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Food reward-sensitive interaction of ghrelin and opioid receptor pathways in mesolimbic dopamine system
Neuropharmacology 67 (2013) 395e402
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Food reward-sensitive interaction of ghrelin and opioid receptor pathways in mesolimbic dopamine system Yukie Kawahara a, *, Fumi Kaneko a, Makiko Yamada a, Yuki Kishikawa a, Hiroshi Kawahara b, Akinori Nishi a a b
Department of Pharmacology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan Department of Dental Anesthesiology, School of Dentistry, Tsurumi University, Yokohama 230-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2012 Received in revised form 22 November 2012 Accepted 24 November 2012
Ghrelin is a stomach-derived orexigenic peptide. The goal of the study was to investigate the roles of mu and kappa opioid receptors in systemic ghrelin-mediated regulation of the mesolimbic dopamine system. To evaluate the interaction of systemic ghrelin with values of food reward, rats were exposed to food removal, regular food or palatable food after systemic ghrelin administration. Extracellular dopamine levels were quantified in the nucleus accumbens (NAc) and receptor-specific compounds were infused into the ventral tegmental area (VTA) using dual-probe microdialysis. Consumption of regular or palatable food without systemic ghrelin administration induced an increase in dopamine levels in the NAc via activation of mu opioid receptors in the VTA. Systemic ghrelin administration (3 nmol, i.v.) followed by no food induced a decrease in dopamine levels via activation of kappa opioid receptors in the VTA. Systemic ghrelin administration followed by consumption of regular food induced an increase in dopamine levels via preferential activation of mu opioid receptors, whereas systemic ghrelin administration followed by consumption of palatable food suppressed the increase in dopamine levels via preferential activation of kappa opioid receptors. Thus, natural food reward and systemic ghrelin activate mu and kappa opioid receptor pathways in the VTA, respectively, resulting in opposite influences on dopamine release in the NAc. Furthermore, systemic ghrelin induces switching of the dominant opioid receptor pathway for highly rewarding food from mu to kappa, resulting in suppression of the mesolimbic dopamine system. These novel findings might provide insights into the neural pathways involved in eating disorders. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Microdialysis Accumbens VTA
1. Introduction Ghrelin is a stomach-derived orexigenic hormone that modulates food intake and energy balance via homeostatic brain circuits that reside in the hypothalamic area (Briggs and Andrews, 2011; Diéguez et al., 2010). Recent evidence suggests a role of ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), in food reward behavior that is tightly associated with the mesolimbic dopamine system (Skibicka and Dickson, 2011; Landgren et al., Abbreviations: NAc, nucleus accumbens; VTA, ventral tegmental area; GHSR, growth hormone secretagogue receptor; HPLC, high performance liquid chromatography. * Corresponding author. Tel.: þ81 942 31 7545; fax: þ81 942 31 7696. E-mail addresses: [email protected] (Y. Kawahara), kaneko_fumi@ kurume-u.ac.jp (F. Kaneko), [email protected] (M. Yamada), [email protected] (Y. Kishikawa), [email protected] (H. Kawahara), [email protected] (A. Nishi). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.11.022
2011). Besides the nutritional state, ghrelin has an impact on the neuronal circuit that signals the reward value of food ingested to dopamine neurons in the ventral tegmental area (VTA) (Abizaid, 2009; Skibicka and Dickson, 2011). In fact, ghrelin secretion is downregulated by high-fat and palatable diets (Beck et al., 2002; Lindqvist et al., 2005), but stimulated before meals and during hunger. The mesolimbic dopamine system from the VTA to the nucleus accumbens (NAc) comprises a core component of the mesolimbic reward circuit. GHSRs are strongly expressed in the VTA, especially in dopamine neurons and GABAergic interneurons (Abizaid, 2009), and circulating ghrelin activates GHSRs in the VTA and hypothalamus (Skibicka and Dickson, 2011). Activation of GHSRs in the VTA by direct application of ghrelin induces dopamine release in the NAc and feeding behavior (Jerlhag et al., 2007; Abizaid et al., 2006; Kawahara et al., 2009). Interestingly, peripherally administered ghrelin also causes feeding behavior, but requires food
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consumption to induce dopamine release in the NAc (Kawahara et al., 2009), suggesting an interaction of ghrelin and food reward in the mesolimbic dopamine system. Ghrelin/GHSR signaling in the VTA has been identified as a crucial component to increase food reward and motivational behavior for food (Skibicka et al., 2011; King et al., 2011), but not homeostatic feeding (Egecioglu et al., 2010; Skibicka et al., 2011). Rewarding aspects of food intake are mediated by the central opioid system, which leads to hedonic consummatory behavior, as shown in many studies (review: Olszewski et al., 2011; Gosnell and Levine, 2009; Tanda and Di Chiara, 1998). Opioids, especially mu opioid receptor agonists, applied to the VTA stimulate feeding behavior (Figlewicz and Sipols, 2010), and palatable food-induced dopamine release in the NAc requires activation of mu opioid receptors in the VTA (Tanda and Di Chiara, 1998). Activation of mu opioid receptors inhibits GABAergic interneurons in the VTA, which results in disinhibition of dopamine neurons and facilitates dopamine release in the NAc (Johnson and North, 1992; Chefer et al., 2009). In contrast, kappa opioid receptors in the VTA inhibit dopamine neurons (Margolis et al., 2003; Ford et al., 2006), suggesting opposing roles of mu and kappa opioid receptors (Pan, 1998; Bruijnzeel, 2009). Thus, opioids and ghrelin act on the mesolimbic dopamine system and may interact to regulate food reward. In fact, a recent study showed that ghrelin induces sucrosemotivated behavior via interaction with mu opioid receptors in the VTA (Skibicka et al., 2012). We have previously shown that peripherally administered ghrelin and GHSRs in the VTA regulate the mesolimbic dopamine system differentially depending on the state of food consumption (Kawahara et al., 2009). In the present study, we examined the role of opioid receptors in regulation of the mesolimbic dopaminergic system by ghrelin associated with food reward. Dual-probe microdialysis experiments revealed the involvement of mu and kappa opioid receptor pathways, which are differentially activated by systemic ghrelin and food consumption depending on the values of food reward.
were allowed to recover for 5e7 days before performance of microdialysis experiments. At 24 h before the start of the experiments, rats were moved to an acrylic box (30 30 40 cm) individually and microdialysis probes were set in the guide cannulas. These probes extended 1 mm beyond the tip of the guide cannulas for both the NAc and VTA. On the same day, novel objects of flavored serial food (Asahi Food & Healthcare Co.) to which rats exhibit palatability were introduced into the acrylic box for 24 h to promote habituation. Flavored serial food was removed before the start of experiments on the day of the experiments, whereas rats had free access to regular food until intravenous administration of ghrelin or saline. During the experimental period, rats were exposed to regular food (3.6 kcal/g) or palatable food (5.0 kcal/g) for 15 min after ghrelin administration after obtaining three stable consecutive samples of dopamine differing by <10%, and thereafter food was removed. In the experimental group that received ghrelin followed by no food, food was removed from the container manually just before ghrelin administration. Rats used for recording a food deprivation-induced eating response received no food for 24 h before exposure to food for a period of 15 min to induce food ingestion. The dose of ghrelin (3 nmol/rat) for intravenous administration was selected based on previous reports (Date et al., 2006; Ukkola, 2004). A polyethylene cannula (PE-10; Becton Dickinson, Franklin Lakes, N.J., USA) filled with heparinized saline (50 IU/mL) was inserted into the external jugular vein. Dialysate fractions were collected every 15 min. Dopamine was quantified by HPLC with electrochemical detection conducted as in our previous study (Kawahara et al., 2009). The average baseline values of dopamine in dialysates from the NAc did not differ significantly among the different experiments. Therefore, these data were grouped and the basal value (S.E.M.) was determined as 11.65 1.06 fmol/sample (n ¼ 84). At the end of the experiments, the rats were given an overdose of chloral hydrate and brains were fixed with 4% paraformaldehyde via intracardiac infusion. Coronal sections (50-mm thick) were cut and dialysis probe placement was localized based on the atlas of Paxinos and Watson (2007). Rats in which dialysis probes and guide cannulas were found to be misplaced were not included in data analysis. 2.4. Expression of data and statistical analysis All values are expressed as percentages of basal values obtained as the average for three baseline samples. Values obtained after drug administration were compared with a basal value of 100%. Repeated measures one-way analysis of variance (ANOVA) and a Dunnett multiple comparison test for post-hoc determination were performed using the SAS mixed procedure (SAS Institute, Cary, N.C., USA). One-way ANOVA and a Scheffe multiple comparison test for post-hoc determination were used for comparison of the amount of food consumption. Repeated measures two-way ANOVA and a Bonferroni multiple comparison test for post-hoc determination were used for comparison between experimental groups (GraphPad Prism, GraphPad Software Inc., San Diego, CA, USA). The level of significance was set at p < 0.05.
2. Materials and methods 2.1. Animals Male Wistar rats (270e340 g, Kyudo, Tosu, Japan) were maintained at 23 2 C under a 12-h lightedark cycle with free access to food and water. All rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the U.S. National Institutes of Health, and the specific protocols were approved by the Committee for Animal Experimentation, Kurume University School of Medicine. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Drugs Rat ghrelin acylated at the serine 3 position with an octanoyl group (Peptide Inc, Osaka, Japan) was dissolved in heparinized saline for intravenous administration. A GHSR antagonist, YIL781 hydrochloride (Tocris, Ellisville, MO, USA), was dissolved in Ringer’s solution containing 0.004% DMSO and 0.002% ethanol for application. A mu1 opioid receptor antagonist, naloxonazine dihydrochloride hydrate (SigmaAldrich, St. Louis, MO, USA), a kappa opioid receptor antagonist, nor-binaltorphimine dihydrochloride (nor-BNI) (Sigma-Aldrich), and a kappa opioid receptor agonist, BRL52537 hydrochloride (Tocris), were dissolved in Ringer’s solution for application by retrograde microdialysis. 2.3. Surgery and brain dialysis A microdialysis probe was implanted in the unilateral accumbens shell region (exposed length 1.0 mm). For dual-probe microdialysis, a second probe was targeted to the ipsilateral VTA (exposed length 1.5 mm) and used to infuse YIL781, naloxonazine or nor-BNI into the VTA. Surgery was conducted under pentobarbital anesthesia (50 mg/kg i.p.) and local application of 10% lidocaine. The coordinates of the implantation of the guide cannula were A/P 1.7 mm, L/M 0.8 mm, V/D 6.2 mm from the bregma and dura for the NAc shell region and A/P 5.3 mm, L/M 2.5 mm, V/ D 7.4 mm at an angle of 12 in the coronal plane for the VTA. After surgery, the rats
3. Results 3.1. Effects of consumption of regular food on dopamine levels in the NAc and the roles of opioid receptors in the VTA Exposure to regular food in satiated rats did not always induce food consumption behavior if ghrelin was not administered. Therefore, rats were deprived of food for 24 h before food presentation. The food-deprived rats consumed 2.24 0.52 g (n ¼ 6) of regular food during 15 min of food presentation. Under these conditions, the effects of consumption of regular food on the extracellular levels of dopamine in the NAc were examined by in vivo microdialysis. Intake of regular food immediately induced an increase in dopamine levels to 150% of basal levels, but the level returned to the basal value within 60 min (Fig. 1A), similarly to the results in our previous report (Kawahara et al., 2009). The roles of mu and kappa opioid receptors in the VTA in the regular food-induced increase in dopamine levels in the NAc were examined. Intrategmental infusion of a mu1 opioid receptor antagonist, naloxonazine (10 mM), or a kappa opioid receptor antagonist, nor-BNI (10 mM), did not affect basal dopamine levels in the ipsilateral NAc (data not shown). The regular food-induced increase in dopamine levels was abolished by naloxonazine (10 mM) (Fig. 1B), but not by nor-BNI (10 mM) (Fig. 1C). These results suggest that intake of regular food in fasted rats induces dopamine release in the NAc via activation of mu opioid receptors in the ipsilateral VTA.
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Fig. 1. Effects of consumption of regular or palatable food on dopamine (DA) contents in the dialysate from the NAc. DA contents were determined after intrategmental infusion of Ringer’s solution (A and D), a mu opioid receptor antagonist, naloxonazine (10 mM) (B and E), or a kappa opioid receptor antagonist, nor-BNI (10 mM) (C and F). For groups that consumed regular food (A, B and C), rats were deprived of food for 24 h before food presentation and the fasted rats were exposed for 15 min to regular food. For groups that consumed palatable food (D, E and F), satiated rats were exposed for 15 min to palatable food. Saline (0.2 mL i.v.) was administered prior to food presentation as a control for groups administered ghrelin in Figs. 2e4. Open and closed squares indicate the periods of regular food and palatable food presentation, respectively. Closed bars indicate the period of Ringer’s solution, naloxonazine or nor-BNI infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B) and (C) (gray circles) and the results of (D) are reproduced for comparison in (E) and (F) (gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 7 (A), 6 (B), 5 (C), 5 (D), 8 (E) and 5 (F) rats. *p < 0.05, **p < 0.01 vs. basal values (repeated measures one-way ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures two-way ANOVA).
3.2. Effects of consumption of palatable food on dopamine levels in the NAc and the roles of opioid receptors in the VTA
receptors in the ipsilateral VTA, as observed with consumption of regular food after food deprivation.
As exposure to palatable food in satiated rats induced food consumption behavior spontaneously, food deprivation was not applied when palatable food was used. The satiated rats consumed 2.00 0.14 g (n ¼ 5) of palatable food intermittently during 15 min of food presentation. Consumption of palatable food in satiated rats induced a transient increase in dopamine levels in the NAc (Fig. 1D). The palatable food-induced increase in dopamine levels was suppressed by intrategmental infusion of naloxonazine (10 mM) (Fig. 1E), but not by intrategmental infusion of nor-BNI (10 mM) (Fig. 1F). These results suggest that consumption of palatable food induces dopamine release in the NAc via activation of mu opioid
3.3. Effects of systemic ghrelin administration followed by no food (ghrelin/no food) on dopamine levels in the NAc and roles of opioid receptors in the VTA We have previously reported that systemic ghrelin administration decreases dopamine levels in the NAc when food is removed after ghrelin administration (Kawahara et al., 2009). In agreement with this finding, systemic administration of ghrelin (3 nmol/ 0.2 mL i.v.) followed by no food induced a sustained decrease in dopamine levels in the NAc (Fig. 2A). The roles of opioid receptors in the VTA in the ghrelin-induced decrease in dopamine levels in
Fig. 2. Effects of systemic ghrelin administration followed by no food on DA contents in the dialysate from the NAc in satiated rats. DA contents were determined with intrategmental infusion of Ringer’s solution (A), naloxonazine (10 mM) (B) or nor-BNI (10 mM) (C). Similarly to other groups that received ghrelin, satiated rats were used. Ghrelin was systemically administered (3.0 nmol/0.2 mL saline, i.v.) where indicated by arrows. Closed bars indicate the period of Ringer’s solution, naloxonazine or nor-BNI infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B) and (C) (gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 5 (A), 5 (B), and 6 (C) rats. *p < 0.05, **p < 0.01 vs. basal values (two-way ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures two-way ANOVA).
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the NAc were investigated under these conditions of elimination of food consumption. The decrease in dopamine levels was not affected by intrategmental infusion of naloxonazine (10 mM) (Fig. 2B), but completely abolished by intrategmental infusion of nor-BNI (10 mM) (Fig. 2C). These results indicate that systemic administration of ghrelin without food consumption induces activation of kappa opioid receptors in the VTA, resulting in a decrease in dopamine levels in the NAc. Furthermore, mu opioid receptors in the VTA are probably activated by food consumption, but not solely by systemic ghrelin. 3.4. Effects of systemic ghrelin administration followed by consumption of regular food (ghrelin/regular food) on dopamine levels in the NAc and roles of opioid receptors in the VTA Systemic administration of ghrelin (3 nmol/0.2 mL i.v.) induced intermittent eating of regular food in all satiated rats. Regular food was presented for 15 min after systemic ghrelin administration and the rats consumed 2.01 0.27 g (n ¼ 7) of regular food. After food consumption, the rats maintained a sleeping position. Systemic ghrelin administration followed by consumption of regular food induced a sustained increase in dopamine levels in the NAc (Fig. 3A), as previously reported (Kawahara et al., 2009). This sustained increase in dopamine levels was completely abolished by intrategmental infusion of naloxonazine (10 mM) (Fig. 3B), but not by intrategmental infusion of nor-BNI (10 mM) (Fig. 3C). Thus, consumption of regular food triggered by ghrelin administration, as well as food deprivation, induces dopamine release in the NAc via mechanisms involving mu opioid receptors. 3.5. Effects of systemic ghrelin administration followed by consumption of palatable food (ghrelin/palatable food) on dopamine levels in the NAc and roles of opioid receptors in the VTA Ghrelin (3 nmol/0.2 mL i.v.) was systemically administered to satiated rats, followed by exposure to palatable food. All satiated rats exhibited food consumption behavior spontaneously and intermittently during 15 min of food presentation. The rats consumed 2.40 0.33 g of palatable food (n ¼ 7). After food consumption, the rats maintained a sleeping position. Systemic ghrelin administration followed by intake of palatable food did not affect dopamine levels in the NAc (Fig. 4A), which was strikingly different from the results obtained under other conditions. When nor-BNI (10 mM) was infused into the ipsilateral VTA, ghrelin/ palatable food induced a sustained increase in dopamine levels in
the NAc (Fig. 4C), whereas infusion of naloxonazine in the ipsilateral VTA (1 and 10 mM) did not affect the dopamine levels (Fig. 4B). The contribution of mu opioid receptors in the VTA to the ghrelin/palatable food-induced increase in dopamine levels was examined under blockade of kappa opioid receptors. Co-infusion of naloxonazine (10 mM) and nor-BNI (10 mM) suppressed the increase in dopamine levels induced by ghrelin/palatable food with nor-BNI infusion (Fig. 4D). This result suggests that both mu and kappa opioid receptors in the VTA are activated under conditions of ghrelin/palatable food, but that the function of kappa opioid receptors is dominant. 3.6. Role of ghrelin in the VTA in suppression of the palatable foodinduced increase in dopamine levels in the NAc The contribution of GHSRs in the VTA to suppression of the palatable food-induced increase in dopamine levels in the NAc by ghrelin administration was examined. Intrategmental infusion of a non-peptidyl GHSR1a receptor antagonist, YIL781 (1 and 10 mM), did not affect basal levels of dopamine in the ipsilateral NAc (data not shown). Intrategmental infusion of YIL781 (1 and 10 mM) dosedependently increased dopamine levels in the ipsilateral NAc under conditions of ghrelin/palatable food (Fig. 4E), suggesting that activation of GHSRs in the VTA by peripherally administered ghrelin suppresses dopamine release stimulated by the palatable food/mu opioid receptor pathway. We next asked whether activation of kappa opioid receptors in the VTA suppresses the palatable food-induced increase in dopamine levels in the absence of ghrelin administration. Intrategmental infusion of a kappa opioid receptor agonist, BRL52537 (10 mM), did not affect basal dopamine levels (data not shown) or the palatable food-induced increase in dopamine levels in the ipsilateral NAc (Fig. 4F). Since activation of kappa opioid receptors in the VTA failed to mimic the effect of ghrelin to suppress dopamine release in the NAc, it is hypothesized that kappa opioid receptors may be functioning as a downstream target of GHSRs or require co-activation of GHSRs. 4. Discussion The present study demonstrates that systemic ghrelin interacts with opioid receptor signaling in the VTA to regulate the activity of the mesolimbic dopamine system. Stimulation of dopamine release in the NAc by natural food consumption is dependent on the mu opioid receptor pathway in the VTA [Fig. 5A, pathway (1)], whereas inhibition of dopamine release by systemic ghrelin without food is
Fig. 3. Effects of systemic ghrelin administration followed by consumption of regular food on DA contents in the dialysate from the NAc in satiated rats. DA contents were determined with intrategmental infusion of Ringer’s solution (A), naloxonazine (10 mM) (B) or nor-BNI (10 mM) (C). Satiated rats were used since they exhibited regular food consumption after ghrelin administration. Ghrelin was systemically administered (3.0 nmol/0.2 mL saline, i.v.) where indicated by arrows. Open squares indicate the period of regular food presentation after systemic ghrelin. Closed bars indicate the period of Ringer’s solution, naloxonazine or nor-BNI infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B) and (C) (gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 7 (A), 5 (B) and 6 (C) rats. *p < 0.05, **p < 0.01 vs. basal values (repeated measures one-way ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures twoway ANOVA).
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Fig. 4. Effects of systemic ghrelin administration followed by consumption of palatable food on DA contents in the dialysate from the NAc in satiated rats. DA contents were determined with intrategmental infusion of Ringer’s solution (A), naloxonazine (1 and 10 mM, indicated by dark gray and closed circles, respectively) (B), nor-BNI (10 mM) (C), norBNI (10 mM) alone followed by nor-BNI plus naloxonazine (10 mM) (D), a GHSR antagonist, YIL781 (1 and 10 mM indicated by dark gray and closed circles, respectively) (E), or a kappa opioid receptor agonist, BRL52537 (10 mM) (F). Satiated rats were used since they exhibited consumption of palatable food regardless of ghrelin administration. Ghrelin (3.0 nmol/ 0.2 mL saline, i.v.) or saline was systemically administered where indicated by arrows. Closed squares indicate the period of palatable food presentation. Closed bars indicate the period of Ringer’s solution, naloxonazine, nor-BNI YIL781 or BRL52537 infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B), (C) and (E) (light gray circles) and the results of (C) are reproduced for comparison in (D) (light gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 7 (A), 6 per group (B), 5 (C), 5 (D), 6 and 5 rats for YIL781 at 1 and 10 mM, respectively (E), and 4 (F) rats. *p < 0.05, **p < 0.01 vs. basal values (repeated measures oneway ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures two-way ANOVA).
Fig. 5. Proposed models of neuronal pathways that mediate the effect of systemic ghrelin administration on the mesolimbic dopamine system in different feeding states. (A) Natural food consumption in the absence of ghrelin administration increases DA release in the NAc via activation of (1) the b-endorphin/mu opioid receptor pathway acting on GABAergic interneurons in the VTA, which results in a decrease in GABA release and disinhibition of DA neurons. (B) Systemic ghrelin administration followed by no food decreases DA release in the NAc via activation of (2) the glutamate/NMDA receptor pathway acting on GABAergic interneurons (Krügel et al., 2001), which results in an increase in GABA release, and (3) the dynorphin A/kappa opioid receptor pathway acting on DA neurons in the VTA. Simultaneous stimulation of GABAA and kappa opioid receptors in DA neurons decreases activity of DA neurons. (C) Systemic ghrelin administration followed by consumption of regular food increases DA release in the NAc via preferential activation of (1) the b-endorphin/mu opioid receptor pathway and (4) the glutamate/NMDA receptor pathway acting on DA neurons (Gronier, 2008). Attenuation of GABAA receptor-mediated inhibition and activation of NMDA receptors increase activity of DA neurons. (D) When regular food is changed to palatable food, systemic ghrelin administration predominantly activates (3) the dynorphin A/ kappa opioid receptor pathway. Activation of kappa opioid receptors in DA neurons counteracts the stimulatory effects of two pathways: (1) the b-endorphin/mu opioid receptor pathway and (4) the glutamate/NMDA receptor pathway acting on DA neurons, resulting in no alteration of DA release in the NAc. The pathways activated by systemic ghrelin : Ghrelin-stimulated neuronal administration in different feeding states [(2), (3) and (4)] are pharmacologically proved to require activation of GHSRs localized in the VTA. : Feeding-stimulated neuronal pathway. pathway;
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dependent on the kappa opioid receptor pathway in the VTA [Fig. 5B, pathway (3)], in addition to NMDA receptor-mediated activation of GABAergic interneurons [pathway (2)] (Kawahara et al., 2009). When systemic ghrelin is combined with food intake, mu and kappa opioid receptor pathways are differentially activated depending on the values of food reward. Systemic ghrelin followed by regular food preferentially activates the mu opioid receptor pathway [Fig. 5C, pathway (1)] in addition to NMDA receptor-mediated activation of dopamine neurons [pathway (4)] (Kawahara et al., 2009). In contrast, systemic ghrelin followed by palatable food predominantly activates the kappa opioid receptor pathway [Fig. 5D, pathway (3)] and masks the effect of the mu opioid receptor pathway [pathway (1)]. Pharmacological studies with intrategmental infusion of GHSR antagonists suggest the involvement of GHSRs in the VTA in the action of systemic ghrelin in all the feeding states [pathways (2), (3) and (4)] (Kawahara et al., 2009). Thus, systemic ghrelin modulates the reward value of food and the tone of the mesolimbic dopamine system by preferential activation of the mu or kappa opioid receptor pathway in the VTA. 4.1. The mu opioid receptor pathway mediates the increase in dopamine release triggered by food reward (Fig. 5A) Natural food consumption, consumption of regular food after food deprivation, and palatable food (without food deprivation) increased dopamine release in the NAc via activation of the mu opioid receptor pathway in the VTA. These results are consistent with previous reports showing that the mu opioid receptor pathway plays a major role in the stimulatory effect of high reward food on the mesolimbic dopamine system (Tanda and Di Chiara, 1998), and that a mu opioid receptor agonist injected into the VTA elicited induction of food intake and dopamine release in the NAc (MacDonald et al., 2004, 2003). Activation of mu opioid receptors has been shown in GABAergic interneurons and inhibits GABA release in the VTA, resulting in disinhibition of dopaminergic neurons (Chefer et al., 2009; Johnson and North, 1992) and an increase in dopamine release in the NAc (Spanagel et al., 1990; Chefer et al., 2009). Such a neural mechanism could underlie the increased dopamine levels in the present findings. It is likely that bendorphin, an endogenous peptide selective for mu opioid receptors, is released in the VTA in response to food reward, as demonstrated in ethanol reinforcement (Jarjour et al., 2009). However, there is currently no direct evidence of a food consumptioninduced release in b-endorphin. 4.2. The kappa opioid receptor pathway mediates the decrease in dopamine release triggered by systemic ghrelin without food (Fig. 5B) The decrease in dopamine release in the NAc elicited by ghrelin without food was mediated through activation of the kappa opioid receptor pathway [pathway (3)]. We previously showed that systemic ghrelin without food activates NMDA receptors and GHSRs localized on GABAergic interneurons, resulting in activation of GABAergic interneurons and inhibition of dopamine neurons in the VTA [pathway (2)] (Kawahara et al., 2009). These two pathways [(2) and (3)] probably coordinate to inhibit dopamine neurons. Kappa opioid receptor agonists inhibit the subpopulation of dopamine neurons that projects to the NAc by direct postsynaptic action (Margolis et al., 2003; Ford et al., 2006) and the corresponding dopamine release in the NAc (Di Chiara and Imperato, 1988; Spanagel et al., 1994; Bruijnzeel, 2009). Such a mechanism could underlie the decreased dopamine release in the NAc. Given the fact that axon terminals containing dynorphin (an endogenous peptide selective for kappa opioid receptors) synapse onto dendrites of
dopamine neurons in the VTA (Fallon et al., 1985; Pickel et al., 1993), it is likely that ghrelin-induced release of dynorphin-like peptides such as dynorphin A mediates the stimulation of kappa opioid receptors in the VTA. However, little is known about the effect of endogenous dynorphin-like peptides in the VTA on food reward. Intrategmental injection of dynorphin is more potent than morphine in producing feeding (Hamilton and Bozarth, 1988) and opioid receptor antagonists selective for kappa, as well as mu, injected into the VTA can attenuate mu opioid receptor agonist-induced feeding (Lamonte et al., 2002). Taken together, the features of kappa opioid receptors in inducing feeding behavior with decreased dopamine release in the NAc are similar to those of systemic ghrelin without food, suggesting a central role of kappa opioid receptors in the VTA in the action of systemic ghrelin without food. 4.3. Values of food reward regulate the balance of activities of mu and kappa opioid receptor pathways activated by systemic ghrelin with food consumption (Fig. 5C and D) We previously showed that systemic ghrelin with consumption of regular food activates NMDA receptors and GHSRs localized on dopamine neurons, resulting in activation of dopamine neurons and the sustained release of dopamine in the NAc [Fig. 5C, pathway (4)] (Kawahara et al., 2009; Jerlhag et al., 2011). Under this condition, the mu opioid receptor pathway (1) played a critical role. Thus, pathways (1) and (4) probably coordinate to activate dopamine neurons and enhance dopamine release in the NAc. When regular food consumed after systemic ghrelin administration was changed to palatable food, dopamine release in the NAc was unaltered. The lack of a change in dopamine release was an unexpected finding, since consumption of palatable food has been consistently shown to increase dopamine release in the NAc (Melis et al., 2007; Rada et al., 2005). The lack of an increase in dopamine release may be due to dominant activation of the kappa opioid receptor pathway [Fig. 5D, pathway (3)] under the control of GHSRs in the VTA. Systemic ghrelin acting on GHSRs in the VTA apparently alters the responses of mesolimbic dopamine neurons to food reward by activating mu and/or kappa opioid receptor pathways. Although regular or palatable food by itself activates the mu opioid receptor pathway (1), systemic ghrelin switches the dominant opioid receptor pathway from mu to kappa only for highly rewarding food, suggesting a pivotal role of ghrelin in regulation of food incentives and hedonics. The mechanism underlying switching of the dominant opioid receptor pathway is unknown and further investigation is needed. It is generally accepted that activation of the kappa opioid receptor system produces negative motivational effects (Tejeda et al., 2012) that are paralleled by a decrease in dopamine release in the NAc (Herz, 1998). In this respect, our findings may contradict these previous reports and lead to the hypothesis that ghrelin functions to suppress excessive food reward and over-eating, because systemic ghrelin combined with highly rewarding food suppresses activation of mesolimbic dopamine system via activation of the kappa opioid receptor pathway. However, ghrelin is thought to increase rewarding behaviors of drugs of abuse and food reward (Dickson et al., 2011; Skibicka et al., 2011). Since palatability is one of the most rewarding aspects of food consumption behavior, it is unlikely that palatable food ingestion induces a negative emotional state. It is of interest to note that the sensitivity of the kappa opioid receptor system and the dynorphin tone in mesolimbic dopamine neurons are upregulated by chronic exposure to drugs of abuse, including alcohol, in humans and rodents (Lindholm et al., 2007; Wee and Koob, 2010), and that, during the withdrawal phase, the upregulated dynorphin system contributes to decreased dopamine release and negative emotional states,
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resulting in craving and increased drug intake (Bruijnzeel, 2009). In the present study, high plasma levels of exogenous ghrelin were shown to switch the dominant opioid receptor pathway for palatable food from mu to kappa, suggesting that overactivation of ghrelin signaling mediates the functional coupling of palatability, hedonic evaluation for food, and the dynorphin/kappa opioid receptor pathway. Eating disorders such as Prader-Willi syndrome, anorexia nervosa and binge eating are associated with hyperghrelinemia, suggesting the possible involvement of the dysregulated dynorphin system in the pathophysiology of eating disorders (Yi et al., 2011). For a better understanding of the conflicting findings, it will be extremely important to study the role of ghrelin in regulating the mesolimbic dopamine system in response to drugs of abuse, alcohol and highly rewarding food, in conjunction with activities of the mu and kappa opioid receptor pathways. 5. Conclusion In conclusion, systemic ghrelin and opioid receptors interact in the VTA to regulate the mesolimbic dopamine system and reward values of food. Systemic ghrelin induces switching of the dominant opioid receptor pathway for highly rewarding food from mu to kappa, resulting in suppression of the mesolimbic dopamine system. The novel findings of food reward-sensitive interaction of ghrelin and opioid receptor pathways in the VTA might provide insights into the neural pathways involved in eating disorders such as food craving, compulsive binge eating, and dependence on palatability of food (Woolley et al., 2007; Olszewski et al., 2011). Disclosure The authors declare no conflict of interest. Acknowledgments This work was supported by a grant from the Kurume University Millennium Box Foundation for the Promotion of Science, Grantsin-Aid for Scientific Research [(C)22590233 and (C)21592580], and the CREST program of JST. References Abizaid, A., 2009. Ghrelin and dopamine: new insights on the peripheral regulation of appetite. J. Neuroendocrinol. 21, 787e793. Abizaid, A., Liu, Z.W., Andrews, Z.B., Shanabrough, M., Borok, E., Elsworth, J.D., 2006. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Invest. 116, 3229e3239. Beck, B., Musse, N., Stricker-Krongrad, A., 2002. Ghrelin, macronutrient intake and dietary preferences in Long-Evans rats. Biochem. Biophys. Res. Commun. 292, 1031e1035. Briggs, D.I., Andrews, Z.B., 2011. Metabolic status regulates ghrelin function on energy homeostasis. Neuroendocrinology 93, 48e57. Bruijnzeel, A.W., 2009. Kappa-opioid receptor signaling and brain reward function. Brain Res. Rev. 62, 127e146. Chefer, V.I., Denoroy, L., Zapata, A., Shippenberg, T.S., 2009. Mu opioid receptor modulation of somatodendritic dopamine overflow: GABAergic and glutamatergic mechanisms. Eur. J. Neurosci. 30, 272e278. Date, Y., Shimbara, T., Koda, S., Toshinai, K., Ida, T., Murakami, N., Miyazato, M., Kokame, K., Ishizuka, Y., Ishida, Y., Kageyama, H., Shioda, S., Kangawa, K., Nakazato, M., 2006. Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell. Metab. 4, 323e331. Di Chiara, G., Imperato, A., 1988. Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J. Pharmacol. Exp. Ther. 244, 1067e1080. Dickson, S.L., Egecioglu, E., Landgren, S., Skibicka, K.P., Engel, J.A., Jerlhag, E., 2011. The role of the central ghrelin system in reward from food and chemical drugs. Mol. Cell. Endocrinol. 340, 80e87. Diéguez, C., da Boit, K., Novelle, M.G., Martínez de Morentin, P.B., Nogueiras, R., López, M., 2010. New insights in ghrelin orexigenic effect. Front. Horm. Res. 38, 196e205.
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Contents lists available at SciVerse ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Food reward-sensitive interaction of ghrelin and opioid receptor pathways in mesolimbic dopamine system Yukie Kawahara a, *, Fumi Kaneko a, Makiko Yamada a, Yuki Kishikawa a, Hiroshi Kawahara b, Akinori Nishi a a b
Department of Pharmacology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan Department of Dental Anesthesiology, School of Dentistry, Tsurumi University, Yokohama 230-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2012 Received in revised form 22 November 2012 Accepted 24 November 2012
Ghrelin is a stomach-derived orexigenic peptide. The goal of the study was to investigate the roles of mu and kappa opioid receptors in systemic ghrelin-mediated regulation of the mesolimbic dopamine system. To evaluate the interaction of systemic ghrelin with values of food reward, rats were exposed to food removal, regular food or palatable food after systemic ghrelin administration. Extracellular dopamine levels were quantified in the nucleus accumbens (NAc) and receptor-specific compounds were infused into the ventral tegmental area (VTA) using dual-probe microdialysis. Consumption of regular or palatable food without systemic ghrelin administration induced an increase in dopamine levels in the NAc via activation of mu opioid receptors in the VTA. Systemic ghrelin administration (3 nmol, i.v.) followed by no food induced a decrease in dopamine levels via activation of kappa opioid receptors in the VTA. Systemic ghrelin administration followed by consumption of regular food induced an increase in dopamine levels via preferential activation of mu opioid receptors, whereas systemic ghrelin administration followed by consumption of palatable food suppressed the increase in dopamine levels via preferential activation of kappa opioid receptors. Thus, natural food reward and systemic ghrelin activate mu and kappa opioid receptor pathways in the VTA, respectively, resulting in opposite influences on dopamine release in the NAc. Furthermore, systemic ghrelin induces switching of the dominant opioid receptor pathway for highly rewarding food from mu to kappa, resulting in suppression of the mesolimbic dopamine system. These novel findings might provide insights into the neural pathways involved in eating disorders. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Microdialysis Accumbens VTA
1. Introduction Ghrelin is a stomach-derived orexigenic hormone that modulates food intake and energy balance via homeostatic brain circuits that reside in the hypothalamic area (Briggs and Andrews, 2011; Diéguez et al., 2010). Recent evidence suggests a role of ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), in food reward behavior that is tightly associated with the mesolimbic dopamine system (Skibicka and Dickson, 2011; Landgren et al., Abbreviations: NAc, nucleus accumbens; VTA, ventral tegmental area; GHSR, growth hormone secretagogue receptor; HPLC, high performance liquid chromatography. * Corresponding author. Tel.: þ81 942 31 7545; fax: þ81 942 31 7696. E-mail addresses: [email protected] (Y. Kawahara), kaneko_fumi@ kurume-u.ac.jp (F. Kaneko), [email protected] (M. Yamada), [email protected] (Y. Kishikawa), [email protected] (H. Kawahara), [email protected] (A. Nishi). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.11.022
2011). Besides the nutritional state, ghrelin has an impact on the neuronal circuit that signals the reward value of food ingested to dopamine neurons in the ventral tegmental area (VTA) (Abizaid, 2009; Skibicka and Dickson, 2011). In fact, ghrelin secretion is downregulated by high-fat and palatable diets (Beck et al., 2002; Lindqvist et al., 2005), but stimulated before meals and during hunger. The mesolimbic dopamine system from the VTA to the nucleus accumbens (NAc) comprises a core component of the mesolimbic reward circuit. GHSRs are strongly expressed in the VTA, especially in dopamine neurons and GABAergic interneurons (Abizaid, 2009), and circulating ghrelin activates GHSRs in the VTA and hypothalamus (Skibicka and Dickson, 2011). Activation of GHSRs in the VTA by direct application of ghrelin induces dopamine release in the NAc and feeding behavior (Jerlhag et al., 2007; Abizaid et al., 2006; Kawahara et al., 2009). Interestingly, peripherally administered ghrelin also causes feeding behavior, but requires food
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consumption to induce dopamine release in the NAc (Kawahara et al., 2009), suggesting an interaction of ghrelin and food reward in the mesolimbic dopamine system. Ghrelin/GHSR signaling in the VTA has been identified as a crucial component to increase food reward and motivational behavior for food (Skibicka et al., 2011; King et al., 2011), but not homeostatic feeding (Egecioglu et al., 2010; Skibicka et al., 2011). Rewarding aspects of food intake are mediated by the central opioid system, which leads to hedonic consummatory behavior, as shown in many studies (review: Olszewski et al., 2011; Gosnell and Levine, 2009; Tanda and Di Chiara, 1998). Opioids, especially mu opioid receptor agonists, applied to the VTA stimulate feeding behavior (Figlewicz and Sipols, 2010), and palatable food-induced dopamine release in the NAc requires activation of mu opioid receptors in the VTA (Tanda and Di Chiara, 1998). Activation of mu opioid receptors inhibits GABAergic interneurons in the VTA, which results in disinhibition of dopamine neurons and facilitates dopamine release in the NAc (Johnson and North, 1992; Chefer et al., 2009). In contrast, kappa opioid receptors in the VTA inhibit dopamine neurons (Margolis et al., 2003; Ford et al., 2006), suggesting opposing roles of mu and kappa opioid receptors (Pan, 1998; Bruijnzeel, 2009). Thus, opioids and ghrelin act on the mesolimbic dopamine system and may interact to regulate food reward. In fact, a recent study showed that ghrelin induces sucrosemotivated behavior via interaction with mu opioid receptors in the VTA (Skibicka et al., 2012). We have previously shown that peripherally administered ghrelin and GHSRs in the VTA regulate the mesolimbic dopamine system differentially depending on the state of food consumption (Kawahara et al., 2009). In the present study, we examined the role of opioid receptors in regulation of the mesolimbic dopaminergic system by ghrelin associated with food reward. Dual-probe microdialysis experiments revealed the involvement of mu and kappa opioid receptor pathways, which are differentially activated by systemic ghrelin and food consumption depending on the values of food reward.
were allowed to recover for 5e7 days before performance of microdialysis experiments. At 24 h before the start of the experiments, rats were moved to an acrylic box (30 30 40 cm) individually and microdialysis probes were set in the guide cannulas. These probes extended 1 mm beyond the tip of the guide cannulas for both the NAc and VTA. On the same day, novel objects of flavored serial food (Asahi Food & Healthcare Co.) to which rats exhibit palatability were introduced into the acrylic box for 24 h to promote habituation. Flavored serial food was removed before the start of experiments on the day of the experiments, whereas rats had free access to regular food until intravenous administration of ghrelin or saline. During the experimental period, rats were exposed to regular food (3.6 kcal/g) or palatable food (5.0 kcal/g) for 15 min after ghrelin administration after obtaining three stable consecutive samples of dopamine differing by <10%, and thereafter food was removed. In the experimental group that received ghrelin followed by no food, food was removed from the container manually just before ghrelin administration. Rats used for recording a food deprivation-induced eating response received no food for 24 h before exposure to food for a period of 15 min to induce food ingestion. The dose of ghrelin (3 nmol/rat) for intravenous administration was selected based on previous reports (Date et al., 2006; Ukkola, 2004). A polyethylene cannula (PE-10; Becton Dickinson, Franklin Lakes, N.J., USA) filled with heparinized saline (50 IU/mL) was inserted into the external jugular vein. Dialysate fractions were collected every 15 min. Dopamine was quantified by HPLC with electrochemical detection conducted as in our previous study (Kawahara et al., 2009). The average baseline values of dopamine in dialysates from the NAc did not differ significantly among the different experiments. Therefore, these data were grouped and the basal value (S.E.M.) was determined as 11.65 1.06 fmol/sample (n ¼ 84). At the end of the experiments, the rats were given an overdose of chloral hydrate and brains were fixed with 4% paraformaldehyde via intracardiac infusion. Coronal sections (50-mm thick) were cut and dialysis probe placement was localized based on the atlas of Paxinos and Watson (2007). Rats in which dialysis probes and guide cannulas were found to be misplaced were not included in data analysis. 2.4. Expression of data and statistical analysis All values are expressed as percentages of basal values obtained as the average for three baseline samples. Values obtained after drug administration were compared with a basal value of 100%. Repeated measures one-way analysis of variance (ANOVA) and a Dunnett multiple comparison test for post-hoc determination were performed using the SAS mixed procedure (SAS Institute, Cary, N.C., USA). One-way ANOVA and a Scheffe multiple comparison test for post-hoc determination were used for comparison of the amount of food consumption. Repeated measures two-way ANOVA and a Bonferroni multiple comparison test for post-hoc determination were used for comparison between experimental groups (GraphPad Prism, GraphPad Software Inc., San Diego, CA, USA). The level of significance was set at p < 0.05.
2. Materials and methods 2.1. Animals Male Wistar rats (270e340 g, Kyudo, Tosu, Japan) were maintained at 23 2 C under a 12-h lightedark cycle with free access to food and water. All rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the U.S. National Institutes of Health, and the specific protocols were approved by the Committee for Animal Experimentation, Kurume University School of Medicine. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Drugs Rat ghrelin acylated at the serine 3 position with an octanoyl group (Peptide Inc, Osaka, Japan) was dissolved in heparinized saline for intravenous administration. A GHSR antagonist, YIL781 hydrochloride (Tocris, Ellisville, MO, USA), was dissolved in Ringer’s solution containing 0.004% DMSO and 0.002% ethanol for application. A mu1 opioid receptor antagonist, naloxonazine dihydrochloride hydrate (SigmaAldrich, St. Louis, MO, USA), a kappa opioid receptor antagonist, nor-binaltorphimine dihydrochloride (nor-BNI) (Sigma-Aldrich), and a kappa opioid receptor agonist, BRL52537 hydrochloride (Tocris), were dissolved in Ringer’s solution for application by retrograde microdialysis. 2.3. Surgery and brain dialysis A microdialysis probe was implanted in the unilateral accumbens shell region (exposed length 1.0 mm). For dual-probe microdialysis, a second probe was targeted to the ipsilateral VTA (exposed length 1.5 mm) and used to infuse YIL781, naloxonazine or nor-BNI into the VTA. Surgery was conducted under pentobarbital anesthesia (50 mg/kg i.p.) and local application of 10% lidocaine. The coordinates of the implantation of the guide cannula were A/P 1.7 mm, L/M 0.8 mm, V/D 6.2 mm from the bregma and dura for the NAc shell region and A/P 5.3 mm, L/M 2.5 mm, V/ D 7.4 mm at an angle of 12 in the coronal plane for the VTA. After surgery, the rats
3. Results 3.1. Effects of consumption of regular food on dopamine levels in the NAc and the roles of opioid receptors in the VTA Exposure to regular food in satiated rats did not always induce food consumption behavior if ghrelin was not administered. Therefore, rats were deprived of food for 24 h before food presentation. The food-deprived rats consumed 2.24 0.52 g (n ¼ 6) of regular food during 15 min of food presentation. Under these conditions, the effects of consumption of regular food on the extracellular levels of dopamine in the NAc were examined by in vivo microdialysis. Intake of regular food immediately induced an increase in dopamine levels to 150% of basal levels, but the level returned to the basal value within 60 min (Fig. 1A), similarly to the results in our previous report (Kawahara et al., 2009). The roles of mu and kappa opioid receptors in the VTA in the regular food-induced increase in dopamine levels in the NAc were examined. Intrategmental infusion of a mu1 opioid receptor antagonist, naloxonazine (10 mM), or a kappa opioid receptor antagonist, nor-BNI (10 mM), did not affect basal dopamine levels in the ipsilateral NAc (data not shown). The regular food-induced increase in dopamine levels was abolished by naloxonazine (10 mM) (Fig. 1B), but not by nor-BNI (10 mM) (Fig. 1C). These results suggest that intake of regular food in fasted rats induces dopamine release in the NAc via activation of mu opioid receptors in the ipsilateral VTA.
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Fig. 1. Effects of consumption of regular or palatable food on dopamine (DA) contents in the dialysate from the NAc. DA contents were determined after intrategmental infusion of Ringer’s solution (A and D), a mu opioid receptor antagonist, naloxonazine (10 mM) (B and E), or a kappa opioid receptor antagonist, nor-BNI (10 mM) (C and F). For groups that consumed regular food (A, B and C), rats were deprived of food for 24 h before food presentation and the fasted rats were exposed for 15 min to regular food. For groups that consumed palatable food (D, E and F), satiated rats were exposed for 15 min to palatable food. Saline (0.2 mL i.v.) was administered prior to food presentation as a control for groups administered ghrelin in Figs. 2e4. Open and closed squares indicate the periods of regular food and palatable food presentation, respectively. Closed bars indicate the period of Ringer’s solution, naloxonazine or nor-BNI infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B) and (C) (gray circles) and the results of (D) are reproduced for comparison in (E) and (F) (gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 7 (A), 6 (B), 5 (C), 5 (D), 8 (E) and 5 (F) rats. *p < 0.05, **p < 0.01 vs. basal values (repeated measures one-way ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures two-way ANOVA).
3.2. Effects of consumption of palatable food on dopamine levels in the NAc and the roles of opioid receptors in the VTA
receptors in the ipsilateral VTA, as observed with consumption of regular food after food deprivation.
As exposure to palatable food in satiated rats induced food consumption behavior spontaneously, food deprivation was not applied when palatable food was used. The satiated rats consumed 2.00 0.14 g (n ¼ 5) of palatable food intermittently during 15 min of food presentation. Consumption of palatable food in satiated rats induced a transient increase in dopamine levels in the NAc (Fig. 1D). The palatable food-induced increase in dopamine levels was suppressed by intrategmental infusion of naloxonazine (10 mM) (Fig. 1E), but not by intrategmental infusion of nor-BNI (10 mM) (Fig. 1F). These results suggest that consumption of palatable food induces dopamine release in the NAc via activation of mu opioid
3.3. Effects of systemic ghrelin administration followed by no food (ghrelin/no food) on dopamine levels in the NAc and roles of opioid receptors in the VTA We have previously reported that systemic ghrelin administration decreases dopamine levels in the NAc when food is removed after ghrelin administration (Kawahara et al., 2009). In agreement with this finding, systemic administration of ghrelin (3 nmol/ 0.2 mL i.v.) followed by no food induced a sustained decrease in dopamine levels in the NAc (Fig. 2A). The roles of opioid receptors in the VTA in the ghrelin-induced decrease in dopamine levels in
Fig. 2. Effects of systemic ghrelin administration followed by no food on DA contents in the dialysate from the NAc in satiated rats. DA contents were determined with intrategmental infusion of Ringer’s solution (A), naloxonazine (10 mM) (B) or nor-BNI (10 mM) (C). Similarly to other groups that received ghrelin, satiated rats were used. Ghrelin was systemically administered (3.0 nmol/0.2 mL saline, i.v.) where indicated by arrows. Closed bars indicate the period of Ringer’s solution, naloxonazine or nor-BNI infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B) and (C) (gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 5 (A), 5 (B), and 6 (C) rats. *p < 0.05, **p < 0.01 vs. basal values (two-way ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures two-way ANOVA).
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the NAc were investigated under these conditions of elimination of food consumption. The decrease in dopamine levels was not affected by intrategmental infusion of naloxonazine (10 mM) (Fig. 2B), but completely abolished by intrategmental infusion of nor-BNI (10 mM) (Fig. 2C). These results indicate that systemic administration of ghrelin without food consumption induces activation of kappa opioid receptors in the VTA, resulting in a decrease in dopamine levels in the NAc. Furthermore, mu opioid receptors in the VTA are probably activated by food consumption, but not solely by systemic ghrelin. 3.4. Effects of systemic ghrelin administration followed by consumption of regular food (ghrelin/regular food) on dopamine levels in the NAc and roles of opioid receptors in the VTA Systemic administration of ghrelin (3 nmol/0.2 mL i.v.) induced intermittent eating of regular food in all satiated rats. Regular food was presented for 15 min after systemic ghrelin administration and the rats consumed 2.01 0.27 g (n ¼ 7) of regular food. After food consumption, the rats maintained a sleeping position. Systemic ghrelin administration followed by consumption of regular food induced a sustained increase in dopamine levels in the NAc (Fig. 3A), as previously reported (Kawahara et al., 2009). This sustained increase in dopamine levels was completely abolished by intrategmental infusion of naloxonazine (10 mM) (Fig. 3B), but not by intrategmental infusion of nor-BNI (10 mM) (Fig. 3C). Thus, consumption of regular food triggered by ghrelin administration, as well as food deprivation, induces dopamine release in the NAc via mechanisms involving mu opioid receptors. 3.5. Effects of systemic ghrelin administration followed by consumption of palatable food (ghrelin/palatable food) on dopamine levels in the NAc and roles of opioid receptors in the VTA Ghrelin (3 nmol/0.2 mL i.v.) was systemically administered to satiated rats, followed by exposure to palatable food. All satiated rats exhibited food consumption behavior spontaneously and intermittently during 15 min of food presentation. The rats consumed 2.40 0.33 g of palatable food (n ¼ 7). After food consumption, the rats maintained a sleeping position. Systemic ghrelin administration followed by intake of palatable food did not affect dopamine levels in the NAc (Fig. 4A), which was strikingly different from the results obtained under other conditions. When nor-BNI (10 mM) was infused into the ipsilateral VTA, ghrelin/ palatable food induced a sustained increase in dopamine levels in
the NAc (Fig. 4C), whereas infusion of naloxonazine in the ipsilateral VTA (1 and 10 mM) did not affect the dopamine levels (Fig. 4B). The contribution of mu opioid receptors in the VTA to the ghrelin/palatable food-induced increase in dopamine levels was examined under blockade of kappa opioid receptors. Co-infusion of naloxonazine (10 mM) and nor-BNI (10 mM) suppressed the increase in dopamine levels induced by ghrelin/palatable food with nor-BNI infusion (Fig. 4D). This result suggests that both mu and kappa opioid receptors in the VTA are activated under conditions of ghrelin/palatable food, but that the function of kappa opioid receptors is dominant. 3.6. Role of ghrelin in the VTA in suppression of the palatable foodinduced increase in dopamine levels in the NAc The contribution of GHSRs in the VTA to suppression of the palatable food-induced increase in dopamine levels in the NAc by ghrelin administration was examined. Intrategmental infusion of a non-peptidyl GHSR1a receptor antagonist, YIL781 (1 and 10 mM), did not affect basal levels of dopamine in the ipsilateral NAc (data not shown). Intrategmental infusion of YIL781 (1 and 10 mM) dosedependently increased dopamine levels in the ipsilateral NAc under conditions of ghrelin/palatable food (Fig. 4E), suggesting that activation of GHSRs in the VTA by peripherally administered ghrelin suppresses dopamine release stimulated by the palatable food/mu opioid receptor pathway. We next asked whether activation of kappa opioid receptors in the VTA suppresses the palatable food-induced increase in dopamine levels in the absence of ghrelin administration. Intrategmental infusion of a kappa opioid receptor agonist, BRL52537 (10 mM), did not affect basal dopamine levels (data not shown) or the palatable food-induced increase in dopamine levels in the ipsilateral NAc (Fig. 4F). Since activation of kappa opioid receptors in the VTA failed to mimic the effect of ghrelin to suppress dopamine release in the NAc, it is hypothesized that kappa opioid receptors may be functioning as a downstream target of GHSRs or require co-activation of GHSRs. 4. Discussion The present study demonstrates that systemic ghrelin interacts with opioid receptor signaling in the VTA to regulate the activity of the mesolimbic dopamine system. Stimulation of dopamine release in the NAc by natural food consumption is dependent on the mu opioid receptor pathway in the VTA [Fig. 5A, pathway (1)], whereas inhibition of dopamine release by systemic ghrelin without food is
Fig. 3. Effects of systemic ghrelin administration followed by consumption of regular food on DA contents in the dialysate from the NAc in satiated rats. DA contents were determined with intrategmental infusion of Ringer’s solution (A), naloxonazine (10 mM) (B) or nor-BNI (10 mM) (C). Satiated rats were used since they exhibited regular food consumption after ghrelin administration. Ghrelin was systemically administered (3.0 nmol/0.2 mL saline, i.v.) where indicated by arrows. Open squares indicate the period of regular food presentation after systemic ghrelin. Closed bars indicate the period of Ringer’s solution, naloxonazine or nor-BNI infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B) and (C) (gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 7 (A), 5 (B) and 6 (C) rats. *p < 0.05, **p < 0.01 vs. basal values (repeated measures one-way ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures twoway ANOVA).
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Fig. 4. Effects of systemic ghrelin administration followed by consumption of palatable food on DA contents in the dialysate from the NAc in satiated rats. DA contents were determined with intrategmental infusion of Ringer’s solution (A), naloxonazine (1 and 10 mM, indicated by dark gray and closed circles, respectively) (B), nor-BNI (10 mM) (C), norBNI (10 mM) alone followed by nor-BNI plus naloxonazine (10 mM) (D), a GHSR antagonist, YIL781 (1 and 10 mM indicated by dark gray and closed circles, respectively) (E), or a kappa opioid receptor agonist, BRL52537 (10 mM) (F). Satiated rats were used since they exhibited consumption of palatable food regardless of ghrelin administration. Ghrelin (3.0 nmol/ 0.2 mL saline, i.v.) or saline was systemically administered where indicated by arrows. Closed squares indicate the period of palatable food presentation. Closed bars indicate the period of Ringer’s solution, naloxonazine, nor-BNI YIL781 or BRL52537 infusion into the VTA, as indicated. The results of (A) are reproduced for comparison in (B), (C) and (E) (light gray circles) and the results of (C) are reproduced for comparison in (D) (light gray circles). Data are expressed as a percentage of basal values S.E.M. Data are an average of values obtained from 7 (A), 6 per group (B), 5 (C), 5 (D), 6 and 5 rats for YIL781 at 1 and 10 mM, respectively (E), and 4 (F) rats. *p < 0.05, **p < 0.01 vs. basal values (repeated measures oneway ANOVA); #p < 0.05, ##p < 0.001 vs. intrategmental infusion of Ringer’s solution (repeated measures two-way ANOVA).
Fig. 5. Proposed models of neuronal pathways that mediate the effect of systemic ghrelin administration on the mesolimbic dopamine system in different feeding states. (A) Natural food consumption in the absence of ghrelin administration increases DA release in the NAc via activation of (1) the b-endorphin/mu opioid receptor pathway acting on GABAergic interneurons in the VTA, which results in a decrease in GABA release and disinhibition of DA neurons. (B) Systemic ghrelin administration followed by no food decreases DA release in the NAc via activation of (2) the glutamate/NMDA receptor pathway acting on GABAergic interneurons (Krügel et al., 2001), which results in an increase in GABA release, and (3) the dynorphin A/kappa opioid receptor pathway acting on DA neurons in the VTA. Simultaneous stimulation of GABAA and kappa opioid receptors in DA neurons decreases activity of DA neurons. (C) Systemic ghrelin administration followed by consumption of regular food increases DA release in the NAc via preferential activation of (1) the b-endorphin/mu opioid receptor pathway and (4) the glutamate/NMDA receptor pathway acting on DA neurons (Gronier, 2008). Attenuation of GABAA receptor-mediated inhibition and activation of NMDA receptors increase activity of DA neurons. (D) When regular food is changed to palatable food, systemic ghrelin administration predominantly activates (3) the dynorphin A/ kappa opioid receptor pathway. Activation of kappa opioid receptors in DA neurons counteracts the stimulatory effects of two pathways: (1) the b-endorphin/mu opioid receptor pathway and (4) the glutamate/NMDA receptor pathway acting on DA neurons, resulting in no alteration of DA release in the NAc. The pathways activated by systemic ghrelin : Ghrelin-stimulated neuronal administration in different feeding states [(2), (3) and (4)] are pharmacologically proved to require activation of GHSRs localized in the VTA. : Feeding-stimulated neuronal pathway. pathway;
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dependent on the kappa opioid receptor pathway in the VTA [Fig. 5B, pathway (3)], in addition to NMDA receptor-mediated activation of GABAergic interneurons [pathway (2)] (Kawahara et al., 2009). When systemic ghrelin is combined with food intake, mu and kappa opioid receptor pathways are differentially activated depending on the values of food reward. Systemic ghrelin followed by regular food preferentially activates the mu opioid receptor pathway [Fig. 5C, pathway (1)] in addition to NMDA receptor-mediated activation of dopamine neurons [pathway (4)] (Kawahara et al., 2009). In contrast, systemic ghrelin followed by palatable food predominantly activates the kappa opioid receptor pathway [Fig. 5D, pathway (3)] and masks the effect of the mu opioid receptor pathway [pathway (1)]. Pharmacological studies with intrategmental infusion of GHSR antagonists suggest the involvement of GHSRs in the VTA in the action of systemic ghrelin in all the feeding states [pathways (2), (3) and (4)] (Kawahara et al., 2009). Thus, systemic ghrelin modulates the reward value of food and the tone of the mesolimbic dopamine system by preferential activation of the mu or kappa opioid receptor pathway in the VTA. 4.1. The mu opioid receptor pathway mediates the increase in dopamine release triggered by food reward (Fig. 5A) Natural food consumption, consumption of regular food after food deprivation, and palatable food (without food deprivation) increased dopamine release in the NAc via activation of the mu opioid receptor pathway in the VTA. These results are consistent with previous reports showing that the mu opioid receptor pathway plays a major role in the stimulatory effect of high reward food on the mesolimbic dopamine system (Tanda and Di Chiara, 1998), and that a mu opioid receptor agonist injected into the VTA elicited induction of food intake and dopamine release in the NAc (MacDonald et al., 2004, 2003). Activation of mu opioid receptors has been shown in GABAergic interneurons and inhibits GABA release in the VTA, resulting in disinhibition of dopaminergic neurons (Chefer et al., 2009; Johnson and North, 1992) and an increase in dopamine release in the NAc (Spanagel et al., 1990; Chefer et al., 2009). Such a neural mechanism could underlie the increased dopamine levels in the present findings. It is likely that bendorphin, an endogenous peptide selective for mu opioid receptors, is released in the VTA in response to food reward, as demonstrated in ethanol reinforcement (Jarjour et al., 2009). However, there is currently no direct evidence of a food consumptioninduced release in b-endorphin. 4.2. The kappa opioid receptor pathway mediates the decrease in dopamine release triggered by systemic ghrelin without food (Fig. 5B) The decrease in dopamine release in the NAc elicited by ghrelin without food was mediated through activation of the kappa opioid receptor pathway [pathway (3)]. We previously showed that systemic ghrelin without food activates NMDA receptors and GHSRs localized on GABAergic interneurons, resulting in activation of GABAergic interneurons and inhibition of dopamine neurons in the VTA [pathway (2)] (Kawahara et al., 2009). These two pathways [(2) and (3)] probably coordinate to inhibit dopamine neurons. Kappa opioid receptor agonists inhibit the subpopulation of dopamine neurons that projects to the NAc by direct postsynaptic action (Margolis et al., 2003; Ford et al., 2006) and the corresponding dopamine release in the NAc (Di Chiara and Imperato, 1988; Spanagel et al., 1994; Bruijnzeel, 2009). Such a mechanism could underlie the decreased dopamine release in the NAc. Given the fact that axon terminals containing dynorphin (an endogenous peptide selective for kappa opioid receptors) synapse onto dendrites of
dopamine neurons in the VTA (Fallon et al., 1985; Pickel et al., 1993), it is likely that ghrelin-induced release of dynorphin-like peptides such as dynorphin A mediates the stimulation of kappa opioid receptors in the VTA. However, little is known about the effect of endogenous dynorphin-like peptides in the VTA on food reward. Intrategmental injection of dynorphin is more potent than morphine in producing feeding (Hamilton and Bozarth, 1988) and opioid receptor antagonists selective for kappa, as well as mu, injected into the VTA can attenuate mu opioid receptor agonist-induced feeding (Lamonte et al., 2002). Taken together, the features of kappa opioid receptors in inducing feeding behavior with decreased dopamine release in the NAc are similar to those of systemic ghrelin without food, suggesting a central role of kappa opioid receptors in the VTA in the action of systemic ghrelin without food. 4.3. Values of food reward regulate the balance of activities of mu and kappa opioid receptor pathways activated by systemic ghrelin with food consumption (Fig. 5C and D) We previously showed that systemic ghrelin with consumption of regular food activates NMDA receptors and GHSRs localized on dopamine neurons, resulting in activation of dopamine neurons and the sustained release of dopamine in the NAc [Fig. 5C, pathway (4)] (Kawahara et al., 2009; Jerlhag et al., 2011). Under this condition, the mu opioid receptor pathway (1) played a critical role. Thus, pathways (1) and (4) probably coordinate to activate dopamine neurons and enhance dopamine release in the NAc. When regular food consumed after systemic ghrelin administration was changed to palatable food, dopamine release in the NAc was unaltered. The lack of a change in dopamine release was an unexpected finding, since consumption of palatable food has been consistently shown to increase dopamine release in the NAc (Melis et al., 2007; Rada et al., 2005). The lack of an increase in dopamine release may be due to dominant activation of the kappa opioid receptor pathway [Fig. 5D, pathway (3)] under the control of GHSRs in the VTA. Systemic ghrelin acting on GHSRs in the VTA apparently alters the responses of mesolimbic dopamine neurons to food reward by activating mu and/or kappa opioid receptor pathways. Although regular or palatable food by itself activates the mu opioid receptor pathway (1), systemic ghrelin switches the dominant opioid receptor pathway from mu to kappa only for highly rewarding food, suggesting a pivotal role of ghrelin in regulation of food incentives and hedonics. The mechanism underlying switching of the dominant opioid receptor pathway is unknown and further investigation is needed. It is generally accepted that activation of the kappa opioid receptor system produces negative motivational effects (Tejeda et al., 2012) that are paralleled by a decrease in dopamine release in the NAc (Herz, 1998). In this respect, our findings may contradict these previous reports and lead to the hypothesis that ghrelin functions to suppress excessive food reward and over-eating, because systemic ghrelin combined with highly rewarding food suppresses activation of mesolimbic dopamine system via activation of the kappa opioid receptor pathway. However, ghrelin is thought to increase rewarding behaviors of drugs of abuse and food reward (Dickson et al., 2011; Skibicka et al., 2011). Since palatability is one of the most rewarding aspects of food consumption behavior, it is unlikely that palatable food ingestion induces a negative emotional state. It is of interest to note that the sensitivity of the kappa opioid receptor system and the dynorphin tone in mesolimbic dopamine neurons are upregulated by chronic exposure to drugs of abuse, including alcohol, in humans and rodents (Lindholm et al., 2007; Wee and Koob, 2010), and that, during the withdrawal phase, the upregulated dynorphin system contributes to decreased dopamine release and negative emotional states,
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resulting in craving and increased drug intake (Bruijnzeel, 2009). In the present study, high plasma levels of exogenous ghrelin were shown to switch the dominant opioid receptor pathway for palatable food from mu to kappa, suggesting that overactivation of ghrelin signaling mediates the functional coupling of palatability, hedonic evaluation for food, and the dynorphin/kappa opioid receptor pathway. Eating disorders such as Prader-Willi syndrome, anorexia nervosa and binge eating are associated with hyperghrelinemia, suggesting the possible involvement of the dysregulated dynorphin system in the pathophysiology of eating disorders (Yi et al., 2011). For a better understanding of the conflicting findings, it will be extremely important to study the role of ghrelin in regulating the mesolimbic dopamine system in response to drugs of abuse, alcohol and highly rewarding food, in conjunction with activities of the mu and kappa opioid receptor pathways. 5. Conclusion In conclusion, systemic ghrelin and opioid receptors interact in the VTA to regulate the mesolimbic dopamine system and reward values of food. Systemic ghrelin induces switching of the dominant opioid receptor pathway for highly rewarding food from mu to kappa, resulting in suppression of the mesolimbic dopamine system. The novel findings of food reward-sensitive interaction of ghrelin and opioid receptor pathways in the VTA might provide insights into the neural pathways involved in eating disorders such as food craving, compulsive binge eating, and dependence on palatability of food (Woolley et al., 2007; Olszewski et al., 2011). Disclosure The authors declare no conflict of interest. Acknowledgments This work was supported by a grant from the Kurume University Millennium Box Foundation for the Promotion of Science, Grantsin-Aid for Scientific Research [(C)22590233 and (C)21592580], and the CREST program of JST. References Abizaid, A., 2009. Ghrelin and dopamine: new insights on the peripheral regulation of appetite. J. Neuroendocrinol. 21, 787e793. Abizaid, A., Liu, Z.W., Andrews, Z.B., Shanabrough, M., Borok, E., Elsworth, J.D., 2006. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Invest. 116, 3229e3239. Beck, B., Musse, N., Stricker-Krongrad, A., 2002. Ghrelin, macronutrient intake and dietary preferences in Long-Evans rats. Biochem. Biophys. Res. Commun. 292, 1031e1035. Briggs, D.I., Andrews, Z.B., 2011. Metabolic status regulates ghrelin function on energy homeostasis. Neuroendocrinology 93, 48e57. Bruijnzeel, A.W., 2009. Kappa-opioid receptor signaling and brain reward function. Brain Res. Rev. 62, 127e146. Chefer, V.I., Denoroy, L., Zapata, A., Shippenberg, T.S., 2009. Mu opioid receptor modulation of somatodendritic dopamine overflow: GABAergic and glutamatergic mechanisms. Eur. J. Neurosci. 30, 272e278. Date, Y., Shimbara, T., Koda, S., Toshinai, K., Ida, T., Murakami, N., Miyazato, M., Kokame, K., Ishizuka, Y., Ishida, Y., Kageyama, H., Shioda, S., Kangawa, K., Nakazato, M., 2006. Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell. Metab. 4, 323e331. Di Chiara, G., Imperato, A., 1988. Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J. Pharmacol. Exp. Ther. 244, 1067e1080. Dickson, S.L., Egecioglu, E., Landgren, S., Skibicka, K.P., Engel, J.A., Jerlhag, E., 2011. The role of the central ghrelin system in reward from food and chemical drugs. Mol. Cell. Endocrinol. 340, 80e87. Diéguez, C., da Boit, K., Novelle, M.G., Martínez de Morentin, P.B., Nogueiras, R., López, M., 2010. New insights in ghrelin orexigenic effect. Front. Horm. Res. 38, 196e205.
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