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Concurrent treatment with benztropine and haloperidol attenuates development of behavioral hypersensitivity but not dopamine receptor proliferation
Pergamn Press
Life Sciences,Vol. 42, pp. 2207-2215 Printed in the U.S.A.
Paul M. Carvey, Ana Hitri*,
Christopher G Goetz, Caroline Harold L. Klawans
M. TWmer and
Dept. of Neurological Sciences, Rush Presbyterian St. Lukes Medical Center, Chicago, IL 60612, *Dept. of Psychiatry, Medical College of Georgia, Augusta, CA (Received
in final
form March 28, 1988)
Groups of male rats (n = 16 each) were treated with normal saline, haloperidol (0.75 mg/kg), benztropine (1.8 mg/kg) or haloperidol and benztropine once a day for 24 dqm. Fol lclwing a 96 hour drug free interval, subsets of these animals were assessed for apcmorphine-indmxd (0.75 mg/kg) stereotypic behavior, sacrificed and analyzed for striatal dqmine biochenistry or sacrificed and analyzed for spimperidol binding sites. Benztropine cotreatnent attenuated the developnent of behavioral hypersensitivity to haloperidol but did not alter either the dopanine receptor proliferation or the striatal domine biochenical Changes itiuced by haloperidol. These results suggest that behavioral @persensitivity is not an autanatic nmifestation of dopmine receptor proliferation but nust depend, in part, on other factors.
In 1972, Klawans and RuboVitS &served that chronic treatment of laboratory animals with the neuroleptic agent chlorprmazine produced an increase in behavioral responsiveness to subsequent dopmine DA) agonist challenge (1). This increased responsiveness, or behavioral hypersensitivity (BH), was thought to reflect an increase in W receptor nunber following a period of pharmacologic denervation (2,3,4,5). Klawans and Rubovits further hypothesized that tardive dyskinesia (!L”D),ostensibly a movement disorder reflecting a state of W hypersensitivity in hmans, might also reflect a pharmacologically-induced W receptor proliferation. This line of reasoning has led to the use of neuroleptic treated animals and their subsequent agonist-induced BH ae an animal model of TD. In prior studies, the degree of BH induced by a neuroleptic agent and the degree of D-2 receptor proliferation were correlated in the animal mrsensitivity model (6,7). This led to the suggestion that D-2 receptor proliferation and BH were related in a cause and effect manner, and, by extrapolation, that ccmparable receptor proliferation related causally to TR Recent post-mortem studies in hmans, however, Challenge the causal relationship between DA receptor proliferation and !l!A Although the brains of patients receiving chronic neuroleptic therapy demmstrate greater D-2 receptor nu&er than control sllbjects, no differences in receptor nunber have been observed betwleen neuroleptic-treated subjects with and without TD (8). These findings suggest that, if BH is a model of WJ, factors in addition to D-2 receptor proliferation must particimte in the expression of BK
0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
2208
Behavioral Hypersensitivity Attenuation
Vol. 42, No. 22, 1988
We previouslyreportedthat ratsor guineapigs chronicallycotreated with antixuscarinic agentsand haloperidoldid not developBH (9). In those studies,the reductionin the degreeof BH was inverselycorrelated with the dose of antinuscarinic agentcoadministered. The presentstudyfocusedon the effectsof &ronic coadministration of benztropine and haloperidolin orderto evaluatethe relationhipbetweenthe preventionof BH by antirfuscarinic agentsand the underlyingstriatalbiochenicaland D-2 receptorchanges. Methods
and Materials:
Sixty-four male, albinoSprague-Bawley rats (Charles River)weighing initiallybetween250 and 275 g. were adaptedto the animalfacilitiesfor at least5 days priorto testing. The facilitywas environmentally regulated with lightson between0600and 1800 hours. All animalshad free accessto food and water duringthe study. &
TreatmentGrouts:
Wo groupsof 32 animalseach were chosento studyeither stereotypic behaviorand striatalW metabolismor striatalspiroperidol bindingsites. The four treatmentgroups (n = 16 in each group)are outlinedin Table I. TABIB I: Experimental Design Treatment Group
1.p.Dose (mg/kg/day)
Saline (NS) __Haloperidol(HAL) 0.75 Benxtropine(BENZ) 1.80 HAL+ BENZ 0.75/1.80
SB&M Biochemistry (n= 1 8 8 8 8
Spiroperidol Binding (n= 1
8 8 8 8
Table depictsthe 4 chronictreatnentgroups,the dosagesadministered and nunberof animals(n= ) canmittedto the biochemicaland binding experirrents respectively.The dose of BIDZ is the ED5b dose for blockadeof oxotrsmorine trgnor(see9). E
Stereotvpic Behavioral Analvsis:
Animalswere ratedfor apcmorphine-induced stereotypic behavior(83) priorto chronictreatment and againfollowingthe last treatmentaccordingto a modifiedErnstbehavioralscale describedin detailelsewhere(9). This methodprimarilyratesthe intensityand frequencyof licking,bitingand gnawingwhich occursin a 30 secondobservationinterval(0 to 5+ scale). Bach animalwas placedin a perspexrat cage that containeda wire mesh grid floor.The cagewas isolatedin an individualdsservation cell equipped with its awn lightingand ventilationsystemand visiblethrougha one-way mirror.The animalswere allwed to adaptto thisenvironment for 45 minutes priorto drug challenge. Sixteenanimalswere evaluatedat a time for their SB responseto 0.75mg/kgfreshlypreparedapanorphine HCl (Sigma) delivered ac as the salt. Bach animalwas ratedfor SB startingat 10 minutesfollawingeach SC injectionand at 5 minuteintervalsthereafter until the completionof the response(65min. pretest;90min. post-test).These interval scoreswere then srrnmed to yield the animalscore (AS.)which represented the SB dependentvariable(9).
Vol. 42, No. 22, 1988
BehavioralHypersensitivityAttenuation
2209
Animals were ranked based on pretest AS and the 4 treatment groups were generated from these ranks so that prior to chronic treatnent each group had the sams group meanAS and standard deviation The animals were then treated daily (ip.) between 1000 and 1400 hours for 24 consecutive days, so as to be ccinparable with previous studies (9). Ninety-sixhours follcwing the last treatment (post-test), the behavioral protocol described above was repeated by the same &server (PMC)who was blinded to treatment history. All the animals tolerated their treatments well. Cne animal in the SAL+ BEN group died of infection unrelated to treabm?nt At sacrifice there were no statistically significant differences in the weight of the animals across the treatment groups * . Q Biochemical Analvsls,
Forty-eight hours following behavioral assesanent each animal was lightly anesthetized (2% halothane for 2 min.), sacrificed by thoracic invasion and the brain perfused with saline for 5 minutes via the amunoncarotid. The brain was removed, then inmersed in liquid freon and stored at -80 degrees C until dissection. Each brain was serially sectioned (4 degrees C) and the axpus striatum was then dissected fran each slice and immediately placed in 1 ml of an antioxidant-protein precipitating solution (0.4N perchlorate, 0.05%bis-matabisulf ite, 0.05%EDTA). The tissue was hartoganizedfor 20 sec. under ice on a Polytron hanogenizer (setting 6). This homogenatewas incubated at roan te perature for 20 minute& Ihe pellet was then spun dawnat 18,000 X g. The supematant was poured off and frozen at -80 degrees C for future analysis. The pellet was resuspended in 2.5 mls of 1 N NaCE-l and analyzed for protein content using the Bio- Bad protein analysis kit. The supernatant was analyzed for dopamine @A), and its metabolites dilydroxy phenyl acetic acid (BOPAC) and hanovanillic acid (BVA)using EPIC coupled to electrochemical detection (Environmsntal Science Assoc Model 51OOA).W and its metabolites were eluted off a C-18 reversed phase colunn (Whatman) using a 0.005 M citrate/phosphate buffer containing 14% nmthanol, 7.5 ng EUPA,50 mg octane sulfonic acid with a pB of 3.25. All sanples were analyzed in tripliate during a a>ntinuous chramtogra@ic run and c-red to standard curves run daily. Q
Snironeridol Bindinq Assavt
As required for the binding analysis protocol, animals were sacrificed 6 days after the last treatment by cervical dislocation, the brains removed and immediately innersed in liquid freon as described above. The dissected striata fran individual brains were haaogenized in 40 volunes of 50 mMTris ionic buffer (pB 7.4) containing 120 mMNaCl, 5 mMKCl, 2 IIWCaCl-2 and 1 mMMgCl-2 with a polytron hanogenimr at setting 6 for 30 sec. The hanoganatewas centrifuged twice at 15,000 EPMfor 20 min. (Sorvall R 28). The resulting pellet was resuspended in 10 vollnnes of the samebuffer and incubated for 5 minutes at 37C Protein content was determined by the method of Lowry (10). The membranepreperatians were incubated for 30 min. at 37 C with various conoantrations of 3-B-Spiroperidol (0.02-0.5 mM)in the presence of 0.1 rs~M (+) or (-) butaclamol. The binding reaction was stopped by rapid ultrafilThe filters were rW 3 times with 2 tration through Whatman GF/B filters. ml ice cold Tris binding buffer. The dried filters were plaoed into scintillation vials and oounti ‘Ihe assay was replicated three timea The conputer analysis of the data was accceplihed using Ligand Softrare,
2210
&
Behavioral Hypersensitivity Attenuation
Vol. 42, No. 22, 1988
Statistical Analvsis:
The behavioralscores,biochemical data and thebindingresultswere analyzedusingTCKYVA.Group differences were determined using the LSD posthcc multiple rangeprocedure(Statistical Packagefor the SocialSciences, SPSS-X).
FIG. 1 depictsthe stereotypic behavioralresponseto apcmorphine following24 treatments with the specifiedagents. The ANWA was statistically significant(F = 8.36,p < 0.001).The LSD multiple rangepost-hoctest revealedthat the BAL treatnentgroupexhibiteda statistically significant increasein behavioralresponsiveness relativeto NS and BENZ (p < 0.05).The SB responseof the RAL + BENZ animalswas not differentfran that of either the controlor BAL animals. Unusualor abnormalstereotypic behaviorswere not observed.ReqXXSe OnSet intensity(defined as sun of intervalsscores fran 10 through25 minutes)was elevatedin BAL treatedanimalsrelativeto both NS and HAL + BEN7,animals.BAL treatedanimalsalso exhibitedan increase inpeak chewingintensity(definedas sunof intervalscoresfrom30through 50 minutes)relativeto NS treatedcontrolsas well as an increasein response duration.BAL + BENZ aninslsexhibiteda peak chewingintensity whichwas reducedrelativeto NS treatedcontrolswhile responsedurationwas increased.
Behavioral Apomorphine in
Response (0.75
to
mg/kg)
Rats
FIG. 1 SB~sponseto~rphineinthespecified~~tgroups~reSsedin of methodsand statisAnimalSoore (A.S.)units. See text for description ticalvalues.
Vol.
Behavioral
42, No. 22, 1988
Hypersensitivity
Attenuation
2211
FIG. 2 depicts the striatal IX, RVAand IiVm levels. A?XXA on the W levels (FIG. 2 top) was statistically significant (F = 12.68; p < 0.001). The post-hoc canparisons revealed that both the BENZand HAL+ BaJz W levels were significantly reduced relative to NS treated animals @ < 0.01 and 0.05 respectively) whereas the BENZW level was significantly reduced relative to all treatment groups. Striatal HVA levels were statistically different among the groups (FIG. 2 middle; F = 9.37: p < 0.001). Post-hoc caqxrisons revealed that both the HAL and HAL+ BENZgroups had significantly reduced RVA levels relative to NS treated animals (p < 0.05). The I-lVlvaA activity ratios were statistically significant among the treatment groups (FIG. 2 bottom; F = 4.29: p < 0.01). The post-hoc comparisons revealed that the HALand SAL + BENZgroups were significantly reduced ( p < 0.05) relative to both the BENZ and NS groups.
Striate1
CDA3
00
N8
BENZ
HAL (0.7Smglkg)
(1.8mglkg)
Striatal c . 6 k
BE;2
FIG. 2
CHVACI
Striatal DA (top), WA (middle) and M activity ratio (bottan) t S.D. f0r the specified treatmznt groups. (* = p < 0.05; ** = p < 0.01 relative to NS treated controls)
8. 6.
%, p
4.
2
2.
i5 NS
HAL
BENZ
HAL IE;(Z
Striatal
HVA /DA
Ratio
l-
NS
HAL
BENZ BE-NZ
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Behavioral Hypersensitivity Attenuation
Vol. 42, No. 22, 1988
Table II depictsthe resultsfrom the best-fitcomputergeneratedestimte of Kd and B mx. Both the HAL andBAL +BEN7d treatment groups exhibited an increasein the nunberof spiroperidol bindingsiteswhile only the HAL + BEW group exhibited any statistically significant alterationin binding affinity. TT+BIEII:
Stereospecific [3-H]spiroperidol Bindingon StriatalSynqtanzs in the TreatmentGroupsSpecified Ns
KD (Fw
(s.d.)
89.25
+11.79
%change vs. NS P<
-
vmax (fM) (s.d.)
19.68 i6.34
% change
vs. NS P<
HAL
105.84 +41.27
BENZ
83.36 +19.27
I-m+~Bmz
122.40 +17.63
+19%
-7%
+37%
ns
ns
0.025
25.89 +4.08
18.27 +4.19
26.50 +5-31
---
+32%
-1%
+35%
-
0.01
ns
0.025
Tabledepictsthedissociationconstant(Kd) andvmax in the treatmentgroupsspecified P valuesare basedon comparisonof treatmentgroupswith normalsaline-treated controls.(ns = not significant, p < 0.05)
The resultsshow a clear dissociation of B-2 reosptorproliferation and BK Whereasall groupstreatedwith neuroleptics develom receptorproliferation,animalstreatedwith the conccenitant antimscsarinic agentdid not have demmstratedthat the developBK In priorstudies,investigators preventionof BH by co-treatment with lithiun(11,12), mantadine (13)or levodopa(14)was associated with the simultaneous preventionof receptorSite proliferation.These studieshave added to the belief that BH is the direct behavioralcorrelateof neuroleptic-induced receptorsitechanges. The current resultsrefutethis -thesis and demonstrate thatBH nust be mediated throughmechanism other than dopmine receptorsite proliferation alone. Thesecould involveboth centralor peripheralchanges. First,it has beensuggestedthatcirculating neurolepticlevels may be reducedby the concurrentadministration of an antinusarinicagent (15). we have previouslydenmstratedthat the administration of scopolamine did not alter the circulatinglevels of haloperidol(9). Besides,our observation of equivalentchangesin receptorsitesand in dopaminemetabolismwould suggest that HAL enteredthe CNS to an equivalentdegreein both the H&L and HAL + BENa treatmnt groups.
Vol. 42, No. 22, 1988
BehavioralHypersensitivityAttenuation
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&cod, it is possible that the co-treatment regimen may have altered apcmoraine levels in the CNSrelative to the other treatxent groupa H* ever, the fact that we have prwiously evaluated 2 other antirmscarinic agents in rats as well as guinea pigs and have observed similar behavioral results reduces the probability that the results reported here can be explained by alterations in CNSapomormine levels. Taken together, the reduction in BH observed in the p?ZeSeIIt study qqears to be the result of a central mechanism. A third possible mechanism for attenlrating BH in an aninel with an increased nunber of b-2 reoeptors would involve a decrease in DA release. If this were the case then the a&ninistered dose of apunorphine would be added to a reduced level of endoqnous DA tone thereby yielding the sams total degree of post-synaptic activation as an animal with both normal DA levels and receptors. The biohemical results reported here give no support to this hypothesia Both the HALand HAL+ BeJz treated animals exhibited similar decreases in striatal DA, HVAand I% activity ([HVA]/[DA]). This profile is similar to that cbservsd by other investigators who have reported similar trends in W metabolism during withdrawal fran haloperidol (16,17,18). Fourth, the present results might also be explained by a sinultaneous up regulation of xuscarinic receptors induced by the antinuscarinic properties of BENZ. A we1 1 established antagonistic balance between dominergic and muscarinic activity exists within the striatum (see 19). If HAL caused upregulation of domine receptors, antinuscarinic agents could counterbalance this by up- regulation of nuscarinic receptors In animals receiving both drugs, W mediated behaviors such as SB would be norxml. We have previously reported behavioral data collected under a similar experimental design to support this hypothesis. In this work trihexyphenidyl or benxtropine cotreatrent both prevented the developrent of HAL-induced BK Such co-treated animals were later acutely challenged with their rapective antinuscarinic agent 60 minutes prior to apanor*ine challenge. Both agents failed to induce the response potentiation that they regularly induced in animals chronically treated with saline or haloperidol alone (9,20). ‘Ihis failure of acute antimuscarinic agents to produce their usual acute behavioral effect following prolonged adninistration suggests that these agents may induce xusaarinic receptor site proliferation Quincylidine binding studies are currently under way to examine this possible mechanism Fifth, Chiodo and Bunney (21) as well as others (22,231 have suggested that the chronic concurrent administration of an antixusoarinic agent and halopzridol prevents the development of depolarization block in the substantia nigra compacta (SW). This hypothesis would suggest that the occurrence of &polarization block in the SNc normally produced by chronic haloperidol adninistration and the associated reduction in striatal DA release this state produces (24) would further enhance the pharmacologic denervation of the striatun and thereby enhance the probability of receptor proliferation. Atypical neuroleptic agents such as cloxapine and thioridaxine, which also possess antinuscarinic activity, prevent depolarization block and thereby reduce the degree of *armacolcgic denervatioh These atypical agents would therefore carry a reduced liability to produce BH or TD. HOWever, the present results would suggest that chronic cotreaunent with BENZdoes not prevent, or even reduce, the W receptor site proliferation as proposed by the depolarization block hypothesis. This is especially true since bsnxtropine also acts as a DA reuptake inhibitor (25) which would be expscted to further enhance the CQIT petition between W and haloperidol for receptor occupancy and thereby reduce receptor proliferation. Taken together, these results would suggest that preventing SNc depolarization block with an antinuscarinic agent does not alter the develomnt of D-2 receptor site proliferation. Of courser the
2214
Behavioral Hypersensitivity Attenuation
Vol. 42, No. 22, 1988
failureto prwent D-2 receptorproliferation observedheremay be the result of the uniguecanbination of antimmarinic activityand DA reuptakeinhibition characteristics of BENZ. Scopolamine cotreatmentsarecurrently underway to evaluatethis possibility. This and other studiessuggestthat BH may still be a usefulmodel of TD We can identifytm distincteffect6of anticholinergic drugson BH: the attenuation effectwhen anticholinergics are usedchronicallyin conjunction with neuroleptics and an augmentation effectwhen theyare used acutelyafter BH has alreadydeveloped(9).This lattereffectis well docunented to occur in hunanswith TD. Whetheranticholinergic drugsprotecthunansfran the risk of TD is still controversial. A recentpopulation-based studysuggeststhat anticholinergic cotreatnmtmay protectagainstthe developmentof l'D(26), althoughseveralmall case controlstudieshave suggestedthe opposite(27, 28).The applicability of theseprevalencestudiesto our BH model and its extrapolation to TD may not howeverbe direct,sinceour data suggestthat the intensityof BH in the populationis attenuated, not necessarilythe prevalence (29).
The authorswish to thankMs. Li ChiungKao for her assistance with this project This work was supportedin part by a grantfran the Boothroyd Foundationand the NIMH (MH41274). References H&.KLAWANS and RRUBOVITS,J. Neural.Trans.33:235-246 (1972). A. HITRI,W.J.WEINER,RL. BORISON,B.I.DIAMOND, P.A.NAUSIEDA and RLKLAWANS, Ann. Neurol.3:134-140(1978). 3. D.R BURT,I.CREESEand S.H.SNYDER,Science 196:326-328(1977). 4. A.CLU&P.JENNER, A DIECEOFW and CD. MARSDEN,Nature 278:59-67 (1979). 5. N. WPNIAK,G.KILPATRICK,M.D.HALL,P. JENNERand CD. m, Psychophmmacol, 84:512519 (1984). 6. HL. KLAWANS,P.M.CARVEY, A. HITRI,P.A.NAUSIEDAandW.J.WEINER, Adv. Biochem 24:569-572(1980) 7. W.C KOLLER,J. CURTIN and J. FIELDS,Sot.Neurosci.Abstr. 9 (1)721 (1983). 8. T.J.CRCW,AJ. CROSS, E.C.JOHNSTGNE,F. CWEN, D. WENS and J. WADDINXON, J.Clin.Psychopharmacol 2:336-340(1982) 9. P.M.CARVEY, L.C.KAO, C.M.TANNER,C.G.GOETZ and H.L.KLAWANS, EuropeanJ. Pharmacol.120:193-199(1986). 10. O.H.LOWRY, N.J. RONEBROUGH, AL. FORR and RS. RANDALL, J. Biol. Chem. 193:265-275(1951). 11. H.L.KLAWANS,W.J.WEINER and P.A.NAUSIEDA,Prog. Neuropsychophamcol.1:53-60 (1977). 12. A. PERT,J.E. R(XmLATT, C. SIVI'IT, C.B. PFRT,W.E. BUNNEX, Science.201:171-173(1978). 13. RALLEN, J.LANEandJ. BRAUCHI.EuropeauJ.Pharmacol. 65:313-320 (1980). 14. S.J. LIST, and P. SEQ4AN.Life Sci. 24:1447-52(1979). 15. L. RIVERA-CALIMLIM, Br. J. Pharmacol. 56:301-309 (1976). 16. B.SCATXW,C.GARRETand L. JIJIGlJ, Nauyn-Schmaid. Arch. Pharmacol.289:419-434(1975). 1% N LINUEFORS,T. SHARPand U. UNZ-, EuropeanJ. Pharmacol.129:401-
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404 (1986). 18. P. LERbER,P. NOSE, E GOWON and W. ILNmFG, Science197:181-183 (1977). 19 H.L. KUMANS, F%armcologyof ExtrapyrmidalMcmment Disorders. Monographsin NeuralScience,Basal,Karger (1973). 20. P.M. (XMTY, C.M. TMlbEZ,C.G.GOEZZ,L.C. RAG and B.L. RLAWANS,Neurology(s) 34:104(1984). 21. LA. CXIODOand B.S. BUNNTY,J. Nsurosci.5:2539-2544(1985). 22. R.J.WHITE and R.Y.MN& science221:1054-1057 (1983). 23. C.D. BLABAand R.F. LUJE,Sot. Neurosci.Abstr. (1) 499 (1985). 24. J.O. SCBENKand B.S. BDNMX, Sot. Neurosci.Abstr.9:1007 (1983). 25. F. SUISERand P.L. KIBLEY,In: Psychotropic Agents,F. Hoffmeister and G. Stille (eds.). Springer, Verlag,99471490 (1980). 26. G. GARDOS,I. S?MJ,M. KALLOSand J.O. OX& PsychoFhamacology 71: 29 - 34 (1980). 27. C.P. CHIm, A. -END and M. -Y, In: Tardive Dyskinesia, W.E. Fahn,RC. Smith,J.M. Davisand E.F.Dunino (eds.).Medicaland Scientific Books,New York, 35-50 (1980). 28. J.G. CSERNANSRY, K.-RI, J.CEmS, J. KAPLANand J.A. YESAVXE, Am J Psychiatry 138: 1362-1365(1981). 29. Addressall reprintrequeststoPaul M. CarveyrDept.of Neurological Sciences;Rush Presbyterian St. Luke'sMedical Center,1753W. BarrisonSt. ; Chicago,IL 60612.
2215
Life Sciences,Vol. 42, pp. 2207-2215 Printed in the U.S.A.
Paul M. Carvey, Ana Hitri*,
Christopher G Goetz, Caroline Harold L. Klawans
M. TWmer and
Dept. of Neurological Sciences, Rush Presbyterian St. Lukes Medical Center, Chicago, IL 60612, *Dept. of Psychiatry, Medical College of Georgia, Augusta, CA (Received
in final
form March 28, 1988)
Groups of male rats (n = 16 each) were treated with normal saline, haloperidol (0.75 mg/kg), benztropine (1.8 mg/kg) or haloperidol and benztropine once a day for 24 dqm. Fol lclwing a 96 hour drug free interval, subsets of these animals were assessed for apcmorphine-indmxd (0.75 mg/kg) stereotypic behavior, sacrificed and analyzed for striatal dqmine biochenistry or sacrificed and analyzed for spimperidol binding sites. Benztropine cotreatnent attenuated the developnent of behavioral hypersensitivity to haloperidol but did not alter either the dopanine receptor proliferation or the striatal domine biochenical Changes itiuced by haloperidol. These results suggest that behavioral @persensitivity is not an autanatic nmifestation of dopmine receptor proliferation but nust depend, in part, on other factors.
In 1972, Klawans and RuboVitS &served that chronic treatment of laboratory animals with the neuroleptic agent chlorprmazine produced an increase in behavioral responsiveness to subsequent dopmine DA) agonist challenge (1). This increased responsiveness, or behavioral hypersensitivity (BH), was thought to reflect an increase in W receptor nunber following a period of pharmacologic denervation (2,3,4,5). Klawans and Rubovits further hypothesized that tardive dyskinesia (!L”D),ostensibly a movement disorder reflecting a state of W hypersensitivity in hmans, might also reflect a pharmacologically-induced W receptor proliferation. This line of reasoning has led to the use of neuroleptic treated animals and their subsequent agonist-induced BH ae an animal model of TD. In prior studies, the degree of BH induced by a neuroleptic agent and the degree of D-2 receptor proliferation were correlated in the animal mrsensitivity model (6,7). This led to the suggestion that D-2 receptor proliferation and BH were related in a cause and effect manner, and, by extrapolation, that ccmparable receptor proliferation related causally to TR Recent post-mortem studies in hmans, however, Challenge the causal relationship between DA receptor proliferation and !l!A Although the brains of patients receiving chronic neuroleptic therapy demmstrate greater D-2 receptor nu&er than control sllbjects, no differences in receptor nunber have been observed betwleen neuroleptic-treated subjects with and without TD (8). These findings suggest that, if BH is a model of WJ, factors in addition to D-2 receptor proliferation must particimte in the expression of BK
0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
2208
Behavioral Hypersensitivity Attenuation
Vol. 42, No. 22, 1988
We previouslyreportedthat ratsor guineapigs chronicallycotreated with antixuscarinic agentsand haloperidoldid not developBH (9). In those studies,the reductionin the degreeof BH was inverselycorrelated with the dose of antinuscarinic agentcoadministered. The presentstudyfocusedon the effectsof &ronic coadministration of benztropine and haloperidolin orderto evaluatethe relationhipbetweenthe preventionof BH by antirfuscarinic agentsand the underlyingstriatalbiochenicaland D-2 receptorchanges. Methods
and Materials:
Sixty-four male, albinoSprague-Bawley rats (Charles River)weighing initiallybetween250 and 275 g. were adaptedto the animalfacilitiesfor at least5 days priorto testing. The facilitywas environmentally regulated with lightson between0600and 1800 hours. All animalshad free accessto food and water duringthe study. &
TreatmentGrouts:
Wo groupsof 32 animalseach were chosento studyeither stereotypic behaviorand striatalW metabolismor striatalspiroperidol bindingsites. The four treatmentgroups (n = 16 in each group)are outlinedin Table I. TABIB I: Experimental Design Treatment Group
1.p.Dose (mg/kg/day)
Saline (NS) __Haloperidol(HAL) 0.75 Benxtropine(BENZ) 1.80 HAL+ BENZ 0.75/1.80
SB&M Biochemistry (n= 1 8 8 8 8
Spiroperidol Binding (n= 1
8 8 8 8
Table depictsthe 4 chronictreatnentgroups,the dosagesadministered and nunberof animals(n= ) canmittedto the biochemicaland binding experirrents respectively.The dose of BIDZ is the ED5b dose for blockadeof oxotrsmorine trgnor(see9). E
Stereotvpic Behavioral Analvsis:
Animalswere ratedfor apcmorphine-induced stereotypic behavior(83) priorto chronictreatment and againfollowingthe last treatmentaccordingto a modifiedErnstbehavioralscale describedin detailelsewhere(9). This methodprimarilyratesthe intensityand frequencyof licking,bitingand gnawingwhich occursin a 30 secondobservationinterval(0 to 5+ scale). Bach animalwas placedin a perspexrat cage that containeda wire mesh grid floor.The cagewas isolatedin an individualdsservation cell equipped with its awn lightingand ventilationsystemand visiblethrougha one-way mirror.The animalswere allwed to adaptto thisenvironment for 45 minutes priorto drug challenge. Sixteenanimalswere evaluatedat a time for their SB responseto 0.75mg/kgfreshlypreparedapanorphine HCl (Sigma) delivered ac as the salt. Bach animalwas ratedfor SB startingat 10 minutesfollawingeach SC injectionand at 5 minuteintervalsthereafter until the completionof the response(65min. pretest;90min. post-test).These interval scoreswere then srrnmed to yield the animalscore (AS.)which represented the SB dependentvariable(9).
Vol. 42, No. 22, 1988
BehavioralHypersensitivityAttenuation
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Animals were ranked based on pretest AS and the 4 treatment groups were generated from these ranks so that prior to chronic treatnent each group had the sams group meanAS and standard deviation The animals were then treated daily (ip.) between 1000 and 1400 hours for 24 consecutive days, so as to be ccinparable with previous studies (9). Ninety-sixhours follcwing the last treatment (post-test), the behavioral protocol described above was repeated by the same &server (PMC)who was blinded to treatment history. All the animals tolerated their treatments well. Cne animal in the SAL+ BEN group died of infection unrelated to treabm?nt At sacrifice there were no statistically significant differences in the weight of the animals across the treatment groups * . Q Biochemical Analvsls,
Forty-eight hours following behavioral assesanent each animal was lightly anesthetized (2% halothane for 2 min.), sacrificed by thoracic invasion and the brain perfused with saline for 5 minutes via the amunoncarotid. The brain was removed, then inmersed in liquid freon and stored at -80 degrees C until dissection. Each brain was serially sectioned (4 degrees C) and the axpus striatum was then dissected fran each slice and immediately placed in 1 ml of an antioxidant-protein precipitating solution (0.4N perchlorate, 0.05%bis-matabisulf ite, 0.05%EDTA). The tissue was hartoganizedfor 20 sec. under ice on a Polytron hanogenizer (setting 6). This homogenatewas incubated at roan te perature for 20 minute& Ihe pellet was then spun dawnat 18,000 X g. The supematant was poured off and frozen at -80 degrees C for future analysis. The pellet was resuspended in 2.5 mls of 1 N NaCE-l and analyzed for protein content using the Bio- Bad protein analysis kit. The supernatant was analyzed for dopamine @A), and its metabolites dilydroxy phenyl acetic acid (BOPAC) and hanovanillic acid (BVA)using EPIC coupled to electrochemical detection (Environmsntal Science Assoc Model 51OOA).W and its metabolites were eluted off a C-18 reversed phase colunn (Whatman) using a 0.005 M citrate/phosphate buffer containing 14% nmthanol, 7.5 ng EUPA,50 mg octane sulfonic acid with a pB of 3.25. All sanples were analyzed in tripliate during a a>ntinuous chramtogra@ic run and c-red to standard curves run daily. Q
Snironeridol Bindinq Assavt
As required for the binding analysis protocol, animals were sacrificed 6 days after the last treatment by cervical dislocation, the brains removed and immediately innersed in liquid freon as described above. The dissected striata fran individual brains were haaogenized in 40 volunes of 50 mMTris ionic buffer (pB 7.4) containing 120 mMNaCl, 5 mMKCl, 2 IIWCaCl-2 and 1 mMMgCl-2 with a polytron hanogenimr at setting 6 for 30 sec. The hanoganatewas centrifuged twice at 15,000 EPMfor 20 min. (Sorvall R 28). The resulting pellet was resuspended in 10 vollnnes of the samebuffer and incubated for 5 minutes at 37C Protein content was determined by the method of Lowry (10). The membranepreperatians were incubated for 30 min. at 37 C with various conoantrations of 3-B-Spiroperidol (0.02-0.5 mM)in the presence of 0.1 rs~M (+) or (-) butaclamol. The binding reaction was stopped by rapid ultrafilThe filters were rW 3 times with 2 tration through Whatman GF/B filters. ml ice cold Tris binding buffer. The dried filters were plaoed into scintillation vials and oounti ‘Ihe assay was replicated three timea The conputer analysis of the data was accceplihed using Ligand Softrare,
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Statistical Analvsis:
The behavioralscores,biochemical data and thebindingresultswere analyzedusingTCKYVA.Group differences were determined using the LSD posthcc multiple rangeprocedure(Statistical Packagefor the SocialSciences, SPSS-X).
FIG. 1 depictsthe stereotypic behavioralresponseto apcmorphine following24 treatments with the specifiedagents. The ANWA was statistically significant(F = 8.36,p < 0.001).The LSD multiple rangepost-hoctest revealedthat the BAL treatnentgroupexhibiteda statistically significant increasein behavioralresponsiveness relativeto NS and BENZ (p < 0.05).The SB responseof the RAL + BENZ animalswas not differentfran that of either the controlor BAL animals. Unusualor abnormalstereotypic behaviorswere not observed.ReqXXSe OnSet intensity(defined as sun of intervalsscores fran 10 through25 minutes)was elevatedin BAL treatedanimalsrelativeto both NS and HAL + BEN7,animals.BAL treatedanimalsalso exhibitedan increase inpeak chewingintensity(definedas sunof intervalscoresfrom30through 50 minutes)relativeto NS treatedcontrolsas well as an increasein response duration.BAL + BENZ aninslsexhibiteda peak chewingintensity whichwas reducedrelativeto NS treatedcontrolswhile responsedurationwas increased.
Behavioral Apomorphine in
Response (0.75
to
mg/kg)
Rats
FIG. 1 SB~sponseto~rphineinthespecified~~tgroups~reSsedin of methodsand statisAnimalSoore (A.S.)units. See text for description ticalvalues.
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FIG. 2 depicts the striatal IX, RVAand IiVm levels. A?XXA on the W levels (FIG. 2 top) was statistically significant (F = 12.68; p < 0.001). The post-hoc canparisons revealed that both the BENZand HAL+ BaJz W levels were significantly reduced relative to NS treated animals @ < 0.01 and 0.05 respectively) whereas the BENZW level was significantly reduced relative to all treatment groups. Striatal HVA levels were statistically different among the groups (FIG. 2 middle; F = 9.37: p < 0.001). Post-hoc caqxrisons revealed that both the HAL and HAL+ BENZgroups had significantly reduced RVA levels relative to NS treated animals (p < 0.05). The I-lVlvaA activity ratios were statistically significant among the treatment groups (FIG. 2 bottom; F = 4.29: p < 0.01). The post-hoc comparisons revealed that the HALand SAL + BENZgroups were significantly reduced ( p < 0.05) relative to both the BENZ and NS groups.
Striate1
CDA3
00
N8
BENZ
HAL (0.7Smglkg)
(1.8mglkg)
Striatal c . 6 k
BE;2
FIG. 2
CHVACI
Striatal DA (top), WA (middle) and M activity ratio (bottan) t S.D. f0r the specified treatmznt groups. (* = p < 0.05; ** = p < 0.01 relative to NS treated controls)
8. 6.
%, p
4.
2
2.
i5 NS
HAL
BENZ
HAL IE;(Z
Striatal
HVA /DA
Ratio
l-
NS
HAL
BENZ BE-NZ
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Table II depictsthe resultsfrom the best-fitcomputergeneratedestimte of Kd and B mx. Both the HAL andBAL +BEN7d treatment groups exhibited an increasein the nunberof spiroperidol bindingsiteswhile only the HAL + BEW group exhibited any statistically significant alterationin binding affinity. TT+BIEII:
Stereospecific [3-H]spiroperidol Bindingon StriatalSynqtanzs in the TreatmentGroupsSpecified Ns
KD (Fw
(s.d.)
89.25
+11.79
%change vs. NS P<
-
vmax (fM) (s.d.)
19.68 i6.34
% change
vs. NS P<
HAL
105.84 +41.27
BENZ
83.36 +19.27
I-m+~Bmz
122.40 +17.63
+19%
-7%
+37%
ns
ns
0.025
25.89 +4.08
18.27 +4.19
26.50 +5-31
---
+32%
-1%
+35%
-
0.01
ns
0.025
Tabledepictsthedissociationconstant(Kd) andvmax in the treatmentgroupsspecified P valuesare basedon comparisonof treatmentgroupswith normalsaline-treated controls.(ns = not significant, p < 0.05)
The resultsshow a clear dissociation of B-2 reosptorproliferation and BK Whereasall groupstreatedwith neuroleptics develom receptorproliferation,animalstreatedwith the conccenitant antimscsarinic agentdid not have demmstratedthat the developBK In priorstudies,investigators preventionof BH by co-treatment with lithiun(11,12), mantadine (13)or levodopa(14)was associated with the simultaneous preventionof receptorSite proliferation.These studieshave added to the belief that BH is the direct behavioralcorrelateof neuroleptic-induced receptorsitechanges. The current resultsrefutethis -thesis and demonstrate thatBH nust be mediated throughmechanism other than dopmine receptorsite proliferation alone. Thesecould involveboth centralor peripheralchanges. First,it has beensuggestedthatcirculating neurolepticlevels may be reducedby the concurrentadministration of an antinusarinicagent (15). we have previouslydenmstratedthat the administration of scopolamine did not alter the circulatinglevels of haloperidol(9). Besides,our observation of equivalentchangesin receptorsitesand in dopaminemetabolismwould suggest that HAL enteredthe CNS to an equivalentdegreein both the H&L and HAL + BENa treatmnt groups.
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&cod, it is possible that the co-treatment regimen may have altered apcmoraine levels in the CNSrelative to the other treatxent groupa H* ever, the fact that we have prwiously evaluated 2 other antirmscarinic agents in rats as well as guinea pigs and have observed similar behavioral results reduces the probability that the results reported here can be explained by alterations in CNSapomormine levels. Taken together, the reduction in BH observed in the p?ZeSeIIt study qqears to be the result of a central mechanism. A third possible mechanism for attenlrating BH in an aninel with an increased nunber of b-2 reoeptors would involve a decrease in DA release. If this were the case then the a&ninistered dose of apunorphine would be added to a reduced level of endoqnous DA tone thereby yielding the sams total degree of post-synaptic activation as an animal with both normal DA levels and receptors. The biohemical results reported here give no support to this hypothesia Both the HALand HAL+ BeJz treated animals exhibited similar decreases in striatal DA, HVAand I% activity ([HVA]/[DA]). This profile is similar to that cbservsd by other investigators who have reported similar trends in W metabolism during withdrawal fran haloperidol (16,17,18). Fourth, the present results might also be explained by a sinultaneous up regulation of xuscarinic receptors induced by the antinuscarinic properties of BENZ. A we1 1 established antagonistic balance between dominergic and muscarinic activity exists within the striatum (see 19). If HAL caused upregulation of domine receptors, antinuscarinic agents could counterbalance this by up- regulation of nuscarinic receptors In animals receiving both drugs, W mediated behaviors such as SB would be norxml. We have previously reported behavioral data collected under a similar experimental design to support this hypothesis. In this work trihexyphenidyl or benxtropine cotreatrent both prevented the developrent of HAL-induced BK Such co-treated animals were later acutely challenged with their rapective antinuscarinic agent 60 minutes prior to apanor*ine challenge. Both agents failed to induce the response potentiation that they regularly induced in animals chronically treated with saline or haloperidol alone (9,20). ‘Ihis failure of acute antimuscarinic agents to produce their usual acute behavioral effect following prolonged adninistration suggests that these agents may induce xusaarinic receptor site proliferation Quincylidine binding studies are currently under way to examine this possible mechanism Fifth, Chiodo and Bunney (21) as well as others (22,231 have suggested that the chronic concurrent administration of an antixusoarinic agent and halopzridol prevents the development of depolarization block in the substantia nigra compacta (SW). This hypothesis would suggest that the occurrence of &polarization block in the SNc normally produced by chronic haloperidol adninistration and the associated reduction in striatal DA release this state produces (24) would further enhance the pharmacologic denervation of the striatun and thereby enhance the probability of receptor proliferation. Atypical neuroleptic agents such as cloxapine and thioridaxine, which also possess antinuscarinic activity, prevent depolarization block and thereby reduce the degree of *armacolcgic denervatioh These atypical agents would therefore carry a reduced liability to produce BH or TD. HOWever, the present results would suggest that chronic cotreaunent with BENZdoes not prevent, or even reduce, the W receptor site proliferation as proposed by the depolarization block hypothesis. This is especially true since bsnxtropine also acts as a DA reuptake inhibitor (25) which would be expscted to further enhance the CQIT petition between W and haloperidol for receptor occupancy and thereby reduce receptor proliferation. Taken together, these results would suggest that preventing SNc depolarization block with an antinuscarinic agent does not alter the develomnt of D-2 receptor site proliferation. Of courser the
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failureto prwent D-2 receptorproliferation observedheremay be the result of the uniguecanbination of antimmarinic activityand DA reuptakeinhibition characteristics of BENZ. Scopolamine cotreatmentsarecurrently underway to evaluatethis possibility. This and other studiessuggestthat BH may still be a usefulmodel of TD We can identifytm distincteffect6of anticholinergic drugson BH: the attenuation effectwhen anticholinergics are usedchronicallyin conjunction with neuroleptics and an augmentation effectwhen theyare used acutelyafter BH has alreadydeveloped(9).This lattereffectis well docunented to occur in hunanswith TD. Whetheranticholinergic drugsprotecthunansfran the risk of TD is still controversial. A recentpopulation-based studysuggeststhat anticholinergic cotreatnmtmay protectagainstthe developmentof l'D(26), althoughseveralmall case controlstudieshave suggestedthe opposite(27, 28).The applicability of theseprevalencestudiesto our BH model and its extrapolation to TD may not howeverbe direct,sinceour data suggestthat the intensityof BH in the populationis attenuated, not necessarilythe prevalence (29).
The authorswish to thankMs. Li ChiungKao for her assistance with this project This work was supportedin part by a grantfran the Boothroyd Foundationand the NIMH (MH41274). References H&.KLAWANS and RRUBOVITS,J. Neural.Trans.33:235-246 (1972). A. HITRI,W.J.WEINER,RL. BORISON,B.I.DIAMOND, P.A.NAUSIEDA and RLKLAWANS, Ann. Neurol.3:134-140(1978). 3. D.R BURT,I.CREESEand S.H.SNYDER,Science 196:326-328(1977). 4. A.CLU&P.JENNER, A DIECEOFW and CD. MARSDEN,Nature 278:59-67 (1979). 5. N. WPNIAK,G.KILPATRICK,M.D.HALL,P. JENNERand CD. m, Psychophmmacol, 84:512519 (1984). 6. HL. KLAWANS,P.M.CARVEY, A. HITRI,P.A.NAUSIEDAandW.J.WEINER, Adv. Biochem 24:569-572(1980) 7. W.C KOLLER,J. CURTIN and J. FIELDS,Sot.Neurosci.Abstr. 9 (1)721 (1983). 8. T.J.CRCW,AJ. CROSS, E.C.JOHNSTGNE,F. CWEN, D. WENS and J. WADDINXON, J.Clin.Psychopharmacol 2:336-340(1982) 9. P.M.CARVEY, L.C.KAO, C.M.TANNER,C.G.GOETZ and H.L.KLAWANS, EuropeanJ. Pharmacol.120:193-199(1986). 10. O.H.LOWRY, N.J. RONEBROUGH, AL. FORR and RS. RANDALL, J. Biol. Chem. 193:265-275(1951). 11. H.L.KLAWANS,W.J.WEINER and P.A.NAUSIEDA,Prog. Neuropsychophamcol.1:53-60 (1977). 12. A. PERT,J.E. R(XmLATT, C. SIVI'IT, C.B. PFRT,W.E. BUNNEX, Science.201:171-173(1978). 13. RALLEN, J.LANEandJ. BRAUCHI.EuropeauJ.Pharmacol. 65:313-320 (1980). 14. S.J. LIST, and P. SEQ4AN.Life Sci. 24:1447-52(1979). 15. L. RIVERA-CALIMLIM, Br. J. Pharmacol. 56:301-309 (1976). 16. B.SCATXW,C.GARRETand L. JIJIGlJ, Nauyn-Schmaid. Arch. Pharmacol.289:419-434(1975). 1% N LINUEFORS,T. SHARPand U. UNZ-, EuropeanJ. Pharmacol.129:401-
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404 (1986). 18. P. LERbER,P. NOSE, E GOWON and W. ILNmFG, Science197:181-183 (1977). 19 H.L. KUMANS, F%armcologyof ExtrapyrmidalMcmment Disorders. Monographsin NeuralScience,Basal,Karger (1973). 20. P.M. (XMTY, C.M. TMlbEZ,C.G.GOEZZ,L.C. RAG and B.L. RLAWANS,Neurology(s) 34:104(1984). 21. LA. CXIODOand B.S. BUNNTY,J. Nsurosci.5:2539-2544(1985). 22. R.J.WHITE and R.Y.MN& science221:1054-1057 (1983). 23. C.D. BLABAand R.F. LUJE,Sot. Neurosci.Abstr. (1) 499 (1985). 24. J.O. SCBENKand B.S. BDNMX, Sot. Neurosci.Abstr.9:1007 (1983). 25. F. SUISERand P.L. KIBLEY,In: Psychotropic Agents,F. Hoffmeister and G. Stille (eds.). Springer, Verlag,99471490 (1980). 26. G. GARDOS,I. S?MJ,M. KALLOSand J.O. OX& PsychoFhamacology 71: 29 - 34 (1980). 27. C.P. CHIm, A. -END and M. -Y, In: Tardive Dyskinesia, W.E. Fahn,RC. Smith,J.M. Davisand E.F.Dunino (eds.).Medicaland Scientific Books,New York, 35-50 (1980). 28. J.G. CSERNANSRY, K.-RI, J.CEmS, J. KAPLANand J.A. YESAVXE, Am J Psychiatry 138: 1362-1365(1981). 29. Addressall reprintrequeststoPaul M. CarveyrDept.of Neurological Sciences;Rush Presbyterian St. Luke'sMedical Center,1753W. BarrisonSt. ; Chicago,IL 60612.
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