César Quiroz

An Update on Adenosine A2A-Dopamine D2 receptor interactions. Implications for the Function of G Protein-Coupled Receptors

Adenosine A(2A)-dopamine D(2) receptor interactions play a very important role in striatal function. A(2A)-D(2) receptor interactions provide an example of the capabilities of information processing by just two different G protein-coupled receptors. Thus, there is evidence for the coexistence of two reciprocal antagonistic interactions between A(2A) and D(2) receptors in the same neurons, the GABAergic enkephalinergic neurons. An antagonistic A(2A)-D(2) intramembrane receptor interaction, which depends on A(2A)-D(2) receptor heteromerization and G(q/11)-PLC signaling, modulates neuronal excitability and neurotransmitter release. On the other hand, an antagonistic A(2A)-D(2) receptor interaction at the adenylyl-cyclase level, which depends on G(s/olf)- and G(i/o)-type V adenylyl-cyclase signaling, modulates protein phosphorylation and gene expression. Finally, under conditions of upregulation of an activator of G protein signaling (AGS3), such as during chronic treatment with addictive drugs, a synergistic A(2A)-D(2) receptor interaction can also be demonstrated. AGS3 facilitates a synergistic interaction between G(s/olf) - and G(i/o)-coupled receptors on the activation of types II/IV adenylyl cyclase, leading to a paradoxical increase in protein phosphorylation and gene expression upon co-activation of A(2A) and D(2) receptors. The analysis of A(2)-D(2) receptor interactions will have implications for the pathophysiology and treatment of basal ganglia disorders and drug addiction.

Key Modulatory Role of Presynaptic Adenosine A2A Receptors in Cortical Neurotransmission to the Striatal Direct Pathway

Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

Striatal Pre- and Postsynaptic Profile of Adenosine A2A Receptor Antagonists

Striatal adenosine A2A receptors (A2ARs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D2 receptors (D2Rs). A2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A1 receptors (A1Rs). It has been hypothesized that postsynaptic A2AR antagonists should be useful in Parkinsons disease, while presynaptic A2AR antagonists could be beneficial in dyskinetic disorders, such as Huntingtons disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A2AR-D2R and A1R-A2AR heteromers to determine possible differences in the affinity of these compounds for different A2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A2AR when co-expressed with D2R than with A1R. KW-6002 showed the best relative affinity for A2AR co-expressed with D2R than co-expressed with A1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively.

Adenosine–cannabinoid receptor interactions. Implications for striatal function

Adenosine and endocannabinoids are very ubiquitous non‐classical neurotransmitters that exert a modulatory role on the transmission of other more ‘classical’ neurotransmitters. In this review we will focus on their common role as modulators of dopamine and glutamate neurotransmission in the striatum, the main input structure of the basal ganglia. We will pay particular attention to the role of adenosine A2A receptors and cannabinoid CB1 receptors. Experimental results suggest that presynaptic CB1 receptors interacting with A2A receptors in cortico‐striatal glutamatergic terminals that make synaptic contact with dynorphinergic medium‐sized spiny neurons (MSNs) are involved in the motor‐depressant and addictive effects of cannabinoids. On the other hand, postsynaptic CB1 receptors interacting with A2A and D2 receptors in the dendritic spines of enkephalinergic MSNs and postsynaptic CB1 receptors in the dendritic spines of dynorphinergic MSN are probably involved in the cataleptogenic effects of cannabinoids. These receptor interactions most probably depend on the existence of a variety of heteromers of A2A, CB1 and D2 receptors in different elements of striatal spine modules. Drugs selective for the different striatal A2A and CB1 receptor heteromers could be used for the treatment of neuropsychiatric disorders and drug addiction and they could provide effective drugs with fewer side effects than currently used drugs.

Adenosine Receptor Heteromers and their Integrative Role in Striatal Function

By analyzing the functional role of adenosine receptor heteromers, we review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS). Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a “biochemical fingerprint” to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as “processors” of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the “local module” (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit), where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module.

Adenosine A2A Receptors and A2A Receptor Heteromers as Key Players in Striatal Function

A very significant density of adenosine A2A receptors (A2ARs) is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs). In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs). In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striatopallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl cyclase (AC). Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striatopallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs) and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders.

Pharmacological evidence for different populations of postsynaptic adenosine A2A receptors in the rat striatum

Adenosine A(2A) receptors (A(2A)Rs) are highly concentrated in the striatum. Two pharmacological different functional populations of A(2A)Rs have been recently described based on their different affinities for the A(2A)R antagonist SCH-442416. This compound has high affinity for A(2A)Rs not forming heteromers or forming heteromers with adenosine A(1) receptors (A(1)Rs) while showing very low affinity for A(2A)Rs forming heteromers with dopamine D(2) receptors (D(2)Rs). It has been widely described that striatal A(1)R-A(2A)R heteromers are preferentially localized presynaptically in the glutamatergic terminals that contact GABAergic dynorphinergic neurons, and that A(2A)R-D(2)R heteromers are localized postsynaptically in GABAergic enkephalinergic neurons. In the present study we provide evidence suggesting that SCH-442416 also targets postsynaptic A(2A)R not forming heteromers with D(2)R, which are involved in the motor depressant effects induced by D(2)R antagonists. SCH-442416 counteracted motor depression in rats induced by the D(2)R antagonist raclopride at a dose that did not produce motor activation or that blocked motor depression induced by an A(2A)R agonist. Furthermore, we re-evaluated the recently suggested key role of cannabinoid CB(1) receptors (CB(1)Rs) in the effects of A(2A)R antagonists acting at postsynaptic A(2A)Rs. By recording locomotor activity and monitoring striatal glutamate release induced by cortical electrical stimulation in rats after administration of A(2A)R and CB(1)R antagonists, we did not find evidence for any significant role of endocannabinoids in the post- or presynaptic effects of A(2A)R antagonists. The present results further suggest the existence of at least two functionally and pharmacologically different populations of striatal postsynaptic A(2A)Rs.

5-HT 1B Receptor-Mediated Serotoninergic Modulation of Methylphenidate-Induced Locomotor Activation in Rats

Previous studies have shown that the dopamine (DA) uptake blocker methylphenidate, a psychostimulant widely used for the treatment of attention-deficit hyperactivity disorder (ADHD), prevents the neurotoxic effects of the highly abused DA releaser methamphetamine. However, there is a lack of information about the pharmacological interactions of these two drugs at the behavioral level. When systemically administered within an interval of 2 h, previous administration of methylphenidate (10 mg/kg, intraperitoneal (i.p.)) did not modify locomotor activation induced by methamphetamine. On the other hand, previous administration of methamphetamine (1 mg/kg, i.p.) markedly potentiated methylphenidate-induced motor activation. With in vivo microdialysis experiments, methamphetamine and methylphenidate were found to increase DA extracellular levels in the nucleus accumbens (NAs). Methamphetamine, but not methylphenidate, significantly increased the extracellular levels of serotonin (5-HT) in the NAs. Methamphetamine-induced 5-HT release remained significantly elevated for more than 2 h after its administration, suggesting that the increased 5-HT could be responsible for the potentiation of methylphenidate-induced locomotor activation. In fact, previous administration of the 5-HT uptake blocker fluoxetine (10 mg/kg, i.p.) also potentiated the motor activation induced by methylphenidate. A selective 5-HT1B receptor antagonist (GR 55562; 1 mg/kg), but not a 5-HT2 receptor antagonist (ritanserin; 2 mg/kg, i.p.), counteracted the effects of methamphetamine and fluoxetine on the motor activation induced by methylphenidate. Furthermore, a 5-HT1B receptor agonist (CP 94253; 1–10 mg/kg, i.p.) strongly and dose-dependently potentiated methylphenidate-induced locomotor activation. The 5-HT1B receptor-mediated modulation of methylphenidate-induced locomotor activation in rat could have implications for the treatment of ADHD.

Orexin–Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking.

Dopamine-galanin receptor heteromers modulate cholinergic neurotransmission in the rat ventral hippocampus

Previous studies have shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little is known about the mechanisms involved and their possible interactions. By using resonance energy transfer techniques in transfected mammalian cells, we demonstrated the existence of heteromers between the dopamine D1-like receptors (D1 and D5) and galanin Gal1, but not Gal2 receptors. Within the D1–Gal1 and D5–Gal1 receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal1 receptors, whereas Gal1 receptor activation or blockade did not modify D1-like receptor-mediated MAPK activation. Ability of a D1-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a “biochemical fingerprint” of D1-like–Gal1 receptor heteromers, allowing their identification in the rat ventral hippocampus. The functional role of D1-like–Gal receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with costimulation of D1-like receptors. Electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission. Thus, a D1-like receptor agonist that was ineffective when administered alone turned an inhibitory effect of galanin into an excitatory effect, an interaction that required cholinergic neurotransmission. Altogether, our results strongly suggest that D1-like–Gal1 receptor heteromers act as processors that integrate signals of two different neurotransmitters, dopamine and galanin, to modulate hippocampal cholinergic neurotransmission.