PARKINSON’S DISEASE is a neurological disorder charac-terized by movement abnormalities such as bradyki-nesia, tremor and rigidity (Factor & Weiner, 1988). Theunderlying neurochemical deficit associated with thiscondition is the loss of greater than 90% of the neuro-transmitter dopamine from the corpus striatum (caudate/putamen) of affected patients (Jellinger,1986). A variety of drugs have been used for the treat-ment of Parkinson’s disease. Among these, L-DOPAhas been the most commonly used. However, follow-

ing long-term treatment with L-DOPA, there is a lossof efficacy with increased fluctuations in mobility,dyskinesia and psychoorganic disturbances which arerelated in part to disease progression and to the therapy (Fahn, 1989; Lesser et al., 1979; Marsden et al., 1982).

Clinical trials to improve treatment include contin-uous delivery of L-DOPA to the proximal small intes-tine (Kurlan et al., 1986), subcutaneous infusion of theD2-dopamine receptor agonist, lisuride (Obeso et al.,1988), and transplantation of adrenal medullary auto-grafts and fetal grafts to the corpus striatum (Freed et al., 1992; Madrazo et al., 1991; Spencer et al., 1992).These procedures ameliorate manifestations ofParkinson’s disease in some patients but the resultsare not totally satisfactory. A proposed alternativeexperimental approach to the treatment of Parkinson’sdisease involves the direct delivery of dopamine, the

161

NEURODEGENERATION, Vol. 4, pp 161–169 (1995)

Intrastriatal Dopamine Infusion ReversesCompensatory Increases in D2-Dopamine Receptors

in the 6-OHDA Lesioned RatChristian Woiciechowsky,1 Tomás R. Guilarte,2 Christiane H. May,1

Jan Vesper,1 Henry N. Wagner Jr.2 and Siegfried Vogel1

1Department of Neurosurgery, Humboldt-University, Medical School (Charité), Berlin,Germany; 2Department of Environmental Health Sciences, The Johns Hopkins University

School of Hygiene and Public Health, Baltimore, Maryland, USA

Direct infusion of dopamine into the corpus striatum has been proposed as a potential approachfor the treatment of Parkinson’s disease. The present study examined the effect of intrastriataldopamine infusion on D2-dopamine receptors in the 6-hydroxydopamine (6-OHDA) lesioned ratbrain. The completeness of the 6-OHDA-induced nigrostriatal injury was confirmed using [3H]-mazindol autoradiography and apomorphine-induced behaviour. Intrastriatal infusion of threedifferent dopamine doses significantly reduced the apomorphine-induced behaviour. [3H]-spiperone autoradiography performed one day after the termination of dopamine infusion intothe striatum revealed a dramatic reduction of D2-dopamine receptor binding. The mean 6 SEMpercent reduction of D2 receptor binding in the affected areas of the striatum was 28.8 6 1.0% for4.74 µg dopamine/h infusion rate, 35.0 6 1.6% for 9.48 µg dopamine/h infusion rate and 33.3 65.0% for 14.22 µg dopamine/h infusion rate when compared to the unlesioned side. Infusion ofvehicle alone did not have any effect. The present results support the concept that intrastriataldopamine infusion may be a useful therapeutic approach for the treatment of Parkinson’s disease.

Key words: Parkinson’s disease, dopamine, D2-dopamine receptors, 6-OHDA, corpus striatum, rat

Correspondence to: Christian Woiciechowsky, MD, Universitätsklinikund Poliklinik für Neurochirurgie, Charité, Humboldt-Universität zuBerlin, Schumannstr. 20/21, 10117 Berlin, Germany.

Received 18 August 1994; revised and accepted for publication 10February 1995

© 1995 Academic Press Limited1055–8330/95/020161 1 9 $08.00/0

neurotransmitter in deficit, or dopamine receptor ago-nists to the affected brain region (De Yébenes et al.,1988; Hargraves & Freed, 1987; Kroin et al., 1991). Thegoal of this approach is to eliminate the undesirablecentral and systemic side effects and fluctuationsencountered with present therapy.

Using unilateral 6-hydroxydopamine (6-OHDA)lesioned rats, Hargraves & Freed (1987) were the firstto show that intrastriatal dopamine administrationimproves the apomorphine-induced behaviouralhypersensitivity in this animal model of Parkinson’sdisease. It is well established that unilateral destruc-tion of dopaminergic neurons projecting to the corpusstriatum by 6-OHDA injection results in an increasednumber of striatal D2-dopamine receptors with docu-mented increases from 25–55% relative to the un-lesioned side (Brené et al., 1990; Cadet & Zhu, 1992;Hargraves & Freed, 1987; LaHoste & Marshall, 1989;Neve et al., 1982). This increase in D2-dopamine recep-tors has been correlated with the behavioural hyper-sensitivity to dopamine receptor agonists observed inthese animals (Neve et al., 1982; Ungerstedt, 1971b).

The goal of this study was to determine the effectsof intrastriatal dopamine infusion on D2-dopaminereceptor upregulation in the 6-OHDA lesioned rat. Itwas found that intrastriatal infusion of dopamineusing minipumps reduced the increased binding of[3H]-spiperone to D2-dopamine receptors in the stria-tum as well as the apomorphine-induced contralateralrotations characteristic of unilateral 6-OHDA lesion ofthe nigrostriatal dopaminergic pathway. Furthermore,intrastriatal infusion of dopamine did not have anysignificant effect on striatal benzodiazepine receptorbinding suggesting a specificity for the D2-dopaminereceptor.

Materials and Methods

6-OHDA lesion of nigrostriatal pathway

Thirty male Long-Evans rats (Charles River Laboratories,Wilmington, MA) weighing 150–175 grams at the time ofsurgery were used. Rats were anaesthetized with sodiumpentobarbital (i.p. 40 mg/kg) and a unilateral lesion of theright ascending nigrostriatal dopaminergic pathway wasproduced by the injection of 1 µL of 6-OHDA (8 µg 6-OHDAin 1 µL 0.1% ascorbate-saline) at a rate of 0.5 µL/min usinga 31 gauge cannula connected via a tubing to a CMA/100Microinjection Pump (Carnegie Medicin, Stockholm,Sweden). The stereotactic coordinates were 4.4 mm poste-rior and 1.1 mm lateral to bregma and 7.8 mm below thedura (König & Klippel, 1963; Ungerstedt, 1971a). This lesioncauses a clearly defined contralateral rotation after the injec-tion of apomorphine (Hargraves & Freed, 1987; Neve et al.,

1982). Rats were treated 0.5 h prior to the infusion of 6-OHDAwith desipramine (i.p. 25 mg/kg) to protect central nor-adrenergic neurons (Jonsson, 1976; LaHoste & Marshall,1989) and with pargyline (i.p. 50 mg/kg) in order to enhancedopamine depletion (Iversen & Kelly, 1975).

Behavioural manifestation of 6-OHDA unilaterallesions

Seven and 14 days after 6-OHDA injection, the developmentof rotational behaviour was monitored by injecting apo-morphine-HCl (i.p. 0.25 mg/kg) and counting manuallytotal contraversive turns in a plastic bowl (33 cm diameter)for 30 min. Only those rats showing more than 8 full con-tralateral rotations per min on day 14 were used for the sub-sequent study. This behavioural outcome representsapproximately a 90% loss of dopaminergic neurons (Creese& Snyder, 1979; Neve et al., 1984).

Implantation of minipumps for dopamine infusion

Twenty animals submitting to the implantation weredivided into four groups of five rats each. The implantationof ALZET-minipumps (Model 2002, Alza Corporation, PaloAlto, CA) was performed as follows: On day 15 after the 6-OHDA lesion, rats were anaesthetized with sodium pento-barbital (i.p. 40 mg/kg) and an L-shaped cannula of theALZET brain infusion kit was placed into the striatum ipsi-lateral to the lesioned side. The coordinates were: 0.2 mmanterior and 3.0 mm lateral to bregma and 4 mm below thedura according to Paxinos & Watson (1982). This atlas hadbeen used instead of that from König & Klippel (1963)because of the appropriate weight of the rats.

The cannula was fixed with two stainless steel screws andmounted to the skull with dental cement. The screws actedas an anchor to secure both the external portion of the can-nula and the dental cement covered and secured the entireimplantation site. The minipump was implanted subcuta-neously in the nape of the neck and attached to the cannulavia a tubing. The pump and tubing were filled with four different solutions: group I (vehicle): 0.1% sodiummetabisulfite in distilled water, group II (low dopamine):9.48 g/l 5 50 mM, 4.74 µg dopamine/h infusion rate, groupIII (medium dopamine): 18.96 g/l 5 100 mM, 9.48 µgdopamine/h infusion rate, group IV (high dopamine) 28.44g/l 5 150 mM, 14.22 µg dopamine/h infusion rate. Thelength of the infusion was 14 days.

During the first week of dopamine infusion all animalswere observed for spontaneous contralateral rotation. Onday 7, apomorphine-induced rotation was evaluated. Oneday after the termination of dopamine infusion, all animalswere euthanized by decapitation. The brains were excisedand immediately frozen on dry ice and stored at 280°C untilused.

Stability of dopamine solutions

The stability of dopamine is limited due to auto-oxidation. Its half-life is approximately 7–8 min. Sodiummetabisulfite is used by some authors as a stabiliser due toits anti-oxidative capacity (Kroin et al., 1991; Hargraves &

162 C. Woiciechowsky et al.

Freed, 1987). In order to verify the stability of dopamine inthe used solutions dopamine contents were measured in0.1% sodium metabisulfite at various concentrations (50mM, 100 mM, 150 mM) at a pH 5.2 for at least 2 weeks. Theampoules were incubated under light and air protected con-ditions to simulate in vivo conditions. Every day thedopamine content was verified comparing the test ampoulewith two standard solutions cooled by 218°C. For thedopamine analysis, HPLC with electrochemical detection(Shimadzu-detector, Japan) was used. The mobile phase con-sisted of 20.5 g sodium sulphate, 370 g bisodium-EDTA, 100mL triethylamine (TEA), 300 mg sodium octansulfonate and30 mL isopropanol per 1 L of double distilled water. The con-centration of dopamine was determined comparing thepeak-areas of the test solution with the standard solutions.Therefore the CR6a Barspec Data System of Shimadzu(Japan) was used. Increase of the dopamine metaboliteshomovanillic acid (HVA) and dihydroxyphenylacetic acid(DOPAC) in the ampoules was controlled regularly toexclude overlapping peaks with dopamine.

Autoradiographic studies

In order to confirm the completeness of the 6-OHDA lesion,dopaminergic terminals in the striatum were visualizedusing [3H]-mazindol autoradiography. Mazindol is a potentinhibitor of neuronal dopamine and norepinephrine highaffinity uptake sites. [3H]-spiperone autoradiography wasused to evaluate D2-dopamine receptors in the striatumunder the different treatment conditions. [3H]-fluni-trazepam autoradiography was performed to assess thespecificity of the dopamine infusion on D2-dopamine recep-tors. Brains were sectioned in a cryostat (222°C) (BrightInstrument Company Ltd, Huntingdon, England), at 20 µmthickness from approximately level A 10 200 µm to 8700 µm,according to Paxinos & Watson (1982) and thaw-mountedonto gelatin coated glass slides. Tissue sections wererefrozen after being dried and remained at 280°C until used(no more than 7 to 14 days). Autoradiographic images wereanalysed using an Image Analyzer system (Image ResearchInc., Ontario, Canada). Images were collected by a videocamera (Nikon, AF, Japan) and a Northern light precisionilluminator, Model C 60 (Image Research Inc., Ontario,Canada) digitized and displayed on an Electrochrome mon-itor Model ECM 1312 with grey values ranging from 0 to 255.A calibration curve was produced by digitizing and mea-suring grey values in tritium containing standards whichwere exposed with the slides. The computer generated a‘look up table’ which was used to convert each 0 to 255 greyvalue scale to fmoles [3H]-ligand per mg wet tissue. Imagesof specific binding were prepared by subtracting corre-sponding pixels of the superimposed linearized images ofnon-specific binding from the linearized images of total-binding.

In [3H]-mazindol autoradiograms the whole striatum wasanalysed. In [3H]-flunitrazepam and [3H]-spiperone autora-diograms the area of change in D2-dopamine receptor bind-ing on the lesioned side was compared with the same areaon the non lesioned side using a mask created by outliningthe area of drastic changes in grey levels on the lesioned sidein [3H]-spiperone autoradiograms.

[3H]-mazindol autoradiography

[3H]-mazindol autoradiography was performed by pre-incubating the tissue slides at 4°C for 5 min in 50 mM Trisbuffer (pH 7.9) containing 300 mM NaCl and 5 mM KCl andthen incubating for 40 min at 4°C in 4 nM [3H]-mazindol(spec. activ. 15.4 Ci/mmol, NEN Dupont, Boston, MA) con-taining 0.3 mM desipramine to eliminate the binding of thetracer to norepinephrine uptake sites. Nonspecific bindingwas determined in the presence of 1 µM unlabelled mazin-dol. After two consecutive washes in the buffer at 4°C andone distilled water rinse the slides were dried at room tem-perature. Autoradiograms were obtained by exposing theslides to a tritium-sensitive film (Amersham, ArlingtonHeights, IL) for 5–6 weeks and developed in Kodak D19(Donnan et al.,1989; Javitch et al., 1985).

[3H]-spiperone autoradiography

[3H]-spiperone autoradiography was performed by pre-incubating the tissue slides at 36°C for 5 min in 50 mM Trisbuffer (pH 7.1) containing 120 mM NaCl, 5 mM KCl, 2 mMCaCl2, 1 mM MgCl2 and then incubating for 30 min at 36°C in the same buffer containing 40 nM ketanserin to block the binding of the tracer to serotonergic receptors and1.4 nM [3H]-spiperone (spec. act. 25.5 Ci/mmol, NENDupont, Boston, MA). To define non-specific binding 1 µM(1)-butaclamol was added. After three washes for 5 sec inbuffer at 4°C, the slides were dried at 37°C for 1 h and desiccated in vacuum at room temperature overnight.Autoradiograms were generated by exposing the slides to atritium-sensitive film for 3 weeks and developed in KodakD19.

[3H]-flunitrazepam autoradiography

[3H]-flunitrazepam autoradiography was performed bythree 5 min preincubations of the tissue slides at 4°C in 50 mM Tris-citrate buffer, pH 7.3. The slides were then incubated for 40 min at 4°C in the same Tris-buffer containing 1 nM [3H]-flunitrazepam (81 Ci/mmol NENDupont, Boston, MA) and 10 µM GABA in the presence and absence of 1 µM unlabelled flunitrazepam. Following incubation, slides were treated with two 1-min rinses of cold buffer and one 1-sec rinse of cold distilled water and dried at 37°C for 1 h and desiccated in vacuum at room temperature overnight. Autoradiograms were generated by exposing the slides to a tritium-sensitive filmfor 2 weeks and developed in Kodak D19 (Robertson et al.,1990).

Histology

In order to relate the structures of the autoradio-graphic images to the anatomy of the brain every 5th tissue section of the brain was mounted onto a glass slide,dried and stained with 0.5% solution of cresyl violet for1 min, followed by rinsing with water, drying, dehydrating,cover slipping and analysing at the light microscopiclevel.

Effects of dopamine infusion on D2 receptors 163

Statistical analysis

The data were analysed by one-way analysis of variance(ANOVA) with Dunnett’s post-hoc test. Differences wereconsidered significant when P , 0.05. All data are shown inmean 6 SEM.

Results

Dopamine stability

The dopamine concentration in the ampoules was stable with only 10–20% degradation occurring during the 14 days observation period (Fig. 1). This10–20% decrease in dopamine concentration is mostlikely due to auto-oxidation. A consequence ofdopamine auto-oxidation is the formation of a blackpigment, most likely melanin. This pigment was alsofound in the corpus striatum around the tip of the can-nula in some of the animals receiving dopamine infu-sion. This observation has also been reported by deYébenes et al. (1988). Additional investigations shoulddetermine how to avoid the production of melanin,since melanin deposits may be neurotoxic (Duff et al.,1988; Mason et al.,1960).

Efficiency of 6-OHDA lesion

Twenty-five of the 30 lesioned rats developed con-tralateral rotation after the injection of apomorphine.The rotation rate on day 7 was 687 6 300 turns/h(314–1536 turns/h) and 821 6 245 turns/h (526–1354turns/h) on day 14. Twenty of these rats had implan-tation of the minipumps. The remaining five animalswere used in subsequent studies (not reported here).

Effect of dopamine infusion on spontaneous andapomorphine-induced rotations

Nineteen of the 20 implanted rats survived the surgeryand subsequent intrastriatal dopamine infusion. The

placement of the cannula was confirmed histologicallyin all animals. The rat that died on the second day afterimplantation had developed a spontaneous rotationrate of 1404 turns/h. Rats receiving dopamine infu-sion directly into the striatum, ipsilateral to the lesion,were observed to have increased contralateral rotationeven without the apomorphine injection (Fig. 2). Thisbehaviour was maximal 24 h following the initiationof intrastriatal dopamine administration and de-creased over the next 7 days. No animals receivingvehicle infusion exhibited spontaneous rotation (Fig. 2). Rats receiving the highest dopamine concen-tration showed the highest rotation rate (783 6 430turns/h). However, this was not statistically signifi-cantly different from the other two dopamine treat-ment groups.

On day 7 of dopamine treatment, the apomorphine-induced rotation test was performed once again. Thedata indicate that intrastriatal infusion of increasingdopamine concentrations resulted in significantreductions in the rotation rate (P 5 0.018; Fig. 3). Thegreatest reduction (64%) of apomorphine-inducedrotation was seen in animals receiving the mediumdopamine dose. This difference was significantly dif-ferent from that of the vehicle-infused lesioned rats (P 5 0.01). The lowest and highest infusion ofdopamine resulted in a reduction of the apomorphineinduced rotation of 33 and 44%, respectively. Thesevalues were not significantly different from vehicle-infused lesioned rats.

164 C. Woiciechowsky et al.

Figure 1. Stability of dopamine concentrations in 0.1%sodium metabisulfite as measured by HPLC coupled to elec-trochemical detection. (d) 50 mM; (j) 100 mM; (m) 150 mM.

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Figure 2. Spontaneous contralateral rotations expressedas turns per hour in rats treated with intrastriatal infusionof vehicle or different dopamine solutions, group I (vehi-cle): 0.1% sodium metabisulfite in distilled water, group II(low dopamine): 9.48 g/l 5 50 mM, 4.74 µg dopamine/hinfusion rate, group III (medium dopamine): 18.96 g/l 5100 mM, 9.48 mg dopamine/h infusion rate, group IV (highdopamine) 28.44 g/l 5 150 mM, 14.22 µg dopamine/h infusion rate. Data are expressed as mean 6 SEM.

[3H]-mazindol autoradiography

To assess the status of pre-synaptic dopaminergic ter-minals, the degree of dopaminergic denervationachieved by unilateral 6-OHDA injection was visual-ized using [3H]-mazindol autoradiography. Theresults revealed that [3H]-mazindol binding wasreduced by 92% on the lesioned side relative to the

non-lesioned side in the same animal. The mean 6SEM of striatal [3H]-mazindol binding in fmoles/mgwet tissue in the lesioned side was 5.8 6 0.6 (n 5 10)and 71.4 6 8.4 (n 5 10) on the unlesioned side.

[3H]-spiperone autoradiography

To assess the status of post-synaptic D2-dopaminereceptors, [3H]-spiperone binding in the striatum ofdopamine and vehicle treated 6-OHDA lesioned ratswas measured in three different slices. The selectedslices included the area at the level of dopamine infu-sion and 0.5 mm anterior and posterior to the site. Thevalue obtained from these slices was averaged for eachanimal. The specific [3H]-spiperone binding measuredin the dopamine deficient (lesioned) striatum was 25.9 6 5.6% (P , 0.001, Table 1, Fig. 4A,B) higher thanthe value obtained in the intact (unlesioned) striatum.Following chronic dopamine infusion into thelesioned striatum, there was a significant reduction in[3H]-spiperone binding on the lesioned side relative to the vehicle treated group (P , 0.005, Table 1).However, the binding was still higher on the lesionedside compared with the unlesioned side resulting in areduced rotational behaviour (Table 1, Fig. 3). Nosignificant differences were observed among the various dopamine treatment groups.

The size of the area with dramatic receptor reduc-tion in striatal [3H]-spiperone binding followingdopamine treatment was measured in the autoradi-ograms. The area in square millimeters was deter-

Effects of dopamine infusion on D2 receptors 165

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Figure 3. Apomorphine-induced contralateral rotationsexpressed as turns per hour in rats treated with intrastriatalinfusion of vehicle or different dopamine solutions, groupI (vehicle): 0.1% sodium metabisulfite in distilled water,group II (low dopamine): 9.48 g/l 5 50 mM, 4.74 µgdopamine/h infusion rate, group III (medium dopamine):18.96 g/l 5 100 mM, 9.48 µg dopamine/h infusion rate,group IV (high dopamine) 28.44 g/l 5 150 mM, 14.22 µgdopamine/h infusion rate. Data expressed as mean 6 SEM.* statistically significant (P 5 0.01) reduction of contra-lateral rotations compared with the vehicle treated group.

Table 1. Effects of intrastriatal dopamine infusion on the specific binding of [3H]-spiperone in the rat striatum

Striatal [3H]-spiperone binding (fmoles/mg wet tissue)

Group I Group II Group III Group IV

A Lesioned side 80.5 6 3.9* 69.9 6 6.3** 69.8 6 3.8** 68.6 6 5.3**

A Control side 64.0 6 2.2 64.5 6 4.1 61.0 6 3.3 61.6 6 2.7

B Area of changein receptor binding 48.6 6 0.87*** 45.3 6 2.6*** 44.3 6 6.8***

B Comparable area oncontrol side 68.3 6 1.7 69.7 6 1.6 66.8 6 3.6

A [3H]-spiperone specific binding was measured in three slices for each rat and averaged to provide a single value. Selected slices werein the area of DA infusion and 0.5 mm posterior and anterior to the infusion site.

B [3H]-spiperone specific binding measured in one slice of each rat at the level of the cannula.* Significant increase of specific [3H]-spiperone binding in the lesioned striatum compared to the unlesioned control side (P , 0.001).** Significant decrease of specific [3H]-spiperone binding in the dopamine striatum compared to the vehicle treated lesioned striatum

(P , 0.01).*** Significant decrease of specific [3H]-spiperone binding in the area of dopamine infusion in the lesioned striatum compared to the

same area on the unlesioned side (P , 0.001).Data are presented as means 6 SD of 4–5 animals.

mined by evaluating the number of pixels inside theoutlined area of drastic changes in grey values andthen converting the amount of pixels to square mil-limeters using 1, 5 and 10 mm2 standards. The size ofthis area at the site of dopamine infusion was 4.51 60.86 mm2 for the low dopamine-group, 5.34 6 0.96mm2 for the medium dopamine-group and 5.16 6 2.58mm2 for the high dopamine group, as also describedby Horne et al. (1992). These results indicate thatdopamine produced a reduction in [3H]-spiperonebinding in an area with a radius of 1.0–1.8 mm fromthe tip of the cannula. The mean 6 SEM percent reduc-tion in [3H]-spiperone binding compared with thesame area on the unlesioned side was 28.8 6 1.0% forthe low dopamine-group, 35.0 6 1.6% for the mediumdopamine-group and 33.3 6 5.0% for the high

dopamine-group. [3H]-spiperone binding remainedhigh in regions of the striatum where dopamine didnot penetrate (Fig. 4C,D). These findings indicate thatthe intrastriatal infusion of dopamine was able toreverse the compensatory ‘upregulation’ of D2-dopamine receptors in rats with pre-synapticdopaminergic neuron damaged by 6-OHDA injection.

[3H]-flunitrazepam autoradiography

In order to assess the specificity of the effects ofdopamine infusion on D2-dopamine receptors, we alsoperformed benzodiazepine receptor autoradiographyusing [3H]-flunitrazepam. The mean 6 SEMfmole/mg tissue [3H]-flunitrazepam specific bindingat the level of the cannula in the lesioned (medium

166 C. Woiciechowsky et al.

Figure 4. Representative autoradiograms of the middle level of the caudate/putamen. A and B represent [3H]-spiperonebinding to D2-dopamine receptors in the vehicle-treated rat showing a marked hemispheric asymmetry in striatal [3H]-spiper-one binding due to lesion-induced upregulation of D2-dopamine binding sites (A is an original autoradiogram of total bind-ing, B is the pseudo-colour autoradiogram after image subtraction). C and D show the dopamine treated rat (mediumdopamine) with a clearly visible area of drastic reduction of [3H]-spiperone binding sites (C is an original autoradiogram oftotal binding, D is the pseudo-colour autoradiogram after image subtraction). Bars are calibrated in fmoles/mg tissue.

dopamine-infused) striatum and unlesioned side from five rats was 32.9 6 0.84 and 35.2 6 0.93, respec-tively. The level of [3H]-flunitrazepam binding in thedopamine infused side was not significantly differentfrom the unlesioned side.

Discussion

Damage to the nigrostriatal dopaminergic system bythe injection of 6-OHDA into the substantia nigra or ventral tegmental area results in long-term im-pairment of striatal neuronal systems involvingdopamine. One of the consequences of the destructionof dopaminergic input to the striatum is the compen-satory increase (upregulation) of striatal D2-dopaminereceptors (Creese et al., 1977, Creese & Snyder, 1979;Neve et al., 1984). This phenomenon is reflected in abehavioural hypersensitivity of lesioned animals tothe injection of apomorphine (Neve et al., 1982;Ungerstedt, 1971b). In the present study, 6-OHDAlesioned rats showed this characteristic rotationalbehaviour in response to apomorphine injection (Fig. 3).

The spontaneous contralateral rotational behaviourobserved one day following dopamine infusion intothe lesioned striatum indicated that dopamine deliv-ered by the minipump stimulates D2-dopamine recep-tors upregulated as the result of the 6-OHDA lesion.This behaviour following infusion of dopaminedirectly into the striatum is in agreement with previ-ous studies (Hargraves et al., 1987; Kroin et al., 1991).The spontaneous rotation rate on the first day ofdopamine infusion was much greater than that previ-ously reported (Hargraves et al., 1987, Kroin et al., 1991)and approached apomorphine-induced rotation rates(Figs 2, 3). During the subsequent 7 days of dopamineinfusion, a gradual decrease of the spontaneous rota-tion was observed suggesting reversal of the upregu-lation of D2-dopamine receptors in the lesionedstriatum (Fig. 2). The reversal of the upregulation ofD2-dopamine receptors in dopamine treated rats wasfurther confirmed by a reduction in the apomorphine-induced rotational behaviour after 7 days of dopaminetreatment (Fig. 3). The reduction in the apomorphine-induced rotation ranged from 33–64% according todopamine dose. Dopamine treatment also decreasedthe grooming and scratching behaviour previouslyobserved in these animals. There was no reduction inapomorphine-induced contralateral behaviour invehicle-treated lesioned rats.

The status of pre-synaptic dopaminergic terminals

following the injection of 6-OHDA was assessed using[3H]-mazindol autoradiography. Mazindol has a highaffinity for the dopamine transporter and has beenused as a neuronal marker for assessing dopamineneuron integrity. The autoradiographic resultsshowed a nearly complete denervation of dopamin-ergic terminals in the lesioned striatum relative to theunlesioned side. Quantitative autoradiography wasalso used to demonstrate increased [3H]-spiperonebinding to D2-dopamine receptors in the 6-OHDAlesioned striatum. Increased binding followed a lat-eral to medial and rostral to caudal gradient in agree-ment with previous studies (Joyce et al., 1985). In thedopamine treated rats there was a dramatic reductionin [3H]-spiperone binding (26–50 %) compared withthe same area on the unlesioned side. These resultssuggest that infused dopamine distributed into thestriatum and reversed the upregulation of D2-dopamine receptors. Other regions of the striatumwhere dopamine did not penetrate still exhibited ahigh level of [3H]-spiperone binding (Fig. 4C,D). Theseregions of the striatum which retained increased bind-ing of [3H]-spiperone may account for the incompleteamelioration of apomorphine-induced rotations insome of the dopamine treated rats. Schneider et al.(1984) described the effects of chronic L-DOPA andbromocriptine treatment in the 6-OHDA lesioned ratmodel. The chronic administration of levodopareversed the D2-dopamine receptors to a value not dif-ferent from the control-side. Chronic administrationof bromocriptine had a more pronounced effect andreversed the D2-dopamine receptors to control values(Schneider et al., 1984). Furthermore Savasta et al.(1992) described a complete reversal of the lesion-induced increase of striatal D2-dopamine receptors 6months after the implantation of embryonic dopamin-ergic neurons. Moreover, this reversal concerned notonly the reinnervated striatal region but also extendedinto non-reinnervated areas of the striatum (Savasta etal., 1992). In the present study we produced, due to ahigh dopamine concentration in the minipumps,down-regulation of D2-dopamine receptors below thelevel of the control-side in a circumscribed striatal area.However, in the whole striatum the D2-dopaminereceptors were still upregulated compared with thecontrol-side, but while we did not determine Bmaxand Kd, so we cannot conclude that the observed plas-ticity reflects changes in receptor density only and notin affinity.

It is likely that the reduction in [3H]-spiperone bind-ing in the striatum of dopamine treated rats was theresult of D2-dopamine receptor down-regulation, and

Effects of dopamine infusion on D2 receptors 167

not due to the residual effects of the infused dopaminefor the following reasons: (1) dopamine is rapidlymetabolized (Spano & Neff, 1972), and is unlikely tobe present in the striatum 1 day after infusion is ter-minated; (2) any remaining dopamine would beremoved since the brain slices were washed prior toexposure with [3H]-spiperone, and (3) the potency ofbinding of spiperone to D2 receptors is significantlyhigher than that of dopamine itself (Lyon et al., 1986).For these reasons, it is unlikely that the reduction in[3H]-spiperone binding in the dopamine treated 6-OHDA-lesioned rat is due to competition for the D2

receptor sites. Although the present data wereobtained using a single ligand concentration, the workof other authors employing Scatchard analyses indi-cates that for D2-dopamine receptors, the plasticityobserved following either 6-OHDA lesion or chronicagonist treatment reflects changes in receptor density,not in affinity (Neve et al., 1984; Schneider et al., 1984).The lower level of [3H]-spiperone binding in the stria-tum of the dopamine-treated rats could also be theresult of injury to striatal neurons from the auto-oxidation of the infused dopamine. In an effort toaddress this possibility, we assessed the integrity ofanother receptor system which should not be phar-macologically affected by the infused dopamine. Thedata show that dopamine treatment resulted in nochange in the binding of [3H]-flunitrazepam to ben-zodiazepine receptors in the dopamine-infused ver-sus the non-infused side, supporting that striatalneurons were undamaged. Furthermore, striatal neu-ronal loss was not observed in histological sections inthis study or in a study by Kroin et al. (1991 ) using acomparable intrastriatal dopamine dose.

The present study is the first to use quantitativeautoradiography to measure the effect of intrastriataldopamine infusion on [3H]-spiperone binding to D2-dopamine receptors. Our results suggest that intra-striatal infusion of dopamine reversed theupregulation of striatal D2-dopamine receptors in the6-OHDA lesioned rat without any effect on benzodi-azepine receptors. Quantitative autoradiography ofD2-dopamine receptors was used to measure theextent of dopamine permeability following intrastri-atal infusion. Studies are currently being extended tonon-human primates with neuroreceptor changesbeing monitored using Positron EmissionTomography (PET).

In conclusion, intrastriatal dopamine infusionreverses the compensatory unilateral increase in D2-dopamine receptor binding and apomorphine-induced rotational behaviour in 6-OHDA lesioned

rats. The results support the concept that intrastriatalinfusion of dopamine or dopamine receptor agonistsmay be useful in the treatment of Parkinson’s diseaseand that the effects of infusion of drugs directly intothe brain can be assessed by autoradiography changesin regional brain chemistry, or by PET and SPECTimaging of intact animals.

Acknowledgments

This work was supported in part by a grant from theBundesministerium für Forschung und Technologie andgrant No. CA 32845 from the United States Public HealthService. The authors thank Miss Renée C. Miceli for techni-cal assistance and Dr Ursula Scheffel for helpful advice.

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