CRF1/CRF2 and MC3/MC4 Receptors Affect Glutamate- Induced Food Intake in Neonatal Meat-Type Chicken

F Ahmadi M Zendehdel V Babapour N Panahi About the authors

ABSTRACT

Central glutamate, melanocortin and corticotropin systems have mediatory role on several physiologic functions in the brain, but their interactions on appetite regulation are not fully elicited. So, the aim of the current study was to determine interaction of the glutamate with melanocortin and corticotropin systems on food intake in 3-h food-deprived (FD3) neonatal meat-type chicken. In experiment 1, chicken intracerebroventricular (ICV) injected (A) phosphate-buffered saline (PBS), (B) glutamate (75 nmol), (C) glutamate (150 nmol) and (D) glutamate (300 nmol). In experiment 2, (A) PBS, (B) astressin-B (CRF1/CRF2 receptors antagonist, 30 µg), (C) glutamate (300 nmol) and (D) astressin-B+glutamate were ICV injected. Experiments 3-5 were similar to experiment 2, except birds were injected with astressin2-B (CRF2 receptor antagonist, 30 µg), SHU9119 (MC3/MC4 receptor antagonist, 0.5 nmol) and MCL0020 (MC4 receptor antagonist, 0.5 nmol) instead of the astressin-B. In experiment 6, the injections were (A) PBS, (B) MTII (MC3/MC4 receptor agonist, 2.5ng), (C) glutamate (75nmol) and (D) MTII+glutamate. Then, cumulative feed intake was recorded at 30, 60 and 120 minutes after injection. According to the results, dose dependent hypophagia observed by ICV injection of the glutamate (75, 150 and 300nmol) compared to control group in neonatal broiler chicken (p<0.05). Co-injection of the astressin-B+glutamate and astressin2-B+glutamate decreased glutamate-induced hypophagia in neonatal broiler chicken (p<0.05). Co-injection of the glutamate+MC3/MC4 receptors antagonist decreased hypophagic effect of the glutamate (p<0.05). These results suggested hypophagic effect of the glutamate mediates via CRF1/CRF2 and MC3/MC4 receptors in chickens.

Keywords:
Glutamate; Melanocortin; Corticotrophin; Food intake; Neonatal chicken

INTRODUCTION

Feed intake, satiety and energy expenditure regulates via diverse signals from central and peripheral tissues (Hassanpour et al., 2015Hassanpour S, Zendehdel M, Babapour V, Charkhkar S. Endocannabinoid and nitric oxide interaction mediates food intake in neonatal chicken. British Poultry Science 2015;56(4):443-451.; Zendehdel et al., 2017Zendehdel M, Tirgari F, Shohre B, Deldar H, Hassanpour S. Involvement of GABA and cannabinoid receptors in central food intake regulation in neonatal layer chicks: role of CB1 and GABAA receptors. British Poultry Science 2017;19(2):51-60.). Neurotransmitters interact by a wide distributed neurological network on feed intake regulation in the central nervous system (CNS) (D’Addario et al., 2014D'Addario C, Micioni Di Bonaventura MV, Puccia M, Romano A, Gaetani S, Ciccocioppo R, et al. Endocannabinoid signaling and food addiction. Neuroscience & Biobehavioral Reviews 2014;47:203-224.). Appetite regulation regulates in several brain areas such as striatum, hypothalamus, amygdala, nucleus tractus solitarius (NTS) and arcuate nucleus (ARC) (D’Addario et al., 2014).

The melanocortin system is one of the central neurotransmitter systems and to date its five subtypes (MC1R-MC5R) have been identified (Alvaro et al., 2003Alvaro JD, Taylor JR, Duman RS. Molecular and behavioral interactions between central melanocortins and cocaine. Journal of Pharmacology and Experimental Therapeutics 2003;304:391-399.). It has prominent role in several physiologic functions e.g. grooming, thermoregulation, learning and energy balance regulation (Schneeberger et al., 2014Schneeberger M, Gomis R, Claret M. Hypothalamic and brainstem neuronal circuits controlling homeostatic energy balance. Journal of Endocrinology 2014;220:T25-T46.). In the brain of the avian Melanocortin receptors have also, been identified (Takeuchi et al., 1998Takeuchi S, Takahashi S. Melanocortin receptor genes in the chicken-tissue distributions. General and Comparative Endocrinology 1998;112:220-231.). Among melanocortin receptors only MC3R and MC4R subtypes are responsible for the central feed intake regulation (Schneeberger et al., 2014). The MC3R and MC4R mainly found in arcuate nucleus (ARC), ventromedial hypothalamus (VMH) and periventricular nucleus (PVN) regions of the hypothalamus (Liu et al., 2003Liu H, Kishi T, Roseberry AG, Cai X, Lee CE, Montez JM, et al. Transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter. Journal of Neuroscience 2003;23:7143-7154.). It is reported ICV injection of the MC3/MC4 receptors agonists decreased feed intake in rats (Strader et al., 2003Strader AD, Schiöth HB, Buntin JD. The role of the melanocortin system and the melanocortin-4 receptor in ring dove (Streptopelia risoria) feeding behavior. Brain Research 2003;960:112-121.).

Corticotrophin-releasing factor (CRF) is a 41 amino acid peptide and has major role in regulating central aspects of the stress response (Silberman and Winder, 2013Silberman Y, Winder DG. Corticotropin releasing factor and catecholamines enhance glutamatergic neurotransmission in the lateral subdivision of the central amygdala. Neuropharmacology 2013;70:316-323.). Corticotrophin receptors (CRF1 and CRF2) are G-protein-coupled receptors and regulators pituitary function in anxiety and stress (Yamada and Bruijnzeel, 2011Yamada H, Bruijnzeel AW. Stimulation of alpha2-adrenergic receptors in the central nucleus of the amygdala attenuates stress-induced reinstatement of nicotine seeking in rats. Neuropharmacology 2011;60:303-311.). Activation of the CRF1 and CRF2 receptors decreases feed intake (Richard et al. 2002Richard D, Lin Q, Timofeeva E. The corticotropin-releasing factor family of peptides and CRF receptors: their roles in the regulation of energy balance. European Journal of Pharmacology 2002;440:189-197.). The ICV injection of the Astressin-B or Astressin2-B decreased feed intake in rats (Stengel et al., 2009Stengel A, Goebel M, Wang L, Rivier J, Kobelt P, Mo¨ nnikes H, et al. Central nesfatin-1 reduces dark-phase food intake and gastric emptying in rats: differential role of corticotropin-releasing factor2 receptor. Endocrinology 2009;150:4911-4919.). Also, Finelli et al., (2014Finelli C, Rossano R, Padula MC, La Sala N, Sommella L, Martelli G. Nesfatin - 1: role as possible new anti obesity treatment. Journal of Obesity and Weight Loss Therapy 2014;4(3):1-4.) reported ICV injection of the Astressin2-B decreased feed intake in rats (Finelli et al., 2014). Recently, Heidarzadeh et al., (2017Heidarzadeh H, Zendehdel M, Babapour V, Gilanpour H. The effect of Nesfatin-1 on food intake in neonatal chicks: role of CRF1 /CRF2 and H1/ H3 receptors. Veterinary Research Communications 2017;42(1):39-47.) reported ICV injection of the Astressin-B affects nesfatin-1 induced hypophagia in FD3 broiler chicken.

Glutamate is the main excitatory neurotransmitter in the CNS and its two classified subtypes, the ionotropic and metabotropic receptors have been identified. N-methyl-D-aspartate receptor (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and Kainate receptors belongs to the ionotropic while mGLUR1, mGLUR2 and mGLUR3 receptors are metabotropic (Baghbanzadeh and Babapour, 2007Baghbanzadeh A, Babapour V. Glutamate ionotropic and metabotropic receptors affect feed intake in broiler cockerels. Journal of Veterinary Research 2007;62(4):125-129.). In several reports, effect of both ionotropic and metabotropic receptors on feeding behavior is identified (Zeni et al., 2000Zeni LA, Seidler HB, De Carvalho NA, Freitas CG, Marino-Neto J, Paschoalini MA. Glutamatergic control of food intake in pigeons: effects of central injections of glutamate, NMDA, and AMPA receptor agonists and antagonists. Pharmacology Biochemistry and Behavior 2000;65(1):67-74.; Baghbanzadeh and Babapour, 2007; Zendehdel et al., 2009Zendehdel M, Baghbanzadeh A, Babapour V, Cheraghi J. The effects of bicuculline and muscimol on glutamate-induced feeding behaviour in broiler cockerels. Journal of Comparative Physiology A 2009;195:715-720.). Hypophagia reported by ICV injection of AMPA receptors agonist into the lateral hypothalamus in mammals (Ciranna, 2006Ciranna L. Serotonin as a Modulator of glutamate- and GABA-Mediated Neuro transmission: implications in physiological functions and in pathology. Current Neuropharmacology 2006;4:101-114.; Hettes et al., 2010Hettes SR, GonzagaWJ, Heyming TW, Nguyen JK, Perez S, Stanley BG. Stimulation of lateral hypothalamic AMPA receptors may induce feeding in rats. Brain Research 2010;1346:112-120.). Also, the ICV injection of the DL-AP5 (NMDA receptor antagonist) increased food consumption in FD3 broiler cockerels (Taati et al., 2011Taati M, Nayebzadeh H, Zendehdel M. The effects of DLAP5 and glutamate on ghrelin-induced feeding behavior in 3- h food-deprived broiler cockerels. Journal of Physiology and Biochemistry 2011;67:217-223.; Mortezaei et al., 2013Mortezaei SS, Zendehdel M, Babapour V, Hasani K. The role of glutamatergic and GABAergic systems on serotonin- induced feeding behavior in chicken. Veterinary Research Communications 2013;37:303-310.). Injection of the AMPA, NMDA and kainate receptors antagonist into the brain increased feeding behavior in pigeon (Da Silva et al., 2003Da Silva AA, Marino-Neto J, Paschoalini MA. Feeding induced by microinjections of NMDA and AMPA-kainite receptor antagonists into ventral striatal and ventral pallidal areas of the pigeon. Brain Reearchs 2003;966:76-83.).

Both melanocortin and glutamatergic neurons are identified in the nucleus of the tractus solitarius (NTS), PVN, amygdala, ARN and area postrema of the brain which engaged with feed intake regulation centers in mammalian (Liu et al., 2003Liu H, Kishi T, Roseberry AG, Cai X, Lee CE, Montez JM, et al. Transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter. Journal of Neuroscience 2003;23:7143-7154.). In rats VTA, excitatory glutamatergic transmission is potentiated through CRF2 modulation of the NMDA transmission (Ungless et al., 2003Ungless MA, Singh V, Crowder TL, Yaka R, Ron D, Bonci A. Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons. Neuron 2003;39:401-407.). Also, CRF increase glutamatergic neurotransmission in the central amygdala (Silberman and Winder, 2013Silberman Y, Winder DG. Corticotropin releasing factor and catecholamines enhance glutamatergic neurotransmission in the lateral subdivision of the central amygdala. Neuropharmacology 2013;70:316-323.). According to the reports there are differences in central food intake regulation mechanisms between mammals and avian (Richards, 2003Richards MP. Genetic regulation of feed intake and energy balance in poultry. Poultry Science 2003;82(6):907-16.). There is no report on the interaction of glutamate with melanocortin and corticotropin systems on appetite regulation in avian. Based on comparative physiology, it is logical to assume the regulatory mechanisms governing these processes in birds (Furuse et al., 2007Furuse M, Yamane H, Tomonaga S, Tsuneyoshi Y, Denbow DM. Neuropeptidergic regulation of food intake in the neonatal chick: a review. The Journal of Poultry Science 2007;44:349-356.). Therefore, the main purpose of the current study was to determine interaction of the glutamate with melanocortin and corticotropin systems on feed intake in FD3 neonatal meat-type chicken.

MATERIAL AND METHOD

Animals

A total of 264 one-day-old male meat- type chickens were purchased from a local hatchery (Mahan Co., Iran). Birds were maintained in stabilizing electrically heated batteries at a temperature of 32 ºC ± 1, kept at 40-50 %relative humidity and 23:1 lighting/dark period (Olanrewaju et al., 2006Olanrewaju HA, Thaxton JP, Dozier WA, Purswell J, Roush WB, Branton SL. A review of lighting programs for broilers production. International Journal of Poultry Science 2006;5(4):301-308.). They were kept for 2 days as flocks and then the birds were randomly allocated and transferred into their individual cages. A commercial starter diet containing 21% crude protein and 2850 kcal/kg metabolizeable energy (Animal Science Research Institute Co. Iran) were provided to the animals (table 1). During the study all birds had ad libitum access to diet and fresh water. 3 h prior to the injections, the birds were food deprived (FD3) but had free access to water. ICV injections were done at 5 days of age. Animal handling and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health, USA (publication No. 85-23, revised 1996) and the current laws of the Iranian government for animal care, and were approved by the Institutional Animal Ethics Committee of Faculty of Veterinary Medicine, University of Tehran.

Table 1
Ingredient and nutrient analysis of experimental diet.

Experimental drugs

Drugs used include glutamate, astressin-B (CRF1/CRF2 receptors antagonist), astressin2-B (CRF2 receptor antagonist), SHU9119 (MC3/MC4 antagonist), MCL0020 (MC4 receptor antagonist), MTII (MC3/MC4 agonist) and Evans blue. They were purchased from Sigma-Aldrich (USA) and Tocris (UK) Co. To remove possible effect of the PBS as well as clarifying the accuracy of injected site, drugs were dissolved in a fresh PBS (phosphate-buffered saline) containing Evan’s blue and the pH was adjusted to 6.5-7.5.

ICV injection protocol

In each experiment, the birds were weighed and based on their body weight allocated into experimental groups so the average weight between treatment groups was as uniform as possible. The chicken was ICV injected once in each experiment using a microsyringe (Hamilton, Switzerland) without anesthesia using the Davis et al., (1979Davis JL, Masuoka DT, Gerbrandt LK, Cherkin A. Autoradiographic distribution of L- proline in chicks after intracerebral injection. Physiology & Behavior 1979;22:693-695.) and Furuse et al., (1997Furuse M, Matsumoto M, Saito N, Sugahara K, Hasegava S. The central corticotropin-releasing factor and glucagon-like peptide -1 in food intake of the neonatal chick. European Journal of Pharmacology 1997;339:211-214.) method. Briefly, the head of the chicken was held with an acrylic device in which the bill holder was 45º and the calvarium was parallel to the surface of the table as explained by Van Tienhoven & Juhasz (1962Van Tienhoven A, Juhasz LP. The chicken telencephalon, diencephalon and mesencephalon in sterotaxic coordinates. Journal of Comparative Neurology 1962;118:185-197.). An orifice was made in a plate over the skull of the right lateral ventricle. A microsyringe was inserted into the ventricle through the orifice in the plate and the tip of the needle perforated only 4 mm below the skin of the skull (Jonaidi & Noori, 2012Jonaidi H, Noori Z. Neuropeptide Y-induced feeding is dependent on GABAA receptors in neonatal chicks. Journal of Comparative Physiology A 2012;198:827-832.). All injections were done in a volume of 10 µL (Furuse et al., 1999). The control group received a control solution (saline containing Evans blue, 10 µL) (Furuse et al., 1999). This technique does not induce any physiological stress in neonatal chicks (Saito et al., 2005Saito ES, Kaiya H, Tachibana T, Denbow DM, Kangawa K, Furuse M. Inhibitory effect of ghrelin on food intake is mediated by the corticotropin-releasing factor system in neonatal chicks. Regulatory Peptides 2005;125:201-208.). At the end of the experiments, to recognize the accuracy of the injection, the chicks were sacrificed by decapitation. Accuracy of placement of the injection in the ventricle was verified by the presence of Evans blue followed by slicing the frozen brain tissue. In each group, 12 birds received the injection, but just the data of those individuals where dye was present in their lateral ventricle were used for analysis (11 chickens per group). All experimental procedures were done from 8:00 A.M. until 3:30 P.M.

Feeding experiments

In this study, six experiments were designed to determine interaction of the glutamate with CRF1/CRF2 and MC3/MC4 receptors in neonatal meat-type chicken. (Each experiment included 4 groups with 11 replicates in each group; n=44). In experiment 1, chicken ICV injected with (A) phosphate-buffered saline (PBS), (B) glutamate (75 nmol), (C) glutamate (150 nmol) and (D) glutamate (300 nmol). In experiment 2, (A) PBS, (B) astressin-B (30 µg), (C) glutamate (300 nmol) and (D) astressin-B + glutamate were ICV injected. In experiment 3, birds ICV injected with (A) phosphate-buffered saline (PBS), (B) astressin2-B (30 µg), (C) glutamate (300 nmol) and (D) astressin2-B + glutamate. In experiment 4, FD3 chicks received ICV injection of (A) PBS, (B) SHU9119 (0.5 nmol), (C) glutamate (300 nmol) and (D) SHU9119 + glutamate. In experimet 5, the ICV injection to the birds were (A) PBS, (B) MCL0020 (0.5 nmol), (C) glutamate (300 nmol) and (D) MCL0020 + glutamate. In experiment 6, the injections were (A) PBS, (B) MTII (2.5 ng), (C) glutamate (75 nmol) and (D) MTII + glutamate. The injection procedure in the experimental procedure is presented in table 2 and in the flow chart. Immediately after the injection feed was provided to the birds and cumulative feed intake (g) was measured at 30, 60 and 120 min after the injection. Food consumption was calculated as a gram of body weight (g/100g BW) to minimize the impact of body weight on the amount of feed intake. These doses of drugs were determined according to the pilot and previous studies (Zeni et al., 2000Zeni LA, Seidler HB, De Carvalho NA, Freitas CG, Marino-Neto J, Paschoalini MA. Glutamatergic control of food intake in pigeons: effects of central injections of glutamate, NMDA, and AMPA receptor agonists and antagonists. Pharmacology Biochemistry and Behavior 2000;65(1):67-74.; Baghbanzadeh & Babapour, 2007Baghbanzadeh A, Babapour V. Glutamate ionotropic and metabotropic receptors affect feed intake in broiler cockerels. Journal of Veterinary Research 2007;62(4):125-129.; Zendehdel et al., 2009Zendehdel M, Baghbanzadeh A, Babapour V, Cheraghi J. The effects of bicuculline and muscimol on glutamate-induced feeding behaviour in broiler cockerels. Journal of Comparative Physiology A 2009;195:715-720.; Ahmadi et al., 2017Ahmadi F, Zendehdel M, Babapour V, Panahi N, Hassanpour S, Khodadadi M. Modulatory function of NMDA glutamate receptor on MC3/MC4 receptors agonist-induced hypophagia in neonatal meat-type chicken. Veterinary Research Communications 2017;41(4):241-248.; Heidarzadeh et al., 2017Heidarzadeh H, Zendehdel M, Babapour V, Gilanpour H. The effect of Nesfatin-1 on food intake in neonatal chicks: role of CRF1 /CRF2 and H1/ H3 receptors. Veterinary Research Communications 2017;42(1):39-47.).

Table 2
Treatments procedure in experiments 1-7

Statistical analysis

Cumulative feed intake was analyzed by repeated measure two-way analysis of variance (ANOVA) and is presented as the mean ± SEM. For treatments found to have an effect according to the ANOVA, mean values were compared with Bonferroni test. P values <0.05 were considered to indicate significant differences between the treatments.

RESULTS

Effects and interactions of central glutamate with CRF1, CRF2, MC3 and MC4 receptors on cumulative feed intake in FD3 neonatal broilers are shown in figures 1- 6. In experiment 1, dose dependent hypophagia was observed by ICV injection of the different doses of the glutamate (75, 150 and 300 nmol) compared to the control group in neonatal broiler chicken [treatment effect: F (3, 80) = 385.1, P<0.0001; time effect: F (2, 80) = 824.7, p<0.0001; treatment and time interaction: F (6, 80) = 73.42; p<0.0001; Fig. 1].

Figure 1
Effects of intracerebroventricular injection of control solution(PBS) and different doses of the glutamate (75, 150 and 300 nmol) on cumulative food intake (gr/100gr BW) in neonatal meat-type chicks. PBS: phosphate- buffered saline. Data are expressed as mean ± SEM. Different letters (a-c) indicate significant differences between treatments at each time (p<0.05).

In experiment 2, ICV injection of the astressin-B (30 µg) had no effect on feed intake (P >0.05). The glutamate injection (300 nmol) significantly decreased feed intake compared to the control group (P<0.05). Co-injection of the astressin-B + glutamate, decreased glutamate-induced hypophagia in neonatal broiler chicken [treatment effect: F (3, 80) = 241.7, p<0.0001; time effect: F (2, 80) = 541.8, p<0.0001; treatment and time interaction: F (6, 80) = 35.18; p<0.0001; Fig. 2].

Figure 2
Effects of intracerebroventricular injection of control solution(PBS), astressin-B (CRF1/ CRF2 receptors antagonist; 30 µg), glutamate (300 nmol) and co-injection of the astressin-B + glutamate on cumulative feed intake (gr/100gr BW) in neonatal meat-type chicks. PBS: phosphate- buffered saline. Data are expressed as mean ± SEM. Different letters (a, b and c) indicate significant differences between treatments at each time (p<0.05).

In experiment 3, ICV administration of the astressin2-B (30 µg) had no effect on feeding behavior (P>0.05) while 300 nmol of the glutamate significantly decreased feed intake compared to the control group (p<0.05). Co-injection of the astressin2-B + glutamate, decreased glutamate-induced hypophagia in neonatal broiler chicken [treatment effect: F (3, 80) = 230.7, p<0.0001; time effect: F (2, 80) = 235.3, p<0.0001; treatment and time interaction: F (6, 80) = 21.6; p<0.0001; Fig. 3].

Figure 3
Effects of intracerebroventricular injection of control solution(PBS), astressin2-B (CRF2 receptor antagonist; 30 µg), glutamate (300 nmol) and co-injection of the astressin2-B + glutamate on cumulative feed intake (gr/100gr BW) in neonatal meat-type chicks. Data are expressed as mean ± SEM. PBS: phosphate- buffered saline. Different letters (a and b) indicate significant differences between treatments at each time (p<0.05).

In experiment 4, ICV administration of the 0.5 nmol MC3/MC4 antagonist (SHU9119) had no effect on feeding behaviour (P >0.05) while the glutamate injection (300 nmol) significantly decreased feed intake compared to control group (p<0.05). Co-injection of the SHU9119 + glutamate decreased the hypophagic effect of the glutamate in neonatal chicks [treatment effect: F (3, 80) = 240.27, p<0.0001; time effect: F (2, 80) = 628.15, p<0.0001; treatment and time interaction: F (6, 80) = 51.17; p<0.0001; Fig. 4].

Figure 4
Effect of ICV injection of control solution(PBS), SHU9119 (MC3/MC4 antagonist, 0.5 nmol), glutamate (300 nmol) and their combination on cumulative feed intake (gr/100gr BW) in neonatal meat-type chicks. PBS: phosphate- buffered saline. Data are expressed as mean ± SEM. Different letters (a, b and c) indicate significant differences between treatments at each time (p<0.05).

In experiment 5, MCL0020 (0.5 nmol) had no effect on feeding behavior in FD3 neonatal broilers compared to the control group (P >0.05) while the glutamate injection (300 nmol) had hypophagic effect in FD3 neonatal broilers compared to the control group (p<0.05). Co- administration of the glutamate + MCL0020 significantly diminished glutamate-induced hypophagia in chicks [treatment effect: F (3, 80) = 163.72, p<0.0001; time effect: F (2, 80) = 407.13, p<0.0001; treatment and time interaction: F (6, 80) = 11.06; p<0.0001; Fig. 5].

Figure 5
Effect of ICV injection of control solution(PBS), MCL0020 (MC4 receptor antagonist, 0.5 nmol), glutamate (300 nmol) and their combination on cumulative feed intake (gr/100gr BW) in neonatal meat-type chicks. PBS: phosphate- buffered saline. Data are expressed as mean ± SEM. Different letters (a and b) indicate significant differences between treatments at each time (p<0.05).

In experiment 6, sole ICV injection of the MTII (2.5 ng) or glutamate (75 nmol) had no effect on feeding behavior in FD3 neonatal broilers compared to the control group (P >0.05). Co-injection of the MTII + glutamate significantly decreased feed intake compared to glutamate or MTII alone [treatment effect: F (3, 80) = 439.52, p<0.0001; time effect: F (2, 80) = 376.08, p<0.0001; treatment and time interaction: F (6, 80) = 6.27; p<0.0001; Fig. 6].

Figure 6
Effect of ICV injection of control solution(PBS), MTII (MC3/MC4 agonist, 2.5 ng), glutamate (75 nmol) and their combination on cumulative feed intake (gr/100gr BW) in neonatal meat-type chicks. PBS: phosphate- buffered saline. Data are expressed as mean ± SEM. Different letters (a and b) indicate significant differences between treatments at each time (p<0.05).

DISCUSSION

To the best of our knowledge, this is the first report on the role of the glutamate with melanocortin and corticotrophin systems on feed intake in FD3 neonatal meat-type chicks. Based on the findings of the current study, dose dependent hypophagia was observed by ICV injection of the glutamate (75, 150 and 300nmol) in neonatal broiler chicken (figure 1). ICV injection of the metabotropic glutamate receptors antagonist increased food intake in broilers (Baghbanzadeh and Babapour, 2007Baghbanzadeh A, Babapour V. Glutamate ionotropic and metabotropic receptors affect feed intake in broiler cockerels. Journal of Veterinary Research 2007;62(4):125-129.). ICV injection of the NMDA receptor antagonist (DL-AP5) increased cumulative feed intake in FD3 cockerels (Taati et al., 2011Taati M, Nayebzadeh H, Zendehdel M. The effects of DLAP5 and glutamate on ghrelin-induced feeding behavior in 3- h food-deprived broiler cockerels. Journal of Physiology and Biochemistry 2011;67:217-223.). Injection of NMDA and AMPA-kainite receptor antagonists into ventral pallidal and ventral striatal nuclei decreased feed intake in the pigeon (Da Silva et al., 2003Da Silva AA, Marino-Neto J, Paschoalini MA. Feeding induced by microinjections of NMDA and AMPA-kainite receptor antagonists into ventral striatal and ventral pallidal areas of the pigeon. Brain Reearchs 2003;966:76-83.). The effect of the glutamatergic system on feed intake is mediated via NMDA, AMPA and mGLU1 receptors in FD3 neonatal chicken (Torkzaban et al., 2017). It seems, glutamate metabotropic receptors have partial interaction with other neurotransmitters in feed intake regulation in broilers (Torkzaban et al., 2017). The mGluR1 mechanically acts via phospholipase C activation which leads to the formation of the IP3 and diacylglycerol, intracellular release of Ca2+ and stimulation of protein kinase C, while mGLUR2 and mGLUR3 coupled to adenylyl cyclase and cyclase respectively (Mortezaei et al., 2013Mortezaei SS, Zendehdel M, Babapour V, Hasani K. The role of glutamatergic and GABAergic systems on serotonin- induced feeding behavior in chicken. Veterinary Research Communications 2013;37:303-310.).

Co-injection of the astressin-B + glutamate and astressin2-B + glutamate decreased glutamate-induced hypophagia in neonatal broiler chicken (figures 2 and 3). The interconnection is reported between CRF1 and CRF2 receptors with glutamatergic system. Activation of the CRF1 and CRF2 receptors have opposed actions on glutamatergic transmission in amygdala and the lateral septum mediolateral nucleus (Liu et al., 2004Liu J, Yu B, Neugebauer V, Grigoriadis DE, Rivier J, Vale WW, et al. Corticotropin-releasing factor and urocortin I modulate excitatory glutamatergic synaptic transmission. Journal of Neuroscience 2004;24(16):4020-4029.). Both CRF1 and CRF2 receptors are highly expressed in amygdala and the lateral septum mediolateral nucleus and influences neuronal properties (Liu et al., 2004). In the VTA, excitatory glutamatergic transmission is potentiated by CRF2 modulation of the NMDA transmission (Liu et al., 2004). In the current study, co-injection of the glutamate + MC3/MC4 receptors antagonist decreased hypophagic effect of the glutamate in chicken (figures 4 and 5). In this regard, Lu et al., (2003Lu XY, Barsh GS, Akil H, Watson SJ. Interaction between a_melanocyte-stimulating hormone and corticotropin-releasing hormone in the regulation of feeding and hypothalamo-pituitary-adrenal responses. Journal of Neuroscience 2003;23(21):7863-7872.) reported MC3 and MC4 receptors decrease feed intake in rats and nonhuman primates. Both CRF and MC4R mRNA are expressed in the PVN. MTII-induced plasma corticosterone was abolished by injection of the HS014 (0.25-1.0 nmol, selective MC4 antagonist) (Lu et al., 2003). In MC4 _/_ mice, because of the inability of MTII to suppress feed intake, MTII-induced anorexia mediates by the MC4R (Marsh et al., 1999Marsh DJ, Hollopeter G, Huszar D, Laufer R, Yagaloff KA, Fisher SL, et al. Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nature Genetics 1999;21:119-122.).

The CRF has two side effects on glutamatergic synaptic transmission in the central nucleus of the amygdala which CRF1 and CRF2 receptors has inhibitory and facilitatory roles, respectively (Liu et al., 2011Liu X, Wellman LL, Yang L, Ambrozewicz MA, Tang X, Sanford LD. Antagonizing corticotropin-releasing factor in the central nucleus of the amygdala attenuates fear-induced reductions in sleep but not freezing. Sleep 2011;34(11):1539-1549.). Mice lacking CRF1 receptors in glutamatergic neurons reduced anxiety and impaired neurotransmission in the hippocampus while increased anxiety-like behavior and reduced dopamine release in the prefrontal cortex (Inda et al., 2017Inda C, Armando NG, dos Santos Claro PA, Silberstein S. Endocrinology and the brain: corticotropin-releasing hormone signaling. Endocrine Connections 2017;6(6):R99-R120). Under physiological conditions CRF1 controlled dopaminergic and glutamatergic systems and could function in an antagonistic manner to retain adaptive responses to stressful situations in balance (Kratzer et al., 2013Kratzer S, Mattusch C, Metzger MW, Dedic N, Noll-Hussong M, Kafitz KW, et al. Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels. Frontiers Cell Neuroscience 2013;7:91.). Presynaptic glutamatergic neurotransmission increases by CRF in the central amygdala (Silberman & Winder, 2013Silberman Y, Winder DG. Corticotropin releasing factor and catecholamines enhance glutamatergic neurotransmission in the lateral subdivision of the central amygdala. Neuropharmacology 2013;70:316-323.). Extracellular glutamate levels increase after ICV injection of CRF into the central amygdala (Skorzewska et al., 2009Skorzewska A, Bidzinski A, Hamed A, Lehner M, Turzynska D, Sobolewska A, et al. The effect of CRF and alpha-helical CRF((9-41)) on rat fear responses and amino acids release in the central nucleus of the amygdala. Neuropharmacology 2009;57:148-156.). CRF1 has a higher affinity for CRF which suggested CRF increases glutamatergic neurotransmission in the central amygdala via CRF2 even after CRF1 receptors become saturated (Silberman & Winder, 2013). Corticotropin-releasing factor neurons express the NMDA receptor and this protein is transported to intracellular locations in dendrites (Beckerman et al., 2013Beckerman MA, Van Kempen TA, Justice NJ, Milner TA, Glass MJ. Corticotropin-releasing factor in the mouse central nucleus of the amygdala: ultrastructural distribution in NMDA-NR1 receptor subunit expressing neurons as well as projection neurons to the bed nucleus of the stria terminalis. Experimental Neurology 2013;239:120-132.).

Co-injection of MC3/MC4 receptors agonist with NMDA glutamate receptors antagonist, decreased melanocortine-induced hypophagia in neonatal chicken (Ahmadi et al., 2017Ahmadi F, Zendehdel M, Babapour V, Panahi N, Hassanpour S, Khodadadi M. Modulatory function of NMDA glutamate receptor on MC3/MC4 receptors agonist-induced hypophagia in neonatal meat-type chicken. Veterinary Research Communications 2017;41(4):241-248.). Hypophagic effect of melanocortin is mediated by glutamatergic system in rats (Campos et al., 2015Campos CA, Ritter RC. NMDA-type Glutamate Receptors Participate in Reduction of Food Intake Following Hindbrain Melanocortin Receptor Activation. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 2015;308(1):R1-9.). ICV administration of the MTII into the NTS decreased feed intake and this effect was weakened by glutamatergic system (Carlos et al., 2015). Activation of the presynaptic MC4 receptors in the central vagal afferent terminals by MTII reduces feed intake in rats (Campos et al., 2014). Glutamate release is required for phosphorylation of synapsin (I) in afferent vagal endings (Carlos et al., 2015). Synapsin (I) activation due to glutamate release is required for MTII-induced hypophagia in the rat (Carlos et al., 2015). Blockade of the glutamate receptor attenuated MTII-induced hypophagia in rats (Carlos et al., 2015). It seems, hypophagic effect of the melanocortin neurons acts by a mediatory role of the pro-opiomelanocortin (POMC) on the glutamate receptors (Dicken et al., 2012Dicken MS, Tooker RE, Hentges ST. Regulation of GABA and glutamate release from proopiomelanocortin neuron terminals in intact hypothalamic networks. Journal of Neuroscience 2012;32:4042-4048). Both POMC and MC3 and MC4 receptors have distribution on ARC and NTS which agouti related protein (AgRP) and neuropeptide Y (NPY) express (Gautron et al., 2010Gautron L, Lee C, Funahashi H, Friedman J, Lee S, Elmquist J. Melanocortin-4 receptor expression in a vago-vagal circuitry involved in postprandial functions. Journal of Comparative Neurology 2010;518:6-24.). The POMC neurons mediate glutamate release in the NTS which contribute to the reduction in food intake in the presence of MC4 receptors agonists (Gautron et al., 2010). POMC and AgRP neurons are located proximate to each other parallel to the glutamate, MC3 and MC4 receptors in the ARC. Based on the literature, there was no previous report on interaction between glutamate with melanocortin and corticotrophin systems on feed intake. So, we were not able to compare our results with it. In conclusion, these results suggested hypophagic effect of the glutamate mediates via CRF1/CRF2 and MC3/MC4 receptors in chickens. However, further investigation is required to elucidate the underlying cellular and molecular signaling pathways in the interconnections between glutamate with melanocortin and corticotrophin systems on food intake in neonatal chicks.


ACKNOWLEDGEMENTS

The authors thank the central laboratory (Dr. Rastegar Lab.) of the Faculty of Veterinary Medicine, University of Tehran for cooperation. This research is conducted as a part of the PhD thesis of the first author.

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  • COMPLIANCE WITH ETHICAL STANDARDS
  • INFORMED CONSENT

    This manuscript does not contain any studies with human subjects performed by any of the authors.
  • HUMAN AND ANIMAL RIGHTS

    All experiments were executed according to the Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal ethics committee.

Publication Dates

  • Publication in this collection
    09 May 2019
  • Date of issue
    Jan-Mar 2019

History

  • Received
    26 June 2018
  • Accepted
    31 Oct 2018
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