabelling in cells.
Finally, cells and processes displaying both specific immuno-
reactivity and NADPH diaphorase activity are termed
immunopositiveidiaphorase-reactive neurons (e.g., CR+i
D + , P V + / I ) + , C B + / D + or GABA+iD+).
RESULTS
Technical considerations
The following variables: ( i ) quality of tissue fixation
(fixative composition, postfixation processing, storage), (ii)
subsequent treatment with either Triton X-100 or freeze-
thawing, (iii)characteristics of the primary antiserum, (iv)
incubation times in sera, and (v) the type of chromagen
used to visualise immunolabelled structures, all affected
the depth and quality of immunoreactivity within the
tissue. In contrast, NADPH diaphorase activity was present
throughout the thickness of tissue sections and diaphorase-
reactive cells were revealed with great morphologcal detail,
rivalling that of cells seen in Golgi preparations (Vincent,
1995).
In this investigation, several chromagens were used
separately to visualise immunoreactive neurons (see Mate-
rials and Methods). Irrespective of the chromagen used, all
the characteristic features of a cell class were present in
immunolabelled neurons; variations were, however, found
in the clarity with which fine morpholoGcal details were
revealed.
As described below, diaphorase-reactive neurons in the
monkey mPFC were present in two separate populations
based on the intensity of somatic staining, either strongly
or weakly labelled cells. The existence of these two separate
populations was not related to methodological factors (e.g.,
incubation time) since both cell classes were consistently
present in material cut at different thicknesses and incu-
bated using substantially different reaction times (45 ver-
sus 120 minutes). Furthermore, the somatic profile-size
distributions of each staining type did not represent two
572
Fig. 2. Unfolded surface map of areas 24a, 24b, 24c, 25 and 32. The
line used to unfold the curvature of the cortex lay at the boundaries
between layers 315 (area 24a,b,c and 25) and layers 314 (area 32). The
unfolded segments were centred on the concavity of the cingulate
sulcus. Orientation markers: R, rostral; C, caudal; V, ventral; and D,
dorsal. Corpus callosum, cc. Calibration bar = 1.75 mm.
discrete parts of a continuum but overlapped markedly
(Fig. 24).
Identification of cortical areas and laminae
Areas 24a, 24b, 24c, 25 and 32 of monkey mPFC were
identified in the coronal and parasagittal Nissl-stained
sections using previous cytoarchitectural criteria (Fig. 1A-C;
see also Walker, 1940; Vogt et al., 1987; Barbas and
Pandya, 1989; Matelli et al., 1991; Dum and Strick, 1991;
Carmichael and Price, 1994; see also Fig. 1A-E in Gabbott
and Bacon, 1996). Of note, is that area 24c was identified as
extending onto the dorsal bank of the cingulate sulcus
where it represented approximately half of the cortical
territory (Fig. 1C). The anterior dorsal borders of area 24c
were not well defined rostral to the genu of the corpus
callosum (Fig. 1C).
Areas 24a, b, c, and 25 were typically agranular cortical
fields with pyramidal shaped neuronal somata located
throughout layers 2-6. Pyramidal cells were abundant in
layers 2 and 3 and comparatively sparse in layer 6. The
largest pyramidal neurons were located in the middle of
layer 5. Area 32 was defined as a granular cortical field
(layer 4) due to the presence of a distinct, numerically large
population of small spherical neurons located above layer 5
(Yeterian and Pandya, 1994).
The distribution of cortical areas 24a,b,c 25 and 32 over
the unfolded surface of the medial wall is shown in Figure 2.
The definition of areal borders is in good agreement with
previous studies (Dum and Strick, 1991; Carmichael and
Price, 1994). The rostrodorsal boundary of area 24c was
difficult to define with certainty (Figs. lC, 2).
Cellular morphology
NADPH diaphorase-reactive neurons
Characteristics of diaphorase labelling i n n P F C neu-
rons. Neurons in the cortex and white matter that were
P.L.A. GABBOTT AND S.J. BACON
histochemically labelled for NADPH diaphorase activity
contained the characteristic dark blueipurple formazan
end-product of the diaphorase reaction. Diaphorase enzyme
activity in the monkey mPFC was present in two separate
types of cell that could be distinguished clearly as either
weakly or strongly diaphorase-reactive on the basis of the
intensity of formazan labelling (Fig. 4c).
In weakly labelled neurons, cytoplasm within the cell
body and in the proximal regions of emerging processes was
faintly discoloured by the formazan end-product. In addi-
tion, fine particles of the formazan precipitate were dis-
persed throughout their somata (Fig. 4C). The staining of
weakly diaphorase-reactive neurons varied considerably
but never reached the intensity nor intracellular distribu-
tion found within strongly diaphorase-reactive cells. Conse-
quently, the morphology of weakly diaphorase-reactive
neurons was not investigated further. By contrast, the
cytoplasm within the cell bodies and cellular processes of
strongly diaphorase-reactive neurons was labelled intensely
and their morphology revealed with great clarity and detail
(Figs. 5-7).
Within the somata of both strongly and weakly diapho-
rase-reactive neurons, cell nuclei were invariably unla-
belled and provided distinctive translucent regions within
the somata of diaphorase-labelled cells. However, in neu-
rons with comparatively large amounts of cytoplasm sur-
rounding centrally placed nuclei, strong diaphorase label-
ling could totally obscure the unstained nuclei (Figs. 3A,B;
4B,D,M; 5E). Nevertheless, the absence of nuclear staining
is a characteristic hallmark of diaphorase-reactive neurons
in the mammalian nervous system that has been reported
in previous studies (Sandell, 1986; Cipolloni and Pandya,
1991; Vincent, 1994).
Qualitatiue distribution. Diaphorase-reactive cells ap-
peared as scattered solitary neurons (Thomas and Pearse,
1964) that were present in all the cortical layers and in the
underlying white matter of the five areas investigated in
this study (Figs. 6-8). The cortical depth distribution of the
diaphorase-reactiveneurons indicated that there were three
peaks in cell density. These peaks occurred in all cortical
areas and were located: (i) in the middle of layer 3, (ii) in a
region extending from very deep layer 5 to upperimidlayer
6, and (iii) within the white matter beneath each cortical
area at a depth of between 100 and 200 pm below layer 6.
The distribution peaks in the cortex produced two tangen-
tially distributed tiers of stained neurons. Quantitative
aspects of the areal and laminar distribution of diaphorase
Fig. 3. A Diaphorase-reactive neurons (1-3) in the superficial
layers of area 24a. Diaphorase reactivity in several smallimedium bore
capillaries is shown (double-headed arrows). c, capillaries. B: Layer 2
diaphorase-reactiveneuron (1 in A) with ascending dendrites (arrows)
that enter layer 1.B: High magnification of the somatic region of cell 1
seen in B. A fine descending axon-like process (arrows) arises proxi-
mally from a reactive dendritic process. C: Radiate multipolar diapho-
rase-reactive neuron (n) in layer 3 of area 25. D: A diaphorase-reactive
neuron in upper layer 3 of area 24c with a descending arcade (small
arrows) of dendrites emerging from the basal pole of the cell body. One
ascending dendritic process curves downwards (double arrow). c,
capillary. E: Beaded dendrites (arrows) from a diaphorase-reactive
neuron. F Diaphorase-reactive neuron ( n ) in layer 5 of area 24b. This
neuron was situated in the centre of a tissue section. All the diaphorase-
reactive processes from this neuron ascended through the cortex (small
arrows). An axon-like process can be seen to emerge from the basal pole
of the soma (double-headed arrow). G: Bitufted diaphorase-reactive
neuron ( n ) in layer 4 of area 32. Calibration bars: A, B, C = 100 pm; D,
F,G = 50 Fm; E = 5 Fm.
Figure 3
Figure 4
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
reactive cells are detailed in the companion paper (Gabbott
and Bacon, 1996).
Diaphorase neurons i n monkey m.PFC. Diaphorase-
reactive neurons composed between 0.20% and 0.32% of all
cortical neurons in monkey mPFC. Within the population
of diaphorase-reactive neurons, strongly reactive cells rep-
resented about 75% of the total cell count. Approximately
60% of weakly stained cells were located in superficial
layers 1 , 2 and 3. (See Table 15 in Gabbott and Bacon, 1996).
Nonpyramidal versus pyramidal. Light microscopic ev-
idence indicates that both weakly and strongly diaphorase-
reactive cells were morphologically diverse types of smooth
and sparsely spinous nonpyramidal cortical neurons (Feld-
man and Peters, 1978; Vogt and Peters, 1981; Peters and
Regidor, 1981; Sandell, 1986; Lewis and Lund, 1990;
Cipolloni and Pandya, 1991; Lund and Lewis, 1993; Jones
et al., 1994). Firstly, their somata did not give rise to true
apical and basal dendrites nor were the cell bodies of the
vast majority of labelled neurons unequivocally pyramidal
in outline. Secondly, the distribution of their dendritic
fields was characteristically nonpyramidal. Thirdly, the
origm and initial course of axon-like processes from these
neurons was highly varied and unlike cortical pyramids.
Finally, spine density counts set diaphorase-reactive neu-
rons apart from spiny stellate and pyramidal neurons (see
below).
Fig. 4. A. Diaphorase-reactive neuron ( D + I situated in layer 1 of
area 24b. A descendingspray of dendritic processes (arrows)can be seen
entering layer 2. Several of these processes descended for over 250 pm
reaching the layer 213 border. B: Diaphorase-reacted and Nissl-
counterstained section. A multipolar diapborase-reactive neuron ( D + I
in layer 3 of area 24b gives rise to a descending axon-like process (ax); than weakly labelled cell bodies in the deep layers of the
part of this process (boxed) is shown a t higher magnification in B’. cortex (Table 2 ) .
Diaphorase-reactive axon fibres and fibre swellings are present in the
neuropil. Some of these labelled axonal processes are aligned vertically
(arrowheads).Several Nissl-stained neurons are indicated ( n ) .B’: High
magnification of the boxed region in B. The identified diaphorase
axon-like process bifurcates (arrowhead),with one segment possessing
several varicosities (small arrows). C: Examples of strongly and weakly
diaphorase-reactive neurons. rJote that there was no gradation in
diaphorase reactivity between t h e two intensities of labelling. A fine
axon-like process (double-headed arrow) can be seen arising from a
labelled dendrite. c, capillary. Clumps of formazan precipitate (small diaphorase-reactive neuron had an ovoid soma giving rise to
thin arrows; end-products of the diaphorase reaction) can be seen in the
soma of the weakly labelled neuron. D: Diaphorase-reactive dendritic
process I D * ) with spine-like protrusions (arrows). One protrusion
[double-headedarrow) shows t h e clear features of a dendritic spine seen
in Gnlg preparations, a spine head and a slender spine neck. Note also,
the occurrence of short stub-like protrusions. E: A labelled diaphorase-
reactive swelling ( D + I comes into close contact (encircled) with a
diaphorase-reactive dendritic process ( D + 1. F: Two diaphorase-reactive
axonal swellings ( D + , arrows) are closely associated with t h e somata of dendritic arbors in layers 2-6 (Figs. 6, cells b,e,f; 7, cells
a Nissl-stained neuron ( n ) .G: Diaphorase-reactive dendrites ( D + 1 with k,i,o; 8, cell a ) , especially within the central territory of the
numerous dendritic spines (arrows) on primary and secondary seg-
ments. Note the variation in spine density between segments. Also note
the predominance of long thin spines. H: Diaphorase-reactive axonal
fibres in layer 3 of area 24c. Labelled fibres surround the somata of
unstained cell bodies (nl. J: Combined GABA immunocytochemistry
and diaphorase enzyme histochemistry. A diaphorase-reactive axonal
swelling iD+ i is associated with the cell body of a GABA-immunoreac-
tive neuron t G + ) . K: Diaphorase-reactive neuron ( D + ) in layer 5 of
area 32 with a lightly labelled axon-like process emerging (arrow)from
the lower part of the cell body. L: Diaphorase-reactive neuron in layer 6
of area 2.5. Labelled processes a r e indicated (arrows).The boxed re&<on (Figs. 3F; 7, cells j,m,o,p); (iv) cells in lower layer 6 and the
is shown enlarged in M. M: A fine axon-like process emerges (arrows) white matter with ovoid cell bodies and long horizontally
from the diaphorase-reactive soma ( D + I and travels horizontally
within layer ti. Along its length the labelled process gave rise to beaded
segments (double-headed arrows). Calibration bars: A = 100 y m ; B,
H = 50 p m ; C, L = 25; D, E, F, J, K, M = 10 ym; G = 5 y m .
57.5
However, despite the foregoing, a few cortical diaphorase-
reactive neurons were encountered that had many (Fig.
3A), but not all, of the characteristic features commonly
ascribed to true cortical pyramidal neurons. The most
extreme example was of inverted pyramidal-like neurons
(Fig. 6, cell IP). These cells closely resembled true cortical
pyramidal neurons but possessed a thick primary process
that descended through the cortex and a tuft of processes
emergmg from the upper surface of the cell body. By
considering the size and shape of somata, dendritic field
structure (tree shape and spine density measurements) and
the origm, course, and morphology of axonal arbors, these
cells could be classified as nonpyramidal neurons represent-
ing extremes of a morphological spectrum.
Cell body shape and size. The shape of diaphorase-reac-
tive neuronal somata commonly ranged from highly ellipti-
cal (Figs. 3G; 6, cells a,dj,q; 7, cells b,d,c,h,m,q; 8, cells b,c)
to nearly circular (Figs. 6, cells e,f,k; 7, cell k,i,p; 8, cell a ) in
outline. However, unusual shapes were frequently encoun-
tered, such as lobulated profiles where cytoplasm appar-
ently blistered from the cell body (Figs. 6, cells i,n,p; 7, cell j )
or somata in which large amounts of cellular cytoplasm
appeared funnelled into exceptionally thick primary pro-
cesses (Figs. 6, cell 0 ; 7, cells c,g,m). Inverted pyramidal-
shaped cell bodies were also observed (Fig. 6, cell IP).
Somatic profile-size distributions of populations of strong
and weakly diaphorase-reactive neurons located in either
layers 213 or layers 516 are given in Figure 24 and data
provided in Table 2. Statistical analyses established that
whereas no somatic size differences occurred between in-
tensely and weakly labelled cells in the superficial layers,
strongly diaphorase-reactive somata were significantly larger
Dendritic morpho1og.y. Two types of diaphorase-reac-
tive neurons were found in layer 1. The first type lay deep in
the molecular layer and gave rise to a spray of descending
beaded processes that ramified in layer 2 and the superficial
part of layer 3 (Fig. 4A). The second type of cell was
comparatively rare, but when present was located immedi-
ately beneath the pial surface (Fig. 6, cell a). This type of
long and relatively thick aspiny processes that were ori-
ented horizontally.
Diaphorase-reactive neurons throughout layers 2-6 usu-
ally possessed multipolar, bipolar or bitufted somata (Figs.
3-71, Although diaphorase-reactive cells in these layers had
diverse morphologies, several overlapping categories were
identified: (i) Diaphorase-reactive neurons with radiate
cortex (lower layer 3, layer 4 in granular area 32, and upper
layer 5; Figs. 3C,G; 5; 6, cells i,m-t; 7, cells b,c,e,f,m,n,o;8,
cell a ) ;(ii)cells located in layers 2 and upper layer 3 that had
radiate tufts of apical processes and long basal processes
aligned vertically (Figs. 3D; 6, cells c,d,gj). The ascending
dendrites from these cells would freely enter layer 1; (iii)
neurons in lower layer 5 and 6 with radiate basal processes
and long processes ascending vertically through the cortex
aligned processes (Figs. 4L,M; 7, cell q; 8, cells b,c); and
finally, (v)a comparatively small group of inverted pyrami-
dal-like neurons (Fig. 6, cell IP).
576 P.L.A. GABBOTT AND SJ. BACON

Figure 5
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 577

Of note is that some diaphorase-reactive neurons had Spine-like protrusions were found emerging from the
processes that travelled for comparatively wide lateral somata of diaphorase-reactive neurons (Feldman and Pe-
extents (see especially cell h in Fig. 6). However, the ters, 1978). These somatic spicules were not uncommon
horizontal spread of diaphorase-reactive processes was not and occurred either singly or in pairs (Fig. 5E; 7, cell b).
a primary feature of these cells, except for the minority of Axonal morphology. Axon-like processes were observed
cells found in upper layer 1, lower layer 6 and the white to emerge from diaphorase-reactive neurons in layers 2-6.
matter. These processes were exceptionally fine and frequently
The dendritic arbors of bitufted diaphorase neurons emerged from labelled somata (Figs. 3F; 4B,B’,K,L,M) or
commonly arose as a spray of processes emerging from from the proximal segments of primary or secondary
thick primary dendrites (see Figs. 6, cells d,g; 7, c). More dendrites (Fig. 3B’; 4C; 5A,B; 6; 7).Occasionally, two fine
distally, these dendritic segments would become wispy and calibre axon-like processes were observed to come from a
sometimes highly beaded (Figs. 3D,E,F; 4A; and cell c in single diaphorase-reactive cell. Usually these processes
Fig. 7). would arise separately from the soma and a proximal
Pyramidal-like diaphorase-reactive neurons were located primary process, and would either ascend or descend
predominantly in superficial layers 2 and 3 (particularly through the cortex. Such neurons were most commonly
layer 3 in caudal area 24c; Fig. 6, cell IP). They possessed a n located in layers 213.
inverted pyramidal-shaped cell body with a thick descend-
No axon-like processes were seen coming from stained
ing process that penetrated into the deeper layers of the
neurons in layer 1. However, diaphorase-reactive varicose
cortex before giving rise to terminal branches. Several
fibres were found coursing horizontally through layer 1 in
dendrites emerged from the upper somatic pole of these
cells in a fashion reminiscent of (but not totally similar to) cingulate areas 24a,b, and c.
basal dendrites arising from the lower surface of true The axonal processes from the inverted pyramidal-like
cortical pyramidal cells. diaphorase neurons would commonly emerge very near to
Dendritic spines: shape and surface density. Spines the soma or from a primary dendrite and course vertically
were present over the dendritic surfaces of diaphorase- upwards before either recurving or giving rise to collaterals
reactive neurons. Spines varied in shape from short stub- (Fig. 6, cell IP).
like protrusions (Fig. 4D) to relatively long thin pedicles Only the initial parts of axonal arbors from identified
with bulbous heads (Fig. 4G; Peters and Kaiserman- diaphorase-reactive neurons could be traced (Figs. 3-7).
Abramof, 1970). One reason for this may be that more distal axon segments
Relative to Golgi-impregnated neurons (DeFelipe and from diaphorase-reactive cells in the cortex contain specifi-
Farinas, 1992) and cells intracellularly filled with HRP cally low or insufficient levels of enzyme activity for histo-
(Larkman, 1991), the dendritic processes of diaphorase- chemical detection. The initial trajectories of identified
reactive neurons ranged from being virtually spine-free axonal arbors were either vertically or tangentially oriented
(smoothiaspiny, Figs. 3E, 4E) to possessing a lowimoder- in the cortex (Figs. 3B’,F; 4B,B’,K,L,M; 5B,F; 6-8). On
ate density of spines (sparsely spiny, Figs. 4D,G; 5C,D). occasions, these arbors could be traced into relatively
Dendritic spines predominantly occurred on second and diffuse arrays of fine, beaded processes ramifying close to
higher order processes (Figs. 4D,G; 5C,D; 8, cell c), and their parent somata (Figs. 4B‘; 5F,G).
spines were found over the dendrites of diaphorase neurons Diaphorase-reactive puncta i n the cortex. Strongly di-
located throughout the cortex, even over processes in lower aphorase-reactive puncta were present uniformly through-
layer 6 that originated from diaphorase-reactive neurons out the cortex (Fig. 4B,B’). These puncta were commonly
situated in the white matter (Fig. 8, cell c). located along interconnected webs of labelled processes
Spine density measurements were calculated for samples (Fig. 4B). Diaphorase-reactive puncta were found closely
of selected secondary and tertiary dendritic processes de- associated with the processes of diaphorase-reactive neu-
rived from neurons situated in layers 213 and 516. The rons (Fig. 4E) and also the somata of counterstained
samples were collected from all the studied cortical areas neurons (Fig. 4F). Indeed diaphorase-reactive puncta in
and the data are presented in Figure 9. No statistical lower layer 3 could be seen to form pericellular arrays, or
differences were detected between diaphorase neurons situ- baskets, partially or completely encircling the somata of
ated in the superficial and deep layers (Fig. 9). The density unlabelled cells (Fig. 4H). These basket-like structures
of spines over the processes of inverted pyramidal-like appeared to be produced by 1-3 varicose fibres. They were
neurons was not significantly different from other diapho- present in all areas of mPFC investigated.
rase-reactive neurons in the cortex. Diaphorase-reactive puncta were not observed to form
the short vertically aligned strings of beads (axon car-
tridges) described below for PV-immunoreactive puncta.
Fig. 5. A A tufted bipolar diaphorase-reactive neuron ( n ) in lower Finally, few diaphorase-reactive axon-like processes and
layer 2 of area 24b. The neuron gave rise to the labelled proximal part of labelled puncta were observed in the white matter under-
an axonal arbor (ax)that ramifies at the layer 2 / 3 border. Parts of the neath the cortical areas investigated.
dendritic arbor (boxed)are shown enlarged in C and D. B: Drawing of NOS-immunoreactiue neurons. Cells in the monkey
the neuron seen in A. The axonal arbor is shown in more detail in F. C:
Part of a distal dendritic segment showingdendritic spines (arrows).D: mPFC were immunolabelled using a n antisera against
Dendritic spines (arrows)arising from a proximal dendritic segment. E: b-NOS (Fig. 10).Although the morphology of these neurons
Soma of the diaphorase-reactive neuron shown in A. A diaphorase- was not revealed in great detail, many cellular characteris-
reactive spicule emerges from the cell body (arrow).F Drawing of the tics of these cells were similar to neurons stained histochemi-
labelled axon arbor (ax) from the cell shown in A. Numerous axonal cally for NADPH diaphorase enzyme activity (Fig. 10. cf
swellings are present over the axon field. One segment of the axon arbor
(boxed) is shown in G. G: Photomicrograph of the axonal swellings Figs. 3-81. In addition, the cortical depth distribution of
along the diaphorase-reactive axonal fibre indicated in F. Calibration bNOS-immunoreactive neurons was essentially similar to
bars: F = 100 p m ; A, B = 50 pm; G = 20 pm; D, E = 10 pm; C = 5 pm. that observed for diaphorase-reactive neurons (see above).
578
Fig. 6. Composite drawing of diaphorase-reactive neurons in layers
1-314 of areas 24a,b,c, 25 and 32. Note the morphology and distribution
of cellular processes as well as the shape and orientation of labelled
somata. Axon-like processes are indicated by arrows. Layout of figure:
The calibration bar applies to all neurons. In order to present neurons
from different cortical areas, where laminar boundaries occur at
different depths, cells have been placed in their corresponding position
P.L.A. GABBOTT AND S.J. BACON
within each lamina although the exact depth of each lamina is not to
scale. Laminar location ofcells (layer/area):a, 1/32; b, 2124a; c, 2/25; d,
212413; e, 2124~;f, 2124a; g, 2124b; h, 3/24a; i, 3124~;j , 3/32; k, 3124b;
; 3124b; p, 3124a; q, 3/25; r, 3/32; s, 3/25; t, 4/32.
m, 3/32; n, 3 1 2 4 ~o,
Inverted pyramidal-like neuron, cell IP from layer 3 in caudal area 24c.
The thick descending process from cell IP is indicated by the curved
open arrow. Calibration bar: 100 km.
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I ,579

Diaphorase t\ \

Fig. 7. Composite drawing illustrating the cellular morphologies of
diaphorase-reactive neurons in layers 5 and 6 of areas 24a,b,c, 2 5 and
32. Axon-like processes are indicated byarrows. T h e layout o f t h e figure
is described in the legend to Figure 6. T h e soma of neuron b is shown
enlarged to illustrate a somatic spicule (arrow).T h e arrows adjacent to
processes a t t h e top of the figure indicate t h a t these processes continued
into superficial cortical layers; the processes from cells j and p both
entered upper layer 3. Laminar location ofcells (1ayer:area): a, 5/24a; b.
5124b; c, 5/32; d, 5/25; ( I , 5 1 2 4 ~ f, ; 5124b; h, 5/24a; i, 6/32; j,
; 5 1 2 4 ~g,
6124b; k, 6/25; m, 6/24b; n , 6 1 2 4 ~ ;0 , 6124a; p. 6 / 3 2 ; q, 6/25.
Calibration bar: 100 kin.
 580 P.L.A. GABBOTT AND S.J. BACON

Diaphorase

I b
f J
,/ /

f
Fig. 8. Composite drawing of the cellular morphologiesof diaphorase-
reactive neurons at the border between layer 6 and the underlying
white matter (wmj of areas 24a,b,c, 25 and 32. Such cells display three
clear morphologies: ( i ) lower layer 6 neurons with processes that enter
the white matter (cell a) (ii) cells in the white matter that are oriented
along the fibre bundles that constitute the white matter (cell b), and (iii)
cells in the white matter that have ascending processes that enter the
cortex (cell b). Cell b has two processes that are beaded (arrows).Part of
one process from cell c (boxed) is shown enlarged to illustrate how
dendritic processes become sparsely spiny (arrow) on entering the
cortex. Laminar location of cells (laymiarea). a, 6/32; b, wmi24c; c,
wmi25. Calibration bar: 100 wm.

TABLE 2. Morphometric Data' Morphology o f calcium binding proteins in mPFC. Neu-

D. circ Range rons immunoreactive for the calcium binding proteins
(pm) (pm) (either CR, PV, or CB) investigated in this study displayed
Calcium binding proteins' strong immunolabelling of their somata and nuclei as well
ICR. PV and CB populations combined) 11.6 2 1.2 4.9-27.8 as their cellular processes. Neurons immunoreactive for a
Calretinin iCRl** 9.0 2 0.8 5.1-13.4
Parvdbumin 1PVI 12 9 2 1.1 6.1-33.2 given CBP displayed consistent morphologies across the
Calbindin ICRI 11 9 i 1.1 5.2-25 3 five cortical areas 24a, 24b, 24c, 25, and 32 of the monkey
GABA 10.8 ? 1.2 5.7-26.3
Diaphorase-reactive
mPFC. However, within each population of CBP-immuno-
Whole population 10.2 ? 0.9 5.617.3 reactive cell, several morphologically distinct subtypes were
Layers 2:3
Strongly reactive 12.7 L 1.2 8.1-16.2
identified. Quantitative aspects of cell densities, cortical
Weakly reactive 1 0 5 ? 1.0 6.0-17.1 and laminar distributions are detailed in the companion
Layers 5 : 6 * * *
Strongly reactive 13.9? 1.1 8.5-19.8
paper (Gabbott and Bacon, 1996).
Weakly reactive 9.8 ? 0.9 5.0-14.7 Calretinin (CR)-immunoreactiue neurons. CR-immuno-
'Mean area equivalent circle diameters !D. circle) of the cell populations investigated in
reactive neurons were found in all layers (1-6) of the areas
this study- a1 CBPs icombined CR, PV and CB populations), CR+,PV+, CB+, and investigated and were also present in the underlying white
GABA+ immunoreactive neurons. Populations were sampled from all layers of the matter. Several classes of CR+ neurons were evident in
cortical arras investigated. bl Strongly and weakly reactive diaphorase-positive cells in
superficial and deep layers within the cortex. These cell populations were composed of each cortical area investigated (Fig. 14). The numerical
neurons taken from layers 213 and 516, respectively, of the corticd areas studied. !Mean depth distribution of CR+ neurons indicated that peak
values ? S D.; n = 3 three data sets. I
Statistical analysis of somatic profile data: density occurred in layer 2 and upper layer 3, with a marked
'CBPs vs. GABA: t = 0.47, df = 4, P = 0.07. Difference not sipificant. decrease in cell numbers through layer 5 and into layer 6.
"CR vs PV: t = 3 9. df = 4, P < 0.05. Difference significant.
***L5;6 strong vs. weakdiaphorase: t = 2.88, df = 4, P < 0.05. Difference significant. The somata and processes of CR+ neurons were strongly
Comparisons between other cell populations were not significant ! P > 0.05). immunoreactive in all cortical layers. CR-immunoreactive
neurons were generally small (9.0 pm in diameter; Fig. 24;
The b-NOS antisera used in this study produced a high Table 2) with ovoid bipolar somata that had either: (i) two
level of background nonspecific staining at all serum dilu- long vertically oriented processes arising from opposite
tions. However, no b-NOS immunostaining was observed in somatic poles (Figs. 1Oc; 14, cells k, n, q, t, u, x,y), or (ii)two
endothelial or glial cells. Conversely, no specific labelling of vertical tufts of processes (Fig. 14, cells j, m, 0,p, s, v, w, z).
cortical neurons was found with a n antiserum directed Although these cells were found throughout all layers of the
against eNOS. mPFC they were most prevalent in middle to upper layer 3.
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 581

Diaphorase Reactive Neurons L 2 / 3 - branches would arise from the thick parent stems and
descend for short distances within layer 1.
The dendritic fields of CR+ neurons in layer 2 were more
radiate than for other CR+ cells. These dendritic fields arose
Mean: 0.60 spines / pm
from multipolar somata and radiated freely into layer 1
SD: 0.26 (Fig. 14, cells d-g, i). Occasionally the cell bodies of CR+
2 neurons in layers 3 and 5 were oriented horizontally, gwing
.d

20 Counts 48
C rise to processes that initially coursed obliquely but then
W Range 0 09 - 1 56 spines I pm adopted vertical orientations within the cortex (Fig. 14, cell r).
a A separate class of bipolaribitufted CR+ neuron in the
&I 15
0 deeper layers of the cortex had triangular or ovoid somata
&
W with long thick ascending processes (Fig. 14, cells z, a') that
P 10 passed through superficial layers, sometimes rising into
Ei upper layer 2 where they tapered gradually (Fig. 14, cell b').
1
C Tufts of basal processes would emerge from these neurons
o i 5 and ramify horizontally in the deep layers of the cortex. The
P lateral spread of the basal processes from these deep lying
4
0
CR+ neurons increased as the position of the parent cell
0 02 04 0'6 08 10 12 14 16 18 body approached the white matter. Layer 6 CR+ neurons
Spine density (spines / pm) lying near the white matter did not have ascending pro-
cesses and were aligned horizontally (Fig. 14, cell c'). The
processes from these latter cells extended for considerable
tangential distances ( < 300 pm) deep within the cortex.
None of the processes from these cells ramified outside the
Diaphorase Reactive Neurons - L 5 / 6 grey matter.
Beaded dendrites were a characteristic structural feature
of all classes of CR+ neurons in the cortex (Figs. 11D, 14,
25 r Mean: 0.49 spines / pm inset). In addition, many of the CR-immunoreactive den-
v) dritic processes bore a sparse number of dendritic spines.
r
W SD 019
.r(
The qualitative appearance of low spine density over the
$
J 20 Counts 51 processes of CR+ neurons was confirmed quantitatively,
with spine density calculated as 0.89 i 0.09 spines per
a
5 Range 0 08 - 0 98 spines / pm
micron (mean 2 S.E.M.: sample ( n ) of 30 primaryisecond-
ru
0
' 5 ary processes. Each process was > 30 pm in length).
k Axon-like processes arose from CR+ neurons located
W throughout the cortex. For CR+ neurons in layers 2-6
P 10
Ei these process would emerge either from upper or lower
1 somatic poles or more commonly from primary or proximal
C
secondary dendrites (or even the intervening dendritic
o i 5
P branch point). They frequently adopted an initial descend-
d ing course but would give rise to ascending branches.
n
I Axonal fields were predominantly oriented vertically within
0 0.2 04 06 08 10 1.2 14 16 18
the cortex and frequently confined within the lateral extent
Spine density (spines / pm) of the dendritic arbor (Fig. 14, cells j, s ) . Although uncom-
mon, notable exceptions were found where the vertically
Fig. 9. Distributions of spine densities over t h e processes of diapho- aligned dendritic and axonal fields were slightly displaced
rase-reactive dcndritic processes in superficial (I, 2 / 3 )and deep ( L 5/61 laterally (Fig. 13). The axon arbors of CR+ neurons gave
layers of monkey mPFC. Data derived from pooled samples from
cortical areas 24a,b,c, 25 and 32 in 4 animals. (Arrows indicate mean rise to swellings or varicosities (Fig. 13B).
values,. Statistical analysis: L213 versus L5/6: t = 0.68, n = 6, P = 0.52. Fine axon-like CR+ processes were also observed to come
Difference not significant. from the somata of labelled layer 1neurons; however, these
processes would course horizontally within layer 1 (Fig.
12A). No collateral branches were observed from these
A common structural feature of these CR+ neurons in latter processes.
layers 2-4, and upper layer 5 was the tight bundling and The overall appearance of immunolabelling in the neuro-
vertical orientation of their dendritic and axon-like pro- pi1 of the five cortical areas clearly showed that within
cesses. layers 2-4, CR+ immunoreactive processes were tightly
CR+ neurons were also found in layer 1 where they were bundled and preferentially aligned along the vertical axis of
commonly encountered at the junction with layer 2. A the cortex (Figs. 11C,D,H; 13; 14). Within layer 5 and
numerically smaller population of CR+ cells was located particularly layer 6, CR+ labelled processes were less
superficially within layer 1 typically beneath the pial sur- strictly oriented vertically.
face (Figs. 12A,B; 14, cells b,c). The cell bodies of these CR-immunoreactive puncta were present throughout all
latter neurons were spindle-shaped and oriented parallel cortical layers. Of particular note is that CR+ puncta were
with the pia (i.e., horizontally). Thick processes emerged found to be closely associated with the somata and initial
from opposite poles of their somata and were aligned dendritic processes of other CR+ neurons. Indeed, on
horizontally within layer 1. Although rare, occasional side occasions strings of CR+ puncta could be found draped
582
Fig. 10. NOS immunocytochemistry. A NOS-immunoreactive neu-
ron in) in layer 3 of area 24a with labelled processes (arrowed). B:
Neuron ( n ) in layer 4 of area 32 immunoreactive for NOS. Immunola-
belled processes extend laterally (arrows) and become beaded (en-
circled). The cell body of another NOS-immunopositive cell is indicated
(double arrow). c, capillary. C: NOS-immunopositive neuron in) in
layer 5 of area 25 with labelled processes (arrows).One fine immunore-
closely over CR-immunoreactive cell bodies (Fig. 26R,S) in
a fashion reminiscent of pericellular baskets (see below).
Parvalbumin (PV)-immunoreactiue neurons. Strongly
immunoreactive PV+ neurons were present in layers 2-6
and in the subjacent white matter of all the cortical areas
investigated (Fig. 15A-F). In these layers immunoreactive
neurons were strongly labelled. Cells in the deep region of
layer 1were also PV-immunoreactive. However, the somata
of these neurons were only weakly labelled and their
dendritic morphology not visible.
The depth distribution of PV+ neurons indicated that
peak density occurred midcortex, in a region centered on
middle to lower layer 3 and upper layer 5 (or layer 4 in the
case of area 32). A decline in PV cell numbers was present in
lower layer 5 and maintained in layer 6.
In general, PV + neurons in layers 2-6 could be divided
into three broad classes on the basis of the size and shape of
their somata and the distribution of their processes (Figs.
15-17). The first class had small bipolaribitufted fusiform
somata (6.1-12.5 km in diameter; Table 2; Fig. 24) with
either vertically aligned processes (Figs. 15C; 16, cell a) or
with small radiate dendritic fields (Fig. 16, cells b-0. This
latter type of PV+ neuron was most common in upper layer
2, but could also be found throughout layer 3. The second
type of PV+ cell had a medium sized fusiform soma
(10-16.5 pm in diameter; Table 2; Fig. 23) that was either
vertically or horizontally disposed (Figs. 15c; 16, cells h, q,
r, s). The processes from these cells radiate outwards along
the axis of their parent somata. Neurons in this second
category were common in lower layer 3, (layer 41, and layers
5 and 6.
The third type of PV+ neuron found in this study of the
mPFC had exceptionally large round multipolar somata
(18.0-30+ km in diameter; Table 2; Fig. 24) with wide
P.L.A. GABBOTT AND SJ. BACON
active process (double arrowhead) emerges from the labelled soma.
(Note the weak immunolabelling of capillaries [cj and compare with
Fig. 3A.) D: Neuron ( n ) and processes (arrows) in layer 3 area 24c
immunoreactive for NOS. E: Multipolar neuron ( n ) with processes
(arrows) immunoreactive for NOS lying in layer 6 of area 24b.
Calibration bars: A-D = 50 pm; E = 25 pm.
radiate dendritic arbors (Figs. 15B, 16).The dendritic fields
were commonly composed of between 3 and 8 thick primary
dendrites that bifurcated proximally with secondary
branches coursing radially outwards from the soma for
considerable distances (Fig. 16, cells g, i, j , p). This third
type of PV+ neuron was less frequent than the former two
types of PV+ cells. However, when set amidst other
PV-immunoreactive neurons, these cells were conspicuous
by size alone (Fig. 15B,E). Indeed, there was a distinct
cortical depth distribution of PV+ somatic profile size, with
a strong tendency for large somatic profiles to occur in the
deeper layers of the cortex, particularly lower layer 5
(Fig. 16).
Fig. 11. Calretinin (CR) immunoreactivity. A Superficial laminae
(1-3) of area 24c showing the distribution of CR-immunoreactive
neurons (arrows).Note presence of labelled neurons in layer 1 tdouble-
headed arrow). B: CR-immunopositive neurons ( n ) in layer 2 of area 25.
A strongly labelled neuron 1 thick arrow) gives rise to numerous radiate
processes (small arrows). C: Lower layer 2 of area 24b. Elongated
CR-immunolabelled neuron (n),oriented vertically in the cortex (arrow-
heads), with labelled processes (arrows). D: CR-immunopositive neu-
ron ( n ) in layer 3 of area 24c. The neuron gives rise to two vertically
oriented tufts of beaded processes (arrows). E: CR-immunopositive
neuron (n)in layer 3 of area 24a. The labelled processes bear spines;
examples are shown in F and G. F: Enlargement of boxed region shown
in E. The immunolabelled process gives rise to many lightly CR-
immunoreactive dendritic spines (arrows). G: Further example of
CR-immunoreactive spines (arrows)arising from the cellular process of
the neuron seen in E. H: CR-immunoreactive neuron in layer 3 of area
32. Note the laterally directed processes of this neuron (arrowheads)
and the oriented immunoreactive processes (arrows)coursing vertically
through the cortex. Calibration bars: A = 100 pm; B, C, H = 50 pm;
D = 25 pm; E = 20 pm; F = 10 pm (G same magnification as F).
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 5x3

Figure 11
584 P.L.A. GABBOTT AND S.J. BACON

The dendritic processes of PV+ neurons were, as far as

could be ascertained, aspiny, and the more distal processes
were frequently beaded in appearance (Figs. 16, cell h; 17).
Dendritic arbors of all types of PV+ cells ramified without
regard for laminar boundaries, type 3 PV+ neurons situ-
ated at the midlevel of the cortex were found to have
processes that ascended into layer 2 and descended through
layer 5 towards layer 6 (e.g., cell j in Fig. 16 and cell m in
layer 4 of area 3 2 ) . Although rare, PV+ cells were encoun-
tered in the white matter (Fig. 15F). These cells had
processes that coursed horizontally for long distances be-
neath the grey matter with occasional side branches that
entered into layer 6 where they bifurcated (Fig. 15F).
PV+ immunoreactive axon-like fibres were seen to emerge
from the somata of labelled cells or from proximal processes
(Fig. 16). In the majority of cases it was impossible to trace
these processes for long distances (Fig. 15). However, on
occasions the initial part of axonal arbors were detailed
(Fig. 17). The principal trunk of these processes (which
were particularly thin over their proximal lengths) would
either ascend or descend vertically through the cortex (Fig.
16). Collateral shoots would then branch and run for short
distances horizontally (Fig. 17); the terminal arbor of axon
collaterals were not identified with certainty.
PV+ immunoreactive processes and puncta were distrib-
uted throughout all layers of the cortex (Fig. 18) but were
most dense in layers 2 and 3. Layer 1,however, contained a
very low density of PV+ puncta and the processes in this
layer could frequently be traced back to P V + neurons in
layer 2. PV+ puncta were also present within the underly-
ing white matter (Fig. 25).
Of particular importance is that some PV+ puncta formed
two morphologically distinct structures, termed axon car-
tridges and pericellular clusters (see Akil and Lewis, 1992).
These two structures were most frequently found within
prelimbic area 32 and cingulate areas 24b and 24c.
Axon cartridges were composed of numerous strongly
immunoreactive PV+ varicosities (akin to strings of dark
beads) that were vertically aligned within the cortex and
situated at variable distances (4-15 Fm) beneath the
somata of unlabelled neurons (Fig. 18A-C,GJ). On occa-
sions the unlabelled somatic profiles were clearly seen to be
pyramidal in shape (Fig. 18C). Axon cartridges were highly
variable in both length and complexity (Fig. 18A-C,G and
H). Their length ranged from 10 pm or so to over 30 Fm; as
a rough first estimate (leaving methodological consider-
ations aside) each cartridge was composed of between 8 and
45 individual PV+ puncta (for example compare Figs. 18A
and 18H).These puncta were either closely apposed to each
other or interconnected by fine immunoreactive strings
(cyto/axoplasmic bridges; DeFelipe et al., 1985; Williams et
al., 1992; see Fig. 18A-C and G J ) .
Axon cartridges were predominantly situated in superfi-
cial cortical layers 213 but were also present (albeit to a

Fig. 12. Calretinin (CRJ immunoreactivity. A: CR-immunopositive

neuron lying very near the pial surface (piaJ of area 24c. A thick labelled
process (thick arrows) emerges from one pole of the soma and courses
horizontally within layer 1. In contrast, a fine immunolabelled process
arose from the opposite pole of the soma and travelled close to the pial
surface for over 200 pm. B: Layer 1 neuron in area 32 displaying CR
immunoreactivity. The neuron lies close to the pia and has processes
(arrows) oriented horizontally beneath the cortical surface. Calibration
bars: A, B = 50 pm.
LOCAL CIRCUIT NEURONS IN MONKEY mI’FC: I 585

much lesser degree) in layer 5 and occasionally in layer 6.

They were frequently encountered in the upper layers of
areas 24b and 24c (Fig. 18A,B,G). Quantitative estimates
indicated that the number of PV+ puncta composing an
A CR individual axon cartridge were more numerous in upper
layers 213 (10-45) than in lower layers 516 (12-33).
Pericellular clusters were identified as numerous PV+
immunolabelled puncta fully or partially encircling the
somata of unlabelled neurons (Fig. 18C-H,J). Similar to
axon cartridges, the unlabelled cells were commonly pyrami-
dal in shape, although circular somatic profiles were also
encountered (Fig. 1 8 - H ) . PV+ puncta were additionally
found around the descending presumed axon hillocks of
pyramidal cells (Fig. 18C,H). Immunoreactive puncta sur-
rounded not only the profiles of unlabelled pyramidal
somata but also surrounded the outlines of their proximal
3 apical (and sometimes basal) dendrites (Fig. 18C).
Of note is the observation that the pericellular clusters
and axon cartridges formed by PV+ puncta could be
present in three types of arrangements around the somata
and presumed descending axonal profiles of unlabelled
neurons: ( 1) PV+ puncta forming pericellular clusters
alone, (2) PV+ puncta forming axon cartridges alone, or (3)
PV+ puncta forming both a pericellular cluster and an axon
cartridge around the same unlabelled cellular profile (Fig.
18A,B,C,H; see also Fig. 27). Figure 18H illustrates a type 3
PV+ puncta formation surrounding the profile of an unla-
belled cell body together with its presumed descending axon
hillock and axon initial segment.
All three types of PV+ puncta arrangements were pre-
sent throughout layers 2-6. However, type 1 arrangements
were predominant in layers 516, whereas arrangements of
types 2 and 3 were most common in layers 213 (Fig. 27).
The occurrence of this tripartite PV+ immunolabelling
surrounding cortical pyramidal neurons occurred most
frequently in areas 24b,c and 32.
It should be pointed out that methodological constraints
may be responsible for these observations and data re-
ported above, for example, whether both the pericellular
cluster and axon cartridge around a given pyramidal cell
were equally available for immunocytochemical identifica-
tion. The depth of the immunoreactive zone and the
orientation of pyramidal cell somata within the tissue
section could select which parts of the cell, and therefore
the number of puncta, were immunolabelled. However, the
observations were too consistent across sections and areas
for such labelling to be related exclusively to methodology.
PV+ puncta were also found closely abutting onto the
somata and processes of PV+ immunoreactive neurons at
midlevels within the cortex.
Thick calibre PV-immunoreactive fibres were found in
the neuropil of lower layer 3 and layer 4 (Fig. 25H).
Similarly large PV-immunoreactive fibres were also present
in the white matter underlying the cortical areas investi-
gated here. These labelled processes coursed for consider-
able distances along the common path of unlabelled fibres
in the white matter (Fig. 245).

Fig. 13. A Drawing of a CR-immunoreactive neuron in layer 3 of

area 24c. T h e cell g v e s rise to a vertically oriented axonal arbor (ax1
part of which (boxed region) is shown in B. B: Photomicrograph of a
segment of t h e axonal region boxed in A. Numerous swellings (arrows)
a r e present along t h e CR-immunolabelled axonal fibre. Calibration
bars: A = 100 p m ; B = 20 pm.
 586
CR
Fig. 14. Composite drawing of calretinin (CR)-immunoreactive
neurons in layers 1-6 ofareas 24a,b,c, 25 and 32. Note the morphology
and distribution of cellular processes as well as the shape and orienta-
tion of labelled somata. Axon-like processes are indicated by arrows.
The boxed inset in the centre of the figure shows the typical beaded
appearance of a CR-immunoreactive dendrite (see also Fig. 12A). wm,
P.L.A. GABBOTT AND S.J. BACON
white matter. The layout of figure is described in the legend to Figure 6.
Laminar location ofcells tlayeriarea): a, 1125; b, 1/32; c, 1 1 2 4 ~d, ; 2/32;
e, 2124a; f, 2124b: g, 2132; h, 2i25;i, 2/24a; j, 3 1 2 4 ~k,
; 3124b; m, 2 1 2 4 ~ ;
; 3124a: r, 3i24b; s, 3125; t, 5 1 2 4 ~U: , 4/32; V,
n, 2/25: 0,3132: P, 2 1 2 4 ~q,
3i24b; w, 5i24c; x, 5124b; y, 5124a; z, 6124a; a ' , 6124c; b', 6i24b; c',
6/32. Calibration bar = 100 pm.
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
Fig. 15. Parvalbumin t PV) immunoreactivity. A: Low power photo-
micrograph showing darkly labelled PV immunopositive neurons in
layer 3 in area 24b. B: A large multipolar PV-immunoreactive neuron
i n ) i n layer 4 of area 32. The cell gives rise to numerous labelled
processes (arrows) radiating outwards for long distances. A fine axon-
like process arises from the cell body (ax).Several other immunoreac-
tive neuron cell bodies are indicated (arrowheads).Compare the sizes of
immunolabelled cells. C: Layer 3 area 25. PV-immunoreactive neuron
i n ) oriented vertically with a spray of labelled processes (arrows)above
the cell body. D: PV-immunolabelled neuron ( n ) in layer 5 of area 24c.
Finally, in all the cortical areas investigated, P V + immu-
noreactivity defined a band of cortex that extended from
lower layer 3 through to upper layer 5 (encompassing layer
4 in area 32). This band was particularly evident due to the
increased frequency of PV+ somata as well as the presence
of immunoreactive puncta.
Calbindin (CB)-immunoreactiue neurons. In the corti-
cal areas studied, CB immunoreactivity was found in
pyramidal and non-pyramidal neurons (Fig 19G). These
two subpopulations-were morphologicallydistinct (Figs.
19G, 20).
Labelled process iarrow).E: Layer 3, area 32. PV-immunolabelled soma
o f a large multipolar neuron (double-headedarrow);compare the size of
this neuron with the somatic sizes of other neighbouring immunola-
belled cells (arrows). F: PV-immunoreactive neuron ( n ) lying in the
white matter ( w m )below layer 6 of area 24aib. Note: immunolabelled
processes elongated along fibres coursing in the white matter (arrows);
process ascending into layer 6 (double-headed arrow); beaded process in
layer 6 (arrowheads).The layer 6iwhite matter boundary is indicated
(dotted line). Calibration bars: A = 100 km: B = 75 p n : F = SO pm: C.
D = 25 km; E = 20 kin.
The majority of CB + immunoreactive pyramidal neurons
were located in lower layer 3 and also present although to a
much lesser extent in layer 5. Both tiers of cells were very
weakly to faintly immunolabelled and possessed character-
istic pyramidal somata with thick processes emerging from
the apical pole of their somata (Figs. 19, 2 0 ) . This staining
pattern was present in all the cortical areas examined.
By comparison, neurons within the nonpyramidal cell
population were strongly immunolabelled for calbindin
(Fig. 19G) and several subtypes were evident (Figs. 19-21).
The cortical depth distribution of the nonpyramidal CB-
588
Fig. 16. Composite drawing of parvalbumin (PV)-immunoreactive
neurons in layers 2-6 of areas 24a,b,c, 25 and 32. Note the morphology
and distribution of cellular processes and size of labelled somata.
Axon-like processes are indicated by arrows. PV-immunoreactive neu-
rons in layer 1 have not been included since only their somata were
lightly stained and no morphological detail of their processes revealed.
P.L.A. GABBOTT AND SJ. BACON
1 ' ......._.
wm, white matter. The layout of this figure is described in the legend to
Figure 6. Laminar location of cells (layerlarea): a, 2124b; b, 2124a; c,
2132; d, 2125; e, 2124~;f, 2124b; g, 3132; h, 3125; i, 3124b; j, 3124b; k,
; 5/32; q, 5124b; r, 5124c; s, 6124a.
5132; m, 4132; n, 512413; 0, 5 1 2 4 ~p,
Calibration bar = 100 Fm.
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
\ immunoreactive cell population was essentially similar to
PV
1
:i-
I+
i
Fig. 17. Drawing of PV-immunoreactive multipolar neuron in layer
4 of area 32. An axonal process emerged (small arrow) from the
proximal part of a dendritic process and gave rise to a vertically and
horizontally disposed axonal field (large arrows) with several identified
collaterals. Calibration bar = 100 pm.
589
that of CR+ neurons, but peak density occurred compar-
atively lower in the cortex, within upper to middle layer 3.
Thereafter CB+ cell density gradually diminished with
increasing cortical depth.
The first two types of CB+ immunoreactive nonpyrami-
dal neurons were located primarily within layers 2 and 3
(Fig. 20). These cell types comprised vertically oriented
bipolaribitufted and radiate multipolar neurons, respec-
tively (Fig. 20, cells a-k). The processes from the bitufted
neurons were confined within narrow, vertically oriented
columns. The somatic profiles of both cell types were small
to medium in size and ranged from 5.2 to 15 pm in diameter
(Fig. 24). Beaded dendrites were a common characteristic of
these CB+ cells (Fig. 19B,F); fine varicose axon-like fibres
were seen to arise from proximal dendrites (Fig. 19F).
Spine-like protrusions were also present over the surfaces
of CB+ neurons in these two classes (Fig. 19E).Mean spine
density for these cell types was calculated as 0.71 2 0.08
spines per micron (mean t S.E.M.: Sample ( n ) of 8 pro-
cesses. Process length ranged from 24 to 46 pm).
The third type of CB+ nonpyramidal neuron was very
distinct and found in layers 2-6 (Fig. 21). Neurons charac-
teristic of this subtype possessed very small round multipo-
lar somata (6-10 Fm in diameter; Fig. 24). The processes
that emerged from these neurons bifurcated readily and
ramified extensively in the vicinity of the cell body. Rarely
did the dendritic territory exceed 200 Frn in diameter. No
axonal processes were seen arising from neurons in this
category of CB+ cell.
Neurons belonging to the fourth category of CB+ neuron
had exceptionally large somata and were regularly located
in the deeper layers of the cortex (Figs. 19D,J; 20). Somatic
measurements ranged from 16 to 25.3 pm in diameter.
Such neurons were multipolar cells with expansive smooth
dendritic arbors that radiated outwards for over 350 km
from the parent somata. The initial segments of fine axonal
processes were seen to emerge from the lower surface of the
cell body (Fig. 19J').
CB+ neurons in the fifth category were located predomi-
nantly in layers 5 and 6 (Fig. 22). Similar to cells in the
previous category, these neurons also had large multipolar
cells (somatic diameter range 15-25 pm) with radiate
dendritic fields. However, a characteristic feature of these
neurons was a well defined single axon-like process that
merged either from the soma or a proximal dendrite and
coursed vertically upwards into superficial layers. Terminal
segments of these axon arbors could not be identified.
The last type of CB+ neuron was located in the white
matter. Like PV+ neurons, the somata and processes of
these cells were aligned horizontally beneath the grey
matter. Occasional side branches would travel obliquely or
perpendicularly into layer 6; these side branches were not
beaded in appearance.
No CB+ cells were found in layer 1 of the cortical areas
investigated. However, CB+ processes were frequent com-
ponents in layer 1 and were commonly derived from CB+
neurons in layers 2 and 3. On occasion, horizontally
oriented CB+ processes were found coursing near the pial
surface. Within the superficial layers 2 and 3, CB+ immuno-
reactive processes were frequently encountered as verti-
cally oriented bundles (fascicles) of labelled processes (simi-
lar to CR+ processes).
CB+ puncta were distributed throughout the cortex,
particularly within layers 2 and 3. In the superficial layers
590 P.L.A. GABBOTT AND S.J. BACON

Figure 18
 LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
strings of immunolabelled puncta were frequently distrib-
uted radially.
CABA-immunoreactiue neurons. The range of mor-
phologes of GABA+ neurons in the monkey mPFC (Fig.
23) overlapped the morphologies of CR+, PV+ and CB+
immunoreactive neurons described in detail above. One
exception, however, was that GABA immunoreactivity was
never found in the somata of pyramidal neurons in layer 3
(cf calbindin immunoreactivity).
The cortical depth distribution of the GABA-immunore-
active cell populations mirrored the combined cortical
depth distribution of CR+, PV+, and CB+ neurons. Peak
cell density for GABA-immunoreactive neurons in monkey
mPFC occurred in layer 2 and diminished gradually to-
wards the white matter without secondary peaks occurring
in lower cortical layers (for quantitative details see Gabbott
and Bacon, 1995).
GABA+ puncta were found throughout the layers of the
mPFC and predominantly in layers 2 and 3. In both layers
213 and 516 GABA+ puncta formed pericellular clusters
(Fig. 18K,I,,M) and axon cartridges (Fig. 18K) similar to
those described above for PV-immunoreactivepuncta. How-
ever, GABA-immunoreactive puncta were not present in
the white matter.
The somatic profile-size distribution of GABA+ neurons
was not significantly different from that of the combined
population of CR+, PV+, and CB+ neurons (calcium
binding proteins, CBPs; Fig. 24). Interestingly, both so-
matic profile-size distributions (GABA+ versus CBPs+ )
Fig. 18. CoinparisonofPV (A-HI and GABA (K-M) immunoreactivi-
ties. A: 1,aye.r 3 , area 24h. Rows of vertically aligned PV-immunorcac- PV+, or CB+ containing NADPH diaphorase-reactive neu-
trings" (arrows)lying beneath unlabelled cell bodies (n; presum-
ably the unlabelled somata of cortical pyramidal neurons). B: Layer 4,
area 32. I n this typical example, a PV-immunopositive string (thick
arrow1 can be seen to be composed of numerous immunoreactive
puncta ismall arrows). C: Layer 3 , area 24c. PV-immunoreactive
puncta are not only clustered around the unlabelled soma and proximal
apical dendritic shaft ( a d ) of a pyramidal neuron (P;small arrows) but
are also clustered around (thick arrow) the basal pole of the neuron,
presumably encasing the axon initial segment. D: PV-immunoreactive
neuron tPV+ I in layer 3 of area 24c. Dark PV-iinmunoreactive puncta
surround the cell bodies of unlabelled neurons ( n ) . E, F: Numerous
PV-immunoreactive puncta (small arrows) encircle t h e unlabelled
somatic profiles of two cells ( n ) .The nuclei and nucleoli of both neurons
are visible. Cell E is located in layer 3 of area 25, while cell F is located in
layer 5 of area 24b. G: Layer 3 , area 24c. The soma of a n unlabelled
neuron ( n ) is surrounded by PV-immunoreactive puncta (example
indicated by thin arrow). PV-immunoreactive puncta also encase (thick CR-, PV-, and CB-immunoreactive structures were investi-
arrows) a thin unlabelled process that emerges from t h e lower surface
of the cell. A neighbouring immunoreactive neuron ( P V + ) is indicated.
!The arrows identify structures shown in H and J.) H: Higher
magnification photomicrograph of unlabelled neuron ( n ) and t h e
perisoinatic immunoreactive puncta (small arrows). The descending
immunoncsgative process remains surrounded by PV-immunoreactive
puncta for ovcr 30 p m (thick arrows). Inset J: Drawing of the
PV-immunoreactive puncta associated with the unlabelled neuron In) structures found in corresponding tissue reacted with only
and the fine process descending from its soma. K GABA immunoreac-
tivity in layer 3 of area 25. The somata of two GABA-immunonegative
pyramidal neurons (PI are covered by GABA-immunopositive puncta
(thin arrows I . The proximal part of the apical dendrite (ad) of one of the
cells is siirrounded by immunoreactive puncta. In addition a descending
string of GABA-immunoreactive puncta is present a t the basal pole of
the same pyramidal neuron (thick arrows). L Layer 4, area 32.
GABA-immunoreactive puncta encircle the soma of a n unlabelled
neuron !nl. M: Soma and proximal apical dendrite ( a d )of an unlabelled
pyramidal neuron (PI in layer 3 of area 24c is surrounded by GABA- H-L). When such interactions occurred, fibre swellings
immunoreactive puncta (arrows).Calibration bars: A, C, D, K,M = 20
p m ;H = 15 pm: J = 12.5 pm; B, E, F, G. L.
.591
showed minor outlying peaks in the range of 19-23 Fm
(Fig. 24), derived from large PV+ and CB+ cells.
NAIIPHdiaphorase histochemistry in comhination with
(peroxidaselllAB) inimunoc.ytochemistry
Cells displaying both N N P H diuphorase activity and
im.munoreactivity. Neurons displaying combined diapho-
rase activity and specific immunoreactivity (for either CR,
PV, CB, or GABA) were readily identified by brown nuclear
labelling contrasted against a blueipurple cytoplasm con-
taining the formazan end-product of the NADPH diapho-
rase reaction (Fig. 25A). Such double-labelled neurons were
uncommon and only encountered within the immunoreac-
tive regions of the tissue. No direct correlation could be
established relating the coexistence of NADPH diaphorase
activity and immunoreactivity (either for CR, PV, or CB)
with staining intensities nor size of labelled somata.
The relative infrequence of diaphorase-reactiveiimmuno-
labelled cells resulted from two confounding factors, the
limited penetration of antisera into tissue sections coupled
to the comparative rarity of diaphorase reactive neurons in
the mPFC. (Comment: diaphorase-reactive neurons consti-
tute 0.20%-0.32% of the cortical neuron population in
monkey mPFC; see Table 15B in Gabbott and Bacon, 1996.)
However, CR, PV, CB and GABA immunoreactivities
were found to be separately colocalised within a proportion
of diaphorase-reactive neurons in layers 2-6 and the under-
lying white matter (Fig. 25A-K; quantitative data are gwen
in Gabbott and Bacon, 1996). Neither the CBPs nor GABA
immunoreactivities were colocalised in layer 1 diaphorase-
reactive cells. The rarity of these neurons together with the
above mentioned technical considerations may have been
responsible for this finding. Furthermore, within the cortex
proper, it was not possible to differentiate between CR+,
rons solely on the basis of either dendritic tree morphology
or specific features (e.g., size and shape) of the cell body
(Fig. 25).
In addition to the distribution of diaphorase-reactive
puncta described above, some puncta were also found
closely apposed to the somata and both proximal and distal
processes from CR-, PV-, CB-, and GABA-immunoreactive
neurons (Figs. 45; 26N,O,P). Conversely, CR+ and CB+
immunoreactive puncta were found in close proximity with
cell bodies and processes of diaphorase reactive neurons.
Dual immunocytochemical labelling: CRIPV, CBIPV,
and CRiCE Using a double immunolabelling technique
(Bevan et al., 1994) the structural interrelationships of
gated in the monkey mPFC.
Comment on methodology. Following specific dual im-
munostaining, DAB-labelled structures appeared brown
and SG-labelled structures grey. No cells were found double-
labelled using this technique. Indeed, immunolabelled neu-
rons and processes were morphologically similar to labelled
one antiserum. It was therefore considered that the chroma-
gens SG and DAB were localised in neurochemically dis-
tinct types of cells and cellular processes.
Double CRIPV immunolabelling. Calretinin-immunore-
active fibres were frequently found lying near proximal
processes and cell bodies of identified PV-immunoreactive
neurons situated in layers 213 of the mPFC (Fig. 26A-E,
would be closely apposed to the proximal processes and
somata of PV-immunoreactive neurons (Fig. 26A-E, H-L).
Figure 19
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
The somata of PV neurons were the common targets of
CR+ puncta; between 1 and 8 CR-immunoreactive fibre
swellings were associated with a given PV+ cell body (Fig.
26E,J,L; see also Fig. 28).
Double CRICB immunolabelling. Similar to CRIPV
interactions described above, CR+ immunoreactive fibres
and fibre swellings were found to be associated with
calbindin-immunoreactive cell bodies, particularly CB +
somata located in layer 3. However, unlike CRiPV interac-
tions there was a distinct tendency for CR+ axonal swell-
ings to be predominantly located away from labelled PV+
somata and preferentially onto primary and secondary
dendritic processes (Fig. 26M,Q; see also Fig. 28).
(Overview. It could not be determined whether the CR+
fibres and puncta associated with PV+ neurons were
derived from the same fibres and puncta contacting CB+
cells. The possibility therefore remains of two distinct
populations of CR+ cell, each preferentially contacting the
somata and processes of either PV+ or CB+ neurons [see
Fig. 281.1
Double CBIPV immunolabelling. No definitive charac-
teristic interactions were observed between CB+ and PV+
immunoreactive structures.
DISCUSSION
The present paper provides a detailed and comprehensive
qualitative description of the distribution and morphology
of neurons in the monkey medial prefrontal cortex (mPFC)
displaying immunoreactivity for the calcium binding pro-
teins calretinin (CR),parvalbumin (PV) and calbindin (CB).
The study also describes the morphology and cortical
distribution of NADPH diaphorase-reactive neurons in the
same region of the monkey cerebral cortex.
The investigation concentrated on the mPFC, a region
located in the anterior medial wall that includes Brodmann
areas 24a, 24b, and 24c (anterior cingulate cortex or
anterior limbic cortex), area 32 (prelimbic cortex) and area
25 (infralimbic cortex; Carmichael and Price, 1994; and see
Fig. 1).This paper directly supports several recent investi-
Fig. 19. Calbindin (CB) immunoreactivity. A Multipolar CB-
immunoreactive neuron ( n ) in lower layer 3 of area 24a. Numerous
labelled processes (arrows) radiate from t h e soma. B: CB neuron in
layer 2 of area 32. Small arrows indicate beaded dendrites. A fine
varicose process arises from the descending process proximal to the
soma (thick arrow). C: Immunoreactive neuron ( n ) with labelled
processes (arrowheads)in upper layer 3 of area 24b. A fine side process
(arrows)emerges from t h e principal ascending dendrite. D: Neighbour-
ing CB-imrnunoreactive cells (arrows) in layer 5 of area 24c. Note size
differences. E: P a r t of a CB-immunopositive dendritic process showing
spine-like protrusions (small arrows). F: A descending beaded (small
arrows) process from a CB-immunolabelled neuron gives rise to a n
exceptionally fine axon-like process (thick arrow). G: Bipolar CB-
immunoreactive neuron ( n ) in superficial layer 5 of area 24c. Labelled
processes (thin arrows) ascend into layer 3 and descend through layer 5.
A faintly CB-immunoreactive layer 3 pyramidal neuron is indicated
(thick arrow). c, capillaries. H: Bipolar CB-immunoreactive neuron ( n )
in layer 6 of area 24b with ascending and descending sprays of
immunolabelled processes (arrows).J: Giant CB-immunoreactive neu-
ron ( n i in layer 5 of area 25. Segments of the dendritic processes of this
cell are beaded (arrowheads). One labelled process (thick arrow)
ascends to superficial layers. An axon-like process (small arrow)
emerges from the lower surface of the cell. Inset J' is an enlargement
of the boxed region in J and shows the axon-like process coursing away
from t h e labelled soma (arrows).Calibration bars: B, C, H = 50 pm; A,
G = 25 pm; J .= 20 pm; D = 15 pm; F, J' = 10 p m ; E = 5 pm.
593
gations studying the regional distributions and morpholo-
gies of neurons displaying CR, PV and CB immunoreactivi-
ties in the monkey prefrontal and cingulate cortices (Hof
and Nimchinsky, 1992; Hofet al., 1993; Conde et al., 1994).
The study of Conde et al. (1994) qualitatively studied
Brodmann areas 9-13, 32 and 46 of the prefrontal cortex
and also provided a quantitative assessment of the relative
densities of CR-, PV-, CB-immunoreactive neurons in areas
9, 11 and 46. The current investigation supplements these
observations by examining pre- and infralimbic cortical
areas as well as the anterior cingulate cortex. Furthermore,
the current paper also extends the cyto- and chemoarchitec-
tural observations of Hof and Nimchinsky (1992) and Hof
et al. (1993) by providing a detailed qualitative and quanti-
tative morphological survey of these neuron populations in
the anterior cingulate cortex (areas 24a, b, and c). In
addition, the present study has provided an extensive
account of NADPH diaphorase-reactive neurons in the
mPFC.
Taken together, this collection of studies gives a compre-
hensive description of the areal and laminar distributions
and cellular morphologies of neurons expressing CR, PV
and CB immunoreactivities and diaphorase enzyme activi-
ties in the prefrontal and cingulate cortices of the adult
macaque monkey.
Qualitative areal and laminar distribution
of CBPs in mPFC
The pattern and intensity of labelling within each cortical
area examined here did not vary significantly across areas.
However, in area 32 there was a distinct band of PV+
immunopositive neurons situated in lower layer 3, layer 4,
and upper layer 5 (see below).
The cortical depth distributions of CR-, PV- and CB-
immunoreactive neurons were similar across the mPFC.
Peak cell densities occurred in layer 2Iupper layer 3 for
CR+ neurons and in upper to middle layer 3 for CB+ cells,
whereas maximum density for PV+ neurons was located in
lower layer 3 to upper layer 5. These findings corroborate
the observations of Hof and Nimchinsky (19921, Hof et al.
(1993) and Conde et al. (1994).
Ferrer et al. (1992) have described the distribution and
morphology of CB+ neurons in the temporal neocortex of
normal humans. Similar to the monkey mPFC, CB+
neurons were predominantly distributed within superficial
layers 2 and 3.
CR-, PV-, CB-immunoreactiue cells: local circuit
GABAergic neurons Convergent evidence derived from a
large number of studies investigating the morphology,
distribution and synaptic connectivity of cortical neurons
displaying immunoreactivities for CBPs, indicate that CR,
PV and CB are expressed in well defined populations of local
circuit GABAergic neurons in the monkey cerebral cortex.
The anatomical evidence stems from the cortical and
laminar distributions, as well as the characteristic morpholo-
gies (somatic size and shape, dendritic and, where possible,
axonal morpholojges) of each immunoreactive cell class
compared with the structure of defined local circuit neu-
rons seen in Golgi specimens and in GABA (and related)
immunocytochemical studies. The morphological evidence
is summarised in Table 3, and discussed in detail by Lund
and Lewis (1993) and Conde et al. (1994).
Although Table 3 provides strong evidence for the similar-
ity between neurons displaying CBPs and specific types of
interneurons in the monkey cortex, it is emphasised that
 594
Fig. 20. Composite drawing of calbindin (CB)-immunoreactiveneu-
rons in layers 2-6 of areas 24a,b,c, 25 and 32. Note the morphology and
distribution of cellular processes as well as the size of labelled somata.
Axon-like processes are indicated by arrows. A population of weakly
immunolabelled layer 3 pyramidal neurons (p) is shown. Note also
CB-immunoredctive neuron in the white matter (wm). The layout of
P.L.A. GABBOTT AND S..J. BACON
the figure is described in the legend to Figure 6. Laminar location of
cells (layer/area):a, 2124~;b, 2124b; c, 2/25; d, 2124a; e, 2/32; f, 2124c;
g, 3/32; h, 3124b; i, 3 1 2 4 ~j,; 3124a; k, 3/25; m, 4 1 2 4 ~n,
; 5124a; 0,5/32;
p, weakly CB+ pyramidal-shaped neurons; q, 5124b; r, 6/24a; s, 6/32; t,
wmi24c; u, wm/25; v, wm132. Calibration bar = 100 pm.
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
Fig. 21. Calhindin (CB)-immunoreactiveneurons (A-F)throughout
the cortex (inset).An initial segment of an “axon-like” process is seen
emerb.ng from cell b (small arrow). Note the compact dendritic arbors
Fig. 22. Large calhindin (CBI-immunoreactive neurons distributed
in the deeper layers (insets) of areas 24a,c, 25, and 32. Identified
immunolahelled “axon-like” processes (small arrows) emerged from
these cells and ascended through the cortex. The somatic profiles of
not all CBP-immunoreactive neurons fit the schemata of
classification. Indeed, several classes of CR+, PV+ and
CB+ neurons were present in the mPFC whose identities
could not be catalogued unequivocally, for example, CR+
neurons with radiate dendritic fields in layer 2 or bipolar
PV neurons in layer 3 (see Fig. 15C; see also Table 2 in
Lund and Lewis, 1993). Without clear descriptions of the
afferent and efferent synaptic connectivity of such neurons
(see WilIiams et al., 1992) further classification of these
.595
ramifying near to the cell bodies. Areal location of cells (layeriareal: A,
32; B, 32,; C, 24c; D, 25; E, 24c; F, 24b. Calibration bar = 100 Fm.
neighhouring CB-immunoreactive neurons are shown. Laminar loca-
tion of cells (layeriareai: A, 5124a; B, 5/32; C, upper 6 1 2 4 ~D,
; 5/25; E,
lower 5/32; F, 6 1 2 4 ~Calibration
. bar = 100 um.
cells is problematic. Nevertheless, spine density measure-
ments along representative CR- and CB-immunoreactive
processes indicate that together with the battery of other
morphological criteria, CR+ and CB+ neurons belong to
aspiny or sparsely spiny cell categories, with PV+ neurons
being predominantly smooth cells (Stichel et al. 1987;
Blumcke et al., 1990; Conde et al., 1994).
Immunoreactivity for CB was also expressed in a popula-
tion of layer 3 pyramidal neurons; the somata of these cells
596 P.L.A. GABBOTT AND S.J. BACON

were lightly CB-immunoreactive. This observation has also 1994).However, the functional significance of this labelling
been reported previously (DeFelipe et al., 1989b; Hof and pattern is unclear but may signify chemically distinct
Morrison, 1991; Hof and Nimchinsky, 1992; Conde et al., populations of pyramidal neurons in layer 3. Such neurons
may receive qualitatively and quantitatively similar sources
of synaptic input and themselves innervate a similar spec-
trum of intracortical targets and subcortical target regions.
The localisation of CB and CR immunoreactivities in
neurons that resemble double bouquet cells (Somogyi and
Cowey, 1981, 1984) suggests that either two separate
populations of these cells are present in monkey mPFC or
that the two calcium binding proteins are differentially
expressed within the same class of local circuit neuron (see
Rogers, 1992; Rogers and Resbois, 1992 ). Immunoreactive
neurons with double bouquet morphologies were much less
frequently encountered in the CB+ cell population than in
the CR+ neuron population (Figs. 14 and 20). Indeed, CB+
neurons with double bouquet morpholoses were located
mainly in layer 2, whereas such CR+ cells were predomi-
nantly present in layer 3 (Figs. 14 and 20).
Although CR and CB antisera may exhibit some cross-
reactivity (since a n 86% amino acid sequence homology in
their molecular structures has been identified; Winsky et
al., 1989), Conde et al. (1994) do not report the colocalisa-
tion of CR and CB in cortical neurons in layers 2-6.
Furthermore, while Conde et al. (1994) report C R + double
bouquet cells, they do not specifically mention CB immuno-
reactivity in double bouquet neurons in prefrontal cortex.
Importantly, DeFelipe et al. (198913, 1990) and DeFelipe
and Jones (1992) provide extensive evidence of CB-
immunopositive double bouquet cells in other areas of
monkey cortex, and Ferrer et al. (1992) also report large
CB+ double bouquet neurons in human temporal cortex. It
therefore seems likely that CR and CB are expressed in two
neurochemically distinct populations of double bouquet cell
in monkey mPFC.
With regard to the postsynaptic targets of double bouquet
cells, DeFelipe and Jones ( 1992) have demonstrated ultra-
structurally that the long, narrow, vertically aligned strings
of CB+ puncta, derived from CB+ double bouquet neurons
in monkey somatosensory cortex, formed symmetric synap-
tic contacts on unlabelled dendritic shafts (62%) and onto
dendritic spines (38%)).However, despite the density and
close proximity of boutons within an individual CB+
bouquet, relatively few immunolabelled boutons coverged
onto the same postsynaptic target. Such targets included
the side branches of pyramidal cell apical and basal den-
drites. DeFelipe and Jones (1992) report that apical den-

Fig. 23. GABA immunoreactivity. A GABA immunopositive neu-

rons (G+I in layer 3 of area 24h. Processes (thin arrows) can be seen
radiating from one of the labelled neurons. B: GABA-immunoreactive
neurons ( G + )in layer 2 of area 24b. Beaded processes (arrows) can be
seen arising from one neuron. Immunoreactive puncta are present in
the neuropil (encircled). GABA immunonegative neuron ( G - 1. C:
GABA-immunoreactive puncta (arrows) surrounding two GABA-
irnmunonegative somata (G- 1. GABA-immunolahelled neurons are
indicated ( G + )and immunopositive puncta are shown in the neuropil
(encircled). D Postemhedding GABA-immunoreacted section from
layer 3 of area 24c showing immunopositive ( G +1 and immunonegative
(G- ) neuronal profiles. Note GABA-immunoreactive puncta closely
apposed (arrows) to the somata of the immunolabelled neuron and the
unlahelled cell. (The structural arrangement of GABA-immunopositive
puncta around GABA-immunoreactive soma correlates with known
synaptic interconnections between GABAergic neurons that mediates
disinhibition within cortical circuits: see Mize et al., 1992; Jones et al..
1994.)Calibration bars: A, B, C = 25 pm; D = 10 p m
 CBPs +ve GABA +ve
150 350

m Mean 11 6 pn 3w

x 2% 5 253
c Counts 1800 c counts 1800
? m
0 Range 4 9 27 8 tm Range 5 7 - 26 3 pm
m
LL 150
1; 150
m a
ul
9 loo Q 1w
% 50

0 .__ 0
0 2 4 6 8 10 12 14 16 18 M ZZ 24 26 28 0 2 4 6 8 10 12 14 16 18 273 22 24 26 28

D Circle (p) D. Circle (pm)

Calret +ve Diaphorase

80 80

70 70
Mean 9 0 pm
60 60
x SD 1 9 1 x

5 % 5 % Counts 400
c7
Counts 300 U
L Q m s o Range 5 4 - 17 3 pm
U Range 5 1 - 13 4 pm U
m 3 0 - 3 0
n
4
n
4
M a
10 10

Y.
0 0
0 2 4 6 8 10 12 14 16 18 M 27 24 26 78 0 2 4 6 8 10 12 14 16 18 M 22 24 26 28
D Circle (pm) D Circle (pm)

Parv +ve Diaphorase Layers 2/3

nu 1M
10 Mean 129 p STRONG

60 SD 3 4 8
1 IKI z Mean 1 2 7 p
SD 155
Counts 400
>. r Range 8 1 - 16 2 pn
z 50
/
Counts 300 C
3
tK)
WEAK
3
Mean l 0 5 p
iJ
? s o
LL
Range 6 1 - > 3 0 p
U
H SD 268
Cowls 400
m 3 0 u) Range 6 0 17 1 prn
n
n
4
70

10 /
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
D Circle (pm) D Circle (p)

Calbin +ve Diaphorase Layers 516

80 liy)

10 Mean 11 9pm H Strong STRONG

Mean 1 3 9 p m
1M) Weak SO 1 8 9
SO 3 4 Counts 400
2
).
80
Range 8 5 19 8 Vrn
x.. Counts 300 m WEAK
U Mean 9 8 p n
Range 5 2 25 3 pm ? 6 0 SD
Counts
2 1 400
U
- 3 0
n
4
:: 40
Range 5 0 1 4 7 p

I,.
4
20
20
10

" 0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 I1 2 4 6 8 10 12 14 16 18 20 22 24 26 28
D Circle (p) D Circle (pm)

Fig. 24. 1)il;trihutions of' somatic profile size iD. Circle, p m ) .
Calcitiin hindiiig protein iCRPsl profile distribution derived from
combined pitpulations ccqually sized; n = 6 0 0 )o f C R + , PV+, and CR+
immunowacttvt~cell profiles. Kotc t h e minor outlying peaks in t h e
ratigt, 19 -2:i p.rn for CRPs+ and CABA+ neurons tarrowed). Distribu-
tions for diaphorasc-rc;ictive neurons have been divided into cells
located in superficial t2it'3) and deep 1516) layers, as well as into strongly
and weakly labelled somata. tAbs. Frequency is absolute number ofcells
occurring within each sizc bin.)
 598
Fig. 25. Photomicrographs of tissue sections reacted for diaphorase
enzyme activity (Dia.)in combination with immunocytochemical label-
ling for either calretinin (CR), parvalbumin (PV), calbindin (CB) or
GABA. A Diaphorase and calretinin, layer 516 area 24b. Three distinct
types of cellular labelling are shown: (i) a cell that is diaphorase-reactive
alone (D+ alone); (ii) a neuron that is CRimmunoreactive (CR+ alone);
and (iii)a cell that displays both diaphorase activity and CR immunore-
activity ID+CR+). Since the nuclei of diaphorase neurons are not
labelled with formazan reaction end-product, the primary feature
identifying the specific immunolabelling of diaphorase-reactive neu-
rons is the presence of brown peroxidase labelling of nuclei in diaphorase-
reactive neurons. Note that specific peroxidaseiDAB immunolabelling
in the nuclei of cells that are either solely immunoreactive (CR+) or
diaphorase reactiveiimmunopositive (D+CR+) is clearly a darker
brown than the general nonspecific DAB peroxidase staining (brown
colouration) of the tissue. Note also that the three neurons shown here
were within the zone of immunolabelling. c, capillaries. B: Diaphorase
and calretinin, layer 3 area 24a. Neuron displaying CR immunoreactiv-
ity (CR+ 1 neighbouringa neuron that displays both diaphorase enzyme
activity and CR immunoreactivity (D+CR+ ). C: Diaphorase and calbin-
din, layer 3 area 24c. A neuron that is solely diaphorase-reactive (D+ 1
lacks brown peroxidase immunolabelling of its nucleus. Compare
staining pattern with local CB-immunopositive neuron (CB+ ). Both
P.L.A. GABBOTT AND SJ. BACON
cells lay within the immunoreactive regions of the tissue section. C':
Enlargement of the boxed region in C showing a fine axon-like process
(arrowhead) emerging from the lower somatic pole of the diaphorase-
reactive neuron. D: Diaphorase reactivity and GABA immunoreactivity
in layer 3 of area 24b. A cell that is solely GABA-immunoreactive ( G + )
lies near to a diaphorase-reactiveIGABA immunopositive neuron
(D+G+ ). Note the brown DAB peroxidase reaction product defining the
nucleus of the double labelled cell. E: Colocalisation of diaphorase and
calbindin within a neuron (D+CB+ 1 in layer 6 of area 32. c, capillary. F:
Neuron lying in the white matter beneath area 24c showing both
diaphorase activity and CB immunoreactivity (D+CB+ 1. G: Diapho-
rase reactivity and calhindin immunolabelling colocalised tD+CB+ 1 in
a neuron in layer 3 of area 25. H Large calibre PV-immunoreactive
fibres (small white arrows) present in the neuropil of layer 4 in area 32.
Diaphorase activity and PV immunolabelling are colocalised in a
neighbouring cell (D+PV+1. J: Neuron lying horizontally in the white
matter (wm) immediately below area 32. The cell displays both
diaphorase activity and PV immunoreactivity (D+PV+ ). Note the large
calibre PV-immunoreactive fibres (small white arrows) coursing in the
white matter. K Weak GABA immunoreactivity colocalised with
diaphorase activity in the cell body of a neuron deep in the white matter
beneath areas 24a,b and c. Calibration bars: A, B = 100 pm; D, E, F, G,
H, J = 20 pm; C, K = 10 pm.
 LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I 599

dritic shafts did not receive input from the CB+ boutons see also Figs. 6 and 8a in Williams et al., 1992). Based on
associated with CB+ double bouquet cells. The above two related assumptions: (i) that all cortical pyramids in
observations on the targets of CB+ double bouquet cells mPFC have pericellular baskets and axon cartridges, and
have been substantiated in a recent report in the human (ii)that the observed PV+ punctate structures (baskets and
temporal neocortex (del Rio and DeFelipe, 1995). The above cartridges) were derived exclusively from PV+ basket and
immuno-cytochemical data support ultrastructural studies PV+ chandelier neurons, respectively (Williams et al.,
investigating the postsynaptic targets of Golgi-impregnated 1992), then the results of this investigation indicate the
double bouquet cells (Somogyi and Cowey, 1981, 1984). presence of two subpopulations of basket and chandelier
Whether the postsynaptic targets of CR+ double bouquet neurons in monkey mPFC (see Fig. 27). This does not
cells in monkey mPFC are similar to those of CB+ double necessarily mean that two of these populations of basket
bouquet is presently unknown (see Fig. 29). If the spec- and chandelier cells were PV-immunonegative (Fig. 27),
trum of postsynaptic targets is significantly different be- since the somata of such cells may be immunopositive but
tween the CB+ and the CR+ double bouquet cell popula- the level of PV in their axon terminals below the level of
tions, then the possibility exists for two separate, vertically immunocytochemical demonstrability (Fig. 29).
oriented and overlapping intracortical circuits. These verti- The study of Lewis and Lund (1990) identified two
cally aligned inhibitory local circuits could differentially andneurochemical types of axon cartridge in the frontal and
significantly influence neuronal operations in a radial cylin- occipital cortices of the monkey; these axon cartridges were
der of cortex extending over several cortical laminae (see either PV+ or immunopositive for corticotrophin releasing
below). factor (CRF). Indeed, the concept of distinct populations of
axoaxonic cells and of basket neurons is further supported
by Akil and Lewis (1992). These authors report that PV+
PV+ immunoreactive pericellular axon cartridges may target small pyramidal neurons in the
clusters and axon cartridges superficial layers that project corticocortically, whereas
PV+ pericellular clusters are located around large pyrami-
Cortical basket and chandelier neurons innervate specific dal neurons deep in the cortex that project subcortically.
parts of cortical pyramidal cells (see references in Table 3; Thus, there may be neurochemically distinct populations of
Douglas and Martin, 1990); basket neurons preferentially axoaxonic neurons and basket cells that specifically inner-
innervate the soma and axon hillock, whereas chandelier vate pyramidal neurons associated with given output path-
cells provide highly selective and exclusive synaptic input to ways from mPFC.
the axon-initial segment. Immunocytochemical evidence A variability in the termination pattern of axoaxonic cells
indicates that although both cell types are considered to be in monkey cortex was described in the GolgiiEM study of
GABAergic, subtypes exist containing either neuropeptides DeFelipe et al. (1985). Pyramidal neurons in layers 213 and
or PV reactivities (Williams et al., 1992; Andressen et al., 516 of the sensory-motor cortices were found to have
1993). different numbers of axoaxonic synaptic contacts; on aver-
The distribution, morphology and synaptic connectivity age, layer 213 pyramids had 2-52 synaptic contacts on their
of PV-irnmunoreactive neurons in area 46 of monkey axon initial segments whereas layer 5 pyramidal neurons
prefrontal cortex have been studied by Williams et al. had lower numbers (2-26). These values correspond with
(1992). These authors described PV+ boutons as heavily the data for presumptive PV+ axoaxonic puncta in monkey
innervating pyramidal neurons in layers 2 and 3 of area 46. mPFC, which showed marked differences in the number of
PV+ boutons formed pericellular clusters and axon car- PV+ puncta associated with presumed pyramidal neurons
tridges resembling those described in mPFC (this study). in the superficial and deep layers of the cortex (see Results).
Williams et al. (1992) report that PV+ boutons established Taken together, the above evidence suggests that PV+
symmetrical (Gray type 2) synapses and that there was no basket and chandelier local circuit neurons preferentially
mixture of PV+ or PV- boutons composing a single axon innervate pyramidal cells in layers 213 of monkey mPFC
cartridge. A more recent study by Wouterlood et al. (1995) compared with pyramidal cells in layers 516. Given the
has investigated PV+ cells in the enthorinal cortex of the tactical siting of such input, PV+ pericellular baskets and
rat. Here again, PV+ neurons were most prevalent in layers axon cartridges could differentially influence the activity
213, as were immunopositive processes and puncta. In the and response characteristics of defined pyramidal cell sub-
rat cortex, PV+ boutons were also found to construct populations in the superficial and deep layers of mPFC.
pericellular baskets and axon cartridges in the superficial
layers, similar to monkey prefrontal cortex.
The overall structural complexity of the PV+ labelled
Neurons in layer I
pericellular baskets and axon cartridges resembled those A characteristic type of neuron in the superficial part of
described in previous GolgiiEM investigations and immuno- layer 1 was labelled by both diaphorase histochemistry and
cytochemical studies in the monkey cortex (Somogyi et al., by CR immunocytochemistry (see Figs. 6 and 12, 14).
1982; DeFelipe et al., 1985; Lund and Lewis, 1993; see also Although infrequent, this type of neuron was present in all
Table 3). Although GolgiiEM studies have provided esti- the cortical areas examined and possessed typically ovoid
mates for the convergence of basket and axoaxonic cells somata with long horizontally aligned processes. In appear-
onto a single postsynaptic cortical pyramidal cell (Somogyi ance and location, these cells resemble the Cajal-Retzius
et al., 1982; DeFelipe et al., 1985), such data are not neurons seen in Golg impregnation studies (Marin-Padilla,
presently available for neurochemically distinct subtypes 1984). Marin-Padilla ( 1990) also describes these neurons in
within these interneuron classes. Golgi preparations of human cortex (see also Meyer and
Of particular interest is that PV+ immunoreactive peri- Gonzalez Hernandez, 1993).
cellular clusters and axon cartridges in mPFC were found Similar to the present study, Conde et al. (1994) found
either together or separately around unlabelled somata cells in layer 1 of the monkey mPFC that were CR-
(Fig. 27), many of which were pyramidal in shape (Fig. 18; immunopositive. Conde et al. (1994) also report that these
Figure 26
LOC.41, CIRCUIT SEURONS IN MONKEY mPFC: I ti01

CR+ cells colocalised CB immunoreactivity. Aside of this,

Huntley and Jones (1990) describe CB, PV, and acetyl
cholinesterase immunoreactivities as being useful markers
for Cajal-Retzius neurons in developing monkey neocortex.
Although this latter study did not investigate whether I-:i
these layer 1 neurons also displayed CR+ immunoreactiv-
ity. the issue has been addressed by Vogt-Weisenhorn et al.
(1994) in the developing cortex of t h e rat. These latter
authors concluded t h a t calretinin is a specific marker
protein for Cajal-Retzius cells throughout the whole period
i li
of corticogenesis into adulthood. Hence several lines of
evidence suggest layer 1 CR+ neurons found in this study
are Cajal-Retzius cells. Indeed, Cajal-Retzius neurons may
express specific combinations of all three calcium binding
proteins a t different functional stages during cortical devel-
opment. Furthermore, t h e morphologcal similarity of these
cells with t h e diaphorase-reactive cells found superficially
2 -i
in laver 1 indicates that they probably also have t h e
potential to synthesise nitric oxide (see below).
Labelled neurons and processes
in the white matter
In addition to a small number of calbindin- and parvalbu-
min-immunoreactiveneurons in the white matter, a numeri-
cally large population of diaphorase-reactive neurons was
located below each of the cortical areas investigated. Lund
2-15
and Lewis (1993) report t h e presence of somatostatin
i6

Fig 26. 1,ight microscope evidence for thc interrelationships of I-:i

calrvtinin i(:R], parvalbumin-t PVJ-, and calbindin (CUI-immunoreac-
tive structurcs in areas 24a,b,c, 25 and 32. A: Area 24a, layer 3 .
(:R-inimunort.activc axonal swellings t C R + ; brown] in proximity with
the soma of a PV-immunorcactive neuronal soma tPV+: g e y l . A
process from the P V t neuron is indicated (double-headed arrow). B:
Enlargement o f t h e boxed area in A. One o f t h e C R t axonal swellings is
close1.v apposed with the soma of the PV-immunorcactive neuron
i PV t I The PV labelled process arising from the cell body is indicated
( a r r o w ) .C: Area 32. layer 4. Calretinin-immunctreactive axonal swell- I
ings i C R i 1 abutting the soma (arrows)of a PV immunoreactive neuron
I P V C D, E: Area 24c. upper layer 2. Corresponding colour and
1.

black white photomicrographs of calretinin immunoreactive puncta

I C R + I in closc contact with the soma (single arrows) and the proximal
dendritic shaft cdouble-headed arrow) of a PV immunolabelled neuron
iPV+ I . F: 'I'wn calretinin~immunoreactivepuncta larrowst lying close
to a prcicess coming from a CR-immunolabelled neuron tCR+I. A
PV-imniunolahelled neuron is indicated. G: Area 24b, layer 2. Calretinin-
iminunortwtive puncta (arrow) in close proximity to a calrctinin-
immunolahellcd cell. H: Low power photomicroLTaph of PV-immunore-
active. neurons and CR-immunolabelled axons. Lower layer 3iarea 25.
J: Drawing of the boxed regon in H showing a CR-immunoreactive
fibre with axon swellings ICR-) in close apposition with the soma o f a
PV-imniunnreactivc iPV+ J cell. The CR+ axon climbs over the pro-
cesses iarrowi and cell body of the PV+ neuron. K, L Corresponding
colour and blackiwhite photomicrog-aphs of calretinin-immunoreac- ( 281-12)-immunoreactiveneurons positioned horizontally in
tive puncta ICR+ 1 in close contact with the soma and the proximal the white matter beneath area 46. The strongly beaded
dendritic shaft of the PV immunolabelled neuron (PV+l seen in H and dendrites of these cells set them apart from the neurons
d. (The two chromagens can be readily differentiated Icfcolour and B/W encountered in this study. Interestingly, Shering and Lowen-
f i p r w I.) M and Q: Layer 3, area 24c. Calretinin-immunoreactivc stein (1994)have recently shown in young kittens a n d adult
axonal swellings t C R + . arrows) engage with the processes of calbindin-
immunoreactive neuron tCR+l. N: Lower layer 6, area 24h. A diapho- cats, t h a t corticoefferent axons e m e r g n g from primary
rase-reactive fibre tD+ 1 gives rise to a fibre swelling (arrow D + 1 that is visual, somatosensory and suprasylvian cortices provide
closely associated with thc soma of a calbindin immunolabelled cell. 0 synaptic input to the shafts and dendritic spines of intersti-
and P: Diaphorase-reactive puncta tD+ I in close contact (whitearrows) tial neurons located in the white matter beneath each area.
with thc somata of PV- and CR-immunoreactive cell bodies tPV+, CR+, The function of neuron populations in the white matter is
respc~tivt.1~-i. R Calretinin-immunoreactive neuron ( C R + ) in layer 5 of
area 24c. S: Enlargement of the boxed reb'lon in R showing calretinin- currently unknown, but may relate to cortical development
immunoreactive puncta ICR+, arrows) closely apposed to the soma of (Nieuwenhuys, 1994).
thecalretinin-immunolabelledcell. Calibration bars: H = 50 p m ; A = 25 Thick calibre PV-immunoreactive fibres were common
p m . hl. N. Q =: 20 p m ;B-F,K,L, 0. P,R = 10 pm; G , S = 5 pm. within the white matter and probably represent cortical
 602
A
1 2 3
9 4,9
ais
B
Fig. 27. A Diagram illustrating the three types of PV immunolabel-
ling observed around the somata and axon initial segments (aisl of
pyramidal neurons (PI in the monkey prefrontal cortex: type 1, PV+
puncta located around both somata and axon initial segments; type 2,
PV+ puncta located around both soma and axon initial segments; and
type 3 , PV+ puncta located around axon initial segments. Type 1
arrangements were most common in layers 5/6, while types 2 and 3
were frequently located in layers 2 and 3. (Note: The study of Williams
et al. 119921 has shown that there is no mixture of PV+ and PV-
boutons composing a single axon cartridge.) B: A schematic illustration
showing how the observed pattern of PV+ puncta surrounding the
somata of pyramidal neurons (PI and/or their axon initial segments
(ais)could be derived from two neurochemically different populations o f
basket cell (Ba) and chandelier (axoaxonic) cell (Chl. These cell
populations would be either PV+ (Ba', Ch' I or PV- (Ba', Ch21).
afferents derived from PV+ projection neurons in subcorti-
cal nuclei (Jones and Hendry, 1989).
Diaphorase-reactive cells
The somatic and dendritic morphologies of strongly
diaphorase-reactive neurons in the mPFC indicate that
these cells are also derived from a morphologically diverse
population of smooth and sparsely spiny cortical interneu-
rons. This conclusion is substantiated by other histochemi-
cal studies investigating diaphorase-reactive neurons in the
P.L.A. GABBOTT AND S.J. BACON
Fig. 28. Diagram illustrating the characteristic structural relation-
ships observed between some CR+ puncta and the processes and
somata of CB+ and PV+ cells in monkey mPFC: (1)CR+ puncta are
predominantly distributed over more distal processes (particularly at
branch points; see Fig. 26M) but also over the soma o f CB+ neurons,
and (21 CR+ puncta were more frequently associated with the somata
of PV+ neurons but also contacted proximal processes. Whether
separate populations of CR+ neurons were responsible for these
characteristic structural arrangements is not known.
visual and auditory cortices of the monkey (Sandell, 1986;
Cipolloni and Pandya, 1991) and in the visual and temporal
cortices of humans (Liith et al., 1994; DeFelipe, 1993).
However, absolute evidence for their status as local circuit
neurons in the mPFC would come from details concerning
their axonal morphologies and spectrum of postsynaptic
target structures.
The studies of Valtschanoff et al. (1993; rat), Hashikawa
et al. (1994; monkey) and Luth et al. (1994; human) have
demonstrated that cortical neurons displaying NADPH
diaphorase activity invariably contain immunoreactivity
for NOS, the biosynthetic enzyme of nitric oxide (NO; see
Vincent, 1995). Importantly, Valtschanoff and her col-
leagues have further shown GABA immunoreactivity to be
localised in NOS-containing, NADPH diaphorase-reactive
neurons in rat neocortex.
In monkey mPFC, diaphorase-reactive neurons are not
all aspiny (c.f., Vincent, 1995). The processes of these
neurons display a gradient of spine density, ranging from
being completely aspiny to bearing moderate numbers of
dendritic spines (Fig. 9). Spine-bearing diaphorase-reactive
neurons have been reported in the visual cortex of humans
(Liith et al., 1994) and are also present in rat mPFC
(Gabbott et al. 1995). It is therefore likely that, similar to
their diverse dendritic morphologies, cortical diaphorase-
reactive neurons also have a range of spine densities over
their dendritic processes. Whether distinct subclasses of
diaphorase-reactive cells can be defined quantitatively on
the basis of spine density remains to be determined.
Similar to the observations of the present study, Liith et
al. (1994) report somatic spicules (spines) arising from the
somata of diaphorase-reactive neurons in human visual
cortex. Aside of species and areal differences, the occur-
LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
pia
1
-
Fig. 29. Summary diagram illustrating the structural relationships
between neurons expressing CR, PV and CB immunoreactivities and
pyramidal neurons situated in layers 213 and 516 of monkey mPFC.
Specific m o r p h o l o g d types of neuron are indicated: M, Martinotti cell;
Ng, neurogliaform cell; DB, double bouquet cell; CR, Cajal-Retzius cell;
and Ba. basket neuron. Of special note are chandelier cells in the deep
layers of the cortex ( C h ) . Results of several studies indicate that the
terminal axon cartridges from these neurons are PV-; however,
rence of these structures appears to be much higher in
humans than monkeys; compare Figure 3B in Luth et al.
( 1994) with Figure 5E in this study.
Sandell ( 1986) reports two tiers of diaphorase-reactive
neurons in the primary visual cortex (area 17) of the rhesus
monkey, a finding also reported here for mPFC (see Fig. 7
in Gabbott and Bacon, 1996). Labelled cells in area 17 were
most common in layers 213 and in layer 6iwhite matter.
The significance of such stratified positioning of diaphorase-
reactive neurons in monkey cortex is unclear but could
relate to the termination pattern of thalamic afferents
which occurs in a region between the two tiers of diaphorase-
reactive somata.
Although the axon-like processes of a few diaphorase-
reactive neurons could be traced for some distances from
their cell bodies, these processes frequently disappeared,
possibly due to decreased levels of enzyme activity. This is,
however, in marked contrast to the strongly diaphorase-
reactive varicose fibres seen throughout the cortex (Fig.
4Bi. The origin of these fibres (intrinsic or extrinsic)
remains to be determined.
The finding that diaphorase-reactive punctate fibres en-
circled the somata of unlabelled cells in monkey mPFC (Fig.
603
mPFC
whether the cell bodies of such neurons are also PV- is unknown (?; see
also Fig. 27). The postsynaptic targets of some neurons in mPFC are
unknown (?I. Note the presence of CR+ and CB+ double bouquet cells
in monkey mPFC. Note also that CR+ double bouquet cell input to
pyramidal cell apical dendrites is currently unknown For clarity,
afferent and efferent pathways have been omitted. I Drawing modified
from Williams et al.. 1992; Andressen ct al., 1993: Nieuwenhuys, 1994;
and Conde et al.. 1994.)
4H) has also been observed in layers 2-6 of the temporal
cortex of humans (see Fig. 2B,C in DeFelipe, 1993). In
addition, basket-like formations of diaphorase-reactive fi-
bres have been reported in layers 4-6 of the human visual
cortex (Luth et al., 1994). However, although these struc-
tures were closely apposed around cellular profiles, their
relative simplicity is in marked contrast to the pericellular
baskets derived from true basket neurons seen in Golgi or
immunocytochemical studies (Table 3). As discussed by
DeFelipe i1993),the diaphorase-reactive basket-like forma-
tions are probably produced by ascending serotonergic
afferents to the cortex rather than being derived from
intrinsic sources.
FUNCTIONAL CONSIDERATIONS
CBPs, nitric oxide, calcium, and neuron
function in mPFC
Colocalisation studies revealed that diaphorase-reactive
neurons in the cortex and white matter also contained
GABA immunolabelling and immunoreactivities for CR,
PV and CB (see Results). One common feature of these cell
604
classes is t,he predominant involvement of Ca2+ in their
functional roles (Andressen et al., 1993).
Although the precise functions of CBPs are poorly under-
stood, they are intimately involved in the intracellular
sequestering, buffering and transport of Ca2+ ions (see
reviews by Baimbridge et al., 1992; Andressen et al., 1993).
Neurons expressing CBPs have high metabolic rates and
slow adaption properties. Changes in Ca2+can affect neu-
ron function by altering the time course of the action
potential, via increasing the probability of bursting activity
(as a result of inhibiting calcium-dependent K+ conduc-
tances) and by allowing Ca2+ entry to contribute to the
overall depolarization of cell membranes (as a result of
inhibiting Ca2+-dependent inactivation of voltage-operated
Ca2+channels; Baimbridge et al., 1992).
Kawaguchi and Kubota (1993) have recently demon-
strated that PV+ and CB+ neurons represent two distinct
physiological subgroupings of nonpyramidal neurons in
layer 5 of rat frontal cortex. PV+ and CB+ cells can be
divided, respectively, into fast spiking (FS) and low thresh-
old spiking (LTS) local circuit neurons. FS cells fire repeti-
tively at depolarised potentials in response to synaptic
excitation with virtually no spike frequency adaption,
whereas LTS nonpyramidal cells produce low threshold
spikes at hyperpolarised potentials. These physiological
properties allow for rapid signal propagation from PV and
CB interneurons to postsynaptic targets in the cortex. The
findings of Kawaguchi and Kubota (1993) may also relate to
other cortical layers and areas as well as to other species.
Further, upon binding Ca2+ PV releases Mg2+ which is a
powerful activator involved in a variety of other intracellu-
lar physiological activities that may also influence the
signalling properties of PV+ interneurons (Celio, 1990).
The physiological characteristics of CR+ neurons in the
cortex are comparatively unknown, but may functionally
define this type of cortical interneuron. Finally, the pres-
ence of CBPs in local circuit neurons may protect cells
against the damaging effects of excessive Ca2+influx during
prolonged periods of high activity (Andressen et al., 1993).
The observations that diaphorase-reactive neurons and
CBPs are colocalised in local circuit neurons may underlie
complementary aspects of their intracortical function. Di-
aphorase activity colocalises with the activity of NOS, the
biosynthetic enzyme for nitric oxide (Bredt et al., 1991;
Hope et al., 1991; Vincent, 1995). Intracellular calcium is
essential for the synthesis of NO. Following elevations in
the local concentration of Ca2++, NO is synthesised inter-
nally and released from NOS-containing neurons. Such
rises in intracellular Ca2+ can occur via N-methyl-D-
aspartate (NMDA) gated channels or by the release of Ca2+
from calcium binding stores within the cell. Nitric oxide is a
freely diffusible signal molecule that can exert its neurophar-
macological actions without the direct involvement of ei-
ther synaptic interactions between neurons or specific
membranes receptors (Vincent, 1994, 1995). From diffu-
sion and decay constants, NO could influence the activity of
target structures up to 100 pm from the site of production
(Vincent, 1995); this may explain the relative paucity of
diaphorase-reactive cells in the cortex. Within neurons, NO
specifically regulates the activity of soluble guanylyl cyclase
which synthesises the second messenger cyclic guanosine
monophosphate (cGMP). Increased levels of cGMP can
exert a variety of short- or long-term neuromodulatory
effects, for example, long-term potentiation (LTP). These
effects may underlie other specific aspects of neuronal
P.L.A. GABBOTT AND SJ. BACON
function, such as during neural development, plasticity,
and in learning and memory, not only in the medial
prefrontal cortex but elsewhere in the cerebral cortex (Vogt
and Gabriel, 1993).
In addition to its actions as a neuromodulator (see
above), NO may also act as a potent vasodilator within the
mPFC (Iadecola, 1993; Vincent, 1995). Recent studies have
shown NO to be the endothelium-derived relaxing factor
released from endothelial cells lining blood vessels that
produces strong vasodilation (Iadecola, 1993). Neurons
synthesising and releasing NO may therefore be operating
at two interrelated levels, firstly by directly affecting the
function of local and projection circuits in the cortex, and
secondly, by altering the biodynamics of blood flow in
response to local neural activity within defined regions of
the mPFC (Iadecola, 1993; Gabbott and Bacon, 1993).
Excitation and disinhibition of local circuits
in mPFC
Thalamic afferents to the visual cortex monosynaptically
innervate both pyramidal and spiny neurons, as well as
GABA-immunoreactive neurons (Freund et al., 1985,1989).
These afferents excite their cortical targets (see review by
Douglas and Martin, 1990). This indicates that local circuit
GABAergic neurons are involved at the earliest stage of
information processing in the visual cortex. Whether tha-
lamic afferents (Ray and Price, 1993) and possibly other
inputs to the monkey mPFC (e.g., from other cortical areas,
amygdala, hippocampus, ventral tegmental area, and brain-
stem structures; see Van Hoesen et al., 1993; Finch, 1993)
also innervate local circuit neurons, thereby directly influ-
encing the functioning of intracortical inhibitory mecha-
nisms, remains to be determined. It can be envisaged that
local inhibitory circuits within the cortex could be strate&-
cally primed by direct feedforward excitation (or inhibition,
see below) following activation of, for example, thalamocor-
tical or hippocampal projection cells (Finch, 1993). Indeed,
the finding that the majority of PV+ cells (basket and
axoaxonic neurons) were found in the principal thalamocor-
tical territories of mPFC supports this idea (Vogt et al.,
1987). Indirect evidence comes from the observations that
afferents to areas 24a, b, and c from other cortical areas
(e.g., areas 25 and 23b of posterior cingulate cortex, and
from regions of the frontal and temporal lobes) terminate
predominantly in layers 2 and 3, where peak cell densities
in both CR+ and CB+ neurons are located.
Of direct significance are the recent observations of Gigg
et al. (1994).These authors show that glutamatergic projec-
tions from the hippocampal formation to the mPFC in the
rat are directly influenced by GABAergic inhibition and
that such inputs converge with glutamatergic projections
from the mediodorsal thalamic nucleus onto individual
cortical neurons. Gigg et al. (1994) speculate that the
source of the GABA-mediated inhibition could be local
circuit neurons in mPFC (see also Finch, 1993).
Pyramidal cells are the predominant targets of local
circuit neurons in the cortex (Douglas and Martin, 1990).
However, synaptic interconnections do occur between inhibi-
tory cortical interneurons (Jones et al., 1994).Such connec-
tions would allow for direct disinhibition within intracorti-
cal networks (see Fig. 23D; Mize et al., 1992). Of particular
interest is the structural relationship between CR+ immu-
noreactive puncta and the somata and processes of PV+ or
CB+ immunoreactive neurons (Fig. 28). In addition, CR+
puncta were found to be closely associated with CR-
 LOCAL CIRCUIT NEURONS IN MONKEY mPFC: I
immunolabelled cell bodies, and PV+ puncta with PV+
somata and processes. These structural relationships could
underlie disinhibitory mechanisms in mPFC (Douglas and
Martin, 1992).One powerful form of disinhibition would be
the CR+ innervation of PV+ basket cells and PV+ axoax-
onic neurons in the superficial layers. Furthermore, the
preferential location of CR+ puncta, more distally on CB+
cellular processes compared with their proximal location on
the somata and initial dendritic processes of PV+ neurons,
indicates selective innervation aimed to differentially affect
the electrotonic properties of the target structures (Douglas
and Martin, 1992). The question arises whether two func-
tionally and morphologically distinct populations of CR+
neurons are involved (see Fig. 28). Conclusive evidence for
this must, however, come from future electrophysiological
and ultrastructural studies.
Local inhibitory circuits within the cortex are directly
innervated by inhibitory afferents ascending from subcorti-
cal sources. Recent studies combining tract-tracing and
immunocytochemistry have shown that different popula-
tions of inhibitory cortical interneurons (identified as con-
taining either somatostatin, parvalbumin or calbindin im-
munoreactivities) are monosynaptically innervated by
afferents from GABA+ neurons in the basal forebrain
(Freund and Gulyas, 1991; Freund and Meskenaite, 1992).
Such innervation, albeit very sparse compared with other
sources of afferentation to local circuit neurons in the
cortex (Vogt and Gabriel, 1993), could underlie defined
functional aspects of information processing within local
cortical networks (see Discussion in Wilson et al., 1994).
The innervation of selected PV+ axoaxonic and PV+
basket cells by ascending afferents to mPFC could influence
widespread populations of pyramidal neurons that are
involved in specific corticocortical and subcortical path-
ways. Of related interest, is that dopamine immunoreactive
boutons, presumably origmating from the dopamine-
containing projection neurons in the ventral tegmental
area, form symmetrical synapticjunctions with the somata,
dendritic shafts and spines of identified pyramidal cells in
the prefrontal, cingulate and motor cortices (Goldman-
Rakic et al., 1989). Goldman-Rakic et al. (1989) also report
dopamine-immunopositive terminals innervating the so-
mata of GABA-containing cortical interneurons. Since inhi-
bition is the most commonly reported effect of dopamine on
the spontaneous activity of neurons in the prefrontal cortex
(Vogt and Gabriel, 19931, such innervation could represent
a further example of ascending feedforward disinhibition of
local circuit neurons in mPFC. The functional significance
of these and other nonspecific afferents to mPFC is poorly
understood (Crino et al., 1993).
As mentioned above, inhibitory mechanisms in the cortex
act in concert to control the output pathways provided by
the pyramidal cell populations in layers 213 and 516. Figure
29 is a summary schematic diagram illustrating the struc-
tural interactions between defined classes of CR+, PV+
and CB+ local circuit neurons and pyramidal cell popula-
tions in the mPFC of the monkey.
Human mPFC and neuropsychiatric disorders
Changes in the functional architecture of local circuits
within the human prefrontal cortex and in the cingulate
cortices (posterior and anterior divisions) have been identi-
fied as underlying neuropsychiatric disorders such as Pick’s
disease (Arai et al., 1991), Alzheimers disease (Braak and
605
Braak, 1993), and in schizophrenia (Vogt and
Gabriel, 1993; Akbarian et al., 1995; Benes, 1995).
Several clinical investigations have clearly indicated that
the pathophysiology of schizophrenia may involve marked
alterations of intrinsic circuits within the anterior cingu-
late cortex (areas 24a, b, and c; Benes et al., 1993). Indeed, a
statistically significant reduction was found in the absolute
number of local circuit cells in layer 2 of area 24 (Benes et
al., 1991).Whether a particular type of local circuit neuron
was affected was not determined. Interestingly, afferents
from the basal nuclei of the amygdala to the areas 24a, b
and c of the cingulate cortex terminate specifically within
deep layer 1 and layer 2 (Vogt and Pandya, 1987), the
cortical zone with peak numbers of CR+ local circuit
neurons. Given the central importance of inhibitory mecha-
nisms in cortical function (Mize et al., 1992; Douglas and
Martin, 19921, future studies should therefore investigate
the extent to which specific types of cortical interneurons,
embedded in functionally strategc local circuits in the
mPFC, are differentially affected by psychiatric disorders
and long-term alterations in emotional behaviour (Neafsey
et a]., 1993; Benes, 1993a,b, 1995; and Braak and Braak,
1993).
CONCLUSIONS
This study has documented the form and distribution of
neurons in the mPFC (areas 24a, b, c, 25 and 3 2 ) of the
monkey that express the calcium binding proteins CR, PV,
and CB. Also investigated were cortical neurons displaying
diaphorase enzyme activity. This enzyme activity coloca-
lises with nitric oxide synthase, the biosynthetic enzyme of
nitric oxide. Morphological evidence from a variety of
sources indicates that these cell populations are local circuit
neurons subserving GABA-mediated inhibitory mecha-
nisms in the monkey mPFC.
Moreover, the neurochemically identified neurons inves-
tigated in this study strongly resemble several classes of
local circuit neurons seen in Golgi preparations of human
prefrontal cortex (Mrzljak et al., 1988, 1990, 1992). Hence
this study, in combination with previous reports (Hof and
Nimchinsky, 1992; Lund and Lewis, 1993; Hof et al., 1993;
Conde et al., 1994; Vogt et al., 1995) provide realistic
structural models with which to assess alterations in the
morphology of local circuit neurons in the prefrontal and
cingulate cortices as a result of psychiatric and emotional
disorders in humans.
The companion paper (Gabbott and Bacon, 1996) pro-
vides data that will allow such structural alterations to be
assessed quantitatively.
ACKNOWLEDGMENTS
The excellent technical assistance of Paul Jays, Tracy-
Ann Warner, Angela Robinson, and Leslie Annetts is
gratefully acknowledged. This work was supported in part
by the MRC (UK). The material used in this study was
kindly provided by Dr. Kevan A.C. Martin (MRC Anatomi-
cal Neuropharmacology Unit, Oxford) and Professor Alan
Cowey (Dept. of Experimental Psychology, Oxford). Paul
Gabbott is a Beit Memorial Research Fellow and Sarah
Bacon is a Bristol-Meyers Squibb Research Fellow.
 606
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