Distribution of the Extracellular Matrix in the Pararubral Area of the Rat

Abstract

Previously we described similarities and differences in the organization and molecular composition of an aggrecan based extracellular matrix (ECM) in three precerebellar nuclei, the inferior olive, the prepositus hypoglossi nucleus and the red nucleus of the rat associated with their specific cytoarchitecture, connection and function in the vestibular system. The aim of present study is to map the ECM pattern in a mesencephalic precerebellar nucleus, the pararubral area, which has a unique function among the precerebellar nuclei with its retinal connection and involvement in the circadian rhythm regulation. Using histochemistry and immunohistochemistry we have described for the first time the presence of major ECM components, the hyaluronan, aggrecan, versican, neurocan, brevican, tenascin-R (TN-R), and the HAPLN1 link protein in the pararubral area. The most common form of the aggrecan based ECM was the diffuse network in the neuropil, but each type of the condensed forms was also recognizable. Characteristic perineuronal nets (PNNs) were only recognizable with Wisteria floribunda agglutinin (WFA) and aggrecan staining around some of the medium-sized neurons, whereas the small cells were rarely surrounded by a weakly stained PNNs. The moderate expression of key molecules of PNN, the hyaluronan (HA) and HAPLN1 suggests that the lesser stability of ECM assembly around the pararubral neurons may allow quicker response to the modified neuronal activity and contributes to the high level of plasticity in the vestibular system.

Full text

Distribution of the Extracellular Matrix in the Pararubral Area of the Rat
Do
´ra Szarvas,
a
Botond Gaa
´l,
a
Clara Matesz
a,b
and E
´va Ra
´cz
a,c
*
a
Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98., Debrecen H-4032, Hungary
b
Division of Oral Anatomy, Faculty of Dentistry, University of Debrecen, Nagyerdei krt. 98., Debrecen H-4032, Hungary
c
MTA-DE Neuroscience Research Group, Nagyerdei krt. 98., Debrecen 4032, Hungary
Abstract—
Previously we described similarities and differences in the organization and molecular composition of
an aggrecan based extracellular matrix (ECM) in three precerebellar nuclei, the inferior olive, the prepositus hypo-
glossi nucleus and the red nucleus of the rat associated with their specific cytoarchitecture, connection and func-
tion in the vestibular system. The aim of present study is to map the ECM pattern in a mesencephalic precerebellar
nucleus, the pararubral area, which has a unique function among the precerebellar nuclei with its retinal connec-
tion and involvement in the circadian rhythm regulation. Using histochemistry and immunohistochemistry we
have described for the first time the presence of major ECM components, the hyaluronan, aggrecan, versican,
neurocan, brevican, tenascin-R (TN-R), and the HAPLN1 link protein in the pararubral area. The most common
form of the aggrecan based ECM was the diffuse network in the neuropil, but each type of the condensed forms
was also recognizable. Characteristic perineuronal nets (PNNs) were only recognizable with Wisteria floribunda
agglutinin (WFA) and aggrecan staining around some of the medium-sized neurons, whereas the small cells were
rarely surrounded by a weakly stained PNNs. The moderate expression of key molecules of PNN, the hyaluronan
(HA) and HAPLN1 suggests that the lesser stability of ECM assembly around the pararubral neurons may allow
quicker response to the modified neuronal activity and contributes to the high level of plasticity in the vestibular
system. Ó2018 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: perineuronal net, hyaluronan, chondroitin sulfate proteoglycans, tenascin-R, link protein, vestibular system.
INTRODUCTION
The intercellular space of central nervous system (CNS)
is filled with the extracellular matrix (ECM). The most
frequently occurring ECM molecules in the CNS are the
hyaluronan (HA), lecticans (aggrecan, brevican,
neurocan and versican), tenascin-R (TN-R) and the
HAPLN1 link protein. Although the majority of these
molecules appear as a diffuse network in the neuropil,
the condensed forms may also present as they
surround the neuronal cell body, dendrites and axon
initial segment as the perineuronal net (PNN), or form
the axonal coat around the presynaptic bouton, or
associated with the node of Ranvier as nodal ECM
(Celio et al., 1998; Carulli et al., 2006; Bruckner et al.,
2008; Bekku et al., 2009; Bekku and Oohashi, 2010;
Dityatev, 2010; Frischknecht and Seidenbecher, 2012;
Lendvai et al., 2012; Blosa et al., 2013). The ECM shows
an area-dependent distribution pattern, and its molecular
and structural heterogeneity is correlated with the mor-
phological and functional properties of the neurons
(Matesz et al., 2005; Szigeti et al., 2006; Meszar et al.,
2008; Morawski et al., 2009; Gati et al., 2010; Lendvai
et al., 2012; Morawski et al., 2012; Jager et al., 2013;
Gaal et al., 2014; Gaati et al., 2014; Racz et al., 2014,
2015b; Kecskes et al., 2015). The ECM molecules are
involved in the synaptic transmission as they are the
fourth components of synaptic machinery besides the
presynaptic and postsynaptic neurons as well as the
astroglia cell (Dityatev and Schachner, 2006; Dityatev
et al., 2006; Faissner et al., 2010; Dityatev and
Rusakov, 2011; Chelini et al., 2018). In addition, the
ECM stabilizes the synaptic connections, creates a
https://doi.org/10.1016/j.neuroscience.2018.10.027
0306-4522/Ó2018 IBRO. Published by Elsevier Ltd. All rights reserved.
*Correspondence to: E
´.Ra
´cz, Department of Anatomy, Histology and
Embryology, Faculty of Medicine, University of Debrecen, Nagyerdei
krt. 98., Debrecen H-4032, Hungary. Fax: +36-52-255-115.
E-mail address: eva@anat.med.unideb.hu (E
´.Ra
´cz).
Abbreviations: AC, axonal coat; bHABP, biotinylated Hyaluronan
Binding Protein; bWFA, biotinylated Wisteria floribunda agglutinin;
BSA, bovine serum albumin; CNS, central nervous system; Cr,
cerebellum; CSPG, chondroitin sulfate proteoglycan; DAPI, 4’,6-
Diamidino-2-Phenylindole; ECM, extracellular matrix; GABA, gamma-
aminobutyric acid; GAD, glutamic acid decarboxylase; HA, hyaluronan;
HAPLN1, hyaluronan and proteoglycan link protein 1; IC, inferior
colliculus; LCN, lateral cerebellar nucleus; MAP2, microtubule-
associated protein 2; ml, medial lemniscus; MCN, medial cerebellar
nucleus; NeuN, neuronal nuclei; NDS, normal donkey serum; NGS,
normal goat serum; NRS, normal rabbit serum; PaR, pararubral
nucleus; PBS, phosphate buffered saline; PNN, perineuronal net;
pRN, parvicellular part of red nucleus; R, red nucleus; RT, room
temperature; SC, superior colliculus; scp, superior cerebellar peduncle;
TN-R, tenascin-R; WFA, Wisteria floribunda agglutinin; IV, fourth
ventricle.
NEUROSCIENCE
RESEARCH ARTICLE
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barrier against the formation of new synaptic contacts,
thereby it restricts the synaptic plasticity of adult CNS.
The molecular composition of ECM is changing parallel
to the neuronal activity in healthy brain and its modifica-
tion was described in different pathophysiological events
(Bradbury et al., 2002; Pizzorusso et al., 2002; Dityatev
and Schachner, 2003; Busch and Silver, 2007; Dityatev
and Fellin, 2008; Kwok et al., 2008; Morita et al., 2010;
Dityatev and Rusakov, 2011; Kwok et al., 2011;
Morawski et al., 2012, 2014; Sorg et al., 2016; Suttkus
et al., 2016; Bozzelli et al., 2018; Chelini et al., 2018;
Ferrer-Ferrer and Dityatev, 2018). In line with these
results it was observed that the lesion of vestibular recep-
tors and subsequent compensatory events resulted in
modification of ECM assembly in the vestibular nuclei of
brainstem suggesting the possible role of ECM in the
vestibular plasticity (Matesz et al., 2005; Deak et al.,
2012; Gaal et al., 2015a; Faralli et al., 2016). Since these
nuclei have widespread reciprocal connections with the
cerebellum, spinal cord and precerebellar nuclei, similar
alteration of ECM is expected in these structures. In order
to detect these possible changes, data on the ECM com-
position are needed in these areas. Previously we have
mapped the distribution and organization of an
aggrecan-based ECM in three precerebellar nuclei of
the vestibular neural circuits, the inferior olive (Kecskes
et al., 2015), the prepositus hypoglossi nucleus (Gaal
et al., 2015b), and the red nucleus (Racz et al., 2016)in
the rat. Besides the similarities we detected a number of
differences between the individual nuclei and their subnu-
clei associated with their specific functions in the vestibu-
lar system. These data are not yet available for the
mesencephalic pararubral area. The pararubral area is
located in the mesencephalic reticular formation, dorsolat-
eral to the red nucleus at the level of its caudal part
(Paxinos and Watson, 1998)(Fig. 1G). The GABA and
parvalbumin-positive small- and medium-sized pararubral
cells are excited by the sensorimotor cortex and terminate
on the rubrospinal neurons thereby they are involved in
the flexor motor execution (Liu et al., 2002). Although
the pararubral area is similar to the adjacent parvicellular
part of the red nucleus regarding the morphology, neuro-
chemistry, physiology, and connections, it has a unique
feature among the members of precerebellar nuclei as it
receives direct projection from the retina and involved in
the circadian rhythm regulation (Cooper et al., 1990;
Morcuende et al., 2002; Horowitz et al., 2004).
In this study we investigated the distribution and
organization of the major types of ECM molecules in the
pararubral nucleus of the rat. Using histochemical and
immunohistochemical methods, we have detected all
major ECM components in the pararubral area and the
staining pattern showed similarity, with some
differences, to the parvocellular part of the red nucleus.
EXPERIMENTAL PROCEDURES
Preparation of the rat brain for the histochemical and
immunohistochemical reactions
The protocol of the experiments was accepted by the
Animal Care Committee of the University of Debrecen,
Debrecen, Hungary and the national laws and EU
regulations (license number: 6/2017/DEMAB).
The study was performed on adult female (12–14-
week old) Wistar rats (n= 5) from Charles River
Laboratory (Strain Crl:WI), weighing from 250 to 300 g.
The animals were deeply anesthetized with
intraperitoneal injection of 10% urethane (1.3 ml/100 g
body weight; Reanal, Budapest, Hungary) and
transcardially perfused with physiological saline. The
brainstem was removed and immersed into Sainte-
Marie’s fixative (99% absolute ethanol and 1% glacial
acetic acid) for one day at 4 °C. The mesencephalon
was embedded in paraffin and sagittal sections were cut
with microtome at a thickness of 8 lm. The sections
were collected on silane-coated slides and left to dry
overnight at 37 °C. Following deparaffination, they were
rehydrated and washed in phosphate-buffered saline,
pH 7.4 (PBS) and treated with 3% H
2
O
2
dissolved in
bidistilled water for 10 min at room temperature (RT).
Histochemistry and immunohistochemistry
Detection of ECM molecules. Prior to histochemical
and immunohistochemical reactions, specimens were
blocked for 60 min at RT in 3% bovine serum albumin
(BSA) (for HA; Wisteria floribunda agglutinin, WFA;
versican), 3% BSA + 10% normal goat serum (NGS)
(for aggrecan), 3% BSA + 10% normal rabbit serum
(NRS) (for chondroitin sulfate proteoglycan, Clone Cat-
301; neurocan), 3% BSA + 10% normal donkey serum
(NDS) (for brevican, TN-R, HAPLN1). Reagents above
were dissolved in PBS.
Histochemical reactions. The distribution of HA was
detected by using biotinylated Hyaluronan Binding
Protein (bHABP; AMS Biotechnology, Abingdon, UK).
WFA histochemistry was carried out using biotinylated
Wisteria floribunda agglutinin (bWFA; Sigma-Aldrich, St.
Louis, MO, USA), a lectin that binds to N-
acetylgalactosamine residues of CSPG-
glycosaminoglycan chains and glycoproteins, as a
marker of PNNs (Hartig et al., 1992). bHABP and bWFA
were dissolved in PBS containing 1% BSA, sections were
incubated overnight at 4 °C. Visualization of labeling was
performed by incubating the samples with Streptavidin
AlexaFluor 555 (Life Technologies, Carlsbad, CA, USA)
for 1 h, diluted in 1:1000, in PBS.
Immunohistochemical reactions. Slides were
incubated in the following primary antibodies: rabbit
polyclonal anti-aggrecan (Merck Millipore, Billerica, MA,
USA), mouse monoclonal anti-chondroitin sulfate
proteoglycan (Clone Cat-301, Sigma-Aldrich), mouse
monoclonal anti-versican (12C5; Developmental Studies
Hybridoma Bank, DSHB, Iowa City, IA, USA), mouse
monoclonal anti-neurocan (1F6; DSHB), sheep
polyclonal anti-brevican (R&D Systems, Minneapolis,
MN, USA), goat polyclonal anti-TN-R (R&D Systems),
goat polyclonal anti-HAPLN1 (R&D Systems). For better
antigen exposure of aggrecan, Cat-301, versican and
brevican molecules, sections were digested with
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Fig. 1. (A, B) Distribution of hyaluronan reaction (red) in the pararubral nucleus. NeuN reaction (green) detects the neurons. Yellow arrow in (B)
shows thin, faintly stained perineuronal net. (C–F) Distribution of Wisteria floribunda agglutinin (WFA) reaction (red) in the pararubral nucleus. NeuN
reaction (green) in (C–F) detects the neurons. MAP2 immunostaining (green) in (F) labels dendrites (white arrowheads). Schematic drawing in (G)
point to the position of the pararubral nucleus in the mesencephalon. SC: superior colliculus; IC: inferior colliculus; Cr: cerebellum; IV: fourth
ventricle; scp: superior cerebellar peduncle; R: red nucleus; PaR: pararubral nucleus; ml: medial lemniscus. (For interpretation of the referencesto
color in this figure legend, the reader is referred to the web version of this article.)
D. Szarvas et al. / Neuroscience 394 (2018) 177–188 179
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chondroitinase ABC (0.02 U/ml; Sigma-Aldrich) in specific
TRIS sodium-acetate buffer, pH 8 for 1 h at 37 °C.
Primary antibodies were diluted in PBS and 1% BSA
+ 3% NGS (aggrecan), 1% BSA (versican), 1% BSA
+ 3% NRS (Cat-301, neurocan), 1% BSA + 3% NDS
(brevican, TN-R, HAPLN1), overnight at 4 °C. The
incubation was followed by repeated rinsing in PBS.
More details of primary reagents are reported in Table 1.
The following secondary antibodies were used: goat-
anti-rabbit IgG AlexaFluor 555 (Life Technologies)
(aggrecan), rabbit-anti-mouse IgG AlexaFluor 555 (Life
Technologies) (Cat-301, versican, neurocan), donkey-
anti-sheep IgG AlexaFluor 555 (Life Technologies)
(brevican), and donkey-anti-goat IgG AlexaFluor 555
(Life Technologies) (TN-R, HAPLN1), for 1 h, all diluted
in 1:1000, in PBS.
Semiquantitative assessment of histochemical and
immunohistochemical reactions. After performing all
ECM reactions, slides containing the identical sagittal
sectional levels were selected from three animals.
Staining intensity of ECM reactions was evaluated on the
images recorded with the microscope using the same
settings for each section. Applying the same computer
screen we used a five-grade scaling (: no staining, +:
weak staining, ++: moderate staining, +++: strong
staining, ++++: very strong staining). The subjective
grading was performed independently by two authors (E
´.
R., B.G.) and checked by the others (C.M., D.Sz.).
Presence of axonal coats was evaluated accordingly:
‘: no ACs’ or ‘+: presence of ACs’ (Table 3).
Double fluorescent labeling. Double fluorescent
labeling was made using neuronal nuclei (NeuN),
neurofilament or microtubule-associated protein 2
(MAP2) antibodies in combination with specific ECM
markers. The following fluorescent labelings were
combined: rabbit polyclonal anti-NeuN (Merck Millipore)
+ bHABP, bWFA, versican, brevican, neurocan, TN-R
or HAPLN1; mouse monoclonal anti-NeuN (Merck
Millipore) + aggrecan; rabbit polyclonal anti-
neurofilament (Sigma-Aldrich) + versican; rabbit
polyclonal anti-MAP2 (Merck Millipore) + bWFA, Cat-
301, brevican or HAPLN1 (Table 2).
Prior to the incubation with NeuN, neurofilament or
MAP2 primary antibodies, specimens were blocked for
60 min at RT in 3% BSA + 10% NDS (for rabbit anti-
NeuN, rabbit anti-neurofilament, rabbit anti-MAP2), in
3% BSA + 10% NGS (for mouse anti-NeuN). Primary
antibodies were diluted in PBS with 1% BSA + 3%
NGS (for mouse anti-NeuN) and 1% BSA + 3% NDS
(for rabbit anti-NeuN, rabbit anti-neurofilament, rabbit
anti-MAP2). Visualization of reactions was by goat anti-
mouse IgG AlexaFluor 488 (mouse anti-NeuN; Life
Technologies) or donkey anti-rabbit IgG AlexaFluor 488
(rabbit anti-NeuN, rabbit anti-neurofilament, rabbit anti-
MAP2; Life Technologies).
Slides were coverslipped with ProLongÒDiamond
Antifade Mountant with DAPI (Life Technologies).
Images were recorded using Olympus CX31
epifluorescent light microscope with DP74 digital
Table 1. Probe, lectin and primary antibodies used for detection of ECM molecules
bHABP
a
bWFA
b
Anti-aggrecan CAT-301
c
Anti-
versican
Anti-
neurocan
Anti-brevican Anti-tenascin-R HAPLN1
d
Supplier,
cat. No.
AMS
Biotechnology;
AMS.HKD-BC41
Sigma-
Aldrich;
L1516
Merck Millipore;
AB1031
Sigma-
Aldrich;
MAB5284
DSHB; 12C5 DSHB; 1F6 R&D Systems;
AF4009
R&D Systems; AF3865 R&D Systems;
AF2608
Species of
origin,
type
Recombinant
human versican
G1 domain
expressed in
E. coli; biotinylated
Lectin
isolated from
Wisteria
floribunda;
biotinylated
Rabbit; polyclonal,
IgG
Mouse;
Monoclonal,
IgG1
Mouse;
monoclonal,
IgG1
Mouse;
monoclonal,
IgG1
Sheep; polyclonal,
IgG
Goat; polyclonal, IgG Goat; polyclonal, IgG
Immunogen – – GST fusion protein
containing amino
acids 1177–1326
of mouse aggrecan
Feline spinal
cord fixed
gray matter
Hyaluronate-
binding
region of
human
versican
PBS-soluble
CSPGs from
rat brain
Mouse myeloma cell
line NS0 derived
recombinant human
Brevican Asp23
Pro911
mouse myloma cell line
NS0-derived
recombinant human
tenascin-R isoform 1,
Glu34-Phe1358
mouse myeloma cell
line NS0-derived
recombinant human
HAPLN1, Asp16-
Asn354
Dilution 1:100 1:500 1:500 1:100 1:100 1:100 1:100 1:300 1:300
a
Biotinylated Hyaluronan Binding Protein.
b
Biotinylated Wisteria floribunda agglutinin.
c
Anti-chondroitin sulfate proteoglycan Clone CAT-301.
d
Hyaluronan and Proteoglycan Link Protein 1.
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camera and processed by Photoshop CS4 v11.0 (Adobe
Systems Inc., San Jose, CA, USA), with minimal
adjustments of contrast and background.
RESULTS
All the ECM reactions studied were positive in the
pararubral area. The staining was detected
predominantly in the neuropil, whereas the presence of
characteristic forms of condensed ECM was variable
with the different reactions.
The HA appeared mostly in diffuse staining in the
neuropil, showing weak intensity. The PNN was
sporadically observed, the other forms of condensed
ECM were not recognizable (Fig. 1A, B). The WFA
staining revealed that some neurons, mostly medium
sized, were surrounded by PNN. WFA-positive PNNs
were rarely shown around the small-sized neurons
(Fig. 1C–F). In case of PNN bearing neurons, the
labeling was also detected around the dendrites as
shown with the combination of WFA and MAP2
reactions (Fig. 1F). The small, ring-like forms of the
condensed ECM, the axonal coats, were also
recognizable. The staining intensity of the neuropil was
the strongest with the WFA among the ECM reactions
studied. The aggrecan reaction was similar to the WFA
staining, regarding the condensed forms of ECM. On
the basis of morphology, both the WFA and aggrecan
reactions revealed the three types of PNNs (Wegner
et al., 2003; Jager et al., 2013). One part of neurons
was surrounded by 1–2 mm clearly contoured PNNs
described as classical or robust type (Fig. 2F), the other
parts were enwrapped by a thin, faintly stained sheath
of ECM (Fig. 2E). The third type of PNNs were not clearly
contoured, the thickness of the lattice-like structure was
about 3–5 mm(Fig. 2C, D). When the aggrecan reaction
labeled the PNN, the labeling was followed into the peri-
dendritic area (Fig. 2B). The staining of neuropil was less
intense than with WFA reaction. Using the versican anti-
body resulted in the characteristic dot like appearance
of versican (Bekku et al., 2009; Bekku and Oohashi,
2010; Racz et al., 2014; Gaal et al., 2015b; Kecskes
et al., 2015). A partial overlap was seen with the combina-
tion of versican and neurofilament reactions indicating the
presence of versican-positive dots around the nodes of
Ranvier (Fig. 2I). Other forms of ECM organization were
not recognizable with versican staining (Fig. 2G–I). The
brevican staining did not reveal PNNs, whereas the other
forms of condensed ECM, the dot like and small, ring like
structures were frequently recognizable indicating the
presence of this molecule at the nodes of Ranvier and
axonal coats. Labelings were shown around the den-
drites, as demonstrated by the MAP2 staining (Fig. 3B,
C). Among the dot and ring like forms of ECM, a weak dif-
fuse staining was observed in the neuropil with brevican
antibody (Fig. 3A–E). Similarly to brevican reaction, the
neurocan labeling indicated its absence in PNNs. In con-
trast, the neuropil was more intensely labeled, whereas
the structures representing the nodal ECM and axonal
coats were rarely observed (Fig. 3F, G). The overall inten-
sity of the tenascin-R immunostaining was very weak,
none of the characteristic forms of ECM were detected
in the lightly stained neuropil (Fig. 4A, B). The HAPLN1
reaction showed patch-like, irregular appearance in the
perisomatic and peridendritic area, however the charac-
teristic form of PNN was rarely identified (Fig. 4D, E).
Axonal coats were also occasionally visible. In the neu-
ropil, darker and lighter HAPLN1-positive irregular areas
were recognizable without showing any regional distribu-
tion in the pararubral area (Fig. 4C–H).
DISCUSSION
Using histochemistry and immunohistochemistry, we
have described for the first time the presence of major
ECM components, the hyaluronan, aggrecan, versican,
Table 2. Primary antibodies used for double labeling with ECM molecules
Anti-NeuN
a
Anti-NeuN
a
Anti-neurofilament Anti-MAP2
b
Supplier, cat. No. Merck Millipore;
ABN78
Merck Millipore;
MAB377
Sigma-Aldrich;
N4142
Merck Millipore;
AB5622
Species of origin,
type
Rabbit;
polyclonal, IgG
Mouse;
monoclonal, IgG
Rabbit;
polyclonal, IgG
Rabbit;
polyclonal, IgG
Immunogen GST-tagged recombinant protein
corresponding to mouse NeuN
Purified cell nuclei
from mouse brain
Purified neurofilament 200
from bovine spinal cord
Purified Microtubule-
associated protein from rat
brain
Dilution 1:1000 1:100 1:80 1:500
a
Neuronal nuclei.
b
Microtubule-associated protein 2.
Table 3. Semiquantitative assessment of the staining intensity of
perineuronal nets (PNN) and neuropil, and presence of axonal coats
(ACs) in the pararubral area of the mesencephalon
PNN Neuropil ACs
Hyaluronan + ++ 
WFA +++ ++++ +
Aggrecan ++++ +++
*
+
Versican #
Brevican +++ +
Neurocan +# +
Tenascin-R +
HAPLN1 +/++
*
+
The staining intensity of perineuronal net and neuropil was scored as :no
staining, +: weak staining, ++: moderate staining, +++: strong staining, ++
++: very strong staining. #: Presence of heavily stained dots in the less intense
neuropil.
ACs: +: presence of ACs; : no ACs.
*
Regional differences in the intensity of reaction.
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neurocan, brevican, TN-R, and the HAPLN1 link protein in
the mesencephalic precerebellar nucleus, the pararubral
area. The most common form of the aggrecan based
ECM was the diffuse network in the neuropil, but each
type of the condensed forms was also recognizable.
The presence of the individual ECM molecules and the
staining intensity of reactions were different in these
compartments of ECM.
Fig. 2. (A–F) Distribution of aggrecan immunoreactivity (red) in the pararubral nucleus. NeuN reaction (green) in (A) and (C–F) detects the neurons.
MAP2 immunostaining (green) in (B) labels dendrites (white arrowheads). Purple arrow in (F) shows robust type of perineuronal net (PNN). The
white arrows in (C, D) indicate lattice-like PNNs, the yellow arrow in (E) shows faintly stained PNN, respectively. (G–I) Distribution of versican
immunoreactivity in the pararubral nucleus. NeuN reaction (green) in (G, H) detects the neurons. Neurofilament immunoreactivity (green) in (I)
represents the axons. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
182 D. Szarvas et al. / Neuroscience 394 (2018) 177–188
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Expression pattern of ECM molecules in the
pararubral area
Characteristic PNNs were only recognizable with WFA
and aggrecan staining around some of the medium-
sized neurons, whereas the small cells were rarely
surrounded by a weakly stained PNN. Besides the WFA
and aggrecan-positive PNNs, the HA and HAPLN1
antibody showed occasionally patch-like staining around
Fig. 3. (A–E) Distribution of brevican immunoreactivity in the pararubral nucleus. NeuN reaction (green) in (A, D and E) detects the neurons. MAP2
immunostaining (green) in (B, C) labels dendrites (white arrowheads). Yellow arrowheads in D show axonal coats. (F-G) Distribution of neurocan
immunoreactivity in the pararubral nucleus. NeuN reaction (green) detects the neurons. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
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the neuronal cell bodies, but no one characteristic form of
PNN was identifiable. These findings indicate the
moderate expression of key molecules of PNN, the HA
and HAPLN1 (Carulli et al., 2010; Kwok et al., 2011), in
the pararubral area. One of the important functions of
the PNN is to restrict the neural plasticity and instead sup-
port the synaptic stability (Happel and Frischknecht,
2016; Bozzelli et al., 2018). The reduced PNN has been
linked to improved cognitive flexibility (Morellini et al.,
2010) and the lack of link protein Crtl1 results in persistent
plasticity in the visual system (Carulli et al., 2010).
Consistent with these results we assume that the lesser
stability of ECM assembly around the pararubral PNNs
may allow quicker response to the modified neuronal
Fig. 4. (A, B) Distribution of TN-R immunoreactivity in the pararubral nucleus. NeuN reaction (green) detects the neurons. (C–H) Distribution of
HAPLN1 immunoreactivity in the pararubral nucleus. NeuN reaction in (C, F–H) (green) detects the neurons. MAP2 immunostaining (green) in (D,
E) labels dendrites (white arrowheads). Yellow arrowheads in (E, H) show axonal coats. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
184 D. Szarvas et al. / Neuroscience 394 (2018) 177–188
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activity and contributes to the high level of plasticity in the
vestibular system.
The presence and molecular composition of PNNs is
highly variable in different regions of CNS, as well as
species and age differences have also been reported
(Wang and Fawcett, 2012; Sorg et al., 2016; Bozzelli
et al., 2018). PNNs were most frequently recognizable
around fast-spiking, parvalbumin-positive GABA-ergic
interneurons in different parts of the brain which are asso-
ciated with low plasticity (Hartig et al., 1992; Bruckner
et al., 1999; Schuppel et al., 2002; Dityatev et al., 2007;
Kwok et al., 2011). Although the neurons of the pararubral
area were also positive for the parvalbumin and GAD (Liu
et al., 2002) and physiological studies also suggested
their GABAergic nature of the pararubral neurons, the
PNNs are only present around some of the pararubral
neurons. This finding is similar to the neuron specific
expression pattern of PNNs in the vestibular nuclei and
related structures in the rat brainstem (Racz et al.,
2014; Gaal et al., 2015a; Racz et al., 2016) and in the
parts of the spinal cord related to the motor function
(Jager et al., 2013). Here, the PNNs are dominant around
the large, excitatory projection neurons, whereas the
majority of small, inhibitory interneurons were not sur-
rounded by ECM. The rare occurrence of PNN around
the inhibitory interneurons seems to be a common feature
of CNS structures which are engaged in the motor coordi-
nation and execution. Accordingly, the majority of small,
inhibitory interneurons are not surrounded by ECM in
the vestibular nuclei and related structures in the rat brain-
stem (Racz et al., 2014; Gaal et al., 2015a; Racz et al.,
2016) and in the parts of the spinal cord related to the
motor function (Jager et al., 2013). On the other hand,
the large, excitatory projection neurons are surrounded
by the PNN in these areas in contrast to the weak or miss-
ing PNNs around the majority of pyramidal cells in the
secondary motor cortex (Alpa
´r et al., 2006). On the basis
of these findings it is tempting to speculate that this
unique organization of the PNN is related to the motor
function of CNS, which needs a continuous balance con-
trol during the body displacement.
The axonal coats stained with aggrecan, brevican and
HAPLN1 antibodies as it was shown in other parts of the
CNS including the vestibular areas of brainstem
(Bruckner et al., 2008; Frischknecht and Seidenbecher,
2012; Lendvai et al., 2012; Blosa et al., 2013; Jager
et al., 2013; Lendvai et al., 2013; Morawski et al., 2014;
Racz et al., 2014; Gaal et al., 2015a; Racz et al., 2016).
The nodal ECM was observed most frequently with the
versican and brevican reactions indicating the presence
of essential molecules of node of Ranvier in the pararu-
bral area (Bekku et al., 2009; Bekku and Oohashi, 2010).
The neuropil was stained with all ECM reactions
studied. The most intense staining was revealed with the
WFA reaction, whereas it was faintly labeled with TN-R.
Comparison between the organization of ECM in the
pararubral area and parvicellular part of red nucleus
(pRN)
Using histochemical and immunohistochemical reactions,
all the ECM molecules studied were present both in the
pararubral area and in the previously studied pRN (Racz
et al., 2016). The expression pattern of ECM was mostly
similar with some differences in the two areas.
Contrary to what its name suggest, the pRN contains
large-sized neurons besides the dominant small- and
medium-sized cells. Since the large neurons are not
present in the pararubral area, in the following part of
discussion the comparison refers to the small- and
medium-sized neurons. Characteristic PNNs were
observed with WFA and aggrecan stainings in both
areas, whereas the HA, brevican, neurocan, TN-R, and
HAPLN1 reactions showed pericellular positivity only
around the small- and medium-sized cells in the pRN
(Racz et al., 2016). The similarity in the ECM staining pat-
tern may be related to their common afferent connections
from the somatosensory cortex and cerebellum and effer-
ent projection to the rubrospinal neurons. The neurons of
sensorimotor cortex and cerebellum excite the GABAer-
gic neurons of pararubral area and pRN (Toyama et al.,
1968; Brown, 1974; Gwyn and Flumerfelt, 1974;
Bernays et al., 1988; Oka, 1988; Billard et al., 1991;
Ruigrok and de Zeeuw, 1993) which inhibit the reticu-
lospinal neurons in the magnocellular part of the red
nucleus. In case of the cerebellum, significant overlap
with some differences were revealed in the origin of pro-
jection fibers by the application of Phaseolus vulgaris leu-
coagglutinin or biotinylated dextran amine into small
areas cerebellar nuclei in the rat (Tenue et al., 2000).
Thus, the pararubral area received fibers only from the
medial cerebellar nucleus (MCN) and all subdivisions of
the lateral cerebellar nucleus (LCN), whereas the labeled
terminals detected in the pRN arrived from all cerebellar
nuclei including their subdivisions. In addition, the density
of labeled cerebellar fibers and terminals were different in
the pararubral area and pRN. Accordingly, the projection
of MCN was stronger in the pararubral area compared
to the density of terminals in the pRN. In case of LCN,
stronger projection was observed from its caudal part to
the pararubral area, whereas the pRN was more abun-
dantly supplied by the rostral part of the LCN. Although
the experiments revealed significant overlap in the termi-
nation areas, the functional differences of the cerebellar
nuclei indicate that the pararubral area is involved mostly
in the visuomotor and vestibular activity due to its stronger
connection with the MCN. The visuomotor function of the
pararubral area was suggested by its polysynaptic projec-
tions to orbicularis oculi muscle (Morcuende et al., 2002).
Moreover, the pararubral nucleus receives substantial
projection form the retina (Cooper et al., 1990) and by
its indirect connection with the suprachiasmatic nucleus
it plays a role in the circadian rhythm regulation
(Horowitz et al., 2004). To the best of our knowledge, nei-
ther the visuomotor of the pRN nor its participation in the
regulation of circadian rhythm was reported.
CONCLUSIONS
Similarly to our previous findings on the mesencephalic
vestibular structure, the parvicellular part of the red
nucleus, we have demonstrated the presence of
aggrecan based ECM in the adjacent, functionally
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related pararubral area. In spite of their common neuronal
architecture, we have found some differences in the ECM
expression pattern related probably to the unique
connection and function of the pararubral area in the
visuomotor system. Our findings support the generally
accepted view that the structural and molecular
organization of the ECM shows regional variation in the
CNS.
ACKNOWLEDGMENT
The authors thank Ms. Timea Horvath for skillful technical
assistance. Grant Sponsors from Hungarian Scientific
Research Fund (OTKA K115471), Hungarian Academy
of Sciences Fund (MTA-TKI 11008).
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
ROLE OF AUTHORS
All authors had full access to all the data in the study and
take responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: E.R., D.Sz.
Acquisition of data: D.Sz; B.G., E.R.
Analysis and interpretation of data: D.Sz, B.G., E.R.,
K.M.
Drafting of the manuscript: E.R.
Critical revision of the manuscript for important
intellectual content: K.M., B.G.
Statistical analysis: -
Obtained funding: E.R., K.M.
Administrative, technical, and material support: E.R.,
B.G., K.M.
Study supervision: E.R.
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(Received 11 July 2018, Accepted 15 October 2018)
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