Brain Research 864 (2000) 163–175

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Research report

Distribution of NADPH-diaphorase cells in visual and somatosensory

cortex in four mammalian species
˜ G. Franca a , Eliane Volchan a , *, Neeraj Jain b , Kenneth C. Catania b , Rita L.S. Oliveira a ,
Joao
Felipe F. Hess a , Marcio Jablonka a , Carlos E. Rocha-Miranda a , Jon H. Kaas b
a
´
Laboratorio de Neurobiologia II, Instituto de Biof ´ısica Carlos Chagas Filho, CCS-Bl. 6, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
21949 -900 RJ, Brazil
b
Department of Psychology, Vanderbilt University, Nashville, TN 37240, USA

Accepted 19 January 2000

Abstract

The distribution of the well-labeled nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) Type I neurons was evaluated
in the isocortex of four mammalian species: the Didelphis opossum, the Monodelphis opossum, the rat and the marmoset. In Didelphis
opossum, laminar distribution was examined in tangential and non-tangential sections. The density increases from superficial to deep
layers of the gray matter. In rats’ tangential sections, infragranular and supragranular layers have higher density than layer IV. Cell density
measurements in the visual and the somatosensory cortices were compared in tangential sections from flattened hemispheres of the four
species. Somatosensory areas were identified histochemically in rat (barrel fields) and marmoset (S1 and S2 / PV). In the opossums, areas
S1 and S2 / PV were identified by multiunit recording. Except in the rat, primary visual cortex (V1) was labeled histochemically by
NADPHd and / or cytochrome oxidase. In the four species, cell density in somatosensory cortex was significantly higher than in visual
cortex. Taken together these results demonstrate that NADPHd Type I neurons are not homogeneously distributed in the isocortex of these
mammals. In conclusion, the tangential distribution of Type I neurons in the sensory areas examined, but not its laminar distribution, was
similar in the four species. Given that rats, marmosets and opossums are distantly related species, and that the latter are considered to have
more ‘generalized’ brains, it is conceivable that this pattern of tangential distribution of Type I neurons is a general feature of mammalian
isocortex.  2000 Elsevier Science B.V. All rights reserved.

Themes: Sensory systems

Topics: Somatosensory cortex and thalamocortical relationships

Keywords: Barrel; Visual cortex; Histochemistry; Marsupial; Primate; Rat

1. Introduction inhibitory and / or modulatory roles. Inhibitory non-pyrami-

The mammalian isocortex contains cellular populations
expressing different morphologies and a variety of neuro-
transmitter molecules. Based on the cell morphology, two
major neuronal populations are identified. Pyramidal cells
represent the majority of the isocortical neurons. Gluta-
mate, the ubiquitous excitatory neurotransmitter, is ex-
pressed in these cells. Non-pyramidal cells, on the other
hand, are more heterogeneous. They are composed of
diverse morphological types [34,40] and play excitatory,
*Corresponding author. Tel.: 155-21-290-6897; fax: 155-21-280-
8193.
E-mail address: evolchan@biof.ufrj.br (E. Volchan)
dal cells use g-aminobutyric acid (GABA) as their neuro-
transmitter and generally co-express molecules with neuro-
modulatory functions [35,38].
One kind of GABAergic non-pyramidal neuron [59] is
currently under intense investigation. It expresses the
enzyme NADPH-diaphorase (NADPHd), and was first
described by Thomas and Pearse in the early 1960s
[57,58]. They revealed this neuronal subpopulation using a
simple histochemical method that was improved by
Scherer-Singler et al. [51] 20 years later.
The interest on NADPHd-expressing neurons increased
after the characterization of this enzyme in fixed brain
tissue [6,30,43] as the nitric oxide synthase (NOS). This
enzyme synthesizes nitric oxide (NO), a gaseous molecule

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02058-8
164 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175

with a very short half-life [7,13]. In the nervous system,
NO acts as an unusual messenger [6,13] that has various
physiological roles, such as synaptic plasticity [4,32,52–
54], control of cerebral blood flow [18,31] and modulation
of neuronal responses in the visual system [11,36]. NO is
also involved in the establishment of synapses during
development [17,63], and might be associated with learn-
ing and memory [19,47].
The neurons labeled by either NADPHd histochemistry
or NOS immunohistochemistry can be further subdivided
in two categories. Type I neurons are intensely labeled.
They present a pattern rich in morphological details
resembling a Golgi impregnation [41,64] and do not
comprise more than 2% of all cortical neurons [26]. Type
II neurons are generally more numerous and are weakly
labeled. They have faint somas and few or no labeled
processes [41,64]. The Type I cell could be identified in all
species so far studied, but the Type II neuron is not found
in lower vertebrates, nor in some mammalian species such
as the mouse and the rat [64].
Although there are various descriptions of the morpholo-
gy of cortical Type I NADPHd / NOS-labeled neurons, few
studies have systematically analyzed the distribution of
these cells. Some qualitative and non-systematic evalua-
tions of the areal distribution of Type I NADPHd / NOS-
positive neurons in the cortical gray matter indicate that
they appear to be equally distributed among the various
areas [16,28,64]. Other studies, however, pointed to the
fact that different cortical areas possess different numbers
of NADPHd / NOS-positive cells [3,15,45,55,60]. Volchan
and Franca [60] were the first to analyze the distribution of
Type I NADPHd-positive neurons in a systematic way.
They demonstrated that the tangential distribution of
intensely labeled NADPHd cells in the opossum (Didelphis
aurita) isocortex is not at all uniform. Further experiments
from our group showed that the isocortex of this marsupial
has actually a high density of NADPHd neurons at the
lateralmost part of the parietal and temporal cortices [22].
This high concentration of labeled neurons seemed to be
localized at the somatosensory representation of the snout
[22]. In contrast, the caudalmost part of the opossum brain,
where the visual areas are located [42,56], has less labeled
neurons [60]. However, a systematic examination of the
distribution of Type I neurons in visual and somatosensory
cortices is still lacking.
The aim of the present work is to compare the dis-
tribution of NADPHd neurons in these sensory areas in
different mammals. The rat and the marmoset were chosen
in this investigation because their somatosensory cortex
can be easily revealed by histological procedures [37,62].
We also studied a smaller opossum, the Monodelphis
domestica, which is about the size of the rat. Therefore, we
used representative species distantly related in the lines of
mammalian evolution (marsupials, rodents and primates),
reasoning that common features in members of these three
groups would likely be widespread among mammals.
Additionally, focusing the investigation of the distribution
of Type I neurons in somatosensory and visual areas could
reveal a heterogeneity not detected in previous studies in
the rodent [3,64] and the primate [16,28]. We also com-
pared two more closely related opossum species to evalu-
ate the possibility of significant variability within tax-
onomic groups. Our preliminary results have been pre-
sented elsewhere [22,23,25].
2. Materials and methods
Adult specimens of four different mammalian species
were used (Table 1): the opossum Didelphis aurita (n56),

Table 1
Cases studied a
Hemisphere Species Section Plane of Histology Electro-
thickness section physiology?
(mm)
DF05 Didelphis 60 Coronal di / nc No
DF04 Didelphis 60 Tangential di No
DF08 Didelphis 60 Tangential di No
DF09 Didelphis 60 Tangential di Yes
9706 Didelphis 30 Tangential dd / co Yes
9708 Didelphis 30 Tangential dd / co Yes
9637 Monodelphis 40 Tangential di / co Yes
9685 Monodelphis 50 Tangential di / dd No
9686 Monodelphis 50 Tangential dd / co Yes
9687 Monodelphis 50 Tangential dd / co Yes
9691 Marmoset 100 Tangential di / co No
9695R Marmoset 60 Tangential di / co No
9695L Marmoset 60 Tangential di / co No
R1 Rat 30 Tangential di No
R2 Rat 60 Tangential di No
a
Abbreviations used: dd, diaphorase ‘direct’ method; di, diaphorase ‘indirect’ method; co, cytochrome oxidase in alternate sections; nc, Nissl
counterstaining in alternate sections. The bar indicates that two different histological protocols were applied to adjacent sections.
 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
the opossum Monodelphis domestica (n54), the marmoset
(Callithrix jacchus: n52) and the Lister Hooded rat (n5
2). One hemisphere of each animal was studied, except in
one marmoset (case 9695) in which both sides were
analyzed. The organization of the somatosensory areas of
three Monodelphis domestica (cases 9637, 9686 and 9687)
are described in detail elsewhere [8].
The hemispheres were used to compare NADPHd cell
labeling in visual versus somatosensory areas of isocortex.
Somatosensory areas were evaluated by electrophysiologi-
cal mapping in the opossums and by histochemical pro-
cedures in the marmoset and the rat. Except for the rat,
primary visual cortex was identified using either cyto-
chrome oxidase or NADPHd histochemistry (see below).
Efforts were made to minimize animal suffering and to
reduce the number of animals used. No alternatives to in
vivo techniques were available to perform this study. The
protocols for these experiments were approved by both the
Institute of Biophysics’ and Vanderbilt University’s
evaluation committees for the care and use of animals in
research and were in accordance with the NIH guidelines.
2.1. Electrophysiology
Somatosensory areas were mapped by multiunit record-
ing in three Didelphis and three Monodelphis (Table 1).
The animals were anesthetized with urethane (125 mg / 100
g initial dose, i.p.). Additional doses were given as needed
during the experiment (usually 85 mg / 100 g, 2 and 4 h
later). The cortex of one hemisphere was partially exposed
and photographed. Recordings were made with low impe-
dance (0.8–1.2 MV) tungsten microelectrodes inserted
perpendicular to the pial surface. Each recording site was
marked on the photograph. Touch, pressure, deflection of
hairs and passive limb manipulation were used to map
multiunit receptive fields [2]. After the recording session,
electrolytic lesions were made at about 400 mm from the
surface. This was performed in three to five selected sites
to help the reconstruction of the maps from histological
sections.
2.2. Perfusion
At the end of the recording session, opossums were
deeply anesthetized with an overdose of urethane. Opos-
sums not recorded were anesthetized with a lethal dose of
pentobarbital. Marmosets were terminally anesthetized
with an intraperitoneal dose of sodium thiopental; and rats,
by inhalation of chloroform.
Perfusion procedures were basically the same in all
cases. It consisted of transcardiac or intraaortic perfusion
with saline, followed by freshly prepared 2–4% parafor-
maldehyde in 0.1 M sodium phosphate buffer (PB), pH
7.4, and by 20 and 30% sucrose phosphate buffer solu-
tions. Brains were removed from the skull and the iso-
cortex was dissected and flattened between glass slides.
165
The flattened cortex was soaked overnight in 30% sucrose
PB. The only exception was the brain of Didelphis DF05,
which was blocked and cut coronally from rostral to caudal
end of the isocortex.
2.3. Histological procedures
Except for animal DF05, all cortices were cut parallel to
the flattened cortical surface on a freezing microtome.
Section thickness ranged from 30 to 100 mm in different
specimens (see Table 1). Sections from all brains were
processed to reveal the NADPHd activity (modified after
Scherer-Singler et al. [51]) In some hemispheres, alternate
free-floating sections were also incubated in histochemical
solution for cytochrome oxidase [61] (Table 1). The brain
of DF05 was cut coronally and every two sections in 10
were collected. Both were reacted for NADPHd, and one
of them was counterstained with cresyl violet. All reagents
were from Sigma. Two different protocols for diaphorase
were applied in different brains (Table 1). For the malic
enzyme ‘indirect’ method, sections were incubated in a
solution containing 0.6% malic acid, 0.03% nitroblue
tetrazolium, 1% dimethylsulfoxide, 0.03% manganese
chloride, 0.1% b-NADP and 1% Triton X-100 in 0.1 M
Tris, pH 8.2. The ‘direct’ method consisted of a solution of
0.1 M Tris, pH 7.4, containing 0.08% b-NADPH, 0.04%
nitroblue tetrazolium, 0.5% Triton X-100 and 1.25%
dimethylsulfoxide. In the Monodelphis 9685, both proto-
cols were tested in alternate sections of the same hemi-
sphere. For cytochrome oxidase, the sections were incu-
bated in 0.1 M PB (pH 7.4) containing 0.05% diaminoben-
zidine, 0.02% catalase and 0.03% cytochrome c. For all
histochemical procedures, reactions were visually moni-
tored during incubation. When a clear pattern of reactive
neuropil appeared, the histochemical reaction for cyto-
chrome oxidase was interrupted. For NADPHd histoch-
emistry, the presence of strongly labeled cells along the
plane of section and across its thickness was required to
interrupt the reaction. The duration of the incubation
ranged from 2 to 6 h for NADPHd, and from 4 to 18 h for
cytochrome oxidase. For both histochemical procedures,
overfixed tissue needed a longer incubation period for
adequate neuropil and cell labeling. Following incubation,
sections were rinsed in PB (pH 7.4), and mounted on
subbed slides. The slides were air-dried, dehydrated and
coverslipped with Entellan (Merck) or DPX (BDH Chemi-
cals).
2.4. Quantification
Selected sections were drawn with the help of an
Axioplan / Zeiss microscope connected to homemade mor-
phometric software running on a personal computer. This
software allowed us to depict relevant anatomical borders
of sections and mark both the position of electrolytic
lesions (in brains from specimens that underwent elec-
166 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
trophysiology) and somas of heavily labeled NADPHd
cells. The number of intensely labeled (Type I) cells in a
given area was counted. This area was measured with the
help of a digitizing table, and the density was calculated in
mm 3 , considering the thickness of the section.
In the coronally cut Didelphis hemisphere (DF05),
laminar distribution of labeled neurons was evaluated in 12
Nissl counterstained sections sampled from stereotaxic
frontal plane A1.8 to A10.2 [48]. All cortical layers,
including the white matter, were analyzed. In the flattened
hemispheres, sections from regularly spaced depths of the
whole cortical thickness were sampled. Cell densities were
estimated in somatosensory and visual areas.
In Didelphis and Monodelphis, the location of somato-
sensory areas was evaluated by multiunit electrophysiolo-
gy. The S1 / S2 border was estimated by reversals in the
progression of receptive field positions representing the
glabrous nose [2]. For Didelphis DF04 and DF08, which
did not undergo recordings, three other regions, besides
V1, were defined based on the anterior peristriate and
orbital fissures: the peristriate region, between V1 and the
anterior peristriate fissure; the parieto-temporal region,
between this and the orbital fissure; and the rostralmost
frontal region.
In marmosets, S1 and S2 / PV stand out in myelin stains
[37]. In the present study, this pattern was best revealed by
cytochrome oxidase histochemistry. As in the opossum
brains, primary visual cortex of marmosets was stained by
both NADPHd and cytochrome oxidase histochemistry
(Fig. 1). In this primate, the analysis was performed in the
opercular region of V1 dorsal to the calcarine fissure.
In the rat, NADPHd histochemistry was used to reveal
barrels [21,24]. The barrel fields were reconstructed from
sections through layer IV and were projected to upper and
lower layers to estimate cell density in primary somato-
sensory cortex (S1). This reconstruction included the
postero-medial barrel subfield (PMBSF), the representation
of the anterior snout and of the fore- and hindlimb. We will
use the terms ‘barrel fields’ and ‘S1’ interchangeably.
Density of labeled neurons were also evaluated in the
caudalmost third of the rat isocortex. We assumed that the
visual areas were represented in this region.
3. Results
3.1. Patterns of neuropil reactivity
The diffuse distribution of the colored histochemical
reaction product corresponds to neuropil labeling (Fig. 1).
This diffuse activity cannot be assigned to any particular
cellular profile. Irrespective of the marker used, the more
intense the fixation the fainter this neuropil labeling. In
alternate sections reacted for NADPHd and cytochrome
oxidase histochemistry, the pattern of neuropil labeling
was similar. Moreover, we could not detect differences in
neuropil labeling obtained with either the ‘direct’ or the
‘indirect’ NADPHd protocols.
In Monodelphis, area V1 was clearly depicted by both
histochemical methods. Its anterior border was very sharp.
Peristriate cortex was paler than V1 (caudally) and parieto-
temporal cortex (rostrally) (Fig. 1).
The pattern observed in Didelphis was very similar to
that described for Monodelphis (Fig. 1), except that, for
the former, the definition of borders was sometimes less
clear, specially in the more fixed brains.
In marmosets, neuropil reactivity to both histochemical
methods stained areas S1 (or area 3b), S2, V1, V2 and MT.
As shown in Fig. 1, the cytochrome oxidase neuropil
labeling was a better marker of somatosensory areas than
the NADPHd one. Modular structures such as blobs in V1
and stripes in V2 could be observed with both methods (not
shown).
In rat, barrels can be revealed by means of NADPHd
histochemistry [21,24] (see Fig. 1). This technique clearly
depicted the postero-medial barrel subfield (PMBSF), and
the barrel fields representing the anterior snout, the inferior
jaw, and the forepaw.
3.2. Types of NADPHd labeled cells
NADPHd histochemistry generally revealed two differ-
ent types of labeled cells in isocortex (Fig. 2). One
subpopulation consisted of small lightly stained cell
bodies. Usually we could not detect any processes associ-
ated with these faint cell bodies. This group of cells
corresponded to Type II NADPHd / NOS-positive neurons
(see Section 1). They were most prominent in less fixed
material, especially in the marmoset, where Type II
neurons predominated in superficial sections (probably
layers 2 and 3). Type II cells were not observed in the rat
and very few of them could be seen in the opossum
isocortex.
The other subpopulation of labeled cells corresponded to
well-impregnated Type I (‘Golgi-like’) neurons. These
cells had large cell bodies and their dendritic trees were
very well depicted, resembling a Golgi impregnation. The
criteria used to distinguish between the two subpopulations
included cell body size, the intensity of the cell labeling
and the presence or absence of labeled primary and
secondary dendrites. Although one of the characteristics of
Type I neurons is its intense labeling, in V1 of both
opossum species (Didelphis and Monodelphis), Type I
neurons usually appeared faint. They were classified as
Type I cells due to the presence of well-labeled dendrites
and due to their soma size. Pale Type I NADPHd neurons
were not observed in the other two species. Because the
pattern of labeling and of spatial distribution of the Type I
neurons seemed rather constant irrespective of variations in
fixation conditions, only this subgroup of NADPHd-
labeled cells was quantified.
In marmosets, we could distinguish a third group of
 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175 167

Fig. 1. Bright-field low-power photomicrographs of representative tangential sections. Top row: adjacent sections from a marmoset reacted for cytochrome
oxidase (left) and NADPHd (right). S1 (dashed contour) is well labeled in the cytochrome-reacted section. V1 is well labeled by both histochemical
methods. The arrows point to the calcarine fissure. The lateralmost part of the sections is not shown. Bottom row: sections reacted for NADPHd from a
Didelphis opossum, a Monodelphis opossum and a rat. V1 is well labeled in the opossums, but not in the rat. In the latter, NAPDHd reveals barrel fields.
The dashed contour depicts part of the barrel field corresponding to the representation of the anterior snout and part of the PMBSF. Scale bars52 mm
(except for the rat, where the bar51 mm).

well-labeled NADPHd cells. They were concentrated in 3.3. Areal distribution of intensely labeled ( Type I)
the medialmost part of the sections and consisted of small NADPHd neurons
but intensely labeled cell bodies without labeled processes.
The distribution of this type of cell was not studied. Distribution of Type I NADPHd cell bodies was evalu-
168 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
Fig. 2. Photomicrograph of opossum NADPH-diaphorase labeled neurons: a Type I neuron is seen next to a Type II cell (arrow). Scale bar550 mm.
ated along selected tangential sections from flattened
isocortex. In all four species, the number of these NADPH-
d-labeled neurons in V1 compared to the rest of the section
was consistently low. This was obvious by simple inspec-
tion of most of the sections obtained from Didelphis,
Monodelphis and Callithrix (see examples in Fig. 3), while
in the rat it could be easily detected only in some sections.
The paucity of labeled neurons in V1 of all species and
its concentration in a region corresponding to the location
of somatosensory cortical areas in marsupials [22] led us to
focus our quantitative analysis in these sensory areas. A
total of 65 tangential sections were analysed from the brain
of marmosets, rats and the opossums submitted to elec-
trophysiology (Didelphis (n517), Monodelphis (n514),
marmoset (n511), rat (n523)). Fig. 4A shows a scatterplot
of the density obtained for V1 compared to that of S1 in all
individual sections analyzed. In 61 out of 65 sections, the
density value measured in S1 was higher than in V1. This
difference between the Type I neuron density in S1 and V1
was statistically significant in all four species (Didelphis,
P,0.001; Monodelphis, P,0.05; rat, P,0.001; marmoset,
P,0.01; Wilcoxon t-test). Fig. 5 shows that, in the
Didelphis opossum and the rat, not only does the somato-
sensory cortex concentrate more Type I cells than the
visual cortex, but also the density values in those sensory
areas are, respectively, higher and lower than the density
averaged for the whole section (‘Total’ values).
In marmosets and opossums, we also compiled the
distribution of Type I NADPHd neurons in the lateral
somatosensory areas (S2 / PV). These areas are well
labeled by cytochrome oxidase histochemistry in mar-
mosets. In opossums, electrophysiology allowed us to
identify the border between S1 and S2 / PV. Density of
labeled neurons measured in area S2 / PV of opossums
tended to be higher than in S1 (Fig. 4B). This confirms our
previous findings in Didelphis aurita where we showed a
trend of NADPHd-positive neurons to concentrate in the
lateral parietal cortex [22]. This trend was not observed in
marmosets.
As will be discussed in the next section, density values
are shown to vary with depth. In the opossum species the
distinct density difference of Type I neurons observed
 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175 169

Fig. 3. Drawings of representative tangential sections of brains from the four species showing the distribution of individual Type I NADPHd-labeled
neurons (dots). The asterisks mark the electrolytic lesions made during electrophysiological experiments in Didelphis and Monodelphis opossum. S1,
S2 / PV, and the barrel subfields are outlined.

between somatosensory and visual areas becomes less
clear in the white matter (Fig. 6).
3.4. Laminar distribution of Type I NADPHd neurons in
Didelphis and rat isocortex
In hemispheres cut in the tangential plane, laminar
distribution of labeled cells cannot be precisely determined
because one can potentially capture different cortical layers
in the same section. However, this fact does not preclude
the comparison between visual and somatosensory areas,
since the difference in density favoring the latter was
found in basically all analysed sections (Fig. 4A).
In the rat and the opossum, it was possible to make
further inferences on laminar distribution. In the rat (Fig.
5A), sections showing barrels were considered as taken
170 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
Fig. 4. (A) Scatterplot of the cell density measured in visual cortex (rat)
or V1 (other species) (ordinate) plotted as a function of cell density in S1
(abscissa). Each point in the graph corresponds to a single histological
section. Different labels represent sections from different species. The
line depicts the points where density in S1 equals density in the visual
cortex (or V1). Sections where density in S1 is higher than in V1 are
plotted below this line. (B) Same as in A, but in this case cell density in
S2 / PV is plotted as a function of density measured in S1 for all species
except the rat.
from layer 4. Density was high in the sections immediately
above the ones with barrels (probably corresponding to
layers 2 and 3), dropped in sections with barrels (layer 4),
and rose again in sections from infragranular layers (i.e.,
between sections showing barrels and the ones from the
white matter). This trend was clearest in S1 (Fig. 5A). In
the opossum gray matter, density values increased from the
more superficial to the deeper sections (Figs. 5B, 6 and 7),
with the sections immediately above the white matter
showing the highest values. This was confirmed in coronal
sections (see data from a coronally cut brain below).
In all species, the white matter had a high number of
NADPHd Type I neurons. In the white matter of mar-
mosets, as in other primates [10,20,50], NADPHd Type I
neurons, although not included in our quantification, are
much more abundant than in the cortical gray matter. This
difference is much less pronounced in the other three
species.
A more precise examination of the laminar distribution
was performed in coronal sections of opossum DF05. Fig.
7 summarizes the results. Data from layers 3–4 and 5–6
were grouped together. These data confirmed the observa-
tions on tangential sections: in Didelphis, the density of
Type I cells increased with depth, peaking at the infrag-
ranular layers (see also Figs. 5B and 6).
3.5. Cell labeling using different NADPHd protocols
NADPHd histochemistry can be performed using slight-
ly different protocols. The so-called ‘direct’ method uses
reduced NADP (NADPH) as a co-factor. The ‘indirect’
method uses NADP (non-reduced form). In this case,
NADP has to be reduced by other endogenous enzymes
before being available for the NADPHd enzymatic re-
action. Use of the ‘indirect’ method could be labeling just
a subset of cells that has the enzymatic battery to reduce
NADP.
In one Monodelphis isocortex, the two protocols were
tested in alternate sections (see Table 1). Results obtained
with either method were similar. No differences were
detected in cell or neuropil labeling. Densities of intensely
labeled cells were also similar using either method.
4. Discussion
The main objective of this study was to compare the
distribution of well-labeled (Type I) NADPHd neurons in
the isocortex of different mammalian species. We looked at
flattened hemispheres of the rat, the marmoset, the Mono-
delphis and Didelphis opossums. In the opossum species,
the spatial distribution of NADPHd Type I neurons has a
more conspicuous pattern [22,60] than the rat and mar-
moset. In the Didelphis opossum, the labeled neurons
concentrate in the lateral part of parietal cortex, extending
laterally to temporal cortex and anteriorly to frontal cortex
(Figs. 3 and 6). As a result of such a distribution,
somatosensory areas are more heavily populated by strong-
ly labeled NADPHd neurons than visual cortex. These
differences were quantified in the two cortical areas.
NADPHd and cytochrome oxidase histochemical methods
allowed the identification of V1 in marmoset and opos-
sums, and also of S1 and S2 / PV in marmosets and of
barrel fields in rats. In the opossum species, the histo-
chemical methods did not allow a reliable identification of
 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175 171

Fig. 5. Cell density of Type I NADPHd neurons (ordinate) plotted as a function of the estimated depth of tangential sections (abscissa) from one rat
(Hemisphere R1) and one Didelphis (Hemisphere DF04). Each point represents density measured in a given region (PT or S1, V1 or Visual, and ‘Total’) of
one single section. Schematic drawings depict the cortical regions referred to in the graphs. (A) Sections presenting barrel fields in rat are between the
vertical lines in the graph. S1, barrel fields; Visual, posterior third of rat isocortex; Total, density measured along the whole section. (B) Data from
Didelphis DF04. Values from the surface to the white matter were obtained from sections a to k, depicted in Fig. 6. Sections deeper than 1 mm (j–k)
include the white matter (arrow in the abscissa). PT, parieto-temporal region; V1, primary visual cortex; Total, density measured along the whole section.

the somatosensory areas. In this case, S1 and S2 / PV were
delimited by means of multiunit electrophysiological re-
cording, following the procedures of Beck et al. [2] for the
Didelphis opossum. Recently, a detailed description of the
organization of the Monodelphis somatosensory cortex was
made by our group [8].
In all four species studied in the present work, the
somatosensory cortex was more densely populated with
Type I NAPDHd-labeled neurons than the visual cortex. In
more than 90% of the tangential sections examined, the
density of Type I neurons was higher in S1 than in V1.
Some previous reports using either the histochemistry for
NADPHd or NOS-immunohistochemistry failed to identify
this trend [16,28,39,64]. In primates, Egberongbe et al.
[16] (humans) and Hashikawa et al. [28] (monkeys)
reported that the distribution of Type I neurons is essential-
ly similar in all the cortical regions examined, but these
authors have not performed a systematic quantification of
the Type I NOS-labeled somata. Studying the distribution
of Type I neurons in the cortical white matter of monkeys,
Smiley et al. [55] showed that these cells are evenly
distributed, except for the medial part of primary visual
cortex where they are less numerous. In the monkey
prefrontal cortex, though, it was found that allocortical
(limbic) areas have two to three times more NADPHd
neurons than isocortical (eulaminate) areas, but no differ-
172 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175

Fig. 6. Drawing of a sequence of horizontal sections from Hemisphere DF04. Type I neurons (dots) increase in number from superficial to deep sections
(a–k). The clear gradient of these cells in the lateral parietal and temporal regions with respect to the posterior region fades in the white matter (sections
j–k).

ences between isocortical areas were reported [15]. Kuch-
iiwa et al. [39] found a homogeneous distribution in the
cat. However, their study was limited to the gray matter /
white matter border where we show that, in the Didelphis
opossum, the distribution becomes more homogeneous
(Fig. 6). Yan and Garey [64] reported no areal differences
in the cortices of different mammalian species, but they
examined only the anterior half of horizontal sections, thus
missing the lower values typical of visual areas.
Using SMI 32 immunohistochemistry to parcel the
cortex of the rat, Bidmon et al. [3] classified the different
areas into three different groups, according to the density
of Type I cells. Nevertheless, they could not find statistical
differences between density measurements in primary
 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
Fig. 7. Laminar cell density (mean and standard error) of Type I
NADPHd neurons in different isocortical layers of a coronally cut
Didelphis hemisphere (DF05, n512 sections). The Nissl counterstained
sections ranged from about stereotaxic coordinates A10.2 to A1.8 [48],
which comprise part of the parieto-temporal region, the peristriate region
and part of the striate area.
visual (areas Oc1M and Oc1B) versus primary somato-
sensory (areas Par1, FL and HL) cortices. In addition, both
cortical regions presented fewer cells than the majority of
the other cortical areas examined. Differences with the
present study might be attributable to the following
differences in methods: (1) while Bidmon et al. [3] made
counts in coronal sections, ours were made in tangential
sections which provided better estimates of the areal
distribution; (2) the former work subdivided the barrel
fields (areas Par1, FL and HL) while we considered them
as single area (i.e., S1); (3) Bidmon et al. [3] parceled the
posterior cortex, making separate counts for the different
areas, while our corresponding counts encompassed not
only the primary visual cortex but probably also areas from
the retrosplenial and temporal cortices [66]; and (4) we
showed that the supra- and infragranular layers are dis-
tinctly more populated in S1 than their counterparts in the
visual cortex, while in the granular layer this difference
vanishes (see Fig. 5A). By sampling the gray matter
altogether, Bidmon et al. [3] may have missed this
difference.
In another study of the rat cortex, Moro et al. [45]
demonstrated that the occipital cortex does have a low
density of NOS-positive neurons. In their study, frontal
and perirhinal cortices showed a high density. They
concluded that this pattern of distribution of NOS-positive
neurons fits well with the pattern of the cortical projection
from the nucleus basalis magnocellularis (NBM) (see also
below). Studies of the distribution of NADPHd / NOS
neurons during development also pointed to a lower
density in the posterior part of the cortex of rodents
[5,14,65]. This might suggest an ontogenetic origin for the
heterogeneous distribution of this cortical neuronal sub-
population.
Work in progress at our laboratory [29] demonstrates
that one month before eye opening in the 40-day-old
173
Didelphis there is already a clear concentration of
NADPH-positive neurons in the parietal cortex as com-
pared with the visual cortex. Interestingly, a study of PET
scanning in newborn humans revealed cortical activation
mainly of the somatosensory areas and the cingulate (see
Ref. [9] for a review). Indeed, according to Damasio [12],
the somatosensory cortices have functional roles in emo-
tion and consciousness not paralleled by the visual cortex.
NO has vasodilating properties and is the putative link
between neuronal activation and the increase in cerebral
blood flow (CBF) [31]. The NBM of rats, the correlate of
Meynert’s nucleus in primates, is a forebrain structure that
sends cholinergic fibers to the cortex and is involved in the
control of cortical CBF [45]. The co-localization of
muscarinic receptors in Type I NADPHd-labeled cortical
neurons suggests that these cells can promote vasodilation
after the release of acetylcholine by the afferent fibers from
the NBM [45,55]. In the intensely labeled Type I neurons,
the co-localization of NOS and neuropeptide Y (NPY), a
vasoconstrictor molecule, suggests that these cells can be
the fine controllers of the CBF at restricted (active) regions
of the brain [18]. While the NO release by the cell body
produces vasodilation, this effect can be restricted distally
by the vasoconstrictive action of NPY released by Type I
neuron axons. By this mechanism, the vasodilation would
be circumscribed to the area around the neuronal soma and
dendrites [18]. Our results suggest that this kind of
regulation of CBF is more likely to occur in somatosensory
areas than in the posterior cortical areas, such as V1. An
interesting finding is that, distinctly from the other mam-
malian species, the terminal blood supply in the capillaries
of opossums forms a peculiar ‘true end-system’, since
arterioles penetrating the brain do not form a capillary
plexus but terminate instead in capillary loops [27]. It
remains to be investigated if this fact has some influence
on the spatial distribution of Type I neurons in this species.
It is important to keep in mind that the relative absence
of NADPHd Type I neurons in V1 does not necessarily
mean that this cortical area synthesizes less nitric oxide
(NO) than the rest of the cortex. In this study, we
demonstrated that NADPHd neuropil labeling in V1 is
present in most of the species examined. This NADPHd-
labeled neuropil might represent dispersed NOS along
axons, dendrites, and even cell bodies with low con-
centration of NOS. In monkey layer IV, Aoki et al. [1]
demonstrated the existence of NOS-immunoreactive neuro-
pil that corresponds predominantly to presynaptic termi-
nals. However, it is possible that, in our sections, part of
the NADPHd-reactive neuropil represents unspecific label-
ing due to the activity of other metabolic diaphorases still
functional after light fixation (see Ref. [43]). The sensitive-
ness of the neuropil reaction to the intensity of the fixation
seems to support this idea. The necessity of lighter fixation
protocols to reveal NADPHd neuropil labeling in the adult
cortex might explain why V1 in the opossum was not
detected in a previous study of our group [60]. This may
174 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
also be the reason why barrel fields were not detected in
rodents in other studies besides Franca and Volchan [24]
and Mitrovic and Schachner [44] in very young mice.
S1 and V1 are primary sensory areas that have many
anatomical and functional aspects in common. Both S1 and
V1 are modularly arranged [46] and they represent the first
cortical relay of incoming sensory information. In both
areas, this sensory information is topographically repre-
sented. Nevertheless, a remarkable difference was pointed
out by Rockel et al. [49]. In their cytoarchitectonic study,
they showed that all areas in mammalian cortex have a
constant number of neurons, except V1 of primates which
presents twice as many neurons. This rules out the
possibility that our findings are due to a general trend of
the total neuronal population and, on the contrary,
strengthens the differences in the cell densities of
NADPHd neurons between S1 and V1. These differences
were found in all four species studied. Since three of these
species (marmosets, rats and opossums) correspond to
taxonomic groups that are not closely related; and given
that opossums are considered to have ‘generalized’ brains
[33], it is possible that the relative paucity of Type I
neurons in the visual cortex and their concentration in
somatosensory areas are general features of mammalian
isocortex.
Acknowledgements
We thank Mr Edimilson A. Pereira, Mr Anderson de O.
Souza and Ms Judy Ives for technical assistance. We also
thank Mr Raymundo F. Bernardes for help with the
photography of living brains and their resulting sections.
Ms Suzana G. Franca provided invaluable help with the
final format of some of the figures. This work was
supported by CNPq / NSF, PRONEX, FINEP and FAPERJ.
References
[1] C. Aoki, S. Fenstemaker, M. Lubin, C.-G. Go, Nitric oxide synthase
in the visual cortex of monocular monkeys as revealed by light and
electron microscopic immunocytochemistry, Brain Res. 620 (1993)
97–113.
[2] P.D. Beck, M.W. Pospichal, J.H. Kaas, Topography, architecture, and
connections of somatosensory cortex in opossums: evidence for five
somatosensory areas, J. Comp. Neurol. 366 (1996) 109–133.
¨
[3] H.-J. Bidmon, J. Wu, A. Godecke, A. Schleicher, B. Mayer, K.
Zilles, Nitric oxide synthase-expressing neurons are area-specifically
distributed within the cerebral cortex of the rat, Neuroscience 81
(1997) 321–330.
[4] G.A. Bohme, C. Bon, J.-M. Stutzmann, A. Doble, J.-C. Blanchard,
Possible involvement of nitric oxide in long-term potentiation, Eur.
J. Pharmacol. 199 (1991) 379–381.
´
[5] H. Bravo, O. Inzunza, V. Fernandez, M. Sanhueza, Distribution of
NADPH-d positive neurons during postnatal development of the rat
somatosensory cortex correlates with gradients of neurogenesis and
development, Neurosci. Lett. 234 (1997) 103–106.
[6] D.S. Bredt, C.E. Glatt, P.M. Hwang, M. Fotuhi, T.M. Dawson, S.H.
Snyder, Nitric oxide synthase protein and mRNA are discretely
localized in neuronal populations of the mammalian CNS together
with NADPH diaphorase, Neuron 7 (1991) 615–624.
[7] D.S. Bredt, S.H. Snyder, Nitric oxide, a novel neuronal messenger,
Neuron 8 (1992) 3–11.
[8] K.C. Catania, Jain, N., Franca, J.G., Volchan, E., Kaas, J.H., The
organization of somatosensory cortex in the short-tailed opossum
(Monodelphis domestica). J. Somatosensory Motor Res., (2000) in
press.
[9] H.T. Chugani, Metabolic imaging: a window on brain development
and plasticity, Neuroscientist 5 (1999) 29–40.
[10] E.T. Costa, J.L.M. do-Nascimento, C.W. Picanço-Diniz, J.A.S.
Quaresma, M. Silva-Filho, Histochemical characterization of
NADPH-diaphorase activity in area 17 of diurnal and nocturnal
primates and rodents, Braz. J. Med. Biol. Res. 29 (1997) 1355–
1362.
[11] J. Cudeiro, C. Rivadulla, Sight and insight-on the physiological role
of nitric oxide in the visual system, Trends Neurosci. 22 (1999)
109–116.
[12] A. Damasio, in: The Feeling of What Happens: Body and Emotion
in the Making of Consciousness, Harcourt Brace Jovanovich, New
York, 1999.
[13] T.M. Dawson, S.H. Snyder, Gases as biological messengers: nitric
oxide and carbon monoxide in the brain, J. Neurosci. 14 (1994)
5147–5159.
[14] P. Derer, M. Derer, Ontogenesis of NADPH-diaphorase neurons in
the mouse forebrain, Neurosci. Lett. 152 (1993) 21–24.
[15] S.M. Dombrowski, H. Barbas, Differential expression of NADPH
diaphorase in functionally distinct prefrontal cortices in the Rhesus
monkey, Neuroscience 72 (1996) 49–62.
[16] Y.I. Egberongbe, S.M. Gentleman, P. Falkai, B. Bogerts, J.M. Polak,
G.W. Roberts, The distribution of nitric oxide synthase immuno-
reactivity in the human brain, Neuroscience 59 (1994) 561–578.
[17] A.F. Ernst, H.H. Wu, E.E. El-Fakahany, S.C. McLoon, NMDA
receptor-mediated refinement of a transient retinotectal projection
during development requires nitric oxide, J. Neurosci. 19 (1999)
229–235.
[18] C. Estrada, J. DeFelipe, Nitric oxide-neurons in the neocortex:
Morphological and functional relationship with intraparenchymal
microvasculature, Cerebral Cortex 8 (1998) 193–203.
[19] ´
V. Fernandez, H. Bravo, M. Sanhueza, O. Inzunza, NADPH-d
positive neurons in the developing somatosensory cortex of the rat:
effects of early and late environmental enrichment, Dev. Brain Res.
107 (1998) 299–307.
[20] J.G. Franca, J.L.M. do-Nascimento, C.W. Picanço-Diniz, J.A.S.
Quaresma, A.L.C. Silva, NADPH-diaphorase activity in area 17 of
the squirrel monkey visual cortex: neuropil pattern, cell morphology
and laminar distribution, Braz. J. Med. Biol. Res. 30 (1997) 1093–
1105.
[21] J.G. Franca, R.L.S. Oliveira, E. Volchan, Nitric oxide synthase
barrels in rat somatosensory cortex, Soc. Neurosci. Abstr. 21 (1995)
121.
[22] J.G. Franca, R.L.S. Oliveira, E. Volchan, Lateral parietal and
temporal neocortex of the opossum have a remarkable concentration
of diaphorase-positive neurons, Soc. Neurosci. Abstr. 22 (1996)
1042.
[23] J.G. Franca, R.L.S. Oliveira, E. Volchan, Diaphorase-positive neu-
rons in the neocortex: comparison of their distribution in opossums
and rats, Rev. Bras. Biol. 56 (1996) 188.
[24] J.G. Franca, E. Volchan, NADPH diaphorase histochemistry as a
marker for barrels in rat somatosensory cortex, Braz. J. Med. Biol.
Res. 28 (1995) 787–790.
[25] J.G. Franca, E. Volchan, K.C. Catania, N. Jain, R.L.S. Oliveira, M.
Jablonka, F.F. Hess, J.H. Kaas, Somatosensory cortex has a higher
density of nitridergic neurons than visual cortex in different mam-
malian species, Soc. Neurosci. Abstr. 23 (1997) 1006.
[26] P.L.A. Gabbott, S.J. Bacon, Co-localisation of NADPH diaphorase
 J.G. Franca et al. / Brain Research 864 (2000) 163 – 175
activity and GABA immunoreactivity in local circuit neurones in the
medial prefrontal cortex (mPFC) of the rat, Brain Res. 699 (1995)
321–328.
[27] L.A. Gillilan, Blood supply to primitive mammalian brains, J.
Comp. Neurol. 145 (1972) 209–222.
[28] T. Hashikawa, M.G. Leggio, R. Hattori, Y. Yui, Nitric oxide synthase
immunoreactivity colocalized with NADPH-diaphorase histochemis-
try in monkey cerebral cortex, Brain Res. 641 (1994) 341–349.
[29] F.F. Hess, Distribution of NADPH positive neurons and neuropile in
the neocortex of juveniles and infants opossums. BSc Dissertation,
Federal University of Rio de Janeiro, Brazil (in Portuguese), 1999
[30] B.T. Hope, G.J. Michael, K.M. Knigge, S.R. Vincent, Neuronal
NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci.
USA 88 (1991) 2811–2814.
[31] C. Iadecola, Regulation of the cerebral microcirculation during
neural activity: is nitric oxide the missing link?, Trends Neurosci. 16
(1993) 206–214.
[32] Y. Izumi, D.B. Clifford, C.F. Zorumski, Inhibition of long-term
potentiation by NMDA-mediated nitric oxide release, Science 257
(1992) 1273–1276.
[33] H.J. Jerinson, Quantitative analysis of evolution of the brain in
mammals, Science 133 (1961) 1012–1014.
[34] E.G. Jones, Varieties and distribution of non-pyramidal cells in the
somatic sensory cortex of the squirrel monkey, J. Comp. Neurol.
160 (1975) 205–267.
[35] E.G. Jones, H.C. Hendry, Co-localization of GABA and neuro-
peptides in neocortical neurons, Trends Neurosci. 9 (1986) 71–76.
[36] P. Kara, M.J. Friedlander, Arginine analogs modify signal detection
by neurons in the visual cortex, J. Neurosci. 19 (1999) 5528–5548.
[37] L.A. Krubitzer, J.H. Kaas, The organization and connections of
somatosensory cortex in marmosets, J. Neurosci. 10 (1990) 952–
974.
[38] Y. Kubota, Y. Kawaguchi, Three classes of GABAergic interneurons
in neocortex and neostriatum, Jpn. J. Physiol. 44 (1994) S 145–S
148.
[39] S. Kuchiiwa, T. Kuchiiwa, S. Mori, S. Nakagawa, NADPH diaphor-
ase neurones are evenly distributed throughout cat neocortex irre-
spective of functional specialization of each region, NeuroReport 5
(1994) 1662–1664.
[40] J.S. Lund, Organization of neurons in the visual cortex, area 17, of
the monkey (Macaca mulatta), J. Comp. Neurol. 147 (1973) 455–
495.
[41] ¨
H.-J. Luth, A. Hedlich, H. Heidegard, E. Winkelmann, B. Mayer,
Morphological analyses of NADPH-diaphorase / nitric oxide synth-
ase positive structures in human visual cortex, J. Neurocytol. 23
(1994) 770–782.
[42] S. Martinich, M.N. Pontes, C.E. Rocha-Miranda, Identification and
characterization of visual specializations in the opossum isocortex.
A study of corticotectal cortocortical and commissural connectivity,
J. Comp. Neurol. 416 (2000) 224–244.
[43] ¨
T. Matsumoto, M. Nakane, J.S. Pollock, J.E. Kuk, U. Forstermann,
A correlation between soluble brain nitric oxide synthase and
NADPH-diaphorase activity is only seen after exposure of the tissue
to fixative, Neurosci. Lett. 155 (1993) 61–64.
[44] N. Mitrovic, M. Schachner, Transient expression of NADPH
diaphorase activity in the mouse whisker to barrel field pathway, J.
Neurocytol. 25 (1996) 429–437.
[45] V. Moro, J. Badaut, V. Springhetti, L. Edvinsson, J. Seylaz, F.
Lasbennes, Regional study of the co-localization of neuronal nitric
oxide synthase with muscarinic receptors in the rat cerebral cortex,
Neuroscience 69 (1995) 797–805.
[46] V.B. Mountcastle, The columnar organization of the neocortex,
Brain 120 (1997) 701–722.
175
[47] E. Oermann, H.-J. Bidmon, A. Schleicher, B. Mayer, H. Schwegler,
K. Zilles, The distribution of nitric oxide synthase-I and NADPH-
diaphorase containing neurons in the cerebral cortex of different
strains of mice and its association with learning and memory, J.
Hirnforsch. 39 (1998) 65–75.
[48] E. Oswaldo-Cruz, C.E. Rocha-Miranda, in: The brain of the
opossum (Didelphis marsupialis), Instituto de Biofısica,´ Univer-
sidade Federal do Rio de Janeiro, Rio de Janeiro, 1968, 99 pp.
[49] A.J. Rockel, R.W. Hiorns, T.P.S. Powell, The basic uniformity in
structure of the neocortex, Brain 103 (1980) 221–244.
[50] J.H. Sandell, NADPH diaphorase histochemistry in the macaque
striate cortex, J. Comp. Neurol. 251 (1986) 388–397.
[51] U. Scherer-Singler, S.R. Vincent, H. Kimura, E.G. McGeer, Demon-
stration of a unique population of neurons with NADPH-diaphorase
histochemistry, J. Neurosci. Methods 9 (1983) 229–234.
[52] E.M. Schuman, D.V. Madison, A requirement for the intracellular
messenger nitric oxide in long-term potentiation, Science 254
(1991) 1503–1506.
[53] E.M. Schuman, D.V. Madison, Nitric oxide and synaptic function,
Annu. Rev. Neurosci. 17 (1994) 153–183.
[54] K. Shibuki, D. Okada, Endogenous nitric oxide release is required
for long-term synaptic depression in the cerebellum, Nature 349
(1991) 326–328.
[55] J.F. Smiley, A.I. Levey, M.-M. Mesulam, Infracortical interstitial
cells concurrently expressing M2-muscarinic receptors, acetylcho-
linesterase and nicotinamide adenine dinucleotide phosphate-
diaphorase in the human and monkey cerebral cortex, Neuroscience
84 (1998) 755–769.
[56] A.P.B. Sousa, R. Gattass, E. Oswaldo-Cruz, The projection of the
opossum’s visual field on the cerebral cortex, J. Comp. Neurol. 177
(1978) 569–588.
[57] E. Thomas, A.G.E. Pearse, The fine localization of dehydrogenases
in the nervous system, Histochemistry 2 (1960) 266–282.
[58] E. Thomas, A.G.E. Pearse, The solitary active cells. Histochemical
demonstration of damage-resistant nerve cells with a TPN-diaphor-
ase reaction, Acta Neuropathologica 3 (1964) 238–249.
[59] J.G. Valtschanoff, R.J. Weinberg, V.N. Kharazia, H.H.H.W. Schmidt,
M. Nakane, A. Rustioni, Neurons in rat cerebral cortex that
synthesize nitric oxide: NADPH diaphorase histochemistry. NOS
immunocytochemistry, and colocalization with GABA, Neurosci.
Lett. 157 (1993) 157–161.
[60] E. Volchan, J.G. Franca, Distribution of NADPH-diaphorase-positive
neurons in the opossum neocortex, Braz. J. Med. Biol. Res. 27
(1994) 2431–2435.
[61] M.T.T. Wong-Riley, Changes in the visual system of monocularly
sutured or enucleated cats demonstrable with cytochrome oxidase
histochemistry, Brain Res. 171 (1979) 11–28.
[62] M.T.T. Wong-Riley, C. Welt, Histochemical changes in cytochrome
oxidase of cortical barrels after vibrissal removal in neonatal and
adult mice, Proc. Natl. Acad. Sci. USA 77 (1980) 2333–2337.
[63] H.H. Wu, C.V. Williams, S.C. McLoon, Involvement of nitric oxide
in the elimination of a transient retinotectal projection in develop-
ment, Science 265 (1994) 1593–1595.
[64] X.X. Yan, L.J. Garey, Morphological diversity of nitric oxide
sythesising neurons in mammalian cerebral cortex, J. Hirnforsch. 38
(1997) 165–172.
[65] X.X. Yan, L.J. Garey, L.S. Jen, Development of NADPH-diaphorase
activity in the rat neocortex, Dev. Brain Res. 79 (1994) 29–38.
[66] K. Zilles, A. Wree, Cortex: areal and laminar structure, in: G.
Paxinos (Ed.), Forebrain and midbrain, Academic Press / Harcourt
Brace Janovich, Sydney, Orlando, San Diego, New York, 1985, pp.
375–415.