Involvement of Gaba and Cannabinoid Receptors in Central Food Intake Regulation in Neonatal Layer Chicks : Role of CB 1 and GABAA Receptors

Feeding behavior is regulated via a complex network which interacts via diverse signals from central and peripheral tissues. Endocannabinoids modulate release of GABA in a variety of regions of the central nervous system. Endocannabinoids and GABAergic system have an important role in the central regulation of appetite. Thus, the present study examines the possible interaction of central canabinoidergic and GABAergic systems on food intake in 3-h food-deprived (FD3) neonatal layer-type chicks. The results of this study showed that intracerebroventricular (ICV) injection of 2-AG (2-Arachidonoylglycerol, selective CB1 receptors agonist, 2μg) significantly increased food intake and this effect of 2-AG was attenuated by Picrotoxin (GABAA antagonist, 0.5μg) (P<0.001); but 21ng CGP54626 (GABAB antagonist) had no effect (p>0.05). Also, hyperphagic effect of CB65 (CB2 receptors agonist, 1.25μg) was not affected by Picrotoxin or CGP54626 (p>0.05). Moreover, the food intake of chicks was significantly increased by ICV injection of GABAA agonist (Gaboxadol, 0.2 μg) and SR141716A (CB1 receptors antagonist, 6.25μg) significantly decreased Gaboxadol-induced hyperphagia (P<0.001) but CB2 receptors antagonist (AM630, 1.25μg) had no effect. In contrast, co-injection of SR141716A or AM630 with GABAB agonist (baclofen, 0.2μg) had no effect on the hyperphagia induced by baclofen (p>0.05). These data showed there might be an interaction between central cannabinoidergic and GABAergic systems via CB1 and GABAA receptors in control of food intake in neonatal layer chicks.


INTRODUCTION
Feeding behavior is a complex physiologic phenomenon which interacts via various signals from central and peripheral tissues.In the central nervous system (CNS), several neurotransmitters interact by a wide distributed neurological network on food intake regulation (Zendehdel et al., 2014;Honda, 2016).γ-aminobutyric acid (GABA) is one of the most abundant neurotransmitter in mammalian brain.The GABAergic is known as an inhibitory neurotransmitter in the CNS (Bonaventura et al., 2012).GABA exerts its effect via two distinct receptors including the bicuculline-sensitive GABA A receptor and the bicuculline-insensitive GABA B receptors (Stratford and Wirtshafter, 2013).The GABA A and GABA C receptors are part of a macromolecular complex coupled to a Cl -ionophore while GABA B is metabotropic receptor belong to G-protein coupled receptors (GPCRs) (Jonaidi et al., 2012).
The appetite-stimulating effects of marijuana (Cannabis sativa) are known in science for the past decades.Animal tissues express at least two cannabinoidergic (CBergic) receptors: cannabinoid type 1 (CB 1 ) and type 2 (CB 2 ) receptors which belong to the GPCRs (Kangas et al., 2013).CB 1 receptors mainly expressed in the presynaptic terminals of the brain (Sharkey et al., 2014) and CB 2 receptors formerly exist on cells and organs of the immune system in the peripheral nervous system (PNS) but they also expressed in the CNS (Onaivi et al., 2008).The endocannabinoids (ECBs) mediates many physiological functions like pain relief, motor control, learning and memory formation as well as appetite regulation (D'Addario et al., 2014).
GABA and CBs involved control of food intake (Volkow et al., 2011).It appears GABA has orexigenic effect in domestic fowls (Jonaidi et al., 2012).ICV injection of Muscimol (a GABA A agonist) and Baclofen (a GABA B agonist) increased food consumption in rats (Ebenezer, 1990), turkeys (Denbow, 1991), meat-type (broiler) and layer-type chicks (hens) (Jonaidi et al., 2002).Both CB 1 and CB 2 receptors have a role in food intake regulation in the rat (Chen et al., 2006).Scarce information exists about the role of ECBs on feeding behavior in domestic fowl.Reports claim just CB 1 receptors have a regulatory role on appetite in broilers (et al., 2011;Novoseletsky et al., 2011).Recent reports revealed both CB 1 and CB 2 receptors have hyperphagic effect in neonatal layer-type chicken (Alizadeh et al., 2015;Hassanpour et al., 2015).
There is central evidence mechanisms for food intake regulation are different between mammalian and birds (Zendehdel & Hassanpour 2014).Comparative physiological studies suggested there are differences award pathways between the meat-type and layertype chicks (Alizadeh et al., 2015).For example, food intake mediates via both CB 1 and CB 2 receptors in layer-type chicken (Alizadeh et al., 2015) but only CB 2 receptors interact on feeding in meat-type chicken (Emadi et al., 2011;Novoseletsky et al., 2011).For instance, ICV injection of Baclofen had no effect on food intake in broilers (Jonaidi et al., 2002;Tajalli et al., 2006;Zendehdel et al., 2008Zendehdel et al., , 2009)).
During the last several years, GABA and ECBs have been the subject of an increasing number of behavioral studies.Several types of researches are done to find the mediatory effect of neurotransmitters on feeding behavior in mammals but aspects of food intake regulation in avian are still unclear (Zendehdel & Hassanpour, 2014).ECBs modulate the release of GABA in a variety of regions of the CNS, including the hippocampus, basal ganglia, cerebellum, and brainstem.Anatomical investigations have shown high levels of CB 1 receptor immune-reactivity and mRNA associated with GABAergic neurons (Irving et al., 2002).In the brain, CB 1 receptors is predominantly expressed presynaptically and modulating the release of neurotransmitters, such as GABA, dopamine, noradrenaline, glutamate and serotonin (Cota et al., 2003).To the best of our knowledge, no report exists on the interconnection of the CBergic and GABAergic systems on food intake regulation and energy balance in neonatal layer type chicken.So, the aim of this study was to investigate the possible involvement of the CBergic and GABAergic systems on feeding behavior in 3 h food deprived (FD 3 ) neonatal layer type chicken.

Animals
In this study, a total 384 one-day-old layer-type chickens (Hy-Line) purchased from a local hatchery (Morghak Co. Iran).Birds were kept as flocks for two 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).All birds received ad libitum food and fresh water during the study.Just three h prior the ICV injections, chicken, were food deprived (FD 3 ) but had free access to water.The injections were applied to all birds at five 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
In this study, eight experiments designed to investigate interconnection of CBergic and GABAergic systems on cumulative food intake in neonatal layertype birds (each includes four groups within 12

Involvement of Gaba and Cannabinoid Receptors in Central Food Intake Regulation in Neonatal Layer
Chicks: Role of CB 1 and GABA A Receptors replicates in each group).Before each treatment, the chicks were weighed and based on their body weight allocated into groups so the average weight between treatment groups was as uniform as possible.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) in which 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 and Juhasz, 1962).In a plate, a hole was drilled in the skull over the right lateral ventricle and immediately overlaid through this plate.A microsyringe inserted into the right ventricle via the hole and 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 bird received an ICV injection (with vehicle or drug solution) in a volume of 10 μL.Right away after the injection, FD 3 birds returned to their individual cages and offered fresh water and food (pre-weighed).Cumulative food intake (gr) was measured at 30, 60 and 120 min after the injection.Food consumption was calculated as a percentage of body weight to minimize the effect of body weight on the amount of food intake.Each bird was just used once in each experimental group.At the end, 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 data of those individuals where the dye was present in their lateral ventricle (12 chickens per group), was used for the analysis.All experimental procedures were done from 8:00 A.M. until 3:30 P.M.Also, the time course for feeding behavior was selected by previous studies (Jonaidi et al., 2012;Zendehdel & Hassanpour 2014;Alimohammadi et al., 2015;Alizadeh et al., 2015;Hassanpour et al., 2015).

Statistical analysis
Data is presented as mean ± SEM (standard error of the mean).Cumulative food intake (as the percentage 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 significant differences between treatments.

RESULTS
Effects and interactions of central CBergic and GABAergic systems on cumulative food intake in FD 3 neonatal layer-type bird are shown in Figures 1-8. In

DISCUSSION
So far, numerous researches were done to investigate the role of neurotransmitters on appetite regulation in mammals, but aspects of food intake regulation in avian are still unclear (Zendehdel & Hassanpour 2014).To the best of our knowledge, there are no studies describing the interaction between CBergic and GABAergic systems on food intake in FD 3 neonatal layer-type chickens.According to the results, ICV injection of CB 1 and CB 2 agonists amplified food consumption in layertype chicks which was similar to mammals (Chen et al., 2006;Wiley et al., 2012) and recent studies on layertype chicks (Alizadeh et al., 2015;Hassanpour et al., 2015).However, these results were dissimilar compared to broilers which only CB 2 receptors were responsible for feeding (Emadi et al., 2011;Novoseletsky et al., 2011).The ECBs is an important modulator in the central and peripheral regulation of energy and

Involvement of Gaba and Cannabinoid Receptors in Central Food Intake Regulation in Neonatal Layer
Chicks: Role of CB 1 and GABA A Receptors metabolism (Bellocchio et al., 2013).Broilers are genetically selected for rapid muscle and body weight gain while layer has indirectly been selected for slow growth and low body weight.So, presumably, genetic selection altered chicken's brain neurological pathways associated with food intake (Denbow 1994;Richards 2003).It is a hypothetical effect of ECBs on appetite regulation which mediates at two levels.First, ECBs reinforces the motivation to find and eating, possibly via interaction with the mesolimbic pathways involved in reward mechanisms.Second, it activated 'on demand' in the hypothalamus after short-term food deprivation, then transiently regulates the action of the other orexigenic and anorectic neurotransmitters.The dual action hypothesis in mesolimbic and hypothalamic regions revealed by the finding that injection of ECBs into these brain areas provoke food intake in rodents (Di Marzo & Matias, 2005).
Based on the results, ICV injection of GABA A and GABA B receptors agonists increased food intake in neonatal layer-type chicks.These results were in agreement with observation in rats (Di Marzo et al., 2004), broilers (Jonaidi et al., 2002), turkeys (Denbow, 1991) sheep (Seone et al., 1984) and pigs (Baldwin et al., 1990).Regarding the site of action of GABA, with rats and sheep suggest GABA might alter food intake on some intra hypothalamic or extra-hypothalamic structures (Jonaidi et al., 2002).Several possible mechanisms introduced the mechanism of action of GABA.For example, it may inhibit the satiety centers e.g.ventromedial hypothalamus (VMH).GABA may act via dis-inhibition of the brain feeding centers by inhibitory GABAergic projection from the medial accumbens to the lateral hypothalamus (LH).Finally, GABA can exert orexigenic effect via a direct excitatory effect in some regions of the brain including the hypothalamus (Jonaidi et al., 2002).
As observed in this study, hyperphagic effects of 2-AG (CB 1 receptors agonist) attenuated by coinjection of 2-AG + Picrotoxin (GABA A receptors antagonist).CB 1 receptors control the activity of many neurotransmitter systems involved in central regulation of food intake (Pagotto et al., 2006).In the CNS, CB 1 is predominantly expressed presynaptically, modulating the release of neurotransmitters, such as GABA, dopamine, noradrenaline, glutamate and serotonin (Cota et al., 2003).These data showed there might be an interaction between central CBergic and GABAergic systems via CB 1 and GABA A receptors.The broad distribution of CB 1 receptors in the CNS, along with its modulatory role on other neurotransmitter systems, complicates the mechanisms underlying its effect on food intake regulation (Rey et al., 2012).
Retrograde signaling by ECBs released from depolarized postsynaptic neurons inhibits presynaptic GABA release in the lateral hypothalamus and arcuate nucleus (ARC) (Di Marzo et al., 2004).The ARC plays a crucial role in the integration of signals regulating appetite.The ARC is accessible to circulating signals of energy balance via the median eminence, this is not protected by the blood-brain barrier.In the ARC two types of neurons identified which integrates signals of nutritional status and influence energy homeostasis: pro-opiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART) which are anorexigenic neuropeptides while another neuronal circuit stimulates feed intake via neuropeptide Y (NPY) and agoutirelated protein (AgRP) (Zendehdel & Hassanpour 2014).The AgRP, POMC and NPY contain the GABA A R alpha (3) subunit in the ventromedial and ventrolateral part of ARC (Jonaidi et al., 2002).ECBs impress their hyperphagic effect by modulating POMC neurons via CB 1 receptors in the ARC (Hentges et al., 2005).Also, ICV injection of the CB 1 receptors agonist increases NPY levels (D'Addario et al., 2014).A strong indication exists for CB 1 receptors on modulating appetite by enhancement of NPY (Cota et al., 2003).One potential target for cannabinoids in the arcuate nucleus is NPY⁄ AgRP ⁄GABA neurons (Menzies et al., 2010).This is most likely a direct inhibitory effect on spontaneous release mediated via pre-synaptic CB 1 receptors on NPY⁄ AgRP ⁄GABA neurons or other GABAergic terminals in the explanation.It is reported that CB 1 receptor mRNA are not expressed by arcuate NPY⁄ AgRP ⁄GABA neurons and target probably underlies the direct effect (Menzies et al., 2010).CB 1 receptors are expressed in presynaptic terminals of excitatory neurons (Sharkey et al., 2014).Functionally, GABA A receptors seem to be located both pre-and post-synapses (Tellez et al., 2012).GABA A receptors are ionotropic receptors which couple to a Cl -ionophore (Jonaidi et al., 2012).ECBs synthesized in postsynaptic neurons and binds to CB receptors on the presynaptic membrane.These receptors belong to G protein, inhibits Ca 2+ influx which decreases the release of other neurotransmitters (Williams and Kirkham 2002).CBs modulate GABA release in a variety of CNS regions, including the hippocampus, basal ganglia, cerebellum, and brainstem.Activation of CB 1 receptors modulates the release of GABA in the brain.However, minute information exists about the mechanism behind the interaction of CB 1 with GABA A receptors.

Involvement of Gaba and Cannabinoid Receptors in Central Food Intake Regulation in Neonatal Layer Chicks: Role of CB 1 and GABA A Receptors
Anatomical investigations have shown high levels of CB 1 receptor immune reactivity and mRNA associated with GABAergic neurons.CBs modulate GABA release by a presynaptic mechanism.Presumably, effect of CB 1 receptors on GABA A release is mediated via inhibition of presynaptic Ca 2+ channels (Irving et al., 2002).
To the best of our knowledge, CB 1 receptors mRNA expressed by POMC neurons and CBs directly inhibit arcuate POMC neurons.In turn, POMC neurons inhibit GABA release from NPY/AgRP neurons (Menzies et al., 2010).As observed in this study, the hyperphagic effect of CB 1 receptors diminished by GABA A receptors antagonist (experiment 1).For instance, hyperphagic effects of GABA A receptors minimized by CB 1 receptors antagonist (experiment 5).Presumably, ECBs, by inhibition of the hypothalamic POMC, leads to increasing GABA release in the CNS of neonatal layer-type chicken.So, it seems both CBergic and GABAergic systems impress their hyperphagic effects via CB 1 receptors in neonatal layer-type chicken.Also, perhaps CBergic and GABAergic systems interact with other systems such as opioidergic system (Menzies et al., 2010).
Most research on central food intake regulation was done on rat models because few investigations were in avian.These results can use as base information on central appetite regulation in layer-type chicken.Finally, the authors recommend merit investigation needed to find direct cellular and molecular signaling pathways of CBergic and GABAergic mechanism with other neurotransmitters in the physiology of eating behavior in poultry.

CONfLICT Of INTEREST
Authors declare that they have no conflict of interest.

INfORMED CONSENT
This manuscript does not contain any studies with human subjects performed by any of the authors.

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