Molecular Brain Research, 12 (1992) 111-119 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$03.50

111

BRESM 70359

Differential regulation of striatal preproenkephalin mRNA by and D 2 dopamine receptors

D 1

Alexia E. Pollack* and G. Frederick Wooten Departments of Neuroscience and Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.)

(Accepted 16 July 1991) Key words: Dopar ~ae receptor; Dopamine D 2 receptor; Dopamine D~ receptor; Preproenkephalin mRNA; mRNA regulation

The effect of administration of subtype selective dopamine (DA) agonists on the 6-hydroxydopamine (6-OHDA) lesion-induced increase of stdatal preproenkephalin (PPE) mRNA was examined by dot-blot hybridization. Eight days following a unilateral 6-OHDA lesion of the substantia nigra pars compacta (SNc), PPE mRNA levels in the ipsilateral striatum were increased approximately two-fold. Administration oi the D 2 DA agonist, quinpirole, dose-dependently attenuated the 6-OHDA lesion-induced increase in striatal PPE mRNA. The effect of quinpirole was blocked by coadministration of the D2 DA antagonist eticlopride. In contrast, administration of the DI DA agonist, SKI: 38393, either dose-dependently augmented or had no effect on the 6-OHDA lesion-induced increase in striatal PPE mRNA. In the contralateral striatum, administration of quinpirole decreased PPE mRNA, while administration of SKF 38393 increased PPE mRNA compared to sham lesioned control levels. These data suggest the action of DA at D t and D2 DA receptors differentially regulates striatal PPE mRNA levels and the apparent inhibition of ENK biosynthesis by DA is mediated via an interaction with D 2 DA receptors. INTRODUCTION The role of dopamine ( D A ) as a trans-synaptic regulator of gene expression has been described for several neuropeptides in the striatum. For example, D A regulated striatal precursor m R N A levels for enkephalin (ENK) 31'37, substance P (SP) 2'37, dynorphin 16'Is, neurotensin 3s, and somatostatin 2s'2s. Following destruction of the nigral D A neurons by a unilateral injection of the neurotoxin 6-hydroxydopamine ( 6 - O H D A ) there was a two-fold increase in striatal P P E m R N A t'37. In addition, chronic (2-3 week) treatment with either the relatively selective D 2 D A antagonist, haloperidol ( H A L ) 31, or the D t D A antagonist, SCH 2339017'2t increased striatal P P E m R N A . Taken together these data suggest that D A tonically inhibits striatal E N K biosynthesis. However, treatment with the D~ D A antagonist, SCH 23390, for only 7 days decreased striatal P P E m R N A ts'21 suggesting that regulation of striatal PPE m R N A by D t D A receptor mechanisms was complex and likely indirect. Using the selective D 1 D A agonist, SKF 3839326, and selective D 2 DA agonist, quinpirole 33, we sought to determine the contribution of D A at each D A receptor subtype to the regulation of striatal P P E m R N A levels. Experiments were designed to assess whether treatment with subtype

selective D A agonists following a unilateral stereotaxic injec~:.on of 6 - O H D A into the left substantia nigra (SN) could attenuate the lesion.induced increase in striatal PPE m R N A . MATERIALS AND METHODS Adult, male Sprague-Dawley rats (Dominion) weighing 225-275 g at the beginning of all experiments were used. Quinpirole (Research Biochemical Inc.) was dissolved in sterile distilled water, and SKI: 38393 (Research Biochemical Inc.) was dissolved in 1% lactic acid. 6-OHDA lesion: time course Rats were anesthetized with ketamine (100 mg/kg)/xylazine (20 mg/kg) and pretreated with desmethylimipramine (DMI) (25 rag/ kg). Rats received a unilateral stereotaxic injection of 6-OHDA (2 /~1, 6-OHDA 4 wg/ml, citric acid 1 mg/mi), coordinates: Bregma -5.0 AP, 4-1.4 ML, -8.0 DV into the left SN. The animals were sacrificed either 2, 4, 8, 14, or 30 days post lesion. RNA was extracted from each striatum separately (left: lesion-sidc, right: control-side, n - 4-9). Drug treated 6-OHDA lesion Rats received a unilateral 6-OHDA lesion as described above. The day following the 6-OHDA injection, rats received a 7 day course of either quinpirole (0.001, 0.01, 0.1, and 1.0 mg/kg/2 × day, s.c.) or SKI: 38393 (0.1, 1.0, 10.0, and 25.0 mg/kg/2 × day, s.c.) (n = 6-10). These animals were sacrificed 8 days post lesion. In a second protocol, the animals received a unilateral 5-OHDA lesion as described above, and beginning on the 7th day post le-

Current address: Laboratory of Molecular Neurobiology, Massachusetts General Hospital East, Building 149, 13th Street, Charlestown, MA 02129, U.S.A. Correspondence: G.F. Wooten, Department of Neurology, Box 394, University of Virginia Health Sciences Center, Charlottesville, VA 229O8, U.S.A. *

112 sion the animals received a 7 day course of either quinpirole (0.1 mg/kg/2 x day, s.c.) or SKF 38393 (10 mg/kg/2 x day). These animals were sacrificed at 14 days post lesion. In all experiments, animals were sacrificed by decapitation 18-24 h after the last injection of drug, and striatal mRNA levels were analyzed separately (!eft: lesion side, right: control side).

Measurement of striatal dopamine levels Nine animals were lesioned unilaterally with 6-OHDA as described above. Eight days post lesion the animals were sacrificed by decapitation, and their striata dissected out separately and stored at -70 °C. Striatal catecholamine levels were measured by HPLC based on the procedure outlined by Che:;z and Wooten s. Each striatum was sonicated at 20 x tissue weight in 0.2 M HCIO4, NaS20 s, centrifuged at 27,000 g at 4 °C. Samples of each supernatant were run through a reverse phase HPLC with electrochemical detection and corrected for recovery by internal standards to determine striatal DA levels.

Histology of 6-OHDA lesions For all 6-OHDA lesion experiments 20 ~m frozen sections through the SNc were stained with Toluidine blue to assess the extent of the destruction of the SN neurons. Only animals which demonstrated a successful lesion by histology were used for RNA analysis.

RNA blots Rats in each treatment group were sacrificed by decapitation. The left and right rostral striata were dissected and individually homogenized in guanidine isothiocyanate solution. Total RNA was extracted by the method of Chirgwin et al. 6. The dot-blot procedure essentially foil/~wed the method described by Romano et al.2~ Serial dilutions of each RNA sample were made from 1-0.5/~g in 10 x SSC/7.4% formaldehyde (SSC/form) (20 x SSC: 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0). The s,,~lales were heated to 65 °C for 10 rain to denature the RNA and then olaced on ice. RNA samples were pipetted onto a Zetabind (AMId:;~ ~'UNO) membrane that had been presoaked in SSC/form, with the me~:~brahe clamped in a dot-blot manifold (Biorad). A gentle vacuum was applied and each well was washed with SSC/form. The membrane was baked in a vacuum oven at 80 °C for 2 h, exposed to UV light (254 nM) for 1 min, and stored in a plastic bag prior to hybridization.

Northern blot RNA gel e!ectrophoresis was performed by the method of Thomas3z. Five ~g of total RNA from rat whole brain, liver and striatum was denatured by heating to 65 *C for 10 min in 2.2 M formaldehyde/50% formamide and electrophoresed on a 1.2% agarose/ 2.2 M formaldehyde gel. After electrophoresis, the RNA was transferred to Zetabind membrane by capillary action overnight. The membrane was baked in a vacuum oven, exposed to UV light ~254 nM) for 1 rain, and stored in a plastic bag until hybridization.

Oligonucleotide probes Two synthetic oligonucleotide probes complementary to PPE mRNA were used in all hybridizations [30mer complementary to bases 7-36 ~ and 48mer complementary to bases 388-43537]. In addition, a synthetic oligo(dT) 30mer was used to 'normalize' the dot-blots following hybridization with PPE probes in order to adjust for RNA sample loading errors. All probes were 5" end-labeled using [a2P]adenosine 5" triphosphate (NEN: 6000 Ci/mmol).and T4 kinase (BRL). To 150 pmol of isotope were added 50 pmol of oligonucleotide, 2 ~! of 10x kinase buffer (0.5 M T~s-HCI, pH 7.6, 0.1 M magnesium chloride, 0.1 M dithiothreitol, 1 mM spermidine, 1 mM EDTA), sterile water, and 20 units of T4 kinase to a total volume of 20 gl and incubated for 90 min at 37 *C. Radioactively labeled oligonucleotide probe was separated from unincorporated [32p]dATP using a NENsorb 20 nucleic acid purification cartridge (NEN; Dupont) according to manufacturer's directions. Radiola-

beled probe was eluted with 50% methanol, and added directly to the hybridization solution. Specific activities were routinely in the range of 106 to l0 T dpm/pmol.

Hybridization Prehybridization and hybridization steps were carried out essentially according to Church and Gilbert ~. For prehybridization, 20 ml of 0.1 x SSPE (20x SSPE: 3 M sodium chloride, 0.02 M EDTA, pH 7.4) containing 1.0% SDS were added to the plastic bag containing the blot, and the blot was incubated for 1 h at 65 °C. The blot was transferred to a new bag and Church hybridization buffer (1% BSA, 0.25 M monobasic sodium phosphate, pH 7.2, 1 mM EDTA, 7% SDS, 100 /~g/ml sheared, denatured herring sperm DNA; 5 ml/100 cm2 blot) was added. After a 5 min incubation at 65 °C, 32P-labeled oligonucleotide probe was added to the bag and the blot was incubated overnight at 62 °C (PPE 48mer), 57 °(2 (PPE 30mer), or 37 °C (oligo(dT) 30mer). The following day, blots were washed at 2 °C below the hybridization temperature for each probe in a solution containing 0.5% BSA, 5% SDS, 40 mM monobasic sodium phosphate, pH 7.2, and 1 mM EDTA (2 x 10 rain) followed by solution of 1% SDS, 40 mM monobasic sodium phosphate, pH 7.2, and 1 mM EDTA (4 x 10 min). The blots were dipped in 40 mM monobasic sodium phosphate, pH 7.2, to remove SDS, wrapped in saran wrap and apposed to X-ray film (Kodak X-AR) at -70 °C. The exposure time varied to insure that all images were within the linear response range of the film. To adjust for small handling errors, all blots were stripped and lehybridized with a 3zP-labeled oligo(dT) 30mer following hybridization to PPE probes. The blots were stripped by incubating them in 5 mM Tris-HCl, 0.5% sodium pyrophosphate, 0.2 mM EDTA, 0.1% Denhardts BSA, pH 8.0 for 90 min at 65 °C. The blots were placed immediately in Church hybridization buffer and incubated overnight with the 3zP-labeled oligo(dT) 30met ~t 37 °(2. The following day the blots were washed as described above and apposed to Kodak X-AR film.

Analysis Dot-blots of experimental groups and controls, as well as a northern blot, containing whole brain, liver and striatal RNA were hybridized together with the same batch of 3zP-labeled PPE probe. The northern blot was included to insure that all hybridizations with oligonucleotides produced the desired specific signal. Dot-blots were analyzed by densitometry of autoradiographs where each dot represented the PPE mRNA from one striata (left or right) from one rat. Densitometry was performed identically for stripped blots that were rehybridized with oligo(dT) 30mer. To normalize the blots, the optical density (O.D.) ~Jalue for each dot hybridized with a PPE oligonucleotide was divided by the O.D. of the corresponding dot hybridized with oligo(dT). The means of normalized O.D. values were calculated for all groups. For the 6-OHDA time course experiment: PPE mRNA from the striatum ipsilateral to the 6-OHDA lesion was expressed as a percent of the respective control (non-lesioned) striatum. Comparisons were made using a matched pair t-test with P < 0.05 considered significant. For the drug treated experiments: the left striatum (ipsilateral to the lesion) from both the lesion control (no drug) as well as the drug-treated lesion animals were expressed as a percentage of the left striatum sham lesion control animals. In addition, the right striatum (contralateral to lesion) from both the lesion control (no drug) and the drug-treated lesion animals were expressed as a percentage of the right striatum of sham lesion control animals. Comparisons were made separately for left and right striata using an ANOVA followed by Duncan's multiple range test with P < 0.05 considered significant. In addition, the differences in striatal PPE mRNA levels between left (ipsilateral) and right (contralateral) striata from sham lesion and untreated lesioned animals were compared to the differences between PPE mRNA levels in left and right striata of drug treated

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Hg. 1. Effect of 6-OHDA lesion on striatal preproenkephalin mRNA levels. A: Northern blot of total striatal RNA (5 gg) at 8 days post 6-OHDA lesion. Lane A: striatum ipsilateral to lesion. Lane B: striatum contralateral to lesion. B: Dot-blot of total striatal RNA (1 and 0.5/~g) at 8 days post 6-OHDA lesion (n - 3). L, striatum ipsilaterai to lesion; R, striatum contralateral to lesion.

Fig. 2. Time course of 6-OHDA lesion effect on striatai preproenkephalin mRNA levels. The substantia nigra of rats was unilaterally lesioned with 6-OHDA as described in Materials and Methods. Animals were sacrificed either 2, 4, 8, 14 or 30 days post lesion (n = 4-9), and their striata processed separately for RNA extraction. PPE mRNA content was determined by densitometry of dotblot autoradiograms. PPE mRNA levels were expressed as a percentage of the control (contralateral to the lesion) striatum S.E.M. *Significantly different from control mean, P < 0.05.

tained this elevation at 14 and 30 days post lesion. lesioned animals by an ANOVA followed by Duncan's multiple range test with P < 0.05 considered significant. RESULTS

Effects of a 6-OHDA lesion At 8 days post lesion, D A concentration in the striarum ipsilateral to a 6 - O H D A lesion decreased an average of 97% -4-- 2 (mean 4-_ S.E.M., n = 9). Toluidine blue-stained coronal sections through the substantia nigra showed that the neurons in left pars compacta were eliminated 8 days following the injection of 6 - O H D A (data not shown). In addition, 8 days post 6 - O H D A lesion there was a two fold increase in PPE m R N A in the striatum ipsilateral to the lesion which was apparent by both northern and dot-blot analyses (Fig. 1).

6-OHDA time course Four days following a 6 - O H D A lesion there was a 50% increase in PPE m R N A in the striatum ipsilateral to the lesion compared to the contralatoral striatum (Fig. 2). By 8 days post lesion PPE m R N A in the ipsilateral striatum increased 100% over control levels and main-

Drug-treated 6-OHDA lesion Eight days post lesion PPE m R N A increased 76% in the ipsilateral striatum compared to control (sham-lesioned) levels, Rats 8 days post lesion which were injected with water for 7 clays expressed striatal PPE m R N A levels in both the ipsi- an,'~ contralateral striatum which were indistinguishable from minjected 8 days post 6 - O H D A lesion animals (data not shown). Therefore, the two groups were pooled to serve as the untreated lesioned control (n = 14).

Effect of quinpirole Treatment of 6-OHDA-lesioned animals with the D2 agonist quinpirole for 7 days dose-dependently attenuated the increase in striatal PPE m R N A ipsilateral to a 6 - O H D A lesion (Fig. 3). All doses of quinpirole used reduced striatal PPE m R N A compared to untreated lesioned animals, although only doses above 0.001 mg/kg/ day attenuated the lesioned induced increase in striatal PPE m R N A to a statistically significant degree. A 7 day course of quinpirole (0.1 and 1.0 mg/kg/2 x day) brought PPE m R N A levels in the striatum ipsilateral to a 6 - O H D A lesion to sham lesioned levels. PPE m R N A

114 levels were elevated only 4 0 ~ over sham lesioned levels with 0.01 mg/kg/2 x day of quinpirole, whereas 0,001 mg/kg 2 x day of quinpirole did not attenuate the increase in PPE m R N A in the striatum ipsilateral to a 6 - O H D A lesion. For all doses of quinpirole PPE m R N A levels in the contralateral striatum decreased 16-25% compared to sham lesioned animals. The difference between ipsi- and contralateral levels of striatal PPE m R N A following administration of 0.1 mg/kg/2 x day of quinpirole was the same as the difference in striatal PPE m R N A levels in sham lesioned animals. The difference in ~triatal PPE m R N A levels following administration of 1.0 mg/kg/2 x day of quinpirole was different from the differences in striatal P P E m R N A levels of both sham lesioned and untreated lesioned animals. However, the d~?ferences in striatal P P E m R N A levels following administration of 0.001 and 0.01 mg/kg/2

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Fig. 4. Effect of chronic coadministration of eticlopride and quinpirole on striatal preproenkephalin mRNA levels in rats with a unilateral 6-OHDA lesion of the substantia mgra. Rats were unilaterally lesioned with 6-OHDA as described in Materials and Methods. Beginning the day following the injection of 6-OHDA rats began receiving a 7 day course of quinpirole (0.1 mg/kg/2 x day, s.c.), quinpirole (0.1 mg/kg/2 x day, s.c.) + eticlopride (2.5 mg/kg 2 x day, s.c.) or eticlopride (2.5 mg/kg/2 x day, s.c.) (n = 5-8). Animals were sacrificed 8 days post lesion, 18-24 h after last drug injection, and their striata processed separately for RNA extraction. PPE mRNA content was determined by densitometry of dot-blot autoradiograms. PPE mRNA levels were expressed as a percentage of control +- S.E.M. (sham lesion, n ffi 9). For 6-OHDAlesioned animals the shaded bars represent striatal PPE mRNA ipsilateral to the 6-OHDA lesion and the open bars represent PPE mRNA in the contralateral striatum. *Significantly different from sham lesion mean, P < 0.05. For comparison of the differences in PPE mRNA levels between ipsi- and contralateral striata see Results.

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Fig. 3. Effec; of chronic treatment with quinpirole on striatal preproenkephalin mRNA levels in rats with a unilateral 6-OHDA lesion of the substantia nigra. Rats were unilaterally lesioned with 6-OHDA as described in Materials and Methods. Beginning the day following the injection of 6-OHDA rats began receiving a 7 day course of quinpirole (0.001, 0.01, 0.1, and 1.0 mg/kgf2 x day, s.c.) (n = 6-9). Animals were sacrificed 8 days post lesion, 18-24 h after last drug injection, and their striata processed separately for RNA extraction. PPE mRNA content was determined by densitomerry of dot-blot autoradiograms. PPE mRNA levels were expressed as a percent of control +- S.E.M. (sham lesion, n ffi 9). For 6-OHDA-lesioned animals the shaded bars represent striatal PPE mRNA ipsilateral to the 6-OHDA lesion and the open bars represent PPE mRNA in the contralateral striatum. *Significantly different from sham lesion mean, P < 0.05. **Significantly different from both sham lesion mean and untreated lesion mean, P < 0.05. For comparison of the differences in PPE mRNA levels between ipsi- and contralateral striata see Results.

x day of quinpirole were the same as the difference in striatal PPE m R N A levels in untreated lesioned animals. The effect of qui~pirole was blocked by coadministration of the D2 D A antagonist eticlopride (Fig. 4). Following coadministration of quinpirole (0.1 mg/kg/2 x day) and eticlopride (5 mg/kg/2 x day), P P E m R N A levels in the striatum ipsilateral to a 6 - O H D A lesion increased 47%, statistically indistinguishable from untreated k'sk,ned animals. In addition, administration of eticlopride (2..5 mg/kg/2 x day) increased P P E m R N A levels in the contralateral striatum about 30% compared to the contralateral striatum of sham lesioned animals. In eticlopride treated lesioned animals, PPE m R N A levels in the ipsi- and contralateral striatum did not differ from one another. W h e n quinpirole administration was delayed 7 days following a 6 - O H D A lesion the drug attenuated, but did

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Fig. 5. Effect of delayed chronic treatment of quinpirole or SKF 38393 on striatal preproenkephalin mRNA levels in rats with a unilateral 6-OHDA lesion of the substantia nigra. Rats were unilaterally lesioned with 6-OHDA as described in Materials and Methods. Beginning on the 7th day following the injection of 6-OHDA rats began receiving a 7 day course of quinpirole (0.1

mg/kg/2 x day, s.c.) or SKF 38393 (10.0 mg/kg/2 x day, s.c.) (n = 9). Animals were sacrificed at 14 days post lesion, 18-24 h after last drug injection, and their striata processed separately for RNA extraction. PPE mRNA content was determined by densitometry of dot-blot autoradiograms. PPE mRNA levels were expressed as a percent of control -+ S.E.M. (sham lesion, n - 9). For 6-OHDAlesioned animals the shaded bars represent striatal PPE mRNA ipsilateral to the 6-OHDA lesion and the open bars represent PPE mRNA in the contralateral striatum. *Significantly different from sham lesion mean, P < 0,05. **Significantly different from both sham lesion mean and untreated 14 day p c ~ lesion mean, P < 0.05. For comparison of the differences in PPE mRNA levels between ipsi- and contralateral striata see Results.

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Fig. 6. Effect of chronic treatment with SKF 38393 on striatal preproenkephalin mRNA levels in rats with a unilateral 6-OI-DA lesion of the substantia nigra. Rats were unilaterally lesioned with 6-OHDA as described in Materials and Methods. Beginning the day following the injection of 6-OHDA rats began receiving a 7 day course of SKF 38393 (0.1, 1.0, 10.0, and 25.0 mg/kg/2 x day, s.c.) (n = 6-9). Animals were sacrificed 8 days post lesion, 18-24 h after last drug injection, and their striata processed separately for RNA extraction. PPE mRNA content was determined by densitometry of dot-blot autoradiograms. PPE mRNA levels were expressed as a percent of control +- S.E.M. (sham lesion, n = 9). For 6-OHDA-lesioned animals the shaded bars represent striatal PPE mRNA ipsilateral to the 6-OHDA lesion and the open bars represent PPE mRNA in the contralateral striatum. *Significantly different from sham lesion mean, P < 0.05. **Significantlydifferent from both sham lesion mean and untreated lesion mean, P < 0.05. For comparison of the differences in PPE mRNA levels between ipsi- and contralateral striata see Results.

Effect of SKF 38393 not entirely reverse, the lesion-induced increase in striataI P P E m R N A (Fig. 5). Administration of a 7 day course of qainpirole (0.1 mg/kg/2 x day) beginning treatment on the 7th day post 6 - O H D A lesion increased PPE m R N A 63% compared to sham lesioned levels. Therefore, administration of quinpirole reversed the increase in striatal PPE m R N A 37% compared to untreated 14 day post lesion animals. In the contralateral striatum PPE m R N A levels decreased 16% following quinpirole treatment compared to the contralateral striatum of sham-lesioned animals. In addition, the differep.ce between ipsi- and contralateral levels of striatal PPE m R N A following administration of 0.1 mg/kg/2 x day of quinpirole was different from the differences in striatal P P E m R N A levels of both sham-lesioned and untreated lesioned animals.

Treating 6-OHDA-lesioned animals with the D1 D A agonist SKF 38393 for 7 days with doses in excess of 10 mg/kg/2 x day augmented the increase in striatal PPE m R N A ipsilateral to a 6 - O H D A lesion (Fig. 6). A 7 day course of SKF 38393 increased PPE m R N A levels in the striatum ipsilaterai to a 6 - O H D A lesion by 129% (10 mg/kg/2 x day) and 115% (25 mg/kg/2 x day) compared to sham-lesioned levels. These values were significantly higher than those detected in the ipsilateral striatum of animals with a 6 - O H D A lesion alone. In addition, a 7 day course of SKF 38393 (25 mg/kg/2 x day) also increased PPE m R N A in the striatum contralateral to a 6 - O H D A by 25% compared to sham lesioned levels. SKF 38393 administered at 0.1 and 1.0 mg/kg/2 x day increased P P E m R N A in the ipsilateral striatum by 58% and 62%, respectively, compared to sham-lesioned levels. However, these values did not differ from PPE

116 mRNA levels in the ipsilateral striatum of untreated 6-OHDA lesioned animals. Following administration of 10 mg/kg/2 x day of SKF 38393 the difference between ipsi- and contralateral levels of striatal PPE mRNA was different from the differences in striatal PPE mRNA levels in both sham lesioned and untreated lesioned animals. However, the differences in striatal PPE mRNA levels with 0.1, 1.0 and 25.0 mg/kg/2 × day of SKF 38393 were the same as the difference, in striatal PPE mRNA in untreated lesioned animals. Administration of SKF 38393 did not alter the lesioninduced increase in striatal PPE rnRNA when drug administration was delayed 7 days following a 6-OHDA lesion (F,:g. 5). Administration of a 7 day course of SKF 38393 (10 mg/kg/2 x day) beginning on the 7th day post 6-OHDA lesion increased PPE mRNA 105% compared to sham lesioned levels. However, this increase in striatal PPE mRNA was indistinguishable from untreated 14 day post lesion control animals. In addition, the difference in PPE mRNA levels between the ipsi- and contralateral stfiatum following administration of 10 mg/kg/2 x day of SKF 38393 was the same as the difference in striatal PPE mR1~IA levels in untreated 14 day post lesion animals. DISCUSSION To optimize the accuracy and consistency of the data, a series of steps were taken to analyze RNA levels following each experimental perturbation. First, all experimental and control blots were hybridized together with the same batch of freshly a2p-labeled oligonucleotide probe to insure that all blots were exposed to identical hybridization conditions. In addition, a northern blot was included with every hybridization to insure that the radioactive 'signal' was specific and labeled only a single band (Fig. 1A). Consequently, dot-blots were only analyzed when the corresponding northern blot gave the desired specific signal. Lastly, all blots were 'normalized' following hybridizations to PPE probes by stripping and rehybridizing with oligo(dT). Since RNA quantitation by blot hybridization was relative to experimental controls and not based on absolute values, it was important to normalize the blots in order to adjust for RNA extraction or handling errors. An oligo(dT) probe, which presumably binds to the poly(A) tail of mRNA, was used to normalize all the blots since there was no difference in poly(A +) RNA levels between 6-OHDA-lesioned or drug-treated animals and sham-lesioned control animals. Following a unilateral 6-OHDA lesion there was a time-dependent two-fold increase in PPE mRNA in the ipsilateral striatum which reached a plateau at 8 days

post lesion (Fig. 2). These changes in PPE mRNA occurred when DA levels in the ipsilateral striatum were 97% depleted and the neurons in the SNc were virtually eliminated. These data were consistent with the direction and magnitude of the change in striatal PPE mRNA levels following a 6-OHDA lesion described by others 1'2~' 37. The lesion-induced increase in PPE mRNA levels was likely due to an increase in PPE gene expression, but could also be due ~o an increase in RNA stability, both of which could reflect an increase in ENK biosynthesis. However, our analysis could not distinguish between these mechanisms. Lastly, analysis by blot hybridization, unlike in situ hybridization, was unable to detect regional changes in striatal PPE mRNA since the entire rostral striatum was homogenized as a whole to extract RNA. However, blot-hybridization analysis should adequately reflect changes in striatal PPE mRNA following a unilateral 6-OHDA lesion since in situ hybridization studies suggest that the lesion-induced increase in PPE mRNA appeared uniformly throughout the striatum 37. Administration of the D 2 DA agonist quinpirole for 7 days attenuated the 6-OHDA lesion-induced increase in striatal PPE mRNA (Fig. 3). The effect of quinpirole was dose-dependent and could be blocked by coadministration of the selective D 2 DA antagonist, eticlopride (Fig. 4) confirming the selectivity of the action of quinpirole on D2 DA receptors. When administration of quinpirole was delayed and begun on the 7th day post lesion, quinpirole attenuated, but did not entirely reverse, the 6-OHDA lesion-induced increase in striatal PPE mRNA (Fig. 5). Recently a D3 DA receptor has been cloned and sequenced 29. Since. quinpirole displays 30- to 100-fold higher affinity for the D3 DA receptor compared to the D 2 DA receptor 29, it is possible that some of the effects of quinpirole on striatal PPE mRNA regulation could be due in part to activation of the D 3 DA receptor. However, since the D 3 DA receptor is expressed at much lower levels in the striatum compared to the D2 DA receptor 29 it is likely that the primary effect of quinpirole in our studies is mediated by occupation of the D 2 DA receptor. It is possible that the inability of quinpirole to fully reverse the lesion-induced increase in striatal PPE mRNA in the delayed administration paradigm is due to a decrease in PPE mRNA turnover as a result of DA deafferentation. We cannot exclude this possibility since the turnover rate for striatal PPE mRNA following a 6-OHDA lesion is not known. The turnover of other eukaryotic mRNA molecules is on the order of minutes to days 4'24, suggesting that if the turnover of PPE mRNA following a 6-OHDA lesion is on the order of days this might affect the ability of quinpirole to reverse the le-

117 sion-induced increase in striatal PPE mRNA. Using a delayed administration paradigm, Gerfen et al. 1~ showed that continuous administration of quinpirole by osmotic minipump (1 mg/kg~day) could fully reverse the increase in PPE mRNA following a 6-OHDA lesion while a once daily injection of quinpirole (1 rag/ kg/day) had no effect. Thus, our data with a twice daily injection of quinpirole could represent a point in the time-action curve between continuous administration by osmotic minipump and once daily injection whereby more frequent dosing increased the ability of quinpirole to bring PPE mRNA to control levels. In the contralateral striatum, adminis~*xation of quinpirole twice daily for 7 days reduced striatal PPE mRNA compared to control levels, suggesting that the action of DA at D 2 DA receptors inhibited ENK biosynthesis in the str~atum. However, chronic constant D2 stimulation must be necessary for this inhibition since 3 h following acute administration of qw.'npirole (1 mg/kg) striatal PPE mRNA levels increased3. On the other hand, our finding of a quinpirole-induced decrease in striatal PPE mRNA levels could be a consequence of our dosing paradigm, since administration of the same dose of quinpirole by osmotic minipump or once daily did not alter PPE mRNA in striata w'~h intact dopamin.:.rgic afferents n. Administration of SKF 38393 dose-dependently augmented the increase in striatal PPE mRNA following a 6-OHDA lesion (Fig. 6). However, there was no effect on the lesion-induced increase in striatal PPE mRNA when treatment of SKF 38393 was delayed and administration begun on the 7th day post lesion (Fig. 5). These data were consistent with those of Gerfen et aI.H who found that delayed administration of SKF 38393 did not alter striatal PPE mRNA levels following a 6-OHDA lesion. In contrast, the findings of Jiang et al. ~4 that SKF 38393, but not quinpirole, reversed the lesioned-induced increase in striatal ME peptide levels were not supported by our data nor by the evidence of Gerfen et al. ~. Administration of a 7 day course of SKF 38393 increased PPE mRNA in the contralateral striatum compared to control levels. These data were consistent with a decrease in striatal PPE mRNA levels following chronic blockade of D~ DA receptors with SCH 23390 ~s'2~. Taken together these data suggested that the action of DA at Ds DA receptors increased ENK biosynthesis. Administration of quinpirole attenuated the 6-OHDA lesion-induced increase in striatal PPE mRNA, whereas, administration of SKF 38393 either augmented or had no effect on the lesion-induced increase in striatal PPE mRNA. These data suggest that striatal ENK biosynthesis is differentially regulated by D 1 and D2 DA receptor effects. Anatomical evidence suggests that D2 receptor mRNA a:ld PPE mRNA are coexpressed in the same

population of striatal medium size spiny neurons i5 whereas D~ DA receptors are selectively expressed by stliatonigral neurons nn3. These data are consistent with a D 2 DA receptor effect on striatal PPE mRNA mediated directly through D 2 DA receptors on striatal ENK neurons. In addition, the 6-OHDA lesion-induced increase in both striatal PPE mRNA and D2 DA receptor mRNA is attenuated by chronic administration of quinpirole,, but not SKF 38393n, supporting a functional colocalization of striatal PPE mRNA and D2 DA receptor mRNA. In c~ntrast, since D~ DA receptors are localized primarily to striatonigral neurons ~a3, it is likely that the effect of SKF 38393 on striatal PPE mRNA levels is not mediated through direct DI DA effects on striatopallidal ENK neurons. The tonic release of DA in the striatum appears to inhibit striatopallidal neurons. Removal of the nigrostriatal DA neurons following a 6-OHDA lesion increased 2-deoxyglucose (2-DG) uptake in the ipsilateral external segment of globus pallidus (GPe) 36. This metabolic marker is thought to reflect increased activity in striatopallidal terminals which can underlie increased transmitter utilization requiring compensatory transmitter biosynthesis. Since both ENK 9'12 and 7-aminobutyric acid (GABA) m neurons project to GPe, removal of the DA input apparently increased the activity of these striatopallidal GABA/ENK neurons. For example, pallidal activity decreased following a 6-OHDA lesion2°, suggesting that when the DA input was removed striatopallidal G A B A neurons were 'disinlfibited' which in turn inhibited pallidal neurons. Consistent with these data, striatal application of a D2 DA agonist inhibited, whereas a DI agonist enhanced the release of GABA in the striatum22. Thus, D1 and D2 DA receptor activation differentially regulated the release of stfiatal GABA. However, only striatal application of a D2 DA agonist reversed the GABA-induced increase in striatal GABA release22, suggesting that D2 DA receptor activation inhibked striatopallidal neurons. Lastly, both striatal PPE mRNA ~' 2~.3,t,37 and glutamic acid decarboxylase (GAD) mRNA (~he synthetic enzyme for GABA) 34 increased in a timedependent fashion following a 6-OHDA lesion, suggesting that there was a compensatory increase in transmitter biosynthesis following a 6-OHDA lesion. Taken together, these data provide further functional evidence for a direct, proximal, selective regulation of PPE mRNA, specifically, and striatopallidal neurons, generally, by D 2 DA receptors. Since D2 DA receptor mRNA is expressed in striatal ENK neurons ~5, it is likely that endogenous DA occupying D2 DA receptors can regulate PPE mRNA levels directly by affecting PPE gene expression in the striaturn. The PPE gene contains DNA regulatory sequences

118 called 'cAMP responsive elements' in the gene promoter region which are essential for both basal and cAMP-induced expression of the PPE gene s. These data suggest that D 2 D A mediated inhibition of striatal PPE m R N A can result from a decrease in PPE gene expression. Since D 2 D A receptor occupation inhibits adenylate cyclase 19' 3o this can reduce PPE transcription by attenuating levels of cAMP generated in ENK neurons by other neu-

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Acknowledgements. This work was supported by Grant NIH DA03787.

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