Topographical Organization of Opioid Peptide Precursor Gene Expression Following Repeated Apomorphine Treatment in the 6-Hydroxydopamine-Lesioned Rat

EXPERIMENTAL NEUROLOGY ARTICLE NO.

150, 223–234 (1998)

EN976771

Topographical Organization of Opioid Peptide Precursor Gene Expression Following Repeated Apomorphine Treatment in the 6-Hydroxydopamine-Lesioned Rat Susan Duty,* Brian Henry, Alan R. Crossman, and Jonathan M. Brotchie Division of Neuroscience, 1.124 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom; and *Neurodegenerative Diseases Research Centre, Pharmacology Group, King’s College London, Manresa Road, London, SW3 6LX, United Kingdom Received September 13, 1997; accepted December 9, 1997

Many studies have previously described changes in preproenkephalin-A (PPE-A) and preproenkephalin-B (PPE-B) gene expression in the striatum of the 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson’s disease (both with or without dopamine replacement treatment). To date, these studies have either taken the striatum as a whole or have focused on a single subregion of the striatum. However, the striatum is organized into anatomically discrete parallel circuits serving different functions (motor, associative, and limbic). We have therefore employed in situ hybridization to examine the detailed topography of changes in opioid precursor expression following dopamine depletion and subsequent treatment with apomorphine (5 mg/kg twice daily for 10 days). In the untreated 6-OHDA-lesioned striatum PPE-A expression was elevated only in the dorsal (sensorimotor) caudate-putamen. Following apomorphine treatment PPE-A mRNA levels were further raised in the sensorimotor striatum (I77%) and approximately doubled and tripled in the ventral caudate-putamen (associative) and nucleus accumbens (limbic), respectively. These subsequent elevations were mostly restricted to rostral portions of the striatum. Although unchanged following vehicle treatment, PPE-B gene expression in the lesioned caudate-putamen (sensorimotor and associative) was elevated some 30-fold by apomorphine treatment. A smaller rise (fivefold) was seen in rostral regions of the lesioned nucleus accumbens. Thus, differential regulation of opioid peptide transmission exists in motor, limbic, and associative regions of the striatum and may contribute to the generation of motor and cognitive disturbances following long-term treatment of the dopamine-depleted striatum. r 1998 Academic Press Key Words: preproenkephalin-A; preproenkephalin-B; in situ hybridization; Parkinson’s disease; LDOPA-induced dyskinesia; enkephalin; dynorphin.

INTRODUCTION

Long-term use of dopamine-replacing agents (e.g., L-DOPA, apomorphine) in the treatment of Parkinson’s

disease is plagued by the appearance of side-effects such as on-off fluctuations, wearing-off of efficacy, hallucinations, and severely debilitating dyskinesias (45). Although these side-effects are believed to result from modification of the activity of striatofugal projection neurons, the underlying molecular mechanisms remain unknown (15). In the 6-hydroxydopamine (6-OHDA)lesioned rat model of Parkinson’s disease, repeated dopamine-replacement therapy also causes pronounced behavioral supersensitivity (hyperkinesia), without significant changes in striatal dopamine receptor populations (8, 11, 20, 38). These results suggest that the postsynaptic modifications resulting from chronic dopamine replacement therapy most likely occur downstream of the dopamine receptors per se. In the rat, GABAergic striatal output neurons project to the globus pallidus, the ‘‘indirect’’ pathway, and the entopeduncular nucleus/substantia nigra pars reticulata (SNr), the ‘‘direct’’ pathway. The indirect pathway predominantly utilizes enkephalin (17, 23) whilst the direct pathway uses dynorphin and substance P as co-transmitters with GABA (23, 62). Two precursor peptides are responsible for the synthesis of enkephalin and dynorphin, preproenkephalin-A (PPEA), and preproenkephalin-B (PPE-B). In animal models of Parkinson’s disease and L-DOPA-induced dyskinesia, alterations in the expression of PPE-A and PPE-B genes have been widely described in both the striatum as a whole and individual subregions of the striatum. For example, when measured in the striatum as a whole, levels of mRNA encoding PPE-A are markedly increased in the 6-OHDAlesioned rat, with corresponding increases in enkephalinlike immunoreactivity in the striatum and globus pallidus (3, 64, 67). Subsequent intermittent dopamine agonist treatment has been reported to produce a small, though nonsignificant, further rise in PPE-A gene expression in a single region of the striatum (24) with either a reduction or no further change in striatal enkephalin-like immunoreactivity (21, 43). Similar changes in PPE-A gene expression have been reported in the caudate nucleus and putamen of the MPTP-treated primate model of Parkinson’s disease

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0014-4886/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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following chronic L-DOPA (32, 37). In comparison, striatal levels of mRNA encoding PPE-B are unaltered (67) or slightly decreased in the 6-OHDA-lesioned rat (24, 43, 64), with subsequent dopamine replacement markedly increasing PPE-B gene expression and dynorphin-like immunoreactivity in both the dopamine-depleted striatum and the substantia nigra (21, 24, 43). We have recently shown that this rise in PPE-B expression has a time course that parallels that of the behavioral supersensitivity associated with repeated dopaminergic therapy (19). The striatum (defined in this context as caudate, putamen, and nucleus accumbens) receives topographically organized dopaminergic and glutamatergic inputs which do not distribute neatly within the classical anatomical boundaries of caudate, putamen, and nucleus accumbens (7, 16, 26, 28, 30, 39, 41, 42, 68). The basal ganglia are organized as a series of functionally distinct parallel circuits which, in simple terms, can be divided into three major categories: sensorimotor, associative, and limbic (1, 54). Different regions of the striatum thus receive inputs from distinct regions of the cerebral cortex such that the more ventral regions receive inputs from limbic and associative cortex while dorsal regions of caudate and putamen are involved in sensorimotor functions. Furthermore, the expression of striatal neuropeptides appears to be differentially regulated both between striatal regions (50, 67) and along the rostrocaudal axis (57, 64). None of the studies to date has examined the distribution of opioid peptide gene expression in basal ganglia dysfunction with respect to a functionally relevant organization which may have important consequences both for movement control and for other basal ganglia functions following dopamine receptor stimulation in the dopaminedepleted striatum. In the present study, we have used in situ hybridization to analyze the expression of PPE-A and PPE-B along both the rostrocaudal and dorsoventral axes of the striatum in the 6-OHDA-lesioned, apomorphinetreated rat. To assess the levels of mRNA semiquantitatively, we have used film autoradiography with comparison to the levels of expression of a reference gene encoding glyceraldehyde 3-phosphate dehydrogenase (G3PDH). The levels of mRNA encoding both PPE-A and PPE-B have thus been topographically mapped throughout the rostrocaudal extent of both the caudate-putamen (dorsal and ventral) and nucleus accumbens (core and shell). This study aims to shed light on the likely functional contribution of the rise in striatal PPE-A and PPE-B expression observed following repeated apomorphine treatment in the 6-OHDA-lesioned rat. MATERIALS AND METHODS

(22 6 1°C), humidity (relative, 30%), 12 h light/dark cycles (light period 8 a.m.–8 p.m.) and were allowed free access to food (Standard rat pellets, B & K Universal) and water. 30 min prior to surgery animals were coinjected intraperitoneally (i.p.) with pargyline (5 mg/kg) and desipramine (25 mg/kg) to maximize selective dopamine depletion by 6-hydroxydopamine (6-OHDA). Under sodium pentobarbitone anesthesia (60 mg/kg, i.p.), rats were positioned in a stereotaxic frame. Each animal received a unilateral injection of 6-OHDA.HBr (12.5 µg in 2.5 µl of sterile water with 0.1% ascorbate) into the right median forebrain bundle at coordinates 22.8 mm from Bregma, 2 mm lateral to the midline, and 9 mm below the skull according to the atlas of Paxinos and Watson (55). The injection was made, by hand, over a 5-min period using a 5-µl Hamilton syringe. Apomorphine Administration and Behavioral Studies Two weeks following the 6-OHDA lesion, rats were divided into two experimental groups (1) apomorphinetreated and (2) vehicle-treated. Animals were injected intraperitoneally twice daily (9 am and 5 pm) for 10 days with either 5 mg/kg apomorphine (dissolved in sterile water) or sterile water (1 ml/kg). Immediately, following injection, animals were placed in stainlesssteel bowls (diameter 40 cm, Amee, Manchester). Locomotor activity of the animals was video-recorded for a 60-min period before the start of dosing (day 0) and then for 60 min immediately following the first injections on days 1, 3, 5, 7, and 10. Net rotations contraversive to the lesion were determined as an index of locomotor activity in these unilateral 6-OHDA-lesioned rats (61). Three hours after the last injection all rats were killed by stunning and cervical dislocation, the brains were removed and rapidly frozen in isopentane cooled to 245°C. Brains were stored desiccated at 270°C until further processing. Tissue Processing Brains were cryostat-sectioned (219°C) at 15 µm, thaw-mounted onto gelatin/chrome-alum-coated slides, and stored desiccated at 270°C until further processing. Sections were obtained throughout the striatum and were further divided into four rostrocaudal levels as follows: Level 1 5 12.0 mm to 11.5 mm from Bregma; Level 2 5 11.5 mm to 11.0 mm from Bregma; Level 3 5 11.0 mm to 10.5 mm from Bregma; Level 4 5 caudal to 10.5 mm from Bregma, as defined by Paxinos and Watson (55). Sections from all four levels from each animal were processed in triplicate.

Animals and Surgery

Lesion Verification/[ 3H]Mazindol Autoradiography

Male Sprague–Dawley rats (260–300 g; Charles Rivers) were used in all experiments. Rats were maintained in standard housing conditions with constant temperature

Mazindol binding to dopamine uptake sites was used as an index of the degree of loss of dopaminergic terminals in the rat striatum following the 6-OHDA

TOPOGRAPHICAL OPIOID PEPTIDE PRECURSOR GENE EXPRESSION

lesion. The method used was identical to that we have previously described (27). Briefly, triplicate sections from each rostrocaudal level of all experimental brains were freeze-dried overnight. The sections were then pre-incubated in 50 mM Tris–HCl (pH 7.9) for 5 min at 4°C followed by 60-min incubation at 4°C in 50 mM Tris–HCl containing 300 mM NaCl, 5 mM KCl, 10 nM [ 3H]mazindol (NEN/DuPont, UK) and 50 nM desipramine (RBI, U.S.A.). Specific binding was defined as that displaced by 100 µM nomifensine. Sections were then washed in ice-cold Tris–HCl (2 3 1 min), dip-rinsed in ice-cold distilled water, dried in a stream of cold air and finally opposed to 3H-sensitive Hyperfilm, alongside 3H standards, for 2 weeks at 4°C. Films were developed using Kodak D-19 and fixed using Kodak Unifix. Average optical density of each of the regions of interest and the standards was determined using a Seescan Image Analysis system (see below) and specific mazindol binding in pmol/mg determined. Only animals showing greater than 90% reduction in striatal [ 3H]mazindol binding were included in the final analyses of locomotor response and opioid peptide gene expression (n 5 5 apomorphine-treated; n 5 6 vehicle-treated). Neuropeptide Expression Studies: In Situ Hybridization Histochemistry The method used for in situ hybridization was essentially that previously described by Gerfen et al. (24). Briefly, sections were firstly warmed to room temperature for 15 min, fixed in 4% paraformaldehyde for 10 min, rinsed and then incubated in a fresh solution of 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. Sections were then dehydrated in a series of ascending concentrations of ethanol, defatted for 5 min in chloroform, air-dried, and then subsequently hybridized with 35S-tailed oligonucleotide probes. Three synthetic oligonucleotide probes were used in these studies. Synthetic oligonucleotide probes (30–45 bases in length) designed to be complementary to mRNa encoding PPE-A and PPE-B were synthesized by Gibco BRL. The primary probe targeted against PPE-A was complementary to nucleotides 343–384 of the rat PPE-A gene (Accession No. M28263: (35)) sequence 58—CTT CAT GAA GCC TCC ATA CCG TTT CAT GAA CCC TCC ATA CTT-38. The primary probe targeted against PPE-B was complementary to nucleotides 754– 798 of the rat PPE-B gene (Accession No. M10088: (14)) sequence 58—GCT CCT CTT GGG GTA TTT GCG CAA AAA GCC GCC ATA GAG TTT GGC-38. The commercially available probe complementary to mRNA encoding the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH, Clontech) was used as a control probe in all in situ hybridizations. This probe is complementary to nucleotides 734–763 of the G3PDH gene (G3PDH), sequence 58—CAC GGA AGG CCA TGC

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CAG TGA GCT TCC CGT-38 (5). Probe sequences were checked using the UK Human Genome Mapping Project Resource Centre database (both EMBL and GenBank databases, BLAST search: (2)) to check that the selected sequences would not hybridize to any other known sequence. Each probe (20 ng in 1 µl) were 38-end tailed with [ 35S]dATP by incubation for 60 min at 37°C in a 35 µl reaction mixture containing (final concentrations): [ 35S]dATP (82.5 µCi), terminal deoxynucleotidyl transferase (36U), CoCl2 (2 mM), sodium cocodylate (120 mM), and dithiothreitol (100 mM) at pH 7.2. [ 35S]dATP and all probe labeling reagents were obtained from NEN/DuPont (UK). The reaction was terminated by the addition of 50 µl ice-cold sterile water and the labeled probe further purified by column chromatography (Biospin 5 columns, Bio-Rad). Hybridization buffer consisted of the 35S-labeled oligonucleotide probe (3 3 106 cpm/ml) in 50% formamide, 43 standard saline citrate (43 SSC) (13 SSC 5 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 10% w/v dextran sulfate, and 10 mM dithiothreitol. Aliquots of hybridization buffer (50 µl) were applied to each of the sections on any one slide. Sections were then covered with parafilm and incubated for 18 h at 42°C in a humid chamber. After incubation, the parafilm coverslips were floated off in 23 SSC and the sections were rinsed in a series of washes; 13 SSC at room temperature for 30 min; 13 SSC at 55°C for 30 min, 0.13 SSC at room temperature for 10 min. Sections were then dehydrated for 3 min in 70 then 95% ethanol and air-dried. The localization of bound probe was revealed by autoradiograms produced by exposure of sections to X-ray film (b-Max Hyperfilm) at 4°C for 7 days. Films were developed using Kodak D-19 and fixed using Kodak Unifix. Densitometric analysis of the autoradiograms was performed using a Seescan Image Analysis system. Film optical density (O.D.) was evaluated in four regions of the striatum; the dorsal and ventral caudate-putamen and the core and shell of the nucleus accumbens (regions defined from Ref. 5). In rostrocaudal levels 1 to 3, values were obtained for all four regions, while only dorsal and ventral caudate-putamen readings were obtainable most caudally in level 4. A total of 14 regions were thus measured both ipsilateral and contralateral to the 6-OHDA lesion for each animal. Nonspecific background signal of the tissue, as defined by the O.D. of the corpus callosum, was subtracted from all O.D. measurements taken for each section. The average of background-corrected O.D. values taken from the three sections for each animal was then determined for all 14 regions. In order to control for nonspecific increases in transcription rate, the O.D. values obtained with the PPE-A and PPE-B probes were finally divided by the O.D. values obtained for the control probe, G3PDH, in total striatum from the

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respective lesioned or intact striatum. The mean level of mRNA encoding PPE-A or PPE-B is therefore expressed as a ratio of the mean test probe O.D. value relative to the mean O.D. value for G3PDH. The O.D. of standards ( 14C Microscales) was used to ensure that the O.D.s measured lay within the range in which the film showed a linear relationship between O.D. and radioactivity. 14C microscales were chosen as they provided a level of radioactivity that was within the range of experimental samples and produced O.D.s similar to experimental samples over the exposure times used in our study. Control Experiments for Hybridization Three sets of experiments were performed to confirm that the hybridization signals obtained represented specific hybridization of the oligonucleotide probes to cellular mRNA encoding the appropriate PPE-A or PPE-B transcripts. Northern analysis was performed to demonstrate the size of the mRNA to which the oligonucleotide probes hybridized. Freshly obtained rat striatal tissue was homogenized and total RNA extracted using RNAzol B (Biogene Ltd.). Ten micrograms of RNA was denatured using formamide, run on a 1.2% agarose gel, and transferred onto nylon membranes (Hybond N) by capillary blotting. Membranes were UV cross-linked and hybridized for 18 h at 42°C with 32P-labeled PPE-A and PPE-B oligonucleotide probes (see above). The membranes were then washed, as described above for the in situ hybridizations, and exposed to X-ray film (Hyperfilm-MP) with intensifying screens at 270°C for 7 days. Control slides containing striatal sections from normal rats were pretreated with Ribonuclease A (20 µg/ml) in phosphate-buffered saline at 37°C for 30 min in order to destroy cellular RNA. Hybridization with the PPE-A and PPE-B test probes was then performed using the protocol detailed above. Additional control sections were hybridized with second probes directed at regions of the PPE-A and PPE-B transcripts starting 10 bases upstream from the probes used in the experimental procedure described above. The striatal distribution of these second probes was compared with that of the test probes.

or region or interactions between treatment and region (Stat-100 1.24, Biosoft). If justified by the two-way ANOVA, significant effects between groups in each region were subsequently tested using a one-way ANOVA followed by Student–Newman–Keuls (SNK) post-hoc analysis (Prism 2.01, GraphPad Software). Differences in locomotor activity between the two treatment groups (apomorphine versus vehicle) were assessed for each time point using an unpaired t test. The effects of different durations of apomorphine treatment alone on locomotor responses were compared using a one-way ANOVA followed by SNK. In all cases P , 0.05 was taken to represent a significant difference (n 5 5 apomorphine; n 5 6 vehicle). RESULTS

Methodological Considerations The specificity of the PPE-A and PPE-B oligonucleotide probes has been confirmed by various means. Northern analysis of striatal RNA detected a single band in each case with sizes corresponding to 1.5 and 2.7 kb, respectively (Fig. 1), as expected for these two transcripts (Howells et al., 1984; Civelli et al., 1985). In addition, the probes used lacked homology with any other sequence submitted to the GenBank and EMBL databases (using MRC-HGMP Resource Centre access). Ribonuclease A pretreatment of sections was found to completely abolish the specific in situ hybridization signal and probes directed at a second region of the cDNA sequences gave the same striatal hybridization patterns as the experimental probes (data not shown). In addition, nonspecific alterations in transcription rate were accounted for during the final analysis by correcting PPE-A and PPE-B signals for any changes seen during parallel hybridizations with the control G3PDH oligoprobe. The importance of this form of analysis is shown by the fact significant differences in

Statistical Analyses In each of the two treatment groups, [ 3H]mazindol binding in each region of the lesioned striatum was compared to that in the intact striatum using nonpaired t tests. For the in situ hybridizations, differences between the PPE-A/G3PDH ratio or PPE-B/G3PDH ratio in the 14 regions across the four treatment groups were evaluated using a two-way analysis of variance (ANOVA) to determine significant effects of treatment

FIG. 1. Northern blot analysis of RNA from rat striatum hybridized with the oligonucleotide probes for (a) PPE-A and (b) PPE-B. Total RNA (10 µg left hand lane, 1 µg right hand lane) was separated on a 1.2% agarose gel, blotted onto nylon filters, and hybridized with the respective probes. The position of size-markers is shown by the black horizontal bars. Bands of (a) 1.5 and (b) 2.7 kb were detected for the PPE-A and PPE-B transcripts, respectively.

TOPOGRAPHICAL OPIOID PEPTIDE PRECURSOR GENE EXPRESSION

the O.D. of the G3PDH signal were observed (1 way ANOVA, F3,18 5 5.25, P , 0.01). In no case were there side-to-side differences within a group of animals (P . 0.05, SNK) although such changes were seen with the O.D. signals for PPE-A and PPE-B probes. However, in apomorphine-treated animals the G3PDH expression was reduced such that it was significantly lower in the apomorphine-treated, unlesioned side than in the vehicle-treated-lesioned side (P , 0.05, SNK) and also in the apomorphine-treated, lesioned side than in the vehicle-treated, lesioned side (P , 0.05 SNK). As we have previously described (27), the 6-OHDAlesioned striata showed an approximate 90–95% reduction in specific [ 3H]mazindol binding compared to the intact striata, at all rostrocaudal levels examined (92.4% 6 0.65, mean 6 SEM, n 5 28 regions). For any striatal region, no significant differences were observed in the loss of specific mazindol binding between the two treatment groups (P . 0.05, n 5 5–6). Effect of Repeated Apomorphine Treatment on Locomotor Activity in 6-OHDA-Lesioned Rats The net rotations contraversive to the lesion, observed before (day 0) and at 1, 3, 5, 7, and 10 days following the commencement of treatment with either apomorphine (5 mg/kg; i.p.) or vehicle are shown in Fig. 2. Animals receiving vehicle injections produced no net contraversive rotations at any time during the course of treatment. In contrast, following the first injection,

FIG. 2. Graph showing the behavioral supersensitivity seen following repeated treatment with apomorphine (5 mg/kg) 23 daily for 10 days (r). Behavioral responses following repeated vehicle treatment are also shown (j). Values shown are means 6 SEM (n 5 5–6). *Represents significant differences between apomorphine and vehicle-treated animals at a given time-point (P , 0.05; unpaired t test); # represents a significant increase in apomorphineinduced contraversive rotations from the previous time point (P , 0.05; one-way ANOVA 1 Student–Newman–Keuls post-hoc test).

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apomorphine-treated rats showed a significant degree of net contraversive rotations/h (299.0 6 28.0; n 5 5) compared to the vehicle-treated group (28.0 6 2.4; n 5 6) (P , 0.01; unpaired t test). Beyond day 1 of treatment, all animals in the apomorphine treatment group showed a significant increase in net contraversive rotations with each consecutive dose (P , 0.01; one-way ANOVA 1 SNK), reaching 2028.0 6 160.8 contraversive rotations/h by day 10. Effect of Apomorphine Treatment on PPE-A Gene Expression in Lesioned and Intact Striata PPE-A gene expression is represented as a ratio of the hybridization signal of PPE-A/G3PDH, as detailed under Materials and Methods. Mean values (6SEM) for the PPE-A/G3PDH ratio in vehicle and apomorphine treatment groups are documented in Table 1. Two way analysis of variance using treatment and region as a factor showed significant effects of treatment and region and a significant interaction between the two for both PPE-A (F(treatment) 5 85.27, P , 0.0001, F(region) 5 8.32, P , 0.0001, F (interaction treatment 3 region) 5 1.6, P , 0.05) and PPE-B (F(treatment) 5 224.7, P , 0.0001, F (region) 5 5.45, P , .0.0001, F (interaction treatment 3 region) 5 4.39, P , 0.0001). Within the vehicle-treatment group, the intact striatum showed significantly higher levels of PPE-A/ G3PDH ratio in the rostral caudate-putamen than in the nucleus accumbens. Additionally, within the vehicletreated group, the PPE-A/G3PDH ratio in dorsal caudate-putamen at rostrocaudal levels 2–4 and in the ventral striatum at rostrocaudal level 2 only (but not in the nucleus accumbens at any level) was higher in the 6-OHDA-lesioned striatum, compared to the intact striatum (see * in Table 1). In the apomorphine-treated group, similar significant rises in PPE-A expression were also seen in the caudate-putamen (dorsally at levels 2–4 and ventrally at level 1 and 2). However, in contrast to the vehicletreated group, PPE-A expression was also elevated, compared to the intact side, in the rostral caudate putamen (i.e., levels 1 and 2) and in the core (levels 1 and 2) and shell (levels 1–3) of the nucleus accumbens. Comparison between the lesioned striata of the two treatment groups (see† in Table 1) revealed many significant differences in the PPE-A/G3PDH ratio. Thus, 9 of 14 regions showed a significantly higher PPE-A/ G3PDH ratio in the lesioned striatum following apomorphine treatment compared to vehicle treatment. These increases were mostly observed rostrally in levels 1 and 2, with only one difference being found at level 3 (core of nucleus accumbens) and no differences being found at level 4. Although there was a tendency for expression levels to decline along the rostrocaudal axes for each of the four striatal areas of the apomorphine-treated

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TABLE 1 Regional Striatal PPE-A/G3PDH mRNA Ratio in 6-OHDA-Lesioned Rat Treated Repeatedly with Vehicle or 5 mg/kg Apomorphine Vehicle treatment

Apomorphine treatment

Striatal Region

Rostrocaudal level

Intact striatum

Lesioned striatum

Intact striatum

Lesioned striatum

Dorsal caudate-putamen

1 2 3 4 1 2 3 4 1 2 3 1 2 3

1.59 6 0.16 1.55 6 0.26 1.18 6 0.21 1.32 6 0.13 1.77 6 0.15 1.37 6 0.18 1.25 6 0.23 1.44 6 0.14 0.89 6 0.15 0.65 6 0.10 0.45 6 0.22 1.08 6 0.18 1.04 6 0.11 0.71 6 0.23

3.5 6 0.70 2.95 6 0.23* 4.75 6 1.21* 2.80 6 0.23* 3.38 6 0.65 2.82 6 0.22* 4.06 6 1.39 2.66 6 0.19 1.47 6 0.23 0.98 6 0.12 0.57 6 0.19 1.91 6 0.29 1.36 6 0.11 1.27 6 0.22

1.77 6 0.27 1.55 6 0.13 1.61 6 0.28 1.73 6 0.39 2.06 6 0.23 1.92 6 0.20 1.71 6 0.30 2.27 6 0.69 1.15 6 0.27 0.87 6 0.23 0.99 6 0.33 1.93 6 0.35 1.79 6 0.34 1.68 6 0.40

5.70 6 0.92*,† 5.21 6 0.55*,† 4.06 6 0.51* 3.69 6 0.73* 6.16 6 0.82*,† 5.67 6 0.61*,† 4.09 6 0.53 3.96 6 1.04 2.98 6 0.60*,† 2.62 6 0.81*,† 1.58 6 0.49 4.46 6 0.51*,† 4.19 6 0.97*,† 2.78 6 0.45*,†

Ventral caudate-putamen

Nucleus accumbens shell

Nucleus accumbens core

Note. Data represent mean 6 SEM (n 5 5–6). * Indicates a significant difference between intact and lesioned striata for a given treatment. † Indicates a significant difference between treatments for either the intact or lesioned striata (P , 0.05; one-way ANOVA 1 SNK post-hoc test).

lesioned striatum, these changes did not reach statistical significance (P . 0.05, 1-way ANOVA). The largest percentage rises were seen in rostrocaudal level 2 and approximated to 77% (dorsal CPu), 101% (ventral CPu), 167% (shell of the nucleus accumbens), and 208%

(core of the nucleus accumbens). The PPE-A hybridization signal obtained in representative sections from rostrocaudal level 2 is shown, for both treatment groups, in Fig. 3A. No mediolateral gradients in PPE-A signal were observed in the lesioned striata of either treat-

FIG. 3. Pseudocolor transformation of autoradiograms of the in situ hybridization signal obtained with the (A) PPE-A and (B) PPE-B oligonucleotide probes in rat striatum. Coronal sections, representative of rostrocaudal level 2, are shown from 6-OHDA-lesioned rats (the lesioned striata are on the right of the image) following treatment with vehicle (upper panel) or apomorphine (5 mg/kg, twice daily for 10 days; lower panel). Increasing intensity of hybridization signal is represented by red.yellow.green.blue.black.

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TABLE 2 Regional Striatal PPE-B/G3PDH mRNA Ratio in 6-OHDA-Lesioned Rat Treated Repeatedly with Vehicle or 5 mg/kg Apomorphine Vehicle treatment

Apomorphine treatment

Striatal region

Rostrocaudal level

Intact striatum

Lesioned striatum

Intact striatum

Lesioned striatum

Dordal caudate-putamen

1 2 3 4 1 2 3 4 1 2 3 1 2 3

0.13 6 0.10 0.12 6 0.11 0.19 6 0.14 0.21 6 0.15 0.24 6 0.13 0.20 6 0.13 0.11 6 0.12 0.10 6 0.10 0.49 6 0.25 0.50 6 0.29 0.48 6 0.30 0.53 6 0.31 0.51 6 0.20 0.42 6 0.20

0.11 6 0.09 0.13 6 0.11 0.21 6 0.15 0.20 6 0.18 0.23 6 0.12 0.21 6 0.20 0.10 6 0.09 0.12 6 0.11 0.52 6 0.30 0.51 6 0.30 0.50 6 0.20 0.52 6 0.30 0.50 6 0.15 0.40 6 0.09

0.10 6 0.09 0.10 6 0.10 0.18 6 0.15 0.19 6 0.12 0.22 6 0.14 0.20 6 0.14 0.12 6 0.10 0.09 6 0.08 0.45 6 0.29 0.41 6 0.30 0.39 6 0.28 0.45 6 0.29 0.40 6 0.24 0.37 6 0.22

3.05 6 0.39*,† 2.03 6 0.41*,† 1.05 6 0.08*,† 1.21 6 0.32*,† 3.41 6 0.40*,† 2.57 6 0.44*,† 1.54 6 0.03*,† 1.68 6 0.49*,† 2.58 6 0.64*,† 1.96 6 0.61 0.92 6 0.09 2.95 6 0.35*,† 1.99 6 0.78 0.95 6 0.22

Ventral caudate-putamen

Nucleus accumbens shell

Nucleus accumbens core

Note. Data represent mean 6 SEM (n 5 5–6). * Indicates a significant difference between intact and lesioned striata for a given treatment. † Indicates a significant difference between treatments for either the intact or lesioned striata (P , 0.05; one-way ANOVA 1 SNK post-hoc test).

ment group. In all regions of the intact striata there were no significant differences in PPE-A/G3PDH ratio between the two treatment groups (P . 0.05, n 5 5–6). Effect of Apomorphine Treatment on PPE-B Gene Expression in Lesioned and Intact Striata PPE-B gene expression is represented as a ratio of the hybridization signal of PPE-B/G3PDH, as detailed under Materials and Methods. Mean values (6SEM) for the PPE-B/G3PDH ratio in vehicle and apomorphine treatment groups are documented in Table 2. Within the vehicle-treatment group, the PPE-B/G3PDH ratio was consistently low in all regions of the striatum. Additionally, in the vehicle-treated group there was no significant difference in PPE-B/G3PDH ratio for any region between the lesioned and intact striata. In contrast, within the apomorphine-treatment group, the PPE-B/G3PDH ratio was significantly higher in the caudate-putamen at all rostrocaudal levels and in the most rostral levels of the nucleus accumbens of the lesioned striatum, compared both to the respective region of the intact striatum (see * in Table 2) and to the respective region of lesioned striatum of the vehicletreatment group (see† in Table 2). The largest increases in the PPE-B/G3PDH ratio (,30-fold) were seen in the dorsal and ventral caudate-putamen at rostrocaudal level 1, while more modest increases (,5-fold) were seen at this level in the core and shell of the nucleus accumbens. Indeed, in all striatal areas the expression was significantly higher in the most rostral level compared to more caudal levels. The hybridization signal obtained with PPE-B at rostrocaudal level 2 is shown

for both treatment groups in Fig. 3B. Within the lesioned striatum of the apomorphine treatment group a significant low-high mediolateral gradient in PPE-B/ G3PDH ratio was detected at all rostrocaudal levels. Mean ratios obtained at rostrocaudal level 2 were: medial, 1.886 6 0.435, and lateral, 4.014 6 0.814 (P , 0.05; n 5 5). No mediolateral gradient was observed in the vehicle-treated lesioned striatum, nor did one exist in the intact striata of either treatment group (data not shown). DISCUSSION

Previous studies have shown that striatal PPE-A expression is elevated following dopamine depletion (e.g., 3, 64, 67). A major finding of this study is that this elevation is confined mainly to dorsal regions of the caudate-putamen and does not affect the nucleus accumbens or ventral parts of the caudate-putamen. The results of this study also show, for the first time, that PPE-A gene expression is increased following dopamine receptor stimulation in the dopamine-depleted rat striatum. This elevation is over and above that seen in the vehicle-treated lesioned striatum and is organized topographically such that it is more prominent rostrally and ventrally. We also confirm previous reports that PPE-B gene expression is increased in the dopamine-depleted rat striatum following repeated dopamine receptor stimulation (21, 24, 43) and show that this rise occurs, with marked mediolateral and rostrocaudal gradients, in all striatal regions.

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Behavioral Supersensitivity The locomotor supersensitivity observed in the present study with repeated apomorphine treatment in the 6-OHDA-lesioned rat is consistent with studies of varying time courses both with apomorphine (8, 19) and with L-DOPA (11, 53). Collectively, these findings show that repeated dopamine receptor stimulation in the 6-OHDA-lesioned rat results in the development of increased locomotor responsiveness or hyperkinesia. Further studies are required to characterize this behavior and assess its relationship to dyskinesias seen in humans and subhuman primates following dopamine replacement therapy of parkinsonian symptoms (6, 15, 45). One possible molecular mechanism proposed to underlie this behavioral change is the alteration in neuropeptide gene expression described previously and extended in the present study (18, 19, 21, 24, 43). Regional Variations in PPE-A Gene Expression From studies in primate and rodent, it is evident that distinct striatal regions receive different patterns of cortical inputs. For example, the dorsolateral caudate nucleus and putamen receive projections from motor and somatosensory regions of cortex (16, 41, 42), while the most ventral parts of the caudate-putamen, together with the nucleus accumbens, receive inputs from limbic and paralimbic cortices, hippocampus, and amygdala (7, 30, 39). The caudate nucleus and most rostral regions of putamen also receive projections from the many association cortices (26, 41). The basal level of expression observed in the present study for PPE-A (caudate-putamen . nucleus accumbens) in the intact, vehicle-treated striatum implies a role for PPE-A in sensorimotor and associative function. In the dopamine-depleted striatum of vehicletreated animals, PPE-A mRNA levels were significantly raised in the dorsal caudate-putamen, less so more ventrally and not at all in the nucleus accumbens, indicating that changes in PPE-A expression brought about by dopamine depletion are again restricted to the sensorimotor and associative regions of striatum. In contrast, since repeated apomorphine administration increased PPE-A expression in both dorsal and ventral caudate-putamen and nucleus accumbens of the lesioned striatum, it seems likely that disturbances within limbic circuits, in addition to sensorimotor and associative circuits may accompany the hyperkinesia observed following long-term apomorphine administration in the 6-OHDA-lesioned rat. Previous studies examining the effects of repeated administration of apomorphine on PPE-A gene expression in the 6-OHDA-lesioned rat striatum have produced data that, at first glance, appear to contradict the data presented here. For example, Gerfen and colleagues (24) found that repeated administration of

apomorphine (5 mg/kg; twice daily for 10 days) produced only a small, nonsignificant rise in PPE-A mRNA expression above that already occurring following lesion alone. However, this previous study measured changes in PPE-A mRNA only in the dorsolateral caudate-putamen and furthermore did not examine along the rostrocaudal axis of the striatum. Since the increased PPE-A gene expression presently observed was significant only in the rostral portion of the striatum, with the largest percentage changes occurring ventrally in the nucleus accumbens, it is easy to see how such changes might have remained undetected in previous studies. It is also easy to envisage how previous studies measuring enkephalin synthesis in whole striatum would not detect topographically organized alterations in synthesis (21). Recently, studies in the MPTP-treated primate model of Parkinson’s disease have shown that chronic levodopa treatment produces no further rise in PPE-A mRNA levels in dorsal and ventral caudate-putamen above those caused by MPTP treatment alone (32, 37). Jolkkonen et al. (37) also found no changes in expression in either rostral or caudal nucleus accumbens. However, these studies did not fully consider the functional organization of the striatum and were unable to provide data comparing PPE-A expression in the association and sensorimotor part of the rostral striatum, so it is difficult to compare these studies with the present one. In agreement with the observations in primates, we detected no further rise in PPE-A gene expression caudally in the dopamine-depleted rat caudate-putamen following repeated dopamine receptor stimulation. Clearly this highlights the need for a rostrocaudal and functionally applicable examination of neuropeptide gene expression in primate studies in order to clarify these issues. The reason behind the subsequent rise in PPE-A expression in 6-OHDA-lesioned striatum following repeated apomorphine treatment remains to be elucidated. One possible explanation centers on the breakdown of inhibitory coupling between dopamine D2 receptors and adenylyl cyclase. cAMP-dependent transcription factors can act as promoters for the PPE-A gene. Thus, D2 receptor stimulation normally provides tonic inhibition of PPE-A gene expression through inhibition of adenylyl cyclase activation (4, 40). However, in the dopamine-depleted striatum, D2 receptors are functionally uncoupled from adenylyl cyclase activation (34, 58); this probably leads to the lesion-induced rise we and others have reported. Subsequent dopamine replacement appears to cause D2 receptors to positively recouple, at least functionally, to adenylyl cyclase (34). This positive recoupling may explain in part the subsequent further rise in PPE-A gene expression seen after dopamine receptor stimulation with apomorphine. Whether the rostrocaudal topography of

TOPOGRAPHICAL OPIOID PEPTIDE PRECURSOR GENE EXPRESSION

this D2/adenylyl cyclase recoupling matches that seen here for the apomorphine-induced rise in PPE-A expression remains to be determined. A more likely candidate influencing the rostrocaudal topography of these PPE-A changes may be the rostrocaudal variation found among the glutamatergic corticostriatal projections. These glutamatergic projections are necessary for maintaining both the high levels of PPE-A mRNA in the intact striatum (57, 60) and the elevated PPE-A gene expression seen following dopamine denervation (10). Interestingly, this glutamatergic regulation of PPE-A gene expression is also most apparent in the rostral portion of the striatum (57). Therefore, glutamatergic cortico-striatal projections, that are thought to be overactive in the 6-OHDAlesioned rat striatum (13, 29), might contribute to the apomorphine-induced increase in PPE-A gene expression seen here in the rostral portion of the dopaminedepleted striatum. Regional Variations in PPE-B Gene Expression In agreement with previous nontopographical investigations, while no significant change in striatal PPE-B mRNA levels was observed following 6-OHDA lesion alone (67), subsequent apomorphine treatment resulted in a pronounced elevation of PPE-B mRNA in the lesioned striatum (21, 24, 36, 43). The relatively global changes in PPE-B expression seen here (all regions except caudal levels of nucleus accumbens) supports the notion of abnormalities in motor, associative, and limbic basal ganglia circuits resulting from this treatment. Comparable studies of PPE-B expression in primates are awaited. Stimulation of dopamine D1 receptors that are reportedly supersensitive in the 6-OHDA-lesioned striatum (34, 48) is the most likely explanation for this increased PPE-B gene expression. An increase in striatal D1 receptor numbers has been reported following 6-OHDA lesioning (9, 22), with the largest increases occuring in lateral aspects of the striatum (22). Although this finding suggests that increased numbers of D1 receptors in lateral striatum per se may contribute to the mediolateral gradient in PPE-B mRNA expression seen in the present study, many other investigations have failed to detect similar changes in D1 receptor binding in the 6-OHDA-lesioned striatum (27, 33, 46). Further studies are clearly required to determine the mechanisms responsible for this mediolateral gradient in PPE-B gene expression. The reason that the largest rise in striatal PPE-B synthesis occurred most rostrally may again reflect corticostriatal glutamatergic influence on neuropeptide synthesis within the rostral striatum which not only influences PPE-A synthesis, as outlined above, but also influences PPE-B synthesis in a similar manner (57, 66). Although PPE-A and PPE-B gene expression were

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both affected to the greatest extent in rostral regions of striatum, their patterns of expression were otherwise very different, suggesting that distinct regulatory mechanisms influence the synthesis of the two neuropeptides. The regional variations in expression are clearly not the result of differences in the degree of dopamine depletion, since the loss of dopamine uptake sites demonstrated by [ 3H]mazindol binding was fairly uniform throughout the striatum. Moreover, different patterns of expression were seen both for the two peptides and after different treatments, again supporting the notion that distinct regulatory mechanisms exist for PPE-A and PPE-B gene expression. Functional Implications of the Alteration in Neuropeptide Gene Expression Increased PPE-A synthesis following dopamine depletion probably leads to enhanced levels of Met- and Leu-enkephalin-mediated transmission (56) in the striato-lateral pallidal pathway. We have previously shown that enkephalins can reduce GABA release in the globus pallidus (44). Since GABA transmission is enhanced in the parkinsonian lateral pallidum (52), this rise in enkephalinergic transmission might act as one of the compensatory mechanisms ensuring that parkinsonian symptoms are not apparent until a high level of dopamine-depletion is attained. Of note is that this enhanced enkephalinergic transmission is restricted to dorsal (sensorimotor) components of the basal ganglia circuitry. Surprisingly, a further rise in enkephalinergic transmission is suggested after repeated apomorphine treatment and the marked elevation of PPE-B gene expression suggests that dynorphinergic transmission may also be enhanced with this treatment. These elevations occur to differing extents in the different striatal regions, such that elevations in PPE-A expression are greatest in associative and limbic circuitry while the elevations in PPE-B are most pronounced in sensorimotor regions. This topographical organization leads one to speculate that changes in transmission by peptides derived from PPE-B (dynorphins, neoendorphins, and Leu-enkephalin: (14)), might be involved in the generation of the motor side-effects of long-term dopamine agonist treatment, e.g., dyskinesia, while the products of PPE-A (Met- and Leu-enkephalin: (56)) might contribute to nonmotor side-effects of treatment. In keeping with this idea are our recent findings that the temporal characteristics of the rise in PPE-B, but not PPE-A, parallel those of the enhanced behavioral response seen after repeated dopamine replacement therapy in the 6-OHDA-lesioned rat (19). The potential importance of enhanced opioid transmission in the generation of this behavioral supersensitivity is supported by the ability of the opioid antagonist naloxone to inhibit the locomotor supersensitivity in L-DOPA-treated 6-OHDA-

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lesioned rats (12). In support of a similar role for the opioids in the generation of dyskinesias following dopamine replacement are early clinical studies, suggesting that naloxone can alleviate levodopa-induced dyskinesias (59). However, injection of a single low dose of naloxone failed to modify dyskinetic symptoms in the MPTP-treated primate (25). We have previously proposed a mechanism by which enhanced opioid transmission might lead to the generation of an hyperkinetic movement disorder (19, 31). Enhanced dynorphin and enkephalin transmission may reduce GABA and glutamate release in the pallidal complex and substantia nigra and may ultimately lead to underactivity of basal ganglia outputs from SNr and medial pallidum. Such a change in activity is associated with the generation of dyskinesias in humans and subhuman primates (e.g., 47, 49, 65). In conclusion, mounting evidence suggests a link between opioid peptides and dyskinesias resulting from long-term dopamine replacement therapy in parkinsonism. Further studies are needed to clarify the nature of this interaction and to assess the benefits of manipulating opioid transmission as a means of limiting dyskinetic side-effects of current antiparkinsonian therapies. ACKNOWLEDGMENTS The authors thank the Medical Research Council, Dystonia Medical Research Foundation, and the Wellcome Foundation for their generous funding and Steven McGuire and Janet Robinson for valuable technical assistance.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

Alexander, G. E., M. D. Crutcher, and M. R. DeLong. 1990. Basal ganglia-thalamocortical circuits: Parallel substrates for motor, oculomotor, ‘prefrontal’ and ‘limbic’ functions. Prog. Brain Res. 85: 119–146. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic logical alignment search tool. J. Mol. Biol. 215: 403–410. Angulo, J., L. Davis, B. Burkhart, and G. Christoph. 1986. Reduction of striatal neurotransmission elevates striatal proenkephalin mRNA. Eur. J. Pharmacol. 130: 341–243. Angulo, J. A. 1992. Involvement of dopamine D1 and D2 receptors in the regulation of preproenkephalin mRNA abundance in the striatum and accumbens of the rat brain. J. Neurochem. 58: 1104–1109. Arcari, P, R. Martinelli, and F. Salvatore. 1984. The complete sequence of a full length cDNA for human liver glyeraldehyde-3phosphate dehydrogenase: Evidence for multiple mRNA species. Nucleic Acids Res. 12(23): 9179–9189. Be´dard, P. J., T. Di Paolo, P. Falardeau, and R. Boucher. 1986. Chronic treatment with L-DOPA, but not with bromocriptine induces dyskinesia in MPTP-parkinsonian monkeys. Correlation with [ 3H]spiperone binding. Brain Res. 379: 294–299. Berendse, H. W., Y. Galis-de Graaf, and H. J. Groenewegen. 1992. Topographical organisation and relationship with ventral striatal compartments of prefrontal corticostriatal projections in the rat. J. Comp. Neurol. 316: 314–347.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Bevan, P. 1983. Repeated apomorphine treatment causes behavioural supersensitivity and dopamine D2 receptor hyposensitivity. Neurosci. Letts. 35: 185–189. Buonamici, M., C. Caccia, M. Carpentieri, L. Pegrassi, A. C. Rossi, and G. DiChiara. 1986. D-1 receptor supersensitivity in the rat striatum after unilateral 6-hydroxydopamine lesions. Eur. J. Pharmacol. 126: 347–348. Campbell, K., and A. Bjo¨rklund. 1994. Prefrontal corticostriatal afferents maintain increased enkephalin gene expression in the dopamine-denervated rat striatum. Eur. J. Neurosci. 6: 1371– 1383. Carey, R. J. 1991a. Chronic L-DOPA treatment in the unilateral 6-OHDA rat: Evidence for behavioural sensitization and biochemical tolerance. Brain Res. 568: 205–214. Carey, R. J. 1991b. Naloxone reverses L-DOPA induced overstimulation effects in a Parkinson’s disease animal model analogue. Life Sci. 48: 1303–1308. Carroll, C. B., V. Holloway, J. M. Brotchie, and I. J. Mitchell. 1995. Neurochemical and behavioural investigations of the NMDA receptor-associated glycine site in the rat striatum: Functional implications for the treatment of parkinsonian symptoms. Psychopharmacology 119: 55–65. Civelli, O., J. Douglass, A. Goldstein, and E. Herbert. 1985. Sequence and expression of the rat prodynorphin gene. Proc. Natl. Acad. Sci. USA 82: 4291–4295. Crossman, A. R. 1990. An hypothesis on the pathophysiological mechanisms that underlie levodopa- or dopamine agonistinduced dyskinesia in Parkinson’s disease: implications for future strategies in treatment. Movement Disorders 5: 100–108. DeLong, M. R. and A. P. Georgopoulos. 1981. Motor functions of the basal ganglia. In Handbook of Physiology, Sect. 1. The Nervous System, Vol. 2, Motor Control, Part 2 (J. M. Brookhart, V. B. Mountcastle, V. B. Brooks, and S. R. Geiger, Eds.), pp. 1017–1061. Am. Phys. Soc., Bethesda, MD. Del Fiacco, M., G. Paxinos, and M. C. Levanti. 1982. Neostriatal enkephalin immunoreactive neurons project to the globus pallidus. Brain Res. 231: 1–17. Duty, S., B. Henry, A. R. Crossman, and J. M. Brotchie. 1995. Speculations on the neural mechanisms underlying the dyskinetic side-effects of dopamine replacement therapy in parkinsonism. Brain Res. Assoc. Abstr. 12: 41.2 Duty, S., and J. M. Brotchie. 1997. Enhancement of the behavioral response to apomorphine administration following repeated treatment in the 6-hydroxydopamine-lesioned rat is temporally correlated with a rise in striatal preproenkephalin-B, but not preproenkephalin-A, gene expression. Exp. Neurol. 144: 423–432. Engber, T. M., Z. Susel, J-L. Juncos, and T. N. Chase. 1989. Continuous and intermittent levodopa differentially affect rotation induced by D-1 and D-2 dopamine agonists. Eur. J. Pharmacol. 168: 291–298. Engber, T. M., Z. Susel, S. Kuo, C. R. Gerfen, and T. N. Chase. 1991. Levodopa replacement therapy alters enzyme activities in striatum and neuropeptide content in striatal output regions of 6-hydroxydopamine lesioned rats. Brain Res. 552: 113–118. Gagnon, C., P. J. Be´dard, L. Rioux, D. Gaudin, M. G. Martinoli, G. Pelletier, and T. Di Paolo. 1991. Regional changes of striatal dopamine receptors following denervation by 6-hydroxydopamine and fetal mesencephalic grafts in the rat. Brain Res. 558: 251–263. Gerfen, C. G., and W. S. Young III. 1988. Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments: An in situ hybridisation histochemistry and fluorescent retrograde tracing study. Brain Res., 460: 161–167.

TOPOGRAPHICAL OPIOID PEPTIDE PRECURSOR GENE EXPRESSION 24.

25.

26.

27.

28. 29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

Gerfen, C. R., J. F. McGinty, and W. S. Young III. 1991. Dopamine differentially regulates dynorphin, substance P and enkephalin expression in striatal neurons: In situ hybridisation histochemical analysis. J. Neurosci. 11: 1016–1031. Gomez-Mancilla, B., and P. J. Be´dard. 1993. Effect of nondopaminergic drugs on L-DOPA-induced dyskinesias in MPTPtreated monkeys. Clin. Neuropharmacol. 16: 418–427. Goldman, P. S., and W. J. H. Nauta. 1977. An intricately patterned prefronto-caudate projection in the rhesus monkey. J. Comp. Neurol. 171: 369–386. Graham, W. C., A. R. Crossman, and G. N. Woodruff. 1990. Autoradiographic studies in animal models of hemi-parkinsonism reveal D2 but not D1 receptor supersensitivity. I. 6-OHDA lesions of ascending mesencephalic dopaminergic pathway in the rat. Brain Res. 514: 93–102. Graybiel, A. M. 1990. Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci. 13: 244–254. Greenamyre, J. T. 1993. Glutamate-dopamine interactions in the basal ganglia: Relationship to Parkinson’s disease. J. Neurotransm. 91: 255–269. Haber, S. N., E. Lynd, C. Klein, and H. J. Groenewegen. 1990. Topographical organisation of the ventral striatal efferent projections in the rhesus monkey: an anterograde tracing study. J. Comp. Neurol. 293: 282–288. Henry, B., and J. M. Brotchie. 1996. Potential of Opioid antagonists in the treatment of levodopa-induced dyskinesias in Parkinson’s disease. Drugs Aging 9: 1–10. Herrero, M-T., S. J. Augood, E. C. Hirsch, F. Javoy-Agid, M. R. Luquin, Y. Agid, J. A. Obeso, and P. C. Emson. 1995. Effects of L-Dopa on preproenkephalin and preprotachykinin gene expression in the MPTP-treated monkey striatum. Neuroscience 86: 1189–1198. Herve´, D., F. Trovero, G. Blanc, A. M. Thierry, J. Glowinski, and J-P. Tassin. 1992. Autoradiographic identification of D1 dopamine receptors labelled with [ 3H] dopamine: Distribution, regulation and relationship to coupling. Neuroscience 46: 687–700. Hossain, M. H., and N. Weiner. 1993. Dopaminergic functional supersensitivity: effects of chronic L-Dopa and carbidopa treatment in an animal model of Parkinson’s disease. J. Pharmacol. Exp. Ther. 267: 1105–1111. Howells, R. D., D. L. Kilpatrick, R. Bhatt, J. J. Monahan, M. Poonian, and S. Udenfriend. 1984. Molecular cloning and sequence determination of rat preproenkephalin cDNA: Sensitive probes for studying transcriptional changes in rat tissues. Proc. Natl. Acad. Sci. USA 81: 7651–7655. Jiang, H. K., J. F. McGinty, and J. S. Hong. 1990. Differential modulation of strionigral dynorphin and enkephalin by dopamine receptor subtypes. Brain Res. 507: 57–64. Jolkkonen, J., P. Jenner, and C. D. Marsden. 1995. L-DOPA reverses altered gene expression of substance P but not enkephalin in the caudate-putamen of common marmosets treated with MPTP. Brain Res. Mol. Brain Res. 32: 297–307. Juncos, J. L., T. M. Engber, R. Raisman, Z. Susel, F. Thibaut, A. Ploska, Y. Agid, and T. N. Chase. 1989. Continuous and intermittent levodopa differentially affect basal ganglia function. Ann. Neurol. 25: 473–478. Kelley, A. E., and V. B. Domesick. 1982. The distribution of the projection from the hippocampal formation to the nucleus accumbens in the rat: an anterograde and retrograde horseradish peroxidase study. Neuroscience 7: 2321–2325. Kowlaski, C., and P. Giraud. 1993. Dopamine decreases striatal enkephalin turnover and proenkephalin messenger RNA abundance via D2 receptor activation in primary cell cultures. Neuroscience 53: 665–672. Kunzle, H. 1975. Bilateral projections from the precentral motor

42.

43.

44.

45. 46.

47. 48.

49.

50.

51.

52.

53.

54.

55. 56.

57.

58.

59.

233

cortex to the putamen and other parts of the basal ganglia. An autoradiographic study in Macaca fascicularis. Brain Res. 88: 195–209. Kunzle, H. 1977. Projections from the primary somatosensory cortex to the basal ganglia and thalamus in the monkey. Exp. Brain Res. 30: 481–492. Li, S. J., H. K. Jiang, M. S. Stachowiak, P. M. Hudson, V. Owyang, K. Nanry, H. A. Tilson, and J. S. Hong. 1990. Influence of nigrostriatal dopaminergic tone on the biosynthesis of dynorphin and enkephalin in rat striatum. Mol. Brain Res. 8: 219–225. Maneuf, Y. P., I. J. Mitchell, A. R. Crossman, and J. M. Brotchie. 1994. On the role of enkephalin in the GABAergic striatal efferents to the globus pallidus. Exp. Neurol. 125: 65–71. Marsden, C. D., and J. D. Parkes. 1977. Success and problems of long term therapy in Parkinson’s disease. Lancet I: 345–349. Marshall, J. F., R. Navarrete, and J. N. Joyce. 1989. Decreased striatal D1 binding density following mesotelencephalic 6hydroxydopamine injections: an autoradiographic analysis. Brain Res. 493: 247–257. Martin, J. P. 1927. Hemichorea resulting from a local lesion of the brain (syndrome of body of Luys). Brain 50: 637–651. Mishra, R. M., A. M. Marshall, and S. L. Varmuza. 1980. Supersensitivity in rat caudate nucleus: effects of 6-hydroxydopamine on the time course of dopamine receptor and cyclic AMP changes. Brain Res. 200: 47–57. Mitchell, I. J., S. Joyce, M. A. Sambrook, and A. R. Crossman. 1992. A 2-deoxyglucose study of the effects of dopamine agonists on the parkinsonian primate brain: implications for the neural mechanisms that mediate dopamine agonist-induced dyskinesia. Brain 115: 809–824. Morris, B. J., V. Ho¨llt, and A. Herz. 1988. Dopaminergic regulation of striatal proenkephalin mRNA and prodynorphin mRNA: Contrasting effects of D1 and D2 antagonists. Neuroscience 25: 525–532. Normand, E., T. Popovici, B. Onteniente, D. Fellmann, D. Piatier-Tonneau, C. Auffray, and B. Bloch. 1988. Dopaminergic neurons of the substantia nigra modulate preproenkephalin A gene expression in rat striatal neurons. Brain Res. 439: 39–46. Pan, H. S., J. B. Penney, and A. B. Young. 1985. GABA and benzodiazepine receptor changes induced by unilateral 6hydroxydopamine lesions of the medial forebrain bundle. J. Neurosci. 45: 1396–1404. Papa, S. M., T. M. Engber, A. M. Kask, and T. N. Chase. 1994. Motor fluctuations in levodopa treated parkinsonian rats: Relation to lesion extent and treatment duration. Brain Res. 662: 69–74. Parent, A., and L-N. Hazrati. 1995. Functional anatomy of the basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res. Rev. 20: 91–127. Paxinos, G., and C. Watson. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, UK. Rosen, H., J. Douglass, and E. Herbert. 1984. Isolation and characterisation of the rat proenkephalin gene. J. Biol. Chem. 259: 14309–14313. Somers, D. L., and R. M. Beckstead. 1992. N-Methyl-DAspartate receptor antagonism alters substance P and met5enkephalin biosynthesis in neurons of the rat striatum. J. Pharmacol. Exp. Ther. 262: 823–833. Thomas, K. L., S. Rose, P. Jenner, and C. D. Marsden. 1992. Dissociation of the striatal D-2 dopamine receptor from adenylyl cyclase following 6-hydroxydopamine-induced denervation. Biochem. Pharmacol. 44: 73–82. Trabucchi, M., S. Bassi, and L. Frattola. 1982. Effect of naloxone

234

60.

61. 62.

63.

64.

DUTY ET AL. on the ‘On-Off’ syndrome in patients receiving long-term levodopa therapy. Arch. Neurol. 39: 120–121. Uhl, G. R., B. Navia, and J. Douglas. 1988. Differential expression of preproenkephalin and preprodynorphin mRNAs in striatal neurons: High levels of preproenkephalin expression depend on cortical afferents. J. Neurosci. 8: 4755–4764. Ungerstedt, U., A. E. Avema, T. Ljundberg, and C. Runge. 1973. Animal models of parkinsonism Adv. Neurol. 3: 257–271. Vincent, S. R., T. Ho¨kfelt, I. Christensson, and L. Terenius. 1982. Immunohistochemical evidence for a dynorphin immunoreactive strionigral pathway. Eur. J. Pharmacol. 85: 251–252. Voorn, P., G. Poest, and H. J. Groenwegen. 1987. Increase of enkephalin and decrease of substance P immunoreactivity in the dorsal and ventral striatum of the rat after midbrain 6-hydroxydopamine lesions. Brain Res. 412: 391–396. Voorn, P., G. J. Docter, A. L. Jongen-Reˆlo A. L., and A. J. Jonker. 1994. Rostrocaudal sub-regional differences in the response of

65.

66.

67.

68.

enkephalin, dynorphin and substance P synthesis in rat nucleus accumbens to dopamine depletion. Eur. J. Neurosci. 6: 486–496. Whittier, J. R., and F. A. Mettler. 1949. Studies on the subthalamus of rhesus monkey. II. Hyperkinesia and other physiological effects of subthalamic lesions, with special reference to the subthalamic nucleus of Luys. J. Comp. Neurol. 90: 319–372. Xie, C.-W., P. H. K. Lee, J. Douglass, B. Crain, and J. S. Hong. 1989. Deep prepyriform cortex kindling differentially alters the levels of prodynorphin mRNA in rat hippocampus and striatum. Brain Res. 495: 156–160. Young III, W. S., T. I. Bonner, and M. R. Brann. 1986. Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83: 9827–9831. Zahm, D. S., and J. S. Brog. 1992. On the significance of subterritories in the ‘accumbens’ part of the ventral striatum. Neuroscience 50: 751–767.