SUPPLEMENTAL MATERIAL The Trace Amine-Associated … · transformed IFIs that fell from e-1. to e. 3. sec (~0.34-20.1 sec), with a bin width of e. 0.2. Total IFI time was calculated

Ferragud, Howell et al, TAAR1 agonism blocks compulsive, binge-like eating

SUPPLEMENTAL MATERIAL

The Trace Amine-Associated Receptor 1 agonist RO5256390 blocks compulsive, binge-like eating

in rats

Antonio Ferragud, Ph.D.1 *; Adam D. Howell, M.S.1 *; Catherine F. Moore, B.A.1; Tina L. Ta1; Marius

C. Hoener, Ph.D.2; Valentina Sabino, Ph.D1.; Pietro Cottone, Ph.D. 1@

1 Laboratory of Addictive Disorders, Departments of Pharmacology and Experimental Therapeutics and

Psychiatry, Boston University School of Medicine, Boston, MA, USA.

2 Neuroscience, Ophthalmology and Rare Diseases Discovery & Translational Area, pRED, Roche

Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland.

* These authors equally contributed to this work

@ Corresponding author ([email protected]) One sentence summary:

TAAR1 agonism blocks compulsive, binge-like eating.

1

mailto:[email protected]


SUPPLEMENTAL MATERIALS AND METHODS

Subjects

164 male Wistar rats, 45-day old, and triple-housed upon arrival (200-225 g) (Charles River,

Wilmington, MA), were housed in a 12:12 hour reverse light cycle (lights off at 11:00am), in a

humidity- and temperature-controlled vivarium. Rats were given access to corn-based chow (Harlan

Teklad LM-485 Diet 7012 (65% (kcal) carbohydrate, 13% fat, 21% protein, 341 cal/100 g); Harlan,

Indianapolis, IN) and water ad libitum, unless otherwise specified. Experimental procedures were

performed during rats’ active cycle (dark cycle). Procedures adhered to the National Institutes of Health

Guide for the Care and Use of Laboratory Animals and were approved by Boston University Medical

Campus Institutional Animal Care and Use Committee (IACUC).

Drugs

The highly selective TAAR1 full agonist RO5256390 (Revel et al, 2012) was synthesized by

F. Hoffman-La Roche. Doses for Experiment 1 (0, 1, 3, 10 mg/kg) were based on a previously

published report (Pei et al, 2014). In the other experiments, the most effective dose from Experiment 1

was used. For intracranial administration, RO5256390 was administered at the doses of 0, 1.5, 5 and 15

µg/side; these doses were chosen based on preliminary observations obtained in our laboratory. For

intraperitoneal administration, RO5256390 was freshly dissolved in 0.3% Tween 80 and 0.9% saline;

for intracranial administration, RO5256390 was freshly dissolved in a mixture of

ethanol:cremophor:saline (2:2:18 ratio). For within-subject drug testing, 2-4 intervening days were

allowed, until subjects’ performance returned to baseline conditions. All drug treatments were

counterbalanced, unless otherwise specified.

Apparatus for self-administration procedures

2


The individual operant test chambers (30×24×29 cm) (Med Associates Inc., St. Albans, VT)

had a grid floor and were located in ventilated, sound-attenuating enclosures (66×56×36 cm) (Cottone et

al, 2012; Smith et al, 2015). Food reinforcers were delivered by a pellet dispenser into a head entry

magazine and water reinforcers by a solenoid into a head entry liquid cup magazine. Two retractable

levers were placed on the opposite wall of the chamber. 28V stimulus cue-lights were located above

each lever and above the food magazine. Responses were recorded automatically by a microcomputer

with 10 ms resolution. In all the procedures, pellet acquisition occurred following a nose poke response,

except for the food seeking experiment in which food pellets delivery occurred following responding on

a lever based on a second order schedule of reinforcement.

Operant binge-like eating procedure in ad libitum-fed rats

Training. As previously described (Blasio et al, 2014; Velazquez-Sanchez et al, 2014), rats

were allowed to self-administer food pellets (45-mg precision pellets, 5TUM: 65.5% (kcal)

carbohydrate, 10.4% fat, 24.1% protein, 330 cal/100 g; TestDiet, Richmond, IN) and water (100 μl)

during daily 1 h experimental sessions under a Fixed Ratio 1 (FR1) schedule of reinforcement in the

operant chambers; head entry within the food magazine was detected by a photobeam, which resulted in

the delivery of a food pellet within the same food magazine. A 1.0 sec timeout period, where no

additional pellets were delivered, was used. Spillage is negligible (4.8% of total responses, n=23)

(Cottone et al, 2012). At the end of the self-administration procedure rats were returned to their home

cages and fed with the same standard chow diet as ~5 g extruded pellets.

Escalation of palatable food intake. After stable baseline performance was achieved in the 1-

h FR1 schedule self-administration sessions, rats were divided into two groups: half of the animals were

assigned to a Chow control group, which received the same 45 mg chow pellets offered during the

training phase, and the other half was assigned to a Palatable group, which received a nutritionally

3


complete, chocolate-flavored, high sucrose (50% kcal) AIN-76A-based diet, comparable in

macronutrient composition and energy density to the chow diet (45-mg precision pellets, chocolate-

flavored, 5TUL: 66.7% (kcal) carbohydrate, 12.7% fat, 20.6% protein, metabolizable energy 344

cal/100 g; formulated as 45-mg precision food pellets; TestDiet). It was previously shown that the

5TUL diet is strongly preferred over the standard chow diet by all rats (M±SEM, 91.2%±3.7 preference)

(Cottone et al, 2008, 2009). Rats underwent 1 h daily sessions until full escalation and stabilization of

food intake.

Experiment 1: effects of the TAAR1 agonist RO5256390 on operant binge-like eating.

Rats in the Chow and Palatable food group (n=12/group) were administered the TAAR1

agonist RO5256390 (0, 1, 3, 10 mg/kg, i.p.) 30 min prior to the operant sessions.

High rate of responding for standard chow induced by food restriction

This task was performed to determine whether the effects of drug treatment on the binge-like

eating procedure were dependent either on palatability or only on the rate of responding (Cottone et al,

2012; Smith et al, 2015). A separate cohort of rats was trained to acquire operant self-administration for

the standard chow diet. To reach a higher rate of responding for chow during the operant self-

administration sessions, rats were food restricted in their home cages. For this purpose, a specific

amount of chow was provided in the home cages at the end of the operant self-administration sessions

so that the total daily intake, including the food consumed during the self-administration session,

equaled 70% of a rat’s daily intake. This expedient was used to: i) ensure that the rats consumed the

entire home cage food before the beginning of the following self-administration session, ii) induce

overeating in the operant self-administration sessions, which was driven by energy homeostatic factors

and, therefore, iii) increase the rate of responding for chow in the operant self-administration sessions

(Cottone et al, 2012; Smith et al, 2015). These experimental conditions make the rate of responding for

4


chow of food-restricted rats comparable to the rate of responding for the highly palatable sugary diet of

ad libitum-fed rats in the Palatable food group. Rats were food restricted as described above for 12 days

before the drug treatment was initiated.

Experiment 2: effects of the TAAR1 agonist RO5256390 on high rate of responding for standard chow

induced by food restriction.

Food-restricted rats (n=9/group) were administered the TAAR1 agonist RO5256390 (0, 10

mg/kg, i.p.) 30 min prior to the operant sessions.

Rate and regularity of sustained eating: inter-feeding intervals and return map analyses

Inter-feeding interval analysis. To identify between group differences of rats in the rate and

regularity of sustained eating, analysis of the ln-transformed duration of consecutive inter-feeding

intervals (IFIs) was performed (Cottone et al, 2012). IFI, defined as the time between two consecutive

nosepoke head entry responses for food, is a variable inversely correlated to eating rate, and it was

automatically recorded by the computer. The mean, total IFI time, entropy, kurtosis, and skewness of

the ln-transformed duration of each subject’s consecutive IFIs were individually determined and then

averaged across subjects. The normalized frequency histogram entropy (H) is a measure of categorical

variability in the rate of ingestion (Shannon, 1997) and was computed as follows:

H is scaled between 0 and 1, with the denominator determined by the number of possible bins in the

histogram (n) and the numerator a function of the proportion of observations that fall within a given

histogram bin (pi). Minimal (H=0) entropy occurs when all observations occur within a single histogram

bin, whereas maximal (H=1) entropy occurs when each histogram bin has an equal probability, or a flat

uniform joint density distribution. For entropy analysis, histograms were constructed from ln-

( )

( )n

ppH

i

n

ii

2

2

log

log∑−=

5


transformed IFIs that fell from e-1 to e3 sec (~0.34-20.1 sec), with a bin width of e0.2. Total IFI time was

calculated as the sum of all the IFIs within a session.

Decreases in the IFI mean indicate a faster pellet-to-pellet responding and therefore, an

increased eating rate, which is an index of food palatability. Decreases in the total IFI time mean a

decrease in the total pellet-to-pellet time. Decreases in the histogram entropy (a measure of categorical

variability, reflected in a decreased number of populated histogram bins, each with less similar event

frequencies) indicate an increased regularity of intake. Conversely, an increase in the kurtosis of the IFI

distribution (a measure of the distribution's 'peakedness', reflected in a more peaked top and smaller tails

of the distribution) is consistent with an increased regularity of pellet-to-pellet responding. Finally, a

significant increase in the skewness (a measure of the distribution’s symmetry, reflected in a selective

increase of the frequency of the IFI falling to the left of the histogram) is consistent with a selective

increase of the fast pellet-to-pellet responding.

Return map analysis. Return maps, or joint inter-event interval plots, are a nonlinear method

of time series analysis that reveals the serial temporal organization of discrete events in time (Cottone et

al, 2007; Dekhuijzen and Bagust, 1996; Iemolo et al, 2015). For return map analysis, each IFI in the

time-series was scatter-plotted against its subsequent IFI (IFI+1) in a Cartesian plane. In such an

analysis, highly regular and rhythmic events are observed as densely focused clusters of points. Joint

probability density distributions were constructed from return maps and appear in gray scale format

with darker shades indicating higher local probabilities (or a color-coded form with warmer colors

(toward red) indicating higher local probabilities in the color figure).

Experiment 3: effects of the TAAR1 agonist RO5256390 on rate and regularity of sustained eating.

Rats in the Chow, food-restricted Chow, and Palatable food group (n=7-10/group) were

administered the TAAR1 agonist RO5256390 (0, 10 mg/kg, i.p.) 30 min prior to the operant sessions.

6


Compulsive-like eating of palatable food: light/dark box conflict test.

Rats in the Chow and Palatable food groups trained in the operant binge-like eating

procedure were subjects of this study. In this test, a light/dark rectangular box (50×100×35 cm) was

used, in which the aversive, bright compartment (50×70×35 cm) was illuminated by a 60 lux light. The

dark compartment (50×30×35 cm) had an opaque cover and ~0 lux of light. The two compartments

were connected by an open doorway which allowed the subjects to move freely between the two. A

shallow, metal cup containing a pre-weighed amount of the same food received during self-

administration (45-mg chow or 45-mg chocolate pellets for rats in the Chow or Palatable food group,

respectively) was positioned in the center of the light compartment. Apparatus-naïve rats were

habituated to an ante-room 2 h prior to testing. White noise was present during both habituation and

testing. On the test day, rats were placed into the light compartment, facing both the food cup and the

doorway; the apparatus was cleaned with water after each subject. Testing lasted 10 min. Behavior was

scored by raters blind to the treatment conditions. Under normal, control conditions, eating behavior is

typically suppressed when a rat is in the aversive, bright compartment; a significant increase in food

intake in spite of the adverse conditions, as compared to control conditions, was operationalized as a

construct of “compulsive-like eating” (Cottone et al, 2012; Smith et al, 2015; Velazquez-Sanchez et al,

2014).

Experiment 4: effects of the TAAR1 agonist RO5256390 on compulsive-like eating.

Rats in the Chow and Palatable food group (n=21-23/group) were administered the TAAR1

agonist RO5256390 (0, 10 mg/kg, i.p.) 30 min prior to the testing session, using a between-subjects

design.

Conditioned food reward: conditioned place preference test

7



procedure were subjects of this study. The conditioned place preference (CPP) apparatus (Med

Associates, Georgia, VT) consisted of two equal sized outer chambers (27.5×20.6×21.5 cm), one black

and one white, separated by a smaller center chamber (11.9×20.6×21.5 cm) with an automatic guillotine

door opening into each chamber and fitted with infra-red photobeam detectors. The CPP procedure

consisted of three phases: pre-conditioning, conditioning, and post-conditioning. During pre-

conditioning, rats were allowed to freely explore the apparatus for 15 min. Conditioning was performed

over 8 consecutive days and lasted 25 min. In this phase, rats were confined on alternate days to either

their initial preferred chamber with an empty food bowl or to their initially non-preferred chamber with

a bowl of the respective food (45-mg chow or 45-mg chocolate pellets for rats in the Chow or Palatable

food group, respectively) (Wang et al, 2014). During post-conditioning, rats were allowed to freely

explore the chamber again for 15 min (as in the pre-conditioning phase). The time spent in each

chamber was recorded, and CPP was determined by calculating a CPP score (time in paired chamber –

time in unpaired chamber) (Velazquez-Sanchez et al, 2015; Wang et al, 2014).

Experiment 5: effects of the TAAR1 agonist RO5256390 on conditioned food reward.

Rats in the Chow and Palatable food group (n=16-22/group) were administered the TAAR1

agonist RO5256390 (0, 10 mg/kg, i.p.) 30 min prior to the post-conditioning phase, using a between-

subjects design.

Food-seeking behavior: second-order schedule of reinforcement


procedure were subjects of this study. Food-seeking behavior under a second-order schedule of

reinforcement is a procedure in which responding is maintained by the contingent presentation of food-

paired stimuli that serve as conditioned reinforcers of instrumental behavior (Everitt and Robbins, 2000;

8


Giuliano et al, 2012). The procedure used here has been described elsewhere (Smith et al, 2015;

Velazquez-Sanchez et al, 2015).

In a first step, rats were first trained to respond on a lever for food pellets (45-mg chow or

45-mg chocolate pellets for rats in the Chow or Palatable food group, respectively) under a fixed ratio 1

(FR1) schedule of reinforcement. Responses on the inactive lever had no programmed consequences but

were recorded to assess discriminated responding and general levels of motor activity. Each lever press

resulted in the delivery of food and illumination of a conditioned stimulus light above the active lever

and the food magazine for 20 s, during which both levers retracted. After 20 sec elapsed, the two levers

were again inserted into the chamber. The session ended after either the delivery of a maximum of 35

pellets or 2h elapsed. A fixed interval (FI) schedule of reinforcement was introduced as the second step

of the training in which the FI increased daily from FI1 min, to FI2, and to FI5 min. During the FI,

active lever pressing was never reinforced. At the end of the FI active lever pressing resulted in the

illumination of a conditioned stimulus light both above the active lever and above the food magazine for

20 sec and in the delivery of 5 pellets during FI1 and FI2 or 10 pellets during FI5. After 20 sec elapsed,

the two levers were again inserted into the chamber. The session ended after either the delivery of a

maximum of 70 pellets or 40 min elapsed. A second-order schedule of reinforcement was introduced

(FI5(FR10:S)) as the third and last step, in which ten active lever presses (FR10) resulted in a brief

illumination of lights above both the active lever and the food magazine for 1 s. Following the 10th

active lever press, after a FI5 min had elapsed, 20 pellets were delivered in the food magazine, both the

active and inactive levers retracted, and the lights above both the active lever and the food magazine

were presented during a 20 s time out. During the FI interval, rats that pressed the active lever did not

receive any pellets. After the time out, the lights above both the active lever and the food magazine

turned off, and the two levers were again extended into the chamber. The session ended after either the

delivery of a maximum of 140 pellets or 40 min elapsed. Criterion for stability was 3 consecutive days

with <10% day-by-day variability.

9


Experiment 6: effects of the TAAR1 agonist RO5256390 on food-seeking.

Rats in the Chow and Palatable food groups (n=6-10/group) were administered the TAAR1

agonist RO5256390 (0, 10 mg/kg, i.p.) 30 min prior to the second-order sessions.

Anxiety-like behavior: defensive withdrawal test


procedure were subjects of this study. The defensive withdrawal test (Cottone et al, 2009; Parylak et al,

2012) apparatus was a walled open field (100×100×35 cm) containing a cylindrical “withdrawal”

chamber (2 liter Pyrex beaker wrapped in black tape). The chamber was located 15 cm from a corner

facing the open arena. Rats were habituated to an ante-room 2 h prior to testing. For the 10-min test, rats

were placed into the withdrawal chamber facing the rear, and testing was performed under a 60 lux

light. Behavior was video recorded and scored by raters blind to the treatment conditions. Latency to

first emerge from the chamber (all four paws in the open field) and withdrawal time were used as

indices of anxiety-like behavior, while the number of entries into the chamber was used as an index of

locomotor activity (Cottone et al, 2009; Parylak et al, 2012).

Experiment 7: effects of the TAAR1 agonist RO5256390 on anxiety-like behavior.


agonist RO5256390 (0, 10 mg/kg, i.p.) 30 min prior to the testing session, using a between-subjects

design.

Depressive-like behavior: forced swim test


procedure were subjects of this study. The forced swim test was performed over 2 consecutive days

10


(pre-test day and test day), as described in the original Porsolt’s procedure (Porsolt et al, 1977), by

which a minimum of two pretest drug administrations is warned to provide more stable pharmacological

results than just a single drug administration (Castagne et al, 2011). On the pre-test session day, rats

were placed into a clear acrylic cylinder (25 cm diameter) filled with water (24±1 °C; 42 cm deep) for

15 min. At the end of the 15-min pre-test session, rats received the first drug treatment. On the test day,

rats received a second drug treatment before being placed in the same glass cylinder for the 5 min test

session. The test was videotaped and later scored by a blind rater for the time rats spent immobile,

swimming or climbing by an experimenter blind to the treatments. Immobility time is a validated

measure of depressive-like behavior and represents the time spent performing the minimum movements

required to keep their head above water, in the absence of other behaviors. Swimming was defined as

horizontal movement throughout the cylinder. Climbing was defined as upward-directed movement of

the forepaws aimed toward the sides of the cylinder. (Revel et al, 2013; Sabino et al, 2009). At the end

of the session, rats were removed from the cylinder, dried, placed into a polycarbonate cage located on a

heating pad for 15 min, and then returned to their home cage.

Experiment 8: effects of the TAAR1 agonist RO5256390 on depressive-like behavior.


agonist RO5256390 (0, 10 mg/kg, i.p.) at the end of the 15-min pre-test session, and again, 30 min prior

to the test, using a between-subjects design.

Western Blot

A different cohort of rats in the Chow and Palatable food groups, which underwent the binge-

like eating procedure, was sacrificed right before the daily binge-like eating session for western blotting

analysis, performed as previously described (Cottone et al, 2012). Rats were euthanized and

decapitated; brains were quickly removed and sliced coronally in a brain matrix. Medial prefrontal

11


cortex (mPFC), dorsal striatum (DS), and nucleus accumbens (NAcc) punches were collected on an ice-

cold stage and stored at -80°C. Punches were collected and homogenized in lysis buffer (20mM

HEPES, pH=7.4, EDTA 2mM, 1% SDS, 10% sucrose) by sonication, and centrifuged at 12,000 g for 10

min at 4°C. The supernatant was transferred to a new tube, and protein concentration in lysates was

determined by the BCA assay (Thermo Fisher Scientific, Inc., Cambridge, MA), using Bovine Serum

Albumin (BSA) as standard. All samples were adjusted to an equal concentration. The lysates were then

diluted with 4X LDS sample buffer (Invitrogen, Carlsbad, CA) and 10X reducing agent (Invitrogen,

Carlsbad, CA), and heated for 10 min at 70°C, then put on ice for 3 min and sonicated for 1 minute at

7,000g. 10 μg of total protein in a volume of 10 μl were run on NuPAGE 4-12% Bis-Tris gradient gel

(Invitrogen, Carlsbad, CA). Chow and Palatable samples of the same area were run in the same gel and

all samples within the given area were run concurrently. After electrophoretic separation, the proteins

were transferred to a PVDF membrane (Bio-Rad, Hercules, CA) at 30 V for 1 h. Membranes were

blocked in 8% BSA for 1.5 h (TAAR1) or 5% milk for 1 h (β-tubulin) in Tris-Buffered Saline (TBS)

buffer (Fisher Scientific, Hampton, NH) with 0.2% Tween-20 (TBST) for 1.5 h. Membranes were then

incubated with the following two primary antibodies overnight at 4°C: anti-TAAR1 mouse polyclonal

antibody (1:5,000; provided by Roche Diagnostics) and anti-β-tubulin mouse monoclonal antibody

(1:30,000; sc-53140, Santa Cruz Biotechnology, Dallas, Texas). The anti-TAAR1 primary antibody was

validated previously (Harmeier et al, 2015). After washing three times with Tween-20 Tris-Buffered

Saline (TBS-T) buffer and TAAR1 membranes were blocked for a second time with 5% BSA in TBS-T

for 40 minutes. Membranes were then incubated with secondary antibody goat anti-mouse IgG-HRP

(1:5,000 or 1:10,000; sc-2004, Santa Cruz Biotechnology, Dallas, Texas for TAAR1 and β-tubulin,

respectively), at room temperature for 1 h. The blots were developed with an enhanced ECL

chemiluminescent substrate according to the manufacturer’s instructions (Thermo Fisher Scientific,

Inc., Cambridge, MA). Blots were exposed to autoradiography film (Denville Scientific, Inc., Holliston,

12


MA) and band densities quantified using the ImageJ software (NIH). TAAR1 expression levels were

normalized to β-tubulin expression and then to Chow controls.

Experiment 9: effects of exposure to palatable food on TAAR1 protein levels in mesocorticolimbic brain

areas.

Medial prefrontal cortex (mPFC), dorsal striatum, and nucleus accumbens from rats in the

Chow and Palatable food groups (n=10-12/group) were compared for TAAR1 protein levels.

Intracranial surgeries, microinfusion procedure, and cannula placement

Intracranial surgeries. Rats in the Chow and Palatable food groups trained in the operant

binge-like eating procedure were subjects of this study. Rats were stereotaxically implanted with

bilateral, intracranial cannulas, as described previously (Dore et al, 2013; Iemolo et al, 2015; Smith et

al, 2015). Briefly, 24 gauge stainless steel guide cannulas (Plastics One, Inc., Roanoke, VA) were

lowered bilaterally 1.6 mm and 1.5 mm above the infralimbic (IL) and prelimbic (PrL) subregions of the

mPFC, respectively. Four stainless steel jeweler’s screws were fastened to the rat’s skull around the

cannula. Dental restorative filled resin (Henry Schein Inc., Melville, NY) and acrylic cement were

applied, forming a pedestal firmly anchoring the cannula. The cannula coordinates from bregma used

for the IL cortex were: A/P +3.2 mm, M/L ± 0.6 mm, D/V -4.0 mm (from skull). The cannula

coordinates from bregma used for the PrL cortex were: A/P +3.2 mm, M/L ±0.6 mm, D/V -2.2 mm

(from skull). Cannula coordinates were calculated according to the Paxinos and Watson atlas (Paxinos

and Watson, 2007), using flat skull. Stainless steel dummy stylets (Plastics One, Inc., Roanoke, VA)

maintained patency of the cannula. After surgery, the rats were allowed a recovery period, during which

they were handled daily.

13


Microinfusion procedure. For intracranial microinfusion, the dummy stylet was removed

from the guide cannula and replaced with a 33-gauge stainless steel injector projecting 1.6 mm and 1.5

mm beyond the tip of the guide cannula for the IL cortex PrL cortex, respectively. The injector was

connected via PE 20 tubing to a Hamilton microsyringe (Hamilton Company, Reno, NV) driven by a

multi-syringe microinfusion pump (Kd Scientifics, Holliston, MA). Microinfusions were performed in

0.5 μl volume per side delivered over 2 mins; injectors were left in place for 1 additional min to

minimize backflow.

Cannula placement. Cannula placement was verified at the conclusion of all testing. Subjects

were anaesthetized (isoflurane, 2–3% in oxygen) and microinfused with India Ink (0.5 μl/side). Brains

were then flash-frozen and stored at -80°C. Coronal sections of 30 µm were collected using a cryostat

and placements were verified under a microscope, following a light cresyl violet staining. 6 subjects

(n=5 for IL cortex, and n=1 for PrL cortex) were excluded from analysis because of incorrect cannula

placement.

Experiments 10-11: effects of microinfusion of the TAAR1 agonist RO5256390 into the IL cortex and

PrL cortex on operant binge-like eating.

Rats in the Chow and Palatable food groups were microinfused with the TAAR1 agonist

RO5256390 (0, 1.5, 5, 15 µg/side) into either the IL (n=8-10/group) or the PrL (n=11-12/group) cortex

10 min prior to the operant sessions.

Statistical analyses

Data were analyzed by simple or factorial ANOVAs. Food intake during palatable food

escalation was analyzed by two-way mixed design ANOVAs, with Diet as a between-subjects factor

and Day as a within-subject factor. The effects of RO5256390 treatment on food intake in the operant

14


binge-like eating procedure, IFI, total IFI time, entropy, kurtosis, skewness, active, and inactive lever

responding in the second-order schedule of reinforcement were analyzed by two-way mixed design

ANOVAs, with Diet as a between-subjects factor and Dose as a within-subject factor. The effects of

RO5256390 treatment on food intake in the food restriction procedure were analyzed by paired

Student’s t-test, with Dose as a within-subject factor. The effects of RO5256390 treatment on food

intake in the light/dark conflict test, CPP scores in pre- and post-conditioning latency, withdrawal time

and entries in the defensive withdrawal test, and immobility time in the forced swim test were analyzed

by two-way between-subjects ANOVAs, with Diet and Dose as between-subjects factors. IFI mean,

entropy, and latency were log-transformed for statistical analysis. Western blot protein levels were

analyzed using unpaired Student’s t-tests. Student’s t-test was used for comparisons between two

means, while the Bonferroni test was used for comparisons among more than two means. Statistical

significance level was set at p≤0.05. The software/graphic packages used were SigmaPlot 11.0,

Statistica 7.0, and Origin 8.5.

15


SUPPLEMENTAL FIGURES

Supplemental Figure S1

Effects of systemic administration of the TAAR1 agonist RO5256390 (0, 10 mg/kg, i.p.) on

(A) skewness and (B) kurtosis, in 1 h self-administration of either a standard chow (in ad libitum or

food restricted regimens) or a highly palatable diet in male Wistar rats (n=7-10/group). Panels represent

M±SEM.

16



Effects of systemic administration of the TAAR1 agonist RO5256390 (0, 10 mg/kg, i.p.) on

food intake during the light/dark conflict test (n=21–23/group). Panels represent M±SEM. # p≤0.05,

vs. Vehicle Chow; * p≤0.05 vs. Vehicle Palatable.

17



(A) Drawing of rat brain slices used for western blotting studies. Circles show brain regions

that were punched: dorsal striatum (DS) and nucleus accumbens (NAcc). (B) Levels of TAAR1

protein levels (n=10–12/group) in (B) DS, and (C) NAcc of male Wistar rats self-administering 1

h/day either a standard chow (Chow) or a highly palatable diet (Palatable). Panels represent

M±SEM.

18



(A) Effects of microinfusion of the TAAR1 agonist RO5256390 (0, 1.5, 5, 15 µg/side) into the PrL

subregion of the prefrontal cortex on 1 h food self-administration of either a standard chow or a

highly palatable diet (n=11-12/group). (B) Drawing of coronal rats’ brain slices. Dots represent the

injection sites in the PrL cortex included in the data analysis. (C) Photomicrograph shows a coronal

section of the brain of a rat with representative injection sites in the PrL cortex. Panels represent

M±SEM.

19


SUPPLEMENTAL REFERENCES

Blasio A, Steardo L, Sabino V, Cottone P (2014). Opioid system in the medial prefrontal cortex mediates binge-like eating. Addict Biol 19(4): 652-662.

Castagne V, Moser P, Roux S, Porsolt RD (2011). Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice. Curr Protoc Neurosci Chapter 8: Unit 8 10A.

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SUPPLEMENTAL MATERIAL The Trace Amine-Associated … · transformed IFIs that fell from e-1. to e. 3. sec (~0.34-20.1 sec), with a bin width of e. 0.2. Total IFI time was calculated