Disruption of a dopamine receptor complex amplifies the actions of cocaine
Introduction
Cocaine addiction is a chronic disorder associated with uncontrollable craving together with compulsive drug-seeking and drug-taking, which persists despite devastatingly negative health, social and economic consequences. It is well established that increased dopamine signaling and activation of dopamine D1 receptors in nucleus accumbens (NAc) plays a significant role in the development and maintenance of cocaine seeking (Anderson and Pierce, 2005, Nestler, 2005). Yet despite this knowledge, therapeutic targeting of specific neuronal pathways in vivo remains difficult to accomplish and, as such, the mainstay of current addiction therapy remains substitution, which has significant limitations, low success rates and high propensity for relapse. Thus the identification of a specific receptor substrate that regulates addiction processes could completely shift drug discovery paradigms.
In the basal ganglia dopamine D1 and D2 receptors are predominantly segregated to distinct neuronal pathways, the direct striatonigral and indirect striatopallidal pathways respectively, and are thought to be the predominant mediators of dopamine effects in this region. However, a fraction of striatal medium spiny neurons (MSNs) express both receptors with colocalization of D1 and D2 receptors occurring predominantly in NAc (Aubert et al., 2000, Bertran-Gonzalez et al., 2008, Deng et al., 2006, Gangarossa et al., 2013, Perreault et al., 2010). Dopamine signaling in NAc has been widely demonstrated to play a pivotal role in cocaine addiction (Anderson and Pierce, 2005, Lammel et al., 2014), yet the contribution of these D1/D2 receptor-coexpressing MSNs in mediating addiction processes has been ignored. As a therapeutic target with proven efficacy in cocaine addiction has yet to be identified, discerning the physiological relevance of these MSNs is imperative to discover novel avenues for drug discovery.
Up to 17–38% of D1-expressing MSNs also express the D2 receptor in specific NAc subregions of the mouse, rat and monkey (Aubert et al., 2000, Bertran-Gonzalez et al., 2008, Deng et al., 2006, Gangarossa et al., 2013, Perreault et al., 2010) whereas in the rodent caudate nucleus (CN) neuronal cell bodies only 1–6% of such MSNs exhibit D1 and D2 receptor colocalization (Bertran-Gonzalez et al., 2008, Perreault et al., 2010). Within most of the D1/D2 receptor-coexpressing MSNs in NAc the receptors exist within a complex forming the D1–D2 receptor heteromer (Perreault et al., 2010). The D1–D2 heteromer was shown to exhibit distinct pharmacological and functional properties (Lee et al., 2004, Rashid et al., 2007) and to induce the activation or expression of proteins known to be highly involved in addiction such as calcium calmodulin kinase II and brain-derived neurotrophic factor (Hasbi et al., 2009, Ng et al., 2010, Perreault et al., 2012a). The role of the D1–D2 heteromer in mediating the behavioral and biochemical effects of cocaine was evaluated.
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Animals
Adult male Sprague-Dawley rats (Charles River, Canada) weighing 300–350 g at the start of each experiment and a total of two adult male Macaca fascicularis primates (body weight 3.8–4.5 kg) were used. Homozygous whole body D1 and D2 receptor gene deleted mice were congenic, backcrossed into a C57BL/6J genetic background.
TAT-D1 and scrambled peptide
The TAT-D1 peptide was generated and its specificity tested as described using different strategies including mutations, truncations, BRET and pharmacological studies (Hasbi et
Dopamine D1–D2 heteromers exist in rodent and monkey striatum
To demonstrate the existence of native D1–D2 heteromer in situ in MSNs in brain two methods were employed, the proximity ligation assay (PLA) and confocal fluorescence resonance energy transfer (FRET). The specificity of the primary antibodies for D1 and D2 receptors was validated by immunochemistry in HEK293T cells individually expressing each of the five dopamine receptor subtypes (Lee et al., 2004) and in striatal sections from D1-/- and D2-/- receptor gene-deleted mice (Appendix A, Figure S1
Discussion
In the present study we showed conclusively that there exists a dopamine receptor complex, the dopamine D1–D2 receptor heteromer, in the NAc of rodents and non-human primates. Pharmacological analysis together with a highly selective interfering peptide further showed that the receptor complex had signaling characteristics distinct from its constituent D1 or D2 receptors, being coupled to the Gq protein with the promotion of intracellular calcium signaling through activation of phospholipase C.
Contributors
MLP, AH, PJF, JLL and SRG designed research; MLP, AH, TF, MS, GN, JLL and RF performed research; MLP, AH, MLP, RF, JLL and SRG analyzed data; and MLP, AH and SRG wrote the paper.
Conflict of interest
The authors declare no conflict of interests.
Acknowledgments
This work was supported by a Grant from the National Institute on Drug Abuse (DA-007223 to S.R.G). S.R.G. holds a Canada Research Chair in Molecular Neuroscience. M.L.P was supported by a NARSAD Young Investigator Award (Brain and Behavior Research Foundation).
Each funding source had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
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2021, Pharmacological ResearchCitation Excerpt :These, the D1R-D2R heterodimers are expressed within striatal and NAc areas, with the highest degree in the NAc shell followed by NAc core, and then putamen and caudate nuclei [32,33,35]. Functionally, D1R-D2R heterodimers were found to signal through the activation of Gαq/11, in so doing, leading to intracellular calcium release, phosphorylation of calcium calmodulin kinase II (CAMKII), and increased brain-derived neurotrophic factor (BDNF) expression in the NAc and VTA [28,31,36,37]. The heterodimer mediated Gαq/11 cascade requires the co-activation of both receptors and the antagonist of either receptor can block this effect [28].
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2020, Neurobiology of DiseaseCitation Excerpt :These data also underlined the difference between NAc and CN in terms of heteromer expression, with the NAc containing a consistently greater number of cells expressing the D1-D2 heteromer compared to the caudate-putamen nuclei throughout the various species investigated. As mentioned, the dopamine D1-D2 heteromer appears to be involved functionally in certain aspects of cocaine addiction as shown in a rat model system (Perreault et al., 2016; Hasbi et al., 2018). The effect(s) of repeated treatment with cocaine (10 mg/kg, daily i.p., for 8 days) on the D1-D2 heteromer and on the colocalization of D1 and D2 mRNAs were investigated.
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2019, Neuroscience and Biobehavioral ReviewsCitation Excerpt :At the molecular level, heteromerization would take place between two adjacent arginine residues located on the D2 third intracellular loop IL3 and two adjacent glutamic acid residues located on the D1 C-terminal tail (O’Dowd et al., 2012). Accordingly, a TAT-D1 peptide encoding the critical portion of the D1 C-terminus disrupted D1–D2 co-immunoprecipitation (Perreault et al., 2016) and abolished the PLA signal (Hasbi et al., 2018) in the rat striatum. Functionally, D1-D2 heteromers would specifically signal through Gαq/11 leading to CAMKII activation, intracellular calcium release and increased BDNF expression when co-activated by the D1 agonist SKF81297 and the D2 agonist quinpirole or when activated by the selective D1–D2 agonist SKF83959 (Hasbi et al., 2009; Lee et al., 2004; Perreault et al., 2016; Rashid et al., 2007).
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Authors contributed equally to the manuscript.