Vol. 273, No. 1Printed in U.S.A.

0022-3565/95/2731-0138$OO.OO/OTHE JoURNAL o� Pa*iu&�cowGY *rm ExpaRwxwr�L Tiias.�i’auricsCopyright C 1995 by The American Society for Pharmacology and Experimental TherapeuticsJPET 273:138-145, 1995

Pharmacological Analysis of the Scratching Produced byDopamine D2 Agonists in Squirrel Monkeys

RICARDO PELLON1, PILAR FLORES2, KEN ALLING, JEFFREY M. WIThIN and JONAThAN L KATZPsychobiology Section, Predinica! Pharmacology Laboratory, National Institute on Drug Abuse, Intramural Research Program, National

Institutes of Health, Baltimore, Maryland

Accepted for publication December 9, 1994

ABSTRACTSeveral dopamine agonists, administered i.m., produced per-sistent, excessive and non-localized scratching in squirrel mon-keys (Saimiri sciureus). Studies were Conducted with a series ofdrugs to determine the pharmacological mechanisms respon-sible for this effect. All of the dopamine D2 agonists studiedprOduCed dose-related increases in scratChing, whereas say-eral dopamine D1 receptor agonists, indirect dopamine ago-fists and drugs acting on other receptors failed to producedose-related increases in scratching. The scratching producedby D2 agonists was stereospecific; (-)-NPA produced scratch-ing whereas its (+)-enantiomer was inactive up to doses 300-fold higher. Scratching induced by quinpirole was attenuatedby both D2 and D1 antagonists, and this antagonism was ste-reospecific, with the D2 antagonist (-)-eticlopnde, but not its

enantiomer, active. Sensitivity developed to the effects of D2agonists with the quinpirole dose-effect curve shifting to the leftby a factor of approximately 64. Two partial D2 receptor ago-nists (SDZ 208-91 1 and SDZ 208-912) had limited efficacy inproducing scratching, however, one partial D2 receptor agonist(tergunde) was fully efficacious, suggesting that there are sparereceptors for this effect. The peripherally active dopamine an-tagonist domperidone and the histamine antagonist diphenhy-dramine also reduced the scratching induced by 02 agonists,but not to the same extent as centrally acting D2 antagonists.Scratching in squirrel monkeys is an effect that appears to bedue to agonist actions at 02 receptors, and may be mediatedby a release of histamine. This behavioral activity may be usefulas an in vivo indication of D2 receptor activity in primates.

Several distinct dopamine receptor subtypes have beenidentified with biochemical methods. Two of these dopaminereceptors, D1 and D2, have been differentiated for their abil-ity to stimulate adenylyl cyclase activity and they have re-ceived extensive study (ci. Kebabian and Calne, 1979; Clarkand White, 1987). Selective agonists at D1 receptors stimu-late adenylyl cyclase activity, whereas D2-receptor agonistshave either no effect, or decrease this activity. Differences inother pharmacological effects ofD1 and D2 agonists have also

been described. For example, D2 agonists produce a hypo-thermia in rats and mice whereas D1 agonists produce ahyperthermia (Barnett et at. , 1972; Sanchez, 1989).

Differences in the behavioral effects of D1 and D2 agonists

have been best described in rodents. Dopamine D2 agonists

Received for publication May 25, 1994.

1 Present address: Departamento de Psicologla Basica, Universidad Nacio-

nal de Educaci#{243}na Distancia, (P.O. Box) Apartado n.#{176}50.487, 28040-Madrid,Spain.

2 Supported by a grant from the Ministerio de Educaci#{243}ny Ciencia (Madrid,Spain). Present address: Departamento de Psicologla B#{225}sica, UniversidadPontificia de Conillas, 28040-Madrid, Spain.

produce locomotor hyperactivity, and at higher doses, stereo-

typies, including gnawing and sniffing (e.g. , Arnt et at. , 1988;Braun and Chase, 1986). Seaman (1980) suggested on thebasis of correlations between binding affinity and in vivo

potency that these effects were due to actions at D2 receptors.Studies of D1 receptor agonists have suggested that the be-havioral effects of these drugs are distinct from those of D2agonists. One prominent effect of D1 receptor agonists in

rodents is a stimulation ofgrooming behavior (see Clark andWhite, 1987, for a review).

In primates, Elsworth et at. (1991) have shown that both

D1 and D2 agonists dose-dependently increased eye-blinkrate, and that these increases were blocked by both selectiveD1 and D2 antagonists. In another study, Loschmann et at.

(1991) showed that the dopamine agonists, quinpirole and

apomorphine, produced dose-dependent increases in motoractivity and stereotypies in marmosets; in contrast, the D1agonist, SKF 38393, only decreased motor activity. Code andTang (1991) reported that some D2 agonists, but not the D1

agonist, SKF 38393, produced yawning in rhesus monkeys.

ABBREVIAT1ONS: j3-CCE: frcarboline-3-carboxylic acid ethyl ester HCI; CL confidence limits; (-)-NPA R(-)-propyl-norapomorphine HCI;(+)-PHNO: (+)-4-propyl-9-hydroxynaphthoxazine; Ru 24213: N-n-propyl-N-phenylethyl-p(3-hydroxyphenyl)ethylamine HCI; SCH 23390: R(+)-7-chloro-8-hydroxy-3-methyl-1 -phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine HCI; SCH 39166: (-)-trans-6,7,7a,8,9,13b-hexahydro-3chloro-2-hy-droxy-N-methyl-5H-benzo(d)naphtho-(2,1-b)azepine; SDZ 208-91 1 : N-[(8-cr)-2,6-dimethylergoline-8-yl)-2,2-dimethylpropanamide; SDZ208-912: N-[(8-a)-2-chloro-6-methylergollne-8-yl]-2,2-dimethylpropanamide; SKF 38393: (±)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tet-rahydro-1 H-3-benzazepine HCI; SKF 75670: 3-methyl-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine HBr; SKF 82958: (±)-6-chloro-7,8-dihydroxy-3-allyl-1 -phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine HBr.

138

1995 Dopaminergic Behavioral EffeCts 139

In addition, a pronounced scratching produced by dopamineagonists has been documented to occur in squirrel monkeys(Pell#{243}nand Katz, 1991; Rosenzweig-Lipson and Bergman,

1993; Rosenzweig-Lipson et at. , 1994). In the studies byPell#{244}nand Katz and Rosenzweig-Lipson et at. , scratching

was produced by dopamine D2 agonists but not D1 agonists orindirectly acting dopamine agonists. Thus, in primates, as inrodents, there appear to be some differences among behav-ioral effects of both D1 and D2 dopamine receptor agonists.

The relation ofthe behavioral effects ofD2 and D1 agoniststo selective activation of dopamine receptor subtypes isclouded by results of studies with antagonists. For example,the motor activating effects of D2 agonists in rodents and

primates can be antagonized by the D1 antagonist SCH

23390 (Clark and White, 1987; Loschmann et at., 1991). Inaddition, grooming in rodents, as well as some other behav-iors induced by administration of D1 agonists, can be antag-onized by D2 antagonists (Molloy and Waddington, 1985;Molloy et at. , 1986; Murray and Waddington, 1989). The

yawning produced by D2 agonists in rhesus monkeys was

antagonized by the D1 antagonist, SCH 2330, but not the D2antagonist, sulpiride (Code and Tang, 1991). Scratching, in-duced by the D2 agonist, (+)-PHNO, was antagonized by bothD2 and D1 antagonists (Rosenzweig-Lipson and Bergman,1993). Despite the often nonselective nature of these antag-

onist results, in vitro data indicate that the antagonists usedare generally highly selective for the subtypes of dopan#{252}nereceptors. As a result, several investigators have concludedthat there is an interaction between D2 and D1 receptors inwhich some D1 receptor activity is necessary for the expres-sion of D2 agonist action (Braun and Chase, 1986; Clark and

White, 1987; Molloy and Waddington, 1984).

Our studies were undertaken to further investigate thescratching response produced by dopaminergic agonists insquirrel monkeys. Studies were initiated to more fully deter-mine the pharmacological specificity ofthe effect with regardto actions at D2 dopamine receptors. Our results suggestedthat this behavior is selectively produced by D2 agonists, butantagonized by both D1 and D2 antagonists. These studiesindicate that scratching in squirrel monkeys may be used asa primate model of in vivo D2 receptor activity.

Methods

Subjects. Four adult male squirrel monkeys (Saimiri sciureus)

with mean body weights of921 to 992 g served as subjects. Each washoused individually in a temperature- and humidity-controlled room

with a 12-hr light/dark cycle (lights on 06:00). Subjects had wire-stricted access to food (Purina Monkey Chow, Purina Mills, Inc., St.Louis, MO) and Teklad (Harlan Teklad Laboratory Diets, Madison,WI), supplemented with fresh fruit) and water. Monkeys had beenstudied previously in various experiments involving acute adminis-tration of drugs or drug self-administration, but none of the subjectshad participated in an experiment or received any drug for at least 3mo before the start of this study.

Procedure. Monkeys were weighed before each experimentalsession, and tasting typically consisted of four successive i.m. injec-tions (thigh muscle), separated by an interval of 15 mm (a cuniula-tive dosing procedure). After each injection, the behavior of themonkeys was observed in their home-cages during three 1-mm pa-

riods to assess if scratching occurred. The first 1-mm observationbegan 5 mm after an injection, the second at 9 mm, and the third at13 mm after the injection, etc.; observations were 3 mm apart.

During each observation, scratching was recorded if a monkey

touched any part of its body with its fingers or toes in short and

repetitive strokes. This behavior differed from grooming primarily in

that the strokes were short and repetitive and occurred in rapid

succession. The behavior was also different from the stereotypiesoften observed with high doses ofstimulants in that the stereotypiestypically involve rapid head movements resembling an intensivevisual scanning (Rosenzweig-Lipson et at. , 1994). Across subjects

there was a maximum of 12 positive scores at each dose. Drugs wereadministered on Tuesdays and Fridays, whereas on Thursdays, ye-

hide (saline or distilled water) was injected. Testing was conductedtypically between 11:00 and 13:00 hr. Observations were made by asingle observer, typically the same individual who was not blinded tothe experimental conditions. On occasions in which different mdi-viduals made observations of the effects of quinpirole, there wasuniformly an extremely close agreement between the repeated as-sessments. Two-way repeated measures analyses ofvariance of three

such comparisons yielded significant effects of dose (F39>5.4878; P< .0202) with neither significant effects of observer (F1,9<.333; P>.6043) nor an interaction ofthe two terms (F39<zl.3197; P > .3273).The scoring procedure allowed a maximum of three positive scoresper subject at each dose.

The drugs studied included several dopaminergic agonists andantagonists, and several compounds with other predominant phar-macological effects. Due to initial changes in sensitivity, some dosesof quinpirole, (-)-NPA and piribedil were tested more than once inour study. For these drugs, the ranges of doses studied on thedifferent occasions overlapped, and the data from the identical doseswere averaged for the combined dose-effect functions presented inthe figures. All other drugs were examined only once at the range ofdoses shown in the figures. For most of the drug interaction studies,doses of the antagonists or distilled water were injected 30 mmbefore the first dose of quinpirole. Because the duration of action ofSCH 23390 is short (Bergman et al. , 1991), it was administered 5 mmbefore quinpirole. Each of two doses of SCH 23390 was studied ontwo occasions. On the first occasion, SCH 23390 was administeredfollowed by cumulative doses of 0.003 and 0.03 mg/kg of quinpirole.On the second occasion, SCH 23390 was administered followed bycumulative doses of0.3 and 3.0 mg/kg ofquinpmrole. Data from thesetwo sessions were combined to produce the full quinpirole dose effectcurve in the presence of SCH 23390.

Drugs. The dopamine D2 agonists studied were quinpirole hydro-chloride (LY 171555; Research Biochemicals Inc., Natick, MA), (-)-

NPA (RB!), RU 24213 (Roussel, Romainville, France), pergolide (LY127809; Eli Lilly and Co., Indianapolis, IN), quinelorane (LY 163502;Eli Lilly and Co.), piribedil (Regis Chemical Co., Martin Grove, IL)and apomorphine hydrochloride (Sigma Chemical Co., St. Louis,MO). The dopamine D1 agonists studied were SKF 38393 hydrochlo-ride (RB!), 6-chloro-APB hydrobromide (SKF 82958; RBI) and SKF

75670 hydrochloride (Smith Kline Beecham, King of Prussia, PA).Other dopaminergic compounds were the ( + )-enantiomer of NPA(RBI), the dopamine uptake inhibitor cocaine hydrochloride (NIDA,Rockville, MD) and the dopamine releaser d-amphetamine sulfate(Sigma). The dopamine D2 antagonists were haloperidol (McNeil,Spring House, NJ), spiperone hydrochloride (RB!), (-)-eticlopridehydrochloride (RB!) and its inactive enantiomer (+)-eticlopride hy-drochloride (RB!). The dopamine D1 antagonist, SCH 23390 maleate(RB!) and the peripheral dopamine antagonist, domperidone (RB!)were also studied, as well as the dopamine partial agonists, terguride(Schering, A.G., Berlin, FRG), SDZ 208-911 (Sandoz, Bern, Switzer-land) and SDZ 208-912 (Sandoz). Other compounds studied were thehistamine H1 receptor antagonist, diphenhydraniine HC1 (Sigma),morphine sulfate (National Institute on Drug Abuse, Rockville, MD),nicotine tartrate (NIDA), quipazine dimaleate (RB!), clonidine hy-drochloride (Sigma), 3-CCE (kindly supplied by J. M. Cook, Milwau-kee, WI) and chlordiazepoxide hydrochloride (Hoffmann-La Roche,Nutley, NJ). Drug doses are expressed as mg or �.mol of the formslisted above per kg body weight.

Quinpirole, RU 24213, piribedil, cocaine, d-amphetamine and mc-

QUINPIROLE

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140 PeIl#{243}nat al. Vol. 273

otine were dissolved in sterile 0.9% NaC1. (-)-NPA, pergolide,

quinelorane, SKF 38393, SKF 82958, SKF 75670, (+)-NPA, spiper-one, (-)-eticlopride, (+)-eticlopride, SCH 23390, diphenhydramine,

morphine, quipazine, clonidine and chiordiazepoxide were dissolvedin sterile water. Apomorphine, SCH 23390 and haloperidol were

dissolved in slightly acidified 0.9% NaCl. Domperidone was dissolvedin distilled water by adding three drops of85% lactic acid. Terguride,

SDZ 208-911, SDZ 208-912 and 13-CCE were dissolved in distilledwater to which one to three drops ofTween 80 were added. All drugswere prepared at concentrations three times higher than the drug

doses to ensure that no more than 1.5 ml was injected to each animal

on each testing session. Vehicle injections were ofa volume similar to

that of the drugs.Data analysis. The percentage ofobservations with scratching at

each dose was obtained by dividing the total number of observations

that contained at least one instance of scratching by the overall

maximum number of observations in all four monkeys (12); thatresult was multiplied by 100. Because there were no differences inthe effects of vehicles, all tests of vehicle administrations were av-eraged, and they are expressed in the same manner. When there wasmore than one determination for a dose of a drug, the average ofthose observations is reported. Dose-effect functions were analyzedusing analysis of variance and linear regression techniques (Snede-

cor and Cochran, 1967). From this analysis, ED� values and their95% confidence limits were derived from data using the linear par-tions ofthe dose-effect curves. A significant difference in ED50 values

is indicated when the 95% confidence limits do not overlap. Datawere analyzed as a two-way analysis ofvariance (subjects X doses) topartial out variance due to between subjects effects. To assess pa-tency of quinpirole treatments relative to a standard, parallel-linebioassay techniques were used as described by Finney (1964). Thevalue for relative patency represents the dose ofquinpirole in mg/kg,e.g, after pretreatment with antagonist, equal to 1 mg/kg of thequinpirole standard. A significant relative patency difference is in-dicated when the 95% confidence limits for that ratio do not include1.0. In these analyses, the data were analyzed as the percentage ofobservations in which scratching occurred, from which the vehicle

percentages were subtracted.

Results

Dose-related effects of D2 receptor agonists. All D2agonists tested produced marked scratching in all four mon-

keys, with gradations in effect as a function ofdose (fig. 1). At

the highest doses, all ofthese drugs produced scratching thatwas persistent and excessive; furthermore, all of the drugsproduced levels of scratching that reached 75% or more. Theopen circles on the left part of figure 1 represent the meansand the S.D. of the scratching produced by consecutive vehi-cle injections. As can be seen, vehicle administrations did notproduce changes in scratching which were related to succes-

sive injections. In addition, although vehicle injections were

followed by occasions in which there was some frequency of

scratching (with average values between 20% and 30%), thisscratching, although meeting the criteria (see above), on vi-

sual observation was distinct from that observed after ad-ministration of the D2 agonists, in that it was occasional and

perfunctory when it occurred. Although all ofthe D2 agonistsproduced similar scratching in terms oftopography and oftenmaximum intensity, the drugs were clearly separated intotwo groups on the basis of potency. Quinelorane and (-)-NPAwere the most potent D2 agonists, whereas all the other

agonists required doses approximating 100-fold higher to

result in similar levels of scratching. The ED50 values and

95% confidence limits for each ofthe drugs are given in table

DOSE (mg/kg)

Fig. 1. Effects of dopamine D2 receptor agonists on frequency ofscratching as a function of dose of drug. Ordinates: frequencyof scratching expressed as the percentage of observations in whichscratching was observed. Abscissae: cumulative dose expressed ascumulative pxnol of drug per kg body weight of subjects. Four subjectswere studied. Because the dose-effect curves were determined bycumulating dose, the effects of four successive vehicle injections werestudied as a control, and are shown to the left, above V, as fourconnected small open circles representing the means and error barsrepresenting the S.D. of those means.Effects of quinpirole, (-)-NPA andpiribedil are averages of, respectively, five, three and two determina-tions of an overlapping series of cumulative doses. All other drugs wereexamined only once at the range of doses shown in the figures.

1. These different ED50 values correspond roughly to the

published affinities for binding to D2 receptors, with quinelo-

rane having a higher potency in vivo than would be expected

based on in vitro binding (e.g. , Izenwasser and C#{244}t#{233},1995).However, because of the small number of agonists examinedin our study, a test ofthe correlation would not have achieved

the power necessary to have confidence in the results.Sensitivity developed to the scratching produced by the D2

receptor agonist quinpirole, which was the first drug to betested. After the initial observations of the effects of quinpi-role, there was a leftward shift in the dose-response curve

determined 24 days later. There were no further changes insensitivity to quinpirole over the 6-mo period during whichthese experiments were conducted. The ED50 value of quin-pirole changed from 0.168 mg/kg (95% CL: 0.066-0.430 mg/

TABLE 1Summary of ED�, values for each of the drugs studled

ED� (�moMcg) 95% Confidence Limits

Quinpirole 0.0469 0.013-0.170(-)-NPA 0.0009 0.0003-0.003Ru 24213 0.1441 0.040-0.518Pergolide 0.1091 0.024-0.493Pinbedil 0.1 760 0.053-0.580Apomorphine 0.0332 b

Quinelorane 0.0026 0.001-0.013Tergunde 0.018 0.009-0.036

a ED� values and their 95% confidence limits are given for each drug, WhiChis the dose produCing 50% observations of scratching. A significant difference

between ED� vaiues is indicated When there is no overlap in the 95% confidencelimits. These values W&� determined from ANOVA and linear regression tech-

niques as described in “Methods.”b The ED� value is only an estimate because the regression did not achieve

statistical significance.

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1995

kg) to 0.012 mg/kg (95% CL: 0.003-0.044 mg/kg), showing arelative potency ratio of 14.3 (95% CL: 1.86 to 97.8) from the

first to the average of later determinations. A quinpiroledose-effect curve determined in these same subjects morethan 1 yr later resulted in an ED50 value ofO.017 mg/kg (95%CL: 0.004 to 0.081 mg/kg).

Stereospecificity of the scratching produced by D2receptor agonists. The stereospecificity of the scratchingproduced by D2 receptor agonists was assessed by comparingthe effects of (-)-NPA and its inactive enantiomer (+)-NPA.

Administrations of ( + )-NPA never produced excessivescratching even at doses 300-fold higher than the lowest dose

of (-)-NPA that produced significant effects. At the highest

dose tested (0.03 mg/kg), (+)-NPA produced scratching dur-ing about 20% of the observations, a result that was within

the range of vehicle administrations (fig. 1).Effects ofpartial dopaminergic agonists. Effects of the

partial D2 agonists terguride, SDZ 208-911 and SDZ 208-.912, are shown in figure 2. Terguride produced dose-relatedincreases in the frequency of scratching with maximal effects

at 0.03 mg/kg. Neither SDZ 208-911 nor SDZ 208-912 pro-

duced comparable effects. SDZ 208-912 did not produce ef-fects that were significantly greater than vehicle, whereas

SDZ 208-911 produced an increase in scratching at the 0.003mg/kg dose.

100�

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Fig. 2. Effects of dopamine D2 receptor partial agonists on frequencyof scratching as a function of dose of drug. Ordinates: frequency ofscratching expressed as the percentage of observations in whichscratching was observed. Abscissae: cumulative dose expressed ascumulative mg of drug per kg body weight of subjects. Four subjectswere studied. Effects of terguride are averages of three determinationsof an overlapping series of cumulative doses. Each of the other drugswas examined only once at the range of doses shown in the figures.

Dopaminergic Behavioral Effects I 41

Effects of dopaminergic compounds other than D2receptor agonists. In contrast to the effects of the D2 do-

pamine receptor agonists, the D1 agonists, SKF 38393, SKF75670 and SKF 82958, produced dose-related decreases in

the percentage oftotal observations that included scratching.These results are represented on the left panel of figure 3.

The lower doses ofthese drugs produced scratching in 40% to50% ofthe observations; however, this level ofscratching was

not comparable to the highest levels obtained by the higher

doses ofthe D2 agonists (see fig. 1). Moreover, the scratching

that was observed was perfunctory and occasional, and sim-

ilar in form to that obtained after vehicle administrations, as

opposed to the rapid and persistent scratching observed after

administrations of D2 receptor agonists. Scratching was not

observed at the highest doses of both SKF 75670 and SKF

82958.

Results similar to those reported for D1 receptor agonistswere also obtained after administration of the indirect dopa-mine agonists, cocaine and d-amphetamine, as can be seen on

the right panel of figure 3. Both of these drugs produceddose-related decreases in scratching; at the lowest doses the

frequency of scratching was within the vehicle range. Themaximum frequency of scratching observed after cocaine ad-

ministration was approximately 40% at doses between 0.1and 1.0 mg/kg, and no scratching was recorded at the 3.0mg/kg dose. The maximum frequency of scratching after d-

amphetamine was 25% at the two lower doses, with scratch-

ing eliminated at the highest doses.

Effects of nondopaminergic compounds. Table 2shows the effects of drugs predominantly acting on systems

other than dopamine. Nicotine, quipazine, clonidine, fJ-CCEand chlordiazepoxide resulted in dose-related decreases in

the frequency ofscratching. As with D1 receptor agonists andindirect dopamine agonists, the scratching that occurred at

the lower doses of these drugs was in form and frequency

similar to that observed after vehicle administrations. Mor-

phine, at 0.3 mg/kg, produced an increase to 58% in the

percentage of observations with scratching. The frequency of

DOSE (mg/kg)

Fig. 3. Effects of dopamine D, receptor agonists (left panel) and mdi-rect agonists (right panel) on frequency of scratching as a function ofdose of drug. The highest dose of SKF 82958 was tested on a separateoccasion from the other doses of this drug. All other drugs wereexamined only once at the range of doses shown in the figures. The

dotted lines in both panels represent the range of scratching producedby four successive vehicle administrations (see fig. 1). Other details areas described in figure 1.

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Fig. 4. Antagonism of the effects of quinpirole by several dopaminereceptor antagonists. Ordinates: frequency of scratching expressed asthe percentage of observations in which scratching was observed.Abscissae: cumulative dose of quinpirole. Filled points represent dose-effect curves for quinpirole 30 mm after an Injection of distilled water.These curves are the averages of three determinations of quinpiroleafter vehicle and are reproduced in each panel. Open points representdose-effect curves for quinpirole after an injection of the designatedantagonist. For all antagonists other than SCH 23390, the quinpiroledose-effect curve was determined 30 mm after the injection of theantagonist. For detalls regarding the study of SCH 23390 see “Math-ods.” Other detalls are as described in figure 1.

142 Pell#{243}net al. Vol. 273

TABLE 2

Effects on the percentage of total observations with scratching of compounds with activity at oplold, cholinergic, serotonsrglc,adrenorgic or b.nzodlazeplne receptors

DrugDose

0.001 0.003 0.01 0.03 0.1 0.3 1.0 3.0 5.6 10.0

Morphine 25.00 58.33 41 .67 8.33Nicotine 41 .67 33.33 16.67 8.33Quipazine 33.33 25.00 0.00 0.00Clonidine 25.00 8.33 8.33 8.33p-CCE 33.33 33.33 16.67 8.33Chiordiazepoxide 1 6.67 25.00 8.33 0.00

scratching observed at this dose ofmorphine was statisticallydifferent from saline (Fisher’s exact test, P = .009). All of

these drugs at their higher doses produced a frequency ofscratching which was below control levels.

Effects of pretreatment with dopamine receptor an-tagonists. Administration of quinpirole, preceded 30 mmbefore by an injection of distilled water, resulted in a dose-response curve similar to that seen in figure 1 for quinpirole

alone. The ED50 value of quinpirole was 0.016 mg/kg (95%CL: 0.004-0.062 mg/kg) and this dose-effect curve is repre-sented by the ifiled circles in all panels offigure 4. As before,high doses of quinpirole produced excessive and persistentscratching in all monkeys. These effects were obtained ap-proximately 3 to 4 mo after the initial determinations of theeffects of quinpirole.

The dose-related increases in frequency of scratching pro-duced by quinpirole were dose-dependently antagonized bythe D2-receptor antagonists, haloperidol, (-)-eticlopride andspiperone (fig. 4, A-C). Pretreatment with 0.03 mg/kg of hal-

operidol (Fig. 4A) resulted in an approximate 100-fold right-ward shift in the dose-response curve ofquinpirole. The ED50value of quinpirole changed to 1.37 mg/kg (95% CL: 0.300-6.22 mg/kg), showing a relative potency ratio of 0.009 (95%CL: 0.001-0.044) compared to quinpirole preceded by vehicle.Pretreatment with lower doses ofhaloperidol (0.003 and 0.01

mg/kg) did not alter the frequency of scratching produced byquinpirole. (-)-Eticlopride also produced dose-dependent

shifts in the quinpirole dose-effect curve (fig. 4B). The lowest

dose studied of this antagonist (0.001 mg/kg) was inactive;however, 0.003 mg/kg of (-)-eticlopride antagonized the ef-fects ofquinpirole, resulting in an ED50 value of0.175 mg/kg

(95% CL: 0.066-0.440 mg/kg), giving a relative potency ratioof 0.100 (95% CL: 0.020-0.493). Pretreatment with 0.01mg/kg of(-)-eticlopride completely attenuated the scratching

produced by all doses of quinpirole studied, showing an in-surmountable antagonism. Pretreatment with spiperone (fig.4C) at the dose of 0.003 mg/kg also resulted in an insur-mountable antagonism of the scratching produced by quin-pirole. At the lower doses of spiperone there was no antago-nism of the effects of quinpirole.

Pretreatment with the D1 receptor antagonist SCH 23390

(fig. 4D) also shifted the quinpirole dose effect curve to theright. This antagonism was dose dependent with the ED50 forquinpirole changed to 0.109 mg/kg (95% CL: 0.038-0.314) at

the dose of 0.01 mg/kg of SCH 23390. The ED50 value forquinpirole at the 0.03 mg/kg dose ofSCH 23390 was changedto 0.521 mg/kg (95% CL: 0.224-1.21).

Possible antagonism of the effects of quinpirole by theinactive (+)-enantiomer of eticlopride (fig. 4E) was assessed

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;�.0003 0.003 0.03 0.3

QUINPIROLE DOSE (mg/kg)

1995 Dopaminergic Behavioral Effects 143

Fig. 5. Attenuation of the effects of quinpirole by the H1 antagonistdiphenhydramine. Other details are as described in figure 4.

to evaluate for stereospecificity in the blockade produced byD2 receptor antagonists. The (+)-enantiomer of eticlopridewas inactive as an antagonist ofthe effects ofquinpirole up to

doses that were 10-fold higher than an active dose of itsenantiomer (fig. 4, middle right panel).

Figure 4F shows the results of pretreating the monkeyswith the peripheral dopamine antagonist domperidone. Atthe doses of 1.0 and 3.0 mg/kg domperidone attenuated theeffects of quinpirole, whereas a lower dose (0. 1 mg/kg) was

inactive.Effects of pretreatment with the H1 receptor antag-

onist diphenhydramine. At the highest doses of D2 ago-nists that produced persistent and excessive scratching, sub-

jects also exhibited a pronounced facial flushing, suggesting

that a release ofhistamine mediated the scratching behavior.Pretreatment with the histamine antagonist diphenhydra-mine at a dose of 1.0 mg/kg did not alter the quinpiroledose-effect curve (fig. 5). At a higher dose (10.0 mg/kg), the

effectiveness of qunipirole was reduced.

Discussion

In our study several dopamine D2 receptor agonists pro-

duced marked and persistent scratching in squirrel monkeys,which appears to be a specific receptor-mediated effect of D2agonist action. Only drugs with direct D2 receptor agonistactions produced this effect; several D1 agonists, indirectdopamine agonists and drugs primarily acting on a variety ofother systems, did not produce persistent scratching. In ad-dition, the scratching was stereospecifically mediated; the(-)-enantiomer of the D2 agonist, NPA, was active, whereas

100 -

its enantiomer was not. Furthermore, the scratching induced

by quinpirole was attenuated by the D2 receptor antagonists,haloperidol, (-)-eticlopride and spiperone, but not by the (+)-

enantiomer ofeticlopride. Moreover, the dopamine D2 partialagonists SDZ 208-911 and SDZ 208-912 had limited efficacyin producing the persistent and excessive scratching behav-ior. These results together suggest that the scratching is

mediated by agonist actions at D2 dopamine receptors.Although the D2 agonists examined in our study were all

effective in producing scratching, quinpirole, which was theprincipal dopaminergic agonist examined, has reported se-

lectivity for D3 over D2 receptors (Sokoloff et al. , 1990). Al-though the selectivity of quinpirole for D3 receptors suggests

that scratching may not be mediated exclusively by D2 re-ceptor activation, a role of D3 receptor activity in mediatingthe scratching response has to be considered cautiously.First, a range of D2 agonists as well as partial agonists

produced scratching that was generally in accord with theirknown D2 pharmacology, such as potency, stereoselectivity

and intrinsic efficacy. In addition, haloperidol, which hassome selectivity for D2 over D3 receptors (Sokoloff et al.,

1990) was an effective antagonist of the scratching inducedby quinpirole. However, very little is known regarding func-tional consequences of D3 receptor activation (Large andStubbs, 1994; Freedman et al. , 1994) although the receptor isfound in somewhat different brain regions, and at densities

considerably lower than those of D2 receptors (Gehlert et al.,

1992). Therefore, definitive information on functional role ofD3 receptor activation in the scratching response will comeonly with the development of a wider variety of more selec-tive agonists and antagonists.

The aminoergolines, SDZ 208-911 and SDZ 208-912, andthe lisuride derivative, terguride, have been characterized asD2 receptor partial agonists. In a variety of pharmacologicaltests, SDZ 208-911 and terguride have displayed greaterefficacy than SDZ 208-912 (Clark et al. , 1991; Coward et al.,1990; Exner and Clark, 1992; Svensson et al., 1991; Izenwas-ser and COt#{233},1994). In our study, both SDZ 208-911 and

terguride increased scratching, whereas SDZ 208-912 didnot. In fact, terguride had maximal effects comparable tothose of any of the other agonists. Our results with these

compounds are consistent with the conclusions that thesecompounds have limited intrinsic activities, and that SDZ208-912 has less activity than the other two partial agonists.In addition, terguride had the greatest degree of activity in

that its maximal effects when given alone were as great asany of the other D2 agonists.

The precipitation ofscratching by the partial agonists, SDZ208-911 and terguride, suggests that the scratching re-sponse can be induced by the production of a relatively smalldegree of pharmacological stimulus. This stimulus might beproduced by less than full occupation of D2 receptors by afully efficacious agonist, or by occupancy of a larger numberof D2 receptors by a partial agonist. Because terguride andSDZ 208-911 often have similar efficacy in pharmacological

�1 assays (see, Svensson et al. , 1991; Coward et al. , 1990), thedifference found between these two compounds indicates that

3.0 a relatively small difference in intrinsic efficacy may be crit-

ical for the observed effects on scratching behavior.Taken as a whole, the data obtained with the partial ago-

nists provide further evidence for a D2 receptor mediation of

scratching in squirrel monkeys. However, the nondopamin-

144 Pell#{243}net al. Vol. 273

ergic actions ofthese compounds may have contributed to the

observed effects of these drugs. These compounds have sig-nificant affinities for a�, a2, 5-liT2 and 5-HT� receptors(Arnt and Hyttel, 1990; Coward et al. , 1990; Svensson et al.,

1991). Any of these actions may have limited the expression

of scratching and lead to incorrect assessments of differencesin efficacy.

Although the excessive scratching we reported appears tobe due to an action at D2 dopamine receptors, other studieshave investigated differing pharmacologies of scratching in

other species. For example, Darmani et al. (1990) showedthat 5-HT2 receptor agonists produced ear scratching inmice. Excessive scratching has been also reported to be in-duced, for example, by TRH in rabbits, and it has beensuggested that such scratching is in part mediated by dopa-mine (Popoli et al., 1991). Schino et al. (1991) have shownthat spontaneous scratching in macaques was reduced byadministrations ofthe benzodiazepine lorazepam. This result

is in keeping with our results with chlordiazepoxide. The

various pharmacological results of studies investigatingscratching suggest that this behavior may have a species-

specific pharmacology and that the pharmacological specific-

ity obtained in our study may not be obtained in those usingdifferent species.

The scratching behavior reported in our study may bemediated by a D2 receptor-induced release ofhistamine. This

claim is supported by our findings that the histamine H1antagonist diphenhydramine reduced the scratching pro-

duced by quinpirole. In addition, all of the D2 agonists pro-duced a marked facial flushing that was suggestive of hista-

mine release, and was alleviated by administration ofdiphenhydramine as well as D2 dopamine receptor antago-nists. The scratching does not appear to be a reflection of ageneral increase in stereotyped behavior because the indirectagomsts that produce stereotyped behavior, such as d-am-

phetamine and cocaine, did not produce scratching. In addi-tion, some of the behavioral correlates that occur with ste-reotyped behavior, such as “visual scanning” did not occur

with the D2 agonists. Although relationships between dopa-mine and histamine systems are not well understood atpresent, interactions between histamine and dopamine re-

ceptor activity have been reported (Dadkar et al. , 1976).Our data indicate that the initiation of the scratching by

D2 receptor agonists may have a peripheral dopaminergiccomponent. The peripherally acting D2 agonist, domperidone(Laduron and Leysen, 1979), attenuated the scratching in-duced by quinpirole. That attenuation was similar to theattenuation produced by diphenhydramine. However, the an-tagonism produced by the centrally acting D2 antagonists,haloperidol, (-)-eticlopride and spiperone, was generallygreater than that seen with the peripherally acting D2 an-tagonists, and was at some doses insurmountable. Thus, itappears that both central and peripheral dopamine receptorsmay regulate scratching in the squirrel monkey.

Sensitization developed to the scratch-inducing actions ofquinpirole. This sensitization was evident as an approximate14-fold shift to the left in the quinpirole dose-effect curveafter initial observations. The sensitization was also longlasting; after the initial changes in the effects of quinpirole,there were no further changes in sensitivity for over a 1-yrperiod during which these experiments were conducted. It islikely that the effects of the other D2 agomsts are influenced

by this sensitivity to quinpirole. Previous studies have alsoindicated sensitization to some effects of D2 receptor ago-

nists. For example, Hoffman and Wise (1992) found thatrepeated administrations of the D2 agonist, bromocriptine,produced successively greater stimulations of locomotor ac-tivity in rats. In addition, vehicle injections produced someelevation in locomotor activity in subjects that had beenexposed previously to bromocriptine. This sensitization oc-curred only in subjects that received the bromocriptine injec-tions in the environment in which the effects ofthe drug were

tested, and it was concluded that the sensitization was due toconditioning of the environmental stimuli uniquely pairedwith the drug effect. The mechanisms for the sensitizationobserved in our study are not readily apparent. A condition-ing mechanism appears unlikely, because the monkeys used

in our study received injections in the environment in whichthey lived. Therefore, the environment in which they re-ceived drugs was not uniquely associated with the drug in-jections. In addition, vehicle injections, which produced stun-

uli that were likely more salient and intimately paired withthe effects of quinpirole, failed to produce excessive scratch-

ing. It remains possible that the stimulus effects of lowerdoses became conditional stimuli for the higher doses thatalways followed under the cumulative dosing procedure (e.g.,

Schindler et al. , 1990); this mechanism should be examinedin further studies of the sensitivity induced to the scratchinginduced by D2 agonists. However, at present, it appears thatconditioning mechanisms likely do not mediate the sensitiv-ity induced by quinpirole, and our procedure may be used tomore fully investigate pharmacological mechanisms under-lying the sensitization to D2 agonists in the absence of envi-ronmental influences.

The antagonism of the effects of quinpirole obtained withD2 antagonists was often insurmountable rather than char-acterized by a progressive shift to the right in the quinpiroledose-effect functions as would be expected if the antagonism

was a classic competitive interaction. The complete antago-nism of the scratching response at particular doses suggeststhat precipitation ofa quantal scratching response requires a

threshold amount of pharmacological stimulus. Under theseconditions, occupancy of only a small fraction of receptors bythe antagonist could completely antagonize the response.That increases in doses of the agonist did not surmount theantagonism may be due to other actions of quinpirole occur-ring at high doses that interfere with scratching. In manyinstances in which quinpirole was examined with an antag-onist, the physical appearance of the subjects suggested thatstudies of the effects of higher doses would adversely affectthe health ofthe subjects. Alternatively, effects ofthe ant.ag-onists mediated by nondopaminergic systems could have in-terfered with surmounting the antagonism. Comparable lim-

itations in the surmounting of opioid antagonism in otherbehavioral procedures have been reported (France andWoods, 1985). These types of limitations may be more prey-alent in behavioral procedures in which the dose range thatcan be studied has stringent limitations.

An alternative explanation for the inability to overcomethe antagonism with higher doses of quinpirole is that theantagonism was nonspecific. High doses of dopamine recep-tor antagonists have sedative and cataleptic effects that mayinterfere with the expression of scratching. Arguing that theantagonism is not entirely nonspecific is the observation that

1995 Dopaminergic Behavioral Effects 145

some antagonist effects of (-)-eticlopride (0.003 mg/kg) and

spiperone (0.003 mg/kg) were obtained at doses that did notproduce grossly observable sedation.

Several previous studies have found that the stimulanteffects of D2 receptor agonists can be antagonized by the D1antagonist, SCH 23390. These results have been interpretedas suggesting that the expression of behavioral effects by D2agonists requires some stimulation ofD1 receptors (see Clarkand White, 1987). Our results are consistent with that sug-gestion in that scratching induced by D2 agonists was antag-onized by pretreatment with SCH 23390. The doses thatantagonized the effects of quinpirole were identical to thosethat have been shown to antagonize behavioral effects of D1

agonists in rats (Terry and Katz, 1992). Similarly, Rosenz-weig-Lipson and Bergman (1993) recently reported thatscratching induced by the D2 agonist, (+)-PHNO, was antag-omzed by the D1 antagonist, SCH 39166, at doses that areactive against D1 agonists. The fact that the antagonism bythe D1 antagonist ofthe effects of a D2 agonist occur at dosesthat are active against D1 agonists suggests that this inter-action is a D1 receptor mediated effect. Taken together, theseresults suggest that the scratching induced by D2 agonists insquirrel monkeys is regulated by D1 receptor activity, andmay be of use in studying the interactions among D1 and D2

receptors.

Acknowledgments

We thank Sharon Rosenzweig-Lipson for comments on a previousversion of this manuscript.

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