Role of Dopaminergic and Cannabinoidergic Receptors on Ghrelin-Induced Hypophagia in 5-Day-Old Broiler Chicken

1. Introduction

Appetite and energy expenditure are controlled using central and peripheral neurons to modulate the ingestion response. The feeding behavior of avians is regulated by several peptides ( 1 ). However, a number of these regulatory mechanisms are different among animals. As a basic catecholamine neurotransmitter within the brain, Dopamine (DA) plays an essential role in appetite regulation. A total of five distinct Dopaminergic (DAergic) receptor subtypes have at least been recognized (D₁-D₅) in the ventral tegmental area, hypothalamus, and substantia nigra ( 2 ). The most abundant DA receptors within the brain are the D₁ and D₂ receptors. The DAergic system engages in numerous physiological functions (e.g. cognition, locomotor activity, food intake, and emotion) ( 3 ). Food intake is reduced in rats by D₁ and D₂ receptors ( 4 ). Moreover, D₁ receptors in chickens mediate DA-induced hypophagia, while other receptors (D₂-D₄) may influence appetite regulation ( 5 ). The central feeding behavior is documented to be not regulated by a single neuropeptide. Furthermore, other neurotransmitters on feeding status are in interaction with a wide distributed neural network ( 6 ).

Ghrelin was detected in the brain, lung, stomach, proventriculus, spleen, ( 7 ), breast muscle, small intestine, and abdominal fat ( 8 ), provoking the release of the growth hormone (GH) via GH secretagogue receptors (GHS-R) ( 9 ). The subtypes of the ghrelin receptor detected in broilers include GHS-R1aV (homologous to mammalian GHS-R1b), GHS-R1tv, and GHS-R1a ad ( 10 ). Ghrelin ICV or systemic injection enhances the feeding behavior of laboratory animals ( 11 ), while it inhibits that of meat-type chicken ( 12 - 14 ) and Japanese quail ( 15 ). It has been 19 years since the discovery of Ghrelin; however, its role has not yet been fully investigated because of the numerous physiological functions in animals. Moreover, the interaction between ghrelin and other neurotransmitters remains yet to be revealed ( 15 ).

The endocannabinoids (ECB) are expressed in the peripheral nervous system, immune cells and tissues, and central nervous system (CNS) ( 16 ). The ICV administration of CB agonists raises the food intake of rodents, whereas selective CB₁ receptor antagonist (SR141716A) diminished that I of rats ( 17 ). The ICV injection of CB₁ or CB₂ cannabinoid receptors agonists (2-AG or JWH015) enhances the feeding behavior in layers ( 18 ), whereas only CB₂ receptors can be effective for broilers ( 19 ). In addition, the injection of the AM251 (inverse CB₁ receptors agonist) attenuates broiler food intake ( 20 ).

The ghrelin and DAergic receptors are co-expressed in the CNS neurons ( 21 ). GHS-R1a forms heteromers with D₁ receptors in hypothalamic neurons and GHS-R1a. D₂ receptor signaling is essential in D₁ receptors agonist-induced hypophagia ( 22 ). GHSR KO mice treated with a selective GHS-R1a antagonist are resistant to the anorexigenic effects of the D₂ receptors agonist ( 22 ). By modulating the DAergic reward pathway from VTA to nucleus accumbens (NAcc), the central ghrelin system engages in food intake ( 23 ). In the mice, the hippocampal D₁ receptor signaling is dependent upon the ghrelin receptor ( 21 ). Ghrelin injection to the VTA and NAcc increases the food intake and extracellular dopamine in mice ( 24 ). The mesolimbic dopamine system’s ghrelin-induced activation in mice is decreased by CB receptor antagonist ICV injection ( 25 ). The hypothalamic ECB content of CB₁ KO or in rimonabant-treated mice is elevated by ghrelin ( 26 ).

To date, several reports have been provided on the neurotransmitter impacts on food intake among mammalian; however, the food intake aspects of avians remain yet to be revealed ( 14 , 20 ). For instance, ghrelin plays an anorexigenic role in rats ( 8 ). For birds, however, ghrelin serves as an anorexigenic neurotransmitter ( 13 , 14 ).

It is essential to understand the interaction between ghrelin in avian and other neurotransmitters. According to the literature, the engagement of DAergic and cannabinoidergic (CBergic) systems in ghrelin-induced hypophagia in broiler avian has not been reported. To shed light on the mechanism that underlies the unique function of ghrelin in chicks, the relationship of ghrelin with the DAergic and CBergic systems is demonstrated for the first time in the present study by investigating the roles of D₁-D₄ and CB₁/CB₂ receptors in the ghrelin-induced food intake suppression of some 5-day-old broiler chickens.

2. Material and Methods

2.1. Animals

In total, 440 one-day-old broiler chickens of a local hatchery (ROSS 308) were obtained (Mahan Co., Iran). The chickens were kept within stabilizing electrically-heated batteries at 32±1ºC and relative humidity of 40%-50 %for a 23:1 light/dark schedule ( 27 ) as flocks for two days. Following that, they were randomly grouped, moved to individual cages, and provided with a commercial diet provided. During the study, the entire broilers were provided with diets and freshwater ad libitum access. The birds were deprived of food (FD₃) three hours before injections; however, water was available. At the age of five days, ICV injections were applied.

2.2. Experimental Drugs

The drugs included ghrelin, AMI-193 (D₂ receptor antagonist), SCH 23390 (D₁ receptor antagonist), L-741,742 (D₄ receptor antagonist), NGB2904 (D₃ receptor antagonist), 6-OHDA (6-hydroxy dopamine), L-DOPA (precursor of dopamine), 6-OHDA (6-hydroxy dopamine), AM630 (CB₂ receptor antagonist), [D-Lys-3]-GHRP-6 (ghrelin antagonist), and Evans blue which was obtained from U.S. Sigma-Aldrich Company and U.K. Tocris Company. The drugs were all dissolved in absolute dimethyl sulfoxide (DMSO) before they were diluted with 0.85% saline with a 1/250 ratio (0.4% DMSO) of the Evans blue (0.1%) content. This DMSO ratio has no cytotoxic impacts ( 28 , 29 ). Evans blue-containing DMSO/Saline was utilized as the control group.

2.3. ICV Injection Protocol

The birds were divided randomly into 10 groups, each of which was composed of four sub-groups (i.e., n=44). In each experiment, an ICV injection was performed on each chicken through a microsyringe (Hamilton, Switzerland) with no anesthesia according to the methods proposed by Davis, Masuoka ( 30 ), ( 31 ). In summary, the chicken heads were held using an acrylic device with a 45-degree bill holder and a parallel calvarium to the table 1 surface according to Van Tienhoven and Juhasz ( 32 ). No physiological stress was induced in neonatal chickens in this technique ( 33 ). Eventually, the chickens were sacrificed in order to measure injection accuracy.

2.4. Feeding Experiments

The present study designed 10 experiments in order to identify the possible impacts of particular dopaminergic receptors (i.e., D₁, D₂, D₃,, and D₄) on the hypophagic impact of ghrelin for FD₃ neonatal broiler chickens. In the first experiment, ICV injections were applied to the chickens with a control solution, ghrelin (6 nmol), SCH 23390+ghrelin, and SCH 23390 (5 nmol). In the second experiment, the control solution, ghrelin (6 nmol), AMI-193 (5 nmol), and their combination were applied by ICV injections. In the third experiment, ICV injections were carried out on the FD₃ birds by using the control solution, ghrelin (6 nmol), NGB2904 (6.4 nmol), and the co-injection of NGB2904+ghrelin. For experiment four, ICV injections were performed on the FD₃ chickens using the control solution, ghrelin (6 nmol), L-741,742 (6 nmol), and the co-injection of L-741,742+ghrelin. Experiment five involved the ICV injection of the control solution, ghrelin (6 nmol), L-DOPA (125 nmol), and their combination into the birds. Experiment six was the ICV injection of the control solution, ghrelin (6 nmol), 6-OHDA+ghrelin, and 6-OHDA (150 nmol). Experiment seven included the ICV injection of the control solution, [D-Lys-3]-GHRP-6 (10 nmol), [D-Lys-3]-GHRP-6 (5 nmol), and [D-Lys-3]-GHRP-6 (20 nmol). In experiment eight, the ICV injection of the control solution, dopamine (40 nmol), [D-Lys-3]-GHRP-6 (10 nmol), and their combination was performed on the FD₃ birds. The control solution, ghrelin+SR141716A, SR141716A (6.25 µg), and ghrelin (6 nmol) were injected in experiment nine. Experiment ten involved the ICV injection of the control solution, AM630 (5 µg), ghrelin (6 nmol), and ghrelin+AM630. The chickens were fed immediately after they were injected, measuring the cumulative food intake (g) when 30, 60, and 120 min elapsed from the injection. The drug doses were determined based on previous studies ( 14 , 34 , 35 ).

2.5. Statistical Analysis

The repeated measure two-way analysis of variance (ANOVA) was used to analyze the cumulative food intake. The results were provided as mean value±SEM. The mean values of the treatments that were found to have posed an effect based on the ANOVA were compared to the Bonferroni test. It was considered that p-values smaller than 0.05 represented significant differences between the treatments. Moreover, the variance analysis was carried out using the following model ( 36 ):

Y_ijk= μ + α_j + β_k + (αβ)_jk + ε_ijk

In which Y_ijk denotes the individual observation variable value, μ is the grand mean, α_j signifies the treatment impact based on time, β_k represents the treatment impact for the drug, (αβ)_jk indicates the interaction impact for time and drugs, and ε_ijk denotes the error.

3. Results

Figures 1- 8 illustrate the central DAergic system’s impacts on the ghrelin-induced hypophagia of the FD₃ neonatal chickens. For the first experiment, the ICV injection of SCH 23390 (5 nmol) had no effects on the cumulative food intake in comparison to the control group at 30, 60, and 120 min post-injections (P>0.05). The ICV injection of ghrelin (6 nmol) considerably diminished food intake in comparison to the control group (P<0.05). Furthermore, the ghrelin-induced hypophagia of the neonatal chickens was considerably attenuated by the co-injection of ghrelin and SCH 23390 (P<0.05), as shown in figure 1.

Figure 1. Effect of ICV injection of SCH 23390 (5 nmol), ghrelin (6 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). SCH 23390: D₁ receptor antagonist; Data are expressed as mean±SEM. Different letters (a, b, and c) indicate significant differences among treatments (P<0.05).

In the second experiment, the AMI-193 (5 nmol) ICV injection had no significant impacts on the cumulative food intake (P>0.05), whereas ghrelin (6 nmol) substantially reduced the cumulative food intake in comparison to the control group (P<0.05). Furthermore, the co-injection of AMI-193 (5 nmol)+ghrelin (6 nmol) had no effects on ghrelin-induced hypophagia in comparison to the control group (P>0.05), as shown in figure 2.

Figure 2. Effect of ICV injection of AMI-193 (5 nmol), ghrelin (6 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). AMI-193: D₂ receptor antagonist; Data are expressed as mean±SEM. Different letters (a and b) indicate significant differences among treatments (P<0.05).

In the third experiment, the NGB2904 (6.4 nmol) ICV injected birds exhibited no significant influence on the cumulative food intake (P>0.05). The ICV injection of ghrelin (6 nmol) considerably induced hypophagia within broilers (P<0.05). In addition, the co-administration of NGB2904+ghrelin showed no significant compact, compared to the control group (P>0.05), as shown in figure 3.

Figure 3. Effect of ICV injection of NGB2904 (6.4 nmol), ghrelin (6 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). NGB2904: D₃ receptor antagonist; Data are expressed as mean±SEM. Different letters (a and b) indicate significant differences among treatments (P<0.05).

In experiment four, the L-741,742 (6 nmol) ICV injection exhibited no impact on the food intake of the neonatal broilers (P>0.05). The ICV injection of ghrelin (6 nmol) substantially reduced the cumulative food intake (P<0.05). Additionally, no significant impacts were induced by the co-injection of L-741,742 (6 nmol) and ghrelin (6 nmol) on ghrelin-induced hypophagia (P>0.05), as shown in figure 4.

Figure 4. Effect of ICV injection of L-741,742 (6 nmol), ghrelin (6 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). L-741,742: D₄ receptor antagonist; Data are expressed as mean±SEM. Different letters (a and b) indicate significant differences among treatments (P<0.05).

For experiment five, the ICV injection of L-DOPA (125 nmol) was observed to have no significant impacts on the cumulative food intake in comparison to the control group (P>0.05). The ICV injection of ghrelin (6 nmol) considerably diminished the cumulative food intake in comparison to the control group (P<0.05). Furthermore, the co-injection of L-DOPA+ghrelin substantially enhanced the ghrelin effect on food consumption (P<0.05), as shown in figure 5.

Figure 5. Effect of ICV injection of L-DOPA (125 nmol), ghrelin (6 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). L-DOPA: precursor of dopamine; Data are expressed as mean±SEM. Different letters (a, b, and c) indicate significant differences among treatments (P<0.05).

For experiment six, the ICV injection of 6-OHDA (150 nmol) (P>0.05) into the birds showed no considerable effects on the cumulative food intake. The ICV injection of ghrelin (6 nmol) substantially diminished the cumulative food intake in comparison to the control group (P<0.05). Furthermore, the co-injection of 6-OHDA (150 nmol) and ghrelin (6 nmol) considerably enhanced the ghrelin hypophagic impact (P<0.05), as shown in figure 6.

Figure 6. Effect of ICV injection of 6-OHDA (150 nmol), ghrelin (6 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). 6-OHDA: 6-hydroxy dopamine; Data are expressed as mean±SEM. Different letters (a, b, and c) indicate significant differences among treatments (P<0.05).

For experiment seven, in comparison to the control group at 30, 60, and 120 min post-injections, the ICV injection of [D-Lys-3]-GHRP-6 (5, 10, and 20 nmol) exhibited a dose-dependent rise in the cumulative food intake (P<0.05), as shown in figure 7.

Figure 7. Effect of ICV injection of [D-Lys-3]-GHRP-6 (5 nmol), [D-Lys-3]-GHRP-6 (10 nmol), and [D-Lys-3]-GHRP-6 (20 nmol) on cumulative food intake in neonatal chicken (n=44). [D-Lys-3]-GHRP-6: ghrelin antagonist; Data are expressed as mean±SEM. Different letters (a, b, and c) indicate significant differences among treatments (P<0.05).

For experiment eight, the ICV injection of dopamine (40 nmol) substantially diminished the food intake (P<0.05), whereas [D-Lys-3]-GHRP-6 (as ghrelin antagonist; 10 nmol) had no effects on the cumulative food intake (P>0.05). The co-administration of [D-Lys-3]-GHRP-6 (10 nmol)+dopamine (40 nmol), however, decreased the dopamine-induced feeding behavior in comparison to the control group (P<0.05), as shown in figure 8.

Figure 8. Effect of ICV injection of [D-Lys-3]-GHRP-6 (10 nmol), dopamine (40 nmol), and their combination on cumulative food intake in neonatal chicken (n=44). [D-Lys-3]-GHRP-6: ghrelin antagonist; Data are expressed as mean±SEM. Different letters (a and b) indicate significant differences among treatments (P < 0.05).

Concerning experiment nine, the ICV injection of ghrelin (6 nmol) considerably reduced the cumulative food intake (P<0.05). Moreover, the ICV injection of SR141716A (6.25 µg) was observed to have no significant impacts on the cumulative food intake (P>0.05), whereas the co-injection of AMI- SR141716A (6.25 µg)+ghrelin (6 nmol) enhanced the ghrelin-induced feeding behavior in comparison to the control group (P<0.05), as shown in figure 9.

Figure 9. Effect of ICV injection of ghrelin (6 nmol), SR141716A (6.25 μg), and their combination on cumulative food intake in neonatal chicken (n=44). SR141716A: CB₁ receptor antagonist; Data are expressed as mean±SEM. Different letters (a, b, and c) indicate significant differences among treatments (P<0.05).

Concerning experiment ten, the ICV injection of ghrelin (6 nmol) considerably induced hypophagia within the birds (P<0.05). Furthermore, the ICV injection of AM630 (5 µg) (P>0.05) into the birds exhibited no significant impact on the cumulative food intake. Eventually, the co-administration of AM630+ghrelin was found to have no considerable effect on ghrelin feeding behavior, compared to the control group (P>0.05), as shown in figure 10.

Figure 10. Effect of ICV injection of ghrelin (6 nmol), AM630 (5 µg), and their combination on cumulative food intake in neonatal chicken (n=44). AM630: CB₂ receptor antagonist; Data are expressed as mean±SEM. Different letters (a and b) indicate significant differences among treatments (P<0.05).

4. Discussion

To the best of the authors’ knowledge, the present study is the first to report the impacts of the DAergic system on ghrelin-induced hypophagia for the broiler chickens. It was observed that the ICV injection of ghrelin (6 nmol) considerably reduced the food intake of the FD₃ chickens. The ICV injection of the ghrelin antagonist into the FD₃ chickens induced a dose-dependent rise in the cumulative food intake ([D-Lys-3]-GHRP-6), which was different from previous studies on mammalian ( 9 , 12 , 15 ). The ghrelin ICV injection reduced the avian food intake ( 13 ). The ghrelin ICV injections at the concentrations of 0.3, 1.1, 4.3, and 6.2 nmol diminished the food intake for 8-week-old broilers ( 37 ). Additionally, the ghrelin ICV injection (1 nmol) reduced the Japanese quail food intake ( 15 ). The ghrelin-induced hyperphagia mediates by activating agouti-related protein (AgRP) and neuropeptide Y (NPY) neurons in mammals, whereas it is completely different in avians ( 14 ). Ghrelin receptors are more expressed in NPY than the pro-opiomelanocortin neurons that are expressed in rats. Ghrelin injection enhanced the dopamine β-hydroxylase expression of the nucleus tractus solitarious ( 38 ).

Our results showed that the co-injection of the SCH 23390 and ghrelin significantly diminished the ghrelin-induced hypophagia, while the ICV injection of L-DOPA considerably enhanced the food intake by ghrelin within the neonatal chickens. In addition, the administration of the 6-OHDA as a dopamine synthesis inhibitor significantly decreased ghrelin-induced food intake. The ICV injection of the ghrelin antagonist diminished dopamine-induced hypophagia in chickens. Based on the previous studies, ghrelin receptors are expressed on VTA dopamine neurons. The ghrelin injection into the VTA increases the dopamine in the NAcc shell ( 39 ). Both D₁ and ghrelin (GHS-R1a) receptors are coexpressed in hippocampal neurons ( 21 ). Ghrelin receptor is quite important in D₁ receptor-induced hippocampal synaptic plasticity and memory formation ( 21 ). G-protein-coupled receptors heteromerization between ghrelin and D₁ receptors initiates a cAMP-independent signaling pathway that regulates DAergic regulation of hippocampal memory ( 21 ).

The administration of the ghrelin into the NAcc increases the dopamine release and induces locomotor activity ( 40 ). The reward system, particularly the mesolimbic dopamine system, enhances food-seeking behaviors ( 40 ). The mesolimbic DAergic pathway is an essential component of the ghrelin-responsive network ( 40 ). Peripherally, the injected ghrelin can access the mesolimbic reward and stimulate the mesolimbic DAergic system ( 41 ). The ICV injection of the GHS-R1A antagonist blocks the hyperphagic effect of the ghrelin, and the dopamine release suggest that the ghrelin activates GHS-R1A expressed on DAergic cell bodies ( 42 ). In mice hippocampal, D₁ receptor signaling is dependent on the ghrelin receptor ( 21 ). Ghrelin injection to the VTA and NAcc increases the food intake and extracellular dopamine in mice ( 24 ). Therefore, the role of ghrelin is undeniable in the midbrain DAergic system in food intake and energy balance to include reward-seeking behavior ( 40 ). Dopamine-containing projection to the dorsal vagal complex has a potent role in ghrelin release in the CNS ( 43 ). Furthermore, it is known that there is a correlation between DA and corticotrophin-releasing hormone (CRH) neurons in the CNS. The possible heteromerization among CRH2 and D₁ receptors is responsible for the synergism between DA and CRH transmission ( 44 ).

In addition, CRH1 and D₁ receptors are expressed in low levels in the VTA ( 45 ). It is known that ghrelin-induced hypophagia mediates via CRH system ( 46 ); moreover, the interaction of DA and CRH plays a mediatory role in ghrelin via CRH system. The ICV injection of the D₂ receptor agonist induced a vagal cholinergic-dependent increase in plasma ghrelin levels ( 43 ). Despite direct cellular and molecular mechanism, the interaction between DAergic and ghrelin is not fully elicited; however, the activation of GHS-R via ghrelin is reported to enhance D₁ receptor-induced cAMP accumulation and change the G protein coupling of the GHS-R from G-11/q into G-i/o through a mechanism that is consistent with the agonist-dependent formation of GHS-R/D1 receptor heterodimers ( 47 ). The VTA dopamine cells are not only sensitive to direct ghrelin but also innervated by ghrelin-sensitive lateral hypothalamic hypocretin/orexin neurons ( 48 ).

As it was observed, the co-injection of the CB₁ receptor antagonist along with the ghrelin enhanced ghrelin-induced hypophagia in comparison to the control group. The CB₁ receptor peripheral blockade diminished food intake. This effect declines gastric ghrelin secretion in food-deprived rats ( 49 ). The observed results are apparently mediated by the mTOR pathway ( 49 ). CB₁ and CB₂ receptor agonists are reported to raise the food intake of neonatal broiler chickens ( 16 ). Furthermore, ghrelin has a hypophagic effect in broilers ( 14 ). However, it seems that receptors have almost no effects on the interactions with the other neurotransmitters in the food intake regulation of broilers ( 16 ). As observed in our findings, CB₁ had a regulatory impact on ghrelin-induced hypophagia, while CB₂ had no interaction with ghrelin on feeding behavior in broiler chicken. Ghrelin had no orexigenic role in CB₁-knockout mice ( 50 ). In our explanations, the CB₁ receptor increased the food intake, while the CB₁ receptor blockade diminished food intake. On the other hand, ghrelin reduced food intake, and hypophagic effect of the ghrelin was enhanced when the CB₁ receptor was blocked. It seems that CB₁ receptor inhibition has yielded to amplify the effect of ghrelin. However, more investigations are required to determine the interactions of these neurons in central food regulation centers in avians.

Hypothalamic AMP-activated protein kinase activity plays a considerable role in the mediating impact of CB₁ receptors on the hypophagic role of ghrelin ( 50 ). Ghrelin hinders the excitatory inputs of CB₁ receptors on the paraventricular nucleus. It is eliminated by the ICV injection of CB₁ antagonists in mice ( 50 ). Ghrelin impresses its impacts via growth hormone secretagogue receptor type-1 and probably elevation on the ECBS synthesis ( 50 ). Rimonabant administration blocks the ghrelin impact stimulatory on 2-AG in mice. Therefore, the interconnection between CBergic and ghrelin mediates through CB₁ receptors on the food intake ( 51 ). Furthermore, major DAergic cell activation within the VTA of the midbrain is reward processing. Such DAergic neurons project the NAc, prefrontal cortex, and other brain areas, such as the hippocampus and hypothalamus ( 52 ). It is worth noting that ghrelin interacting with ECB and GABA also has an interaction with ECBs and dopamine ( 21 ).

In conclusion, this study demonstrated that the ICV injection of ghrelin suppressed food intake in chicks and was probably mediated by the DAergic and CBergic systems within the brain. The co-administration of the ghrelin antagonist+dopamine diminished the dopamine-induced feeding behavior. Therefore, D₁ and CB₁ receptors may be involved in the inhibitory impact of ghrelin on food intake in the brain feeding centers of neonatal broiler chickens. Accordingly, ghrelin-induced anorexia is mediated by the D₁ and CB₁ receptors of the broiler chickens. Furthermore, ghrelin, dopamine, cannabinoids, and their receptors have been demonstrated to exist in several areas of the chicken brain (5, 6, 18, 20). However, the precise sites in the chicken brain are engaged in the action of ghrelin and dopamine or in that of ghrelin and cannabinoids. As a result, their interaction is yet to be revealed.

Authors' Contribution

Study concept and design: R. F. and V. B.

Acquisition of data: R. F. and V. B.

Analysis and interpretation of data: M. Z.

Drafting of the manuscript: A. A.

Critical revision of the manuscript for important intellectual content: V. B.

Statistical analysis: H. G

Administrative, technical, and material support: R. F. and V. B.

Ethics

This manuscript did not contain any studies with human subjects performed by any of the authors. All experiments were executed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Ethics Committee.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgement

The authors are thankful to the central laboratory (Dr. Rastegar Lab.) of the Faculty of Veterinary Medicine, the University of Tehran for their cooperation. This study was conducted as a part of the Ph.D. thesis of the first author.

References

1. Kaneko K, Yoshikawa M, Ohinata K. Novel orexigenic pathway prostaglandin D2-NPY system--involvement in orally active orexigenic delta opioid peptide. Neuropeptides. 2012; 46(6):353-7.
2. Cadet JL, Jayanthi S, McCoy MT, Beauvais G, Cai NS. Dopamine D1 receptors, regulation of gene expression in the brain, and neurodegeneration. CNS Neurol Disord Drug Targets. 2010; 9(5):526-38.
3. Madhavan A, Argilli E, Bonci A, Whistler JL. Loss of D2 dopamine receptor function modulates cocaine-induced glutamatergic synaptic potentiation in the ventral tegmental area. J Neurosci. 2013; 33(30):12329-36.
4. Volkow ND, Wang GJ, Baler RD. Reward, dopamine and the control of food intake: implications for obesity. Trends Cogn Sci. 2011; 15(1):37-46.
5. Zendehdel M, Ghashghayi E, Hassanpour S, Baghbanzadeh A, Jonaidi H. Interaction Between Opioidergic and Dopaminergic Systems on Food Intake in Neonatal Layer Type Chicken. Int J Pept Res Ther. 2016; 22(1):83-92.
6. Irwin N, Hunter K, Frizzell N, Flatt PR. Antidiabetic effects of sub-chronic administration of the cannabinoid receptor (CB1) antagonist, AM251, in obese diabetic (ob/ob) mice. Eur J Pharmacol. 2008; 581(1- 2):226-33.
7. Xu P, Siegel PB, Denbow DM. Genetic selection for body weight in chickens has altered responses of the brain&#039;s AMPK system to food intake regulation effect of ghrelin, but not obestatin. Behav Brain Res. 2011; 221(1):216-26.
8. Kaiya H, Miyazato M, Kangawa K. Recent advances in the phylogenetic study of ghrelin. Peptides. 2011; 32(11):2155-74.
9. Richards MP. Genetic regulation of feed intake and energy balance in poultry. Poult Sci. 2003; 82(6):907-16.
10. Jonaidi H, Babapour V, Denbow DM. GABAergic control of food intake in the meat-type chickens. Physiol Behav. 2002; 76(4-5):465-8.
11. Kaiya H, Kangawa K, Miyazato M. What is the general action of ghrelin for vertebrates? - comparisons of ghrelin&#039;s effects across vertebrates. Gen Comp Endocrinol. 2013; 181:187-91.
12. Furuse M, Tachibana T, Ohgushi A, Ando R, Yoshimatsu T, Denbow DM. Intracerebroventricular injection of ghrelin and growth hormone releasing factor inhibits food intake in neonatal chicks. Neurosci Lett. 2001; 301(2):123-6.
13. Saito ES, Kaiya H, Tachibana T, Tomonaga S, Denbow DM, Kangawa K, et al. Inhibitory effect of ghrelin on food intake is mediated by the corticotropin-releasing factor system in neonatal chicks. Regul Pept. 2005; 125(1-3):201-8.
14. Zendehdel M, Hassanpour S. Ghrelin-induced hypophagia is mediated by the beta2 adrenergic receptor in chicken. J Physiol Sci. 2014; 64(5):383-91.
15. Shousha S, Nakahara K, Kojima M, Miyazato M, Hosoda H, Kangawa K, et al. Different effects of peripheral and central ghrelin on regulation of food intake in the Japanese quail. Gen Comp Endocrinol. 2005; 141(2):178-83.
16. Hassanpour S, Zendehdel M, Babapour V, Charkhkar S. Endocannabinoid and nitric oxide interaction mediates food intake in neonatal chicken. Br Poult Sci. 2015; 56(4):443-51.
17. Chen RZ, Frassetto A, Fong TM. Effects of the CB1 cannabinoid receptor inverse agonist AM251 on food intake and body weight in mice lacking mu-opioid receptors. Brain Res. 2006; 1108(1):176-8.
18. Alizadeh A, Zendehdel M, Babapour V, Charkhkar S, Hassanpour S. Role of cannabinoidergic system on food intake in neonatal layer-type chicken. Vet Res Commun. 2015; 39(2):151-7.
19. Emadi L, Jonaidi H, Hosseini Amir Abad E. The role of central CB2 cannabinoid receptors on food intake in neonatal chicks. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2011; 197(12):1143-7.
20. Novoseletsky N, Nussinovitch A, Friedman-Einat M. Attenuation of food intake in chicks by an inverse agonist of cannabinoid receptor 1 administered by either injection or ingestion in hydrocolloid carriers. Gen Comp Endocrinol. 2011; 170(3):522-7.
21. Kern A, Mavrikaki M, Ullrich C, Albarran-Zeckler R, Brantley AF, Smith RG. Hippocampal Dopamine/DRD1 Signaling Dependent on the Ghrelin Receptor. Cell. 2015; 163(5):1176-90.
22. Kern A, Albarran-Zeckler R, Walsh HE, Smith RG. Apo-ghrelin receptor forms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of DRD2 agonism. Neuron. 2012; 73(2):317-32.
23. Salahpour A, Caron MG. Food for thought: the physiological relevance of ghrelin and dopamine D2 receptor heterodimerization in the regulation of appetite. Neuron. 2012; 73(2):210-1.
24. Skibicka KP, Shirazi RH, Hansson C, Dickson SL. Ghrelin interacts with neuropeptide Y Y1 and opioid receptors to increase food reward. Endocrinology. 2012; 153(3):1194-205.
25. Kalafateli AL, Vallof D, Jornulf JW, Heilig M, Jerlhag E. A cannabinoid receptor antagonist attenuates ghrelin-induced activation of the mesolimbic dopamine system in mice. Physiol Behav. 2018; 184:211-9.
26. Lim CT, Kola B, Feltrin D, Perez-Tilve D, Tschop MH, Grossman AB, et al. Ghrelin and cannabinoids require the ghrelin receptor to affect cellular energy metabolism. Mol Cell Endocrinol. 2013; 365(2):303-8.
27. Olanrewaju H, J.P T, Dozier W, Purswell J, W.B R, S.L B. Review of Lighting Programs for Broiler Production. Int J Poult Sci. 2006; 5
28. Blevins JE, Stanley BG, Reidelberger RD. DMSO as a vehicle for central injections: tests with feeding elicited by norepinephrine injected into the paraventricular nucleus. Pharmacol Biochem Behav. 2002; 71(1):277-82.
29. Qi W, Ding D, Salvi RJ. Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures. Hear Res. 2008; 236(1-2):52-60.
30. Davis JL, Masuoka DT, Gerbrandt LK, Cherkin A. Autoradiographic distribution of L-proline in chicks after intracerebral injection. Physiol Behav. 1979; 22(4):693-5.
31. Furuse M, Matsumoto M, Saito N, Sugahara K, Hasegawa S. The central corticotropin-releasing factor and glucagon-like peptide-1 in food intake of the neonatal chick. Eur J Pharmacol. 1997; 339(2):211-3.
32. Van Tienhoven A, Juhasz LP. The chicken telencephalon, diencephalon and mesencephalon in sterotaxic coordinates. J Comp Neurol. 1962; 118:185-97.
33. Furuse M, Ando R, Bungo T, Ao R, Shimojo M, Masuda Y. Intracerebroventricular injection of orexins does not stimulate food intake in neonatal chicks. Br Poult Sci. 1999; 40(5):698-700.
34. Asakawa A, Inui A, Kaga T, Katsuura G, Fujimiya M, Fujino MA, et al. Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut. 2003; 52(7):947-52.
35. Jonaidi H, Noori Z. Neuropeptide Y-induced feeding is dependent on GABAA receptors in neonatal chicks. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2012; 198(11):827-32.
36. Chatfield C. The analysis of time series: an introduction. Chapman and Hall: CRC; 2003.
37. Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev. 2005; 85(2):495-522.
38. Date Y, Shimbara T, Koda S, Toshinai K, Ida T, Murakami N, et al. Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell Metab. 2006; 4(4):323-31.
39. Cone JJ, McCutcheon JE, Roitman MF. Ghrelin acts as an interface between physiological state and phasic dopamine signaling. J Neurosci. 2014; 34(14):4905-13.
40. Dickson SL, Egecioglu E, Landgren S, Skibicka KP, Engel JA, Jerlhag E. The role of the central ghrelin system in reward from food and chemical drugs. Mol Cell Endocrinol. 2011; 340(1):80-7.
41. Quarta D, Di Francesco C, Melotto S, Mangiarini L, Heidbreder C, Hedou G. Systemic administration of ghrelin increases extracellular dopamine in the shell but not the core subdivision of the nucleus accumbens. Neurochem Int. 2009; 54(2):89-94.
42. Jerlhag E, Egecioglu E, Dickson SL, Engel JA. Glutamatergic regulation of ghrelin-induced activation of the mesolimbic dopamine system. Addict Biol. 2011; 16(1):82-91.
43. Maedeh G, Mehranfard N, Eliassi A, Alaei H. Modulation of the Plasma Ghrelin Level by Intraventromedial Hypothalamic Microinjection of D2 Receptors Agonist and Antagonist. Neuropsychiatry. 2018; 08
44. Fuenzalida J, Galaz P, Araya KA, Slater PG, Blanco EH, Campusano JM, et al. Dopamine D1 and corticotrophin-releasing hormone type-2alpha receptors assemble into functionally interacting complexes in living cells. Br J Pharmacol. 2014; 171(24):5650-64.
45. Bernardi RE, Broccoli L, Hirth N, Justice NJ, Deussing JM, Hansson AC, et al. Dissociable Role of Corticotropin Releasing Hormone Receptor Subtype 1 on Dopaminergic and D1 Dopaminoceptive Neurons in Cocaine Seeking Behavior. Front Behav Neurosci. 2017; 11:221.
46. Diz-Chaves Y. Ghrelin, appetite regulation, and food reward: interaction with chronic stress. Int J Pept. 2011; 2011:898450.
47. Jiang H, Betancourt L, Smith RG. Ghrelin amplifies dopamine signaling by cross talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype 1 heterodimers. Mol Endocrinol. 2006; 20(8):1772-85.
48. Abizaid A. Ghrelin and dopamine: new insights on the peripheral regulation of appetite. J Neuroendocrinol. 2009; 21(9):787-93.
49. Senin LL, Al-Massadi O, Folgueira C, Castelao C, Pardo M, Barja-Fernandez S, et al. The gastric CB1 receptor modulates ghrelin production through the mTOR pathway to regulate food intake. PLoS One. 2013; 8(11):e80339.
50. Kola B, Farkas I, Christ-Crain M, Wittmann G, Lolli F, Amin F, et al. The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system. PLoS One. 2008; 3(3):e1797.
51. Turu G, Simon A, Gyombolai P, Szidonya L, Bagdy G, Lenkei Z, et al. The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptor-stimulated cannabinoid CB1 receptor activity. J Biol Chem. 2007; 282(11):7753-7.
52. Perello M, Dickson SL. Ghrelin signalling on food reward: a salient link between the gut and the mesolimbic system. J Neuroendocrinol. 2015; 27(6):424-34.