Study of GABA A receptors on the sleep-like behavior in Coturnix japonica (Temminck Schlegel, 1849) (Galliformes: Aves)

J Comp Physiol A (2009) 195:247–252 DOI 10.1007/s00359-008-0402-7 ORIGINAL PAPER Study of GABAA receptors on the sleep-like behavior in Coturnix jap...
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J Comp Physiol A (2009) 195:247–252 DOI 10.1007/s00359-008-0402-7

ORIGINAL PAPER

Study of GABAA receptors on the sleep-like behavior in Coturnix japonica (Temminck Schlegel, 1849) (Galliformes: Aves) Patrícia Almeida Polo · André Souza Mecawi · Marco Antonio Pires Camilo Lapa · Wellington Silva Côrtes · Luis Carlos Reis

Received: 17 September 2008 / Revised: 22 November 2008 / Accepted: 24 November 2008 / Published online: 9 December 2008 © Springer-Verlag 2008

Abstract The present study was carried out to investigate the inXuence of GABAA signaling on sleep-like behaviors through systemic administration of bicuculline and picrotoxin (GABAA antagonists) and thiopental (an allosterical modulator). A thiopental (20 mg/kg) injection increased the eye closure frequency compared to the control group. The birds quickly became sleepy with a low frequency of early behavioral stages, such as rapid oral movement (ROM), feather ruZing and blinking. A bicuculline administration (1 and 4 mg/kg) did not modify the frequency of feather ruZing, ROM, eye closure or blinking responses. A lower dose of picrotoxin (2 mg/kg) stimulated an active awakening status, while an intermediate dose (4 mg/kg) elicited a moderate awakening status, which was associated with an increase in the frequency of ROM, blinking and eye closure. At the higher dose (8 mg/kg), the birds exhibited thermoregulatory-like behaviors and convulsions immediately after the injection. Interestingly, picrotoxin (4 mg/kg) intensiWed the eye closures when given in combination with thiopental (20 mg/kg). Both barbiturate and picrotoxininduced sleep-like responses have the same behavioral neuropharmacological properties, conceivably because they are correlated with action at an identical site on the GABAA receptor. Keywords Sleep-like behavior · Thiopental · GABAergic antagonist · GABAA receptors · Quails

P. A. Polo · A. S. Mecawi · M. A. P. C. Lapa · W. S. Côrtes · L. C. Reis (&) Department of Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, BR465, Km07, Seropédica, RJ 23890-000, Brazil e-mail: [email protected]

Abbreviations ROM Rapid oral movements BCC Bicuculline PTX Picrotoxin THIO Thiopental

Introduction Sleep is a homeostatically regulated behavior, highly complex and has some components that are still not fully understood (Taheri and Mignot 2002; Rattenborg et al. 2008). A physiological role for sleep has been widely established through studies that showed that animals that are completely deprived of sleep have several pathophysiological disturbances (RechtschaVen and Bergmann 2002). In addition, according to Taheri and Mignot (2002), the conservation of this behavior during the course of evolution indicates that sleep is an adaptive behavior. Sleep in vertebrates exhibits a continuous phylogenic development, which is expressed through behavioral and electrophysiological patterns according to evolutionary proWle acquired by the brain (Ayala-Guerrero et al. 2003; Campbell and Tobler 1984). Yawning is a behavioral component that is associated with sleep and is highly preserved in vertebrata, particularly the mandibular appearance (Lehmann 1979; Argiolas and Melis 1998). Its role is still not understood; however, this semi-voluntary phenomenon seems to constitute a behavioral arousal when sleep is imminent (Daquin et al. 2001). Despite numerous studies implicating that sleep is a repair mechanism for brain metabolism or memory maintenance, its precise physiological role remains unclear (Taheri and Mignot 2002; Cirelli and Tononi 2008). From a neurochemical viewpoint, the gamma amino butyric

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acid (GABA) represents the main inhibitory neurotransmitter in the brain (Gottesmann 2002; Sanford et al. 2002). Takagi et al. (2001; 2003) have demonstrated in Gallus domesticus that intracerebroventricular injection of GABAA or GABAB receptor antagonists attenuates food ingestion and sleeping-like behavior elicited by L-pipecolic acid, a GABA releaser produced in the brain following conversion from L-lysine. In this context, paralleled Wndings have settled that GABAergic mechanism through GABAA receptor stimulates feeding in Meleagris gallopavo and Gallus domesticus (Denbow 1991; Jonaidi et al. 2002). GABA acts through two diVerent pharmacologically identiWed receptors: GABAA receptor, which is bicuculline sensitive, and GABAB receptor, which is insensitive to bicuculline (MacDonald and Twyman 1992; Bormann 2000; Jonaidi et al. 2002; Glykys and Mody 2007). The GABAA receptor complex is located mainly at postsynaptic sites, where it mediates the most inhibitory transmissions (Adachi et al. 2006). It contains a channel for chloride ions (whose permeability is activated by GABA) and recognition sites for barbiturates, benzodiazepine, neurosteroids and picrotoxin (Gottesmann 2002). There are some diVerences between GABAergic receptors of mammals and birds. In mammals, pharmacological GABAergic activation produces clear-cut hypnogenic eVects; in birds, however, it has not been elucidated in detail, especially the early components of sleep (Gottesmann 2002; Takagi et al. 2003; Polo et al. 2007). GABA neurotransmitter and GABAergic receptors have been identiWed in the brain of chickens (Van Luijtelaar et al. 1987; Liu et al. 2000); probably, their distribution in Galliformes exhibits a similar pattern. Other brain mediators have been reported to be hypnogenic when systemically administered. Here, we aimed to assess the involvement of GABAA receptors in sleep-like behaviors, particularly their preliminary components, through the systemic administration of bicuculline and picrotoxin (GABAA antagonists) and sodium thiopental, a typical barbiturate (allosterical modulator).

Materials and methods Animals Male quails (Coturnix japonica) weighting 130–160 g (3– 4 months old) were used. Birds were housed in individual cages under a 12/12 h light/dark cycle at 25–30°C ambient temperature, with water and food rations (Purina for quails, 16% crude protein) available ad libitum throughout the period of adaptation to laboratory conditions.

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Drugs preparation Picrotoxin (PTX, Sigma, St Louis, USA) was diluted in 0.9% NaCl plus 0.1 N NaOH and the pH was adjusted to 7.0 with the addition of 0.1 N HCl for subcutaneous (sc) injection (Khisti et al. 1998). Bicuculline (BCC, Biosynth Chemistry & Biology, Staad, Switzerland) was diluted in dimethyl sulfoxide plus 0.9% NaCl (1:9, v/v) for sc injection, and sodium thiopental (THIO, Cristália, Itapira, SP, Brazil) was diluted in 0.9% saline for sc and intracoelomic (ic) administration. Experimental procedures Four series of studies were undertaken: 1. THIO (20 mg/kg), an allosterical agonist of GABAA receptors, or 0.9% saline was administered to evaluate the sleep-like behaviors produced by a barbiturate (barbituric sleep) (N = 6 for each group), 2. BCC (1.0 and 4.0 mg/kg, N = 5 and 11, respectively,), an antagonist of GABAA receptors, or vehicle (N = 11) was injected to assess the inXuence of GABAA on arousal behavior. 3. PTX (2, 4 and 8 mg/kg, N = 4, 9 and 9, respectively), an antagonist of GABAA receptors, or vehicle (N = 8) was administered to evaluate the inXuence of GABAA on arousal behavior. 4. Vehicle, BCC (4 mg/kg) or PTX (4 mg/kg) was administered 10 min before the THIO (20 mg/kg) as follows: saline plus THIO (N = 6), PTX plus THIO (N = 9) and BCC plus THIO (N = 11). This protocol was designed to assess the allosterical action of the GABAA receptor antagonists PTX and BCC and their eVects on the barbiturate sleep-like reactions. All experimental sessions were conducted between 8:00 and 11:00 a.m. Assessment of sleep behaviors Behavioral observations were made during the Wrst 20 min following drug administration. Analyses were performed in sessions of 10 min (0–10 min and subsequent 10–20 min). The following behavioral responses were observed: feather ruZing, rapid oral movements (ROM), yawning, blinking and closure of the eyes (as reported by Polo et al. 2007). These reactions are typical of the sleepiness stage at the beginning of the sleep in quails, and they predict the development of sleep behavior. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni test. Unpaired “t”

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Student test was used to compare two groups. The analyses were performed with the statistical GraphPad Prism software (version 4, Inc., San Diego, USA). In all comparisons, the level of signiWcance was set at P < 0.05.

Results Administration of thiopental increased eye closure frequency compared to a saline control (0.0 § 0.0 vs. 8.2 § 1.9, P < 0.001, at 0–10 min; Fig. 1). The birds quickly became sleepy (in less than 1 min), and there was a low frequency of preliminary behavioral stages, such as ROM, feather ruZing and blinking.

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Neither dose of BCC administration changed the frequency of ruZing, ROM, blinking or closure of eyes responses (Fig. 2). The low dose of PTX (2 mg/kg) provoked an active awakening status characterized by behaviors such as pecking, ambulation, head outside the cages and continuously stretched body and neck (data not shown). The intermediate PTX dose (4 mg/kg) caused the birds to exhibit a moderate awakening status associated with increase in ROM (1.5 § 0.6 vs. 7.7 § 1.5, P < 0.05), blinking (2.2 § 1.6 vs. 17.7 § 3.5, P < 0.01) and closure of eyes (0.0 § 0.0 vs. 7.0 § 2.1, P < 0.05) after 0–10 min of evaluation (Fig. 3). Similar responses were observed at 10–20 min of evaluation.

Fig. 1 EVect of thiopental treatment (20 mg/kg, sc) on the frequency of sleep-like behaviors. Data are presented as mean § SEM. * P < 0.05 and ** P < 0.01 compared to control group (Unpaired “t” Student test)

Fig. 2 EVect of bicuculline treatment (BCC, 1 and 4 mg/kg, sc) on the frequency of sleeplike behaviors. Data are presented as mean § SEM. No signiWcance diVerences were found between the groups (one-way ANOVA)

Fig. 3 EVect of picrotoxin treatment (PTX, 2, 4 and 8 mg/kg, sc) on the frequency of sleep-like behaviors. Data are presented as mean § SEM. * P < 0.05 and ** P < 0.01 compared to control group (one-way ANOVA)

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With a higher dose (8 mg/kg), the birds presented thermoregulatory-like behaviors immediately after injection, such as open beak, tachypnea and epileptic crisis. Approximately three minutes after PTX injection, the birds fell on the cage grid and presented a limping walk followed by intense tremors. Seizures were easily elicited by noise stimuli. A pretreatment with BCC (4 mg/kg) did not modify ruZing, ROM or blinking responses following THIO administration (Fig. 4a–c). BCC attenuated the closure of eyes induced by THIO administration at 0–10 min (8.2 § 1.0 vs. 3.0 § 1.0, P < 0.05). PTX did not modify ruZing, ROM or blinking responses following THIO administration (Fig. 4a–c). Curiously, PTX intensiWed the closure of eyes when combined with a THIO administration at 0–10 min (8.2 § 1.0 vs. 17.8 § 1.5, P < 0.01) and at 10–20 min (2.3 § 1.3 vs. 12.4 § 2.7, P < 0.01) (Fig. 4d). A high frequency of yawning was observed throughout the hypnogenic actions of THIO and after the intermediate dose of PTX (data not shown).

Discussion Studies of the eVect of GABA signaling on sleep behaviors in birds have frequently employed the antagonist bicuculline and the agonist muscimol. Most of these studies have reported on electromyography and electroencephalography parameters (Colom and Saggau 1994; Bormann 2000; Gottesmann 2002). In our study, we studied the role of GABAA receptor on sleep-like behaviors by using an allosterical modulator (thiopental that potentiates the of Fig. 4 EVect of bicuculline (BCC, 4 mg/kg, sc) or picrotoxin (PTX, 4 mg/kg, sc) treatments on the frequency of sleep-like behaviors induced by thiopental (20 mg/kg, ic). Data are presented as mean § SEM. ** P < 0.01 compared to control group (one-way ANOVA)

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GABAergic transmission) and antagonists (picrotoxin and bicuculline, silent GABA-A receptor antagonists) in quails. Thiopental has been conWrmed to induce hypnogenic eVects in quails, similar to what has been observed in mammals (Radil-Weiss and Stýblová 1967; Yamamoto 2005). At the dose employed, the barbiturate was found to induce an increased frequency of eye closure with rare preliminary sleep-associated behaviors, such as those provoked by serotonin, a biogenic amine that crosses the blood-brain barrier in quails following peripheral administration (Polo et al. 2007). Throughout diurnal resting state, quails exhibit some components of sleep, such as ROM and blinking, at regular intervals. Drowsiness has also been reported in other diurnal birds (Fuchs et al. 2006; Malleau et al. 2007). BCC treatment did not signiWcantly aVect these brief episodes of a preliminary stage of sleep in the present study. Therefore, it seems that in diurnal resting, bicuculline does not perform an intrinsic GABAA antagonist action in quails. Bicuculline combined with thiopental, did not aVect the responses achieved by the barbiturate in ROM and closure of eyes. In domestic fowl, a comparable dose of bicuculline antagonized the GABA and benzodiazepine-induced sleep. In addition, the GABA-induced decrease in electromyography activity and synchronization of the electroencephalography in chickens were antagonized by bicuculline (Wambebe 1983). Picrotoxin (a receptor GABAA antagonist) treatment exhibited intriguing and seemingly paradoxical results. A convulsive eVect was observed at a high dose, as reported for rats and chickens (Jayakumar et al. 1999), while an intermediate dose produced sleep-like behaviors.

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These puzzling eVects of picrotoxin probably indicate that there are two diVerent GABAA receptor sites in the brain of quails with distinct threshold levels. In this context we can not discard the hypothesis that picrotoxin acts on two diVerent brain areas or neuronal circuitries implied in opposite control mechanism. Concerning the thiopental results, it is interesting to note that the dose used (20 mg/kg) caused hypnogenic eVects only in the Wrst 10 min, while this same dose is an anesthetic for chickens when administered by an intra-osseous route (Valverde et al. 1993). In another gallinaceous animal, the turkey, thiopental injection by an intracoelomic route demands a higher dose (50 mg/kg) to cause an anesthetic eVect (Ayala-Guerrero et al. 2003). Takagi et al. (2003) investigated the interactions between pipecolic acid (a major metabolic intermediate of lysine that enhances the release and inhibits the uptake of GABA) and picrotoxin in neonate chickens. These authors showed that pipecolic acid stimulates the closure of eyes while the birds maintain a squatting position and decrease the frequency of open eyes in an upstanding position (related to sleep and awakening, respectively). Picrotoxin administration signiWcantly attenuates the sleep-like behavioral response. In our study, the picrotoxin and thiopental interaction was examined by observing other behavioral categories. In contrast to the results of Takagi et al. (2003), we observed that a higher dose of picrotoxin acts as an excitatory agent. There was a critical dose range for picrotoxin protocols. A high dose was clearly excitatory, exempliWed by seizure; this convulsing dose of picrotoxin resulted in an increase in wakefulness and a concomitant decrease in all sleep states (Langebartels et al. 2001), in accordance with previous data from Ali et al. (1999) and Lancel et al. (1999). Our study has shown, for the Wrst time, that in quails sleep-like behaviors are expressed upon GABAA receptor activation. On the other hand, we can not discard the probability that other hypnogenic mediators without GABAergic properties could induce sleep-like behaviors in quails. Thiopental induced sleep-like behavior resembled that raised by peripheral serotonin administration (Polo et al. 2007). It remains to be determined how the thiopental action, GABAergic activity and serotonergic stimulation interact and further how an individual pharmacological blockade inXuences the eVect of a following activation of either serotonin or GABAA receptors. Taken together, we deduced from behavior that GABAA receptors in quails may express diVerent sites for picrotoxin that have not yet been reported for mammals. Both barbiturate and picrotoxin-induced sleep-like responses have similar behavioral neuropharmacological properties, perhaps because they share an identical site on a GABAA receptor.

251 Acknowledgments The authors are grateful to Mr. Ipojucan P. Souza for animal care and technical assistance. This research was in part, supported by Conselho Nacional de Desenvolvimento CientíWco (CNPq). Animal handling and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996) and conforms to pertinent Brazilian legislations and was approved by the institutional animal bioethics and welfare committee. Finally we are indebted to American Journal Experts for reviewing the manuscript.

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