Numerous data reported in lampreys, which belong to the phylogenetically oldest branch of vertebrates, show that the dopaminergic system was already well developed at the dawn of vertebrate evolution. The expression of dopamine in the lamprey brain is well conserved when compared to other vertebrates, and this is also true for the D2 receptor. Additionally, the key role of dopamine in the striatum, modulating the excitability in the direct and indirect pathways through the D1 and D2 receptors, has also been recently reported in these animals. The moment of divergence regarding the two whole genome duplications occurred in vertebrates suggests that additional receptors, apart from the D1 and D2 previously reported, could be present in lampreys. We used in situ hybridization to characterize the expression of a novel dopamine receptor, which we have identified as a D4 receptor according to the phylogenetic analysis. The D4 receptor shows in the sea lamprey a more restricted expression pattern than the D2 subtype, as reported in mammals. Its main expression areas are the striatum, lateral and ventral pallial sectors, several hypothalamic regions, habenula, and mesencephalic and rhombencephalic motoneurons. Some expression areas are well conserved through vertebrate evolution, as is the case of the striatum or the habenula, but the controversies regarding the D4 receptor expression in other vertebrates hampers for a complete comparison, especially in rhombencephalic regions. Our results further support that the dopaminergic system in vertebrates is well conserved and suggest that at least some functions of the D4 receptor were already present before the divergence of lampreys.

Dopamine depletion in experimental models of Parkinson's disease promotes burst firing of neurons in the subthalamic nucleus (STN) and substantia nigra zona reticulata (SNR). A synaptically generated form of burst firing has been shown to arise from complex excitatory postsynaptic currents (EPSCs) that are evoked in SNR neurons by STN stimulation. The present experiments were designed to characterize actions of dopamine on complex EPSCs in slices of rat brain. Using patch pipettes to record whole-cell currents under voltage clamp, dopamine (30 μm) caused a reversible 64% reduction in complex EPSC charge. This effect was partially mimicked by D(2), D(3) and D(4) receptor agonists, and the action of dopamine could be nearly completely blocked by the combined effects of the D(2/3) antagonist sulpiride and the D(4) antagonist L-745,870. Local application of dopamine to the STN caused a larger inhibition of the complex EPSC (55% reduction) than did dopamine application to the SNR (15% reduction). Simple, monophasic EPSCs, which were evoked in SNR neurons by stimulating the SNR close to the recording pipette, were inhibited to a smaller extent compared to complex EPSCs. Bursts of action potentials evoked in SNR neurons by STN stimulation were inhibited by dopamine to a greater extent than was spontaneous firing. These results show that dopamine D(2)-like receptors inhibit complex EPSCs and burst discharges in the SNR by acting within the STN to suppress transmission in the subthalamonigral pathway. Dopamine receptor-mediated inhibition of polysynaptic connections in the STN might be beneficial in the treatment of Parkinson's disease.

Prior studies have found that dopamine (DA), acting at D4 receptors, and neuregulin (NRG), likely acting at ErbB4 receptors, are involved in a form of depotentiation of long-term potentiation (LTP) at Schaffer collateral (SC) synapses in the hippocampus. Furthermore, DA and NRG actions are intertwined in that NRG induces DA release. We previously found that low-frequency stimulation (LFS) of temperoammonic (TA) inputs to area CA1 also depotentiates previously established SC LTP through a complex signaling pathway involving endocannabinoids, GABA, adenosine, and mitogen-activated protein kinases (MAPKs), but not glutamate. In the present studies, we found that TA-induced SC depotentiation in hippocampal slices from Sprague-Dawley albino rats also involves activation of both D4 receptors and NRG-activated ErbB receptors, but that the roles of these two modulator systems are independent with D4 receptor antagonism failing to alter chemical depotentiation by NRG1β. Furthermore, a selective D4 receptor agonist was unable to depotentiate SC LTP when administered alone, suggesting that D4 receptor activation is necessary but not sufficient for TA-induced SC depotentiation. Chemical depotentiation by NRG1β was inhibited by a Pan-ErbB antagonist and by picrotoxin (PTX), an antagonist of GABA-A receptors (GABAARs), indicating that NRG likely promotes SC depotentiation via effects on GABA and interneurons. These findings have implications for understanding the role of DA and NRG in cognitive dysfunction associated with neuropsychiatric illnesses.

Although multiple studies report that unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) in rats induce depressive-like behaviors and hyperactivity of the lateral habenula (LHb), effects of dopamine (DA) D4 receptors in the LHb on depressive-like behaviors are unclear. Here we found that intra-LHb injection of the different doses of D4 receptor agonist A412997 and antagonist L741742 produced the different behavioral responses in SNc sham-lesioned rats, and only the high doses of A412997 and L741742 increased the expression of depressive-like behaviors or produced antidepressant-like effects in SNc-lesioned rats. The low doses of A412997 and L741742 altered the firing rate of LHb neurons and release of DA, GABA and glutamate in the LHb via the GABAergic rostromedial tegmental nucleus (RMTg) in SNc sham-lesioned rats, but not in SNc-lesioned rats. The high doses of A412997 and L741742 also altered the firing rate and release of the transmitters in both SNc sham-lesioned and SNc-lesioned rats, whereas these effects were not involved in the RMTg. Lesions of the SNc shortened the duration of significant effects on the firing rate and release of the transmitters induced by the high doses of A412997 and L741742. These findings suggest that D4 receptors in the LHb are involved in depression-like behaviors via the pre- and post-synaptic mechanisms and depletion of DA decreases the function and/or the expression of both pre- and post-synaptic D4 receptors. This study also points to the importance of the pre-synaptic D4 receptors in the regulation of Parkinson's disease-related depression.

The dopamine D4 receptor (D4R) has attracted considerable attention as potential target for the treatment of a broad range of central nervous system disorders. Although many efforts have been made to improve the performance of putative radioligand candidates, there is still a lack of D4R selective tracers suitable for in vivo PET imaging. Thus, the objective of this work was to develop a D4-selective PET ligand for clinical applications.

D4 dopamine receptor (D4R) activation uniquely promotes methylation of plasma membrane phospholipids, utilizing folate-derived methyl groups provided by methionine synthase (MS). We evaluated the impact of D4R expression on folate-dependent phospholipid methylation (PLM) and MS activity, as well as cellular redox and methylation status, in transfected CHO cells expressing human D4R variants containing 2, 4, or 7 exon III repeats (D4.2R, D4.4R, D4.7R). Dopamine had no effect in non-transfected CHO cells, but increased PLM to a similar extent for both D4.2R- and D4.4R-expressing cells, while the maximal increase was for D4.7R was significantly lower. D4R expression in CHO cells decreased basal MS activity for all receptor subtypes and conferred dopamine-sensitive MS activity, which was greater with a higher number of repeats. Consistent with decreased MS activity, D4R expression decreased basal levels of methylation cycle intermediates methionine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), as well as cysteine and glutathione (GSH). Conversely, dopamine stimulation increased GSH, SAM, and the SAM/SAH ratio, which was associated with a more than 2-fold increase in global DNA methylation. Our findings illustrate a profound influence of D4R expression and activation on MS activity, coupled with the ability of dopamine to modulate cellular redox and methylation status. These previously unrecognized signaling activities of the D4R provide a unique link between neurotransmission and metabolism.

Using a rodent slot machine task (rSMT), we have previously shown that rats, like humans, are susceptible to the reinforcing effects of winning signals presented within a compound stimulus array, even when the pattern generated predicts a negative rather than a positive outcome such as during a "near-miss". The dopamine D4 receptor critically mediates the erroneous reward expectancy generated on such trials. D4 receptors are particularly enriched within frontal and limbic areas activated during slot machine play, such as the anterior cingulate cortex (ACC). We therefore selectively inactivated the ACC to confirm involvement of this region in rSMT performance, and subsequently examined the specific contribution of local D4 receptors. ACC inactivations generally impaired animals' ability to optimally differentiate winning from losing outcomes. Local administration of the D4 agonist PD168077 had a qualitatively similar effect, but increased reward expectancy was only evident on archetypal "near-miss" trials i.e. when the first two of three stimuli in the array were concordant with a rewarding outcome, and only the last stimulus critically signalled a non-win. These data indicate that the ACC is critically involved in parsing the appropriate response when competing stimulus-outcome associations are activated, and that signalling via D4 receptors may play a particularly important role in gating the temporal and spatial summation of salient events. Such findings provide novel insights into the mechanism underlying the erroneous expectations of reward generated when playing slot machines, and suggest a mechanism by which D4 receptor antagonists may be effective in treating gambling disorder.

Growth factor-induced receptor dimerization and cross-phosphorylation are hallmarks of signal transduction via receptor tyrosine kinases (RTKs). G protein-coupled receptors (GPCRs) can activate RTKs through a process known as transactivation. The prototypical model of RTK transactivation involves ligand-mediated RTK dimerization and cross-phosphorylation. Here, we show that the platelet-derived growth factor receptor beta (PDGFRbeta) transactivation by the dopamine receptor D4 (DRD4) is not dependent on ligands for PDGFRbeta. Furthermore, when PDGFRbeta dimerization is inhibited and receptor phosphorylation is suppressed to near basal levels, the receptor maintains its ability to be transactivated and is still effective in signaling to ERK1/2. Hence, the DRD4-PDGFRbeta-ERK1/2 pathway can occur independently of a PDGF-like ligand, PDGFRbeta cross-phosphorylation and dimerization, which is distinct from other known forms of transactivation of RTKs by GPCRs.

Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications. Dopamine receptors is known to play an important protective role in diabetes. However, the protective effect of dopamine receptors against hyperglycemia-induced endothelial damage in diabetic rats is still unknown. In the present study, we established a cell model of hyperglycemia-induced endothelial dysfunction by treating human umbilical vein endothelial cells (HUVEC) with high glucose. MTT and lactate dehydrogenase assays results showed that high glucose treatment significantly reduced the cell viability and down-regulated dopamine D4 receptor. Pre-treatment with PD168077, a specific D4 receptor agonist, greatly improved endothelial cell viability and decreased apoptosis. Furthermore, pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and endothelial nitric oxide synthase (eNOS) eliminated the protective effect of D4 receptor against endothelial injury. More importantly, the expression level of D4 receptor was also dramatically down-regulated in the arterial endothelium of rats with streptozotocin-(STZ)-induced diabetes, and the STZ-induced impairment of acetylcholine-induced vasodilation was reversed by activation of D4 receptor. In conclusion, our results indicated that dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via the PI3K/eNOS pathway, which may provide a novel strategy in the treatment of diabetes.

Multiple subtypes of dopamine receptors within the GPCR superfamily regulate neurological processes through various downstream signaling pathways. A crucial question about the dopamine receptor family is what structural features determine the subtype-selectivity of potential drugs. Here, we report the 3.5-angstrom crystal structure of mouse dopamine receptor D4 (DRD4) complexed with a subtype-selective antagonist, L745870. Our structure reveals a secondary binding pocket extended from the orthosteric ligand-binding pocket to a DRD4-specific crevice located between transmembrane helices 2 and 3. Additional mutagenesis studies suggest that the antagonist L745870 prevents DRD4 activation by blocking the relative movement between transmembrane helices 2 and 3. These results expand our knowledge of the molecular basis for the physiological functions of DRD4 and assist new drug design.

The dopamine D4 receptors (DRD4) play a key role in numerous brain functions and are involved in the pathogenesis of various psychiatric disorders. DRD4 ligands have been shown to moderate anxiety, reward and depression-like behaviours, and cognitive impairments. Despite a series of promising but ambiguous findings, the therapeutic advantages of DRD4 stimulation remain elusive.

Gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility. The release of GnRH peptide regulates the synthesis and release of both luteinizing hormone (LH) and Follicle stimulation hormone (FSH) from the anterior pituitary. While it is known that dopamine regulates GnRH neurons, the specific dopamine receptor subtype(s) involved remain unclear. Previous studies in adult rodents have reported juxtaposition of fibers containing tyrosine hydroxylase (TH), a marker of catecholaminergic cells, onto GnRH neurons and that exogenous dopamine inhibits GnRH neurons postsynaptically through dopamine D1-like and/or D2-like receptors. Our microarray data from GnRH neurons revealed a high level of Drd4 transcripts [i.e., dopamine D4 receptor (D4R)]. Single-cell RT-PCR and immunocytochemistry confirmed GnRH cells express the Drd4 transcript and protein, respectively. Calcium imaging identified changes in GnRH neuronal activity during application of subtype-specific dopamine receptor agonists and antagonists when GABAergic and glutamatergic transmission was blocked. Dopamine, dopamine with D1/5R-specific or D2/3R-specific antagonists or D4R-specific agonists decreased the frequency of calcium oscillations. In contrast, D1/5R-specific agonists increased the frequency of calcium oscillations. The D4R-mediated inhibition was dependent on Gαi/o protein coupling, while the D1/5R-mediated excitation required Gαs protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons.

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer's disease and substance use disorders. To identify new D4R-selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A-412997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)- N-( m-tolyl)acetamide). Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cyclic AMP inhibition assays, and molecular dynamics computational modeling. We identified several novel D4R-selective ( Ki ≤ 4.3 nM and >100-fold vs other D2-like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. These compounds highlight receptor-ligand interactions that control efficacy at D2-like receptors and may provide insights into targeted drug discovery, leading to a better understanding of the role of D4Rs in neuropsychiatric disorders.

Gambling is an addictive disorder with serious societal and personal costs. To-date, there are no approved pharmacological treatments for gambling disorder. Evidence suggests a role for dopamine in gambling disorder and thus may provide a therapeutic target. The present study therefore aimed to investigate the effects of selective antagonists and agonists of D2, D3 and D4 receptors in a rodent analogue of the Iowa gambling task used clinically. In this rat gambling task (rGT), animals are trained to associate different response holes with different magnitudes and probabilities of food pellet rewards and punishing time-out periods. As in the Iowa gambling task, the optimal strategy is to avoid the tempting high-risk high-reward options, and instead favor those linked to smaller per-trial rewards but also lower punishments, thereby maximizing the amount of reward earned over time. Administration of those selective ligands did not affect decision making under the rGT. Only the D4 drug had modest effects on latency measures suggesting that D4 may contribute in some ways to decision making under this task.

Long-term exposition to morphine elicits structural and synaptic plasticity in reward-related regions of the brain, playing a critical role in addiction. However, morphine-induced neuroadaptations in the dorsal striatum have been poorly studied despite its key function in drug-related habit learning. Here, we show that prolonged treatment with morphine triggered the retraction of the dendritic arbor and the loss of dendritic spines in the dorsal striatal projection neurons (MSNs). In an attempt to extend previous findings, we also explored whether the dopamine D4 receptor (D4R) could modulate striatal morphine-induced plasticity. The combined treatment of morphine with the D4R agonist PD168,077 produced an expansion of the MSNs dendritic arbors and restored dendritic spine density. At the electrophysiological level, PD168,077 in combination with morphine altered the electrical properties of the MSNs and decreased their excitability. Finally, results from the sustantia nigra showed that PD168,077 counteracted morphine-induced upregulation of μ opioid receptors (MOR) in striatonigral projections and downregulation of G protein-gated inward rectifier K+ channels (GIRK1 and GIRK2) in dopaminergic cells. The present results highlight the key function of D4R modulating morphine-induced plasticity in the dorsal striatum. Thus, D4R could represent a valuable pharmacological target for the safety use of morphine in pain management.

Polymorphic variants of the dopamine D(4) receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D(4.4)) and the 2-repeat (D(4.2)) variants form functional heteromers with the short isoform of the dopamine D(2) receptor (D(2S)), the 7-repeat risk allele (D(4.7)) does not. D(2) receptor activation in the D(2S)-D(4) receptor heteromer potentiates D(4) receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D(4.7) or in the striatum of knockin mutant mice carrying the 7 repeats of the human D(4.7) in the third intracellular loop of the D(4) receptor. In the striatum, D(4) receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D(2S) receptors. This interaction shows the same qualitative characteristics than the D(2S)-D(4) receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D(2S) receptor activation potentiates D(4) receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D(2S)-D(4.7) heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD.

Gamma oscillations are electric activity patterns of the mammalian brain hypothesized to serve attention, sensory perception, working memory and memory encoding. They are disrupted or altered in schizophrenic patients with associated cognitive deficits, which persist in spite of treatment with antipsychotics. Because cognitive symptoms are a core feature of schizophrenia it is relevant to explore signaling pathways that potentially regulate gamma oscillations. Dopamine has been reported to decrease gamma oscillation power via D1-like receptors. Based on the expression pattern of D4 receptors (D4R) in hippocampus, and pharmacological effects of D4R ligands in animals, we hypothesize that they are in a position to regulate gamma oscillations as well.

The variety of physiological functions controlled by dopamine in the brain and periphery is mediated by the D1, D2, D3, D4 and D5 dopamine GPCRs. Drugs acting on dopamine receptors are significant tools for the management of several neuropsychiatric disorders including schizophrenia, bipolar disorder, depression and Parkinson's disease. Recent investigations of dopamine receptor signalling have shown that dopamine receptors, apart from their canonical action on cAMP-mediated signalling, can regulate a myriad of cellular responses to fine-tune the expression of dopamine-associated behaviours and functions. Such signalling mechanisms may involve alternate G protein coupling or non-G protein mechanisms involving ion channels, receptor tyrosine kinases or proteins such as β-arrestins that are classically involved in GPCR desensitization. Another level of complexity is the growing appreciation of the physiological roles played by dopamine receptor heteromers. Applications of new in vivo techniques have significantly furthered the understanding of the physiological functions played by dopamine receptors. Here we provide an update of the current knowledge regarding the complex biology, signalling, physiology and pharmacology of dopamine receptors.

Recent data indicate that cortical dopamine denervation results in dystrophic changes in the dendrites of pyramidal cells, including decreases in dendritic spine density and length. However, it is not known if the loss of signaling through specific dopamine receptors subserves these dendritic changes. We examined the dendritic structure of layer V pyramidal cells in the prefrontal cortex of D(1), D(2), and D(4) dopamine receptor null mutant mice and their wild-type littermates. Decreased basal dendritic length and spine density were observed in the D(1) knockout mice. Similarly, a decrease in basal dendritic spine density was uncovered in the D(2) knockout mice relative to wild-type littermates. No changes in any dendritic parameter were observed in the D(4) knockout mice. These observations suggest that the dystrophic changes observed in prefrontal cortical pyramidal cell dendrites are due to loss of signaling through D(1) and possibly D(2) receptors. The current data also suggest that caution should be exercised in the interpretation of behavioral, physiological, and biochemical studies of the prefrontal cortex in dopamine receptor knockout mice.

Dopamine is a key neuromodulatory transmitter in the brain. It acts through dopamine receptors to affect changes in neural activity, gene expression, and behavior. In songbirds, dopamine is released into the striatal song nucleus Area X, and the levels depend on social contexts of undirected and directed singing. This differential release is associated with differential expression of activity-dependent genes, such as egr1 (avian zenk), which in mammalian brain are modulated by dopamine receptors. Here we cloned from zebra finch brain cDNAs of all avian dopamine receptors: the D1 (D1A, D1B, D1D) and D2 (D2, D3, D4) families. Comparative sequence analyses of predicted proteins revealed expected phylogenetic relationships, in which the D1 family exists as single exon and the D2 family exists as spliced exon genes. In both zebra finch and chicken, the D1A, D1B, and D2 receptors were highly expressed in the striatum, the D1D and D3 throughout the pallium and within the mesopallium, respectively, and the D4 mainly in the cerebellum. Furthermore, within the zebra finch, all receptors, except for D4, showed differential expression in song nuclei relative to the surrounding regions and developmentally regulated expression that decreased for most receptors during the sensory acquisition and sensorimotor phases of song learning. Within Area X, half of the cells expressed both D1A and D2 receptors, and a higher proportion of the D1A-only-containing neurons expressed egr1 during undirected but not during directed singing. Our findings are consistent with hypotheses that dopamine receptors may be involved in song development and social context-dependent behaviors.