Department of Physics, Chemistry and Biology
Master Thesis
Validation of a transgenic mouse line with knock
down of mGluR5 selectively in dopamine D1
receptor expressing neurons
Ali Nasr Esfahani
LITH-IFM-A-EX---10/2317-SE
Supervisor: David Engblom, (IKE) Linköpings universitet
Rapporttyp Report category Licentiatavhandling x Examensarbete C-uppsats x D-uppsats Övrig rapport _______________ Språk Language Svenska/Swedish X Engelska/English ________________ Titel
Validation of a transgenic mouse line with knock down of mGluR5 selectively in dopamine D1 receptor expressing neurons
Författare
Ali Nasr Esfahani
Sammanfattning
One of the main difficulties of addiction treatment is the high risk of relapse even after a long abstinence and fully detoxification. Therefore, discovering the underlying molecular principles of relapse is essential. The metabotropic glutamate receptor, mGluR5, is considered to be involved in this aspect. One of the brain structures expressing mGluR5 is the striatum, an area with well-established role in addiction which is largely composed of medium-sized spiny neurons (MSNs). These neurons are basically divided into two major subpopulations characterized based on their projections and protein properties. It is known that the mGluR5 receptor is expressed on both subpopulations of MSNs. Consequently, it can be used to establish the proportional contribution of each of MSNs subpopulations in relapse to addiction. In our constellation, we have generated a mouse line designed to have a selective mGluR5 knock-down in one of these subpopulations – the dopamine D1 receptor (D1R) expressing neurons. It has however been unclear if the expression of the transgene is indeed limited to only D1R-expressing neurons. By immunofluorescence technique, I here show that the construct is expressed only in MSNs and is restricted to the D1R-expressing cell population in the striatum. Thus the transgenic mouse line is a good tool for the study of mGluR5 selectively in D1R expressing neurons.
ISBN
__________________________________________________ ISRN
LITH-IFM-A-EX---10/2317-SE
Serietitel och serienummer ISSN
Title of series, numbering Handledare:
Supervisor: David Engblom
Ort:
Linköping
Nyckelord
Cocaine, DARPP-32, Enkephalin, GFP, mGluR5, MSNs, Relapse, Striatum
Datum
Date 2010-06-04
URL för elektronisk version
Avdelning, Institution
Division of Biology
Content
1. Abstract……….………...1
2. List of abbreviation...………...1
3. Introduction…………...…………...…………...…...1
4. Materials and Methods………….……...…...…..…...………...3
4.1. Animal Housing………...….…...……...3
4.2. Tissue Processing and Preparation....……….……...…………3
4.3. Tissue Sectioning...…………...3
4.4. Immunohistochemistry (IHC)...3
4.5. Immunofluorescence (IFC) ………...…….…….…..…….…..…….4
4.6. Light Microscopy ………...….………...………4
5. Results……….………..…...………4
5.1. MSNs markers are detectable by DAB Staining...4
5.2. The mGluR5KD-D1 is only expressed in D1-R expressing neurons...5
6. Discussion...6
7. Acknowledgment...7
1 Abstract
One of the main difficulties of addiction treatment is the high risk of relapse even after a long abstinence and fully detoxification. Therefore, discovering the underlying molecular principles of relapse is essential. The metabotropic glutamate receptor, mGluR5, is considered to be involved in this aspect. One of the brain structures expressing mGluR5 is the striatum, an area with well-established role in addiction which is largely composed of medium-sized spiny neurons (MSNs). These neurons are basically divided into two major subpopulations characterized based on their projections and protein properties. It is known that the mGluR5 receptor is expressed on both subpopulations of MSNs. Consequently, it can be used to establish the proportional contribution of each of MSNs subpopulations in relapse to addiction. In our constellation, we have generated a mouse line designed to have a selective mGluR5 knock-down in one of these subpopulations – the dopamine D1 receptor (D1R) expressing neurons. It has however been unclear if the expression of the transgene is indeed limited to only D1R-expressing neurons. By immunofluorescence technique, I here show that the construct is expressed only in MSNs and is restricted to the D1R-expressing cell population in the striatum. Thus the transgenic mouse line is a good tool for the study of mGluR5 selectively in D1R expressing neurons.
Keywords: Cocaine, DARPP-32, Enkephalin, GFP, MSNs, mGluR5, Relapse, Striatum
2 List of abbreviations
D1R – Dopamine Receptor D1 D2R – Dopamine Receptor D2 DA – Dopamine
DARPP-32 - Dopamine And cAMP Regulated Phosphoprotein of 32kDa GFP- Green Fluorescent Protein
IFC- Immunofluorescence IHC- Immunohistocehmistry mGluR5- Metabotropic Glutamate Receptor5
MSNs- Medium Spiny Neurons ppENK - pre-pro Enkephalin
3 Introduction:
Addiction is a major burden on every society. It is a complicated phenomenon caused by various psychological and social consequences, and several biological processes underlie it. Drug addiction is classically defined as a chronically relapsing disorder characterized by repeating of compulsive drug seeking and taking, despite potential harms (Koob, and Le Moal, 1997). Moreover, addiction is recognized as a neuroadaptation disorder characterized by dysregulation of the mesocorticolimbic dopamine (DA) reward system (Nestler et al., 1996).
Cocaine addiction, one of the most destructive forms of drugs abuse has a high risk of relapse (Carroll et al., 1994) that can appear even after a long period of abstinence of drug administration and fully detoxification (Meyer, 1988; Jaffe, 1990; De Vos et al., 1996; Horns et
al., 1975). Numerous experimental studies on both animals and humans reveal that relapse to
cocaine abuse occurs from extreme craving that may start from exposure to different environmental stimuli including drug-associated cues (Worley et al.,1994; De Wit H and Stewart J ,1981; Ludwig et al.,1974; Childress et al.,1988; Wikler, 1971). There are several indications showing the role of neuroadaptations related to behavioural sensitization which is participating in addiction relapse (Kalivas and Stewart, 1991; Robinson and Berridge, 2000; Robinson and Berridge, 1993). These lasting effects of cocaine stimuli are caused by the ability of the drug to dysregulate reward-related associative learning and memory processes.
Mesocorticolimbic DA reward pathways contribute to various drug-induced conditions including physical dependence, craving and relapse (Kauer and Malenka, 2007; Wise, 2002; Thomas et al., 2008; Hyman and Malenka, 2001). The striatum, including the nucleus accumbens, is one of the main anatomical parts of this circuit that receives a large number of DA and glutamatergice projections participating in reward and relapse (Wolf, 2002; Childress et al., 1999; Grant et al., 1996; Koob, 1992; Koob, 1992; Wise and Hoffman, 1992; Valjent et al., 2009; Tzschentke and Schmidt, 2003; Karler et al., 1989; Wolf and Khansa, 1991; Tallaksen et al., 1998). It has been demonstrated that 95% of the striatal neurons consist of medium spiny neurons (MSNs) (Tepper and Bolam, 2004) which are mainly divided into two subclasses base on their projections and peptide content (Beckstead and Cruz, 1986; Gerfen and Young, 1988; Kawaguchi et al., 1990). One population expresses D1Rs (D1-MSNs) projecting to the substantia nigra and co-expresses the neuropeptide dynorphin while the other population expresses D2Rs (D2-MSNs) projecting to the globus pallidus and co-expresses the neuropeptide enkephalin (Sibley and Monsma, 1992). Although the role of the striatum/nucleus accumbens in addiction is well established, the proportional contribution of each D1 and D2 MSNs, and the specific underlying neurobiological mechanisms of plasticity changes in the striatal neurons after cocaine administrations that underlie addiction relapse are poorly understood. In this perspective, the role of the metabotropic glutamate receptor, mGluR5, which is a glutamatergic receptor expressed on both MSN populations is potentially interesting (Testa et al.,1995; Conn and Pin, 1997; Anwyl ,2009; Lu, X.Y. et al., 1999; Kenny and Markou, 2004; McGeehan and Olive ,2003; Chiamulera et
al.,2001; Backstrom and Hyytia,2007; Albin et al.,1992; Tallaksen et al.,1992).
We aimed to point out more specific and restricted targets of the addiction relapse mechanisms within the reward pathway. For this purpose, a novel mouse line has been generated in our constellation in which mGluR5 is selectively knocked-down in dopamine D1 receptor (D1R) expressing neurons. These mice (called mGluR5KD-D1 mice) show reduced levels of mGluR5 in the striatum and a strongly reduced reinstatement of cocaine seeking, a model for relapse. In
addition they show deficiencies in specific aspects of reward-related learning that may explain this reduced relapse tendency.
To link the function of mGluR5 in D1R neurons to these behavioural changes, it is important to test if our construct is expressed in the accurate location which is D1-R expressing neurons. Consequently, the aim of this study was to show if the construct is expressed only in D1R- and not in D2R-expressed neurons in the striatum.
4 Materials and Methods: 4.1 Animal Housing:
Transgenic mice and control mice on C57BL6 background were used for this study. Animals were all males aged 6-15 weeks old. They were housed in standard cages at a steady room temperature (20 ºC) on a regulated 12 h light/dark cycle (light was available at 7 a.m.) and provided with ad libitum food and water. All experiment procedures were performed in compliance with the Swedish national guidelines and confirmed by the local Animal Care and Use Committee.
4.2 Tissue Processing and Preparation:
Mice were deeply anesthetized with CO2 respiration and transcardially perfused with 20 mL of 0.9% saline followed by 50 mL of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PBS) (pH 7.4). The whole brain was excised from the skull and post-fixed in 4% paraformaldehyde in PBS for 3 h and then incubated in 30% sucrose in 0.1 M PBS overnight at 4 °C.
4.3 Tissue Sectioning:
Brains were coronally cut into 30 µm sections using a freezing microtome (1320 Leitz) and stored for later usage in sterile bins containing cold cryoprotectant (0.1 M phosphate buffer, 30% ethylene glycol, 20% glycerol) in -20 ºC.
4.4 Immunohistochemistry (IHC):
Free floating immunohistochemistry was performed to achieve good specificity.
Sections were rinsed in PBS for 10 m and then bathed in 600 µL block solution (PBS (1X) + 1% bovine albumin + 0.3% Triton) for 45 min.
The first antibody was used against dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32) (Walaas et al.,1983), which is a regulatory protein enriched in cytoplasm of all MSNs (Ouimet et al.,1984; Ouimet and Greengard ,1990; Stipanovich et al.,2008) and works as a marker of both D1-R and D2-R expressing neurons, while the second antibody was against pro-pre enkephaline (ppENK) that is pro-present only in D2-R expro-pressing neurons (Le Moine et
al.,1990).
Thus, the sections were then incubated in primary antibody including mouse polyclonal anti-DARPP-32 (1:500; BD Biosciences cat: 611520, lot: 58902) or rabbit anti-enkephalin (1:500; Neuromics, cat: RA14124-50) overnight at room temperature.
After three times rinsing with PBS for 10 min, sections were incubated in 600 µL of 0.3% H2O2 for 30 min. After rinsing 3X10 min with 1000 µL PBS, sections were incubated for 2 h in biotinylated anti mouse or anti rabbit (1:1000; Vector laboratories) secondary antibodies. After rinsing, sections were put in Avidin Biotin Complex (ABC) solution (1XPBS concentration for both A and B 1:1000) for 2 h and then washed with PBS for 20 min. Color was developed by keeping sections in DAB solution (1 tablet of DAB (3.3 diaminobenzidine) in 15 ml of Tris-buffered saline, PH 7.6, added to 12 µL of fresh 30% hydrogen peroxide; 1:1000) for 2-5 min in ventilation bench and then rinsed 2X10 min in PBS and finally mounted on slides and dried overnight. The glasses were put in xylene for about 4 h and then mounted with DPX and left in a flow bench to be dried. All the processes were performed at room temperature.
4.5 Immunofluorescence (IFC):
To detect colocalization of the proteins of interest, free floating triple staining was performed. After rinsing with PBS for 10 min the brain sections were incubated with a mixture of mouse polyclonal DARPP-32 (1:500; BD Biosciences cat: 611520, lot: 58902), rabbit anti-enkephalin (1:500; Neuromics, cat: RA14124-50) and chicken polyclonal anti-GFP (1:1000; abcam, cat: ab13970-100, lot: 660556) overnight at room temperature. Next day sections were rinsed in PBS 6X10 min and then incubated in the presence of anti-mouse Cy5 (1:500; Jackson Immunoresearch) and anti-rabbit Alexa568 (1:1000; Invitrogen) and anti-chicken Alexa488 (1:1000; Invitrogen) secondary antibodies for 2 h at room temperature. Following three washes in PBS, the sections were immediately moved to slides and mounted with Fluoromount. Then the slides were kept in 4 ºC and protected from direct light.
4.6 Microscopy:
Slides obtained from IHC were observed with a Nikon MD105 light microscope. Brain areas were identified using a mouse brain atlas (Paxinos and Franklin, 2001).
Confocal microscope images from IFC were acquired with a Nikon Eclipse E600 confocal microscope. Images were captured and auto-averaged to reduce the noise. The images were taken from different locations which had the best appearance and most cellular density.
5 Results:
5.1 MSNs markers are detectable by DAB Staining:
This step was done to confirm that the expression of markers for different MSN populations in the striatum is detectable. Images collected from the sections after DAB staining are shown in the Figure 1. As it is shown in Figure 1, both antibodies work properly and both markers are expressed in many striatal neurons whereas they are not expressed in the cortical neurons.
Figure 1. Expression of the MSNs markers in the striatum of an mGluR5KD-D1 mouse. A) Immunohistochemical detection of DARPP-32, a protein present in all MSNs. B) Labeling of pre-pro Enkephalin (ppENK), a protein expressed in D2- but not D1-medium spiny neurons. (►)
Examples of DARPP-32 and ppENk positive cells.
5.2 The mGluR5KD-D1 is only expressed in D1-R expressing neurons:
As the main aim of this study was to localize the expression of the transgene to be able to interpret the behavioural findings in a correct way, I focused on the expression of the construct in the striatum. Anti DARPP-32 antibody was used for DARPP32, which is abundant in both D1-R and D2-R MSNs (Ouimet CC and Greengard P; 1990) to mark out all the cell bodies of MSNs. This should label all MSNs, as shown by Matamales et al. In the confocal microscope, I observed DARPP-32-positive cells in all sections (Fig.2 A; blue cells). These cells consist of two MSNs subpopulations, D1-R and D-2 expressing neurons that comprise 95% of striatal neurons (Tepper and Bolam, 2004). The next step was to see whether two subpopulations of MSN can be separately marked in our generated mice.
In the transgenic mice, the coding sequence for green fluorescent protein (GFP) was introduced to facilitate tracing the construct expression of interest. Since GFP sequence was under the control of D1-R promoters, I anticipated that it will be only expressed on D1-R expressed neurons. Eventually, I identified GFP–positive cells by Immunofluorescence and merged the confocal images. Consequently, it was shown that all GFP cells co-express DARPP-32 (Fig.2 B). However, around half of the DARPP-32-positive cells did not co-express GFP. This shows that in the striatum the construct is exclusively expressed in MSNs, but only in a sub-population of them (Matamales et al, 2009).
To distinguish D1-R from D2-R expressed neurons, I looked for pre-pro Enkephalin (ppENK), a protein which is only expressed in D2-R expression neurons of the striatum (Gerfen CR and Young WS, 1988; Hong JS et al, 1977; Gerfen C.R. et al, 1990; Yung, K. K. et al, 1995; Le Moine, C. et al, 1990). Merging the images, ppENK expression (red in Fig.2) was seen in around
half of the MSNs while the rest were expressing GFP (Fig.2C). I found no co-localization between ppENK and GFP, showing that the construct is not expressed in D2R-expressing MSNs. Since all MSNs showed either GFP or ppENK labelling (Fig.2D), I can conclude that the construct is expressed selectively in D1R-expressing cells.
Figure 2. Immunoflourescent labeling showing that the expression of the transgene is selective
to D1-MSNs. (A) Immunofluorescent labeling for DARPP-32 (blue) identifying medium spiny neurons (MSNs; DARPP-32; blue). (B) The expression is restricted to MSNs and involves around half of them (GFP; green). (C) GFP is not expressed in D2-MSNs (ppENK; red). (D) All the MSNs (blue) express either GFP or ppENK. Examples of GFP-expressing (
→
) andnon-GFP-expressing (►) MSNs. Scale bar 20µm.
6 Discussion:
One of the major obstacles in addiction treatment is the high tendency of relapse, particularly after exposure to environmental stimuli associated with previous drug-use. This project is a part of an ongoing study observing the role of mGluR5 receptors on dopamine D1 receptor- expressing neurons in incentive learning underlying cocaine relapse. However, in order to interpret the behavioral experiments we had to be confident that our mGluR5KD-D1 construct was properly located and expressed in the desired site. In the present study we show that the
transgene, identified by immunofluorescence, is indeed expressed in D1R-expressed neurons of the striatum.
It is generally accepted that relapse to drug addiction involves the dopamine system in the striatum (Self DW et al, 1998); however, the deficiency of dopaminergic therapies on relapse highlights the probable existence of other neurotransmitters in the relapse process (Spealman RD
et al, 1999; Gerrits M.A.F.M. and Van Ree J. M, 1996; McFarland K. and Ettenberg A., 1997).
One of these candidate neurotransmitters is glutamate (Goto Y & Grace AA, 2008; Kauer JA and Malenka RC, 2007). There are various studies emphasizing the possible roles of glutamatergic circuit, particularly mGluRs in relapse to drug addiction, and different forms of plasticity in the striatum (Bellone C et al. 2008; Grueter BA et al. 2007; Tallaksen-Greene SJ et al, 1998; Anwyl R, 2009; Kauer JA and Malenka RC, 2007).
The impairment of cocaine self-administration seen in mGluR5 -/- mice and the fact that pharmacological blockade of mGluR5 inhibits aspects of cocaine’s stimulant and rewarding effects suggest that mGluR5 signaling may be critical for drug-seeking behavior (Chiamulera, C
et al., 2001; Kenny, P.J. et al., 2005; Backstrom, P. et al. , 2004; Cosford ND et al. 2003; Kumaresan, V et al. 2009).
According to my results, all the blue MSNs are expressing either ppEnkephalin or GFP (Fig2. D), and there is no co-expression of them. This reveals that the expression of the mGluR5KD-D1 construct is limited to D1-R expressing neurons as we desired.
Considering the significance of both dopaminergic and glutamatergic systems, we focused on the role of mGluR5 in the striatum expressed on the D1-R subpopulation of MSNs which is separate from D2-R expressing neuron subclass. However, there is some evidence indicating that a small population of MSNs expresses both D1 and D2 types of receptor. This third subpopulation of MSNs is also expressing ppENK together with both D1 and D2 receptors (Surmeier DJ et al, 1996; Aizman O et al., 2000). Looking at our captured images, there was no cell body containing both GFP and ppEnkephalin. This could be due to that this subpopulation is either very small, thus we did not have enough images from different regions of the striatum or that the construct is not expressed in this population. However, this contrast does not question our results since the majority of MSNs are D1R and D2R neurons and the impact of the third group may not be relatively significant.
To conclude, our experiment shows that the construct in the mGluR5KD-D1 mice is expressed accurately. Therefore we can draw firm conclusions from the behavioral studies and say that mGluR5 on D1R-expressing cells is important for incentive learning processes underlying relapse. Moreover, our results show that the mGluR5KD-D1 mouse line is a good tool for further studies of mGluR5 on D1R-expressing cells.
7 Acknowledgements:
I would like to express my deepest gratitude to my supervisor David Engblom whose help, guidance and encouragement were always motivating to me. I am also indebted to Milen Kirlov Anna Eskilsson, Anna Nilsson, Daniel Björk and Nina Ottosson for their always help and support. Last but not least, I would like to thank my wife and my family for the encouraging environment they have provided me during my education.
8 References:
Aizman, O., Brismar, H., Uhlén, P., Zettergren, E., Levey, A.I., Forssberg, H., Greengard, P., Aperia, A. (2000) Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons. Nat Neurosci 3 (3): 226 - 230
Albin R.L., Makowiec R.L., Hollingsworth Z., Dure L.S., Penney J.B., Young A.B. (1992) Excitatory amino acid binding sites in the basal ganglia of the rat: a quantitative autoradiographic study,Neuroscience (46): 35–48
Anwyl R. (2009) Metabotropic glutamate receptor-dependent long-term potentiation.
Neuropharmacology 56(4):735-740
Backstrom, P., Bachteler, D., Koch, S., Hyytia, P., Spanagel, R. (2004) mGluR5 antagonist MPEP reduces ethanol-seeking and relapse behavior. Neuropsychopharmacology (29): 921–928 Backstrom, P., & Hyytia, P. (2007) Involvement of AMPA/kainate, NMDA, and mGlu5 receptors in the nucleus accumbens core in cue-induced reinstatement of cocaine seeking in rats. Psychopharmacology (Berl) 192(4), 571−580
Beckstead RM , Cruz CJ (1986) Striatal axons to the globus pallidus, entopeduncular nucleus and substantia nigra come mainly from separate cell populations in cat. Neuroscience. 19: 147 – 158
Bellone C, Luscher C, Mameli M. (2008) Mechanisms of synaptic depression triggered by metabotropic glutamate receptors. Cell Mol Life Sci (65): 2913–23
Carroll KM, Rounsaville BJ, Gordon LT, Nich C, Jatlow P, Bisighini RM, Gawin FH (1994) Psychotherapy and pharmacotherapy for ambulatory cocaine abusers. Arch Gen Psychiatry 51:177–187
Chiamulera C, Epping-Jordan M, Zocchi A, Marcon C, Cottiny C, Tacconi S, Corsi M, Orzi F, Conquiet F (2001) Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice. Nat Neurosci, (4):873-874
Childress, A. R., McLellan, A. T., Ehrman, R., O’Brien, C. P. (1988) Classically conditioned responses in opioid and cocaine dependence: A role in relapse? In: Ray, B. A., ed. Learning factors in substance abuse. NIDA Res. Monogr. 84:25–43
Childress, A.R, Mozley PD, McElgin W, Fitzgerald J, Reivich M, O’Brien CP (1999) Limbic activation during cue-induced cocaine craving. Am J Psychiatry 156:11–18
Conn, P.J., Pin, J.P. (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205–237
Cosford ND, Roppe J, Tehrani L, Schweiger EJ, Seiders TJ, Chaudary A (2003)[3H]-methoxymethyl-MTEP and [3H]-methoxy-PEPy: potent and selective radioligands for the metabotropic glutamate subtype 5 (mGlu5) receptor. Bioorg Med Chem Lett (13):351–4
De Vos, J.W., Ufkes, J.G.R., Van Brussel, G.H.A., Van Den Brink, W. (1996) Craving despite extremely high methadone dosage , Drug and Alcohol Dependence 40 (3), pp. 181-184
De Wit H, Stewart J (1981) Reinstatement of cocaine-reinforced responding in the rat. Psychopharmacology 75:134 –143
Gerfen, C. R. Gerfen, C.R., Engber, T.M., Mahan, L.C., Susel, Z., Chase, T.N., Monsma Jr., F.J., Sibley, D.R. (1990) D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science 250, 1429–1432
Gerfen CR , Young WS (1988) Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments: an in situ hybridization histochemistry and fl uorescent retrograde tracing study. Brain Res. 460 : 161 – 167
Gerrits M. A. F. M. and Van Ree J. M. (1996) Effect of nucleus accumbens dopamine depletion on motivational aspects involved in initiation of cocaine and heroin self-administration in rats. Brain Res. 713, 114–124
Goto Y & Grace AA (2008) Limbic and cortical information processing in the nucleus accumbens. Trends Neurosci 31(11):552-558
Grant S, London ED, Newlin DB, Villemagne VL, Liu X, Contoreggi C, Phillips RL, Kimes AS, Margolin A (1996) Activation of memory circuits during cue-elicited cocaine craving. Proc Natl Acad Sci USA 93:12040–12045
Grueter BA, McElligott ZA, Winder DG. (2007) Group I mGluRs and long-term depression: potential roles in addiction? Mol Neurobiol (36) :232–44
Hong JS, Yang HY, Costa E (1977) On the location of methionine enkephalin neurons in rat striatum. Neuropharmacology 16: 451–453
Horns, W.H., Rado, M., Goldstein, A. (1975) Plasma levels and symptom complaints in patients maintained on daily dosage of methadone hydrochloride. Clin. Pharm. Ther. 17, 636–649
Hyman, S.E., Malenka, R.C. (2001) , addiction and the brain- The neurobiology of compulsion and its persistence, Nature Reviews Neuroscience 2 (10), pp. 695-703
Jaffe JH (1990) Drug addiction and drug abuse. In: Goodman and Gilman’s the pharmacological basis of therapeutics (Gilman AG, Rall TW, Nies AS, Taylor P, eds), pp 522–557
Kalivas P. W. and Stewart J. (1991) Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res. Rev. 16, 223–244
Karler, R., Calder, L.D., Chaudhry, I.A., and Turkanis, S.A. (1989) Blockade of “reverse tolerance” to cocaine and amphetamine by MK-801. Life Sci. 45, 599–606
Kauer, J.A., and Malenka, R.C. (2007) Synaptic plasticity and addiction. Nat. Rev. Neurosci. 8, 844–858
Kawaguchi Y , Wilson CJ , Emson PC (1990) Projection subtypes of rat neostriatal matrix cells revealed by intracellular injection of biocytin. J Neurosci (10), 3421–3438
Kenny, P.J., Markou, A. (2004) The ups and downs of addiction: role of metabotropic glutamate receptors. Trends Pharmacol. Sci. (25), 265–272
Kenny, P. J., Boutrel B., Gasparini, F., Koob G.F, Markou A. (2005) Metabotropic glutamate 5 receptor blockade may attenuate cocaine self-administration by decreasing brain reward function in rats. Psychopharmacology 179 (1), 247–254
Koob G. F. (1992) Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends pharmac. Sci. 13, 177–184
Koob G. F. (1992) Neural mechanisms of drug reinforcement. Ann. N. Y. Acad. Sci. 654, 171– 191
Koob, G. F., & Le Moal, M. (1997) Drug abuse: hedonic homeostatic dysregulation. Science 278(5335), 52−58
Kumaresan, V., Yuan, M., Yee, J., Famous, K.R., Anderson, S.M., Schmidt, H.D., Pierce, R.C. (2009) Metabotropic glutamate receptor 5 (mGluR5) antagonists attenuate cocaine priming- and cue-induced reinstatement of cocaine seeking,Behavioural Brain Research 202 (2) : 238-244
Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R., Bloch, B. (1990) Dopamine receptor gene expression by enkephalin neurons in rat forebrain, Proceedings of the National Academy of Sciences of the United States of America 87 (1), pp. 230-234
Lu, X.Y., Ghasemzadeh, M.B., Kalivas, P.W.(1999) Expression of glutamate receptor subunit/subtype messenger RNAs for NMDARI, GLuRl, GLuR2 and mGLuR5 by accumbal projection neurons. Brain Res. Mol. Brain Res (63): 287–296
Ludwig AM, Wikler A, Stark LH (1974) The first drink: psychobiological aspects of craving. Arch Gen Psychiatry 30:539–547
Matamales, M., Bertran-Gonzalez, J., Salomon, L., Degos, B., Deniau, J.-M., Valjent, E., Hervé, D., Girault, J.-A. (2009) Striatal medium-sized spiny neurons: Identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice ,PLoS ONE (4) 3: 4770
McFarland K. and Ettenberg A. (1997) Reinstatement of drug-seeking behavior produced by heroin-predictive environmental stimuli. Psychopharmacology 131, 86–92
McGeehan AJ, Olive MF (2003) The mGluR5 antagonist MPEP reduces the conditioned rewarding effects of cocaine but not other drugs of abuse. Synapse (47):240-242
Meyer RE (1988) Conditioning phenomena and the problem of relapse in opioid addicts and alcoholics. NIDA Res Monogr 84:161–179
Nestler EJ, Berhow MT, Brodkin ES (1996) Molecular mechanisms of drug addiction: adaptations in signal transduction pathways. Mol Psychiatry 1:190–199
Ouimet CC, Greengard P (1990) Distribution of DARPP-32 in the basal ganglia: an electron microscopic study. J Neurocytol 19: 39–52
Ouimet C. C., Miller P. E., Hemmings H. C. Jr, Walaas S. I. and Greengard P. (1984) DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. III. Immunocytochemical localization. J. Neurosci. 4, 111– 124
Paxinos G. and Franklin K.B.J, 2001, The Mouse Brain in Sterotaxic Coordinates, 2nd edition, Academic Press.
Robinson, T. E. & Berridge, K. C. (1993) The neural basis of drug craving: an incentive– sensitization theory of addiction. Brain Res. Brain Res. Rev. 18, 247–291
Robinson, T. E. & Berridge, K. C. (2000) The psychology and neurobiology of addiction: an incentive-sensitization view. Addiction 95, S91–117
Self DW, Genova LM, Hope BT, Barnhart WJ, Spencer JJ, Nestler EJ (1998) Involvement of cAMP-dependent protein kinase in the nucleus accumbens in cocaine self-administration and relapse of cocaineseeking behavior. J Neurosci 18:1848 –1859
Sibley, D.R., and Monsma, F.J., Jr. (1992). Molecular biology of dopamine receptors. Trends Pharmacol. Sci. 13, 61–69
Spealman RD, Barret-Larimore RL, Rowlett JK, Platt DM, Khroyan TV (1999) Pharmacological and environmental determinants of relapse to cocaine-seeking behavior. Pharmacol Biochem Behav 64:327–336
Stipanovich A, Valjent E, Matamales M, Nishi A, Ahn JH, et al. (2008) A phosphatase cascade by which rewarding stimuli control nucleosomal response.Nature 453: 879–884
Surmeier DJ , Song WJ , Yan Z (1996) Coordinated expression of dopamine receptors in neostriatal medium spiny neurons. J Neurosci . (16) : 6579 – 6591
Tallaksen-Greene SJ , Kaatz KW , Romano C , Albin RL. (1998) Localization of mGluR1a-like immunoreactivity and mGluR5-like immunoreactivity in identifi ed populations of striatal neurons. Brain Res . 780 : 210 – 217
Tallaksen -Greene S.J., Wiley R.G, Albin R.L. (1992) Localization of striatal excitatory amino acid binding site subtypes to striatonigral projection neurons, Brain Res. 594. 165–170
Tepper JM, Bolam JP (2004) Functional diversity and specificity of neostriatal interneurons. Curr Opin Neurobiol 14: 685–692
Testa C. M., Standaert D. G., Landwehrmeyer G. B., Penney J. B. Jr., and Young A. B. (1995) Differential expression of mGluR5 metabotropic glutamate receptor mRNA by rat striatal neurons. J. Comp. Neurol. 354, 241–252
Thomas, M.J., Kalivas, P.W., and Shaham, Y. (2008) Neuroplasticity in the mesolimbic dopamine system and cocaine addiction. Br. J. Pharmacol. 154, 327–342
Tzschentke TM, Schmidt WJ (2003) Glutamatergic mechanisms in addiction. Mol Psychiatry 8:373–382
Valjent, E., Bertran-Gonzalez, J., Hervé, D., Fisone, G., Girault, J.-A. (2009) Looking BAC at striatal signaling: cell-specific analysis in new transgenic mice ,Trends in Neurosciences 32 (10), pp. 538-547
Walaas S. I., Aswad D. W. and Greengard P. (1983) A dopamine- and cyclic AMP regulated phosphoprotein enriched in dopamineinnervated brain regions. Nature 301, 69–71
Wikler, A. (1971) Some implications of conditioning theory for problems of drug abuse. Behav. Sci. 16:92–97
Wise R. A. and Hoffman D. C. (1992) Localization of drug reward mechanisms by intracranial injections. Synapse 10, 247–263
Wise, R.A., 2002. Brain reward circuitry: insights from unsensed incentives. Neuron 36, 229– 240
Wolf, M.E. (2002) Addiction: making the connection between behavioral changes and neuronal plasticity in specific pathways. Mol Interv 2 (3), pp. 146-157
Wolf, M.E. and Khansa, M.R. (1991) Repeated administration of MK-801 produces sensitization to its own locomotor stimulant effects but blocks sensitization to amphetamine. Brain Res. 562, 164–168
Worley CM, Valadez A, Shenk S (1994) Reinstatement of extinguished cocaine-taking behavior by cocaine and caffeine. Pharmacol Biochem Behav 48:217–221
Yung, K. K. (1995). Immunocytochemical localization of D1 and D2 dopamine receptors in the basal ganglia of the rat: light and electron microscopy.Neuroscience 65, 709–730
