Kabai Met Amphetamine Chick Dec Ere Brat Ion

8/6/2019 Kabai Met Amphetamine Chick Dec Ere Brat Ion

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Neurochemical Research, Vol.24, No. 12, 1999, pp . 1563-1569

M ethamphetamine-Induced Stereotypies in Newly-HatchedDecerebrated Domestic ChicksPeter Kabai,1,3 Andras Liker,1 and Andras Csillag2

(Accepted June 19 , 1999)

Metam phetamine in high dose has been reported to induce stereotypic behavior of abnorm al formin the pigeon and dom estic chick. A numb er of reports suggested that the target of metamp heta-mine was the paleostr iatal com plex, the highest motor center of the avian brain. The present studytested this hypo thesis by treat ing newly-hatched domestic chicks with high dose of metamp het-amine (10 mg/kg b.w.)after complete decerebrat ion or sham operat ion. Stereotypic mandibula-t ions were observed both in sham-operated and in decerebrated birds in similar form followingmethamphetamine treatment. The results suggested that brainstem pattern generators remain re -sponsive to dopaminergic s t imul i in the absence of the main telencephalic (striatal) targets.

K E Y W O R D S : Domestic chick; metham phe tam ine; dopam ine; basal ganglia; paleostriatum; stereotypicbehavior .

IN TR ODU C TIONCatecho laminerg ic sys tems know n to be involved

in attention and arousal are highly conserved in ver-tebral evolution. Of these, the dopaminergic systempredominantly controls motor activit ies includinghighly stereotypic movements. Particular attention hasbeen paid to the ascending pathways ar ising fromth e substantia nigra and ventral tegmentum and termi-nating in the striatum and nucleus accumbens, respec-t ively. Birds have been demonstrated to possessdopaminergic perikarya and fibers (1-5) and dopamineD1 and D2 receptors, highly enriched in the str ia talregions. D 1 receptor binding in the medial striatalregion, lobus parolfactor ius (LPO), was altered as aresult of passive avoidance learning (6).In most studies1 Behav iora l Biology Research Group, Department of Ecology, Un i-

versity of Veter inary Science , Bud apest , H unga ry .2 Department of Ana tom y, H is to logy an d Emb ryology, SemmelweisUnivers i ty of Medicine , Budapest , Hu ngary.3 Address reprint requests to: Peter Kabai, Department of Ecology,Univers i ty of Veterinary Science, 1400 Budapest , P.O.Box 2. ,Hungar y , e-mail: pka ba i @ ns .un i ve t .hu tel.: (36) 1-3222-660.

stimulation of dopaminergic activity was achievedby agents which block the reuptake of dopamine and,to some extent, other catecholamines. Such agents(amphetamine derivates, apomorphine) have beenshown to enhance attention to stimuli (7) and motoractivity (8) of domestic chickens. Dopamine was alsoimplicated in various forms of stereotyped behaviorof birds (9,10,11,12). Kainic acid lesions of the pale-ostriatum in pigeon resulted in similar behavior def i-ciencies (circling, postural disturbances etc.) as inmammals (13).Young domestic chicks failed to per-form imprinting related approach response followingthe removal of the paleostriatal complex (14).Suchbehavioral and anatomical evidence confirmed thenotion that the paleostriatal complex is the highestmotor center of the avian brain (15) and is critical incontrolling behavior patterns mediated by dopam inergicsystems.On th e other hand, Kabai an d Kovach demon-strated, that contrary to previous f indings (14), com-plete decerebration, including the removal of the pale-ostr ia tal complex did not diminish imprinting relatedapproach response nor affected open-f ield activity or

15630364-3190/99/1200-1563$16.00/0 1999 Plenum Publishing Corporation


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1564 Kabai, Liker, an d Csillag

tonic imm obili ty (16) , and even color and pattern dis-cr imination w as spared (17) in Japanese quail chicks,wh en thalamic/hypothalamic structures were notdamaged by the operation. Moreover , at least in onestudy, the predominantly dopaminergic agent am-phetamine was effective in increasing forced loco-motion, head shakes and wing drooping though notpecking in decerebrated domestic chicks (18) .The question rises whether these observed dopa-mine related effects are dependent on the integrityof tegmentostr ia tal pathways or else dopaminergiceffects on avian behavior may be elicited also in theabsence of striatum. One possible approach to thisproblem, overcoming the difficulty in achieving iso-lated yet complete striatal lesions, is total telecephalicablation, which in contrary to mammals, leaves th emajori ty of sensory and locomotor functions intact, atleast in youn g nidifugous birds (16-18). Should a con-siderable portion of dopaminergic response persist indecerebrated birds, it could be concluded that dopa-mine fibers terminatin g in subtelencephalic areas areinstrumental in that particular task. Decerebration istechnically feasible in chicks at a very early age, sur-vival rates being considerably greater than in adults.Dopamine receptors are present in post-hatch chicks(6,19,20), and D2 sites did not show significantchanges within the first four posthatch days (21). Fora relevant comparison w ith previous ex perimentsperformed on young birds, in the present study, weinvestigated the effect of the predominantly dopamin-ergic st imulant methamphetamine on simple behav-ioral events of normal an d decerebrated domestic chicksat the age of 3 days.E XPE R IME N TAL PR OC E DU R E

Animals. One day old Hunnia broiler chicks were purchasedfrom a commercia l ha tchery. The chicks were kept in groups of 15under warm lamps an d were provided with water an d pelleted foodad libitum. Fourty chicks were random ly assigned to two group s oneof which was sham operated and the other one was decerebrated.Twen ty chicks were anesthetized by IM injection of ketamine-xylazine mixtu re (3 mg-0.02 mg/10 g. b.wt), and a circular incision(approx. 5 mm in diameter) was made on the frontal bone of theskull above th e ventral portion of the telencephalon. In the shamoperated group th e cutaneous incision w as closed by tissue adhesivean d chicks were allowed to recover from anesthesia.Surgery. For decerebration the telencephalic brain tissue wascompletely removed rostral and dorsal to the tractus septomesen-cephal icus except for an approx. 1 mm thick layer of forebrain tis-sue spared at the telencephalon/diencephalo-mesencephalic jun ctionto prevent damage to the underlying hypothalamic/thalamic struc-tures. Our previous studies showed, that the spared tissue layer aswell as the tractus septomesencephalicus degenerates rapidly after

such surgery (16). The extent of the lesions was checked w ith post-mortem craniotomy and macroscopic inspection (Fig. 1). Followingperfusion with 4% buffered paraformaldehyde, horizontal sectionsof 10 um w ere cut with a freezing microtom e and stained with cresyl-violet (Fig. 2 and 3). The telencephalic hemispheres including thestriatum an d pallidum were absent after th e lesions, only part of theseptum remaining intact. The diencephalon and brainstem weremacroscopically intact, however, considerable retrograde degenera-tion in some subtelencephalic areas fol lowing such ablations wasreported in a previous s tudy (16).Behavioral Test. Open-field test was performed one day afterthe surgery. The open-field arena was a 40 x 40 x 30 cm tan card-board box. The floor of the box w as divided into nine, 13 x 13 cmsquares by white masking tape. Chicks were pu t individually in themiddle of the box, and behavioral m easuremen ts started 20 sec. afterthe placement of the chick. Occurrence of 15behavior elements andmotor activity w ere recorded for 20 m in. by an observer sitting 2 maway from the box. The observer were blind to the drug treatment.Behavio ral categories to be recorded and the dose of drug to beapplied were established during a preliminary experiment using dif-ferent doses of metamphetamine (0.1, 1, and 10 mg/kg body weight)in 5-5 unoperated chicks. Metha mph etamine in lower doses resultedin some increase in locomotion and pecking, but caused no alterationof the form of any behavior . Following the injection of 10 mg/kgb.w. methamphetamine behaviors of abnormal form appeared (open-pecking, upw ard movement of body , etc.). Since our aim was to testany specific effects of the dopaminergic drug on telencephalec-tomized birds we used methamphetamine only in the higher dose(10 mg/kg b.w.) in the main experiment.Some of the behaviors such as "preening or wiping the beak","body or wing shaking", "scratching", "pecking wall or floor","defecation", "eye closure" an d "head shaking" occurred in a regu-lar pattern and were identical to categories traditionaly used open-field studies in chicks (9,16).However , fol lowig metamphetamine trea tment , behaviors ofabnormal forms similar to drug induced orofacial movemen ts in mam-mals also occured, which need further explanation. "Yawning" w asdefined as opening the beaks wide. "Vacuum preening" occurred inform similar to normal preening but witho ut the beaks touching thebody. "Upward movement of the body" was scored when the birdsstretched their body vertically. This m ovemen t sometimes ended withquick steps to one side ("loosing balance"). Methamph etamine treatedbirds often made movements which started similar to pecking butwithout the beaks closing ("open-pecking"). Such open-pecking waseither oriented to the wall or floor ("oriented open-pecking") orhappened in the air without touching an y surface ("unoriented open-pecking"). One behavior characterized by quick, alternative openingand closing the beaks w ithout head m ovement ("chewing") was omit-ted from analyses because it occurred only in a few birds with lo w fre-quency. Emittance of any sound was scored as "vocalization", be-cause we did not categorize peeps, trills etc. into different classes.Locomotor activity was m easured by the num ber of squares entered.

Drug t reatment . Decerebrated and unoperated birds (D and Utrea tment , respect ive ly) were injec ted with methamphetamine(1 0 mg/kg b.w.) or saline intraperitoneously (M and S trea tment ,respectively) 1 min. before th e open-field test (1 0 chicks in eachtreatment groups, DS, DM, US, UM).Statistical Analysis. Treatment effects were analyzed statisti-

cally by comparing frequencies of each behavior category amongthe experimental groups. Nonparametric tests were used because thedistribution of behavior variables violated the normality assumptionof parametric tests (22). We tested for overall differences among


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Methamphetamine-Induced Behavior in Newly Hatched Decerebrated Chicks 1565

Figs. 1-3. Representat ive photographs demonstrat ing the extent of brain t i ssue lesion fol lowing telencephal ic ablat ion in day-olddomestic chicks. Fig. 1. M acroscopic ph otograph sho wing the anatom ical lesion site one day after surgery, s - septum, t - thala mu s,o - optic lobe, c - cerebel lum, m - medulla oblongata. Fig. 2. Microphotograph of Nissl stained horizontal section showing th e lesionone day after surgery. The su rface of lesion is marked by arrowhe ads, v - third ventricle, rot - nuc leus rotund us, Ipc - nucleus isthm iparvocellularis, vt - tectal ventricle, ot - optic tectum, flm - fasciculus longi tudinalis m edial is . Bar : 1 m m . Fig. 3. Microphotograph ofNissl stained horizontal section showing the lesion five days after surgery. The surface of lesion is marked by arrowheads. Note thepromi ne nt distensio n of the ventricles , s - septum , v - third ventricle, t - thalam us, pt - pretectum , vt - tectal ventricle, ot - optic tectum.Bar: 1 m m

t reatment groups by Kr uska l - Wal l i s one- way A NOVA . If a signifi-cant overall effect w as found w e conducted Mann-Whitney U- testfo r 4 combinat ions of the groups (US/DS, UM/DM, US/UM andDS /DM) to detect significant differences related to surgery or drugtreatment. As "familywise" error may inflate the true level of sig-nificance in such post-hoc mu l t ip le comparisons (23), we identifieda (c = 4) = 0.0127 as a critical level of significance for the pa i r wi secomparisons. One chick represents one data point in each test. Mean SE and two-tailed probabilities are given.

RESULTSWe found significant differences among treat-ment groups in al l behaviors (Kruskal-Wall is tests :

x2 = 7.909 - 27.098, df = 3, P < 0.05) except in the

f requency of yawning (x2 = 4.194, df = 3, P > 0.2). A l-though there were significant differences among thegroups in the frequency of vacuum preening (x 2 =12.955, P < 0.01), loosing balance (x2 = 16.619, P 0.241 an d UM/DM:z = 2.234 ,P > 0.025, respectively, Table I), because oflarge i ndi vid ual var iations and the inf la t ion of signif i-cance after correction for pairw ise comparison.

DISCUSSIONSA variety of dopaminergic responses have beenreported in birds, including an increase of locomotion,

pecking and vocalization (7), as well as init ia tion ofstereotyped behaviors (9). Some of these effects arelikely to depend on the birds ' attention to stimuli (7).Similarly, an impairment of mesotelencephalic dopa-mine i nput resulted in a sensory inattentio n in rats (24).The dopaminergic st imulant apomorphine w as found toenhance motor activity and to decrease th e duration oftonic immobility in 3 to 5-week-old chickens (8).T he present findings indicate that th e metham-phetamine administered ip. is able to elicit markedchanges of stereotyped behavior even in the absenceof telencephalic, in particular striatal structures. Thisobservation is in agreement with one previous study(18) according to which amphetamine-induced forcedlocomotion an d head shakes were still present after ab -lation of the cerebral hemispheres. At the concentrationof methamphetamine applied in our study we found typ-ical drug induced mandibulations, such as open peck-ing, both in the nonoperated and decerebrated birds.


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1568 Kabai, Liker, and Csillag

Although some areas controlling elementary be-havioural responses, such as gaze stabilisation havebeen localised (Burns, wallman 261. old.,) in the brain -stem, areas organising pecking have not yet beenanatomically identif ied. Our f inding that a funct ionaltegmento-str ia tal pathway is not essential for metam-phetamine induced stereotypies, might focus the at-tent ion on catecholaminoceptive, more specificallydopaminoceptive subtelencephalic regions. There isconsiderable projection from nigral and ventral tegmen-tal areas to subtelencephalic targets in birds. In thepigeon, efferent fibers from A V T neurons reach th ecentral gray, pretectal areas, th e lateral habenular nu-cleus, dorsomedial an d dorsolateral thalamus as wellas the lateral hypothalamus, whereas those from th enigra (TPc) innervate, among others, th e central gray,nucleus intercollicularis, reticular formation, dorsalthalamus, lateral hypothalamus an d pretectum (25).The presence of TH-positive fibers in subtelencephalicregions has also been reported in pigeon (26), in zebrafinch (3), in quail (2), and in domestic fowl (27). BothD1 and D2 receptors are detectable at the subtelen-cephalic level, in particular in the tegmentum of themidbrain (Stewart, Kabai and Csillag, unpublished),although the studies know n to us have focused attentionon the forebrain (19,28,6,20). Thus, available anatom-ical and functional evidences seem to support that themetamphetamine sensitive pattern generators are oper-ational independent of the striatal system, in the meso-diencephalic region.I t should be noted that the different repeti t ivebehavioral elements were differentially affected bothby ablation and by methamphetamine in the presentstudy. Stereotypies occurring in regular form such aspreening, wing shaking, scratching, vocalization orpecking were enhanced only by detelencephalizationan d not by drug treatment. Conversely, typical "orofa-cial" and postural methamphetamine induced behav-iors, such as open-pecking or upward movement of thebody were increased by methamphetamine but not bysurgery i tself. Thus methamph etamine had an unques-tionable effect on both intact and decerebrated birds,how ever, the results are inconsistent as to w hether sucheffects were quantitatively different in the two groups.Our f indings, that following methamphetamine treat-ment , decerebrated birds tended to target their peck-ing, whereas intact birds performed more non-directedbeak movements is ambiguous, because this differencedi d not reach the level of statistical significance in thefour way comparisons. However, this question is ofsome importance, because cholinergic st imulation ofth e ventrolateral striatum in rodents has been shown

to elic it non-directed mouth movements, quite dis-t inguishable from stimulus-directed, amphetamine-induced biting (29). T he possible difference betweenintact and decerebrated birds in their response tomethamphetamine treatment could be ascribed to thefact, that methamphetamine also affects muscariniccholinergic receptors, abundant in the avian striatum(28,30) in the non-operated birds. However, this doesnot invalidate the basic f indin g that certain b rainstempattern generators remain responsive to cat cholamin-ergic , predominantly dopaminergic , st imuli in theabsence of the main telencephalic (striatal) targets ofthese ascending activator systems. This may be atransient phenomenon typical for the young bird andsuperseded later by the activity of the fully maturetegmentostriatal pathway. In contrast with newbornmamm als, however , most neural sytems of nidifugo usbirds are anatomically and functionaly mature alreadyat the time of hatching. Thus, an alternative hypothesisis that the findings reflect a more lasting but normallylatent feature of motor organization.

AC KN OWLE DG ME N TSThis s tudy w as supported by H unga r i a n Research Fund grants(No. T021008 and No. TO 17754), an d partly by grants from tw o

Hungar ian minis tries (MKM 649 and ETT-500).

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Kabai Met Amphetamine Chick Dec Ere Brat Ion

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