Regulation of appetite is among the complex aspects of animals’ life and is modulated through both the central nervous system (CNS) and peripheral nervous system (PNS). In the brain, appetite is regulated by diverse neurotransmitters in hypothalamic areas, including the arcuate nucleus, nucleus tractus solitarius, and amygdala ( 1 ). Noradrenalin is a catecholamine neurotransmitter in the CNS. The norepinephrine (NE) has two major receptors, including α adrenergic (including α1 and α2) and β adrenergic (including β1, β2, and β3). Based on the evidence, ICV injection of the norepinephrine or clonidine (as an α2-receptor agonist) increases food intake, which yohimbine (as an α2 receptor antagonist) inhibited food intake ( 2 ). ICV injection of clonidine raised the broilers’ food intake as well ( 3 ). However, ICV injection of salbutamol (β2 adrenergic receptor agonist) reduced the rats’ cumulative food intake ( 4 ), and ICV injection of isoproterenol (β1 and β2 adrenergic receptor agonist) reduced chicken’s food and water intake, respectively ( 5 ).
Moreover, the feeding behavior is not mediated by a single neuropeptide. Various neurotransmitters interact through a widely distributed neural network for the regulation of food intake in both animals and humans ( 6 ). Histaminergic (HAergic) neurons are among the most impressive neurons in the brain and seem to play a vital role in controlling food intake. Central HAergic neurons were found in the tuberomammillary nucleus with axon projects branched to various brain areas ( 7 ). Brain histamine is of high importance in determining feeding behavior. Consequently, histamine administration through the ICV route reduced food intake, while food intake was elevated under the influence of chlorpheniramine, as an antagonist of H1 receptor, and α-FMH, as a selective inhibitor of the histamine-synthesizing enzyme histidine decarboxylase ( 8 ).
Previously, interaction between central HAergic and adrenergic neurons on physiological function has been reported. The majority of the H1 receptor antagonist antipsychotics and antidepressants considerably changed the sleep-wake cycle through adrenergic receptors. Moreover, central H2 and α2 adrenergic receptors are involved in crocetin-induced antinociception ( 9 ). Investigations have shown that the antinociceptive effect of intra-peritoneal administration of xylazine (α2 adrenergic receptor agonist) is antagonized by yohimbine but not by naloxone (an opioid receptor antagonist). Microinjection of ranitidine (H2 receptor blocker) prevented histamine-induced anti-nociception in orofacial formalin pain ( 10 ). The adrenal medulla H1 receptor elicits the release of adrenaline and noradrenaline. Histamine can stimulate phosphorylation of the tyrosine hydroxylase enzyme by intracellular calcium release from chromaffin cells of the adrenal gland ( 11 ).
The noradrenergic (NAergic) and HAergic systems play a vital role in food intake control in birds and mammals. In a similar report, Mirnaghizadeh, Zendehdel ( 12 ) reported that oxytocin-induced hypophagia is possibly mediated in broiler chickens through H1 and H3 histaminergic and β2 NAergic receptors. ICV co-injection of histamine and NA results in systemic and intranuclear elevation of oxytocin release in rats ( 13 ). Existing literature reported no interaction between these systems in the broilers’ feeding behaviors. Therefore, the present study aimed to investigate the potential interaction of central histaminergic and adrenergic systems in food intake regulation in broiler chickens.
2. Materials and Methods
The current study was performed on 396 one-day-old broiler chickens (ROSS 308) supplied by a local hatchery (Morghak Co., Iran). They were kept in stabilizing electrically-heated batteries at the temperature of 32±1ºC, relative humidity of 40%-50%, and lighting/dark period of 23:1 ( 14 ). The subjects were taken care of at the mentioned conditions for two days as flocks, and then they were randomly allocated and transferred to the individual cages. Moreover, the broiler chickens were provided with a commercial diet containing 2,850 kcal/kg metabolizable energy and 21% crude protein during the study (Chineh Co., Iran) (Table 1). The birds had free access to fresh water and food. The subjects were food-deprived for 3 h (FD3) before injections; however, they were allowed to drink water. The five-day-old birds underwent ICV injections.
|Soybean meal, 48% CP||31.57||Crude protein (%)||21|
|Wheat||5||Linoleic acid (%)||1.69|
|Gluten meal, 61% CP||2.50||Crude fiber (%)||3.55|
|Wheat bran||2.47||Calcium (%)||1|
|Di-calcium phosphate||1.92||Available phosphorus (%)||0. 5|
|Oyster shell||1.23||Sodium (%)||0.15|
|Soybean oil||1.00||Potassium (%)||0.96|
|Mineral premix||0.25||Chlorine (%)||0.17|
|Vitamin premix||0.25||Choline (%)||1.30|
|Sodium bicarbonate||0.21||Arginine (%)||1.14|
|Sodium chloride||0.20||Isoleucine (%)||0.73|
|Toxin binder||0.10||Methionine+cystine (%)||0.83|
|L-Lysine HCl||0.05||Threonine (%)||0.70|
|Vitamin D3||0.1||Tryptophan (%)||0.20|
|Multi enzyme||0.05||Valine (%)||0.78|
2.2. Experimental Medications
The administered medications in the present study included histamine prazosin (an α1 receptor antagonist), metoprolol (a β1 adrenergic receptor antagonist), yohimbine (an α2 receptor antagonist), SR 59230R (a β3 adrenergic receptor antagonist), ICI 118,551 (a β2 adrenergic receptor antagonist), chlorpheniramine (an H2 receptor antagonist), noradrenaline, thioperamide (an H3 receptor antagonist), famotidine (an H2 receptor antagonist), α-FMH (an alpha fluoromethyl histidine), and Evans blue. All the medications were supplied from Sigma-Aldrich (USA) and Tocris Co. (UK), which were then dissolved in an absolute solution of dimethyl sulfoxide (DMSO). Afterward, the medicines were diluted using 0.85% saline, which contained Evans Blue at a 1:250 ratio (0.4% DMSO). No cytotoxic effect was found for DMSO at this ratio. The DMSO/saline mixture containing Evans blue was utilized for the control group.
2.3. ICV Injections
The subjects were randomly assigned to nine experimental groups, including four sub-groups (n=44). Initially, the birds were weighed and accordingly allocated to the test groups so that the mean body weight of different treatment groups was similar. The ICV injections were performed once for each group, without anesthesia, using a microsyringe (Hamilton, Switzerland) following the techniques adopted by Davis, Masuoka ( 15 ). In summary, the head of the chicken was held using an acrylic device and a bill holder at an angle of 45º. Calvarium was parallel to table surface, according to van Tienhoven and Juhász ( 16 ). Subsequently, an orifice was made in a plate over the skull surrounding the right lateral ventricle, which was then used to insert the microsyringe. The needle tip perforated 4 mm under the skull skin and the 10 μL of the solutions were injected in all groups ( 17 ). Moreover, animals in the control group were injected with the control solution (10 μL). It should be noted that the mentioned method did not cause physiological stress for the newly hatched chickens ( 17 ). Decapitation was carried out using ketamine overdose to ascertain injection accuracy at the end of the experiments. Injection site accuracy in the ventricle was confirmed by the presence of Evans blue and slicing the frozen brain tissues. All birds in the intervention groups received injections. However, only the data from 11 birds in each group were analyzed in which dye was present in the lateral ventricle. All testing procedures were carried out from 8 am to 3 pm.
2.4. Feeding Experiments
In the first experiment, the control solution, including 10 nmol of prazosin (an α1-receptor antagonist), 300 nmol of histamine, as well as prazosin and histamine were ICV injected into the FD3 birds. Experiments two to five were conducted similar to the first experiment, in which FD3 birds were ICV injected with 13 nmol of yohimbine (an α2-receptor antagonist), 24 nmol of metoprolol (a β1 adrenergic receptor antagonist), 5 nmol of ICI 118,551 (a β2 adrenergic receptor antagonist), and 20 nmol of SR 59230R (a β3 adrenergic receptor antagonist). In the sixth experiment, control solution, including 300 nmol of noradrenaline, 250 nmol of α-FMH, and noradrenaline and α-FMH were injected into the chickens. Seventh to ninth experiments were similar to the sixth experiment, except that the FD3 birds were ICV injected with 300 nmol of chlorpheniramine (a histamine H1 receptors antagonist), 82 nmol of famotidine (a histamine H2 receptors antagonist), and 300 nmol of thioperamide (a histamine H3 receptors antagonist) rather than α-FMH (Table 2). Following the completion of injections, the birds were fed, and cumulative food intake was quantified 30 min, 60 min, and 120 min following the injection. The food consumption was recorded as percent of body weight (g/100g BW) to overcome the body weight’s effect on food intake. (g/100g BW) to overcome the body weight’s effect on food intake.
|Exp. 1||ICV Injection|
|B||Prazosin (10 nmol)|
|C||Histamine (300 nmol)|
|D||Prazosin + Histamine|
|Exp. 2||ICV Injection|
|B||Yohimbine (13 nmol)|
|C||Histamine (300 nmol)|
|D||Yohimbine + histamine|
|Exp. 3||ICV Injection|
|B||Metoprolol (24 nmol)|
|C||Histamine (300 nmol)|
|D||Metoprolol + histamine|
|Exp. 4||ICV Injection|
|B||ICI 118,551 (5 nmol)|
|C||Histamine (300 nmol)|
|D||ICI 118,551 + histamine|
|Exp. 5||ICV Injection|
|B||SR 59230R (20 nmol)|
|C||Histamine (300 nmol)|
|D||SR 59230R + histamine|
|Exp. 6||ICV Injection|
|B||α-FMH (250 nmol)|
|C||NA (300 nmol)|
|D||α-FMH + NA|
|Exp. 7||ICV Injection|
|B||Chlorpheniramine (300 nmol)|
|C||NA (300 nmol)|
|D||Chlorpheniramine + NA|
|Exp. 8||ICV Injection|
|B||Famotidine (82 nmol)|
|C||NA (300 nmol)|
|D||Famotidine + NA|
|Exp. 9||ICV Injection|
|B||Thioperamide (300 nmol)|
|C||NA (300 nmol)|
|D||Thioperamide + NA|
2.5. Statistical Analysis
The current study included nine experimental groups. Each test group included four subgroups (I-IV). Only one injection was performed in each group. Cumulative food intake was presented as g/100g BW for the analysis of each intervention group using two-way repeated-measures analysis of variance (ANOVA). Data were analyzed using SPSS software (Version 16) (IBM, Chicago, Il., USA). The Tukey test (P<0.05) was used to compare means, and the descriptive statistics were reported as mean±SEM.
In the first experiment, hypophagia was observed after ICV injection of histamine (300 nmol) (P<0.05). However, prazosin (10 nmol) injection did not affect the cumulative food intake (P>0.05). Moreover, the co-injection of prazosin and histamine had no impact on hypophagia due to histamine in chickens (P>0.05) (Figure 1).
In the second experiment, hypophagia was observed after ICV injection of histamine (300 nmol) (P<0.05). However, yohimbine (13 nmol) did not affect the cumulative food intake (P>0.05) in chickens. Co-injection of yohimbine and histamine had no impact on hypophagia due to histamine in chickens (P>0.05) (Figure 2).
In the third experiment, ICV injection of histamine (300 nmol) reduced food intake, compared to the control group (P<0.05). Metoprolol (24 nmol) did not affect the cumulative food intake (P>0.05), and co-injection of metoprolol and histamine had no impact on hypophagia due to histamine in chickens (P>0.05) (Figure 3).
In the fourth experiment, ICV injection of histamine (300 nmol) reduced food intake in comparison with the control group (P<0.05). However, ICI 118,551 (5 nmol) did not significantly affect the cumulative food intake (P>0.05). Co-injection of the ICI 118,551 and histamine significantly reduced histamine-induced hypophagia in comparison with the control group (P<0.05) (Figure 4).
In the fifth experiment, hypophagia was observed following ICV injection of histamine (300 nmol) (P<0.05). Cumulative food intake did not change after the injection of SR 59230R (20 nmol) (P>0.05). Co-injection of SR 59230R and histamine exerted no impact on hypophagia due to histamine in chickens (P>0.05) (Figure 5).
In the sixth experiment, ICV injection of NA (300 nmol) reduced food intake in comparison with the control group (P<0.05). However, α-FMH (250 nmol) did not significantly affect the cumulative food intake (P>0.05). Co-injecting of the α-FMH and NA reduced NA-induced hypophagia, compared to the control group (P<0.05) (Figure 6).
In the seventh experiment, hypophagia was observed following ICV injection of NA (300 nmol) (P<0.05). However, Chlorpheniramine (300 nmol) did not significantly affect the cumulative food intake (P>0.05). Co-injection of the chlorpheniramine and NA reduced NA-induced hypophagia, compared to the control group (P<0.05) (Figure 7).
In the eighth experiment, ICV injection of NA (300 nmol) reduced food intake in comparison with the control group (P<0.05). Famotidine (82 nmol) did not significantly affect the cumulative food intake (P>0.05). Co-injection of NA and famotidine did not significantly affect hypophagia due to the NA in chickens (P>0.05) (Figure 8).
In the ninth experiment, ICV injection of NA (300 nmol) reduced food intake, in comparison with the control group (P<0.05). Thioperamide (300 nmol) did not significantly affect the cumulative food intake (P>0.05). ICV injection of the thioperamide and NA intensified NA-induced hypophagia, in comparison with the control group (P<0.05) (Figure 9).
To the best of the authors’ knowledge, the present study was the first report regarding the interconnection between HAergic and adrenergic systems in the regulation of food intake in broiler chickens. Based on the obtained results, ICV injection of histamine (300 nmol) reduced food intake. H1 receptors are considered hypophagic receptors in broiler chickens and rats ( 18 ). In broilers, the anorexic effects were reported for the H2 receptors, and thioperamide reduced cumulative food intake in the broilers ( 18 ). Although the H1 receptors are used to mediate the effects of histamine in poultry ( 19 ), controversial debates exist regarding the role of H3 receptors. It was reported that ICV injection of thioperamide (300 and 600 nmol) reduced the food intake in the deprived-food broilers ( 18 ). Limited information is available regarding H4 receptors in the poultry brain ( 19 ). ICV injection of thioperamide did not significantly affect feeding behavior in the food-deprived or non-deprived rats in the lighting period ( 20 ); however, it reduced their appetite in the dark period in which the central levels of histamine was low. This indicated the impact of histamine on the low activity of the histaminergic system by H3 presynaptic autoreceptor ( 20 ). The H3 receptors blockade reduced food intake among rats, while H1 receptor antagonist injection attenuated H3 antagonist effects among rats ( 20 ).
Results of this study suggested that ICV injection of NA (300 nmol) reduced food intake. Baghbanzadeh, Hamidiya ( 5 ) reported that ICV injection of β adrenergic receptor antagonists diminished food and water intake in broilers. Moreover, ICV injection of 5 nmol of ICI 118,551 (a β2 adrenergic receptor antagonist) or 20 nmol of SR 59230R (a β3 adrenergic receptor antagonist) improved broilers’ cumulative food intake ( 3 ).
Based on the findings of the present study, co-injection of histamine and a β2 adrenergic receptor antagonist and the co-injection of the NA and histamine H1 receptor antagonist decreased food intake. Moreover, co-injection of NA and histamine H3 receptor antagonists intensified the hypophagic effects of NA in neonatal chickens. These findings demonstrated that structurally the H1 receptor was much similar to β1- and β2-adrenoceptors as well as the dopamine D3 receptor ( 21 ), while it was considerably different from the chemokine receptor CXCR4 and the adenosine A2A receptor ( 21 ). Activation of brain HAergic and NAergic neurons induces the release of neurohypophysial hormones, including oxytocin and arginine vasopressin that are involved in the are involved in the hormone responses by physiological stimuli, including suckling and dehydration ( 22 ). Activation of H1 receptor leads to excitation in the majority of brain sites (including hypothalamus, brainstem, thalamus, striatum, cortex, amygdala) via Gq protein as well as direct blockade of a potassium leak conductance or inositol trisphosphate (IP3), diacylglycerol (DAG), and phospholipase C mediation ( 22 ). The histaminergic neurons in TMN are projected to the rest of brain areas in addition to the rest of hypothalamic sites, such as SON (Supra optic nucleus) and PVN (Paraventricular nucleus). Moreover, noradrenergic neurons originating from the brain stem are spread across the PVN and SON. HAergic, adrenergic, and NAergic fibers contact the oxytocinergic neurons in SON and PVN in the hypothalamus ( 12 ). ICV injection of NE into the PVN improves food intake in the domestic fowls ( 23 ). ICV injection of the clonidine (an α2 receptor agonist) or NE increases food intake, which is inhibited by yohimbine (an α2 receptor antagonist), not by prazosin (an α1 receptor antagonist). ICV injection of clonidine improved broilers’ food intake ( 4 ), while ICV administration of NE did not affect the feeding behavior in layers ( 24 ). ICV injection of non-selective isoproterenol (a β adrenergic receptor agonist) reduced food intake in rats, while the anorexigenic effect was observed by β3 adrenergic receptor agonist.
Several studies have been conducted on the central regulation of food intake in rat models. It is known that the central regulation of food intake is not similar in birds and mammals ( 3 ). Therefore, it is rational to investigate the regulatory mechanisms of food intake in birds. It was not possible to compare the results of the present study with other researches due to limited information on the interconnection of HAergic and ADergic receptors and the food intake processes In conclusion, the results of the present study suggest that the interconnection of the adrenergic and histaminergic systems is mediated through β2 adrenergic, H1, and H3 histaminergic receptors on food intake in broiler chicken.
Study concept and design: M. Z.
Acquisition of data: M. D.
Analysis and interpretation of data: B. V.
Drafting of the manuscript: M. D.
Critical revision of the manuscript for important intellectual content: M. Z.
Statistical analysis: A. A.
Administrative, technical, and material support: M. Z.
All experimental procedures were approved by the Faculty of Veterinary Medicine, Islamic Azad University, Science and Research Branch, Tehran, Iran.
Conflict of Interest
The authors declare that they have no conflict of interest.
This study was supported by Science and Research Branch, Islamic Azad University, Tehran, Iran.
The authors would like to express their sincere regards to the authorities and staff in the central laboratory (Dr. Rastegar Lab) in the Veterinary Medicine Faculty, University of Tehran, Tehran, Iran, for their cooperation. This study was performed as a part of the first author’s Ph.D. thesis.
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