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Coupling Between D1 and D2 Receptors Implicated in Depression

8 December 2010. Coupling between two different types of dopamine receptor appears enhanced in human depression, and interfering with this interaction has antidepressant-like effects in rats, according to a report in Nature Medicine published online on November 28.

In experimental preparations spanning postmortem brain tissue from humans, non-neuronal cell cultures, and rats, Fang Liu and colleagues at the University of Toronto in Canada examined coupling between dopamine D1 and D2 receptors. Previous work had shown that D1 and D2 could form a complex which activates a G-protein pathway that is not recruited when either receptor is activated alone (see SRF related news story). This suggested that abnormal coupling between D1 and D2 could shift cell signaling into pathological states related to psychiatric disease, and that finding ways to normalize coupling could be a new strategy for treatment.

The new study bolsters these ideas by finding that a direct interaction between D1 and D2 is increased in the brains of people who had major depression. Similarly, using a peptide to interfere with this D1-D2 interaction decreased depression-like behaviors in rats.

The D1-D2 interface
First author Lin Pei and colleagues looked at D1-D2 coupling in postmortem tissue taken from the striatum using a D2-specific antibody. In co-immunoprecipitation experiments, this antibody pulled down about 30 percent more D1 in tissue from 15 people who had had severe depression than from age- and sex-matched controls. Because amounts of D2 bound by the antibody did not differ between the two groups, this suggested that the antibody was snaring more D1 in complex with D2. Consistent with this, over twice as much of the total pool of D1 was in complex with D2 in depression than in controls.

The researchers then identified the regions of the D1 and D2 receptors that were essential for coupling. By engineering fusion peptides containing different parts of each receptor type, they found that the carboxyl tail of D1 directly interacted with the third intracellular loop of D2. Further experiments narrowed in on a 29 amino acid-stretch within this loop: one half of this section bound D1 and the other half did not. The researchers developed a blocking peptide consisting of the D1-binding half to disrupt coupling between endogenous D1 and D2. Treating non-neuronal cells expressing D1 and D2 with this peptide blocked the activation of D1- and D2-induced calcium release in the cells.

Uncoupling depression
To see if interfering with D1-D2 coupling could alleviate depression-like behaviors, the researchers turned to a forced swim test, in which rats are placed in a water bath and the amount of time they spend swimming, climbing, or floating passively—considered a depression-like behavior—is tallied. Rats that had the blocking peptide infused into their prefrontal cortex exhibited less of the passive, immobile behavior than did controls, though overall, their locomotion was normal. The effect size was similar to that obtained by infusing the antidepressant imipramine. The researchers verified that the blocking peptide was working as it should by examining the degree of D1-D2 coupling with co-immunoprecipitation experiments on brain tissue from the different groups of rats subjected to the forced swim test.

The researchers then tested the antidepressant-like activity of this blocking peptide in a learned helplessness paradigm. In the first stage, rats were exposed to the stress of an inescapable foot shock; this elevated levels of coupling between D1 and D2 receptors, which could be mitigated by imipramine. In the second stage three days later, the rats were tested for their reaction to a tone warning of imminent shock in a chamber with two rooms—one that delivered a shock, and one that offered a shock-free retreat. Rats that received the blocking peptide or imipramine after the first stage more frequently escaped the shock than did rats that had received a peptide derived from a section of the D2 receptor that did not disrupt D1-D2 coupling. Rats with the non-disrupting peptide on board seemed stuck in a learned helplessness mode, almost never escaping the shock, whereas rats with the blocking peptide escaped shock in nearly half of the trials, an effect similar to that seen with imipramine.

Figuring out how interfering with D1-D2 coupling alters dopamine signaling will require more experiments. But these results suggest that manipulating the association between these two receptors may be a useful avenue for developing effective treatments for depression that may come with fewer side effects than do current antidepressants, which typically act on D2 receptors.—Michele Solis.

Reference:
Pei L, Li S, Wang M, Diwan M, Anisman H, Fletcher PJ, Nobrega JN, Liu F. Uncoupling the dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med. 2010 Nov 28. Abstract

Comments on News and Primary Papers
Comment by:  Christoph Kellendonk
Submitted 14 December 2010
Posted 14 December 2010

Heterodimerization between D1 and D2 receptors is a recently discovered, novel mechanism by which D1 and D2 receptors activate Gq-mediated signaling in the brain. Although first met with skepticism, evidence for the existence of functional D1/D2 heterodimers under physiological conditions has become more and more convincing.

Heterodimerization between D1 and D2 receptors is linked to their coexpression in the same cell. The localization of D1 and D2 receptors has been extensively studied in the striatum. After D1 and D2 receptors were cloned 20 years ago, in situ hybridization studies suggested that there are two main populations of neurons in the striatum: one population that predominantly expresses D1 receptors and projects mono-synaptically to the substantia nigra (called the striato-nigral or direct pathway), and the other population that expresses D2 receptors and projects over several synapses to the substantia nigra (called the striato-pallidal or indirect pathway).

When these studies were followed up using single cell PCR and immunohistochemistry (IHC) using antibodies against D1 and D2 receptors, the percentage of neurons coexpressing both receptors increased to 15-30 percent, or even 100 percent for some IHC studies. One argument for the inconsistency between the early in situ hybridization studies and the newer studies had been that in situ hybridization may not be as sensitive as single cell PCR or IHC. However, the main problem with IHC is that different antibodies were used in different studies, and it is known that not all available antibodies against D2 receptors are really specific.

Recently, mice that express green fluorescent protein under the control of either the D1 or the D2 promoter have been developed that allow for selective labeling of D1- and D2-positive MSNs, respectively. The findings with D1- and D2-GFP mice are more in line with the original in situ hybridization studies showing relatively low overlap of expression in the dorsal striatum (5-7 percent of MSNs) and higher overlap in the ventral striatum (around 20 percent).

The problem of antibody specificity has also been a problem for studying heterodimers in the striatum. Therefore, in a recent study, Susan George's laboratory at the University of Toronto took great effort in testing the specificity of their antibodies (Perreault et al., 2010). One important control they included was knockout mice in which the D1 or the D2 receptor gene was inactivated. Immunohistochemistry for D1 and D2 did not show any signal in these mice, indicating specificity of the employed antibodies. Moreover, colocalization studies with these antibodies showed a degree of overlap that was comparable to what had been observed in the classical in situ hybridization studies and the recent studies using D1- and D2-GFP mice. Last, the authors used FRET technology and demonstrated coexpression at a spatial resolution that supports a direct interaction between both receptors in vivo.

The laboratory of Fang Liu, also at the University of Toronto, has now found that heterodimerization may be increased under pathological conditions. Using immunoprecipitation experiments, Pei et al. found increased coupling between D1Rs and D2Rs in the striatum and the cortex of patients with major depression (Pei et al., 2010). Perreault et al. found increased affinity for SKF83959, a heterodimer specific dopamine receptor agonist in the globus pallidus of patients with schizophrenia (Perreault et al., 2010). Since the globus pallidus is the main output structure of the indirect pathway of the striatum, it raises the question whether the degree of D1 and D2 receptor coexpression may be increased under pathological conditions

Obviously, both postmortem findings will need replication using higher subject numbers. Due to the confounding effects of postmortem tissue analysis and medication, PET imaging studies could greatly benefit this analysis. Imaging could be done earlier in the disease process and under drug-na´ve conditions. The challenge here may be the development of appropriate tracers that are both suitable for PET imaging and specific for detecting heterodimers.

That SKF83959 selectively activates heterodimers raises the possibility for the development of selective antagonists. If increased heterodimers are indeed involved in the pathophysiology of depression and schizophrenia, they may be good targets for treating negative symptoms such as anhedonia and avolition that are associated with both disorders. They may also help against psychosis, though we would then expect that D1 receptor antagonists, which block heterodimer-mediated signaling, would be effective antipsychotics.

References:

Perreault ML, Hasbi A, Alijaniaram M, Fan T, Varghese G, Fletcher PJ, Seeman P, O'Dowd BF, George SR The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons: increased high affinity state following amphetamine and in schizophrenia. J Biol Chem 285:36625-36634. Abstract

Pei L, Li S, Wang M, Diwan M, Anisman H, Fletcher PJ, Nobrega JN, Liu F Uncoupling the dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med 16:1393-1395. Abstract

View all comments by Christoph KellendonkComment by:  Jeremy Seamans
Submitted 23 December 2010
Posted 23 December 2010

Christoph nicely summarized key aspects of the paper in the context of the relevant literature. In addition, I feel the paper makes an important contribution because it draws attention to a signaling mechanism that may help explain some of the more contentious effects of dopamine.

View all comments by Jeremy Seamans

Comments on Related News


Related News: Dopamine Receptors: The Right Combination Unlocks Calcium Release

Comment by:  Christoph Kellendonk
Submitted 29 January 2007
Posted 30 January 2007
  I recommend the Primary Papers

The paper by Rashid et al. presents yet another interesting example of how dopamine D2 receptors may activate signaling pathways independent of the classical cAMP pathway, a finding that may have potential therapeutic implications. Most antipsychotic drugs that ameliorate positive symptoms antagonize D2 receptors, which may be also at the origin of many of the side effects associated with these medications. But, if antipsychotic action utilizes signaling pathways that are distinct from those responsible for the side effects we may have the chance to develop new compounds with higher specificity and reduced side effects. Observations such as those made in Rashid et al. are essential steps in this direction.

View all comments by Christoph Kellendonk

Related News: Dopamine Receptors: The Right Combination Unlocks Calcium Release

Comment by:  Eleanor Simpson
Submitted 29 January 2007
Posted 30 January 2007
  I recommend the Primary Papers

This is a very exciting paper. The concept of D1 and D2 cellular coexpression had been debated for a long time; with limited antibodies for these receptors available, investigators had found conflicting results, dependent on the method of detection used.

The authors recently described the existence of D1-D2 hetero-oligomers. Here they elucidate a possible function of such a complex. The authors begin with a very thorough biochemical characterization in HEK cells stably expressing either D1, D2, or both receptors, concluding that SKF83959 is a specific agonist for Gq/11 coupled D1-D2 receptor hetero-oligomers. By using striatal membrane preparations from wild-type, D1 mutant, or D2 mutant mice, the authors identify a D1-D2 Gq11 complex in the brains of mature mice.

The authors conclude by suggesting that D1-D2 receptor signaling may be altered in neuropsychiatric disease and that this should be explored. This may be a little premature, and perhaps some more fundamental characterization of this newly discovered complex should first be undertaken. The increase in GTPgS incorporation by 100 uM dopamine is modest compared to the increase observed with 100 uM SKF+Quin treatment. Since none of these experiments are under in vivo physiological conditions, it would be reassuring to see that this modest DA response is also blocked by SCH or RAC.

The fact that the D1-D2 Gq/11 complex was detected in 8-month-old mice but not 3-month-old mice is fascinating and begs the questions, when do these complexes form? How and why do they form? Both RT-PCR and primary culture experiments suggest that at least a fraction of neurons in the striatum coexpress D1 and D2 receptors in young adult mice. Does the number of coexpressing neurons increase with age? Or does hetero-oligomer coupling to Gq/11 increase with age? There is evidence that D1 receptor-Gs protein coupling is reduced in very old rats (Sugawa et al., 1996). Is the appearance of D1-D2 Gq/11 complexes in the striatum relevant to brain maturation, or does it relate to a decline in DA signaling efficiency?

View all comments by Eleanor Simpson

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Susan George
Submitted 15 January 2015
Posted 15 January 2015

The paper by Frederick et al. is presented as definitive evidence against the existence of the dopamine D1-D2 receptor heteromer and attempts to convey the impression that all the conclusions from the work conducted in my laboratory or those of other scientific colleagues who have worked on this complex are wrong.

This paper simply cannot be accepted at face value as definitive, as we and others have extremely robust evidence of D1 and D2 receptors forming complexes, not just in heterologously expressing HEK cells, but also in rat brain and in human brain (Lee et al., 2004; Pei et al., 2010). If the receptors were not within a complex, they would not co-immunoprecipitate with each other. Furthermore, direct receptor-receptor interaction has been shown in cultured rat striatal neurons and rat nucleus accumbens in situ by confocal FRET and FRET photobleaching (Hasbi et al., 2009; Perreault et al., 2010) and from human striatal tissue by GST pulldown and human prefrontal cortex by co-immunoprecipitation (Pei et al., 2010). These facts are overwhelming proof that the D1 and D2 receptors directly interact and exist within a complex in brain.

With respect to the calcium signal, the paper contradicts recent data from one of the co-authors, Dr. Sibley, who previously reported, "When cells were transfected with D1R and D2LR or D1R with D2SR, a clear dose-dependent activation of calcium mobilization was observed in response to DA," with the conclusion that "These data suggest that expression and activation of both the D1R and D2R are essential for coupling to calcium mobilization and signaling" (Chun et al., 2013). We and others have obtained a calcium signal from cell lines expressing sub-picomolar densities of both receptors (Rashid et al., 2007) and we have demonstrated a calcium signal by D1-D2 heteromer activation using nanomolar concentrations of DA and other agonists in early postnatal cultured striatal neurons (Hasbi et al., 2009; Verma et al., 2010).

Regarding the involvement of Gq in the signaling of this complex, we showed this by selectively immunoprecipitating 35S-GTPγS-labeled Gq from striatum (Rashid et al., 2007) and by use of a selective Gq inhibitor in cultured striatal neurons (Hasbi et al., 2009). We have never attributed the Gq pathway as being involved in the behavioral effects of SKF 83959 such as locomotion or grooming, nor have we suggested that the only mechanism by which D1-D2 heteromer signals is through Gq, so the parts of the paper showing lack of involvement of Gq or CaMKII in the behavioral studies are not necessarily surprising. We have evidence for other signaling pathways activated by the heteromer, and it remains to be analyzed what the precise mechanism for these behavioral effects might be.

Our data show that the highest expression of the D1-D2 complex in rat striatum is in nucleus accumbens shell, where we estimate 20-30 percent of medium spiny neurons expressing the D1 receptor also express the D2 receptor. Of these neurons, approximately 90 percent had evidence for heteromers by confocal FRET. In contrast, in dorsal striatum, only 5-6 percent of D1 neurons also expressed the D2 receptor, and only 20 percent of these neurons had evidence for heteromer formation. Furthermore, these neurons had a distinctive and unique phenotype in expressing both of the neuropeptides that are considered exclusive markers of D1- or D2-receptor-expressing medium spiny neurons, namely dynorphin and enkephalin. Our numbers from rat striatum are consistent with what has been reported for D1 and D2 receptor colocalization in BAC transgenic mice by numerous groups, ranging about 17 percent in nucleus accumbens (Bertran-Gonzalez et al., 2008; Matamales et al., 2009). A detailed study of subregions of the nucleus accumbens in these mice found D1 and D2 receptor colocalization in the medial bundle region of nucleus accumbens to be as high as 38 percent (Gangarossa et al., 2013). Thus, the paper by Frederick et al. reporting colocalization of D1 and D2 receptors in the nucleus accumbens shell of BAC-Tg mice of 2-5 percent is distinctly out of the range compared to what other groups have reported.

Further, the PLA technique used in the paper is another method by which protein complexes may be detected in situ. It is also based on immunohistochemistry, and the signals generated critically depend on the performance and affinities of the antibodies used. Moreover, it seems unwarranted to disregard the D1 and D2 receptors co-expressed on the neuronal cell bodies and only analyze presence of heteromer in the neuropil, as there is precedent for important extrasynaptic functions that can be regulated by receptors from the cell soma. We did demonstrate D1-D2 heteromer colocalized with synaptophysin in the dendritic field, indicating presynaptic localization in intrastriatal projections (Perreault et al., 2010). Regardless of the number of nucleus accumbens neurons expressing the D1-D2 receptor heteromer, it is still the functional importance that needs to be analyzed. There is no question of the immense importance of interneurons that only comprise 1-2 percent of neurons in striatum, in spite of the very low number.

We have used SKF 83959 to activate the D1-D2 receptor complex in our studies, fully cognizant of the fact that this atypical agonist is an agonist at the D5 receptor, has little ability to activate D1 receptor mediated adenylyl cyclase, and has several other unwanted cross-reactivities. However, in cultured striatal neurons, where there is no D5 receptor, the use of D1 and D2 receptor antagonists has ascertained the fidelity of the responses we were studying. To transition our studies into the whole animal, we have delineated the specific residues mediating a critical interaction interface between D1 and D2 receptors (two amino acids in each receptor), and have generated a specific peptide based on the D1 receptor sequence and relied on the use of this peptide in vivo to dissect out specific heteromer mediated functions. We have some very exciting results emerging regarding the physiological functions and certain pathophysiological roles of the D1-D2 heteromer, so stay tuned. The chapter has not ended.

References:

Lee I, Skinner MA, Guo HB, Sujan A, Pierce M. Expression of the vacuolar H+-ATPase 16-kDa subunit results in the Triton X-100-insoluble aggregation of beta1 integrin and reduction of its cell surface expression. J Biol Chem. 2004 Dec 17; 279(51):53007-14. Abstract

Pei L, Li S, Wang M, Diwan M, Anisman H, Fletcher PJ, Nobrega JN, Liu F. Uncoupling the dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med. 2010 Dec; 16(12):1393-5. Abstract

Hasbi A, Fan T, Alijaniaram M, Nguyen T, Perreault ML, O'Dowd BF, George SR. Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth. Proc Natl Acad Sci U S A. 2009 Dec 15; 106(50):21377-82. Abstract

Perreault ML, Hasbi A, Alijaniaram M, Fan T, Varghese G, Fletcher PJ, Seeman P, O'Dowd BF, George SR. The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons: increased high affinity state following amphetamine and in schizophrenia. J Biol Chem. 2010 Nov 19; 285(47):36625-34. Abstract

Chun LS, Free RB, Doyle TB, Huang XP, Rankin ML, Sibley DR. D1-D2 dopamine receptor synergy promotes calcium signaling via multiple mechanisms. Mol Pharmacol. 2013 Aug; 84(2):190-200. Abstract

Rashid AJ, So CH, Kong MM, Furtak T, El-Ghundi M, Cheng R, O'Dowd BF, George SR. D1-D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum. Proc Natl Acad Sci U S A. 2007 Jan 9; 104(2):654-9. Abstract

Verma V, Hasbi A, O'Dowd BF, George SR. 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. J Biol Chem. 2010 Nov 5; 285(45):35092-103. Abstract

Bertran-Gonzalez J, Bosch C, Maroteaux M, Matamales M, Herve, Valjent E, Girault JA. Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol. J Neurosci. 2008 May 28; 28(22):5671-85. Abstract

Matamales M, Bertran-Gonzalez J, Salomon L, Degos B, Deniau JM, Valjent E, Herve, Girault JA. Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice. PLoS One. 2009; 4(3):e4770. Abstract

Gangarossa G, Espallergues J, Mailly P, De Bundel D, de Kerchove d'Exaerde A, Herve D, Girault JA, Valjent E, Krieger P. Spatial distribution of D1R- and D2R-expressing medium-sized spiny neurons differs along the rostro-caudal axis of the mouse dorsal striatum. Front Neural Circuits. 2013; 7():124. Abstract

View all comments by Susan George

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Richard Mailman (Disclosure)
Submitted 16 January 2015
Posted 20 January 2015
  I recommend the Primary Papers

Dr. George's response ignores two papers recently published by my group. In Lee et al. (2014a), we offered experimental evidence challenging much of the foundation on which the D1/D2 heterodimer hypothesis rests. Again in Lee et al. (2014b), we critically reviewed the relevant literature. Although this was some time prior to the current controversy, we concluded: "The ... hypothesis [of] a critical role for D1-D2 heterodimers .... is the one of the greatest of current interest because of the novel underlying molecular mechanism....

Although it was based originally on the D1-PLC mechanism and the use of SKF-83959, it has now taken on a life of its own because of the hypothesized unique role of D1-D2 heterodimers in subsets of co-expressing striatal neurons (Perreault et al., 2012; 2014). We have reviewed the available data that we feel can be construed as weakening this hypothesis. Above and beyond the experimental issues (including our failure to be able to see evidence of PLC signaling in D1-D2-co-transfected HEK293 cells [Lee et al., 2014a]), there is the question of the degree to which D1-D2-co-localization occurs in the mature nervous system. Thus, our view is that [this] hypothesis is also false."

We urge those who still feel that D1-D2 dimers are important to read our review and then consider the elegant aforementioned studies of Javitch, Stanwood, and their colleagues. Together, the weight of the evidence suggests strongly that this hypothesis should be given a long overdue retirement.

References:

Lee SM, Kant A, Blake D, Murthy V, Boyd K, Wyrick SJ, Mailman RB. SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C. Neuropharmacology. 2014a Jun 12. S0028-3908(14)00218-4. Abstract

Lee SM, Yang Y, Mailman RB. (2014) Dopamine D1 receptor signaling: Does GQ-phospholipase C actually play a role? J Pharmacol Exp Ther. 2014b Jul 22. Abstract

Perreault ML, Fan T, Alijaniaram M, O'Dowd BF, George SR. Dopamine D1-D2 receptor heteromer in dual phenotype GABA/glutamate-coexpressing striatal medium spiny neurons: regulation of BDNF, GAD67 and VGLUT1/2. PLoS One. 2012; 7(3):e33348. Abstract

Perreault ML, Hasbi A, O'Dowd BF, George SR. Heteromeric dopamine receptor signaling complexes: emerging neurobiology and disease relevance. Neuropsychopharmacology. 2014 Jan; 39(1):156-68. Abstract Paper ID:

View all comments by Richard Mailman

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Philip Seeman (Disclosure)
Submitted 16 January 2015
Posted 20 January 2015
  I recommend the Primary Papers

Numerous papers cited by the other authors in this discussion have already confirmed that D1 and D2 receptors can be immunoprecipitated together from human brain, indicating that an interaction can occur between these two receptors. The extent of immunoprecipitation may vary depending on the brain region. The single paper by Frederick et al. is out of line and does not reflect the true situation.

View all comments by Philip Seeman

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Bryan Roth, SRF Advisor
Submitted 22 January 2015
Posted 22 January 2015

How might this controversy be settled?

As the original in vitro signaling assays were key to the genesis of the model, I would recommend that independent labs attempt to replicate the in vitro findings—ideally using cell lines supplied by both groups. As one of the key original findings was an apparent Ca++ mobilization signal, this should be the easiest to quantify and replicate.

Going forward, what would be needed would be selective and potent agonists for the D1/D2 complex with minimal off-target actions. The test agent SKF 83959 has always been problematic, as it has quite high potency at Gq-coupled serotonin receptors and other GPCRs—a point made by others (see Professor Mailman's recent review: Lee et al., 2014). Assuming D1/D2 heteromer selective compounds could be identified, they would be exceedingly illuminating for the field.

References:

Lee SM, Yang Y, Mailman RB. Dopamine D1 receptor signaling: does GaQ-phospholipase C actually play a role? J Pharmacol Exp Ther. 2014 Oct; 351(1):9-17. Abstract

View all comments by Bryan Roth