Expression of calcium-binding proteins in the mouse claustrum

Ma Angeles Real, Jose Carlos Davila, Salvador Guirado *

Departamento de Biologıa Celular, Genetica y Fisiologıa, Universidad de Malaga, Campus de Teatinos, 29071 Malaga, Spain

Received 12 July 2002; received in revised form 29 October 2002; accepted 1 December 2002

Abstract

The present paper describes the distribution of three calcium-binding proteins (calbindin D28k, calretinin, and parvalbumin) in

the mouse dorsal claustrum and endopiriform nucleus. The three calcium-binding proteins were distinctly expressed in structures of

both the claustrum and the endopiriform nucleus. Calbindin was the calcium-binding protein showing the highest expression in the

claustrum and the endopiriform nucleus. In contrast, calretinin-immunoreactive structures, particularly cell bodies, were very scarce

in these regions. Both calbindin-immunoreactive and parvalbumin-immunoreactive neurons were more abundant in the claustrum

than in the endopiriform nucleus, and more in rostral than in caudal levels. Nevertheless, calcium-binding protein immunoreactive

neurons constitute a minority population of claustral neurons. The colocalization study of calbindin and parvalbumin

immunoreactivities has demonstrated that both calcium-binding proteins are mostly expressed by separate claustral neurons in

the mouse. On the other hand, our results on parvalbumin and calretinin immunoreactivity match a novel subdivision of the mouse

claustrum mostly based on the pattern of cadherin expression [Neuroscience 106 (2001) 505]. In this sense, we propose that a specific

zone of the dorsal claustrum with cell bodies that strongly express Rcad and cadherin-8 would be the selective target for

parvalbumin-expressing fibers, and that they would be mostly avoided by calretinin-expressing axons.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Calbindin; Parvalbumin; Calretinin; Lateral pallium; Ventral pallium

1. Introduction

The claustrum is a pallial subcortical region present in

all mammals. Two parts of the claustrum can be

distinguished: the dorsal part is usually called claustrum

proper and it is located deep to the insular cortex

(therefore it is also named dorsal or insular claustrum);

the ventral part is called endopiriform nucleus and is

located deep to the piriform cortex (Druga, 1966; Sherk,

1988; Dinopoulos et al., 1992). Krettek and Price (1977)

considered a ventral division of the endopiriform

nucleus located deep to the periamygdaloid cortex and

adjoining ventral part of the middle region of piriform

cortex. For the sake of clarity, from now on the dorsal

claustrum will be referred to as claustrum whereas the

ventral claustrum will be referred to as endopiriform

nucleus.

The claustrum has extensive connections with the

neocortex (Pearson et al., 1982; Macchi et al., 1983;

Markowitsch et al., 1984; Li et al., 1986; Sloniewski et

al., 1986; Sherk, 1988; Sadowski et al., 1997; Kowianski

et al., 1998; Majak et al., 2000), whereas the endopiri-

form nucleus is hodologicaly related with the prepiri-

form and entorhinal cortices (Druga, 1971;

Markowitsch et al., 1984; Witter et al., 1988).

Despite the evidence for hodologicaly different zones

of the claustrum, previous cyto and chemoarchitectonic

studies show an overall uniform structure (Sherk, 1988;

Reynhout and Baizer, 1999). Recently, however, three

subdivisions within the claustrum have been distin-

guished on the basis of specific cadherin expression

patterns (Obst-Pernberg et al., 2001). It is not known

whether these claustral subdivisions have different

connections or play different functions.

In this context, the expression of calcium-binding

proteins has probed to be very useful in revealing

subdivisions in different regions of the central nervous

system, notably the thalamus (Jones and Hendry, 1989;

Davila et al., 2000). Therefore, here we studied the

* Corresponding author. Tel.: �/34-952-13-1961; fax: �/34-952-13-

2000.

E-mail address: guirado@uma.es (S. Guirado).

Journal of Chemical Neuroanatomy 25 (2003) 151�/160

www.elsevier.com/locate/jchemneu

0891-0618/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0891-0618(02)00104-7

distribution of three calcium-binding proteins (calbindin

D28k, calretinin, and parvalbumin) in the mouse

claustrum searching for specific distribution patterns

putatively related either to hodologicaly-based subdivi-sions or cadherin expression patterns.

2. Material and methods

Fifteen adult OF1 mice (42�/46 g body weight) were

used in the present study. Throughout the experimental

work animals were treated according to the EuropeanCommunities Council directive (86/609/EEC) on treat-

ment of experimental animals.

Mice were deeply anesthetized with sodium pentothal

(65 mg/kg; Abbott Laboratories) and transcardially

perfused with 0.1 M phosphate-buffered saline (PBS),

pH 7.4, followed by 4% paraformaldehyde, 0.1%

glutaraldehyde and 0.2% picric acid in PBS at room

temperature for 30 min. The brains were then removedand stored in 4% paraformaldehyde and 0.2% picric acid

in PBS at 4 8C overnight; afterwards they were

embedded in 4% agar and cut into 50-mm-thick frontal

sections, using a vibratome. The sections were washed

extensively in PBS prior to immunocytochemical stain-

ing with the peroxidase�/antiperoxidase method.

Free-floating sections were first incubated in 2%

normal goat serum and 0.3% Triton X-100 in PBS atroom temperature for 1 h, to block nonspecific binding

of the antibodies and permeate the tissues, respectively,

and then were transferred to the primary antibody. The

three polyclonal antibodies, anti-parvalbumin, anti-

calretinin, and anti-calbindin, were raised in rabbits

(SWant, Bellinzona, Switzerland) and used at a dilution

of 1:2000 for 18 h. After three washes in PBS for 45 min,

the sections were incubated in goat anti-rabbit IgGdiluted 1:35 for 1 h, washed again in PBS for 45 min,

and incubated in peroxidase�/antiperoxidase diluted

1:100 for 1 h. The immunolabeling was revealed with

0.05% diaminobenzidine (DAB; Sigma Chemical Co, St.

Louis, MO), 0.05% nickel ammonium sulfate and 0.03%

hydrogen peroxide (H2O2) in PBS. All steps were carried

out at room temperature with gentle agitation. After a

thorough wash in PBS, the sections were mounted on

gelatinized slides, air dried, dehydrated in ethanol,

cleared in xylene and coverslipped with DPX (BDH,

Poole, UK).

In order to provide some quantitative data about the

populations of calcium-binding protein immunoreactive

neurons as well as the putative colocalization of these

proteins in the same neuron, we carried out post-

embedding immunocytochemistry on semithin sections

of the claustrum in one mouse. After fixation, brain

slices 500-mm-thick, obtained on a vibratome, were

collected in PBS, dehydrated in acetone, and flat

embedded in Araldite 502 (Sigma). Resin-embedded

slices containing the claustrum were glued onto poly-

merized blocks, trimmed and then sectioned on an

ultramicrotome. Series of three sections of 1.5 mm were

made. The first series was stained with toluidine blue,

whereas the second and third series were stained with

anti-calbindin and anti-parvalbumin, respectively. After

removing the resin with sodium ethoxide, sections were

processed according to an immunohistochemical proce-

dure similar to that described above, except for that the

primary antibodies were used at a higher concentration

(anti-CB: 1/1000; anti-PV: 1/500). To determine coex-

istence, we took into consideration only those cells

clearly positive to calbindin or parvalbumin and dis-

playing a wide nuclear profile.

Controls: as controls of the immunohistochemical

method used in the present study, sections were pro-

cessed as indicated but the corresponding primary

antiserum was replaced by rabbit nonimmune serum

(1:500). No immunostaining could be detected under

these conditions. In addition, as a control of the

specificity of the different primary antisera under the

experimental conditions used in this work, we incubated

control sections in the primary antibody preadsorbed

with the corresponding protein (1 mg/ml of the diluted

antibody). As a result, specific immunostaining was

completely abolished.

Fig. 1. Calbindin-immunoreactivity. (A) Panoramic view of the claustrum (Cl) and dorsal endopiriform nucleus (DEn) at a rostral level (see Fig. 17

of Paxinos and Franklin, 2001). Transverse section. Scale bar: 1 mm. (B) Detail of the claustrum and dorsal endopiriform nucleus at an intermediate

rostrocaudal level. The claustrum is characterized by the presence of a number of immunoreactive cells within a moderately immunostained neuropil,

similarly to the adjacent cortical areas and dorsal endopiriform nucleus. Scale bar: 500 mm. (C) Most neuronal cell bodies in the claustrum are devoid

of calbindin immunoreactivity and appear as white profiles surrounded by an immunostained neuropil. Scale bar: 100 mm. (D) Morphological

features of calbindin immunoreactive neurons. Most stained neurons are multipolar cells with aspiny dendrites. Scale bar: 100 mm. (E) Pairs of

calbindin immunostained neurons with apposed cell bodies are sometimes observed in the claustrum (arrow). A number of lightly stained somata can

also be observed. Scale bar: 100 mm. (F) Panoramic view of the endopiriform region at a caudal level (see Fig. 41 of Paxinos and Franklin, 2001).

Both the number of immunoreactive neurons and the intensity of neuropil staining decrease at these levels. Scale bar: 1 mm. aca, Anterior

commissure, anterior; AID, agranular insular cortex, dorsal; AIP, agranular insular cortex, posterior; AIV, agranular insular cortex, ventral; BLA,

basolateral amygdaloid nucleus, anterior; Cl, claustrum; CPu, caudate putamen; DEn, dorsal endopiriform nucleus; DI, dysgranular insular cortex;

ec, external capsule; La, lateral amygdaloid nucleus; lo, lateral olfactory tract; Pir, piriform cortex; Ven, ventral endopiriform nucleus.

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160152

Fig. 1

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160 153

Light microscopic images were photographed by

using a Leica microscope equipped with a Nikon

DXM1200 digital camera. Digital images were loaded

into ADOBE PHOTOSHOP software and converted tograyscale images. Brightness and contrast were adjusted

for the final images. No additional filtering or manip-

ulation of the images was performed. The final figures

were composed and labeled with ADOBE PAGEMAKER

software and printed with an Epson Photo 750 printer.

3. Results

The three calcium-binding proteins studied in this

work were distinctly expressed in structures of both theclaustrum and the endopiriform nucleus. We will next

describe separately calbindin-immunoreactivity in the

claustrum and in the endopiriform nucleus, and then

calretinin- and parvalbumin-immunoreactivities, respec-

tively.

3.1. Calbindin-immunoreactivity

3.1.1. Claustrum

Sections immunostained for calbindin showed an

overall stronger staining of both neurons and neuropil

than those stained for either of the other antibodies;

accordingly numerous calbindin-immunoreactive (ir)neurons embedded in a moderately immunostained

neuropil were found from rostral to caudal levels of

the claustrum (Fig. 1A and B). Nevertheless, the number

of immunostained cells decreased slightly at caudal

levels. As in other parts of the telencephalon, two cell-

staining patterns for calbindin were observed through-

out the claustrum: darkly stained neurons with a Golgi-

like appearance, and lightly stained cell bodies withbarely visible processes (Fig. 1D and E). Cell morphol-

ogies varied from small, round or elongated neurons to

medium-sized multipolar neurons (Fig. 1C and D).

These distinct cell types appeared intermingled through-

out the claustrum. Multipolar calbindin-ir neurons

displayed typically three to five processes splitting into

a few secondary aspiny dendrites. Sometimes, pairs of

calbindin-ir multipolar neurons with apposed somatawere observed within the claustrum (Fig. 1E).

The uniform background staining of the claustrum

and adjacent cortical areas made it difficult to delineate

the borders of the claustrum in calbindin immunos-

tained sections (Fig. 1B). A moderately stained neuropil

extended over the whole anterior�/posterior extent of the

claustrum. Neuropil staining consisted of varicose axons

oriented in all directions and puncta surroundingnumerous immunonegative cell profiles (Fig. 1C).

Although many perisomatic calbindin-positive terminals

were observed, they did not form pericellular baskets.

3.1.2. Endopiriform nucleus

As in the claustrum, calbindin-ir neurons in the

endopiriform nucleus exhibited a variety of staining

intensities, sizes, and morphologies. Small or medium-sized multipolar cells were the most common types,

although pyramidal-shaped cell bodies were also found

at intermediate levels of the dorsal endopiriform nu-

cleus. In the dorsal endopiriform nucleus, the number of

calbindin immunostained neurons was highest at rostral

levels (Fig. 1A), decreasing at intermediate and caudal

levels (Fig. 1F). On the other hand, the number of

calbindin immunoreactive neurons in the ventral en-dopiriform nucleus was similar to that found in the

caudal part of the dorsal endopiriform nucleus. Pairs of

calbindin immunostained cells with apposed somata

were also found in the endopiriform nucleus.

A uniform, moderately stained, neuropil extended

throughout both the dorsal and ventral regions of the

endopiriform nucleus (Fig. 1F). Calbindin-positive ax-

ons bearing varicosities were observed in this neuropil.

3.2. Calretinin-immunoreactivity

3.2.1. Claustrum

Calretinin immunostaining in the claustrum consistedof a few scattered positive cells against a background of

stained fibers and boutons. Most calretinin-ir neurons

were densely stained and exhibited small, round or

elongated cell bodies (Fig. 2C), from which two or three

aspiny dendrites arose. Some immunostained bipolar

neurons located near the external capsule had their cell

bodies and dendrites oriented parallel to it whereas

other calretinin-ir neurons extended their dendrites,perpendicular to the external capsule and to the cortical

surface.

In contrast to the piriform cortex and to the subjacent

striatum, the claustrum is characterized by the presence

of a dense calretinin-ir neuropil consisting of both

stained fibers and puncta (Fig. 2A and B). Nevertheless,

the neuropil immunostaining was unevenly distributed

within the claustrum. A calretinin-negative neuropilzone, virtually devoid of immunoreactive fibers or

puncta, appeared as an oval region in the core of the

claustrum (Fig. 2B and C). This oval region was also

largely devoid of calretinin-ir neurons, which, if present,

were found at the periphery of the region (Fig. 2C). The

calretinin-negative region was clearly visible from inter-

mediate to caudal levels of the claustrum since it was

surrounded by immunopositive structures, including athin deep layer of neuropil that separates it from the

fibers of the external capsule (Fig. 2C), and a superficial

layer of neuropil continuous with the deep layers of the

neighboring insular cortex.

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160154

3.2.2. Endopiriform nucleus

The overall calretinin immunostaining in the endo-

piriform nucleus was similar to that of the claustrum

and the insular cortex. Few scattered neurons embedded

in a moderate-to-dense immunoreactive neuropil were

found in the endopiriform nucleus. The most abundant

calretinin-ir cell type consisted of small bipolar cells with

the elongated cell body and processes oriented parallel

to the external capsule. Bipolar neurons were observed

in both the dorsal and ventral endopiriform nuclei.

Neuropil staining was highest at intermediate levels in

the region of the dorsal endopiriform nucleus adjacent

to the claustrum (Fig. 2B). For the rest of the dorsal

endopiriform nucleus and the whole ventral endopiri-

Fig. 2. Calretinin-immunoreactivity. (A) Transverse section at a rostral level of the mouse telencephalon (see Fig. 16 of Paxinos and Franklin, 2001)

showing the claustrum (Cl) and the dorsal endopiriform nucleus (DEn). Scale bar: 1 mm. (B) At intermediate rostrocaudal levels (see Fig. 31 of

Paxinos and Franklin, 2001), the claustrum, the dorsal endopiriform nucleus, and the insular cortex are characterized by a moderately

immunoreactive neuropil. An oval immunonegative area can be recognized within the claustrum. Scale bar: 1 mm. (C) Detail of the boxed area in B.

This oval area is virtually devoid of immunoreactive fibers. A small stained neuron can be observed in the periphery of the negative region (arrow).

Scale bar: 100 mm. (D) Panoramic view of the endopiriform region at a caudal level (see Fig. 41 of Paxinos and Franklin, 2001). Scale bar: 1 mm. aca,

anterior commissure, anterior; acp, anterior commissure, posterior; AID, agranular insular cortex, dorsal; AIP, agranular insular cortex, posterior;

AIV, agranular insular cortex, ventral; BLA, basolateral amygdaloid nucleus, anterior; Cl, claustrum; CPu, caudate putamen; DEn, dorsal

endopiriform nucleus; DI, dysgranular insular cortex; ec, external capsule; GI, granular insular cortex; La, lateral amygdaloid nucleus; Pir, piriform

cortex; VEn, ventral endopiriform nucleus.

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160 155

form nucleus, the neuropil was moderately immunos-

tained for calretinin (Fig. 2D). Calretinin immunoreac-

tive axons were fine and displayed small varicosities.

3.3. Parvalbumin-immunoreactivity

3.3.1. Claustrum

Parvalbumin immunostaining in the claustrum con-

sisted of a number of immunoreactive neurons sur-

rounded by a dense meshwork of stained fibers andpuncta (Fig. 3C and D). Both darkly and lightly stained

neurons were found throughout the claustrum but they

were less abundant at the caudal part of the claustrum.

Most parvalbumin-ir cells were medium in size and had

multipolar cell bodies (Fig. 3C) with several thin, beaded

dendrites that extended for long distances.

Parvalbumin-ir varicose axons were oriented in all

directions within the claustrum (Fig. 3C and D).However, it was unclear whether these axons formed

perisomatic contacts.

In sections immunostained for parvalbumin, the

claustrum was characterized by the presence of a sharply

defined, moderately stained patch of neuropil particu-

larly evident at intermediate and caudal levels of the

claustrum (Fig. 3B). The deep layers of the adjacent

agranular insular cortex presented also a patch ofmoderately stained neuropil, and between these two

neuropil patches there was an intervening immunone-

gative cell-poor zone that was most apparent at inter-

mediate levels of the claustrum (Fig. 3B).

The patch of parvalbumin immunopositive neuropil

in the claustrum roughly corresponds to the calretinin-

negative oval area described above.

3.3.2. Endopiriform nucleus

A number of parvalbumin-ir neurons embedded in aplexus of immunostained varicose axons and processes

were found at rostral levels of the dorsal endopiriform

nucleus (Fig. 3A). At intermediate and caudal levels of

the dorsal endopiriform nucleus, the number of positive

neurons and axons decreased dramatically: very few

immunoreactive neurons were observed, and the neuro-

pil was almost negative at these levels (Fig. 3E). The

parvalbumin immunoreactivity pattern in the ventral

endopiriform nucleus was similar to that of the caudal

part of the dorsal endopiriform nucleus.

Most parvalbumin stained neurons located in the

rostral part of the dorsal endopiriform nucleus were

medium in size and exhibited multipolar morphologies,

without a specific orientation of their cell processes. In

the ventral endopiriform nucleus, some multipolar

immunoreactive neurons with the cell body and main

dendrites oriented parallel to the pial surface were

observed (Fig. 3F).

3.4. Colocalization study

We carried out a colocalization study for the two

major calcium-binding proteins present in neurons of

the mouse claustrum, i.e. calbindin and parvalbumin,

using post-embedding immunocytochemistry on adja-

cent semithin sections. The analysis was made in the

dorsal claustrum, since it was the claustral region with

more positive neurons for either calcium-binding pro-

tein.

In addition, by comparing immunostained semithin

sections with the adjacent Nissl-stained semithin section,

we obtained quantitative data about the relative density

of calbindin- and parvalbumin-immunoreactive popula-

tions.

Calbindin- and parvalbumin-immunoreactive neu-

rons constituted a minority of the claustral neurons,

accounting for only 5.4% (n�/122) and 7.9% (n�/178)

of the total number of counted cells (n�/2260), respec-

tively. Neurons expressing either of the two proteins

represent 12.3% of the claustral cells. In addition,

calbindin- and parvalbumin-immunoreactive cells are

mostly segregated populations (Fig. 4): more than 87%

of parvalbumin-ir cells do not express calbindin (156 out

of 178) and, conversely, 82% of calbindin-ir cells do not

express parvalbumin (100 out of 122), whereas only 22

out of 2260 cells coexpress both proteins.

Fig. 3. Parvalbumin-immunoreactivity. (A) Low power photomicrograph of a transverse section of the rostral telencephalon (see Fig. 17 of Paxinos

and Franklin, 2001). At this level, the dorsal endopiriform nucleus (DEn) presents its highest number of immunoreactive neurons. Scale bar: 1 mm.

(B) Detail of the claustrum at an intermediate level. A central region with a moderately stained neuropil, just deep to the ventral part of the agranular

insular cortex can be easily recognized (arrows). Scale bar: 500 mm. (C) Two multipolar medium-size neurons can be observed in the central region of

the claustrum. Scale bar: 100 mm. (D) Neuropil immunostaining consists of a dense meshwork of axons and terminals, although no pericellular

baskets can be distinguished. Scale bar: 50 mm. (E) Detail of the endopiriform region at a caudal level (see Fig. 38 of Paxinos and Franklin, 2001).

Both the dorsal and ventral parts of the endopiriform nucleus are characterized by an immunonegative neuropil. Scale bar: 1 mm. (F) Multipolar

stained neurons with their elongated cell bodies and dendrites oriented parallel to the pial surface are observed in the ventral endopiriform nucleus

(VEn). Scale bar: 100 mm. aca, Anterior commissure, anterior; AID, agranular insular cortex, dorsal; AIP, agranular insular cortex, posterior; AIV,

agranular insular cortex, ventral; BLA, basolateral amygdaloid nucleus, anterior; Cl, claustrum; CPu, caudate putamen; DEn, dorsal endopiriform

nucleus; DI, dysgranular insular cortex; ec, external capsule; La, lateral amygdaloid nucleus; lo, lateral olfactory tract; Pir, piriform cortex; VEn,

ventral endopiriform nucleus.

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160156

Fig. 3

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160 157

4. Discussion

Although little is known about the exact functions of

the calcium-binding proteins parvalbumin, calbindinand calretinin, they have provided useful markers of

specific neuronal subpopulations in studies of the

neuronal circuitry of the cerebral cortex and other brain

regions, and they reveal chemoarchitectonic subdivi-

sions in different regions of the central nervous system.

Results of the present study indicate the existence of

distinct immunoreactivity patterns for each of the three

calcium-binding proteins in both the claustrum and theendopiriform nucleus as it has been reported in other

mammals, and also reveal details not described pre-

viously. We will next discuss our results in comparison

with other mammals and then analyze the calcium-

binding protein expression patterns in the light of

recently described subdivisions within the mouse claus-

trum.

Calbindin and parvalbumin immunoreactivities havebeen reported in the claustrum and endopiriform

nucleus in other mammals but inter-specific differences

exist regarding the expression pattern of each protein. In

the rat, parvalbumin and calbindin exhibit a largely

complementary distribution pattern: parvalbumin-im-

munoreactivity is concentrated in the claustrum,

whereas calbindin-immunoreactivity prevails in the en-

dopiriform nucleus (Celio, 1990; Druga et al., 1993).From our results in mouse, we cannot demonstrate this

complementary distribution pattern between calbindin

and parvalbumin; instead, we showed that for either of

these calcium-binding proteins, more immunostained

neurons were observed in the claustrum than in the

endopiriform nucleus, and more in rostral than in

caudal levels.

Our colocalization analysis has demonstrated that, inspite of a similar distribution pattern, parvalbumin and

calbindin are mostly expressed by separate claustral

neurons in the mouse. In this context, is to be noted that

in the rat cortex parvalbumin- and calbindin-ir cells

represent two classes of GABAergic interneurons dis-

playing morphological and neurochemical specificity

(Celio, 1986; Hendry et al., 1989; Kosaka and Heiz-

mann, 1989), and indirect evidence suggests that cal-cium-binding protein immunoreactive neurons may also

represent, at least in part, inhibitory local circuit

neurons in the rat claustrum (Druga et al., 1993).

With regards to calretinin, it has been claimed that the

rat claustrum is demarcated by the absence of calretinin

staining (Paxinos et al., 1998). However, calretinin

immunoreactive structures have been reported in the

monkey claustrum (Reynhout and Baizer, 1999) and inthe endopiriform nucleus of the guinea pig (Frassoni et

al., 1998). Our results in the mouse showed that

calretinin staining consisted of a few positive cell bodies

embedded in a moderate neuropil both in the claustrum

and the endopiriform nucleus. Calretinin is present in

the claustral regions of monkeys, guinea pigs, and mice.

Thus, the reported absence of calretinin in the rat

claustrum could be due to inter-specific differences orto a differential sensitivity of the immunocytochemical

method.

Calcium-binding protein immunoreactive neurons

constitute together a small subset (12.3%) of the

claustral neurons in the mouse, as demonstrated by

our study on semithin sections. This low incidence of

calcium-binding protein immunoreactive neurons in the

claustrum was to be expected if they represent differentsubpopulations of GABAergic interneurons, as sug-

gested above, since GABAergic neurons in the rabbit

claustrum accounted only for 12% of all neurons in the

same claustral samples (Gomez-Urquijo et al., 2000).

These data on the low incidence of inhibitory claustral

neurons are also in agreement with the presence in the

rat claustrum of numerous latexin-immunoreactive

neurons (Arimatsu et al., 1992), most of which areglutamate positive (and GABA negative), suggesting

that they are excitatory projection neurons (Arimatsu et

al., 1999).

4.1. Different zones within the dorsal claustrum

Recently, three novel subdivisions within the mouse

claustrum based on the pattern of cadherin expression

and cytoarchitecture have been distinguished (Obst-Pernberg et al., 2001): a superior, an intermediate and

an inferior part. The superior and intermediate parts are

located deep to the insular cortex whereas the inferior

part is located deep to the dorsal part of the piriform

cortex. Cytoarchitectonically the intermediate part is

characterized by the presence of cell aggregates. The

superior and intermediate parts contain densely packed

cell bodies that strongly express R-cadherin (Rcad;Obst-Pernberg et al., 2001) and cadherin-8 mRNA

(Korematsu and Redies, 1997), whereas the inferior

part shows weak expression of Rcad, and only contain

scattered cell bodies expressing cadherin-8 mRNA. In

addition, the superior part shows a very strongly

immunoreactive neuropil for Rcad and moderately

labeled for cadherin-N (Ncad); the intermediate part is

strongly immunoreactive for Rcad and moderately tostrongly labeled for Ncad; and the inferior part is

weakly labeled for Rcad and moderately labeled for

Ncad (Obst-Pernberg et al., 2001).

Our results on parvalbumin and calretinin immunor-

eactivity match these novel subdivisions of the mouse

claustrum. Thus, in parvalbumin-immunostained sec-

tions a patch of moderately immunoreactive neuropil

occupies a position just deep to the ventral part of theagranular insular cortex. This parvalbumin positive

zone is bordered dorsal and ventrally by claustral zones

virtually devoid of immunostained neuropil. The dorsal

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160158

negative zone lies deep to the dorsal part of the

agranular insular cortex and to the dysgranular cortex,

whereas the ventral zone negative for parvalbumin is

continuous with the endopiriform nucleus. In calretinin

immunostained sections, on the other hand, a comple-

mentary pattern was observed: the intermediate region

was virtually devoid of calretinin immunoreactive fibers,

and dorsal and ventral calretinin-positive zones bor-

dered it. It is tempting to relate these three zones of the

dorsal claustrum showing a distinct calcium-binding

protein expression pattern to the three subdivisions

described by Obst-Pernberg and coworkers on the basis

of cadherin expression patterns (Obst-Pernberg et al.,

2001). These authors suggest that the selective adhesion

of neural structures that express the same types of

cadherin contribute to the formation of gray matter

areas, neural circuits and functional connections in the

postnatal forebrain of the mouse. In this sense, we

propose that the cell aggregates of the intermediate zone

of the dorsal claustrum with cell bodies that strongly

express Rcad and cadherin-8 would be the selective

target for parvalbumin-expressing fibers, and that they

would be mostly avoided by calretinin-expressing axons.

We cannot conclude whether these fibers have an

intrinsic or extrinsic origin.

While these novel subdivisions of the mouse dorsal

claustrum appear to be well documented from a cyto

and neurochemical point of view, it is difficult to relate

them to the different functional zones that have been

described in the mammalian claustrum on the basis of its

connections. In mammals with a well developed claus-

trum the claustro-neocortical connections are topogra-

phically organized, with an anterior part of the

claustrum linked mainly with motor and prefrontal

cortices, a central part linked with somatosensory

cortex, a posterior claustrum related to visual cortex,

and a ventral zone connected with auditory cortex

(Pearson et al., 1982; Macchi et al., 1983; Sherk, 1988;

Morys et al., 1996). In the rat claustrum, however, two

main cortico-related zones have been described, an

anterodorsal sensorimotor and a posteroventral vi-

suoauditory zones (Sadowski et al., 1997).

Our results indicate that the incidence of calcium-

binding protein-expressing neurons is higher in rostral

than caudal levels of the mouse dorsal claustrum. Since

all hodological studies confirm at least two different

functional zones in the claustrum, an anterior and a

posterior region, it is likely that calcium-binding pro-

tein-expressing neurons, putatively GABAergic cells, are

more represented in claustral intrinsic circuits related

with the limbic and motor cortices rather than with the

visuoauditory regions. As suggested for somatostatin-,

neuropeptide Y-, and vasoactive intestinal peptide-ir

neurons in the rat claustrum (Kowianski et al., 2001),

calcium-binding protein-expressing neurons do not

appear to play a significant role in the claustro-cortical

projection but are most probably involved in modula-

tion and information transfer in the claustrum.

It is to be noted that the complementary expression of

calretinin and parvalbumin in patches of neuropil in the

mouse claustrum is best seen at intermediate/posterior

levels in the rostrocaudal axis of the claustrum. It

Fig. 4. Photomicrographs of two adjacent semithin sections immunostained for calbindin (A) and parvalbumin (B). Only one neuron in this field is

immunoreactive for both calcium-binding proteins (arrow). Capillaries (c) were used as landmarks. Scale bar: 30 mm.

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160 159

cannot be determined, however, whether these claustral

zones correspond with claustral regions with specific

cortical connections. In this sense, it would be of interest

to perform combined immunohistochemical-fiber tra-cing experiments in order to study the specific connec-

tions of the chemoarchitectonic subdivisions of the

dorsal claustrum.

Acknowledgements

We would like to thank Luis Olmos for his excellent

technical assistance. This work was supported bySpanish DGI grant BFI2000-1359-C02-01, as well as

by Spanish FIS grant 01-0057-01.

References

Arimatsu, Y., Miyamoto, M., Nihonmatsu, I., Hirata, K., Uratani, Y.,

Hatanaka, Y., Takiguchi-Hayashi, K., 1992. Early regional speci-

fication for a molecular neuronal phenotype in the rat neocortex.

Proc. Natl. Acad. Sci. USA 89, 8879�/8883.

Arimatsu, Y., Ishida, M., Sato, M., Kojima, M., 1999. Corticocortical

associative neurons expressing latexin: specific cortical connectivity

formed in vivo and in vitro. Cereb. Cortex 9, 569�/576.

Celio, M.R., 1986. Parvalbumin in most gamma-aminobutyric acid-

containing neurons of the rat cerebral cortex. Science 231, 995�/

997.

Celio, M.R., 1990. Calbindin D-28k and parvalbumin in the rat

nervous system. Neuroscience 35, 375�/475.

Davila, J.C., Guirado, S., Puelles, L., 2000. Expression of calcium-

binding proteins in the diencephalon of the lizard Psammodromus

algirus . J. Comp. Neurol. 427, 67�/92.

Dinopoulos, A., Papadopoulos, G.C., Michaloudi, H., Parnavelas,

J.G., Uylings, H.B., Karamanlidis, A.N., 1992. Claustrum in the

hedgehog (Erinaceus europaeus ) brain: cytoarchitecture and con-

nections with cortical and subcortical structures. J. Comp. Neurol.

316, 187�/205.

Druga, R., 1966. The claustrum of the cat (Felis domestica ). Folia

Morphol. (Praha) 14, 7�/16.

Druga, R., 1971. Projection of prepyriform cortex into claustrum.

Folia Morphol. (Praha) 19, 405�/410.

Druga, R., Chen, S., Bentivoglio, M., 1993. Parvalbumin and

calbindin in the rat claustrum: an immunocytochemical study

combined with retrograde tracing frontoparietal cortex. J. Chem.

Neuroanat. 6, 399�/406.

Frassoni, C., Radici, C., Spreafico, R., de Curtis, M., 1998. Calcium-

binding protein immunoreactivity in the piriform cortex of the

guinea-pig: selective staining of subsets of non-GABAergic neurons

by calretinin. Neuroscience 83, 229�/237.

Gomez-Urquijo, S.M., Gutierrez-Ibarluzea, I., Bueno-Lopez, J.L.,

Reblet, C., 2000. Percentage incidence of gamma-aminobutyric

acid neurons in the claustrum of the rabbit and comparison with

the cortex and putamen. Neurosci. Lett. 282, 177�/180.

Hendry, S.H., Jones, E.G., Emson, P.C., Lawson, D.E., Heizmann,

C.W., Streit, P., 1989. Two classes of cortical GABA neurons

defined by differential calcium binding protein immunoreactivities.

Exp. Brain Res. 76, 467�/472.

Jones, E.G., Hendry, S.H.C., 1989. Differential calcium-binding

protein immunoreactivity distinguishes classes of relay neurons in

monkey thalamic nuclei. Eur. J. Neurosci. 1, 222�/245.

Korematsu, K., Redies, C., 1997. Restricted expression of cadherin-8

in segmental and functional subdivisions of the embryonic mouse

brain. Dev. Dynam. 208, 178�/189.

Kosaka, T., Heizmann, C.W., 1989. Selective staining of a population

of parvalbumin-containing GABAergic neurons in the rat cerebral

cortex by lectins with specific affinity for terminal N -acetylgalac-

tosamine. Brain Res. 483, 158�/163.

Kowianski, P., Morys, J., Karwacki, Z., Dziewiatkowski, J., Narkie-

wicz, O., 1998. The cortico-related zones of the rabbit claustrum-

study of the claustrocortical connections based on the retrograde

axonal transport of fluorescent tracers. Brain Res. 784, 199�/209.

Kowianski, P., Timmermans, J.P., Morys, J., 2001. Differentiation in

the immunocytochemical features of intrinsic and cortically

projecting neurons in the rat claustrum*/combined immunocyto-

chemical and axonal transport study. Brain Res. 905, 63�/71.

Krettek, J.E., Price, J.L., 1977. Projections from the amygdaloid

complex to the cerebral cortex and thalamus in the rat and cat. J.

Comp. Neurol. 172, 687�/722.

Li, Z.K., Takada, M., Hattori, T., 1986. Topographic organization

and collateralization of claustrocortical projections in the rat. Brain

Res. Bull. 17, 529�/532.

Macchi, G., Bentivoglio, M., Minciacchi, D., Molinari, M., 1983.

Claustroneocortical projections studied in the cat by means of

multiple retrograde fluorescent tracing. J. Comp. Neurol. 215,

121�/134.

Majak, K., Kowianski, P., Morys, J., Spodnik, J., Karwacki, Z.,

Wisniewski, H.M., 2000. The limbic zone of the rabbit and rat

claustrum: a study of the claustrocingulate connections based on

the retrograde axonal transport of fluorescent tracers. Anat.

Embryol. 201, 15�/25.

Markowitsch, H.J., Irle, E., Bang-Olsen, R., Flindt-Egebak, P., 1984.

Claustral efferents to the cat’s limbic cortex studied with retrograde

and anterograde tracing techniques. Neuroscience 12, 409�/425.

Morys, J., Berdel, B., Maciejewska, B., Sadowski, M., Sidorowicz, M.,

Kowianska, J., Narkiewicz, O., 1996. Division of the human

claustrum according to its architectonics, morphometric para-

meters and cortical connections. Folia Morphol. (Warsz) 55, 69�/

82.

Obst-Pernberg, K., Medina, L., Redies, C., 2001. Expression of R -

cadherin and N -cadherin by cell groups and fiber tracts in the

developing mouse forebrain: relation to the formation of functional

circuits. Neuroscience 106, 505�/533.

Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic

Coordinates. Academic Press, New York.

Paxinos, G., Kus, L., Ashwell, K.W.S., Watson, C., 1998. Chemoarch-

itectonic Atlas of the Rat Forebrain. Academic Press, New York.

Pearson, R.C., Brodal, P., Gatter, K.C., Powell, T.P., 1982. The

organization of the connections between the cortex and the

claustrum in the monkey. Brain Res. 234, 435�/441.

Reynhout, K., Baizer, J.S., 1999. Immunoreactivity for calcium-

binding proteins in the claustrum of the monkey. Anat. Embryol.

199, 75�/83.

Sadowski, M., Morys, J., Jakubowska-Sadowska, K., Narkiewicz, O.,

1997. Rat’s claustrum shows two main cortico-related zones. Brain

Res. 756, 147�/152.

Sherk, H., 1988. The claustrum and the cerebral cortex. In: Jones,

E.G., Peters, A. (Eds.), Sensory-Motor Areas and Aspects of

Cortical Connectivity, vol. 5. Plenum Press, New York, London,

pp. 467�/499.

Sloniewski, P., Usunoff, K.G., Pilgrim, C., 1986. Retrograde transport

of fluorescent tracers reveals extensive ipsi- and contralateral

claustrocortical connections in the rat. J. Comp. Neurol. 246,

467�/477.

Witter, M.P., Room, P., Groenewegen, H.J., Lohman, A.H., 1988.

Reciprocal connections of the insular and piriform claustrum with

limbic cortex: an anatomical study in the cat. Neuroscience 24,

519�/539.

M.A. Real et al. / Journal of Chemical Neuroanatomy 25 (2003) 151�/160160