Organization of brain extracellular matrix in the Chilean fat-tailed mouse opossum Thylamys elegans (Waterhouse, 1839)

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Journal of Chemical Neuroanatomy 32 (2006) 143–158

Organization of brain extracellular matrix in the Chilean fat-tailed

mouse opossum Thylamys elegans (Waterhouse, 1839)

Gert Bruckner a,*, Sanja Pavlica a, Markus Morawski a,Adrian G. Palacios b, Andreas Reichenbach a

a Paul Flechsig Institute for Brain Research, Department of Neurochemistry, University of Leipzig, Jahnalle 59, D-04109 Leipzig, Germanyb Centro de Neurociencia de Valparaıso (CNV), Facultad de Ciencias, Universidad de Valparaıso, Gran Bretana 1111, Playa Ancha, Valparaıso, Chile

Received 13 July 2006; received in revised form 8 August 2006; accepted 15 August 2006

Available online 25 September 2006

Abstract

We investigated the structural and molecular organization of the extracellular matrix in Thylamys elegans, a marsupial representative of the

mammalian order Didelphimorphia. Perineuronal nets (PNs) associated with distinct types of neurons were visualized by detection of chondroitin

sulfate proteoglycans and hyaluronan, and by labeling with Wisteria floribunda agglutinin (WFA), a marker for PNs in the mammalian brain. In the

neocortex of Thylamys, these methods revealed PNs on pyramidal cells. In contrast, parvalbumin-immunoreactive interneurons in the neocortex

and hippocampal formation (displaying robust, WFA-labeled PNs in placental mammals) were ensheathed only with a delicate rim of hyaluronan

and proteoglycans not detectable with WFA. The absence of WFA staining was characteristic also of some subcortical regions which contained PNs

intensely labeled for chondroitin sulfate proteoglycan and hyaluronan. However, corresponding to placental mammals, numerous subcortical

nuclei showed clearly WFA-stained PNs. Similar as in placental mammals, cholinergic basal forebrain neurons and tyrosine hydroxylase-

immunoreactive neurons of the substantia nigra and locus coeruleus were devoid of PNs. Together with our earlier study on Monodelphis, the

present results reveal that South American opossums show either a particular ‘‘marsupial’’ or ‘‘Didelphid’’ type of extracellular matrix

chemoarchitecture, supporting the view that these components may vary phylogenetically as integral parts of neuronal physiology at the systems

and single cell level.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Perineuronal nets; Chondroitin sulfate proteoglycans; Hyaluronan; Cerebral cortex; Brain evolution; Marsupials

1. Introduction

The extracellular matrix specifically contributes to func-

tional characteristics of cells, tissues and organs in develop-

mental and adult stages (for reviews see Comper, 1996). In the

mammalian brain, the spatial and molecular organization of the

extracellular matrix shows a great diversity that obviously

mirrors the complexity of neuronal functions at the single cell

and systemic level (Bruckner et al., 1996; Carlson and

Hockfield, 1996; Matthews et al., 2002). It can be assumed

that brain evolution is accompanied by a corresponding

evolution of the extracellular matrix.

Since perineuronal nets (PNs) have been shown to be a basic

form of extracellular matrix organization in the central nervous

* Corresponding author. Tel.: +49 341 9725 732; fax: +49 341 9725 749.

E-mail address: [email protected] (G. Bruckner).

0891-0618/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.jchemneu.2006.08.002

system (Brauer et al., 1984; Hendry et al., 1988; Delpech et al.,

1989; Bruckner et al., 1993, 1994, 1996; Murakami et al., 1994;

for reviews see Celio and Blumcke, 1994; Carlson and

Hockfield, 1996; Celio et al., 1998; Yamaguchi, 2000), they

appear as appropriate subjects for studies focused on

evolutionary aspects. In the various vertebrate species

investigated so far, PNs are composed of aggregating

chondroitin sulfate proteoglycans, connected with hyaluronan,

link proteins and tenascin glycoproteins (Yamaguchi, 2000;

Bruckner et al., 2003; Dityatev and Schachner, 2003; Carulli

et al., 2006). These major components may form complexes in

variable proportions resulting in distinct molecular properties,

such as hydration and viscosity, or electrical load of the cellular

microenvironment. Especially, the proteoglycans are assumed

to have a great potential of molecular variation, by formation of

splice variants of their core proteins and by variable

glycosylation by the attached glycosaminoglycan chains

(Matthews et al., 2002; Rauch, 2004). It is conceivable that

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http://dx.doi.org/10.1016/j.jchemneu.2006.08.002

G. Bruckner et al. / Journal of Chemical Neuroanatomy 32 (2006) 143–158144

distinct characteristics of the extracellular matrix persist from

ancient to recent animal species in association with neuronal

systems conserved during brain evolution. Correspondingly, the

extracellular matrix may undergo evolutionary alterations

related to functional changes of anatomically defined neuronal

systems. However, the great variability of extracellular matrix

components also inherits the spontaneous formation of multiple

new molecular combinations, which are endowed with

analogous functional properties.

The present study was undertaken to investigate the

association of PNs with defined types of neurons in the South

American mouse opossum (Thylamys elegans), a marsupial

representative of the phylogenetically ancient mammalian

order Didelphimorphia (Steiner et al., 2005). Complementary

to our previous study focussed on the cerebral cortex of the

related species Monodelphis domestica (Bruckner et al., 1998),

we performed a detailed analysis of subcortical regions in the

present investigation. Since, we used cytochemical methods

established in extracellular matrix research, the results are

comparable with data obtained from phylogenetically distant

mammalian, including the human, brain. Our data suggest that

there exist unique marsupial as well as common mammalian

features of extracellular matrix organization.

2. Materials and methods

2.1. Animals and tissue processing

Four adult male mouse opossums (T. elegans) were captured in the wild and

kept in a standard animal facility at the Universidad de Valparaiso (Chile).

Permission to work on collected specimens was under authorization #3014 from

Chilean Servicio Agricola y Ganadero (SAG). Animal care and experimental

procedures complied with international regulations (NIH publications No. 80-

23). For histochemical procedures, animals were euthanized by intraperitoneal

lethal dose of ketamine hydrochloride (1 g/kg body weight) and xylazine

hydrochloride (0.5 g/kg body weight). The brains were removed from the skull

and placed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS),

pH 7.4 for 14 days, cryoprotected in 30% sucrose, frozen and cut into a series of

30 mm thick sections. All sections were collected and extensively rinsed in

0.1 M Tris-buffered saline, pH 7.4 (TBS).

2.2. Anatomical mapping of brain regions and associated

extracellular matrix

Every sixth section of one series of frontal and one series of sagittal sections

were stained with a modified Nissl technique using 0.1% toluidine blue stem

solution diluted 1:200 in 0.1 M acetic acid buffer, pH 4.6, overnight at room

temperature (RT). Alternate sections of these series were used for extracellular

matrix staining with biotinylated Wisteria floribunda agglutinin (Bio-WFA;

Table 1

Cytochemical detection of extracellular matrix components

Detected components Marker Dilution

N-acetylgalactosamine Bio-WFAa 2.5 mg/ml

Chondroitin sulfate

proteoglycan core

protein

Rabbit anti-CSPGb 1:800

Hyaluronan BHABPc 10 mg/ml

a Biotinylated Wisteria floribunda agglutinin, reduced form.b Antigen from chondroitinase ABC-digested bovine nasal cartilage proteoglycac Biotinylated hyaluronic acid binding protein, isolated from bovine nasal cartila

Sigma, Deisenhofen) that was visualized with a standard streptavidin/perox-

idase technique and nickel-enhanced diaminobenzidine as a chromogen. The

treatment of the tissue and the staining followed the previously published

protocol (Hartig et al., 1992, 1994).

Since no cytoarchitectonic brain atlas is available for Thylamys, brain

regions were identified using the stereotaxic atlas of the brain of the opossum

Didelphis marsupialis (Oswaldo-Cruz and Rocha-Miranda, 1968). Structural

details of individual brain regions were identified with the help of studies

describing the anatomy of the cholinergic basal forebrain nuclei (Semba, 2004),

midbrain catecholaminergic regions (Hazlett et al., 1991), red nucleus (King

et al., 1971), cerebellar nuclei (Martin et al., 1974), as well as cortical areas and

hippocampus (Benevento and Ebner, 1971; Hamel, 1967, 1982; Rowe, 1990;

Beck et al., 1996; Huffman et al., 1999; Frost et al., 2000) in American

marsupials.

Most of structural abbreviations used in the present paper were adopted

from the nomenclature applied for mice (Franklin and Paxinos, 1997) and rats

(Paxinos and Watson, 1998).

2.3. Cytochemistry of extracellular matrix components

The reagents used for the fluorescence microscopic analysis of extracellular

matrix components in the present study are specified in Table 1. Tissue sections

showed no labeling when staining procedures were performed without the use

of primary antibodies or WFA.

Antibodies proved to be non-reactive for Thylamys brain were mouse anti-

tenascin-R (Mab 619 and Mab 596; M. Schachner, Hamburg) and rabbit anti-

mouse aggrecan (AB1031; Chemicon, Temecula).

Lectin staining of N-acetylgalactosamine-containing components: Preced-

ing the labeling with Bio-WFA, free-floating sections were treated for 1 h with a

blocking solution consisting of 5% normal goat serum in 0.1 M Tris-buffered

saline with 0.3% Triton X-100 (NGS-TBS-T). The sections were incubated with

Bio-WFA (2.5 mg/ml NGS-TBS-T), rinsed with TBS and processed for 1 h with

a second solution containing streptavidin conjugated to the red fluorescent Cy3

or green fluorescent Cy2 (Dianova, Hamburg; 20 mg/ml TBS containing 2%

bovine serum albumin, BSA).

Immunoreaction for chondroitin sulfate proteoglycan components: Non-

specific binding sites for subsequently applied immunoreagents were blocked

with NGS-TBS-T for 1 h. Free-floating sections were then incubated overnight

at room temperature with solutions containing the primary anti-chondroitin

sulfate proteoglycan (CSPG) antibodies specified in Table 1. The sections were

then rinsed with TBS and processed for 1 h with a solution containing Cy3-goat

anti-rabbit IgG or Cy3-goat anti-mouse IgG (Dianova, Hamburg; 20 mg/ml TBS

containing 2% BSA) as secondary antibodies.

Detection of hyaluronan: The presence and distribution of hyaluronan was

detected by using biotinylated hyaluronic acid-binding protein (BHABP,

1 mg/ml TBS containing 2% BSA) overnight at room temperature. The

labeling was then visualized by Cy3-streptavidin as described for WFA-

staining.

To test the specificity of the BHABP binding, free-floating sections were

pretreated with hyaluronidase from Streptomyces hyalurolyticus (50 U/ml

0.1 M PBS, pH 5.0; Sigma H1136; Koppe et al., 1997) for 4 and 16 h at

37 8C. Hyaluronidase treatment of the tissue sections for 4 h resulted in

decreased staining intensity. Binding of BHABP was at the background level

after 16 h of enzymatic treatment.

Source References

Sigma (Deisenhofen) Hartig et al. (1992, 1994)

Quartett (Berlin) Bertolotto et al. (1986)

Seikagaku America

n.

ge proteoglycan.

G. Bruckner et al. / Journal of Chemical Neuroanatomy 32 (2006) 143–158 145

Table 2

Association of different types of neurons with perineuronal nets revealed by

WFA staining, CSPG immunoreaction and detection of hyaluronan using

BHABP in Thylamys elegans

WFA CSPG BHABP

Cortex, primary sensory areas

Pyramidal cells, layer 5 + + +

Interneurons, layers 2–6a � � �

Subiculum

Pyramidal cells, deep layers + + +

Interneurons, deep layersa �/� ++ +/�

Hippocampus, CA1 region

SO and SP interneuronsa � � �

2.4. Cytochemistry of neuronal markers

Parvalbumin (PARV): Immunoreaction was performed by incubation of

sections after blocking in 5% GNS and 0.3% Triton for 1 h with rabbit anti-

parvalbumin (PV 28; antigen from rat muscle; Swant, Belinzona, dilution

1:250) overnight at RT. After rinsing, sections were processed for 1 h with a

second solution containing Cy2-goat anti-rabbit IgG (Dianova, Hamburg;

20 mg/ml) and streptavidin conjugated to Cy3 or Cy2 (Dianova, Hamburg;

20 mg/ml TBS containing 2% bovine serum albumin, BSA).

Choline acetyltransferase (ChAT): The sections were incubated in blocking

solution containing 5% donkey normal serum in TBS (D-TBS) and 0.3% Triton,

and were then incubated with goat anti-choline acetyltransferase (ChAT)

antibody (Chemicon AB144P; dilution 1:50) in 5% D-TBS and 0.1% Triton,

overnight at room temperature. Thereafter, sections were rinsed in TBS and

incubated with Cy3-donkey anti-goat IgG (20 mg/ml) in TBS supplemented

with 2% BSA for 60 min.

Tyrosine hydroxylase (TH): After blocking in 5% normal goat serum in

TBS containing 0.3% Triton (NGS-TBS-T), the sections were incubated

with rabbit anti-tyrosine hydroxylase (AB152, Chemicon, Temecula; dilu-

tion 1:200). The sections were further processed as described for PARV

immunoreaction.

2.5. Double labeling of neurons and extracellular matrix components

The immunoreactions for the detection of PARV, ChAT and TH were used

for double labeling with markers detecting extracellular matrix components.

Mixtures of primary and secondary reagents were applied according to the

reagents and the subsequent treatment of sections as described for single

labeling.

2.6. Light microscopy, confocal laser scanning microscopy and

image processing

After carbocyanine staining, the sections were extensively washed with

TBS, briefly placed in distilled water, mounted on fluorescence-free slides, air-

dried and coverslipped with Entellan (dissolved in toluene; Merck, Darmstadt).

Tissue sections were examined with a Zeiss Axioplan-AxioVision micro-

scope and a Zeiss confocal laser scanning microscope (LSM 510). Confocal

images of Cy2 fluorescence were obtained with the Argon laser (488 nm) and

emission filter BP 505-530. The HeNe 1 laser (543 nm) and the emission filter

BP 560–615 were used to detect Cy3 fluorescence. Photoshop 5.0 (Adobe

Systems, Mountain View, CA) was used to process the confocal images with

minimal alterations to the contrast and background.

Abbreviations

Subcortical forebrain
aca A nterior commissure M o M olecular layer
Medial septum/diagonal band (ChAT�) + + +
Aq A quaeduct (Sylvius) M o5 M otor trigeminal nu
Globus pallidus, external parta + + +
Au P rimary auditory cortex M S M edial septal nu
Reticular thalamic nua � + +/++
BMAB asomedial amygdala, anterior n u N ucleus
Zona incerta + + +
BLP B asolateral amygdala, posterior O B O lfactory bulb
Cpu C
audate putamen o MolO uter molecular layer Brainstem CA1 C A1 field, hippocampus o pt O ptic tract
Substantia nigra, reticular part (TH�)a + + +
CA3 C A3 field, hippocampus p c P osterior commissure Red nu ++ ++ ++ CbC C erebellar cortex P e P eriventricular hypothalamic area Medial nu trapezoid bodya � +/++ ++ CbN C erebellar nu P ir P iriform cortex Gigantocellular reticular nu ++ ++ ++ Cg C ingulate cortex P o P olymorph layer Motor trigeminal nu �/� + ++ Cl C laustrum R MCR ed nu, magnocellular part
Cerebellar cortex
cp C erebral peduncle R S R etrosplenial cortex
Golgi neurons + � �
DG D entate gyrus R t R eticular thalamic nu
Basket neuronsa � + +
DLG D orsolateral geniculate nu S S ubiculum
Ent E
ntorhinal cortex S 1 P rimary somatosensory cortex Deep cerebellar nuclei � ++ ++ Fr F rontal cortex S C S uperior colliculus
a Clearly immunoreactive for parvalbumin in WFA or BHABP double labeling
GP G lobus pallidus S N S ubstantia nigra
Gr G
ranule cell layer S O S tratum oriens experiments. Symbols indicate staining intensity of perineuronal nets:�/� = near
H H
ippocampus S P S tratum pyramidale background level, � = very low, + = low, +/++ = moderate, ++ = high; � = no
�
hc H ippocampal commissure T h T halamus staining detectable; ChAT = choline acetyltransferase-immunonegative neu-
�
HDB N u horizontal limb diagonal bandT u O lfactory tubercle
ic I

rons;

nternal capsule T

TH = tyrosine hydroxylase-immu

z N

noneg

u trapezoid body

Icj I
sland of Caleja V 1 P rimary visual cortex
iMol I
nner molecular layer V DBN u vertical limb diagonal band
lo L
ateral olfactory tract V LG V entrolateral geniculate nu
LC L
ocus coeruleus V TA V entral tegmental area
LV L
ateral ventricle Z I Z ona incerta
Med M
edial cerebellar nu 3 O culomotor nu
MG M
edial geniculate nu 4 V F ourth ventricle
7 F
acial nu
3V T
hird ventricle
3. Results

The results presented in this study were selected with respect

to anatomical regions (Table 2) that express clear patterns of

extracellular matrix organization in M. domestica, the only

marsupial investigated so far (Bruckner et al., 1998), as well as

regional patterns known from the rat (Seeger et al., 1994) and

murine brain (Bruckner et al., 2000, 2003). The location of the

analyzed regions in the Thylamys brain is indicated in selected

Nissl-stained sections in Fig. 1. Since our study is focussed on

PNs, the neuropil zones are described only if particular region-

specific properties were apparent.

ative neurons.


Fig. 1. Structural features of the brain of Thylamys elegans shown in Nissl-stained sections. (A) Parasagittal plane. (B–F) Frontal sections selected approximately at the

rostro-caudal position indicated in (A). For explanation of abbreviations see list of abbreviations. Scale bars: 1 mm in (A); 1 mm in (B) and (F), also applies to (C)–(E).

3.1. Phenotypes of perineuronal nets

WFA-staining of PNs revealed a great divergence in the

staining intensity between different regions, and even within

individual nuclei. Three major structural phenotypes may be

defined (Fig. 2). In the cerebral cortex, WFA-labeled PNs were

associated exclusively with pyramidal cells and showed a very

faint morphology; this will be called the ‘‘pyramidal type’’ of

PNs (Fig. 2A). By contrast, the well-known robust PN

phenotype expressing a clearly contoured, lattice-like structure

was widely distributed in subcortical regions (Fig. 2B). The

soma and the proximal parts of dendrites as well as the axon

initial segment of the subcortical neurons were covered by

matrix aggregates. The third PN type is characterized by a

diffuse perineuronal accumulation of WFA-stained extracel-

lular matrix components (Fig. 2C). Such ‘‘diffuse’’ PNs


Fig. 1. (Continued ).


Fig. 2. Different structural phenotypes of perineuronal nets revealed by WFA-DABNi staining in a frontal section located close to section C in Fig. 1. (A) Pyramidal cell

type frequently seen in layer 5 of primary sensory areas and the retrosplenial cortex. WFA-binding extracellular matrix faintly covers the soma, proximal parts of apical

dendrites and the axon initial segments of pyramidal cells in the S1 cortex. (B) Robust lattice-like type of PNs, predominating in many subcortical regions. The image

shows perineuronal nets in the magnocellular preoptic nucleus of the basal forebrain. (C) Diffuse type of PNs, characteristic of the periventricular hypothalamic area.

Extracellular matrix components infiltrate the adjacent pericellular neuropil. Arrows in (A) and (B) indicate the axon initial segments. Scale bar: 20 mm (applies to all).

(Wegner et al., 2003) were frequently found in the periven-

tricular area of the hypothalamus.

In addition to the variable structural appearance, a great

heterogeneity in the molecular composition of PNs is indicated

by combined application of WFA staining, CSPG immunor-

eaction and detection of hyaluronan by BHABP. Perineuronal

matrix components were labeled with BHABP and CSPG

antibodies in the cerebral cortex and many subcortical nuclei.

However, PNs detected with these stainings were devoid of

WFA labeling in several other regions (Table 2), thereby

contrasting with the patterns known from other mammalian

species.

3.2. Cerebral cortex and hippocampal formation

In the neocortex, clearly stained, robust PNs were not

detected. WFA-staining revealed PNs associated with layer 5

pyramidal cells in primary sensory areas (Fig. 3A and A0) and

with layer 2/3 pyramidal cells in the perirhinal cortex located

dorsal to the rhinal fissure (not shown). The pyramidal PNs

were also labeled with the CSPG (Fig. 3B0) immunoreaction.

Non-pyramidal neurons frequently showed a CSPG-immunor-

eactive perisomatic rim (Fig. 3B0). BHABP staining usually

revealed the perisomatic rim around layer 5 pyramidal cells and

some PARV-positive interneurons, however, its intensity barely

exceeded that of the surrounding neuropil (Fig. 3C and C0).WFA-labeled PNs associated with PARV-negative neurons

were found in the claustrum (not shown).

The hippocampus was characterized by intensely CSPG-

immunoreactive neuropil in the CA1–3 regions (Fig. 4A)

indicating a sharp border to the retrosplenial cortex. In the

subiculum, interneurons were associated with PNs clearly

positive for CSPG (Fig. 3A and A0). WFA-stained pyramidal

PNs were located in a particular cell layer extending from the

subiculum into the CA1 region (Fig. 3A and A0). PARV-positive

interneurons were negative for WFA staining in the hippo-

campus and dentate gyrus, such as found in the neocortex.

However, BHABP-staining (Fig. 4B, B0 and B00) revealed

perisomatic rims around PARV-positive neurons in the dentate

gyrus (Fig. 4B00).

3.3. Subcortical forebrain

The medial septum-diagonal band nuclei, well differentiated

in marsupials (Semba, 2004), contained densely packed PNs

embedded in a diffusely stained neuropil (Fig. 5A). ChAT-

positive neurons in the medial septum-diagonal band complex,

ventral pallidum and substantia innominata, as well as striatal

interneurons were not associated with PNs. However, clearly

contoured, intensely WFA-stained PNs were distributed in the

ventral striatopallidal region of the forebrain (not shown). In the

globus pallidus, the net-associated neurons expressed PARV

immunoreactivity (not shown). The distribution pattern of

WFA-stained PNs was largely corresponding with the patterns

detected by CSPG immunoreaction and BHABP staining in the

basal forebrain. As in other subcortical regions, BHABP

indicated an ubiqitous occurrence of hyaluronan (Fig. 5C), as

well as clearly contoured PNs (Fig. 5C0). In the hypothalamus

diffuse WFA-stained PNs occurred in the close proximity to the

third ventricle (Fig. 5B).

Special features of extracellular matrix organization were

detected in the ventral thalamus. In contrast to the pattern

known from placental mammals, the reticular thalamic nucleus

failed to display WFA staining (Fig. 6A, A00 and B) and was

only weakly labeled by the CSPG immunoreaction (Fig. 6B).

The PARV-positive neurons of this nucleus were ensheathed by


Fig. 3. Characteristics of extracellular matrix in the neocortex. Parvalbumin (PARV) expressing interneurons are labeled by immunofluorescence in the S1

somatosensory cortex. For correlation of anatomical regions see Fig. 1A–C. (A) WFA-staining reveals faint PNs only around layer five pyramidal cells (L5). (A0)Subcellular domains of WFA-stained pyramidal PNs include proximal parts of apical dendrites, somata and axon initial segments (arrows). (B and B0) Weak CSPG

immunoreactivity reveals PNs associated with an interneuron (arrow head) devoid of WFA staining whereas pyramidal PNs are double labeled. (C) Detection of

hyaluronan with BHABP shows uniform distribuion pattern of neuropil staining at low magnification. (C0) Staining intensity of BHABP-labeled perisomatic rims

around layer 5 pyramidal cells (asterisks) is similar to neuropil staining. Scale bars: 100 mm in (A) and (C); 20 mm in (A0), (B), (B0) and (C0).

a faint rim of CSPG immunoreactivity and BHABPstaining

(Fig. 6B and C). However, PNs in the zona incerta (Fig. 6A, A0

and B) and the ventral lateral geniculate nucleus were clearly

stained by WFA.

In the dorsal thalamus the primary relay nuclei were

virtually devoid of clearly labeled PNs (not shown).

3.4. Brainstem

The basic distribution patterns of PNs in the brainstem

largely corresponded with the patterns known from placental

mammals. Typically, the red nucleus was the subcortical region

most prominently stained by WFA, CSPG immunoreaction and

BHABP (Fig. 7A, B, B0 and C). In contrast, tyrosine

hydroxylase-immunoreactive neurons in the substantia nigra,

ventral tegmental area and locus coeruleus were devoid of PNs

(Fig. 7A, A0, C, C0 and D).

As an example of motor nuclei, Fig. 8 shows the motor

trigeminal nucleus (Mo5). Motor neurons immunoreactive for

choline acetyltransferase were ensheathed by PNs immunor-

eactive for CSPG, whereas WFA binding was not detectable. In

contrast to CSPG, the intense BHABP staining of PNs was a


Fig. 4. Characteristics of extracellular matrix in the hippocampal formation. Clearly contoured CSPG-immunoreactive PNs around interneurons occur in the

subiculum together with WFA-stained pyramidal PNs. For correlation of anatomical regions see Fig. 1D. (A) CSPG immunoreaction combined with WFA staining

showing the transition zone of the subiculum (S) to the CA1 region of the hippocampus as well as the dorsal dentate gyrus (DG). Intense CSPG immunoreaction is a

characteristic of CA1 neuropil zones. (A0) Deep pyramidal cell layer of the subiculum containing a CSPG-immunoreactive PN associated with an interneuron and

numerous WFA-stained PNs surrounding pyramidal cells (asterisks). The faint WFA label also covers axon initial segments (arrows). (B) BHABP staining of

hyaluronan combined with parvalbumin (PARV) immunoreaction showing the transition zone of the subiculum (S) to the CA1 region of the hippocampus as well as

the dorsal dentate gyrus (DG). Different sublayers can be identified by their neuropil staining especially in the DG, such as outer molecular layer (oMol), inner

molecular layer (iMol) and polymorph layer (Po). (B0) Transition zone of the subiculum and the medial CA1 region. A PARV-positive interneuron, indicated by arrow

in image (B), is located at the CA1 pyramidal cell layer. BAHBP-stained perisomatic rims can be seen around PARV-negative neurons. (B0 0) BHABP-stained

perisomatic rim associated with PARV-positive interneurons in the dentate gyrus indicated by open arrow in image (B). Scale bars: 100 mm in (A) and (B); 20 mm in

(A0), (B0) and (B0 0).

feature also of the interposed neuropil. In contrast to the motor

nucleus proper, the peritrigeminal area was characterized

by numerous intensely WFA-stained PNs (Fig. 8A, A0, B, C

and C0).

3.5. Cerebellum

In the cerebellar cortex, PNs associated with neurons located

mainly in the inner third of the molecular layer were clearly


Fig. 5. Characteristics of extracellular matrix in the basal forebrain. Cholinergic neurons detected by ChAT immunoreaction are devoid of PNs. For correlation of

anatomical regions see Fig. 1B and C. (A) The medial septum (MS)-diagonal band (DB) region is characterized by neighboured clusters of WFA-stained PNs and

ChAT-immunoreactive neurons. (A0) Higher magnification of the DB region. (B) Periventricular hypothalamic region (Pe) characterized by diffuse PNs and

interposed WFA-stained neuropil. ChAT-immunoreactive neurons are devoid of PNs. (C) Distribution pattern of hyaluronan revealed by BHABP staining. The

external part of the globus pallidus (GP) shows moderate staining intensity related to numerous PNs. ChAT-immunoreactive neurons in the ventral striatopallidal

region (VP) are intermingled with PN-associated neurons. (C0) BHABP-stained PNs in the ventral pallidum are associated with non-cholinergic neurons. Scale bars:

100 mm in (A)–(C); 20 mm in (A0) and (C0).


Fig. 6. Characteristics of extracellular matrix in the ventral thalamus. Ventral to the zona incerta (ZI) the parvalbumin immunoreactive neurons delineate the oval

reticular thalamic nucleus (Rt) located in close proximity to cerebral peduncle (cp). For correlation of anatomical regions see Fig. 1D. (A and A0) WFA-stained PNs

occur only in the ZI. (B) CSPG immunoreaction combined with WFA staining reveals double-labeling and molecular heterogeneity of the ZI. Rt is CSPG-

immunoreactive with low intensity. (C) Moderate BHABP staining of PNs is shown in ZI and Rt. Scale bars: 100 mm in (A)–(C); 20 mm in (A0).

labeled by CSPG immunoreaction (Fig. 9A). The position of

these neurons is suggestive of deep stellate cells or basket cells

(Palay and Chan-Palay, 1974). BHABP staining revealed a

perineuronal rim around PARV-immunoreactive neurons in the

inner molecular layer (Fig. 9B). PNs were not detectable with

WFA in the molecular layer and around Purkinje cells. Golgi

neurons in the granule cell layer were ensheathed by extremely

faintly WFA-binding PNs virtually not detectable by CSPG

immunoreaction (not shown). PNs of Golgi neurons could not

be differentiated from adjacent BHABP-stained matrix

components.

PNs in the cerebellar nuclei (lateral, interposed, medial)

were clearly labeled by CSPG immunoreaction (Fig. 9C and C0)and BHABP (Fig. 9D and D0) but were not stained by WFA. The

net-associated neurons were densely covered by PARV-positive

synaptic profiles (Fig. 9D0).

4. Discussion

The spatial and molecular organization of extracellular

matrix components was revealed in the present study by lectin

(WFA) staining, CSPG immunoreaction and detection of


Fig. 7. Characteristics of extracellular matrix in the substantia nigra and locus coeruleus. Dopaminergic and noradrenergic neurons detected by tyrosine hydroxylase

immunoreaction are devoid of PNs. For correlation of anatomical regions see Fig. 1A (sagittal plane) and Fig. 1F. (A) Overview showing the red nucleus (RMC)

associated with intensely WFA-stained PNs, the substantia nigra (SN) and the ventral tegmental area (VTA) crossed by the oculomotor nerve (3n). (A0) Lateral part of

the reticular substantia nigra. Clearly contoured PNs ensheath the soma, proximal parts of dendrites and the axon initial segment (arrows) around tyrosine hydroxylase

immunonegative neurons. (B, B0 and B0 0) CSPG immunoreaction combined with WFA staining reveal a large degree of double labeling of PNs in the midbrain.

Molecular heterogeneity of PNs is indicated by different proportions of red and green fluorescence. (C) Detection of hyaluronan by BHABP indicates the red nucleus

as a prevalently stained midbrain region corresponding with the distribution patterns of PNs. (C0) In the substantia nigra PNs appear as BHABP-stained perisomatic

rims around tyrosine hydroxylase-immunonegative neurons. (D) Tyrosine hydroxylase immunoreaction showing the locus coeruleus (LC) and the subcoeruleus area

in the close proximity to brainstem neurons associated with intensely WFA-stained PNs in a sagittal section. Caudal is to the right. Scale bars: 100 mm in (A), (B),

(B0), (B0 0), (C), (D); 20 mm in (A0), (C0).


Fig. 8. Characteristics of extracellular matrix in the motor trigeminal nucleus (Mo5). Cholinergic neurons were labeled by ChAT immunoreaction. The nucleus

is characterized by CSPG-immunoreactive and BHABP-stained perisomatic rims and neuropil zones. Clearly contoured WFA-stained PNs only occur in the

peritrigeminal region. For correlation of anatomical regions see Fig. 1G. (A and A0) WFA-staining reveals the motor neurons devoid of label. (B and B0) CSPG-


hyaluronan by affinity binding of BHABP in the brain of the

South American mouse opossum T. elegans. This indicated the

existence of important specific characteristics, in parallel with

features shared with many placental mammalian species. Thus,

two basic trends were identified: (i) there are clear changes

during the evolution of cortical and subcortical neuronal

systems, as revealed by the quantity of perineuronal deposition

and by the molecular properties of extracellular matrix

components and (ii) however, distinct architectural patterns

of extracellular matrix organization at the systemic and single

cell level, as well as the structural characteristics of subcellular

PN domains, appear to be conserved during mammalian

phylogeny for millions of years.

4.1. Modified versus conserved properties of perineuronal

nets

On the one hand, there emerges the picture of several

‘‘marsupial features’’ of the extracellular matrix. One feature

clearly differentiating the extracellular matrix in the brain of

South American marsupials (Thylamys, present study, Mono-

delphis, Bruckner et al., 1998) from that of placental mammals

represents a quantitative phenomenon, indicated especially by

the absence of robust PNs around parvalbumin-immunoreactive

interneurons in the neocortex and hippocampus. These PNs

only weakly accumulate CSPGs and hyaluronan, and are

virtually not detectable with WFA known to label PNs of

placental mammalian cortical interneurons with high intensity

(Hartig et al., 1992). A similar phenomenon is apparent also in

the reticular thalamic nucleus characterized by strong

accumulation of CSPGs in placental mammalian species

(Bruckner et al., 1996).

As another hallmark of the extracellular matrix in Thylamys,

clearly CSPG-immunoreactive PNs are devoid of WFA-

staining in a number of subcortical regions that are

characterized by strong WFA-binding in placental mammals,

e.g. the cerebellar nuclei (Seeger et al., 1994; Bruckner et al.,

2000). This observation confirms the view that extracellular

matrix organization in the mammalian brain can be varied not

only quantitatively but also qualitatively (Matthews et al.,

2002). There is more than ‘‘presence/lack’’ or ‘‘dominant/

faint’’; rather, the molecular composition of CSPGs is

differentially regulated, and appears to play a major role in

phylogenetic and/or functional adaptation.

On the other hand, features indicating conserved patterns of

extracellular matrix organization are expressed in different

subcortical regions in Thylamys, e.g. the red nucleus, sharing

major structural and molecular properties with regions mapped

in the rat and murine brain (Seeger et al., 1994; Bruckner et al.,

2000). Likewise, the absence of PNs around certain types of

neurons, such as cholinergic neurons in the basal forebrain

(Brauer et al., 1993; Adams et al., 2001) and aminergic neurons

immunoreactive PNs appear as perisomatic rims around ChAT-positive neurons.

double labeling. PNs in the peritrigeminal regions show molecular diversity in

Detection of hyaluronan by BHABP. The motor nucleus is intensely labeled by sta

20 mm in (A0)–(D0).

of the substantia nigra and the locus coeruleus (Hobohm et al.,

1998) also appears to be a principle conserved during

mammalian evolution.

4.2. Functional significance of phylogenetic differences in

the extracellular matrix

Although the functional role of PNs has not yet been

elucidated, more than a single function may be considered.

Their activity-dependent formation during postnatal devel-

opment (Guimaraes et al., 1990), as well as the reactivation of

ocular dominance plasticity (Pizzorusso et al., 2002) and the

induction of axonal sprouting (Corvetti and Rossi, 2005) after

enzymatic decomposition of extracellular matrix CSPGs

indicate an involvement in the regulation of PNs in neuronal

plasticity (Hockfield, 1993; Rhodes and Fawcett, 2004). The

polyanionic character of many PNs is suggestive of a role in

regulation of the ionic micromilieu in analogy to the

perinodal gap substance at the nodes of Ranvier (Bruckner

et al., 1993, 2006; Hartig et al., 1999; Horn et al., 2003).

Macromolecular matrix complexes may significantly modify

the hydration and the diffusion parameters of the extracelluar

space (Sykova, 1997). Finally, supplementing rather than

replacing other roles, a neuroprotective effect of perineuronal

matrix components is reasonable to assume (Bruckner et al.,

1999; Morawski et al., 2004).

Considering the special pattern of extracellular matrix

organization in South American marsupials, notably the

absence of robust PNs around interneurons in the cerebral

cortex, it may be concluded that the discussed functions of PNs

are not relevant in the cerebral cortex of these species.

However, since numerous PNs occurred in association with

layer 5 pyramidal cells in an area-specific distribution in the

two opossum species investigated so far, the extracellular

matrix appears to mirror a special type of cortical structure and

function in marsupials, contrasting with that of placental

mammals (Bruckner et al., 1998). To our knowledge, studies

investigating electrophysiological characteristics of the par-

valbumin-expressing interneurons have not been performed in

South American opossums. It would be interesting to know

whether these neurons belong to the fast-spiking type of

cortical interneurons, which is predominantly associated with

PNs in placental mammals (Kawaguchi et al., 1987; Kosaka

and Heizmann, 1989; Naegele and Barnstable, 1989; Hartig

et al., 1992, 1994; Celio, 1993). Regarding the possible

involvement of PNs in the regulation of synaptic plasticity,

especially the critical period-plasticity of local cortical circuits

(Hensch, 2005), the cortex of opossums may be used as an

alternative model. Studies in Monodelphis showed that massive

cross-modal cortical plasticity occurs after enucleation at early

developmental stages (Kahn and Krubitzer, 2002), whereas

such plasticity was previously demonstrated in macaque

Neuropil zones are stained with moderate intensity. (C and C0) WFA/CSPG

dicated by different proportions of red and green fluorescence. (D and D0)ining of perisomatic rims and neuropil zones. Scale bars: 100 mm in (A)–(D);


Fig. 9. Characteristics of extracellular matrix in the cerebellar cortex and deep cerebellar nuclei. Parvalbumin immunoreaction (PARV) indicates clearly labeled

Purkinje cells inclusive axonal projections in sagittal sections (for comparison see Fig. 1A). (A) CSPG/WFA double staining reveals CSPG-immunoreactive PNs in

the lower molecular layer (m) devoid of WFA staining. Asterisks indicate the Purkinje cell layer. (B) Detection of hyaluronan by BHABP shows PNs as perisomatic


monkeys (Dehay et al., 1996) and rodents (Olavarria and Li,

1995) to occur at later stages of brain development.

Acknowledgements

The authors wish to thank Mrs. Margit Schmidt for excellent

technical assistance, her last contribution to our research after

three decades of intelligent, accurate and patient professional

work. This work was supported by the Deutsche Forschungs-

gemeinschaft, grant number BR 1208/3-3 and the Interdiszi-

plinares Zentrum fur Klinische Forschung (IZKF) in the course

of the MD/PhD program at the University of Leipzig.

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Documents

Organization of brain extracellular matrix in the Chilean fat-tailed mouse opossum Thylamys elegans (Waterhouse, 1839)