Download - Area-specific substratification of deep layer neurons in the rat cortex

Area-Specific Substratification of Deep Layer Neuronsin the Rat Cortex

Akiya Watakabe,1* Junya Hirokawa,1 Noritaka Ichinohe,2 Sonoko Ohsawa,1 Takeshi Kaneko,3

Kathleen S. Rockland,4 and Tetsuo Yamamori1

1Division of Brain Biology, National Institute for Basic Biology, Graduate University for Advanced Studies (SOKENDAI), Okazaki,

Japan2Department of Ultrastructural Research, National Institute of Neuroscience, NCNP, Tokyo, Japan3Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan4Picower Institute of Learning and Memory, RIKEN-MIT Center for Neural Circuit Genetics, Cambridge, Massachusetts, 02139-4307

ABSTRACTGene markers are useful tools to identify cell types for

fine mapping of neuronal circuits. Here we report area-

specific sublamina structure of the rat cerebral cortex

using cholecystokinin (cck) and purkinje cell protein4

(pcp4) mRNAs as the markers for excitatory neuron

subtypes in layers 5 and 6. We found a segregated

expression, especially pronounced in layer 6, where cor-

ticothalamic and corticocortical projecting neurons re-

side. To examine the relationship between gene

expression and projection target, we injected retro-

grade tracers into several thalamic subnuclei, ventral

posterior (VP), posterior (PO), mediodorsal (MD), medial

and lateral geniculate nuclei (MGN and LGN); as well as

into two cortical areas (M1 and V1). This combination

of tracer-in situ hybridization (ISH) experiments

revealed that corticocortical neurons predominantly

express cck and corticothalamic neurons predominantly

express pcp4 mRNAs in all areas tested. In general,

cck(þ) and pcp4(þ) cells occupied the upper and lower

compartment of layer 6a, respectively. However, the

sublaminar distribution and the relative abundance of

cck(þ) and pcp4(þ) cells were quite distinctive across

areas. For example, layer 6 of the prelimbic cortex was

almost devoid of cck(þ) neurons, and was occupied

instead by corticothalamic pcp4(þ) neurons. In the lat-

eral areas, such as S2, there was an additional layer of

cck(þ) cells positioned below the pcp4(þ) compart-

ment. The claustrum, which has a tight relationship

with the cortex, mostly consisted of cck(þ)/pcp4(�)

cells. In summary, the combination of gene markers

and retrograde tracers revealed a distinct sublaminar

organization, with conspicuous cross-area variation in

the arrangement and relative density of corticothalamic

connections. J. Comp. Neurol. 520:3553–3573, 2012.

VC 2012 Wiley Periodicals, Inc.

INDEXING TERMS: corticothalamic; corticocortical; pcp4; cck; tracer; in situ hybridization

It has long been known that the excitatory neurons

that constitute 80–90% of the cortical neurons differenti-

ate into highly heterogeneous populations that have dis-

tinct morphology, physiological properties, and specificity

of projections and gene expression (Gong et al., 2003;

Nelson et al., 2006; Lein et al., 2007; Thomson and Lamy,

2007; Belgard et al., 2011). Recent studies revealed that

the specification of such cortical projection neurons

occurs through a cascade of gene regulatory events. That

is, the expression of particular genes (e.g., ctip2, fezl, and

satb2) during development determines the future fate of

a given cell by turning on and off the expression of down-

stream genes (Molyneaux et al., 2007; Britanova et al.,

2008; Leone et al., 2008).

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Japan Society for the Promotion of Science (JSPS); Grantnumbers: KAKENHI19500304 and 22500300 (to A.W.); Grant sponsor:Ministry of Education, Culture, Sports, Science, and Technology of Japan,‘‘Scientific Research on Innovative Areas (Neocortical Organization)’’; Grantnumber: 22123009; Grant sponsor: Ministry of Education, Culture, Sports,Science, and Technology of Japan, ‘‘Strategic Research Program for BrainSciences (Highly Creative Animal Model Development for Brain Science)’’;Grant sponser: JSPS; Grant number: Grants-in-Aid for Scientific ResearchA 20240030 (to T.Y.). Current affiliations for the authors are as follows:J.H., Cold Spring Harbor Laboratory.

*CORRESPONDENCE TO: Akiya Watakabe, Division of Brain Biology,National Institute for Basic Biology, Graduate University for AdvancedStudies (SOKENDAI), 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585,Japan. E-mail: [email protected]

VC 2012 Wiley Periodicals, Inc.

Received February 1, 2012; Revised April 16, 2012; Accepted June 1,2012

DOI 10.1002/cne.23160

Published online June 8, 2012 in Wiley Online Library (wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 520:3553–3573 (2012) 3553

RESEARCH ARTICLE

Although fate determination occurs early in develop-

ment, some genes exhibit apparently cell-type specific

expression even in adults. For example, Arimatsu and co-

workers found that a unique class of corticocortically pro-

jecting cells expresses latexin and nurr1 gene products in

the lateral cortical areas of rats (Arimatsu et al., 2003;

Bai et al., 2004). Correlation between gene expression

and projection phenotypes is also reported for layer 5

neuron subtypes (Molnar and Cheung, 2006; Yoneshima

et al., 2006; Groh et al., 2010) or for callosal projection

neurons (Molyneaux et al., 2009). These studies among

others have shown the usefulness of gene markers in

identifying known and unknown categories of projection

neurons.

Using several selected layer-specific genes, we previ-

ously reported the existence of excitatory neuronal sub-

populations that exhibit conserved lamina distributions in

monkeys and rodents (Watakabe et al., 2007, 2009; Hiro-

kawa et al., 2008). Similar cross-species analyses have

been carried out for the ferret cortex (Rowell et al.,

2010). The common theme that appeared in these

reports is the prevalence of area-specific sublamination

in deep layers 5 and 6, which likely reflects the distribu-

tion of particular projection neuron subtypes. Although

anatomical segregation of functionally distinct cell types

into sublayers have been repeatedly reported (Lund et al.,

1975; Killackey et al., 1989; Zhang and Deschenes,

1997; Prieto and Winer, 1999; Killackey and Sherman,

2003; Anderson et al., 2010; Mao et al., 2011), correla-

tive cross-area gene expression analyses have been more

limited. How the sublamina-specific gene expression pat-

terns correlate with specific anatomical areas, in terms of

relative abundance and topology, is therefore still not

clear.

In this article we demonstrate area-specific sublamina

organization of deep layers 5 and 6 using cholecystokinin

(cck) and purkinje cell protein 4 (pcp4) genes as excita-

tory subtype markers. Although CCK is well known as a

marker for inhibitory neuronal subtypes (Kawaguchi and

Kubota, 2007), its mRNA is actually expressed widely by

the excitatory neurons in layers 2–6 (Ingram et al., 1989;

Burgunder and Young, 1990). When we performed in situ

hybridization (ISH) of the cck gene, however, we noticed

a conspicuous absence of expression in the lower part of

layer 6a in rat V1. This finding led us to search through

the literature and databases for a gene marker that could

fill this gap. By testing the expression of several candi-

date genes (e.g., Molyneaux et al., 2007; Allen Brain

Atlas: http://www.brain-map.org/; Lein et al., 2005) by

double-ISH with cck probe, we found that pcp4 mRNA

exactly fills the cck-empty sublayer of area V1. Impor-

tantly, cck and pcp4 genes were expressed in distinct

subpopulations of layer 6, even in areas where the posi-

tive cells for each gene intermingled extensively. This ob-

servation suggested that layer 6 excitatory neurons may

be generally categorized into two broad subgroups based

on their expression of these two genes. Combining gene

expression analysis with retrograde tracing, we found

that corticothalamic and corticocortical projection pheno-

types are specifically correlated with expression of these

genes. Nurr1(þ) neurons, a corticocortical subtype,

belonged to the cck(þ) subgroup. Based on these obser-

vations, we propose 1) that there exist two basic sub-

types of layer 6 neurons that are aligned in distinct sub-

layers, and 2) that their relative abundance and topology

differs greatly across areas. We believe that our data con-

tribute a framework toward understanding the cortical or-

ganization as a whole.

MATERIALS AND METHODS

Animals and tissue preparationThirty-one adult male Wistar rats (15–17 weeks) were

purchased from Japan SLC (Hamamatsu, Japan): 24 for

tracer experiments and seven as normal samples. For the

preparation of the brain samples, rats were perfused

through the heart under deep anesthesia induced by

Nembutal (50 mg/kg body weight, intraperitoneally [i.p.])

either with 4% paraformaldehyde in 0.1 M phosphate

buffer (pH 7.4) or with 4% paraformaldehyde, 0.2% picric

acid, 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH

7.4). The rat brains were dissected out and cut into sev-

eral blocks. After immersion in 30% sucrose / phosphate-

buffered saline (PBS), the blocks were cut into 15, 20, or

40-lm thick sections on a freezing microtome and stored

in antifreeze solution (30% glycerol, 30% ethylene glycol,

40% 0.1 M PBS) at�30�C until use.

All the experiments were conducted in accordance

with the Guide for the Care and Use of Laboratory Animals

(National Institutes of Health, Bethesda, MD: publication

number 86-23, 1985) and the guidelines of the Okazaki

National Research Institutes in Japan. We made all efforts

to minimize the number of animals used and their

suffering.

Antibody characterizationThe antiserum to the rat prepro-CCK was made by

immunizing guinea pigs with the synthetic peptide that

was conjugated to the maleimide-activated keyhole lim-

pet hemocyanin (Pierce, Rockford, IL) as the antigen. As

the immunizing peptide, the C-terminal 20 amino acid

residues of prepro-CCK, ‘‘DYMGWMDFGRRSAEDYEYPS’’

was used. The same peptide was used to affinity purify

the antibody as reported previously (Lee et al., 1997;

Kaneko et al., 1998). The specificity of the antibody was

confirmed by the absence of staining on the brain tissue

Watakabe et al.

3554 The Journal of Comparative Neurology |Research in Systems Neuroscience

after absorption of the antibody with the antigen peptide.

The antiserum to PCP4 (PEP-19) was a generous gift of

Dr. James I. Morgan (Ziai et al., 1988). The specificity of

detection for these antibodies were confirmed by colocal-

ization of ISH signals as well.

Tracer injectionRats were anesthetized by ether inhalation followed by

administration of chloral hydrate (35 mg/100 g body

weight, i.p.). A small hole was made through the skull

using a dental drill. 4% Fast Blue (Dr. Illing Plastics, Breu-

berg, Germany) in PBS or 2% FluoroGold (Biotium, Hay-

ward, CA) in water was injected into the thalamus or the

cortex by pressure through a glass micropipette attached

to a Hamilton syringe (10 ll). The injection was per-

formed by KDS310 nano pump (KD Scientific, Holliston,

MA) at a rate of 0.01 ll/min for 5 minutes (0.05 ll/injec-tion in total). After survival for 2–3 days the rats were per-

fused transcardially as described above.

ISH probesThe cDNA fragments of cck and pcp4 genes were

obtained by polymerase chain reaction (PCR) using the

following primer sets on mouse cDNA. For cck, 50-aaagc-

catgaanagcggcgt-30 and 50-ggcagaakgaaatcwctttaatmgc-

30; for pcp4, 50-acaacatcaaggaaaatagttgc-30 and 50-gccaa-

catgagtgagmgacaa-30. The probe for VGluT1 gene was as

described previously (Watakabe et al., 2006). The digoxi-

genin (DIG)- and fluorescein (FITC)-labeled riboprobes

were produced by in vitro transcription using these plas-

mids as templates. The riboprobes were purified using

ProbeQuant 50 spin column (Amersham Biosciences, Lit-

tle Chalfont, UK).

ISHCoronal sections were cut at 40 lm thickness for sin-

gle-color ISH and 15–20 lm for double-ISH, immuno-ISH,

and tracer-ISH using a freezing microtome. A detailed

description of these methods can be found elsewhere

(Watakabe et al., 2006, 2010).

For immuno-ISH, hybridization was performed in the

same way as the standard ISH. After hybridization, the

washed sections were blocked with immersion buffer

(10% normal goat serum, 2% bovine serum albumin, and

0.5% Triton X-100 in TBS, pH 7.4). The antibody to prepro-

CCK (produced by T.K.; Table 1) was diluted to 1 lg/ml

in immersion buffer and used for incubating sections at

4�C overnight. After washing, the sections were incu-

bated with biotin-conjugated anti-guinea pig antibody, fol-

lowed by Cy2-conjugated streptavidin and anti-DIG anti-

body, washed, and processed for HNPP/Fast Red (Roche

Diagnostics, Basel, Switzerland) reaction. For immuno-

ISH of PCP4, the antiserum to PCP4/PEP19 (generous

gift of Dr. Morgan) was diluted to 1:2,000 and used for

incubating sections at 4�C overnight. After washing, the

sections were incubated with Cy2-conjugated antirabbit

antibody together with anti-DIG antibody in 1% blocking

reagents (Roche Diagnostics), washed, and processed for

HNPP/Fast Red (Roche Diagnostics) reaction. The fluo-

rescent images were captured with an Olympus DP70 dig-

ital camera and processed by Adobe Photoshop (San

Jose, CA) for proper contrast for presentation.

Data analysesThe cortical box method was performed as previously

described using the ISH data of four rats (Hirokawa et al.,

2008). Briefly, the sections of the posterior part of the rat

cortex (approximately with 280-lm interval) were proc-

essed for single-color ISH. The cortical regions defined by

the medial and lateral ends were cut out from the images

and transformed to rectangles. The intensities of ISH sig-

nals were normalized according to the means and stand-

ard deviations of all the pixel values for the ISH images of

one dataset, which were then used for further image

processing as can be seen in Figure 5. The colocalization

analyses for double-ISH and tracer-ISH were performed

as follows. First, a vertical strip of 430 lm width was cut

out from the photos of the double-stained sections. The

layer 5/6 portion was cut out from this strip and the size

was adjusted to 500 � 1,000 pixel rectangle. The original

images in red and green channels were each converted to

TABLE 1.

Antibodies Used in This Study

Name Immunogen Provider Species

Anti-prepro-CCK 20 a.a. peptide of prepro-cckDYMGWMDFGRRSAEDYEYPS

Dr. Takeshi Kaneko Guinea pig polyclonal

Anti-PCP4 13 a.a. peptide of PCP4VAIQSQFRKFQKK Dr. James I. Morgan Rabbit polyclonal, serumAnti-Fluorogold Fluorogold ChemiconAB153 Rabbit polyclonal, serumAnti-DIG-AP Digoxigenin Roche Diagnostics11 093 274 910 Sheep polyclonal, Fab fragmentsAnti-FITC-HRP Fluorescein (FITC) Jackson ImmunoResearch

laboratory#200-032-037Mouse monoclonal, IgG

Anti-DNP Alexa488 Dinitrophenyl (DNP) Molecular Probes#A-11097 Rabbit polyclonal, IgG

Stratification of Cortical Deep Layers

The Journal of Comparative Neurology | Research in Systems Neuroscience 3555

a binary image by ImageJ (http://imagej.nih.gov/ij/)

using a custom-made macro incorporating the following

procedures. There were two key points in this conversion.

First, to enable observer-independent thresholding we

utilized the ‘‘Otsu thresholding’’ plug-in. We used the

built-in ‘‘FFT bandpass filter’’ plug-in before this threshold-

ing to extract ‘‘cells’’ out of tissue background more con-

sistently. Second, to despeckle and for smoothing we

sandwiched the built-in ‘‘Gaussian blur’’ filter between

two Otsu thresholding procedures. This was very impor-

tant because of the granular nature of the ISH signals.

Also, this procedure was helpful in converting the donut-

like shape of the cytoplasmic mRNA distribution into oval

shapes. After this conversion the colocalization of the red

and green signals was examined with the ‘‘colocalization’’

plug-in. The positions and the areas occupied by the posi-

tive signals were measured and assigned to 20 subdivi-

sions of layers to make lamina profiles in Figures 3 and

10. To smooth the graphs, half values of the neighboring

sublayers were added to each sublayer of interest.

Identification of areas and layersCortical areas of rats were identified and designated in

reference to the stereotaxic atlas of Paxinos and Watson

(2007). Cortical layers were determined based on the

expression patterns of VGluT1 mRNA and Hoechst nu-

clear staining. In this article we defined layer 5 as the

layer with the least neuronal density based on these

stains. When VGluT1 pattern was not available, cck and

pcp4 mRNA patterns were also used as reference. In all

the areas examined we were able to unambiguously

determine the layer 4/5 border. ‘‘Layer 6b’’ in this study

is a thin layer above the white matter, which is also

termed subgriseal layer or layer 7 (Clancy and Cauller,

1999). The bulk of layer 6 above this layer is called layer

6a in this study.

RESULTS

Cell-type specific expression of cck andpcp4 mRNAs in rat V1

Figure 1 (Supporting Fig. S1) shows the double ISH of

cck and pcp4 mRNAs in V1 of the rat cortex. Cck mRNA

was expressed strongly in layers 2/3, 5, and 6 (Fig. 1A).

Cck mRNA was also expressed in layer 4, but the level

per cell was lower than that in layers 2/3. On the other

hand, the pcp4 mRNA was expressed in layers 5 and 6

(Fig. 1B). As shown in this figure, these mRNAs exhibited

overall complementary sublamina patterns within the

deep layers: cck mRNA was most abundantly expressed

in the upper part of layer 6a, whereas pcp4 mRNA was

abundant in the lower part of layer 6a (Fig. 1A, arrow).

Importantly, these two mRNAs exhibited complementary

patterns in layer 6a, even at the cellular level. As shown

in the magnified view, two populations of neurons were

clearly distinguished based on the expression of these

genes even when they intermingled. The merged view

shows distinct segregation of two colors: yellow staining

at several locations is due to physical overlap of two cells

(Fig. 1G–I). This observation strongly suggested that there

are at least two types of layer 6a neurons that differen-

tially express the cck and pcp4 mRNAs.

Outside layer 6a, cck and pcp4 mRNA expression was

not necessarily mutually exclusive. For example, in layer

5, pcp4 mRNA was expressed at low levels in many cells

and, thus, it was often coexpressed with cck mRNA,

although it may appear subtle in the figure (Fig. 1D–F).

There was also coexpression in layer 6b. In addition to

the deep layer laminar expression, we observed that scat-

tered cells in layer 1 express cck mRNA and scattered

cells in layers 2–4 express pcp4 mRNA. By double-ISH,

we identified these as the GAD67-positive inhibitory neu-

rons (data not shown).

In previous studies, CCK-8 immunostaining was used as

a marker for a subclass of inhibitory neurons in the cere-

bral cortex (Hendry et al., 1984; Kawaguchi and Kubota,

1997). This apparent discrepancy with the mRNA distribu-

tion pattern shown here is explicable by the localization of

CCK peptides in the presynaptic projection neurons (Mor-

ino et al., 1992). To confirm that the prepro-CCK protein

exhibits the same cell-type specificity as the cck mRNA,

we performed immuno-ISH double staining using prepro-

CCK antibody and cck mRNA probe and confirmed their

matching (Fig. 1J,K). We also performed the immuno-ISH

with PCP4 antibody and cck mRNA probe and confirmed

that PCP4 protein exhibits the same cell type preference

as its mRNA (Fig. 1L,M). PCP4 immunoreactivity was

observed mostly in the cell body as shown in the figure,

but was also detected in large dendrites (data not shown).

There are at least three types of projection neurons in

layer 6, corticothalamic, corticocortical, and corticoclaus-

tral neurons. Of these, corticoclaustral cells are expected

to be only a small population (Katz, 1987). To test

whether gene expression correlates with extrinsic projec-

tions, we injected a fluorescent retrograde tracer,

FastBlue, into the lateral geniculate nucleus (LGN) and

performed ISH. As shown in Figure 1O, the corticothala-

mic neurons that incorporated FastBlue were tightly

packed in layer 6a. Double staining with ISH showed that

this corticothalamic population aligned exactly to the cck-

low/pcp4-high sublayer in the lower part of layer 6a (Fig.

1N,S). At higher magnification, the correlation of gene

expression and projection specificity was even more

striking. As in Figure 1P–R, cck mRNA was not expressed

in the FastBlue-labeled corticothalamic cells, whereas

pcp4 mRNA(þ) cells matched the FastBlue-labeled cells

Watakabe et al.


very well (Fig. 1U–W). Thus, pcp4 and cck mRNAs appear

to represent corticothalamic and noncorticothalamic neu-

rons, respectively, at least in this sublayer.

Taking advantage of such correlation between gene

expression and projections, we aimed to map the projec-

tion profiles of layer 6 across different areas.

Figure 1. Expression patterns of cck and pcp4 genes in rat V1. A–I: Double-ISH of cck (red) and pcp4 (green) mRNAs in rat V1. The areas

in the white boxes in layers 5 (D–F) and 6 (G–I) are magnified on the right panels. Note that these two genes are coexpressed in layer 5 but

not in layer 6. The white arrow in panel A indicates the cck-empty sublayer where the pcp4(þ) cells cluster. J,K: Immunofluorescence-ISH of

cck mRNA (red) and prepro-CCK (green). Panel J shows only the cck immunofluorescence, whereas panel K shows the colocalization of pro-

tein and mRNA signals for cck gene. L,M: Immunofluorescence-ISH of cck mRNA (red) and PCP4 (green). N–W: Fluorescent ISH of cck

mRNA (red) was performed on sections in which the corticogeniculate neurons were retrogradely labeled by Fast Blue (O,T). The white boxes

in panels N,O, and S,T are magnified below (P–R and U–W, respectively). A magenta-green version of this figure has been provided as an

online supporting file for the assistance of color-blind readers. Scale bars ¼ 200 lm in A–C; 20 lm in D–I; 25 lm in J–M; 100 lm in N–W.



Unfortunately, we cannot simply equate pcp4 mRNA

expression with corticothalamic projection, because of

the presence of the pcp4(þ) cells that are not corticotha-

lamic (e.g., layers 5 and 6b). Therefore, we first analyzed

detailed sublamina expressions of cck and pcp4 mRNAs

across various cortical areas by the double-ISH and corti-

cal box method (Hirokawa et al., 2008) and then com-

pared such profiles with the patterns of retrograde label-

ing by a series of thalamic and cortical injections on a

semiquantitative basis.

Area-specific sublamina profiles of cck andpcp4 mRNAs

In Figure 2A (Supporting Fig. S2A), double-ISH images

of cck and pcp4 mRNAs for the rat coronal sections are

Figure 2. Expression patterns of cck and pcp4 mRNAs in various cortical areas. A: Low magnified views of the double-ISH of cck (red)

and pcp4 (green) mRNAs in the rat brain. PL, prelimbic cortex; Cla, claustrum; M1, primary motor cortex; S1Tr, primary somatosensory

cortex, trunk region; S1BF, primary somatosensory cortex, barrel field; S2, secondary somatosensory cortex; V1, primary visual cortex;

V2L, secondary visual cortex lateral area. B–G: The magnified cortical areas labeled in panel A. Note the overall complementary lamina

patterns of cck and pcp4 signals. A magenta-green version of this figure has been provided as an online supporting file for the assistance

of color-blind readers. Scale bars ¼ 1 mm in A; 200 lm in B–G.

Watakabe et al.


shown at low magnification. Although their lamina pat-

terns appeared consistent across areas at a first glance,

there in fact existed considerable area differences. Of

particular note were the two areas at the medial and lat-

eral edges of the frontal sections, the prelimbic cortex

(PL) and the insular cortex/claustrum (Fig. 2A, left).

These two areas exhibited two extreme patterns of lamina

differentiation. In the prelimbic cortex, layer 6 was almost

devoid of cck mRNA (red) and was filled with pcp4(þ)

cells (Fig. 2A, PL, and Fig. 2E). In contrast, lateral to the

striatum, in what corresponds to the claustrum, there

were alternate layers of cck and pcp4(þ) cells, in which

the cck(þ) cells occupied the bottom sublayer (Fig. 2A,

Cla).

The pcp4/cck patterns in various areas may by and

large be regarded as variations of the above two patterns.

For example, in V1 (Fig. 1A), V2L (Fig. 2G), and S1Tr

(trunk region of S1: Fig. 2F), the pcp4(þ) cells occupied

the bottom part of layer 6a, as in the prelimbic cortex. In

S2 (Fig. 2D), which lies close to the insular cortex/claus-

trum, the bottom part of layer 6a was occupied by the

cck(þ) cells. In M1 (Fig. 2B) and S1BF (barrel field: Fig.

2C), the pcp4 and cck(þ) cells extensively intermingled to

make sublamina distinction difficult. In addition to the

area differences observed for layer 6, there were conspic-

uous area differences in layer 5 as well. In S1Tr, for exam-

ple, we observed large pyramidal cells in the lower part of

layer 5 that intensely expressed pcp4 mRNA (Fig. 2F). In

this area, pcp4 mRNA signal in layer 5 appeared to be

more abundant than that in layer 6 compared with other

areas. Finally, we refer to the entorhinal cortex as exhibit-

ing an exceptional pattern. In this area as well, cck mRNA

was expressed widely across layers and pcp4 mRNA was

expressed in the deep layers. Unlike other areas, how-

ever, these two mRNAs were extensively coexpressed,

the rhinal fissure being a clear-cut border for transition

(Fig. 2A, right).

Proportions of cck(1) and pcp4(1) cellsamong the excitatory neurons vary greatlyacross areas and layers

To describe the area differences of pcp4/cck mRNA

expression in more detail, we quantitated the ISH signal

(Fig. 3; Fig. S3). In the first set of experiments, we per-

formed double-ISH of pcp4/cck mRNA and VGluT1

mRNA. The idea was to calculate the ratio of pcp4/

cck(þ) cells among the VGluT1(þ) cells in different sub-

layers of layers 5 and 6. One example is shown in Figure

3A–C. Briefly, the ISH signals for pcp4 or cck and VGluT1

mRNAs were each transformed to a binary image by a se-

ries of despeckling/smoothing and thresholding (Fig. 3C).

This procedure turned the granular ISH signals to a more

smooth shape of cells and the colocalization was effi-

ciently detected. This analysis was done for the layer 5/6

portion that was divided into 20 subdivisions. In each sub-

division, we calculated the ratio of the double-positive

area (Fig. 3C, right panel, white) among the VGluT1-posi-

tive area. Summation of the 20 subdivisions resulted in

the lamina profiles as shown in Figure 3D (see Materials

and Methods for details of this analysis). Thus, the rough

estimate of the ratio of the cell numbers was obtained by

the ratio of area size, although this measurement should

be considered semiquantitative (see Discussion).

From these graphs, the high proportion of pcp4 or

cck(þ) neurons among the excitatory neurons was imme-

diately clear. Depending on layers and areas, the ratio

reached as high as 70–80%. Consistent with our qualita-

tive observations, pcp4 and cck mRNAs were generally

opposite in their lamina patterns. For example, cck mRNA

showed the highest peak around the border between

layers 5 and 6, where the ratio of pcp4 mRNA to VGluT1

mRNA was lowest. In the case of S2, cck mRNA showed

two peaks that exactly matched the low ends of the pcp4

signals. In S2, the sum of the two mRNAs was 80–100%

anywhere in layer 6a, suggesting their mutually exclusive

expressions. To test this point directly, we performed the

double-ISH of pcp4 and cck mRNAs and calculated the

coexpression ratio (double-positive area compared to the

summated area for pcp4 and cck(þ) area). As shown in

the overlaid graphs in Figure 3D (green line), the coex-

pression ratio of these two mRNAs were 10–20% in layer

6a and approached 30–40% in layer 5 in some areas. This

is partly explained by an inadvertent overlap of the neigh-

boring cells (e.g., Fig. 3B, see Materials and Methods). In

many cases, the expression of one gene was predominant

over the other, even if both genes were counted as ‘‘posi-

tive.’’ But there did exist neurons that expressed both

these mRNAs at high levels, and double-positive cells

were constantly noted (Fig. 3D). From this, we suggest

that it is the ratio of the single positive cells that varies

greatly across layers and areas.

Despite technical limitations, the above quantification

suggests that layer 6 neurons may be generally catego-

rized into cck(þ) and pcp4(þ) cells. To test this, we exam-

ined the coexpression of another layer 6 marker, the

nurr1 gene with cck gene. In the rat cortex, nurr1 mRNA

is abundantly expressed in layer 6 of the lateral area such

as S2 (Watakabe et al., 2007; Hirokawa et al., 2008). The

double-ISH of cck and nurr1 mRNAs in S2 revealed strik-

ing colocalization of these two mRNAs (Fig. 4). The cck

and nurr1 mRNAs were also coexpressed in the claustrum

(data not shown). It thus appears that the nurr1(þ) neu-

rons are a subpopulation of cck(þ) neurons.

The graphs in Figure 3D provide quantitative evidence

for the sublamination of the two neuronal populations



within layer 6a. Generally speaking, the cck(þ) cells were

more abundant around the border between layers 5 and

6, leaving the lower part of layer 6a empty (see Fig. 3A,C

as well). The pcp4(þ) cells, on the other hand, occupied

this cck-empty sublayer. The only exception was area S2,

where cck signals showed an additional peak below the

pcp4 peak. This sublayer, however, was not found in

S1BF. It is quite likely that this cck peak represents the

nurr1-expressing subpopulation of the cck(þ) neurons.

Comparison of S1BF and S2 also showed the quantitative

Figure 3. Quantification of cck and pcp4 mRNA expressions among the VGluT1-positive excitatory neurons in the rat cortical areas. A:

Double-ISH of cck (red) and VGluT1 (green) mRNAs in S1. The images for layers 5 and 6 were cut out for quantification of coexpression.

B: An example of a VGluT1(�) neuron expressing cck mRNA. The white box in panel A is magnified. Note that a part of cck mRNA signals

overlap with VGluT1 mRNA signals, which leads to overestimation of the coexpression in the following quantification method. C: The binary

images of panel A used for quantification. The original images were processed for several steps of despeckling/smoothing and threshold-

ing for cck (left panel) and VGluT1 (center panel) signals and overlaid for quantification (right panel; red for cck only, green for VGluT1 only

and white for the overlaps). The areas occupied by the positive signals were summated for each of the 20 subdivisions to make graphs

shown in panel D. For details, see Materials and Methods. D: The ratio of the cck (red) and pcp4 (blue)-positive areas among the VGluT1-

positive areas in 20 subdivisions of layers 5 and 6 are plotted for six cortical areas. The green plots indicate the cck/pcp4-double positive

areas among the sum of these two. The averages and standard errors (n ¼ 3) of three independent section data each for cck/VGluT1,

pcp4/VGluT1, double/(cckþpcp4) are shown. The border between layers 5 and 6 was determined on the basis of the VGluT1 signal pat-

terns, being the average of six VGluT1 data (three each for cck/VGluT1 and pcp4/VGluT1). A magenta-green version of this figure has

been provided as an online supporting file for the assistance of color-blind readers. Scale bars ¼ 200 lm in A; 20 lm in B.

Watakabe et al.


difference of the pcp4 and cck ratio. In S1BF, the pcp4(þ)

cells were generally more abundant than the cck(þ) cells

regardless of sublayers. In contrast, the cck(þ) cells

tended to dominate the excitatory populations in S2. This

observation is consistent with the double-ISH photos

shown in Figure 2. The pcp4/cck ratio was quite variable

across areas. For example, comparison of areas V1, V2L,

and A1 showed that the pcp4/cck ratio in layer 6a varies

across areas, despite similar lamina profiles. Among the

areas that we examined, the general rule was that the

pcp4(þ) cells dominate in the primary sensory areas

(S1BF, V1, A1), while the cck(þ) cells dominate in the

association area (S2, V2L).

Cortical box method reveals the area-specific distribution of cck and pcp4(1)cells

To analyze the area-specific differences of gene

expression in a more systematic way, we used the corti-

cal box method (Hirokawa et al., 2008), which transforms

a series of coronal section data into a single flat map (see

Fig. 5A; Fig. S4). By this strategy, we can have a spatial

overview of gene expression at a defined lamina position.

In Figure 5B, we showed the layer 4 map of RORbeta

mRNA (in heat map) as well as the layer 6 maps of pcp4

and cck mRNAs (in grayscale), which were each con-

structed by averaging the coronal section data of four

rats. In this panel, the RORbeta mRNA map served as a

marker to delineate area borders (shown by contours).

Compared with the RORbeta pattern, the area differences

of the pcp4 and cck mRNAs were less conspicuous, but

still evident: consistent with the double-ISH experiments

described above, the pcp4 mRNA was more abundant in

the primary sensory areas, while the cck mRNA was more

abundant in the association areas between the primary

sensory areas. Such complementarity was clearly shown

in the merged view of the two maps. Because these

mRNAs exhibit large sublayer differences, we next di-

vided layers 5 and 6 into 10 subdivisions and compared

the area distributions of each mRNA in a heatmap (Fig.

5D).

In Figure 5D, the sublamina map of L6_7 best repre-

sents the complementarity of the pcp4 and cck mRNA

distributions. In this sublayer, cck mRNA was abundant

(shown by red color) in the medial and lateral ends as

well as in a laterocaudal area denoted by the purple

circle. The pcp4 mRNA, on the other hand, exhibited the

opposite pattern. At first sight, the low level of pcp4

mRNA in the caudal part of A1 in L6_7 (purple circle) is

against the sensory-enriched expression of the pcp4

mRNA. In the lower L6_8 and L6_9 sublayer, however,

pcp4 mRNA exhibited high level in this caudal part of A1.

When combined together, the pcp4 mRNA level was high

throughout area A1. Similarly, the pcp4 mRNA was abun-

dant in V1 and S1 in the sublayers L6_7 to L6_10. The

cck mRNA expression tended to be low in these primary

sensory areas in these sublayers. Thus, the ‘‘primary

Figure 4. Double-ISH of nurr1 and cck mRNAs in the lateral cortical areas. Double-ISH of nurr1 and cck mRNAs were performed in the

lateral cortical areas including S1 and S2. A: ISH of nurr1 mRNA shows lateral area specific expression. The pia is to the left of the figure.

B: A merged view of nurr1 (green) and cck (magenta) mRNAs. C–E: Magnified images for the white boxes in panels A,B. Note that nurr1(þ)

cells (D) are a subpopulation of cck(þ) cells (C), as is clear from the merged view (E). Scale bars ¼ 200 lm in A,B; 20 lm in C–E.



Figure 5. Cortical box method for quantitation of the area and sublamina differences of cck and pcp4 gene expressions. The schematic

view of cortical box method. The photos show the original ISH data for pcp4 mRNA. Sixteen coronal sections that cover the posterior part

of the rat brain were processed to make a flat map of the cortical layers. The areas denoted by the red line were cut out from the photos,

transformed to rectangles, and merged to make a cortical box. B: The cortical box shown in A was cut tangentially to display flat maps of

different genes. The data from four rats were averaged to demonstrate the area differences of gene expression. The strength of expression

is shown in heat map for RORbeta mRNA, and in single color scales for cck and pcp4 mRNAs. The cck and pcp4 data are pseudocolored

for overlaying. C: Lamina profiles of RORbeta, cck, and pcp4 mRNAs. Each profile is the averaged value of all the areas examined. Despite

area differences in the thickness of layers and sublayers, the averaged data exhibit clear sublamina structures. D: The sublamina flatmaps

for cck and pcp4 mRNAs in layers 5 and 6. The area borders defined by RORbeta mRNA in layer 4 are shown in all the panels and anno-

tated in the bottom panels. The purple circle in panel L6_7 indicates the location where low expression of pcp4 mRNA and high expres-

sion of cck mRNA matches. The yellow circle in panel L5_4 indicates the area with high pcp4 mRNA in layer 5. A magenta-green version

of this figure has been provided as an online supporting file for the assistance of color-blind readers.

Watakabe et al.


sensory-association’’ was one axis of distribution rule. In

addition to this rule, this sublamina analysis revealed rich

distribution of the cck mRNA signal in the laterocaudal

areas. The area denoted by the purple circle in L6_7 is

considered the bottom of the cck-rich subcompartment.

Upward into L6_6 and L5_5, the cck mRNA signal spread

beyond the association areas and was observed in the

caudal part of A1.

We have described above that the area S1Tr exhibits

particularly intense expression of the pcp4 mRNA in the

pyramidal cells of layer 5 (Fig. 2F). Figure 5D map shows

that the layer 5 expression of the pcp4 mRNA is outstand-

ing in this relatively small area (the yellow circle). From

location and cell morphology (Fig. 2F), this is possibly a

cluster of large pyramidal cells projecting to the dorsal

column nuclei of the spinal cord.

Thalamic and cortical projections correlatewith pcp4 and cck mRNA expressions

We have so far described area differences in the subla-

mina architectures of pcp4 and cck mRNA expressions in

the deep layers. To test whether such patterns correlate

with corticothalamic projections beyond V1, we per-

formed a series of tracer-ISH experiments. In these

experiments, we used FluoroGold instead of FastBlue as

the retrograde tracer, so as to increase the sensitivity of

tracer detection by using anti-FluoroGold antibody for

detection. As the targets, we injected FluoroGold into

specific sensory thalamus (VP, PO, and MG) and associa-

tion thalamus (MD and LD) (Fig. 6; Fig. S5). In all these

experiments, we found positive and negative correlations

of pcp4 and cck mRNA expressions, respectively, with the

thalamic projections. We also performed injections into

cortical areas (M1 and V1) and found the opposite corre-

lations. Below, we first provide qualitative accounts of

representative tracer-ISH data and then follow this by

semiquantitative analyses.

FluoroGold injection into thalamusIn Figure 7 (Fig. S6), we presented the data for the Flu-

oroGold injection into somatosensory nuclei VP and PO.

In the barrel cortex, the neurons projecting to these

nuclei are localized in different sublayers: the VP-projec-

ting neurons in layer 6a, and the PO-projecting neurons in

layers 5 and the lower part of layer 6a (Killackey and

Sherman, 2003). We tested whether these neurons

express cck and pcp4 mRNAs. In case R30, we suc-

ceeded in making a relatively small injection into VP

(Figs. 6, 7B). This injection resulted in a focal labeling of

the corticothalamic neurons in layer 6a of S1 (Fig. 7A).

The tracer-ISH experiment showed that the Fluo-

roGold(þ) cells expressed pcp4 but not cck mRNA (Fig.

7C–J). After tracer injection into PO (case R22), on the

other hand, retrograde label was located in layer 5 and

the lower part of layer 6a in S1 (Fig. 7K). The FluoroGold-

labeled cells in layer 6a were located in the sublayer that

showed low pcp4 ISH signals (Fig. 7Q,R). Despite gener-

ally low pcp4 mRNA level per cell, these PO-projecting

neurons clearly expressed pcp4 mRNA (Fig. 7S,T). We

also observed that a very low level of cck mRNA is coex-

pressed in these cells (Fig. 7O,P).

We have shown in Figure 2 that the lamina differentia-

tion of pcp4 and cck mRNAs is most conspicuous at two

limbic areas, the prelimbic and the insular cortex and

claustrum. The FluoroGold injections into different parts

of MD nuclei of the thalamus resulted in the retrograde

labeling of neurons in these areas (Fig. 8; Fig. S7), con-

sistent with previous studies (Reep and Winans, 1982;

Sesack et al., 1989; Gabbott et al., 2005). In both cases

we observed striking correlations between the cck/pcp4

mRNA expression and MD-projection neurons. In case

R16, the injection size was large and centered around

paratenial, anteroventral, and anteromedial thalamic

nuclei, as well as the rostral MD (Fig. 8A), which are

Figure 6. Injection sites of FluoroGold. The fluorescent images

of FluoroGold around the injection sites in rat R30, R22, R16,

and R09 are shown. The location of these FluoroGold deposition

sites are specified in reference to the Paxinos and Watson (2007)

atlas in Figures 7 and 8. A Color version of this figure has been

provided as an online supporting file.



known to receive corticothalamic projections from the

prelimbic cortex (Sesack et al., 1989). In this injection,

the corticothalamic cells in the prelimbic cortex were

localized to layer 6, where there were very few cck(þ)

neurons (Fig. 8D–G). These neurons were positive for the

pcp4 mRNA (Fig. 8H–K). In case R09 (Fig. 8B), the injec-

tion was in the caudal part of MD and the retrograde

labeling was observed in the insular cortex (Fig. 8L). The

corticothalamic cells in this area were localized to the

cck-poor layer, between the cck-rich layer 5 and the

claustrum (Cla). This sublayer exactly matched the pcp4-

rich layer, where almost all the FluoroGold-positive cells

Figure 7. Tracer-ISH analyses of the corticothalmic cells projecting to VP and PO. FluoroGold was injected into VP (A–J) and PO (K–T) to

retrogradely label the corticothalamic cells in area S1 for tracer-ISH analyses. A,K: The distributions of the FluoroGold-labeled cells are

shown in low magnification. B,L: The locations of the FluoroGold depositions in rat R30 (B) and R22 (L) are shown in the coordinates of

Paxinos and Watson (2007). The original images for these injection sites are shown in Figure S5. C,D: Double staining of FluoroGold

detected by antibody (green) and cck mRNA (red) in rat R30. Taken from the red box in panel B. E,F: Magnified images of the white boxes

in panels C and D. G,H: Double staining of FluoroGold detected by antibody (green) and pcp4 mRNA (red) in rat R30. White boxes are

magnified in I,J. M–T: Same as C–J. The samples were from the rat R22. A magenta-green version of this figure has been provided as an

online supporting file for the assistance of color-blind readers. Scale bars ¼ 500 lm for A,K; 100 lm for C,D,G,H,M,N,Q and R; 20 lmfor E,F,I,J,O,P,S and T.

Watakabe et al.


Figure 8. Tracer-ISH analyses of the corticothalmic cells in the prelimbic and insular cortices, projecting to MD. FluoroGold was injected

into medial thalamus including MD, which resulted in the labeling of the corticothalamic cells in the prelimbic (R16) and the insular (R09) cor-

tices. A–C: The locations of the injection sites are shown in A,B. The analyzed areas are shown by the red boxes in C. PL, prelimbic cortex;

Cla claustrum. D: Double staining of FluoroGold detected by antibody (green) and cck mRNA (red) in the prelimbic cortex of rat R16. Taken

from the red box in C. E–G: Magnified images of the white box in D. H: Double staining of FluoroGold (green) and pcp4 mRNA (red) of in the

prelimbic cortex of rat R16. I–K: Magnified images of the white box in H. L: Double staining of FluoroGold (green) and cck mRNA (red) in the

insular cortex of rat R09. Taken from the red box in C. N,O: Magnified images of the white box in L. P: Double staining of FluoroGold (green)

and pcp4 mRNA (red) in the insular cortex of rat R09. R,S: Magnified images of the white box in L. A magenta-green version of this figure

has been provided as an online supporting file for the assistance of color-blind readers. Scale bars ¼ 100 lm in H (applies to D); 15 lm in

K (applies to E–G, I–K); 200 lm in L (applies to M,P,Q); 25 lm in O,S (applies to N,R).



were also positive for the pcp4 mRNA and negative for

the cck mRNA (Fig. 8P–S). We performed similar tracer-

ISH experiments in A1 (projecting to MG), S2 (projecting

to PO), and V2 (projecting to LD) and observed high pcp4

and low cck mRNA expressions in the corticothalamic

cells in these areas, too (see below).

FluoroGold injection into the cortexIn Figure 9 (Fig. S8), we present data of a FluoroGold

injection into M1. Although the injection was relatively

large, it appeared to be mostly restricted to M1 (Fig. 9B).

Consistent with previous studies (Donoghue and Parham,

1983; Mitchell and Macklis, 2005), we observed extensive

labeling of the contralateral cortex. We also observed Flu-

oroGold-positive cells in the claustrum and the insular cor-

tex in the ipsilateral side (Fig. 9C–E). The correlation of

gene expression and projection was obvious in this lateral

region. As shown in Figure 9C–E, FluoroGold-positive cells

distributed exactly to the pcp4-poor regions in the claus-

trum and the insular cortex. In Figure 9F–Q, we show the

magnified view of these tracer-ISH experiments. In the ip-

silateral side (Fig. 9F–K), FluoroGold label was mainly

observed in lower layer 5 and upper layer 6a. The Fluoro-

Gold labeling in this sublayer was very similar to the cck

mRNA pattern at the cellular level (Fig. 8F–H). Cck mRNA

was also expressed in the lower part of layer 6a in this

area, but FluoroGold did not label these populations of the

cck(þ) neurons. Regarding this population, we believe

that it represents a nurr1(þ) subtype of cortically projec-

ting neurons (Arimatsu et al., 2003). This statement is sup-

ported by double-ISH of cck and nurr1 mRNAs (Fig. 4). In

contrast, the FluoroGold pattern was different from that of

the pcp4 mRNA. Notably, the sublayer of the FluoroGold

label corresponded exactly to the pcp4-poor sublayer

(Fig. 9I–K). Such correlations were essentially the same in

lower layer 5 / upper layer 6a of the contralateral side

(Fig. 9L–Q). In the contralateral side, the FluoroGold label-

ing was also found in layers 2/3, consistent with previous

studies (Donoghue and Parham, 1983; Mitchell and

Macklis, 2005). In layers 2/3, almost all the excitatory

neurons expressed cck mRNA and no excitatory neurons

expressed pcp4mRNA (data not shown). Thus, the cortical

projection showed positive and negative correlations with

cck and pcp4mRNA expressions, respectively.

Sublamina distribution of the layer 6projection neurons in different areas

So far, we have observed the coincidence of cck/pcp4

mRNA expressions and corticocortical/corticothalamic

projections qualitatively. Next, we quantified their subla-

mina distributions by the same overlap measurement

strategy as we took in Figure 3. Each of the graphs in Fig-

ure 10 indicates the ISH signals (red line), FluoroGold sig-

nals (green line), and the double-positive signals (blue

line) in layers 5 and 6 (upper side is to the left). The Y-axis

of this graph indicates the area occupied by the positive

signals. To estimate the accuracy of measurement, we

compared the result of the overlap measurement and

manual counting. In the peak area for the FluoroGold

labeling, 95% of the FluoroGold-positive cells were also

positive for pcp4 mRNA by manual counting, whereas the

ISH signals covered 73% of the FluoroGold signal areas

(data not shown). This discrepancy can be attributed to

the different shapes of the signals for ISH and the tracer.

On closer inspection, we confirmed that the two methods

detected essentially the same cell populations. We con-

clude that the overlap measurement provides semiquanti-

tative but reliable estimation of the relative abundance of

costaining by ISH and tracers.

By our method, the expression of pcp4 but not cckmRNA

in the corticothalamic cells was obvious at first glance (Fig.

10A–G, blue line). The ratios of the pcp4(þ) cells among the

FluoroGold-labeled cells were �75–83%, while those of the

cck(þ) cells were 9–18%. This cck(þ) populations generally

showed a low level of cck mRNA expression, even when

judged to be positive. The PO-projecting populations in S1

(Fig. 10G) showed a lower ratio of the pcp4(þ) cells (62%),

which reflects a lower level of pcp4 mRNA expression in the

lower part of layer 6a (Fig. 7T). Overall, this quantitation was

consistent with the qualitative images for the tracer-ISH

experiments we have described so far.

Next, we examined the ratios of the FluoroGold-labeled

corticothalamic cells among the pcp4 and cck(þ) cells in

each area. Generally speaking, the pcp4 plots consisted

of three peaks. These peaks represent neurons in layer 5,

layer 6a (the bulk of layer 6), and layer 6b. Comparison

with the FluoroGold plots for the corticothalamic projec-

tions showed that it corresponded exactly to the second

peak of the pcp4 plots, (Fig. 10A–F, right panels). In each

case of injections, the double-positive signals accounted

for 42–60% of the pcp4 positive signals in layer 6a, but

only 1–17% in layer 5. This observation suggests that,

regardless of areas, there are two distinct pcp4(þ) popu-

lations in layers 5 and 6a, and that the distribution of the

latter population corresponds to that of the corticothala-

mic cells. In contrast to the case in pcp4 ISH, we

observed very small overlap (7–11%) between the Fluoro-

Gold and cck signals in layer 6a (Fig. 10A–F, left panels).

In the case of corticocortical projections, the Fluoro-

Gold plots exhibited a rather broad lamina profile that has

peaks in layer 5 and the upper part of layer 6a (Fig. 10H–

J). The cck signals showed good correlations with the Flu-

oroGold signals across layers (Fig. 10H–J). The cck(þ)

cells among the FluoroGold positive cells were 71–81%,

while the FluoroGold positive cells among the cck(þ) cells

were 45–57% across layers 5 and 6. Although we have

Watakabe et al.


Figure 9. Distribution patterns of the corticocortical cells labeled by FluoroGold injection into M1. A: The location of the FluoroGold depo-

sition in M1 is shown by purple filling. The two green lined boxes indicate the locations for the low-magnification photos around the injec-

tion site and the retrogradely labeled lateral areas (shown in B,D). The red line boxes indicate the locations for the high magnification

photos (F–Q). B: Low-magnification photo around the injection site. FluoroGold deposited in M1 were immunodetected after ISH. C,D: Flu-

oroGold-labeled cells (green) in the ipsilateral side are shown in reference to the pcp4 ISH (red). Note that the clusters of the Fluo-

roGold(þ) cells exactly fill the pcp4-low regions, including the claustrum. E: The white box in C is magnified. St, striatum; Ins, insular

cortex; Cla, Claustrum. F–H: Double staining of FluoroGold (green) and cck mRNA (red) in the ipsilateral insular cortex of rat R24. Taken

from the red box in A,f. The white boxes are magnified in the lower panels. I–K: Double staining of FluoroGold (green) and pcp4 mRNA

(red) in the ipsilateral insular cortex of rat R24. L–N: Double staining of FluoroGold (green) and cck mRNA (red) in the contralateral cortex

of rat R24. Taken from the red box in A,l. The white boxes are magnified in the lower panels. O–Q: Double staining of FluoroGold (green)

and pcp4 mRNA (red) in the contralateral cortex of rat R24. A magenta-green version of this figure has been provided as an online sup-

porting file for the assistance of color-blind readers. Scale bars ¼ 1 mm in B–D; 200 lm in E; 100 lm in F–K,L–Q; 25 lm in lower

panels.



described reverse lamina profiles for pcp4 mRNA and cor-

ticocortical cells (Fig. 9), there was considerable overlap

between these two (28–55% for pcp4/FluoroGold and

12–22% for FluoroGold/pcp4). One possible scenario is

that the ‘‘corticocortical’’ cells in this experiment may

include various subtypes of projection neurons that have

collaterals to M1. Technically, we cannot exclude the pos-

sibility that the FluoroGold may have invaded the white

matter (see Fig. 9B). Nevertheless, the labeling patterns

were consistent with the previous reports of cortical

injections and distinct from the thalamic ones. Overall,

our results support our hypothesis that the distribution

patterns of pcp4 and cck mRNAs reflect the thalamic and

cortical projections in layer 6a.

Figure 10. Lamina profiles of FluoroGold/ISH colocalization in six thalamic and two cortical injections. Tracer-ISH experiments qualitatively

described in Figures 6–8 were quantified. To obtain these lamina profiles, the ISH signals (for cck or pcp4 mRNA) and FluoroGold signals

were converted to binary images. The relative strength of expression in 20 sublamina was estimated as the sum of the areas occupied by

these signals in each sublamina (see Fig. 3B). The red, green, and blue plots represent the ISH, FluoroGold, and the overlap of these two sig-

nals, respectively. The averages and standard errors of three independent section data are shown (n ¼ 3). A: The tracer-ISH of area S1 in

rat R07, with FluoroGold injection into VP. B: Second case of VP injection (rat R30) examined in S1. C: Tracer-ISH of A1 in rat R06 with MG

injection. D: Tracer-ISH of S2 in rat R22 with PO injection. This is the same rat used to label the corticothalamic cells in the lower part of

layer 6a (see panel G). E: Tracer-ISH of the insular cortex in rat R09 with MD injection. In this analysis, claustrum (Cla) was included as a

part of the lamina structure to show the colocalization of the pcp4 and FluoroGold signals within in this lateral architecture. F: Tracer-ISH of

V2 in rat R05 with LD injection. In this rat and rat R22 the corticothalamic cells in layer 5 were labeled. G: Tracer-ISH of S1BF in rat R22

with PO injection. H: Tracer-ISH of M1 in rat R24, which received FluoroGold injection into the contralateral side of M1. I: Tracer-ISH of the

insular cortex in rat R24 with M1 injection of the ipsilateral side. J: Tracer-ISH of V2L in rat R11 with V1 injection.

Watakabe et al.


DISCUSSION

The main finding of this study is the identification of

distinct pcp4(þ) and cck(þ) neuronal subpopulations in

layers 5 and 6. In general, the pcp4(þ) cells formed three

sublayers in layers 5, 6a, and 6b, whereas the cck(þ)

cells formed one or two sublayers in between. The posi-

tion of the pcp4 sublayer in layer 6a and the cck sublayer

at the layer 5/6 border matched exactly with that of the

corticothalamic and corticocortical cells in all the areas we

tested (Fig. 10). Importantly, there was a very high correla-

tion between gene expression and corticothalamic/corti-

cocortical projections in layer 6a even when the two cell

types intermingled. Based on these data, we conclude that

area-specific sublamination of pcp4 and cck mRNAs

reflects the distribution of corticothalamic and corticocorti-

cal projection neurons. Specifically, we observed extensive

intermingling of pcp4(þ) and cck(þ) cells in areas M1 and

S1BF, in contrast to almost complete segregation in pre-

limbic cortex and claustrum/insular areas. In areas such

as V2L and V1, sublamina segregation was also conspicu-

ous. In S2, we observed two peaks of cck(þ) neurons,

which we believe to represent distinct subpopulations of

corticocortical neurons, with differential nurr1 expression

and distinct cortical targets. We believe that such subla-

mina distributions are related to the functional organization

of each area; but, ultimately, specializations of cell type,

layer, and area must need to be coordinated as a system.

Below, we discuss these issues based on the findings of

current and past studies.

Cell types of deep layers in relation to cckand pcp4 mRNA expression

Layer 6 has been historically viewed as a polymorphic

layer containing neurons with highly heterogeneous mor-

phology (Ferrer et al., 1986a,b; Zhang and Deschenes,

1997; Chen et al., 2009). Phenotypic diversity is not lim-

ited to morphology but is also found in intrinsic connectiv-

ity (Briggs and Callaway, 2001; Zarrinpar and Callaway,

2006; Thomson and Lamy, 2007), electrophysiology

(Brumberg et al., 2003), and gene expression (Kaneko

et al., 1995; Arimatsu et al., 2003; Bai et al., 2004; Wata-

kabe et al., 2007). One of the key features to classify

these heterogeneous groups of cells is the extrinsic con-

nectivity. Three major classes of projection neurons, cor-

ticothalamic, corticocortical, and corticoclaustral cells

each exhibit characteristic morphology (Katz, 1987;

Zhang and Deschenes, 1997; Veinante et al., 2000;

Briggs, 2010; Thomson, 2010) and are considered to

form subclasses of pyramidal cells. Since, as shown by

our data and other studies, gene expression correlates

with projections, gene expression profiles can, with some

caution, be used for cell type identification.

One advantage of gene expression analysis over other

methods of cell identification is that the expression speci-

ficity of various genes can be compared by double stain-

ing. For example, we showed in this study that a nurr1(þ)

subpopulation is included in the cck(þ) cells. This finding

not only supports the validity of the cck-pcp4 classifica-

tion, but also suggests further subtype differentiation of

‘‘corticocortical cells.’’ We tested the expression of

ZFPM2 (FOG2) and SatB2 genes, which are considered to

correlate with corticothalamic and corticocortical projec-

tions, respectively (Leone et al., 2008; Han et al., 2011).

As expected, ZFPM2 mRNA was expressed in pcp4 but

not in cck mRNA-positive cells in the adult rat cortex,

while SatB2 mRNA expression coincided with the cck

mRNA expression in rat V1 (data not shown). On the other

hand, the expression of some previously reported layer 6

marker genes, such as Tbr1 (Bedogni et al., 2010), foxp2

(Ferland et al., 2003, Rowell et al., 2010) and sema3E

(Watakabe et al., 2006) were observed in both cck(þ)

and cck(�) cells. That is, these probes have low specific-

ity for the different projectionally defined cell types.

Compared to layer 6 cell populations, the cell types

expressing cck and pcp4 genes in layer 5 are less clearly

delineated. There are two basic types of layer 5 neurons,

which occupy distinct sublaminae: those including cortico-

striatal cells and those including corticotectal and cortico-

pontine cells (Kasper et al., 1994; Molnar and Cheung,

2006; Morishima and Kawaguchi, 2006; Groh et al., 2010,

Mao et al., 2011). In our data, pcp4 mRNA was expressed

by the corticothalamic cells in layer 5, which are considered

similar in kind to the corticopontine cells (Veinante et al.,

2000). However, unlike the projectionally defined cortico-

thalamic and corticopontine cells, which occupy the lower

sublayers of layer 5 (Molnar and Cheung, 2006; Morishima

and Kawaguchi, 2006; Mao et al., 2011), cells expressing

pcp4 mRNA were located in both layers 5a and 5b. ER81

mRNA, an established marker of layer 5 (Hevner et al.,

2003; Yoneshima et al., 2006), was coexpressed with cck

or pcp4 mRNAs in layer 5 (A.W., data not shown). We con-

sider that the cck and pcp4(þ) cells in layer 5 are distinct

cell types from those in layer 6, although they probably

share some common features related to gene expression.

Two previous reports have investigated the correlation

of cck mRNA expression and corticothalamic projections.

These reached the same conclusions as ours (Burgunder

and Young, 1990) or opposite (Senatorov et al., 1995).

One possibility for the differences is the use of oligonu-

cleotide probes for ISH, which are less sensitive and spe-

cific than the RNA probe. In this regard, we are confident

of the specificity of our ISH probe, because we found the

same result using prepro-CCK antibody (Fig. 1B). Addi-

tionally supporting our conclusions, we used two antago-

nistic gene probes to show the positive and negative



correlations with projections. We must, however, keep in

mind that there are still mismatches in the cell identifica-

tion by the four criteria proposed in this article; that is,

cck and pcp4 gene expression, and corticothalamic and

corticocortical projections. The mismatch could be partly

explained by technical problems, such as insufficient

labeling by histological procedures or inability to distin-

guish functional and nonfunctional levels of expression. A

more likely cause of mismatch is the phenotypic diver-

sions that occurred during development and in adult. Due

to extensive previous studies, phenotypic complexity of

inhibitory neurons is now well recognized (Markram et al.,

2004). Comparable studies need to address the full com-

plexity of excitatory cells as well. With the advent of opto-

genetic technologies, the issue of cell type is becoming

more important than ever (for a review, see Fenno et al.,

2011). The kind of approach we took in this study should

be useful in implementing such knowledge toward gener-

ating cell type-targeted mouse lines for cortical studies.

Potential roles of cck and pcp4 geneproducts in cortical functions

The cck gene is posttranslationally processed to

encode an 8 amino acid neuropeptide, CCK-8, in the

brain. Pharmacological studies as well as studies in the

knockout mouse for cck, and its receptor, CCKR-A or B,

suggest the involvement of the CCK system in anxiety-

related behavior and satiety, as well as learning and mem-

ory (Miyasaka et al., 2002; Lo et al., 2008). The precise

role of the CCK system in the cortex, however, is not very

well known at present. It is reported that CCK octapep-

tide has depolarizing effects on most layer 6b neurons

and on a subpopulation of the cells in layer 5 and 6a

(Chung et al., 2009). It is therefore possible that the CCK

system plays a crucial role in aspects of associative learn-

ing that require corticocortical interactions.

The pcp4 gene, encoding a 61 amino acid polypeptide,

has been thought to play a role in regulation of calmodulin

signaling (Johanson et al., 2000). In the hippocampus,

pcp4 mRNA is specifically expressed in CA2 pyramidal

cells (e.g., see Fig. 2A) and is considered to limit plasticity

by regulation of the postsynaptic calcium dynamics

(Simons et al., 2009). Deficiency of the pcp4 gene leads

to modified plasticity of the Purkinje cells and deficits in

locomotor learning (Wei et al., 2011). The expression of

PCP4 in the corticothalamic cells may affect the plasticity

of this class of cortical neurons.

Area differences of sublamina structure inlayer 6

Three characteristic features of the rat layer 6 emerged

clearly from our study. First, layer 6a of the rat cortex con-

sists of two or three sublayers, delineated by cck and pcp4

mRNA expression. Second, the extent of cck and pcp4 seg-

regation differs greatly across areas. For example, segre-

gation was almost complete in the prelimbic and the insu-

lar/claustrum cortex (Fig. 8), very pronounced in A1, V2L,

or V1 (Fig. 3), but not so much in M1 or S1BF (Figs. 2, 3).

These patterns are likely associated with the sublamina

segregation and intermixing of the corticothalamic and cor-

ticocortical cells in these areas. Third, the semiquantitative

data showed that the relative abundance of the two subpo-

pulations differs across areas. Generally speaking, the

pcp4(þ) cells were more abundant in the sensory areas

than in the association areas, whereas the cck(þ) cells

showed the opposite pattern (Figs. 3, 5). We believe that

this distribution preference applies to the corticothalamic

and corticocorticortical cells.

Sublamina stratification is discernible by anatomical

tract tracing and even by conventional cytoarchitectonic

staining (e.g., Nissl staining or immunocytochemistry).

However, it has been difficult to ascertain that sublamina

structure is homologous across areas and species, due

to the lack of a reliable definition for commonality. The

use of various gene markers is beginning to change this.

As we have shown in this and a previous study (Watakabe

et al., 2007), similar sublaminar structure can be defined

on the basis of gene expression. Rowell et al. (2010) also

reported fine sublaminar stratification, in mouse and fer-

ret cortex, by using a panel of layer-enriched genes. We

speculate that the genetic program that specified various

projection neuron types still operates in the adult brain

for maintenance of subtype identity.

Our data raise several issues of area-specific variation

across limbic, primary, and associational cortices. One is

that the relative density of corticothalamic cells may be

regulated in balance with the thalamocortical inputs, in

an area-specific manner. A related point is that the segre-

gation and intermixing of the pcp4 and cck(þ) cells may

reflect thalamocortical and corticocortical innervations,

in the framework of a wider area-specific or region-spe-

cific microcircuitry. Finally, since the area-specific pat-

terns of layer 6 organization can be correlated with a

medial-lateral topography, future studies might address

the influence of early developmental gradients (Altman

and Bayer, 1991; Rash and Grove, 2006) in establishing

these anatomical and functional specializations.

ACKNOWLEDGMENT

We thank Dr. James I. Morgan at the Department of De-

velopmental Neurobiology, St. Jude Children’s Research

Hospital for his kind gift of the anti-PEP 19 (PCP4) serum.

CONFLICT OF INTERESTNone.

Watakabe et al.


ROLE OF AUTHORSAll authors had full access to all the data in the study and

take responsibility for the integrity of the data and the

accuracy of the data analysis. Study concept and design:

A.W. Acquisition of data: A.W., S.O.; Analysis and

interpretation of data: A.W., J.H. Drafting of the article:

A.W., K.S.R. Obtained funding: A.W., T.Y. Administrative,

technical, andmaterial support: N.I, T.K. Study supervision:

K.S.R. and T.Y.

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