- Email: [email protected]
Acute orexigenic effect of agmatine involves interaction between central α2-adrenergic and GABAergic receptors
Biomedicine & Pharmacotherapy 93 (2017) 939–947
Available online at
ScienceDirect www.sciencedirect.com
Original article
Acute orexigenic effect of agmatine involves interaction between central a2-adrenergic and GABAergic receptors Brijesh Gulabrao Taksandea , Omi Sharmaa , Manish Manohar Aglawea , Mayur Bhimrao Kalea , Dinesh Yugraj Gawandea , Milind Janraoji Umekara , Nandkishor Ramdas Kotagalea,b,* a b
Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, M.S., 441 002, India Government College of Pharmacy, Kathora Naka, Amravati 444604, Maharashtra, India
A R T I C L E I N F O
Article history: Received 18 March 2017 Received in revised form 14 June 2017 Accepted 3 July 2017 Keywords: Agmatine a2-adrenoceptors GABA Food intake Satiated rats
A B S T R A C T
Agmatine and GABA have been abundantly expressed in brain nuclei involved in regulation of energy homeostasis and promoting stimulation of food intake in rodents. However, their mutual interaction, if any, in the elicitation of feeding behavior is largely remains unclear. The current study provides experimental evidence for the possible interaction of agmatine, adrenergic and GABAergic systems in stimulation of feeding in satiated rats. Satiated rats fitted with intracerebroventricular (i.c.v.) cannulae and were administered agmatine, alone or jointly with (a) GABAA receptor agonist, muscimol, diazepam or antagonist bicuculline and flumazenil, GABAA positive modulator, allopregnanolone or negative modulator of GABAA receptor, dehydroepiandrosterone (b) In view of the high affinity of agmatine for a2-adrenoceptors and the close association between a2-adrenoceptors and GABAergic system, the effect of their modulators on feeding elicited by agmatine/GABAergic agonists were also examined. I.c.v. administration of agmatine (40– 80 mg/rat) induces the significant orexigenic effect in satiated rats. The orexigenic effect of agmatine was potentiated by muscimol (25 ng/rat, i.c.v.); diazepam (0.5 mg/kg, i.p.); allopregnanolone (0.5 mg/kg, s.c.) and blocked by bicuculline (1 mg/kg, i.p.) and dehydroepiandrosterone (4 mg/kg, s.c.). However, it remained unaffected in presence of flumazenil (25 ng/rat, i.c.v.). The orexigenic effect of agmatine and GABAergic agonists was potentiated by a a2-adrenoceptors agonist, clonidine (10 ng/rat, i.c.v.) and blocked by its antagonist, yohimbine (5 mg/rat, i.c.v.). Yohimbine also blocked the hyperphagic effect elicited by ineffective dose combination of agmatine (5 mg/rat, i.c.v.) with muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.). The results of the present study suggest that agmatine induced a2-adrenoceptors activation might facilitate GABAergic activity to stimulate food intake in satiated rats. © 2017 Published by Elsevier Masson SAS.
1. Introduction Agmatine is an endogenous amine synthesized from an amino acid, L-arginine by arginine decarboxylase. It is stored in synaptic
Abbreviations: aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; DHEA, dehydroepiandrosterone; GABA, gamma-aminobutyric acid; i.c.v., intracerebroventricular; i.p., intraperitoneally; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; NPY, neuropeptide Y; PVN, paraventricular nucleus; s.c., subcutaneous. * Corresponding author at: Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, Maharashtra, 441 002 India. E-mail address: [email protected] (N.R. Kotagale). http://dx.doi.org/10.1016/j.biopha.2017.07.004 0753-3322/© 2017 Published by Elsevier Masson SAS.
vesicles, accumulated by uptake, released by depolarization and consequently proposed as a new neuromodulator in the mammalian brain [1,2]. Agmatine exhibits interesting pharmacological profile in several neuropsychiatric disorders including depression [3,4], anxiety [5,6], epilepsy [7], psychosis [8,9], nociception [10], inflammatory cachexia [11] and is also implicated in the modulation of addictive behavior [12,13]. It activates a2-adrenoceptors [14] and imidazoline receptors [15], antagonizes NMDA receptors [16] and inhibits nitric oxide (NO) synthase [17]. Agmatine and a2-adrenoceptors have important functional interactions, including the inhibitory effect on nicotine-induced behavioral sensitization in mice [12] and potentiating effect on morphine-induced analgesia, conditioned place preference and anticonvulsant effects in rats [18–21]. We have recently
940
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
demonstrated the involvement of a2-adrenoceptors within the hypothalamic paraventricular nucleus (PVN) in orexigenic effect of agmatine in satiated rats [22]. Gamma-aminobutyric acid (GABA), a predominant inhibitory neurotransmitter in the central nervous system, plays a key role in the regulation of feeding behavior [23,24]. The administration of GABA agonists produces a powerful feeding response in satiated rats [25,26]. The physiological interaction between a2-adrenoceptors and GABA receptors within the central nervous system has been widely accepted and the expression of a2-adrenoceptors has been confirmed in the presynaptic nerve terminal of GABAergic neurons projecting to the PVN [27]. Additionally, it has been reported that the input of GABAergic neurons to PVN neurons is inhibited by stimulation of a2-adrenoceptors in the PVN, resulting in an increase in the excitability of PVN neurons [28,29]. Moreover, a2-adrenoceptors activation increases the release of GABA in several brain regions [30–34]. In view of this background, we speculate that agmatine and a2-adrenoceptors interaction might facilitate the GABAergic system in the brain to influence feeding behaviors. Interestingly, GABA receptors and agmatine immunoreactivity have been abundantly expressed in hypothalamic PVN and critically involved in the control of ingestive behavior. However, their mutual interaction if any, in reference to feeding behavior largely remains unclear. Therefore, it appears necessary to determine the precise role of GABAergic system in orexigenic effect of agmatine in satiated rats. Therefore, we examined the effect of agmatine on spontaneous food intake and its modulation by GABAergic agonist and antagonist as well as GABAA receptor modulators. Subsequently, the influence of a2-adrenoceptor modulators on feeding elicited by agmatine/GABAergic agonists was also observed. 2. Materials and methods 2.1. Subjects Adult male Sprague-Dawley rats (240–260 g) were group housed (4 per cage) in acrylic cages (24 17 12 cm) under standard laboratory conditions and, maintained at 12:12 h darklight cycle (lights on at 07:00 h). Food and water were available ad libitum. All the experimental procedures were approved by Institutional Animal Ethics Committee and carried out according to Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India. 2.2. Drugs and administration Agmatine sulfate, muscimol, bicuculline, flumazenil, allopregnanolone, dehydroepiandrosterone, clonidine, and yohimbine were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). Diazepam was obtained from Ranbaxy Laboratories Ltd., New Delhi, India. Agmatine, clonidine, muscimol, yohimbine, flumazenil were administered by intracerebroventricular (5 ml/rat, i.c.v.) route and diazepam and bicuculline which were dissolved in saline and injected by intraperitoneally (i.p.). Allopregnanolone and dehydroepiandrosterone were dissolved in saline and injected by subcutaneous (s.c.) route. For i.c.v. administrations, dilutions were made with artificial cerebrospinal fluid (aCSF) of following composition 0.2 M NaCl, 0.02 M NaH2CO3, 2 mM KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mM Na2SO4, and 5.8 mM D-glucose. 2.3. Intracerebroventricular (i.c.v.) cannula implantation The procedure of the cannulation has been standardized in our laboratory [8]. Briefly, rats were anesthetized with combination of
ketamine (80 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.) and cannulated with 22-gauge stainless steel guide cannulae (C313G/Spc, plastic UK) into the right lateral ventricle (surgical coordinates 0.8 mm posterior, +1.2 mm lateral to midline and 3.5 mm ventral to bregma). The guide cannulae were then fixed to the skull with dental cement (DPI-RR cold cure, acrylic powder, Dental Product of India, Mumbai, India) and stainless steel screws. A 28-gauge stainless steel dummy cannula was used to occlude the guide cannula when not in use. Animals were housed individually and allowed to recover for 7 days. During this period, rats were injected with cefotaxime sodium (50 mg/kg, s.c.; Cefantral1, Lupin Pharmaceuticals, Ankhaleshwar, Gujarat, India) to prevent infection. Moreover, buprenorphine (0.05 mg/kg, s.c.; Tidigesic1, Sun Pharmaceuticals, Vadodara, Gujarat, India) was administered to alleviate post surgery pain. Antibiotic ointment (Neosporin-H1, GlaxoSmithKline Pharmaceuticals, Nashik, Maharashtra, India) was applied twice daily to prevent any local infection. The bedding in the cages was changed every alternate day. Those rats losing more than 10% of their body weights during the recovery period were excluded from the study [19]. Drugs were injected (5 ml/rat) into the right ventricle with a microliter syringe (Hamilton, Reno, NV, USA) connected to PE-10 polyethylene tubing with a 28-gauge internal cannula (C313I/Spc, plastic one, internal diameter0.18 mm, outer diameter- 0.20 mm). The internal cannula was kept in a position for another 60 s after each injection to facilitate proper diffusion and to prevent backflow. 2.4. Food intake studies After a recovery period from surgery, animals showing stable baseline intake (18–20 g) were selected and assigned to different treatment groups (n = 5–8). Rats usually show a peak feeding activity during the dark phase [35], hence, drug treatments were offered at the onset of the dark phase. Animals were injected with either vehicle or different drugs 10 min prior to the onset of the dark phase and rat was placed individually in a cage with an arrangement of grid floor. Pre-weighted (30 g) food pellets were placed in the hopper. The food intake was monitored (g) manually by weighing the leftover food 2 h after drug administration. Food spillage was collected and it was subtracted from the total food consumed by each animal [26,36]. 2.5. Dose specific effect of agmatine and GABAA receptors ligands on food intake in satiated rats This experiment examined the dose dependent effect of agmatine, GABAA receptors agonist, muscimol; benzodiazepine binding site agonist, diazepam; positive modulator of GABAA receptors, allopregnanolone; GABAA receptors antagonist, bicuculline; benzodiazepine binding site antagonist, flumazenil and negative modulator of GABAA receptors, dehydroepiandrosterone (DHEA) on food intake in satiated rats. Different group of rats (n = 5–17) were administered with different doses of either agmatine (20, 40 and 80 mg/rat, i.c.v.) or muscimol (25, 50 and 100 ng/rat, i.c.v.) or diazepam (0.5, 1 and 1.5 mg/kg, i.p.) or allopregnanolone (0.5, 1 and 2 mg/kg, s.c.) or clonidine (10, 25, 50 ng/rat, icv) or bicuculline (1, 1.5 and 2 mg/kg, i.p.) or flumazenil (25, 50 and 100 ng/rat, i.c.v.) or DHEA (2, 4 and 6 mg/kg, s.c.) or yohimbine (5, 10, 20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Immediately after injections, the rats were returned to their home cages and preweighed pellets of food were placed in cages. Food intake was measured manually at 2 h post injection time point by weighing the leftover food in hoppers. The food spillage from the tray positioned beneath the grid floor was subtracted from the food consumed at 2 h time point.
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
941
The doses of agmatine, muscimol, diazepam, allopregnanolone, bicuculline, clonidine, yohimbine, flumazenil, and DHEA employed in the present study were selected on the basis of our preliminary experiments and available literature [22,25]. This dose-response information was subsequently used to select drug doses for the interaction experiments.
effective doses of agmatine (20 mg/rat, i.c.v.) or muscimol (25 ng/ rat, i.c.v.), diazepam (0.5 mg/kg, i.p.), allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/ kg, s.c.). Immediately after injections, the rats were returned to their home cages and preweighed food pallets were placed in their cages. Food intake was measured as described earlier.
2.6. Influence of GABAA receptors agonist on orexigenic effect of agmatine
2.9. Influence of a2-adrenoceptor antagonist on orexigenic effect of agmatine and GABAergic agonist
This experiment examined the orexigenic effect of agmatine in animals pretreated with GABAA receptors agonist, muscimol; benzodiazepine binding site agonist, diazepam and positive modulator of GABAA receptors, allopregnanolone. Different groups of rats (n = 5–16) were pretreated with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/ kg, s.c.). Fifteen min following these treatments, all animals received sub effective dose of either agmatine (20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.). Immediately after injections, the rats were returned to their home cages containing pre-weighed food pallets and food intake was measured 2 h post injection time point by weighing the leftover food in hoppers.
Separate group of rats (n = 5–14) were pretreated with a2adrenoceptors antagonist, yohimbine (5 mg/rat, i.c.v.) or aCSF (5 ml/ rat, i.c.v.) 15 min before the administration of effective doses of agmatine (40 mg/rat, icv) or muscimol (50 ng/rat, i.c.v.) or diazepam (1 mg/kg, i.p.) or allopregnanolone (1 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Rats were returned to their home cages containing preweighed food pallets immediately after the injections. Food intake was measured as described earlier. In separate group, yohimbine (5 mg/rat, i.c.v.) or aCSF (2 ml/rat, i. c.v.) was injected 15 min before sub effective dose combination of agmatine (20 mg/rat, icv) with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Immediately after injections, the rats were returned to their home cages and food intake was measured as described earlier.
2.7. Influence of GABAA receptors antagonists on orexigenic effect of agmatine Rats (n = 5–13) were injected with GABAA receptor antagonist, bicuculline (1 mg/kg, i.p.), benzodiazepine binding site antagonist, flumazenil (25 ng/rat, i.c.v.) and negative modulator of GABAA receptors, DHEA (4 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) 15 min prior to administration of effective dose of agmatine (40 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c. v.). Immediately after injections, the rats were returned to their home cages and pre-weighed food pallets were placed in their cages. Food intake was measured 2 h post injection time point by weighing the leftover food in hoppers. 2.8. Influence of a2-adrenoceptor agonist on orexigenic effect of agmatine and GABAergic agonists Separate group of rats (n = 5–14) were pretreated with a2adrenergic receptors agonist, clonidine (10 ng/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) 15 min before the administration of per se non
2.10. Cannula placement verification At the end of all experiments employing i.c.v. administration, dilute India ink was injected by icv route and the animals were euthanized in the anesthetic chamber by an overdose of thiopentone sodium (65 mg/kg, i.p.). Immediately the brain of rat was dissected out and cut in coronal plane to verify the placement of the guide cannula and distribution of ink in the ventricles. The data of only those animals that showed a uniform distribution of ink in the ventricles were considered for statistical analysis. 2.11. Data analysis The data are presented as mean SEM. Statistical significance was determined using one or two-way analysis of variance (ANOVA) followed by the post hoc analysis of means by Dunnett
Fig. 1. Effect of agmatine on food intake. Different group of rats (n = 5–7) were administered with different doses of either agmatine (20–80 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c. v.) and food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.05, **P < 0.01 vs aCSF treated animals [One way ANOVA post hoc Dunnett mean comparisons].
942
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
or Bonferroni multiple-comparison test. The criterion for statistical significance was considered at P < 0.05. 3. Results 3.1. Effects of agmatine and GABAergic agents on food intake As shown in Fig. 1, administration of agmatine (40 and 80 mg/ rat, i.c.v.) resulted into dose dependent increase in food consumption in first 2 h after treatment [F(3, 22) = 6.17, P < 0.01]. Post hoc Dunnet mean comparisons demonstrated significant orexogenic effect following agmatine treatment [40 (P < 0.05) and 80 mg/rat (P < 0.01)]. Similarly, administrations of muscimol [50 (P < 0.05) and 100 (P < 0.01) (ng/rat, i.c.v.)] [F(3, 26) = 6.45, P < 0.01]; diazepam [1 (P < 0.05) and 1.5 (P < 0.05) (mg/kg, i.p.)] [F(3, 27) = 5.07, P < 0.01] and allopregnanolone [1 (P < 0.05) and 2 (P < 0.01) (mg/kg, s.c.)] [F(3, 29) = 5.04, P < 0.01] produced significant increase in food intake in satiated rats (Fig. 2). At higher doses muscimol (100 ng/rat, i.c.v.), diazepam (1.5 mg/kg, i.p.) and allopregnanolone (2 mg/kg, s.c.) increased the food intake by 101%, 64% and 78% respectively as compared to aCSF treated control animals. Their respective lower doses however, did not significantly affect the feeding behavior in animals. On the contrary, injection of bicuculline [1.5 (P < 0.05) and 2 (P < 0.01) (mg/kg, i.p.)] [F(3, 28) = 6.08, P < 0.01] and DHEA [6 mg/ kg, s.c. (P < 0.05)] [F(3, 29) = 3.18, P < 0.05], exhibited significant anorexia in satiated rats. Their respective lower doses however, did not significantly affect the feeding behavior in animals. Similarly, benzodiazepine receptor agonist, flumazenil did not alter the food intake in animals at any of the doses used in this study. 3.2. GABAergic agents potentiated the orexigenic effect of agmatine Fig. 3 depicts the effects of combination of agmatine and GABAergic agents, muscimol or diazepam or allopregnanolone on
food intake in satiated rats. At the given doses, combination of the agmatine (20 mg/rat, i.c.v.) with muscimol (25 ng/rat, i.c.v.) [FAgmatineTreatment (1, 31) = 12.79, P < 0.01; FMuscimolTreatment (1, 31) = 15.81, P < 0.001; FAgmatine MuscimolTreatment (1, 31) = 4.22, P < 0.05] or diazepam (0.5 mg/kg, i.p.) [FAgmatineTreatment (1, 31) = 13.58, P < 0.001; FDiazepamTreatment (1, 31) = 11.20, P < 0.01; FAgmatine DiazepamTreatment (1, 31) = 4.75, P < 0.05] or allopregnanolone (0.5 mg/kg, s.c.) [FAgmatineTreatment (1, 29) = 12.96, P < 0.001; FAllopregnanoloneTreatment (1, 29) = 26.32, P < 0.001; FAgmatine AllopregnanoloneTreatment (1, 29) = 4.39, P < 0.05] resulted in a significant elevation in food consumption of satiated animals as compared to their respective individual effect. The doses of agmatine, muscimol or diazepam or allopregnanolone used in this experiment per se did not influence food intake in satiated rats. 3.3. GABAA receptors antagonist blocked the orexigenic effect of agmatine As depicted in Fig. 4, pretreatment of rats with bicuculline (1 mg/ kg, i.p.) completely attenuated the hyperphagic effect elicited by effective doses of agmatine (40 mg/rat, i.c.v.) [FAgmatineTreatment (1, 27) = 20.13, P < 0.001; FBicucullineTreatment (1, 27) = 7.83, P < 0.01; FAgmatine BicucullineTreatment (1, 27) = 5.38, P < 0.05]. Similarly, negative GABAA receptor modulator, DHEA (4 mg/kg, s.c.) also attenuated the orexigenic effect of agmatine (40 mg/rat, i.c.v.) [FAgmatineTreatment (1, 29) = 20.10, P < 0.001; FDHEATreatment (1, 29) = 4.20, P < 0.015; FAgmatine DHEATreatment (1, 29) = 9.49, P < 0.01]. Post hoc Bonferroni mean comparison demonstrated the significant attenuation of orexigenic effect of agmatine in bicuculline (P < 0.01) and DHEA (P < 0.01) pretreated animals. Administration of bicuculline or DHEA to aCSF treated animals at the dose used here did not evoke any response on feeding behavior. On the other hand, the orexigenic effect induced by agmatine remained unaffected by prior treatment of benzodiazepine receptors agonist, flumazenil.
Fig. 2. Effect of GABAergic agents and a2–adrenoceptor ligands on food intake. Different group of rats (n = 5–17) were administered with different doses of either muscimol (25–100 ng/rat, i.c.v.) or diazepam (0.5–1.5 mg/kg, i.p.) or allopregnanolone (0.5–2 mg/kg, s.c.) or clonidine (10–50 ng/rat, icv) or bicuculline (1–2 mg/kg, i.p.) or flumazenil (25–100 ng/rat, i.c.v.) or DHEA (2–6 mg/kg, s.c.) or yohimbine (5–20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) and food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle treated control animals [One way ANOVA post hoc Dunnett mean comparisons].
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
943
Fig. 3. Effect of GABAergic agents and agmatine on food intake. Different groups of rats (n = 5–16) were pretreated with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/ kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) and agmatine (20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) was administered 15 min after the earlier injections. Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. $P < 0.001 vs vehicle treated control animals; *P < 0.01, **P < 0.001 vs agmatine treatment; #P < 0.01, ##P < 0.001 vs respective GABAergic agent treated animals [Two way ANOVA post hoc Bonferroni mean comparisons].
3.4. Influence of a2-adrenoceptor agonist and antagonist on orexigenic effect of agmatine and GABAergic agonist As shown in Fig. 5, pretreatment of animals with subeffective dose of a2-adrenergic receptors agonist, clonidine (10 ng/rat, i.c. v.) significantly potentiated the orexigenic effect of agmatine
(20 mg/rat, i.c.v.) [FClonidineTreatment (1, 27) = 19.97, P < 0.001; FAgmatineTreatment (1, 27) = 10.66, P < 0.01; FClonidine AgmatineTreatment (1, 27) = 9.32, P < 0.01], muscimol (25 ng/rat, i.c.v.) [FClonidineTreatment (1, 28) = 4.24, P < 0.05; FMuscimolTreatment (1, 28) = 13.80, P < 0.01; FClonidine MuscimolTreatment (1, 28) = 11.58, P < 0.01], diazepam (0.5 mg/kg, i.p.) [FClonidineTreatment (1, 29) = 11.60, P < 0.01;
Fig. 4. Effect of GABAergic antagonists in combination with agmatine on food intake. Different groups of rats (n = 5–13) were pretreated with either GABAA receptor antagonist, bicuculline (1 mg/kg, i.p.), benzodiazepine binding site antagonist, flumazenil (25 ng/rat, i.c.v.) and negative modulator of GABAA receptors, DHEA (4 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) 15 min prior to administration of effective dose of agmatine (40 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. $P < 0.001 vs vehicle treated control animals; *P < 0.01 vs agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
944
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
Fig. 5. Effect of a2-adrenoceptor agonist and agmatine or GABAergic agents on food intake. Different groups of rats (n = 5–14) were pretreated with a2-adrenoceptor agonist, clonidine (10 ng/rat, i.c.v.), or aCSF (2 ml/rat, i.c.v.) ng/rat, i.c.v.) 15 min prior to administration of sub-effective dose administration of agmatine (20 mg/rat, i.c.v.) or muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.001 vs vehicle treated control animals; $P < 0.01, $$P < 0.001 vs respective GABAergic agent/agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
FDiazepamTreatment (1, 29) = 24.39, P < 0.001; FClonidine Diazepam (1, 29) = 4.62, P < 0.05] and allopregnanolone (0.5 mg/ kg, s.c.) [FClonidineTreatment (1, 28) = 15.59, P < 0.01; FAllopregnanoloneTreatment (1, 28) = 24.25, P < 0.001; FClonidine AllopregnanoloneTreatment (1, 28) = 6.24, P < 0.05]. The doses of clonidine, agmatine, muscimol or diazepam or allopregnanolone did not influence food intake in satiated rats.
Treatment
In contrast pretreatment of rats with a2-adrenergic receptors antagonist, yohimbine (5 mg/rat, i.c.v.) completely attenuated the hyperphagic effect elicited by effective doses of agmatine (40 g/rat, icv) [FYohimbineTreatment (1, 28) = 7.00, P < 0.05; FAgmatineTreatment (1, 28) = 12.75, P < 0.01; FYohimbine AgmatineTreatment (1, 28) = 4.47, P < 0.05], muscimol (50 ng/rat, i.c.v.) [FYohimbineTreatment (1, 25) = 10.03, P < 0.01; FMuscimolTreatment (1, 25) = 21.11, P < 0.001;
Fig. 6. Effect of a2-adrenoceptor antagonist and agmatine or GABAergic agents on food intake. Separate group of rats (n = 5–14) were pretreated with a2-adrenoceptors antagonist, yohimbine (5 mg/rat, i.c.v.) or aCSF (5 ml/rat, i.c.v.) 15 min before the administration of effective doses of agmatine (40 mg/rat, icv) or muscimol (50 ng/rat, i.c.v.) or diazepam (1 mg/kg, i.p.) or allopregnanolone (1 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. $P < 0.01, $$P < 0.001 vs vehicle treated control animals; *P < 0.05, **P < 0.01, ***P < 0.001vs respective GABAergic agent/agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
FYohimbine MuscimolTreatment (1, 25) = 7.12, P < 0.05], diazepam (1 mg/ kg, i.p.) [FYohimbineTreatment (1, 28) = 6.15, P < 0.05; F DiazepamTreatment (1, 28) = 10.30, P < 0.01; FYohimbine DiazepamTreatment (1, 28) = 4.35, P < 0.05] or alloprenelonone (1 mg/kg, s.c.) [FYohimbineTreatment (1, 26) = 7.45, P < 0.01; F AllopregnanoloneTreatment (1, 26) = 11.60, P < 0.01; FYohimbine AllopregnanoloneTreatment (1, 26) = 4.86, P < 0.05] (Fig. 6). Similarly, yohimbine (5 mg/rat, i.c.v.) also blocked the hyperphagic effect elicited by ineffective dose combination of agmatine (20 mg/rat, i.c.v.) with muscimol (25 ng/rat, i.c.v.) [FYohimbineTreatment (1, 29) = 6.31, P < 0.05; FCombinationTreatment (1, 29) = 20.01, P < 0.01; FYohimbine CombinationTreatment (1, 29) = 4.43, P < 0.05] or diazepam (0.5 mg/kg, i.p.) [FYohimbineTreatment (1, 29) = 5.95, P < 0.05; FCombinationTreatment (1, 29) = 11.89, P < 0.01; FYohimbine CombinationTreatment (1, 29) = 4.36, P < 0.05] or alloprenelonone (0.5 mg/kg, s.c.) [FYohimbineTreatment (1, 27) = 6.29, P < 0.05; FCombinationTreatment (1, 27) = 18.05, P < 0.01; FYohimbine CombinationTreatment (1, 27) = 4.30, P < 0.05] (Fig. 7). Administration of yohimbine to aCSF treated animals at the dose used here did not evoke any response on food consumption. 4. Discussion The presence of agmatine in the PVN and its ability to enhance caloric intake [22] suggests that agmatine may be an additional regulator of feeding behavior. In replication of our earlier findings, the results of the present study showed that i.c.v. administration of agmatine dose-dependently increased the food intake in satiated rats. Animals showed maximum food intake within first 2 h following agmatine administration; hence food intake was monitored at this time point only. Although agmatine and GABA are widely distributed throughout the brain including hypothalamic PVN [37,38] their interaction pertaining to the regulation of energy homeostasis largely remained unexplored. In the present study, the orexigenic effect of agmatine dose-dependently enhanced by GABAA agonist, muscimol and benzodiazepine
945
binding site agonist, diazepam. Although, diazepam [39] or muscimol [40] are well known for their hyperphagic effect at lower doses these agents [diazepam (0.5 mg/kg, i.p.) or muscimol (25 ng/rat, i.c.v.)] showed no effect on feeding behavior per se. However, in the present study, at these doses, they significantly potentiated agmatine induced hyperphagia. In contrast, it was completely abolished in presence of GABAA antagonist, bicuculline. Antagonistic interactions of bicuculline with muscimol or diazepam in anxiety and feeding behavior have already been documented [41,42,25]. However, the orexigenic effect of agmatine remains unaffected in presence of benzodiazepine receptors antagonist, flumazenil. This showed that agmatine and diazepam although produced a synergistic effect on food intake they might act through different intracellular signal transduction mechanism which promotes stimulation of food intake. Interestingly the orexigenic effect of agmatine also potentiated by the positive modulator of GABAA receptor, allopregnanolone and blocked by its negative modulator, DHEA. Allopregnanolone is abundantly expressed in hypothalamic nuclei [43] that regulate feeding and energy balance. Indeed, several preclinical studies have demonstrated the orexigenic potential of allopregnanolone in food deprived [44] and satiated animals [45,46]. Further, its repeated administration also promotes an increase in body weights and its concentration was found to be elevated in the serum of obese humans [47]. GABA type A receptors seem to mediate the effect of allopregnanolone on feeding behavior, as allopregnanolone positively modulate GABAA receptors [48]. In view of these findings, we suggest that agmatine may stimulate feeding by direct or indirect activation of GABAA receptors. However, the direct relationship between endogenous agmatine and the GABAergic system is still unclear. The involvement of a2-adrenergic receptors in agmatine induced feeding has been already demonstrated in our earlier finding [22]. Several preclinical evidences are available for a2adrenoceptor and GABA receptor interaction within hypothalamic
Fig. 7. Effect of a2-adrenoceptor antagonist and agmatine combination with GABAergic agents on food intake. Separate group of rats (n = 5–15) were pretreated with a2adrenoceptors antagonist, yohimbine (5 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) was injected 15 min before subeffective dose combination of agmatine (20 mg/rat, icv) with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.01 vs vehicle treated control animals; $P < 0.05 vs respective GABAergic agent and agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
946
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
PVN in the regulation of physiological behavior [49,50]. Alpha2adrenoceptor agonists promote the release of GABA from the nerve terminals in the hippocampus, nucleus accumbens, striatum and the cortex of animals [30,31,33,34]. It seems likely that a2adrenoceptor activation may be coupled to GABA release since GABAergic neurons express a2-adrenoceptor [33]. The expression of a2-adrenoceptors has been confirmed in the presynaptic nerve terminal of GABAergic neurons projecting to the PVN [27]. Moreover, the input of GABAergic neurons to PVN neurons is inhibited by stimulation of a2-adrenoceptors in the PVN, resulting in an increase in the excitability of PVN neurons [28,29]. In view of this background, we designed an additional protocol to verify whether modulatory effect GABAergic agents on agmatine stimulated food consumption involves a2-adrenoceptors. Interestingly, the orexigenic effect of agmatine, muscimol, diazepam and alloprenelonone significantly potentiated by a a2adrenoceptor agonist, clonidine at its sub effective dose and blocked by a2-antagonist, yohimbine. The data are in agreement with previous investigations, indicating an interaction between the GABAergic system and a2-adrenoceptors in the laboratory animals [30–32,51]. In fact, a2-adrenoceptor activation modulates the release of other neurotransmitters in several areas of the brain [33,52]. Extensive evidence indicates that a2-adrenoceptors activation increases the basal release of GABA in rat hippocampus, cerebral cortex, and striatum [30–34]. Thus agmatine induced a2adrenoceptors activation might increase endogenous GABA release to enhance food intake. Consequently, the direct activation of GABAA receptors in agmatine induced stimulation of food intake cannot be completely ruled out at present. The a2-adrenoceptors activation by agmatine increases neuropeptide Y (NPY) immunoreactivity in hypothalamic arcuate nucleus and PVN to induce orexigenic effect [22]. Interestingly, a subpopulation of NPYproducing neurons in the arcuate nucleus coproduces GABA [53]. Further, NPY is colocalized with GABA in a subpopulation of axon terminals in the PVN and produce an additive orexigenic effect when combined with GABAA receptor agonist, muscimol [54]. Thus, the possibility exists that agmatine might influence both NPYergic and GABAergic system in hypothalamic PVN to induce stimulation of food intake. More region-specific studies are warranted to clarify this issue. It is important to note that agmatine possesses multireceptorial affinity. Apart from, a2-adrenoceptors, agmatine also interacts with other neurotransmitter systems like imidazoline receptors [15], NMDA receptors [16], and nitric oxide [17] involved in regulation of energy homeostasis. Therefore, their participation in the orexigenic effects of agmatine wants further investigation. This study clearly demonstrated the role of agmatine in normal homeostatic food consumptions. Earlier, we have also demonstrated the role of agmatine altered feeding behavior in several pathological conditions like bacterial infections, stress, anorexia nervosa, obesity and cachexia [11,19,55,56]. In clinical context, this study suggests that agmatinergic pathways can be targeted to develop novel therapeutic agents for the treatment of feedingrelated disorders like anorexia nervosa and other pathological conditions associated with altered food intake. In conclusion, the current study provides the first functional evidence for the involvement of a2-adrenoceptors and GABAA receptors in the agmatine induced stimulation of food intake and suggest that agmatine induced a2-adrenoceptors activation might facilitate GABA activity to stimulate food intake in satiated rats. Conflict of interest The author(s) declare(s) that there is no conflict of interest.
Acknowledgement This project was sponsored under the head of institutional research grants. References [1] W. Raasch, S. Regunathan, G. Li, D.J. Reis, Agmatine the bacterial amine, is widely distributed in mammalian tissues, Life Sci. 56 (1995) 2319–2330. [2] D.J. Reis, S. Regunathan, Is agmatine a novel neurotransmitter in brain? Trends Pharmacol. Sci. 21 (2000) 187–193. [3] A.D. Zomkowski, L. Hammes, J. Lin, J.B. Calixto, A.R.S. Santos, A.L. Rodrigues, Agmatine produces antidepressant-like effects in two models of depression in mice, Neuroreport 13 (2002) 387–391. [4] B.G. Taksande, N.R. Kotagale, S.J. Tripathi, R.R. Ugale, C.T. Chopde, Antidepressant like effect of selective serotonin reuptake inhibitors involve modulation of imidazoline receptors by agmatine, Neuropharmacology 57 (2009) 415–424. [5] D. Lavinsky, N.S. Arteni, C.A. Netto, Agmatine induces anxiolysis in the elevated plus maze task in adult rats, Behav. Brain Res. 141 (2003) 19–24. [6] B.G. Taksande, N.R. Kotagale, D.Y. Gawande, A.P. Bharne, C.T. Chopde, D.M. Kokare, Neuropeptide Y in the central nucleus of amygdala regulates the anxiolytic effect of agmatine in rats, Eur. Neuropsychopharmacol. 24 (2014) 955–963. [7] T. Singh, N. Bagga, A. Kaur, N. Kaur, D.Y. Gawande, R.K. Goel, Agmatine for combined treatment of epilepsy, depression and cognitive impairment in chronic epileptic animals, Biomed. Pharmacother. 92 (2017) 720–725. [8] N.R. Kotagale, B.G. Taksande, P.J. Wadhwani, M.W. Palhade, S.M. Mendhi, D.Y. Gawande, P.N. Hadole, C.T. Chopde, Psychopharmacological study of agmatine in behavioral tests of schizophrenia in rodents, Pharmacol. Biochem. Behav. 100 (3) (2012) 398–403. [9] T. Uzbay, G. Goktalay, H. Kayir, S.S. Eker, A. Sarandol, S. Oral, L. Buyukuysal, G. Ulusoy, S. Kirli, Increased plasma agmatine levels in patients with schizophrenia, J. Psychiatr. Res. 47 (8) (2013) 1054–1060. [10] M.M. Aglawe, B.G. Taksande, S.S. Kuldhariya, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Participation of central imidazoline binding sites in antinociceptive effect of ethanol and nicotine in rats, Fundam. Clin. Pharmacol. 28 (3) (2014) 284–293. [11] B.G. Taksande, D.Y. Gawande, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Agmatine ameliorates adjuvant induced arthritis and inflammatory cachexia in rats, Biomed. Pharmacother. 86 (2016) 271–278. [12] N.R. Kotagale, B.G. Taksande, A.Y. Gahane, R.R. Ugale, C.T. Chopde, Repeated agmatine treatment attenuates nicotine sensitization in mice: modulation by a2-adrenoceptors, Behav. Brain Res. 213 (2010) 161–174. [13] B.G. Taksande, N.R. Kotagale, M.R. Patel, G.P. Shelkar, R.R. Ugale, C.T. Chopde, Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats, Eur. J. Pharmacol. 637 (2010) 89– 101. [14] J.E. Piletz, D.N. Chikkala, P. Ernsberger, Comparison of the properties of agmatine and endogenous clonidine-displacing substance at imidazoline and alpha-2 adrenergic receptors, J. Pharmacol. Exp. Ther. 272 (1995) 581–587. [15] D.J. Reis, G. Li, S. Regunathan, Endogenous ligands of imidazoline receptors: classic and immunoreactive clonidine displacing substance and agmatine, Ann. N. Y. Acad. Sci. 763 (1995) 295–313. [16] X.C. Yang, D.J. Reis, Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons, J. Pharmacol. Exp. Ther. 288 (1999) 544–549. [17] M. Auguet, I. Viossat, J.G. Marin, P.E. Chabrier, Selective inhibition of inducible nitric oxide synthase by agmatine, Jpn. J. Pharmacol. 69 (1995) 285–287. [18] C.A. Fairbanks, K.L. Schreiber, K.L. Brewer, C.G. Yu, L.S. Stone, K.F. Kitto, H.O. Nguyen, B.M. Grocholski, D.W. Shoeman, L.J. Kehl, S. Regunathan, D.J. Reis, R.P. Yezierski, G.L. Wilcox, Agmatine reverses pain induced by inflammation neuropathy, and spinal cord injury, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 10584–10589. [19] O. Yesilyurt, I.T. Uzbay, Agmatine potentiates the analgesic effect of morphine by an alpha(2)-adrenoceptor-mediated mechanism in mice, Neuropsychopharmacology 25 (2001) 98–103. [20] K. Riazi, H. Honar, H. Homayoun, N. Rashidi, S. Kiani, M.R. Ebrahimkhani, A.R. Noorian, K. Ghaffari, A. Jannati, A.R. Dehpour, The synergistic anticonvulsant effect of agmatine and morphine: possible role of alpha 2-adrenoceptors, Epilepsy Res. 65 (1–2) (2005) 33–40. [21] P. Tahsili-Fahadan, N. Yahyavi-Firouz-Abadi, M.A. Khoshnoodi, R. MotieiLangroudi, S.A. Tahaei, M.H. Ghahremani, A.R. Dehpour, Agmatine potentiates morphine-induced conditioned place preference in mice: modulation by alpha (2)-adrenoceptors, Neuropsychopharmacology 31 (2006) 1722–1732. [22] B.G. Taksande, N.R. Kotagale, K.T. Nakhate, P.D. Mali, D.M. Kokare, K. Hirani, N. K. Subhedar, C.T. Chopde, R.R. Ugale, Agmatine in the hypothalamic paraventricular nucleus stimulates feeding in rats: involvement of neuropeptide Y, Br. J. Pharmacol. 164 (2011) 704–718. [23] J. Kelly, G.F. Alheid, A. Newberg, S.P. Grossman, GABA stimulation and blockade in the hypothalamus and midbrain: effects on feeding and locomotor activity, Pharmacol. Biochem. Behav. 7 (1977) 537–541.
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947 [24] V.R. Olgiati, C. Netti, F. Guidobono, A. Pecile, The central GABAergic system and control of food intake under different experimental conditions, Psychopharmacology (Berl) 68 (1980) 163–167. [25] T.L. Rao, D.M. Kokare, S. Sarkar, R.T. Khisti, C.T. Chopde, N. Subhedar, GABAergic agents prevent alpha-melanocyte stimulating hormone induced anxiety and anorexia in rats, Pharmacol. Biochem. Behav. 76 (3–4) (2003) 417–423. [26] D.M. Kokare, A.M. Patole, A. Carta, C.T. Chopde, N.K. Subhedar, GABA(A) receptors mediate orexin-A induced stimulation of food intake, Neuropharmacology 50 (1) (2006) 16–24. [27] D.P. Li, L.M. Atnip, S.R. Chen, H.L. Pan, Regulation of synaptic inputs to paraventricular-spinal output neurons by alpha2 adrenergic receptors, J. Neurophysiol. 93 (2005) 393–402. [28] S.K. Han, W. Chong, L.H. Li, I.S. Lee, K. Murase, P.D. Ryu, Noradrenaline excites and inhibits GABAergic transmission in parvocellular neurons of rat hypothalamic paraventricular nucleus, J. Neurophysiol. 87 (2002) 2287–2296. [29] W. Chong, L.H. Li, K. Lee, M.H. Lee, J.B. Park, P.D. Ryu, Subtypes of alpha1- and alpha2-adrenoceptors mediating noradrenergic modulation of spontaneous inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus, J. Neuroendocrinol. 16 (2004) 450–457. [30] A. Pittaluga, M. Raiteri, GABAergic nerve terminals in rat hippocampus possess alpha-2 adrenoceptors regulating GABA release, Neurosci. Lett. 76 (1987) 363– 367. [31] G. Maura, A. Pittaluga, M. Ulivi, M. Raiteri, Enhancement of endogenous GABA release from rat synaptosomal preparations is mediated by alpha-2 adrenoceptors pharmacologically different from alpha-2 autoreceptors, Eur. J. Pharmacol. 157 (1988) 23–29. [32] L. Ciranna, F. Licata, G. Li Volsi, F. Santangelo, Alpha 2- and beta-adrenoceptors differentially modulate GABAA- and GABAB-mediated inhibition of red nucleus neuronal firing, Exp. Neurol. 185 (2004) 297–304. [33] W. Zhang, G.A. Ordway, The alpha2C-adrenoceptor modulates GABA release in mouse striatum, Brain Res. Mol. Brain Res. 112 (2003) 24–32. [34] A. Alachkar, J. Brotchie, O.T. Jones, Alpha2-adrenoceptor-mediated modulation of the release of GABA and noradrenaline in the rat substantia nigra parsreticulata, Neurosci. Lett. 395 (2006) 138–142. [35] T. Kimura, T. Maji, K. Ashida, Periodicity of food intake and lipogenesis in rats subjected to two different feeding plans, J. Nutr. 100 (1970) 691–697. [36] A. Kask, L. Rägo, J. Harro, Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226, Br. J. Pharmacol. 124 (7) (1998) 1507–1515. [37] K. Otake, D.A. Ruggiero, S. Regunathan, H. Wang, T.A. Milner, D.J. Reis, Regional localization of agmatine in the rat brain: an immunocytochemical study, Brain Res. 787 (1998) 1–14. [38] H. Hörtnagl, R.O. Tasan, A. Wieselthaler, E. Kirchmair, W. Sieghart, G. Sperk, Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain, Neuroscience 236 (2013) 345–372. [39] A.H. Soderpalm, K.C. Berridge, Food intake after diazepam, morphine or muscimol: microinjections in the nucleus accumbens shell, Pharmacol. Biochem. Behav. 66 (2000) 429–434. [40] E. Khaimova, Y. Kandov, Y. Israel, G. Cataldo, M.M. Hadjimarkou, R.J. Bodnar, Opioid receptor subtype antagonists differentially alter GABA agonist-induced feeding elicited from either the nucleus accumbens shell or ventral tegmental area regions in rats, Brain Res. 1026 (2004) 284– 294.
947
[41] A.P. Lopes, L. Ganzer, A.C. Borges, L. Kochenborger, A.C. Januário, M.S. Faria, J. Marino-Neto, M.A. Paschoalini, Effects of GABA ligands injected into the nucleus accumbens shell on fear/anxiety-like and feeding behaviours in fooddeprived rats, Pharmacol. Biochem. Behav. 101 (1) (2012) 41–48. [42] V. Znamensky, J.A. Echo, N. Lamonte, G. Christian, A. Ragnauth, R.J. Bodnar, Gamma-aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats, Brain Res. 906 (2001) 84–91. [43] E. Holmberg, M. Johansson, T. Bäckström, D. Haage, Allopregnanolone preferentially induces energy-rich food intake in male Wistar rats, Physiol. Rep. 2 (12) (2014) e12190. [44] D.S. Reddy, S.K. Kulkarni, Sex and estrous cycle-dependent changes in neurosteroid and benzodiazepine effects on food consumption and plus-maze learning behaviors in rats, Pharmacol. Biochem. Behav. 62 (1999) 53–60. [45] E. Holmberg, T. Bäckström, M. Johansson, M. Löfgren, D. Haage, Allopregnanolone induces a diurnally dependent hyperphagic effect and alters feeding latency and duration in male Wistar rats, Acta Physiol. 208 (4) (2013) 400–409. [46] K.T. Nakhate, N.K. Subhedar, A.P. Bharne, P.S. Singru, D.M. Kokare, Involvement of cocaine- and amphetamine-regulated transcript peptide in the hyperphagic and body weight promoting effects of allopregnanolone in rats, Brain Res. 1532 (2013) 44–55. [47] R. Menozzi, P. Florio, M. Bondi, S. Luisi, L. Cobellis, A.R. Genazzani, G. Del Rio, F. Petraglia, Increased response of plasma allopregnanolone to corticotropinreleasing hormone in obese patients, Neuroendocrinology 75 (2002) 124–129. [48] J.J. Lambert, D. Belelli, D.R. Peden, A.W. Vardy, J.A. Peter, Neurosteroid modulation of GABAA receptors, Prog. Neurobiol. 71 (2003) 67–80. [49] Y. Li, A.N. van denPol, Direct and indirect inhibition by catecholamines of hypocretin/orexin neurons, J. Neurosci. 25 (1) (2005) 173–183. [50] E.R. Samuels, E. Szabadi, Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organization, Curr. Neuropharmacol. 6 (3) (2008) 235– 253. [51] Y. Yamama, K. Nishikawa, T. Funao, T. Mori, A. Asada, Intrathecal gabapentin and clonidine synergistically inhibit allodynia in spinal nerve-ligated rats, Life Sci. 87 (17–18) (2010) 565–571. [52] Y. Wang, J. Liu, Z.H. Gui, U. Ali, L.L. Fan, C. Hou, T. Wang, L. Chen, Q. Li, a2Adrenoceptor regulates the spontaneous and the GABA/glutamate modulated firing activity of the rat medial prefrontal cortex pyramidal neurons, Neuroscience 182 (2011) 193–202. [53] T.L. Horvath, I. Bechmann, F. Naftolin, S.P. Kalra, C. Leranth, Heterogeneity in the neuropeptide Y-containing neurons of the rat arcuate nucleus: GABAergic and non-GABAergic subpopulations, Brain Res. 756 (1997) 283–286. [54] S. Pu, M.R. Jain, T.L. Horvath, S. Diano, P.S. Kalra, S.P. Kalra, Interactions between neuropeptide Y and g-aminobutyric acid in stimulation of feeding: a morphological and pharmacological analysis, Endocrinology 140 (1999) 933– 940. [55] B.G. Taksande, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Agmatine attenuates hyperactivity and weight loss associated with activity-based anorexia in female rats, Pharmacol. Biochem. Behav. 132 (2015) 136–141. [56] B.G. Taksande, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Agmatine attenuates lipopolysaccharide induced anorexia and sickness behavior in rats, Pharmacol. Biochem. Behav. 132 (2015) 108–114.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Acute orexigenic effect of agmatine involves interaction between central a2-adrenergic and GABAergic receptors Brijesh Gulabrao Taksandea , Omi Sharmaa , Manish Manohar Aglawea , Mayur Bhimrao Kalea , Dinesh Yugraj Gawandea , Milind Janraoji Umekara , Nandkishor Ramdas Kotagalea,b,* a b
Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, M.S., 441 002, India Government College of Pharmacy, Kathora Naka, Amravati 444604, Maharashtra, India
A R T I C L E I N F O
Article history: Received 18 March 2017 Received in revised form 14 June 2017 Accepted 3 July 2017 Keywords: Agmatine a2-adrenoceptors GABA Food intake Satiated rats
A B S T R A C T
Agmatine and GABA have been abundantly expressed in brain nuclei involved in regulation of energy homeostasis and promoting stimulation of food intake in rodents. However, their mutual interaction, if any, in the elicitation of feeding behavior is largely remains unclear. The current study provides experimental evidence for the possible interaction of agmatine, adrenergic and GABAergic systems in stimulation of feeding in satiated rats. Satiated rats fitted with intracerebroventricular (i.c.v.) cannulae and were administered agmatine, alone or jointly with (a) GABAA receptor agonist, muscimol, diazepam or antagonist bicuculline and flumazenil, GABAA positive modulator, allopregnanolone or negative modulator of GABAA receptor, dehydroepiandrosterone (b) In view of the high affinity of agmatine for a2-adrenoceptors and the close association between a2-adrenoceptors and GABAergic system, the effect of their modulators on feeding elicited by agmatine/GABAergic agonists were also examined. I.c.v. administration of agmatine (40– 80 mg/rat) induces the significant orexigenic effect in satiated rats. The orexigenic effect of agmatine was potentiated by muscimol (25 ng/rat, i.c.v.); diazepam (0.5 mg/kg, i.p.); allopregnanolone (0.5 mg/kg, s.c.) and blocked by bicuculline (1 mg/kg, i.p.) and dehydroepiandrosterone (4 mg/kg, s.c.). However, it remained unaffected in presence of flumazenil (25 ng/rat, i.c.v.). The orexigenic effect of agmatine and GABAergic agonists was potentiated by a a2-adrenoceptors agonist, clonidine (10 ng/rat, i.c.v.) and blocked by its antagonist, yohimbine (5 mg/rat, i.c.v.). Yohimbine also blocked the hyperphagic effect elicited by ineffective dose combination of agmatine (5 mg/rat, i.c.v.) with muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.). The results of the present study suggest that agmatine induced a2-adrenoceptors activation might facilitate GABAergic activity to stimulate food intake in satiated rats. © 2017 Published by Elsevier Masson SAS.
1. Introduction Agmatine is an endogenous amine synthesized from an amino acid, L-arginine by arginine decarboxylase. It is stored in synaptic
Abbreviations: aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; DHEA, dehydroepiandrosterone; GABA, gamma-aminobutyric acid; i.c.v., intracerebroventricular; i.p., intraperitoneally; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; NPY, neuropeptide Y; PVN, paraventricular nucleus; s.c., subcutaneous. * Corresponding author at: Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, Maharashtra, 441 002 India. E-mail address: [email protected] (N.R. Kotagale). http://dx.doi.org/10.1016/j.biopha.2017.07.004 0753-3322/© 2017 Published by Elsevier Masson SAS.
vesicles, accumulated by uptake, released by depolarization and consequently proposed as a new neuromodulator in the mammalian brain [1,2]. Agmatine exhibits interesting pharmacological profile in several neuropsychiatric disorders including depression [3,4], anxiety [5,6], epilepsy [7], psychosis [8,9], nociception [10], inflammatory cachexia [11] and is also implicated in the modulation of addictive behavior [12,13]. It activates a2-adrenoceptors [14] and imidazoline receptors [15], antagonizes NMDA receptors [16] and inhibits nitric oxide (NO) synthase [17]. Agmatine and a2-adrenoceptors have important functional interactions, including the inhibitory effect on nicotine-induced behavioral sensitization in mice [12] and potentiating effect on morphine-induced analgesia, conditioned place preference and anticonvulsant effects in rats [18–21]. We have recently
940
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
demonstrated the involvement of a2-adrenoceptors within the hypothalamic paraventricular nucleus (PVN) in orexigenic effect of agmatine in satiated rats [22]. Gamma-aminobutyric acid (GABA), a predominant inhibitory neurotransmitter in the central nervous system, plays a key role in the regulation of feeding behavior [23,24]. The administration of GABA agonists produces a powerful feeding response in satiated rats [25,26]. The physiological interaction between a2-adrenoceptors and GABA receptors within the central nervous system has been widely accepted and the expression of a2-adrenoceptors has been confirmed in the presynaptic nerve terminal of GABAergic neurons projecting to the PVN [27]. Additionally, it has been reported that the input of GABAergic neurons to PVN neurons is inhibited by stimulation of a2-adrenoceptors in the PVN, resulting in an increase in the excitability of PVN neurons [28,29]. Moreover, a2-adrenoceptors activation increases the release of GABA in several brain regions [30–34]. In view of this background, we speculate that agmatine and a2-adrenoceptors interaction might facilitate the GABAergic system in the brain to influence feeding behaviors. Interestingly, GABA receptors and agmatine immunoreactivity have been abundantly expressed in hypothalamic PVN and critically involved in the control of ingestive behavior. However, their mutual interaction if any, in reference to feeding behavior largely remains unclear. Therefore, it appears necessary to determine the precise role of GABAergic system in orexigenic effect of agmatine in satiated rats. Therefore, we examined the effect of agmatine on spontaneous food intake and its modulation by GABAergic agonist and antagonist as well as GABAA receptor modulators. Subsequently, the influence of a2-adrenoceptor modulators on feeding elicited by agmatine/GABAergic agonists was also observed. 2. Materials and methods 2.1. Subjects Adult male Sprague-Dawley rats (240–260 g) were group housed (4 per cage) in acrylic cages (24 17 12 cm) under standard laboratory conditions and, maintained at 12:12 h darklight cycle (lights on at 07:00 h). Food and water were available ad libitum. All the experimental procedures were approved by Institutional Animal Ethics Committee and carried out according to Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India. 2.2. Drugs and administration Agmatine sulfate, muscimol, bicuculline, flumazenil, allopregnanolone, dehydroepiandrosterone, clonidine, and yohimbine were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). Diazepam was obtained from Ranbaxy Laboratories Ltd., New Delhi, India. Agmatine, clonidine, muscimol, yohimbine, flumazenil were administered by intracerebroventricular (5 ml/rat, i.c.v.) route and diazepam and bicuculline which were dissolved in saline and injected by intraperitoneally (i.p.). Allopregnanolone and dehydroepiandrosterone were dissolved in saline and injected by subcutaneous (s.c.) route. For i.c.v. administrations, dilutions were made with artificial cerebrospinal fluid (aCSF) of following composition 0.2 M NaCl, 0.02 M NaH2CO3, 2 mM KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mM Na2SO4, and 5.8 mM D-glucose. 2.3. Intracerebroventricular (i.c.v.) cannula implantation The procedure of the cannulation has been standardized in our laboratory [8]. Briefly, rats were anesthetized with combination of
ketamine (80 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.) and cannulated with 22-gauge stainless steel guide cannulae (C313G/Spc, plastic UK) into the right lateral ventricle (surgical coordinates 0.8 mm posterior, +1.2 mm lateral to midline and 3.5 mm ventral to bregma). The guide cannulae were then fixed to the skull with dental cement (DPI-RR cold cure, acrylic powder, Dental Product of India, Mumbai, India) and stainless steel screws. A 28-gauge stainless steel dummy cannula was used to occlude the guide cannula when not in use. Animals were housed individually and allowed to recover for 7 days. During this period, rats were injected with cefotaxime sodium (50 mg/kg, s.c.; Cefantral1, Lupin Pharmaceuticals, Ankhaleshwar, Gujarat, India) to prevent infection. Moreover, buprenorphine (0.05 mg/kg, s.c.; Tidigesic1, Sun Pharmaceuticals, Vadodara, Gujarat, India) was administered to alleviate post surgery pain. Antibiotic ointment (Neosporin-H1, GlaxoSmithKline Pharmaceuticals, Nashik, Maharashtra, India) was applied twice daily to prevent any local infection. The bedding in the cages was changed every alternate day. Those rats losing more than 10% of their body weights during the recovery period were excluded from the study [19]. Drugs were injected (5 ml/rat) into the right ventricle with a microliter syringe (Hamilton, Reno, NV, USA) connected to PE-10 polyethylene tubing with a 28-gauge internal cannula (C313I/Spc, plastic one, internal diameter0.18 mm, outer diameter- 0.20 mm). The internal cannula was kept in a position for another 60 s after each injection to facilitate proper diffusion and to prevent backflow. 2.4. Food intake studies After a recovery period from surgery, animals showing stable baseline intake (18–20 g) were selected and assigned to different treatment groups (n = 5–8). Rats usually show a peak feeding activity during the dark phase [35], hence, drug treatments were offered at the onset of the dark phase. Animals were injected with either vehicle or different drugs 10 min prior to the onset of the dark phase and rat was placed individually in a cage with an arrangement of grid floor. Pre-weighted (30 g) food pellets were placed in the hopper. The food intake was monitored (g) manually by weighing the leftover food 2 h after drug administration. Food spillage was collected and it was subtracted from the total food consumed by each animal [26,36]. 2.5. Dose specific effect of agmatine and GABAA receptors ligands on food intake in satiated rats This experiment examined the dose dependent effect of agmatine, GABAA receptors agonist, muscimol; benzodiazepine binding site agonist, diazepam; positive modulator of GABAA receptors, allopregnanolone; GABAA receptors antagonist, bicuculline; benzodiazepine binding site antagonist, flumazenil and negative modulator of GABAA receptors, dehydroepiandrosterone (DHEA) on food intake in satiated rats. Different group of rats (n = 5–17) were administered with different doses of either agmatine (20, 40 and 80 mg/rat, i.c.v.) or muscimol (25, 50 and 100 ng/rat, i.c.v.) or diazepam (0.5, 1 and 1.5 mg/kg, i.p.) or allopregnanolone (0.5, 1 and 2 mg/kg, s.c.) or clonidine (10, 25, 50 ng/rat, icv) or bicuculline (1, 1.5 and 2 mg/kg, i.p.) or flumazenil (25, 50 and 100 ng/rat, i.c.v.) or DHEA (2, 4 and 6 mg/kg, s.c.) or yohimbine (5, 10, 20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Immediately after injections, the rats were returned to their home cages and preweighed pellets of food were placed in cages. Food intake was measured manually at 2 h post injection time point by weighing the leftover food in hoppers. The food spillage from the tray positioned beneath the grid floor was subtracted from the food consumed at 2 h time point.
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
941
The doses of agmatine, muscimol, diazepam, allopregnanolone, bicuculline, clonidine, yohimbine, flumazenil, and DHEA employed in the present study were selected on the basis of our preliminary experiments and available literature [22,25]. This dose-response information was subsequently used to select drug doses for the interaction experiments.
effective doses of agmatine (20 mg/rat, i.c.v.) or muscimol (25 ng/ rat, i.c.v.), diazepam (0.5 mg/kg, i.p.), allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/ kg, s.c.). Immediately after injections, the rats were returned to their home cages and preweighed food pallets were placed in their cages. Food intake was measured as described earlier.
2.6. Influence of GABAA receptors agonist on orexigenic effect of agmatine
2.9. Influence of a2-adrenoceptor antagonist on orexigenic effect of agmatine and GABAergic agonist
This experiment examined the orexigenic effect of agmatine in animals pretreated with GABAA receptors agonist, muscimol; benzodiazepine binding site agonist, diazepam and positive modulator of GABAA receptors, allopregnanolone. Different groups of rats (n = 5–16) were pretreated with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/ kg, s.c.). Fifteen min following these treatments, all animals received sub effective dose of either agmatine (20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.). Immediately after injections, the rats were returned to their home cages containing pre-weighed food pallets and food intake was measured 2 h post injection time point by weighing the leftover food in hoppers.
Separate group of rats (n = 5–14) were pretreated with a2adrenoceptors antagonist, yohimbine (5 mg/rat, i.c.v.) or aCSF (5 ml/ rat, i.c.v.) 15 min before the administration of effective doses of agmatine (40 mg/rat, icv) or muscimol (50 ng/rat, i.c.v.) or diazepam (1 mg/kg, i.p.) or allopregnanolone (1 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Rats were returned to their home cages containing preweighed food pallets immediately after the injections. Food intake was measured as described earlier. In separate group, yohimbine (5 mg/rat, i.c.v.) or aCSF (2 ml/rat, i. c.v.) was injected 15 min before sub effective dose combination of agmatine (20 mg/rat, icv) with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Immediately after injections, the rats were returned to their home cages and food intake was measured as described earlier.
2.7. Influence of GABAA receptors antagonists on orexigenic effect of agmatine Rats (n = 5–13) were injected with GABAA receptor antagonist, bicuculline (1 mg/kg, i.p.), benzodiazepine binding site antagonist, flumazenil (25 ng/rat, i.c.v.) and negative modulator of GABAA receptors, DHEA (4 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) 15 min prior to administration of effective dose of agmatine (40 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c. v.). Immediately after injections, the rats were returned to their home cages and pre-weighed food pallets were placed in their cages. Food intake was measured 2 h post injection time point by weighing the leftover food in hoppers. 2.8. Influence of a2-adrenoceptor agonist on orexigenic effect of agmatine and GABAergic agonists Separate group of rats (n = 5–14) were pretreated with a2adrenergic receptors agonist, clonidine (10 ng/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) 15 min before the administration of per se non
2.10. Cannula placement verification At the end of all experiments employing i.c.v. administration, dilute India ink was injected by icv route and the animals were euthanized in the anesthetic chamber by an overdose of thiopentone sodium (65 mg/kg, i.p.). Immediately the brain of rat was dissected out and cut in coronal plane to verify the placement of the guide cannula and distribution of ink in the ventricles. The data of only those animals that showed a uniform distribution of ink in the ventricles were considered for statistical analysis. 2.11. Data analysis The data are presented as mean SEM. Statistical significance was determined using one or two-way analysis of variance (ANOVA) followed by the post hoc analysis of means by Dunnett
Fig. 1. Effect of agmatine on food intake. Different group of rats (n = 5–7) were administered with different doses of either agmatine (20–80 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c. v.) and food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.05, **P < 0.01 vs aCSF treated animals [One way ANOVA post hoc Dunnett mean comparisons].
942
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
or Bonferroni multiple-comparison test. The criterion for statistical significance was considered at P < 0.05. 3. Results 3.1. Effects of agmatine and GABAergic agents on food intake As shown in Fig. 1, administration of agmatine (40 and 80 mg/ rat, i.c.v.) resulted into dose dependent increase in food consumption in first 2 h after treatment [F(3, 22) = 6.17, P < 0.01]. Post hoc Dunnet mean comparisons demonstrated significant orexogenic effect following agmatine treatment [40 (P < 0.05) and 80 mg/rat (P < 0.01)]. Similarly, administrations of muscimol [50 (P < 0.05) and 100 (P < 0.01) (ng/rat, i.c.v.)] [F(3, 26) = 6.45, P < 0.01]; diazepam [1 (P < 0.05) and 1.5 (P < 0.05) (mg/kg, i.p.)] [F(3, 27) = 5.07, P < 0.01] and allopregnanolone [1 (P < 0.05) and 2 (P < 0.01) (mg/kg, s.c.)] [F(3, 29) = 5.04, P < 0.01] produced significant increase in food intake in satiated rats (Fig. 2). At higher doses muscimol (100 ng/rat, i.c.v.), diazepam (1.5 mg/kg, i.p.) and allopregnanolone (2 mg/kg, s.c.) increased the food intake by 101%, 64% and 78% respectively as compared to aCSF treated control animals. Their respective lower doses however, did not significantly affect the feeding behavior in animals. On the contrary, injection of bicuculline [1.5 (P < 0.05) and 2 (P < 0.01) (mg/kg, i.p.)] [F(3, 28) = 6.08, P < 0.01] and DHEA [6 mg/ kg, s.c. (P < 0.05)] [F(3, 29) = 3.18, P < 0.05], exhibited significant anorexia in satiated rats. Their respective lower doses however, did not significantly affect the feeding behavior in animals. Similarly, benzodiazepine receptor agonist, flumazenil did not alter the food intake in animals at any of the doses used in this study. 3.2. GABAergic agents potentiated the orexigenic effect of agmatine Fig. 3 depicts the effects of combination of agmatine and GABAergic agents, muscimol or diazepam or allopregnanolone on
food intake in satiated rats. At the given doses, combination of the agmatine (20 mg/rat, i.c.v.) with muscimol (25 ng/rat, i.c.v.) [FAgmatineTreatment (1, 31) = 12.79, P < 0.01; FMuscimolTreatment (1, 31) = 15.81, P < 0.001; FAgmatine MuscimolTreatment (1, 31) = 4.22, P < 0.05] or diazepam (0.5 mg/kg, i.p.) [FAgmatineTreatment (1, 31) = 13.58, P < 0.001; FDiazepamTreatment (1, 31) = 11.20, P < 0.01; FAgmatine DiazepamTreatment (1, 31) = 4.75, P < 0.05] or allopregnanolone (0.5 mg/kg, s.c.) [FAgmatineTreatment (1, 29) = 12.96, P < 0.001; FAllopregnanoloneTreatment (1, 29) = 26.32, P < 0.001; FAgmatine AllopregnanoloneTreatment (1, 29) = 4.39, P < 0.05] resulted in a significant elevation in food consumption of satiated animals as compared to their respective individual effect. The doses of agmatine, muscimol or diazepam or allopregnanolone used in this experiment per se did not influence food intake in satiated rats. 3.3. GABAA receptors antagonist blocked the orexigenic effect of agmatine As depicted in Fig. 4, pretreatment of rats with bicuculline (1 mg/ kg, i.p.) completely attenuated the hyperphagic effect elicited by effective doses of agmatine (40 mg/rat, i.c.v.) [FAgmatineTreatment (1, 27) = 20.13, P < 0.001; FBicucullineTreatment (1, 27) = 7.83, P < 0.01; FAgmatine BicucullineTreatment (1, 27) = 5.38, P < 0.05]. Similarly, negative GABAA receptor modulator, DHEA (4 mg/kg, s.c.) also attenuated the orexigenic effect of agmatine (40 mg/rat, i.c.v.) [FAgmatineTreatment (1, 29) = 20.10, P < 0.001; FDHEATreatment (1, 29) = 4.20, P < 0.015; FAgmatine DHEATreatment (1, 29) = 9.49, P < 0.01]. Post hoc Bonferroni mean comparison demonstrated the significant attenuation of orexigenic effect of agmatine in bicuculline (P < 0.01) and DHEA (P < 0.01) pretreated animals. Administration of bicuculline or DHEA to aCSF treated animals at the dose used here did not evoke any response on feeding behavior. On the other hand, the orexigenic effect induced by agmatine remained unaffected by prior treatment of benzodiazepine receptors agonist, flumazenil.
Fig. 2. Effect of GABAergic agents and a2–adrenoceptor ligands on food intake. Different group of rats (n = 5–17) were administered with different doses of either muscimol (25–100 ng/rat, i.c.v.) or diazepam (0.5–1.5 mg/kg, i.p.) or allopregnanolone (0.5–2 mg/kg, s.c.) or clonidine (10–50 ng/rat, icv) or bicuculline (1–2 mg/kg, i.p.) or flumazenil (25–100 ng/rat, i.c.v.) or DHEA (2–6 mg/kg, s.c.) or yohimbine (5–20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) and food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle treated control animals [One way ANOVA post hoc Dunnett mean comparisons].
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
943
Fig. 3. Effect of GABAergic agents and agmatine on food intake. Different groups of rats (n = 5–16) were pretreated with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/ kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) and agmatine (20 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) was administered 15 min after the earlier injections. Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. $P < 0.001 vs vehicle treated control animals; *P < 0.01, **P < 0.001 vs agmatine treatment; #P < 0.01, ##P < 0.001 vs respective GABAergic agent treated animals [Two way ANOVA post hoc Bonferroni mean comparisons].
3.4. Influence of a2-adrenoceptor agonist and antagonist on orexigenic effect of agmatine and GABAergic agonist As shown in Fig. 5, pretreatment of animals with subeffective dose of a2-adrenergic receptors agonist, clonidine (10 ng/rat, i.c. v.) significantly potentiated the orexigenic effect of agmatine
(20 mg/rat, i.c.v.) [FClonidineTreatment (1, 27) = 19.97, P < 0.001; FAgmatineTreatment (1, 27) = 10.66, P < 0.01; FClonidine AgmatineTreatment (1, 27) = 9.32, P < 0.01], muscimol (25 ng/rat, i.c.v.) [FClonidineTreatment (1, 28) = 4.24, P < 0.05; FMuscimolTreatment (1, 28) = 13.80, P < 0.01; FClonidine MuscimolTreatment (1, 28) = 11.58, P < 0.01], diazepam (0.5 mg/kg, i.p.) [FClonidineTreatment (1, 29) = 11.60, P < 0.01;
Fig. 4. Effect of GABAergic antagonists in combination with agmatine on food intake. Different groups of rats (n = 5–13) were pretreated with either GABAA receptor antagonist, bicuculline (1 mg/kg, i.p.), benzodiazepine binding site antagonist, flumazenil (25 ng/rat, i.c.v.) and negative modulator of GABAA receptors, DHEA (4 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.) 15 min prior to administration of effective dose of agmatine (40 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. $P < 0.001 vs vehicle treated control animals; *P < 0.01 vs agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
944
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
Fig. 5. Effect of a2-adrenoceptor agonist and agmatine or GABAergic agents on food intake. Different groups of rats (n = 5–14) were pretreated with a2-adrenoceptor agonist, clonidine (10 ng/rat, i.c.v.), or aCSF (2 ml/rat, i.c.v.) ng/rat, i.c.v.) 15 min prior to administration of sub-effective dose administration of agmatine (20 mg/rat, i.c.v.) or muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.001 vs vehicle treated control animals; $P < 0.01, $$P < 0.001 vs respective GABAergic agent/agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
FDiazepamTreatment (1, 29) = 24.39, P < 0.001; FClonidine Diazepam (1, 29) = 4.62, P < 0.05] and allopregnanolone (0.5 mg/ kg, s.c.) [FClonidineTreatment (1, 28) = 15.59, P < 0.01; FAllopregnanoloneTreatment (1, 28) = 24.25, P < 0.001; FClonidine AllopregnanoloneTreatment (1, 28) = 6.24, P < 0.05]. The doses of clonidine, agmatine, muscimol or diazepam or allopregnanolone did not influence food intake in satiated rats.
Treatment
In contrast pretreatment of rats with a2-adrenergic receptors antagonist, yohimbine (5 mg/rat, i.c.v.) completely attenuated the hyperphagic effect elicited by effective doses of agmatine (40 g/rat, icv) [FYohimbineTreatment (1, 28) = 7.00, P < 0.05; FAgmatineTreatment (1, 28) = 12.75, P < 0.01; FYohimbine AgmatineTreatment (1, 28) = 4.47, P < 0.05], muscimol (50 ng/rat, i.c.v.) [FYohimbineTreatment (1, 25) = 10.03, P < 0.01; FMuscimolTreatment (1, 25) = 21.11, P < 0.001;
Fig. 6. Effect of a2-adrenoceptor antagonist and agmatine or GABAergic agents on food intake. Separate group of rats (n = 5–14) were pretreated with a2-adrenoceptors antagonist, yohimbine (5 mg/rat, i.c.v.) or aCSF (5 ml/rat, i.c.v.) 15 min before the administration of effective doses of agmatine (40 mg/rat, icv) or muscimol (50 ng/rat, i.c.v.) or diazepam (1 mg/kg, i.p.) or allopregnanolone (1 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. $P < 0.01, $$P < 0.001 vs vehicle treated control animals; *P < 0.05, **P < 0.01, ***P < 0.001vs respective GABAergic agent/agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
FYohimbine MuscimolTreatment (1, 25) = 7.12, P < 0.05], diazepam (1 mg/ kg, i.p.) [FYohimbineTreatment (1, 28) = 6.15, P < 0.05; F DiazepamTreatment (1, 28) = 10.30, P < 0.01; FYohimbine DiazepamTreatment (1, 28) = 4.35, P < 0.05] or alloprenelonone (1 mg/kg, s.c.) [FYohimbineTreatment (1, 26) = 7.45, P < 0.01; F AllopregnanoloneTreatment (1, 26) = 11.60, P < 0.01; FYohimbine AllopregnanoloneTreatment (1, 26) = 4.86, P < 0.05] (Fig. 6). Similarly, yohimbine (5 mg/rat, i.c.v.) also blocked the hyperphagic effect elicited by ineffective dose combination of agmatine (20 mg/rat, i.c.v.) with muscimol (25 ng/rat, i.c.v.) [FYohimbineTreatment (1, 29) = 6.31, P < 0.05; FCombinationTreatment (1, 29) = 20.01, P < 0.01; FYohimbine CombinationTreatment (1, 29) = 4.43, P < 0.05] or diazepam (0.5 mg/kg, i.p.) [FYohimbineTreatment (1, 29) = 5.95, P < 0.05; FCombinationTreatment (1, 29) = 11.89, P < 0.01; FYohimbine CombinationTreatment (1, 29) = 4.36, P < 0.05] or alloprenelonone (0.5 mg/kg, s.c.) [FYohimbineTreatment (1, 27) = 6.29, P < 0.05; FCombinationTreatment (1, 27) = 18.05, P < 0.01; FYohimbine CombinationTreatment (1, 27) = 4.30, P < 0.05] (Fig. 7). Administration of yohimbine to aCSF treated animals at the dose used here did not evoke any response on food consumption. 4. Discussion The presence of agmatine in the PVN and its ability to enhance caloric intake [22] suggests that agmatine may be an additional regulator of feeding behavior. In replication of our earlier findings, the results of the present study showed that i.c.v. administration of agmatine dose-dependently increased the food intake in satiated rats. Animals showed maximum food intake within first 2 h following agmatine administration; hence food intake was monitored at this time point only. Although agmatine and GABA are widely distributed throughout the brain including hypothalamic PVN [37,38] their interaction pertaining to the regulation of energy homeostasis largely remained unexplored. In the present study, the orexigenic effect of agmatine dose-dependently enhanced by GABAA agonist, muscimol and benzodiazepine
945
binding site agonist, diazepam. Although, diazepam [39] or muscimol [40] are well known for their hyperphagic effect at lower doses these agents [diazepam (0.5 mg/kg, i.p.) or muscimol (25 ng/rat, i.c.v.)] showed no effect on feeding behavior per se. However, in the present study, at these doses, they significantly potentiated agmatine induced hyperphagia. In contrast, it was completely abolished in presence of GABAA antagonist, bicuculline. Antagonistic interactions of bicuculline with muscimol or diazepam in anxiety and feeding behavior have already been documented [41,42,25]. However, the orexigenic effect of agmatine remains unaffected in presence of benzodiazepine receptors antagonist, flumazenil. This showed that agmatine and diazepam although produced a synergistic effect on food intake they might act through different intracellular signal transduction mechanism which promotes stimulation of food intake. Interestingly the orexigenic effect of agmatine also potentiated by the positive modulator of GABAA receptor, allopregnanolone and blocked by its negative modulator, DHEA. Allopregnanolone is abundantly expressed in hypothalamic nuclei [43] that regulate feeding and energy balance. Indeed, several preclinical studies have demonstrated the orexigenic potential of allopregnanolone in food deprived [44] and satiated animals [45,46]. Further, its repeated administration also promotes an increase in body weights and its concentration was found to be elevated in the serum of obese humans [47]. GABA type A receptors seem to mediate the effect of allopregnanolone on feeding behavior, as allopregnanolone positively modulate GABAA receptors [48]. In view of these findings, we suggest that agmatine may stimulate feeding by direct or indirect activation of GABAA receptors. However, the direct relationship between endogenous agmatine and the GABAergic system is still unclear. The involvement of a2-adrenergic receptors in agmatine induced feeding has been already demonstrated in our earlier finding [22]. Several preclinical evidences are available for a2adrenoceptor and GABA receptor interaction within hypothalamic
Fig. 7. Effect of a2-adrenoceptor antagonist and agmatine combination with GABAergic agents on food intake. Separate group of rats (n = 5–15) were pretreated with a2adrenoceptors antagonist, yohimbine (5 mg/rat, i.c.v.) or aCSF (2 ml/rat, i.c.v.) was injected 15 min before subeffective dose combination of agmatine (20 mg/rat, icv) with either muscimol (25 ng/rat, i.c.v.) or diazepam (0.5 mg/kg, i.p.) or allopregnanolone (0.5 mg/kg, s.c.) or aCSF (2 ml/rat, i.c.v.) or saline (1 ml/kg, i.p.) or saline (1 ml/kg, s.c.). Food intake (g) was determined 2 h after drug administrations. Each column represents food intake (g) SEM. *P < 0.01 vs vehicle treated control animals; $P < 0.05 vs respective GABAergic agent and agmatine treatment [Two way ANOVA post hoc Bonferroni mean comparisons].
946
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947
PVN in the regulation of physiological behavior [49,50]. Alpha2adrenoceptor agonists promote the release of GABA from the nerve terminals in the hippocampus, nucleus accumbens, striatum and the cortex of animals [30,31,33,34]. It seems likely that a2adrenoceptor activation may be coupled to GABA release since GABAergic neurons express a2-adrenoceptor [33]. The expression of a2-adrenoceptors has been confirmed in the presynaptic nerve terminal of GABAergic neurons projecting to the PVN [27]. Moreover, the input of GABAergic neurons to PVN neurons is inhibited by stimulation of a2-adrenoceptors in the PVN, resulting in an increase in the excitability of PVN neurons [28,29]. In view of this background, we designed an additional protocol to verify whether modulatory effect GABAergic agents on agmatine stimulated food consumption involves a2-adrenoceptors. Interestingly, the orexigenic effect of agmatine, muscimol, diazepam and alloprenelonone significantly potentiated by a a2adrenoceptor agonist, clonidine at its sub effective dose and blocked by a2-antagonist, yohimbine. The data are in agreement with previous investigations, indicating an interaction between the GABAergic system and a2-adrenoceptors in the laboratory animals [30–32,51]. In fact, a2-adrenoceptor activation modulates the release of other neurotransmitters in several areas of the brain [33,52]. Extensive evidence indicates that a2-adrenoceptors activation increases the basal release of GABA in rat hippocampus, cerebral cortex, and striatum [30–34]. Thus agmatine induced a2adrenoceptors activation might increase endogenous GABA release to enhance food intake. Consequently, the direct activation of GABAA receptors in agmatine induced stimulation of food intake cannot be completely ruled out at present. The a2-adrenoceptors activation by agmatine increases neuropeptide Y (NPY) immunoreactivity in hypothalamic arcuate nucleus and PVN to induce orexigenic effect [22]. Interestingly, a subpopulation of NPYproducing neurons in the arcuate nucleus coproduces GABA [53]. Further, NPY is colocalized with GABA in a subpopulation of axon terminals in the PVN and produce an additive orexigenic effect when combined with GABAA receptor agonist, muscimol [54]. Thus, the possibility exists that agmatine might influence both NPYergic and GABAergic system in hypothalamic PVN to induce stimulation of food intake. More region-specific studies are warranted to clarify this issue. It is important to note that agmatine possesses multireceptorial affinity. Apart from, a2-adrenoceptors, agmatine also interacts with other neurotransmitter systems like imidazoline receptors [15], NMDA receptors [16], and nitric oxide [17] involved in regulation of energy homeostasis. Therefore, their participation in the orexigenic effects of agmatine wants further investigation. This study clearly demonstrated the role of agmatine in normal homeostatic food consumptions. Earlier, we have also demonstrated the role of agmatine altered feeding behavior in several pathological conditions like bacterial infections, stress, anorexia nervosa, obesity and cachexia [11,19,55,56]. In clinical context, this study suggests that agmatinergic pathways can be targeted to develop novel therapeutic agents for the treatment of feedingrelated disorders like anorexia nervosa and other pathological conditions associated with altered food intake. In conclusion, the current study provides the first functional evidence for the involvement of a2-adrenoceptors and GABAA receptors in the agmatine induced stimulation of food intake and suggest that agmatine induced a2-adrenoceptors activation might facilitate GABA activity to stimulate food intake in satiated rats. Conflict of interest The author(s) declare(s) that there is no conflict of interest.
Acknowledgement This project was sponsored under the head of institutional research grants. References [1] W. Raasch, S. Regunathan, G. Li, D.J. Reis, Agmatine the bacterial amine, is widely distributed in mammalian tissues, Life Sci. 56 (1995) 2319–2330. [2] D.J. Reis, S. Regunathan, Is agmatine a novel neurotransmitter in brain? Trends Pharmacol. Sci. 21 (2000) 187–193. [3] A.D. Zomkowski, L. Hammes, J. Lin, J.B. Calixto, A.R.S. Santos, A.L. Rodrigues, Agmatine produces antidepressant-like effects in two models of depression in mice, Neuroreport 13 (2002) 387–391. [4] B.G. Taksande, N.R. Kotagale, S.J. Tripathi, R.R. Ugale, C.T. Chopde, Antidepressant like effect of selective serotonin reuptake inhibitors involve modulation of imidazoline receptors by agmatine, Neuropharmacology 57 (2009) 415–424. [5] D. Lavinsky, N.S. Arteni, C.A. Netto, Agmatine induces anxiolysis in the elevated plus maze task in adult rats, Behav. Brain Res. 141 (2003) 19–24. [6] B.G. Taksande, N.R. Kotagale, D.Y. Gawande, A.P. Bharne, C.T. Chopde, D.M. Kokare, Neuropeptide Y in the central nucleus of amygdala regulates the anxiolytic effect of agmatine in rats, Eur. Neuropsychopharmacol. 24 (2014) 955–963. [7] T. Singh, N. Bagga, A. Kaur, N. Kaur, D.Y. Gawande, R.K. Goel, Agmatine for combined treatment of epilepsy, depression and cognitive impairment in chronic epileptic animals, Biomed. Pharmacother. 92 (2017) 720–725. [8] N.R. Kotagale, B.G. Taksande, P.J. Wadhwani, M.W. Palhade, S.M. Mendhi, D.Y. Gawande, P.N. Hadole, C.T. Chopde, Psychopharmacological study of agmatine in behavioral tests of schizophrenia in rodents, Pharmacol. Biochem. Behav. 100 (3) (2012) 398–403. [9] T. Uzbay, G. Goktalay, H. Kayir, S.S. Eker, A. Sarandol, S. Oral, L. Buyukuysal, G. Ulusoy, S. Kirli, Increased plasma agmatine levels in patients with schizophrenia, J. Psychiatr. Res. 47 (8) (2013) 1054–1060. [10] M.M. Aglawe, B.G. Taksande, S.S. Kuldhariya, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Participation of central imidazoline binding sites in antinociceptive effect of ethanol and nicotine in rats, Fundam. Clin. Pharmacol. 28 (3) (2014) 284–293. [11] B.G. Taksande, D.Y. Gawande, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Agmatine ameliorates adjuvant induced arthritis and inflammatory cachexia in rats, Biomed. Pharmacother. 86 (2016) 271–278. [12] N.R. Kotagale, B.G. Taksande, A.Y. Gahane, R.R. Ugale, C.T. Chopde, Repeated agmatine treatment attenuates nicotine sensitization in mice: modulation by a2-adrenoceptors, Behav. Brain Res. 213 (2010) 161–174. [13] B.G. Taksande, N.R. Kotagale, M.R. Patel, G.P. Shelkar, R.R. Ugale, C.T. Chopde, Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats, Eur. J. Pharmacol. 637 (2010) 89– 101. [14] J.E. Piletz, D.N. Chikkala, P. Ernsberger, Comparison of the properties of agmatine and endogenous clonidine-displacing substance at imidazoline and alpha-2 adrenergic receptors, J. Pharmacol. Exp. Ther. 272 (1995) 581–587. [15] D.J. Reis, G. Li, S. Regunathan, Endogenous ligands of imidazoline receptors: classic and immunoreactive clonidine displacing substance and agmatine, Ann. N. Y. Acad. Sci. 763 (1995) 295–313. [16] X.C. Yang, D.J. Reis, Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons, J. Pharmacol. Exp. Ther. 288 (1999) 544–549. [17] M. Auguet, I. Viossat, J.G. Marin, P.E. Chabrier, Selective inhibition of inducible nitric oxide synthase by agmatine, Jpn. J. Pharmacol. 69 (1995) 285–287. [18] C.A. Fairbanks, K.L. Schreiber, K.L. Brewer, C.G. Yu, L.S. Stone, K.F. Kitto, H.O. Nguyen, B.M. Grocholski, D.W. Shoeman, L.J. Kehl, S. Regunathan, D.J. Reis, R.P. Yezierski, G.L. Wilcox, Agmatine reverses pain induced by inflammation neuropathy, and spinal cord injury, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 10584–10589. [19] O. Yesilyurt, I.T. Uzbay, Agmatine potentiates the analgesic effect of morphine by an alpha(2)-adrenoceptor-mediated mechanism in mice, Neuropsychopharmacology 25 (2001) 98–103. [20] K. Riazi, H. Honar, H. Homayoun, N. Rashidi, S. Kiani, M.R. Ebrahimkhani, A.R. Noorian, K. Ghaffari, A. Jannati, A.R. Dehpour, The synergistic anticonvulsant effect of agmatine and morphine: possible role of alpha 2-adrenoceptors, Epilepsy Res. 65 (1–2) (2005) 33–40. [21] P. Tahsili-Fahadan, N. Yahyavi-Firouz-Abadi, M.A. Khoshnoodi, R. MotieiLangroudi, S.A. Tahaei, M.H. Ghahremani, A.R. Dehpour, Agmatine potentiates morphine-induced conditioned place preference in mice: modulation by alpha (2)-adrenoceptors, Neuropsychopharmacology 31 (2006) 1722–1732. [22] B.G. Taksande, N.R. Kotagale, K.T. Nakhate, P.D. Mali, D.M. Kokare, K. Hirani, N. K. Subhedar, C.T. Chopde, R.R. Ugale, Agmatine in the hypothalamic paraventricular nucleus stimulates feeding in rats: involvement of neuropeptide Y, Br. J. Pharmacol. 164 (2011) 704–718. [23] J. Kelly, G.F. Alheid, A. Newberg, S.P. Grossman, GABA stimulation and blockade in the hypothalamus and midbrain: effects on feeding and locomotor activity, Pharmacol. Biochem. Behav. 7 (1977) 537–541.
B.G. Taksande et al. / Biomedicine & Pharmacotherapy 93 (2017) 939–947 [24] V.R. Olgiati, C. Netti, F. Guidobono, A. Pecile, The central GABAergic system and control of food intake under different experimental conditions, Psychopharmacology (Berl) 68 (1980) 163–167. [25] T.L. Rao, D.M. Kokare, S. Sarkar, R.T. Khisti, C.T. Chopde, N. Subhedar, GABAergic agents prevent alpha-melanocyte stimulating hormone induced anxiety and anorexia in rats, Pharmacol. Biochem. Behav. 76 (3–4) (2003) 417–423. [26] D.M. Kokare, A.M. Patole, A. Carta, C.T. Chopde, N.K. Subhedar, GABA(A) receptors mediate orexin-A induced stimulation of food intake, Neuropharmacology 50 (1) (2006) 16–24. [27] D.P. Li, L.M. Atnip, S.R. Chen, H.L. Pan, Regulation of synaptic inputs to paraventricular-spinal output neurons by alpha2 adrenergic receptors, J. Neurophysiol. 93 (2005) 393–402. [28] S.K. Han, W. Chong, L.H. Li, I.S. Lee, K. Murase, P.D. Ryu, Noradrenaline excites and inhibits GABAergic transmission in parvocellular neurons of rat hypothalamic paraventricular nucleus, J. Neurophysiol. 87 (2002) 2287–2296. [29] W. Chong, L.H. Li, K. Lee, M.H. Lee, J.B. Park, P.D. Ryu, Subtypes of alpha1- and alpha2-adrenoceptors mediating noradrenergic modulation of spontaneous inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus, J. Neuroendocrinol. 16 (2004) 450–457. [30] A. Pittaluga, M. Raiteri, GABAergic nerve terminals in rat hippocampus possess alpha-2 adrenoceptors regulating GABA release, Neurosci. Lett. 76 (1987) 363– 367. [31] G. Maura, A. Pittaluga, M. Ulivi, M. Raiteri, Enhancement of endogenous GABA release from rat synaptosomal preparations is mediated by alpha-2 adrenoceptors pharmacologically different from alpha-2 autoreceptors, Eur. J. Pharmacol. 157 (1988) 23–29. [32] L. Ciranna, F. Licata, G. Li Volsi, F. Santangelo, Alpha 2- and beta-adrenoceptors differentially modulate GABAA- and GABAB-mediated inhibition of red nucleus neuronal firing, Exp. Neurol. 185 (2004) 297–304. [33] W. Zhang, G.A. Ordway, The alpha2C-adrenoceptor modulates GABA release in mouse striatum, Brain Res. Mol. Brain Res. 112 (2003) 24–32. [34] A. Alachkar, J. Brotchie, O.T. Jones, Alpha2-adrenoceptor-mediated modulation of the release of GABA and noradrenaline in the rat substantia nigra parsreticulata, Neurosci. Lett. 395 (2006) 138–142. [35] T. Kimura, T. Maji, K. Ashida, Periodicity of food intake and lipogenesis in rats subjected to two different feeding plans, J. Nutr. 100 (1970) 691–697. [36] A. Kask, L. Rägo, J. Harro, Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226, Br. J. Pharmacol. 124 (7) (1998) 1507–1515. [37] K. Otake, D.A. Ruggiero, S. Regunathan, H. Wang, T.A. Milner, D.J. Reis, Regional localization of agmatine in the rat brain: an immunocytochemical study, Brain Res. 787 (1998) 1–14. [38] H. Hörtnagl, R.O. Tasan, A. Wieselthaler, E. Kirchmair, W. Sieghart, G. Sperk, Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain, Neuroscience 236 (2013) 345–372. [39] A.H. Soderpalm, K.C. Berridge, Food intake after diazepam, morphine or muscimol: microinjections in the nucleus accumbens shell, Pharmacol. Biochem. Behav. 66 (2000) 429–434. [40] E. Khaimova, Y. Kandov, Y. Israel, G. Cataldo, M.M. Hadjimarkou, R.J. Bodnar, Opioid receptor subtype antagonists differentially alter GABA agonist-induced feeding elicited from either the nucleus accumbens shell or ventral tegmental area regions in rats, Brain Res. 1026 (2004) 284– 294.
947
[41] A.P. Lopes, L. Ganzer, A.C. Borges, L. Kochenborger, A.C. Januário, M.S. Faria, J. Marino-Neto, M.A. Paschoalini, Effects of GABA ligands injected into the nucleus accumbens shell on fear/anxiety-like and feeding behaviours in fooddeprived rats, Pharmacol. Biochem. Behav. 101 (1) (2012) 41–48. [42] V. Znamensky, J.A. Echo, N. Lamonte, G. Christian, A. Ragnauth, R.J. Bodnar, Gamma-aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats, Brain Res. 906 (2001) 84–91. [43] E. Holmberg, M. Johansson, T. Bäckström, D. Haage, Allopregnanolone preferentially induces energy-rich food intake in male Wistar rats, Physiol. Rep. 2 (12) (2014) e12190. [44] D.S. Reddy, S.K. Kulkarni, Sex and estrous cycle-dependent changes in neurosteroid and benzodiazepine effects on food consumption and plus-maze learning behaviors in rats, Pharmacol. Biochem. Behav. 62 (1999) 53–60. [45] E. Holmberg, T. Bäckström, M. Johansson, M. Löfgren, D. Haage, Allopregnanolone induces a diurnally dependent hyperphagic effect and alters feeding latency and duration in male Wistar rats, Acta Physiol. 208 (4) (2013) 400–409. [46] K.T. Nakhate, N.K. Subhedar, A.P. Bharne, P.S. Singru, D.M. Kokare, Involvement of cocaine- and amphetamine-regulated transcript peptide in the hyperphagic and body weight promoting effects of allopregnanolone in rats, Brain Res. 1532 (2013) 44–55. [47] R. Menozzi, P. Florio, M. Bondi, S. Luisi, L. Cobellis, A.R. Genazzani, G. Del Rio, F. Petraglia, Increased response of plasma allopregnanolone to corticotropinreleasing hormone in obese patients, Neuroendocrinology 75 (2002) 124–129. [48] J.J. Lambert, D. Belelli, D.R. Peden, A.W. Vardy, J.A. Peter, Neurosteroid modulation of GABAA receptors, Prog. Neurobiol. 71 (2003) 67–80. [49] Y. Li, A.N. van denPol, Direct and indirect inhibition by catecholamines of hypocretin/orexin neurons, J. Neurosci. 25 (1) (2005) 173–183. [50] E.R. Samuels, E. Szabadi, Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organization, Curr. Neuropharmacol. 6 (3) (2008) 235– 253. [51] Y. Yamama, K. Nishikawa, T. Funao, T. Mori, A. Asada, Intrathecal gabapentin and clonidine synergistically inhibit allodynia in spinal nerve-ligated rats, Life Sci. 87 (17–18) (2010) 565–571. [52] Y. Wang, J. Liu, Z.H. Gui, U. Ali, L.L. Fan, C. Hou, T. Wang, L. Chen, Q. Li, a2Adrenoceptor regulates the spontaneous and the GABA/glutamate modulated firing activity of the rat medial prefrontal cortex pyramidal neurons, Neuroscience 182 (2011) 193–202. [53] T.L. Horvath, I. Bechmann, F. Naftolin, S.P. Kalra, C. Leranth, Heterogeneity in the neuropeptide Y-containing neurons of the rat arcuate nucleus: GABAergic and non-GABAergic subpopulations, Brain Res. 756 (1997) 283–286. [54] S. Pu, M.R. Jain, T.L. Horvath, S. Diano, P.S. Kalra, S.P. Kalra, Interactions between neuropeptide Y and g-aminobutyric acid in stimulation of feeding: a morphological and pharmacological analysis, Endocrinology 140 (1999) 933– 940. [55] B.G. Taksande, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Agmatine attenuates hyperactivity and weight loss associated with activity-based anorexia in female rats, Pharmacol. Biochem. Behav. 132 (2015) 136–141. [56] B.G. Taksande, C.T. Chopde, M.J. Umekar, N.R. Kotagale, Agmatine attenuates lipopolysaccharide induced anorexia and sickness behavior in rats, Pharmacol. Biochem. Behav. 132 (2015) 108–114.