Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB.

A significant subpopulation of neurons in rat nucleus accumbens (NAc) coexpress dopamine D1 and D2 receptors, which can form a D1-D2 receptor complex, but their relevance in addiction is not known. The existence of the D1-D2 heteromer in the striatum of rat and monkey was established using in situ PLA, in situ FRET and co-immunoprecipitation. In rat, D1-D2 receptor heteromer activation led to place aversion and abolished cocaine CPP and locomotor sensitization, cocaine intravenous self-administration and reinstatement of cocaine seeking, as well as inhibited sucrose preference and abolished the motivation to seek palatable food. Selective disruption of this heteromer by a specific interfering peptide induced reward-like effects and enhanced the above cocaine-induced effects, including at a subthreshold dose of cocaine. The D1-D2 heteromer activated Cdk5/Thr75-DARPP-32 and attenuated cocaine-induced pERK and ΔFosB accumulation, together with inhibition of cocaine-enhanced local field potentials in NAc, blocking thus the signaling pathway activated by cocaine: D1R/cAMP/PKA/Thr34-DARPP-32/pERK with ΔFosB accumulation. In conclusion, our results show that the D1-D2 heteromer exerted tonic inhibitory control of basal natural and cocaine reward, and therefore initiates a fundamental physiologic function that limits the liability to develop cocaine addiction.

A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism.

Although the dopamine D1-D2 receptor heteromer has emerging physiological relevance and a postulated role in different neuropsychiatric disorders, such as drug addiction, depression, and schizophrenia, there is a need for pharmacological tools that selectively target such receptor complexes in order to analyze their biological and pathophysiological functions. Since no selective antagonists for the D1-D2 heteromer are available, serial deletions and point mutations were used to precisely identify the amino acids involved in an interaction interface between the receptors, residing within the carboxyl tail of the D1 receptor that interacted with the D2 receptor to form the D1-D2 receptor heteromer. It was determined that D1 receptor carboxyl tail residues (404)Glu and (405)Glu were critical in mediating the interaction with the D2 receptor. Isolated mutation of these residues in the D1 receptor resulted in the loss of agonist activation of the calcium signaling pathway mediated through the D1-D2 receptor heteromer. The physical interaction between the D1 and D2 receptor could be disrupted, as shown by coimmunoprecipitation and BRET analysis, by a small peptide generated from the D1 receptor sequence that contained these amino acids, leading to a switch in G-protein affinities and loss of calcium signaling, resulting in the inhibition of D1-D2 heteromer function. The use of the D1-D2 heteromer-disrupting peptide in vivo revealed a pathophysiological role for the D1-D2 heteromer in the modulation of behavioral despair. This peptide may represent a novel pharmacological tool with potential therapeutic benefits in depression treatment.

µ-δ opioid receptor heteromer-specific signaling in the striatum and hippocampus.

The µ-δ opioid receptor heteromer activates the pertussis toxin-resistant Gαz GTP-binding protein following stimulation by the δ-agonist deltorphin-II whereas µ- and δ-receptors activate the pertussis toxin-sensitive Gαi3 protein following stimulation by µ- and δ-agonists, respectively. Although the regulation of the µ-δ heteromer is being investigated extensively in vitro, its physiological relevance remains elusive owing to a lack of available molecular tools. We investigated µ-δ heteromer signaling under basal conditions and following prolonged morphine treatment in rodent brain regions highly co-expressing µ- and δ-receptors and Gαz. Deltorphin-II induced Gαz activation in the striatum and hippocampus, demonstrating the presence of µ-δ heteromer signaling in these brain regions. Prolonged morphine treatment, which desensitizes µ- and δ-receptor function, had no effect on µ-δ heteromer signaling in the brain. Our data demonstrate that µ-δ heteromer signaling does not desensitize and is regulated differently from µ- and δ-receptor signaling following prolonged morphine treatment.

Dopamine D1-D2 receptor heteromer regulates signaling cascades involved in addiction: potential relevance to adolescent drug susceptibility.

Adolescence is a developmental period that has been associated with heightened sensitivity to psychostimulant-induced reward, thus placing adolescents at increased risk to develop drug addiction. Although alterations in dopamine-induced synaptic plasticity are perhaps the most critical factor in mediating addiction processes, developmental differences in the cell signaling mechanisms that contribute to synaptic plasticity, and their contribution to adolescent reward sensitivity, has been grossly understudied. The most abundant dopamine receptors, the D1 and D2 receptors, as well as the dopamine D1-D2 receptor heteromer, exhibit age-dependent and brain region-specific changes in their expression and function and are responsible for regulating cell signaling pathways known to significantly contribute to the neurobiological mechanisms underlying addiction. The D1-D2 receptor heteromer, for instance, has been associated with calcium calmodulin kinase IIα, brain-derived neurotrophic factor and glycogen synthase kinase 3 (GSK-3) signaling, three proteins highly implicated in the regulation of glutamate transmission and synaptic plasticity and which regulate addiction to amphetamine, opioids and cocaine. Therefore, in this review the importance of these signaling proteins as potential mediators of addiction susceptibility in adolescence will be highlighted, and the therapeutic potential of the D1-D2 receptor heteromer in addiction will be discussed. It is the overall goal of this review to draw attention to the research gap in dopamine-induced cell signaling in the adolescent brain--knowledge that would provide much-needed insights into adolescent addiction vulnerability.

Heteromeric dopamine receptor signaling complexes: emerging neurobiology and disease relevance.

The pharmacological modification of dopamine transmission has long been employed as a therapeutic tool in the treatment of many mental health disorders. However, as many of the pharmacotherapies today are not without significant side effects, or they alleviate only a particular subset of symptoms, the identification of novel therapeutic targets is imperative. In light of these challenges, the recognition that dopamine receptors can form heteromers has significantly expanded the range of physiologically relevant signaling complexes as well as potential drug targets. Furthermore, as the physiology and disease relevance of these receptor heteromers is further understood, their ability to exhibit pharmacological and functional properties distinct from their constituent receptors, or modulate the function of endogenous homomeric receptor complexes, may allow for the development of alternate therapeutic strategies and provide new avenues for drug design. In this review, we describe the emerging neurobiology of the known dopamine receptor heteromers, their physiological relevance in brain, and discuss the potential role of these receptor complexes in neuropsychiatric disease. We highlight their value as targets for future drug development and discuss innovative research strategies designed to selectively target these dopamine receptor heteromers in the search for novel and clinically efficacious pharmacotherapies.

Enhanced brain-derived neurotrophic factor signaling in the nucleus accumbens of juvenile rats.

Brain-derived neurotrophic factor (BDNF) signaling through its receptor, tropomyosin receptor kinase B (TrkB), plays a critical role in neural plasticity and its dysregulation in striatum and prefrontal cortex (PFC) has been implicated in the etiology of mental health disorders such schizophrenia and drug addiction. In the present study, we characterized age-dependent differences in BDNF signaling and TrkB expression within the nucleus accumbens (NAc), caudate putamen (CP) and PFC in rats and determined the effects of administration of the dopamine agonist, SKF 83959, which activates the Gq-coupled dopamine receptors, the dopamine D5 receptor and the D1-D2 receptor heteromer. As proBDNF binds with high affinity to the p75 neurotrophin receptor (p75NTR), expression levels of these proteins were also assessed. The present findings showed that juvenile rats (aged 26-28 days) exhibited significantly elevated basal BDNF expression and activation of full-length TrkB (TrkBfull) in NAc compared to their adult counterparts, as evidenced by increased TrkBfull phosphorylation. These changes were concomitant with an increase in the relative expression of TrkBfull compared to the truncated isoform, TrkB.T1, in NAc and CP. Conversely, in PFC the basal expression of BDNF in juvenile rats was significantly lower than in adult rats with an elevated relative expression of TrkBfull. Acute administration of SKF 83959 to juvenile rats abolished the age-dependent differences in BDNF expression in NAc and PFC, and in the relative expression of TrkBfull in NAc and CP. Together these findings indicate that the expression and/or signaling of BDNF and TrkB in striatum and PFC of juvenile rats is fundamentally different from that of adult rats, a finding that may have implications in neuropsychiatric disorders that exhibit age-dependent susceptibility such as schizophrenia and drug addiction.

Postnatal maternal deprivation and pubertal stress have additive effects on dopamine D2 receptor and CaMKII beta expression in the striatum.

The goal of this study was to determine whether two stressors commonly used to model aspects of neuropsychiatric disease in rats have an additive effect on striatal dopamine type 2 receptor (D2R) expression, a key player in the etiology of neuropsychiatric disease. Animals subjected to early postnatal stress show alterations in function of the dopaminergic system thought to be mediated by stress-induced glucocorticoid release. Subsequent stress during puberty is known to further impact the dopaminergic system and result in dopaminergic hyperactivity analogous to schizophrenia. We exposed rats to maternal deprivation (MD) during the second postnatal week, a time of active striatal development. A subset of these animals were then subjected to pubertal stress induced by immobilization. Both procedures are know to induce glucocorticoid release. At the conclusion of the MD protocol, we observed upregulation in the expression of D2R and of dopamine- and cAMP-regulated phosphoprotein 32-KD (DARPP-32; PPP1R1B), but not of D1R, calcium/calmodulin-dependent protein kinase II beta (CaMKIIß), CaMKIIα or neurokinin B (NKB). Animals exposed to pubertal stress showed upregulation in expression of both D2R and CaMKIIß. Furthermore, rats previously exposed to MD showed a much greater upregulation in CaMKIIß expression, than animals only exposed to pubertal stress. These results support the two-hit hypothesis, indicating that such stressors have an additive effect. The main targets appear to be the D2R and the CaMKIIß, the latter being an important member of the DR signalling pathway, both of which are associated with schizophrenia.

D5 dopamine receptor carboxyl tail involved in D5-D2 heteromer formation.

We have demonstrated that D(5) and D(2) dopamine receptors exist as heteromers in cells, and determined these receptor interact through amino acids in the cytoplasmic regions of each receptor. Specifically involved in heteromer formation we identified in the carboxyl tail of the D(5) receptor three adjacent glutamic acid residues, and in intracellular loop 3 of the D(2) receptor two adjacent arginine residues. Any pairing of these three D(5) receptor glutamic acids were sufficient for heteromer formation. These identified residues in D(5) and D(2) receptors are oppositely charged and likely interact by electrostatic interactions.

A physiological role for the dopamine D5 receptor as a regulator of BDNF and Akt signalling in rodent prefrontal cortex.

The dopamine D5 receptor (D5R) exhibits a wide distribution in prefrontal cortex (PFC) but its role in this region has not yet been elucidated. In the present study, we identified a novel physiological function for the D(5)R as a regulator of brain-derived neurotrophic factor (BDNF) and Akt signalling in PFC. Specifically, acute activation of the D(5)R by the dopamine agonist SKF 83959 enhanced BDNF expression and signalling through its receptor, tropomyosin receptor kinase B (TrkB), in rats and in mice gene-deleted for the D1 receptor but not the D(5)R. These changes were concomitant with increased expression of GAD67, a protein whose down-regulation has been implicated in the aetiology of schizophrenia. Furthermore, D(5)R activation increased phosphorylation of Akt at the Ser(473) site, consequently decreasing the activity of its substrate GSK-3ß. These findings could have wide-reaching implications given evidence showing activation of these pathways in PFC has therapeutic effects in neuropsychiatric disorders such as drug addiction, schizophrenia and depression.

Striatal development involves a switch in gene expression networks, followed by a myelination event: implications for neuropsychiatric disease.

Because abnormal development of striatal neurons is thought to be the part of pathology underlying major psychiatric illnesses, we studied the expression pattern of genes involved in striatal development and of genes comprising key striatal-specific pathways, during an active striatal maturation period, the first two postnatal weeks in rat. This period parallels human striatal development during the second trimester, when prenatal stress is though to lead to increased risk for neuropsychiatric disorders. To identify genes involved in this developmental process, we used subtractive hybridization, followed by quantitative real-time PCR, which allowed us to characterize the developmental expression of over 60 genes, many not previously known to play a role in neuromaturation. Of these 12 were novel transcripts, which did not match known genes, but which showed strict developmental expression and may play a role in striatal neurodevelopment. An additional 89 genes were identified as strong candidates for involvement in this neurodevelopmental process. We show that during the first two postnatal weeks in rat, an early gene expression network, still lacking key striatal-specific signaling pathways, is downregulated and replaced by a mature gene expression network, containing key striatal-specific genes including the dopamine D1 and D2 receptors, conferring to these neurons their functional identity. Therefore, before this developmental switch, striatal neurons lack many of their key phenotypic characteristics. This maturation process is followed by a striking rise in expression of myelination genes, indicating a striatal-specific myelination event. Such strictly controlled developmental program has the potential to be a point of susceptibility to disruption by external factors. Indeed, this period is known to be a susceptibility period in both humans and rats.

Disruption of the mu-delta opioid receptor heteromer.

The crystal structure of the mu and kappa opioid receptors has revealed dimeric structural arrangements. Mu-delta receptors heteromers also exist and we have identified discrete cytoplasmic regions in each receptor required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In intracellular loop 3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation.

Dopamine D1-D2 receptor heteromer in dual phenotype GABA/glutamate-coexpressing striatal medium spiny neurons: regulation of BDNF, GAD67 and VGLUT1/2.

In basal ganglia a significant subset of GABAergic medium spiny neurons (MSNs) coexpress D1 and D2 receptors (D1R and D2R) along with the neuropeptides dynorphin (DYN) and enkephalin (ENK). These coexpressing neurons have been recently shown to have a region-specific distribution throughout the mesolimbic and basal ganglia circuits. While the functional relevance of these MSNs remains relatively unexplored, they have been shown to exhibit the unique property of expressing the dopamine D1-D2 receptor heteromer, a novel receptor complex with distinct pharmacology and cell signaling properties. Here we showed that MSNs coexpressing the D1R and D2R also exhibited a dual GABA/glutamate phenotype. Activation of the D1R-D2R heteromer in these neurons resulted in the simultaneous, but differential regulation of proteins involved in GABA and glutamate production or vesicular uptake in the nucleus accumbens (NAc), ventral tegmental area (VTA), caudate putamen and substantia nigra (SN). Additionally, activation of the D1R-D2R heteromer in NAc shell, but not NAc core, differentially altered protein expression in VTA and SN, regions rich in dopamine cell bodies. The identification of a MSN with dual inhibitory and excitatory intrinsic functions provides new insights into the neuroanatomy of the basal ganglia and demonstrates a novel source of glutamate in this circuit. Furthermore, the demonstration of a dopamine receptor complex with the potential to differentially regulate the expression of proteins directly involved in GABAergic inhibitory or glutamatergic excitatory activation in VTA and SN may potentially provide new insights into the regulation of dopamine neuron activity. This could have broad implications in understanding how dysregulation of neurotransmission within basal ganglia contributes to dopamine neuronal dysfunction.

Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1-D2 heteromer formation.

D(1) and D(2) dopamine receptors exist as heteromers in cells and brain tissue and are dynamically regulated and separated by agonist concentrations at the cell surface. We determined that these receptor pairs interact primarily through discrete amino acids in the cytoplasmic regions of each receptor, with no evidence of any D(1)-D(2) receptor transmembrane interaction found. Specifically involved in heteromer formation we identified, in intracellular loop 3 of the D(2) receptor, two adjacent arginine residues. Substitution of one of the arginine pair prevented heteromer formation. Also involved in heteromer formation we identified, in the carboxyl tail of the D(1) receptor, two adjacent glutamic acid residues. Substitution of one of the glutamic acid pair prevented heteromer formation. These amino acid pairs in D(1) and D(2) receptors are oppositely charged, and presumably interact directly by electrostatic interactions.

The dopamine d1-d2 receptor heteromer in striatal medium spiny neurons: evidence for a third distinct neuronal pathway in Basal Ganglia.

Dopaminergic signaling within the basal ganglia has classically been thought to occur within two distinct neuronal pathways; the direct striatonigral pathway which contains the dopamine D1 receptor and the neuropeptides dynorphin (DYN) and substance P, and the indirect striatopallidal pathway which expresses the dopamine D2 receptor and enkephalin (ENK). A number of studies have also shown, however, that D1 and D2 receptors can co-exist within the same medium spiny neuron and emerging evidence indicates that these D1/D2-coexpressing neurons, which also express DYN and ENK, may comprise a third neuronal pathway, with representation in both the striatonigral and striatopallidal projections of the basal ganglia. Furthermore, within these coexpressing neurons it has been shown that the dopamine D1 and D2 receptor can form a novel and pharmacologically distinct receptor complex, the dopamine D1-D2 receptor heteromer, with unique signaling properties. This is indicative of a functionally unique role for these neurons in brain. The aim of this review is to discuss the evidence in support of a novel third pathway coexpressing the D1 and D2 receptor, to discuss the potential relevance of this pathway to basal ganglia signaling, and to address its potential value, and that of the dopamine D1-D2 receptor heteromer, in the search for new therapeutic strategies for disorders involving dopamine neurotransmission.

Dopamine D1-D2 receptor heteromer signaling pathway in the brain: emerging physiological relevance.

Dopamine is an important catecholamine neurotransmitter modulating many physiological functions, and is linked to psychopathology of many diseases such as schizophrenia and drug addiction. Dopamine D1 and D2 receptors are the most abundant dopaminergic receptors in the striatum, and although a clear segregation between the pathways expressing these two receptors has been reported in certain subregions, the presence of D1-D2 receptor heteromers within a unique subset of neurons, forming a novel signaling transducing functional entity has been shown. Recently, significant progress has been made in elucidating the signaling pathways activated by the D1-D2 receptor heteromer and their potential physiological relevance.

Separation and reformation of cell surface dopamine receptor oligomers visualized in cells.

We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR-NLS is retained at the cell surface, and following ligand removal, the GPCR-NLS translocated from the cell surface. The D(1) dopamine receptor expressed with either D(2)-NLS or D(1-)NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D(1)-D(2) dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D(1) receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D(1)-D(2) heteromer. Dopamine D(1) or D(2) receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D(1)-D(2) heterooligomer contains D(1) homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation.

The role of palmitoylation in directing dopamine D1 receptor internalization through selective endocytic routes.

We previously determined that D1 receptors can endocytose through caveolae, a subset of lipid rafts, in addition to internalization via a clathrin-dependent pathway. In this report, we investigated the potential role that palmitoylation might have on directing D1 receptor internalization through either a clathrin or caveolar-dependent route. Through whole cell binding analysis and sucrose gradient fractionation studies, we demonstrated that although palmitoylation of the D1 receptor was not required for agonist-independent localization to caveolae, agonist induced internalization kinetics of a de-palmitoylated D1 receptor were accelerated ∼8-fold in comparison to wild-type D1 receptor and were very similar to that observed for clathrin-dependent D1 receptor internalization. Additionally, inhibition of the clathrin mediated pathway led to significant attenuation in the extent of agonist induced internalization of the de-palmitoylated D1 receptor, suggesting the de-palmitoylated D1 receptor was directed to a clathrin-dependent internalization pathway. Taken together, these data suggest that palmitoylation may be involved in directing agonist-dependent D1 receptor internalization through selective endocytic routes.

Dopamine receptor homooligomers and heterooligomers in schizophrenia.

Over the past two decades the dopamine D2 receptor has been undoubtedly the most widely studied dopamine receptor for the therapeutic treatment of schizophrenia, as the majority of antipsychotics exhibit antagonism at this receptor. However, the cognitive symptoms of the disorder are mostly resistant to the majority of available antipsychotic treatments and, as a result, there is a critical need to develop novel therapies that ameliorate all symptoms. The recognition that dopamine receptors, such as all G protein-coupled receptors (GPCRs), exist as oligomeric complexes has provided new avenues for drug design in the search for novel therapies. Furthermore, that it is now known that dopamine receptors can form heteromers, such as the dopamine D1-D2 receptor heteromer, with pharmacology and function distinct from its constituent receptors, has significantly expanded the range of potential drug targets. The aim of this review is to discuss the therapeutic relevance of these dopamine receptor oligomers to schizophrenia and to address the potential value of dopamine receptor heteromers in the search for new therapeutic strategies.

Dopamine D1-D2 receptor Heteromer-mediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2.

We identified that activation of the G(q)-linked dopamine D1-D2 receptor hetero-oligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pretreatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822, which only activates the D1 receptor homo-oligomer, led to rapid desensitization of the D1-D2 receptor heteromer-mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. Bioluminescence resonance energy transfer and co-immunoprecipitation studies indicated an agonist-induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pretreatment with either agonist. Expression of the catalytically inactive and RGS (regulator of G protein signaling)-mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer-mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.

The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons: increased high affinity state following amphetamine and in schizophrenia.

The distribution and function of neurons coexpressing the dopamine D1 and D2 receptors in the basal ganglia and mesolimbic system are unknown. We found a subset of medium spiny neurons coexpressing D1 and D2 receptors in varying densities throughout the basal ganglia, with the highest incidence in nucleus accumbens and globus pallidus and the lowest incidence in caudate putamen. These receptors formed D1-D2 receptor heteromers that were localized to cell bodies and presynaptic terminals. In rats, selective activation of D1-D2 heteromers increased grooming behavior and attenuated AMPA receptor GluR1 phosphorylation by calcium/calmodulin kinase IIα in nucleus accumbens, implying a role in reward pathways. D1-D2 heteromer sensitivity and functional activity was up-regulated in rat striatum by chronic amphetamine treatment and in globus pallidus from schizophrenia patients, indicating that the dopamine D1-D2 heteromer may contribute to psychopathologies of drug abuse, schizophrenia, or other disorders involving elevated dopamine transmission.