Functional and pharmacological role of the dopamine D4 receptor and its polymorphic variants.

The functional and pharmacological significance of the dopamine D4 receptor (D4R) has remained the least well understood of all the dopamine receptor subtypes. Even more enigmatic has been the role of the very prevalent human DRD4 gene polymorphisms in the region that encodes the third intracellular loop of the receptor. The most common polymorphisms encode a D4R with 4 or 7 repeats of a proline-rich sequence of 16 amino acids (D4.4R and D4.7R). DRD4 polymorphisms have been associated with individual differences linked to impulse control-related neuropsychiatric disorders, with the most consistent associations established between the gene encoding D4.7R and attention-deficit hyperactivity disorder (ADHD) and substance use disorders. The function of D4R and its polymorphic variants is being revealed by addressing the role of receptor heteromerization and the relatively avidity of norepinephrine for D4R. We review the evidence conveying a significant and differential role of D4.4R and D4.7R in the dopaminergic and noradrenergic modulation of the frontal cortico-striatal pyramidal neuron, with implications for the moderation of constructs of impulsivity as personality traits. This differential role depends on their ability to confer different properties to adrenergic α2A receptor (α2AR)-D4R heteromers and dopamine D2 receptor (D2R)-D4R heteromers, preferentially localized in the perisomatic region of the frontal cortical pyramidal neuron and its striatal terminals, respectively. We also review the evidence to support the D4R as a therapeutic target for ADHD and other impulse-control disorders, as well as for restless legs syndrome.

1 Introduction

Discovered 30 years ago, the dopamine D4 receptor (D4R) initially drew attention due to its strong affinity for the atypical antipsychotic clozapine, an affinity found to be significantly higher than for the previously discovered D1, D2 and D3 receptors (D1R, D2R and D3R) (1). At the time, it was assumed that this high affinity could underlie clozapine’s unique clinical efficacy, which led to a very intense search for selective D4R antagonists. Unfortunately, the newly discovered D4R antagonists that followed were ineffective as antipsychotic drugs in clinical trials and the interest in D4R as a target for drug development waned [reviewed in ref. (2)].

Not only the pharmacological, but also the functional significance of the D4R has remained the most enigmatic of all the dopamine receptor subtypes. The human D4R gene (DRD4) displays a high number of polymorphisms in its coding sequence. The most extensive polymorphism is found in exon 3, a region that encodes the third intracellular loop (3IL) of the receptor (3–5). This polymorphism comprises a variable number of tandem repeats of a 48-base pair sequence, from 2- to 11 repeats. The most common polymorphisms contain 2, 4 or 7 repeats (with allelic frequencies of about 8%, 60% and 20%, respectively) (4), which encode a D4R with the respective number of repeats of a proline-rich sequence of 16 amino acids (D4.2R, D4.4R and D4.7R) (3–5). DRD4 polymorphisms have been associated with individual differences in impulse control-related neuropsychiatric disorders, with the most consistent associations found between the gene encoding D4.7R and attention-deficit hyperactivity disorder (ADHD) (3, 6–8) and substance use disorders (SUDs) (9). It follows that these significant associations should provide clues about the functional and pharmacological significance of the D4R. The main functional and pharmacological properties of the D4R and its different D4R polymorphic variants have been elucidated only recently with studies addressing the role of receptor heteromerization and the relatively avidity of norepinephrine for D4R. This includes the role of pineal D4R in the circadian noradrenergic modulation of melatonin synthesis and release (10), as well as the role of D4R in the inhibitory dopaminergic modulation of frontal cortico-striatal glutamatergic neurotransmission (11). Here we review the evidence to support the role of frontal cortical D4Rs in the moderation of constructs of impulsivity as personality traits. Based on the available evidence, we submit that the D4R should be exploited as a therapeutic target for impulse control-related neuropsychiatric disorders (primarily, ADHD), as well as for restless legs syndrome (RLS).

2 The D4R-modulated frontal-cortico-striatal neuron

D4R is highly expressed in the prefrontal cortex of mammals, including rodents and human and non-human primates, particularly in deep layer neurons (1, 12–14). In contrast with other dopamine receptors (D1R, D2R and in the ventral striatum, D3R), striatal mRNA expression of D4R is much lower (1, 12). However, immunohistochemical studies using different antibodies against different epitopes of the rodent and human D4R produced incongruent results, particularly in relation to its striatal density, indicating a lack of antibody specificity (15–19). Further studies, using a transgenic mouse expressing a fluorescent protein under the transcriptional control of the mouse dopamine D4R gene (Drd4), confirmed its predominant expression in the deep layer neurons of the prefrontal cortex and its lack of expression in the striatum (20). These results agreed with those obtained using in situ hybridization studies and indicate that striatal D4Rs are localized in striatal nerve terminals and, most probably in the terminals from glutamatergic neurons originated in the deep cortical layers.

In fact, significant evidence for the existence of D4Rs in the prefrontal cortex and the striatum of rats and humans has been obtained by radioligand binding experiments (21–25). The D4R labeling strategies included radioligands with significant selectivity for D4R or non-selective D2-like receptor radioligands in the presence of the D2R-D3R antagonist raclopride, which demonstrates a specific low affinity for the D4R (1). The results showed a disproportionally high density of striatal D4R binding sites when compared with the striatal expression of D4R mRNA, in agreement with their putative localization in cortico-striatal terminals. This was confirmed with experiments with frontal cortex ablation, which produced a significant reduction of striatal D4R binding sites (24).

The neocortex is composed of two major neuronal populations: glutamatergic pyramidal (P) neurons (70-80%) and GABAergic interneurons (20%-30%). There are several subtypes of GABAergic interneurons which have been grouped in three main classes, based on their transcriptional similarities and the expression of selective markers (26, 27). The largest class of cortical interneuron is characterized by the expression of the calcium-binding protein parvalbumin (PV). PV neurons target the perisomatic region of P neurons and control their spiking output. The second class expresses the neuropeptide somatostatin, and these neurons preferentially target the dendritic region of P neurons. And the third class expresses the 5-HT3a serotonin receptor, with a common subclass also expressing the calcium binding protein calretinin and the vasoactive intestinal peptide. These are mainly disinhibitory neurons that preferentially target PV+ and somatostatin+ interneurons. It has been postulated that the three subclasses of GABAergic interneurons establish a local cortical circuit that is critically involved in working memory, by which stimulus tuning of persistent activity arises from the concerted action of widespread inhibition mediated by PV+ interneurons and localized disinhibition of P neurons mediated by calretinin-containing interneurons (27, 28). In non-human primates, most P neurons and nearly half of the GABAergic interneurons express D4R. Among the interneurons, D4R are particularly expressed in PV+ interneurons (13, 14).

D4Rs are therefore positioned to exert a significant modulatory influence on frontal cortico-striatal P neurons. Considering the Gi-coupled D4R as mostly inhibitory, those localized in their cortical perisomatic region and striatal terminals should be expected to mediate an inhibitory effect of dopamine, while those localized in PV+ GABAergic interneurons should be expected to produce disinhibition. Nevertheless, a recent electrophysiological study on mouse cortical slices showed that D4R activation induces a more complex set of effects, with a direct slow decreasing effect on the excitability of P neurons and a fast and transient increase followed by a delayed decrease of the excitability of PV+ interneurons (29). The initial effect on PV+ interneurons should in fact lead to an initial potent suppression of PFC network activity and output signal. The effect of the delayed decrease of the excitability of PV+ interneurons on P neurons should be masked by the direct D4R-mediated decrease excitability of P neurons. In summary, activation of frontal cortical D4R should mostly keep a low output signal of the PFC network (29). In addition, a recent study using a combined optogenetic-microdialysis technique demonstrated the ability of D4Rs localized in frontal cortico-striatal terminals to exert a significant inhibitory role of striatal glutamate release (11). Overall, these studies suggest that D4Rs are instrumental for the dopamine-mediated functional inhibition of frontal cortico-striatal neurotransmission, in agreement with the hyperexcitability observed on frontal cortical P neurons in D4R-deficient mice (30). However, as reviewed below, several studies indicate a more complex picture, where D4Rs can also directly mediate the effects of norepinephrine and indirectly modulate the function of adrenoceptors and other dopamine receptor subtypes by heteromerization.

3 Lessons from the pineal gland. D4R heteromerization and D4R as a target for norepinephrine

Apart from the frontal cortex, D4Rs are highly expressed in the retina (31) and by the pinealocytes of the pineal gland (32), which main function is the circadian secretion of the hormone melatonin. This circadian control is mediated by a neuronal circuit that includes the suprachiasmatic and paraventricular nuclei of the hypothalamus, the intermediolateral column of the spinal cord and the superior cervical ganglion, which sends noradrenergic afferents to the pineal gland (33, 34). Darkness-induced norepinephrine release in the pineal gland activates pinealocyte Gs-coupled β1 and Gq-coupled α1B adrenergic receptors (β1R and α1BR), which promotes the synthesis and release of melatonin and its precursor serotonin (33, 34). Several experimental observations indicate that D4Rs play a fundamental role in the circadian adrenergic control of melatonin synthesis by pinealocytes and that this role depends on their circadian expression, their heteromerization-dependent ability to inhibit β1R and α1BR function and their ability to bind and be activated by norepinephrine (10, 32, 35).

D4R expression in the retina and pineal gland varies significantly in a circadian manner, being particularly elevated during the second half of the dark period (32). This increased expression is under the control of pineal β1R and α1BR and requires thyroid hormone (32). We found evidence indicating that D4R can form heteromers with both β1R and α1BR in mammalian transfected cells and in rat pinealocytes, and that the expression of β1R-D4R and α1B-D4R heteromers follows the circadian expression of D4R, being maximal at sunrise (10). In these heteromers, activation of D4R leads to a significant decrease in the ability of its partner adrenoceptor to signal (10), apparently suggesting that, in the pineal gland, dopamine inhibits the effect of norepinephrine. However, there is no clear evidence for a functional significant dopamine release in the pineal gland, where the catecholaminergic input is largely noradrenergic, the noradrenergic afferents from the superior cervical ganglion (33, 34).

A similar situation can be found in the cerebral cortex, particularly of rodents, which receives dense and widespread noradrenergic innervation, whereas dopaminergic terminals are restricted to the prefrontal cortex (36, 37). However, dopamine receptors are expressed throughout the cortex, and their localization exceeds that of the dopaminergic terminals (38–41). It has therefore been suggested that dopamine is either a co-neurotransmitter in noradrenergic neurons (42) or that there is a long-range volume-transmission of catecholamines (43). In fact, asynaptic varicosities represent most cortical norepinephrine releasing sites (37). A more parsimonious mechanism is the possibility that endogenous norepinephrine can also be an endogenous ligand of D4R. Thus, as demonstrated with in vitro experiments, norepinephrine binds and activates D4Rs at submicromolar concentrations (35, 44–46), up to ten times higher than the concentration able to activate β1R or α1BR in pineal cell preparations or pineal tissue (47, 48). Neverthelss, the potency of norepinephrine at activating pineal D4Rs needs to be determined. Altogether, a very plausible mechanism by which pineal D4Rs control melatonin synthesis and release emerges: at the beginning of the dark period, the initial noradrenergic activation of the pineal gland targets β1R and α1BR, favoring a progressive increase in melatonin synthesis, but also D4R expression; whereas at the end of the dark period, the increased expression of D4Rs leads to the formation of β1R-D4R and α1B-D4R heteromers, which allows norepinephrine to preclude β1R and α1BR signaling within the heteromer, thus dwindling melatonin synthesis and release (10).

4 The enigmatic role of DRD4 polymorphic variants. GPCR oligomerization comes to the rescue

The question of the functional significance of the different receptor isoforms encoded by DRD4 polymorphisms has remained enigmatic until recently. Those include electrophysiological and biochemical experiments in frontal cortical slices from D4R knockout mice with rescued expression of human D4.4R and D4.7R by viral transduction (49, 50), and immunohistochemical and in vivo optogenetic-microdialysis experiments in D4.7R knock-in mice expressing a humanized D4R with the 3IL of the human D4.7R (11). Electrophysiological experiments in frontal cortical slices from D4R knockout mice showed an increased ability of a D4R agonist to suppress network bursts and NMDA receptor-mediated excitatory postsynaptic currents from P neurons after viral transduction of human D4.7R, as compared with human D4.4R receptor cDNA (49, 50). Interestingly, biochemical experiments in the same slice preparations revealed that these differences correlated with a more profound D4R agonist-mediated downregulation of NMDA receptor surface expression (i.e., NR1 subunit) in frontal cortical slices virally transduced with D4.7R than with D4.4R (49). The in vivo experiments in D4.7R knock-in mice showed a blunting of methamphetamine-induced cortical activation and ontogenetically and methamphetamine-induced frontal cortico-striatal glutamate release (11). In summary, these studies showed a pronounced gain of function of D4.7R, as compared to D4.4R, in its ability to mediate the inhibitory influence of dopamine on frontal cortico-striatal neurotransmission.

The next enigma to solve was the mechanism behind the functional differences of the products of DRD4 polymorphisms. Thus, in mammalian transfected cells, D4.2R, D4.4R and D4.7R did not show clear pharmacological differences in response to endogenous or exogenous ligands, although an earlier study seemed to indicate that D4.7R signals with less efficiency than D4.4R (51). Nevertheless, by using a functional bioluminescence resonance energy transfer (BRET) technique, we could not find significant differences in the ability of the D4.2R, D4.4R or D4.7R polymorphic variants to promote dopamine-induced activation of any of the five Gi/o protein subtypes (35). Importantly, differences emerged when studying their ability to form heteromers, more specifically with the Gi-coupled D2R and α2AR, preferentially localized in the perisomatic region and nerve terminals of prefrontal cortical P neurons, respectively (52–54).

Apart from D4Rs, a significant proportion of frontal cortical P neurons from layer V also express D2Rs (in monkeys, about 75% and 55%, respectively) (14). Furthermore, in vitro experiments in striatal slices (52) and in vivo optogenetic-microdialysis experiments in rodents (11, 55) have provided evidence for the presence of functional D2R and D4R receptors in cortico-striatal glutamatergic terminals. This provides the framework for the existence of D2R-D4R heteromers in striatal terminals and, possibly, in the perisomatic region of P neurons. The first studies on heteromers of the D4R polymorphic variants, based on BRET and co-immunoprecipitation techniques in transfected mammalian cells, suggested that the D4.7R establishes weaker intermolecular and functional interactions with the D2R (both isoforms, D2LR and D2SR) than the D4.4R (52, 56, 57). At the functional level, co-transfection of D2Rs with D4.4Rs, but not D4.7Rs, led to an increase in MAPK signaling (52, 57), which would seem opposite to the expected D4.7R gain of function. Nevertheless, a dependence on heteromerization of these differences in MAPK signaling was not clearly established. On the other hand, a more recent study using the complemented donor acceptor bioluminescence resonance energy transfer (CODA-RET) technique demonstrated a D4.7R heteromerization-dependent gain of function of the D2R (53).

CODA-RET is a BRET assay that allows the measurement of ligand-induced changes in the interaction between G protein-coupled receptors (GPCRs) forming specific homomers or heteromers with transducer proteins, including G proteins (58). In the CODA-RET assay, two complementary halves of a bioluminescent chromophore, such as Renilla Luciferase are separately fused to two different receptor molecules putatively able to oligomerize and a fluorescent chromophore (such as yellow fluorescent protein) can be fused to a G protein subunit. Ligand-induced changes in CODA-RET measurements imply, first, a successful complementation of Renilla Luciferase and, therefore, oligomerization of the corresponding GPCR units. Second, although CODA-RET does not provide estimates of the degree of oligomerization, such as the affinity of the intermolecular interactions between GPCR subunits or the proportion of subunits forming oligomers, this technique does allow a qualitative measure of the ability of a specific endogenous or exogenous ligand to activate a GPCR homodimer or heterodimer and signal through a specific G protein (58, 59). Thus, using CODA-RET, we were able to disclose, for the first time, a different qualitative profile of several D2-like receptor ligands for D4.4R and D4.7R, but only when forming heteromers with D2R or α2AR (53, 54).

Using CODA-RET, we found two possible mechanisms for the D4.7R gain of function as mediator of an inhibition of frontal cortico-striatal neurotransmission ( ): first, a specific increase in the potency of dopamine for the D2R-D4.7R, but not for D2R-D4.4R heteromer, as compared with the D2R homomer (53); second, a divergent decrease or increase in the constitutive activity of D2R when it was forming D2R-D4.4R or D2R-D4.7R heteromers, respectively (53). In addition, a fraction of D4R should not be expected to form heteromers, but homomers, which should be more prevalent with D4.7R than with D4.4R. In fact, as compared with D4.4R, not only D4.7R seems to be less able to heteromerize with D2R (52, 56, 57), but it also seems to be more able to homomerize (56). Since CODA-RET experiments also demonstrated a significantly higher potency of dopamine for D4.4R-D4.4R and D4.7R-D4.7R homomers than for D2R-D4.4R and the D2R-D4.7R heteromers (53), the expected larger population of D4.7R-D4.7R versus D4.4R-D4.4R homomers should represent a third mechanism involved in the gain of function of D4.7R ( ).

Figure 1

Schematic representation of cortico-striatal glutamatergic terminals and their modulatory D4.4Rs (left terminal) and D4.7Rs (right terminal), which form heteromers with D2Rs. Heteromerization of D2R with D4.7R promotes a gain of function of the dopaminergic-mediated inhibition of glutamate (GLU) release, as compared with heteromerization with D4.4R, since it increases the potency for dopamine (DA) and the constitutive activity of the D2R. In addition, the population of D4.7Rs not forming heteromers with D2Rs is larger than with D4.4Rs, which increases the potency of DA to inhibit GLU release, because of the higher affinity of DA for D4Rs versus D2Rs. The scheme is based on results obtained from experiments in mammalian transfected cells and from in vitro and in vivo experiments in rodents (see text).

In view of the involvement of D4R and α2AR in impulsivity and ADHD (see next section), their concurrent localization in frontal cortical P neurons (see below) and the demonstrated ability of D4R to form functional heteromers with other adrenoceptors in the pineal gland (see above), we investigated the possible existence of functionally significant α2AR-D4.4R and α2AR-D4.7R heteromers in the brain. Using several biophysical and biochemical techniques and using heteromer-specific disruptive peptides, we demonstrated the ability of both D4.4R and D4.7R to heteromerize with α2AR both in mammalian transfected cells and in the mouse cerebral cortex (54). The results of BRET experiments indicated that, akin to D2R-D4R heteromers, the D4.7R variant was less able to heteromerize with α2AR than D4.4R. Furthermore, results from radioligand-binding, CODA-RET and signaling experiments indicated that heteromerization with D4.7R, but not with D4.4R, increases the potency of norepinephrine at activating α2AR. Furthermore, D4.4R, but not D4.7R activation, allosterically inhibited α2AR-mediated signaling in their respective heteromers (54). Thus, dopamine should be able to promote a significant inhibitory effect of α2AR signaling though α2AR-D4.4R, but not α2AR-D4.7R heteromers. Furthermore, as elaborated below, D4R can also be activated by endogenous norepinephrine in the cerebral cortex, and high concentrations should determine a significant inhibition of α2AR signaling by the α2AR-D4.4R, but not by the α2AR-D4.7R heteromer. If the main functional output of α2AR-D4R heteromers is a decrease in excitability of P neurons (see below, section 6) this could provide an additional mechanism for the gain of function of D4.7R in its inhibitory control of frontal cortico-striatal neurotransmission ( ).

Figure 2

Schematic representation of the perisomatic region of frontal cortical P neurons and their modulatory D4.4Rs (left neuron) and D4.7Rs (right neuron), which form heteromers with α2ARs. Heteromerization of α2AR with D4.7R promotes a gain of function of the noradrenergic-mediated inhibition of neuronal excitability, as compared with heteromerization with D4.4R, since it increases the potency of norepinephrine (NE). In addition, high concentrations of NE promote a decrease in the potency of NE in the α2AR-D4.4R but not in the α2AR-D4.7R heteromer (see text). Furthermore, the population of D4.7Rs not forming heteromers with α2AR is larger than with D4.4Rs, which also increases the population of α2AR not forming heteromers and therefore the potency of NE to decrease neuronal excitability. The increased proportion of D4.7Rs not forming heteromers additionally increases the ability of DA to decrease neuronal excitability. The scheme is based on results obtained from experiments in mammalian transfected cells and from in vitro experiments in rodents (see text).

5 D4R as a moderator of the personality traits action and choice impulsivity

The most popular models of personality are the Big-Three and the Big-Five models, operationalized by the Multidimensional Personality Questionnaire (MPQ) (60) and the NEO Personality Inventory-Revised (NEO-PI-R) psychometric tests (61), respectively. The MPQ measures three orthogonal traits: positive emotionality (PEM), negative emotionality (NEM) and constraint (CON) (60, 62). PEM and NEM incorporate dispositions toward positive and negative emotions, respectively, and are linked conceptually to the brain systems underlying appetitive-approach and defensive-withdrawal behaviors. Constraint (CON) encompasses dimensions related to behavioral restraint, the opposite end of which implies disinhibition. NEO-PI-R (61) measures neuroticism (N), extraversion (E), openness (O), agreeableness (A) and conscientiousness (C). N and E highly correlate with NEM and PEM respectively, generally constituting the same personality constructs (63, 64). O captures interest toward experience, and A implies an empathic personality. Finally, C is a spectrum of constructs that describes individual differences in the propensity to be self-controlled, responsible to others, hardworking, orderly, and rule abiding (65).

Impulsivity is defined as a predisposition toward rapid, unplanned reactions to internal or external stimuli with little regard for the negative consequences to the individual or others (66). Impulsivity has been decomposed into “rapid-response” or “action” impulsivity, and “cognitive” or “choice” impulsivity (67–69). Action impulsivity (AI) is defined as a diminished ability to inhibit prepotent responses, or a failure of volitional motor inhibition or disinhibition (68). Choice impulsivity (CI) implies a tendency to accept small immediate or likely rewards at the expense of large delayed or unlikely rewards (69). Excessive CI overlaps conceptually with impairment in decision-making and particularly with temporal or delay discounting (70). Delay discounting is the phenomenon by which a delayed outcome of a choice reduces the subjective value of a reward and constitutes an operational measure of the degree of CI (71, 72). We have previously maintained that AI constitutes the same concept as strong disinhibition, the opposite end of the personality trait CON (9), and that the trait C encapsulates both dimensions of impulsivity (AI and CI) (73). In fact, a significant correlation between measures of CON and C has been reported (63, 64). We have also argued that the same as AI, CI fulfills the criteria to be considered as a personality trait (73). The substantial overlap of several of these personality traits with maladaptive behaviors and specifically, with mental health disorders, justifies the sustained search for their neural underpinnings as therapeutic targets. But this has been a significant challenge for the field of neuropsychiatry.

The ‘endophenotype’ concept has provided an invaluable approach for the identification of genes that predispose or indemnify individuals from mental and psychiatric disorders. The endophenotype concept is understood as simpler clues to genetic underpinnings than the disease syndrome itself and involves the genetic analysis of any of a variety of biological markers (cognitive, neurophysiological, anatomical, biochemical, etc.) of the disease. The concept promotes the view that psychiatric diagnoses can be decomposed or deconstructed into more tractable intermediate phenotypes by virtue of their assumed proximity to the genetic antecedents of the disease (74, 75). Based on the results of studies that have linked the structure of psychopathology to the structure of personality, as defined by the MPQ or NEO-PI-R (76), we argued previously that specific personality traits constitute endophenotypes of mental health disorders, such as SUD (9).

We initially identified PEM/E (from the MPQ and NEO-PI-R assessments, respectively), NEM/N (again, from the two assessments, respectively), and CON (the inverse measure of AI), as tied to specific brain circuits and genes (9). PEM/E is modulated by the function of the central dopaminergic system and is moderated by the D2R gene. NEM/N is modulated by the glutamatergic outputs from the right anterior cingulate cortex and ventromedial prefrontal cortex to the amygdala and insula and is moderated by the serotonin transporter gene. AI is modulated by a circuit including the glutamatergic neurons arising in the pre-supplementary motor area and right inferior frontal gyrus and innervating the dorsal striatum and the subthalamic nucleus and is moderated by the genes of the D4R and the dopamine transporter (9) ( ). Individuals with low PEM/E, high NEM/N and high AI would be most vulnerable (least resilient) to develop SUD. Conversely, individuals with high PEM/E, low NEM/N and low AI would be least vulnerable (most resilient) to SUD (9).

Figure 3

Scheme of personality traits as endophenotypes of SUD, with their linkage to specific brain circuits and genes. Positive emotionality/extroversion (PEM/E) is modulated by the central dopaminergic (DA) system and is moderated by the D2R gene. Negative emotionality/neuroticism (NEM/N) is modulated by a circuit that involves the right anterior cingulate cortex (rACC), ventromedial prefrontal cortex (vmPFC) and the amygdala and is moderated by the gene of the serotonin transporter (5-HTT). Action impulsivity (AI) is modulated by a circuit that includes the pre-supplementary motor area (preSMA), the right inferior frontal gyrus (rIFG), the striatum and the subthalamic nucleus (STN). Choice impulsivity (CI) is modulated by a circuit that includes the ventromedial prefrontal cortex (vmPFC), the posterior cingular cortex (pCC) and the nucleus accumbens (NAc). AI and CI are moderated by the genes of the dopamine transported (DAT) and the enzyme cathecol-O-methyltransferase (COMT), respectively. In addition, AI and CI are both moderated by the genes of D4R and α2AR. “+” and “-” indicate that individuals with low PEM, high NEM, high AI and high CI are most vulnerable (least resilient) to develop SUD [modified from ref (71)].

To our initial analysis, we added CI as an additional personality trait, modulated by a circuit including glutamatergic neurons from the ventromedial prefrontal cortex and the posterior cingular cortex that innervate the ventral striatum (nucleus accumbens) and, as with AI, is also moderated by the D4R gene, and by the gene of the enzyme catechol-O-methyltransferase (73) ( ). The fact that D4R moderates both AI and CI provide a clue for the apparent orthogonality of the C trait, as it encapsulates both traits (see above), which constitute endophenotypes for ADHD (76–80) and SUD (9, 73). Not surprisingly, as for SUD, low C is a consistent finding in ADHD (81), which if left untreated, constitutes a risk factor for SUD (82–84).

The same as for the D4R gene, polymorphisms of the α2AR gene may confer vulnerability to developing ADHD as well as symptoms of impulse control disorders (85, 86). However, a large meta-analysis did not find a consistent significant association (87). Yet, when studied at the intermediate phenotype level, as an endophenotype, a clear significant association was established between α2AR gene polymorphisms and AI (88). Additionally, the α2AR agonist guanfacine, which is currently used in the symptomatic treatment of ADHD (89), significantly decreases delay discounting in nonhuman primates (90). Our recent study on α2AR-D4R heteromers brings together two key receptors involved in the pathogenesis and treatment of ADHD, since α2AR-D4R heteromers represent a significant population of both catecholaminergic receptors in the mouse cerebral cortex (54).

6 D4R antagonists as a plausible treatment for ADHD and other impulse-control disorders

Apart from the association with D4.7R, several other preclinical and clinical findings converge on the involvement of D4R and its heteromers in ADHD and other impulse-control disorders and, therefore, on its possible utility as a therapeutic target for those disorders. At the preclinical level, several animal models of ADHD with varied face, construct, and predictive validity have been developed, particularly in rodents (91). The complexity of the clinical symptoms and pathology of ADHD has been very challenging for the development of those models and, unfortunately, the lack of knowledge about the etiology and pathogenetic mechanisms of the disorder hampers their construct validity.

The most consistent pathogenetic finding in ADHD is a frontal cortical hypoactivity (92, 93), for which there is not yet a clearly accepted explanation. Although it might seem counterintuitive that a decrease and not an increase in cortico-striatal glutamatergic transmission is associated with impulsivity and ADHD, this could probably be explained by considering the well-established differential effects of the activation of the direct and indirect striatal efferent pathways. Classically, the direct and indirect pathways have been conceptualized as gas and brake pedals of the output signals of the basal ganglia (“Go” and “NoGo” pathways), respectively (94). It therefore seems that a sufficient decrease in the activation of the indirect pathway (releasing the gas pedal) determines an increase in the basal ganglia output, irrespective of a concomitant decreased activation of the direct pathway. Considering a more cognitive conceptualization, which includes the processing of information of relevant and irrelevant stimuli (95), a blunted cortico-striatal neurotransmission affecting the activity of both the direct and indirect striatal efferent pathways should decrease their respective ability to increase the reactivity to reward-related stimuli and to suppress the reactivity to nonrewarded- or aversive-related stimuli. As previously proposed (11), the outcome should be an increased “interest” for irrelevant stimuli and a reduced inhibition of irrelevant responses, which could be important in explaining not only the impulsivity, but also the attentional deficit of ADHD.

Genetic manipulations of the D4R in the experimental animal have provided significant correlative information supporting the role of a decrease in striatal glutamatergic transmission in ADHD. As mentioned before, D4R-knockout mice showed hyperexcitability of frontal cortical P neurons (30), while the gain of function provided by D4.7R induced the opposite effect, with a decrease in cortico-striatal glutamatergic transmission (11). In addition, one of the classical animal models of ADHD, the rodent with neonatal lesions with 6-OH-dopamine, showed an ADHD-like phenotype, including locomotor hyperactivity, paradoxical hypolocomotor response to amphetamine and methylphenidate and poor behavioral inhibition, which was counteracted by genetic or pharmacological blockade of the D4R (96, 97). Furthermore, it was also shown that locomotor hyperactivity in 6-OH-dopamine-lesioned rats correlate with increases in the striatal density of D4R (97).

The 6-OH-dopamine ADHD rodent model also demonstrated predictive value, since apart from amphetamine and methylphenidate, selective norepinephrine uptake inhibitors were effective at counteracting locomotor hyperactivity (98). In fact, apart from amphetamine and methylphenidate, the inhibitor of the norepinephrine transporter atomoxetine and the α2AR agonists guanfacine and clonidine are the most accepted pharmacological treatments for ADHD (89). In the cortex, α2ARs are preferentially localized postsynaptically, in P neurons of the deep layers (99, 100), therefore, potentially co-localized with D4R. In fact, as mentioned before, we have recently provided experimental evidence indicating that α2AR-D4R heteromers represent a significant population of both receptors in the mouse cerebral cortex (54). Two different and opposite neuronal effects, both dependent on Gi protein-mediated decrease in cAMP formation, have been described upon activation of cortical postsynaptic α2AR: an excitatory effect, dependent on the inactivation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (101), and an inhibitory effect, dependent on the inactivation of AMPA receptors (102, 103). These results indicate the existence of two different functional populations of α2AR. It is assumed that the excitatory effect mediates the therapeutic effect of α2AR agonists, by counteracting the cortical frontal hypoactivity of ADHD (100). It was then suggested that the inhibitory effect would be mediated by a different population of α2AR, which could provide a protective mechanism upon overstimulation by high levels of norepinephrine released under stress conditions (102).

It is plausible, and experimentally testable, that D4Rs mostly heteromerize and modulate the population of α2ARs that mediate the inhibitory effect of P neuronal function. This could explain at least part of the protective effect of D4.4R in ADHD, as well as the D4.7R-mediated increased vulnerability to develop ADHD. Thus, the same as with pineal β1R-D4R and α1B-D4R heteromers, the cortical α2AR-D4.4R heteromer could function as a norepinephrine concentration-sensing device, where high concentrations activate D4.4R and counteract the effect of the activation of the α2AR in the heteromer, diminishing the α2AR-mediated inhibitory effect on P neuronal function. On the other hand, the increase in the potency of norepinephrine for α2AR in the α2AR-D4.7R heteromer would facilitate the α2AR-mediated inhibitory effect on P neuronal function ( ). As in cortico-striatal terminals, activation of D4R localized in the perisomatic region of P neurons not forming heteromers (or possible forming heteromers with D2R) should directly promote an inhibition of the activity of P neurons ( ). D4R antagonists should then be considered as possible therapeutic targets for ADHD, as predicted by the positive results obtained in rodents with neonatal 6-OH-dopamine lesions (96, 97), specially, when considering their additional ability to counteract the dopamine-mediated inhibition of glutamate release by cortico-striatal terminals.

However, a randomized, double-blind, crossover study with the selective D4R antagonist L-745,870 (also named MK-0929) in adults with ADHD did not demonstrate a significant effect over placebo (104). To our knowledge, this is the only published study addressing the possible clinical efficacy of D4R antagonists in ADHD. Obvious limitations, such as the small number of patients, short duration of each treatment period and lack of quantitative cognitive assessments of inhibitory control and other executive functions cannot be ignored. Importantly, D4.7R polymorphism characterization was not included in this study, which may also explain the negative findings. Apart from the above reviewed preclinical role of D4R as a very significant mediator of the effect of dopamine and norepinephrine on the function of frontal cortical P neuronal function, there is the unequivocal association of D4.7R to ADHD. It would therefore be important to carry out more clinical studies with D4R antagonists, which should also include addressing their ability to modify the underlying endophenotypes AI and CI (9, 73).

7 D4R agonists as plausible treatment of restless legs syndrome

The term akathisia is used to define an urgent need to move. Thus, the primary component of akathisia is a sensory experience which acts as a “drive” or “motivational state” that compels the subject to move. This is objectively perceived by an observer as restlessness or motor hyperactivity (105). Akathisia is also implicit in the description of the symptoms of the very prevalent sensorimotor disorder RLS, where, more often, the sensory experience is an urgent need to specifically move one’s legs (106). Brain iron deficiency (BID), more often without concomitant peripheral iron deficiency, is recognized as the main initial pathogenetic mechanism in RLS (107, 108). BID seems then to trigger a series of pathogenetic mechanisms, including an increase in the motor-cortical and thalamic excitability, conceptualized as a hyperglutamatergic state, that seems to underly RLS symptomatology (108).

BID in rodents is a well-accepted animal model of RLS, which can be induced by providing a severe iron-deficient diet during the postweaning period. The model has both construct and face validity since it recapitulates several biochemical and behavioral findings of RLS (109, 110). Using the optogenetic-microdialysis method, an increase in the sensitivity of cortico-striatal terminals to release glutamate could be demonstrated in the rat with BID, since a lower frequency of optogenetic stimulation was necessary to induce striatal glutamate release as compared to controls (55). This increased sensitivity seems to be related to a BID-induced alteration in the expression of adenosine receptor subtypes in the cortico-striatal glutamatergic terminals, with a downregulation of adenosine A1 receptors (A1Rs) and a relative upregulation of adenosine A2A receptors (A2ARs) (111, 112). Using the optogenetic-microdialysis method, evidence was also obtained for the ability of the equilibrative nucleoside transporter dipyridamole, which increases the extracellular levels of adenosine, to significantly inhibit cortico-striatal glutamate in control rats and in rats with BID (113). The results of these experiments indicated a possible therapeutic effect of dipyridamole, which was recently demonstrated by two clinical studies, an open trial, and a randomized, placebo-controlled crossover study (114, 115). These studies provided a new therapeutic approach for RLS and significantly validated the cortico-striatal glutamatergic terminals as targets for the treatment of RLS.

Using the same model, perfusion of the most prescribed drugs in RLS, pramipexole, ropinirole and gabapentin, all counteracted the ability of optogenetic stimulation to induce glutamate release, both in controls and in rats with BID (55). By binding to the α2δ subunit of voltage-dependent calcium channels localized in glutamatergic terminals, gabapentin reduces their function and trafficking, therefore decreasing striatal glutamate release (116). Pramipexole and ropinirole are non-selective D2-like receptor agonists with a slightly higher affinity for D3R. It has therefore been suggested that their therapeutic effect is related to their preferential affinity for D3R (117). However, the effect of pramipexole on the optogenetically induced glutamate release was not antagonized by a selective D3R antagonist, but by D2R and D4R antagonists (55). These results, therefore, provided a significant support to the key mediation of D2R and D4R in the local striatal inhibitory control of dopamine on cortico-striatal glutamate release.

The predictive value offered by the conceptual framework of an increased sensitivity of cortico-striatal terminals in RLS points to D4R agonists as a possible new treatment for RLS. This could possibly avoid secondary effects of the currently used dopaminergic compounds, which very commonly lead not only to disappearance of their therapeutic effect, but to an increase in the RLS symptoms, known as “augmentation” (118). Thus, it is conceivable that augmentation is secondary to activation of postsynaptic dopamine receptors, similar to the mechanism involved in L-DOPA-induced dyskinesia, a common complication of the treatment with dopaminergic compounds in Parkinson’s disease (119).

8 Conclusions

We reviewed the evidence conveying a significant role of D4R in the dopaminergic and noradrenergic modulation of the frontal cortico-striatal pyramidal neuron, with implications for the moderation of constructs of impulsivity as personality traits. We also reviewed the evidence strongly supporting that these D4Rs should be exploited as therapeutic targets for ADHD and other impulse-control disorders and for RLS. Special emphasis was placed on the concept of receptor heteromerization, which has played a fundamental role in the understanding of D4R function and in the understanding of the different functional differences between D4R polymorphic variants.

Particularly striking is the fact that the most common polymorphic variants, D4.4R and D4.7R (with allelic frequencies of about 60% and 20%, respectively) (4), confer significantly different functional and pharmacological properties to α2AR-D4R and D2R-D4R heteromers, which mediate a dopamine- and norepinephrine-dependent fine-tune modulation of the frontal cortico-striatal glutamatergic neuronal function. This can explain the differential effect of D4R polymorphisms in the moderation of the personality traits AI and CI and their role as endophenotypes of impulse-control disorders, including ADHD and SUD. More specifically, it can explain the association of D4.7R with impulse-control disorders. The demonstrated mediation of a stronger inhibition of cortico-striatal glutamatergic transmission mediated by D4.7R (in D4.7R knock-in mouse expressing a humanized D4R with the 3IL of the human D4.7R) (11), would then disclose a mechanism determining an increase in AI and CI.

Reviewing the results from experimental models of ADHD and RLS, it becomes evident that selective D4R antagonists and agonists could be respectively effective. Although a single clinical study with a D4R antagonist resulted negative in the treatment of ADHD (104), we believe that the reviewed preclinical evidence calls for additional clinical studies. Apart from addressing the ability of D4R antagonists to modify the underlying endophenotypes AI and CI, the role of D4R polymorphisms should also be addressed. Similarly, to our knowledge, there are no studies concerning the frequency of the different D4R polymorphic variants in RLS patients, and a prediction could be made about an expected lower frequency of D4.7R. As mentioned above, we also found a different qualitative profile of several D2-like receptor ligands for D4.4R and D4.7R, but only when forming heteromers with D2R or αAR (52, 53). Therefore, when searching for new D4R ligands, D4R polymorphisms and D4R heteromers (D2R-D4R and α2AR-D4R heteromers) should be considered as targets, which could provide a more effective and individualized treatment. Importantly, in this review we only considered the most prevalent and studied D4R polymorphisms, and more studies need to be performed to evaluate the specific properties of less common yet prevalent polymorphisms.

Finally, in this review we have not discussed the role of D4R localized in brain areas other than the frontal cortico-striatal pyramidal neuron and the pinealocytes. The globus pallidus and the lateral habenula are additional regions where D4Rs have been shown to play a significant role in the mediation of inhibitory transmission by dopamine or norepinephrine, respectively (120–122). The present review emphasizes the need to find the heteromeric partners of the D4R and to establish the differential functional and pharmacological role of its polymorphic variants.

Author contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Funding

S.F. is supported by the intramural funds of the National Institute on Drug Abuse. E. M., V.C.-A. and V.C. are supported by “Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación’ (MCIN/AEI/10.13039/501100011033) and FEDER (SAF2017–87629-R and PID2020–113938RB-I00). V.C.-A also acknowledges the Spanish MCIN/AEI/10.13039/501100011033 for a ‘Juan de la Cierva’ fellowship (FJC2019-041020-I).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.