Enkephalin regulates acute D2 dopamine receptor antagonist-induced immediate-early gene expression in striatal neurons

Pergamon

PII:

Neuroscience Vol. 88, No. 3, pp. 795–810, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00241-3

ENKEPHALIN REGULATES ACUTE D2 DOPAMINE RECEPTOR ANTAGONIST-INDUCED IMMEDIATE-EARLY GENE EXPRESSION IN STRIATAL NEURONS H. STEINER†* and C. R. GERFEN‡ †Department of Anatomy and Neurobiology, University of Tennessee, College of Medicine, Memphis, TN 38163, U.S.A. ‡Laboratory of Neurophysiology, National Institute of Mental Health, Bethesda, MD 20892, U.S.A. Abstract––Projection neurons of the striatum release opioid peptides in addition to GABA. Our previous studies showed that the opioid peptide dynorphin regulates that subtype of projection neurons which sends axons to the substantia nigra/entopeduncular nucleus, as indicated by an inhibitory action of dynorphin/agonists on D1 dopamine receptor-mediated immediate-early gene induction in these neurons. The other subtype of striatal projection neurons projects to the globus pallidus and contains the opioid peptide enkephalin. Here, we investigated whether enkephalin regulates the function of striatopallidal neurons, by analysing opioid effects on immediate-early gene induction by D2 dopamine receptor blockade that occurs in these neurons. Thus, the effects of systemic and intrastriatal administration of various opioid receptor agonists and antagonists on immediate-early gene expression (c-fos, zif 268) induced by the D2 receptor antagonist eticlopride were examined with in situ hybridization histochemistry. Intrastriatal infusion of enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed immediate-early gene induction by eticlopride in a dose-dependent manner. This suppression was blocked by the opioid receptor antagonist naloxone, confirming the involvement of opioid receptors. Repeated treatment with D2 receptor antagonists produces increased enkephalin expression and diminished immediate-early gene inducibility in striatopallidal neurons, as well as behavioral effects that are attenuated compared to those of acute treatment (e.g., reduced akinesia). Naloxone reversed such behavioral recovery (i.e. reinstated akinesia), but did not significantly affect suppressed immediate-early gene induction. Our results indicate that enkephalin acts, via mu and delta receptors in the striatum, to inhibit acute effects of D2 receptor blockade in striatopallidal neurons. Moreover, the present findings suggest that increased enkephalin expression after repeated D2 receptor antagonist treatment is an adaptive response that counteracts functional consequences of D2 receptor blockade, but is not involved in suppressed immediate-early gene induction. Together with our earlier findings of the role of dynorphin, these results indicate that opioid peptides in the striatum serve as negative feedback systems to regulate the striatal output pathways in which they are expressed.  1998 IBRO. Published by Elsevier Science Ltd. Key words: enkephalin, delta and mu opioids, immediate-early genes, D2 dopamine receptor, striatum.

Projection neurons of the striatum contain opioid peptides in addition to the inhibitory neurotransmitter GABA. These neurons can be divided into two subtypes based on their projection targets,28 and these subtypes also differ in the opioid peptides they express. Striatonigral neurons, which provide projections to the substantia nigra and/or the entopeduncular nucleus (the internal part of the globus pallidus in primates), and form the so-called ‘‘direct’’ striatal output pathway, mostly contain the opioid peptide dynorphin.15,20,44,62 The other subtype of projection neurons sends an axon to the globus pallidus (the external part of the globus pallidus in primates), forming the initial segment of the ‘‘indirect’’ pathway; these striatopallidal neurons contain the opioid *To whom correspondence should be addressed. Abbreviations: DADLE, [-Ala2,-Leu5]enkephalin; DAMGO, [-Ala2,N-Me-Phe4,Gly5-ol]enkephalin.

peptide enkephalin.1,6,8,20 Both subtypes also feature extensive local axon collaterals within the striatum,28,65 which may mediate local neuronal interactions. In these neurons, dopamine receptors regulate gene expression, including that of opioid peptides (for reviews, see Refs 18 and 56). Such gene regulation is distinct and opposite for striatonigral and striatopallidal neurons, based on the dopamine receptor subtypes these neurons express. Striatonigral neurons principally express D1 receptors7,16,17,32,34 and display, for the most part, elevated levels of gene expression following stimulation of D1 receptors. An opposite pattern of gene regulation is found in striatopallidal neurons, which express the D2 receptor subtype.7,16,17,19,32,35 Gene expression in these neurons is increased by blockade of D2 receptors. After pharmacological treatments targeting dopamine receptors, two general types of gene regulation

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H. Steiner and C. R. Gerfen Table 1. Experimental design

Experiment 1 2

Treatment Acute (time-course) Repeated (time-course)

D2 antagonist eticlopride (i.p.)

Opioid agonist (intrastriatal)

Opioid antagonist (s.c. or intrastriatal)

Figures

5 mg/kg

—

—

1

—

—

2

DADLE (0.03–3 µg) DADLE (0.06 µg)

— Naloxone (1–10 mg/kg or 20 µg) —

3, 4 5

Naloxone (10–50 mg/kg or 20–50 µg) on day 4

7

3 4

Acute Acute

5 mg/kg, once per day, one to five days 1 mg/kg 5 mg/kg

5

Acute

5 mg/kg

6

Repeated

5 mg/kg, once per day, four days

Deltorphin (2 µg), DAMGO (0.5 µg), U-50488 (10 µg) —

response have been described.18,56 One type, longerterm adaptive responses, is typified by changes in expression of neuropeptides such as dynorphin and substance P in striatonigral neurons and enkephalin in striatopallidal neurons. The other type, short-term responses, is exemplified by the rapid and transient induction of immediate-early genes, including c-fos, zif 268 and others. We have used such acute immediate-early gene induction as a measure of a cellular response to investigate the functional role of normal and up-regulated levels of opioid peptides in the striatum. In previous studies, we described a functional relationship between dynorphin in striatonigral neurons and the acute gene regulation response to D1 receptor stimulation in these neurons. Thus, the magnitude of immediate-early gene induction by cocaine, which is mediated by D1 receptors (e.g., Refs 5, 11, 21, 54, 66) in striatonigral neurons,4,26,30 is negatively correlated with the level of local dynorphin expression.53 This is the case in normal animals, as well as after repeated cocaine treatment, which produces up-regulated dynorphin expression in striatonigral neurons.53 Moreover, systemic or intrastriatal administration of a dynorphin (kappa) receptor agonist suppresses cocaine-induced immediate-early gene expression.54 Further studies indicated that presynaptic kappa receptors on dopamine terminals, as well as postsynaptic kappa receptors on (ventral) striatal neurons, mediate this dynorphin regulation.55 Based on these results, we have proposed that dynorphin functions as a negative feedback mechanism to regulate the responsiveness of striatonigral neurons to D1 receptor stimulation. Furthermore, our studies indicate that increased dynorphin expression is an adaptive response to repeated excessive D1 receptor stimulation that counteracts the effects of dopamine input to these neurons.53,56 In the present studies, we investigated whether the opioid peptide contained in striatopallidal neurons, enkephalin, has a similar autoregulatory function. In these neurons, treatment with D2 receptor

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antagonists results in acute induction of immediateearly genes.12,48 We used this immediate-early gene response to examine whether enkephalin regulates the responsiveness of striatopallidal neurons, and whether increased enkephalin expression seen after repeated D2 receptor blockade could also be an adaptive response to compensate for the effects of lost D2 receptor activation. EXPERIMENTAL PROCEDURES

Subjects Male Sprague–Dawley rats (Taconic, Germantown, NY), 200–300 g at the beginning of the experiments, were housed in groups of three to four under standard laboratory conditions. Animals had free access to food and water and were maintained under a 12-h/12-h light/dark cycle. Experiments were carried out between 1.00 p.m. and 5.00 p.m. All experiments were performed in accordance with the National Institutes of Health guidelines for laboratory animal care. All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques when available. Implantation of guide cannulae Rats were anesthetized with sodium pentobarbital (80 mg/kg) and placed in a David Kopf stereotaxic frame. A guide cannula (26-gauge, stainless steel; Plastics One, Roanoke, VA) was lowered into the right striatum and fixed to the skull with acrylic cement. The coordinates used for the tip of the guide cannula were (relative to bregma): A +0.4, L 3.0, V
4.0.46 The guide cannula was closed with a ‘‘dummy cannula’’ of the same length. Rats were then allowed to recover for one week. One day before the infusion, the dummy cannula was replaced with a longer dummy cannula that protruded 2.5 mm beyond the tip of the guide cannula. This procedure reduced the probability of acute damage by the infusion cannula (31-gauge, 1–2 mm longer than the guide cannula), which can cause massive induction of immediate-early genes in the cortex and striatum. Drugs and injection procedures In experiment 1 (Table 1), the D2 receptor antagonist eticlopride (eticlopride hydrochloride; Research Biochemicals, Natick, MA; in 0.02% ascorbic acid, 1 ml/kg, i.p.) was administered in a dose of 5 mg/kg, and rats were killed 0, 20, 40, 60 min, 4 or 24 h after the injection (n=5

Opioid regulation of striatopallidal neurons each). In experiment 2, eticlopride (5 mg/kg) was administered once daily for 0, one, three or five days (n=5–7 each). All animals received a total of five injections (drug or vehicle). In experiment 3, the delta opioid receptor agonist [-Ala2,-Leu5]enkephalin [DADLE; Research Biochemicals; 0, 0.03, 0.3, 3 µg (as peptide) in 0.5 µl of saline; n=5–6 each] was infused into the striatum in freely moving animals. DADLE infusions were performed with a pump at a rate of 0.05 µl/min. After the infusion, the cannula was left in place for an additional 2.5 min to allow for diffusion of the drug. The rat was then returned to the home cage. Fifteen minutes after beginning the intrastriatal infusion, the animals received a systemic injection of eticlopride (1 mg/kg). Controls received an infusion of DADLE (3 µg) followed by a vehicle injection (n=3). In experiment 4, naloxone (naloxone hydrochloride, Research Biochemicals; 0, 1, 10 mg/kg, s.c.; n=6 each) was injected 15 min before the start of the intrastriatal infusion of DADLE (0.06 µg in 1 µl of saline, over 10 min) and 30 min before the eticlopride injection (5 mg/kg). Other rats had naloxone (20 µg) coinfused into the striatum with DADLE before the eticlopride injection (n=4) or received an intrastriatal infusion of naloxone only (n=3). In experiment 5, the delta receptor agonist deltorphin ([-Ala2]deltorphin-II; Sigma, St Louis, MO; 2 µg in 1 µl of saline), the mu receptor agonist [-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO; Sigma; 0.5 µg), the kappa agonist U-50488 [()-trans-U-50488, Research Biochemicals; 10 µg] or saline (n=6–8 each) was infused into the striatum over 10 min, starting 15 min before the eticlopride injection (5 mg/kg). In experiment 6, rats were treated with eticlopride (5 mg/kg) or vehicle once daily for three days and, on day 4, received an injection of naloxone (0, 10, 50 mg/kg; n=5–6 each) 15 min prior to the eticlopride administration. Controls received vehicle injections only, or vehicle plus naloxone (n=3 each). In other groups, naloxone (0, 20, 50 µg in 1 µl; n=4–5 each) was infused into the striatum prior to the eticlopride injection on day 4. Drug-induced behavior was assessed during the intrastriatal infusion and subsequently in the home cage. In addition, behavioral effects were measured in an open-field apparatus (60 cm60 cm40 cm, with lines dividing the floor into 33 squares) by an experimenter who was unaware of the pharmacological treatment. Thus, in experiment 2, locomotor activity (i.e. the number of lines crossed with four feet, crossings) and rearing behavior were assessed during minutes 41–44 after eticlopride administration. In experiment 3, the number of full turns and crossings were measured during minutes 40 and 41, and in experiments 4 and 5 during minutes 41–44 of the survival time. In experiment 6, line crossings were counted during minutes 41–43. In experiments 2–6, animals were killed with CO2 45 min after D2 antagonist administration. Tissue preparation The brains were rapidly removed, frozen in isopentane cooled on dry ice, and stored at
20C until cryostat sectioning. Coronal sections (12 µm) were thaw-mounted on to glass slides twice coated with gelatin, dried on a warm plate and stored at
20C. For further processing, the slides were first warmed to room temperature, then fixed in a 4% paraformaldehyde solution (in 0.9% saline) for 10 min, and incubated in a fresh solution of 0.25% acetic anhydride in 0.1 M triethanolamine and 0.9% saline (pH 8.0) for 10 min. The slide-mounted sections were then dehydrated, defatted for 25 min in chloroform, rehydrated and air dried. The sections were stored at
20C until hybridization. In situ hybridization histochemistry Oligonucleotide probes (48-base cDNAs) were labeled with [35S]dATP as described earlier.53 The sequence of the c-fos probe was: 5 -CAG-GGC-TAG-CAG-TGT-GGG-

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CGA-GCT-CAG-TGA-GTC-AGA-GGA-GGG-CTCGTT-3 ; those of the zif 268,54 substance P and enkephalin probes67 have been described before. Labeled probe (about 0.5106 c.p.m.) in 25 µl of hybridization buffer67 was added to each brain section. The sections were coverslipped and incubated overnight at 37C. After incubation, the slidemounted sections were rinsed in four washes of 1saline citrate (150 mM sodium chloride, 15 mM sodium citrate). The slides were then washed for 320 min in 2saline citrate/50% formamide at 40C, followed by two 30-min washes in 1saline citrate at room temperature. After a brief water rinse, the sections were air dried and then apposed to X-ray film (X-Omat, Kodak) for two to three weeks. Analysis of autoradiograms Coronal sections collected at three rostrocaudal levels were examined: rostral striatal (approximately 10.5 mm rostral to the interaural line46), mid-striatal (9.5 mm) and caudal striatal (8.0 mm) levels. In experiments with systemic drug administration only, gene expression was measured either across the whole striatum or in the medial and the lateral one-thirds of the striatum, at the mid-striatal level. In experiments with intrastriatal infusions, sections containing the cannula track, in most cases from the mid-striatal region, were also collected and analysed. On these sections, gene expression was measured across the whole striatum. In addition, the size of the area in which gene expression was suppressed by the infused drug (affected area) was measured in some experiments. Levels of gene expression were determined on film autoradiograms with a Macintosh-based image analysis system (IMAGE, Wayne Rasband, NIMH). For each region, mean density values were measured in both hemispheres. In experiments with systemic drug treatments only, values from the two hemispheres were averaged. Density values presented are background corrected (mean density of gray matter
mean density of film). Drug effects were determined with one-factor ANOVA followed by post hoc Dunnett or Tukey–Kramer tests. All P values are two-tailed. The illustrations of film autoradiograms displayed in Figs 2–7 are computer-generated images in which the maximal hybridization signal is black and lack of a signal is white. RESULTS

Time-course of changes in immediate-early gene and enkephalin expression after a single injection of the D2 receptor antagonist eticlopride Acute administration of the D2 antagonist eticlopride (5 mg/kg) resulted in rapid induction of immediate-early genes in the striatum (Figs 1, 2). The levels of both c-fos and zif 268 mRNAs increased significantly within 20 min after injection of the drug (Fig. 1; results for zif 268 not shown). While zif 268 mRNA levels remained elevated at near-peak level for a somewhat longer period than those of c-fos (see Ref. 56), both mRNA levels returned to baseline by about 4 h. The immediate-early gene response was greater in the lateral striatum than in the medial striatum (P<0.0001, two-factor ANOVA; Figs 1, 2). Moreover, the time-course also differed between these two striatal regions. In the lateral striatum, immediate-early gene induction peaked 40–60 min after drug administration, while in the medial striatum, the highest mRNA levels were observed 20 min after eticlopride injection. Consequently, the

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H. Steiner and C. R. Gerfen Table 2. Locomotor activity (crossings) and rearing behavior during repeated eticlopride treatment 0

Duration of treatment (days) 1 3

5

Crossings 34.82.9 1.31.1** 3.81.6** 7.73.1** Rearings 12.22.0 0.40.3** 2.50.7** 3.01.0** Rats received eticlopride injections (5 mg/kg) once daily for one, three or five days. Controls (0 days) were injected with vehicle. Behavior (meanS.E.M.) was measured in an open field apparatus (60 cm60 cm) during minutes 41–44 after drug administration. **P<0.01, vs 0-day group, ANOVA, Tukey–Kramer.

Fig. 1. Time-course of changes in c-fos and enkephalin expression in the striatum after a single injection of the D2 dopamine receptor antagonist eticlopride. Levels of c-fos mRNA (A) and enkephalin mRNA (B) (mean density, meanS.E.M., arbitrary units) at different time-points after an injection of eticlopride (5 mg/kg, i.p.) measured in the lateral one-third and in the medial one-third of the striatum are shown. *P<0.05, **P<0.01, vs 0 min.

distinctive medial–lateral gradient in the hybridization signal (Fig. 2) was minimal initially and became more pronounced at later time-points. In contrast to these immediate-early genes, changes in enkephalin expression occurred with a delayed onset (Fig. 1). Significantly increased enkephalin mRNA levels were first seen 1–4 h after the drug treatment. These mRNA levels remained elevated through 24 h. However, similar to c-fos and zif 268 expression, changes in enkephalin expression were greater in the lateral striatum than in the medial striatum (P<0.0001, two-factor ANOVA). Immediate-early gene induction is suppressed and enkephalin expression is increased after repeated D2 antagonist treatment Animals were treated with eticlopride (5 mg/kg) once daily for one to five days, and mRNA levels

were determined 45 min after the last injection. Induction of immediate-early genes by the D2 antagonist was maximal after the first drug injection (day 1) and was suppressed thereafter (Fig. 2). Thus, on treatment days 3 and 5, c-fos and zif 268 mRNA levels, as measured across the whole striatum, were not significantly increased over basal levels (day 0). However, some minimal gene induction could still be seen (c-fos; Fig. 2A, day 5). In contrast, enkephalin mRNA levels changed with an inverse time-course. They were not significantly altered after the first drug administration, but increased with repeated treatment (Fig. 2). These changes could be measured throughout the striatum (Fig. 2), but were significantly greater in lateral parts than in medial parts (P<0.0001; data not shown). Thus, in medial and lateral striatal regions, the magnitude of changes in enkephalin expression was positively related to that of the initial immediate-early gene response. Such a relationship could also be seen in the ventral striatum (nucleus accumbens), with minimal acute immediateearly gene induction and minimal subsequent changes in enkephalin expression (Fig. 2A). Administration of the D2 receptor antagonist produced akinesia, expressed as reduced locomotion and rearing behavior in the open-field test. These behavioral effects also changed during repeated eticlopride treatment (Table 2). The first eticlopride injection resulted in maximal suppression of locomotor activity and rearing. By day 5, these behavioral measures were still significantly affected by the D2 antagonist treatment, but to a somewhat reduced extent. The delta opioid receptor agonist DADLE suppresses immediate-early gene induction by the D2 antagonist To test whether enkephalin receptors in the striatum regulate the immediate-early gene response to D2 antagonist treatment, the delta opioid receptorpreferring agonist DADLE was infused into the striatum prior to systemic injection of eticlopride (1 mg/kg). Intrastriatal administration of DADLE (0.03–3 µg) suppressed eticlopride-induced c-fos and zif 268 expression in a dose-dependent manner

Opioid regulation of striatopallidal neurons

A

eticlopride / day 1

eticlopride / day 5

enkephalin

zif 268

c-fos

basal

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0

1

enkephalin

80

**

60

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zif 268

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mean density

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Fig. 2. Time-course of changes in immediate-early gene and enkephalin expression in the striatum during repeated (once daily) treatment with the D2 receptor antagonist eticlopride (5 mg/kg). (A) Illustrations of film autoradiograms for c-fos (top), zif 268 (middle) and enkephalin expression (bottom) in coronal sections from the rostral striatum depict basal expression (i.e. after a vehicle injection; left) and eticlopride-induced expression assessed 45 min after the first drug injection (day 1; middle), or after the last injection of a five-day treatment (day 5; right). Maximal hybridization signal is black, lack of a signal is white. (B) Time-course of changes in c-fos (left), zif 268 (middle) and enkephalin mRNA levels (right) during repeated eticlopride treatment. Mean density values (meanS.E.M.) were measured across the whole striatum after a vehicle injection (day 0), or after eticlopride administration once (day 1), or once daily for three or five days. Immediate-early gene expression is maximal after the first D2 antagonist treatment and is suppressed thereafter. In contrast, enkephalin mRNA levels increase with repeated treatment. *P<0.05, **P<0.01, vs 0 days.

(Fig. 3). Thus, increasing doses of DADLE produced an increasing area around the infusion cannula where immediate-early gene induction was suppressed, which resulted in decreasing overall mean density levels (Fig. 3B). DADLE (3 µg) also significantly reduced basal levels of zif 268 mRNA (in vehicleinjected controls; mean density on infused side vs

mean density on non-infused side, 31.70.6 vs 43.61.3, meanS.E.M.; P<0.01). Neither intrastriatal infusion of DADLE (3 µg) alone (data not shown), nor DADLE (0.03–3 µg) followed by eticlopride administration (Fig. 4), significantly affected enkephalin mRNA levels in the striatum. The expression of substance P in striato-

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nigral neurons can be enhanced within 30 min by cocaine administration.11,53 In contrast, neither eticlopride administration alone (data not shown), nor intrastriatal DADLE alone (data not shown), nor DADLE in combination with eticlopride (Fig. 4) produced changes in substance P mRNA levels. Behavioral effects were measured in the open-field test. DADLE infusion produced, in a dose-dependent manner, turning behavior in the direction away from the infused side (Fig. 3C). DADLE-induced turning typically appeared towards the end of the infusion period and lasted until the animal was killed. D2 antagonist treatment after DADLE infusion did not reduce turning behavior, as turning rates in the open field produced by 3 µg of DADLE did not differ significantly between animals with subsequent vehicle injections (9.72.3, meanS.E.M.) and those with subsequent eticlopride injections (15.72.0) (P>0.05). Naloxone blocks DADLE-induced suppression of immediate-early gene induction and turning behavior Involvement of opioid receptors in the DADLEinduced suppression of immediate-early gene induction by eticlopride was confirmed with systemic and intrastriatal administration of the non-selective opioid receptor antagonist naloxone. Systemic administration of naloxone (1–10 mg/kg) prior to intrastriatal infusion of DADLE (0.06 µg) dosedependently inhibited the suppressive effect of the delta receptor agonist on c-fos and zif 268 induction by a subsequent eticlopride injection (Fig. 5). Also, co-infusion of naloxone (20 µg) together with DADLE into the striatum reduced the delta agonist effects on gene induction (Fig. 5A). In controls, intrastriatal naloxone (20 µg) alone did not affect striatal immediate-early gene expression (Fig. 5A). However, the effects of systemic and intrastriatal administration of naloxone differed in that systemic naloxone shrank the area around the cannula track where immediate-early gene induction was suppressed (i.e. the strongest naloxone effects occurred distal to the track, probably reflecting the diffusion gradient of DADLE). In contrast, the effects of intrastriatal naloxone were strongest immediately around the track (Fig. 5A). Systemic administration of naloxone also inhibited DADLE-induced turning behavior in a dosedependent manner (Fig. 5C). Similarly, intrastriatal administration of naloxone (20 µg) blocked DADLEinduced turning (not shown). Intrastriatal naloxone alone was without effect on turning. Delta and mu, but not kappa, opioid receptor agonists suppress immediate-early gene induction by the D2 antagonist The receptors involved in the opioid regulation of eticlopride-induced gene expression were further

investigated by comparing the effects of high doses of a more selective delta receptor agonist, a mu receptor agonist and a kappa receptor agonist. Intrastriatal infusion of the selective delta receptor agonist deltorphin (2 µg) or the mu receptor agonist DAMGO (0.5 µg) prior to eticlopride injection significantly reduced c-fos and zif 268 induction by the D2 antagonist (Fig. 6). Both drugs had similar effects on mRNA levels measured across the whole striatum, as well as on the size of the area where gene induction was suppressed (affected area). In contrast, the kappa agonist U-50488 (10 µg) did not reduce gene induction by the D2 antagonist (Fig. 6). Contraversive turning was observed towards the end of or shortly after intrastriatal infusion of both delta and mu agonists. However, while deltorphininduced turning was still present during the openfield test (i.e. during minutes 41–44 after eticlopride injection), DAMGO-induced turning was blocked by the subsequent D2 antagonist treatment (Fig. 6C). Infusion of the kappa agonist U-50488 did not induce turning behavior at any time. In the open field, the number of crossings did not differ significantly between the groups that received opioid agonist infusions or vehicle prior to eticlopride injection (data not shown). Effects of naloxone on immediate-early gene inducibility and behavior after repeated D2 receptor antagonist treatment The preceding experiments demonstrated that striatal enkephalin receptors inhibit immediate-early gene induction by acute D2 antagonist treatment. To investigate whether the suppression of the immediateearly gene response after repeated D2 antagonist treatment is mediated by increased enkephalin function in the striatum, rats were treated with eticlopride daily for three days and, on day 4, the effects of systemic and intrastriatal administration of naloxone on immediate-early gene induction by eticlopride were examined. Repeated treatment with the D2 antagonist for four days resulted in suppressed induction of c-fos and zif 268 mRNAs (Fig. 7). Systemic administration of naloxone prior to the eticlopride injection on the last day had minimal or no effects on this suppression. Neither the relatively high dose of naloxone (10 mg/kg), which attenuated the DADLEinduced suppression of the immediate-early gene response in the acute situation (see Fig. 5), nor an even higher dose (50 mg/kg) significantly affected c-fos and zif 268 mRNA levels (Fig. 7). The same results were obtained with intrastriatal infusion of naloxone. Neither 20 nor 50 µg had a significant effect on the suppressed immediate-early gene induction (data not shown). In contrast to the immediate-early gene response, systemic administration of naloxone prior to eticlopride affected locomotor activity in the open-field test (Fig. 7C). Acute injection of eticlopride produced

Opioid regulation of striatopallidal neurons DADLE 0 µg (i.s.) + eticlopride (i.p.)

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turning behavior 20 contraversive turns

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15 10 5 0

0

Fig. 3. D2 receptor antagonist-induced immediate-early gene expression in the striatum is suppressed by intrastriatal infusion of an enkephalin (delta) receptor agonist. (A) Illustrations of film autoradiograms for c-fos (top) and zif 268 expression (bottom) in coronal sections from the middle striatum show the effects of unilateral intrastriatal infusion of the delta receptor-preferring agonist DADLE in doses of 0 µg (vehicle; left), 0.03 µg (middle) or 0.3 µg (right) prior to the administration of eticlopride (1 mg/kg, i.p.). Animals were killed 45 min after the D2 antagonist injection. Maximal hybridization signal is black. (B) Dose-dependent suppression of eticlopride-induced c-fos (left) and zif 268 mRNA levels (right) by the delta agonist DADLE. Mean density values (meanS.E.M.) were measured across the whole striatum at the infusion site and are expressed as percentages of the values on the non-infused side. Animals received an intrastriatal infusion of DADLE (0–3 µg) prior to the eticlopride injection and were killed 45 min later. (C) Intrastriatal infusion of DADLE produces contraversive turning behavior in a dose-dependent manner. Full turns (meanS.E.M.) were measured in an open-field apparatus during minutes 40 and 41 after eticlopride injection. **P<0.01, vs 0 µg.

severe akinesia. Rats treated with eticlopride daily for four days showed significantly more crossings (i.e. less akinesia) than rats that received the first injection. Naloxone reversed this behavioral recovery in a dose-dependent manner. Thus, on day 4, rats that received 50 mg/kg of naloxone before the eticlopride injection were almost completely inactive. This dose of naloxone had minimal effects on locomotion in vehicle-treated controls (not shown). Intrastriatal administration of naloxone produced a similar tendency that did not, however, reach statistical significance (not shown). DISCUSSION

Our previous studies indicate that dynorphin acts in the striatum to regulate the activation of striato-

nigral neurons by other neurotransmitter systems, and that repeated excessive D1 receptor stimulation results in a compensatory or adaptive increase in dynorphin function that dampens such activation.53–56 Here, we extended our analysis of opioid regulation of striatal projection neurons to determine whether enkephalin plays a similar autoregulatory role for striatopallidal neurons. The present findings indicate that enkephalin does serve such a function. Our results (summarized in Fig. 8) show that acute stimulation of enkephalin (delta and mu) receptors in the striatum inhibits D2 receptor antagonist-induced immediate-early gene expression that occurs in striatopallidal neurons. Our previous findings on gene regulation in striatonigral neurons demonstrated that dynorphin expression increases as a consequence of repeated D1 receptor activation,

802

DADLE 0.3 µg (i.s.) + eticlopride (i.p.)

substance P

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enkephalin

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B 120 100 80 60 40 20

substance P mean density (% of noninfused side)

mean density (% of noninfused side)

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DADLE (i.s.) DADLE

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Fig. 4. DADLE infusion into the striatum did not affect enkephalin and substance P mRNA levels. (A) Illustrations of film autoradiograms show enkephalin (left) and substance P expression (right) in coronal sections at the level of the cannula track in the middle striatum. For comparison with gene expression depicted in Fig. 3, examples of rats that received an intrastriatal infusion of 0.3 µg DADLE prior to the eticlopride injection (1 mg/kg, i.p.) are shown. Maximal hybridization signal is black. (B) Enkephalin (left) and substance P mRNA levels (right) in the striatum after intrastriatal infusion of DADLE (0–3 µg), followed by a systemic injection of eticlopride. Mean density values (meanS.E.M.) measured on the infused side are expressed relative to those on the non-infused side.

and indicated that this response serves to downregulate the effects of subsequent D1 receptor activation. Repeated treatment with D2 receptor antagonists increases enkephalin expression in striatopallidal neurons and suppresses immediateearly gene inducibility in these neurons. The present behavioral results suggest that increased enkephalin expression is also an adaptive response that mediates recovery of function after repeated D2 antagonist treatment. Our present gene regulation data indicate that enkephalin inhibits immediate-early gene induction by acute D2 antagonist treatment, but do not support a role for this opioid peptide in the suppressed immediate-early gene response after repeated D2 antagonist treatment. Opioid peptides as negative feedback systems to regulate striatal projection neurons Acute treatment with D2 receptor antagonists induces immediate-early gene expression in striatopallidal neurons.12,48 This effect is, at least in part, dependent on cortical input or glutamate (N-methyl-aspartate) receptor activation,2,12,61,69 and is thus

considered to reflect disinhibition of striatopallidal neurons due to blockade of inhibitory D2 receptors. In the present study, this ‘‘D2 receptor response’’ in striatopallidal neurons was employed as a functional marker to investigate the effects of enkephalin and agonists on these neurons. Enkephalin peptides are endogenous ligands of delta and mu opioid receptor subtypes,45 which are highly expressed in the striatum.38 Our present findings provide evidence that enkephalin regulates striatopallidal neuron function by interacting with delta and mu receptors in the striatum. Results obtained with intrastriatal infusion of the delta receptor-preferring agonist DADLE suggested that delta receptors inhibit D2 antagonist-induced immediate-early gene expression in a very potent manner. Systemic and intrastriatal administration of the non-selective opioid receptor antagonist naloxone attenuated this DADLE action, as well as DADLEinduced turning behavior, demonstrating that these effects were indeed mediated by opioid receptors. Similar effects on gene expression and behavior were produced by deltorphin, a very delta-selective opioid agonist,31 confirming the involvement of delta

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receptors. However, intrastriatal infusion of the mu agonist DAMGO also reduced immediate-early gene induction by eticlopride. Thus, our results indicate that both enkephalin receptor subtypes are involved in the regulation of gene expression in striatopallidal neurons. In contrast, dynorphin (kappa) receptors do not appear to play a role. A high dose of the kappa

agonist U-50488 did not reduce D2 antagonistinduced immediate-early gene expression. In our earlier studies on the function of dynorphin, we have shown that intrastriatal infusion of a kappa agonist inhibits cocaine-induced immediate-early gene expression,54 a response that occurs in striatonigral neurons4,26,30 and is mediated by D1 receptors in the striatum.54 Therefore, whereas dynorphin

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Fig. 6. Opioid receptors involved in the regulation of D2 receptor antagonist-induced immediate-early gene expression in the striatum. (A) Illustrations of film autoradiograms depict c-fos (top) and zif 268 expression (bottom) in coronal sections at the mid-striatal level in animals that received an intrastriatal infusion of vehicle (left), the selective delta receptor agonist deltorphin (2 µg; second from left), the mu receptor agonist DAMGO (0.5 µg; second from right) or the kappa receptor agonist U-50488 (10 µg; right), starting 15 min before the eticlopride injection (5 mg/kg, i.p.). Maximal hybridization signal is black. (B) Effects of different opioid receptor agonists on eticlopride-induced c-fos (top) and zif 268 mRNA levels (bottom) in the striatum. Mean density values (meanS.E.M., percentage of non-infused side) measured across the whole striatum at the level of the cannula track (left) and size of area where mRNA levels were reduced (affected area; right) are shown for rats that received an intrastriatal infusion of vehicle, deltorphin, DAMGO or U-50488, and a subsequent injection of eticlopride. (C) Turning behavior after intrastriatal infusion of different opioid receptor agonists followed by D2 antagonist treatment. Contraversive full turns (meanS.E.M.) measured in the open field during minutes 41–44 after the eticlopride injection are depicted for rats that received intrastriatal administration of vehicle, deltorphin, DAMGO or U-50488. **P<0.01, vs vehicle.

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Fig. 7. Effects of naloxone on D2 receptor antagonist-induced immediate-early gene expression in the striatum and behavior after repeated D2 antagonist treatment. (A) Film autoradiograms show examples of c-fos (top) and zif 268 expression (bottom) in coronal sections at mid-striatal levels in rats that received vehicle injections (left) or eticlopride (5 mg/kg; middle) once daily for 3 days, followed, on day 4, by a vehicle injection (naloxone 0 mg/kg) prior to the injection of eticlopride (5 mg/kg), or in rats that received repeated eticlopride treatment followed by naloxone (50 mg/kg) plus eticlopride on day 4 (right). Maximal hybridization signal is black. (B) Effects of naloxone on c-fos (left) and zif 268 mRNA levels (right). Mean density values (meanS.E.M.) measured across the whole striatum at the mid-striatal level are shown for rats that were pretreated with vehicle (veh.) or eticlopride (etic.) for three days and received, on day 4, naloxone (nal.; 0–50 mg/kg) prior to the eticlopride injection. The broken lines indicate mRNA levels in controls that received vehicle injections only. (C) Effects of naloxone on locomotor activity in the open-field test during minutes 41–43 after the last eticlopride injection. The number (meanS.E.M.) of line crossings expressed as percentage of crossings in vehicle-injected controls is presented. *P<0.05, **P<0.01, vs eticlopride once (first group) or as indicated.

(kappa) receptors inhibit immediate-early gene induction in striatonigral neurons, enkephalin receptors regulate gene induction in striatopallidal neurons. Our findings suggest that enkephalin acts as a negative feedback mechanism to regulate striatopallidal neuron function. Behavioral effects of delta and mu receptor stimulation in the striatum In these experiments, we have also described behavioral effects of the pharmacological treatments to further characterize the actions of opioid drugs in the striatum (Fig. 8). Previous studies have shown

that bilateral intrastriatal administration of delta agonists stimulates locomotor activity.27,36,47 In the present studies, unilateral intrastriatal infusion of the two delta agonists and the mu agonist, but not of the kappa agonist, resulted in turning behavior that was directed away from the side of infusion, thus reflecting lateralized stimulatory effects of the opioid agonists on behavior. DADLE- and deltorphininduced turning was not reduced by the subsequent systemic D2 antagonist treatment, consistent with previous observations.27,36 Thus, delta receptor stimulation also prevented D2 antagonist-induced akinesia. In contrast, turning behavior induced by the mu agonist DAMGO was attenuated by the D2

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Fig. 8. Schematic summary of the effects of opioid receptor agonists and antagonists on alterations in striatal enkephalin and immediate-early gene expression (IEG response) and behavior, produced by acute and repeated treatment with the D2 receptor antagonist eticlopride. Enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed acute immediate-early gene induction by eticlopride. This effect was reversed by the opioid antagonist naloxone. In contrast, only delta, but not mu, receptor agonists prevented eticlopride-induced akinesia. After repeated D2 receptor antagonist treatment for three to five days, enkephalin expression is increased, the immediate-early gene response is suppressed and the behavioral inhibition (akinesia) is reduced (recovery of function). Whereas naloxone did not affect the suppressed immediate-early gene response, the behavioral recovery was reversed by this opioid antagonist. It is concluded that enkephalin, by acting on delta and mu receptors in the striatum, exerts an autoregulatory function in striatopallidal neurons (as indicated by the inhibitory effects on D2 receptor antagonistinduced immediate-early gene expression), but also affects other striatal mechanisms (as indicated by the dissociation in gene regulation and behavioral effects of delta and mu agonists). Moreover, increased enkephalin expression seems to be an adaptive response to repeated D2 receptor blockade, which mediates recovery of function, but is not involved in suppressed immediate-early gene induction.

antagonist, and rats were as akinetic as controls that had received eticlopride only. Therefore, while both delta and mu receptor agonists prevented D2 antagonist-induced immediate-early gene expression in striatopallidal neurons, behavioral effects of the D2 antagonist were differentially affected by these opioid agonists. This dissociation in effects on gene regulation and behavior allows several conclusions. First, akinesia produced by D2 receptor antagonist treatment is not critically related to effects of D2 receptor blockade in striatopallidal neurons, but also involves other D2 receptor-regulated mechanisms in the striatum. Secondly, such mechanisms are also under control of enkephalin, but are differentially affected by delta and mu receptors. Such differential effects of these two receptor subtypes are not surprising given their distribution in the striatum. For example, whereas delta receptor expression and binding are relatively uniform throughout the striatum, mu receptor expression and binding are highly enriched in the patch compartment.9,39,40,41 Also, cholinergic interneurons seem to express high levels of delta, but not mu, receptors,33,39 and acetylcholine release has been shown to be regulated by delta, but not mu, receptor agonists (e.g., Ref. 51). How-

ever, the neuronal basis for the differential behavioral effects of these opioid agonists remains to be demonstrated. Potential mechanisms that mediate enkephalin actions in the striatum Our results demonstrate that mu and delta receptors located within the striatum are involved in the opioid regulation of striatopallidal neurons. The exact underlying mechanisms are unknown. For one, the phenotypes of striatal neurons that express the different opioid receptor subtypes remain to be fully established. However, an association between some opioid receptors and certain neuronal types has emerged in recent studies. Based on these studies, there is a number of potential mechanisms by which enkephalin could exert such negative feedback. Such mechanisms include the following. (1) Enkephalin released from local axon collaterals could act directly on inhibitory opioid receptors on striatopallidal neurons. Delta and mu receptor agonists have been shown to inhibit postsynaptic potentials in presumed striatal projection neurons,25,68 although the phenotype of these neurons has not been identified.

Opioid regulation of striatopallidal neurons

Support for a direct interaction between enkephalin and striatopallidal neurons is provided by recent ultrastructural immunocytochemical studies that colocalized enkephalin and delta or mu receptors in the dorsal or ventral striatum.58,59,64 Also, mu receptor mRNA has been demonstrated in a portion of enkephalin neurons.22 However, the ultrastructural studies58,59,64 also indicated that some enkephalinnegative neurons contain these opioid receptor subtypes as well, suggesting that additional, indirect mechanisms may exist. Indirect mechanisms may contribute to the suppressive effect of the mu agonist DAMGO on D2 antagonist-induced immediate-early gene expression in the matrix compartment (Fig. 6), given the relative absence of mu receptor expression and binding in this compartment.9,39,40 Indirect mechanisms may include the following. (2) Actions mediated by striatal interneurons; for example, cholinergic interneurons express delta receptors.33,39 Inhibition of acetylcholine release in the striatum by enkephalin and delta receptor agonists is well established.43,49,51 In a recent study, we investigated the effects of inhibiting cholinergic activity in the striatum on gene induction in striatopallidal neurons. Our results showed that, although intrastriatal infusion of the muscarinic antagonist scopolamine affected gene expression in striatonigral neurons and behavior, D2 antagonist-induced immediate-early gene expression was not suppressed by this treatment.57 These findings suggest that cholinergic interneurons do not mediate the inhibitory effects of opioid receptor stimulation on immediate-early gene induction in striatopallidal neurons. (3) Based on electrophysiological investigations, it has also been proposed that enkephalin may inhibit glutamate release from corticostriatal terminals by interacting with presynaptic opioid receptors (e.g., Ref. 25). Local inhibition of glutamate input would likely attenuate immediate-early gene induction by the D2 antagonist.2,12,61,69 Delta receptors are expressed throughout the cerebral cortex, whereas mu receptor expression is minimal or absent in most cortical areas.9,39,40,41 However, it is presently unknown whether corticostriatal neurons express opioid receptors. (4) Studies also demonstrated that D1 receptor-stimulated adenylate cyclase activity can be inhibited by enkephalins, mu and delta agonists,49,50,51 providing indirect evidence for mu and delta receptors in striatonigral neurons. A recent double labeling study confirmed the presence of mu receptor mRNA in a subset of striatonigral (dynorphin-positive) neurons.22 The present results also showed that the delta agonist DADLE considerably decreased basal levels of zif 268 mRNA in the striatum. Basal zif 268 mRNA levels mostly reflect gene expression in striatonigral neurons.17 Thus, these findings indicate that enkephalin can also affect the function of striatonigral neurons. It remains to be seen whether long-loop regulation involving striatonigral neurons and nigro-

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thalamo-cortico-striatal or nigro-thalamo-striatal connections also plays a role in striatopallidal neuron function. Taken together, several potential mechanisms exist by which enkephalin could affect striatopallidal neurons. Future studies will have to elucidate the functional significance of these mechanisms. Increased enkephalin expression as an adaptive response? Chronic blockade of D2 receptors (e.g., Refs 24, 60) or dopamine depletion (e.g., Refs 13, 16, 19, 55, 63 and 67) results in increased levels of enkephalin mRNA and peptides in the striatum. It is conceivable that up-regulated enkephalin synthesis after a chronic lack of D2 receptor stimulation is an adaptive response to counteract increased activation of striatopallidal neurons, similar to increased dynorphin expression in striatonigral neurons. Consistently, repeated treatment with the D2 receptor antagonist haloperidol has also been reported to result in suppressed inducibility of c-fos and other immediateearly genes at time-points when enkephalin expression is increased.29,52 Here, we confirmed and extended these findings with the more selective D2 receptor antagonist eticlopride. After having demonstrated that enkephalin receptor stimulation in the striatum inhibits the acute immediate-early gene response to D2 receptor blockade, and that this inhibition can be blocked with the opioid receptor antagonist naloxone, we tested whether increased enkephalin expression could be responsible for the suppressed immediate-early gene induction after repeated D2 antagonist treatment. Our results do not support such a function. Naloxone, in doses that attenuated the acute opioid agonist effects, and in even higher doses, given systemically or intrastriatally, did not reverse the suppressed immediate-early gene induction. The observed behavioral consequences of naloxone treatments (i.e. reversal of recovery from akinesia; see below) show that the opioid antagonist was effective. Therefore, the suppressed immediate-early gene response after repeated D2 antagonist treatment is apparently not simply attributable to increased enkephalin expression in the striatum. On the other hand, evidence for a compensatory role of increased enkephalin function is provided by our behavioral results (Fig. 8). Acute injection of eticlopride produced severe akinesia. Repeated D2 receptor antagonist treatment resulted in diminished inhibition of behavior, an effect that is well known (e.g., Refs 3, 14, 23 and 42). Naloxone, given after repeated eticlopride pretreatment, which increased enkephalin expression, reversed this behavioral recovery (i.e. reinstated akinesia). These findings suggest that increased enkephalin levels may act as an adaptive mechanism to ‘‘normalize’’ basal ganglia function in other ways; for example, by influencing

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other striatal neuron populations (see above) or by acting in the target region of the striatopallidal projection (e.g., by regulating GABA release from striatopallidal terminals10,37). CONCLUSIONS

Our earlier studies demonstrated that the opioid peptide dynorphin regulates the function of striatonigral neurons which express this neuropeptide, and indicated that increased dynorphin expression after repeated D1 receptor stimulation is an adaptive response that serves to counteract excessive activation. Striatopallidal neurons contain the opioid peptide enkephalin. In the present series of studies, we examined whether enkephalin affects the function of striatopallidal neurons by investigating the influence of enkephalin agonists on a cellular response in these neurons, immediate-early gene induction by D2 receptor blockade. Our results indicate that enkephalin acts as a negative feedback mechanism to

regulate the function of striatopallidal neurons, similar to the role of dynorphin in striatonigral neurons. Moreover, the current findings indicate that increased enkephalin expression after repeated D2 receptor blockade is also an adaptive response to counteract functional consequences of lost D2 receptor stimulation; however, our results do not support a role of enkephalin in suppressed immediate-early gene induction after repeated D2 antagonist treatment. Taken together, these results further indicate that opioid peptides in the striatum function, at least in part, as autoregulatory mechanisms for striatal output pathways.

Acknowledgements—We thank Dawn AnuszkiewiczLundgren, Ron Paletzki, Sharon Frase and Guiyuan Sun for excellent technical assistance. This work was supported in part by NIMH/DIRP and USPHS Grants NS26473 and NS20702.

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