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Supplemental Methods
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Cocaine-induced conditioned place preference
A three-chamber CPP apparatus was used. The two-compartments (15  13  12 cm)
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had either a grid floor with black walls or a mesh floor with white walls that were separated by a
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gray chamber (6  13  12 cm) with guillotine doors. The compartments were dimly illuminated.
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The procedure consisted of habituation, a pre-test, conditioning, and a post-test. During
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habituation, the mice were allowed to freely explore all of the compartments for 30 min for 3
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days. On the pre-test day, the amount of time spent in each compartment was recorded for 20
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min for Fev-Cre transgenic mice and their littermate mice or 30 min for 5-HT1A autoKD and their
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control mice. Conditioning for 5-HT1A autoKD mice and their control mice was conducted using a
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biased subject assignment procedure. The selection of the cocaine-paired compartment (CS+
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or positive conditioned stimulus), a distinctive environmental cue, is based on the subject’s
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unlearned least-preference in the pre-test (Cunningham et al, 2006). Half of the animals
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received a cocaine injection (7.5 mg/kg, i.p.) and were immediately confined to the least-
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preferred compartment (CS+) for 40 min on the first conditioning day. The other half of the
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animals received saline (i.p.) and was confined to the preferred compartment (CS-) for 40 min
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on the first conditioning day. Conditioning for Fev-Cre transgenic mice was conducted using an
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unbiased and counter-balanced procedure in which the CS+ is assigned randomly, regardless
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of the subject’s unlearned preference/ least-preference (Cunningham et al, 2006). Half of the
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animals received a cocaine injection (7.5 mg/kg, i.p. or 4 mg/kg, i.p. for mice injected with AAV-
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hM3D(Gq)-mCherry or AAV-hM4D(Gi)-mCherry, respectively) and were randomly and
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immediately confined to the least-preferred or preferred compartment (CS+) for 40 min on the
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first conditioning day. The other half of the animals received saline (i.p.) and was confined to the
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remaining least-preferred or preferred compartment (CS-) for 40 min on the first conditioning
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day. Conditioning with cocaine (C) and saline (S) alternated daily for 6 days (C-S-C-S-C-S or S-
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C-S-C-S-C). On the day following the last conditioning session, mice were given free access to
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all of the compartments for 20 min for Fev-Cre transgenic mice and their littermate mice or 30
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min for 5-HT1A autoKD and their control mice. The time spent in each compartment was then
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recorded.
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Cocaine self-administration
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Rats were catheterized with indwelling intravenous catheters (inner diameter, 0.3 mm;
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outer diameter, 0.64 mm; Dow Corning, Midland, MI, USA) as described previously (Wee et al,
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2007) and trained to self-administer cocaine (0.5 mg/kg/infusion) under a fixed-ratio one (FR1)
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schedule of reinforcement, with one lever press producing one drug injection, for 1 h per day.
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After 10 days of cocaine self-administration with daily 1-h access (baseline sessions), the
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animals were divided into two groups balanced by cocaine self-administration in the last
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baseline session. One group continued to self-administer cocaine with 1-h access (short access
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[ShA]), whereas the other group was allowed to self-administer cocaine for 6 h per day (long
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access [LgA]). After 14 sessions of cocaine self-administration with extended access, the effect
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of pretreatment with WAY100635, a selective 5-HT1A receptor antagonist, on cocaine self-
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administration was examined under a progressive-ratio (PR) schedule in the rats. For the PR
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schedule, the response requirement began at one response per injection and increased
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according to the following equation: responses/injection = [5  e(injection number  0.2)] - 5 (Richardson
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and Roberts, 1996). When a rat failed to achieve the response requirement within 1 h, the
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session ended. This PR schedule measures how much work an animal will expend for a drug
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injection. The test sessions were separated by at least two or three FR sessions with either 1-h
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or 6-h access, depending on the group.
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Microinjection in the DRN on cocaine self-administration
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Male Wistar rats were catheterized with indwelling catheters and trained to self-
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administer cocaine (0.5 mg/kg/infusion) under an FR schedule for 1 h per day. After a minimum
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of 8 days of cocaine self-administration with daily 1 h access, the rats were implanted with
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intracranial cannulae that ended 2 mm dorsal to the DRN (anterior/posterior, -7.4 mm; lateral,
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+1.4 mm; dorsal/ventral, -6mm; with a lateral angle of 12°; Paxinos and Watson, 1998). After
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recovery, the rats were again allowed to self-administer cocaine for six more days and divided
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into two groups balanced by cocaine intake in the last baseline session. One group of rats
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continued to self-administer cocaine with 1-h access (ShA), whereas the other group was
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allowed to self-administer cocaine for 6 h per day (LgA). After 13 sessions of cocaine self-
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administration with either 1-h or 6-h access, the effect of WAY100635 (75, 150 µg) in the DRN
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on cocaine self-administration was determined under a PR schedule. On the test day, the
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obturator was removed, and a 33-gauge microinjector was inserted into the guide cannula. The
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microinjector extended 2 mm below the end of the guide cannula to reach the DRN. The drug
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was infused in a volume of 0.5 l over 2 min, and the microinjector was left in place for an
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additional 2 min after the infusion to allow the drug to diffuse completely. Immediately after the
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injection, the rats were placed in self-administration chambers prior to the start of the PR
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session.
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Drug
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WAY100635 was administered subcutaneously. The WAY100635 pretreatment time was
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30 min. The drug doses were tested in a counter-balanced manner across rats. For
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microinjections, WAY100635 was dissolved in 0.5 X PBS (pH 7.4).
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Supplemental Results
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Effect of cocaine injection on time spent in the CS- and CS+ of the pre-test and post-test in WT
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and 5-HT1A autoKD mice
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WT mice showed a difference in both the time spent in the CS- and CS+ of pre-test
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versus post-test (Fig. 1b; Test  Stimulus interaction, F1,16 = 28.68, p < 0.001; Stimulus F1,16 =
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0.18, p =0.68, Test F1,16 = 0.19, p =0.67); whereas 5-HT1A autoKD mice did not significantly
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differ in time spent in either the CS- and CS+ of pre-test compared with post-test (Fig. 1c; Test 
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Stimulus interaction, F1,8 = 5.21, p =0.05; Stimulus F1,8 = 2.86, p =0.13, Test F1,8 = 0.71, p
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=0.43).
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Effect of WAY100635 microinjection in the DRN on cocaine self-administration under a PR
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schedule
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Cocaine self-administration increased with daily 6-h access in rats, whereas it remained
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constant with daily 1-h access (Fig. S4A, Session  Access interaction, F11,187 = 5.91, p < 0.001;
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Session, F11,187 = 11.48, p < 0.001; Access, F1,17 = 63.92, p < 0.001).
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Supplemental Figure Legends
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Fig. S1. Excitation and inhibition of the DRN→NAc 5-HT projection by DREADDs did not affect
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anxiety-like behavior. Effect of CNO injection (1 mg/kg, i.p.) on the time spent in the open arms
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of the elevated plus maze (EPM) in Cre+ and Cre- mice expressing Gq-DREADD (a), Gi-
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DREADD (b), and blank-DREADD (c). Effect of CNO injection (1 mg/kg, i.p.) on the entries into
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the open arms of the EPM in Cre+ and Cre- mice expressing Gq-DREADD (d), Gi-DREADD (e),
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and blank-DREADD (f).
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Fig. S2. Excitation and inhibition of the DRN→NAc 5-HT projection by DREADDs did not affect
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the time spent immobile for the first 2 min. Effect of CNO injection (1 mg/kg, i.p.) on the time
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spent immobile for the first 2 min after CNO injection in Cre+ and Cre- mice expressing Gq-
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DREADD (a), Gi-DREADD (b), and blank-DREADD (c).
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Fig. S3. Excitation and inhibition of the DRN→NAc 5-HT projection by DREADDs did not affect
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motor-behaviors. Effect of CNO injection (1 mg/kg, i.p.) on distance travelled in a 30-min and
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20-min locomotor activity session after CNO injection in Cre+ and Cre- mice expressing Gq-
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DREADD (a), Gi-DREADD (b), and blank-DREADD (c).
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Fig. S4. Cocaine self-administration in catheterized ShA and LgA rats that were tested with
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intra-DRN WAY100635 administration. (a) Entire-session cocaine self-administration in short
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access (ShA) and extended access (LgA) rats. ***p < 0.001, compared with Session 1. (b)
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Representative images of the drug injector location, verified by an ink injection at the end of the
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study. (c) Injection sites of WAY100635 in the DRN in all of the animals that were included in
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the data analysis.
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References
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Cunningham CL, Gremel CM, Groblewski PA (2006). Drug-induced conditioned place
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preference and aversion in mice. Nature protocols 1(4): 1662-1670.
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Richardson NR, Roberts DC (1996). Progressive ratio schedules in drug self-administration
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studies in rats: a method to evaluate reinforcing efficacy. J Neurosci Methods 66(1): 1-11.
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Wee S, Specio SE, Koob GF (2007). Effects of dose and session duration on cocaine self-
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administration in rats. J Pharmacol Exp Ther 320(3): 1134-1143.
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