Intracerebroventricular Administration ofGABA-A and GABA-B Receptor AntagonistsAttenuate Feeding and Sleeping-LikeBehavior Induced by L-Pipecolic Acid inNeonatal Chicks

T. Takagi,1 T. Bungo,2 T. Tachibana,1 E-S. Saito,1 S. Saito,1 I. Yamasaki,1

S. Tomonaga,1 D.M. Denbow,3 and M. Furuse1*1Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource andBioenvironmental Sciences, Kyushu University, Fukuoka, Japan2Laboratory of Animal Science, Department of Agrobiological Science, Faculty of Agriculture, EhimeUniversity, Matsuyama, Japan3Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University,Blacksburg, Virginia

It has been demonstrated that L-pipecolic acid (L-PA), amajor metabolic intermediate of L-lysine (L-Lys) in themammalian and chicken brain, is involved in the func-tioning of the GABAergic system. A previous study hasshown that intracerebroventricular (i.c.v.) injection ofL-PA suppressed feeding and induced sleep-like behav-ior in neonatal chicks; however, the precise relationshipbetween the GABAergic system and L-PA has not beenclarified. In the present study, the role of the GABA-A orGABA-B receptors in the suppression of food intake andinduction of sleeping-like behavior by L-PA was investi-gated. Chicks were injected i.c.v. with the GABA-A an-tagonist picrotoxin or GABA-B antagonist CGP54626along with L-PA. Although suppression of food intake byL-PA was restored partially by co-injection withCGP54626, but not picrotoxin, sleep-like behavior in-duced by L-PA was suppressed significantly by bothantagonists. These results suggested that L-PA activatedboth GABA-A and GABA-B receptors, and GABA-B re-ceptors alone contributed to food intake whereas bothreceptors contributed to sleep-like behavior.© 2003 Wiley-Liss, Inc.

Key words: L-pipecolic acid; intracerebroventricular in-jection; GABA receptor; food intake; sleeping-like behav-ior

L-pipecolic acid (L-PA) is a major metabolic inter-mediate of L-lysine (L-Lys) in the mammalian (Schmidt-Glenewinkel et al., 1977; Giacobini et al., 1980) and chickbrain (Nomura et al., 1978). The major pathway of L-Lysmetabolism in peripheral tissue is through the intermediatesaccharopine (�-N-[glutaryl-2]-L-lysine), but this path-way is not as active in the brain (Hutzler and Dancis,

1968). L-PA is incorporated into the crude synaptosomalfraction (Meek, 1974), and stimulates some brain sites inthe rat (Kase et al., 1980). Therefore, L-PA itself, as well asthe L-PA pathway, may be particularly important in thebrain, and be involved in the feeding regulation as one ofthe physiological functions of L-Lys. We demonstratedpreviously that L-PA injected intracerebroventricularly(i.c.v.) had several physiological actions, including reduc-tion of food intake and induction of sleep-like behavior inthe neonatal chick (Takagi et al., 2001). In addition, L-PAconcentration in several sites in mouse brain fluctuateddepending on feeding conditions (Nishio et al., 1986).Thus, endogenous L-PA, at least, may function as suppres-sive factor for food intake.

It has been reported also that L-PA was able toincrease the release of �-aminobutyric acid (GABA) stim-ulated by mild depolarization with potassium from brainslices and decrease the uptake of GABA by brain (Guti-errez and Delgado-Coello, 1989). In mammals, i.c.v. in-jection of GABA or inhibition of GABA-transaminasedecreased food intake (Olgiati et al., 1980) and increasedslow-wave sleep with no rebound effect (Karadzic, 1966;Holmes and Sugden, 1975). Therefore, the decrease infood intake and induction of sleep-like behavior by L-PA

Contract grant sponsor: Japan Society for the Promotion of Science.

*Correspondence to: Dr. Mitsuhiro Furuse, Laboratory of Advanced An-imal and Marine Bioresources, Graduate School of Bioresource and Bio-environmental Sciences, Kyushu University, Fukuoka 812-8581, Japan.E-mail: furuse@brs.kyushu-u.ac.jp

Received 7 January 2003; Revised 25 February 2003; Accepted 15 March2003

Journal of Neuroscience Research 73:270–275 (2003)

© 2003 Wiley-Liss, Inc.

may have resulted from augmentation of GABA action. Inthe case of feeding behavior, however, the i.c.v. admin-istration of muscimol, a GABA-A receptor agonist, andbaclofen, a GABA-B agonist, inconsistently increased foodintake (Olgiati et al., 1980; Baldwin et al., 1990; Ebenezer,1990; Ebenezer and Baldwin, 1990). In chickens, musci-mol also increased food intake (Jonaidi et al., 2002). More-over, the differences in functions of the GABA-A receptorand GABA-B receptor have also been demonstrated. Forexample, muscimol injected into the amygdala decreasedfood intake, whereas baclofen increased it (Minano et al.,1992). Baclofen had no effect whereas muscimol decreaseddesynchronized sleep when administered into the nucleusbasalis of Meynert (Manfridi et al., 2001). Currently, thereare no studies investigating the relationship between L-PAand the GABAergic system on ingestive and sleep behav-ior in neonatal chicks. Therefore, the aim of present studywas to clarify whether the reduction in food intake andinduction of sleep-like behaviors by L-PA are associatedwith GABAergic neurotransmission, and if so, whichGABA subtype receptor activation is concerned with be-havior.

MATERIALS AND METHODS

Animals and Food

One-day-old male broiler and layer chicks were pur-chased from a local hatchery (Mori Hatchery or Murata Hatch-ery, Fukuoka, Japan) and housed in a windowless room at aconstant temperature of 29 � 1°C. Continuous lighting wasprovided. The birds were given free access to a commercialstarter diet (Toyohashi Feed and Mills, Aichi, Japan) and water.Chicks were reared individually beginning one day before theexperiment. On the day of the experiment, chicks were distrib-uted into groups based on body weight such that the averagebody weight between groups was as uniform as possible withinthe same experiment. Experimental procedures followed theguidance for Animal Experiments in Faculty of Agriculture andin the Graduate Course of Kyushu University and the Law (No.105) and Notification (No. 6) of the Government.

Preparation of Drugs

L-PA was purchased from Bachem (Bubendolf, Switzer-land). The GABA-A receptor antagonist picrotoxin was pur-chased from Wako Pure Chemical Industries (Osaka, Japan), andthe GABA-B receptor antagonist CGP54626 was purchasedfrom Tocris (Bristol, UK). Drugs were dissolved in 0.85% salinecontaining 0.1% Evans Blue solution and administered i.c.v. intothe right lateral ventricle of the birds using a microsyringeaccording to the method of Davis et al. (1979). The injectionwas administered over 30 sec, and the volume was 10 �l.Control groups were given the saline solution containing EvansBlue.

Experiment 1: Effect of Picrotoxin on Decreased FoodIntake Induced by L-PA in Neonatal Chicks

This experiment was done using broiler and layer chicks.The birds (2-day-old broiler or 5-day-old layer chicks) werefasted for 3 hr but given free access to water. Chicks were

injected i.c.v. with a mixture of L-PA (1.0 mg) and two levels(0.9 or 1.8 �g) of picrotoxin, L-PA alone, or saline as a control.In layer chicks, the levels of picrotoxin were 0.5 or 1.0 �g.Chicks were given access to food post-injection, and intake wasmeasured at 30, 60, and 120 min for broilers and at 30, 60, 90,and 120 min for layer chicks. At the end of experiment, the birdswere decapitated after an overdose of sodium pentobarbital.

Experiment 2: Effect of CGP54626 on Decreased FoodIntake Induced by L-PA in Neonatal Chicks

This experiment was similar to Experiment 1 except thatCGP54626 (10.5 and 21.0 ng) was substituted for picrotoxin. Inaddition, 3- and 6-day-old broilers and layers, respectively, wereused in this experiment.

Experiment 3: Effect of Picrotoxin on Sleep-LikeBehavior Induced by L-PA in Neonatal Layer Chicks

The birds (3- and 4-day-old) were divided into fourgroups that received either saline, 1.0 mg of L-PA, 1.0 �g ofpicrotoxin, or L-PA plus picrotoxin. The birds were fasted for3 hr but given free access to water. They were then injected(i.c.v.), returned to their original cage for 10 min post-injectionto exclude possible convulsions caused by picrotoxin, and thenplaced in a monitoring cage (40 cm � 30 cm � 20 cm). Thebehavior of each bird was recorded for 10 min using a videorecording set. At the end of observation, the birds were sacri-ficed as described above. The behaviors were classified into thefollowing four behavioral categories: (1) standing with eyesopened; (2) sitting with eyes opened; (3) standing with eyesclosed; (4) sitting with eyes closed.

Experiment 4: Effect of CGP54626 on Sleep-LikeBehavior Induced by L-PA in Neonatal Layer Chicks

This experiment was similar to Experiment 3 except thatpicrotoxin was replaced with CGP54626 (10.5 ng).

Statistical Analysis

Data were deleted for those individuals that were notverified as having Evans Blue dye present in the lateral ventricle.Chick food intake data were subjected to repeated measuretwo-way analysis of variance (ANOVA) using a commerciallyavailable package (StatView, version 5, SAS institute, Cary,NC). Significant alternations of L-PA-suppressed feeding rela-tive to corresponding saline control were detected using theTukey-Kramer test (P � 0.05). Dunnett comparisons (P� 0.05)were employed to examine significant alterations induced byco-injection of GABA antagonists compared to L-PA-suppressed food intake. A two-way ANOVA was applied toassess the effects of L-PA, GABA antagonist, or interactionsbetween L-PA and GABA antagonists on behavior of chicks, andcomparisons between means of L-PA and L-PA plus GABAantagonist group were carried out by t-test (P� 0.05) in cate-gories where significant effects were determined for interactionsbetween L-PA and GABA antagonists. In all experiments, out-liers were removed from data by Thompson’s test at P� 0.01.

RESULTSFigure 1 shows the effect of i.c.v. administration of

picrotoxin on reduced food intake induced by L-PA in the

L-Pipecolic Acid and GABAergic System 271

neonatal broiler (top) or layer (bottom) chicks. The effectof treatments was significant for food intake in the neo-natal broiler chicks (F[3,28] � 8.162; P� 0.001). A sig-nificant (F[6,56] � 4.471; P� 0.001) interaction betweendrug and time was detected. Food intake at 30 and 60 minwas suppressed by the i.c.v. injection of L-PA, but nosignificant effects were observed at 120 min. The effect ofL-PA was not attenuated by co-injection of picrotoxin. A

similar trend was observed in layer chicks. The effect ofdrug (F[3,24] � 9.827; P� 0.001) was significant. Asignificant (F[9,72] � 2.893; P� 0.01) interaction be-tween drug and time was also detected. Food intake of thelayer chicks at 30, 60, and 90 min was suppressed by L-PAinjection, but this suppression was not affected by co-injection with picrotoxicin.

Figure 2 shows the effect of i.c.v. administration ofCGP54626 on reduced food intake induced by L-PA inthe neonatal broiler (top) or layer (bottom) chicks. Themain effects of treatments were significant in the neonatalbroiler (F[3,44] � 7.246; P� 0.001) and layer (F[3,25] �11.029; P� 0.0001) chicks. A significant (F[9,75] �2.961; P� 0.01) interaction was detected between drugand time in layer, but not broiler, chicks. The effect ofL-PA was attenuated significantly by the co-administrationof GABA-B antagonist at 30 and 60 min in the broilerchicks. This effect was not observed in the layer chicks,although the food intake of the L-PA plus CGP54626(10.5 ng) group at 90 min tended to be increased whencompared to the L-PA group.

The effect of i.c.v. administration of picrotoxin onbehavior induced by L-PA in neonatal chicks is shown inTable I. Interactions between L-PA and picrotoxin werestatistically significant in the following categories: standingwith eyes opened, standing with eyes closed, and sittingwith eyes closed (F[1,25] � 4.765, 6.851, and 14.174,respectively; P� 0.05, P� 0.05, P� 0.001, respectively).L-PA particularly increased the behaviors sitting with eyesclosed and decreased standing with eyes opened (regardedas sleeping posture and waking posture, respectively), andpicrotoxin attenuated the effects. Because picrotoxinhardly affected the two behavioral categories under thecontrol condition, it indicated that picrotoxin altered be-haviors induced by L-PA without altering behavior per se.Taken together, sleeping posture induced by L-PA wasattenuated by co-injection with picrotoxin and conse-quently, the behavior of chicks tended toward wakeful-ness.

Table II shows the effect of CGP54626 on behaviorcaused by L-PA. Significant interactions between L-PAand CGP54626 were detected in the behaviors of standingwith eyes closed and sitting with eyes closed (F[1,23] �10.794 and 11.142, respectively; P� 0.01 and P� 0.01,respectively), and a nearly significant interaction for stand-ing with eyes opened (P� 0.059). L-PA strongly affectedbehaviors of sitting with eyes opened and standing witheyes opened, and CGP54626 attenuated the effects asobserved with picrotoxin. These results were similar tothose in Experiment 3, and implied that sleep-like behav-ior induced by L-PA was attenuated partially byCGP54626.

DISCUSSIONThis study provides evidence that suppression of

food intake and induction of sleep-like behavior by L-PAinvolved the GABAergic system in neonatal chicks. It hasbeen demonstrated that i.c.v. injection of L-PA suppressedfood intake and induced sleep-like behavior in the neo-

Fig. 1. Cumulative food intake of the neonatal broiler or layer chicksintracerebroventricularly injected with saline, a mixture of picrotoxin(broiler: 0.9 or 1.8 �g, layer: 0.5 or 1.0 �g) and L-PA (1.0 mg), or L-PAalone after a 3-hr fast. Means with an asterisk at each time are signifi-cantly different at P � 0.05 compared to the saline group. Values aremeans � SEM. The numbers of birds used were broiler: saline, 10;L-PA, 8; L-PA � picrotoxin 0.9 �g, 6; L-PA � picrotoxin 1.8 �g, 8;layer: saline, 8; L-PA, 8; L-PA � picrotoxin 0.5 �g, 5; L-PA �picrotoxin 1.0 �g, 7.

272 Takagi et al.

natal chick (Takagi et al., 2001). The previous results wereobtained using broiler chicks. Broiler-type chickens havebeen selected intensively for rapid body weight gain andhigh meat yield. Consequently, food intake and growthrate are two- to threefold greater in broilers than in layers.In Experiments 1 and 2, we confirmed that the action ofL-PA was similar in broiler and layer chicks. Induction of

sleep-like behavior by L-PA was also confirmed in layerchicks in Experiments 3 and 4. These results indicated thatthe effect of L-PA was not influenced by genetic selection.

L-PA enhances GABA release and inhibits reuptakein vitro (Gutierrez and Delgado-Coello, 1989), suggestingthat the effect of L-PA might be involved in the GABAer-gic systems. We therefore investigated the effect of co-injection of L-PA with either picrotoxin or CGP54626 onfood intake in Experiments 1 and 2. Intracerebroventric-ular administration of GABA receptor subtype antagonistsexerted differential actions upon food intake reduced byL-PA. Injection of picrotoxin did not attenuate L-PA-suppressed food intake; however, the i.c.v. injection ofCGP54626 partially attenuated the decrease in food intakecaused by L-PA. Preliminary experiments confirmed thati.c.v. administration of CGP54626 alone did not influencefood intake (unpublished data). These results indicatedthat GABA-B receptors are involved in the suppression offood intake caused by L-PA. The following two hypoth-eses may be considered as the functional mechanism. First,although both GABA-A and B receptors were activated bythe increased release and decreased reuptake of GABA byL-PA (Gutierrez and Delgado-Coello, 1989), the activa-tion of GABA-B receptor alone might be responsible forthe reduced food intake. Second, because it was demon-strated that L-PA had no affinity for GABA-A binding sitesbut could displace the specific binding of 3H-baclofen, aGABA-B receptor agonist (Bernasconi et al., 1986), L-PAmight suppress food intake due to an agonistic functionafter binding to GABA-B receptor; however, we couldnot verify which hypothesis is applicable for food intakefrom the results mentioned above.

Subsequently, we investigated the effect of picro-toxin or CGP54626 on sleep-like behavior caused byL-PA in chicks. These experiments indicated that blockingof either the GABA-A or GABA-B receptor could atten-uate the behavioral alterations induced by L-PA (particu-larly an increase of sitting with eyes closed). Because thetwo receptor blockers produced similar effects on theL-PA-induced sleep-like behavior, the effect seems medi-ated by both GABA-A B receptors. These responses weresomewhat different from the response to feeding behavior.These results implied that suppression of food intake byL-PA did not necessarily result from induction of sleep-likebehavior, and the action site of L-PA might vary accordingto each behavior. Moreover, these results also suggestedthat i.c.v. injection of L-PA led to the activation of bothGABA-A and GABA-B receptors. GABA-A andGABA-B receptors were distributed extensively withinthe brain (Chu et al., 1990). Consequently, L-PA mayactivate both GABA-A B receptors in many brain sites.Thus, the first hypothesis described above may be themain mechanism for suppression of food intake by L-PA.Indeed, the i.c.v. injection of baclofen, a GABA-B recep-tor agonist, reduced food intake in the neonatal broiler andlayer chicks, but muscimol, a GABA-A receptor agonist,had little effect on feeding (unpublished data). This impliesthat direct activation of the GABA-B, but not the

Fig. 2. Cumulative food intake of the neonatal broiler or layer chicksinjected intracerebroventricularly with saline, a mixture of CGP54626(10.5 or 21.0 ng) and L-PA (1.0 mg), or L-PA alone after a 3-hr fast.Means with asterisks at each time are significantly different at P � 0.05compared to the saline group, and daggers indicate that CGP54626co-injection groups were significantly different at P � 0.05 comparedto only L-PA group. Values are means � SEM. The number of birdsused: broiler: saline, 10; L-PA, 14; L-PA � CGP54626 10.5 ng, 8;L-PA � picrotoxin 21.0 ng, 16; layer: saline, 7; L-PA, 9; L-PA �CGP54626 10.5 ng, 6; L-PA � CGP54626 21.0 ng, 7.

L-Pipecolic Acid and GABAergic System 273

GABA-A receptor, was involved in the decrease of foodintake in chicks. Consequently, the activation of GABA-Breceptor should be responsible for the decrease in foodintake induced by L-PA. Taken together, L-PA activatedboth GABA-A and GABA-B receptors, but GABA-Breceptors alone contributed to the decrease in food intake,whereas both receptors mediated sleep-like behavior inchicks.

In mammals, GABA receptors are associated withfeeding behavior, and have hypnotic effects when stimu-lated (Gottesmann, 2002). The i.c.v.-injected GABA-Aand GABA-B receptor agonists increased food intake (Ol-giati et al., 1980; Baldwin et al., 1990; Ebenezer, 1990;Ebenezer and Baldwin, 1990). In addition, inhibition ofGABA-B receptor decrease wakefulness, and increasenon-REM and REM sleep (Puigcerver et al., 1996).Therefore, the actions of GABA receptors might varysomewhat between mammals and birds. Compared tomammals, however, it has not been fully elucidated howGABA receptors are involved in the behavior (especiallysleep) of the bird. Thus, further studies will be necessary toclarify the functions of GABA receptors in chicks.

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TABLE I. Influence of Intracerebroventricular Administration of Picrotoxin and L-PA on Various Behaviorsof Neonatal Layer Chicks†

Picrotoxin

Saline L-PA P

0 1.0 0 1.0 L-PA PicrotoxinL-PA �

picrotoxin

Standing with eyes opened 600 � 0 571 � 16 0 � 0 151 � 83 �0.001 NS0.149

�0.05

Sitting with eyes opened 0 � 0 29 � 16 138 � 24 162 � 64 �0.001 NS0.445

NS0.941

Standing with eyes closed 0 � 0 0 � 0 0 � 0 122 � 48* �0.05 �0.05 �0.05Sitting with eyes closed 0 � 0 0 � 0 462 � 24 165 � 78** �0.001 �0.001 �0.001Total 600 600 600 600†The numbers of birds used were: saline, 9; picrotoxin, 7, L-PA, 6; L-PA � picrotoxin, 7. Values (second) are means � SEM. NS, not significant.*Significant difference between L-PA and L-PA plus picrotoxin at P � 0.05.**Significant difference between L-PA and L-PA plus picrotoxin at P � 0.01.

TABLE II. Influence of Intracerebroventricular Administration of CGP54626 and L-PA on Various Behaviorsof Neonatal Layer Chicks†

CGP54626

Saline L-PA P

0 10.5 0 10.5 L-PA CGP54626L-PA �

CGP54626

Standing with eyes opened 563 � 19 600 � 0 0 � 0 173 � 74 �0.001 �0.01 NS0.059

Sitting with eyes opened 2 � 2 0 � 0 58 � 15 47 � 29 �0.01 NS0.705

NS0.780

Standing with eyes closed 35 � 18 0 � 0 0 � 0 137 � 55* NS0.065

NS0.065

�0.01

Sitting with eyes closed 0 � 0 0 � 0 543 � 15 243 � 99** �0.001 �0.01 �0.01Total 600 600 600 600†The numbers of birds used were: saline, 7; CGP54626, 6; L-PA, 8; L-PA � CGP54626, 6. Values (second) are means � SEM. NS, not significant.*Significant difference between L-PA and L-PA plus CGP54626 at P � 0.05.**Significant difference between L-PA and L-PA plus CGP54626 at P � 0.01.

274 Takagi et al.

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