Supporting InformationSchweizer et al. 10.1073/pnas.1323499111SI Materials and MethodsAnimal Housing. All mice used in the study were housed in theanimal facility at the Biomedical Centre, Uppsala University, inaccordance with the Swedish regulation guidelines (Animal Wel-fare Act SFS 1998:56) and European Union legislation (Con-vention ETS123 and Directive 2010/63/EU), and ethical approvalwas obtained from the Uppsala Animal Ethical Committee.

Generation of Transgenic Mice. The Vglut2f/f;Pitx2 mouse line wasproduced by using the breeding procedure established for con-ditional knockout (cKO) mice to ensure identical genetic back-ground (1) by breeding the Pitx2-Cre mice (2) to Vglut2f/f (3)mice, thereby generating cKO (Vglut2f/f;Pitx2-Cre+) and controls(Vglut2f/f;Pitx2-Cre- and Vglut2wt/wt;Pitx2-Cre+) as littermates, whichallows for behavioral phenotyping and comparison betweengenotype groups (4). For immunohistochemistry and RT-PCRanalyses, the TaumGFP Cre-reporter mouse, which enables visu-alization of Cre-expressing cell nuclei by detection of β-gal pro-tein and projections of the corresponding cells by GFP, was bredinto the mouse line. In all experiments, littermates have beenused to ensure that the specificity of observed phenotypes is onlydependent on genotype. Also, the observers were blind to thegenotype until the stage of analysis.

Immunohistochemistry.The protocols described previously (3) wereused for detection of rabbit β-gal (ICN/Cappel) at 1:2,000 andchicken GFP (Abcam) at 1:200 on mouse coronal cryosections.

Single-Cell RT-PCR. Brains were obtained from postnatal day (P) 1and P20 cKO and their littermate controls. Single cells pickedfrom each of the subthalamic nucleus (STN) (P1/P20), mamillarynucleus (MN), and posterior hypothalamus (PH) (P20) werecollected and cDNA prepared for multiplex RT-PCR analysis aspreviously described (5). Vglut1-, 2-, and 3-specific primers weredesigned based upon sequences deposited in the GenBank da-tabase (www.ncbi.nlm.nih.gov/nucleotide). The Vglut2 primerswere designed around exons 4, 5, and 6 to detect both the WTand knockout allele.

In Situ Hybridization.Quantitative radioactive in situ hybridizationwas performed as described using antisense oligonucleotideprobes for detection of Pitx2 [mix of three oligonucleotides(NM_001042504.1: bases 1091–1125, 1277–1310, and 1641–1680)]and Vglut2 [two independent probes, one composed of a mix ofthree oligonucleotides (NM_080853.3: bases 13–47 [exon1], bases872–908 [exon 1,2], and bases 3220–3254 [exon 12]) and a secondprobe specific for exon 5 (bases 1432–1464) mRNA, respectively],TH [mix of three oligonucleotides (NM_009377.1: bases 272–305,774–807, and 1621–1655)], Vglut1 (NM_182993.2: 1046–1080,1496–1530, and 2634–2670), and GAD (NM_008077.4: 629–663,1936–1970, and 2308–2342). Overlaid images (Fujifilm BioImag-ing Analyzer BAS-5000) corresponding to adjacent sections hy-bridized with Pitx2 and Vglut2 probes were displayed; the contoursof STN, MN, and PH, respectively, were based on Pitx2 expres-sion. The levels of Vglut2 mRNA were defined as means, dis-tributions of signal intensity, and correlation between Pitx2 andVglut2 mRNA labeling. To evaluate the difference of Vglut2expression between control and cKO brains, mRNA ratios ofVglut2 in STN, MN, and PH vs. Vglut2 in thalamus were es-tablished, the thalamus serving as control reference. The basisof the quantitative analysis is the strength of the signal forevery single pixel, an integer ranging from 0 to 255. For signal

distribution, histograms of both analyzed and background signalswere displayed and the signal for each mRNA were analyzedfurther by quintiles. Quantitative data were analyzed by a univer-sal t test. For overlaid images, a pixelwise comparison of Pitx2 andVglut2 signals was established; standard Pearson correlation co-efficient was calculated locally (i.e., samples were seven-by-sevenpixel squares around every single pixel, represented by heat-maplevel lines) and globally (i.e., the whole selected region was con-sidered as a sample). Global correlations as well as regressionlines are displayed on scatter plots.

Slice Electrophysiology.Mice (P17–P35) were decapitated and thebrains placed in ice-cold artificial cerebrospinal fluid (ACSF) (6)and 400-μm-thick parasagittal slices containing the basal ganglia(7) were obtained. Slices were kept in a submerged holdingchamber containing ACSF, constantly bubbled with 95% O2 and5% CO2, incubated at 35 °C for 1 h, and then allowed to cool toroom temperature (6). A concentric stimulating electrode wasplaced on the STN. Recording pipettes (3–5 MΩ) were filledwith a K-gluconate–based internal solution. Inhibitory postsynapticcurrents were blocked by the application of 50 μM picrotoxin(Sigma) and the application of NMDA and AMPA blockers(dAP5 and CNQX; Tocris) eliminated all detectable excitatorypostsynaptic currents (EPSCs), indicating the purely glutamatergicnature of EPSCs. Data were acquired using an Axopatch 200Bor Multiclamp 700B amplifiers (Molecular Devices) and digitizedby a National Instruments DAQ card using the winWCP software(Dr. John Dempster, University of Strathclyde, Glasgow, Scot-land). EPCS recordings were performed from visually identifiedcells in the substantia nigra pars reticulata (SNr) and en-topeduncular nucleus (EP). The voltage amplitude of the shockwas gradually (1V steps) increased until ESPCs were detectedpostsynaptically (threshold). EPSCs were isolated by the applica-tion of 10 μM picrotoxin (Tocris). Measurements of EPSC currentdensity, first peak amplitude, and rise time were obtained withsingle shocks with stimulation amplitudes 10% greater thanthreshold. It was not possible to compare EPSC decay timeowing to the appearance of compound EPSCs.

Virus Injection and in Vivo Electrophysiology. For virus injections,Pitx2-Cre–expressing littermate mice (controls, Vglut2wt/wt;Pitx2-Cre+

and cKO, Vglut2f/f;Pitx2-Cre+) were anesthetized with isofluorane(0.5–2%) and restrained in a stereotaxic apparatus. Injections ofAAV9-ChR2-EYFP-DIO virus (UNC Vector Core) were doneat a concentration of 1 × 1012 at two dorsal-ventral (DV) levelsat the following coordinates: −1.90 anterior-posterior (AP), −1.70medial-lateral (ML), −4.75 and –4.25 DV (750 nL at each site)according to ChR2-injection procedures described previously (6).The animals were allowed to rest for 3 wk before testing. In vivounit recording was performed in ChR2-injected control and cKOanimals anesthetized with ketamine and midazolam (80 and75 mg/kg, respectively). An optical fiber (200 μm/0.39 N.A.;Thorlabs) was placed at the vicinity of the STN (stereotaxic co-ordinates in millimeters: −2.18AP, 1.5 ML, and 4.3 DV) and acollimated laser generated ∼10 mW (at the fiber tip) of 473-nmlight. A multisite (16 channels, 50-μm interchannel space, ∼1.5 MΩimpedance) silicon substrate recording probe (Neuronexus) wasinitially lowered in the EP region to record postsynaptic spikes(stereotaxic coordinates in millimeters: −1.34 AP, 1.75 ML, and4.5 DV). Local field potential was amplified, multiplexed, anddigitized by an Intan amplifier (Intan Technologies). Single unitswere isolated using the unsupervised MATLAB package for

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 1 of 8

spike sorting Wave_Clus (8). Both in vitro and in vivo data wereanalyzed in MATLAB 2011b (The MathWorks). Electrophysi-ology data are presented as mean ± SEM and significance wasset to P < 0.05, two-tailed unpaired student t test.

Behavioral Analyses, Procedure, and Number of Animals. Protocolsfor beam walking (n = 33; 17 controls, 8 males and 9 females;16 cKO, 6 males and 10 females), rotarod (n = 41; 20 controls,8 males and 12 females; 21 cKO, 8 males and 13 females), elevatedplus maze (n = 40; 20 cKO, 8 males and 13 females; 20 controls,8 males and 12 females), forced swim test (n = 24; 12 controlsand 12 cKO all females), basal (n = 31; 17 controls, 14 cKO, allfemales) and amphetamine-induced (n = 22; 11 controls,11 cKO) locomotor activity have been described previously (5, 9).Gait analysis was performed on an automated treadmill (TreadScan;CleverSys) (n = 14; 6 controls, 2 males and 4 females; 8 cKO,3 males and 5 females). Analysis of GBR12783-induced andreserpine-suppressed locomotor activity were performed in asimilar way as for amphetamine. The radial maze is describedbelow (n = 40; 20 controls, 8 males and 12 females; 20 cKO,8 males and 12 females), as is the delay discounting analysis (n = 22;8 controls, 14 cKO, only males).

The Radial-Arm Maze. Working and reference memory were mea-sured by the baited radial-arm maze (10). The maze consisted ofeight walled arms connected by a central platform, from whereeach mouse started the trial. Each arm had a hidden food re-ceptacle at the distal end. The same four out of eight arms werebaited during each trial. Animals were food-restricted 3 d beforescheduled maze performance (access to food 6 h/d) and kept onfood restriction throughout the test (access to food 2 h/d). For taskacquisition, the animals were placed individually in the center ofthe maze once each day for 5 d. The animals explored the mazefor 6 min on the first trial day. Thereafter, the animals were al-lowed to remain in the apparatus until all pellets were obtained oruntil the maximum time of 6 min was reached.

The Delay Discounting Paradigm.Behavioral testing was carried outin self-administration chambers (15.9 cm × 14.0 cm × 12.7 cm;Med Associates) enclosed in sound-attenuating boxes and equip-ped with a fan to provide airflow and to mask exterior sounds. Thechambers were fitted with two feeders, each consisting of a feederlight providing the visual cue, a food receptacle for the delivery ofthe reinforcement (20-mg sucrose pellets, 5TUL TestDiet), anda photobeam sensor for registering head entry into the feederopening. Chambers were illuminated throughout the session by

a house light on the top center of the wall opposite the feeders.Mice were food-restricted for 2 d to 3 g standard chow per mouseper day and food restriction was maintained throughout the ex-perimental procedures, monitored by weighing (90% of initialbody weight per mouse). Experimental procedures were composedof the following steps: habituation (day 1), training steps 1 (days2–5, food collection), 2 (days 6–15, preference instatement), and3 (days 16–26, choice making), testing (days 27–42), extinction(days 43–46), reinstatement (day 47), and reversal (days 48 and49), of which the habituation to testing steps assess impulsivity(11, 12) whereas the extinction, reinstatement, and reversal stepsassess task relearning, a measure of cognitive flexibility. Duringall experimental days, except habituation, mice were left in cham-bers until 30 food collections were achieved or until trial timeended. Trial time was 30 min for days 1–32 and 43–49, 40 minfor days 33–36, and 45 min for days 37–42.

Dopamine Biochemistry Assays. Brains (n = 14; 7 controls, 7 cKO)were rapidly removed upon cervical dislocation and processed byautoradiography for dopamine transporter (DAT) expression byspecific binding of the DAT-selective ligand [125I]RTI-121 (ref.13 with minor modifications), [3H]SCH23390 binding to D1 re-ceptors (D1R) (14), and [125I]iodosulpride binding to D2 re-ceptors (D2R) (ref. 14 with minor modifications).

High-Speed Dopamine Chronoamperometry in Vivo. High-speed invivo chronoamperometric measurements were performed inurethane-anesthetized mice using the FAST-16 system (Quan-teon), as previously described (14, 15). KCl-evoked endogenousdopamine (14) (n = 23; 11 controls, 12 cKO) and exogenouslyapplied dopamine (200 μM; n = 13; 5 controls, 8 cKO) peakswere analyzed during 100 ms, 0- to 550-mV square-wave pulses(5 Hz). Recordings by nafion-coated single carbon fiber elec-trodes (SF1A; 30 μm outer diameter, 100- to 200-μm length;Quanteon) were performed in the dorsal striatum (AP, +1.1;ML, 1.5; and DV, −3.2 in millimeters from bregma; same co-ordinates as for local application of exogenous dopamine). Pa-rameters examined were amplitude, defined as the peak dopamineconcentration (micromolar) from baseline; peak area, area underthe curve (micromolar × s); T80, the time (seconds) from maximumpeak concentration until 80% decrease of the maximum ampli-tude, as a measure of dopamine clearance; and clearance rate(micromoles per second), defined as the maximum amplitude(2 μM in this case) multiplied with the rate of decay of theelectrochemical dopamine signal.

1. Wolfer DP, Crusio WE, Lipp HP (2002) Knockout mice: Simple solutions to theproblems of genetic background and flanking genes. Trends Neurosci 25(7):336–340.

2. Martin DM, et al. (2004) PITX2 is required for normal development of neurons in themouse subthalamic nucleus and midbrain. Dev Biol 267(1):93–108.

3. Wallén-Mackenzie Å, et al. (2006) Vesicular glutamate transporter 2 is required forcentral respiratory rhythm generation but not for locomotor central pattern generation.J Neurosci 26(47):12294–12307.

4. Crusio WE (2004) Flanking gene and genetic background problems in geneticallymanipulated mice. Biol Psychiatry 56(6):381–385.

5. Birgner C, et al. (2010) VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation. Proc Natl Acad Sci USA 107(1):389–394.

6. Leão RN, et al. (2012) OLM interneurons differentially modulate CA3 and entorhinalinputs to hippocampal CA1 neurons. Nat Neurosci 15(11):1524–1530.

7. Hashimoto T, Elder CM, Okun MS, Patrick SK, Vitek JL (2003) Stimulation of thesubthalamic nucleus changes the firing pattern of pallidal neurons. J Neurosci 23(5):1916–1923.

8. Quiroga RQ, Nadasdy Z, Ben-Shaul Y (2004) Unsupervised spike detection andsorting with wavelets and superparamagnetic clustering. Neural Comput 16(8):1661–1687.

9. Wallén-Mackenzie A, et al. (2009) Restricted cortical and amygdaloid removal ofvesicular glutamate transporter 2 in preadolescent mice impacts dopaminergicactivity and neuronal circuitry of higher brain function. J Neurosci 29(7):2238–2251.

10. Olton DS, Papas BC (1979) Spatial memory and hippocampal function.Neuropsychologia17(6):669–682.

11. Winstanley CA, Baunez C, Theobald DEH, Robbins TW (2005) Lesions to thesubthalamic nucleus decrease impulsive choice but impair autoshaping in rats: Theimportance of the basal ganglia in Pavlovian conditioning and impulse control. Eur JNeurosci 21(11):3107–3116.

12. Koot S, van den Bos R, Adriani W, Laviola G (2009) Gender differences in delay-discounting under mild food restriction. Behav Brain Res 200(1):134–143.

13. Boja JW, et al. (1995) Selective labeling of the dopamine transporter by the highaffinity ligand 3 beta-(4-[125I]iodophenyl)tropane-2 beta-carboxylic acid isopropylester. Mol Pharmacol 47(4):779–786.

14. Alsiö J, et al. (2011) Enhanced sucrose and cocaine self-administration and cue-induced drug seeking after loss of VGLUT2 in midbrain dopamine neurons in mice. JNeurosci 31(35):12593–12603.

15. Gerhardt GA, Hoffman AF (2001) Effects of recording media composition on theresponses of Nafion-coated carbon fiber microelectrodes measured using high-speedchronoamperometry. J Neurosci Methods 109(1):13–21.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 2 of 8

1 2 3 45 6 7 8 9 1011 12

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 7 8 9 10 11 12

662bp product506bp product

372bp product216bp product

Ctrl (Vglut2 )

Vglut2

First RoundSecond Round

First RoundSecond Round

Vglut2.1-fwV

glut2.2-fw

Vglut2.2-rev

Vglut2.1-rev

Vglut2.1-fwV

glut2.2-fw

Vglut2.2-rev

Vglut2.1-rev

P P

f/f

f/f;Pitx2-Cre

DNA

mRNA

mRNA

A

BExpression STN MN PH

Vglut2

Vglut1

Vglut1 + Vglut2

no Vglut1 or 2

Vglut3

cKO Ctrl cKO Ctrl cKO Ctrl

5WT, 3KO 5WT, 0KO 5WT, 1KO 12WT 4WT, 1WT/KO 6WT

7 6 3 1 5 5

3 (2WT, 1KO) 2(WT) 1(WT) 4(WT) 0 1(WT)

22 12 25 11 24 21

0 0 2 1 0 0

total 4040 40 39 39 40

CSTN/TH MN/TH PH/TH

cKO

Ctrl

0.94 0.12 2.50 0.13 1.41 0.09

1.57 0.16 2.43 0.09 1.54 0.09+-

+-

+-

+-

+-

+-

CtrlcKO

3.0

2.5

2.0

1.5

1.0

0.5

0STN MN PH

*

Fig. S1. Single cell multiplex RT-PCR and quantitative in situ hybridization. (A) DNA sequence of the WT Vglut2 allele (Top), followed by the mRNA transcriptgenerated from the WT floxed allele (Middle), and the knockout allele (Bottom). Primer positions for nested RT-PCR are indicated below the transcript (firstround, Upper row; second round, Lower row). (B) Results from single-cell RT-PCR analysis. Vglut2was detected as WT allele only in the control mice, but as bothknockout (KO) and WT allele in the cKO mice at both P1 and P20, shown here. A number of cells contained Vglut1, or a combination of Vglut1 and Vglut2, butno STN cells contained Vglut3. A mixture of Vglut2-only, Vglut1-only, and Vglut1/Vglut2 cells were also found in the MN and PH, and the MN containeda couple of Vglut3-expressing cells. (C) Quantitative radioactive in situ hybridization results showing intensity ratio between thalamus and STN, MN, and PH,respectively, in control (white bars) and cKO (gray bars) mice. For mean values and SD, see table (Right). cKO, conditional knockout; Ctrl, control.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 3 of 8

cKO

Ctrl

Vglut1 Vglut2

STN STN

STN STN

cKO

S4 S5

S3 S4

Ctrl

TH GAD

S1S2

S3S4

S5S6

S7S8

S9S10

S11S12

S1S2

S3S4

S5S6

S7S8

S9S10

S11S12

S1S2

S3S4

S5S6

S7S8

S9S10

S11S12

S1S2

S3S4

S5S6

S7S8

S9S10

S11S12

A

B

Fig. S2. In situ hybridizations for Vglut1, Vglut2, TH, and GAD. (A and B) The expression of vesicular glutamate transporter (Vglut)1 and Vglut2 (A) andtyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) (B) was analyzed by radioactive in situ hybridization in control and cKO animals to assess foralterations in excitatory, dopaminergic, and inhibitory populations, respectively. Two panels of adjacent sections are presented for Vglut1 and Vglut2 (A) andGAD (B). For TH, one panel comprising the VTA and SNc is shown (B). Section (S) 3 and S4 (control) and S4 and S5 (cKO), respectively, represent overlaysof GAD (red) and TH (green) staining for the selected sections. The SNr is marked in red on adjacent sections analyzed for GAD. cKO, conditionalknockout; Ctrl, control.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 4 of 8

Stimulus amplitude (V)

140120100

80604020

0

160180

15 20 25 30 35 40 45

A

Elevated plus maze Swim test

Tim

e sp

ent i

mob

ile (s

)Trial

0

3

4

2

1

*

Late

ncy

(s)

CenterOpen arm

0

300

400

200

100

inner outer

*

0

100

150

1

50

2

*

B C

Input-Output

Fig. S3. Electrophysiology of STN target area and affective behavior analyses. (A) Input–output (stimulus intensity vs. absolute peak EPSC amplitude) re-lationship of entopeduncular cells from control (black circles) and cKO (red circles) mice (n = 20 cells). The average correlation coefficient (stimulus intensity vs.absolute peak EPSC amplitude) was equal to 0.40 ± 0.04 for control and 0.29 ± 0.03 for cKO mice (n = 20 cells, P = 0.03) (supplemental information to Fig. 2).Dashed lines represent a linear fit of stimulus intensity vs. absolute peak EPSC amplitude in control (black) and cKO (red) mice cells. (B) In the elevated plusmaze, the time spent in each arm [closed arm (CA): P = 0.5458, inner open arm (IOA): P = 0.5133, and outer open arm (OOA): P = 0.1738] as well as the frequency ofvisits (CA: P = 0.7044, IOA: P = 0.7219, and OOA: P = 0.5520) was similar between cKO and control mice. However, when addressing how fast the mice start movingwhen placed in this arena, it was discovered that the latency with which cKO mice moved out from the center and the latency to enter the OOA of the maze wassignificantly shorter for the cKO mice (P = 0.0115 for center and P = 0.0280 for OOA). (C) In the forced swim test, the time spent immobile during the second trialwas significantly shorter for the cKO mice than for control littermates (P = 0.0449), showing an increase in overall swimming activity in the cKO group. *P < 0.05.cKO, conditional knockout; Ctrl, control.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 5 of 8

***CtrlcKO

Pho

tobe

am in

terr

uptio

ns 300

200

100

030 60 90 120

time (min.)

A

B

15000

10000

5000

07.50 15.0

Pho

tobe

am in

terr

uptio

ns

mg/kg

CtrlcKO

GBR12783-induced locomotion

Reserpine-suppressed locomotion

Fig. S4. GBR12783-induced and reserpine-suppressed locomotion. (A) Mice were challenged with 7.5 and 15 mg/kg of GBR12783, a DAT-selective blocker, andanalyzed in activity chambers as previously described (1); both doses led to a heightened response in cKO compared with control mice; however, the differencefailed to reach statistical significance (P = 0.1138 for 7.5 mg/kg and P = 0.1138 for 15 mg/kg). (B) After an injection of reserpine (2 mg/kg), a potent blocker ofvesicular monoamine packaging by VMAT that causes catalepsy, a separate batch of mice showed no difference between controls and cKOs (P = 0.1247, F =2.867, df = 1). Both groups show a similar successive decrease in locomotion after reserpine injection (for change over time: P = >0.0001, F = 26.16, df = 11).Thus, cKO mice show a normal response to amphetamine (Fig. 3), GBR12783, and reserpine; however, a slightly increased response was observed with the DAT-selective blocker GBR12783. cKO, conditional knockout; Ctrl, control.

1. Birgner C, et al. (2010) VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation. Proc Natl Acad Sci USA 107(1):389–394.

CtrlcKO

DM DL

VM VL

SN

r

150

100

50

0[ H]S

CH

2339

0 sp

ecifi

c b

indi

ng (%

of c

ontro

l) 3

D1R150

100

50

0[ I]

iodo

sulp

rid s

peci

fic b

indi

ng (%

of c

ontro

l)12

5

D2R

DM DL

VM VL

SN

c

B CA

D1R

D2R

Fig. S5. Radioactive ligand binding assay for dopamine D1 and D2 receptors. (A) Representative examples of serial striatal sections analyzed for D1R (Upper)and D2R (Lower) ligand binding. (B and C) Specific binding capacity levels expressed as percent of control for D1R-specific [3H]SCH23390 binding (B) and D2R-specific [125I]iodosulpride binding (C) in the following areas (illustrated in Fig. 4A): dorsomedial (DM), dorsolateral (DL), ventromedial (VM), ventrolateral (VL)striatal areas, substantia nigra pars reticulata (SNr), and substantia nigra pars compacts (SNc). No significant differences were detected between Ctrl and cKO.cKO, conditional knockout; Ctrl, control.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 6 of 8

Num

ber o

f err

ors

Radial eight arm maze

0

20

30

10

Num

ber o

f ani

mal

s

15

25*

Days

Ctrl

cKO

completeincomplete

completeincomplete

ACorrect choice

12

3

45

6

7

8

Referencememory error (RME)

Working memory error (WME)

0

4

6

2

8

2 4 5 121 30

4

6

2

8

2 4 5 121 3

B CCtrlcKO

WME RME

Fig. S6. Radial maze analysis of spatial memory capacity. (A) For each trial, a correct choice was defined as entry into a baited arm and collection of the foodreward, a working memory error (WME) was defined as a reentry into an arm in which the reward was already obtained during the session and a referencememory error (RME) was defined as a visit into an unbaited or incorrect arm. Eight days after the last acquisition session (day 12), a retention trial for memoryfunction was performed. (B) Number of animals that achieved or did not achieve completion of task. (C) WME (Left) and RME (Right) over all five training daysand testing at day 12. All data are presented as mean ± SEM. B was analyzed by two-sided χ2 test and C was analyzed by repeated measures ANOVA andBonferroni post hoc test when appropriate. *P < 0.05. cKO, conditional knockout; Ctrl, control.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 7 of 8

Average latencyto choose small reward to choose large reward to collect small reward to collect large reward

0 2 4 8 14 30 60120 (s)

*

num

ber

session

Omissions to collect Inappropriate head entryat small reward side at large reward sideat inactive holes

sessionlate

ncy

(s)

E

G

F

H

I

Omissions to chooseRatio large/small reward Reinstatement and reversal

0 2 4 8 14 30 60120 (s) 0 2 4 8 14 30 60120 (s)0 2 4 8 14 30 60120 (s)

Average latency

0 2 4 8 14 30 60120 (s) 0 2 4 8 14 30 60120 (s) 0 2 4 8 14 30 60120 (s) 0 2 4 8 14 30 60120 (s)

0

108642

0

108642

0

108642

0

108642

0

108642

0

108642

0

400

300

200

100

0

5040302010

CtrlcKO

Parameter P-value F-value significance

Ratio large reward to small rewardOmissions to choose side

Omissions to collect large rewardInappropriate HE at large reward sideInappropriate HE at small reward side

Average latency to choose large rewardAverage latency to choose small rewardAveragre latency to collect large rewardAverage latency to choose small reward

0.7000.0440.6880.2420.0380.7930.1040.5940.225

0.1534.6140.1671.4565.0020.0712.9060.2941.576

n.s.

n.s.n.s.

n.s.

n.s.

n.s.

n.s.*

*

*

Cho

ices

in p

erce

nt

Num

ber o

f om

issi

ons

Num

ber o

f cho

ices

A

Program start

40s both feeder lights on

choose large reward

choose small reward

0-120s

0s

5 pellets,light onfor 10s

1 pellet,light onfor 10s

light off

light off

no choice for 10s omission counted,lights off

session

n.s.

B D

Ctrl small rewardcKO small rewardCtrl large rewardcKO large reward

0 2 4 8 14 30 60120 (s) 0 2 4 8 14 30 60120 (s)

CtrlcKO

reinst. rev. 1 rev. 20

30

20

10

0

30

20

10

0

10080604020

C

Fig. S7. The delay discounting paradigm for impulsive choice analysis. (A) The delay discounting task design: format of a single free-choice trial. The delay onthe large reward side was increased from 0 to 120 s, and the average of two sessions at the same delay time was calculated. (B) Large reward chosen over smallreward in percent of choices. (C) Omissions to choose between large and small reward feeder (lower graph) for each delay time. (D) Number of choices uponreinstatement (reinst.) after extinction, and subsequent reversal of feeder sites (rev. 1, reversal day 1; rev. 2, reversal day 2). (E) Omissions to collect the reward.(F) Inappropriate head entries at inactive holes (Left), small reward (Center), and large reward (Right) side. (G and H) Average latency to choose a side (G) andto collect a reward (H). A mean value of two sessions at the same delay for the large reward is represented by 0–120 s. Data are presented as mean ± SEM andwere analyzed with repeated measures ANOVA and Tukey post hoc test, when appropriate. *P < 0.05. (I) Statistical overview of all parameters analyzed in theexperiment. HE, head entry. cKO, conditional knockout; Ctrl, control.

Schweizer et al. www.pnas.org/cgi/content/short/1323499111 8 of 8