Abstract: In this study, the effect of intracerebroventricular (icv) injection of H1, H2 and H3 antagonists on feed intake induced by GABAA agonist was evaluated. In Experiment 1, the animals received chloropheniramine, a H1 antagonist and then muscimol, a GABAA agonist. In Experiment 2, chickens received famotidine, a H2 receptor antagonist, prior to injection of muscimol. Finally in Experiment 3, the birds were injected with thioperamide, a H3 receptor antagonist and muscimol. Cumulative food intake was measured 15, 30, 45, 60, 90, 120, 150 and 180 min after injections. The results of this study indicated that effects of muscimol on food intake inhibited by pretreatment with chloropheneramine maleate (p<=0.05), significantly, while the famotidine and thioperamide were ineffective. These results suggest the existence of H1-receptor mediated histamine-GABAA receptor interaction on food intake in broiler cockerels
INTRODUCTION
Histamine regulates neurotransmission in the brain through multiple specific G-protein coupled receptors and N-methyl-D-aspartate (NMDA) ion channels (Gantz et al., 1991; Yamashita et al., 1991; Lovenberg et al., 1999; Haas and Panula, 2003). Neurons, which synthesize histamine, are found exclusively in the tuberomamillary nucleus (TM) of the posterior hypothalamus (Schwartz et al., 1991; Brown et al., 2001; Haas and Panula, 2003).
Histamine is synthesized from L-histidine by histidine decarboxylase (HDC) (Brown et al., 2001). Histamine localized within the brain must be synthesized via this pathway since peripheral histamine dose not cross the blood brain-barrier (Mercer et al., 1994).
There are three central histamine receptors, H1, H2 and H3, that are involved in regulatory processes such as food intake, sleep-wakefulness and thermoregulation (Brown et al., 2001). Histamine acts within the central nervous system to suppress food intake through H1 receptors in rats (Lecklin et al., 1998), mice (Masaki et al., 2001) and chickens (Meade and Denbow, 2001). Feeding was induced by injection of histamine H1, but not H2-antagonists in the ventromedial hypothalamus (VMH) (Lecklin et al., 1998; Morimoto et al., 2001; Mercer et al., 1994; Scharonda and Denbow, 2001).
The γ-aminobutyric acid (GABA) ergic system is the major contributor of the inhibitory tone throughout the mammalian central nervous system (Jonaidi et al., 2002). GABA mediates its effects by activating two types of receptors; the GABAA receptor and the GABAB receptor. GABAA Rs are chloride ion channels that mediate fast synaptic transmission and belong to a superfamily of pentameric ligand-gated ion channels (Unwin, 1993). Whereas GABAB Rs belong to the family of seven-transmembrane receptors (Bowery, 1993).
It has been reported that the intracerebroventricular injection of muscimol, a GABAA agonist, in rat (Pu et al., 1999), sheep (Seoane et al., 1984), pig (Baldwin et al., 1990), Turkey (Denbow, 1990) and chicken (Jonaidi et al., 2002; Tajalli et al., 2006) and baclofen, a GABAB agonist, in rat (Ebenezer, 1990) and pig (Ebenezer and Baldwin, 1990) but not in chicken (Jonaidi et al., 2002) increased food consumption.
GABA has been localized to the tuberomamillary neurons (Ericson et al., 1991). Also the GABA-synthesizing enzyme Glutamic Acid Decarboxylase (GAD) (Sherin et al., 1998) and its mRNA (Esclapez et al., 1994) have been localized in histaminergic neurons, indicating that GABA is actively synthesized by these cells. The aim of the present study was to characterize the interaction between histaminergic and GABAergic systems in stimulation of feeding in chickens.
MATERIALS AND METHODS
Animals: Broiler cockerels (Iran-Germany Co.) were reared in heated batteries with continuous lighting (one 40 watt light bulb per 200 square feet of floor space) until 3 weeks of age. Birds were provided a mash diet (21% protein and 2869 kcal kg-1 of metabolizable energy) and water for ad libitum consumption. At approximately 2 weeks of age, the birds were transferred to individual cages. Each cages was supplied with individual feeders and waterers with continuous lighting. The room temperature and relative humidity were maintained at 22±1°C and 50%, respectively.
Surgical preparation: At 3 weeks of age, broilers were anesthetized with sodium pentobarbital (Sagatal, Rhone Merieux) (25 mg kg-1 body weight, (iv) and a 23-gauge thin-walled stainless steel guide cannula was stereotaxically implanted into the right lateral ventricle, according to the technique described previously by Denbow et al. (1981). The stereotaxic coordinates were AP = 6.7, L = 0.7, H = 3.5-4 mm below the dura matter with the head oriented as described by Van Tienhoven and Juhaz (1962). The cannula was secured with 3 stainless steel screws placed in the calvaria surrounding each guide cannula and acrylic dental cement (Pars acryl) was applied to the screws and guide cannula. An orthodontic #014 wire (American orthodontics), trimmed to the exact length of the guide cannula, was inserted into the guide cannula while the chicks were not being used for experiments. Lincospectine (Razak) was applied to the incision to prevent infection. The birds were allowed a minimum of 5 days recovery prior to injection.
Drugs: Muscimol (a, GABAA agonist) (Sigma), Chloropheneramine maleate (a, histamine H1-receptor antagonist) (Sigma), Famotidine (a, histamine H2-receptor antagonist) (Merck) and thioperamide (a, histamine H3-receptor antagonist) (Sigma) were used in this experiment. All solutions were prepared in pyrogen-free 0.9% NaCl solution that served as the control.
Experiments: To determine the involvement of histamine H1-H2 and H3-receptors in the brain in GABAA-induced hyperphagia, effects of centrally administered chloropheneramine, famotidine and thioperamide on muscimol-induced hyperphagia were determined in chickens. Injections were made with a 29-gauge, thin-walled stainless steel injection cannula which extends 1.0 mm beyond the guide cannula. This injection cannula was connected to a 10 μL Hamilton syringe connected to a 60 cm length of PE-20 tubing. Before the injections, the birds were removed from their individual cages and were restrained by hand, then they were put back into their cages after injections. Birds were handled and mock injected daily during the 5 days recovery period to habituate them to the injection procedure. On the day of experiment, food was removed 1 h before central injection at 09:00 h. Placement of the guide cannula into the ventricle was verified by the presence of cerebrospinal fluid and intracerebroventricular injection of methylene blue and anatomically slicing the frozen brain tissue at the end of the experiments.
In experiment 1, each bird received two injections in each treatment. The first injection consisted of either 0 or 100 μg chloropheneramine in a volume of 5 μL. The second injection consisted of either 0 or 20 nm muscimol in a volume of 5 μL, 15 min after the first injection as described in Table 1 (n = 8 for each treatment). Fresh food was supplied at the time of injection and food intake (g) was recorded at 15, 30, 45, 60, 90, 120, 150 and 180 min after second injection.
Experiment 2 was conducted similarly to the first experiment except that the chicks were injected with 0 or 25 μg/5 μL of famotidine, instead of chloropheneramine.
Experiment 3, was similar to experiment 2 except thioperamide at doses of 0 or 150 μg was used instead of famotidine.
The dosages of drugs were selected based on previous studies (Meade and Denbow, 2001; Tajalli et al., 2006; Itoh et al., 1999) and preliminary experiments. During the injection of higher doses of famotidine, the birds were very active, excited, vocal and sometimes convulsive.
All of experiments was conducted in the Physiological Laboratory of Veterinary Faculty of Tehran University (Iran) from April 2006 to August 2007.
Statistical analysis: Cumulative food intake (g) was analysed using an analysis of variance at each time period. Bonferroni test was used for obtaining all pair wise comparisons among sample means at each time period. Significant differences imply to p<=0.05.
RESULTS AND DISCUSSION
There is evidence to suggest that the central histaminergic system influences gamma-aminobutyric acid (GABA) neurotransmission (Kamei and Okuma, 2001; Korotkova et al., 2002) and that H2-receptor subtype is implicated in this process (Philippu et al., 1999; Prast et al., 1999). The intracerebroventricular injection 100 μg of chloropheneramine maleate and 20 nm of muscimol elicited submaximal food intake from 15-180 min after injection but famotidine (25 μg) and thioperamide (150 μg) had no effect on food intake in broiler cockerels, these doses were selected for evaluation of effect of icv injections of chloropheneramine maleate, famotidine and thioperamide followed by muscimol on food consumption (Table 2-7). It has been reported that the icv injection of muscimol in rat (Pu et al., 1999), sheep (Seoane et al., 1984), pig (Baldwin et al., 1990), Turkey
(Denbow, 1990) and chicken (Jonaidi et al., 2002; Tajalli et al., 2006) and chloropheneramine in rat (Sakata et al., 1988) and chicken (Meade and Denbow, 2001) increased food consumption.
| Table 1: | Procedure of receiving treatments by chickens in experiment 1 |
| S: Saline, CHP: Chloropheneramine maleate, M: Muscimol | |
| Table 2: | Mean (±SEM)
cumulative food intake (g) following intracerebroventricular injections
of Chloropheneramine maleate (CHP) (100 μg) followed by muscimol
(M) (20 nm) in broiler cockerels |
| S: Saline, Control, SEM: Standard Error of mean | |
| Table 3: | Significant difference between different treatments in experiment 1 |
| A: S+S, B: CHP+S, C: S+M, D: CHP+M (+: p<=0.05, -: p>=0.05) | |
| Table 4: | Mean (±SEM) cumulative food intake (g) following intracerebroventricular injections of famotidine (F) (25 μg), followed by muscimol (M) (20 nm) in broiler cockerels |
| S: Saline, Control, SEM: Standard Error of Mean | |
| Table 5: | Significant difference between different treatments in experiment 2 |
| A: S+S, B: F+S, C: S+M, D: F+M (+: p<=0.05, -: p>=0.05) | |
| Table 6: | Mean (±SEM) cumulative food intake (g) following intracerebroventricular injection of thioperamide (THP) (150 μg), followed by muscimol (M)(20 nm) in broiler cockerels |
| S: Saline, Control, SEM: Standard Error of mean | |
| Table 7: | Significant difference between different treatments in experiment 3 |
| A: S+S, B: THP+S, C: S+M, D: THP+M, (+: p<=0.05, -: p>=0.05) | |
In this experiment, effects of muscimol on food intake significantly, inhibited by pretreatment with chloropheneramine maleate (p<=0.05) (Table 2, 3), however, we were estimating that coadministration of chloropheneramine and muscimol intracerebroventricaly amplified the feeding response over that evoked by chloropheneramine or muscimol alone. In this regard, Pu et al. (1999) reported that combination of NPY (0.47 nm) and muscimol (0.44 nm) stimulated a feeding response that was markedly higher than that elicited by these compounds administered alone. Coadministration of either a higher dose of muscimol (0.88 nm) with the same dose of NPY (0.47 nm) or a higher dose of NPY (2.0 nm) with the same dose of muscimol (0.44 nm) did not improve the feeding response evoked by individual treatments. Also, coinjection of higher dose of muscimol (1.76 nm) with 2.0 nm NPY did not further augment the feeding response (Pu et al., 1999). Aside from stimulation of feed intake, injection of chloropheneramine and muscimol at used dosages in this research is likely to simultaneously stimulates and/or inhibit several neuroendocrine and behavioral responses and thereby at these dosages did not show synergistic effect on ingestive behavior and may be allow only a narrow range of dose-dependent coactions for example in lower doses of these drugs.
In experiment 2, famotidine at dose of 25 μg had no effect on feed intake and using of higher doses, birds were very activity, excited, vocal and some times convulsive (Table 4, 5). previous studies showed, administration of either cimetidine (Schentag., 1980) or famotidine (Yoshimoto et al., 1994) in patients has been reported to cause convulsions related to inhibition of GABA neurotransmission. But this interaction between GABAA receptor and H2-receptor antagonist did not alter food intake in chickens, because, H2-receptor do not have any role in ingestive behavior. Additionally, Morimoto et al. (2001), Scharonda and Denbow (2001) and Lecklin et al. (1998) reported, feeding was induced by injection of histamine H1, but not H2-antagonist.
Present data in experiment 3 showed that thioperamide on feeding are consistent with the report of Naruse and Ishii (1995) that no feeding-inhibitory effect was found under fasting and nonfasting conditions during the light period; however, Ookuma et al. (1993) reported that thioperamide decreased the food intake in the nonfasted state in rats. Thioperamide maybe effective only when the activity of histaminergic neurons is low during the dark (Itoh et al., 1999). In this study chickens were under continuous lighting and maybe to this reason thioperamide did not effect on ingestive behavior in chickens. In our research icv administration of thioperamide prior to icv administration of muscimol was ineffective (Table 6, 7).
In summary, these results suggest the existence of H1-receptor mediated histamine-GABAA receptor interaction on food intake in broiler cockerels. Moreover, histamine and GABA systems can function independently or together by mobilizing common or differing intracellular signal transduction pathways in the target sites. It is also possible that each of these neurotransmitters may activate a distinct orexigenic or anorexigenic networks within the hypothalamus to stimulate or inhibit feeding and using of lower dosages for chloropheneramine and muscimol may resulted in augmented responses. Elucidation of the underlying cellular and molecular events involved in the interaction of histamine and GABA in the hypothalamus warrants further investigation.
ACKNOWLEDGMENT
This study was supported by a grant from the Research Council, Veterinary Faculty, University of Tehran, Iran.