Impaired hippocampus-dependent and facilitated striatum-dependent behaviors in mice lacking the delta opioid receptor

Julie Le Merrer, Xavier Rezai, Grégory Scherrer, Jérôme A.J. Becker and Brigitte L. Kieffer

Supplementary information

Supplementary Material and Methods

Behavioral experiments

Experiments designed to compare Oprm1+/+ and Oprm1-/- mice were performed on

independent cohorts of naïve animals, except for the runway test that was performed in the

same cohort as rotarod experiment (easy condition). Rotarod testing was performed when the

animals were aged 7 weeks. After completion of this test (1 week), the animals were given a

1-week recovery period before food restriction started in view of runway training.

Elevated plus-maze (EPM): preliminary experiment. The EPM was a plus-shaped maze

elevated 52cm from base, with black Plexiglas floor, consisting of two open and two closed

arms (37x6 cm each) connected by a central platform (6x6 cm). The walls of the closed arms

were made of 18 cm-high clear acrylic. Light intensity in open arms was set at 15 lx. The

apparatus was placed over an infrared-lit platform. The movement and location of the mice

were analyzed by an automated tracking system equipped with an infrared-sensitive camera

(Videotrack; View Point, Lyon, France). All sessions were videotaped for further analyses.

The test started when the mouse was placed on the central platform facing a closed arm and

lasted 5 minutes. The distance travelled, time spent and entries in different parts of the

apparatus were counted automatically (videotracking). Head dips were scored manually on

video recordings to assess risk taking behavior. Three ratios were calculated as measures of

anxiety: an entry ratio (entries in open arms/total entries in arms), a distance ratio (distance

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travelled in open arms/total distance travelled in arms) and a time ratio (time spent in open

arms/total time spent in arms). Total distance travelled in the apparatus (including central

platform) was used as an index of forward activity.

Novel object recognition: experiments 1 and 2. The experiments were conducted in 4 equal

square arenas (50x50 cm) separated by 35 cm-high opaque grey Plexiglas walls. Light

intensity of the room was set at 15 lx to facilitate exploration and minimize anxiety levels,

previously shown to be high in Oprd1-/- mice (Filliol et al. 2000) and naltrindole-treated

animals (Perrine et al. 2006; Saitoh et al. 2005). These conditions were chosen based on the

results of a preliminary experiment showing that Oprd1-/- mice do not display signs of

increased anxiety in the EPM under such light conditions (see above). The floor was a white

Plexiglas platform (View Point, Lyon, France), spread with sawdust. The room was equipped

with an overhead video camera connected to a computerized interface, allowing visualization

and recording of behavioral sessions on a computer screen in the adjacent room.

The experimental paradigm was adapted from (Carey et al. 2009), and lasted for 2 days. On

day 1, the animals were placed in an arena for a 15 min-habituation session with two copies of

an unfamiliar object (T-shaped plastic tubing, 1.5x3.5 cm). These objects were not used later

for recognition test. On day 2, the recognition test was performed. In experiment 1, this test

consisted of 3 trials of 10 minutes separated by 2 intertrial intervals of 5 minutes, during

which the animals were returned to their home cage (see Figure 1). In experiment 2, the third

trial (object phase) was omitted. Indeed, pilot studies revealed that, due to anxiogenic effects

of naltrindole (Perrine et al. 2006), exploration of the objects during this third phase was not

sufficient to allow a reliable measure of object recognition. On the first trial, or familiarization

phase, the mice were presented with two copies of an unfamiliar object. On the second trial,

or place phase, one of the two copies was displaced to a novel location in the arena. Finally,

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on the third trial, or object phase, the copy that had not been moved on previous trial was

replaced by a novel object. Stimuli objects used in all previous experiments were Lego bricks,

plastic rings, dices or marbles (size 1.5-3x2-3 cm). The identity of the objects as well as the

spatial location in which these objects were positioned was balanced between subjects. The

number of visits and the time spent to explore each object were scored manually on video

recordings. A visit was acknowledged when the nose of the mouse came in direct contact with

an object. A percentage of discrimination was calculated for number of visits and time

exploring the objects as following: exploration of displaced or novel object / total exploration

* 100. The percentage of discrimination during familiarization phase was arbitrary calculated

for the object located in the right up corner of the arena. Animals that failed to explore the

objects more than 2 sec during familiarization phase were excluded from further analysis, as

well as mice that failed to explore one of the objects during place and object phases

(Experiment 1: 5 WT and 3 Oprd1-/- mice were excluded; Experiment 2: 4 WT and 4 Oprd1-/-

mice were excluded).

Place and response learning: experiments 3 and 4. Experiments were run in a cross-shaped

maze, adapted from (Passino et al. 2002). Elevated 40 cm above the floor, the maze consisted

of four arms (35 cm x 8 cm) with black Plexiglas floors enclosed in transparent Plexiglas

walls (15 cm), except for the terminal half of west and east arms. Removable sliding doors

made of black opaque Plexiglas delimited two starting boxes (10 x 8 cm) at the end of south

and north arms. Four more identical sliding doors separated each arm from the central

platform (8 x 8 cm). A food well (2 cm diameter) was inserted into the floor at 1 cm from the

distal end of east and west arms. The maze was located in a testing room that contained

several extra-maze visual cues, and maintained in a constant orientation during the

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experiment. Light intensity in the room was set at 15 lx. The floor and walls were cleaned

daily to limit intra-maze olfactory cues.

Place learning was evaluated in experiment 3 using a dual-solution cross-maze task, whereas

response learning was measured in experiment 4 using a single-solution cross-maze task (see

Packard 2009). In both experiments, mice were reduced to 85% of their ad lib feeding weights

over 7 days before maze training and maintained at this weight throughout the experiment.

The animals received sucrose reward tablets (5-10 per mouse; Formula 5TUT-formerly

PJFSC-20 mg, TestDiet, Richmond, USA) in their home cage for three consecutive days

before maze habituation. Habituation lasted three days. In experiment 3, access to the north

arm of the cross maze was blocked with a sliding door during this phase. In experiment 4, all

arms were accessible. The mice were placed in the south start box (experiment 3) or in the

south or north start box (counterbalanced, experiment4) and allowed to explore the maze for 5

min. On day 1, sucrose tablets were available throughout the apparatus. On day 2, a trail of

five tablets leading to the food cup was placed along the length of west and east arms. On day

3, tablets were present only in the food well at the end of baited arms.

In experiment 3, we adapted a classical dual-solution cross-maze protocol to assess spatial

strategy in rodents (Deipolyi et al. 2008; Packard 1999; Passino et al. 2002). Training (4 trials

per day) started immediately after habituation. The north arm was closed and mice were

released from the south arm, after a 15 s-confinement in the start box (see Figure 2). For half

of the animals, single food-pellet bait was located in the east arm, while the other half of the

animals received food in the west arm. After entering an arm, the door was closed and mice

were confined for at least 20 s or until food was consumed. If a mouse failed to eat the food

within 5 min, the trial was terminated. A correction procedure was used during the first two

training sessions only: mice making an incorrect response were allowed to trace back to the

baited maze arm and consume the food pellet. On probe trials (days 5, 12 and 19), mice were

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released from the north arm and had access to the previously baited arm (place learning) or to

the opposed arm (response learning). Food was available in both arms (see Figure 2). Two

parameters were recorded: choice accuracy was expressed as the percentage of entries in the

baited arm during each session, and choice latency was recorded as the latency to enter an arm

of the maze (baited or not).

In experiment 4, a single-solution response learning task was designed based on (Packard

2009). For two days after habituation, mice received pre-training sessions of two trials per day

during which they were released (after a 15 s-confinement in start box) alternatively from

north and south arm (see Figure 2). During these sessions, mice were allowed to correct a

wrong choice and retrieve food in the baited arm. Then training sessions of 5 trials per day

started. Mice were released (after a 15 s-confinement in the start box) from north or south

arm, following a random sequence (for example: north-north-south-north-south). Each mutant

animal was matched to a WT mouse for the release sequence. North arm was closed when the

animals were released from south arm. Conversely, south arm was not available when mice

were released from north arm. Half of the animals received food when turning left, and the

other half received food when turning right. After entering an arm, the door was closed and

mice were confined for at least 20 s or until food was consumed. If a mouse failed to eat the

food within 5 min, the trial was terminated. A correction procedure was used during the two

pre-training sessions and the first training session: mice making an incorrect response were

allowed to trace back to the baited maze arm and consume the food pellet. Two parameters

were recorded: choice accuracy was expressed as the percentage of entries in the baited arm

during each session, and choice latency was recorded as the latency to enter an arm of the

maze (baited or not). In this experiment, one mutant male mouse failed to eat the sucrose

reward pellets and was hence excluded from testing.

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Skill motor learning: experiment 5. Mice were aged 7 weeks at the beginning of this

experiment, to limit weight bias. The animals were placed on a rotarod apparatus (Bioseb,

Valbonne, France) accelerating from 4 to 40 rpm in 5 min. In the “easy” condition, the rod

was covered with insulation tubing, which external perimeter was 5 cm. In the “hard”

condition, tubing was removed and external perimeter of the rod was 3 cm. Light intensity in

the room was 40 lx.

Behavioral testing lasted five days. On day 1, mice were habituated to rotation on the rod

under a constant speed of 4 rpm, until they were able to stay on the rod more than 180 s under

the “easy” condition or 90 s under the “hard” condition. From day 2 to day 5, mice were

tested for three trials a day (60-s intertrial interval) on consecutive days. Each trial started by

placing the mice on the rod and beginning rotation at constant 4 rpm-speed for 60 s. Then the

accelerating program was launched, and trial ended for a particular mouse when falling off the

rod. Time stayed on the rod was automatically recorded.

Locomotor activity under pharmacological challenge: experiment 6. Locomotor activity

was assessed in clear Plexiglas boxes (21 × 11× 17 cm) placed over a white Plexiglas

infrared-lit platform. Light intensity of the room was set at 15 lx. The trajectories of the mice

were analyzed and recorded via an automated tracking system equipped with an infrared-

sensitive camera (Videotrack; View Point, Lyon, France). To focus on forward activity, only

movements which speed was over 6 cm/s were taken into account for the measure of

locomotor activity.

Behavioral testing started when the animals were placed in the activity boxes for a 60 min-

habituation period. Habituation allowed the animals to reach a low and stable level of basal

activity and ensured reliable subsequent measure of drug-induced locomotor effects. Mice

were injected with either vehicle, the D1/D3 dopamine receptor agonist SKF-81297 (0.5, 1 or

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2.5 mg/kg, s.c.) or the D2/D5 dopamine receptor agonist quinpirole (0.05, 0.1 or 0.5 mg/kg,

s.c.), and locomotor activity was monitored for further 60 min. Doses were chosen based on a

review of literature (Guzman et al. 2011; Napolitano et al. 2010; Smith et al. 2005).

Food seeking: experiments 7 and 8. In experiment 7, food seeking was assessed in a runway

(70 x 8 cm) with black Plexiglas floor enclosed in transparent Plexiglas walls (15 cm) and

elevated 40 cm above the floor. Removable sliding doors made of black opaque Plexiglas

delimited two boxes (10 x 8 cm), a start box and a goal box, at each end of the alley. A food

well (2 cm diameter) was inserted into the floor at 1 cm from the end of the goal box. Light

intensity in the room was set at 15 lx.

We adapted a runway task (Barbano et al. 2009; Pecina et al. 2003) to match as closely as

possible the conditions of the cross-maze tasks that we used in experiments 3 and 4. The

animals (WT – n=5 females, 5 males; Oprd1-/- - n=5 females, 5 males) were reduced to 85%

of their ad lib feeding weights over 7 days before runway training and maintained at this

weight throughout the experiment. They received sucrose reward tablets (5-10 per mouse;

Formula 5TUT-formerly PJFSC-20 mg, TestDiet, Richmond, USA) in their home cage for

three consecutive days before habituation. Habituation lasted three days. The mice were

placed in the start box and allowed to explore the alley for 5 min. On day 1, sucrose pellets

were available throughout the apparatus. On day 2, a trail of five pellets leading to the food

cup was placed along the alley. On day 3, pellets were present only in the food well. Training

(4 trials per day) started immediately after habituation. Mice were released after a 15 s-

confinement in the start box. A single food-pellet bait was located in the food well of the goal

box. After entering this goal box, the door was closed and mice were confined for at least 20 s

or until food was consumed. If a mouse failed to eat the food within 5 min, the trial was

terminated. An observer recorded the amount of time needed for each mouse to reach the goal

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box (running latency). Observation of retreat behavior in Oprd1-/- mice (not scored) evoked an

approach-avoidance conflict in these animals (Geist and Ettenberg 1997), consistent with their

high levels of anxiety (Filliol et al. 2000). To reduce anxiety levels in this task, we thus

abolished confinement in end box during two additional “challenge” sessions. Under these

novel conditions, the animals performed as many trials as they could (starting with 15 s-

confinement in the start box) in a fixed delay of 4 min.

In experiment 8, food seeking was assessed in 4 equal square arenas (50x50 cm) separated by

35 cm-high opaque grey Plexiglas walls. The floor was a white Plexiglas platform, covered

with 5 cm of fresh sawdust. Three pellets of ordinary lab chow were placed on a white square

tissue in the center of the arena. Light intensity in the center of the arenas was set at 60 lx.

A novelty-suppressed feeding protocol was adapted from (Zhou et al. 2010). Mice (Exposed

to arena: WT – n=5 females, 4 males; Oprd1-/- - n=4 females, 4 males; control: WT – n=6

females, 8 males; Oprd1-/- - n=6 females, 8 males) were food-deprived (no food - water only)

for 24 h before the test, and isolated in a standard housing cage for 30 min before testing.

Animals were tested one at a time. The test started when a mouse was placed in a corner of

one of the arenas and allowed to explore for a maximum of 15 min. A food approach was

counted each time the nose of the mouse came in contact with the food pellets. Latency to

feed was measured as the time necessary to bite a food pellet. Immediately after an eating

event, the mouse was transferred back to home cage (free from other cage-mates) and allowed

to feed on lab chow for 5 min. Food consumption in the home cage was measured. Non-

exposed control mice undergone 24-h fasting and 30 min isolation in a standard housing cage

but were not introduced into the testing arenas. They were instead immediately placed back in

their home cage (free from other cage-mates) and allowed to feed on lab chow for 5 min.

Gene expression

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Tissue collection. As previously described (Goeldner et al. 2011; Le Merrer et al. 2012),

brains were removed and placed into a matrix with 1 mm division (ASI Instruments, Warren,

MI, USA) cooled on ice. Slices were rapidly collected and structures were dissected based on

the stereotaxic atlas of mouse brain (Paxinos and Franklin 2001). Bilateral punches were

taken from two consecutive slices of the caudate putamen (CPu) and nucleus accumbens

(NAc). Dorsal hippocampus was dissected bilaterally from two consecutive brain slices

(HPC). These tissues were immediately frozen on dry ice and kept at -80°C until use.

Real-time quantitative PCR analysis. For each structure of interest and each genotype

(n=10 mice per genotype), tissue collected bilaterally from one male and one female mouse

was pooled in the same sample (n=5 samples per genotype). Therefore, real-time quantitative

PCR (qRT-PCR) was performed on 5 independent samples per genotype. RNA was extracted

and purified using the MIRNeasy mini-kit (Qiagen, Courtaboeuf, France) according to the

manufacturer's instructions. Total RNA (2.5 μg) was treated for 30 min at 37 °C by DNase I

RNase-free (5 U; Invitrogen, Carlsbad, USA) in first-strand Superscript buffer (Invitrogen)

and the reaction was stopped by incubating the mix for 5 min at 75 °C. RNA was pre-

incubated with anchored-oligo-dT primer (8 µM), random hexamer (16 µM) and dNTPs

(500 μM each) in a volume of 30 μL for 5 min at 65 °C. First-strand Superscript buffer,

dithiothreitol (0.01 M) and Superscript II (400 U; Invitrogen) were added in a final volume of

50 μL for 50 min at 42 °C. The reaction was stopped by 15 min incubation at 70 °C. qRT-

PCR was performed in quadruplets on a LightCycler 480 Real-Time PCR (Roche, Manheim,

Germany) using iQ SYBR Green supermix (Bio-Rad, Marnes-la-Coquette, France) kit with

0.25 µl cDNA in a 12.5 µl final volume. Gene-specific primers were designed using primer3

(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to obtain a 100- to 150-bp

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product. Each primer pair was validated for specificity and checked for unique melting curve

of each amplicon (see Table S1 for primer sequences). Thermal cycling parameters were

5 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 15 s at 60 °C and 30 s at 72 °C.

Relative expression ratios were normalized to the level of actin as the reference gene and the

2−ΔΔCt method was applied to evaluate differential expression level (Livak and Schmittgen

2001).

Drugs

The nonpeptide antagonist of delta opioid receptors naltrindole hydrochloride was obtained

from Sigma (Sigma-Aldrich, Lyon, France). The D1/D3 dopamine receptor agonist SKF-

81297 (2,3,4,5-tetrahydro-6-chloro-7,8-dihydroxy-phenyl-1H-3-benzazepine) and the D2/D5

dopamine receptor agonist quinpirole were purchased from Tocris (Tocris Bioscience, Bristol,

UK). Compounds were dissolved in sterile isotonic saline solution (NaCl 0.9%) and injected

s.c. in a volume of 10 ml/kg. Doses refer to salt weight.

Statistical analyses

Behavioral experiments. Results from preliminary EPM experiment

Novel object recognition data (experiments 1-2) were expressed as percentage of time spent

exploring or number of visits to the displaced or novel object. During the first phase, this

percentage was arbitrary calculated for the object located in the right and/or top half of the

arena. These data were analyzed using three-way ANOVA, with genotype and gender as

between-subject factors and phase as within-subject factor. Data from cross-maze and runway

tasks (experiment 3, 4 and 7) were analyzed using three-way ANOVA with genotype and

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gender as between-subject factors and session as within-subject factor. Pearson’s 2 test was

used to compare the percentage of place learners between Oprd1-/- and WT mice in

experiment 3. Data from the skill motor learning task (experiment 5) were analyzed using

five-way ANOVA with genotype, gender and condition (“hard” versus “easy”) as between

subject factors and trials and sessions as within-subject factors. Locomotor activity data

(experiment 6) were analysed using three-way ANOVA, with genotype, gender and dose of

dopamine agonist as between-subject factors. Finally, data from novelty-suppressed feeding

experiment (experiment 8) were analyzed using two-way ANOVA with genotype and gender

as between factors (latency to first eat, approaches) or three-way ANOVA with genotype,

gender and condition (arena exposure or not) as between factors. Statistical significance was

set at p<0.05 for all tests (Statistica 9.0, StatSoft, Maisons Alfort).

Gene expression. qRT-PCR data were transformed prior to statistical analysis to obtain a

symmetrical distribution centered on 0 (corresponding to no change in gene expression) using

the following formula: if x<1, y=1-1/x; if x>1, y=x-1 (x: qPCR data; y: transformed data) (Le

Merrer et al. 2012). A Student’s t test was then performed to assess their statistical

significance. Calculated p values indicated probability for a regulation to differ from 0. Only

regulations over +1.20 or below -1.20 (fold change) were retained as significant.

Supplementary Results

Oprd1-/- mice do not display signs of elevated anxiety when tested under low light

conditions in the EPM

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In a preliminary experiment, we evaluated signs of anxiety in mutant mice and their controls

using the EPM test under low light conditions (15 lx).

At 15 lx, WT and Oprd1-/- mice displayed similar numbers of entries (genotype: F1,12<1;

gender: F1,12<1; arm: F1,12<1; arm x gender: F1,12=12.91, p<0.01), similar distance travelled

(genotype: F1,12=3.84, NS; gender: F1,12=6.37, p<0.05; arm: F1,12=14.49, p<0.0001; arm x

gender: F1,12=10.18, p<0.001) and time spent (genotype: F1,12<1; gender: F1,12<1; arm:

F1,12=7.36, p<0.05; arm x gender: F1,12=8.59, p<0.05) in the open and closed arms of the EPM

(see Figure S1a,b and c), resulting in similar entry (genotype: F1,12<1; gender: F1,12=13.86,

p<0.001), distance (genotype: F1,12<1; gender: F1,12=8.17, p<0.05) and time (genotype: F1,12<1;

gender: F1,12=8.68, p<0.05) ratios (Figure S1d). Females appeared more anxious than males,

independently from the genotype. Control and mutant mice travelled the same distance in the

apparatus (genotype: F1,12=3.10, NS; gender: F1,12=9.16, p<0.05), demonstrating similar

forward activity in this test (females in both genotype being less active than males). Finally,

delta opioid receptor null mice performed significantly more head dips than WT animals

(genotype: F1,12=9.24, p<0.05; gender: F1,12<1), suggesting that risk taking behavior may be

increased in these animals.

Increased food seeking in mice lacking delta opioid receptors

In experiments 7 and 8, we evaluated whether motivation to obtain a food reward was

modified delta opioid receptor null mice.

In a runway task (experiment 7), running latencies were similar across training sessions in

Oprd1-/- and WT mice (Figure S3A), and were slower to decrease in females than in males

(Three-way ANOVA, genotype: F1,12<1; gender: F1,12=11.14, p<0.01; session: F16,192=6.71,

p<0.0001; session x gender: F16,192=3.03, p<0.001). We then performed two consecutive

challenge sessions, during which the animals were allowed to retrieve as many food pellets as

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they could in 4 min, and were no longer confined in goal arm. Under these conditions, mutant

mice obtained significantly more pellets (genotype: F1,12=40.59, p<0.0001) and reached the

food quicker (genotype: F1,12=5.91, p<0.05) than their WT counterparts.

In a novelty-suppressed feeding task (experiment 8), Oprd1-/- and WT mice displayed

equivalent latencies to start feeding in the center of the arena (two-way ANOVA, genotype:

F1,13<1) (Figure S3B). Mutant mice, however, approached the food more often than WT

controls (genotype: F1,13=36.65, p<0.0001). Back in their home cage, mice exposed to the

bright arena ate less lab chow than non-exposed controls (three-way ANOVA; genotype:

F1,37<1; arena exposure: F1,37=4.39, p<0.05). Subsequent two-way ANOVA revealed that

mutant mice consumed less food than their WT counterparts when placed back in their home

cage immediately after the novelty-suppressed feeding test (genotype: F1,13=6.86, p<0.05).

Data from experiments 7 and 8 indicate that motivation for food is not reduced in Oprd1-/-

mice, but instead appears increased, as suggested by facilitated performance in challenge

sessions of experiment 7 and increased number of food approaches in experiment 8.

Transcriptional regulations in the ventral striatum of Oprd1-/- animals

In the nucleus accumbens (NAc), delta receptor deletion modified the expression of 10 genes,

coding for actors of GABA signaling (up-regulation: Slc6a1; down-regulation: Gabra4;

Gabbr2), a glutamate transporter and an element of the post-synaptic densities (up-regulation:

Slc1a6; down-regulation: Dlg2), enzymes involved in monoamine metabolism (up-regulation:

Maoa, Ache) and MSN markers (up-regulation: Pde10a; down-regulation: Adora2a, Cnr1).

The expression of only 3 genes was found regulated in both the CPu and NAc: Grm4, Slc6a11

(up-regulated) and Slc6a4 (down-regulated in the CPu, up-regulated in the NAc).

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Discussion experiments 7 and 8

Increased food seeking in mice lacking delta opioid receptors

In the runway task (Figure S2a), latencies to reach the food were not modified in mutant

animals as compared to WT controls when experimental conditions matched these of the

cross-maze tasks (confinement in end box). However, under challenge conditions (no

confinement), Oprd1-/- mice obtained more food pellets than WT mice, suggesting increased

food seeking. This result may reflect competing high levels of anxiety (avoidance behavior)

and high motivation for food (approach behavior) in mutant animals (Aupperle and Paulus

2010; Montgomery 1955; Powell et al. 2004). By omitting confinement in the runway

experiment, we aimed at reducing anxiety levels, which are elevated in Oprd1-/- mice (Filliol

et al. 2000). This manipulation may have unmasked high motivation for food in mutants.

Consistent with this, in the novelty-suppressed feeding experiment (Figure S2b), these

animals took as long as WT controls to start eating in the arena, but approached food pellets

more often (and retreated), revealing conflicting avoidance and approach behaviors (Powell et

al. 2004). Decreased food intake of mutant animals in their home cage was likely due to high

anxiety levels after arena exposure, since not observed when this exposure was omitted. From

food-seeking experiments, therefore, we cannot exclude that motivation to obtain food is

increased in Oprd1-/- mice. Increased motivation for food may have contributed to ameliorate

response learning in a single-solution response cross-maze task (experiment 4), but failed to

ameliorate place learning in a dual-solution task (experiment 3).

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Supplementary figure legends

Figure S1. Oprd1-/- and WT mice show similar levels of anxiety when tested in the elevated

plus-maze under low light conditions (15 lx). Control and mutant animals display similar (a)

number of entries, (b) distance travelled and (c) time spent in open and closed arms of the

EPM. (d) Entry, distance and time ratios are equivalent in Oprd1-/- and WT mice. (e) WT and

mutant mice travel the same distance in the EPM, (f) but the later perform more head dips

than their WT counterparts. Data are expressed as mean ± esm. Genotype effect: one solid

star: p<0.05 (two-way ANOVA); Arm effect: one open star: p<0.05, two open stars:

p<0.01(three-way ANOVA).

Figure S2. Choice latency decreases over sessions in cross-maze tasks. (a) In the dual

solution cross-maze task, Oprd1-/- and WT mice display progressively shorter choice latencies

as they learned the task, females being slower than males in both mouse lines. (b) In the single

solution response task, choice latency is shorter to decrease over sessions in WT than in

mutant animals, females displaying longer latencies than males. Data are expressed as mean

(± esm) choice latency over 4 (a) or 5 (b) daily trials.

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Figure S3. Food seeking appears increased in Oprd1-/- mice. (a) In the runway task, running

latencies decrease similarly over training sessions in mutant and WT animals, females being

slower than males in both lines. Under challenge conditions, however, Oprd1-/- mice gain

more food pellets and displayed shorter feeding latencies than WT mice. (b) In the novelty-

suppressed feeding task, latencies to feed are equivalent between WT and mutant mice. The

later, however, approach the food more often than their WT counterparts. Back in their home

cage immediately after the test, Oprd1-/- mice eat less than WT mice. This effect likely reflects

high anxiety levels in mutant animals, as it disappears when the animals are not exposed to

the test arena (control condition). Data are expressed as mean ± esm. Genotype effect: one

solid star: p<0.05, three solid stars: p<0.001 (two-way ANOVA); one open star: p<0.05, three

open stars: p<0.001(one-way ANOVA).

Figure S4. Genetic or pharmacological inactivation of delta opioid receptors results in

increased sensitivity to the locomotor effects of the D1/D5 agonist SKF-81297. This effect or

a trend towards this effect is seen both in male and female mice, except for the highest dose of

SKF-81297 in Oprd1-/- mice, at which only males displayed higher locomotor response as

compared to WT animals. Genotype/treatment effect: one solid star: p<0.05, two solid stars:

p<0.01; gender effect: one open star: p<0.05, two open stars: p<0.01(one- or two-way

ANOVA).

Supplementary table legends

Table S1. List of primers used for qRT-PCR.

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Table S2. Transcription of genes coding for a set of 67 genes, including actors of GABA,

glutamate or monoamine signaling and neuronal markers, was evaluated in the hippocampus

(HPC), caudate putamen (CPu, dorsal striatum) and nucleus accumbens (NAc, ventral

striatum) of Oprd1-/- mice.

Data are presented as fold-change Oprd1-/- versus WT mice (median ± sem). Student’s t-tests

were performed on transformed data (see Material and Methods) to determine whether fold

changes differed from 0 (no regulation: corresponds to ±1 in table). Significant regulations are

highlighted in bold.

Table S3. Animal numbers used in experiment 6: effects of dopamine receptor agonists on

locomotor activity.

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