Central Opioidergic System Interplay with Histamine on Food Intake in Neonatal Chicks : Role of μ-Opioid and H 1 / H 3 Receptors

The present study was designed to examine the role of Opioidergic and Histaminergic systems on feeding behavior in 3-hour food deprived neonatal meattype chicks. In experiment 1, chicks received intracerebroventricular (ICV) injection of (A) control solution, (B) α-FMH (alpha fluoromethyl histidine; 250 nmol), (C) DAMGO (μ-opioid receptor agonist, 125 pmol) and (D) α-FMH + DAMGO. Experiments 2-4 were similar to experiment 1, except chicken ICV injected with Chlorpheniramine (histamine H1 receptors antagonist; 300 nmol), famotidine (histamine H2 receptors antagonist; 82 nmol) and Thioperamide (histamine H3 receptors antagonist; 300 nmol) instead of the α-FMH. In experiments 5-8, birds ICV injected with the same procedure as experiments 1-4, except they were injected with DPDPE (δ-opioid receptor agonist, 40 nmol) instead of DAMGO. Experiments 9-12 were similar to the experiments 1-4, except neonatal broilers ICV were injected with U-50488H (κ-opioid receptor agonist, 30 nmol) instead of DAMGO. Then the cumulative food intake was measured until 120 min post injection. According to the results, ICV injection of DAMGO, significantly decreased food intake (p<0.05) while DPDPE and U-50488H increased feeding behavior compared to the control group (p<0.05). Co-administration of the α-FMH and DAMGO significantly inhibited hypophagic effect of the DAMGO in neonatal broilers (p<0.05). Also, Chlorpheniramine significantly inhibited DAMGOinduced feeding behavior in neonatal chicks (p<0.05). In addition, coadministration of the Thioperamide + DAMGO significantly amplified the hypophagic effect of the DAMGO in neonatal chicks (p<0.05). However, famotidine had no effect on food intake induced by DAMGO (p>0.05). Also, the hyperphagic effect of DPDPE and U-50488 had no affect by α-FMH, Chlorpheniramine, famotidine and Thioperamide (p>0.05). These results suggested that an interconnection between central opioidergic and histaminergic systems on feeding behavior is mediated via μ-opioid and H1/H3 receptors in neonatal broilers.


IntRoduCtIon
Feeding behavior is a very complex neurochemical pathway which is regulated hierarchically from the central nervous system (CNS) and the peripheral nervous system (PNS) (Jonaidi et al., 2012).Complex physiological interaction exists on food intake and expenditure by afferent signals and efferent effectors between the CNS and gastrointestinal tract (Hassanpour et al., 2015).In the CNS, this process is regulated by complex neurochemical mechanisms in the hypothalamic nuclei, striatum, amygdala and arcuate nucleus (ARC) (D'Addario et al., 2014).Several neurotransmitters in the CNS have been identified where food intake is regulated (Ladepeche et al., 2013).
Histamine is one of the main neurotransmitters which express in the paraventricular nucleus (PVN) and ventromedial hypothalamus (VMH) of the brain (Giannoni et al., 2009;Blandina et al., 2012).To date, 4 subtypes of histamine receptors have been identified including H 1 , H 2 , H 3 and H 4 in the several parts of CNS (Schneider et al., 2014).The central histaminergic (HAergic) system has the key role in feeding behavior (Rozov et al., 2014) where the ICV injection of histamine decreases food intake whereas ICV injection of alphafluoromethylhistidine (α-FMH, selective inhibitor of the histidine decarboxylase as histamine synthesizing enzyme) or chlorpheniramine (histamine H 1 receptor antagonist) increases food intake in rats (Morimoto et al., 2001) and chicken (Kawakami et al., 2000).It is well documented that appetite is regulated by the interaction of various neurotransmitters and complex network (Branch et al., 2013).
Based on the literature, an interconnection exists between central HAergic and opioidergic systems in some areas in the brain.Endomorphins have the highest affinity in the amygdala, PVN and dorsomedial hypothalamus with histaminergic neurons and might regulate arousal and sedative behaviors (Koneru et al., 2009).It is reported that Naloxoneinduced water intake decreased by blockade of H 1 and H 2 receptors in male rats (Oryan et al. 2004).The interaction of the histaminergic and opioidergic systems in the hippocampus mediates pain from originating (Mojtahedin et al., 2008).Co-injection of the histamine H 1 receptor antagonists and morphine, increased analgesic activity in the acute trigeminal model of pain in rats (Khalilzadeh et al., 2017).Despite the researches being done on interaction of the central HAergic and opioidergic systems, there is no report on interaction of these two systems on feeding behavior in mammals and poultry.It is known that central food intake regulation is dissimilar between mammals and birds (Zendehdel & Hassanpour, 2014).It is logical to assume the regulatory mechanisms governing these processes in birds (Hassanpour et al., 2015).Therefore, the current study was designed for the first time to determine the possible interconnection of the central opioidergic and HAergic systems on feeding behavior in neonatal meat-type chicks.

Animals
A total 528 male meat-type one-day-old chickens (Ross 308) were purchased from a local hatchery (Mahan Co. Iran).Birds were kept as flocks for 2 days then randomly transferred into individual cages at a temperature of 30 ± 1ºC with 50 ± 2 percent humidity (Olanrewaju et al., 2006).A commercial diet provided during the study containing 21% crude protein and 2850 kcal/kg of metabolizable energy (Chineh Co. Iran) (table).All birds had free access to food and fresh water during the study.Just 3 hours prior the ICV injections, chicken were food deprived (FD 3 ) but had free access to water.The injections were applied to all birds 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) and the current laws of the Iranian government for animal care.

ICV injection procedures
The birds were randomly allocated into 12 experimental groups (each experiment includes 4 groups, n=11 in each group).Prior to each experiment, the chicks were weighed and based on their body weight, divided into experimental groups so the average weight between treatment groups was as uniform as possible.The ICV injection was applied using a microsyringe (Hamilton, Switzerland) without anesthesia according to the technique previously described by Davis et al., (1979) and Furuse et al., (1997) where the head of the birds was held with an acrylic device while the bill holder was 45º and calvarium parallel to the surface of table (Van Tienhoven & Juhasz, 1962).A hole was drilled in a plate where the skull overlaid immediately over the right lateral ventricle.A microsyringe was inserted into the right ventricle via the hole and the tip of the needle penetrated 4 mm beneath the skin of the skull.It is revealed that, there is no injection-induced physiological stress using this method in neonatal chicks (Saito et al., 2005).Each chick received an ICV injection (with vehicle or drug solution) in a volume of 10 μL (Furuse et al., 1999).The control group received a control solution (DMSO/ saline mixture containing Evan's blue, 10 μL) (Furuse et al., 1999).Right away after the injection, FD 3 the birds returned to their individual cages and supplied fresh water and food (pre-weighed).Cumulative food intake (gr) was measured at 30, 60 and 120 minutes post the injection.Food consumption was calculated as a percentage of body weight to minimize the impact of the body weight on the amount of food intake.Each bird was used just once in each experimental group.At the end of the experiments, the accuracy of the placement of the injection in the ventricle was verified by presence of Evans blue followed by slicing the frozen brain tissue.All experimental procedures were done from 8:00 A.M. until 3:30 P.M.

Statistical analysis
Data is presented as mean ± SEM (standard error of the mean).Cumulative food intake (as percent of body weight) was analyzed by repeated measure two-way analysis of variance (ANOVA) using SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL, USA).For treatment showing a main effect by ANOVA, means were compared by Tukey-Kramer test.p<0.05 was considered as significant differences between treatments.

ReSultS
Effects and interactions of central HAergic and opioidergic systems on cumulative food intake in FD 3 neonatal meat-type chicks are shown in figures 1-12.In this study to examine the possible interaction between these two systems, effective and sub-effective doses of pharmacological agents were administered to confront nullifying effects of the agents.In experiment 1, ICV injection of the DAMGO (µ opioid receptors agonist, 125 pmol) significantly decreased food intake until 120 min post injection compared to the control group (p<0.05).The ICV injection of the sub effective dose of the α-FMH (alpha fluoromethyl histidine; 250 nmol) had no effect on cumulative food intake compared to the control group (p>0.In experiment 2, hypophagia was observed after the ICV injection of DAMGO (125 pmol) in FD 3 neonatal chicken, compared to the control group (p<0.05).The ICV injection of the chlorpheniramine In experiment 3, significant decrease in food intake was observed after the ICV injection of DAMGO (125 pmol) in birds compared to the control group (p<0.05).The ICV injection of the famotidine (histamine H 2 receptors antagonist; 82 nmol) had no effect on food intake in comparison to the control group (p>0.05).Co-injection of the famotidine + DAMGO was not able to change DAMGO-induced hypophagia [treatment effect: F (3, 80) = 89.35,p<0.0001; time effect: F (2, 80) = 549.7,p<0.0001; treatment and time interaction: F (6, 80) = 9.17; p<0.0001;Fig. 3].

Central Opioidergic System Interplay with Histamine on Food Intake in Neonatal Chicks: Role of µ-Opioid and H1/H3 Receptors
In experiment 8, no significant effect was observed on food intake after the ICV injection of the thioperamide (300 nmol) (p>0.05).ICV injection of the DPDPE (40 pmol) significantly increased food intake in FD 3 neonatal birds compared to the control group (p<0.05).While, the ICV injection of the DPDPE + famotidine was not able to affect hyperphagic effect of the DPDPE in FD 3 neonatal birds [treatment effect: F (3, 80) = 39.14, p<0.0001; time effect: F (2, 80) = 548.15,p<0.0001; treatment and time interaction: F (6, 80) = 5.17; p<0.001;Fig. 8].In experiment 9, ICV administration of the U-50488H (κ-opioid receptor agonist, 30 nmol) significantly increased food intake in FD 3 neonatal broilers compared to the control group (p<0.05).ICV injection of the α-FMH (250 nmol) had no effect on cumulative food intake compared to the control group (p>0.05).Also, Co-administration of the α-FMH and U-50488H had no effect on U-50488H-induced hyperphagia in neonatal broilers injection [treatment effect: F (3, 80) = 63.08,p<0.0001; time effect: F (2, 80) = 682.1,p<0.0001; treatment and time interaction: F (6, 80) = 4.53; p<0.0001;Fig. 9].In experiment 10, the ICV injection of the chlorpheniramine (300 nmol) had no effect on cumulative food intake compared to the control group (p>0.05).ICV administration of the U-50488H (30 nmol) had hyperphagic effect compared to control group (p<0.05).Co-administration of the Chlorpheniramine + U-50488H had no effect on κ-opioid receptor agonist-induced hyperphagia in neonatal broilers In experiment 11, ICV administration of the 82 nmol of the famotidine had no effect on feeding behavior compared to the control group (p>0.05).ICV administration of the 30 nmol of the U-50488H increased cumulative food intake compared to the control group (p<0.05).Co-administration of the famotidine + U-50488H had no effect on κ-opioid receptor agonistinduced hyperphagia in neonatal broilers dISCuSSIon The present study was designed for the first time to investigate the possible interconnection of the opioidergic system with histamine on food intake in neonatal broiler chicks.To the best of our knowledge, this is the first report on the interaction of the central HAergic and opioidergic systems on appetite regulation in FD 3 neonatal broiler chicks.In this study we used sub effective doses of the HAergic antagonists and effective doses of the opioid receptors agonists to determine possible interconnection between HAergic and opioidergic systems on food intake in FD 3 broiler chicks.The results obtained imply that the ICV injection of DAMGO decreased food intake while DPDPE and U-50488H increased feeding behavior in FD 3 neonatal broilers.µ-opioid receptors act as orexigenic neurotransmitter on feeding behavior via nucleus accumbens (NAc) and nucleus tractus solitaries (NTS) in rodents (Zheng et al., 2007).The central food intake regulation is also different among avian species.ICV injection of DAMGO inhibits food intake in neonatal broiler (Bungo et al., 2004;2005) while Khan et al., (2009) reported µ-opioid receptors have an orexigenic role in broilers.Perhaps genetic selection for growth in broilers and layers for egg production altered their central appetite regulation mechanisms (Denbow, 1994).
H 1 receptors are known as hypophagic receptors in rats (Morimoto et al., 2001) and broiler chickens (Taati et al., 2010).Anorexic effects reported for H 2 receptors in broilers (Meade and Denbow, 2001) and thioperamide decreases cumulative food intake in broilers (Taati et al., 2010).In poultry, histamine mediates its effect via H 1 receptors (Zendehdel et al., 2015) but controversial reports exists for H 3 receptors.Taati et al., (2009) reported that ICV injection of the thioperamide(300 and 600 nmol) decreased food intake in food-deprived broilers (Taati et al., 2009).Scarce informtion exists about expression of the H 4 receptors in poultry brain (Zendehdel et al., 2015).The ICV injection of thioperamide had no effect on feeding behavior in fasted or non-deprived rats in the lighting period (Passani et al., 2011) while decreased appetite in the dark period when central histamine is at low levels.Perhaps, it affects when histaminergic system activity is low (Passani et al., 2011).Blockade of the H 3 receptors decreases food intake in rats (Chiba et al., 2009) and injection of the H 1 receptor antagonists attenuated effects of the H 3 antagonists in rats (Hancock & Brune, 2005).
As observed, α-FMH and chlorpheniramine inhibited hypophagic effect of the DAMGO in neonatal broilers.Additionally, co-administration of the histamine H 3 receptors antagonist (Thioperamide) with DAMGO significantly amplified hypophagic effect of the DAMGO in neonatal chicks.It is reported that Thioperamide induced anti-nociception mediates via the endogenous opioid system (Khalilzadeh et al., 2010).ICV injection of thioperamide increased the nociceptive threshold at supraspinal level in a rat (Mobarakeh et al., 2009).Also, Hough et al., (1997) reported that the ICV injection of the thioperamide had no analgesic activities in nociception tests in rats while analgesic and hyperalgesic effects reported by ICV administration of the thioperamide and R-αmethylhistamine in rats (Malmberg-Aiello et al., 1994).The H 3 receptors, exerted inhibitory effects on the morphine-induced anti-nociception at the spinal level (Mobarakeh et al., 2009).The anti-nociceptive effect of the histamine was reversed by the ICV injection of the naloxone into periaqueductal gray (Khalilzadeh et al., 2010).A close relationship reported between H 1 receptor and μ-opioid receptor in scratching behavior in mice, where co-injection of the histamine and morphine caused scratching and simultaneous

(Figure 2 -
Figure 2 -Effects of intracerebroventricular injection of control solution, chlorpheniramine (histamine H1 receptors antagonist; 300 nmol), DAMGO (µ-opioid receptor agonist, 125 pmol) and combination of chlorpheniramine plus DAMGO on cumulative food intake (gr/100gr BW) in neonatal chicks.Data are expressed as mean ± SEM.Different letters (a and b) indicate significant differences between treatments at each time (p<0.05).

Figure 3 -
Figure 3 -Effects of intracerebroventricular injection of control solution, famotidine (histamine H2 receptors antagonist; 82 nmol), DAMGO (µ-opioid receptor agonist; 125 pmol) and combination of famotidine plus DAMGO on cumulative food intake (gr/100gr BW) in neonatal chicks.Data are expressed as mean ± SEM.Different letters (a and b) indicate significant differences between treatments at each time (p<0.05).

Figure 10 -
Figure 10 -Effects of intracerebroventricular injection of control solution, chlorpheniramine (histamine H1 receptors antagonist; 300 nmol), U-50488H (κ-opioid receptor agonist; 30 nmol) and combination of chlorpheniramine plus U-50488H on cumulative food intake (gr/100gr BW) in neonatal chicks.Data are expressed as mean ± SEM.Different letters (a and b) indicate significant differences between treatments at each time (p<0.05).

Table - Ingredient
and nutrient analysis of experimental diet