D2 dopamine receptor messenger RNA is altered to a greater extent by blockade of glutamate receptors than by blockade of dopamine receptors

Neuroscience Vol. 60, No. I, pp. 97-114,

Pergamon

0306-4522(93)E0078-5

1994

ElsevierScienceLtd Copyright 0 1994IBRO Printed in Great Britain. All rights reserved 0306-4522/94

.S6.00 + 0.00

D2 DOPAMINE RECEPTOR MESSENGER RNA IS ALTERED TO A GREATER EXTENT BY BLOCKADE OF GLUTAMATE RECEPTORS THAN BY BLOCKADE OF DOPAMINE RECEPTORS Z.-H. QIN, L.-W. ZHOU and B. WEISS* Department of Pharmacology, Division of Neuropsychopharmacology, Medical College of Pennsylvania at EPPI, 3200 Henry Avenue, Philadelphia, PA 19129, U.S.A. Abstract-To study further the molecular mechanisms by which glutamate and dopamine interact to regulate the functions of the basal ganglia, the effects of persistently inhibiting dopamine receptors and glutamate N-methyl-D-aspartate receptors on the density of D, and D, dopamine receptors and on the level of their transcripts were examined in mouse brain. To block dopamine receptors, mice were treated with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline once daily for two and six days, or were treated with fluphenazine-N-mustard once daily for five days. To block N-methyl-D-aspartate receptors, mice were treated with dizocilpine by continuous infusion with osmotic mini-pumps for two and six days. The density of D, and D, dopamine receptors was measured by receptor autoradiography, and the level of D, and D, dopamine receptor messenger RNA was measured by in situ hybridization histochemistry. The results showed that N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline blocked about 90% of both D, and D, dopamine receptors, but had no significant effect on the level of either D, or D, dopamine receptor messenger RNA. Fluphenazine-N-mustard, which was as effective as N-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline in blocking D, dopamine receptors but had little effect on D, dopamine receptors, also had no significant effect on the level of D, and D, dopamine receptor messenger RNAs. By contrast, continuously infusing dizocilpine significantly decreased the levels of D, dopamine receptor messenger RNA in striatum, nucleus accumbens and olfactory tubercle. Dizocilpine also caused small decreases in the density of D, dopamine receptors, but only in posterior striatum was this decrease statistically significant. Dizocilpine slightly and transiently decreased the levels of D, dopamine receptor messenger RNA in striatum but had no significant effect on the density of D, dopamine receptors in any region examined. This study demonstrates that persistent blockade of D, and D, dopamine receptors has relatively little effect on the levels of D, and D, dopamine receptor messenger RNA, but that blockade of N-methyl-uaspartate receptors produces a rapid and profound decrease in the levels of D, dopamine receptor messenger RNA and a smaller decrease in the density of D, dopamine receptors. These results suggest that N-methyl-o-aspartate receptors play an important role in the expression of D, dopamine receptors in basal ganglia.

Recent cloning of the transcripts of dopamine receptors has revealed at least five subtypes in mammalian

The level of dopamine receptors and their transcripts as well as the biological responses that these

brain.51,58 Among these, the D, and D2 dopamine receptors have been most intensively studied because of the availability of selective ligands for these receptors, and because of their relatively high expression in discrete brain areas. Of these brain areas, the basal ganglia and mesolimbic regions have attracted particular interest since D, and D, dopamine receptors are thought to play important roles in regulating motor and cognitive behaviors.22 Indeed, it is now established that dysfunction and dysregulation of dopamine receptors in these brain regions are associated with several neuropsychological disorders such as Parkinsonism and schizophrenia.’

receptors subserve can be altered through various pharmacological manipulations. Increasing the input to D, dopamine receptors has been shown to decrease the levels of D, dopamine receptors and D, dopamine receptor mRNA in striatum.‘3*64Decreasing the dopamine input by destroying dopaminergic pathways with neurotoxins, or by blocking postsynaptic dopamine receptors with D, and D, receptor antagonists also has been reported to alter the levels of dopamine receptors. Chronic blockade of D, dopamine receptors with selective antagonists8J4J5*29or lesioning dopaminergic pathway with 6-hydroxydopamine30.47.48.55 increases the density of D, dopamine receptors. Similarly chronic blockade of D, dopamine receptors with selective antagonists increases the density of D, dopamine receptors.16 However, lesioning dopaminergic pathways with 6-hydroxydopamine decreases the density of D, dopamine

*To whom correspondence should be addressed. Abbreoiufions:EEDQ, 2-ethoxy-I-ethoxycarbonyl-1,2-dihy-

droquinoline; FNM, fluphenazine-N-mustard; MK-801, dizocilpine maleate; NMDA, N-methyl-o-aspartate. 97

98

Z.-H. QIN et ~1.

receptors, 3.3o,39X53 although these results are still somewhat controversial.26~52 To uncover the molecular bases for the upregulation of D, and D, dopamine receptors, alterations in the levels of D, and D, dopamine receptor mRNAs have been studied following 6-hydroxydopamine-induced striatal lesions or chronic treatment with dopaminergic antagonists. These studies showed that 6-hydroxydopamine lesions increase the level of DZ dopamine receptor mRNA2,23,50 but decrease the level of D, dopamine receptor mRNA.25.S3 Consistent with this finding are the results showing that 6-hydroxydopamine lesions increase the rate of synthesis of D, dopamine receptors49 but decrease the rate of synthesis of D, dopamine receptorss3 Results of studies on the effects of chronic treatment with D, dopamine receptor antagonists on the level of D, dopamine receptor mRNA were inconsistent, since both increases4s5.7.‘9*33,40 and no change’7,25,57,62.“8 in the level of D, dopamine receptor mRNA have been reported. Many factors may contribute to this apparent discrepancy. Among them is the magnitude and persistence of blockade of dopamine receptors which in many studies is uncertain. Reversible antagonists are often administered by intermittent injection (once or twice a day). This drug regime results in large fluctuations in the concentration of drug at receptor sites. The fluctuation of input to receptors may cause fluctuations in gene transcription, and alterations in the steady-state levels of D, dopamine receptor mRNA may not become evident, particularly if its turnover rate is rapid.” Although it is generally accepted that D, and D, dopamine receptors are involved in regulating diverse psychomotor behaviors, these behaviors appear to be influenced by other neurotransmitter systems as well.” In recent years, there has been growing interest in the possibility that some dopamine-associated responses are influenced by interactions between dopamine and glutamate systems in basal ganglia. That there are such interactions at a presynaptic level is shown by studies indicating that D, dopamine receptors are present in the glutamatergic terminals and that N-methyl-D-aspartate (NMDA) receptors, one of the subtypes of glutamate receptors, are present on dopaminergic terminals in striatum.‘0.63 These presynaptic receptors appear to exert mutual regulatory effects on the release of the respective there are some neurotransmitters,36~3’~65~67 although inconsistent results which remain to be clarified.3’,43,44 Interactions between dopamine and glutamate may also exist at the postsynaptic level since both systems send efferent projections to the same target neurons in basal ganglia, especially in striatum and nucleus accumbens.6~56 In behavioral studies of rodents, blockade of NMDA receptors with dizocilpine (MK801) a non-competitive NMDA receptor antagonist, produces locomotion and stereotypy which, in many aspects, are similar to those produced by D2 receptor agonists. These effects can be antagonized by pre-

treatment with D, receptor antagonists, suggesting that these two systems have opposite effects on the function of basal ganglia.35.‘“.59.“‘.66 Since the molecular consequences of the interactions between dopamine and glutamate at the postsynaptic level are still poorly understood, in the present studies we examined the effects of blocking dopamine and NMDA receptors on the expression of D, and D, dopamine receptors in basal ganglia. To achieve persistent blockade of D, and DZ dopamine receptors, two irreversible dopamine receptor antagonists, N-ethoxycarbonyl-2-dihydroquinoline (EEDQ), which blocks both D, and D, receptors4’ and fluphenazine-N-mustard (FNM), which preferentially blocks D, dopamine receptors,60,67 were used. To achieve persistent blockade of NMDA receptors, a non-competitive NMDA receptor antagonist, MK-801, was continuously infused with osmotic mini-pumps. The effects of these treatments on D, and D, dopamine receptors and D, and D, dopamine receptor mRNAs were then measured. These studies revealed that blockade of NMDA receptors has a greater effect on the level of D, dopamine receptor mRNA than blockade of dopamine receptors. These results may explain some of the recently described behavioral effects of MK-801.

EXPERIMENTAL

PROCEDURES

Animals

Male Swiss Webster mice weighing 22-24 g were purchased from ACE Animals Inc. (Boyertown, PA). The mice were housed in groups of 10 per cage in a temperaturecontrolled room (22°C) with a 12-h light/dark cycle. All animals had free access to food and water. Chemiculs

EEDQ was purchased from Aldrich Chemical Co. (Milwaukee, WI). FNM and MK-801 were purchased from Research Biochemicals Inc (IBI, Natick, MA). [‘H]SCH23390 (specific activity = 86.5 Ci/mmol) and [‘HIraclopride (specific activity = 80.3 Ci/mmol) were purchased from New England Nuclear (NEN, Boston). A 36-mer oligonucleotide probe complementary to the D, dopamine receptor mRNA and a 39-mer oligonucleotide probe complementary to the D, dopamine receptor mRNA were designed and synthesized as described earlier.‘3.64 Drug administration

EEDQ and FNM were dissolved in absolute alcohol and diluted with 0.9% NaCl (alcohol :0.9%NaCl= 1: 2). EEDQ was administered to mice subcutaneously daily for two and six days (60pmol/kg on the first day and 30pmol/kg thereafter). FNM was administered daily (20 nmoI/kg, i.p.) for t&e days. All mice were killed 24-h after the last injection. Control groups received vehicle injections only (0.1 ml/20 g body weight). MK-801 (in 0.9% NaCl with 1% ascorbic acid) was administered either by acute injections (1.2 pmol/kg, i.p.; equivalent to 0.4 mg/kg, i.p.). and mice were killed 2 h after the injection, or by continuous infusion, using Alzet osmotic minipumps (1.2 pmol/kg per h) for two and six days. The dose used in the acute experiments was similar to that used in rodents by other investigators.‘0.3’ Control groups were treated with vehicle only. To minimize weight loss, animals received oral injections of concentrated milk.

MK-801 decreases Dr receptor mRNA and D, receptors RESULTS

Preparation of brain sections

Mice were decapitated and the brains dissected on a precooled plate. The brains were quickly frozen on powdered

dry-ice and stored at -80°C. Coronal brain sections (12pm) were cut on a cryostat at - 20°C and thaw-mounted on to gelatin-coated microscope slides. The sections were stored at -80°C until processing for receptor autoradiography or in situ hybridization histochemistry. Receptor autoradiography

Receptor autoradiography was performed by a modification of the method described by Kohler and Radesater” and McCabe et aL4’ Briefly, sections were dried under vacuum and pre-incubated in 50 mM Tris-HCl buffer twice for Smin. For D, dopamine receptor assays, the sections were incubated in 50 mM Tris-HCl buffer, containing 2 nM [‘H]SCH23390, for 60 min at 22°C. Nonspecific binding was determined by incubating anatomically adjacent sections in the presence of 2 PM SCH23390. For Dz dopamine receptor assays, the sections were incubated in 50 Tris-HC1 buffer, containing 2 nM [3H]raclopride, for 60 min at 22°C. Nonspecific binding was determined by incubating anatomically adjacent sections in the presence of 2 PM sulpiride. After incubation with radioactive ligands, all sections were rinsed, dried and exposed to X-ray film (Hyperfilm-3H, Amersham). Tritium standards (American Radiolabeled Chemicals Inc., St Louis, MO) and brain mash standards were co-exposed to the same X-ray film. Automdio~ams were quantified with an image analyzer system (DUMAS, Drexel University), and the densities of receptors were expressed as fmol/mg protein, In situ hybridization histochemistry The radioactive oligodeoxynucleotide probes were prepared by end-la~lling with [3sS]-dATP (NEN) using terminal deoxynucleotidyl transferase and were purified with NENSORB@” 20 nucleic acid purification cartridges, The hybridization procedure was performed according to procedures described earlier.‘* Briefly, the sections were fixed, rinsed, dehydrated and air-dried. Hybridization with 35Sm labeled oligodeoxynucleotide probes was achieved by incubating tissue sections with a hybridization solution containing 50% formamide, 4 x SSC, Denhard’s solution, 10% dextran sulfate, 0.25mgjml yeast tRNA, 0.5 mg/ml salmon sperm DNA, 10 nM dithiothreitol and 5 x lo6 c.p.m./ml 35S-labeled oligodeoxynucleotide nrobes. After 20 h of incubation, the sections were washed: dehydrated, dried and exposed to X-ray film (Hyperfilm$ max, Amersham) for seven to 10 days (D2 receptor mRNA) or 30 days (D, receptor mRNA). The resulting autoradiograms were quantified with an image analyser system (DUMAS, Drexel University). The levels of D, and D, dopamine receptor mRNAs were expressed as relative optical densities. Emulsion autoradiography

After hybridizing with the [35S]dATP-labe1edoligodeoxynucleotide probes, tissue sections were coated with autoradiographic emulsion (NTB-2, Kodak) diluted with distilled water (1: 1) and warmed to 32°C. The microscope slides were slowly dipped into emulsion and dried vertically overnight in the dark. The slides were stored for 30 days (D2 receptor mRNA) or 60 days (D, receptor mRNA) at 4°C. The slides were then developed, fixed and stained with hematoxylin and eosin. The mRNA levels in individual neurons were analysed with a Bioquant image analyser system. Statistical analysis

A one-way ANOVA followed by a Dunnett r-test was used when multiple means were compared. A two-group unpaired Student’s r-test was used when only two means were compared.

99

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N-ethoxycurbonyl-2-ethoxy-1,2-dihydroon D, and Dz dopam~e receptors in mouse

The effects of EEDQ treatment on D, and D, dopamine receptors in mouse brain as determined by receptor autoradiography are shown in Fig. 1. As may be seen, 24 h after the last injection of EEDQ there was a marked reduction in the binding of [3H]SCH23390 (Fig. IA) and [3H]raclopride (Fig. 2B). Profound inhibition was seen after two and six days of treatment. A quantitative analysis of the results of similar experiments from eight mice per group demonstrated that repeated treatment with EEDQ produced about a 90% reduction in D, (Fig. 1C) or Dz (Fig. ID) dopamine receptors in striatum, nucleus accumbens and olfactory tubercle. These results indicate that the dosage regimen of EEDG employed caused a profound and irreversible blockade of D, and D, dopamine receptors in these three brain areas. Efiet of N-ethoxycarbonyI-2-ethoxy-1,2-dihydruquinoline on D, and Dz dopamine receptor mRNA in mouse brain

The effects of EEDQ treatment on D, and D, dopamine receptor mRNA, as determined by in situ hybrid~ation hist~hemist~, are presented in Fig. 2. There was no apparent change in the levels of either D, (Fig. 2A) or D, (Fig. 2B) dopamine receptor mRNA in mouse brain. In confirmation of the conclusion drawn from visual observations, a quantitative analysis of the results of similar experiments from eight mice per group failed to show any significant effects of EEDQ on the levels of either D, (Fig. 2C) or D, (Fig. 2D) dopamine receptor mRNA in striatum, nucleus accumbens and olfactory tubercle. Eficts o~~uphenazine-N-mustard on D, and Dz dopa mine receptors in mouse brain The effects of FNM treatment with that of EEDQ on D, (Fig. 3A, C) and D, (Fig. 3B, D) dopamine receptors in mouse brain as determined by receptor autoradiography are compared in Fig. 3. The level of D, dopamine receptors as assessed from the binding of [3H]raclop~de was greatly reduced by treatment with either FNM or EEDQ. Unlike EEDQ, FNM only slightly decreased the binding of [‘H]SCH23390, suggesting that FNM was less effective in blocking D, dopamine receptors than in blocking D, dopamine receptors. A quantitative analysis of the results of similar experiments from eight mice per group revealed that both EEDQ and FNM inhibited about 90% of the D, dopamine receptors (Fig. 3D). However, whereas EEDQ treatment blocked about 90% of the D, dopamine receptors, FNM blocked only about 20% of D, dopamine receptors (Fig. 3C).

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Fig. 1. Effects of EEDQ on D~ and D 2 dopamine receptors in mouse brain as determined by receptor autoradiography. Mice were treated with EEDQ once daily (60 #mol/kg on the first day and 30 #mol on the second to sixth day, s.c.) for two and six days. Animals were killed 24 h after the last injection, and D~ and D 2 dopamine receptors in brain sections were measured by receptor autoradiography as described in Experimental Procedures. D~ and D 2 dopamine receptors were quantified using an image analyser (DUMAS). Each bar represents the mean value from eight mice. Vertical brackets indicate the standard error. Statistical comparisons between control and drug-treated groups were carried out by one-way ANOVA followed by a Dunnett t-test. ***P < 0.001 compared with vehicle-treated control samples. (A, C) D~ dopamine receptors; (B, D) D2 dopamine receptors. ST, corpus striatum, Acb, nucleus accumbens, OT, olfactory tubercle.

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Fig. 2. Effects of EEDQ on D~ and D 2 dopamine receptor mRNAs in mouse brain as determined by in situ hybridization histochemistry. Mice were treated with EEDQ as described in the legend to Fig. I and D] and D 2 dopamine receptor mRNAs were determined by in situ hybridization histochemistry as described in Experimental Procedures. D~ and D 2 dopamine receptor m R N A were quantified using an image analyser (DUMAS). Each bar represents the mean value from eight mice. Vertical brackets indicate the standard error. Statistical comparisons between control and drug-treated groups were carried out by one-way ANOVA followed by a Dunnett t-test. (A, C) D~ dopamine receptor mRNA; (B, D) D 2 dopamine receptor mRNA. ST, corpus striatum; Acb, nucleus accumbens; OT, olfactory tubercle. NSC 601

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Fig. 4. Effects of F N M on D~ and D 2 dopamine receptor m R N A s in mouse brain as determined by in situ hybridization histochemistry. Mice were treated with F N M or E E D Q for five days as described in the legend to Fig. 3, and D~ and D 2 dopamine receptor m R N A s were measured by in situ hybridization histochemistry as described in Experimental Procedures. D~ and D 2 dopamine receptor m R N A s were quantified using an image analyser (DUMAS)• Each bar represents the mean value from eight mice. Vertical brackets indicate the standard error. Statistical comparisons between control and drug-treated groups were carried out by one-way A N O V A followed by a Dunnett t-test. (A, C) D~ dopamine receptor m R N A ; (B, D) D 2 dopamine receptor m R N A . ST, corpus striatum; Acb, nucleus accumbens; OT, olfactory tubercle•

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Fig. 5. Effects of MK-801 on D, dopamine receptor mRNA in mouse brain as determined by in situ hyb~di~tion hist~hemist~. Mice were treated ~ntinuously with MK-801 (1.2 pmol/kg per h, equivalent to 0.4 m&kg/h) for two and six days, or were acutely injected with MK-801 (I 2 pmol/kg, i.p.) and were killed 2 h after the acute injection D, dopamine receptor mRNA was determined by in situ hybridization histochemistry as described in Experimental Procedures.

MK-801 decreases D, receptor mRNA and D, receptors

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Fig. 6. Effects of MK-801 on D, dopamine receptor mRNA in mouse brain as determined by in siru hybridization histochemistry. Mice were treated continuously with MK-801 (1.2 pmol/kg per h) for two and six days, or were acutely injected with MK-801 (1.2 .nmol/kg, i.p.) and were killed 2 h after the acute injection. D, dopamine receptor mRNA was determined by itp situ hyb~dization hist~hemistry as described in Experimental Procedures.

105

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Effect o f fluphenazine-N-mustard on D l and D2 dopamine receptor m R N A s in mouse brain

A comparison of the effects of FNM treatment with that of EEDQ on D~ and D 2 dopamine receptor mRNAs in mouse brain is shown in Fig. 4. From the representative autoradiograms generated following in situ hybridization histochemistry it may be seen that, like EEDQ, FNM also failed to change the levels of either D1 (Fig. 4A) or D 2 (Fig. 4B) dopamine receptor mRNA. A quantitative analysis of the results of similar experiments from eight mice per group confirmed these visual observations. Again, treatment with either FNM or EEDQ failed to significantly alter the level of D~ (Fig. 4C) or D 2 (Fig. 4D) dopamine receptor mRNA. Effects o f dizocilpine on DI and D2 dopamine receptor m R N A s in mouse brain

Representive autoradiograms of the effects of MK801 on D~ and D2 dopamine receptor mRNA are shown in Figs 5 and 6. As may be seen, acute injections of MK-801 failed to change the level of either D~ or D2 dopamine receptor mRNA in any region examined. A small decrease in the levels of D~ dopamine receptor mRNA was evident in striatum at two days but not at six days of continuous treatment with MK-801, and there was no apparent effect of MK-801 on D~ dopamine receptor mRNA in other brain regions examined. By contrast, a large reduction in the levels of D2 dopamine receptor mRNA was seen in striatum, nucleus accumbens and olfactory tubercle after two and six days of treatment with

MK-801. A quantitative analysis of the results of similar experiments from six to seven mice per group showed that continuous treatment with MK-801 significantly decreased the levels of D 1 dopamine receptor mRNA only in striatum after two days of treatment and no effect was seen after six days of treatment (Fig. 7A). MK-801 induced a highly significant decrease in the levels of D 2 dopamine receptor mRNA in striatum, nucleus accumbens and olfactory tubercle after two and six days treatment (Fig. 7B). Quantitative analysis of the effects of acute injection of MK-801 showed no statistically significant changes in D~ and D 2 dopamine receptor mRNA in any brain region examined (data not shown). Effects o f dizocilpine on D 1 and D 2 dopamine receptors in mouse brain

Representative autoradiograms showing the effects of MK-801 on D 1 and D 2 dopamine receptors are presented in Figs 8 and 9. Neither acute (2 h) nor continuous treatment with MK-801 changed the density of D~ dopamine receptors in mouse brain. However, continuous, but not acute, treatment with MK-801 produced a small decrease in the density of Dz dopamine receptors in posterior striatum. A quantitative analysis of the results of similar experiments from six to seven mice per group demonstrated that continuous treatment with MK-801 had no significant effect on the density of D~ dopamine receptors in any brain region examined after two and six days of treatment (Fig. 10A). By contrast, continuous treatment with MK-801 significantly decreased the

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Fig. 7. Quantitative analysis of the effects of MK-801 on D~ and D 2 receptor mRNA in mouse brain. Mice were treated with MK-801, and D~ and D2 dopamine receptor mRNAs were measured as described in the legends to Figs 5 and 6. D I (Fig. 7A) and D2 (Fig. 7B) dopamine receptor mRNAs were quantified using an image analyser (DUMAS) as described in Experimental Procedures. Each bar represents the mean value from six to seven mice. Vertical brackets indicate the standard error. Statistical comparisons were carried out by one-way ANOVA followed by a Dunnett t-test. *P < 0.05, ***P < 0.001 compared with vehicle-treated control samples. ST(A), anterior corpus striatum; ST(P), posterior corpus striatum; Acb, nucleus accumbens; OT, olfactory tubercle.

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Fig. 8. Effects of MK-801 on D, dopamine receptors in mouse striatum as determined by receptor autoradiography. Mice were treated continuously with MK-801 (1.2 pmol/kg per h) for two and six days, or were acutely injected with MK-801 (1.2 pmol/kg, i.p.) and were killed 2 h after the acute injection. D, dopamine receptors were determined by receptor autoradiography as described in Experimental Procedures.

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D2 RECEPTOR ANTEWOR

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STRIATUM

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of MK-801 on D, dopamine receptors in mouse brain as determined by receptor Mice were treated as described in Fig. 8, and D, dopamine receptors were determined by receptor autoradiography as described in Experimental Procedures.

density of D, dopamine receptors in posterior striaturn. There were also smaller decreases in the densities of D, dopamine receptors in other brain areas examined, although these changes were not statisti-

tally significant (Fig. 10B). There were no significant effects of acute injection of MK-801 on either D, or D, receptors in any brain region examined by quantitative analysis (data not shown).

109

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Fig. 10. Quantitative analysis of the effects of MK-80I on D~ and D, dopamine receptors in mouse brain. Mice were treated with MK-801 and D~ and D e dopamine receptors were measured as described in the legends to Figs 8 and 9. D~ (A) and D 2 (B) dopamine receptors were quantified using an image analyser (DUMAS) as described in Experimental Procedures. Each bar represents the mean value from six to seven mice. Vertical brackets indicate the standard error. Statistical comparisons were carried by one-way ANOVA followed by a Dunnett t-test. *P < 0.05 compared with vehicle-treated control samples. ST(A), anterior corpus striatum; ST(P), posterior corpus striatum: Acb, nucleus accumbens: OT, olfactory tubercle.

Cellular ana@sis of the c~bcts qf continuous treatment with dizocilpine on D, doparnine receptor rnRNA in mouse striatum Representative emulsion autoradiograms of experiments performed above are shown in Fig. 11. As may be seen, MK-801 markedly reduced the level of D2 dopamine receptor m R N A in individual striatal neurons. A quantitative analysis of the results of similar experiments from five mice per group, performed by counting the number of radioactive grains in the cells, showed that the number of cells in anterior striatum (Fig. t2A) and posterior striatum (Fig. 12B) expressing high levels of D 2 dopamine receptor m R N A was reduced by continuous treatment with MK-801. The average level of D: dopamine receptor m R N A expressed in individual striatal neurons was also significantly reduced in both anterior (Fig. 12A, inset) and posterior (Fig. 12B, inset) striatum by MK-801 treatment. DISCUSSION

This paper compares the effects of persistently blocking dopaminergic input with the effects of persistently blocking glutamatergic input on dopamine receptors and their respective m R N A s . To persistently block Dj and D, dopamine receptors, the irreversible dopamine receptor antagonists E E D Q and F N M were used. That E E D Q irreversibly blocks both D~ and D~ dopamine receptors is supported by the data showing that more than 90% of the D~ and D: dopamine receptor binding sites are lost 24 h after administering EEDQ. Moreover, since E E D Q was administered daily, and since the recovery of D~

dopamine receptors after E E D Q treatment requires the synthesis of new receptors, 3~ blockade of D l and D: dopamine receptors should be well maintained during the course of the treatment. Based on previous studies on the regulation of receptors, one would predict that persistent and profound irreversible blockade of dopamine receptors should increase the rate of synthesis of new receptor molecules and that this may require an increase in the levels of dopamine receptor messenger RNAs. However, in this study, we did not find any significant change in the levels of either D~ and D 2 dopamine receptor m R N A s in striatum, nucleus accumbens and olfactory tubercle with up to 6 days of E E D Q treatment. Increases in the levels of D l and D e dopamine receptor m R N A were also not seen during four days of a drug wash-out period after six days of treatment with E E D Q (not shown). The pharmacological effects of E E D Q may be complicated by the fact that E E D Q blocks both D~ and D 2 dopamine receptors as well as some other neurotransmitter receptors, l For these reasons, we included in this study another, more selective irreversible dopamine receptor antagonist, F N M . F N M has been shown to be a relatively selective irreversible Dz receptor antagonist with few effects on D~ dopamine receptors and little or no effects on other neurotransmitter receptors. 6°67 The present results support this conclusion as F N M was as effective as E E D Q in blocking D e dopamine receptors but only slightly blocked Dt dopamine receptors. Like E E D Q , F N M did not produce any significant effect on the levels of either D l or D: dopamine receptor m R N A in striatum, nucleus accumbens or olfactory tubercle.

MK-801 2d

MK-801 6d

Fig. Il. Cellular analysis of the effects of continuous treatment with MK-801 on Dz dopamine receptor mRNA in mouse striatum. Mice were treated and D, receptor mRNA was determined by in sita hybridization histochemistry as described in the legend to Fig. 6. Brain sections were coated with autoradiographic emulsion and analysed as described in Experimental Procedures. Representative autoradiograms of anterior corpus striatum are shown. Scale bar = 40 ~lrn.

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mRNA in mouse striatal cells. Mice were treated and the cellular analysis of D 2 dopamine receptor mRNA was determined by in situ hybridization histochemistry as described in the legend to Fig. I 1. D 2 dopamine receptor mRNA in individual striatal neurons was quantitatively analysed using a Bioquant image analyser as described in Experimental Procedures. The radioactive grains were counted in each cell under light microscopy. Each bar represents the mean value (after subtracting the background) from five mice. Vertical brackets indicate the standard error. Statistical comparisons were carried out by one-way ANOVA followed by a Dunnett t-test. ***P < 0.001 compared with vehicle-treated control samples. A, anterior corpus striatum; B, posterior corpus striatum.

This study, therefore, provides additional evidence in support of results of other investigators which showed that blockade of dopaminergic receptors produces little or no increase in D2 dopamine receptor m R N A . ]7'57'6z'6sThis issue is complex, however, as other studies have shown that acute treatment with haloperidol produces a small and transient decrease in D2 dopamine receptor m R N A in rat striatum, ~ whereas chronic treatment with haloperidol produces a slight increase in D2 dopamine receptor m R N A in rat striatum? '7"]933"4°Chronic treatment of mice with haloperidol failed to show an increase in D 2 dopamine receptor m R N A in striatum. 25 The apparent

discrepancies among the various studies may be due to different species used, different lengths of treatments and different dosage employed, and to the fact that persistent stimulation or inhibition of dopaminergic receptors produces a relatively small effect on the dopamine receptors transcripts. The present studies lend further support to the concept that important and complex interactions exist between glutamate and dopamine systems. However, the molecular bases for these interactions are still poorly understood. Biochemical studies showed that glutamate increases the release of dopamine in vitro via N M D A receptors, ~7"6~ but the

I12

Z.-H. QW et nl.

physiological role of glutamate in the release of dopamine in oivo is not clear.3’.43Behavioral studies indicate that blockade of NMDA receptors with MK-801 increases locomotor activity and stereotypy and that these effects are antagonized by pretreatment with D, antagonist haloperido1.24.3s~3B~59.6’.66 In an attempt to characterize the interactions between NMDA receptors and.different subtypes ofdopamine receptors, investigators found that MK-801 potentiated the behavioral effects of D, dopamine agonists but inhibited the behavioral effects of Dz dopamine agonists.24+5,46These results suggested that NMDA receptors have differential effects on D, and D? dopamine receptor-mediated behaviors. Other recent studies showed that chronic treatment with MK-801 decreased the behavioral responses to acute injections of the D, dopamine agonist quinpirole but failed to alter the behavioral responses to the D, dopamine agonist SKF38393.1H These investigators also found a 30% reduction in the density of D, dopamine receptors but no change in the density of D, dopamine receptors in mouse striatum.2* Taken together, these results imply that the giutamatergic input, through interaction with NMDA receptors, may increase the steady-state level of D, dopamine receptors in striaturn and thereby increase D, dopamine receptormediated behaviors. In the present studies, we found that continuous infusion of MK-801 produced a rapid and profound reduction in the levels of D, dopamine receptor mRNA in striatum, nucleus accumbens and olfactory tubercle. This decrease in the levels of D, dopamine receptor mRNA was also shown at the cellular level. MK-801 significantly reduced the level of D, dopamine receptor mRNA in individual striatal neurons and in the number of neurons expressing a high level of D2 dopamine receptor mRNA. Treatment with MK-801 also produced a small decrease in the density of Dz dopamine receptors in all brain areas examined, but only in posterior striatum did this difference reach statistically significant levels. The relatively small changes in the densities of D, dopamine receptors could be explained by the relatively slow turnover rate of D, dopamine receptors.53 In contrast to its effect on D? dopamine receptors, MK-801 had little effect on D, dopamine receptor mRNA and had no effect on the density of D, dopamine receptors. These results are consistent with the results of DaIl’Olio et ~1.‘~ and Gandohi and Dall’Olio” who showed that MK-801 decreased D2 dopamine receptors but had no effect on D, dopamine receptors, and suggest that MK-801 selectively down-regulates the expression of D, dopamine receptors.

Acute administration of MK-801 failed to alter the mRNA levels or receptor densities of either D, or D, dopamine receptors as measured 2 h after the injection, indicating that the effects of MK-801 on the level of Dz dopamine receptors and their transcripts are long-term effects mediated by NMDA receptors. The molecular link between NMDA receptors and the expression of D2 dopamine receptor gene is still unknown. Some studies indicate that NMDA receptor agonists increase the release of dopamine in vitro.3i.65 However, there is also evidence that the NMDA antagonist MK-801 increases the release of dopamine in mice.37*63.65 Further, MK-801 induces locomotion and stereotypy in rodents, and these behavioral effects are blocked by D, receptor antagonists, suggesting that MK-801 may release endogenous dopamine.“8~s9~6’Carlsson and Carlsson” suggested that the increase in dopamine release could result from an inhibition of glutamatergic transmission in striatum. From these studies, one might predict that MK-801 would downregulate dopamine receptors. Alternatively, MK-801 may decrease the level of D, dopamine receptor mRNA by interfering with glutamatergic transmission in basal ganglia. This notion is supported by other studies showing that repeated administration of MK-801 decreased the density of D, dopamine receptors and decreased the behavioral responses to the D, receptor agonist quinpirole. i*,21Regardless of the specific mechanisms involved, the present findings offer additional molecular evidence illustrating the interactions between glutamate and D, and D, dopamine receptors in basal ganglia.

CONCLUSIONS

The present results show that continuous blockade of D, dopamine receptors or of Dr and D, dopamine receptors had no significant effect on the levels of either D, or DZ dopamine receptor mRNA in mouse brain. By contrast, continuous biockade of NMDA receptors produced rapid and profound decreases in the levels of D, dopamine receptor mRNA and small decreases in the density of D, dopamine receptors in basal ganglia. This study suggests that glutamate plays an important regulatory role on the expression of D2 receptors and, therefore, on D, dopamine receptor-associated behaviors.

Acknowledgement-This

MH42148 awarded Health.

work was by the National

supported by Grant Institute of Mental

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