Download Region Specific Micromodularity in the Uppermost Layers in Primate

Region Specific Micromodularity in the
Uppermost Layers in Primate Cerebral
Cortex
Noritaka Ichinohe and Kathleen S. Rock land
A micromodularity specific to the uppermost cortical layers, layers 1
and 2, is demonstrated in primates. This is most pronounced as
patches of zinc-positive (Zn+) terminations, preferentially in the preRolandic and limbic areas. The upper layer modularity can frequently
be demonstrated by parvalbumin-immunoreactive (PV-ir) GABAergic
terminations and by bundles of apical dendrites. Double-labeling or
alternate section analysis shows that PV-ir and Zn+ terminations comingle at the layer 1,2 border and appear to coincide with dendritic
bundles, proposed to originate from layer 2 pyramidal neurons. This
model is basically similar to the prominent wall-and-hollow honeycomb organization in rat visual cortex. The organization of the PV-ir
and dendritic components, however, is more difficult to define in
primate than in rat. Moreover, the micromodularity is not uniform
across areas. In some areas (motor and limbic), the modularity can
be visualized by both zinc and PV. In other areas (i.e. primary sensory,
sensory associational and prefrontal areas 46 and 8), although PV
immunohistochemisty shows a periodic distribution, there is no
detectable Zn+ modularity. These results add to the evidence for the
complexity of layers 1 and 2 and raise the possibility that patches of
Zn+ terminations correspond to zones of area-specific zinc-related
plasticity. This might figure in the context of top-down or feedback
influences, as often associated with layer 1.
Those deriving from layer 2 pyramidal neurons, in contrast, are
located preferentially in the walls and are hypothesized to form
a specialized complex with Zn+ and PV-ir terminations.
Preliminary screening with PV revealed a honeycomb configuration in several other areas in rat, as well as in several areas
in cat and monkey (Ichinohe et al., 2003b). As a next step, it
seemed important to assess more widely the specific features
of this organization. We have accordingly carried out more
extensive analysis of cortical areas in the macaque. This further
investigation has confirmed the finding of a modularity of the
uppermost layers. In particular, histochemistry for zinc reliably
demonstrates a small-scale upper layer modularity that is
pronounced in the pre-Rolandic motor and limbic areas.
Although our first impression with PV had suggested a honeycomb pattern, the modularity is not uniform. Rather, it shows
a high degree of regional variability in both size and shape,
ranging from small-scale honeycomb or reticulum, to largerscale patches.
The upper layer modularity can also be demonstrated by
immunohistochemistry for PV or for dendritic bundles labeled
by microtubule-associated protein 2 (MAP2), but this tends to
be more subtle (see ‘Single and Double Labeling’ in Materials
and Methods). PV modularity is located more at the border
between layers 1 and 2, while MAP2 labeling extends into layer
1. In areas of larger scale modules (such as the orbitofrontal
and parahippocampal), the Zn+, PV-ir and MAP2-ir densities
can be shown to co-mingle, as in the rat area V1. PV-ir modularity may also occur independently of Zn+ modularity.
In this report, we concentrate on four regions with pronounced modularity: parahippocampal, orbitofrontal, rostral
cingulate and motor and dorsolateral prefrontal regions. The
main result concerns zinc modularity. This is followed by sections on dendritic bundles, cell clusters in layer 2 and PV modularity.
Keywords: apical dendritic tufts, dendritic bundles, gradients, layer 1, topdown, zinc
Introduction
The most superficial cortical layer, layer 1, has long been both
under-investigated and puzzling (‘mysterious’; Mountcastle,
2003). This may be in part because of its thinness and location
immediately beneath the pia surface, which has made it paradoxically inaccessible to conventional physiological techniques. Recently, we reported an unsuspected stratification
and modularity in layers 1 and 2. The main components, investigated in detail in rat visual cortex (Ichinohe et al., 2003b),
consist of a complex organization of different afferent and
dendritic systems. In layer 1a, a uniform zone of thalamocortical (TC) terminations can be visualized by immunohistochemistry for vesicular glutamate transporter 2 (VGluT2); but
this gives way in layer 1b to interdigitated patches of TC and
zinc-enriched (Zn+) corticocortical terminations. In the upper
part of layer 2, the Zn+ terminations co-mingle with parvalbumin-immunoreactive (PV-ir) GABAergic terminations and
form the walls of a small-scale wall-and-hollow honeycomb
mosaic. In layers 1b and 2, there is also a mix of apical dendritic
components. These relate in an orderly way to the honeycomb
mosaic. That is, bundles of apical dendrites deriving from
deeper pyramidal neurons are concentrated in the honeycomb
hollows, where they are likely to receive dense TC inputs.
Cerebral Cortex V 14 N 11 © Oxford University Press 2004; all rights reserved
Laboratory for Cortical Organization and Systematics, Brain
Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama
351-0198, Japan
Materials and Methods
Subjects
Twelve adult macaque monkeys (Macaca mulatta and M. fuscata)
were used in this study. All experimental protocols were approved by
the Experimental Animal Committee of the RIKEN Institute and were
carried out in accordance with the guidelines published in the NIH
Guide for the Care and Use of Laboratory Animals (NIH publication
No. 86-23, revised 1987).
Fixation and Tissue Preparation
Animals were anesthetized with ketamine (11 mg/kg, i.m.) and Nembutal (overdose, 75 mg/kg, i.p.). Six animals used for zinc histochemistry were perfused transcardially, in sequence, with saline containing
0.1% sodium sulfide for 5 min and then 0.1% sodium sulfide and 4%
paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 30 min.
The brains were removed from the skulls, trimmed and postfixed for
Cerebral Cortex November 2004;14:1173–1184; doi:10.1093/cercor/bhh077
12–15 h in 0.1 M PB containing 4% paraformaldehyde. Then, brains
were immersed into 30% sucrose in 0.1 M PB. Another six animals,
used only for immunohistochemistry, were perfused transcardially, in
sequence, with saline containing 0.5% sodium nitrite; 4% paraformaldehyde in 0.1 M PB for 30 min; and chilled 0.1 M PB with 10, 20 and
30% sucrose. The brains were removed from the skulls and were
immersed into 30% sucrose in 0.1 M PB. After the brains sank (for both
series), some blocks were frozen with dry ice for future processing.
The remaining blocks were cut serially in either the coronal or tangential plane by frozen microtomy (at 40–50 µm thickness).
Single and Double Labeling
In our preceeding rodent study (Ichinohe et al., 2003b), in order to
visualize micromodularity at the border of layers 1 and 2, we used
zinc, PV, MAP2 (for dendrites) and GABA receptor type A α1 subunit
(GABAaα1; also for dendrites), VGluT2 (for TC terminations), glutamate receptor 2 and 3 (GluR2/3), glutamate receptor 5, 6 and 7
(GluR5/6/7), NMDAR1, calbindin (CB) and cytochrome oxidase. The
present results are mainly based on the distribution pattern of zinc,
PV, MAP2 and GABAaα1. In the monkey, VGluT2 has been less
successful than in the rodent, where a good correspondence has been
shown for the antibody and TC terminals by both electron microscopic and thalamic lesion studies (Fujiyama et al., 2001). In monkey,
GluR2/3, GluR5/6/7 and NMDAR1 tended to stain only cell bodies and
thick apical dendrites, with weak staining of neuropil. Calbindin is a
marker for both pyramidal neurons and GABAergic interneurons in
the monkey and rat; but in the monkey, CB-ir pyramidal neurons in
the upper layers and their apical dendritic components are much
fewer than in rat. More particularly, preliminary screening of CB and
calretinin in the motor and parahippocampal areas did not show any
discernible pattern at the border between layers 1 and 2 and the
results are therefore not further described. Finally, cytochrome
oxidase was not effective for examining the most superficial layers in
the monkey, perhaps because of the frequency of edge artifact.
In rat V1, double immunofluorescence, with PV as one of the labels,
was particularly useful in ascertaining spatial relationships of overlap
or interdigitation. In monkey, however, labeling of PV-ir terminals
was better achieved by the enhanced DAB method, rather than by
immunofluorecence. In the present study, we therefore used a double
labeling method combining zinc histochemistry and immunofluorescence for MAP2 or GABAaα1 (see below), but not PV. For analysis of
PV-ir terminal distribution, we employed alternate tangential sections
stained by the immunoperoxidase technique and compared PV and
MAP2 (see below). This approach was not used for direct comparison
of PV and zinc distributions because the perfusion method needed for
zinc histochemistry (with sodium sulfide) weakened the PV-ir intensity
Zinc Histochemistry
Sections perfused with a solution containing sodium sulfide were
washed thoroughly with 0.1 M PB, followed by 0.01M PB. The IntenSE
M silver Enhancement kit (Amersham International, Little Chalfont,
Bucks, UK) was used to intensify zinc signals (Danscher et al., 1987;
De Biasi and Bendotti, 1988; Akagi et al., 2001). A one-to-one cocktail
of the IntenSE M kit solution and 33% gum arabic solution was used as
a reagent. Development of reaction products was checked under a
microscope and terminated by rinsing the sections in 0.01 M PB and,
subsequently, several times in 0.1 M PB. Staining intensity was carefully controlled by periodic visual inspection under a low power
microscope to avoid either over- or under-staining, which might
obscure any modularity. Selected sections were further processed for
Nissl substrate using NeuroTrace 500/525 green fluorescent Nissl
stain (Molecular Probes, Eugene, OR) according to the company’s
protocol. Other sections were further processed for double labeling
with immunohistochemistry for MAP2 or GABAaα1 (see below).
Double Labeling Combining Zinc Histochemistry and
Immunohistof luorescence for MA P2 and GABAaα 1
After finishing the silver intensification, sections were immunoblocked with 0.1 M phosphate buffered saline (PBS, pH 7.3)
containing 0.5% Triton X-100 and 5% normal goat serum (PBS-TG) for
1 h at room temperature and subsequently incubated for 40–48 h at
1174 Micromodularity in the Uppermost Cortical Layers • Ichinohe and Rockland
4°C in PBS-TG containing anti-MAP2 monoclonal mouse antibody
(1:2000; Chemicon, Temecula, CA) or anti-GABAaα1 polyclonal rabbit
antibody (1:5000; Chemicon, Temecular, CA). After rinsing, the
sections were placed in PBS-TG containing Alexa Fluor 488 conjugated anti-mouse IgG polyclonal goat antibody (1:200; Molecular
Probes, Eugene, OR) or Alexa Fluor 488 conjugated anti-rabbit IgG
polyclonal goat antibody (1:200; Molecular Probes, Eugene, OR) for
1.5 h.
Immunoperoxidase Staining for MAP2 and PV
Sections were incubated for 1 h with PBS-TG at room temperature and
then 40–48 h at 4°C with PBS-TG containing anti-MAP2 monoclonal
mouse antibody (1:8000; Chemicon; Temecula, CA) or anti-PV monoclonal mouse antibody (1:50 000; Swant, Bellinzona, Switzerland).
After rinsing, the sections were placed in PBS-TG containing biotinylated anti-mouse IgG polyclonal goat antibody (1:200; Vector,
Burlingame, CA) for 1.5 h at room temperature. Immunoreactivity was
visualized by ABC incubation (one drop of reagents per 7 ml 0.1 M PB;
ABC Elite kits; Vector, Burlingame, CA) followed by diaminobenzidine
histochemistry with 0.03% nickel ammonium sulfate. In three of six
animals, MAP2 immunohistochemistry showed clear dendritic aggregation in layers 1 and 2 with very low interbundle background staining.
In one monkey, dendritic clusters were detectable in layers 1 and 2,
but the interbundle spaces also had background-like staining. The
remaining two animals had weaker MAP2 staining in layers 1 and 2.
In order to visualize PV-ir neurons together with Nissl-stained
neurons, we reacted some tissue by double fluorescent labeling for PV
and Nissl substrate. Sections were immunoblocked with 0.1 M phosphate buffered saline (PBS, pH 7.3) containing 0.5% Triton X-100 and
5% normal goat serum (PBS-TG) for 1 h at room temperature and
subsequently incubated 40–48 h at 4°C in PBS-TG containing anti-PV
monoclonal mouse antibody (1:5000; Swant, Bellinzona, Switzerland).
After rinsing, the sections were placed in PBS-TG containing Alexa
Fluor 594 conjugated anti-mouse IgG polyclonal goat antibody (1:200;
Molecular Probes, Eugene, OR) and NeuroTrace 500/525 green fluorescent Nissl stain (Molecular Probes, Eugene, OR) according to the
company’s protocol for 1.5 h. Immunofluorescence for PV was
ordinarily much weaker in our hands than the immunoperoxidase
technique (however, see Fig. 5C).
Measurements and Analysis
In the first step of this investigation, we scanned a through-brain
series of coronal sections (spaced at 250 µm) stained for zinc to ascertain the areas with zinc modularity. Four regions were chosen for
more detailed analysis based on the occurrence of upper layer
modularity (i.e. parahippocampal region, orbitofrontal region, rostral
cingulate region and motor and dorsolateral prefrontal areas; Fig. 2).
Quantitative analysis of Zn+ patches was achieved with the aid of a
Neurolucida System (MicroBrightField, Colchester, VT). First,
multiple tangential blocks were prepared through architectonic areas
of regions I–IV of Figure 2. For parahippocampal and orbitofrontal
regions, two or three blocks were made and six or seven for the
medial and dorsolateral regions (III and IV of Fig. 2). Secondly, the
centers of Zn+ patches were identified and plotted in an area of
0.36–1.49 mm2, using a ×20 objective lens. The size of the area examined was constrained by the extent of flatness within the region. This
was frequently too small to allow for random sampling with a grid.
Therefore, rather than employ an unbiased sampling procedure, we
instead measured all the available flat area from the four regions of
interest from two animals. Then, we obtained the mean value of these
stacked data. The nearest center-to-center distance (CCD) was
obtained by using NeuroExplorer analysis software (MicroBrightField,
Colchester, VT). In regions of large patch size, averages were based
on 20–30 CCDs; in regions of smaller patch size, on 40–70. To assure
that change in size is real and is not influenced by a variation in the
plane of section, we were careful to scan through several serial
sections (five sections on average).
Light and fluorescent images were photographed with an Olympus
DP50 digital camera mounted on an Olympus BX60 microscope with
an appropriate filter for green fluorescence (BA515IF; Olympus,
Tokyo, Japan) and with a Zeiss LSM 5 Pascal confocal microscope
(Jena, Germany).
Nomenclature
Cortical areas were identified by reference to sulcal landmarks, by
comparison with published maps and by architectonic analysis of
selected histological sections stained for cell bodies. Where several
nomenclatures are current, we usually adopted those using broader
categories. We followed most closely: for prefrontal cortex, the
nomenclature proposed by Barbas and Pandya (1989), with reference
to that of Preuss and Goldman-Rakic (1991) and Carmichael and Price
(1994); for motor cortices, that of Brodmann (1909), with reference
to that of Matelli et al. (1985, 1991) and Dum and Strick (1991); for the
parahippocampal and inferotemporal cortex, that of Yukie et al.
(1990) and Saleem and Tanaka (1996), with reference to that of Suzuki
and Amaral (2003); for visual cortices, that of Felleman and Van Essen
(1991); and for parietal and cingulate cortex, that of Brodmann
(1909).
In referring to zinc-enriched terminals and modular patches, we
have for convenience used the shorter designation Zn+ (zincpositive).
Results
Modularity in the uppermost cortical stratum can be demonstrated in several areas by different marker substances, but the
organization is complex and characterized by areal variability.
We report the main features of this modularity as seen by zinc
histochemistry, because this is so far the most reliable marker
in primate. We concentrate on four regions with pronounced
Zn+ modularity: parahippocampal, orbitofrontal, rostral cingulate and motor and dorsolateral prefrontal regions. We also
report the modular distribution of MAP2 (for thicker
dendrites), GABAaα1 (for thinner dendrites), Nissl (for cell
bodies) and PV (showing terminals, as well as dendrites and
cell bodies of some GABAergic neurons).
Zinc
The demonstration of vesicular zinc requires a modified
perfusion procedure, involving sodium sulfide; and relatively
few studies have so far been carried out on the distribution of
Zn+ terminations in monkey cortical regions. In terms of
overall density, we found considerable regional variation. As a
trend, zinc density is higher in agranular or dysgranular, and
limbic cortices, including perirhinal and parahippocampal
cortices; the temporal pole; agranular insular, rostral cingulate
and caudal orbitofrontal cortices. There are generally two
dense bands of Zn+ terminations, in the superficial (layers 1b,
2 and upper layer 3) and deep layers (layers 5 and 6). The
middle layers (layer 4 and adjacent layer 3) and layer 1a have
lower levels of zinc (see also Carmichael and Price, 1994;
Franco-Pons et al., 2000). The detailed laminar distribution
varies in relation to overall zinc density. In areas where zinc
density is low [e.g. primary motor cortex (area 4), Fig. 1G], the
infragranular band tends to be less dense in layer 5 than in layer
6, but the reverse occurs in zinc dense areas (e.g. parahippocampal cortex, Fig. 1E; rostral cingulate cortex, Fig. 1J). In zinc
weak areas, the supragranular band in layer 3 is narrower and
remains confined to the upper part of this layer.
Inspection of coronal and tangential sections reveals a region
specific trend in both distinctness and size of Zn+ modularity at
the border of layers 1 and 2. In some areas, this superficial Zn+
modularity is conspicuous and can easily be recognized in
coronal as well as tangential sections [e.g. areas 4 and 6, caudal
orbitofrontal cortex and parahippocampal area, just caudal to
the entorhinal cortex (EC); see Fig. 1A–I, and yellow shading in
Fig. 2]. Other areas have a more subtle modularity, which is
obvious only in tangential sections (e.g. rostral cingulate
cortex, Fig. 1J,K: gray shading in Fig. 2). In ‘conspicuous’
regions, one module can be identified through 2–3 serial
sections (80–120 µm thickness), but in less conspicuous
regions, a given module is usually limited to only a single
section (40 µm thickness, with commensurate width). Finally,
some areas, especially those with a cell-dense layer 4 (‘eulaminate’), do not exhibit modularity in the uppermost layers. These
include most of the dorsolateral post-Rolandic cortex (primary
and paraprimary sensory areas) and dorsolateral prefrontal
areas, adjacent to the principal sulcus (areas 46 and 8; white
regions in Fig. 2). Areas 46 and 8 thus form a small zone
without Zn+ modularity, surrounded by regions with modularity (but see ‘PV’ section below).
In coronal sections, the patches of Zn+ terminations often
appear dome-like, rising into layer 1b from a base in layer 2
(Fig. 1A,E). In areas of lesser density, they tend to be thinner
and more delicate (Fig. 1B,G). In the tangential plane, the
larger patches form a conspicuous grid in layer 1b (Fig. 1D,F),
while the thinner pattern is rather chevron, reticular, or
‘honeycomb’ in shape (Fig. 1C,H,I,K). It is worth emphasizing
that the details of shape are complicated. That is, even a clearly
patchy pattern frequently looks reticular or honeycomb at the
lower border of the patches, as these merge into a more continuous distribution.
Size differences in Zn+ modules are easily recognized in
tangential sections (measured as CCD of Zn+ patches; see
‘Measurements and Analysis’ in Materials and Methods). Four
regions are distinguishable on the basis of what appears to be a
coherent pattern of size gradation.
1. In the parahippocampal region, at the border of caudal EC
and medial parahippocampal cortex (area TH), the average
CCD is 217 µm. Proceeding caudally, at the mid-portion of
area TH (1 mm caudal to the EC), the average CCD decreases
to 140 µm and still more caudally (2 mm from EC), the average CCD is 110 µm (Figs 1F and 2C). The caudalmost part of
area TH (3 mm or more from EC) has high levels of zinc, but
no distinguishable periodicity. A second, mediolateral size
gradient is also apparent in the parahippocampal region,
where medial patches (mean CCD = 198 µm, in area TH, just
lateral to caudal EC) are larger than lateral patches (mean
CCD = 108 µm, in area TF in the lateral parahippocampal
region, 2 mm from the EC/TH border; see also Figs 1E and
2C). With zinc histochemistry, the EC itself, well-known to
have conspicuous cell islands in layer 2, shows a complex
subregion dependent modular organization and will be
treated elsewhere in more detail.
2. In the orbitofrontal region, there is a similar size gradient as
in the parahippocampal region, but reversed, in that patch
size decreases rostrally. That is, Zn+ patches are large (mean
CCD = 300 µm) in the proisocortical zone of orbitofrontal
cortex (area OPro), at the rostral edge of the lateral fissure;
smaller (mean CCD = 180 µm) in area 13, 5 mm rostral from
the lateral fissure and even smaller more rostrally (mean CCD
= 102 µm), in the caudal part of area 11, located 10 mm
rostral from the lateral fissure (Figs 1D and 2C). Size
gradation is obvious in coronal sections through the caudal
orbitofrontal cortex (Figs 1A and 2C) and, as in the parahippocampal region, more lateral patches are smaller.
3. In the rostral cingulate gyrus, patches are less conspicuous
(Fig. 1J; gray shading in Fig. 2E), but still easily detectable in
tangential section (Fig. 1K). Patches are larger (mean CCD =
171 µm) surrounding the genu of the corpus callosum (area
Cerebral Cortex November 2004, V 14 N 11 1175
Figure 1. Micromodular organization at the border of layers 1 and 2 visualized by zinc histochemistry. (A) Caudal part of orbitofrontal cortex [rostrocaudal (RC) level is indicated by
line in Fig. 2D]. The largest modules occur in proisocortex (OPro) and the module size becomes smaller laterally, at left. (B) Higher magnification of ventral area 6 (premotor cortex)
on the lateral surface (coronal section). Arrowheads point to several small patches of Zn+ terminations protruding into layer 1. (C) Tangential section of thin patches of Zn+
terminations in ventral area 6. (D) Tangential section of Zn+ patches in the caudal part of orbitofrontal cortex. The arrow points to large patches in OPro and the arrowhead to a
field of smaller patches in area 13. (E) Coronal section of the parahippocampal area just caudal to entorhinal cortex (RC level is indicated by line in Fig. 2F). (F) Tangential section
through the parahippocampal area. Large patches (arrows) occur in area TH immediately caudal to the EC, but further caudally these grade into a smaller scale pattern (arrowheads).
(G) Coronal section of primary motor cortex (area 4). (RC level is indicated by short line in Fig. 2D.) (H, I) Serial tangential sections of small scale pattern in area 4. (J) Coronal section
of the rostral cingulate cortex. (RC level is indicated by short line in Fig. 2E.) (K) Tangential sections of a small scale periodicity in the rostral cingulate cortex. Abbreviations: AON,
anterior olfactory nucleus; cir; circular sulcus; EC, entorhinal cortex; HF, hippocampal formation; OPAll, orbitofrontal periallocortex; OTS, occipitotemporal sulcus; PH,
parahippocampal cortex; ProM, area ProM. Scale bars = 1 mm (A, D); 500 µm (B, C, G–K); 220 µm (E, F).
24) and then decrease in size both caudally and rostrally.
Caudally, the mean CCD = 150 µm at the area 24/23 border
and rostrally, this is 110 µm, in area 32, 4 mm rostral to the
genu of corpus callosum (Fig. 2B).
4. For most of the motor-related areas (areas 4 and 6) and
dorsolateral prefrontal cortex, there is modularity in the
uppermost layers, but of a more uniform small scale (mean
CCD = 108 – 124 µm; Figs 1A–C,G–I and 2A,B).
1176 Micromodularity in the Uppermost Cortical Layers • Ichinohe and Rockland
MAP2
MAP-2 staining was investigated most closely for those four
regions showing strong Zn+ modularity. In our previous investigation of rat visual cortex, Zn+ patches were found to interdigitate with MAP2-ir thick dendritic bundles from layer 3 and
5 pyramidal neurons, as these pass through layer 2. The
patches co-localized, instead, with more weakly labeled
dendrites, interpreted as originating from layer 2 pyramidal
Figure 2. Schematic brain diagrams (A, D, lateral view; B, E, medial view; C, F, ventral view). (A–C) Size distribution of Zn+ modules according to large, medium and smaller size
(large filled circle = CCD > 170 µm; medium sized filled circle = 170 µm > CCD > 135 µm; small dots = CCD < 135 µm). (D–F) Areas with a conspicuous modularity of Zn+
terminations are indicated in yellow shading and those where the modularity is more subtle are in gray. Subtle modularity may only be detected in tangential sections. Regions
without perceptible Zn+ modularity are in white. In the lateral part of gyrus rectus, high curvature (owing to its location at the hemispheric edge) hindered confident assessment
of periodicity (marked by ?). The full and short lines in D–F show RC level of coronal section in Fig. 1A, E, G and J. Roman numbers I–IV indicate the four regions described in detail
in the Results. Abbreviations: CS, central sulcus; EC, entorhinal cortex; LF, lateral fissure; RS, rhinal sulcus; STS, superior temporal sulcus.
neurons. In the monkey cortex, in contrast with rat, we found
that MAP2 does not strongly stain the distal portions of apical
dendrites of layer 3 and 5 pyramidal neurons (see also Peters
and Sethares, 1991a). Conversely, at least in some areas, MAP2
staining of apical dendrites of the layer 2 pyramidal neurons is
stronger in monkey (Peters and Sethares, 1991a). According to
our model, we would expect these to co-localize with Zn+
patches.
In coronal sections of both the caudal orbitofrontal cortex
and the parahippocampal area just caudal to the EC, MAP2
immunohistochemistry
demonstrates
distinct dendritic
bundles rising into layer 1 from layer 2. These have a predominantly oblique orientation, and can often be followed back to
pyramidal cells in layer 2 (Fig. 3A–C,F). Double labeling for
MAP2 and zinc shows that the MAP2 bundles in fact co-localize
with zinc patches, except that they tend to extend slightly
higher into layer 1a (Fig. 3D–G). In motor-related and dorsolateral prefrontal cortices, MAP2 also demonstrates dendritic
patches and, in double-labeled sections, these can again be
seen to co-localize with Zn+ terminations (Fig. 3H,I). However,
in these areas the MAP2 modularity is weak, even in tangential
sections, and often not detectable in coronal sections, so that
MAP2-ir dendrites are more difficult to trace back to their cell
bodies. For areas with subtle or no zinc pattern (such as area 8,
rostral cingulate and somatosensory areas), we could not find
evidence for MAP2-ir dendritic bundles in layer 1 (data are not
shown).
Dendritic bundles in layer 1 exhibit size differences in
parallel with the gradations of the Zn+ terminations (CCD of
100–300 µm; see Figs 1A and 3A). It is worth noting that not all
dendrites can be labeled with MAP2 immunohistochemisty
(Peters and Sethares, 1996). Strongly MAP2-ir dendrites, on
which our observations are based, are a subpopulation.
GABAaα 1
In order to investigate the organization of dendrites from
deeper neurons, we used GABAaα1, which frequently yields an
image of vertically oriented, presumably dendritic processes,
especially in layers 1b–upper 3 (Fig. 3J). In monkey visual
cortex, Hendry et al. (1994) have reported that densely
GABAaα1-ir dendrites are mainly thin dendrites and that these
frequently form vertical bundles. Double labeling for GABAaα1
and zinc shows that bundles of thin GABAaα1-ir dendrites at
the layer 1, 2 border preferentially avoid Zn+ patches and
instead occupy the interpatch zones (Fig. 3J–M). These
bundles can be traced back to the middle of layer 3, before
blending into densely immunoreactive neuropil (areas examined were areas 4 and 6, and orbitofrontal and parahippocampal areas; for area 4, Fig. 3J–M). These may be the tapered
distal ends of apical dendrites from neurons deeper in layer 3
and possibly layer 5. Again, there is a good match between the
spacing of GABAaα1 bundles and the scale of Zn+ modularities
at the border between layers 1 and 2.
Nissl
Cell clusters in layer 2 and/or a cell-dense ‘accentuated’ layer 2
have been reported in several cortical areas in Nissl stained
material of several species (e.g. Sanides, 1970; DeFelipe et al.,
2002; Suzuki and Amaral, 2003). These might be considered as
a basis for micromodularity, but the relationship between cell
clusters and micromodularity is not strict. In some areas with
large zinc modules and dendritic bundles (i.e. caudal orbitofrontal cortex and parahippocampal cortex, just caudal to the
EC; Fig. 4A,B), cell clusters at a comparable spatial scale can be
discerned in layer 2. In contrast, in the perirhinal cortex, lateral
to the rostral part of the EC (gray shading in Fig. 2), there is a
very conspicuous clustering of cell bodies in layer 2 (especially
in area 36 and, somewhat less, in area 35; see also Suzuki and
Amaral, 2003; Fig. 4C), but only a weak manifestation of MAP2ir dendritic or Zn+ modularity. In other areas, including motorrelated and cingulate cortices, there are clear zinc modules,
but no obvious cell aggregation. Motor but not cingulate
cortex, as described above, has demonstrable dendritic
bundles in layer 1.
Cerebral Cortex November 2004, V 14 N 11 1177
Figure 3. Micromodular organization at the border of layers 1 and 2 visualized by dendritic markers (MAP2 and GABAaα1). (A–C) Coronal sections showing MAP2 positive dendritic
clusters (arrowheads in B) at the caudal part of orbitofrontal cortex (the section in A is adjacent to that illustrated in Fig. 1A). C is higher magnification of B. (D, E) Tangential section
from area OPro, double labeled with MAP2 (D) and zinc (E). (F, G) Coronal section through area TH, double-labeled for MAP2(F) and zinc (G). (H, I) Semi-tangential section through
area 4, double labeled for MAP2 (H) and zinc (I). (J–M) Coronal section through area 4 double-labeled for zinc and GABAaα1. For convenience, in K, zinc images were converted
to pseudo-red-colour. In D–M, arrowheads point to the same locations in the pairs of double-labeled images. Scale bars = 1 mm (A); 160 µm (B, D–M), 80 µm (C).
PV
In the upper layers in the monkey cerebral cortex, PV is
present in subtypes of GABAergic, aspiny, non-pyramidal cells
and their terminations (Hendry et al., 1989; Blumcke et al.,
1990; Williams et al., 1992; Melchitzky et al., 1999). PV
immunohistochemistry in monkey showed a honeycomb-like
1178 Micromodularity in the Uppermost Cortical Layers • Ichinohe and Rockland
modularity (Fig. 5A,B,G–I) or occasionally a larger, patch-like
pattern (Fig. 5D). This was usually situated in a thin stratum at
the border between layers 1 and 2, but the larger scale modularity dipped further into layer 2. PV-ir terminations, as in rat
V1, are dense (Fig. 5C) and can be taken as a main source of the
modularity; but the distinction between walls and hollows
(parahippocampal case) or rostrally (orbitofrontal case). This is
in parallel with the change in scale in these same regions of the
Zn+ and MAP2-ir patches (Fig. 5A, for parahippocampal area).
Alternate section analysis showed that the PV-ir terminations
co-localize, but at a slightly deeper level (i.e. more at the
border between layers 1 and 2) with the MAP2-ir dendritic
bundles (Fig. 5D,E). Since the MAP2-ir and Zn+ patches overlap
in these areas, we suggest that the PV-ir pattern also overlaps
with the Zn+ pattern (direct PV–zinc comparison was not
undertaken; see ‘Single and Double Labeling’ in Materials and
Methods).
In areas 4 and 6, the dorsolateral prefrontal area and the
rostral cingulate, a PV-ir small scale pattern, more reminiscent
of a honeycomb-like organization, is apparent (Fig. 5G).
Center-to-center distance is ∼100 µm in areas 4 and 6 and in the
dorsolateral prefrontal area. This is similar to the size of the Zn+
patches in these areas. In the rostral cingulate areas, size gradation is similar for PV-ir and zinc patches. The thinness and small
size of both the PV-ir and MAP2-ir pattern in these areas again
hindered direct comparison of spatial overlap.
Most of the post-Rolandic areas (e.g. primary auditory and
somatosensory cortices), exhibited some degree of PV-ir
honeycomb-like pattern (Fig. 5I; see also fig. 11D in Ichinohe
et al., 2003b). Interestingly, prefrontal areas 8 and 46 also have
this feature (Fig. 5H). The CCD of the honeycomb in these
areas is again ∼100 µm. None of these areas, as reported above,
exhibited any clear Zn+ modularity in layers 1 and 2.
In the parahippocampal region, just caudal to EC, we also
identified a separate mosaic of PV-ir fibers, of unknown origin,
in a thin stratum within layer 1 (border of layers 1a and b; Fig.
5F). The CCD was ∼200 µm. Comparison of alternate sections
stained for PV or MAP2 showed that these PV-ir fibers surround
clusters of MAP2 labeled dendrites at this level, in the middle of
layer 1 (Fig. 5E,F). As this fiber mosaic is comparatively narrow
and easily obscured by PV-ir dendrites and other dense PV-ir
fiber systems in layer 1a, we do not know whether it is widespread in other areas.
Figure 4. Three areas with prominent aggregations of cell bodies in layer 2. Coronal
section outlines in the right column show the areas (arrows) from which the pictures
were taken. (A) OPro. (B) Perirhinal cortex (area 36) adjacent to the caudal border of the
entorhinal cortex (EC). An arrow (left column) points to the caudal tip of the rhinal
sulcus. (C) Rostral perirhinal cortex (area 36). For all three images in the left column,
medial is to the left and dorsal at the top (as in the section outlines). Arrowheads point
to cell clusters. Abbreviations: AMT, anterior middle temporal sulcus; AON, anterior
olfactory nucleus; OT, occipitotemporal sulcus; Rh, rhinal sulcus. Scale bars = 1 mm
(left column); 5 mm (right column).
(Fig. 5B) does not seem as sharp in the monkey as in rat V1.
Another important difference between rat and monkey relates
to PV-ir dendrites from layers 2 and 3. In monkeys, robust
vertically oriented dendritic arbors reach up to the middle of
layer 1 or higher (Blumcke et al., 1990; Williams et al., 1992;
Gabbott and Bacon, 1996). They frequently penetrate through
PV-ir weak hollows as well as PV-ir dense walls and obscure or
confound any periodicity. As a consequence, PV-ir periodicity
can more easily be evaluated in areas with weakly PV-ir
neuropil and fewer PV-ir cell bodies.
Thus, in the orbitofrontal and parahippocampal regions,
where overall PV density is lower, PV-ir patchness is readily
apparent (Fig. 5A,B,D for parahippocampal area). The PV
modularity shows a distinct size gradation in these regions; that
is, it progressively decreases in size laterally and either caudally
Discussion
These results provide further evidence for a general stratified
and modular specialization specific to the uppermost cortical
layers. As in the rat visual cortex, the anatomic components
involve Zn+ terminations, PV-ir terminations and portions of
distal apical dendrites. In areas where the modularity is
conspicuous and large scale (i.e. caudal orbitofrontal and parahippocampal areas), the Zn+ patches can be verified to match
well with MAP2-ir dendritic bundles in layer 1, thought to originate from pyramidal neurons in layer 2. In turn, by analysis of
alternate sections, MAP2-ir bundles can be shown to co-localize
with patches of PV-ir terminations at the border of layers 1 and
2. GABAaα1-ir dendritic bundles, presumed to be distal
dendritic portions from deeper neurons, avoid Zn+ patches
(see Fig. 6). This model is basically similar to the wall-andhollow honeycomb organization demonstrated by several overlapping or complementary components in rat visual cortex
(Ichinohe et al., 2003b).
The organization of the components is more difficult to
define in primate than in rat, however; and the formulation of
general principles is complicated by the fact that the modularity is not uniform across areas. For example, in some areas
(motor and limbic areas), the modularity can be visualized by
Cerebral Cortex November 2004, V 14 N 11 1179
Figure 5. Micromodularity in layers 1 and 2, visualized by PV and MAP2. Images are from the border between layers 1 and 2, except for F, which does not involve layer 2. (A)
Tangential section through the parahippocampal area (PH) stained for PV. Rostral is to left, and lateral at the top. Low magnification is intended to show size gradation. (B) Higher
magnification of tangential section of the PH to show PV-ir elements. (C) Merged confocal images from the border between layers 1 and 2 of PH (red, PV; green, neurotrace labeled
neuronal somata). Arrowheads point to PV-ir basket-like terminals surrounding somata. A vertically ascending PV-ir dendrite is visible at left. (D–F) Serial tangential sections of PH
to show spatial relationship of PV-ir and MAP2-ir modular organization. Rostral is to the left and lateral at the top. Two tangential sections are stained for PV (D, F) and the intervening
section for MAP2 (E). D is deepest (at the uppermost part of layer 2) and F is more superficial (middle of layer 1). Arrowheads point to corresponding patches in all three sections.
Note correspondence of PV (D) and MAP2 (E) and interdigitation of MAP2 dendrites (E) with PV-ir fiber plexus (F). (G–I) Tangential sections through area 4 (G), area 8 (H) and primary
auditory cortex (I). Abbreviation: EC, entorhinal cortex; PH, parahippocampal area. Scale bars = 300 µm (A); 100 µm (B); 20 µm (C); 200 µm (D–F); 160 µm (G–I).
both zinc and PV. In other areas (i.e. primary and associational
sensory post-Rolandic cortices and prefrontal areas 46 and 8),
although PV immunohistochemisty shows a periodic distribution, there is no detectable Zn+ modularity. In monkey primary
visual cortex, Zn+ patches have indeed been reported, but
these are in layers 3 and 4A, complementary to cytochrome
oxidase patches (Dyck and Cynader, 1993).
By way of comparison, areal specific differences occur in rat
cortex as well. In the rat, areas with discernible upper layer
Zn+ modules, in addition to V1, include V2, the caudal part of
retrosplenial agranular cortex, and medial prefrontal cortex
(fig. 2 in Ichinohe and Rockland, 2003a; fig. 1 in Mengual et al.,
1995). Neither motor nor barrel cortices show modularity in
zinc (unpublished observation), but do so with PV immunohistochemistry (Ichinohe et al., 2003b).
1180 Micromodularity in the Uppermost Cortical Layers • Ichinohe and Rockland
Areal Variability in Modular Size and Shape
One striking result in this study is the difference in size and
shape of the upper layer modularity. The issue of shape is
particularly complex, as this varies from an obviously patchlike to an obviously ‘honeycomb’ configuration. In areas with
smaller-scale modularity (e.g. area 4), the module shape visualized by histochemistry for zinc, PV and MAP2 tends to
resemble more a reticulum or wall-and-hollow honeycomb; but
in areas with large-scale modularity (e.g. parahippocampal
area), it tends to be more patchy. The ‘honeycomb’ itself can
also be viewed as consisting of patches (‘hollows’) embedded
in a thinner matrix. Thus, in the rat V1, the honeycomb could
be described as ‘patches’ of TC connections surrounded by a
thin matrix (‘wall’) of Zn+ and PV-ir terminations (Ichinohe et
al., 2003b).
Figure 6. Highly schematic summary of the micromodularity of layers 1 and 2 of the monkey cerebral cortex. Cubes in A and B represent coronal views (en face) and tangential
views (top surface) of two areas, with larger-scale (A) and smaller-scale (B) modularity. The laminar distribution of Zn+ terminals is represented at the right edge of each cube in
solid blue. An important factor in the size and shape of the modularity may be the arrangement of the apical dendritic bundles of layer 2 pyramidal neurons, which are suggested to
be a segregated system. This may range from conspicuous aggregation (in A) to a semi-covert aggregation (in B). Dendrites, labeled by GABAaα1 and arising from layer 3 (and/or
layer 5, indicated by ?), may also be segregated. In the figure, PV-ir and Zn+ modularity are shown as co-localized, although this is not always the case. The PV-ir modularity is
comprised largely of GABAergic terminations. In the monkey cortex, it is frequently obscured by interpenetrating vertically-oriented dendrites from the deeper layers (neurons at
right in A, in red). As shown at higher magnification in C, a dendritic compartmentalization of layer 2 pyramidal neurons is suggested, where Zn+ and PV-ir terminations avoid the
most distal apical dendrite (targeted instead by TC and other terminations) and partially co-mingle in the more proximal dendritic portion. There, a single spine (arrow) may be
targeted by both Zn+ and PV-ir terminals.
The issue of complementarity is better understood in the
middle layers of primary sensory areas. In primate V1, the
patch and matrix organization of layer 3 consists of CO ‘blobs’,
corresponding to a subpopulation of TC connections,
embedded in a matrix of Zn+ corticocortical terminations
(Dyck and Cynader, 1993). A similar complementary pattern of
Zn+ and TC terminals occurs in layer 4 of rodent somatosensory barrel cortex (Czupryn and Skangiel-Kramska, 1997).
In layer 4A of monkey V1, a thin ‘wall’ of TC terminals
surrounds zinc dense hollows (Dyck and Cynader, 1993). It
would be helpful, in further interpreting our results, to know
the distribution of a general marker for TC connections in the
primate.
The difference in size is somewhat easier to see. For most
areas, the mean CCD of Zn+ modularity is 110 µm. This is close
to the size of honeycomb walls in rat cortex (mean CCD = 80
µm; Ichinohe et al., 2003b), as well as to that of the classic
honeycomb in layer 4A of monkey V1 (mean CCD = 100 µm;
Peters and Sethares, 1991b). In three regions, however, there
is a marked size gradation; that is, in the orbitofrontal, para-
hippocampal and anterior cingulate regions. In the orbitofrontal zone, the change in size is particularly drastic, ranging
from 100 to 300 µm over an expanse of 10 mm. For MAP2-ir
dendritic bundles or PV patches, the same size differential is
observed.
The factors underlying this variation in size and shape are not
clear (but see next section). Other trendwise gradations within
and across areas, however, have been frequently observed.
Classic architectonic studies noted differences in the degree of
development of cortical lamination and distinguished granular,
dysgranular and agranular cortical types. These have been
related to distance of particular areas from allocortex (Sanides,
1970; Barbas and Pandya, 1989; Dombrowski et al., 2001).
Other differences have been observed in pyramidal dendritic
architecture. Apical dendritic bundles are more prominent in
some areas (Del Rio and DeFelipe, 1994) and the average size
and complexity of basal dendritic arbors, as well as the degree
of spininess, increase in prefrontal areas in comparison with
early association or primary visual areas (for a review, see
Elston, 2002). Gradient-like gene expression and protein distri-
Cerebral Cortex November 2004, V 14 N 11 1181
butions in developing and adult cortex have attracted
increasing attention as influences on areal size and regionalization (Donoghue and Rakic, 1999; O’Leary and Nakagawa,
2002; Job and Tan, 2003). Of particular interest with regard to
the uppermost layers, the monoclonal antibody 8B3, that
recognizes a chondroitin sulfate proteoglycan, is expressed in
a rostrocaudal gradient in monkey cortex. More specifically,
8B3 is concentrated in three bands, including a row of neurons
at the border of layers 1 and 2 (Pimenta et al., 2001).
Composition of Dendritic Bundles
The issue of dendritic bundles is complex. Previous investigations have reported the occurrence of apical dendritic
bundles in several neocortical areas and further suggested that
these may be a general feature of cortical organization (e.g.
Fleischhauer et al., 1972; Peters and Sethares, 1991a;
Mountcastle, 1997). These investigations have also shown that
apical dendrites of layer 2 pyramidal neurons may not join
dendritic bundles arising from deeper pyramidal neurons
(either from layer 3 or from layers 3 and 5; Schmolke and
Viebahn, 1986; Peters and Sethares, 1991a; Peters et al., 1997).
Our results from MAP2 and GABAaα1 labeling, in both rat and
primate, are consistent with the interpretation that apical
dendrites from layer 2 pyramidal neurons form a separate
system of bundles in at least some cortical areas (see Fig. 6). A
clear example of this type of dendritic formation is in rat granular retrosplenial cortex. Here the apical dendrites of layer 2
pyramidal neurons form distinct bundles in layer 1, which comingle with clusters of PV-ir dendrites (Wyss et al., 1990;
Ichinohe and Rockland, 2002). Dendritic tufts from pyramidal
neurons in layers 3 and 5, in contrast, co-localize with patches
of calretinin-ir terminal-like puncta and with patches of corticocortical terminals (Wyss et al., 1990; Ichinohe and Rockland,
2003a; Ichinohe et al., 2003a).
Our results in monkey suggest that patches of dense Zn+
terminations selectively target dendrites belonging to layer 2
pyramidal neurons. In orbitofrontal and parahippocampal
areas, distinct dendritic bundles can be seen to originate from
pyramidal neurons in layer 2 and double-labeling further indicates that these co-localize with Zn+ terminals. In other areas,
such as areas 4 and 6 and dorsolateral prefrontal areas, the
source of the MAP2-ir dendritic patches is less obvious. Area
differences in the organization of apical dendrites may be
related to variations in the size and shape of upper layer modularity.
The Origin of Zn+ Terminations
Zinc is known to distinguish a subpopulation of corticocortical
excitatory terminals (Perez-Clausell and Danscher, 1985; Beaulieu et al., 1992). In rodents, this has been demonstrated by
several workers using intra-cerebral or i.p. injections of sodium
selenite (Slomianka et al., 1990; Christensen et al., 1992;
Casanovas-Aguilar et al., 1998, 2002; Brown and Dyck, 2003).
Injected Se2– forms reaction product with vesicular zinc which
is transported retrogradely to cell somata. Notably, zincenriched neurons have not been reported in the thalamus.
Sodium selenite injections in several areas in monkey confirm a
cortical location, although some zinc-enriched neurons also
can occur in the claustrum and amygdala (Ichinohe and Rockland, 2003b, 2004).
The amygdala projects widely to cortical areas. As amygdalocortical terminations target in layers 1 and 2 (Amaral and Price,
1182 Micromodularity in the Uppermost Cortical Layers • Ichinohe and Rockland
1984; Stefanacci et al., 1996), this is further evidence that the
amygdala could contribute to Zn+ patches. However, in this
matter also, area specializations are likely to be significant. For
example, primary motor cortex exhibits Zn+ modularity, but
does not receive projections from the amygdala (Avendano et
al., 1983).
Dendritic Localization of Zn+ and PV-ir Terminals
In regions where there is spatial overlap of the PV-ir, MAP2-ir
and Zn+ patches, PV-ir and Zn+ terminals might be supposed to
share the same target dendrites (see Fig. 6). Several inferences
follow.
A marked decrease of PV-ir terminals in layer 1 (Williams et
al., 1992; Melchitzky et al., 1999; the present study) and of Zn+
terminals in layer 1a suggests that both probably avoid distalmost apical dendrites. As Zn+ terminals seem to continue
higher into layer 1, they probably occur independently of PV-ir
terminals in this portion of the apical dendrites. Another observation, from electron microscopic studies of monkey
prefrontal cortex (Williams et al., 1992; Melchitzky et al.,
1999), is that PV-ir terminals in layers 2 and 3a make symmetrical (presumably GABAergic) synaptic contact onto dendritic
spines (44%) and shafts (39%). Thus, it is possible that Zn+ and
PV-ir terminals can terminate even on the same spines in these
layers (see Fig. 6C).
The putative close association of Zn+ and PV-ir terminations
is important in the context of interactions between zinc and
GABAergic synapses. Both pre- and post-synaptic effects of zinc
on GABAergic transmission have been described. Bath application of zinc is known to enhance GABA release (Zhou and
Hablitz, 1993; Smart et al., 1994). Smart et al. (1994) have
proposed a possible mechanism of increasing excitability of
GABAergic terminals through a direct effect of zinc on voltagegated ion channels. Other studies show that zinc antagonizes
the GABAa receptor complex (for a review, see Smart et al.,
1994). Importantly, our results on the areal variability of Zn+
and PV-ir modularity suggest area-specific effects.
Importance of Layer 1
Our results support ongoing re-appraisals of the complexity of
the uppermost cortical stratum (e.g. Rakic and Zecevic, 2003;
Zhu and Zhu, 2004). This zone has attracted considerable
interest by virtue of its high density of apical dendritic tufts.
These are now known to have a potentially powerful impact
on the generation of action potentials, owing to several mechanisms that counteract distance from the axon hillock/initial
segment ‘trigger’ site (Magee, 2000; Zhu, 2000; Williams and
Stuart, 2003). In addition, field potential recordings in mouse
neocortex have demonstrated a significant contribution of
layer 1 to the balance between excitation and inhibition in the
underlying layers (Shlosberg et al., 2003).
Layer 1 in many areas receives cortical feedback and TC
connections and, as noted above, amygdalo-cortical projections terminate widely at the border of layers 1 and 2 (Amaral
and Price, 1984; Stefanacci et al., 1996). On the basis of single
axon analysis, as well as retrograde tracer experiments, these
projections to layer 1 have been considered as characteristically divergent, but the present results demonstrate that the
subpopulation of Zn+ corticocortical terminations is frequently
patchy.
An interesting possibility is that the patches of Zn+ terminations reported here may correspond to zones of elevated zinc-
related plasticity in the upper layers. This is suggested by the
reported action of zinc in primate visual and rat somatosensory
cortices, where levels of synaptic zinc are rapidly and dynamically regulated in conditions of sensory deprivation (Brown and
Dyck, 2002; Dyck et al., 2003). Activity dependent zinc regulation may be a general phenomenon involved in long-term
potentiation (as in the hippocampus; Li et al., 2001a,b), or in
other processes associated with plasticity (for a review, see
Frederickson and Bush, 2001). In this case, the pronounced
regional variation in modularity of Zn+ terminations may indicate areal differences in the potential for functional and/or
structural remodeling. A similar suggestion has been made
concerning the plasticity-related growth-associated phosphoprotein GAP43, which is preferentially distributed in layer 1 of
association cortical areas (Benowitz et al., 1989). Some form of
activity-dependent regulation may be significant for what are
considered ‘top-down’ (or ‘feedback’) influences and further
work on specialized dendritic and circuitry properties may
help in further elucidating these contextual and attentional
effects.
Notes
This work was supported by the Brain Science Institute, RIKEN. We
would like to thank Ms Kyoko Shirasawa, Miyoko Bellinger, Yoshiko
Abe, Hiromi Mashiko and Mr Adrian Knight for their excellent technical help.
Address correspondence to Noritaka Ichinohe, Laboratory for
Cortical Organization and Systematics, Brain Science Institute,
RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Email:
[email protected].
References
Akagi T, Kaneda M, Ishii K, Hashikawa T (2001) Differential subcellular localization of zinc in the rat retina. J Histochem Cytochem
49:87–96.
Amaral DG, Price JL (1984) Amygdalo-cortical projections in the
monkey (Macaca fascicularis). J Comp Neurol 230:465–496.
Avendano C, Price JL, Amaral DG (1983) Evidence for an amygdaloid
projection to premotor cortex but not to motor cortex in the
monkey. Brain Res 264:111–117.
Barbas H, Pandya DN (1989) Architecture and intrinsic connections of
the prefrontal cortex in the rhesus monkey. J Comp Neurol
286:353–375.
Beaulieu C, Dyck R, Cynader M (1992) Enrichment of glutamate in
zinc-containing terminals of the cat visual cortex. Neuroreport
3:861–864.
Benowitz LI, Perrone-Bizzozero NI, Finklestein SP, Bird ED (1989)
Localization of the growth-associated phosphoprotein GAP-43 (B50, F1) in the human cerebral cortex. J Neurosci 9:990–995.
Blumcke I, Hof PR, Morrison JH, Celio MR (1990) Distribution of
parvalbumin immunoreactivity in the visual cortex of Old World
monkeys and humans. J Comp Neurol 301:417–432.
Brodmann K (1909) Vergleichende Lokalisationslehre der Grosshirnrinde. Leipzig: Barth.
Brown CE, Dyck RH (2002) Rapid, experience-dependent changes in
levels of synaptic zinc in primary somatosensory cortex of the
adult mouse. J Neurosci 22:2617–2625.
Brown CE, Dyck RH (2003) An improved method for visualizing the
cell bodies of zincergic neurons. J Neurosci Methods 129:41–47.
Carmichael ST, Price JL (1994) Architectonic subdivision of the orbital
and medial prefrontal cortex in the macaque monkey. J Comp
Neurol 346:366–402.
Casanovas-Aguilar C, Reblet C, Perez-Clausell J, Bueno-Lopez JL (1998)
Zinc-rich afferents to the rat neocortex: projections to the visual
cortex traced with intracerebral selenite injections. J Chem Neuroanat 15:97–109.
Casanovas-Aguilar C, Miro-Bernie N, Perez-Clausell J (2002) Zinc-rich
neurones in the rat visual cortex give rise to two laminar segregated systems of connections. Neuroscience 110:445–458.
Christensen MK, Frederickson CJ, Danscher G (1992) Retrograde
tracing of zinc-containing neurons by selenide ions: a survey of
seven selenium compounds. J Histochem Cytochem 40:575–579.
Czupryn A, Skangiel-Kramska J (1997) Distribution of synaptic zinc in
the developing mouse somatosensory barrel cortex. J Comp Neurol
386:652–660.
Danscher G, Norgaard JO, Baatrup E (1987) Autometallography: tissue
metals demonstrated by a silver enhancement kit. Histochemistry
86:465–469.
De Biasi S, Bendotti C (1988) A simplified procedure for the physical
development of the sulphide silver method to reveal synaptic zinc
in combination with immunocytochemistry at light and electron
microscopy. J Neurosci Methods 79:87–96.
DeFelipe J, Alonso-Nanclares L, Arellano JI (2002) Microstructure of
the neocortex: comparative aspects. J Neurocytol 31:299–316.
Del Rio MR, DeFelipe J (1994) A study of SMI 32-stained pyramidal
cells, parvalbumin-immunoreactive chandelier cells, and presumptive thalamocortical axons in the human temporal neocortex. J
Comp Neurol 342:389–408.
Dombrowski SM, Hilgetag CC, Barbas H (2001) Quantitative architecture distinguishes prefrontal cortical systems in the rhesus
monkey. Cereb Cortex 11:975–988.
Donoghue MJ, Rakic P (1999) Molecular gradients and compartments
in the embryonic primate cerebral cortex. Cereb Cortex
9:586–600.
Dum RP, Strick PL (1991) Premotor areas: nodal points for parallel
efferent systems involved in the central control of movement. In:
Motor control: concepts and issues (Humphrey DR, Freund H-J,
eds), pp. 383–397. London: Wiley.
Dyck RH, Cynader MS (1993) An interdigitated columnar mosaic of
cytochrome oxidase, zinc, and neurotransmitter-related molecules
in cat and monkey visual cortex. Proc Natl Acad Sci USA
90:9066–9069.
Dyck RH, Chaudhuri A, Cynader MS (2003) Experience-dependent
regulation of the zincergic innervation of visual cortex in adult
monkeys. Cereb Cortex 13:1094–1109.
Elston GN (2002) Cortical heterogeneity: implications for visual
processing and polysensory integration. J Neurocytol 31:317–335.
Felleman DJ, Van Essen DC (1991) Distributed hierarchical processing
in the primate cerebral cortex. Cereb Cortex 1:1–47.
Fleischhauer K, Petsche H, Wittkowski (1972) Vertical bundles of
dendrites in the neocortex. Z Anat Entwickl Gesch 136:213–223.
Franco-Pons N, Casanovas-Aguilar C, Arroyo S, Rumia J, Perez-Clausell
J, Danscher G (2000) Zinc-rich synaptic boutons in human
temporal cortex biopsies. Neuroscience 98:429–435.
Frederickson CJ, Bush AI (2001) Synaptically released zinc: physiological functions and pathological effects. Biometals 14:353–366.
Fujiyama F, Furuta T, Kaneko T (2001) Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat
cerebral cortex. J Comp Neurol 435:379–387.
Gabbott PL, Bacon SJ (1996) Local circuit neurons in the medial
prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: I. Cell
morphology and morphometrics. J Comp Neurol 364:567–608.
Hendry SH, Jones EG, Emson PC, Lawson DE, Heizmann CW, Streit P
(1989) Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities. Exp Brain Res
76:467–472.
Hendry SH, Huntsman MM, Vinuela A, Mohler H, de Blas AL, Jones EG
(1994) GABAA receptor subunit immunoreactivity in primate
visual cortex: distribution in macaques and humans and regulation
by visual input in adulthood. J Neurosci 14:2383–2401.
Ichinohe N, Rockland KS (2002) Parvalbumin positive dendrites colocalize with apical dendritic bundles in rat retrosplenial cortex.
Neuroreport 13:757–761.
Ichinohe N, Rockland KS (2003a) Interactive vision: a new columnar
system in layer 2. In: The neural basis of early vision (Kaneko A,
ed.), pp. 199–203. Tokyo: Springer.
Cerebral Cortex November 2004, V 14 N 11 1183
Ichinohe N, Rockland KS (2003b) Zinc-enriched neural system in the
monkey cortex. Sixth IBRO World Congress of Neuroscience,
abstract, p. 314.
Ichinohe N, Rockland KS (2004) Distribution of vesicular zinc in the
monkey amygdala. Fourth Forum of European Neuroscience,
abstract, in press.
Ichinohe N, Yoshihara Y, Hashikawa T, Rockland KS (2003a) Developmental study of dendritic bundles in layer 1 of the rat granular
retrosplenial cortex with special reference to a cell adhesion molecule, OCAM. Eur J Neurosci 18:1764–1774.
Ichinohe N, Fujiyama F, Kaneko T., Rockland KS (2003b) Honeycomblike mosaic at the border of layers 1 and 2 in the cerebral cortex. J
Neurosci 23:1372–1382.
Job C, Tan SS (2003) Constructing the mammalian neocortex: the role
of intrinsic factors. Dev Biol 257:221–232.
Li Y, Hough CJ, Suh SW, Sarvey JM, Frederickson CJ (2001a) Rapid
translocation of Zn(2+) from presynaptic terminals into postsynaptic hippocampal neurons after physiological stimulation. J
Neurophysiol 86:2597–2604.
Li Y, Hough CJ, Frederickson CJ, Sarvey JM., (2001b) Induction of
mossy fiber → CA3 long-term potentiation requires translocation
of synaptically released Zn2+ . J Neurosci 21:8015–8025.
Magee JC (2000) Dendritic integration of excitatory synaptic input.
Nat Rev Neurosci 1:181–190.
Matelli M, Luppino G, Rizzolatti G (1985) Patterns of cytochrome
oxidase activity in the frontal agranular cortex of the macaque
monkey. Behav Brain Res 18:125–136.
Matelli M, Luppino G, Rizzolatti G (1991) Architecture of superior and
mesial area 6 and the adjacent cingulate cortex in the macaque
monkey. J Comp Neurol 311:445–462.
Melchitzky DS, Sesack SR, Lewis DA (1999) Parvalbumin-immunoreactive axon terminals in macaque monkey and human prefrontal
cortex: laminar, regional, and target specificity of type I and type
II synapses. J Comp Neurol 408:11–22.
Mengual E, Casanovas-Aguilar C, Perez-Clausell J, Gimenez-Amaya JM
(1995) Heterogeneous and compartmental distribution of zinc in
the striatum and globus pallidus of the rat. Neuroscience
66:523–537.
Mountcastle VB (1997) The columnar organization of the neocortex.
Brain 120:701–722.
Mountcastle VB (2003) Introduction. Computation in cortical
columns. Cereb Cortex 13:2–4.
O’Leary DD, Nakagawa Y (2002) Patterning centers, regulatory genes
and extrinsic mechanisms controlling arealization of the
neocortex. Curr Opin Neurobiol 12:14–25.
Perez-Clausell J, Danscher G (1985) Intravesicular localization of zinc
in rat telencephalic boutons. A histochemical study. Brain Res
337:91–98.
Peters A, Sethares C (1991a) Organization of pyramidal neurons in
area 17 of monkey visual cortex. J Comp Neurol 306:1–23.
Peters A, Sethares C (1991b) Layer IVA of rhesus monkey primary
visual cortex. Cereb Cortex 1:445–462.
Peters A, Sethares C (1996) Myelinated axons and the pyramidal cell
modules in monkey primary visual cortex. J Comp Neurol
365:232–255.
Peters A, Cifuentes JM, Sethares C (1997) The organization of pyramidal cells in area 18 of the rhesus monkey. Cereb Cortex 7:405–421.
1184 Micromodularity in the Uppermost Cortical Layers • Ichinohe and Rockland
Pimenta AF, Strick PL, Levitt P (2001) Novel proteoglycan epitope
expressed in functionally discrete patterns in primate cortical and
subcortical regions. J Comp Neurol 430:369–388.
Preuss TM, Goldman-Rakic PS (1991) Architectonics of the parietal
and temporal association cortex in the strepsirhine primate
Galago compared to the anthropoid primate Macaca. J Comp
Neurol 310:475–506.
Rakic S, Zecevic N (2003) Emerging complexity of layer I in human
cerebral cortex. Cereb Cortex 13:1072–1083.
Saleem KS, Tanaka K (1996) Divergent projections from the anterior
inferotemporal area TE to the perirhinal and entorhinal cortices in
the macaque monkey. J Neurosci 16:4757–4775.
Sanides F (1970) Functional architecture of motor and sensory
cortices in primates in the light of a new concept of neocortex
evolution. In: The primate brain: advances in primatology (Noback
CR, Montagna W, eds), pp. 137–208. New York: AppletonCentury-Crofts.
Schmolke C, Viebahn C (1986) Dendrite bundles in lamina II/III of the
rabbit neocortex. Anat Embryol 173:343–348.
Shlosberg D, Patrick SL, Buskila Y, Amitai Y (2003) Inhibitory effect of
mouse neocortex layer I on the underlying cellular network. Eur J
Neurosci 18:2751–2759.
Slomianka L, Danscher G, Frederickson CJ (1990) Labeling of the
neurons of origin of zinc-containing pathways by intraperitoneal
injections of sodium selenite. Neuroscience 38:843–854.
Smart TG, Xie X, Krishek BJ (1994) Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Prog Neurobiol
42:393–341.
Stefanacci L, Suzuki WA, Amaral DG (1996) Organization of connections between the amygdaloid complex and the perirhinal and
parahippocampal cortices in macaque monkeys. J Comp Neurol
375:552–582.
Suzuki WA, Amaral DG (2003) Perirhinal and parahippocampal
cortices of the macaque monkey: cytoarchitectonic and chemoarchitectonic organization. J Comp Neurol 463:67–91.
Williams SM, Goldman-Rakic PS, Leranth C (1992) The synaptology of
parvalbumin-immunoreactive neurons in the primate prefrontal
cortex. J Comp Neurol 320:353–369.
Williams SR, Stuart GJ (2003) Role of dendritic synapse location in the
control of action potential output. Trends Neurosci 26:147–154.
Wyss JM, van Groen T, Sripanidkulchai K (1990) Dendritic bundling in
layer I of granular retrosplenial cortex: intracellular labeling and
selectivity of innervation. J Comp Neurol 295:33–42.
Yukie M, Takeuchi H, Hasegawa Y, Iwai E (1990) Differential connectivity of inferotemporal area TE with the amygdala and the hippocampus in the monkey. In: Vision, memory and temporal lobe
(Iwai E, Mishkin M, eds), pp. 129–135. New York: Elsevier.
Zhou FM, Hablitz JJ (1993) Zinc enhances GABAergic transmission in
rat neocortical neurons. J Neurophysiol 70:1264–1269.
Zhu JJ (2000) Maturation of layer 5 neocortical pyramidal neurons:
amplifying salient layer I and layer 4 inputs by Ca2+ action potentials in adult rat tuft dendrites. J Physiol 526:571–587.
Zhu Y, Zhu JJ (2004) Rapid arrival and integration of ascending
sensory information in layer 1 nonpyramidal neurons and tuft
dendrites of layer 5 pyramidal neurons of the neocortex. J Neurosci
24:1272–1279.