Neuroscience Vol. 49, No. 2, pp. 285 296, 1992 Printed in Great Britain

0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd (c) 1992 IBRO

D1 AND D 2 DOPAMINE RECEPTORS DIFFERENTIALLY REGULATE c-fos EXPRESSION IN STRIATONIGRAL AND STRIATOPALLIDAL NEURONS G. S. ROBERTSON, S. R. VINCENT and H. C. FIBIGER Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver B.C., Canada V6T 1Z3 Abstract--The expression of Fos, the product of the proto-oncogene c-fos, is thought to be a marker of neuronal activity. D~, but not D2, dopamine receptor agonists have previously been shown to increase Fos immunoreactivity in striatonigral neurons ipsilateral to a 6-hydroxydopamine lesion of the nigrostriatal pathway. In the present study, it was demonstrated that the D~ receptor agonist SKF 38393 rarely increased Fos in striatopallidal neurons of the 6-hydroxydopamine denervated striatum. Conversely, in the intact striatum, the D 2 receptor antagonist haloperidol enhanced Fos expression predominantly in striatopallidal neurons labelled retrogradely from the globus pallidus or with an oligonucleotide probe complementary to mRNA encoding enkephalin. These results are consistent with studies suggesting that D l receptors are located predominantly on striatonigral neurons and that D 2 receptors reside principally on enkephalin-containing striatopallidal neurons. They also provide a neuroanatomical basis for neurochemical and neurophysiological observations indicating that dopamine facilitates the activity of striatonigral neurons but inhibits striatopallidal neurons. In another experiment the selective D 2 receptor agonist quinpirole was found to increase Fos immunoreactivity in the globus pallidus ipsilateral to a 6-hydroxydopamine lesion, It is proposed that this may have been due to a D 2 receptor-mediated inhibition of enkephalin and GABA release from striatopallidal terminals that in turn disinhibited the pallidal neurons. In a final series of experiments, brain microdialysis was used to determine the location of dopamine receptors regulating striatal Fos expression. Local application of the selective D~ receptor agonist CY 208-243 in the 6-hydroxydopamine-denervated striatum, or of haloperidol in the intact striatum via the dialysis probe increased Fos immunoreactivity in the immediate vicinity of the probe. Hence, the inductive effects of these systematically administered compounds on F0s expression in the striatum are mediated at least partly by local dopamine receptors in the striatum. Taken together, these results suggest that the differential regulation of striatonigral and striatopallidal activity by dopamine is mediated by the largely separate location of DI and D 2 receptors on these outputs.

The two major output systems of the striatum are to the globus pallidus (GP) and substantia nigra (SN). 5,6 Each of these projections arises primarily from separate populations of striatal neurons. 5,32 Both utilize the inhibitory neurotransmitter G A B A but each contains different neuropeptides. 2'6 Striatopallidal neurons contain enkephalin while striatonigral neurons are thought to utilize both dynorphin and substance P as neurotransmitters. 2'6'22'33'5s'58 On the basis of lesion studies, it has been suggested that striatopallidal and striatonigral neurons also express different dopamine receptors, with D 1 recep-

Abbreviations: ABC, Avidin biotin complex; CY 208-243, ( - )-4,6,6a,7,8,12b-hexahydro-7-methyl-indolo[4,3-ab]phenanthidine; DEP, diethylpyrocarbonate; FG, FluoroGold; GP, globus pallidus; MFB, medial forebrain bundle; PBS, phosphate-buffered saline; 6-OHDA, 6hydroxydopamine; SCH 23390, (R)-(+)-8-chloro2,3,4, 5- tetrahydro - 3- methyl- 5- phenyl- 1H - 3-benzazepin7-ol; SKF 38393, 2,3,4,5,-tetrahydro-7,8-dihydroxy-1phenyl-lH-3-benzazepines; SN, substantia nigra; SSC, sodium citrate buffer; TBS, Tris-buffered saline; YM 09151-2; cis-N-(t -benzyl-2-methylpyrrolidin-3-yl)-5chloro-2-methoxy-4-(methylamino)benzamide. 285

tors being located predominantly on striatonigral neurons and D 2 receptors being expressed mainly by striatopallidal neurons. 4,26 Recent in situ hybridization studies have confirmed this organization by demonstrating that D1 receptor and substance P m R N A s are co-expressed by striatonigral neurons while D 2 and enkephalin m R N A s are co-expressed by striatopallidal neurons. 23,33'34 The proto-oncogene c-fos encodes a 380-amino acid phosphoprotein, Fos. Neuronal expression of Fos is increased in the central nervous system by a host of pharmacological and physiological treatments (for review, see Refs 41-43). F o r example, pentylenetetrazol- and kindling-induced seizures produce a marked elevation of Fos immunoreactivity in the pyrifrom cortex, amygdala, septum, olfactory bulb and granule cells of the hippocampus, 14'4° Caffeine and morphine increase c-fos expression in the striatum, H,44 whereas opiate withdrawal does so in the locus coeruleus. 27 The ability of such a wide variety of stimuli to increase c-fos expression has led to the suggestion that Fos may be a marker of neuronal activity and that Fos immunohistochem-

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istry might be used to map functional pathways in the central nervous system. 15'52 Stimulants such as cocaine, d-amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine produce large increases in striatal c-fos expression which is potently blocked by the selective D 1 receptor antagonist SCH 23390.1°'17,24,56,62 In contrast, directly acting D1 receptor agonists such as S K F 38393 and C Y 208-243 only weakly increase Fos immunoreactivity in the intact striatum. 47,5°However, after 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway, S K F 38393 and CY 208-243 produce dramatic increases in Fos expression in the ipsilateral striatum. 47,5° Using retrograde tracing techniques, we have recently demonstrated that S K F 38393-induced increases occur in striatonigral neurons. 48 This result is consistent with 2-deoxyglucose uptake studies showing that D1 receptor agonists activate striatonigral neurons ipsilateral to the 6-OHDA-lesioned SN. 59 The preferential localization of D1 receptors on striatonigral neurons suggests that D 1 receptor-activated Fos may be expressed only by striatonigral neurons. Consequently, one of the major objectives of the present study was to determine whether D1 receptor agonists also increased Fos expression in striatopallidal neurons ipsilateral to a 6 - O H D A lesion of the nigrostriatal pathway. D2 receptor antagonists such as haloperidol and raclopride increase c-fos expression in the intact striatum. 16'39'49The distribution of D 2 receptor antagonist-induced Fos very closely matches the distribution of striatal D 2 receptors. 16,49 The close topographical relationship between D 2 antagonistinduced Fos immunoreactivity and D 2 receptors suggests that haloperidol and raclopride increase Fos in striatal neurons which express D 2 receptors, i.e. in striatopallidal neurons. 23,33,34 In addition, because enkephalin is utilized principally by striatopallidal neurons as a neurotransmitter, it should also be possible to label striatal neurons which express D 2 antagonist-induced Fos with an oligonucleotide probe complementary to m R N A encoding enkephalin. Consequently, we have used retrograde tracing and in situ hybridization methodologies to determine whether the D~ receptor agonist, S K F 38393 and the D 2 receptor antagonist, haloperidol, differentially increase Fos immunoreactivity in striatonigral and striatopallidal neurons, respectively. D~ and D 2 dopamine receptors are also found in other brain regions such as the nucleus accumbens, cortex, olfactory tubercle, amygdala, thalamus and septum. 21,37,6° It is therefore possible that the actions of D~ agonists and D 2 antagonists on striatal c-fos expression are not mediated by dopamine receptors located in the striatum. In order to test this hypothesis, we studied the effects of local applications of the selective D l agonist C Y 208-24336 and the D 2 antagonist haloperidol on c-fos expression in the striatum.

EXPERIMENTAL PROCEDURES

6-Hydroxydopamine lesions Rats weighing 300-325 g were injected i.p. with desmethylimipramine (25 mg/kg) and then anesthetized 30 min later with sodium pentobarbital (50 mg/kg, i.p.). Unilateral medial forebrain bundle (MFB) lesions were made by injection 11.4 #g of 6-OHDA HBr (Sigma) dissolved in 4 #1 saline containing 0.05% ascorbic acid. The solution was injected over 10 min into the left MFB at the coordinates (in ram): AP 4.0, ML 1.3 and DV 1.6 from interaural zero according to the atlas of Paxinos and Watson. 46 Behavioural screening was carried out four weeks after surgery. Animals that turned at a rate of at least nine times/min after an apomorphine (0.2 mg/kg, s.c.) injection were used in subsequent experiments. Retrograde labeling of striatonigral and striatopallidal with Fluoro-Gold Rats were anaesthetized with pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic frame. Striatonigral neurons were retrogradely labeled by pressure injections of FluoroGold (FG) into the SN. 48 Striatopallidal neurons were retrogradely labeled in a second group of intact animals by iontophoretic application of FG into the GP. This approach was chosen in order to minimize damage to striatonigral fibres of passage coursing through the GP. FG was applied iontophoretically through a glass pipette with a tip diameter of 40 pm by a 5-#A current applied in pulses (6 s on/off) for 8 min into the GP at the coordinates (in mm): AP -0.9, ML -2.8 and DV --5.0 from bregma according to the atlas of Paxinos and Watson. 46 Some animals with unilateral 6OHDA lesions received iontophoretic injections of FG into the ipsilateral GP. Experimental protocol for drug studies Intact rats which had received unilateral FG injections in either the SN or GP were injected with haloperidol (1.0 mg/kg; dissolved in 1 ml of saline containing 40 #1 20% acetic acid). Rats with unilateral 6-OHDA lesions received a single injection of SKF 38393 (4 mg/kg, i.p.) or quinpirole (0.1 mg/kg, i.p.). Two hours later, all of the animals were deeply anaesthetized with pentobarbital (100 mg/kg, i.p.) and perfused with saline (200 ml) followed by 200 ml of 4% paraformaldehyde in phosphate-buffered (0.1 M) saline (PBS). Each brain was removed immediately after perfusion and allowed to postfix in fresh fixative for at least 12 h. lntrastriatal application of haloperidol and CY 208-243 via a microdialysis membrane Nine intact and three rats with unilateral 6-OHDA lesions of the nigrostriatal pathway (275-300 g) were anaesthetized with pentobarbital (50~50 mg/kg, i.p.) and implanted with a horizontal microdialysis probe in both striata as described in detail elsewhere. 12 Three days after surgery the microdialysis membrane in the intact animals was perfused at 5 #l/min with a 1 mM sodium phosphate-buffered solution (147 mM NaC1, 3raM KC1, 1.2mM CaC12, 1 mM MgC12) containing one of the following: vehicle (0.002% acetic acid); haloperidol (10 #M) or CY 208-243 (10 #M). The microdialysis membrane implanted in the striata of 6-OHDA-lesioned rats was perfused with the same perfusion solution except that it contained CY 208-243 (10 #M). All the animals were anaesthetized and perfused with 4% paraformaldehyde 2 h after the onset of perfusion. Fos immunohistochemistry was performed on coronal sections adjacent to, and through, the implantation site. Fos immunohistochemistry After postfixation, 20- and 30-#m sections were cut from the striatum using a Vibratome. Antisera from two different suppliers was used to detect Fos. The majority of exper-

D 1 and

D z receptor regulation of c-fos expression

iments were performed using a sheep polyclonal antibody (Cambridge Research Biochemicals, CRB OA-11-823) directed against residues 2-16 of N-terminal region of the Fos molecule. A second sheep polyclonal antibody (Serotec, PEPA 53) which also recognizes amino acids 2-16 of the N-terminal region of Fos was used to verify results obtained with the CRB antibody. Both antibodies produced similar results. Fos immunohistochemistry was performed according to Robertson et al.48 on sections (20 pm) from the unilaterally lesioned rats which had received FG injections in the ipsilateral GP. Briefly, striatal sections were washed three times (20 min/wash) with 0.02 M PBS and incubated for 48 h with primary antisera (diluted 1:200). Sections were then washed three times in PBS and incubated for 1 h in PBS containing Texas Red-labeled anti-sheep secondary (diluted 1:200) and 0.3% Triton X-100. Sections were washed three times with PBS, mounted and coverslipped with Immersionoel 518C (Zeiss). The slides were observed using a Leitz fluorescence microscope either under UV light (340-380 nm) to examine FG, or green light (530-560 nm) to look at Texas Red fluorescence. Haloperidol-induced Fos immunoreactivity could not be detected using a fluorescent secondary. Consequently, it was necessary to use the more sensitive avidin-biotin complex (ABC) immunoperoxidase method to visualize Fos immunoreactivity in the striatum after haloperidol administration. Sections (30#m) from 6-OHDA-lesioned rats which had received either SKF 38393 (4mg/kg, i.p.) or quinpirole (0.1 mg/kg, i.p.) were also processed using this method. Sections were washed three times with PBS and then incubated in PBS containing 0.3% hydrogen peroxide for 10min to block endogenous peroxidase activity. Sections were then washed three times in PBS and incubated in PBS containing 0.3% Triton X-100, 0.02% azide and Fos primary antisera (diluted 1 : 2000) for 48 h. The sections were then washed three times with PBS and incubated with a biotinylated rabbit anti-sheep secondary antibody (diluted 1:500) for 1 h. The sections were washed three times with PBS and incubated for 1 h with PBS containing 0.3% Triton X-100 and 0.5% (1:200 dilution) avidin biotinylated horseradish peroxidase complex (Vector Laboratories). After three washes in PBS the sections were rinsed in 0.1 M acetate buffer, pH 6.0. The reaction was visualized using a glucose oxidase~liaminobenzidine--nickel method described previously. 53 The reaction was terminated by washing in acetate buffer, and the sections were mounted on chrome-alum-coated slides. Sections from haloperidoltreated animals were coverslipped with Immersionoel 518C (Zeiss) and prepared for microscopic observation. Striatal sections from the 6-OHDA lesioned animals were dehydrated through a graded series of alcohols, and two changes of xylene, and coverslipped for microscopic observation.

Fos immunohistochemistry and in situ hybridization histochemistry Solutions were pretreated with 0.1% diethylpyrocarbonate (DEP) and autoclaved where appropriate prior to use in order to block RNAase activity. After postfixation, 20-pm sections were cut from each brain using a Vibratome and collected in sterile saline at 4°C. These sections were processed for Fos immunohistochemistry using an ABC method. Briefly, free-floating sections were incubated in Tris-buffered saline (TBS) containing 0.3% Triton X-100, 0.02% azide and Fos primary antisera (diluted 1:2000) for 48 h. The sections were then washed with TBS (3 × 20 min) and incubated with a biotinylated rabbit anti-sheep secondary antibody (Vector Laboratories; diluted 1:500) for 1 h. The sections were washed with TBS (3 × 20 min) and incubated for 1 h with TBS containing 0.3% Triton X-100 and 0.5% avidin-biotinylated horseradish peroxidase complex (Vector Laboratories). After washes in TBS (3 × 20 min) the reaction was visualized using 0.025% 3,3'-

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diaminobenzidine, 0.01 M imidazole and 0.0075% H202 in 50 mM Tris buffer. The reaction was terminated by washing in TBS. The sections were mounted onto slides that had been coated with 3-aminopropyltriethoxysilane and allowed to dry before processing for in situ hybridization. In situ hybridization was performed using the standard procedure of Lewis et al. 35 as previously describedJ 6 Mounted sections were immersed into 4% paraformaldehyde for 5 min at 21°C, rinsed 2 × 5 min in water and then allowed to dry. Next, the sections were hybridized overnight at 37°C in 4 × sodium citrate buffer (SSC; 0.6 M NaC1, 0.06 M sodium citrate, pH 7.0), 50% deionized formamide, 1 x Denhardt's (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin), 0.25mg/ml tRNA, 10% dextran sulphate, 40mM b-mercaptoethanol, and the 35S-labeled probe (1 × 10 6 d.p.m./200/~1 per slide). After hybridization, the sections were washed at 21°C ( 4 x 15min) with 2 x SSC, then with 0.5x SSC (4 x 15 min), followed by 4 x 15 min at 50°C in 0.5 x SSC. After drying, the slides were dipped in Kodak NTB-2 emulsion (diluted 1:1 with 0.6 M ammonium acetate) at 42°C, dried and exposed for two to four weeks at 4°C. The slides were developed with Kodak D-19 for 3 min at 16°C, rinsed with water, fixed, and washed in water. The sections were then dehydrated though a graded series of alcohols, and two changes of xylene, and coverslipped for microscopic observation.

Oligonucleotide probe The enkephalin oligodeoxyribonucleotide probe was 39 bases in length and complementary to bases 382~420 of p r e p r o e n k e p h a l i n A . 61 This probe has been characterized previously in neuroanatomical studies. 22,63 The enkephalin probe was tailed with [35S]dATP for in situ hybridization by terminal deoxynucleotidyl transferase using a standard oligonucleotide labeling kit and purified using a NENSORB column (NEN Research Products, Boston, MA). RESULTS

Effects o f selective D~ and D 2 agonists on Fos immunoreactivity in the striatum and globus pallidus ipsilateral to a 6-hydroxydopamine lesion o f the nigrostriatal pathway In agreement with previous findings, the selective agonist S K F 38393 ( 4 m g / k g , i.p.) p r o d u c e d a high rate o f contralateral circling (12-15 t u r n s / m i n ) a n d a d r a m a t i c increase in Fos i m m u n o r e a c t i v i t y in the s t r i a t u m ipsilateral, b u t n o t contralateral, to a 6 - O H D A lesion o f the nigrostriatal p a t h w a y (Fig. IA,B). 47'5° However, this t r e a t m e n t h a d little effect o n Fos i m m u n o r e a c t i v i t y in either the ipsilateral or contralateral GP. In contrast, the selective D2 receptor agonist quinpirole (0.1 mg/kg, i.p.) which also p r o d u c e d vigorous contralateral circling (10-12 t u r n s / m i n ) e n h a n c e d F o s expression in the ipsilateral, b u t n o t contralateral, G P (Fig. 1C,D). A s reported earlier, 5° quinpirole did n o t increase F o s i m m u n o r e activity in the s t r i a t u m (Fig. 1C,D). D 1

Overlap between retrogradely labeled striatopallidal neurons and D1 receptor-activated Fos I o n t o p h o r e t i c application o f F G into the G P retrogradely labeled medium-sized cell bodies (12-18 # m ) in the ipsilateral s t r i a t u m (Fig. 2A). E x a m i n a t i o n of the injection site for each of the four cases revealed t h a t diffusion o f F G was limited to the G P (results

Fig. 1 288

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B

Fig. 2. (A) Striatopallidal neurons retrogradely labeled with FG from the GP ipsilateral to a 6-OHDA lesion of the MFB. (B) D~ receptor-activated Fos in the same region of the ipsilateral shown in A. Scale bar = 75 # M. (C) Double exposure of A and B illustrating the lack of overlap between retrogradely labeled striatopallidal neurons and Fos-positive nuclei. Scale bar = 25 #m. not shown). Fos immunoreactivity was visualized with a fluorescent secondary antibody. S K F 38393 (4 mg/kg, i.p.) increased Fos immunoreactivity in the

6-OHDA-denervated striatum (Fig. 2B). Fos-positive nuclei were rarely located in striatal neurons retrogradely labeled from the G P (Fig. 2A-C). The results

Fig. 1. Effects of the selective D l receptor agonist SKF 38393 (4 mg/kg, i.p., A, B) and the selective D 2 receptor agonist quinpirole (0.1 mg/kg, i.p., C, D) on Fos immunoreactivity in the striatum and GP ipsilateral (B, D) and contralateral (A, C) to a 6-OHDA lesion of the nigrostriatal pathway. CP, caudate-putamen. Scale bar = 225/~m.

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shown in Fig. 2A-C are representative of the four cases examined. Overlap between retrogradely labeled striatonigral and striatopallidal neurons and D 2 antagonist-induced Fos Injection of F G into the SN retrogradely labeled medium-sized cell bodies (12-18/~ m) in the ipsilateral striatum (Fig. 3A). Examination of the injection site for each of the four cases revealed that diffusion of F G was limited to the SN (results not shown). Fos immunoreactivity, visualized by the ABC method, was increased in the striatum by haloperidol (1 mg/kg, i.p.; Fig. 3A,B). Fos-positive nuclei (Fig. 3A) were rarely located in striatal neurons retrogradely labeled from the SN (Fig. 3A). However, haloperidolinduced Fos-positive nuclei were frequently found in striatal neurons retrogradely labeled from the GP (Fig. 3B). The results shown in Fig. 3A,B are representative of the results from four animals each. Overlap between enkephalin neurons identified by in situ hybridization and D1 agonist- and D 2 antagonistinduced Fos D l receptor-activated Fos was infrequently found in enkephalin neurons identified with the oligonucleotide probe (Fig. 4A). Only 30 out of 201 Fos-positive neurons examined in the dorsal lateral striatum coincided with neurons autoradiographically labeled with the oligonucleotide probe. In contrast, haloperidol often increased Fos immunoreactivity in striatal neurons labeled with the enkephalin probe (Fig. 4B). Thus the autoradiographically labeled enkephalin probe was contained in 176 of the 208 Fos-immunoreactive neurons examined in the dorsal lateral striaturn. The results presented in Fig. 4A,B are typical of the three animals examined. Effects o f local application o f C Y 208-243 and haloperidol and striatal Fos immunoreactivity Fos immunoreactivity was slightly enhanced by local application of CY 208-243 (10 #M) in the intact striatum (Fig. 5A). In contrast, local delivery of the DI agonist in the 6-OHDA-denervated striatum produced striking increases in Fos immunoreactivity in the vicinity of the dialysis membrane (Fig. 5B). The vehicle used to dissolve the CY 208-243 and haloperidol in the perfusion solution did not increase Fos immunoreactivity in the intact (Fig. 5C) or denervated striatum (not shown). Addition of haloperidol to the perfusion solution for 2 h elevated Fos expression in the vicinity of the dialysis membrane in the striatum of intact animals (Fig. 5D). Results presented in Fig. 5 are representative of all of the animals examined. DISCUSSION

As reported previously, the selective D 1 receptor agonist S K F 38393 (4 mg/kg, i.p.) produced a large

increase in Fos immunoreactivity in the striatum ipsilateral to a 6-OHDA lesion of the nigrostriatal pathwayY '48'5° In contrast, the selective O 2 receptor agonist quinpirole, at a dose that induced vigorous rotation, failed to increase Fos expression in the 6-OHDA-denervated striatum. 48 However, unlike SKF 38393, quinpirole enhanced Fos immunoreactivity in the ipsilateral GP. Electrophysiological studies have shown that D2 receptor activation increases the activity of pallidal neurons and that 6-OHDA lesions of the nigrostriatal projection potentiate this increase. 9 The increase in Fos immunoreactivity in the GP ipsilateral to the 6-OHDAdenervated striatum is consistent with the enhanced ability of quinpirole to activate pallidal neurons. Since D 2 receptors are expressed by both striatal and pallidal neurons, 37'6° the quinpirole-induced increases in Fos expression in the GP may have been mediated indirectly via actions in the striatum or via direct effects of the agonist on the pallidal neurons themselves. We have previously shown that SKF 38393 elevates Fos expression in striatonigral neurons. 5° In the present study, striatopallidal neurons were retrogradely labeled by iontophoretic application of F G to the GP ipsilateral to the 6-OHDA-lesioned side. In contrast to the situation with retrogradely identified striatonigral neurons, S K F 38393-induced Fospositive nuclei were seldom found in retrogradely labeled striatopallidal neurons. Similarly, SKF 38393-induced Fos was infrequently found in striatopallidal neurons identified with an enkephalin oligonucleotide probe. Hence, D1 receptor agonists preferentially increase c-fos expression in striatonigral neurons. This finding is in agreement with autoradiographic and in situ hybridization studies which report that D 1 receptors are expressed predominantly by striatonigral neurons. 23,26,33 Haloperidol and selective D 2 receptor antagonists such as raclopride and YM 09151-2 increase Fos expression in the intact striatum) 6,49 Furthermore, haloperidol and raclopride produce very similar patterns of Fos immunoreactivity in the striatum. In the anterior striatum, haloperidol- and raclopride-induced Fos immunoreactivity is concentrated in the dorsolateral region, in the middle portion Fos-immunoreactive neurons are concentrated in the lateral rim, and caudally they are positioned ventrally.49 This distribution of U 2 antagonist-induced Fos immunoreactivity is very similar to that of striatal D 2 receptors, 8'31,37,38 and this suggests that these D2 antagonists increase Fos expression in striatal neurons that strongly express the D 2 receptorfl The present results are consistent with this proposal. Thus, haloperidol frequently increased Fos in striatal neurons retrogradely labeled from the GP but not from the SN. In addition, haloperidol-induced Fos was often found in neurons labeled with an oligonucleotide probe complementary to m R N A encoding enkephalin.

D~ and

D2

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291

Fig. 3. A shows that haloperidol does not increase Fos immunoreactivity (dark nuclei, open arrows) in retrogradely labeled striatonigral neurons (dark arrows). The bottom panel (B) illustrates the high degree of overlap between haloperidol-induced Fos (dark nuclei) and retrogradely labeled striatopallidalneurons (white arrows). Scale bar = 30/~m. These results suggest that haloperidol preferentially activates D2- and enkephalin-containing striatopallidal neurons.

Direct application of haloperidol in the striatum via a microdialysis probe increased Fos immunoreactivity in the vicinity of the probe. Similarly, local

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Fig. 4. A shows silver grains (open arrows) produced by a 35S-labeled enkephalin probe and D 1 receptor-activated Fos (dark arrows) in the 6-OHDA-lesioned striatum. B shows enkephalin neurons identified with the oligonucleotideprobe (open arrows) and haloperidol-induced Fos (dark arrows). Note that in contrast to the situation in the top panel (A), silver grains from the enkephalin probe frequently label Fos-positive nuclei after haloperidol. Scale bar = 15 #m. application of the selective D l receptor agonist CY - CY 208-243 and haloperidol did not increase Fos 208-243 in the 6-OHDA-denervated striatum in- expression, perfusion p e r se was n o t sufficient to creased Fos expression. These results suggest that the activate Fos expression. In addition, local application effects of peripherally administered haloperidol and of CY 208-243 (10 #M) in the intact striatum proCY 208-243 are mediated by dopamine receptors in duced only a slight elevation of Fos immunoreactivity the striatum. Since just the vehicle used to dissolve the and this is consistent with previous studies which

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suggest that the activation of c-fos by selective 9 1 receptor agonists is dependent on the development of D 1 receptor supersensitivity. The present results suggest that striatonigral and striatopallidal neurons are differentially regulated by D 1 and D 2 receptors and this may have implications for the neuroantomical basis of rotational behavior produced by selective D 1 and D 2 receptor agonists in rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. Herrera-Marschitz and Ungerstedt28'29 have hypothesized that turning produced by the mixed D~/D 2 receptor agonist apomorphine was mediated by the striatonigral pathway, while circling produced by the D2 receptor agonist pergolide was mediated by striatopallidal outputs. These proposals were challenged by Fletcher and Starr ~9'2° who demonstrated that circling produced by both D~ and D 2 receptor agonists is abolished by kainic acid lesions of either the SN or GP ipsilateral to 6-OHDA lesions of the MFB. Hence, Fletcher and Starr 2° concluded that dopamine D 1and D2 receptor-mediated turning is not separately organized by striatonigral and striatopallidal pathways. The present data are more consistant with HerreraMarschitz and Ungerstedt's27,29hypothesis in that D 2 receptor-induced turning was associated with increased e-fos expression in the GP while D~-mediated circling was associated with changes in striatonigral neurons. Fos contains a heptad repeat of five leucine residues which enables it to form heterodimer complexes with a variety of other transcriptional regulating factors which share this motif such as Jun and ATF-4? ,12,25 These complexes are thought to influence transcription by binding to a region of DNA known as the AP-1 site. ~'7 Cell culture studies suggest that the proenkephalin gene, which expresses an AP-1 site, may be a physiological target for Fos and Jun. 54 Chronic administration of haloperidol increases enkephalin expression in striatopallidal neurons. 3°'33'51 The fact that haloperidol-induced Fos was located predominantly in striatopallidal neurons, raises the possibility that the increase in Fos expression observed in the present study may be

responsible for the elevation of striatal enkephalin transcription. Fos may also regulate dynorphin transcription. 18,44 D 1 receptor stimulation produces a dramatic increase in striatal dynorphin mRNA after 6-OHDA lesions of the nigro-striatal pathway. 23 The present study suggests that DI receptor Fos is localized predominantly in striatonigral neurons which are the chief source of striatal dynorphin. Hence, DI receptor-activated c-fos expression may also participate in the regulation of dynorphin transcription. CONCLUSION In summary, the present results are consistent with autoradiographic and in situ hybridization studies which have demonstrated that D 1 receptors are located principally on striatonigral neurons and that D 2 receptors reside predominantly on striatopallidal neurons. Furthermore, the preferential localization of D 1 agonist-induced Fos in striatonigral neurons suggests that dopamine selectively facilitates the activity of this pathway. In contrast, haloperidol selectively increased Fos expression in striatopallidal neurons suggesting that D2 receptor blockade may disinhibit this output. Conversely, D 2 receptor agonists are thought to inhibit striatopallidal neurons. Hence, the increase in pallidal Fos immunoreactivity produced by the selective D2 agonist quinpirole may reflect a disinhibition of these neurons. This result is consistent with eleetrophysiological studies which have demonstrated that 6-OHDA lesions enhance the ability of D2 receptor agonists to increase the discharge rate of pallidal neurons. Finally, the ability of locally applied haloperidol and CY 208-243 to increase striatal Fos expression suggests that when given systemically, the effects of these drugs on Fos expression are mediated by striatal dopamine receptors. Acknowledgements--We thank H. Matsumura for excel-

lent technical assistance. This work was supported by the Medical Research Council of Canada, Grant PG-23 and the Parkinson Foundation of Canada. G.S.R. is an M.R.C. Fellow, and S.R.V. an M.R.C. Scientist.

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D1 and D2 dopamine receptors differentially regulate c-fos expression in striatonigral and striatopallidal neurons.

The expression of Fos, the product of the proto-oncogene c-fos, is thought to be a marker of neuronal activity. D1, but not D2, dopamine receptor agon...
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