Retinol-binding Protein Gene is Highly Expressed in Higher-order Association Areas of the Primate...

Retinol-binding Protein Gene is HighlyExpressed in Higher-order AssociationAreas of the Primate Neocortex

Yusuke Komatsu1, Akiya Watakabe1, Tsutomu Hashikawa2,

Shiro Tochitani1,3 and Tetsuo Yamamori1

1Division of Speciation Mechanisms 1, National Institute for

Basic Biology, Aichi 444-8585, Japan and 2Laboratory for Neural

Architecture, Brain Science Institute, RIKEN, Wako, Saitama

351-0198, Japan 3Present address: The Picower Center for

Learning and Memory, Massachusetts Institute of Technology,

Cambridge, MA 02139, USA

The neocortex consists of histochemically, connectionally, andfunctionally distinguishable areas. Recently, molecular biologicaltechniques have enabled us to find rare types of genes expressedin specific neocortical areas. We previously reported occ1 gene aspreferentially expressed in the primary visual cortex (V1), using thedifferential display method. Here, by differential display, we foundselective and strong expression of the serum retinol-binding protein(RBP) gene, in higher-order association areas. In V1, RBP mRNAwas expressed only in the superficial part of layer II, but its ex-pression increased, involving deeper layers, along the visual path-way. In visual association areas such as TE, RBPmRNAwas stronglyexpressed in both supra- and infragranular layers. In primary audi-tory and somatosensory areas, as in V1, RBP expression waslow, and restricted to the upper part of the supragranular layers.The laminar pattern of RBP expression is in marked contrast withthat of occ1; and in early visual areas where both genes are ex-pressed, these occur in distinct sublayers within the supragranularlayers. In neonatal monkeys, the area-specific expression patternof RBP was less distinct, suggesting that the characteristic expres-sion of RBP in higher-order association areas is mainly establishedpostnatally.

Keywords: area formation, cortical layer, differential display, macaquemonkey, RBP, thalamic projection

Introduction

Recent advances in molecular biological techniques allow us to

screen thousands of genes for differential expression; and it is

evident that this tool provides a potentially important new

approach for investigating the classic questions of cortical

structure, function, and arealization (Hendry et al., 1984; Levitt,

1984; Arimatsu et al., 1994). Using the differential display

method, our strategy has been to identify genes that were

differentially expressed in neocortical areas of adult macaque

monkeys, in order to compare differences in level of expression

and laminar distribution across the systems.

By this method, we previously identified one gene, gdf7,

which was enriched in the primary motor cortex (Watakabe

et al., 2001) and another gene, occ1, which was preferentially

expressed in V1 (Tochitani et al., 2001, 2003). Within V1, the

expression of occ1 was concentrated in layer 4Cb, which

receives strong direct input from the lateral geniculate nucleus.

Furthermore, we demonstrated that occ1 is expressed in an

activity-dependent and a developmentally regulated manner

(Tochitani et al., 2001, 2003). These results are consistent with

the importance of thalamic projections in establishing the func-

tional architecture of the primary sensory cortex, in addition to

the geneticaly programmed mechanisms (Rakic, 1988; O’Leary,

1989; Katz and Shatz, 1996; Donoghue and Rakic, 1999a,b;

Sestan et al., 2001; O’Leary and Nakagawa, 2002).

Here, we report that serum retinol-binding protein (RBP) is

highly expressed in cerebral cortex, preferentially in association

areas. RBP is abundantly synthesized in liver and is secreted as

a complex with retinol and transthyretin into plasma (Good-

man, 1980; Blaner, 1989), but its function and distribution in the

cerebral cortex has not previously been recognized.

By in situ hybridization, our study indicated that, in associ-

ation and limbic cortices, RBP gene was intensely expressed

in layers II, III and V, mainly in excitatory neurons. In primary

sensory areas, by contrast, the expression was low and re-

stricted to the upper part of the supragranular layers. Inspection

of the well-defined visual pathway showed that neurons ex-

pressing RBPmRNA became progressively more numerous with

distance from V1, and gradually extended to the deeper part of

layer III and infragranular layers. The laminar distribution of RBP

expression was complementary to that of occ1.

We also compared the expression of RBP with the distribu-

tion of thalamocortical terminations, and found a reverse cor-

relation in the early sensory areas. The influence of thalamic

projections is thus likely to be more indirect than for occ1. We

conclude that RBP is a useful marker gene for studying the func-

tion and formation of association areas and their related subcor-

tical structures. With further characterization, it may provide

clues as to area and laminar specializations of the primate

neocortex.

Materials and Methods

AnimalsTwo young adult crab-eating monkeys (Macaca fascicularis) were used

for the differential display screening. Three young adult Japanese

monkeys (Macaca fuscata) were used for histological analysis (body

weight 2.9--4.5 kg). Three neonatal macaques (postnatal day 1, n =2 and

postnatal day 2, n = 1) were used as previously reported (Tochitani et al.,

2003). Frozen sections from two young adult animals, monocular-

deprived by TTX-injection for 7 or 14 days, previously reported

(Tochitani et al., 2001), were available for processing. All experiments

followed the animal care guidelines of Okazaki National Research

Institute, Japan, and the NIH, USA.

Differential DisplayFive neocortical areas were sampled for the fluorescent differential

display--polymerase chain reaction (PCR; Fig. 1A). The abbreviations for

the neocortical areas are according to Von Bonin and Bailey (1947):

FDD, both banks of the principal sulcus including areas 9 and 46; FA,

primary motor cortex in the anterior bank of the central sulcus; PC,

primary somatosensory area, in the posterior bank of the central sulcus;

TE, visual association cortex, located in the superior temporal sulcus

(middle of the ventral bank); OC, primary visual cortex, posterior to the

lunate sulcus.

Cerebral Cortex V 15 N 1 � Oxford University Press 2005; all rights reserved

Cerebral Cortex January 2005;15:96--108

doi:10.1093/cercor/bhh112

Advance Access publication June 24, 2004

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Total RNA was extracted by acid guanidinium thiocyanate--phenol--

chloroform extraction method (Chomczynski and Sacchi, 1987). Re-

verse transcription was carried out following the protocol from the

supplier (GIBCO BRL/Invitrogen: Life Technologies, San Diego, CA).

The fluorescent differential display with a carboxy-X-rhodamine (ROX)-

labeled anchor primer was performed as previously reported (Yoshi-

kawa et al., 1998; Tochitani et al., 2001; Watakabe et al., 2001). The

ROX-labeled anchor primer was obtained from Takara (Otsu, Shiga,

Japan): 59-AAGCTTTTTTTTTTT(C, G or A)-39. Thirty-seven oligonucleo-

tides of 13-mer were synthesized (GenHunter Corporation, Nashville,

TN; GIBCO BRL/Invitrogen: Life Technologies, San Diego, CA). Each 13-

mer oligonucleotide was used for an arbitrary primer with the anchor

primer. A target band was reamplified by the same PCR primer set and

inserted into the Eco RV site of pBluescript II (KS+) vector (Stratagene,

La Jolla, CA) for cloning of the DNA fragment (the primer set of 59-

AAGCTTTTTTTTTTTG-39 and 59-AAGCTTGATCGTC-39 was used for

RBP cDNA cloning).

PCR Primers and Probe SynthesisAll PCR primers except for occ1 were designed based on the human

sequence database. The DNA fragments were produced by RT (reverse

transcription)--PCR from African green monkey cDNA: (1) monkey RBP

gene (corresponding to GenBank X00129, nt 133--862); (2) monkey

glutamate decarboxylase 1, 67kDa (GAD67; corresponding to GenBank

BC037780, nt 422--1051 ); (3) monkey vesicular glutamate transporter 1

(VGluT1; corresponding to GenBank: AB032436 nt 204--1093 ). PCR

fragments were ligated into Eco RV site of the pBluescript II (KS+) vector

and transfected into E. coli. The plasmids were extracted and linearized

by Eco RI or Sal I before being used for the template of antisense or

sense probes. The occ1 probe was derived from GenBank AB039661

(Tochitani et al., 2001). The digoxigenin (DIG)-UTP labeling kit (Roche

Diagnostics, Basel, Switzerland) was used for in situ hybridization. The

fluorescein (FITC)-UTP labeling kit (Roche Diagnostics) was also used

to produce RNA probe for double in situ hybridization. RT-PCR for

tissue from the five neocortical areas was performed by using RBP

primer sets as above and African green monkey cDNA in order to

confirm the differential display result.

Tissue PreparationMonkeys were deeply anesthetized with nembutal (60 mg/kg body wt,

i.p. injection) following ketamine pretreatment (16 mg/kg body wt, i.m.

injection) and perfused through the heart with warmed 0.9% NaCl

containing 2 U/ml heparin followed by ice cold 4% paraformaldehyde in

0.1 M phosphate buffer (pH 7.4). The brains were postfixed for 4--6 h

at room temperature and then cryoprotected in 30% sucrose in 0.1 M

phosphate buffer at 4�C. Sections at 40 lm thickness were sliced from

a frozen block of tissue. Serial sections were used for the thionin

staining, parvalbumin immunostaining, in situ hybridization and histo-

chemistry for cytochrome oxidase (Wong-Riley, 1979). in situ hybrid-

ization was performed for RBP and occ1 probes using adjacent sections.

Mousemonoclonal anti-parvalbumin antibody (Sigma-Aldrich, St Louis,

MO) was used for parvalbumin immunostaining. The sections were pre-

incubated for 1 h in 0.1 M phosphate buffer (pH 7.4) containing 0.3%

Triton X-100 and 5% normal donkey serum, then incubated in

a solution of the primary antibody (1:5000) in 0.1 M phosphate buffer,

1% normal donkey serum, and 0.3% Triton X-100 for 12 h at 4�C. Thesections were then washed thoroughly and incubated in 0.1 M

phosphate buffer, containing 1% normal donkey serum, and 0.3%

Triton X-100, a donkey anti-mouse IgG antibody (diluted 1:500; Jackson

Immunoresearch Laboratories, West Grove, PA) for 2 h at room tem-

perature. The reaction was completed by the avidin-biotin-peroxidase

method (Vectastain ABC reagents; Vector Laboratories, Burlingame,

CA).

In Situ HybridizationIn situ hybridization was carried out as previously described (Liang

et al., 2000). Briefly, free-floating sections were treated with 1--6 lg/ml

proteinase K for 30 min at 37�C, acetylated, then incubated in hy-

bridization buffer containing 0.5--1.0 lg/ml DIG-labeled riboprobes at

60�C. The sections were sequentially treated in 2 3 SSC/50% forma-

mide/0.1% N-lauroylsarcosine for 15 min at 55�C, two times; 30 min

at 37�C in RNase buffer (10 mM Tris--HCl, pH 8.0, 1 mM EDTA, 500 mM

NaCl) containing 20 lg/ml RNase A (Sigma-Aldrich); 15 min at 37�C in

2 3 SSC/0.1% N-lauroylsarcosine, two times; 15 min at 37�C in 0.2 3 SSC/

0.1% N-lauroylsarcosine, two times. The hybridized probe was detected

by alkaline phosphatase conjugated anti-DIG antibody using DIG nucleic

acid detection kit (Roche Diagnostics). There were no apparent signals

detected in control sections with the sense probes throughout the brain

areas examined in macaque monkeys.

Double In Situ HybridizationSections (15 lm thickness) were used for double in situ hybridization.

Pre-hybridization and washing were the same as the single hybridization

protocol. Hybridization was performed at 65�C in buffer containing 500

ng/ml of DIG-labeled and FITC-labeled probes. The immunodetection

by anti-DIG antibody conjugated with horseradish peroxidase (Roche

Figure 1. Differential display for five neocortical areas. (A) Lateral surface view of themonkey cerebral cortex. Samples for differential display screening were taken amongfive areas as schematically shown by different colors. FDD (red): from both banks ofthe principal sulcus (ps). OC (blue): from posterior to the lunate sulcus (lu) and superiorto the inferior occipital sulcus (ios). FA (purple) and PC (green): from the anterior andthe posterior banks of the central sulcus (cs), respectively. TE (yellow): from the regionventral to the superior temporal sulcus (sts). Other abbreviations are as follows: ar,arcuate sulcus; lf, lateral fissure; ips, inferior parietal sulcus; D, dorsal; V, ventral; A,anterior; P, posterior. (B) Differential display showed a band that is thick in the FDDand TE lanes, thin in PC and very faint in OC lane. Arrowheads indicate the DNA bandscorresponding to RBP mRNA. Two young adult crab-eating macaques (monkeys A andB) were used.

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Diagnostics) was enhanced with TSA Plus DNP System (PerkinElmer Life

Sciences, Boston, MA). The fluorodetection was done by anti-DNP

antibody conjugated with Alexa 488 (Amersham Biosciences, Piscat-

away, NJ). The FITC probe was detected by using Fast Red (Roche

Diagnostics) after immunolabeling by anti-FITC antibody conjugated

with alkaline phosphatase.

Results

Isolation of RBP cDNA by Differential Display

We compared mRNA expression profiles of five distinct neo-

cortical areas (Fig. 1A; see also Materials and Methods) by

differential display--PCR. The differential display--PCRwas simul-

taneously carried out using cDNA from two different monkeys

(Fig. 1B). We found one band that was abundant in lanes of

FDD (prefrontal association areas) and TE (visual association

areas),moderate in FA (primarymotor area),weak in PC (primary

somatosensory areas) and very faint in OC (primary visual area).

The DNA fragment derived from this band was purified and

cloned. The sequence analysis revealed that the fragment was

highly homologous to the39 regionof human serumretinol-binding

protein (RBP) sequence (GenBank accession No. X00129 nt

599--879, with the 10 bp gap sequence between nt 817 and 818).

Based on this sequence, we designed the PCR primers; and

the differential expression of RBP was confirmed by reverse

transcription-PCR (data not shown).

RBP Expression in the Cortical Areas of Monkeys

Next, in order to examine the expression pattern of RBP in

more detail, we performed in situ hybridization using the entire

cerebral hemisphere. The in situ hybridization experiments

basically confirmed the differential display results, showing high

expression in association areas and low expression in primary

sensory areas. The sites of high RBP expression generally

corresponded to association areas as follows: the prefrontal

higher-order association areas located in sections 1 and 2 of

Figure 2 (e.g. square 3A in section 1 is enlarged in Fig. 3A), the

dysgranular insular cortex located in the bottom of the anterior

part of the lateral fissure (Fig. 2, sections 5--8, e.g. square 3C

in section 7), the anterior part of the inferior temporal cortex

which contains visual association (TE) and limbic areas (Fig. 2,

sections 4--10; e.g. square 3I in section 7), and the dorsal bank

of the superior temporal sulcus corresponding to the superior

temporal polysensory area (STP; Fig. 2, sections 4--12; e.g. square

3B in section 7). In addition, the parietal association areas (PG)

showed a high level of RBP expression (Fig. 2, sections 12--15;

e.g. square 3E in section 12). Intense expression was also

observed in the limbic cortices, for example the cingulate

cortex and the entorhinal cortex (Fig. 2, sections 1--12; e.g.

square 3D in section 7).

In these areas, strong RBP expression was observed in layers

II, III and V (Fig. 3A--D). Although this pattern was typical of

various association and limbic cortices, we noticed area-specific

differences. For example, in PG and TE (Fig. 3E,I), RBP signals

were more obvious in layer VI than in other association and

limbic cortices (Fig. 3A--D).

In contrast, RBP expression was very weak and restricted to

the upper part of the supragranular layers in primary sensory

areas. Expression in V1 showed the lowest level of any cortical

area (Fig. 3M). In V1, expression was mostly restricted to

a narrow region at the top of layer II, with a few very weak

signals in layer VI. Area 3b which is part of the primary

somatosensory area also showed a low level of RBP expression

(Fig. 3G). As in V1, the signals in the supragranular layers were

mostly restricted to layer II, but weak signals were observed in

infragranular layers. The primary auditory area, AI, showed

similar laminar distributions as area 3b (Fig. 3H).

Along the sensory pathway progressing from the primary

sensory toward the sensory association areas, we observed an

increase of RBP expression in both intensity and width of

laminar distribution. This was obvious along the well-charac-

terized ventral visual pathway. RBP expression was restricted to

the upper part of the supragranular layers in V2 (Fig. 3L), but

extended to the deeper part of layer III in V4 (Fig. 3K). In TEO,

RBP expression was observed in both supra- and infragranular

layers although its expression in the infragranular layers was

much weaker than that in the supragranular layers (Fig. 3J). In

TE, as described above, intense signals extended throughout the

supragranular layers; and the expression in the infragranular

layers was much higher than that in TEO (Fig. 3I). The change in

the laminar distribution within the supragranular layers was

gradual and did not observe any sharp border between layers II

and III (also see next section).

In the primary motor cortex (area 4), intense signals were

located in the upper part of the supragranular layers (Fig. 3F). In

addition, RBP signals in giant pyramidal cells were conspicuous in

layer V. In the premotor areas, compared to the primary motor

area, RBP expression extended more deeply into the supragra-

nular layers and was more apparent in layer V (data not shown).

In summary, RBP expression was high in association and

limbic cortices, with strong expression in layers II, III and V, and

was low in primary sensory areas, with a restricted distribution

in the supragranular layers. Furthermore, the laminar distribu-

tion increased from the primary areas toward association areas,

as was most obvious in the visual pathway. Additionally, we

noted scattered cells that showed relatively strong RBP expres-

sion in the white matter under every cortical area.

Selective Expression of RBP in other Brain Regions

High expression of RBP was observed in subcortical limbic

structures and striatum (Fig. 4). In the amygdala, the dorsal

intermediate subdivision of the lateral nucleus showed a rela-

tively low level of RBP expression (Fig.4A), while this was

relatively high in the central nucleus (compare Fig. 4A with

Fig. 4B). The hippocampal formation also showed high RBP ex-

pression (Fig.4C). Althoughweobservedweaker senseprobe sig-

nals in the dentate gyrus, the intense labeling in this region is

specific to the antisense probe of RBPmRNA. CA2, where there

is occ1 expression (Tochitani et al., 2003), showed a relatively

lower expression of RBP among the hippocampal subdivisions

(Fig.4C). The caudate and putamen showed equal expression

levels of RBP anteriorly (Fig.4D), but in the posterior part,

expression in the caudate was higher than that of the putamen

(Fig. 4E,F). In posterior striatum we also noticed that the ventral

region of the putamen showed more intense expression than

did the dorsal region (data not shown).

Choroid plexus is reported to express RBP mRNA in rodents,

ruminants and carnivores (Duan and Schreiber, 1992; Aldred

et al., 1995). We also detected some expression of RBP in the

choroid plexus of monkeys, but the level was much fainter

compared to the expression described here (data not shown).

Complementary Expression of RBP and occ1

Previously, we identified the occ1 gene that is preferentially

expressed in area V1 (Tochitani et al., 2001). When we

98 RBP Expression in Association Areas d Komatsu et al.



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compared the expression of RBP with that of occ1 in the

adjacent sections, the distribution of these two genes showed

striking complementarity (Fig. 5A). occ1 expression appeared

almost absent or negligible in the prefrontal cortex and anterior

part of the temporal cortices where RBP expression was high

(Fig. 5A and area 46 in Fig. 5B). In the posterior region, occ1was

expressed heavily in V1 and moderately in extrastriate areas,

with gradual decrease toward the anterior region, along the

dorsal and ventral visual pathways (Fig. 5A, inset). In adjacent

sections, RBP mRNA by contrast was almost absent in V1, and

gradually increased along both the visual pathways (Fig. 5A).

In these occipital regions, both dorsal and ventral, RBP and

occ1 were expressed in separate sublayers. For example, in V2,

occ1 expression was relatively strong in layer III, whereas RBP

expression was mostly restricted to the uppermost part of the

supragranular layers (Fig. 5D).

In the more anterior regions, occ1 expression gradually de-

creased in intensity and width, and was restricted to the very

Figure 2. Distribution of RBP signals shown by in situ hybridizain. Coronal sections arranged in anteroposterior orientation (sections 1--17). RBP expression was observed in allcortical areas, but at variable levels. Boxed regions are shown at higher magnification in Figure 3. Sulcus abbreviations are as follows: cg, cingulate sulcus; sar, superior arcuatesulcus; iar, inferior arcuate sulcus; cal, calcarine sulcus. Other abbreviations are the same as in Figure 1. Orientation of each section is indicated: D, dorsal; V, ventral; L, lateral; M,medial. Scale bar ¼ 20 mm.




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bottomof layer III (Fig. 5B, lower panels a-3--a-1). Conversely, the

intensity of RBP signals increased (Fig. 5C) and the expression

extended deeper into layer III (Fig. 5B, upper panels a-3--a-1).

Although RBP and occ1 signals remained largely segregated in

the upper and lower compartments of the supragranular layers,

in a complementary fashion, our double in situ hybridization

showed that, in the deeper part of layer III in V2, occ1 positive

cells expressed RBPmRNA at a low level (data not shown).

In the infragranular layers of the posterior regions, we ob-

served that a few intense signals of occ1 existed predominantly in

layerV, and that the signals seemed invariant across areas (Fig. 5B,

lower panels a-3--a-1). In contrast,weobserved a gradual increase

of RBP signals in the infragranular layers (Fig. 5B, a-3--a-1) in both

layers V and VI from posterior (Fig. 4B, a-3) to anterior regions

(Fig. 5B, a-1). We did not see clear laminar segregation between

RBP and occ1 signals in the infragranular layers.

In addition to visual related areas, occ1 is shown to be

preferentially expressed in the middle layers of AI and area 3b

compared with the surrounding areas (Tochitani et al., 2001).

In these sensory areas, we observed complementarity in the

layer distribution between RBP and occ1 signals. Consistent

with complementary expression in other areas, occ1 showed

much weaker expression in AI and SI, than in V1, whereas RBP

expression was higher in AI and area 3b than in V1 (Fig.

3G,H,M).

Complementary Expression of RBP and ThalamicTerminations in Primary Sensory Areas

We previously found that occ1 expression in V1 is dependent on

retinal activity and thalamocortical connections (Tochitani

et al., 2001, 2003). To investigate the relationship between

RBP expression and the thalamocortical projection, we com-

pared the RBP expression around primary sensory areas, V1, AI

and SI, with two other markers. One was parvalbumin immu-

noreactivity (PV-IR), which demonstrates dense thalamic ter-

minals in the middle layers of sensory cortex (Steriade et al.,

1997). The other was cytochrome oxidase (CO) whose activity

is high in primary sensory cortices (Wong-Riley, 1989; Morel

et al., 1993; Jones et al., 1995, 2002) and which is related to

sensory input from specific thalamic nuclei in V1 and SI (Wong-

Riley, 1989, Jones et al., 2002). Since a subset of cortical

interneurons also expresses PV, we aimed to distinguish the

thalamic fibers from the intrinsic PV-IR by using a short incuba-

tion and reaction time. We felt that this approach was successful

in delimiting the primary auditory and somatosensory areas, and

our data are limited to these regions.

Within somatosensory areas, area 3b and area 1 have both

been considered as primary areas. Area 3b received denser PV-

IR fibers in the middle layers and exhibited higher CO activity

than area 1 (Fig. 6B,C,D). In comparison, RBP expression was

lower and more restricted in layers in area 3b than in area 1

(Fig. 6A).Weobserved a similar relationship fromAI to secondary

auditory cortex; namely, a decrease of PV-IR and CO staining,

and an increase of RBP mRNA (Fig. 6F,G,H). In both these

regions, the laminar expression of RBP was complementary to

that of the PV-IR plexus in the middle layers (Fig. 6F,G). Thus,

the laminar distribution of RBP mRNA seemed to be reversely

correlated to that of PV-IR in both auditory and somatosensory

cortices. We also observed abundant PV-IR fibers in the middle

layers of other areas; for example, area 5 (Fig. 6E) and area 9

Figure 3. Layer distribution of RBP mRNA among cortical areas. A--E correspond to association and limbic cortices. (A) area 9, (B) STP (superior temporal area), (C) dysgranularinsular cortex, (D) entorhinal cortex, (E) PG (parietal association cortex), (F) area 4 (primary motor cortex), (G) area 3b (primary somatosensory area), (H) AI is primary auditorycortex. The bottom row shows areas corresponding to the visual ventral pathway; (I) TE, (J) TEO, (K) V4, (L) V2 and (M) V1. Scale bar ¼ 500 lm.




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(data not shown). However, a complementarity to RBP signals

was not clear in these higher-order sensory and association

areas.

RBP expression is complementary with PV-IR fibers and occ1

expression in the early sensory areas. In view of this result, we

wanted to investigate the relationship between RBP expression

and thalamic activity, as this is known to be important for occ1

expression. To test this possibility, we examined RBP expres-

sion in V1 of monocularly deprived monkeys which were re-

peatedly injected with TTX into one eye for 7 or 14 days. As we

reported previously, monocular deprivation leads to a clear

reduction of occ1 in the ocular dominance columns of the

deprived eye. This did not occur for RBP expression which,

in contrast, was restricted to a narrow layer at the top of

layer II in both deprived and non-deprived columns (data not

shown). We observed little difference if any in this thin layer or

in any other layer in V1. Thus, the low level of RBP expression in

V1 did not seem to be attributable to an influence of thalamic

activity.

Cell Types that Express RBP in Supragranular Layers

In order to examine the cell types that express RBP mRNA, we

performed double in situ hybridization using cell-type specific

markers. We used VGluT1 as a glutamatergic neuronal marker

(Fujiyama et al., 2001) and GAD 67 as a GABAergic neuronal

marker. In our experimental condition, these two probes

Figure 4. Subcortical distribution of RBP mRNA expression. RBP mRNA expression (A) and thionine stain (B) in the amygdala complex. RBP expression in the hippocampalformation (C), anterior (D) and posterior (E, F) striatum. Abbreviations: CE, central nucleus; Ldi, dorsal intermediate subdivision of the lateral nucleus; Lvi, ventral intermediate of thelateral nucleus; Lv, ventral subdivision of the lateral nucleus; Bmc, magnocellular subdivision of the basal nucleus; Bi, intermediate subdivision of the basal nucleus; Bpc, parvicellularsubdivision of the basal nucleus; ME, medial nucleus; Cop, posterior cortical nucleus; AHA, amygdalohippocampal area; DG, dentate gyrus; Cd, caudate nucleus; Pu, putamen; Cl,posterior claustrum; L, lateral ventricle. Scale bar ¼ 2 mm.




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labeled distinct populations (data not shown). Here we illus-

trate examples from two different areas (TE and area 1), in the

same section, where the signals were processed exactly in the

same condition. In TE, the majority of RBP signals within

supragranular layers colocalized with VGluT1 mRNA signals

(Fig. 7A); but only a restricted population of GAD67 mRNA

positive cells expressed RBP signals (Fig. 7B). This bias was also

observed in area 1 where the band of RBP mRNA positive

neurons was thinner than in TE: most of the RBPmRNA positive

cells were VGluT1 mRNA positive (Fig. 7C), while only some

were GAD67 mRNA positive (Fig. 7D). We confirmed essentially

the same results in V1, V2, STP and area 4 as well (data not

shown).

RBP Expression in Neonatal Monkey

Our next question was whether the graded pattern of RBP

expression along the anteroposterior axis might already be

present during development (Donoghue and Rakic, 1999a,b)

and retained throughout maturation. Thus, we examined the

expression pattern of RBP in newborn monkeys (P1 and P2

monkeys) and compared it with that of the adult.

In neonatal monkeys, the V1--V2 border was clearly visible

because the RBP expression in V1 was lower than that in V2

(Fig. 8A). However, RBP expression in both V1 and V2 of

neonatal monkeys was broader than that in the adult monkey

(Fig. 8A, upper panels). Intense RBP signals in V2 were widely

distributed within supragranular layers, including the deeper

part of layer III, whereas RBP signals were almost restricted to

layer II in the adult. Similarly, in V1, the signal, even though

weaker than in V2, extended beyond layer II. In contrast with

RBP expression, however, the occ1 expression was low in V1

and was not observed in V2 of newborn monkeys (Fig. 8A, lower

panels), as previously reported (Tochitani et al., 2003).

In the prefrontal cortex (area 11) of neonatal monkeys, RBP

signals were mostly observed in layers II and III, and only weakly

expressed in infragranular layers (Fig. 8B, P1). This is in contrast

with adult monkeys, where the RBP expression was widely

present in layers II, III and V (Fig. 8B, adult). In newborns, the

layer distribution of RBP signals in area 11 was similar to that

in area V2; in other words, without the characteristic adult re-

gional gradation. These results suggested that the graded

pattern of RBP expression in adult monkeys is mainly estab-

lished during postnatal development by processes that enhance

its laminar distribution in higher-order association areas but

diminish this in early sensory areas.

Discussion

Using the differential display method, we found that the RBP

gene was differentially expressed in neocortical areas, being

strongly expressed in higher-order association and limbic areas

and only slightly in primary sensory areas. This differential

mapping reinforces the traditional distinction between sensory

and association areas, from a new perspective which may allow

a further means for investigating mechanisms of area speciali-

zation. The differential expression levels among cortical areas

were also associated with differences in laminar distributions.

Beyond the primary sensory areas, there was a gradual recruit-

ment of neurons through the supragranular and then infragra-

nular layers.

In the early sensory areas, RBP expression was complemen-

tary to that of occ1 and to PV-IR fibers in the middle layers. From

this, we infer that it does not seem to be directly related to the

thalamocortical projection. Consistent with this, its distribution

in area V1 is not sensitive to monocular deprivation. Finally,

the area-specificity of RBP expression is likely to result from

maturational processes during the postnatal period. This is

suggested by the developmental shift in laminar distributions. In

neonates, RBP mRNA is distributed in a similar laminar pattern

in higher-order association and early visual areas, but this is not

the case in adults.

In addition, to our knowledge, this is the first report of RBP

mRNA expression in characterized neuronal populations in the

primate central nervous system (CNS).

Figure 5. RBP and occ1 expression are complementary. (A) RBP mRNA signals(upper) and occ1 signals (lower) in a parasagittal section. Boxed regions were enlargedin right panels at higher magnification (a-1--3 are shown at further highermagnifications in B). Scale bar ¼ 10 mm. Small arrows indicate the V1--V2 borders.Scale bar ¼ 5 mm. (B) Layer distribution of RBP signals. With anterior progression,RBP signals gradually extended from layer II deeper into layer III (V1, a-1--3 upperpanels). In layers V--VI, RBP signals increased with a scattered pattern (V1, a-1--3upper panels). In area 46, many signals were observed throughout all layers except forlayer I. Especially intense signals were observed in layers II, III and V. The lower rowshows the occ1 expression in the sections adjacent to the just above sectionsexamined for RBP expression. Scale bar ¼ 150 lm. (C) Different expression levels incells in layers II and III. Signals were more intense in anterior areas than in posteriorareas (e.g. a-1[ a-3). Scale bar ¼ 100 lm. (D) RBP and occ1 expression in layers IIand III of V2. Scale bar ¼ 150 lm.




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Possible Relations of Retinoic Acid and RBP in the Brain

Vitamin A (retinol) is dietarily obtained as b-carotene in plants

and retinyl esters in animal tissues and stored in liver (Goodman,

1984; Malik et al., 2000a). For delivery to peripheral tissues,

vitamin A is mobilized from storage and effectively transported

into the plasma as retinol bound to RBP (Ong, 1994; Malik et al.,

2000a). Once retinol is received by the cell, alcohol dehydro-

genase can catalyze the oxidation of retinol into retinaldehyde,

abbreviated retinal (Duester, 2000, 2001). Retinal is used for

visual pigments (Applebury and Hargrave, 1986) and as the

substrate of retinal aldehyde dehydrogenase (RALDH) which

irreversibly converts retinal into retinoic acid (RA) (Duester,

2000, 2001). Both ADH and RALDH are expressed in neural

tissues (Wagner et al., 2002; Galter et al., 2003).

RA is a biologically active metabolite of retinoid, and trans-

duces its effect through the RA receptor (RAR a, b and c) andthe retinoid X receptor (RXR a, b and c) which act as RA-

dependent transcriptional regulators. The binding of these re-

ceptors to the RA response element (RARE) in promoter regions

activates a large number of gene transcriptions (Gudas et al.,

1994; Clagett-Dame and Plum, 1997). Thus, RA is important

for morphogenesis and differentiation in a number of tissues

including the CNS; for example, for hindbrain segmentation

(Gavalas, 2002) and possibly for regionalization of the forebrain

(LaMantia et al., 1993; Smith et al., 2001).

While these reports focus on embryonic development, a re-

cent study suggests that RALDH is transiently expressed even in

the postnatal brain (Wagner et al., 2002). In mice, RALDH3, one

of the isotypes of RALDH, is transiently expressed in the nuclear

accumbens, the olfactory bulb and the cerebral cortex during

the early postnatal period (Wagner et al., 2002). Interestingly,

RALDH3 expression in the cerebral cortex resembles the RBP

expression in neonatal monkeys. That is, RALDH3 is differen-

tially expressed in layers II and III of cortical regions; and its

expression is high in V2 (both medial and lateral regions), but

appears to be absent in V1. Since RBP has the RARE sequence in

the promoter region and its transcription could be strongly

regulated by RA (Mourey et al., 1994; Panariello et al., 1996;

Clagett-Dame and Plum, 1997), RBP mRNA expression could

depend on RA activity.

Figure 6. RBP mRNA signal distribution around the primary somatosensory area (SI) and primary auditory area (AI). (A) The RBP expression level of area 3b is lower than that ofarea1 (B, C). The lateral border of area 3b (shown by the arrow) was visualized by parvalubumin immunoreactive (PV-IR) fibers and histochemistry for cytochrome oxidase (CO).Boxed regions in (A) and (B) are enlarged in (D). cs, central sulcus. Scale bar ¼ 2.5 mm. (D) RBP mRNA signals were weak and restricted to layer II in area 3b. PV-IR fibers in themiddle layer were abundant in the adjacent section. In area 1, RBPmRNA signals were intense in layer II and moderate in layers III and V. PV-IR fibers in layers III to IV in area 1 wereapparently lesser than in area 3b. (E) RBP expression was complementary to that of PV-IR fibers in the middle layers in SI but much less in area 5. RBP expression extended intodeeper layer III and layer V. Side bars: the extent of intense RBP signals in the supragranular layers. Arrowheads, RBP mRNA signals of layer V. Scale bar ¼ 350 lm. (F) RBPexpression level was also low in AI and gradually increased towards the adjacent areas. (G, H) PV-IR fibers in the middle layers and CO staining indicated extent of AI. The RBPexpression was complementary to the PV-IR fibers in the middle layers of AI. Arrows indicate the border between AI and belt region. lf: lateral fissure. Scale bar ¼ 2 mm.




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The postnatal remodeling of the cortex is considered to be

necessary in order for neuronal circuits in the brain to adapt to

sensory stimuli (Rakic, 1988; Batardiere et al., 2002). The

significant differences in expression level among early postnatal

cortical regions suggests that RA transcriptional activity may

play a role in the postnatal differentiation of higher-order

sensory and association cortices, in response to environmental

inputs.

The characteristic RBP mRNA distribution shown in this

study overlaps some of adult brain regions in which RA

transcriptional activity may be involved in neuronal plasticity.

One such region is the hippocampus where RBP mRNA

expression is high. In rodents, RAR and RXR are highly ex-

pressed in the hippocampus (Krezel et al., 1999; Zetterstrom

et al., 1999); and RALDH 1 and 2 are expressed in hippocampal

meninges. Vitamin A deficiency in adult rodents lowers or

eliminates synaptic plasticity of CA1 (Misner et al., 2001) and

results in a relational memory deficit (Cocco et al., 2002;

Etchamendy et al., 2003).

The activity of RA in the mature CNS is more directly shown

using RA-reporter mice (Haskell GT et al., 2002). A neuronal

population presumably activated by RA can be visualized in

transgenic mice that are introduced with lacZ gene fused to

minimally required promoter at the down stream of five direct

Figure 7. RBP mRNA expressed among the large population of excitatory neurons within supragranular layers. (A) RBP mRNA (left, Alexa 488, green) was intensely expressedamong large population of VGluT1 positive cells (middle, Fast Red, red) within the sublayer labeled with RBP signals in layers II and III of TE (overlaid in the right panel). (B) The RBPexpression (left, Alexa 488, green) was observed in the restricted population of GAD 67 positive neurons (middle, Fast Red, red; overlaid in right panels). (C, D) This bias was alsoobserved in area 1 where the layer labeled with RBP signals was thinner than that of TE. (C) Compared to VGluT1, (D) Compared to GAD 67. Scale bar ¼ 150 lm.




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repeats of the RARE sequence. These transgene-expressing cells

are located in the dorsal horn of the spinal cord, the olfactory

bulb, habenular complex, the amygdala and cerebral cortex. In

our material, RBP expression in the spinal cord and the olfactory

bulb remains to be studied, but we confirmed RBP expression in

the amygdala (Fig. 4A) and the lateral habenular nucleus (data

not shown).

It should be noted that there is some discrepancy between

the RA-active cells in mice and the RBP-mRNA-positive cells in

monkeys. The study using the RA-reporter mice indicated that

the RA active cells are fusiform or possibly stellate cells in the

neocortex (Haskell et al., 2002); but our in situ hybridization

results showed that RBP expression is usually observed in

pyramidal cells in the primate. Future studies will be needed to

determine what accounts for the discrepancy.

Roles of RBP and RA in the Brain Suggested byPathological Studies

Human neuropathologies provide further evidence that the RBP-

mediated RA synthesis pathway may play a role in maintaining

higher brain function. Possible disease links include Alzheimer’s

disease (see below), schizophrenia (see below), and fronto

temporal dementia (Davidsson et al., 2002a). In Alzheimer’s

disease patients, RBP secretion into cerebrospinal fluid is re-

duced (Davidsson et al., 2002b; Puchades et al., 2003) and its

immunoreactivity is observed in amyloid extracts (Maury and

Teppo, 1987). In addition, the human RBP locus in chromosome

10q24 is near to a marker sequence whose recombination

correlates with Alzheimer’s disease onset (Bertram et al., 2000;

Goodman and Pardee, 2003). Further analysis of the loci of

retinoid-related genes and those related to late-onset Alzhei-

mer’s disease suggests that retinoid metabolism is implicated in

the disease (Goodman and Pardee, 2003). RA synthesis from

retinaldehyde is higher in the hippocampus and parietal cortices

of the Alzheimer’s disease cohort than in controls (Conner and

Sidell, 1997).

The pathology of Alzheimer’s disease suggests a further re-

lationship with RA. That is, Alzheimer’s disease involves the

degeneration of cholinergic neurons whose differentiation and

maintenance can be induced by RA. RA promotes expression of

choline acetyltransferase (ChAT) and the vesicular acetyl-

choline transporter, ChAT activity and intracellular ACh con-

centration in vitro studies (Berse and Blusztajn, 1995; Pedersen

et al., 1995; Malik et al., 2000b). The cholinergic neurons and

Figure 8. Neonatal laminar distribution of RBP mRNA signals. (A) Upper panel: RBP expression in V2 of a monkey at postnatal day 1 (left) and in an adult monkey (right). Lowerpanel: the occ1 expression in the visual areas of the P1 monkey (left) was much weaker than that in the adult monkey (right), although the V1--V2 border is already apparent at birthas previously reported (Tochitani et al., 2003). Arrowheads indicate the V1--V2 border. Scale bar ¼ 1 mm (B) RBP expression is shown in the left panels of P1 and adult sections.Nissl staining is shown in each right panel. Scale bar ¼ 250 lm.




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innervations that are affected in Alzheimer’s disease patients are

distributed in basal forebrain, limbic and association cortex (De

Lacalle and Saper, 1997; Dickson, 1997; Wisniewski et al., 1997;

Clippingdale et al., 2001; Selkoe, 2001; Kar and Quirion, 2004).

Given the apparent expression of RBP in these areas (Fig. 2), cell

sorting by using RBP expression may provide further linkage of

retinoid metabolism with Alzheimer’s disease and cholinergic

subpopulations.

The relationship between schizophrenia and RA is somewhat

controversial. Macroarray analysis suggests that there is no

significant difference in the expression of genes related to

retinoid metabolism (Middleton et al., 2002). However, three

independent lines of evidence suggest some involvement of RA

in schizophrenia (Goodman, 1998; Citver et al., 2002): (i)

congenital anomalies similar to those caused by retinoid dys-

function are found in schizophrenics and their relatives; (ii)

those loci suggested as linked to schizophrenia are also the loci

of the genes of the retinoid cascade (convergent loci); and (iii)

transcription of the dopamine D2 receptor and numerous

schizophrenia candidate genes is regulated by RA. Assuming

there to be a high expression of RBP mRNA in human associ-

ation cortices, disruption of RBP-mediated retinoid metabolism

might specifically damage neuronal circuits of prefrontal and

other association and limbic cortices due to the strong effects

of RA transcriptional activity (Weiberger and Berman, 1998;

Buchsbaum et al., 2002; Kurachi, 2003).

Implication of RBP Distribution in the Cerebral Cortex

A striking characteristic of RBP expression in the supragranular

layers is its expansion from early visual areas (V1 and V2) to

higher visual areas. The interpretation of this pattern is not

clear; but the concentration of RBP-mRNA-positive neurons in

the upper layers might suggest a preferential association with

corticocortical projections.

Layer II in visual and other areas is one source of feedback

projections; and the dendrites of layer II neurons are among

the likely targets of feedback and other terminations layer I

(Rockland and Pandya, 1979; Felleman and Van Essen, 1991). In

this regard, it is interesting that the laminar distribution of both

feedback terminations and cells of origin broadens in anterior

areas (i.e. TEO to V4; Rockland et al., 1994, fig. 21), a broadening

which parallels the shift in laminar expression of RBP. In

addition, layer II neurons are involved in horizontal intrinsic

connections (Lund et al., 1981; Yoshioka et al., 1992; Fujita and

Fujita, 1996). These connectivity features are suggestive of

some involvement in integrative processing.

A distinctive role for layer II is suggested by recent electro-

physiological results of Shipp and Zeki (2002). That is, their

study indicates that, in each type of CO stripe (thick, thin and

pale), the specificity of cell response for visual stimuli was to

some degree laminar-specific; and in particular, the responses in

layer II were more generalized, in terms of directional, orien-

tation, and spectral sensitivity, than those deeper in layer III

within V2.

The laminar expansion of RBP expression occurs gradient-

wise, seemingly across area borders. Other features, such as

pyramidal neuron dendritic architecture, are known to system-

atically change along the visual and somatosensory pathway.

Increases in the size of the basal dendritic field, in the degree of

branching, and in the spine density have been demonstrated

by intracellular injection of Lucifer yellow, and interpreted as

signifying differential integrative capacity of neurons in early

and higher-order visual areas (Elston et al., 1999; Elston, 2001).

Further morphological elaboration is reported for neurons in

the prefrontal cortex (Elston, 2000; Elston et al., 2001), a region

with strong RBP expression.

In the amygdala complex, which is heavily connected with

cortical areas, RBPmRNA is expressed at low levels in the lateral

nuclei and higher levels in the accessory and central nuclei

(Fig. 4). These nuclei are organized in a largely unidirectional

intrinsic circuit and have distinctive connections with cortical

areas: the lateral nucleus is preferentially innervated by visual

cortices and projects to basal accessory nuclei (Stefanacci and

Amaral, 2002). The basal accessory nucleus is densely intercon-

nected with frontal and limbic cortices and projects to the

central nuclei (Stefanacci and Amaral, 2002). Amaral and his

coworkers hypothesize that the sequential processing of these

nuclei is a substrate for the contextual fear response for novel or

potentially dangerous objects (Stefanacci and Amaral, 2002). An

intriguing possibility is that RBP mRNA selective expression in

the amygdala is related to an anatomical and functional hier-

archywithin the amygdala and its connectivitywith cortical areas.

RBP mRNA is preferentially distributed in the caudate and

ventral putamen in the posterior striatum (Fig. 4). The caudate

and ventral putamen are connected to higher-order and visual

association areas (Alexander et al., 1986; Webster et al., 1993;

Yeterian and Pandya, 1994), whereas the dorsal putamen is

more directly connected to the motor cortex (Alexander et al.,

1986).

In summary, the expression of RBP mRNA in primate cortical

structures, as well as its complementary expression with occ1

and PV-IR in the cerebral cortex, suggest that RBP mRNA is

preferentially distributed in association areas and related sub-

cortical structures rather than the early sensory and motor

areas. This further suggests that RBP and RA may play important

roles in selectively influencing executive functions and social

behavior in primates. We hope that further studies on the

function of RBP and retinoids in the brain may provide clues to

the organization of association areas at the molecular level.

Notes

We thank Drs Fumiko Ono of the Corporation for Production and

Research of Laboratory Primates, Keiji Terao, Tsukuba Primate Center,

National Institute of Infectious Diseases, H. Horie, S. Abe and S.

Hashizume of the Japan Poliomyelitis Research Institute for supplying

monkey tissues, and Kathleen Rockland for critical reading and valuable

discussions. This research was support by a Grant-in-Aid for Scientific

Research on Priority Areas (A) from the Ministry of Education, Culture,

Sports, Science and Technology of Japan (to T.Y.).

Address correspondence to Tetsuo Yamamori, Division of Speciation

Mechanisms 1, National Institute for Basic Biology, Aichi 444-8585,

Japan. Email: [email protected].

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