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Downregulation of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens
Neuroscience Letters 275 (1999) 1±4 www.elsevier.com/locate/neulet
Downregulation of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens AÊsa Rosin a,*, Sara Lindholm a, Johan Franck a, Jeanette Georgieva a, b a
Department of Clinical Neuroscience, Experimental and Clinical Drug Addiction Research Sections, CMM L8:01, Karolinska Institutet, S-171 76 Stockholm, Sweden b Department of Clinical Pharmacology, Karolinska Hospital, S-171 76 Stockholm, Sweden Received 7 April 1999; received in revised form 6 August 1999; accepted 6 August 1999
Abstract The combination of ethanol and cocaine is commonly abused by human addicts which has serious clinical consequences. Here, the effects of separately and concurrently administered ethanol and `binge' cocaine on kappa opioid receptor (KOR) mRNA in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of rats were studied. KOR mRNA was down-regulated in both brain regions during concurrent as well as separate treatment with these drugs. In the VTA, the most pronounced decrease was obtained following combined treatment with ethanol and `binge' cocaine. In the NAc, the strongest decrease was observed in the `binge' cocaine group. This profound decrease of KOR mRNA in regions important for brain reward suggests a potential role of the KOR system in the abuse of cocaine and ethanol. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kappa opioid receptor mRNA; `Binge' cocaine; Ethanol; Competitive polymerase chain reaction; Rat; Ventral tegmental area; Nucleus accumbens
Concurrent abuse of ethanol and cocaine is common among human addicts. This drug combination has been reported to increase the risk of morbidity and mortality as well as compulsive drug seeking behaviour [13]. Although the clinical consequences of dual substance abuse receive increasing attention, the reasons for its high prevalence are still poorly understood. Important progress has been made in recent years in understanding the pharmacodynamics of either drug alone. Thus, abuse and dependence on cocaine or ethanol may be related to the dysregulation of a common set of neurochemical pathways. A feature shared by both drugs is the targeting of nerve cells primarily in mesocorticolimbic and nigrostriatal brain dopaminergic systems to produce an altered behaviour. The mesolimbic dopaminergic neurons with cell bodies in the ventral tegmental area (VTA) projecting to receptive ®elds in the nucleus accumbens (NAc) and prefrontal cortex, are considered to mediate the reinforcing effects of both cocaine and ethanol [3]. Administration of cocaine or ethanol to rats stimulates dopa* Corresponding author. Tel.: 146-8-517-74692; fax 146-8517-76180. E-mail address: [email protected] (AÊ. Rosin)
mine (DA) release in the NAc. It is widely accepted that cocaine binds to and inactivates dopamine transporters (DATs) thereby enhancing dopaminergic transmission [5]. For ethanol, no speci®c receptor has been identi®ed but it is known that signal transmission through GABA and glutamate (NMDA) receptor is affected [4]. Closely connected to the DA system and involved in the effects of ethanol as well as cocaine is the endogenous opioid system [9]. In the NAc and dorsal striatum, DAergic neurotransmission is modulated by the opioid peptides dynorphins and enkephalins. The functional effects of dynorphin peptides are mediated by kappa opioid receptors (KORs). The present study was conducted to assess the effects of separate and combined exposure to cocaine and ethanol on KOR mRNA levels in the mesolimbic dopamine system using a newly developed quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. RTPCR represents a highly sensitive method for analyzing RNA levels [1] and allows the detection of very small mRNA amounts as well as their changes, in a single region. Male Sprague±Dawley rats weighing approximately 250-g (60 days old, purchased from B&K Universal, Stockholm, Sweden) were caged in groups of six, with free access
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 67 5- 8
AÊ. Rosin et al. / Neuroscience Letters 275 (1999) 1±4
2 Table 1 Drug treatment scheme Group
Day 1±12
Day 13
Day 14
A (n 6)
Ethanol 2 g/kg 2£ day
B (n 6)
Ethanol 2 g/kg 2£ day
C (n 6)
Saline 1 ml/kg 2£ day
D (n 6)
Saline 1 ml/kg 2£ day
Ethanol 2 g/kg 2£ day `Binge' cocaine 15 mg/kg £3 Ethanol 2 g/kg 2£ day `Binge' saline 1 ml/kg £3 Saline 1 ml/kg 2£ day `Binge' cocaine 15 mg/kg £3 Saline 1 ml/kg 2£ day `Binge' saline 1 ml/kg £3
Ethanol 2 g/kg 2£ day `Binge' cocaine 15 mg/kg £3 Ethanol 2 g/kg 2£ day `Binge' saline 1 ml/kg £3 Saline 1 ml/kg 2£ day `Binge' cocaine 15 mg/kg £3 Saline 1 ml/kg 2£ day `Binge' saline 1 ml/kg £3
to food and water, in a stress-minimized environment. Animals were kept on a 12:12 h light/dark cycle with lights on at 06:00 h. After an adjustment period of 1 week, rats received during the following 14 days two i.p. injections per day (at 09:00 and 16:00 h) of 1 ml/kg, of either ethanol (2 g/ kg in a 18% v/v saline solution; group A and B) or saline (group C and D) (Table 1). On days 13 and 14, rats of groups A and C were subjected to a `binge' cocaine administration (three consecutive i.p. doses of 15 mg/kg cocaine HCl at 1-h intervals, to mimic the common abuse pattern observed in human cocaine addicts [15]), beginning 30 min after the ®rst ethanol/saline injection. Rats in groups B and D were treated with saline `binges'. Animals were decapitated on day 14, 30 min after the ®nal cocaine/saline injection and the VTA and the NAc were dissected on ice, in a rodent brain matrix (Activational Systems Inc., Warren, MI, USA). All animal housing and experimental procedures were performed according to the relevant provisions and general recommendations in the Swedish animal protection legislation and all measures were taken to avoid rat's suffering. Total RNA was prepared from VTA and NAc using RNAzol B (Biotecx Laboratories; Houston, TX, USA). In
Fig. 1. PCR ampli®cation of the 475 bp KOR target and the 159 bp KOR internal standard separated on a 2% agarose gel. Lanes 1±7 indicate co-ampli®cation of a ®xed volume of target KOR and decreasing amounts of KOR internal standard.
short, the tissues were suspended in 1 ml of ice cold RNAzol B and total RNA was extracted according to the manufacturer's instructions. The ®nal RNA pellet was resuspended in 40 ml diethyl-pyrocarbonate-treated water, followed by spectrophotometric measurement of the RNA amount and storage at 2808C. First strand cDNA synthesis of 0.16±1.32 mg total RNA from VTA and NAc was performed by using random hexamer primers pd(N)6 (Pharmacia Biotech, Uppsala, Sweden) as described elsewhere [8]. Primers for the rat KOR and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were designed using the OLIGO 4.0 primer analysis software. The 5 0 - and 3 0 -primers for KOR corresponded to bases 261±282 and 716±736, respectively, of the KOR sequence published in the GenBank (AC U00442), generating a 475 bp KOR cDNA fragment. The KOR internal standard was constructed by deletion of an internal AccI-AccI (159 bp) fragment as described previously [2]. Speci®c 5 0 - and 3 0 oligonucleotide primers for G3PDH were designed according to the rat sequence (AC X02231 in the GenBank) and corresponded to bases 225±245 and 674±695, respectively. The 470 bp long G3PDH cDNA fragment was subjected to a restriction enzyme cleavage with StyI (191 bp) to produce a 299 bp long G3PDH internal standard. Competitive polymerase chain reaction (PCR) ampli®cation was performed in a total volume of 25 ml, including 2 ml of cDNA and 1 ml of internal standard in six different concentrations (0.0625± 1 attomole/ ml for KOR and 0.625±10 attomole/ ml for G3PDH). The PCR was conducted in 35 cycles using a Perking Elmer Thermocycler 480 (Perkin Elmer, Norwalk, USA) under the following temperature conditions: denaturation at 958C for 1 min, annealing at 608C for 1 min and elongation at 728C for 1 min and ®nal extension at 728C for 5 min. The PCR products were separated on an ethidium bromide stained 2% agarose gel (Life Technologies, Paisley, Scotland) (Fig. 1) and the ratio between the mRNA for KOR and G3PDH, respectively, was determined for each specimen, as described elsewhere [16]. Data are presented as group means (^SEM) and analyzed for overall treatment effect using Kruskal±Wallis ANOVA by ranks. Statistical signi®cance of differences between individual treatment groups was tested using Mann±Whit-
AÊ. Rosin et al. / Neuroscience Letters 275 (1999) 1±4
Fig. 2. Levels of KOR mRNA in rats treated with ethanol and `binge' cocaine, separately or in combination. Bars represent the means ^ SEM of six rats for each group. Data are expressed as KOR mRNA/ G3PDH mRNA. Differences among treatments were estimated by Kruskal±Wallis ANOVA. (a) VTA; (b) NA; *A signi®cant difference (P 0:004) from the saline control (group D) by Mann±Whitney's U test. **A signi®cant difference (P 0:005) between group B and C by Mann±Whitney's U test.
ney's U test. A probability (P) level of ,0.05 was considered signi®cant. Quantitative effects on KOR mRNA expression in the VTA and the NAc after various treatments are shown in Fig.2a,b, respectively. In the VTA, a signi®cant down-regulation of KOR mRNA levels (P 0:00075, Kruskal±Wallis ANOVA) was found in all drug-treated groups (Fig. 2a), with a slight tendency for group A to show a stronger decrease of KOR mRNA levels (88% as compared with saline) than either group B (85% decrease) or C (82% decrease). This decrease was however essentially the same and the difference between groups was not statistically signi®cant. In the NAc of the same rats (Fig. 2b), there was a profound decrease of KOR mRNA in all drug-treated groups (P 0:0007, Kruskal±Wallis ANOVA). The strongest decrease of KOR mRNA levels was observed in rats treated with `binge' cocaine alone (group C, 88% decrease; P 0:006 as compared with saline, Mann±Whitney's U test). The mRNA levels in group C differed statistically signi®cant from group B, i.e. ethanol 14 days (71%; P 0:015, Mann±Whitney's U test). The decrease of
3
KOR mRNA did not vary between groups A (76%) and B (71%). The study provided additional information about the relative amounts of KOR mRNA in the mesolimbic DAergic system. Thus, KOR mRNA levels in the VTA of saline treated rats were approximately 20-fold higher than in the NAc, i.e. molar ratios KOR/G3PDH of 0.1 vs. 0.024 were observed. The present study was designed and performed to elucidate the effects of separate and dual administration of cocaine and ethanol on KOR mRNA in brain regions critical for reward, such as the VTA and NAc. To our knowledge, this is the ®rst report analyzing the consequences of combined ethanol/cocaine treatment on the expression of the KOR gene. Our hypothesis was that the combined administration would result in a stronger effect than the corresponding separate treatments. The data showed that each treatment alone gave a large (more than 80%) reduction in KOR mRNA levels. The fact that the administration of two pharmacologically different drugs such as cocaine (a central nervous system (CNS) stimulant) and ethanol (a CNS depressant, in the doses used in this study), produced a similar qualitative and quantitative effect on KOR mRNA was an interesting observation. Although this was not tested, it could be possible that lower doses of both ethanol and cocaine would result in synergy. Clearly, this was indicated since both treatments alone caused effects in the same direction, e.g. a decrease. However, since we used rather high doses of both ethanol and cocaine it was not possible to detect any further changes after the combination of the drugs. KOR mRNA was downregulated in both VTA and NAc, following separate as well as combined administration. Although the downregulation of KOR mRNA was extensive in all treatment groups, there were some statistically signi®cant differences between them. In the NAc, the largest decrease of KOR mRNA occurred in rats treated with `binge' pattern cocaine for 2 days. In the VTA, the effect was similar following separate and combined treatment, with a tendency for the combination ethanol/ cocaine to show a stronger decrease. The dramatic decrease in KOR mRNA levels in VTANAc supports a role of the KOR system in the abuse to ethanol and cocaine. The anatomical structure of the mesolimbic DAergic pathway suggests an interaction between the DAergic activity and KOR gene expression. Earlier studies have demonstrated an abrupt elevation of DA release in NAc in response to either cocaine or ethanol alone [13]. Prodynorphin gene expression in NAc also increases following chronic ethanol or `binge' cocaine administration [10,14]. Thus, the downregulation of KOR mRNA in the mesolimbic DAergic system observed by us, could act to oppose the increased levels of the endogenous agonists dynorphin peptides as an adaptive response. Indeed, sustained elevations in dynorphin levels have been suggested to cause a compensatory downregulation of the number of kappa binding sites [11]. However, dynorphin
4
AÊ. Rosin et al. / Neuroscience Letters 275 (1999) 1±4
peptides have been shown to tonically inhibit DA release in NAc and thus blunt the neuronal response to DA input [12]. In that way, the downregulation of the KOR gene expression would affect DAergic activity. Another aspect of the present work deals with the relative amounts of KOR mRNA in the VTA and NA. KOR mRNA was present in higher amounts in the VTA than NA in saline treated rats. This ®nding is in agreement with other studies [6,7] demonstrating an intense expression of KOR mRNA in the VTA but very low KOR binding. It was suggested that VTA is the primary site of KOR synthesis from where they are axonally transported to the NAc. However, a portion of KORs is synthesized by cells in the NAc and is postsynaptic. In conclusion, ethanol and `binge' cocaine administration profoundly downregulated KOR mRNA levels in the VTA and NAc of rats. In the VTA, the effect was similar following separate and combined treatment, with a tendency for the combination ethanol/ cocaine to show a stronger decrease. In the NAc, `binge' cocaine treatment during 2 consecutive days produced the largest decrease in KOR mRNA. Clearly, the use of lower doses of both ethanol and cocaine is warranted in order to address their potential interaction. The present data indicate that alteration in KOR gene expression in the mesolimbic DA system may contribute to the neuroadaptation which characterizes ethanol and cocaine dependence. We are grateful to Professor Lars Terenius for his critical reading of the manuscript and Dr. Ingeborg van der Ploeg for her expert help with the RT-PCR method. This work was supported by grants from the Swedish Medical Research Ê ke Wibergs Stiftelse, StiftelCouncil (Project No. 12212), A sen Lars Hiertas Minne, Karolinska Institute's Research Funds, the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly (Projects No. 98/17:1 and 94/30:5) and The National Institute on Drug Abuse, Rockville, MD. [1] Freeman, W.M., Walker, S.J. and Vrana, K.E., Quantitative RT-PCR: pitfalls and potential. Biotechniques, 26 (1999) 112±125. [2] Huang, S.K., Xiao, H.Q., Kleine-Tebbe, J., Paciotti, G., Marsh, D.G., Lichtenstein, L.M. and Liu, M.C., IL-13 expression at the sites of allergen challenge in patients with asthma. J. Immunol., 155 (1995) 2688±2694. [3] Koob, G.F. and Bloom, F.E., Cellular and molecular mechanisms of drug dependence. Science, 242 (1988) 715±723.
[4] Koob, G.F. and Weiss, F., Neuropharmacology of cocaine and ethanol dependence. Recent Dev. Alcohol, 10 (1992) 201±233. [5] Kuhar, M.J., Recent biochemical studies of the dopamine transporter ± a CNS drug target. Life Sci., 62 (1998) 1573± 1575. [6] Mansour, A., Fox, C.A., Meng, F., Akil, H. and Watson, S.J., Kappa 1 receptor mRNA distribution in the rat CNS: comparison to kappa receptor binding and prodynorphin mRNA. Mol. Cell. Neurosci., 5 (1994) 124±144. [7] Minami, M., Hosoi, Y., Toya, T., Katao, Y., Maekawa, K., Katsumata, S., Yabuuchi, K., Onogi, T. and Satoh, M., In situ hybridization study of kappa-opioid receptor mRNA in the rat brain. Neurosci. Lett., 162 (1993) 161±164. [8] Pisa, E.K., Pisa, P., Hansson, M. and Wigzell, H., OKT3induced cytokine mRNA expression in human peripheral blood mononuclear cells measured by polymerase chain reaction. Scand. J. Immunol., 36 (1992) 745±749. [9] Spanagel, R., Herz, A. and Shippenberg, T.S., Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc. Natl. Acad. Sci., USA, 89 (1992) 2046±2050. [10] Spangler, R., Zhou, Y., Maggos, C.E., Schlussman, S.D., Ho, A. and Kreek, M.J., Prodynorphin, proenkephalin and kappa opioid receptor mRNA responses to acute `binge' cocaine. Brain Res. Mol. Brain Res., 44 (1997) 139±142. [11] Staley, J.K., Rothman, R.B., Rice, K.C., Partilla, J. and Mash, D.C., Kappa2 opioid receptors in limbic areas of the human brain are upregulated by cocaine in fatal overdose victims. J. Neurosci., 17 (1997) 8225±8233. [12] Steiner, H. and Gerfen, C.R., Dynorphin regulates D1 dopamine receptor-mediated responses in the striatum: relative contributions of pre- and postsynaptic mechanisms in dorsal and ventral striatum demonstrated by altered immediate-early gene induction. J. Comp. Neurol., 376 (1996) 530±541. [13] Torres, G. and Horowitz, J.M., Individual and combined effects of ethanol and cocaine on intracellular signals and gene expression. Prog. Neuropsychopharmacol. Biol. Psychiatry, 20 (1996) 561±596. [14] Turchan, J., Przewlocka, B., Lason, W. and Przewlocki, R., Effects of repeated psychostimulant administration on the prodynorphin system activity and kappa opioid receptor density in the rat brain. Neuroscience, 85 (1998) 1051± 1059. [15] Unterwald, E.M., Rubenfeld, J.M. and Kreek, M.J., Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors. NeuroReport, 5 (1994) 1613±1616. [16] Van der Ploeg, I., Jeddi Tehrani, M., Matuseviciene, G., Wahlgren, C.F., Fransson, J. and Scheynius, A., IL-13 over-expression in skin is not con®ned to IgE-mediated skin in¯ammation. Clin. Exp. Immunol., 109 (1997) 526±532.
Downregulation of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens AÊsa Rosin a,*, Sara Lindholm a, Johan Franck a, Jeanette Georgieva a, b a
Department of Clinical Neuroscience, Experimental and Clinical Drug Addiction Research Sections, CMM L8:01, Karolinska Institutet, S-171 76 Stockholm, Sweden b Department of Clinical Pharmacology, Karolinska Hospital, S-171 76 Stockholm, Sweden Received 7 April 1999; received in revised form 6 August 1999; accepted 6 August 1999
Abstract The combination of ethanol and cocaine is commonly abused by human addicts which has serious clinical consequences. Here, the effects of separately and concurrently administered ethanol and `binge' cocaine on kappa opioid receptor (KOR) mRNA in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of rats were studied. KOR mRNA was down-regulated in both brain regions during concurrent as well as separate treatment with these drugs. In the VTA, the most pronounced decrease was obtained following combined treatment with ethanol and `binge' cocaine. In the NAc, the strongest decrease was observed in the `binge' cocaine group. This profound decrease of KOR mRNA in regions important for brain reward suggests a potential role of the KOR system in the abuse of cocaine and ethanol. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kappa opioid receptor mRNA; `Binge' cocaine; Ethanol; Competitive polymerase chain reaction; Rat; Ventral tegmental area; Nucleus accumbens
Concurrent abuse of ethanol and cocaine is common among human addicts. This drug combination has been reported to increase the risk of morbidity and mortality as well as compulsive drug seeking behaviour [13]. Although the clinical consequences of dual substance abuse receive increasing attention, the reasons for its high prevalence are still poorly understood. Important progress has been made in recent years in understanding the pharmacodynamics of either drug alone. Thus, abuse and dependence on cocaine or ethanol may be related to the dysregulation of a common set of neurochemical pathways. A feature shared by both drugs is the targeting of nerve cells primarily in mesocorticolimbic and nigrostriatal brain dopaminergic systems to produce an altered behaviour. The mesolimbic dopaminergic neurons with cell bodies in the ventral tegmental area (VTA) projecting to receptive ®elds in the nucleus accumbens (NAc) and prefrontal cortex, are considered to mediate the reinforcing effects of both cocaine and ethanol [3]. Administration of cocaine or ethanol to rats stimulates dopa* Corresponding author. Tel.: 146-8-517-74692; fax 146-8517-76180. E-mail address: [email protected] (AÊ. Rosin)
mine (DA) release in the NAc. It is widely accepted that cocaine binds to and inactivates dopamine transporters (DATs) thereby enhancing dopaminergic transmission [5]. For ethanol, no speci®c receptor has been identi®ed but it is known that signal transmission through GABA and glutamate (NMDA) receptor is affected [4]. Closely connected to the DA system and involved in the effects of ethanol as well as cocaine is the endogenous opioid system [9]. In the NAc and dorsal striatum, DAergic neurotransmission is modulated by the opioid peptides dynorphins and enkephalins. The functional effects of dynorphin peptides are mediated by kappa opioid receptors (KORs). The present study was conducted to assess the effects of separate and combined exposure to cocaine and ethanol on KOR mRNA levels in the mesolimbic dopamine system using a newly developed quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. RTPCR represents a highly sensitive method for analyzing RNA levels [1] and allows the detection of very small mRNA amounts as well as their changes, in a single region. Male Sprague±Dawley rats weighing approximately 250-g (60 days old, purchased from B&K Universal, Stockholm, Sweden) were caged in groups of six, with free access
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 67 5- 8
AÊ. Rosin et al. / Neuroscience Letters 275 (1999) 1±4
2 Table 1 Drug treatment scheme Group
Day 1±12
Day 13
Day 14
A (n 6)
Ethanol 2 g/kg 2£ day
B (n 6)
Ethanol 2 g/kg 2£ day
C (n 6)
Saline 1 ml/kg 2£ day
D (n 6)
Saline 1 ml/kg 2£ day
Ethanol 2 g/kg 2£ day `Binge' cocaine 15 mg/kg £3 Ethanol 2 g/kg 2£ day `Binge' saline 1 ml/kg £3 Saline 1 ml/kg 2£ day `Binge' cocaine 15 mg/kg £3 Saline 1 ml/kg 2£ day `Binge' saline 1 ml/kg £3
Ethanol 2 g/kg 2£ day `Binge' cocaine 15 mg/kg £3 Ethanol 2 g/kg 2£ day `Binge' saline 1 ml/kg £3 Saline 1 ml/kg 2£ day `Binge' cocaine 15 mg/kg £3 Saline 1 ml/kg 2£ day `Binge' saline 1 ml/kg £3
to food and water, in a stress-minimized environment. Animals were kept on a 12:12 h light/dark cycle with lights on at 06:00 h. After an adjustment period of 1 week, rats received during the following 14 days two i.p. injections per day (at 09:00 and 16:00 h) of 1 ml/kg, of either ethanol (2 g/ kg in a 18% v/v saline solution; group A and B) or saline (group C and D) (Table 1). On days 13 and 14, rats of groups A and C were subjected to a `binge' cocaine administration (three consecutive i.p. doses of 15 mg/kg cocaine HCl at 1-h intervals, to mimic the common abuse pattern observed in human cocaine addicts [15]), beginning 30 min after the ®rst ethanol/saline injection. Rats in groups B and D were treated with saline `binges'. Animals were decapitated on day 14, 30 min after the ®nal cocaine/saline injection and the VTA and the NAc were dissected on ice, in a rodent brain matrix (Activational Systems Inc., Warren, MI, USA). All animal housing and experimental procedures were performed according to the relevant provisions and general recommendations in the Swedish animal protection legislation and all measures were taken to avoid rat's suffering. Total RNA was prepared from VTA and NAc using RNAzol B (Biotecx Laboratories; Houston, TX, USA). In
Fig. 1. PCR ampli®cation of the 475 bp KOR target and the 159 bp KOR internal standard separated on a 2% agarose gel. Lanes 1±7 indicate co-ampli®cation of a ®xed volume of target KOR and decreasing amounts of KOR internal standard.
short, the tissues were suspended in 1 ml of ice cold RNAzol B and total RNA was extracted according to the manufacturer's instructions. The ®nal RNA pellet was resuspended in 40 ml diethyl-pyrocarbonate-treated water, followed by spectrophotometric measurement of the RNA amount and storage at 2808C. First strand cDNA synthesis of 0.16±1.32 mg total RNA from VTA and NAc was performed by using random hexamer primers pd(N)6 (Pharmacia Biotech, Uppsala, Sweden) as described elsewhere [8]. Primers for the rat KOR and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were designed using the OLIGO 4.0 primer analysis software. The 5 0 - and 3 0 -primers for KOR corresponded to bases 261±282 and 716±736, respectively, of the KOR sequence published in the GenBank (AC U00442), generating a 475 bp KOR cDNA fragment. The KOR internal standard was constructed by deletion of an internal AccI-AccI (159 bp) fragment as described previously [2]. Speci®c 5 0 - and 3 0 oligonucleotide primers for G3PDH were designed according to the rat sequence (AC X02231 in the GenBank) and corresponded to bases 225±245 and 674±695, respectively. The 470 bp long G3PDH cDNA fragment was subjected to a restriction enzyme cleavage with StyI (191 bp) to produce a 299 bp long G3PDH internal standard. Competitive polymerase chain reaction (PCR) ampli®cation was performed in a total volume of 25 ml, including 2 ml of cDNA and 1 ml of internal standard in six different concentrations (0.0625± 1 attomole/ ml for KOR and 0.625±10 attomole/ ml for G3PDH). The PCR was conducted in 35 cycles using a Perking Elmer Thermocycler 480 (Perkin Elmer, Norwalk, USA) under the following temperature conditions: denaturation at 958C for 1 min, annealing at 608C for 1 min and elongation at 728C for 1 min and ®nal extension at 728C for 5 min. The PCR products were separated on an ethidium bromide stained 2% agarose gel (Life Technologies, Paisley, Scotland) (Fig. 1) and the ratio between the mRNA for KOR and G3PDH, respectively, was determined for each specimen, as described elsewhere [16]. Data are presented as group means (^SEM) and analyzed for overall treatment effect using Kruskal±Wallis ANOVA by ranks. Statistical signi®cance of differences between individual treatment groups was tested using Mann±Whit-
AÊ. Rosin et al. / Neuroscience Letters 275 (1999) 1±4
Fig. 2. Levels of KOR mRNA in rats treated with ethanol and `binge' cocaine, separately or in combination. Bars represent the means ^ SEM of six rats for each group. Data are expressed as KOR mRNA/ G3PDH mRNA. Differences among treatments were estimated by Kruskal±Wallis ANOVA. (a) VTA; (b) NA; *A signi®cant difference (P 0:004) from the saline control (group D) by Mann±Whitney's U test. **A signi®cant difference (P 0:005) between group B and C by Mann±Whitney's U test.
ney's U test. A probability (P) level of ,0.05 was considered signi®cant. Quantitative effects on KOR mRNA expression in the VTA and the NAc after various treatments are shown in Fig.2a,b, respectively. In the VTA, a signi®cant down-regulation of KOR mRNA levels (P 0:00075, Kruskal±Wallis ANOVA) was found in all drug-treated groups (Fig. 2a), with a slight tendency for group A to show a stronger decrease of KOR mRNA levels (88% as compared with saline) than either group B (85% decrease) or C (82% decrease). This decrease was however essentially the same and the difference between groups was not statistically signi®cant. In the NAc of the same rats (Fig. 2b), there was a profound decrease of KOR mRNA in all drug-treated groups (P 0:0007, Kruskal±Wallis ANOVA). The strongest decrease of KOR mRNA levels was observed in rats treated with `binge' cocaine alone (group C, 88% decrease; P 0:006 as compared with saline, Mann±Whitney's U test). The mRNA levels in group C differed statistically signi®cant from group B, i.e. ethanol 14 days (71%; P 0:015, Mann±Whitney's U test). The decrease of
3
KOR mRNA did not vary between groups A (76%) and B (71%). The study provided additional information about the relative amounts of KOR mRNA in the mesolimbic DAergic system. Thus, KOR mRNA levels in the VTA of saline treated rats were approximately 20-fold higher than in the NAc, i.e. molar ratios KOR/G3PDH of 0.1 vs. 0.024 were observed. The present study was designed and performed to elucidate the effects of separate and dual administration of cocaine and ethanol on KOR mRNA in brain regions critical for reward, such as the VTA and NAc. To our knowledge, this is the ®rst report analyzing the consequences of combined ethanol/cocaine treatment on the expression of the KOR gene. Our hypothesis was that the combined administration would result in a stronger effect than the corresponding separate treatments. The data showed that each treatment alone gave a large (more than 80%) reduction in KOR mRNA levels. The fact that the administration of two pharmacologically different drugs such as cocaine (a central nervous system (CNS) stimulant) and ethanol (a CNS depressant, in the doses used in this study), produced a similar qualitative and quantitative effect on KOR mRNA was an interesting observation. Although this was not tested, it could be possible that lower doses of both ethanol and cocaine would result in synergy. Clearly, this was indicated since both treatments alone caused effects in the same direction, e.g. a decrease. However, since we used rather high doses of both ethanol and cocaine it was not possible to detect any further changes after the combination of the drugs. KOR mRNA was downregulated in both VTA and NAc, following separate as well as combined administration. Although the downregulation of KOR mRNA was extensive in all treatment groups, there were some statistically signi®cant differences between them. In the NAc, the largest decrease of KOR mRNA occurred in rats treated with `binge' pattern cocaine for 2 days. In the VTA, the effect was similar following separate and combined treatment, with a tendency for the combination ethanol/ cocaine to show a stronger decrease. The dramatic decrease in KOR mRNA levels in VTANAc supports a role of the KOR system in the abuse to ethanol and cocaine. The anatomical structure of the mesolimbic DAergic pathway suggests an interaction between the DAergic activity and KOR gene expression. Earlier studies have demonstrated an abrupt elevation of DA release in NAc in response to either cocaine or ethanol alone [13]. Prodynorphin gene expression in NAc also increases following chronic ethanol or `binge' cocaine administration [10,14]. Thus, the downregulation of KOR mRNA in the mesolimbic DAergic system observed by us, could act to oppose the increased levels of the endogenous agonists dynorphin peptides as an adaptive response. Indeed, sustained elevations in dynorphin levels have been suggested to cause a compensatory downregulation of the number of kappa binding sites [11]. However, dynorphin
4
AÊ. Rosin et al. / Neuroscience Letters 275 (1999) 1±4
peptides have been shown to tonically inhibit DA release in NAc and thus blunt the neuronal response to DA input [12]. In that way, the downregulation of the KOR gene expression would affect DAergic activity. Another aspect of the present work deals with the relative amounts of KOR mRNA in the VTA and NA. KOR mRNA was present in higher amounts in the VTA than NA in saline treated rats. This ®nding is in agreement with other studies [6,7] demonstrating an intense expression of KOR mRNA in the VTA but very low KOR binding. It was suggested that VTA is the primary site of KOR synthesis from where they are axonally transported to the NAc. However, a portion of KORs is synthesized by cells in the NAc and is postsynaptic. In conclusion, ethanol and `binge' cocaine administration profoundly downregulated KOR mRNA levels in the VTA and NAc of rats. In the VTA, the effect was similar following separate and combined treatment, with a tendency for the combination ethanol/ cocaine to show a stronger decrease. In the NAc, `binge' cocaine treatment during 2 consecutive days produced the largest decrease in KOR mRNA. Clearly, the use of lower doses of both ethanol and cocaine is warranted in order to address their potential interaction. The present data indicate that alteration in KOR gene expression in the mesolimbic DA system may contribute to the neuroadaptation which characterizes ethanol and cocaine dependence. We are grateful to Professor Lars Terenius for his critical reading of the manuscript and Dr. Ingeborg van der Ploeg for her expert help with the RT-PCR method. This work was supported by grants from the Swedish Medical Research Ê ke Wibergs Stiftelse, StiftelCouncil (Project No. 12212), A sen Lars Hiertas Minne, Karolinska Institute's Research Funds, the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly (Projects No. 98/17:1 and 94/30:5) and The National Institute on Drug Abuse, Rockville, MD. [1] Freeman, W.M., Walker, S.J. and Vrana, K.E., Quantitative RT-PCR: pitfalls and potential. Biotechniques, 26 (1999) 112±125. [2] Huang, S.K., Xiao, H.Q., Kleine-Tebbe, J., Paciotti, G., Marsh, D.G., Lichtenstein, L.M. and Liu, M.C., IL-13 expression at the sites of allergen challenge in patients with asthma. J. Immunol., 155 (1995) 2688±2694. [3] Koob, G.F. and Bloom, F.E., Cellular and molecular mechanisms of drug dependence. Science, 242 (1988) 715±723.
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