- Email: [email protected]
Quantification of dopamine D3 receptor mRNA level associated with the development of amphetamine-induced behavioral sensitization in the rat brain
Neuroscience Letters 264 (1999) 69–72
Quantification of dopamine D3 receptor mRNA level associated with the development of amphetamine-induced behavioral sensitization in the rat brain Hisao Hondo a, Rebecca H. Spitzer a, Brone Grinius a, Neil M. Richtand a , b ,* a
Department of Psychiatry, University of Cincinnati College of Medicine, 231 Bethesda Avenue, ML 559, Cincinnati, OH 45267-0559, USA b Cincinnati Veterans Affairs Medical Center, Psychiatry Service, V-116A 3200 Vine Street, Cincinnati, OH 45220, USA Received 12 March 1998; received in revised form 9 February 1999; accepted 10 February 1999
Abstract We hypothesized that changes in expression of dopamine (DA) D3 receptor gene in the rat brain would correlate with the behavioral sensitization induced by amphetamine (AMPH). In order to test this hypothesis, we measured D3 receptor mRNA levels in the striatum, nucleus accumbens and prefrontal cortex, in individual rats following AMPH treatment (2.5 mg/kg s.c., for 5 consecutive days) using a ribonuclease protection assay method. We observed similar levels of D3 receptor mRNA in saline and AMPH treated animals in each brain region examined. These results suggest behavioral sensitization to AMPH is not mediated through postsynaptic transcriptional regulation of D3 receptor. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Dopamine D3 receptor; Behavioral sensitization; Amphetamine; Ribonuclease protection assay
Repeated intermittent administration of amphetamine (AMPH) to rats produces progressive and enduring enhancement of responses, such as hyperactivity and stereotyped behavior, called behavioral sensitization (see Ref. [13] for a review). It has been suggested that behavioral sensitization is a useful animal model of amphetamine psychosis and schizophrenia [13]. Converging evidence indicates a critical role for dopaminergic pathways in the phenomenon. In rats, behaviors which exhibit sensitization include locomotor and stereotyped behaviors. Lesion of the mesolimbic dopamine (DA) system attenuates the locomotor stimulant actions of AMPH [3], while the stereotypy producing effects can be blocked by lesion of the nigro-striatal DA pathway [4]. AMPH has indirect DA agonist properties and pretreatment with DA antagonist blocks development of behavioral sensitization [8]. Previous studies of DA receptor binding following treatment with AMPH have shown inconsistent results [13]. * Corresponding author. Tel.: +1-513-558-6657; fax: +1-513-5584805; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(99)001 63-9
These ligand binding studies, performed prior to the molecular cloning of five different types of DA receptors, could not distinguish among DA receptor subtypes. More recently, several research groups have investigated the effects of chronic AMPH treatment on levels of DA receptor mRNA in experimental animals. However, these investigations have been limited to the D1 and D2 receptors [7,11]. The predominant limbic localization of D3 receptor [1, 12] suggests that D3 receptor could be an important mediator of behavioral sensitization. The behavioral and pharmacological effects of the relatively D3 selective antagonist U-99194A [20], as well as studies with D3 receptor knockout mice (reviewed in [21]), suggest an inhibitory role of the D3 receptor in mediating the effects of concurrent D1 and D2 receptor stimulation. In order to test the hypothesis that decreased gene expression of the D3 receptor underlies behavioral sensitization, we measured D3 receptor mRNA levels in the rat striatum and nucleus accumbens after repeated administration of AMPH using a ribonuclease protection assay (RPA) method. In addition, mRNA level was also measured in the prefrontal cortex, since much evidence suggests that this region contributes to striatal and nucleus
1999 Elsevier Science Ireland Ltd. All rights reserved.
70
H. Hondo et al. / Neuroscience Letters 264 (1999) 69–72
accumbens DA transmission as well as locomotor activity [2,18]. Male Sprague–Dawley rats (350–400 g) were housed individually and habituated for 3 days to injection by saline in their home cages and then received either saline (s.c.) or AMPH (2.5 mg/kg, s.c.) for 5 additional days. This AMPH treatment regimen produces pronounced sensitization characterized by a progressively more rapid onset and intensification of stereotypy, and enhanced post-stereotypy hyperactivity [15]. Six days after the last injection, rats were sacrificed by decapitation and brains were quickly removed and placed on ice. Dissection was performed using a coronal rodent brain matrix as previously described [12]. Briefly, the most anterior brain region dissected was used for assay of prefrontal cortex after peeling away olfactory bulb. Striatum was taken as a circular punch 3 mm in diameter. Nucleus accumbens was taken as the portion of the same slice ventral to an extension of the anterior commisure. Following dissection, brain regions were frozen in liquid nitrogen and stored at −80°C prior to use. Total RNA was extracted from the rat striatum, nucleus accumbens and prefrontal cortex with TRI REAGENT (Molecular Research Center, Cincinnati, OH) following the manufacturer’s instruction. Radiolabeled riboprobes were produced by in vitro transcription of linearized plasmids containing fragments of the D3 receptor cDNA using a MAXI script kit (Ambion, Austin, TX). Plasmid D3 receptor construct was prepared by polymerase chain reaction amplification of rat brain cDNA and linearized by digestion with Eco RI as previously described [12]. Reaction contained 0.5 mg linearized plasmid; transcription buffer (as supplied by manufacturer); 0.5 mM ATP, CTP and GTP; 3.125 mM of 32P-UTP (800 Ci/ mmol, NEN Life Science Products, Boston, MA); and 20 units T7 RNA polymerase and 10 units Ribonuclease inhibitor, in a volume of 20 ml. The mixture was incubated for 1 h at 37°C, then treated with 1 ml of DNase I (2 units/l) for 15 min at 37°C. The labeled antisense RNA probe for cyclophilin mRNA was prepared using 1 mg vector obtained from Ambion. The protocol for its synthesis was similar to that described above. The exceptions were that in this case, 1.0 mM 32P-UTP and 50 mM cold UTP were used. Full length riboprobes were gel purified, then eluted from the gel at 37°C for 6 h with buffer containing 0.5 M ammonium acetate, 1 mM EDTA and 0.2% SDS. The RPA was performed using an RPA II Kit (Ambion, Austin, TX). For the assay of D3 mRNA in striatum, approximately 200 000 cpm of D3 32P-riboprobe and 20 000 cpm cyclophilin 32P-riboprobe were added to 18 mg total RNA in water. For the assay of D3 mRNA in nucleus accumbens, approximately 330 000 cpm of D3 32P-riboprobe and 10 000 cpm cyclophilin 32P-riboprobe were added to 18 mg total RNA in water. For the assay of D3 mRNA in prefrontal cortex, approximately 300 000 cpm of D3 32P-riboprobe and 40 000 cpm cyclophilin 32P-riboprobe were added to 40 mg total RNA in water.
The mixtures were precipitated by adding 0.1 volume of 5.0 mM ammonium acetate and 2.5 volumes of ethanol at −20°C for 30 min and centrifuged at 14 000 rpm at 4°C for 15 min. The pellets were hybridized in 20 ml buffer containing 80% deionized formamide, 100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4) and 1 mM EDTA for 16 h at 45°C. After hybridization, 1 unit RNase A and 40 units RNase T1 were added. Samples were incubated at 37°C for 30 min to digest unhybridized RNA. The protected RNA fragments were precipitated, denatured by heating at 90°C for 4 min and separated on a 6% polyacrylamide/8 M urea gel. The negative control for each run consisted of 20 mg of yeast RNA processed as described above. Gels were dried and placed in a Phosphorimager GS-525 (BIO-RAD, Hercules, CA), and image intensity of bands corresponding to 396 base pairs (protected by D3 receptor mRNA) and 103 base pairs (protected by the cyclophilin mRNA) determined. To control for differences in the RNA concentration of each sample, the results were expressed as a ratio of D3 receptor mRNA densities to cyclophilin mRNA densities. Differences between AMPH-treated rats and saline-treated control rats were tested by Mann–Whitney Rank Sum test. Relative level of D3 receptor mRNA was measured in striatum, nucleus accumbens and prefrontal cortex of saline and AMPH treated rats. Pilot studies were performed to ensure that concentrations of probes used were in great excess (≥10-fold) of measured mRNAs in each brain region (data not shown). The radiolabeled probes were fully degraded in blank assays in which sample RNA was replaced with yeast transfer RNA. Fig. 1 shows a representative autoradiogram of D3 receptor and cyclophilin mRNA detected by RPA in each brain region. The bands for D3 receptor and cyclophilin mRNA migrated to the positions corresponding to 396 and 103 bases, respectively. As previously reported [1,12], D3 receptor mRNA was most abundant in the nucleus accumbens, however, reliable signals for the D3 receptor mRNA were also detected in the striatum
Fig. 1. A representative autoradiogram of D3 receptor and cyclophilin mRNA detected by ribonuclease protection assay. Total RNA from the rat striatum (18 mg, ST), nucleus accumbens (18 mg, NAc) or prefrontal cortex (40 mg, PFC) was hybridized with antisense cRNA probes, digested with RNase A/T1 and loaded on polyacrylamide/ urea gel. Upper panel, D3 receptor mRNA; lower panel, cyclophilin mRNA.
H. Hondo et al. / Neuroscience Letters 264 (1999) 69–72 Table 1 D3 receptor mRNA levels in the rat brain after chronic AMPH treatment Brain region
Striatum Nucleus accumbens Prefrontal cortex
D3 mRNA level Saline
AMPH
100 ± 12.6 100 ± 8.5 100 ± 13.3
101 ± 11.6 95 ± 7.3 89 ± 9.1
Results shown are ratios of D3 mRNA to cyclophilin mRNA in the same sample. Data are expressed as percent of saline treated control group and are means ± SEM for 9–10 samples.
and prefrontal cortex. In all brain regions examined, D3 receptor mRNA levels did not change significantly following chronic AMPH treatment (Table 1). Acute AMPH administration elevates extracellular DA content through action at the DA transporter in terminal fields including the striatum, nucleus accumbens and prefrontal cortex [8,9]. Some findings suggest that the increase in extracellular DA is augmented in the nucleus accumbens and striatum following repeated AMPH treatment ([8], see also Ref. [14]). However, attempts to find changes in DA receptors in the striatum using in vitro binding assays in animals showing sensitized responses to AMPH have been inconclusive: increases, decreases and no change in DA receptors have all been reported [13]. One possible reason for the observed inconsistencies is that these studies had been done using ligands which do not have selectivity between five different types of DA receptors. Since the cloning and identification of the dopamine D3 receptor in 1990 [17], studies to date indicate that D3 receptor is localized preferentially in limbic brain areas [1,12], and stimulation of D3 receptor appears to inhibit hyperactivity induced by AMPH. [5,19–21]. The dopamine D3 receptor may act as an autoreceptor exerting an inhibitory effect on presynaptic DA function [6]. However, there have also been several reports suggesting the existence of postsynaptic D3 receptor sites that directly inhibit locomotor activity after stimulation [5,19–21]. Blockade of D3 receptors with the relatively D3 selective antagonist U-99194A results in increased c-fos expression in medial prefrontal cortex [10], suggesting a role for D3 mediated pathways in regulating gene expression in this brain region. Recent studies examining expression of D3 receptor mRNA in human brain following chronic cocaine use, report an increase in the nucleus accumbens [16] but no change in the striatum [10]. Cocaine is another powerful psychostimulant and shares indirect DA agonist properties with AMPH. These observations led us to hypothesize that decreased D3 receptor transcription may be involved in AMPHinduced behavioral sensitization. Therefore, we examined D3 receptor mRNA levels after chronic AMPH administration. Despite these considerations, we failed to find evidence that behavioral sensitization to AMPH is mediated through
71
transcriptional regulation of D3 receptor mRNA levels in the striatum, nucleus accumbens or prefrontal cortex. Although RPA is a specific and sensitive method to identify mRNA, care should be taken to interpret our results. Since RPA requires the dissection and subsequent homogenization of brain areas, fine anatomical resolution is lost. Consequently it is possible that changes in the level of mRNA in more discrete brain regions (e.g. the core and shell of the nucleus accumbens) might be masked. It is possible that the D3 receptor may play a role in behavioral sensitization through mechanisms other than postsynaptic transcriptional regulation. Further studies investigating the effects of more selective manipulation of D3 receptor signaling on behavioral sensitization should be done. Recently developed technologies such as antisense knockdown or knockout animals may be suitable for these purposes. In conclusion, we observed similar levels of D3 receptor mRNA in saline and AMPH treated animals. These results suggest postsynaptic transcriptional regulation of D3 mRNA level in the brain is most likely not involved in the development of behavioral sensitization to AMPH. This work was supported by a NARSAD Young Investigator Award to N.M.R. [1] Bouthenet, M.-L., Souil, E., Matres, M.-P., Sokoloff, P., Giros, B. and Schwartz, J.-C., Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA, Brain Res., 564 (1991) 203–219. [2] Carter, C.J. and Pycock, C.J., Behavioral and biochemical effects of dopamine and noradrenalin depletion within the medial prefrontal cortex, Brain Res., 192 (1980) 163–169. [3] Costall, B. and Naylor, R.J., Extrapyramidal and mesolimbic involvement with the stereotypic activity of D- and L-amphetamine, Eur. J. Pharmacol., 25 (1974) 121–129. [4] Creese, I. and Iversen, S.D., The pharmacological and anatomical substrates of amphetamine response in the rat, Brain Res., 83 (1975) 419–436. [5] De Boer, P., Enrico, P., Wright, J., Wise, L.D., Timmerman, W., Moor, E., Dijkstra, D., Wikstrom, H.V. and Westerink, B.H.C., Characterization of the effect of dopamine D3 receptor stimulation on locomotion and striatal dopamine levels, Brain Res., 758 (1997) 83–91. [6] Gilbert, D.B. and Cooper, S.J., 7-OH-DPAT injected into the accumbens reduces locomotion and sucrose ingestion: D3 autoreceptor-mediated effects? Pharmacol. Biochem. Behav., 52 (1995) 275–280. [7] Jaber, M., Cador, M., Dumartin, B., Normand, E., Stinus, L. and Bloch, B., Acute and chronic amphetamine treatments differentially regulate neuropeptide messenger RNA levels and Fos immunoreactivity in rat striatal neurons, Neuroscience, 65 (1995) 1041–1050. [8] Kalivas, P.W. and Stewart, J., Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity, Brain Res. Rev., 16 (1991) 223– 244. [9] Meador-Woodruff, J.H., Little, K.Y., Damask, S.P. and Watson, S.J., Effects of cocaine on D3 and D4 receptor expression in the human striatum, Biol. Psychatry, 38 (1995) 263–266. [10] Merchant, K.M., Figur, L.M. and Evans, D.L., Induction of c-fos mRNA in rat medial prefrontal cortex by antipsychotic drugs:
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H. Hondo et al. / Neuroscience Letters 264 (1999) 69–72 role of dopamine D2 and D3 receptors, Cereb. Cortex, 6 (1996) 561–570. Richtand, N.M., Kelsoe, J.R., Kuczenski, R. and Segal, D.S., Quantification of dopamine D1 and D2 receptor mRNA levels associated with the development of behavioral sensitization in amphetamine treated rats, Neurochem. Int., 31 (1997) 131– 137. Richtand, N.M., Kelsoe, J.R., Segal, D.S. and Kuczenski, R., Regional quantification of D1, D2, and D3 dopamine receptor mRNA in rat brain using a ribonuclease protection assay, Mol. Brain Res., 33 (1995) 97–103. Robinson, T.E. and Becker, J.B., Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev., 11 (1986) 157–198. Segal, D.S. and Kuczenski, R., In vivo microdialysis reveals a diminished amphetamine-induced DA response corresponding to behavioral sensitization produced by repeated amphetamine pretreatment, Brain Res., 571 (1992) 330–337. Segal, D.S. and Kuczenski, R., Behavioral pharmacology of amphetamine. In A. Cho and D.S. Segal (Eds.), Amphetamine and Its Analogues: Neuropharmacology, Toxicology and Abuse, Academic Press, Orlando, FL, 1994, pp. 81–115. Segal, D.M., Moraes, C.T. and Mash, D.C., Up-regulation of D3
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dopamine receptor mRNA in the nucleus accumbens of human cocaine fatalities, Mol. Brain Res., 45 (1997) 335–339. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. and Schwartz, J.-C., Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics, Nature, 347 (1990) 146–151. Tassin, J.P., Stinus, L., Simon, H., Blanc, G., Thierry, A.M., LeMoal, M., Cardo, B. and Glowinski, J., Relationship between the locomotor hyperactivity induced by A10 lesions and the destruction of the fronto-cortical dopaminergic innervation in the rat, Brain Res., 141 (1978) 267–281. Thorn, L., Ashmeade, T.E., Storey, V.J., Routledge, C. and Reavill, C., Evidence to suggest that agonist modulation of hyperlocomotion is via post-synaptic dopamine D2 or D3 receptors, Neuropharmacology, 35 (1997) 787–792. Waters, N., Lofberg, L., Haadsma-Svensson, S., Svensson, K., Sonesson, C. and Carlsson, A., Differential effects of dopamine D2 and D3 receptor antagonists in regard to dopamine release, in vivo receptor displacement and behaviour, J. Neural Transm. Gen. Sect., 98 (1994) 39–55. Xu, M., Unraveling dopamine D3 receptor function in response to psychostimulants using a genetic approach, Ann. N.Y. Acad. Sci., 844 (1998) 27–39.
Quantification of dopamine D3 receptor mRNA level associated with the development of amphetamine-induced behavioral sensitization in the rat brain Hisao Hondo a, Rebecca H. Spitzer a, Brone Grinius a, Neil M. Richtand a , b ,* a
Department of Psychiatry, University of Cincinnati College of Medicine, 231 Bethesda Avenue, ML 559, Cincinnati, OH 45267-0559, USA b Cincinnati Veterans Affairs Medical Center, Psychiatry Service, V-116A 3200 Vine Street, Cincinnati, OH 45220, USA Received 12 March 1998; received in revised form 9 February 1999; accepted 10 February 1999
Abstract We hypothesized that changes in expression of dopamine (DA) D3 receptor gene in the rat brain would correlate with the behavioral sensitization induced by amphetamine (AMPH). In order to test this hypothesis, we measured D3 receptor mRNA levels in the striatum, nucleus accumbens and prefrontal cortex, in individual rats following AMPH treatment (2.5 mg/kg s.c., for 5 consecutive days) using a ribonuclease protection assay method. We observed similar levels of D3 receptor mRNA in saline and AMPH treated animals in each brain region examined. These results suggest behavioral sensitization to AMPH is not mediated through postsynaptic transcriptional regulation of D3 receptor. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Dopamine D3 receptor; Behavioral sensitization; Amphetamine; Ribonuclease protection assay
Repeated intermittent administration of amphetamine (AMPH) to rats produces progressive and enduring enhancement of responses, such as hyperactivity and stereotyped behavior, called behavioral sensitization (see Ref. [13] for a review). It has been suggested that behavioral sensitization is a useful animal model of amphetamine psychosis and schizophrenia [13]. Converging evidence indicates a critical role for dopaminergic pathways in the phenomenon. In rats, behaviors which exhibit sensitization include locomotor and stereotyped behaviors. Lesion of the mesolimbic dopamine (DA) system attenuates the locomotor stimulant actions of AMPH [3], while the stereotypy producing effects can be blocked by lesion of the nigro-striatal DA pathway [4]. AMPH has indirect DA agonist properties and pretreatment with DA antagonist blocks development of behavioral sensitization [8]. Previous studies of DA receptor binding following treatment with AMPH have shown inconsistent results [13]. * Corresponding author. Tel.: +1-513-558-6657; fax: +1-513-5584805; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(99)001 63-9
These ligand binding studies, performed prior to the molecular cloning of five different types of DA receptors, could not distinguish among DA receptor subtypes. More recently, several research groups have investigated the effects of chronic AMPH treatment on levels of DA receptor mRNA in experimental animals. However, these investigations have been limited to the D1 and D2 receptors [7,11]. The predominant limbic localization of D3 receptor [1, 12] suggests that D3 receptor could be an important mediator of behavioral sensitization. The behavioral and pharmacological effects of the relatively D3 selective antagonist U-99194A [20], as well as studies with D3 receptor knockout mice (reviewed in [21]), suggest an inhibitory role of the D3 receptor in mediating the effects of concurrent D1 and D2 receptor stimulation. In order to test the hypothesis that decreased gene expression of the D3 receptor underlies behavioral sensitization, we measured D3 receptor mRNA levels in the rat striatum and nucleus accumbens after repeated administration of AMPH using a ribonuclease protection assay (RPA) method. In addition, mRNA level was also measured in the prefrontal cortex, since much evidence suggests that this region contributes to striatal and nucleus
1999 Elsevier Science Ireland Ltd. All rights reserved.
70
H. Hondo et al. / Neuroscience Letters 264 (1999) 69–72
accumbens DA transmission as well as locomotor activity [2,18]. Male Sprague–Dawley rats (350–400 g) were housed individually and habituated for 3 days to injection by saline in their home cages and then received either saline (s.c.) or AMPH (2.5 mg/kg, s.c.) for 5 additional days. This AMPH treatment regimen produces pronounced sensitization characterized by a progressively more rapid onset and intensification of stereotypy, and enhanced post-stereotypy hyperactivity [15]. Six days after the last injection, rats were sacrificed by decapitation and brains were quickly removed and placed on ice. Dissection was performed using a coronal rodent brain matrix as previously described [12]. Briefly, the most anterior brain region dissected was used for assay of prefrontal cortex after peeling away olfactory bulb. Striatum was taken as a circular punch 3 mm in diameter. Nucleus accumbens was taken as the portion of the same slice ventral to an extension of the anterior commisure. Following dissection, brain regions were frozen in liquid nitrogen and stored at −80°C prior to use. Total RNA was extracted from the rat striatum, nucleus accumbens and prefrontal cortex with TRI REAGENT (Molecular Research Center, Cincinnati, OH) following the manufacturer’s instruction. Radiolabeled riboprobes were produced by in vitro transcription of linearized plasmids containing fragments of the D3 receptor cDNA using a MAXI script kit (Ambion, Austin, TX). Plasmid D3 receptor construct was prepared by polymerase chain reaction amplification of rat brain cDNA and linearized by digestion with Eco RI as previously described [12]. Reaction contained 0.5 mg linearized plasmid; transcription buffer (as supplied by manufacturer); 0.5 mM ATP, CTP and GTP; 3.125 mM of 32P-UTP (800 Ci/ mmol, NEN Life Science Products, Boston, MA); and 20 units T7 RNA polymerase and 10 units Ribonuclease inhibitor, in a volume of 20 ml. The mixture was incubated for 1 h at 37°C, then treated with 1 ml of DNase I (2 units/l) for 15 min at 37°C. The labeled antisense RNA probe for cyclophilin mRNA was prepared using 1 mg vector obtained from Ambion. The protocol for its synthesis was similar to that described above. The exceptions were that in this case, 1.0 mM 32P-UTP and 50 mM cold UTP were used. Full length riboprobes were gel purified, then eluted from the gel at 37°C for 6 h with buffer containing 0.5 M ammonium acetate, 1 mM EDTA and 0.2% SDS. The RPA was performed using an RPA II Kit (Ambion, Austin, TX). For the assay of D3 mRNA in striatum, approximately 200 000 cpm of D3 32P-riboprobe and 20 000 cpm cyclophilin 32P-riboprobe were added to 18 mg total RNA in water. For the assay of D3 mRNA in nucleus accumbens, approximately 330 000 cpm of D3 32P-riboprobe and 10 000 cpm cyclophilin 32P-riboprobe were added to 18 mg total RNA in water. For the assay of D3 mRNA in prefrontal cortex, approximately 300 000 cpm of D3 32P-riboprobe and 40 000 cpm cyclophilin 32P-riboprobe were added to 40 mg total RNA in water.
The mixtures were precipitated by adding 0.1 volume of 5.0 mM ammonium acetate and 2.5 volumes of ethanol at −20°C for 30 min and centrifuged at 14 000 rpm at 4°C for 15 min. The pellets were hybridized in 20 ml buffer containing 80% deionized formamide, 100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4) and 1 mM EDTA for 16 h at 45°C. After hybridization, 1 unit RNase A and 40 units RNase T1 were added. Samples were incubated at 37°C for 30 min to digest unhybridized RNA. The protected RNA fragments were precipitated, denatured by heating at 90°C for 4 min and separated on a 6% polyacrylamide/8 M urea gel. The negative control for each run consisted of 20 mg of yeast RNA processed as described above. Gels were dried and placed in a Phosphorimager GS-525 (BIO-RAD, Hercules, CA), and image intensity of bands corresponding to 396 base pairs (protected by D3 receptor mRNA) and 103 base pairs (protected by the cyclophilin mRNA) determined. To control for differences in the RNA concentration of each sample, the results were expressed as a ratio of D3 receptor mRNA densities to cyclophilin mRNA densities. Differences between AMPH-treated rats and saline-treated control rats were tested by Mann–Whitney Rank Sum test. Relative level of D3 receptor mRNA was measured in striatum, nucleus accumbens and prefrontal cortex of saline and AMPH treated rats. Pilot studies were performed to ensure that concentrations of probes used were in great excess (≥10-fold) of measured mRNAs in each brain region (data not shown). The radiolabeled probes were fully degraded in blank assays in which sample RNA was replaced with yeast transfer RNA. Fig. 1 shows a representative autoradiogram of D3 receptor and cyclophilin mRNA detected by RPA in each brain region. The bands for D3 receptor and cyclophilin mRNA migrated to the positions corresponding to 396 and 103 bases, respectively. As previously reported [1,12], D3 receptor mRNA was most abundant in the nucleus accumbens, however, reliable signals for the D3 receptor mRNA were also detected in the striatum
Fig. 1. A representative autoradiogram of D3 receptor and cyclophilin mRNA detected by ribonuclease protection assay. Total RNA from the rat striatum (18 mg, ST), nucleus accumbens (18 mg, NAc) or prefrontal cortex (40 mg, PFC) was hybridized with antisense cRNA probes, digested with RNase A/T1 and loaded on polyacrylamide/ urea gel. Upper panel, D3 receptor mRNA; lower panel, cyclophilin mRNA.
H. Hondo et al. / Neuroscience Letters 264 (1999) 69–72 Table 1 D3 receptor mRNA levels in the rat brain after chronic AMPH treatment Brain region
Striatum Nucleus accumbens Prefrontal cortex
D3 mRNA level Saline
AMPH
100 ± 12.6 100 ± 8.5 100 ± 13.3
101 ± 11.6 95 ± 7.3 89 ± 9.1
Results shown are ratios of D3 mRNA to cyclophilin mRNA in the same sample. Data are expressed as percent of saline treated control group and are means ± SEM for 9–10 samples.
and prefrontal cortex. In all brain regions examined, D3 receptor mRNA levels did not change significantly following chronic AMPH treatment (Table 1). Acute AMPH administration elevates extracellular DA content through action at the DA transporter in terminal fields including the striatum, nucleus accumbens and prefrontal cortex [8,9]. Some findings suggest that the increase in extracellular DA is augmented in the nucleus accumbens and striatum following repeated AMPH treatment ([8], see also Ref. [14]). However, attempts to find changes in DA receptors in the striatum using in vitro binding assays in animals showing sensitized responses to AMPH have been inconclusive: increases, decreases and no change in DA receptors have all been reported [13]. One possible reason for the observed inconsistencies is that these studies had been done using ligands which do not have selectivity between five different types of DA receptors. Since the cloning and identification of the dopamine D3 receptor in 1990 [17], studies to date indicate that D3 receptor is localized preferentially in limbic brain areas [1,12], and stimulation of D3 receptor appears to inhibit hyperactivity induced by AMPH. [5,19–21]. The dopamine D3 receptor may act as an autoreceptor exerting an inhibitory effect on presynaptic DA function [6]. However, there have also been several reports suggesting the existence of postsynaptic D3 receptor sites that directly inhibit locomotor activity after stimulation [5,19–21]. Blockade of D3 receptors with the relatively D3 selective antagonist U-99194A results in increased c-fos expression in medial prefrontal cortex [10], suggesting a role for D3 mediated pathways in regulating gene expression in this brain region. Recent studies examining expression of D3 receptor mRNA in human brain following chronic cocaine use, report an increase in the nucleus accumbens [16] but no change in the striatum [10]. Cocaine is another powerful psychostimulant and shares indirect DA agonist properties with AMPH. These observations led us to hypothesize that decreased D3 receptor transcription may be involved in AMPHinduced behavioral sensitization. Therefore, we examined D3 receptor mRNA levels after chronic AMPH administration. Despite these considerations, we failed to find evidence that behavioral sensitization to AMPH is mediated through
71
transcriptional regulation of D3 receptor mRNA levels in the striatum, nucleus accumbens or prefrontal cortex. Although RPA is a specific and sensitive method to identify mRNA, care should be taken to interpret our results. Since RPA requires the dissection and subsequent homogenization of brain areas, fine anatomical resolution is lost. Consequently it is possible that changes in the level of mRNA in more discrete brain regions (e.g. the core and shell of the nucleus accumbens) might be masked. It is possible that the D3 receptor may play a role in behavioral sensitization through mechanisms other than postsynaptic transcriptional regulation. Further studies investigating the effects of more selective manipulation of D3 receptor signaling on behavioral sensitization should be done. Recently developed technologies such as antisense knockdown or knockout animals may be suitable for these purposes. In conclusion, we observed similar levels of D3 receptor mRNA in saline and AMPH treated animals. These results suggest postsynaptic transcriptional regulation of D3 mRNA level in the brain is most likely not involved in the development of behavioral sensitization to AMPH. This work was supported by a NARSAD Young Investigator Award to N.M.R. [1] Bouthenet, M.-L., Souil, E., Matres, M.-P., Sokoloff, P., Giros, B. and Schwartz, J.-C., Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA, Brain Res., 564 (1991) 203–219. [2] Carter, C.J. and Pycock, C.J., Behavioral and biochemical effects of dopamine and noradrenalin depletion within the medial prefrontal cortex, Brain Res., 192 (1980) 163–169. [3] Costall, B. and Naylor, R.J., Extrapyramidal and mesolimbic involvement with the stereotypic activity of D- and L-amphetamine, Eur. J. Pharmacol., 25 (1974) 121–129. [4] Creese, I. and Iversen, S.D., The pharmacological and anatomical substrates of amphetamine response in the rat, Brain Res., 83 (1975) 419–436. [5] De Boer, P., Enrico, P., Wright, J., Wise, L.D., Timmerman, W., Moor, E., Dijkstra, D., Wikstrom, H.V. and Westerink, B.H.C., Characterization of the effect of dopamine D3 receptor stimulation on locomotion and striatal dopamine levels, Brain Res., 758 (1997) 83–91. [6] Gilbert, D.B. and Cooper, S.J., 7-OH-DPAT injected into the accumbens reduces locomotion and sucrose ingestion: D3 autoreceptor-mediated effects? Pharmacol. Biochem. Behav., 52 (1995) 275–280. [7] Jaber, M., Cador, M., Dumartin, B., Normand, E., Stinus, L. and Bloch, B., Acute and chronic amphetamine treatments differentially regulate neuropeptide messenger RNA levels and Fos immunoreactivity in rat striatal neurons, Neuroscience, 65 (1995) 1041–1050. [8] Kalivas, P.W. and Stewart, J., Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity, Brain Res. Rev., 16 (1991) 223– 244. [9] Meador-Woodruff, J.H., Little, K.Y., Damask, S.P. and Watson, S.J., Effects of cocaine on D3 and D4 receptor expression in the human striatum, Biol. Psychatry, 38 (1995) 263–266. [10] Merchant, K.M., Figur, L.M. and Evans, D.L., Induction of c-fos mRNA in rat medial prefrontal cortex by antipsychotic drugs:
72
[11]
[12]
[13]
[14]
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