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Supplementary material
Supplementary Figure Legends
Fig. S1. Characterization of the NestinfloxGFPflox-TK-IRES-LacZ and NestinTK
mouse lines.
(A) Schematic representation of the NestinTK double transgenic mouse line.
(B to G) Representative confocal microscopy images of NestinfloxGFPfloxTK mouse coronal
brain sections at the level of the SVZ (B, D, E, F and G) showing that GFP positive cells (in
green in B to G) express Nestin (red in D), Id1 (red in E), GFAP (red in F) and DCX (red in
G). Note that GFP positive cells are not present within the subgranular zone of the
hippocampus (C). DAPI stained nuclei are in blue. LV, lateral ventricle; DG, dentate gyrus.
Scale bar, 50μm.
(H) Electron Microscopy of the C57Bl/6-GCV+ (black bars), NestinTK-GCV- (grey bars) and
NestinTK-GCV+ (white bars) SVZ. The number of ependymal cells (type E), astrocytes
(including both B+ and B- cells), neuroblasts (type A) and transit amplifying cells (type C)
was counted and expressed as number of cells per mm. n=3 mice per group.
Fig. S2. GCV treatment does not affect neurogenesis in the dentate gyrus and
hippocampal functions.
(A) Representative confocal images of BrdU positive cells (in red) in the dentate gyrus (DG)
of an untreated control (upper image) and of a NestinTK-GCV+ mouse (lower image). Transit
amplifying cells (BrdU cell) in the dentate gyrus are not reduced by GCV treatment. Nuclei
stained by DAPI are in blue.
(B) Quantification of BrdU positive cells of the dentate gyrus (DG) of control (black bar) and
GCV treated NestinTK mice (white bar).
(C and D) Testing episodic-like memory on the Morris water maze on control (black circles
and bars, n=37) and NestinTK-GCV+ (white circles and bars, n=19).
(C) Mice were subjected to 3 days of 6 training trials per day with the platform in the same
position (acquisition phase). On the fourth day the platform was moved to a different position
and mice were subjected to 2 days of 6 trials per day (reversal phase). The animals of the
different treatment groups (control mice in black; NestinTK-GCV+ mice in white) showed no
difference regarding the escape latency to locate the new platform position (reversal phase
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from trial 23 to 28. Values are presented as means ± SEM. One-way ANOVA by repeated
exposures F[1;53]=2.40, p=0.13.
(D) No difference between the treatment groups was observed on the percentage of time spent
in a narrow zone around the previous target location during the probe trial. A high preference
for the trained goal quadrant (og) compared to the averaged time in the three control zones
(oo+ol+or) was observed in both treatment groups
The dashed line represents the 25% chance level of swimming in a particular zone. Values are
presented as means ± SEM. One-way ANOVA, F[1,53]=1.27, p=0.27.
(E and G) Testing of associative memory in auditory fear conditioning on control (black
circles and bars, n=37) and NestinTK-GCV+ (white circles and bars, n=19).
NestinTK-GCV+ mice showed intact associative fear-related memory: no significant
differences were observed compared to control mice in the percentage of freezing over the
five tone presentation during the conditioning session (E) (One-Way ANOVA,
F[1,270]=1.52, p=0.22), as well as testing animals 24 hours later for context memory (F)
(F[1,54]=0.002, P=0.97) and cue memory test (G) (genotype effect: F[1,54]=0.25, P=0.62).
Values are presented as means ± SEM. BI, CS, conditioning stimulus and BI.
Fig. S3. Behavioural tests specific for the striatum.
(A-B) Balance and motor coordination on the rotarod. C57Bl/6-GCV+ (n=9, black bars) and
NestinTK-GCV+ (n=10, white bar) mice were subjected to 5 trials (every 30 minutes) in
accelerating speed (A) at the day 1. Latency to fall didn’t show any difference between the
two groups. (B) On the second day C57Bl/6-GCV+ (n=9, black bars) and NestinTK-GCV+
(n=10, white bar) mice were tested for 5 trials using as rod velocity the average of maximum
speed reached on day 1. The latency to fall didn’t was not different between the two groups.
Values are shown as mean ± SEM.
Fig.S4. Neurospheres from NestinTK mice are sensitive to GCV treatment.
(A
to
C)
Representative
microphotographs
of
neurospheres
derived
from
a
NestinfloxGFPfloxTK mouse (A and B) and from a NestinTK mouse (C). Cells in the sphere
express the GFP protein (green in A and B) that co-localizes with Nestin (red in B). Cells of
NestinTK derived neurosphere express the LacZ (C) (x-gal stained cells in blue). Scale bar,
20 μm.
(D) Dose survival curve for GCV-treated cultured aNPCs. Concentration higher than 0.1μM
reduce the survival of NestinTK derived aNPCs down to 5.1%, wheras concentrations higher
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than 100μM become toxic, as displayed by the reduction of survival of the C57Bl/6 and
NestinfloxGFPfloxTK derived aNPCs. *P<0.05; one-way ANOVA followed by Bonferroni
post-hoc test.
(E) The presence of 0.1μM and 1μM GCV in neural stem cell culture medium affects the
expansion rates of NestinTK- derived aNPCs, but not that of C57Bl/6 and not that of
NestinfloxGFPfloxTK derived aNPCs. Values in D and E are reported as means ± SEM from
three independent biological replicates.
(F) Representative immunofluorescence image of cultured aNPCs stained for TLR-4 (green)
and Nestin (red) showing that aNPCs express the TLR-4. (G) Inset shown in panel F. Nuclei
were counterstained by DAPI. Scale bar 50μm.
(H and I) Growth curve and differentiation of aNPCs used in electrophysiological
experiments. They showed a normal growth and they were able to differentiate in neuron
(8%), oligodendrocytes (2%) and astrocytes (90%).
For all experiments we used the same line of aNPCs at P4-P6.
Fig. S5. AraC-treated mice show increased susceptibility to excitotoxic damage.
(A,
B
and
D,
E)
Representative
confocal
images
of
C57Bl/6-AraC-
(PBS1x
intracerebroventricular (icv), A and D) and C57Bl/6-AraC+ (2% in 0.9% saline icv for 7 days,
B and E). Id1+ (green in A and B), DCX+ and BrdU+ (respectively green and red in D and E)
cells. Nuclei in blue were stained by DAPI. Scale bars, 50 μm. (C and F) The number of Id1 +
(C), BrdU+ and DCX+ (F) cells of the dorsal SVZ expressed as number of positive on the total
number of cells (four sections comprehending the SVZ, n=3 animals per group). Values are
reported as means ± SEM. ***P<0.001; unpaired, two-tailed t-test.
(G) Quantification of maximum seizure severity by the Racine scale and survival recorded
over a 90 minutes in C57Bl/6 AraC- (n=6) and C57Bl/6 AraC+ (n=16) mice treated with 4AP. Values shown as means ± SEM. *P<0.05, unpaired, two-tailed t-test. #P<0.05, Log-rank
test.
(H) Modified neurological severity score and survival at 3 days after 45 minutes of MCAO in
C57Bl/6 AraC- (n=8) and C57Bl/6 AraC+ (n=5). **P<0.05, Mann-Whitney.
##
P<0.01, Log-
rank test.
Fig. S6. Characterization of the striatum after 4-AP treatment.
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Representative confocal microscopy images of coronal brain sections of the striatum showing
(A-B) c-fos activation in a 4-AP treated C57Bl/6 mouse (A) but not in the untreated mouse
(B). Nuclei stained by DAPI are in blue. Scale bar, 50μm.
Fig. S7. C57Bl/6-GCV+ and NestinTK-GCV- have a comparable outcome after epilepsy
and stroke.
(A-B) At 90 minutes after 4-AP administration the survival (A) and the quantification of
maximum seizure severity by the modified Racine scale (B) showed no difference between
C57Bl/6-GCV+ (n=15) and NestinTK-GCV- (n=17) mice.
(C) The percentage of survival recorded in C57Bl/6-GCV+ (n=12) and NestinTK-GCV(n=23) mice subjected to 45’ MCAO was similar in the two groups.
(D) Neurological deficit quantified by the modified Neurological Severity Score (mNSS) 7
days after 45’ MCAO showed no differences between C57Bl/6-GCV+ (n=7) and NestinTKGCV- (n=9).
(E) Body weight was not different between C57Bl/6-GCV+ (n=7) and NestinTK-GCV- (n=9).
(F) Lesion volume quantified using cresyl violet on coronal sections (from +2 to -4 mm from
bregma, interval between sections 600 μm) obtained from C57Bl/6-GCV+ (n=7) and
NestinTK-GCV- (n=8) 3 days after 45’ MCAO showed no differences between the two
groups.
Fig. S8. Neurogenesis 72 hours after induction of ischemia.
(A-D) Representative confocal microscopy images of coronal brain sections of the SVZ
(lateral ventricle, LV) of a C57Bl/6-GCV+ (A and B) and a NestinTK-GCV+ mice 72 hours
after MCAo, displaying BrdU (white), DCX (green) and GFAP (red) cells in the
contralesional (A and C) and ipsilesional (B and D) hemisphere. Note the absolute reduction
of BrdU+ and DCX+ in NestinTK-GCV+ compared to C57Bl/6-GCV+ mice. Nuclei stained by
DAPI are in blue. Scale bar, 25μm.
(E and F) Number of BrdU (E) and DCX (F) positive cells expressed as number of positive
cells on the total number of cells (by counting nuclei in Dapi) in the same region of interest.
‘contra.’ indicates the contralesional while ‘ipsi’ indicates the ipsilesional hekisphere. Values
in E and F are reported as means ± SEM. *P<0.05, unpaired, two-tail T-test, n=5 mice per
group.
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Supplementary Material and Methods
Generation of transgenic mouse lines
We used a third generation SIN lentiviral vector (Follenzi et al.,2000) to generate a
NestinfloxGFPfloxTK targeting lentivirus. A conserved 1.8 Kb second intronic region of the
rat Nestin gene (Zimmerman et al., 1994) was cut out (XbaI, HindIII) from the p401ZgII
plasmid (gift from Dr. McMahon, Harvard University, Cambridge, MA) and sub-cloned
upstream to the minimal promoter of the Hsp68 gene. The loxP sites were synthetically
produced and were cloned upstream and downstream to the EGFP coding region. EGFP was
obtained from the BamHI-SalI fragment of the #277 PGK-GFP lentivirus construct (gift from
Dr. Naldini, San Raffaele Hospital, Milan, Italy). Downstream to the EGFP sequence we sub
cloned the suicide gene Thymidine Kinase (TK). BamHI-XbaI fragment TK coding sequence
was cut out from the pBSIISK-TK construct. Finally, we inserted the IRES-lacZ fragment
obtained from the pMODLacZnls plasmid downstream the TK gene. Nestin-floxGFPfloxTK
founder mice were generated by the NestinfloxGFPfloxTK vector injection into the
perivitelline space of the C57Bl/6 zygote. Mice were genotyped by PCR using genomic DNA
and the following primers:
FW: 5’-AACTTTCCCCGGAGAGCATCCACGC-3’;
Rev1: 5’- TAGGTCAGGGTGGTCACGAGGGT-3’;
Rev2: 5’-TGTTGATGGCAGGGGTACGAAGC-3’.
Pharmacological treatments
Ganciclovir and AraC administration
All pumps and of the icv cannula implantations were performed on anesthetized mice (130
mg/kg ketamine (Ketavet 100, Intervet, Italy), and 20 mg/kg xylazine (Rompun, Bayer,
Germany)). C57Bl/6 mice treated with AraC are named as C57Bl/6 -AraC+, while PBS
treated C57Bl/6 mice are named as C57Bl/6-AraC-.
To study the proliferation of aNPCs in the SVZ and in the hippocampus of NestinTK mice or
of controls after GCV or after AraC treatment, BrdU (Sigma) was administered using two
different protocols. To label the transit amplifying cells in the SVZ 100 mg/kg of BrdU
dissolved in saline were intraperitoneally given every two hours for 10 hours followed after 2
hours by sacrifice of the animals. To label proliferating cells in the hippocampus we injected
the 100 mg/Kg of BrdU (Sigma) dissolved in saline every 12 hours for 5 days followed by
sacrifice of the animals.
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Evaluation of toxicity of GCV treatment
Serum electrolytes (Na+:139.0±0.58, 141.4±0.68, 140±1.41; K+: 6.49±0.3, 6.16±0.22,
6.76±0.24; Cl-: 103±1.93, 103.8±0.7, 104.6±0.8; values are in mmol/l in NestinTK-GCV(n=3), WT-GCV+ (n=5) and NestinTK-GCV+(n=4) respectively), serum glucose (fasting
glucose: 90.5±3.13, 92.78±5.86; non fasting glucose 133.3±6.04, 136.6±4.55; values are in
mg/dL in WT-GCV+ (n=9) and NestinTK-GCV+(n=9) respectively) and complete blood
counts (Red cells: 7.8±0.68, 7.26±0.15, 7.27±0.08 values as 106 cells per µl; White cells:
9±1.7, 9±1.1, 7.45±0.75 values as 103 cells per µl; Hemoglobin: 12.5±1.25, 11.4±0.65,
10.9±0.1 values as g per dl; Hematocrit: 36±3.5, 33±1.6, 33±0.7 values in %; Platelets:
771±131, 747.6±42.6, 624±67 values as 103 cells per µl; in WT-GCV- (n=3), WT-GCV+
(n=3) and NestinTK-GCV+(n=3) respectively) did not differ between treatment groups.
NestinTK-GCV+mice showed over the 4 weeks of GCV treatment less weight increase when
compared to control groups (121.3±2.6, 114.4±1.8, 115.7±1.8, 105.8±1.5, values are the
weight increase in percentage compared to baseline values in WT-GCV- (n=13), NestinTKGCV- (n=6), WT-GCV+ (n=18) and NestinTK-GCV+(n=23) respectively, p<0.0001).
Whole blood (180μL) was collected from the tail of mice and added of the anticoagulant
citrate-phosphate-dextrose (1:10, Sigma). Platelets, white cells, red blood cells, hemoglobin
and hematocrit values were counted with an automated cell counter (System 9000, SeronoBaker Diagnostics). The electrolytes were quantified by indirect potentiometry <Iannacone,
Sitia et al., 2008>.
Blood glucose levels were measured using a Glucometer Elite (Bayer Canada, Toronto,
Ontario, Canada) in the morning after either overnight deprivation of food (fasting) or no food
deprivation (non fasting). 15, 30 and 60 minutes after the end of surgical procedure, daily for
the first week and then every second day post-transplantation.
Weight increase was evaluated at baseline and than every week up to the end of the 4 weeks
of GCV or sham treatment in WT and NestinTK mice. Weight increase was calculated as
percentage of weight at the end of treatment over baseline values.
Histology and pathological analysis
For immunohistochemistry/fluorescence studies adult mice were sacrificed after anaesthetic
overdose of ketamine and xylazine and transcardially perfused with ice-cold 4%
paraformaldehyde (Sigma) in PBS 1x pH 7.2. Dissected brains were post-fixed in the same
PFA4% solution for 12 hours at +4 °C and then cryoprotected for at least 48 h in 30% sucrose
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(Sigma) in PBS 1x at +4 °C. Coronal 10 m -thick cryostat sections were cut from the entire
forebrain (i.e. starting from olfactory bulbs to the cerebellum). For immunofluorescence
stainings, coronal brain sections (10 µm) were incubated with blocking solution (FBS 10%,
BSA 1 mg/ml and Triton 0.1% in PBS 1x, Sigma), for 1 hour and then primary antibodies
were applied in the same solution overnight at +4°C.
For signal amplification, when necessary (i.e. for the Id1 staining) biotinylated rabbit
secondary antibodies (1:200, Vector laboratories) were used. The biotinylated secondary
antibody was further reacted with avidin and biotinylated HRP complex (1:250, TSAfluorescent system Perkin Elmer, Massachusetts 02451 USA). HRP activities were revealed
with Tyramide-Alexa 488 or Tyramide-Cy5 (1:100, TSA-fluorescent system Perkin Elmer).
Omission of the primary antibodies showed no specific staining. Nuclei were counterstained
with 4′-6-diamidino-2-phenylindole, DAPI (Roche).
Light (Olympus, BX51, equipped with 4× and 20× objectives, Japan) and confocal (Leica,
SP5 equipped with 40X and 63X objectives, Germany) microscopy images were obtained to
analyse tissue stainings. Analyses of images were performed by using Leica LCS lite or
Adobe Photoshop CS software (Adobe Systems Incorporated, CA 95110, USA).
X-Gal staining
To detect Nestin positive cells in NestinTK mice, frozen coronal sections (10 μm) were
incubated overnight at 37 °C in 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal, Roche)
solution for detecting nuclear -gal activity.
Quantification of cells in the subventricular zone (SVZ)
From the 10μm thick cryostat coronal sections of the NestinTK-GCV+ and NestinTK-GCVmice (see above), one systematic random series of sections per mice was stained (i.e. for all
abovementioned antibodies), so that sections were spaced at 28 section intervals (280 μm)
from each other. The section series represented a systematic random sample of sections that
covered the entire extent of the forebrain. Images were first acquired using a confocal
microscope with a CCD-IRIS color video camera (Leica, SP5 equipped with 40X and 63X
objectives). Cell numbers in the right and left dorsal subventricular zone (Pluchino et al.,
2008) were manually quantified using Adobe Photoshop CS software (Adobe Systems
Incorporated) on at least 4 sections per mouse containing the SVZ. Where indicated cell
numbers were calculated as ratio over the total cell number obtained by counting all DAPI
positive nuclei in the same region of interest (dSVZ).
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In situ- hybridization
In situ hybridizations were performed as previously described (Centonze et al., 2007; Muzio
et al., 2009). Briefly, 10 μm-thick brain sections were post-fixed 15 min in 4% PFA, then
washed three times in PBS 1x. Slides were incubated in 0.5 mg/ml of Proteinase K (Roche) in
100 mM Tris-HCl (pH 8, Sigma), 50 mM EDTA (Sigma) for 10 min at 30°C. This was
followed by 15 min in 4% PFA. Slices were then washed three times in PBS 1x then washed
in H2O. Sections were incubated in triethanolamine (Merk, Germany) 0.1 M (pH 8) for 5 min,
and then 400 ml of acetic anhydride (Sigma) was added two times for 5 min each. Finally,
sections were rinsed in H2O for 2 min and air-dried. Hybridization was performed overnight
at 60° C with a-UTP-P33 (GE, USA) riboprobes at a concentration ranging from 106 to 107
counts per minute (cpm). The following day, sections were rinsed in SSC 5 X (Sigma) for 5
min then washed in formamide 50% (Sigma)-SSC 2 X for 30 min at 60 °C. Then slides were
incubated in ribonuclease-A (Roche) 20 mg/ml in 0.5 M NaCl, 10 mM Tris-HCl (pH 8), 5
mM EDTA 30 min at 37°C. Sections were washed in Formamide 50% SSC 2X for 30 min at
60°C then slides were rinsed two times in SSC 2X. Finally, slides were dried by using ethanol
series. Lm1 (GE) emulsion was applied in dark room, following manufacturer instructions.
After 10 days, sections were developed in dark room, counterstained with DAPI and mounted
with DPX (BDH, UK) mounting solution. The following probes were used: mouse Dlx2
riboprobe (gift from Dr. Vania Broccoli, San Raffaele Hospital, Milan, Italy).
Microphotographs of sections were digitalized in dark field light microscopy (Olympus
BX51, and 46 objective) by using a CCD camera (Leica). To confirm the specificity of the
different RNA probes, sense strand RNA probes (showing no signal) were used as negative
controls.
Ischemic volume measurement and representation
To measure the ischemic lesion volumes, coronal 30 m-thick coronal cryostat sections were
prepared from 2 mm rostral to 4 mm caudal to the bregma (Paxinos and Franklin, 2000). One
systematic random series of sections per mouse was stained for cresyl violet (Sigma), so that
sections were spaced at 20 section intervals (total of 10 sections per mouse, distance between
sections 600μm). The section series thus represented a systematic random sample of sections
that covered the entire extent of the forebrain. Sections were digitalized and analysed with
Image J (NIH, USA) image analysis system. The lesion area was measured for each reference
level by the ‘indirect method’, which corrects for brain oedema (Lin et al., 1993). The lesion
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volume was determined by integrating the mean lesion areas of the ten levels. Data were
expressed as lesion volume in mm3. For the representative 3D volume rendering image
(Figure 1 I and J), a representative brain was traced using the assistance of the Stereo
Investigator v 3.0 software (MicroBrightField, Inc., Colchester, VT and a personal computer
running the software connected to a color video camera mounted on a Leica microscope)
(Bacigaluppi et al., 2009; West et al., 1991). The motorized stage of the microscope, which
was controlled by the software, allowed precise and well-defined movements along the x-, yand z-axes. Images were first acquired with a CCD-IRIS color video camera and the cerebral
hemispheres, lesioned area and ventricles were interactively delineated at low magnification
on a video image of the section.
EM quantification of aNPCs in the SVZ
For EM studies, ketamine/xylazine anesthetised mice were perfused transcardially with 0.9%
saline, followed by Karnovsky’s fixative (2% PFA and 2.5% glutaraldehyde, Sigma). The
brains were removed and post-fixed in the same fixative overnight. Then, the brains were
washed in 0.1 M phosphate buffer pH 7.2 (Sigma). Transverse 200 μm-thick brain sections
were cut on a vibratome, post-fixed in 2% osmium for 2 hours, rinsed, dehydrated, and
embedded in Durcupan araldite (Fluka Biochemika, Ronkokoma, NY USA). To study the
organization of the SVZ, 1.5 μm semithin sections were cut with a diamond knife and stained
with 1% toluidine blue (Sigma). Three different antero-posterior SVZ levels were selected
and photographed for each sample. Both the dorsal, medial and ventral regions of the
subependymal layer of the rostral portion of the lateral ventricle were included in the
analyses. To identify individual cell types, ultrathin (70 nm-thick) sections were cut with a
diamond knife, stained with lead citrate, and examined under a Fei Tecnai Spirit electron
microscope (Fei Tecnai, Hillsboro, OR, USA). The number of profiles corresponding to the
different cell types along the ventricular wall of the anterior horn was quantified in a fixed
region in six to eight ultrathin sections for each group of mice under the electron microscope
(Doetsch et al., 1997). Cells with only small fragments of cytoplasm or nucleus in a given
section were classified as unidentified. All quantifications were performed blinded. For cell
counts, type A cells were identified by the small size, scanty and dark cytoplasm, dark
chromatine and fusiform appearance. They show dense contacts surrounded by intercellular
spaces. Type B cells were identified as large, less electrondense cells, rich of intermediate
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filaments. Although they occasionally had a darker cytoplasm, other unequivocal
ultrastructural features such as nuclear invaginations and an irregular cell contour (noticeable
at higher magnification) allowed a clear detection of this cell type. Type C cells were
characterized by the presence of large cytoplasm and nuclei, lack of intermediate filaments
and abundant cytoplasmic organelles.
Electrophysiology
WT (C57/Bl6 mice) mice were sacrificed by cervical dislocation under halothane anaesthesia,
and coronal slices at the level of the Bregma (200 m) were prepared from fresh tissue blocks
of the brain using a vibratome (Centonze et al., 2007; Centonze et al., 2005). A single slice
was then transferred to a recording chamber and submerged in a continuously flowing
artificial cerebrospinal fluid (CSF, 34°C, 2–3 ml/min) gassed with 95% O2–5% CO2. The
composition of the control solution contained the following solutes: 126mM NaCl, 2.5mM
KCl, 1.2mM MgCl2, 1.2mM NaH2PO4, 2.4mM CaCl2, 11mM glucose, and 25mM NaHCO3
(all Sigma). Whole-cell patch-clamp recordings were made with borosilicate glass pipettes
(1.8 mm outer diameter; 2–4 M), in voltage-clamp mode, at the holding potential of -80
mV. The recording pipettes were filled with an internal solution composed of: 125mM K+gluconate, 10mM NaCl, 1.0mM CaCl2, 2.0mM MgCl2, 0.5mM BAPTA, 19mM HEPES,
0.3mM GTP, and 1.0mM Mg-ATP, adjusted to pH 7.3 with KOH. 10 M Bicuculline (from
Sigma/RBI) was added to the perfusing solution to block GABAA-mediated transmission.
Generation and maintenance of aNPC cultures
aNPC cultures were obtained from 6 weeks old C57Bl/6, NestinfloxGFPfloxTK and
NestinTK mice, as previously described (Pluchino et al., 2008). Briefly, three mm-thick
coronal sections were obtained from the anterior forebrain of 6 weeks old mice (2 mm from
the anterior pole of the brain). Dorsal SVZs were carefully dissected by using a fine scissor in
the following dissociating medium: Earl's Balanced Salt Solution (Gibco, Invitrogen)
supplemented with 1 mg/ml Papain 27 U/mg (Sigma), 0.2 mg/ml Cysteine (Sigma) and 0.2
mg/ml EDTA (Sigma). Then, dissected tissue was incubated in the same solution for 30 min
at 37°C on a rocking platform. Finally, dissociated cells were plated in standard neurosphere
growth medium Neurocult proliferation medium (Stem cell Technology, BC, CA)
supplemented with EGF (20 ng/ml) and FGF2 (10 ng/ml). For each in vitro passage, single
cells were obtained by incubating neurospeheres in Accumax (Sigma) for 10 minutes, and
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then 8000 cells/mm2 were plated on T75 plastic flasks (Nunc, Rochester, USA). Neurospheres
were propagated in vitro and assayed for self-renewal and cell differentiation after 6 passages,
as previously described (Pluchino et al., 2008). To induce LPS stimulation, aNPCs were
dissociated, stimulated with 100 ng/ml of LPS (026:B6, Sigma) and then collected after 16
hours, thus obtaining (LPS+aNPCs). To test the aNPCs sensitivity to GCV treatment, 5.000
cells per well were plated in a 96-well plate and the day after they were treated with
increasing concentrations (0.001, 0.01, 0.1, 1, 10, 30, 100 and 300 M) of GCV (Roche).
After a week, 50 l of MTT (2.5 mg/ml, Sigma) followed after 2 hours by 50 l of SDS/DMF
(0.6g/ml in DMF, Sigma) were added to the cells. Optical density at 570-650 nm was read 18
hours later with the Elisa-Plate reader (Biorad).
Differantiation in vitro of aNPC cultures
To induce the differentiation of NPCs in vitro, single-cell dissociated NPC suspensions were
plated on MATRIGEL (Growth Factor Reduced Matrigel, Becton Dickinson Labware) coated
round 12-mm coverslips in a 24-well plate (40000 cells/well) in Neurocult differentiation
medium (Stem cell Technology), as previously described (Pluchino et al., 2008). The cells
were cultured for 8 days in vitro before processing for immunofluorescence.
Video-EEG recordings in epilepsy
To investigate drug-induced seizures a subset of animals used in 4-AP experiments was
examined with video-EEG recordings. Epidural stainless-steel screw electrodes (0.9 mm
diameter, 2 mm length) were surgically implanted under sevoflurane (Sevorane™, Abbott
S.p.a. Campoverde, Italy) anaesthesia and secured using cyanoacrylate and dental cement
(Ketac Cem, ESPE Dental AG, Seefeld, Germany) (Cambiaghi et al., 2011; Chabrol et al.,
2010). Active electrodes were placed over parietal areas [2mm lateral to midline, 1mm
posterior to bregma (Paxinos and Franklin)] and a common reference was fixed over the
cerebellum (1mm posterior to lambda). After 24 hours recovery, unrestrained mice were
monitored by video-EEG in recording sessions of 90 minutes in a Faraday cage. For
experiments with HU-210, electrodes and Alzet minipumps were implanted in the same
surgery session. During EEG recording, mice were connected via a flexible cable to an
amplifier and EEG data were recorded and digitally saved (0.15 – 100 Hz filtered; 256
sampling frequency; 16-bit resolution) using a video-EEG equipment (System Plus device;
Micromed, Mogliano Veneto, Italy). Tracings were filtered between 1 and 10 Hz.
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Simultaneous video data were acquired with a Canon MV550I camera connected to the EEG
system device via firewire. To detect spontaneous seizures duration and morphology, videoEEG recordings were visually inspected.
Induction of transient MCAO
Transient, 45 minutes long MCAO, was induced as previously described (Bacigaluppi et al.,
2009). Briefly, animals were anaesthetized with 1% isoflurane (Merial, Assago, Italy) in 30%
O2 (remainder N2O). Rectal temperature was maintained constant during the experiment
(between 36.5 and 37°C) using a feedback-controlled heating system (Harvard Instruments,
Holliston, MA, USA). During the surgery, blood flow in the territory of the left MCA was
monitored by means of laser Doppler flowmetry (LDF). A flexible 0.5 mm fiberoptic probe
(Perimed, Stockholm, Sweden) was attached to the intact skull overlying the core region of
the left MCA territory. Changes of LDF were recorded before, during and up to 15 minutes
after reperfusion. Focal cerebral ischemia was induced with a silicon-coated (Xantopren,
Bayer Dental, Osaka, Japan) 8-0 nylon filament (Ethilon, Ethicon, Norderstedt, Germany) that
was inserted into the left common carotid artery and was advanced through the internal
carotid artery to the origin of the middle cerebral artery, as described (Hata et al., 2000;
Hermann et al., 2001).
Following 45 minutes of MCAO, reperfusion was induced by withdrawing the filament. Only
animals showing on laser Doppler flowmetry a fall of blood flow during MCAO down to 30%
of baseline values and subsequent reperfusion were considered for the study. Wounds were
carefully sutured, anaesthesia was discontinued and mice were put back in their cages to
allow recovery. To correct fluid loss during the surgical intervention, 0.5 ml of sterile isotonic
saline was intra-peritoneally injected in each animal (once a day for the first three days). At
baseline and on the subsequent days, body weight was daily measured until the end of the
experiments.
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Behavioural tests
Hippocampal functions learning assessment
Water maze task. The standard hidden-platform version of the water maze was done as
previously described (D'Adamo et al., 2002). Briefly, the test included an acquisition phase
(18 trials, six/day, inter-trial time 30–40 min) followed by a reversal phase during which the
platform was moved to the opposite position (12 trials, six/day). For the analyses the trials
were averaged in blocks of two trials. The first 30 sec of trial 19 (first reversal trial) were
considered as probe trial. For the analysis the trials were averaged in blocks of two trials. The
following measures were calculated to assess acquisition: escape latency, swim speed, time
floating, wall hugging and the percentage of time in the current quadrant goal (excluding
episodes of floating). Spatial selectivity during the probe trial was quantified using the
following parameters: percentage of time in the trained quadrant, percentage of time in a
circular target zone comprising one-eighth of the pool surface and the annulus crossings. To
assess platform reversal learning the following measures were calculated: escape latency and
the percentage of time in current quadrant goal (excluding episodes of floating).
Fear conditioning test. Auditory trace fear conditioning was performed as previously
described (D'Adamo et al., 2002). All mice were pre-exposed to the test chamber for 10
minutes on the two days preceding conditioning. The fear conditioning trace trial started with
the presentation of the conditioning stimulus, CS (15 s), followed 15 s later by the
presentation of the shock for 2 s. This procedure was repeated 5 times with a 60 seconds intertrial intervals (ITI). Twenty-four hours after training, mice were placed in the conditioning
box again, and their freezing behaviour in the context test and in the tone test was measured.
Context testing consisted of 2 min without CS (“contextual freezing”), tone testing consisted
of 1 min without CS followed by 1 min with the CS turned on.
Video tracking, data collection and statistical analysis
During the water maze test, animals were video-tracked using the EthoVision 2.3 system
(Noldus Information Technology, Wageningen, the Netherlands, http://www.noldus.com)
using an image frequency of 4.2/s. Raw data were transferred to Wintrack 2.4
(http://www.dpwolfer.ch/wintrack) (Wolfer and Lipp, 1992) for off-line analyses.
Rotarod
Five mice are simultaneously placed on the rotarod apparatus with the rod rotating at 4 rpm
(rotations/minute) during the first minute. Then rotation speed is increased every 30 sec by 4
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rpm. A trial ends for a mouse when it falls down or when 5 min are completed. Each mouse is
submitted to 5 trials on day 1 with an intertrial interval of 30 min. On day 2, all mice are
tested again for 5 min at a constant speed (average of maximum speed reached from all mice
on day 1). Some mice cling to the rod and ride a full circle without falling down. Passive rides
are recorded separately.
Behavioural analysis for epilepsy
Clonic and tonic convulsions were induced in mice by i.p. injection of 8mg/kg 4-AP
(Yamaguchi and Rogawski, 1992). After receiving 4-AP, mice were observed for 90 minutes
and evaluated by a modified Racine scale (McCord et al., 2008). Briefly, mice were scored
each 5 minutes for 90 minutes as follows: score 1, immobility; 2, forelimb and/or extension,
rigid posture; 3, repetitive movements, head bobbing, forepaw shaking; 4, rearing and falling;
5, continuous rearing and falling; 6, severe tonic-clonic seizures. When animals died after a
seizure a score of 6 was assigned. Trained observers blinded to genotype and drug treatment
quantified scores. Maximum score and number of seizures were analysed.
Behavioural analysis for cerebral ischemia
On ischemic mice the modified Neurological Severity Score (mNSS) - a motor and
coordination test battery assessing the severity of the neurological deficits on a graded scale
ranging from 0 to 14, where 0 represents normal function and 14 maximal deficits – was
evaluated by trained observers blind to genotype and drug treatment groups, at baseline, on
the day of surgery and on a daily base up to the end of the study (Jackson and Sudlow, 2005).
Measurement of endogenous cannabinoids
Endogenous levels of AEA and 2-AG were measured on tissue of GCV- treated C57Bl/6 and
NestinTK mice. Mice were sacrificed 30 minutes after the injection of saline or of LPS.
Brains were quickly removed and snap-frozen in isopentane/dry ice. Striata were punched
from the frozen brain using a cylindric brain puncher (Fine Science Tools, Science Tools
Foster City, CA, USA, internal diameter 3.0mm). Length of punches was approximately 3mm
(start: bregma 1.5). Brain tissue of 2 mice were pooled to obtain a single data point
(Marsicano et al.,2002). To measure endogenous levels of AEA and 2-AG on aNPCs, the
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cells were either stimulated for 16h with LPS or not and then washed, centrifuged, snapfrozen
in liquid nitrogen and stored at –80°C.
[3H]AEA (205 Ci/mmol) was from Perkin Elmer Life Sciences (Boston, MA, USA). [3H]NArachidonoyl-phosphatidylethanolamine ([3H]NArPE, 200 Ci/mmol), d8-AEA and d8-2-AG
standards were from Cayman Chemicals (Ann Arbor, MI, USA).
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