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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 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 OFT 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

animals 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 5 x 5 cm

opening in the wall between them. The apparatus was cleaned with a dilute ethanol

solution (10%) between animals 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 animals 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 (0.5-

1.2 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

treatments, in addition to genotype, treatment effect was analyzed as a factor for

two-way multifactorial analysis of variance (ANOVA test). Where significant effects

were detected, Fisher’s LSD post hoc analysis indicated significant differences

between individual groups. False discovery rate (FDR) correction of the p-values

was performed for multiple testing.

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 to 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 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 sub-

populations, 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 control 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 meta-

analysis 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). The original downloaded datasets are available in the

supplementary datasets folder. 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 or

not 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-

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step permutation was conducted in order to adjust the empirical P-values for testing

multiple candidate target sets.

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, **p<0.01,

***p<0.001

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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*TreatmentF1,36=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). (d) 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).

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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

suggest that En1+/Otx2 mutants on a Black Swiss genetic background maintained the

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Jukic et al.

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 t-

test: *p<0.05, **p<0.01, ***p<0.001

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Jukic et al.

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