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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