Download Jukic et al. SUPPLEMANTARY SUPLEMENTARY METHODS En1+/

Jukic et al.
SUPPLEMANTARY
SUPLEMENTARY METHODS
En1+/Otx2 mutants
The generation and genotyping of En1+/Otx2 mice has been reported earlier (Broccoli,
et al 1999; Brodski, et al 2003). Mutants were kept on a CD-1 genetic background.
For lithium and carbamazepine repetitive OFT experiments mutants were
backcrossed for 6 generations to a Black Swiss genetic background. Mice were kept
in a temperature-controlled (21–22 °C) room under reversed 12 h light/dark cycle
(lights were turned off at 10:00 a.m.), with free access to food and water. After
weaning, animals were kept as groups of 4-6 in a cage. All experiments were
performed on 3-6 months old males, which were single caged one week prior to any
behavioral test. OFT, EPM, LDB, and social interaction tests were all conducted
between 12:00 and 17:00 in the separate behavior testing room. Mice were
transferred to the behavior testing room 30 min prior to the beginning of the above
mentioned behavioral tests in order to habituate to the new environment. In case that
the same animals were used repetitively in more than one behavioral test
(Supplementary figure 2a, d, e), tests were conducted from the least invasive to the
most invasive test: OFT, LDB, and EPM with at least 72 h break between the tests
(Flaisher-Grinberg and Einat, 2010).
Home cage locomotor activity
Prior to the testing, animals were anesthetized with an intraperitoneal injection of a
ketamine-xylazine mixture (100 mg/kg and 10 mg/kg, respectively) and chronically
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implanted with DSI TA-F10 telemetry probes. The transmitter was able to freely move
among the peritoneal organs because it was not attached to the peritoneum. After ten
days of recovery, locomotor activity of animals was recorded and analyzed with DSI
DataQuest ART 4.3 software. Locomotor activity (counts) was obtained by counting
the number of impulses, detected by changes in signal strength, per time unit. The
signal was received by an antenna under each animal’s cage and transferred to a
peripheral processor connected to a personal computer. All transmitters were
calibrated before surgery and at the completion of experimentation to ensure the
validity of biotelemetry measurements. Locomotor activity was calculated as an hourly
or daily average of the raw data, collected at 1 s intervals by counting the number of
impulses, detected by changes signal strength. (Tarasiuk, et al 2014).
Open Field Test
Mice were placed in the open fields made of Plexiglas and measuring 40 x 40 x 30
cm under red light. The box was cleaned with a dilute ethanol solution (10%)
between trials and carefully dried. For locomotor activity, animals were videotaped
and recordings were analyzed off-line using Noldus EthoVision® 9 program. In order
to define the center/periphery exploration, we defined the center zone in Noldus
Ethovision® 9 program (20 x 20 cm). In order to study intra-session habituation of
En1+/Otx2 mice in more detail, we quantified the level of activity change ratios (ACR).
Values derived with the formula ACR=b/(a+b), were computed for each trial of each
individual mice as described previously (Bothe, et al 2004; Leussis and Bolivar
2006). In this formula a is the activity during the first 5 minutes, and b is the
averaged value of the 5 min intervals after 15 minutes of habituation. Values below
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0.5 indicate a decrease, values above 0.5 indicate an increase and values closely
around 0.5 indicate no changes in activity over time.
Light-Dark Box
The experiments were conducted in a well-lit behavioral testing room. The Plexiglas
apparatus was constructed from a 13 x 40 x 30 cm dark compartment closed with a
lid and a 27 x 40 x 30 cm light compartment. Those two were joined by a 5 x 5 cm
opening in the wall between them. The apparatus was cleaned with a dilute ethanol
solution (10%) between trials and carefully dried. Mice were placed in the light
compartment, videotaped for 10 min, and recordings were analyzed off-line using the
Noldus EthoVision® 9 program.
Elevated Plus Maze
The experiments were conducted in a well-lit behavioral testing room. The apparatus
was constructed out of transparent Plexiglas and consisted of two opposing open
arms and two perpendicular opposing closed arms. The arms were 50 cm in length
and 5 cm wide. The closed arms had walls that were 40 cm high, whereas the open
arms had a half cm ledge to prevent falling. The EPM was wiped clean with a dilute
ethanol solution (10%) between trials and carefully dried. At the start of the trial, the
mouse was placed in the center of the maze, facing a closed arm and videotaped for
5 min. The number of entries and time spent in each of the arms were analyzed off
line using the Noldus Ethovision® 9 program. We measured the number of entries
and the time spent in the open arms as a percentage of the total number of entries
and the time spent in all arms.
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Chronic sucrose preference
Prior to the experiment animals were exposed for two weeks to two bottles of tap
water. During the following 6 weeks animals received a choice between water and a
2% or 5% sucrose solution and sucrose preference was calculated for the last four
weeks according to the weekly protocol. Every week, mice were given access to two
drinking bottles positioned one next to the other. Twenty-four hour fluid consumption
was measured by daily weighing the bottles during the last hour before the onset of
the dark phase (9:00-10:00 AM). All interaction with the animals was conducted
during this period. Sucrose solution (2% and 5% prepared in tap water) was placed
in one bottle and the tap water in the other. In order to study fluctuations in sucrose
preference over time, we designed a one week protocol according to a previously
described method (Taliaz, et al 2011). For day one and two of the week, the sucrose
solution was placed on the left side of the cage and water on the right. On day three
of the week, only tap water was administered on both sides and all animals were
weighed. On day four and five the sucrose solution and water were placed in reverse
to control for side preference. On day six and seven of the week animals received
two tap water bottles. Sucrose preference for each mouse was defined as the
average percentage of sucrose consumption of the total liquid consumption. Sucrose
and total liquid intake were calculated by normalizing the volume of consumed
sucrose solution or total liquid intake with the animal weight for that week. Only day
two and day five were included in the analysis. This procedure was repeated for six
weeks, out of which the first two weeks represent the adjustment period and were
not taken into the analysis.
Social interaction test
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Two mice of the same age, weight and genotype were placed in the opposite corners
of the open-field under the red light as previously described (Shaltiel, et al 2008).
Their activities were videotaped for 5 min, and the time spent in social activity was
manually scored using the Noldus EthoVision® program. The open field was wiped
clean between trials with a 10% alcohol solution.
Acute (IP) injections
Quipazine maleate (5mg/kg, purchased from Sigma-Aldrich Israel) and CP-809101
(5mg/kg, purchased from Tocris Bioscience) were both dissolved in saline and
injected IP into test animals 15 minutes prior to behavioral tests. These doses were
chosen to robustly increase serotonin receptor activation in the brain as previously
described (Hutchinson, et al 2012; Kaur and Kulkarni 2002; Siuciak, et al 2007).
Olanzapine (purchased from Sigma-Aldrich Israel) was dissolved in 0.1 M HCl,
diluted with water, and titrated with 0.1 M NaOH towards neutrality (pH higher than
5) without any precipitation. Vehicle and olanzapine solutions (1 mg/kg) were
injected IP into test animals one hour before behavioral tests. This protocol was
chosen according to the previously reported anti-manic effect in mice (Engel, et al,
2010). Animals were tested in the OFT, LDB, and EPM in this order for the animals
treated with olanzapine (figures S4, 3a, b) and quipazine (figure 4a, b), with a distinct
cohort used for each drug. Established wash-out periods of 14 days were used
between repetitive acute IP injections followed by behavioral tests (Mattiuz et al,
1997; Valencia-Flores et al, 1990). The sequence of the tests was conducted from
the least invasive to the most invasive test (Flaisher-Grinberg and Einat, 2010).
Chronic lithium and carbamazepine treatment
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Lithium carbonate and carbamazepine (purchased from Sigma-Adrich Israel) were
mixed into powdered food. For the lithium experiments, an additional bottle of saline
was added to each cage in order to compensate for the electrolyte loss in lithium
treated animals. After one week of food containing 0.12% lithium, lithium
concentrations were raised to 0.24%, for four weeks before the start and throughout
the testing (Einat, et al 2003). After the experiment lithium serum concentrations
were measured and only animals having lithium levels in the therapeutic range (0.51.2 mEq/l) were included in the analysis. Six OFTs were conducted during a period
of three weeks. For carbamazepine treatment mice received for one week powdered
food containing carbamazepine at a concentration of 0.25%. Two weeks before the
start and throughout the testing, carbamazepine concentrations in the food were
0.5% as previously suggested (Kara, et al 2014). Four OFTs were conducted during
a period of two weeks.
Statistical analysis:
All results are expressed as mean ± SE. IBM® SPSS® Statistics 21 software was
used for the statistical analysis. A two-tailed unpaired Student's t-test was used for
the comparative analyses. For the experiments that included pharmacological
treatment, in addition to genotype, treatment effect was analyzed as a factor for twoway multifactorial analysis of variance (ANOVA test). Where significant effects were
detected, Fisher’s LSD post hoc analysis indicated significant differences between
individual groups.
INRICH Pathway analysis
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1. Compilation of gene lists: Lists of genes specifying DA and 5HT neurons were
compiled
in
a
two-step
process.
In
the
first
step,
a
http://www.ncbi.nlm.nih.gov/pubmed search was performed using the search terms
“dopamine and development”, “dopamine and embryo”, as well as “serotonin and
development” and “serotonin and embryo”, including studies published up until May
2014. In a second step, the published studies obtained from this search were
screened according to the following criteria: 1. Studies performed in vivo 2. Studies
performed in mammals 3. Studies related to midbrain dopaminergic or rostral
serotonergic neurons. 4. Studies related to the specification of monoaminergic
neurons. We choose these criteria according to the following rationales. 1: In vitro
studies are not necessarily relevant in vivo (Hegarthy et al., 2013). 2: Only
experiments performed in mammals were considered, since pathways for
monoaminergic neuronal specification in non-mammals differ substantially from
mammalian pathways (Hegarthy et al., 2013). 3. Only data relevant for midbrain DA
or rostral 5HT sub-populations were considered, since they are affected in En1+/Otx2
mutants and relevant to psychiatric disorders. In contrast, other DA and 5HT subpopulations, not affected in En1+/Otx2 mutants, e.g. olfactory DA or caudal hindbrain
5HT neurons projecting to the spinal cord, are less likely to be involved in these
disorders. 4. Since En1+/Otx2 mutants exhibit alterations in the specification of
monoaminergic neurons rather than in their migration or projection (Brodski, et al
2003), only studies that report this aspect of development were taken into
consideration. Based on these publications a gene list was compiled for DA as well
as for 5-HT neurons.
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2. INRICH Analysis In order to test if the sequence variants of selected gene sets
are correlated with psychiatric disorders we used INRICH, an interval-based
enrichment analysis tool for GWAS (Lee, et al 2012). INRICH evaluates each target
set by considering the number of intervals that contain at least one target gene
sequence. The data used for the statistical analysis was obtained from the metaanalysis study of bipolar disorders conducted by the Psychiatric Genomics
Consortium (PGC) (Psychiatric GWAS Consortium Bipolar Disorder Working Group
2011). Associated LD-independent genomic intervals were based on the LD pruned
SNP dataset downloaded from PGC website (http://pgc.unc.edu) on 10.11.2013 for
each disease (Supplementary files: pgc.bip.clump for BPD, pgc.scz.clump for SCZ,
and pgc.mdd.clump for MDD). Intervals were spanning 250kbp up/downstream on
variants attaining statistical significance for relationships at P≤0.001. We defined
gene regions as 100 kb up/downstream of the transcription starting/ending sites for
all genes on autosomal chromosomes based on the ENTREZ gene map hg 18
(Maglott,
et
al
2005)
downloaded
from
the
INRICH
website
(atgu.mgh.harvard.edu/inrich/downloads.html) on 10.11.2013 (Supplementary file
entrez.gene.map). Significance was assessed through 100,000 permutations in
which randomly selected genomic regions matched for gene and SNP density were
compared with the target interval set. In order to test whether there is a degree of
correspondence between the test intervals and the target sets, we calculated global
enrichment. It indicates whether more targets than expected by chance are within
one of the associated intervals and within at least one set that is nominally significant
at P≤0.01. Finally, resampling-based 10000 second-step permutation was conducted
in order to adjust the empirical P-values for testing multiple candidate target sets.
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Since we repeatedly tested the hypothesis that sequence variants of genes
directing the development of DA neurons are associated with BPD using different
data sets, we corrected for multiple testing. We used the p-values obtained from the
INRICH analysis using the lists of genes directing the specification of dopaminergic
neurons compiled by us and by Hegarthy et al, for the FDR correction, according to
SanGiovanni and Lee, 2013.
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SUPPLEMENTARY FIGURE 1
En1+/Otx2 mutants show increased spontaneous fluctuations in their locomotor
activity in their home cage under constant darkness
After five days in the normal light/dark cycle, the animal activity was recorded daily
for 33 days under constant darkness, normalized to the average activity level of that
animal during the entire period, and shown as a function of day in separate plots for
each of the nine (a) WT and eleven (b) En1+/Otx2 mutant animals. (c) En1+/Otx2
mutants showed increased intra-individual fluctuation as indicated by an increased
CV (t18=2.157, p=0.047). Two-tailed unpaired Student's t-test: *p<0.05
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Jukic et al.
SUPPLEMENTARY FIGURE 2
En1+/Otx2 mutants show increased levels of locomotor activity, risk taking
behavior and reduced levels of hedonic-like and social behavior.
(a) In the OFT, mutants were more active (t16=2.972, p=0.003) and entered more into
the center than controls (t16=3.572, p=0.001), while the percentage of the distance
traveled in the center did not significantly differ between genotypes (t16=0.997,
p=0.452). (b) The time mutants spent in social interaction was decreased compared
to the WTs (t16=2.945, p=0.011). (c) The sucrose preference in the mutant mice was
lower compared to WTs (t18=3.459, p=0.003), while the sucrose (t18=0.631, p=0.536)
and total liquid intake (t18=1.476, p=0.157) did not differ between genotypes. (d) In
the EPM, mutants showed more entries into (t18=3.583, p=0.001) and time spent
(t18=2.149, p=0.036) in the open arms compared to controls, while number of entries
into all arms did not differ between groups (t18=0.684, p=0.497). Mutants spent
significantly more time in the light compartment in the LDB than WTs (t18=5.149,
p<0.001). Two-tailed unpaired Student's t-test: *p<0.05, **p<0.01, ***p<0.001
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SUPPLEMENTARY FIGURE 3
The activity of En1+/Otx2 mutants correlates with their risk-taking behavior and
habituation, but not with their fluctuations in activity
Locomotor activity, ACRs, the number of entries into and the percentage of distance
traveled in the center of the OFT were analyzed for the 45 minute OFT for each
animal. (a) The frequency of entries to the center correlated with activity levels for
both mutants (r27=0.882, p<0.001) and controls (r29=0.772, p<0.001), while the
distance traveled in the center correlated with activity levels only in En1+/Otx2
(r27=0.447, p=0.015) and not in WT animals (r29=0.089, p=0.633). (b) Activity levels
and ACRs were correlated for both WTs (r28=0.699, p<0.001) and mutants
(r27=0.661, p<0.001). (c) The activity of the animals was not correlated with the
coefficients of variance for both the mutants (r27=0.187, p<0.418) and the controls
(r27=0.015, p<0.953) in six repetitive one hour OFTs.
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Jukic et al.
SUPPLEMENTARY FIGURE 4
Olanzapine reduces activity and risk-taking behavior in both mutants and
controls
Vehicle and olanzapine solutions (1 mg/kg) were injected IP into animals one hour
before they were tested in the OFT for 60 minutes. Olanzapine decreased the (a)
activity (TreatmentF1,36=4.834, p=0.034) and the (b) number of entries into the center
(TreatmentF1,36=8.092, p=0.007) of both WT and En1+/Otx2 mutants, without a differential
effect on genotypes (Genotype*TreatmentF1,36=0.588, p=0.448;
Genotype*TreatmentF
1,36=0.313,
p=0.580). There was no significant interaction between the genotype and the
treatment for the percentage of distance traveled in the center of the OFT
(Genotype*TreatmentF1,36=1.023, p=0.319). (c) ACR was increased after the acute
olanzapine treatment (TreatmentF1,36=6.829, p=0.013) with no selective effect to neither
of the genotypes (Genotype*TreatmentF1,36=0.338, p=0.564).
20
Jukic et al.
SUPPLEMENTARY FIGURE 5
En1+/Otx2 mutants maintained on a BS genetic background show increased
fluctuations in their locomotor activity and risk-taking behavior
En1+/Otx2 mutants were exposed to the repetitive OFT, the values for the distance
traveled were normalized by the individual animal average, and were depicted by
different colors for the each animal as the function of the trial. En1+/Otx2 mutants
showed an increase in the CV in their activity levels in the repetitive OFT compared
to WTs when maintained on the CD1 genetic background (t18=3.912, p=0.001) and
on the BS genetic background (t18=4.143, p=0.001). The number of entries and the
percentage of the distance (center vs total) traveled in the center was also measured
in animals on both backgrounds. In contrast to (c) the CD1 genetic background
where only number of entries into (t18=4.32, p<0.001) and not the percentage of
distance traveled in the center (t18=0.648, p=0.52) was increased in mutants, (d)
mutants on BS background entered more into (t18=4.939, p<0.001) and traveled
more distance in the center (t18=7.537, p<0.001). Taken together, these results
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Jukic et al.
suggest that En1+/Otx2 mutants on a Black Swiss genetic background maintained the
intra-individual differences observed on a CD-1 genetic background and that they
showed a stronger manic-like phenotype in the OFT. Two-tailed unpaired Student's ttest: ***p<0.001
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Jukic et al.
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