Decreased hippocampal volume and increased anxiety in a
transgenic mouse model expressing the human CYP2C19
gene
Supplementary Information 1: Detailed methodological descriptions
Supplementary Figure 1: Distribution of CYP2C19 mRNA expression in the
brain
Supplementary Figure 2: Size measurements in brain sections from
CYP2C19Tg-Hom and Wt mice at PND0
Supplementary Figure 3: Brain weights
Supplementary Figure 4: BrdU and Ki-67 positive cells in the hippocampus
Supplementary Figure 5: Number of BrdU, Ki-67, DCX, and PA positive cells in
the hippocampus corrected by hippocampal size
Supplementary Figure 6: Immobility time in the Morris Water Maze
Supplementary Figure 7: Plasma corticosterone levels before and after acute
restraint stress
Supplementary Information 1: Detailed methodological descriptions
Transgenic mice
Mice (C57BL/6) transgenic for a BAC insert containing the human CYP2C18 and CYP2C19 genes,
which express both mRNA’s but only the CYP2C19 enzyme protein, have previously been produced
and characterized with regards to basic phenotype.1, 2 In line with the inability of detecting CYP2C18
protein in the transgenic mice is the absence of the CYP2C18 enzyme also in human tissues. 3, 4
Hemizygous transgenic mice (CYP2C19Tg-Hem) display a specific motion phenotype, where one or
both hind paws are lifted very high when walking. However, this locomotion pattern does not appear
to present an obstacle for walking as we have not observed reduced locomotor activity in
CYP2C19Tg-Hem compared to Wt mice.
Mice were group-housed with a 12-h light/dark cycle and ad libitum access to food and water. All
experiments were approved by the local ethical committee; Stockholm Northern Ethics Board of
Animal Experimentation.
CYP2C19Tg-Hem mice carry an estimated number of 12 copies of the human CYP2C18/19 genes 2 and
CYP2C19Tg-Hem mice used in the current study were generated by crossing CYP2C19Tg-Hem with
wild-type (Wt) mice. Wt litter mates were always used as controls. Homozygous (CYP2C19Tg-Hom)
mice were only bred for investigations of embryonic and fetal development as these mice do not
survive past post natal day 3 (PND3). Tail or ear biopsies were used for DNA extraction and
genotyping analysis was performed by Transnetyx, Inc (Cordova, Tennessee, US) using primers
designed before.2
CYP2C18 and CYP2C19 expression in transgenic mice and human fetal brain
CYP2C19 and CYP2C18 mRNA expression was investigated in brain and liver tissue during embryonic
development (embryonic day 14 (E14) and E18), early postnatal days (PND0 and PND7) and at 7
weeks of age. Brain and liver tissues were isolated and immediately placed in RNAlater RNA
stabilizing solution (Qiagen). Human brain tissue was obtained from the NICHD Brain and Tissue Bank
for Developmental Disorders at the University of Maryland, Baltimore, MD. Brain tissue samples
were collected 1-12 hours post-mortem and frozen in isopentane/dry-ice and stored at -85°C. Brain
tissues were obtained from 3 different female Caucasian donors; gestational week 19, 24 and 39. 2-3
cerebrum tissue pieces were obtained from each donor. All mouse and human tissues were
homogenized using Zirconium oxide beads (0.5 mm) using the Bullet Blender™ (Next Advance, Inc.,
USA). RNA isolation and cDNA synthesis was performed according to the manufacturer´s
recommendations using the RNeasy Mini Kit (Qiagen) and SuperScript III Reverse Transcriptase
(Invitrogen). Real-time PCR protocol and primers were obtained from Löfgren et al,2 except for the
human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were the following
primers were used: GAPDH- reverse: 5´-GAT GGG ATT TCC ATT GAT GAC A and GAPDH- forward 5´CCA CCC ATG GCA AAT TCC. Data were analyzed using the 7500 Fast System software (Applied
Biosystems).
Brain morphology
Immediately after birth, PND0 pups were anesthetized with Ketamine/Xylacine (80/10 mg/kg,
Intervet International/Bayer Healthcare) and perfused transcardially with phosphate buffered saline
(PBS; 0.1 M, pH 7.4) with 5 IE/ml Heparin followed by 4% paraformaldehyde in 0.2 M sodium
phosphate buffer, pH 7.2. Mouse brains were removed and post-fixed over night at 4° C and
cryoprotected in 20% and 30% sucrose for a total of 24 hours. Brains were snap-frozen and coronally
sectioned at 40 µm using a cryostat (MICROM International GmbH, Walldorf, Germany). All sections
were stained with cresyl violet and images were obtained using a Zeiss Axioskop 2 plus microscope
with an Axiocam (Carl Zeiss HB, Stockholm, Sweden). Images were merged into whole brain images
using the Photo merge function in Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Sections
were visually assessed to examine morphological changes, with the genotypes blinded to the
investigator. For area measurements of different brain regions at PND0, sections from 3 CYP2C19TgHom and 3 Wt mice were scanned with an Epson perfection V750 Pro scanner and the cortex,
hippocampus and central regions were manually outlined in all sections using the pen tablet, Intuos4
(Wacom, Tokyo, Japan). The total area of each area was calculated in Adobe Photoshop as the
number of pixels within the area selected.
To further investigate brain morphology, brains from 7 and 15 weeks old mice were prepared in the
same way as described above for the PND0 pups. Every 7th section, starting approximately at Bregma
-0.94 mm, was stained with crystal violet. A total of 12 coronal sections were stained in each animal,
thus covering most of the hippocampal formation. All sections were scanned with an Epson
perfection V750 Pro scanner and the hippocampi were manually outlined in all sections using the
Lasso tool in Adobe Photoshop and the pen tablet, Intuos4 (Wacom, Tokyo, Japan). The total area of
each hippocampus was calculated in Adobe Photoshop as the number of pixels within the area
selected. Brain and body weights were recorded in all perfused mice.
Magnetic resonance imaging
In vivo magnetic resonance imaging (MRI) experiments were conducted on CYP2C19Tg-Hem and Wt
adult (15-weeks old) male mice using a horizontal 9.4 T Varian magnet equipped with a 12 cm inner
diameter gradient system with a maximum gradient strength of 600 mT/m. A volume coil with an
inner diameter of 30 mm and a resonator length of 40 mm of the millipede design was used (Varian,
Inc. Abdington, UK). Animals were anaesthetized by isoflurane (5% for induction and then maintained
at 1.25-1.75%) in a 1:9 mixture of O2 and air. Mice were placed in prone position in a MR compatible
animal holder with the head firmly fixed. Respiration rate was monitored and the body temperature
was maintained at 37±0.5° C throughout the MRI experiment using a feedback controlled air-heater
system (SA Instruments). The volumetric images were acquired using a 3D Inversion Recovery Fast
Spin-Echo Sequence (Echo Train Length=4, kzero=1, inversion time=500 ms, repetition time/echo
time=2.5s/5.46 ms, matrix size=128 x 128 x 64 covering a Field Of View of 20 x 18 x 10 mm3 in the
caudal-cranial read out, left-right phase encode and inferior-superior phase encode 2 directions,
respectively). The total scan time was of 1 h and 25 min per mouse. The data were zero filled by a
factor of two in each dimension before Fourier transformed. Measurements of hippocampus and
whole brain volumes (including cerebellum) were manually outlined using the image analysis
software ITK-SNAP (www.itksnap.org).5 Mouse genotypes were blinded during the analysis.
BrdU injections and immunohistochemistry analyses of the hippocampus
For investigations of cell proliferation and cell survival in the hippocampus,6 7 weeks old mice were
intraperitoneally injected with bromodeoxyuridine (BrdU, 75mg/kg). For proliferation assessments,
mice were injected twice, with a 16 hours gap, and sacrificed by perfusion 2 hours after the last
injection as described before. To investigate survival rates of proliferating cells, a second group of
mice were injected once a day for 3 consecutive days and sacrificed by perfusion 4 weeks after the
first injection. Brains and sections were prepared in the same way as described above. Every 7th
section, starting approximately from Bregma -1.46 mm and totaling 8 sections per animal covering
the whole hippocampal formation, was stained using a BrdU antibody. Staining for the proliferation
marker Ki-67 was used as a control to validate BrdU stainings in 15 weeks old mice. For BrdU
stainings, sections were pre-treated, to denature DNA with 1 % Triton X-100 in PBS for 15 minutes, 2
M Hydrochloric acid for 30 min at 37°C, and 0.1 M Boric acid (pH 8.5) for 10 min. For all stainings,
sections were then incubated for 2 hours in blocking solution (3 % Bovine Serum Albumin (Sigma),
0.25 % Triton X-100 (Sigma), 0.01 % NaN3 (Merck) in 0.1 M PBS) followed by over-night incubation
with primary antibody at 4° C. Primary antibodies used were rat anti-BrdU (1:500; Accurate Chemical;
Westbury, NY, USA) and rabbit anti-Ki-67 (1:500; Novocastra, Newcastle upon Tyne, UK). Sections
were further incubated with fluorescent secondary antibodies (1:500) for 1.5 hours; anti-rat IgG
Alexa Fluor™555 and anti-rabbit IgG Alexa Fluor™488 (Invitrogen). All immunohistochemical steps
were performed free-floating and on 5-7 brains/genotype. Sections were mounted, dried and finally
covered with ProLong® Gold Antifade with DAPI (Invitrogen).
For quantification of BrdU and Ki-67 positive cells, images were obtained using a Zeiss 710LSM laser
scanning microscope (Carl Zeiss HB, Stockholm, Sweden). Positive cells were counted manually in all
8 hippocampal sections from each mouse using the count tool in Adobe Photoshop. All
quantifications were performed by an investigator blind to mouse genotypes. To assess the number
and structure of immature neurons in the DG of adult 15-week-old mice, sections were stained using
a rabbit anti-double-cortin (DCX) ( 1:500 dilution; Abcam, Cambridge, UK) antibody and the
ImmPRESS anti-rabbit Ig (peroxidase) polymer detection kit (Vector Laboratories, USA). Images were
obtained using a Zeiss Axioskop 2 Plus microscope with an Axiocam (Carl Zeiss HB, Stockholm,
Sweden) and cells were manually counted.
To further investigate hippocampal function,7, 8 parvalbumin (PA) positive γ-aminobutyric acid (GABA)
interneurons were calculated using IHC. Sections from 15-week-old mice were incubated with a
primary mouse anti-PA antibody (1:1000 dilution; Swant, Bellinzona, Switzerland) using the protocol
described for Ki-67 stainings above. Quantifications were performed by manual counting in the DG,
CA1+2 and the CA3 regions of the mouse hippocampus. For all IHC markers, total numbers were also
corrected for total hippocampal volumes as measured by MRI.
Behavioral tests
Behavioral tests were applied to Wt and CYP2C19Tg-Hem male mice at two different ages;
adolescent (7-week-old) and adult (15-week-old) mice. All mice were handled by the experimenter
once a day for four consecutive days before a specific test or test battery described below. Animals
were always acclimatized to the test room at least one hour before behavioral sessions and tested
during the light phase by an experimenter blind to their genotype.
The tail-suspension test (TST) was evaluated on two sets of CYP2C19Tg-Hem and Wt mice that were
either only evaluated in this test (n=6-10 per group) or after exposure to the Morris water maze
(MWM). MWM was only employed on 15-week-old mice (Wt: n=10; CYP2C19Tg-Hem: n=9). The
open-field (OF) and the light-dark box (LDB) tests were performed consecutively on the same group
of mice at both 7 and 15 weeks of age with at least three days between the tests (n=15 per group).
Tail-suspension test
Mice were suspended by the tail on a Plexiglas table with their head approximately 15 cm above the
ground using an adhesive tape placed roughly 0.5 cm from the tip of the tail. The sessions were
recorded using a video camera and immobility time was calculated manually for the whole 6-minute
session. Mice were considered immobile when they hanged passively without moving.
Morris water maze
To asses spatial learning abilities of the CYP2C19Tg-Hem mice the MWM was employed.9 The water
maze had a diameter of 120 cm with a platform diameter of 10 cm and the water temperature was
23-25 °C. Four visual cues were placed around the pool. Mice were evaluated in a pre-training
session with a visible platform to study motivation and swim capacity. Three days after the pretraining session, mice were trained for five consecutive days with four trials per day with an intertrial interval of at least 20 minutes. Training sessions were performed with a hidden (transparent)
platform in the north-west quadrant of the pool and the mice were randomly placed at four different
starting positions per day; north, west, south and east. Three seconds on the platform was
considered a successful response during the training sessions. If the platform was not located during
the 90 second session time the mice were gently helped to the platform and required to stay for 10
seconds. In the retention test, performed without the platform one day after the last training day,
mice were placed in the south-east quadrant and left in the water for 60 seconds. Spatial learning
was evaluated by measuring time to first platform crossing, number of total platform crossings and
total time spent in the platform quadrant.
Open-field
To investigate spontaneous motor activity and response to a novel open space, mice were placed in
the middle of a square open-field opaque Plexiglas box (50 cm3). No bedding was used and boxes
were cleaned with 70 % ethanol between each trial. Each mouse was video-recorded for 30 minutes
and videos were analyzed using the top-view based behavior analysis software TopScan Lite (Clever
Sys Inc, Reston, Virginia). The open-field was divided into peripheral, intermediary and central
regions. Distances traveled and time spent in each delimited area was recorded in 5-minute bouts.
Light-dark box
The light-dark box was used for assessing anxious behavior and consisted of two compartments: one
dark, closed compartment (black Plexiglas) and one illuminated, open compartment (white Plexiglas).
Both compartments measured 25 cm3 and were connected by an opening (10 cm wide and 5 cm tall)
to allow animals to freely move from one compartment to the other.
Each individual mouse was gently placed in the light box facing away from the dark compartment and
video-recorded for 5 minutes. Videos were analyzed using the top-view based behavior analysis
software TopScan Lite (Clever Sys Inc, Reston, Virginia). Time spent in light and dark compartments
and number of transitions between the compartments was recorded.
Effect of acute restraint stress on serum corticosterone levels and expression of c-fos in the
hippocampus
Both 7- and 15-week-old mice were either decapitated directly without any prior stressor (control
samples) or subjected to restraint stress for 30 minutes by use of ventilated 50 ml Falcon tubes. Mice
subjected to restraint stress were removed from the tubes and either immediately decapitated or
allowed to recover in their home cages for 30 minutes, after which they were decapitated.
Corticosterone (CORT) quantifications were performed by an enzyme-linked immunosorbent assay
specific for mouse/rat CORT according to the manufacturer’s instructions (ELISA, MBS494312,
MyBiosource).
Hippocampi were dissected from all decapitated mice and placed in RNAlater solution (Qiagen). RNA
isolation and cDNA synthesis was performed in the same way as described above. For mRNA
quantification of the immediate-early gene (IEG) c-fos a Taqman assay from Applied Biosystems was
employed (Cat No: Mm00487425_m1).
Supplementary Figure 1: Distribution of CYP2C19 mRNA expression in
the brain
Relative CYP2C19 mRNA expression levels in three different brain regions at embryonic day 18 (E18)
in 4 CYP2C19Tg-Hem embryos. To investigate the distribution of CYP2C19 mRNA expression during
the embryonic peak at E18, the hippocampus, cortex and the rest of the cerebrum were dissected
and processed for mRNA expression. The hippocampi were pooled from the four embryos due to
their low weight. No significant differences in mRNA expression patterns were seen between the
three regions. HC= hippocampi, *Cerebrum without cortex and hippocampus
Supplementary Figure 2: Size measurements in brain sections from
CYP2C19Tg-Hom and Wt mice at PND0
Area measurements of cortex and hippocampus in brain sections of CYP2C19Tg-Hom and Wt mice in
relation to whole brain area. Consecutive sections of brains from three Wt and three CYP2C19TgHom mice were scanned with an Epson perfection V750 Pro scanner and the cortex and
hippocampus were manually outlined in all sections using the pen tablet, Intuos4 (Wacom, Tokyo,
Japan). The total area was calculated in Adobe Photoshop as the number of pixels within the selected
area. The data are shown as in relation to whole brain volume and with mean Wt values set as 100%.
Relative size reduction was 15% (p=0.029) and 61%(p=0.014) for the cortex and hippocampus,
respectively.*p<0.05
Supplementary Figure 3: Brain weights of CYP2C19Tg-Hem mice and
Wt controls
Brain weights of CYP2C19Tg-Hem mice and Wt controls. Brain weights were significantly lower in
CYP2C19Tg-Hem mice compared to Wt controls at both 7 (5.4 %; p=0.007) and 15 (5.6 %; p=0.0004)
weeks of age. Brain weights include the cerebellum and data are presented as mean with S.E.M.
**p<0.01; ***p<0.001
Supplementary Figure 4: BrdU and Ki-67 positive cells in the
hippocampus
Seven-week-old mice were intraperitoneally injected with 75mg/kg bromodeoxyuridine (BrdU);
either twice, with a 16 hours gap, and sacrificed 2 hours after the last injection (a), or injected once a
day for 3 consecutive days and sacrificed 4 weeks after the first injection (b). Brain sections were
stained by immunohistochemistry using Ki-67 or BrdU antibodies. (a) No difference in cell
proliferation, as measured by BrdU and Ki-67 positive cells in the DG of the hippocampus were
observed in 7- or 15-week-old mice. (b) There was furthermore no difference in number of surviving
BrdU positive cells at 4 weeks after the final injection. Data are presented as mean with S.E.M.
Supplementary Figure 5: Number of BrdU, Ki-67, DCX, and PA positive
cells in the hippocampus corrected by hippocampal size
In Supplementary Figure 4 and in Figure 4, absolute values of number of positive cells are presented.
Since the hippocampal formation is 7.11 % smaller in CYP2C19Tg-Hem mice, as calculated from the
magnetic resonance images absolute numbers have here been corrected for this size reduction. (a)
No differences in cell proliferation, as measured by neither BrdU and Ki-67 positive cells nor DCX
positive cells were seen after size corrections. As for the calculations of total numbers, DCX positive
cells are the only marker with a significant difference between CYP2C19Tg-Hem and Wt mice. (b)
There was furthermore no difference in number of surviving BrdU positive cells at 4 weeks after the
final injection. The difference previously seen in PA positive cells in the DG of the hippocampus is
now border-line significant when the total number is corrected for size (p= 0.071). Data are
presented as mean with S.E.M. ***p<0.001
Supplementary Figure 6: Immobility time in the Morris Water Maze
Immobility time at training day 1 and 5 in the Morris Water Maze (MWM). CYP2C19Tg-Hem mice and
Wt controls were subjected to the MWM, including 5 training days with 4 sessions per day and one
retention test performed on the day after the last training session (data not shown). CYP2C19Tg-Hem
mice developed a despair (floating) behavior during the training week as seen above.
Immobility/floating time was measured manually in the first session of training day 1 and 5. At 15
weeks of age immobility time was significantly higher in CYP2C19Tg-Hem mice compared to Wt
controls (p=0.018). Due to the discrepancy in immobility time, memory function could not be
evaluated. Data are presented as mean with S.E.M. *p<0.05
Supplementary Figure 7: Plasma corticosterone levels before and after
acute restraint stress
Plasma corticosterone levels before and after acute restraint stress. CYP2C19Tg-Hem and Wt mice
were subjected to acute restraint stress by use of a 50 ml ventilated Falcon tube. Both 7- and 15week-old mice were either decapitated directly without any prior stressor (0 min) or subjected to
restraint stress. Mice subjected to restraint stress were either immediately decapitated (30 min) or
allowed to recover in their home cages for 30 minutes, after which they were decapitated (60 min).
Corticosterone quantifications were performed by an enzyme-linked immunosorbent assay according
to the manufacturer’s instructions (ELISA, MBS494312, MyBiosource). No differences in plasma
corticosterone levels were detected between CYP2C19Tg-Hem mice and Wt controls within
respective age group. Data are presented as mean with S.E.M.
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