Divergent circuitry underlying food reward and intake effects of ghrelin: dopaminergic VTA-accumbens projection mediates ghrelin’s effect on food reward but not food intake (2013)

Keywords

• Ghrelin;
• Food motivation;
• Food intake;
• Overeating;
• Operant conditioning;
• Dopamine;
• D1;
• D2

1. Introduction

The circulating hormone ghrelin and the neural circuits through which it operates are well researched in the context of obesity and appetite control (Skibicka and Dickson, 2011), motivated also by therapeutic opportunities in this disease area (Cardona Cano et al., 2012). Ghrelin is unique amongst the circulating gut peptides in that it increases food intake (Wren et al., 2000, Inui, 2001, Shintani et al., 2001 and Kojima and Kangawa, 2002) a CNS effect mediated by dedicated receptors, GHS-R1A (Salome et al., 2009 and Skibicka et al., 2011) notably those located in brain areas involved in “homeostatic feeding” (i.e. feeding linked to energy deficit), the hypothalamus and brainstem (Melis et al., 2002, Faulconbridge et al., 2003 and Olszewski et al., 2003). Recently, however, a role for ghrelin outside of these homeostatic regions has emerged. GHS-R1A is also present in key nodes of the mesolimbic reward system, in areas such as the ventral tegmental area (VTA) and the nucleus accumbens (NAc) (Zigman et al., 2006 and Skibicka et al., 2011), areas involved in incentive motivated behavior that have also been linked to “hedonic feeding” (i.e. food intake coupled to its rewarding properties). Ghrelin is able to drive food intake from both of these sites and this effect is likely linked to its action to increase the incentive and motivational reward value of foods (Naleid et al., 2005, Abizaid et al., 2006 and Skibicka et al., 2011). Thus, in fully satiated rats or mice, ghrelin applied peripherally or centrally (including directly into the VTA) leads to an increased food intake and also food reward behavior (Naleid et al., 2005, Perello et al., 2010, Skibicka et al., 2011 and Skibicka et al., 2012b) reflected, for example, by increased lever-pressing for a sugar reward in a progressive ratio operant schedule. This action reflects an emerging role for ghrelin within the mesolimbic reward system to enhance reward behavior, not only for food but also for alcohol and drugs of abuse (Dickson et al., 2011). Importantly, this effect of ghrelin on food motivation over-rides satiety signals, as ghrelin elicits food reward behavior in satiated animals to a level comparable to that detected in food-deprived rats. Furthermore, the fact that blockade of the ghrelin signal, not only systemically but also selectively within the VTA (Skibicka et al., 2011), results in a potent suppression of food reward behavior underscores the importance and necessity of the ghrelin signal in food reward.

Ghrelin action at the level of the VTA is sufficient to drive food intake and motivated behavior, effects that appear to require signaling via GHS-R1A (Abizaid et al., 2006 and Skibicka et al., 2011). Surprisingly, the circuitry downstream of ghrelin’s reward-promoting actions in the VTA remains largely unresolved. Within the VTA, ghrelin engages opioid, NPY and GABAergic signaling (Abizaid et al., 2006 and Skibicka et al., 2012a). Nonetheless, VTA dopamine neurons, shown previously to express ghrelin receptors (Abizaid et al., 2006), may be the final VTA target for ghrelin’s effects on food reward. Palatable/rewarding foods engage the VTA dopamine neurons and the dopamine signal in select CNS areas such as the NAc, thereby stimulating food reward behavior (Hernandez and Hoebel, 1988 and Joseph and Hodges, 1990). It should be noted, however, that although dopamine release has been strongly linked to motivated behavior for food, it is also necessary for basic feeding as mice which are unable to synthesize dopamine die of starvation (Cannon et al., 2004). A functional link between ghrelin and dopamine is suggested by the effects of ghrelin on VTA dopamine neuron activity and also by the fact that intact VTA dopaminergic neurons are needed for ghrelin’s effects on food reward (Abizaid et al., 2006 and Weinberg et al., 2011). However, the VTA dopamine neurons project to a number of sites and it remains completely unexplored whether dopamine signaling in the NAc is required for VTA-driven effects of ghrelin on food-motivated behavior. Furthermore, ghrelin is involved in the control of behaviors other than food intake or motivation, namely novelty-seeking, which have also been linked to dopamine release in the NAc (Bardo et al., 1996 and Hansson et al., 2012).

In the present study, we tested the hypothesis that the effects of ghrelin on food motivated behavior and/or food intake exerted at the level of the VTA require dopamine receptor signaling in the NAc. To this end, food intake and food motivated behavior induced by VTA ghrelin was assessed in the progressive ratio lever-pressing for sucrose paradigm along with simultaneous NAc dopamine signaling blockade. In separate studies we tested the individual contribution of dopamine 1 (D1) like receptors and dopamine 2 receptors (D2). Furthermore, in order to explore the contribution of endogenous ghrelin to the NAc dopamine signal, we determined whether these dopamine receptors play a role in hunger-driven enhancement of food reward behavior. Finally, in order to evaluate the molecular consequences of endogenously elevated ghrelin in NAc dopamine signaling, we determined the effect of hunger/food deprivation on mRNA expression of NAc dopamine receptors and enzymes.

2. Materials and methods

Animals: Adult male Sprague-Dawley rats (200–250 g, Charles River, Germany) were housed in a 12-h light/dark cycle (lights on at 6 am) with regular chow and water available ad libitum in their home cages. All animal procedures were carried out with ethical permission and in accordance with the University of Gothenburg Institutional Animal Care and Use Committee guidelines.

Surgery: All rats in the behavioral studies were implanted with a guide cannula (26 gauge; Plastics One, Roanoke, VA), targeting the VTA and the NAc shell for subsequent unilateral, ipsilateral injections. Ketamine anesthesia was used. Cannulae were placed 1.5 mm above the target site, and an injector extending 1.5 mm from guide cannulae was used for microinjections. To target the VTA, the following coordinates were chosen from Skibicka et al. (2011): ±0.75 from the midline, 5.7 mm posterior to bregma, and 6.5 mm ventral from the surface of the skull, with injector aimed 8.0 mm ventral to skull. For the NAc shell, the following coordinates were used (modified from Quarta et al. (2009): ±0.75 from the midline, 1.7 mm anterior to bregma, and 6.0 mm ventral to skull, with injector aimed 7.5 mm ventral). Cannulae were attached to the skull with dental acrylic cement and jeweler’s screws and closed with an obturator, as described previously (Skibicka et al., 2009). In all rats, the microinjection site for both VTA and NAc was verified post mortem, by microinjection of india-ink at the same microinjection volume (0.5 μl) used throughout the study. Only subjects with the correct placement (Fig. 2) were included in the data analysis.

2.1. Operant conditioning procedure

Operant conditioning experiments took place in rat operant conditioning chambers (30.5 × 24.1 × 21.0 cm; Med-Associates, Georgia, VT, USA). The training procedure used for operant conditioning was adapted from previous studies (la Fleur et al., 2007 and Hansson et al., 2012). To facilitate operant training for sucrose, all rats were subjected to a mild food restriction during which their initial body weight was gradually reduced to 90% over a period of one week. Prior to placement in the operant boxes, rats were exposed to the sucrose pellets (45 mg sucrose pellets; test Diet, Richmond, IN, USA) in the home cage environment on at least two occasions. Next, rats learned to lever press for sucrose pellets under a fixed ratio FR1 schedule, with 2 sessions/day. In FR1, a single press on the active lever resulted in the delivery of one sucrose pellet. All FR sessions lasted 30 min or until the rats earned 50 pellets, whichever occurred first. Most rats achieved the 50 pellets per session criterion after 5–7 days. Presses on the inactive lever were recorded, but had no programmed consequence. FR1 schedule sessions were followed by FR3 and FR5 (i.e. 3 and 5 presses per pellet respectively). The FR5 schedule was followed by the progressive ratio (PR) schedule during which the cost of a reward was progressively increased for each following reward, in order to determine the amount of work the rat is willing to put into obtaining the reward. The response requirement increased according to the following equation: response ratio=(5e(0.2 × infusion number)) – 5 through the following series: 1, 2, 4, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328. The PR session ended when the rat had failed to earn a reward within 60 min. Responding was considered stable when the number of food pellets earned per session did not differ more than 15% for three consecutive sessions. In most cases, responding stabilized within 5 sessions. Those rats that did not reach the required criteria in that amount of time were trained in additional sessions. The PR test was carried out on 1 session/day. Rats were subsequently transferred to their home cages for 1 h chow intake measurement. At the end of training and prior to surgery and testing, rats had ad libitum access to normal chow.

2.2. Drugs

Acylated rat ghrelin (Tocris, Bristol, UK) was administered to the VTA at a dose of 1.0 μg with artificial cerebrospinal fluid (aCSF) as vehicle (and control). The 1.0 μg dose of ghrelin has previously been shown to increase operant responding for sugar and to induce an orexigenic response when delivered to the VTA (Naleid et al., 2005 and Skibicka et al., 2011). The D1-like receptor antagonist, SCH-23390, was administered to the NAc at a dose of 0.3 μg (Tocris), with aCSF as vehicle (control). For the food deprivation study, the dose was increased to 0.5 μg due to lack of effect of the original 0.3 μg dose. SCH-23390 is a potent and selective antagonist of D1-like dopamine receptors with >1000-fold affinity for D1-like versus D2-like dopamine receptors (Barnett et al., 1986). It has a similar affinity for D1 and D5 receptors (Barnett et al., 1992) hence throughout the study we will refer to its ability to block D1-like receptors, a term encompassing both D1 and D5 receptors. The initial 0.3 μg dose of SCH-23390 was chosen based on (Grimm et al., 2011). This dose injected into the shell of NAc was shown to be effective at reducing lever pressing for a cue previously paired to the delivery of a sucrose solution without affecting performance at the inactive lever. The dopamine D2 receptor antagonist, eticlopride hydrochloride (Tocris), was administered to the NAc with aCSF as vehicle (control). The initial dose of eticlopride chosen (1.0 μg) was based on (Laviolette et al., 2008) but was increased to 1.5 μg in the food deprivation study. All drugs were delivered in a 0.5 μl volume of aCSF.

2.3. Experimental design

All rats received NAc and VTA directed injections early in the light cycle, with the second injection at 10 min prior to the start of operant testing. All conditions were separated by a minimum of 48 h and run in a counterbalanced manner, such that each rat received all four conditions: first vehicle or dopamine receptor antagonist to the NAc and then, 10 min later, vehicle or ghrelin to the VTA. For each rat the ipsilateral VTA and NAc were targeted. Details of each experiment are also illustrated in Fig. 1.

2.3.1. Effect of D1-like receptor blockade on ghrelin-induced food reward and chow intake

Responses were examined after targeted VTA and NAc (n = 12–14) drug delivery after four conditions as follows: 1) control condition (vehicle solutions to the NAc and VTA), 2) NAc vehicle + VTA 1.0 μg ghrelin, 3) NAc 0.3 μg SCH-23390 + VTA vehicle, 4) NAc 0.3 μg SCH-23390 + VTA 1.0 μg ghrelin. Testing was performed in the satiated state (after the dark cycle period of feeding). On experimental days rats were returned to their home cages after 120 min of operant testing and chow intake was measured during 1 h in the home cage environment (as in schedule 1, Fig. 1). This time point corresponds to the third hour after VTA ghrelin injection, during which an orexigenic response would be expected to continue, based on previous studies exploring the time course of action of ghrelin, administered centrally or peripherally ( Wren et al., 2000 and Faulconbridge et al., 2003) and our previous studies that utilized a similar experimental setup.

2.3.2. Effect of D2 receptor blockade on ghrelin-induced food reward and chow intake

Responses were examined after targeted VTA and NAc (n = 7) drug delivery in four conditions as follows: 1) control condition (vehicle solutions to the NAc and VTA), 2) NAc vehicle + VTA 1.0 μg ghrelin, 3) NAc 1 μg eticlopride hydrochloride + VTA vehicle, 4) NAc 1 μg eticlopride hydrochloride + VTA 1.0 μg ghrelin. Testing was performed in the satiated state (after the dark cycle period of feeding). Rats were returned to their home cages after 120 min of operant testing and chow intake was measured during 1 h in the home cage environment (as in schedule 1, Fig. 1) as ghrelin-mediated orexigenic effect is still present after a delayed placement of chow pellets (after 2 h).

2.3.3. Effects of D1-like and D2 receptor blockade (separate or combined) on ghrelin-induced chow intake alone

In order to confirm that the results obtained on chow intake in the previous experiments were not confounded by the prior exposure to the sucrose in the operant paradigm or the 2 h time delay, in a separate study, we explored the effects of NAc delivery of the two dopamine receptor antagonists alone or in combination on VTA ghrelin-induced 2 and 3 h food intake in satiated rats (n = 10–11; as in schedule 2, Fig. 1). In this case the rats were not exposed to the operant conditioning paradigm prior to chow measurement. Thus, food intake was measured after targeted VTA and NAc drug delivery after four conditions as follows: 1) control condition (vehicle solutions to the NAc and VTA), 2) NAc vehicle + VTA 1.0 μg ghrelin, 3) NAc dopamine receptor antagonist + VTA vehicle, 4) NAc dopamine receptor antagonist + VTA 1.0 μg ghrelin. First we explored the two dopamine receptor antagonists separately such that, in conditions 3 and 4, one group of rats received 0.3 μg SCH-23390 and the other group received 1 μg eticlopride hydrochloride. After recovery for 3 days, approximately half of the rats from each group were retested, this time with a combination of the two antagonists in conditions 3 and 4. In each of these 3 experiments a counterbalanced design was used between treatments, as before (all rats received all conditions in each experiment for within subject comparison of effect). The position of the cannulae was verified post-mortem as before. Data shown include only rats with injection placement confirmed to reach the VTA and NAc.

2.3.4. Effect of D1-like and D2 receptor blockade on food deprivation-induced food reward and chow intake

The dopamine receptor antagonists were tested in 2 different experiments. In the first experiment, responses were examined after targeted NAc (n = 20) delivery of either vehicle or the D1-like receptor antagonist (0.5 μg SCH-23390). Testing was performed in the fasted state (after food has been restricted for the duration of the dark cycle period). In the second experiment responses were examined after targeted NAc (n = 7) delivery of either vehicle or 1.5 μg NAc eticlopride hydrochloride. Testing was performed in the fasted state (after food has been restricted for the duration of the dark cycle period; as illustrated in schedule 3, Fig. 1).

2.3.5. Food deprivation-induced changes in dopamine related gene expression in NAc

Food deprivation-driven changes in gene expression of key selected dopamine-related genes [dopamine receptors D1A, D2, D3, D5, catechol-O-methyltransferase (COMT), and monoamine oxidase A (MAO)] were measured in the NAc.

2.3.6. RNA isolation and mRNA expression

Brains were rapidly removed and the NAc was dissected using a brain matrice, frozen in liquid nitrogen and stored at −80 °C for later determination of mRNA expression. Individual brain samples were homogenized in Qiazol (Qiagen, Hilden, Germany) using a Tissue Lyser (Qiagen). Total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen) with additional DNAse treatment (Qiagen). RNA quality and quantity were assessed by spectrophotometric measurements (Nanodrop 1000, NanoDrop Technologies, USA). For cDNA synthesis iScript cDNA Synthesis kit (BioRad) was used. Real-time RT PCR was performed using TaqMan^® probe and primer sets for target genes chosen from an on-line catalog (Applied Biosystems). Gene expression values were calculated based on the C_t method ( Livak and Schmittgen, 2001), where the ad libitum fed group was designated the calibrator. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference gene.

2.3.7. Statistical analysis

All behavioral parameters were analyzed by repeated measures analysis of variance (ANOVA) followed by post hoc Tukey HSD test as appropriate or student’s t test where only two conditions were compared. All statistical analyses were conducted using the GraphPad software. Differences were considered significant at p < 0.05.

3. Results

3.1. Effect of D1-like receptor blockade (NAc) on VTA ghrelin-induced food reward and chow intake

To determine whether activity at the D1-like receptors is necessary for the VTA ghrelin-induced increase in food reward behavior the impact of pretreatment with a D1-like antagonist (SCH-23390) on ghrelin-induced operant responding for sucrose was tested. A post hoc Tukey test following a one way ANOVA (F_(3,33) = 11.1, p < 0.0005; F_(3,33) = 3.7, p < 0.01; F_(3,39) = 3.6, p < 0.05 for rewards, active lever and chow respectively) revealed a significant effect of ghrelin to increase the number of rewards earned (p < 0.0005; Fig. 3A), the number of active lever presses (p < 0.05; Fig. 3B), and chow intake (p < 0.05; Fig. 3C). Reward behavior-associated parameters, the rewards earned and active lever presses, were clearly blocked by SCH-23390 pretreatment ( Fig. 3A, B). Activity at the inactive lever was minor and did not differ significantly between the different treatment groups ( Fig. 3B) suggesting that treatment does not produce unspecific non-goal directed changes in activity. Chow hyperphagia observed after ghrelin was microinjected into the VTA was not altered by SCH-23390 pretreatment ( Fig. 3C). These data demonstrate that dopamine and D1-like receptors in the NAc shell are downstream of ghrelin and are necessary for VTA administered ghrelin to exert its effects on food reward behavior. They are not, however, essential for ghrelin’s ability to increase chow intake. NAc treatment with SCH-23390 had no effect per se on either operant responding for food or chow intake ( Fig. 3).

3.2. Effect of D2 blockade (NAc) on VTA ghrelin-induced food reward and chow intake

To determine whether activity at the D2s is necessary for expression of the VTA ghrelin-induced elevation of food reward behavior, the impact of pretreatment with a selective D2 antagonist (eticlopride hydrochloride) on ghrelin-induced increase in sucrose operant behavior was tested. One way ANOVA demonstrated a significant effect of drug treatment (F_(3,18) = 9.5, p < 0.0005; F_(3,18) = 8.1, p < 0.001; F_(3,39) = 3.8, p < 0.05 for rewards, active lever and chow respectively). A post hoc Tukey test indicated a significant increase in rewards earned (p < 0.01; Fig. 4A) and active lever presses (p < 0.01; Fig. 4B) after ghrelin treatment that were blocked with eticlopride pretreatment. Activity at the inactive lever was minor and did not differ significantly between the different treatment groups ( Fig. 4B). In contrast to the operant responding data, eticlopride pretreatment did not alter the ghrelin-induced increase in chow intake (p < 0.05; Fig. 4C). In this combination study the interaction was confirmed by two-way ANOVA between pretreatment × ghrelin in rewards earned: F_(1,24) = 4.8, p < 0.05; active lever presses: F_(1,24) = 4.7, p < 0.05 but not chow intake. Thus D2 receptors may be utilized by ghrelin to induce changes in reward-related behaviors but not chow consumption.

3.3. Effect of D1-like and/or D2 receptor blockade (NAc) on VTA ghrelin-induced chow intake

To seek further validation of the lack of effect of the two dopamine antagonists on chow feeding, we repeated the study, this time in rats never exposed to the operant conditioning paradigm. This validation study was extended to include a third test in which we explored the effects of co-delivery of the D1-like and D2 receptor antagonists to the NAc on VTA ghrelin-driven food intake. Chow intake was significantly increased by VTA ghrelin at 2 h after injection (one way ANOVA: F_(3,30) = 6.4, p < 0.005 and F_(3,27) = 9.0, p < 0.0005 for the D1 and D2 receptor study respectively) and this was unaffected by pretreatment with either the D1-like ( Fig. 5A) or the D2 receptor antagonist ( Fig. 5B). In the final test, exploring the combined effect of the two dopamine receptor antagonists, we could not detect a significant effect of VTA ghrelin until the 3 h time point, perhaps reflecting the impact of the triple parenchymal injection needed in this study. One way ANOVA indicated a significant effect of treatment (F_(3,30) = 9.6, p < 0.0005). Food intake after VTA ghrelin delivery reached significance at the 3 h time point, however, this was again not suppressed by co-application of the dopamine receptor antagonists to the NAc ( Fig. 5C). Note that the combined application of both dopamine receptor antagonists to the NAc had no effect per se on food intake.

3.4. Effect of D1-like and D2 receptor blockade on food deprivation-induced food reward and chow intake

Food deprivation elevates both operant responding and 1 h chow intake; rats pressed the active lever nearly twice as much when hungry and three to six times more chow at the 1 h measurement point (compare vehicle condition in Figs. 3 and 4). Blockade of D1-like receptors in the NAc shell significantly reduced the food deprivation-induced elevation in food reward behavior when assessed as a reduction in food rewards earned (p < 0.01; Fig. 6A) and a reduction in active lever presses (p < 0.01; Fig. 6B). This treatment did not have any significant effects on food-deprivation-induced chow intake ( Fig. 6C). Infusion of a D2 antagonist into the NAc shell significantly reduced food deprivation-induced elevation in food reward behavior when assessed as a reduction in food rewards earned (p < 0.01; Fig. 7A). Even though every rat reduced its active lever pressing after D2 blockade in the NAc the effect resulted in a trend (p = 0.08; Fig. 7B) likely due to high baseline variability in lever pressing (standard error = 86 for vehicle and 41 for drug conditions, range of active lever pressing on vehicle from 57 to 707 presses). Removal of the highest responding rat from the data set results in p = 0.001. Notably the removed rat showed 707 presses on vehicle and only 303 on drug, thus also supporting the overall conclusion. Neither dopamine receptor antagonist altered lever pressing at the inactive lever. Chow intake was not altered by the D2 blockade in the NAc ( Fig. 7C).

3.5. Food deprivation-induced changes in dopamine-related gene expression in NAc

Overnight fasting had a significant impact on the mRNA expression of several dopamine related genes in the NAc. Expression of mRNA of dopamine receptor D2 was significantly reduced while dopamine receptor D5 mRNA was elevated. Dopamine receptor D1, D3, COMT and MAO mRNAs were not altered by the overnight fasting (Fig. 8). D1 and D2 receptors are considered the most abundant dopamine receptor in the brain while D3 and D5 presence in CNS is a lot more restricted. We therefore compared the mRNA levels in the accumbens of D5 receptors to D1 and arrived at 2%; a similar relationship was detected for D3 and D2 (data not shown). Thus here we confirm that within NAc the majority of dopamine receptor mRNA is made up of that of the D1 and D2 receptors while D3 and D5 receptors represent only a small fraction of the total dopamine receptor mRNA detected in the NAc.

4. Discussion

The major findings of the current study indicate that dopamine signaling in the shell of the NAc is a necessary downstream mediator of ghrelin’s effects on food reward. The results indicate that D1-like and D2 receptors in the shell of NAc are key components of the ghrelin-activated circuitry and are essential for VTA applied ghrelin to exert its effects on food reward behavior. D1-like and D2 receptor signaling in the NAc (shell) are not, however, essential for ghrelin’s ability to increase chow intake. These data suggest a divergence in the neural targets for ghrelin that control food reinforcement vs. food intake. Finally our findings indicate that this circuitry is also engaged by endogenous ghrelin as, in a state of hunger, when circulating ghrelin levels are elevated, dopamine signaling in the NAc is required for the increased food reward behavior.

Surprisingly, while it is clear that ghrelin has an impact on the dopaminergic system (Abizaid et al., 2006, Jerlhag et al., 2007, Kawahara et al., 2009 and Weinberg et al., 2011), this is the first study to demonstrate that ghrelin’s effects on food reward require NAc dopamine receptor signaling (in this case, D1-like and D2 signaling). This emerged as an important question as other hormones or neuropeptides linked to appetite control have recently been shown to have a rather unexpected relationship with the mesolimbic dopamine system. Leptin, for example, like ghrelin, has receptors on the dopamine neurons in the VTA; most of these leptin-sensitive dopaminergic neurons, however, do not project to the striatum but instead innervate the amygdala (Hommel et al., 2006 and Leshan et al., 2010). Melanocortin, a potent anorexigenic neuropeptide with receptors in the VTA, in contrast to what may be predicted for an anorexic agent, actually increases dopaminergic activity and dopamine release in the striatum, while clearly reducing food intake behavior (Torre and Celis, 1988, Lindblom et al., 2001 and Cone, 2005). Another layer of complexity is added by data indicating that the dopamine- releasing effect of ghrelin appears to be dependent on the availability of food: NAc dopamine levels detected by microdialysis were only increased by peripherally applied ghrelin in rats that were allowed to eat after ghrelin administration (as in the experimental conditions used in the present study) and were even suppressed by ghrelin in those that were denied access to food (Kawahara et al., 2009), an effect recently shown to involve differential opioid signaling pathways in the VTA (Kawahara et al., 2013). These two examples emphasize the complexity in the relationship between feeding peptides, food availability and dopamine and highlight the importance of studies exploring the utility of ghrelin’s effects on the dopamine system in food reward behavior.

An interesting aspect of the results is the contrasting effect of NAc dopamine receptor blockade on food motivation vs. food intake. Notably, we confirmed the lack of effect of suppressed NAc dopamine signaling on VTA ghrelin-induced food intake in 2 independent studies: in one paradigm the food intake measurement was made immediately after the operant responding test (for which eating sugar rewards could have altered subsequent chow intake) and, in the other, only food intake was measured in the animals without prior operant testing. Additionally, in the second experiment we were able to show that co-application of both dopamine receptor antagonists to the NAc had no effect on VTA ghrelin-induced food intake, increasing support for the hypothesis that NAc dopamine signaling via D1-like and D2 receptors is not required for ghrelin hyperphagia. Taken together with the fact that the antagonists interrupt VTA ghrelin-induced food motivated behavior, these collective results suggest a divergence of neuro-circuitry downstream of VTA ghrelin, with one branch controlling food intake and the other food motivation/reward. It appears that ghrelin utilizes dopamine to alter food motivation but not intake. Previously, we showed that VTA ghrelin engages neuropeptide Y in the VTA selectively to control food intake and opioids in an opposite manner (Skibicka et al., 2012a). Thus, there already exists precedence for a divergence in the circuitry engaged by ghrelin for food intake versus food-motivated behavior.

Accumbal D1-like receptors have a well-established role in both drug and food reinforcement with an array of previous evidence indicating that intra-NAc D1-like antagonist infusion reduces goal-oriented behavior toward food. Systemic D1-like receptor antagonists reduce cue- or context-induced self-administration of cocaine, heroin, nicotine and alcohol [for example (Weissenborn et al., 1996, Liu and Weiss, 2002, Bossert et al., 2007 and Liu et al., 2010)], highlighting the key role of these receptors in reward-oriented processes. The present data indicate that NAc D1-like receptors are an essential element of the circuitry activated by VTA-acting ghrelin. Supportively, peripheral application of this D1 antagonist has also been shown to reduce ghrelin-enhanced object recognition (Jacoby and Currie, 2011). However, considering that peripheral application targets all D1-expressing neuronal populations in the brain and that populations outside of the NAc (for example, in the hippocampus) can have a major role in learning and memory, it is not clear whether the NAc population examined here contribute to the memory enhancing effects of ghrelin.

D2 receptors often act in concert with D1; thus many studies indicate a role of D2 receptors in aspects of reward processing and reward oriented behavior. However, it is noteworthy that D1 and D2 receptors do not always act in the same way w.r.t. reward function. In the amygdala, for example, blockade of D1 receptors attenuates reinstatement to cue-induced cocaine seeking, while D2 antagonists can actually enhance this behavior (Berglind et al., 2006). This functional dissociation may also have a neuroanatomical contribution, as D2 receptors in NAc appear to serve a rather opposite function to those in the hypothalamus. While in the NAc stimulation of D2 receptors can increase food motivation, making an animal more likely to exert effort to obtain food, in the hypothalamus stimulation of D2 receptors is clearly anorexic (Leibowitz and Rossakis, 1979 and Nowend et al., 2001). It follows that it can be difficult to interpret results after peripheral application of D2-targeting drugs for which the target receptor populations are linked to opposing function. This might be one of the reasons that explain why, in a previous study, peripheral injection of a D2 antagonist had no effect on ghrelin-induced responding for a sucrose solution. Another possible explanation is that D2 is an autoreceptor on the dopamine-producing neurons in the substantia nigra and VTA, where its activation can lead to a suppression of dopaminergic activity (Lacey et al., 1987). Thus, when injected peripherally, D2-targeting drugs could potentially gain access to this receptor population, while in our study only the NAc shell D2 receptor were targeted. Notably, the net effect of systemic D1-like receptor blockade did block the responding for a sucrose drink in the same paradigm (Overduin et al., 2012). Furthermore, systemic, subcutaneous injection of a D1 agonist appears to enhance the preference for palatable food while systemic injection of a D2 agonist reduces it (Cooper and Al-Naser, 2006). Thus, it seems that our data indicating a suppressing effect of D1 antagonists on ghrelin-induced food motivation is in line with the overall net (suppressive) effect of stimulating D1 receptors on reward function. By contrast, the net effect of D2 receptor population follows more closely with that what is known about the hypothalamic D2 receptors, than the data presented here for the NAc.

In the present study both D1-like and D2 antagonists were able to block operant behavior for sucrose after VTA ghrelin administration and after food deprivation suggesting that a cooperative action at both receptors in the NAc is needed for ghrelin to exert its effects. This makes sense when considering the endogenous situation in which VTA-derived dopaminergic terminals release dopamine in the NAc shell simultaneously activating all accessible dopamine receptors. The need for simultaneous activation of both D1-like and D2 receptors has already been reported for other behaviors including reinforcement (Ikemoto et al., 1997) and locomotor activity (Plaznik et al., 1989) as well as neuronal firing (White, 1987). The results of the present study indicate that blockade of only one of the two dopaminergic receptors was sufficient to reduce those behaviors just as blockade of either one of those receptors was sufficient to reduce ghrelin-driven sucrose operant behavior. The mechanism behind this interaction is unclear. Some neurons in the NAc coexpress both D1 and D2 receptors. One possibility is the involvement of heterodimers is required for the reward response, the formation of heterodimers by the D1 and D2 receptors was reported recently and this coupling was shown to contribute to depression-like behavior (Pei et al., 2010). Nevertheless, our results indicate that D1 and D2 signal in the NAc is not redundant, and each receptor is needed in order to transmit the ghrelin effect on food reward since individual blockade was effective in attenuating the reward response. Additionally, since individual blockade was not effective for ghrelin hyperphagia, we have separately evaluated the possibility whether the D1 and D2 signal was redundant for chow intake, i.e. simultaneous blockade of both would be needed to eliminate the response. This, however, was not the case as ghrelin hyperphagia was not affected by the simultaneous blockade of D1 and D2 receptors in the NAc. Thus alone or in combination the NAc shell D1 and D2 receptor signaling is not utilized by ghrelin to increase chow intake.

Here, we targeted the D1-like and D2 receptors in the shell of the NAc. The function of shell and core of the NAc seems to be dissociable to some degree especially with the core underlying changes in drug self-administration linked to discrete cue and the shell being more influential in context dependent drug self-administration (Bossert et al., 2007). This functional dissociation is supported by the neuroanatomical connections, where the core receives more input from the amygdala and the shell is more densely innervated by the hippocampus (Groenewegen et al., 1999 and Floresco et al., 2001). Rats will also self-administer the combination of D1 and D2 receptor agonists only in the shell of NAc and not in the core (Ikemoto et al., 1997), indicating that their cooperative action on reward is primarily linked to the shell region targeted here.

In the present study, we explored specifically, the impact of suppressed NAc dopamine signaling on food intake and food motivated behavior driven by VTA-applied ghrelin. It should be noted, however, that ghrelin may also drive feeding behaviors by activating afferent pathways to the VTA. For example, ghrelin has been shown to enhance food-reinforced behaviors by activating orexin neurons in the lateral hypothalamus (Perello et al., 2010), an orexinergic cell group that projects to the VTA and stimulate dopamine release (Narita et al., 2006). While our study using neuroanatomy and neuropharmacology specifically dissects the VTA-NAc pathway, in an endogenous situation ghrelin released in the circulation likely stimulates the VTA as wells as other brain nuclei expressing ghrelin receptor with efferent projections to the VTA. Thus, in a physiological situation, the impact of ghrelin is distributed over many sites in the brain which likely act in concert. The concept of a hormone or a neuropeptide acting on many distributed sites in the brain from which it can elicit a similar outcome, for example a change in food intake, is not novel and has already being proposed and evaluated for leptin and melanocortin (Grill, 2006, Leinninger et al., 2009, Skibicka and Grill, 2009 and Faulconbridge and Hayes, 2011).

Food deprivation is associated with high levels of circulating ghrelin. In conditions of food deprivation food presentation elicits a dopamine release in the NAc (Kawahara et al., 2013). It follows that nutritional state, may also influence dopamine signaling in the NAc, the impact of food deprivation on mRNA expression of dopamine receptors (D1-like receptors (D1, D5) and D2-like receptors (D2, D3)) and dopamine degrading enzymes (MAO, COMT) evaluated in the present study. While food deprivation did not alter the mRNA expression of any of the dopamine degrading enzymes measured, we did see a differential regulation of D5 vs. D2 receptors. The expression of D5 receptors was increased by nearly 30% while the D2 receptor mRNA was reduced by about 20%. Consistent with this divergence, simultaneous application of D1-like and D2 receptor agonists has been previously shown to down-regulate D2 receptors but to upregulate D1 receptors in the substantia nigra (and with a similar trend in the NAc) (Subramaniam et al., 1992). Interestingly, the effects of food deprivation on NAc dopamine receptor expression converge with our data demonstrating a role for D1-like (that include D5) and D2 receptors in fasting-induced motivation for food.

One caveat of our study is that food deprivation increases circulating ghrelin levels so that other ghrelin receptors populations outside of the VTA can potentially be activated. Thus, while food deprivation is an endogenous and more physiologically relevant way to increase ghrelin, it does not allow for selective VTA stimulation. We cannot therefore eliminate the possibility that the dopamine receptor changes detected in the NAc are a result of ghrelin activity in areas outside of the VTA with an indirect influence on the NAc. Finally, it should be noted that our data link fasting to changes in NAc dopamine receptor expression but further experiments would be required to show mediation of the (ghrelin-stimulated) VTA-NAc dopaminergic projection in this effect and, indeed, to explore the role of other pathways and transmitter systems in this effect, like the lateral hypothalamus (as discussed above).

Since many of the neurobiological substrates are common to both drug addiction and disordered eating, it is possible that present findings are indicative of a role of D1-like and D2 receptors in drug and alcohol reinforcing effects of ghrelin (Dickson et al., 2011). Both food and cocaine reward lead to a release of dopamine in the NAc (Hernandez and Hoebel, 1988). Blockade of D1 or D2 receptors reduces reward behavior for drugs of abuse, alcohol and nicotine. Since a considerable contribution of ghrelin to intake or reward behavior for all these substances has been reported previously, it is rather likely that the ghrelin-VTA-dopamine-NAc circuitry described here is relevant for an array of reward behaviors and not exclusively for food. Preliminary support for this idea can be drawn from data demonstrating that food deprivation can reinstate heroin seeking that is blocked by blockade of D1-like receptors (Tobin et al., 2009).

Our data provide new knowledge about an integration of two key food reward-linked signaling systems: the VTA-driven circuits that are responsive to the orexigenic hormone, ghrelin, and the NAc dopamine-responsive circuits. In particular we show that ghrelin’s well-documented VTA-linked effects on food-motivated behavior require D1 and D2 signaling in the NAc. Our data also indicate that the VTA-driven (D1/D2-dependent) effects of ghrelin on food reward involve divergent circuitry to those important for food intake, as neither antagonist affected ghrelin-induced food intake when delivered to the NAc. Finally, studies in hungry (overnight fasted and hence, hyperghrelinemic) rats implicate NAc D1/D2 signaling in the effects of endogenous ghrelin on food-motivated behavior. Thus, mechanisms and therapies interfering with dopamine signaling in the NAc appear to have relevance for ghrelin-mediated effects on the reward system, including those linked to feeding control and hence, obesity and its treatment.

Disclosure statement

The authors have nothing to disclose.

References

• Weissenborn et al., 1996
• R. Weissenborn, V. Deroche, G.F. Koob, F. Weiss
• Effects of dopamine agonists and antagonists on cocaine-induced operant responding for a cocaine-associated stimulus
• Psychopharmacology (Berl), 126 (1996), pp. 311–322
• View Record in Scopus

   | 

  Citing articles (74)
•  
• Zigman et al., 2006
• J.M. Zigman, J.E. Jones, C.E. Lee, C.B. Saper, J.K. Elmquist
• Expression of ghrelin receptor mRNA in the rat and the mouse brain
• J. Comp. Neurol., 494 (2006), pp. 528–548
• View Record in Scopus

  |

  Full Text via CrossRef

   | 

  Citing articles (383)
• Wren et al., 2000
• A.M. Wren, C.J. Small, H.L. Ward, K.G. Murphy, C.L. Dakin, S. Taheri, A.R. Kennedy, G.H. Roberts, D.G. Morgan, M.A. Ghatei, S.R. Bloom
• The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion
• Endocrinology, 141 (2000), pp. 4325–4328
• View Record in Scopus

  |

  Full Text via CrossRef

   | 

  Citing articles (1108)
• White, 1987
• F.J. White
• D-1 dopamine receptor stimulation enables the inhibition of nucleus accumbens neurons by a D-2 receptor agonist
• Eur. J. Pharmacol., 135 (1987), pp. 101–105
• Article

  |

   PDF (342 K)

  |

  View Record in Scopus

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  Citing articles (88)
•  
•  

•  
• Corresponding author. Department of Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Medicinaregatan 11, PO Box 434, SE-405 30 Gothenburg, Sweden. Tel.: +46 31 786 3818 (office); fax: +46 31 786 3512.