Supporting Information - PNAS

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Jun 29, 2009 - ulation and dopamine release alcohol (VWR International) was diluted in saline (0.9% NaCl) to 15% vol/vol for i.p. injections. For NMRI and ...
Supporting Information Jerlhag et al. 10.1073/pnas.0812809106 SI Materials and Methods Animals. The results from experiments on 3 different strains of mice have led to similar conclusions in this study. All experiments were performed in adult postpubertal age-matched male mice (8–12 weeks old). GHS-R1A knockout mice on a mixed 129 Sv/Evbrd(LEX1)/C57BL/6 background (backcrossed 3 times and on average 87.5% C57BL/6 mice (1) and their corresponding wild-type littermates, generated through heterozygous breeding, were used in all experimental protocols (locomotor activity, microdialysis, CPP, and alcohol consumption experiments). The age of GHS-R1A knockout mice and littermates was 8–12 weeks. Genotyping of all mice were performed to confirm knockouts, wild-type, and heterozygous animals. Complementary experiments were conducted in outbred NMRI mice and inbred C57BL/6 mice. NMRI mice (25–40 g body weight; B & K Universal) were used for studies of locomotor activity, dopamine release, and CPP testing as such studies are well documented in this strain (2–5). Alcohol consumption experiments, however, were performed only on C57BL/6 mice (23–36 g body weight; B & K Universal) that are generally considered to be alcohol preferring and therefore have a higher level of spontaneous alcohol intake in comparison to other mouse strains (6), an advantage when attempting to suppress intake. All mice were maintained at 20 °C with 50% humidity and a 12/12 h light/dark cycle (lights on at 7 a.m.). Tap water and food (normal chow; Harlan Teklad) were supplied ad libitum, except following drug/vehicle administration. Studies were approved by the Ethics Committee for Animal Experiments in Gothenburg, Sweden. Drugs. For studies investigating alcohol-induced locomotor stim-

ulation and dopamine release alcohol (VWR International) was diluted in saline (0.9% NaCl) to 15% vol/vol for i.p. injections. For NMRI and GHS-R1A knockout mice the dose of alcohol injected i.p. was predetermined. The dose of 1.75 g/kg (i.p) was used for the NMRI mice in all locomotor activity, dopamine release, and CPP studies, based on previous experiments (7). As different mouse strains show different sensitivity to alcohol in behavioral and neurochemical studies, it was important to establish the correct dose to use for the GHS-R1A knockout mice. Thus, for these mice, the dose used (1.0 g/kg, i.p.) was determined from dose–response studies in wild-type littermates. For alcohol consumption experiments the percentage of alcohol (diluted in tap water) tested was predetermined for both strains of mice using an established protocol, as described below. Acylated ghrelin (Bionuclear) was administered bilaterally into the VTA or LDTg at a dose of 2 ␮g/mouse. This dose has been shown to activate the mesolimbic dopamine system, specifically the cholinergic–dopaminergic reward link (3) and was selected from a previous dose–response study in which it emerged as the lowest dose to increase the locomotor activity. Here we also tested 2 doses of ghrelin (1 or 2 ␮g/mouse) in the i.c.v. ghrelininduced alcohol consumption studies. Ghrelin was administered 10 min before the initiation of the experiment. The dose of BIM28163 (Ipsen Biomeasure), a GHS-R1A antagonist, has been determined previously (8) and in a dose–response study (Fig. S4), where 5 ␮g i.c.v. was the highest dose not to affect locomotor activity per se. This dose was used in all experiments and was always administered 40 min before alcohol exposure. Previous studies have established that this compound is a GHS-R1A antagonist and fully inhibits ghrelin-induced GHSR1A activation. In vivo, BIM28163 fully blocked ghrelin-induced growth hormone secretion but unexpectedly acted as a full Jerlhag et al. www.pnas.org/cgi/content/short/0812809106

agonist with regard to ghrelin-induced weight gain and food intake (8). Acylated ghrelin and BIM28163 were diluted in Ringer vehicle (Merck) and were administered in a volume of 1 ␮L via chronically implanted catheters, over 60 s. The cannula was left in place for a further 60 s to facilitate diffusion. The volume administered during intrategmental injections could raise concerns about specificity because of potential leakage into neighboring structures. Previously, however, we only observed ghrelin-induced locomotor stimulation and dopamine release using this volume for mice in which the cannula location was within the VTA or LDTg, and not when located in closely adjacent sites (3). Supportively, in the present study ghrelin did not increase alcohol consumption in mice in which the canulae were misplaced in neighboring structures (data not shown). The selected dose of JMV2959 [synthesized at the Institut des Biomole´cules Max Mousseron (IBMM), UMR5247, Centre National de la Recherche Scientifique, Montpellier 1 and 2 Universities, France], a second GHS-R1A antagonist, was determined in a dose–response study where 6 mg/kg (i.p.) was the highest dose not to affect locomotor activity per se (Fig. S5). This dose was used in all studies and was always administered 20 min before alcohol exposure. A second antagonist (JMV2959) was tested, not only to verify effects observed with the first antagonist (BIM28163) but also to extend our studies to show effects independent of the route of administration. Indeed, previous studies have established that this compound, when administered peripherally, is a GHS-R1A antagonist and suppresses food intake induced by the GHS-R1A agonist, hexarelin (9). The peripheral administration of JMV2959 also made possible studies showing repeated effects over several days. JMV2959 was dissolved in vehicle. A balanced design was used in all drug challenges. All peripheral injections were administered by the i.p. route. Locomotor Activity Experiments. We measured alcohol-induced locomotor activity as most drugs of abuse (including alcohol) cause locomotor stimulation, an effect mediated, at least in part, by their ability to enhance the extracellular concentration of accumbal dopamine. Such parameters have been suggested to be homologous effects evolving from a common mechanism involving the dopaminergic reward system, implying that these parameters reflect reward induced by drugs of abuse (10–12). Whereas CPP-testing and self-administration approaches demonstrate alcohol reward more directly, locomotor stimulation provides a more indirect yet supportive measure. Locomotor activity was recorded as described previously (2). Mice were allowed to habituate to the locomotor activity box 1 hour before drug challenge. Alcohol-induced locomotor stimulation was investigated in GHS-R1A knockout mice and in their littermate controls following i.p. administration of alcohol or an equal volume of vehicle. Alcohol-induced locomotor stimulation was further investigated following administration of BIM28163 (i.c.v.) or JMV2959 (i.p.) to NMRI mice. In Vivo Microdialysis and Dopamine Release Measurements. For measurements of extracellular dopamine levels (that reflect dopamine release), GHS-R1A knockout mice and NMRI mice were implanted unilaterally with a microdialysis probe positioned in the nucleus accumbens. For studies involving i.c.v. GHS-R1A antagonist administration, NMRI mice were also implanted with an ipsilateral guide cannula in the third ventricle. The surgery was preformed as previously described (2). 1 of 9

The effects of i.p. administered alcohol on accumbal dopamine release using microdialysis was investigated in freely moving GHS-R1A knockout mice, their littermate controls and in NMRI mice pretreated with BIM28163 (i.c.v.) or with JMV2959 (i.p.). After 1 hour of habituation to the microdialysis setup, perfusion samples were collected every 20 min. The baseline dopamine level was defined as the average of 3 consecutive samples before the first drug/vehicle challenge. For GHS-R1A knockout mice, baseline samples were followed by an i.p. injection of vehicle. In the experiments evaluating the effect of BIM28163 or JMV2959 on alcohol-induced dopamine release, NMRI mice were administered an initial dose of either alcohol or vehicle i.p. followed by BIM28163 (i.c.v.), or JMV2959 (i.p.), or vehicle. A second i.p. injection of either alcohol or vehicle was thereafter delivered and 4 subsequent perfusion samples were collected. The dopamine levels in the dialysates were determined by HPLC with electrochemical detection as described previously (2). Conditioned Place Preference. To further evaluate the impact of

ghrelin on the rewarding effects of alcohol, CPP tests were performed, both in NMRI mice (GHS-R1A antagonist studies) and also in GHS-R1A knockout mice and their littermates. A 2-chambered CPP apparatus, with distinct visual and tactile cues was used (5, 13). One compartment was defined by black and white striped walls and by a dark laminated floor whereas the other had a white unlaminated floor and walls of wooden texture. Compartments were illuminated by 45 lx. The procedure consisted of preconditioning (day 1), conditioning (days 2–5), and postconditioning (day 6). On day 1 (preconditioning), mice were i.p. injected with vehicle and initial place preference was determined during 20 min, to determine which of the 2 compartments could be labeled ‘‘least preferred’’ for each mouse. Conditioning (20 min per session) was done using a biased procedure in which alcohol was paired to the least preferred compartment. In this biased procedure, it should be more difficult to obtain a positive CPP response. Mice received a total of 2 i.p. injections of alcohol per day (1.75 g/kg in NMRI mice and 1.0 g/kg to GHS-R1A knockout mice) in the morning and vehicle conditioning in the afternoon, or vice versa. After injection the mice were placed in the appropriate compartment. On day 6 mice were given free access to both compartments for 20 min. Before the test session NMRI mice were acutely injected with either of the 2 GHS-R1A antagonists (BIM28163 i.c.v. or JMV2959 i.p.) or vehicle, whereas the GHS-R1A knockout mice were untreated this day. CPP was calculated as the difference in percentage of total time spent in the alcohol-paired (i.e., least preferred) compartment during the postconditioning and the preconditioning session. Alcohol Consumption Using a Limited Access Paradigm in C57BL/6 Mice and GHS-R1A Knockout Mice. C57BL/6 mice, known to consume

high amounts of alcohol compared to other strains of mice, and GHS-R1A knockout mice were used in alcohol consumption experiments. Initially, all mice were group housed and had continuous access to both tap water and increasing concentration of alcohol (2, 4, 6, 8, and 10%) over a 2-week period (approximately 3 days at each percentage of alcohol). Thereafter, they were housed individually for 9 weeks with continuous access to tap water and alcohol solution (10%). Following this free choice continuous paradigm, water and alcohol intake were limited to the first 90 min of the dark period (i.e., a limited access paradigm) (6). In studies using JMV2959 and the GHS-R1A knockout mice, access to alcohol solution was limited to the first 90 min of the dark period and is considered a more conservative protocol. This modified protocol was used because of changes in ethical permission. These 2-bottle (alcohol/water) free choice limited access paradigms were maintained for 2 weeks before Jerlhag et al. www.pnas.org/cgi/content/short/0812809106

ghrelin/GHS-R1A antagonist treatment. Drugs were administered before lights off. Four days before treatment mice were inserted with an i.c.v. guide cannula, as previously described (2), for central administration of either ghrelin (experiment using both GHS-R1A knockout and C57BL/6 mice) or BIM28163 (C57BL/6 mice) or bilateral guide cannulae into the VTA or LDTg for local injection of ghrelin (C57BL/6 mice) as previously described (3). The mice received either drug (ghrelin or BIM28163) or vehicle on day 1 and the reverse treatment on day 2, according to a balanced design. The same experimental setups were used both days (vide infra). In subsequent experiments ghrelin or vehicle were administered bilaterally into the VTA or LDTg. These latter alcohol consumption experiments involved a single test on each mouse because of experimental limitations of repeated intranuclear injection via bilateral cannulae. Either the GHSR1A antagonist, JMV2959, or vehicle was subsequently administered by repeated i.p. injections over a period of 5 days, because of advantages with peripheral administrations. These data were analyzed as the average 90-min intake over the 5 treatment days. In all experiments the intake of alcohol, water, and food were measured throughout the 90-min drinking session. Twenty-fourhour food intake was also measured. The measurements of alcohol consumption are expressed per gram of body weight. Verification of Probe and/or Guide Cannula Placement. After com-

pletion of locomotor activity measurements, microdialysis, and alcohol consumption experiments, the locations of the probe and/or cannulae were verified as previously described (2). Only mice with guide cannula placement in the third ventricle, VTA or LDTg and/or probe placement in the nucleus accumbens were included in the statistical analysis (Fig. S3). Statistical Analyses. All locomotor activity and CPP data were evaluated by a 2-way ANOVA followed by Tukey/Kramer (for locomotor activity in GHS-R1A knockout mice) or Bonferroni post hoc tests comparing treatments. The microdialysis experiments in GHS-R1A knockout mice were evaluated by a 2-way ANOVA followed by Bonferroni post hoc test for comparisons between different treatments at given time points. In microdialysis experiments using GHS-R1A antagonist, data were evaluated by a 2-way ANOVA followed by an analysis of repeated measures in 2 parts. The first part (⫺40 min to 160 min) analyzes the effects of alcohol on accumbal dopamine release and the second part (160 min to 240 min) investigates the effects of GHS-R1A antagonists or vehicle on alcohol-induced accumbal dopamine release. The limited access drinking data were evaluated by paired t test (ghrelin and BIM28163 i.c.v.) in both C57BL/6 and GHS-R1A knockout mice as the individual mice serve as their own controls. An unpaired t test was used when ghrelin was administered into the VTA or LDTg as the constraints of this procedure made it possible to perform only 1 bilateral injection to each animal. To test the differences in the magnitude of the drinking response to ghrelin treatment between the different administration routes (i.c.v., VTA, or LDTg) a z-score was calculated for the delta difference of the paired and unpaired experiments. The P value was then calculated and it was based on the assumption of an approximate normal distribution (according to the central limit theorem). An unpaired t test was also used in GHS-R1A knockout studies when comparing, in different genotypes, the effect of vehicle treatment on alcohol consumption and spontaneous alcohol intake before treatment. As JMV2959 or vehicle were administered on 5 consecutive days, a 2-way ANOVA followed by the Bonferroni post hoc comparisons between different treatments was used, allowing detection in differences over time. Data are presented as mean ⫾ SEM. A probability value of P ⬍ 0.05 was considered as statistically significant. 2 of 9

GHS-R1A knockout mice were developed in collaboration with Lexicon Genetics. Using a PCR probe, genomic clones were isolated by screening of the 129SvEvBrd-derived lambda pKOS genomic library (14). A 9.5-kb genomic clone spanning exon 1 and exon 2 was used to generate the targeting vector via yeast-mediated homologous recombination. In this vector a 1325-bp genomic fragment, spanning exon 1 and exon 2, was replaced by a floxed version of exon 1 and exon 2 including a 1.7-kb PGK-neo selection cassette flanked by 2 Frt sites. The NotI-linearized vector was electroporated into 129 Sv/ Evbrd(LEX1) embryonic stem (ES) cells and G418-fialuridine (FIAU)-resistant ES cell clones were isolated and analyzed for homologous recombination by Southern blot analysis. Targeted ES cell clones were injected into C57BL/6 (albino) blastocysts, and the resulting chimeras were mated to C57BL/6 (albino) females to generate animals heterozygote for the floxed GHSR1A allele. These were subsequently crossed to protamine-Cre mice (15) and male descendants heterozygote for both the floxed GHS-R1A allele and the protamine Cre transgene were crossed to C57BL/6 females to obtain heterozygote GHS-R1A knockout animals. These were subsequently crossed to generate all 3 genotypes used in the reported studies. PCR was used to screen genotypes of all animals by using DNA isolated from mouse tail biopsy samples. DNA was extracted using Qiagen DNeasy Tissue kit (Qiagen). The DNA was diluted 1:5 and then amplified by PCR on a PTC-200 Thermal Cycler (Bio-Rad) using the follow-

ing conditions: 95 °C 15 min, 95 °C 30 s, 56 °C 45 s, and 72 °C 60 s for 33 cycles followed by 72 °C for 10 min. Primers 5⬘TGGGGGTGCGA ACAT TAGC3⬘ and 5⬘CTGA AGGCATCTTTCACTACG⬘ amplified a 412-bp band from the wild-type allele and primers 5⬘ACATATTCTATGTGAGGCACC3⬘ and 5⬘CTGAAGGCATCTTTCACTACG3⬘ amplified a 469-bp band from the knockout allele. The PCR product was analyzed on a 1.5% agarose gel. Quantitative RT-PCR analysis was used to show expression or absence of the GHSR1A transcript. Total RNA was isolated from brain and pituitary using TRIzol (Invitrogen) and first strand cDNA synthesis was performed on 0.5 ␮g of total RNA using random hexamer primers and Superscript II RT (Invitrogen). Quantitative PCR was performed on a ABI Prism 7700 cycler (Applied Biosystems) using a Taqman PCR kit. Serial dilutions of cDNA were used to generate standard curves of threshold cycles vs. the logarithms of concentration for ␤-actin and GHS-R1A. A linear regression line calculated from the standard curves allowed the determination of transcript levels in RNA samples from mice. The GHS-R1A primer-probe pair (primer 5⬘CCGCCTCTGGCAGTATCG3⬘ primer 5⬘GCTGACAAACTGGAAGAGTTTGC3⬘, probe 5⬘CCCTGGA ACT TCGGCGACCTGC3⬘ [5⬘]FA M [3⬘]TAMRA) relative to ␤-actin (primer 5⬘CATCTTGGCCTCACTGTCCAC3⬘, primer 5⬘GGGCCGGACTCATCGTACT3⬘, probe 5⬘TGCTTGCTGATCCACATCTGCTGGA3⬘ [5⬘]FAM [3⬘]TAMRA) was used to asses expression levels.

1. Verhulst PJ, et al. (2008) Role of ghrelin in the relation between hyperphagia and accelerated gastric emptying in mice with streptozotocin-induced diabetes. Gastroenterology 134:A49 –A49. 2. Jerlhag E, et al. (2006) Ghrelin stimulates locomotor activity and accumbal dopamineoverflow via central cholinergic systems in mice: implications for its involvement in brain reward. Addict Biol 11:45–54. 3. Jerlhag E, et al. (2007) Ghrelin administration into tegmental areas stimulates locomotor activity and increases extracellular concentration of dopamine in the nucleus accumbens. Addict Biol 12:6 –16. 4. Jerlhag E, Egecioglu E, Dickson SL, Svensson L, Engel JA (2008) Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelininduced locomotor stimulation and dopamine overflow in nucleus accumbens. Eur Neuropsychopharm 18:508 –518. 5. Jerlhag E (2008) Systemic administration of ghrelin induces conditioned place preference and stimulates accumbal dopamine. Addict Biol 13:358 –363. 6. Rhodes JS, Best K, Belknap JK, Finn DA, Crabbe JC (2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav 84:53– 63. 7. Jerlhag E, Grotli M, Luthman K, Svensson L, Engel JA (2006) Role of the subunit composition of central nicotinic acetylcholine receptors for the stimulatory and dopamine-enhancing effects of ethanol. Alcohol and Alcohol 41:486 – 493.

8. Halem HA, et al. (2004) Novel analogs of ghrelin: Physiological and clinical implications. Eur J Endocrinol 151:S71–S75. 9. Moulin A, et al. (2007) Toward potent ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure. 2. Synthesis and pharmacological in vitro and in vivo evaluations. J Med Chem 50:5790 –5806. 10. Engel JA, et al. (1988) Biochemical and behavioral evidence for an interaction between ethanol and calcium-channel antagonists. Alcohol and Alcohol 23:A13–A13. 11. Imperato A, Dichiara G (1986) Preferential stimulation of dopamine release in the nucleus-accumbens of freely moving rats by ethanol. J Pharmacol Exp Ther 239:219 – 228. 12. Wise RA, Bozarth MA (1987) A psychomotor stimulant theory of addiction. Psychol Rev 94:469 – 492. 13. Sanchis-Segura C, Spanagel R (2006) Behavioural assessment of drug reinforcement and addictive features in rodents: An overview. Addict Biol 11:2–38. 14. Wattler S, Kelly M, Nehls M (1999) Construction of gene targeting vectors from lambda KOS genomic libraries. Biotechniques 26:1150 – 6, 1158, 1160. 15. O’Gorman S, Dagenais NA, Qian M, Marchuk Y (1997) Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells. Proc Natl Acad Sci USA 94:14602–14607

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2.0

Alc-Veh-Alc Alc-BIM-Alc Dopamine % baseline

*

1.5 g/kg/90 min

B. Dopamine release

1.0

0.5

C. Locomotor activity Veh-BIM-Veh Alc-Veh-Veh



* * * * * * *

***

***

Counts/60min

A. Alcohol intake

#

Alc/Veh

↑ ↑ Alc/Veh

BIM/Veh

0.0 Vehicle

BIM

Veh/Veh

BIM/Veh

Veh/Alc

BIM/Alc

Fig. S1. Central (i.c.v.) administration of a ghrelin receptor (GHS-R1A) antagonist (BIM28163) suppressed alcohol reward in NMRI or C57BL/6 mice. (A) Alcohol consumption was decreased by BIM28163 (i.c.v.), when compared to vehicle treatment, in a 2-bottle (alcohol/water) free choice limited access paradigm in C57BL/6 mice (n ⫽ 22 in each group; *, P ⫽ 0.01, paired t test). (B) The alcohol-induced increase in accumbal dopamine release was absent in antagonist-treated but not in vehicle-treated NMRI mice (n ⫽ 8 for Alc–Veh–Alc, Veh–BIM–Veh, and Alc–Veh–Veh; n ⫽ 10 for Alc–BIM–Alc). In B we first demonstrated an increase of alcohol on dopamine release in comparison to vehicle control NMRI mice [F (1, 16) ⫽ 0.01, P ⫽ 0.92; time F (11, 176) ⫽ 9.43, P ⫽ 0.001; treatment ⫻ time interaction F (11, 176) ⫽ 1.44, P ⫽ 0.16]. Second, we showed that pretreatment of BIM28163 (i.c.v.) attenuated the alcohol-induced enhanced dopamine release compared to vehicle pretreatment [treatment F (1, 16) ⫽ 9.33, P ⫽ 0.01; time F (4, 64) ⫽ 10.22, P ⫽ 0.001; treatment ⫻ time interaction F (4, 64) ⫽ 7.20, P ⫽ 0.001]. This difference (P ⬍ 0.01) was evident at time intervals 200, 220, and 240 min (**, P ⬍ 0.01; ***, P ⬍ 0.001, Bonferroni post hoc test). (C) The alcohol-induced locomotor stimulation was attenuated by acute i.c.v. administration of BIM28163 in NMRI mice [F (3, 28) ⫽ 10.15, P ⫽ 0.001] (n ⫽ 8 in each group; **, P ⬍ 0.001; #, P ⫽ n.s. for Veh–Veh vs. BIM–Alc, Bonferroni post hoc test). All values represent mean ⫾ SEM.

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1

A.

2 FRT PGK-neo FRT

Targetting vector LoxP

LoxP

1

2

1

2

3

Wild type locus 3 FRT PGK-neo FRT

Targetted locus LoxP

LoxP

3 Cre excised locus

Lox P

B.

Fig. S2. (A) GHS-R1A knockout mice were generated in collaboration with Lexicon Genetics. The strategy applied yield deletion of the first 2 out of the 3 exons that encode GHSR1 (ENSEMBL: ENSMUSG00000051136). Correct targeting in ES cells was confirmed by Southern blot analysis (results not shown). In mice, loss of the wild-type GHS-R1A allele was confirmed by PCR analysis (results not shown). (B) Loss of expression of the GHS-R1A transcript in the knockout mice was confirmed by quantitative RT-PCR performed on total RNA, isolated from brain and pituitary from wild type (⫹/⫹) and homozygote (⫺/⫺) GHS-R1A knockout animals (n ⫽ 6 in each group). The GHSR1 transcript was absent in all tissues derived from the homozygote GHS-R1A knockout.

.

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C.

A.

Bregma -3.4 mm

Bregma +1.5 mm

B.

D.

Bregma -0.9 mm

Bregma -5.0 mm

Fig. S3. A coronal mouse brain section showing 10 representative probe placements (illustrated by vertical lines) in the nucleus accumbens (A), i.c.v. (B), VTA (C), or LDTg (D) of mice used in the present study (1). The number given in each brain section indicates millimetres anterior (⫹) and posterior (⫺) from bregma.

1. Franklin KBJ, Paxinos G (1996) In: The Mouse Brain in Stereotaxic Coordinates. Academic Press, New York.

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Locomotor activity

Counts/60 min

***

Veh

BIM 20

BIM 5

BIM 1.25

Fig. S4. Dose–response effects of i.c.v. injection of the GHS-R1A antagonist, BIM28163, on locomotor activity in NMRI mice. A statistically significant overall effect of treatment on locomotor activity [F (3, 28) ⫽ 6.30, P ⫽ 0.002, 1-way ANOVA, n ⫽ 8 in each group] was observed. BIM28163 increased the locomotor activity at a dose of 20 ␮g/1 ␮l (P ⫽ 0.001, Bonferroni post hoc test). Neither 5 ␮g/1 ␮l nor 1.25 ␮g/1 ␮l (P ⫽ 0.206, P ⫽ 0.299, respectively, Bonferroni post hoc test) had an effect on locomotor activity per se.

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Locomotor activity

Counts/60 min

***

***

Veh

JMV 24

JMV 12

JMV 6

Fig. S5. Dose–response effects of i.p. injection of the GHS-R1A antagonist, JMV2959, on locomotor activity in NMRI mice. A statistically significant overall effect of treatment on locomotor activity [F (3, 28) ⫽ 20.11, P ⫽ 0.001, 1-way ANOVA, n ⫽ 8 in each group] was observed. JMV2959 reduced the locomotor activity at the doses 24 mg/kg and 12 mg/kg (both P ⫽ 0.001, Bonferroni post hoc test). Six milligrams/kilogram had no effect on locomotor activity per se (P ⫽ 0.905, Bonferroni post hoc test).

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Table S1. Alcohol, water, and food intake in GHS-R1A knockout mice following i.c.v. ghrelin treatment

n

Treatment

Alcohol intake (g/kg/90 min)

Wt/wt (GHS-R1A)

6

Wt/⫺ (GHS-R1A)

12

⫺/⫺ (GHS-R1A)

8

Vehicle Ghrelin Vehicle Ghrelin Vehicle Ghrelin

0.40 ⫾ 0.02 0.58 ⫾ 0.04* 0.42 ⫾ 0.04 0.50 ⫾ 0.09 0.55 ⫾ 0.10 0.56 ⫾ 0.14

Genotype

Water intake (g/90 min)

Total fluid intake (g/90 min)

Food intake (g/90 min)

0.22 ⫾ 0.07 0.27 ⫾ 0.04 0.38 ⫾ 0.06 0.33 ⫾ 0.04 0.38 ⫾ 0.08 0.34 ⫾ 0.04

0.45 ⫾ 0.09 0.58 ⫾ 0.03 0.62 ⫾ 0.07 0.59 ⫾ 0.07 0.68 ⫾ 0.11 0.65 ⫾ 0.09

0.01 ⫾ 0.01 0.73 ⫾ 0.18* 0.11 ⫾ 0.10 0.65 ⫾ 0.26* 0.15 ⫾ 0.10 0.29 ⫾ 0.11

The effects of i.c.v. ghrelin on alcohol intake and food consumption were absent in GHS-R1A knockout mice but not in wild-type littermates, indicating that GHS-R1A is required for ghrelin-induced alcohol intake. I.c.v. injection of ghrelin increased food intake but not alcohol intake in heterozygote mice. No significant effect on water or total fluid intake was seen in any genotype following ghrelin treatment (n ⫽ 6 –12; *, P ⬍ 0.05 vs. vehicle treatment, paired t test). No differences in alcohol, water, total fluid, and food intake were observed between any genotype (unpaired t test). Any apparent difference in response to vehicle treatment between genotype did not give a statistical difference (P ⬎ 0.05, unpaired t test). All values represent mean ⫾ SEM.

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