Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Supplementary Material and Methods
Animals
Generation of tn-r
-/-
mice has been described [1]. The tn-r
-/-
mice (n = 27) used for the organotypic
slice cultures derived from heterozygous breeding pairs on the C57BL/6 background and C57BL/6
wild-type mice were used as control animals (n =30), as appearance of PNs in cultures from these
animals was not different from those in cultures of tn-r
+/+
mice obtained from the heterozygous
breeding. Dissociated cell cultures were obtained from mice derived from heterozygous breedings. In
animals obtained from heterozygous parents, the tn-r
genotyping
and
confirmed
by
TN-R
-/-
and tn-r
+/+
genotypes were detected by PCR
immunocytochemistry
(see
Cytochemistry
and
immunocytochemistry). No difference was seen between cultures of male and female mice.
All animals used in this study were treated in agreement with the German law on the use of
laboratory animals, following the ethical guidelines of the laboratory animal care and use committee at
the University of Leipzig and the Food and Veterinary Office in a governmental body for Social
Affairs, Family, Health and Consumer Protection in Hamburg.
Preparation of organotypic slice culture
Two- to five-day-old mice (P2-5) were sacrificed by decapitation. Brains were removed from the skull
and briefly washed in sterile-filtered, ice-cold Ringer solution containing (in mM): 2.5 KCl, 2 CaCl2, 1
MgCl2, 260 D-glucose, 26 NaHCO3, 1.25 NaH2PO4, 2 Na-pyruvate, 3 myo-inositol, 1 kynurenic acid,
pH 7.4. Brains were then embedded in agar (1.5%, gelling temperature 34-38°C; Serva, Heidelberg,
Germany) and cut into slices (350µm) in the frontal plane with a vibrating microtome (Vibratome
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
3000, TPI, St. Louis, MO, USA) in Ringer solution oxygenated with Carbogen gas (95% O2 / 5% CO2)
at 4°C.
The static slice culture method [2] was used as described [3, 4]. Slices were placed on Millicell
CM membranes (Millipore, Billerica, MA, USA) in six-well plates (Greiner Bio-One GmbH,
Frickenhausen, Germany). Culture medium (1 ml) was added to each well, and the cultures were
incubated at 36.5°C for 3 weeks in a humified atmosphere containing 5% CO2. The standard culture
medium consisted of 71% DMEM/HAM’S F-12, 24% horse serum (Biochrom AG, Berlin, Germany),
1.5% HEPES (unless specified otherwise all reagents were from Sigma-Aldrich, Munich, Germany)
supplemented with 2% D-glucose (200g/l), 1% L-glutamine (200mM) and 0.5% gentamycin (10mg/ml,
Biochrom). The medium was changed 3 times per week.
Organotypic slice co-cultures
To investigate the influence of organotypic slice cultures from tn-r +/+ mice on the formation of PNs in
the organotypic slice cultures from tn-r
-/-
mice, slices of both genotypes were placed on the same
culture membrane in two modes: 1) One tn-r
+/+
-/-
slice was surrounded at a distance (1-2 mm) by 4 tn-r
slices to allow diffusible factors to spread via the culture medium; 2) slices of both genotypes were
placed on the membrane with overlapping areas of the cortical hemispheres (Fig. 1), thereby creating a
zone of direct tissue contact.
Preparation of monovalent WFA
Five mg WFA (Sigma L-8258) were dissolved in 2 ml 0.75 M Tris-HCl pH 8.8. After addition of 80
mg DTT and incubation at room temperature for 20 min sulfhydryl residues were blocked by reaction
2
Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
with 160 mg iodoacetamide in the dark for 20 min at room temperature. Monovalent WFA was purified
by desalting on NAP25 columns equilibrated with PBS. Representative results of the preparations are
presented in Suppl. Fig. 6.
Western blotting for TN-R detection
Brains of tn-r
-/-
and a control tn-r
+/+
mice were used. Animals were anesthetized with carbon dioxide
and decapitated. Brains were removed immediately and quick-frozen in liquid nitrogen. Frozen brains
were cut coronally on dry ice into 2 mm sections and the cortex was dissected out from corresponding
cortical areas from each genotype slice and homogenized in lysis buffer [20 mM Tris-HCl, pH 7.4, 2
mM MgCl2, 150 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 5% glycerol, 1% NP-40, 1mM AEBSF, 1 mM
DTT, 2 mM EDTA, 2 mM EGTA, 2 μg/ml leupeptin, Complete Protease Inhibitor Cocktail (Roche,
ratio of tissue to buffer: 1:5)]. After centrifugation (10.000 x g, 15 min, 4°C) protein content was
determined via Bradford assay. For SDS-PAGE 30 µg protein per lane was applied.
Organotypic slice culture supernatants from wild-type slices were collected after 10 days of
incubation. AlbuminOUTTM columns (G-Biosciences, St. Louis, MO, USA) were used according to the
manufacturer´s protocol to remove albumin from the supernatant.
Brain homogenates and organotypic slice culture supernatants were separated via SDS-PAGE
(8% polyacrylamide), blotted onto PVDF membrane and analyzed by immunostaining with mouse antitenascin-R monoclonal antibody 619 (1 µg/ml, R&D Systems, Wiesbaden, Germany, [5]) and sheep
anti-non-immune mouse IgG linked to peroxidase (GE Healthcare, Freiburg, Germany).
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Cytochemistry and immunocytochemistry on organotypic slice cultures
Cultures were fixed on the CM membranes with 4% paraformaldehyde in 0.1 M phosphate buffer (PB,
pH 7.4) containing 2% saccharose for 24 h at 4°C. The slices were then rinsed 3 times in Tris-buffered
saline (TBS, pH 7.4) for 20 min each and processed as whole-mounts for cytochemistry and
immunocytochemistry. PNs were detected using the biotinylated lectin WFA (20 µg/ml, Bio-WFA L1766, Sigma-Aldrich; [6]) recognizing N-acetylgalactosamine on glycosaminoglycan side chains
(GAG) of the aggrecan core protein [7]. To visualize the development of different subpopulations of
neurons in relation to the distribution patterns of extracellular matrix components, the WFA staining
was combined with the immunocytochemical detection of parvalbumin (PARV) typically expressed in
many PN-associated neurons [6] and of choline acetyltransferase (ChAT) to detect cholinergic
forebrain neurons which are devoid of PNs in different species [8-10] and which develop robustly in
organotypic slice cultures from basal forebrain [3, 11, 12].
The cultures were incubated in blocking solution containing 5% normal serum related to the
secondary antibodies in TBS and 0.3% Triton-X 100 for 60 min at room temperature. For double
immunofluorescence labelling the first staining cocktail consisted of Bio-WFA and a monoclonal
antibody to PARV (1:400, PV-28, Swant, Marly, Switzerland) or a polyclonal goat anti-ChAT antibody
(1:200, AB144P, Millipore) in TBS containing 5% blocking serum and 0.1% Triton-X 100, applied
overnight at room temperature. After rinsing in TBS, a second cocktail was applied that consisted of
carbocyanine (Cy2, Cy3)-tagged streptavidin (20 µg/ml, Dianova, Hamburg, Germany) and
carbocyanine-tagged goat anti-mouse IgG or donkey anti-goat IgG (20 µg/ml, Dianova) for 60 min at
room temperature.
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
To confirm the genotype of the cultures, the monoclonal antibody 619 to TN-R (1:5 diluted
hybridoma culture supernatant; [13], recognizing its protein backbone (Xiao et al. 1996)) was used in
double- or triple-labelling experiments. TN-R immunoreactivity was visualized with carbocyaninetagged goat anti-mouse IgG (20µg/ml, Dianova).
After staining, the cultures were extensively washed with TBS, briefly rinsed in distilled water,
mounted on fluorescence-free slides and embedded with Aqua-Poly/Mount medium (Polysciences
Europe, Eppelheim, Germany).
Preparation of dissociated hippocampal cell cultures
Dissociated cell cultures were prepared as described [14]. Hippocampi of 1- to 3-day-old mice were
isolated, cut into small pieces and treated with trypsin (6 mg/1.8 ml) and DNase I (1.5 mg/1.8 ml) in
Ca2+ and Mg2+ free Hanks’ balanced salt solution. After mechanical dissociation, cells were plated into
cloning cylinders, 6 mm in diameter, at a density of 1500 cells/mm2 on glass (Assistent, Sondheim,
Germany) or plastic (Eppendorf, Hamburg, Germany) coverslips coated with poly-L-lysine (100
g/ml) and Matrigel (20 g/ml; Becton Dickinson, Bedford, MA, USA). Cultures were maintained for
up to three weeks in defined Neurobasal-A medium (Invitrogen) supplemented with 2% B-27, Lglutamine (0.5 mM), b-FGF (10 ng/ml), and from day 3 in vitro with cytosine--arabinofuranoside (2.5
M). Half of the culture medium was replaced every second day.
Cytochemistry and immunocytochemistry with dissociated cell cultures
Cells were briefly washed in phosphate-buffered saline, pH 7.3, and fixed in 4% formaldehyde at 37°C
for 5 min and kept in this fixative at +4°C for several days. Before staining, fixed cultures were rinsed
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
three times in Tris-buffered saline (pH 7.4) for 20 min each. After blocking in TBS containing 5%
normal goat serum and 0.3 % Triton-X 100 for 1 h, cells were incubated with cocktails of 2-3 primary
reagents, including Bio-WFA (20 µg/ml, Sigma-Aldrich) and the following antibodies: monoclonal
mouse anti-neuron-specific nuclear protein, NeuN (Chemicon, Temecula, California, USA, 1:100),
polyclonal rabbit anti-parvalbumin (PV-28, Swant, 1:250), polyclonal rabbit anti-vesicular GABA
transporter (VGAT; Synaptic Systems, Göttingen, Germany, 1:300), polyclonal rabbit anti-aggrecan
antibodies (Chemicon, AB1031, 1:50) and monoclonal mouse anti-tenascin-R (monoclonal antibody
596, 1:1, [15]). The primary reagents were diluted in TBS containing 5% normal goat serum and 0.1 %
Triton-X100. In experiments, where live cells were treated with primary antibodies to aggrecan to
visualize incorporation of antibodies into PNs, only the corresponding secondary antibodies were
applied to the fixed cultures. After incubation overnight at room temperature, cells were rinsed three
times with TBS and reacted with a cocktail containing the carbocyanine-tagged secondary reagents
(Cy3 streptavidin, Cy2 goat anti-mouse IgG and Cy5 goat anti-rabbit IgG, 20µg/ml, Dianova) for 2
hours at room temperature. Cells were washed with TBS, either embedded using Aqua-Poly/Mount
(Polysciences Europe) or dehydrated through graded concentrations of ethanol, dipped into toluene,
and mounted on fluorescence-free slides with Entellan® (Merck, Darmstadt, Germany).
Quantification of perisomatic GABAergic synaptic profiles and WFA-labelled PNs
The quantification was performed as described [14] using five tn-r +/+ cultures and five tn-r -/- cultures
maintained for 16-20 days in vitro and stained with WFA (Cy3) and VGAT antibody (Cy2). WFAlabelled neurons showing a basket cell-like morphology (i.e. large and multipolar cells) were selected
for image analysis (control cultures, n=25 neurons; cultures from tn-r
-/-
mice, n=15 neurons). Five
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
planes of images were selected for the analysis of each cell from z-stacks of images, which covered the
total thickness of neuronal somata: bottom (first section on which the soma was visible), near bottom
(1-3 optical sections above the bottom), periphery (an optical section with the maximal soma diameter),
near apex (1-3 optical sections below the apex), and apex (last section on which the soma was visible).
If clearly identifiable, the axon initial segment was also analyzed.
Image data acquired using a Zeiss Laser Scanning Microscope (LSM 510, Zeiss, Göttingen,
Germany) under constant parameters were transformed into 8 bit two channel red/green information. A
custom made Delphi 6 (Borland Inc., Rockville, MD, USA) program was used for analysis. Cellular
borders were marked manually using each selected image. Grey levels of both channels (red and green,
representing the intensity values of fluorescence) were measured for pixels in contact with the analyzed
line. If channel data of a single pixel were beyond half range limits, the dot was counted into the group
of pixels with signal in only one channel (red, green) or no staining. Two-way ANOVA (with synaptic
area in subdomains/planes of image as a repeated measure) was used to compare the distribution of
synaptic contacts in cultures from tn-r -/- and tn-r +/+ mice.
Microscopic examination
Qualitative evaluation of stained cultures were performed with a Zeiss Axioplan fluorescence
microscope equipped with appropriate filter combinations for red fluorescent dye Cy3 (filter set no. 15)
and for green fluorescent dyes Cy2 and Alexa-488 (filter set no. 09). Labelling was further examined
with a Zeiss Laser Scanning Microscope (LSM 510). An argon laser (488 nm) was used for
fluorescence detection of Cy2 and Alexa-488, and a helium/neon laser for Cy3 (543 nm) and Cy5 (633
7
Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
nm) . Photoshop 9.0 (Adobe Systems, Mountain View, CA, USA) was used to process the confocal
images with minimal alterations to the brightness, sharpness, colour saturation and contrast.
References
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Shrager P., Montag D., et al. 1999 Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased
axonal conduction velocities in the CNS. Journal of Neuroscience 19(11), 4245-4262.
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Stoppini L., Buchs P.A., Muller D., N1-Department of Pharmacology C.M.U.G.S.L. 1991 A simple method for
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Brückner G., Grosche J. 2001 Perineuronal nets show intrinsic patterns of extracellular matrix differentiation in
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Härtig W., Brauer K., Brückner G. 1992 Wisteria floribunda agglutinin-labelled nets surround parvalbumincontaining neurons. Neuroreport 3(10), 869–872.
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Giamanco K.A., Morawski M., Matthews R.T. 2010 Perineuronal net formation and structure in aggrecan
knockout mice. Neuroscience 170(4), 1314–1327. (doi:10.1016/j.neuroscience.2010.08.032).
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Bruckner G., Brauer K., Hartig W., Wolff J.R., Rickmann M.J., Derouiche A., Delpech B., Girard N., Oertel W.H.,
Reichenbach A. 1993 Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain
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Adams I., Brauer K., Arélin C., Härtig W., Fine A., Mäder M., Arendt T., Brückner G. 2001 Perineuronal nets in
the rhesus monkey and human basal forebrain including basal ganglia. Neuroscience 108(2), 285–298.
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Brückner G., Pavlica S., Morawski M., Palacios A.G., Reichenbach A. 2006 Organization of brain extracellular
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32(2-4), 143–158. (doi:10.1016/j.jchemneu.2006.08.002).
11.
Baratta J., Marienhagen J.W., Ha D., Yu J., Robertson R.T. 1996 Cholinergic innervation of cerebral cortex in
organotypic slice cultures: sustained basal forebrain and transient striatal cholinergic projections. Neuroscience 72(4),
1117–1132.
12.
Distler P.G., Robertson R.T. 1993 Formation of synapses between basal forebrain afferents and cerebral cortex
neurons: an electron microscopic study in organotypic slice cultures. Journal of neurocytology 22(8), 627–643.
13.
Fuss B., Pott U., Fischer P., Schwab M.E., Schachner M. 1991 Identification of a cDNA clone specific for the
oligodendrocyte-derived repulsive extracellular matrix molecule J1-160/180. Journal of Neuroscience Research 29(3), 299–
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14.
Dityatev A., Brückner G., Dityateva G., Grosche J., Kleene R., Schachner M. 2007 Activity-dependent formation
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15.
Pesheva P., Spiess E., Schachner M. 1989 J1-160 and J1-180 are oligodendrocyte-secreted nonpermissive
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Suppl. Fig. 1. Morphological characteristics of WFA-stained perineuronal nets are clearly visible
around large mesencephalic neurons in deep layers of the superior colliculus after maintenance in slice
cultures for 21 days. (A) A lattice-like net in a wild-type (tn-r
+/+
) culture. (B) A granular matrix
structure typical for tenascin-R-deficient (tn-r -/-) mice is seen in all parts of PNs at cell body, dendrites
and axon initial segment (arrowhead). Scale bars: 20 µm in B also applies for A.
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Suppl. Fig. 2. Western blot analysis of TN-R expression in brain homogenates (BH) of a tn-r
+/+
(wt)
and a tn-r -/- (ko) mouse and supernatant (SU) of a tn-r +/+ organotypic slice culture (OSC). In the tn-r
+/+
BH two immunoreactive bands at 160 and 180kDa appear, which correspond to the two TN-R
isoforms. In the tn-r -/- BH lane no TN-R immunoreactivity is detected. In the supernatant of the tn-r +/+
organotypic slice culture a band at 160kDa is detected, which corresponds to the smaller isoform of
TN-R.
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Suppl. Fig. 3. PNs in organotypic forebrain slice cultures from TN-R-deficient (tn-r
-/-
) mice
maintained for 21 days in culture medium containing 10 mM KCl. Immunostaining of cortical neurons
for parvalbumin (A) is combined with Wisteria floribunda agglutinin (WFA) fluorescence labelling
(B), an overlay is shown in (C). Scale bars: 20 µm in C also applies for A and B.
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Suppl. Fig. 4. Distribution of VGAT-immunoreactive synaptic profiles (red) on hippocampal
interneurons associated with WFA-stained PNs (green) in dissociated cell cultures. (A) A wild-type (tnr
+/+
) neuron. (B) A neuron from a TN-R-deficient (tn-r -/-) mutant. Arrowheads indicate axon initial
segment. (C) Quantification of VGAT-immunoreactive presynaptic structures on neuronal somata and
axon initial segment showing no difference between wild-type and mutant mice. Scale bars: 20 µm in B
also applies for A.
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Suppl. Fig. 5. No restoration of WFA-positive perisomatic PNs is observed in dissociated cell cultures
from tn-r
-/-
mice after treatment with TN-R protein, and some clustering of WFA is observed after
treatment with aggrecan antibodies. Scale bar: 20µm applies for all images.
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Supplementary Data for the article “Tenascin-R promotes assembly of the extracellular matrix of
perineuronal nets via clustering of aggrecan“ by Markus Morawski, Alexander Dityatev et al.
Suppl. Fig. 6. Coomassie blue staining of purified WFA before reduction (lane 2) and after reduction
with DTT (lane 3). Two µg of each protein fraction were resolved on 10% SDS-PAGE under nonreducing conditions. Lane 1 protein standards, representing 26, 35, 43, 55, 72 (red band), 95, 130 and
170 kDa.
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