Physiological specialization of the brain in bumble bee castes: Roles of dopamine in mating-related behaviors in female bumble bees.

We aimed to investigate the roles of dopamine in regulating caste-specific behaviors in bumble bees and mating-related behaviors in bumble bee gynes. We examined caste differences in behaviors, biogenic amine levels, and expression levels of genes encoding dopamine receptors in the brains of bumble bees, and analyzed the effects of dopamine-related drugs on bumble bee behavior. Locomotor and flight activities were significantly higher in 8-day-old gynes and light avoidance was significantly lower in 4-8-day-old gynes than in same-aged workers. Brain levels of dopamine and octopamine were significantly higher in 8-day-old gynes than in same-aged workers, but tyramine and serotonin levels did not differ between the castes. Relative expression levels of the dopamine receptor gene BigDop1 were significantly lower in 8-day-old gynes than in same-aged workers, but expression levels of other dopamine receptor genes did not differ between castes. Dopamine significantly enhanced locomotor and flight activities in 7-9-day-old workers, whereas the dopamine receptor antagonist flupentixol inhibited flight activity and mating acceptance in same-aged gynes. These results suggest that dopamine plays important roles in gyne-specific behavior in bumble bees and has a common dopaminergic function in female eusocial bees.

A Combination of a Dopamine Receptor 2 Agonist and a Kappa Opioid Receptor Antagonist Synergistically Reduces Weight in Diet-Induced Obese Rodents.

As the global obesity rate increases, so does the urgency to find effective anti-obesity drugs. In the search for therapeutic targets, central nervous system (CNS) mechanisms engaged in the regulation of energy expenditure and food intake, such as the opioid and dopamine systems, are crucial. In this study, we examined the effect on body weight of two drugs: bromocriptine (BC), a D2R receptor agonist, and PF-04455242, a selective κ opioid receptor (KOR) antagonist. Using diet-induced obese (DIO) rats, we aimed to ascertain whether the administration of BC and PF-04455242, independently or in combination, could enhance body weight loss. Furthermore, the present work demonstrates that the peripheral coadministration of BC and PF-04455242 enhances the reduction of weight in DIO rats and leads to a decrease in adiposity in a food-intake-independent manner. These effects were based on heightened energy expenditure, particularly through the activation of brown adipose tissue (BAT) thermogenesis. Overall, our findings indicate that the combination of BC and PF-04455242 effectively induces body weight loss through increased energy expenditure by increasing thermogenic activity and highlight the importance of the combined use of drugs to combat obesity.

Loss-of-function of GNAL dystonia gene impairs striatal dopamine receptors-mediated adenylyl cyclase/ cyclic AMP signaling pathway.

Loss-of-function mutations in the GNAL gene are responsible for DYT-GNAL dystonia. However, how GNAL mutations contribute to synaptic dysfunction is still unclear. The GNAL gene encodes the Gαolf protein, an isoform of stimulatory Gαs enriched in the striatum, with a key role in the regulation of cAMP signaling. Here, we used a combined biochemical and electrophysiological approach to study GPCR-mediated AC-cAMP cascade in the striatum of the heterozygous GNAL (GNAL+/-) rat model. We first analyzed adenosine type 2 (A2AR), and dopamine type 1 (D1R) receptors, which are directly coupled to Gαolf, and observed that the total levels of A2AR were increased, whereas D1R level was unaltered in GNAL+/- rats. In addition, the striatal isoform of adenylyl cyclase (AC5) was reduced, despite unaltered basal cAMP levels. Notably, the protein expression level of dopamine type 2 receptor (D2R), that inhibits the AC5-cAMP signaling pathway, was also reduced, similar to what observed in different DYT-TOR1A dystonia models. Accordingly, in the GNAL+/- rat striatum we found altered levels of the D2R regulatory proteins, RGS9-2, spinophilin, Gß5 and ß-arrestin2, suggesting a downregulation of D2R signaling cascade. Additionally, by analyzing the responses of striatal cholinergic interneurons to D2R activation, we found that the receptor-mediated inhibitory effect is significantly attenuated in GNAL+/- interneurons. Altogether, our findings demonstrate a profound alteration in the A2AR/D2R-AC-cAMP cascade in the striatum of the rat DYT-GNAL dystonia model, and provide a plausible explanation for our previous findings on the loss of dopamine D2R-dependent corticostriatal long-term depression.

Dopamine receptor D2 regulates genes involved in germ cell movement and sperm motility in rat testes†.

The function of dopamine receptor D2 (D2R) is well associated with sperm motility; however, the physiological role of D2R present on testicular cells remains elusive. The aim of the present study is to delineate the function of testicular D2R. Serum dopamine levels were found to decrease with age, whereas testicular D2R expression increased. In rat testicular sections, D2R immunolabeling was observed in interstitial cells, spermatogonia, spermatocytes and mature elongated spermatids, whereas tyrosine hydroxylase immunolabeling was selectively detected in Leydig cells. In vitro seminiferous tubule culture following bromocriptine (D2R agonist) treatment resulted in decreased cAMP levels. Microarray identified 1077 differentially expressed genes (511 up-regulated, 566 down-regulated). The majority of differentially expressed genes were present in post-meiotic cells including early and late spermatids, and sperm. Gene ontology elucidated processes related to extra-cellular matrix to be enriched and was supported by differential expression of various collagens and laminins, thereby indicating a role of dopamine in extra-cellular matrix integrity and transport of spermatids across the seminiferous epithelium. Gene ontology and enrichment map also highlighted cell/sperm motility to be significantly enriched. Therefore, genes involved in sperm motility functions were further validated by RT-qPCR. Seven genes (Akap4, Ccnyl1, Iqcf1, Klc3, Prss55, Tbc1d21, Tl18) were significantly up-regulated, whereas four genes (Dnah1, Dnah5, Clxn, Fsip2) were significantly down-regulated by bromocriptine treatment. The bromocriptine-stimulated reduction in seminiferous tubule cyclic AMP and associated changes in spermatid gene expression suggests that dopamine regulates both spermatogenesis and spermiogenesis within the seminiferous epithelium, and spermatozoa motility following spermiation, as essential processes for fertility.

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors.

Neuromodulatory signaling via G protein-coupled receptors (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. The recent development of optogenetic tools to induce G protein-mediated signaling provides the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimize the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). These optoDopRs exhibit high signaling specificity and light sensitivity in vitro. In vivo, we show receptor and cell type-specific effects of dopaminergic signaling in various behaviors, including the ability of optoDopRs to rescue the loss of the endogenous receptors. This work demonstrates that optoXRs can enable optical control of neuromodulatory receptor-specific signaling in functional and behavioral studies.

Voluntary alcohol intake alters the motivation to seek intravenous oxycodone and neuronal activation during the reinstatement of oxycodone and sucrose seeking.

Opioid-alcohol polysubstance use is prevalent and worsens treatment outcomes. Here we assessed whether co-consumption of oxycodone and alcohol influence the intake of one another, demand for oxycodone, and the neurocircuitry underlying cue-primed reinstatement of oxycodone-seeking. Male and female rats underwent oxycodone intravenous self-administration (IVSA) with homecage access to alcohol (20% v/v) and/or water immediately after the IVSA session. Next, economic demand for intravenous oxycodone was assessed while access to alcohol and/or water continued. Control rats self-administered sucrose followed by access to alcohol and/or water. Rats underwent a cue-primed reinstatement test and brains were processed for c-fos mRNA expression. While both sexes decreased oxycodone intake if they had access to alcohol, and decreased alcohol intake if they had access to oxycodone, only female oxycodone + alcohol rats exhibited decreased demand elasticity and increased cue-primed reinstatement. Alcohol consumption increased the number of basolateral and central amygdala neurons activated during sucrose and oxycodone reinstatement and the number of ventral and dorsal striatum neurons engaged by sucrose reinstatement. Nucleus accumbens shell dopamine 1 receptor expressing neurons displayed activation patterns consistent with oxycodone reinstatement. Thus, alcohol alters the motivation to seek oxycodone in a sex-dependent manner and the neural circuitry engaged by cue-primed reinstatement of sucrose and oxycodone-seeking.

Boolean analysis shows a high proportion of dopamine D2 receptors interacting with adenosine A2A receptors in striatal medium spiny neurons of mouse and non-human primate models of Parkinson's disease.

The antagonistic effect of adenosine on dopaminergic transmission in the basal ganglia indirect motor control pathway is mediated by dopamine D2 (D2R) and adenosine A2A (A2AR) receptors co-expressed on medium spiny striatal neurons. The pathway is unbalanced in Parkinson's disease (PD) and an A2AR blocker has been approved for use with levodopa in the therapy of the disease. However, it is not known whether the therapy is acting on individually expressed receptors or in receptors forming A2A-D2 receptor heteromers, whose functionality is unique. For two proteins prone to interact, a very recently developed technique, MolBoolean, allows to determine the number of proteins that are either non-interacting or interacting. After checking the feasibility of the technique and reliability of data in transfected cells and in striatal primary neurons, the Boolean analysis of receptors in the striatum of rats and monkeys showed a high percentage of D2 receptors interacting with the adenosine receptor, while, on the contrary, a significant proportion of A2A receptors do not interact with dopamine receptors. The number of interacting receptors increased when rats and monkeys were lesioned to become a PD model. The use of a tracer of the indirect pathway in monkeys confirmed that the data was restricted to the population of striatal neurons projecting to the GPe. The results are not only relevant for being the first study quantifying individual versus interacting G protein-coupled receptors, but also for showing that the D2R in these specific neurons, in both control and PD animals, is under the control of the A2AR. The tight adenosine/dopamine receptor coupling suggest benefits of early antiparkinsonian treatment with adenosine receptor blockers.

G Protein-Coupled Receptor Kinase 2 Selectively Enhances ß-Arrestin Recruitment to the D2 Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation.

The D2 dopamine receptor (D2R) signals through both G proteins and ß-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. ß-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for ß-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for ß-arrestin recruitment to the D2R, and the role of GRKs in D2R-ß-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which ß-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated ß-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased ß-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-ß-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance ß-arrestin recruitment and activation independently of receptor phosphorylation.

Regulation Mechanism of Dopamine Receptor 1 in Low Temperature Response of Marsupenaeus japonicus.

Dopamine receptors (DARs) are important transmembrane receptors responsible for receiving extracellular signals in the DAR-mediated signaling pathway, and are involved in a variety of physiological functions. Herein, the D1 DAR gene from Marsupenaeus japonicus (MjDAD1) was identified and characterized. The protein encoded by MjDAD1 has the typical structure and functional domains of the G-protein coupled receptor family. MjDAD1 expression was significantly upregulated in the gills and hepatopancreas after low temperature stress. Moreover, double-stranded RNA-mediated silencing of MjDAD1 significantly changed the levels of protein kinases (PKA and PKC), second messengers (cyclic AMP (cAMP), cyclic cGMP, calmodulin, and diacyl glycerol), and G-protein effectors (adenylate cyclase and phospholipase C). Furthermore, MjDAD1 silencing increased the apoptosis rate of gill and hepatopancreas cells. Thus, following binding to their specific receptors, G-protein effectors are activated by MjDAD1, leading to DAD1-cAMP/PKA pathway-mediated regulation of caspase-dependent mitochondrial apoptosis. We suggest that MjDAD1 is indispensable for the environmental adaptation of M. japonicus.

Anxiety, aggression, reward sensitivity, and forebrain dopamine receptor expression in a laboratory rat model of early-life disadvantage.

Despite early-life disadvantage (ELD) in humans being a highly heterogenous construct, it consistently predicts negative neurobehavioral outcomes. The numerous environmental contributors and neural mechanisms underlying ELD remain unclear, though. We used a laboratory rat model to evaluate the effects of limited resources and/or heavy metal exposure on mothers and their adult male and female offspring. Dams and litters were chronically exposed to restricted (1-cm deep) or ample (4-cm deep) home cage bedding postpartum, with or without lead acetate (0.1%) in their drinking water from insemination through 1-week postweaning. Restricted-bedding mothers showed more pup-directed behaviors and behavioral fragmentation, while lead-exposed mothers showed more nestbuilding. Restricted bedding-raised male offspring showed higher anxiety and aggression. Either restricted bedding or lead exposure impaired goal-directed performance in a reinforcer devaluation task in females, whereas restricted bedding alone disrupted it in males. Lead exposure, but not limited bedding, also reduced sucrose reward sensitivity in a progressive ratio task in females. D1 and D2 receptor mRNA in the medial prefrontal cortex and nucleus accumbens (NAc) were each affected by the early-life treatments and differently between the sexes. Most notably, adult males (but not females) exposed to both early-life treatments had greatly increased D1 receptor mRNA in the NAc core. These results illuminate neural mechanisms through which ELD threatens neurobehavioral development and highlight forebrain dopamine as a factor.

Fluoroalkoxylated C-3 and C-9 (S)-12-bromostepholidine analogues with D1R antagonist activity.

To illuminate the tolerance of fluoroalkoxylated groups at the C-3 and C-9 positions of tetrahydroprotoberberines (THPBs) on D1R activity, C-3 and C-9 fluoroalkoxylated analogues of (S)-12-bromostepholidine were prepared and evaluated. All compounds examined were D1R antagonists as measured by a cAMP assay. Our structure-activity studies herein indicate that the C-3 position tolerates a 1,1-difluoroethoxy substituent for D1R antagonist activity. Compound 13a was the most potent cAMP-based D1R antagonist identified and was also found to antagonize ß-arrestin translocation in a TANGO assay. Affinity assessments at other dopamine receptors revealed that 13a is selective for D1R and unlike other naturally-occurring THPBs such as (S)-stepholidine, lacks D2R affinity. In preliminary biopharmaceutical assays, excellent BBB permeation was observed for 13a. Further pharmacological studies are warranted on (S)-stepholidine congeners to harvest their potential as a source of novel, druggable D1R-targeted agents.

Comparative study of the molecular mechanisms underlying the G protein and ß-arrestin-dependent pathways that lead to ERKs activation upon stimulation by dopamine D2 receptor.

Dopamine D2 receptor (D2 R) has been shown to activate extracellular signal-regulated kinases (ERKs) via distinct pathways dependent on either G-protein or ß-arrestin. However, there has not been a systematic study of the regulatory process of D2 R-mediated ERKs activation by G protein- versus ß-arrestin-dependent signaling since D2 R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2 R-mediated ERKs activation via these two pathways. Our results showed that G protein-dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, ß-arrestin-dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein- and ß-arrestin-dependent pathway-mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2-CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3-kinase inhibitor all blocked G protein-dependent ERKs activation. In contrast, GRK2 and ß-Arr2 played a main role in ß-arrestin-dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho-ERKs, ß-Arr2, and importinß1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes.

Structural and expression analysis of the dopamine receptors reveals their crucial roles in regulating the insulin signaling pathway in oysters.

Dopamine performs its critical role upon binding to receptors. Since dopamine receptors are numerous and versatile, understanding their protein structures and evolution status, and identifying the key receptors involved in the modulation of insulin signaling will provide essential clues to investigate the molecular mechanism of neuroendocrine regulating the growth in invertebrates. In this study, seven dopamine receptors were identified in the Pacific oysters (Crassostrea gigas) and were classified into four subtypes according to their protein secondary and tertiary structures, and ligand-binding activities. Of which, DR2 (dopamine receptor 2) and D(2)RA-like (D(2) dopamine receptor A-like) were considered the invertebrate-specific type 1 and type 2 dopamine receptors, respectively. Expression analysis indicated that the DR2 and D(2)RA-like were highly expressed in the fast-growing oyster "Haida No.1". After in vitro incubation of ganglia and adductor muscle with exogenous dopamine and dopamine receptor antagonists, the expression of these two dopamine receptors and ILPs (insulin-like peptides) was also significantly affected. Dual-fluorescence in situ hybridization results showed that D(2)RA-like and DR2 were co-localized with MIRP3 (molluscan insulin-related peptide 3) and MIRP3-like (molluscan insulin-related peptide 3-like) in the visceral ganglia, and were co-localized with ILP (insulin-like peptide) in the adductor muscle. Furthermore, the downstream components of dopamine signaling, including PKA, ERK, CREB, CaMKK1, AKT, and GSK3ß were also significantly affected by the exogenous dopamine and dopamine receptor antagonists. These findings confirmed that dopamine might affect the secretion of ILPs through the invertebrate-specific dopamine receptors D(2)RA-like and DR2, and thus played crucial roles in the growth regulation of the Pacific oysters. Our study establishes the potential regulatory relationship between the dopaminergic system and insulin-like signaling pathway in marine invertebrates.

Gradients of neurotransmitter receptor expression in the macaque cortex.

Dynamics and functions of neural circuits depend on interactions mediated by receptors. Therefore, a comprehensive map of receptor organization across cortical regions is needed. In this study, we used in vitro receptor autoradiography to measure the density of 14 neurotransmitter receptor types in 109 areas of macaque cortex. We integrated the receptor data with anatomical, genetic and functional connectivity data into a common cortical space. We uncovered a principal gradient of receptor expression per neuron. This aligns with the cortical hierarchy from sensory cortex to higher cognitive areas. A second gradient, driven by serotonin 5-HT1A receptors, peaks in the anterior cingulate, default mode and salience networks. We found a similar pattern of 5-HT1A expression in the human brain. Thus, the macaque may be a promising translational model of serotonergic processing and disorders. The receptor gradients may enable rapid, reliable information processing in sensory cortical areas and slow, flexible integration in higher cognitive areas.

A single-cell trajectory atlas of striatal development.

The striatum integrates dense neuromodulatory inputs from many brain regions to coordinate complex behaviors. This integration relies on the coordinated responses from distinct striatal cell types. While previous studies have characterized the cellular and molecular composition of the striatum using single-cell RNA-sequencing at distinct developmental timepoints, the molecular changes spanning embryonic through postnatal development at the single-cell level have not been examined. Here, we combine published mouse striatal single-cell datasets from both embryonic and postnatal timepoints to analyze the developmental trajectory patterns and transcription factor regulatory networks within striatal cell types. Using this integrated dataset, we found that dopamine receptor-1 expressing spiny projection neurons have an extended period of transcriptional dynamics and greater transcriptional complexity over postnatal development compared to dopamine receptor-2 expressing neurons. Moreover, we found the transcription factor, FOXP1, exerts indirect changes to oligodendrocytes. These data can be accessed and further analyzed through an interactive website ( https://mouse-striatal-dev.cells.ucsc.edu ).

Neonatal olfactory bulbectomy induces anxiety-related behavior and modifies dopaminergic receptor expression in post-pubertal rats.

Olfaction is a complex physiological process producing effects in the central nervous system (CNS) and implicated in emotional processes. Indeed, the olfactory bulbs (OB) send projections to various CNS regions including the nucleus accumbens (NAcc) and caudate-putamen (CPu). Both the NAcc and CPu receive important dopaminergic input. Emerging evidence suggests that dopamine (DA) is related to anxiety-related behaviors. Therefore, we aimed to investigate the consequences of neonatal olfactory bulbectomy (nOBX) to anxiety-related behavior as assayed in the elevated plus maze (EPM) as well as the expression of dopaminergic receptors (D1-like, D2-like, and D3) in the NAcc and CPu at pre- and post-pubertal ages in the rat. The results show that nOBX increased the number of entries in the open arm of the EPM post-pubertally, suggesting an anxiolytic-related effect. nOBX increased the D2-like binding in the NAcc shell and D3 binding in the NAcc core pre-pubertally. At post-pubertal ages, the D3 binding was reduced at the olfactory tubercle and islands of Calleja in nOBX rats. Alterations in the DA receptor expression may be one mechanism responsible for the observed behavioral modifications in nOBX rats.

Structural genomics of the human dopamine receptor system.

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson's Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

Dopamine Receptor D1 Is Exempt from Transforming Growth Factor ß-Mediated Antifibrotic G Protein-Coupled Receptor Landscape Tampering in Lung Fibroblasts.

Pulmonary fibroblasts are the primary producers of extracellular matrix (ECM) in the lungs, and their pathogenic activation drives scarring and loss of lung function in idiopathic pulmonary fibrosis (IPF). This uncontrolled production of ECM is stimulated by mechanosignaling and transforming growth factor beta 1 (TGF-ß1) signaling that together promote transcriptional programs including Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). G protein-coupled receptors (GPCRs) that couple to G α s have emerged as pharmacological targets to inactivate YAP/TAZ signaling and promote lung fibrosis resolution. Previous studies have shown a loss of expression of "antifibrotic GPCRs"-receptors that couple to G α s, in IPF patient-derived fibroblasts compared with non-IPF samples. Of the 14 G α s GPCRs we found to be expressed in lung fibroblasts, the dopamine receptor D1 (DRD1) was one of only two not repressed by TGF-ß1 signaling, with the ß2-adrenergic receptor being the most repressed. We compared the potency and efficacy of multiple D1 and ß2 receptor agonists +/- TGF-ß1 treatment in vitro for their ability to elevate cAMP, inhibit nuclear localization of YAP/TAZ, regulate expression of profibrotic and antifibrotic genes, and inhibit cellular proliferation and collagen deposition. Consistently, the activity of ß2 receptor agonists was lost, whereas D1 receptor agonists was maintained, after stimulating cultured lung fibroblasts with TGF-ß1. These data further support the therapeutic potential of the dopamine receptor D1 and highlight an orchestrated and pervasive loss of antifibrotic GPCRs mediated by TGF-ß1 signaling. SIGNIFICANCE STATEMENT: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with limited therapies. GPCRs have emerged as a primary target for the development of novel antifibrotic drugs; however, a challenge to this approach is the dramatic changes in GPCR expression in response to profibrotic stimuli. Here, we investigate the impact of TGF-ß1 on the expression of antifibrotic GPCRs and show the D1 dopamine receptor expression is uniquely maintained in response to TGF-ß1, further implicating it as a compelling target to treat IPF.

Dopamine receptor D4 (DRD4) negatively regulates UCP1- and ATP-dependent thermogenesis in 3T3-L1 adipocytes and C2C12 muscle cells.

The activation of beige fat and muscle tissues is an interesting and encouraging target for therapeutic intervention in obesity owing to their remarkable lipolytic activity and energy-consuming futile cycles. This study examined the effect of dopamine receptor D4 (DRD4) on lipid metabolisms as well as UCP1- and ATP-dependent thermogenesis in Drd4-silenced 3T3-L1 adipocytes and C2C12 muscle cells. Silencing of Drd4, followed by quantitative real-time PCR, immunoblot analysis, immunofluorescence, and staining methods, were applied to evaluate the effects of DRD4 on diverse target genes and proteins of both cells. The findings showed that DRD4 was expressed in the adipose and muscle tissues of normal and obese mice. Furthermore, the knockdown of Drd4 upregulated the expression of brown adipocyte-specific genes and proteins while downregulating lipogenesis and the adipogenesis marker proteins. Drd4 silencing also upregulated the expression of key signaling molecules involved in ATP-dependent thermogenesis in both cells. This was further elucidated by mechanistic studies showing that a Drd4 knockdown mediates UCP1-dependent thermogenesis via the cAMP/PKA/p38MAPK pathway in 3T3-L1 adipocytes and UCP1-independent thermogenesis via the cAMP/SLN/SERCA2a pathway in C2C12 muscle cells. In addition, siDrd4 also mediates myogenesis via the cAMP/PKA/ERK1/2/Cyclin D3 pathway in C2C12 muscle cells. Silencing of Drd4 promotes ß3-AR-dependent browning in 3T3-L1 adipocytes and α1-AR/SERCA-based thermogenesis through an ATP-consuming futile process in C2C12 muscle cells. Understanding the novel functions of DRD4 on adipose and muscle tissues in terms of its ability to enhance energy expenditure and regulate whole-body energy metabolism will aid in developing novel obesity intervention techniques.

Membrane excitability of nucleus accumbens neurons gates the incubation of cocaine craving.

After drug withdrawal, a key factor triggering relapse is progressively intensified cue-associated drug craving, termed incubation of drug craving. After withdrawal from cocaine self-administration, incubation of cocaine craving develops more reliably in rats compared to mice. This species difference provides an opportunity to determine rat-specific cellular adaptations, which may constitute the critical mechanisms that contribute to incubated cocaine craving in humans. Expression of incubated cocaine seeking is mediated, in part, by cocaine-induced cellular adaptations in medium spiny neurons (MSNs) within the nucleus accumbens (NAc). In rats, decreased membrane excitability in NAc MSNs is a prominent cellular adaptation, which is induced after cocaine self-administration and lasts throughout prolonged drug withdrawal. Here, we show that, similar to rats, mice exhibit decreased membrane excitability of dopamine D1 receptor (D1)-, but not D2 (D2)-, expressing MSNs within the NAc shell (NAcSh) after 1 d withdrawal from cocaine self-administration. However, in contrast to rats, this membrane adaptation does not persist in mice, diminishing after 45-d withdrawal. We also find that restoring the membrane excitability of NAcSh MSNs after cocaine withdrawal decreases cocaine seeking in rats. This suggests that drug-induced membrane adaptations are essential for behavioral expression of incubated cocaine craving. In mice, however, experimentally inducing hypoactivity of D1 NAcSh MSNs after cocaine withdrawal does not alter cocaine seeking, suggesting that MSN hypo-excitability alone is insufficient to increase cocaine seeking. Together, our results demonstrate an overall permissive role of cocaine-induced hypoactivity of NAcSh MSNs in gating increased cocaine seeking after prolonged cocaine withdrawal.