INTRASTRIATAL ADMINISTRATION OF AN OLIGODEOXYNUCLEOTIDE ANTISENSE TO THE D2 DOPAMINE RECEPTOR mRNA INHIBITS D2 DOPAMINE RECEPTOR-MEDIATED BEHAVIOR AND D2 DOPAMINE RECEPTORS IN NORMAL MICE AND IN MICE LESIONED WITH 6-HYDROXYDOPAMINE

P

Neurochem.Int.Vol.29,No. 6, pp. 583-595,1996 Copyright01996 ElsevierScienceLtd PII: S0197-0186(96)000642 Printedin Great Britain.All rightsreserved 01974186/96$15.00+0.00

INTRASTRIATAL ADMINISTRATION OF AN OLIGODEOXYNUCLEOTIDE ANTISENSE TO THE D2 DOPAMINE RECEPTOR mRNA INHIBITS D2 DOPAMINE RECEPTOR-MEDIATED BEHAVIOR AND D2 DOPAMINE RECEPTORS IN NORMAL MICE AND IN MICE LESIONED WITH 6-HYDROXYDOPAMINE LONG-WU ZHOU, SUI-POZHANG and BENJAMIN WEISS* Department of Pharmacology, Medical College of Pennsylvania, 3200 Henry Avenue, Philadelphia, PA 19129, U.S.A. (Received 25 March 1996; accepted 13 June 1996) Abstract—Previous studies have shown that the intracerebroventricular injection of antisense oligodeoxynucleotides targeted to themRNAsencoding the different subtypes ofdopamine receptors inhibited behaviors mediated by these receptors. The present studies were designed to determine whether such antisense oligodeoxynucleotides could produce similar effects when injected into a discrete brain area. A Dz dopamine receptor antisense oligodeoxynucleotide (Dz antisense) was repeatedly injected into one corpus striatum of either normal mice or mice with unilateral lesions of the striatum induced by 6-

hydroxydopamine.In the latter, intrastriatal injectionof D2antisenseblockedthe contralateral rotational behavior induced by the parenteral administration of the D2 dopamine receptor agonist quinpirole. The inhibitory effect of Dz antisense was dose- and time-related and was reversed upon cessation of Dz antisense treatment. This inhibitory effect was also selective in that Dz antisense treatment inhibited the rotational behavior induced by quinpirole but not that induced by the D, dopamine receptor agonist SKF 38393 or by the muscarinic cholinergic agonist oxotremorine. Following repeated intrastriatal injections of D, antisense into normal mice, parenteral administration of quinpirole caused rotational behavior ipsilateral to the side in which the Dz antisense was injected. No such rotational behavior was seen when similarly treated mice were challenged with SKF 38393 or oxotremorine. The quinpirole-induced rotational behavior in mice given intrastriatal injections of Dz antisense disappeared upon cessation of D2 antisense treatment. Repeated intrastriatal administration of Dj antisense also caused a significant reduction in the levels of D~, but not D,, dopamine receptors in striatum, as determined by receptor autoradiography. The levels of D, dopamine receptors returned to normal upon cessation of Dz antisense treatment. Intrastriatal administration of an oligodeoxynucleotide with randomly placed nucleotides failed to alter the rotational response to quinpirole in either 6-hydroxydopamine-lesioned or normal mice and failed to alter the levels of D~ dopamine receptors in striatum. These results show that selective inhibition of behavioral responses mediated by Dz dopamine receptors can be achieved by the direct injection of a Dz antisense oligodeoxynucleotide into a discrete brain area. Copyright ~ 1996 Elsevier Science Ltd

is a large body of evidence suggesting that schizophrenia is associated with an increased level of the D2 subtype of dopamine receptors in certain brain regions (Seeman et al., 1987; Wong et al., 1986) and that the therapeutic effect of antipsychotic drugs is related to their ability to antagonize the action of dopamine at these central dopamine receptors (Carlsson and Lindqvist, 1963; Seeman et al., 1976). Although the clinically available antipsychotic drugs

There

*To whomall correspondenceshould be addressed.

are effective in relieving many of the symptoms of schizophrenia, there are several problems associated with their use. Namely, they block dopamine receptors not only in the mesolimbic regions, which appear to be responsible for their therapeutic antipsychotic effects (Seeman and Ulpian, 1983; Angulo et al., 1991), but also in the nigrostriatal region of the brain, which may explain their extrapyramidal motor effects (Seeman and Ulpian, 1983; Baldessarini and Tarsy, 1980). Moreover, although many antipsychotics were thought to be relatively selective for the D2 dopamine

583

584

Long-Wu Zhou et al.

receptors, recent studies show that they bind to several subtypes of dopamine receptor (Schoots et al., 1995; Buckland et al., 1992) and to receptors for other neurotransmitters as well (Gattaz et al., 1994; Sumiyoshi et al., 1994; Qin and Weiss, 1994; See et al., 1990). Further, long treatment with these agents often leads to an upregulation of the very receptors they inhibit (Rupniak et al., 1983; See et al., 1990), a biochemical event that may explain the debilitative tardive dyskinesia which is often associated with their use (Marsden and Jenner, 1980).Clearly, the discovery of more effective and selective dopamine receptor antagonists may provide a better therapeutic regimen for treating disorders associated with dopaminergic hyperactivity. Recent molecular biological studies have revealed that dopamine is capable of acting through at least five distinct subtypes of dopamine receptors. These receptors can be classified pharmacologically and structurally into a D1-like family (Dl and D~) and a D,-like family (Dz, D3 and Dd) (Seeman, 1992; Gingrich and Caron, 1993; Civelli et al., 1991). These receptor subtypes or their respective transcripts are unevenly distributed in brain (Sunahara et al., 1991; Van Tol et al., 1991; Weiss et al., 1990; Zhang and Creese, 1993), change at different rates in different brain areas during ontogeny and aging (Mesco et al., 1993; Weiss et al., 1992), and are differentially modulated both acutely (Hillefors-Berglund and von Euler, 1994) and chronically (Chen et al., 1993; Weiss et al., 1990; Buckland et al., 1992) by pharmacological agents. As a result of these new studies, agents that were once thought to be relatively selective for a certain subtype of dopamine receptor have now been shown to bind to several dopamine receptor subtypes. For example, many neuroleptics, such as haloperidol, eticlopride and clozapine not only interact with D2 receptors but also with Da and Dd dopamine receptors (Boundy et al., 1993; Schoots et al., 1995), and quinpirole, one of the classic Dz dopamine receptor agonists, has been shown to have a high affinity for both Dz and D3 dopamine receptors. In order to uncover the biological functions of these subtypes of dopamine receptors, new and more selective agents which act on these receptors must be developed. In the past few years, antisense oligodeoxynucleotides have been shown to interfere with the expression of receptors and behaviors mediated by receptors for several brain proteins, including neuropeptide Y (Wahlestedt et al., 1993a), glutamate receptors (Wahlestedt et al., 1993),opioid receptors (Standifer et al., 1994), and D1 (Zhang et al., 1994) and Dz (Weiss

et al., 1993;Zhou et al., 1994; Silvia et al., 1994;Zhang and Creese, 1993) dopamine receptors. Studies from our laboratory showed that a phosphorothioatemodified oligodeoxynucleotide antisense to the D2 dopamine receptor mRNA (Dz antisense), when administered intracerebroventricularly (i.c.v.) into mice with unilateral 6-hydroxydopamine (6-OHDA) lesions of the corpus striatum, inhibited rotational behavior induced by a Dz dopamine agonist but not that induced by a D1 dopamine agonist or by a muscarinic cholinergic agonist. Treatment with the D2 antisense decreased the number of Dz dopamine receptors and the level of Dz dopamine receptor mRNA in the corpus striatum but did not alter the density of D, dopamine receptors or the level of D, receptormRNA in striatum (Weiss et al., 1993; Zhou et al., 1994). In analogous experiments, the i.c.v. administration of a D, antisense oligodeoxynucleotide (Dl antisense) to normal mice inhibited grooming behavior induced by a D, dopamine agonist but not the stereotype produced by a D2 dopamine agonist. Similarly, in 6OHDA-lesioned mice the D1 antisense inhibited rotational behavior induced by a D, dopamine agonist but not that produced by a D, dopamine agonist or by a muscarinic cholinergic agonist (Zhang et al., 1994). Since it would be desirable to reduce the function of specific subtypes of dopamine receptor not only in areas immediately accessible to the cerebral ventricles but in other brain areas as well, the present studies were designed to determine whether antisense oligodeoxynucleotides, when injected into a discrete brain region, can produce a blockade of selective dopamine receptor subtypes. Accordingly, in this study we examined the behavioral and biochemical effects of a D2 antisense oligodeoxynucleotide administered unilaterally directly into the corpus striatum of normal mice and of mice with unilateral 6-OHDAinduced lesions of the striatum. The results showed that the unilateral, intrastriatal administration of D2 AS resulted in an ipsilateral rotational behavior in response to a Dz dopamine agonist in normal mice, and inhibited the contralateral rotational behavior induced by a D2 dopamine agonist (but not that induced by a D1 dopamine agonist or by a muscarinic cholinergic agonist) in mice with 6-OHDA lesions. D1 AS treatment also reduced the levels of D2, but not D,, dopamine receptors in striatum. MATERIALSAND METHODS

Animals Male Swiss–Webster mice (2W24 g), purchased from Ace Animals Inc. (Boyertown, PA, U.S.A.), were

Intrastriatal administration of an oligodeoxynucleotide antisense used throughout these studies. All mice were housed in groups of 10 in plastic cages with wood chip bed-

ding and were provided free access to food and water. The mice were maintained in a temperature- and humidity-controlled room with a 12-h light+ark cycle. Administrationof drugs Quinpirole [lrans<-)4,4a,5,6,7,8,8a,9-octahydro5-propyl-lH(or 2H)-pyrazolo-(3,4g)-quinoline dihydrochloride] and (+)-SKF 38393 [l-phenyl-2,3,4,5tetrahydro-l H-3-benzazepine-7,8 -diol hydrochloride] were purchased from Research Biochemical Inc. (Natick, MA, U.S.A.). Oxotremorine was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Drugs for acute injections were prepared fresh, dissolved in 0.90/. NaCl containing 0.10/0ascorbic acid, and were administered subcutaneously. Design and synthesis of nucleotides

antisense oligodeoxy-

Based on the cDNA sequence for the D2 dopamine receptor (Mack et al., 1991; Bunzow et al., 1988), a 20-mer phosphorothioate oligodeoxynucleotide was designed and synthesized (Weiss et al., 1993; Zhou et al., 1994). The antisense was targeted to the area of the Dz dopamine receptor cDNA sequence that bridges the initiation codon (from –10 to + 10; 5’-GTGGATCCATTGGGGCAGTG-3’). This selected target sequence has relatively low homology with any of the other known cDNA sequences found in the Gene Bank data base (Bainbridge Island, WA, U.S.A.). As a control, a phosphorothioatesame modified oligodeoxynucleotide with the

proportion of bases as the Dz antisense, but having a random sequence (random oligo) (5’-GTGCTTGACGCGGATGGTGA-3’), was similarly prepared. The oligodeoxynucleotides were dissolved in sterilized vehicle (cerebrospinal fluid; CSF) and stored at –20”C until use. Intrastriatal injection of nucleotides

antisense oligodeoxy-

Mice were anesthetized with halothane and injected with antisense oligodeoxynucleotides into the right lateral striatum using a plastic mold (Goodale et al., 1985). Injections (2 pl) were made with a 30 gauge needle, administering the antisense oligodeoxynucleotides at a rate of 1 pl/min, followed by an additional 1.5 min period before removing the needle. Lesion techniques An adaptation (Von Voigtlander et al., 1973) of the Ungerstedt (1971) model of dopaminergic super-

585

sensitivity was used in these studies. Mice were anesthetized with halothane and injected with 2 pl of 6OHDA (100 nmol in 0.05% ascorbic acid) into the right corpus striatum by the technique of Goodale et al. (1985) as described previously (Winkler and Weiss, 1986). Ten days after the 6-OHDA lesion was made, all animals were examined for the presence of dopaminergic supersensitivity. Mice were considered to exhibit a supersensitive response if they evidenced at least 10 rotations per 5 min in response to acute injections of the D2 dopamine receptor agonist quinpirole (5 pmol/kg, subcutaneously). Rotationalbehavior Mice were placed in individual rectangular plastic cages (17 x 28 cm), and were allowed at least 20 min to acclimatize to that environment. Rotations were defined as tight 360° turns (with radii no greater than one body length) in a direction contralateral to the side of the lesion. Rotational behavior was scored as the number of rotations produced during a 5-rein period measured at the peak activity of each drug; i.e. 5 min after quinpirole, 15 min after SKF 38393 and 5 min after oxotremorine. D2andD, dopaminereceptor autoradiography The methods used to measure the D1 and Dz dopamine receptors were modified from those described by Kohler and Radesater (1986) for the D, dopamine receptor and by Boyson et al. (1986) for the D1 dopa-

mine receptor. Dried tissue sections were preincubated twice for 5 min in cold 50 mM Tris–HCl buffer (pH 7.4), which contained 1 mM MgCl,, 2 mM CaCl,, 5 mM KC1 and 120 mM NaC1. Specific Dz dopamine receptor binding was defined as the binding of 2 nM [3H]-raclopride in the absence and presence of 2 PM sulpiride. Specific D, dopamine receptor binding was defined as the binding of 2 nM [3H]-SCH 23390 in the absence and presence of 2 PM SCH 23390. Incubations were performed in 50 mM Tris–HCl buffer for 60 min at 25°C. The tissue sections were washed twice with cold Tris–HCl buffer and dried with a cool air stream. The sections were then exposed to tritiumsensitive Hyperfilm (Amersham, Arlington Heights, IL, U. S.A.) for either 30 days (for Dz dopamine receptors) or 5 days (for D1 dopamine receptors). The optical densities of the selected brain areas on the film were determined using a Drexel UNIX-based Microcomputer Analysis System (DUMAS software, Drexel University Image Processing Center, Philadelphia, PA, U. S.A.; Feingold et al., 1987). The resultant autoradiograms were quantified by converting optical density readings to femtomoles of

586

Long-Wu Zhou et al.

ligand bound per milligram of protein tissue by using tritium brain mash standards. Statistics Statistical analyses were performed by a one- or two-way analysis of variance followed by a post-hoc Dunnett’s or Newman–Keul’s test, as appropriate. RESULTS

Effect of intrastriataladministration of D2antisenseon rotationalbehaviorinducedby quinpirolein 6-OHDAIesionedmice Figure 1 shows the effects of varying doses of D2 antisense, given intrastriatally every 12 h for four injections, on the contralateral rotational behavior induced by the Dz dopamine agonist quinpirole. Clearly, the D, antisense produced a dose-related decrease in rotational behavior induced by quinpirole with an ID50value of approximately 1 nmol/injection.

0.0

0.5

Compared to previous studies, in which the Dz antisense and the random oligomer were injected i.c.v. (Weiss et al., 1993; Zhou et al., 1994), intrastriatal injections of the D2 antisense and the random oligomer produced a less intense and shorter duration of abnormal motor activity (locomotion and barrelrolling behavior). To avoid interference of these nonspecific effects with the behaviors to be measured, rotational behavior induced by the oligomers was determined 10 h after the injections, at which time no obvious abnormal behaviors were apparent. Comparisonof theeffects of theD2antisensetreatment on rotational behavior inducedby quinpirole,SKF 38393andoxotremorinein6-OHDA lesionedmice To determine the specificity by which the D2 antisense inhibited D2 dopamine receptor-mediated rotational behavior, mice with unilateral 6-OHDA lesions were administered D, antisense, and were then challenged with the D2 dopamine agonist quinpirole,

1.0

D2 ANTISENSE

1.5

(nmol/2

2.0

2.5

uI)

Fig, 1. Effect of repeated intrastriatal administration of D, antisense on quinpirole-induced rotational behavior in 6-OHDA lesioned mice. Mice with unilateral 6-OHDA lesions received intrastriatal injections of various doses of Dz antisense twice daily for 2 days. Rotational behavior induced by quinpirole (5 ~mol/kg, s.c., given 10 h after the fourth injection of D, antisense) was measured during a 5-rein period 5–10 min after the injection of quinpirole. Each point represents the mean value from four to five mice. The vertical brackets indicate the SE. Statistical comparisons between control and each antisense-treated group were carried out by one-way analysis of variance followed by a Dunnett test. “p< O.05; ““p
Intrastriatal administration of an oligodeoxynucleotide antisense the D, dopamine agonist SKF 38393, and the mus-

carinic cholinergic agonist oxotremorine. Figure 2 shows that direct, intrastriatal injections of Dz antisense (2.5 nmol/2 pl) almost completely inhibited rotational behavior induced by quinpirole. By contrast, Dz antisense treatment elicited no significant inhibition of rotations induced by SKF 38393 or oxotremorine. Similar intrastriatal injections of the vehicle or the random oligomer failed to produce significant inhibition of quinpirole-induced rotations. Recovery of’ quinpirole-induced rotational behavior after cessation of d2antisensetreatmentin 6-OHDA lesionedmice To determine whether the effect of D2 antisense on quinpirole-induced rotational behavior was reversible, mice with unilateral 6-OHDA lesions were treated with 2.5 nmol of Dz antisense every 12 h for four injections, and rotational behavior induced by quinpirole was measured at varying times after the

last injection of D, antisense. Figure 3 shows that there was a significant reduction of quinpiroleinduced rotations 0.5, 1, and 1.5 days after the fourth injection of Dz antisense. However, at 2 and 4 days after the last injection, the effect of quinpirole was not significantly different when compared to that found in vehicle-treated animals. Intrastriatalinjectionsof D2 antisenseresults in ipsilaleral rotations in response to quinpirolein normal mice To determine further the effect of injecting D2 antisense into the striatum of normal mice, mice treated with D2 antisense were challenged with quinpirole, SKF 38393 and oxotremorine. The results shows that in mice treated with D2 antisense, quinpirole produced a significant ipsilateral rotational behavior (Fig. 4). By contrast, no significant rotations were induced by SKF 38393 or by oxotremorine. Since the same mice were challenged with each of these agents, quinpirole

30

❑

VEHICLE

H

D2ANTISENSE

❑

RANDOMOLIGO

20

TT

I

SKF

38393

-rTT

10

0

587

— OXOTREMORINE

Fie. 2. Effect of re~eated intrastriatal administration of D, antisense on rotational behavior induced bv qu%pirole, SKF 38393 and oxotremorinein 6-OHDAlesion~d mice. Mice with unilatera16-OHDA-induced lesions received intrastriatal injections of D, antisense (2.5 nmol/2 Yl), random oligodeoxynucleotide (2.5 nmol/2 ~1) or vehicle (2 pl of artificial CSF) twice daily for 4 days. The mice were challenged SC. with quinpirole (5 ymol/kg), SKF 38393 (40 Lmol/kg) and oxotremorine (5 ~mol/kg), and rotational behavior was assessed. The dopaminergic and cholinergic agonists were administered to these different groups of mice 10 h after the fourth (for quinpirole), 6th (for SKF 38393) and 8th (for oxotremorine) injection of oligomers or vehicle. Each point represents the mean value from 5 to 15mice. The vertical brackets indicate the S.E. “’”p<0.01 compared with vehicle- or random-oligo-treated mice.

Long-WuZhou et al.

588 40

30

~

VEHICLE

~

D2 ANTISENSE

I

20

10

0

1

n

1

0 A ‘A A 2A 3

1

4

I

1

1

1

1

(

5

6

7

8

9

‘0

DAYS Fig, 3. Recovery of quinpirole-induced rotational behavior after cessation of D2 antisense treatment in 6-OHDA lesioned mice. Mice with 6-OHDA-induced lesions received intrastriatal injections of D2 antisense (2.5 nmol/2 pl) or vehicle (2 pl of artificial CSF) four times at 12 h intervals (shown by the arrowheads). Rotational behavior induced by quinpirole (5 pmol/kg, s.c.) was measured 10 h after the last injection of vehicle or Dz antisense. The mice were challenged again with quinpirole at 1, 1.5, 2, 4, and 8 days after the last injection of vehicle or D, antisense. Each point represents the mean value from five mice. The vertical braCkets indicate the S.E. ‘p< O.05, “p
was injected at two different times after D2 antisense treatment, both before and after challenge doses of SKF38393 and oxotremorine. Similar treatment of mice with the vehicle or random-oligo failed to produce ipsilateral rotational behavior in response to any of the agonists (Fig. 4). Quinpirole-induced rotationalbehaviorcausedby intrastriatalinjectionsof D2antisensedisappearsuponcessationof D2antisensetreatment To determine whether the ipsilateral rotational behavior induced by quinpirole in mice given intrastriatal injections of D2 antisense was reversible, mice were treated with Dz antisense every 12 h for 2 days and then once a day for 4 days. Rotational behavior induced by quinpirole was measured 10 h after 2, 4 and 6 days of injection with D2 antisense, and then at varying times after the last injection of D2 antisense.

Figure 5 shows that there was a significant ipsilateral rotational behavior induced by quinpirole at 2, 4 and 6 days after the injection of D, antisense. At 2 and 3 days after the last injection of D2 antisense, quinpirole continued to produce significant rotational behavior. However, 5 days after the last injection of D2 antisense, the ipsilateral rotational behavior induced by quinpirole ceased (Fig. 5). Effect of intrastriatalinjectionsof D, antisenseon the levelsof D2dopaminereceptors inmousestriatum To determine whether the effect of D2 antisense treatment on DJ dopamine receptor-mediated rotational behavior was associated with changes in the levels of D, dopamine receptors, mice were injected intrastriatally with Dz antisense once daily for 6 days. The brains were then sectioned, and the levels of D, and Dz dopamine receptors were analysed and quant-

Intrastriatal administration of an oligodeoxynucleotide antisense 12

*

❑ VEHICLE ❑ D2ANTISENSE ❑ RANDOM OI-11=

T 1

10

589

* 1-

I

8

6

4

2

0

-2

1

QUINPIROLE DAYS OF TREATMENT

2

1

SKF

1

38393 4

1

4

OXOTREMORINE QUINPIROLE 6

5

Fig. 4. Intrastriatal injection of D, antisense results in ipsilateral rotations in response to quinpirole but not in response to SKF 38393 or oxotremorine in mice. Mice were administered unilateral intrastriatal injections of vehicle, Dz antisense (2.5 nmol/2 Ml),or random-oligo (2.5 nmol/2 pl) twice daily for 2 days, then once daily for 4 days. Rotational behavior induced by challenge injections of qninpirole (5 #mol/kg, s.c., measured at 2 and 6 days after antisense treatment), SKF 38393 (40 pmol/kg, s.c., measured at 4 days after antisense treatment), and oxotremorine (5 ~mol/kg, s.c., measured at 5 days after antisense treatment) was determined 10 h after the last injection of vehicle or oligomer. Each column represents the mean value from five to seven mice. Vertical brackets indicate the S.E. “p
ified using receptor autoradiographic techniques. Figure 6 shows that repeated intrastriatal injections of mice with Dz antisense reduced the number of Dz dopamine receptors in the injected striatum. By contrast, there were no apparent changes in the levels of D1 dopamine receptors in the injected striatum. Table 1 shows a quantitative analysis of the results of these treatments from 5 animals per group. The number of Dz dopamine receptors in the Dz antisense injected striata was significantly decreased at 6 days after treatment with Dz antisense; the levels of D2 dopamine receptors recovered to normal values by 4 days after the last injection of D2 antisense. Similar treatment of mice with the vehicle or random oligomer failed to alter the levels of Dj dopamine receptors at any time point studied. Neither Dz antisense nor the random oligomer significantly altered the levels of D1 dopamine receptors in the striatum.

DISCUSS1ON

The present data show that the function of D, dopamine receptors can be inhibited by injecting an antisense oligodeoxynucleotide targeted to the D2 dopamine receptor mRNA directly into a discrete area of the brain. The overall results of these experiments are similar to those which the Dz antisense was administered i.c.v. in animals unilaterally lesioned with 6hydroxydopamine. That is, in both cases the inhibitory effect of Dz antisense treatment on D2-mediated behaviors was dependent upon the dose of Dz antisense and on the length of time it was administered. Also in both cases these effects were reversible upon cessation of D2 antisense treatment. Similarly, in 6hydroxydopamine lesioned mice i.c.v. administered and direct intrastriatal injections of D2 antisense resulted in an inhibition of the contralateral rotational

590

Long-Wu Zhou et al. 12 * 10

**

*

~

VEHICLE

~

D2 ANTISENSE

--- -o---

RANDOMOLIGO

8

6

4

* 2

0

‘A A A 2A

A

4A

A ‘

8

10

DAYS Fig. 5. Recovery of quinpirole-induced rotational behavior after cessation of intrastriatal injections of D2 antisense in mice, Mice were given intrastriatal injections of D2 antisense (2.5 nmol/2 pi), random-oligo (2.5 nmol/2 pl) or vehicle (2 pl of artificial CSF) twice daily for 1 day, then once daily for 5 days (shown by the arrowheads). Ipsilateral rotational behavior induced by quinpirole (5 pmol/kg, s.c.) was measured 20 h after the 3rd, 5th and 6th injection of vehicle or oligomer, and at 2,3 and 5 days after the last injection of vehicle or oligomer. Each point represents the mean value from five mice. Vertical brackets indicate the SE. “p< O.05,“p
Table 1,Effectof intrastriatalinjectionof D, antisenseon Dzand D1receptorsin mousestriatum Treatment

D, receptors(fmol/mgprotein) Controlside

Vehicle(2 days) Vehicle(6 days) Vehicle(6 days)+no vehicle(4 days) AS (2days) AS (6 days) AS (6 days)+no AS (4 days) RND (2 days) RND (6 days) RND (6days)+no RND (4 days)

460+ 10 490f 10 440+ 10 460t 10 450+ 10 460+20 460+5 450f 10 460+ 10

D, receptors(fmol/mgprotein)

Injectedside

Control side

Injectedside

460+ 10 480+ 10 490+ 10 450+ 10 360+ 10”’ 480+29 460i20 440+20 460+20

2500~ 100 2500+80 2600~70 2500t80 2300~80 2500+60 2600+ 100 2300+ 100 2300+70

2400+ 100 2400+ 100 2500+90 2500+80 2100+80 2400+80 2500+ 100 21OO*100 2200* 100

Micereceivedunilateralintrastriatal injectionsof vehicle(2 Ml),D, antisense(2.5nmol/2vl) or rarrdom-oligo(2.5nmol/2jd) oncedaily for either2 or 6 days.The brainswereremoved2 h after the last injection.In a third groupof mice,animalsweregivenintrastriatalinjections of the oligomersfor 6 days,and the brainswereremoved4 days after the last injection,D2and DI dopaminereceptorswereanalysedby receptorautoradiographyusing [3H]-racloprideand [3H]-SCH23390as ligandsfor Dzand D1receptors,respectively.The signalsfrom the striata wereanalysedusing a DUMAS imageanalyser,Each valuerepresentsthe mean+S,E. from 5 mice,two sectionsfrom each animal.*p< O.001comparedwith valuesfrom the non-injectedside;‘p< O.01comparedwith the valuefrom the samesideof vehicle-or random-oligo-treatedmice.

Intrastriatal administration of an oligodeoxynucleotide antisense

D, RECEPTOR

D, RECEPTOR Non-injected

591

Injected

Non-injected

Injected

,..,

Vehicle

D2 Antisense

Random Oligo

*::* Fig. 6. Effect of intrastriatal injections of D2 antisense on D2 and D, dopamine receptors in mouse brain. Mice received unilateral intrastriatal injections of vehicle (2 pl of artificial CSF), D, antisense (2.5 nmol/ 2 @) and random-oligo (2.5 nmol/2 yl) once daily for 6 days, The brains were removed 2 h after the last injection, and 16 pm coronal sections were prepared for receptor autoradiography, The sections were incubated with [3H]-raclopride and [3H]-SCH 23390 to visualize the D2 and D1 receptors, respectively, The signals were detected using tritium-sensitive film. Arrow points to needle track.

behavior induced by a DJ dopamine receptor agonist but not that induced by a D, dopamine agonist or by a cholinergic agonist. In the present study we showed in addition that direct injection of a D2 antisense into one striatum causes ipsilateral rotations when challenged with the Dz agonist quinpirole but not when challenged with the D, agonist SKF38393 or with the cholinergic agonist oxotremorine. This is in agreement with the conclusion that D2 antisense treatment reduced the level of functional DJ dopamine receptors in the injected striatum (Qin et al., 1995). The phosphorothioate-modi fied antisense sequence used in this study was targeted to –10 to + 10 of

the dopamine sequence of the Dz dopamine receptor mRNA and was the same as that employed in previous studies in which the oligodeoxynucleotide was injected intracerebroventricularly (Zhou et al., 1994). This action was likely due to a true antisense effect, since another phosphorothioate-modified sequence targeted to the +12 to +31 sequence of the D2 mRNA and an amidate-modified D2 antisense oligodeoxynucleotide targeted to the –10 to +10 site on the Dz mRNA also inhibited quinpirole-induced rotational behavior in this model system. Since it has been reported that four consecutive Gs in an oligodeoxynucleotide sometimes produce nonspecific

592

Long-Wu Zhou et al.

effects (Yaswen et al., 1993), we have examined the actions of an oligodeoxynucleotide with randomly placed nucleotides containing four consecutive guanine nucleotides. This compound failed to significantly inhibit quinpirole-induced rotations (Weiss et al., 1996), indicating once more that the antisense oligodeoxynucleotide used in our study was selective and due to an antisense action. The conclusion that specific effects of antisense oligodeoxynucleotides can be produced by injecting them directly into discrete brain regions is consistent with those of Silvia et al. (1994) who showed that direct injection of a Dz antisense oligodeoxynucleotide into the substantial nigra of rats caused contralateral rotational behavior in response to parenterally administered cocaine. The present results also support the conclusions of previous studies, which showed that the inhibitory effects ofi.c.v. administered D2 antisense on Dz-mediated behaviors was caused by an inhibition of the synthesis of D2 dopamine receptors in the corpus striatum (Qin et al., 1995). That similar behavioral effects were produced by D2 antisense when given either i.c.v. or directly into the striatum was not surprising since D2 antisense administered i.c.v. readily penetrates the corpus striatum (Zhang et al., 1995). That repeated injections of Dz antisense into the striatum did not cause a permanent destruction of the striatum is supported by the evidence that the Dzmediated behaviors returned to normal upon cessation of treatment with Dz antisense. Moreover, the levels of Dz dopamine receptors in the striatum also returned to normal after cessation of Dz antisense treatment. The advantage of direct injection of antisense oligodeoxynucleotide into discrete brain regions is the potential for more selective localization of effect. For example, although it is generally believed that the motor effects mediated by DJ dopamine receptors involve the nigrostriatal system (LaHoste and Marshall, 1990; Barone et al., 1986), whereas the effects of dopamine on processes related to learning and motivation are associated with the mesolimbic or mesocortical areas of brain (van den Bos et al., 1991; Beninger, 1983), this has not yet been definitely proven. Moreover, which specific subtype of dopamine receptor is responsible for each of these effects is still uncertain. The use of specific antisense oligodeoxynucleotides targeted to the individual dopamine receptor mRNAs and injected directly into discrete brain regions may help resolve these outstanding questions. The data showing that direct intrastriatal injections of D2 antisense into mice with unilateral lesions of

the striatum resulted in a reversal of contralateral behaviors in response to treatment with the D2 dopamine agonist quinpirole supports the long-held view that 6-hydroxydopamine treatment causes an upregulation of postsynaptic D2 dopamine receptors (Arnt, 1985) and that D, antisense treatment reduces the levels of these receptors (Zhou et al., 1994). Similarly, the data showing that unilateral intrastriatal injections of Dj antisense into normal mice results in an ipsilateral rotational response to parenterally administered quinpirole supports the view that animals rotate in a direction opposite from the side of greater Dz dopaminergic activity (Miller and Beninger, 1991). Our studies showed that intrastriatal injection of Dz antisense in mice produced about a 20% reduction in the levels of Dz dopamine receptors. Although relatively large changes in the density of D2 receptors following continuous D2 antisense treatment in rats have been reported (Zhang and Creese, 1993), most investigators have found relatively modest reductions in receptors in response to antisense treatment (Standifer et al., 1994; Karle et al., 1995; Bourson et al., 1995). In fact, Standaert et al. (1996) recently reported that intrastriatal injection of an NMDAR1 antisense oligodeoxynucleotide produced rotational behaviors but had little or no effect on the levels of NMDA receptors. Examination of the time course of the effects of D2 antisense treatment on D2 dopamine receptormediated behaviors and on the levels of Dz dopamine receptors, as measured by receptor autoradiography, showed that Dz dopamine mediated behaviors were reduced before there was a discernible reduction in the levels of D2 dopamine receptors in striatum. That is, after 2 days of treatment with DJ antisense there was a significant reduction in D2 dopamine receptormediated turning behavior but no significant reduction in the levels of D2 dopamine receptors. Only after longer term treatment with Dz antisense (i.e. after 6 days) was there a statistically significant reduction in the level of D2 dopamine receptors. This apparent discrepancy between dopamine receptor-mediated behaviors and the level of dopamine receptors may be explained by the existence of functional and nonfunctional pools of dopamine receptors (Thermos et al., 1987; van den Bos et al., 1991; Qin et al., 1995). According to this hypothesis, the newly synthesized dopamine receptors constitute a relatively small, functionally active pool of receptors, which is inhibited by the antisense oligodeoxynucleotides. Therefore, a reduction in the synthesis of these receptors, although causing a relatively small reduction in the total pool

Intrastriatal administration of an oligodeoxynucleotide antisense

of receptors, would cause a large reduction in the functional pool. This hypothesis is supported by recent studies in which the total pool of D2 dopamine receptors was inhibited by treating mice with the selective, irreversible Dj dopamine antagonist fluphenazine-N-mustard, after which the Dj antisense caused a relatively large reduction in the rate of restoration of the receptors. The restoration of this hypothesized functional pool of D2 dopamine receptors correlated well with the restoration of D2 dopamine receptor mediated behaviors (Qin ei al., 1995). In conclusion, the present results show that direct oliintrastriatal injection of an antisense godeoxynucleotide targeted to the D2dopaminereceptor mRNA results in a reduction in the levels of D2 dopamine receptors in corpus striatum and an inhibition of behaviors that are mediated by D, dopamine receptors in the corpus striatum. These studies suggest the possibility of uncovering not only the function but also the specific brain regions responsible for the functions mediated by other subtypes of receptor for neurotransmitters. Acknowledgements—This work was supported by Grant MH42148 awarded by the National Institute of Mental Health. REFERENCES

Angulo J. A., Coirini H., Ledoux M. and Schumacher M. (1991) Regulation by dopaminergic neurotransmission of dopamine D2 mRNA and receptor levels in the striatum and nucleus accumbens of the rat, Mol. Brain Res. 11, 161-166. Arnt J. (1985) Hyperactivity induced by stimulation of separate dopamine D, and D2 receptors in rats with bilateral 6-OHDA lesions. Lije Sci. 37,717-723. Baldessarini R. and Tarsy D. (1980) Dopamine and the pathophysiology of dyskinesias induced by antipsychotic drugs. Ann. Rev. Neurosci. 3, 23W11. Barone P., Davis T., Braun A. R. and Chase T. N. (1986) Dopaminergic mechanisms and motor function: characterization of D, and Dz dopamine receptor interactions. Eur. J. Pharmacol. 123, 109–114. Beninger R. J. (1983) The role of dopamine in locomotor activity and learning. Brain Res. Rev. 6, 173–196. Boundy V. A., Luedtke R. R., Gallitano A. L., Smith J. E., Filtz T. M., Kallen R. G. and Molinoff P. B. (1993) Expression and characterization of the rat D3 dopamine receptor: pharmacologic properties and development of antibodies. J. Pharmacol. Exp. Ther. 264, 1002–1011. Bourson A., Borroni E., Austin R. H., Monsma F, J. Jr and Sleight A. J. (1995) Determination of the role of the S-ht, receptor in the rat brain: A study using antisense oligonucleotides. J. Pharmacol. Exp. Ther. 274, 173–180. Boyson S. J., McGonigle P. and Molinoff P. B. (1986) Quantitative autoradiographic localization of the D-1 and D-2 ., ... --.. .-—. ,- ___!--..:- T-.,.. ,. ”..; of D2 antisense in~o mice with unilateral lesions of

593

Buckland P. R., O’Donovan M. C. and McGuffin P. (1992) Changes in dopamine D,, Dz and D, receptor mRNA levels in rat brain following antipsychotic treatment. Psychopharmacology 106, 479483. Bunzow J. R., Van Tol H. H. M., Grandy D. K., Albert P., Salon J., Macdonald C., Machida C, A., Neve K. A. and Civelli O. (1988) Cloning and expression of a rat DZ dopamine receptor cDNA. Nature 336,783-787. Carlsson A. and Lindqvist M. (1963) Effect of chlorpromazine or haloperidol on formation of 3-methoxytyramine and normetanephrine in mouse brain. ,4cta Pharmacol. Toxicol. 20, 14&144. Chen J. F., Aloyo V. J. and Weiss B. (1993) Continuous treatment with the Dz dopamine receptor agonist quinpirole decreases Dz dopamine receptors, Dz dopamine receptor messenger RNA and proenkephalin messenger RNA, and increases mu opioid receptors in mouse striatum. Neuroscience 54, 669–680. Civelli O., Bunzow J. R., Grandy D. K., Zhou Q.-Y. and Van Tol H, H. M. (1991) Molecular biology of the dopamine receptors. Eur. J. Pharmacol. Mol. Pharmacol, 207, 277– 286. Feingold, E., Seshadri, S.B. and Tretiak, O. (1987) Hardware and software design consideration in engineering an image processing workstation: autoradiographic analysis with DUMAS and the brain autoradiography analysis software package. In Experimental Biology and Medicine, Vol. 11, FunctionalMapping in Biology and Medicine, McEachron, cd., pp. 175–203. Karger, Basel. Gattaz W. F., Schummer B. and Behrens S. (1994) Effects of zotepine, haloperidol and clozapine on MK-801-induced stereotype and locomotionin rats. J. Neural T’ransm.96, 227-232. Gingrich J. A. and Caron M. G. (1993) Recent advances in the molecular biology of dopamine receptors. Ann. Reu. Neurosci. 16, 299–321. Goodale D. B., Jacobi A. G. M., Winkler J. D. and Weiss B. (1985) Rapid stereotaxic injections into mouse striatum using a plastic mold. Soc. Neurosci. 11, 772. Hillefors-Berglund M. W. and von Euler G. (1994) Pharmacology of dopamine Dj receptors in the islands of CalIeja of the rat using quantitative receptor autoradiography. Eur. J. Pharmacol. 261, 179-183. Karle J., Witt M.-R. and Nielsen M. (1995) Antisense oligonucleotide to GABA~ receptor 72 subunit induces loss of neurons in rat hippocampus. Neurosci. Lett. 202, 97–

100,

Kohler C. and Radesater A.-C. (1986) Autoradiographic visualization of dopamine D-2 receptors in the monkey brain using the selective benzamide drug [3H]raclopnde. Neurosci. Lett. 66, 85–90. LaHoste G. J. and Marshall J. F. (1990) Nigral D, and striatal D2 receptors mediate the behavioral effects of dopamine agonists. Behav. Brain. Res. 38,233-242. Mack K. J., Todd R. D. and O’Malley K. L. (1991) The mouse dopamine Dz receptor gene: sequence homology with the rat and human genes and expression of alternative transcripts. J. Neurochem. 57, 795–801. Marsden C. D. and Jenner P. (1980) The pathophysiology of extrapyramidal side effects of neuroleptic drugs. Psychol. Med. 10, 55–72. Mesco E. R., Carlson S. G. and Joseph J. A., Roth G. S. (1993) Decreased striatal D, dopamine receptor mRNA e.r..+l..=o; . Allm;”m..”;”” a,fn/ R.flkl R@.,.,17. JfjoA67. -.=--

causing a relatively small reduction in the total pool

594

Long-Wu Zhou et al.

srsymmetries of posture and locomotion produced with dopamine agonists in animals with unilateral depletion of striatal dopamine. Progr. Neurobird. 36, 229-256. Qin Z.-H,, Zhou L.-W,, Zhang S.-P., Wang Y. and Weiss B. (1995) D, dopamine receptor antisense oligodeoxynucleotide inhibits the synthesis of a functional pool of D1 dopamine receptors. Mol. Pharmocol. 48, 73& 737’. Qin Z.-H. and Weiss B. (1994) Dopamine receptor blockade increases dopamine D2 receptor and glutamic acid decarboxylase mRNAs in mouse substantial nigra. Eur. J. Pharmacol. Mo[. Pharmacol. 269, 25–33. Rupniak N., Jenner P. and Marsden C. (1983) The effect of chronic neuroleptic administration on cerebral dopamine receptor function, Lye SCi.32, 2289-231 I. Schouk 0., Seeman P., Guan H.-C,, Paterson A, D. and Van ToI H, H, M, (’995) Long-term haloperidol elevates dopaminc D, receptors by 2-fold in rats. Eur.J. Pharmacol. 289,67-72. See R. E., Toga A. W. and Ellison G. (1990) Autoradiographic analysis of regional alterations in brain receptors following chronic administration and withdrawal of typical and atypical nerrroleptics in rats. J. Neural Transm. Cien.Sect, 82, 93–109. Seeman P. (1992) Dopaminereceptor sequences. Therapeutic levels of neuroleptics occupy D, receptors, clozapine occupies D,. Neuropsychopharmacology7,261-284. Seeman P., Bzowej N. H., Guan H.-C., Bergeron C., Reynolds G. P., Bird E. D., Riederer P,, Jellinger K. and Tourtellotte W. W. (1987) Human brain D, and Dz dopamine receptors in schizophrenia, Alzheimer’s, Parkinson’s, and Huntington’s diseases. Neuropsychopharmacology 1, 5– 15. Seeman P., Lee T., Chau-Wong M. and Wong K. (1976) Antipsychotic drug doses and neuroleptic/dopamine receptors. Nafure 261, 717–719. Seeman P. and Ulpian C. (1983) Neuroleptics have identical potencies in human brain limbic and putamen regions. Eur. J. Pharmaco[. 94, 145–148. Silvia C. P., King G. R., Lee T. H., Xue Z.-Y., Caron M. G. and Ellinwood E. H, (1994) Intranigral administration of Dj dopamine receptor antisense oligodeoxynucleotides establishes a role for nigrostriatal D2 autoreceptors in the motor actions of cocaine. Mol. Pharmacol. 46, 51–57, Standaert D. G., Testa C. M., Rudolf G. D. and HolIingsworth Z. R. (1996) Inhibition of N-methyl-D-aspartate glutamate receptor subunit expression by antisense oligonucleotides reveals their role in striatal motor regulation. J. Pharmacol. Exp. Ther. 276, 342–352. Standifer K. M., Chien C.-C., Wahlestedt C., Brown G. P. and Pasternak G, W. (1994) Selective loss of 6 opioid analgesia and binding by antisense oligodeoxynucleotides to a ~ opioid receptor. Neuron 12, 805–810. Sumiyoshi T., Kido H,, Sakamoto H., Urasaki K., Suzuki K., Yamaguchi N,, Mori H. and Shiba K. (1994) Time course of dopamine,,j and serotoninz receptor binding of antipsychotics in vivo. Pharmacol. Biochem. Behau. 49, 165-169. Sunahara R. K., Guan H. C., O’Dowd B. F., Seeman P., Laurier L. G., Ng G., George S. R., Torchia J., Van Tol H. H. and Niznik H. B. (1991) Cloning of the gene for a human dopamine D~ receptor with higher affinity for dopamine than D,. Nature 350,614-619. Thermos K., Winkler J. D. and Weiss B. (1987) Comparison of the effects of fluphenazine-N-mustard on dopamine

binding sites and on behavior induced by apomorphine in supersensitive mice. Neuropharmacology 26, 1473–1480. Ungerstedt U. (1971) Postsynaptic supersensitivity after 6hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acfa Psychiafr. Scand. Suppl, 367, 69–93. van den Bos R., Charria Ortiz G. A., Bergmans A, C. and Cools A, R. (1991) Evidence that dopamine in the nucleus accumbens is involved in the ability of rats to switcb to cue-directed behaviors. BehaL!Brain. Res. 42, 107–114. Van Tol H. H,, Bunzow J. R., Gum H. C., Sunahara R. K., Seeman P., Niznik H. B. and Civelli 0, (1991) Cloning of the gene for a human dopamine Dt receptor with high affinity for the antipsychotic clozapine. Nature 350, 610614. Von Voigtlander P. P,, Boukma S, J, and Johnson S. J. (1973) Dopatnincrgic denervation supersensitivity and rfopamine 12, stimulated adenyl cyclase activity. Neuropharmueology 1081–1086. Wahlestedt C., Golanov E,, Yamamoto S,, Yee F,, Ericson H., Yoo H., Inturrisi C. E. and Reis D. J. (1993) Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions. Nafure 363, 26&263. Wahlestedt C., Pich E. M., Koob G. F., Yee F. and Heilig M. (1993) Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science 259, 528-531. Weiss B., Chen J. F., Zhang S. and Zhou L.-W. (1992) Developmental and age-related changes in the D, dopamine receptor mRNA subtypes in rat brain. Nerrrochem. 49S-58S. Int. 20 (SuppL), Weiss, B., Zhang, S.-P. and Zhou, L.-W. (1996) Antisense strategies in dopamine receptor pharmacology. L~~eSci., in press.. Weiss B., Zhou L.-W., Zhang S.-P. and Qin Z.-H. (1993) Antisense oligodeoxynucleotide inhibits Dz dopamine receptor-mediated behavior and Dz messenger RNA. Neurosci. 55, 607–612. Weiss B., Zhou L. W., Chen J. F., Szele F. and Bai G. (1990) Distribution and modulation of the Dz dopamine receptor mRNA in mouse brain: molecular and behavioral correlates. Adv. Biosci. 77, 9–25. Winkler J. D. and Weiss B, (1986) Reversal of supersensitive apomorphine-induced rotational behavior in mice by continuous exposure to apomorphine. J, Pharmacol. Exp. Ther. 238,242-247. Wong D., Wagner J. H., Tune L., Dannals R,, Pearlson G., Links J., Tamminga C., BroussoOe E., Ravert H., Wilson A., Toung J., Malat J., Williams J., O’Tuama L., Snyder S., Kuhar M. and Gjedde A. (1986) Positron emission tomography reveals elevated D2 dopamine receptors in drug-naive schizophrenics. Sci, 234, 1558–1563. Yaswen P., Stampfer M. R., Gosh K. and Cohen J. (1993) Effects of sequence of thioated oligonucleotides on cultured human mammary epithelial cells. Anlisense Res. Deu. 3,67-77. Zhang M, and Creese I. (1993) Antisense oligodeoxynucleotide reduces brain dopamine Dz receptors: behavioral correlates. Neurosci. Left. 161, 223–226. Zhang, S.-P., Zhou, L.-W., Morabito, M,, Lin, R.C.S. and Weiss, B. (1996) Uptake and distribution of fluoresceinlabeied Dz dopamine antisense oligodeoxynucleotide in mouse brain. J. Mol. Neurosci., in press. Zhang S.-P., Zhou L.-W. and Weiss B. (1994) Oli-

Intrastriatal administration of an oligodeoxynucleotide antisense godeoxynucleotide antisense to the D1 dopamine receptor mRNA inhibits D1 dopamine recefitor-mediated behaviors in normal mice and in mice lesioned with 6hydroxydopamine. J. Pharmacol.Exp. Ther.271, 1462–1470. Zhou L.-W,, Zhang S.-P., Qin Z.-H. and Weiss B. (1994) In

595

vim administration of an oligodeoxynucleotide antisense to the Dz dopamine receptor mRNA inhibits Dz dopamine receptor-mediated behavior and the expression of DZ dopamine receptors in mouse striatum. J. Pharmacol. Exp. Ther. 268, 1015–1023.