Structural basis of psychedelic LSD recognition at dopamine D1 receptor.

Understanding the kinetics of LSD in receptors and subsequent induced signaling is crucial for comprehending both the psychoactive and therapeutic effects of LSD. Despite extensive research on LSD's interactions with serotonin 2A and 2B receptors, its behavior on other targets, including dopamine receptors, has remained elusive. Here, we present cryo-EM structures of LSD/PF6142-bound dopamine D1 receptor (DRD1)-legobody complexes, accompanied by a ß-arrestin-mimicking nanobody, NBA3, shedding light on the determinants of G protein coupling versus ß-arrestin coupling. Structural analysis unveils a distinctive binding mode of LSD in DRD1, particularly with the ergoline moiety oriented toward TM4. Kinetic investigations uncover an exceptionally rapid dissociation rate of LSD in DRD1, attributed to the flexibility of extracellular loop 2 (ECL2). Moreover, G protein can stabilize ECL2 conformation, leading to a significant slowdown in ligand's dissociation rate. These findings establish a solid foundation for further exploration of G protein-coupled receptor (GPCR) dynamics and their relevance to signal transduction.

The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta.

BACKGROUND: We recently reported that the dopamine (DA) analogue CA140 modulates neuroinflammatory responses in lipopolysaccharide-injected wild-type (WT) mice and in 3-month-old 5xFAD mice, a model of Alzheimer's disease (AD). However, the effects of CA140 on Aß/tau pathology and synaptic/cognitive function and its molecular mechanisms of action are unknown. METHODS: To investigate the effects of CA140 on cognitive and synaptic function and AD pathology, 3-month-old WT mice or 8-month-old (aged) 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10, 14, or 17 days. Behavioral tests, ELISA, electrophysiology, RNA sequencing, real-time PCR, Golgi staining, immunofluorescence staining, and western blotting were conducted. RESULTS: In aged 5xFAD mice, a model of AD pathology, CA140 treatment significantly reduced Aß/tau fibrillation, Aß plaque number, tau hyperphosphorylation, and neuroinflammation by inhibiting NLRP3 activation. In addition, CA140 treatment downregulated the expression of cxcl10, a marker of AD-associated reactive astrocytes (RAs), and c1qa, a marker of the interaction of RAs with disease-associated microglia (DAMs) in 5xFAD mice. CA140 treatment also suppressed the mRNA levels of s100ß and cxcl10, markers of AD-associated RAs, in primary astrocytes from 5xFAD mice. In primary microglial cells from 5xFAD mice, CA140 treatment increased the mRNA levels of markers of homeostatic microglia (cx3cr1 and p2ry12) and decreased the mRNA levels of a marker of proliferative region-associated microglia (gpnmb) and a marker of lipid-droplet-accumulating microglia (cln3). Importantly, CA140 treatment rescued scopolamine (SCO)-mediated deficits in long-term memory, dendritic spine number, and LTP impairment. In aged 5xFAD mice, these effects of CA140 treatment on cognitive/synaptic function and AD pathology were regulated by dopamine D1 receptor (DRD1)/Elk1 signaling. In primary hippocampal neurons and WT mice, CA140 treatment promoted long-term memory and dendritic spine formation via effects on DRD1/CaMKIIα and/or ERK signaling. CONCLUSIONS: Our results indicate that CA140 improves neuronal/synaptic/cognitive function and ameliorates Aß/tau pathology and neuroinflammation by modulating DRD1 signaling in primary hippocampal neurons, primary astrocytes/microglia, WT mice, and aged 5xFAD mice.

The role of heterodimers formed by histamine H3 receptors and dopamine D1 receptors on the methamphetamine-induced conditioned place preference.

RATIONALE: The rewarding effect of Methamphetamine (METH) is commonly believed to play an important role in METH use disorder. The altered expression of dopamine D1 receptor (D1R) has been suggested to be essential to the rewarding effect of METH. Notably, D1R could interact with histamine H3 receptors (H3R) by forming a H3R-D1R heteromer (H3R-D1R). OBJECTIVES: This study was designed to specifically investigate the involvement of H3R-D1R in the rewarding effect of METH. METHODS: C57BL/6 mice were treated with intraperitoneal injections of a selective H3R antagonist (Thioperamide, THIO; 20 mg/kg), an H1R antagonist (Pyrilamine, PYRI; 10 mg/kg), or microinjections of cytomegalovirus (CMV)-transmembrane domain 5 (TM5) into the nucleus accumbens (NAc). The animal model of Conditioned Place Preference (CPP) was applied to determine the impact of H3R-D1R on the rewarding effect of METH. RESULTS: METH resulted in a significant preference for the drug-associated chamber, in conjunction with increased H3R and decreased D1R expression in both NAc and the ventral tegmental area (VTA). THIO significantly attenuated the rewarding effect of METH, accompanied by decreased H3R and increased D1R expression. In contrast, pyrilamine failed to produce the similar effects. Moreover, the inhibitory effect of THIO on METH-induced CPP was reversed by SKF38393, a D1R agonist. Furthermore, SCH23390, a D1R antagonist, counteracted the ameliorative effect of SKF38393 on THIO. Co-immunoprecipitation (CO-IP) experiments further demonstrated the specific interaction between H3R and D1R in METH CPP mice. The rewarding effect of METH was also significantly blocked by the interruption of CMV-transmembrane domain 5 (TM5), but not CMV-transmembrane domain 7 (TM7) in NAc. CONCLUSION: These results suggest that modulating the activity of H3R-D1R complex holds promise for regulating METH use disorder and serves as a potential drug target for its treatment.

Sex and Age Differences in Ontogeny of Alloparenting: A Relation to Forebrain DRD1, DRD2, and HTR2A mRNA Expression?

Alloparenting refers to the practice of caring for the young by individuals other than their biological parents. The relationship between the dynamic changes in psychological functions underlying alloparenting and the development of specific neuroreceptors remains unclear. Using a classic 10-day pup sensitization procedure, together with a pup preference and pup retrieval test on the EPM (elevated plus maze), we showed that both male and female adolescent rats (24 days old) had significantly shorter latency than adult rats (65 days old) to be alloparental, and their motivation levels for pups and objects were also significantly higher. In contrast, adult rats retrieved more pups than adolescent rats even though they appeared to be more anxious on the EPM. Analysis of mRNA expression using real-time-PCR revealed a higher dopamine D2 receptor (DRD2) receptor expression in adult hippocampus, amygdala, and ventral striatum, along with higher dopamine D1 receptor (DRD1) receptor expression in ventral striatum compared to adolescent rats. Adult rats also showed significantly higher levels of 5-hydroxytryptamine receptor 2A (HTR2A) receptor expression in the medial prefrontal cortex, amygdala, ventral striatum, and hypothalamus. These results suggest that the faster onset of alloparenting in adolescent rats compared to adult rats, along with the psychological functions involved, may be mediated by varying levels of dopamine DRD1, DRD2, and HTR2A in different forebrain regions.

Electrical stimulation of the ventral tegmental area restores consciousness from sevoflurane-, dexmedetomidine-, and fentanyl-induced unconsciousness in rats.

BACKGROUND: Dopaminergic neurons in the ventral tegmental area (VTA) are crucially involved in regulating arousal, making them a potential target for reversing general anesthesia. Electrical deep brain stimulation (DBS) of the VTA restores consciousness in animals anesthetized with drugs that primarily enhance GABAA receptors. However, it is unknown if VTA DBS restores consciousness in animals anesthetized with drugs that target other receptors. OBJECTIVE: To evaluate the efficacy of VTA DBS in restoring consciousness after exposure to four anesthetics with distinct receptor targets. METHODS: Sixteen adult Sprague-Dawley rats (8 female, 8 male) with bipolar electrodes implanted in the VTA were exposed to dexmedetomidine, fentanyl, ketamine, or sevoflurane to produce loss of righting, a proxy for unconsciousness. After receiving the dopamine D1 receptor antagonist, SCH-23390, or saline (vehicle), DBS was initiated at 30 µA and increased by 10 µA until reaching a maximum of 100 µA. The current that evoked behavioral arousal and restored righting was recorded for each anesthetic and compared across drug (saline/SCH-23390) condition. Electroencephalogram, heart rate and pulse oximetry were recorded continuously. RESULTS: VTA DBS restored righting after sevoflurane, dexmedetomidine, and fentanyl-induced unconsciousness, but not ketamine-induced unconsciousness. D1 receptor antagonism diminished the efficacy of VTA stimulation following sevoflurane and fentanyl, but not dexmedetomidine. CONCLUSIONS: Electrical DBS of the VTA restores consciousness in animals anesthetized with mechanistically distinct drugs, excluding ketamine. The involvement of the D1 receptor in mediating this effect is anesthetic-specific.

Striatal parvalbumin interneurons are activated in a mouse model of cerebellar dystonia.

Dystonia is thought to arise from abnormalities in the motor loop of the basal ganglia; however, there is an ongoing debate regarding cerebellar involvement. We adopted an established cerebellar dystonia mouse model by injecting ouabain to examine the contribution of the cerebellum. Initially, we examined whether the entopeduncular nucleus (EPN), substantia nigra pars reticulata (SNr), globus pallidus externus (GPe) and striatal neurons were activated in the model. Next, we examined whether administration of a dopamine D1 receptor agonist and dopamine D2 receptor antagonist or selective ablation of striatal parvalbumin (PV, encoded by Pvalb)-expressing interneurons could modulate the involuntary movements of the mice. The cerebellar dystonia mice had a higher number of cells positive for c-fos (encoded by Fos) in the EPN, SNr and GPe, as well as a higher positive ratio of c-fos in striatal PV interneurons, than those in control mice. Furthermore, systemic administration of combined D1 receptor agonist and D2 receptor antagonist and selective ablation of striatal PV interneurons relieved the involuntary movements of the mice. Abnormalities in the motor loop of the basal ganglia could be crucially involved in cerebellar dystonia, and modulating PV interneurons might provide a novel treatment strategy.

The novel psychoactive substance 25E-NBOMe induces reward-related behaviors via dopamine D1 receptor signaling in male rodents.

Novel psychoactive substances (NPSs) are new psychotropic drugs designed to evade substance regulatory policies. 25E-NBOMe (2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine) has recently been identified as an NPS, and its recreational misuse has been reported to be rapidly increasing. However, the psychopharmacological effects and mechanisms of 25E-NBOMe have not been studied. We examined the abuse potential of 25E-NBOMe using the conditioned place preference in male mice and self-administration paradigms in male rats. Additionally, immunoblot assay, enzyme-linked immunosorbent assay, and microdialysis were used to determine the molecular effects of 25E-NBOMe in the nucleus accumbens (NAc). Our data demonstrated that 25E-NBOMe induces conditioned place preference, and the dopaminergic signaling in the NAc mediates these. Following 25E-NBOMe administration, expression of dopamine transporter and dopamine D1 receptor (D1DR) were enhanced in the NAc of male mice, and NAc dopamine levels were reduced in both male mice and rats. Induction of intracellular dopaminergic pathways, DARPP32, and phosphorylation of CREB in the NAc of male mice was also observed. Significantly, pharmacological blockade of D1DR or chemogenetic inhibition of D1DR-expressing medium spiny neurons in the NAc attenuated 25E-NBOMe-induced conditioned place preference in male mice. We also examined the hallucinogenic properties of 25E-NBOMe using the head twitch response test in male mice and found that this behavior was mediated by serotonin 2A receptor activity. Our findings demonstrate that D1DR signaling may govern the addictive potential of 25E-NBOMe. Moreover, our study provides new insights into the potential mechanisms of substance use disorder and the improvement of controlled substance management.

Anterior cingulate cortex projections to the dorsal medial striatum underlie insomnia associated with chronic pain.

Chronic pain often leads to the development of sleep disturbances. However, the precise neural circuit mechanisms responsible for sleep disorders in chronic pain have remained largely unknown. Here, we present compelling evidence that hyperactivity of pyramidal neurons (PNs) in the anterior cingulate cortex (ACC) drives insomnia in a mouse model of nerve-injury-induced chronic pain. After nerve injury, ACC PNs displayed spontaneous hyperactivity selectively in periods of insomnia. We then show that ACC PNs were both necessary for developing chronic-pain-induced insomnia and sufficient to mimic sleep loss in naive mice. Importantly, combining optogenetics and electrophysiological recordings, we found that the ACC projection to the dorsal medial striatum (DMS) underlies chronic-pain-induced insomnia through enhanced activity and plasticity of ACC-DMS dopamine D1R neuron synapses. Our findings shed light on the pivotal role of ACC PNs in developing chronic-pain-induced sleep disorders.

Optogenetic activation of the lateral habenulaD1R-ventral tegmental area circuit induces depression-like behavior in mice.

A number of different receptors are distributed in glutamatergic neurons of the lateral habenula (LHb). These glutamatergic neurons are involved in different neural pathways, which may identify how the LHb regulates various physiological functions. However, the role of dopamine D1 receptor (D1R)-expressing habenular neurons projecting to the ventral tegmental area (VTA) (LHbD1R-VTA) remains not well understood. In the current study, to determine the activity of D1R-expressing neurons in LHb, D1R-Cre mice were used to establish the chronic restraint stress (CRS) depression model. Adeno-associated virus was injected into bilateral LHb in D1R-Cre mice to examine whether optogenetic activation of the LHb D1R-expressing neurons and their projections could induce depression-like behavior. Optical fibers were implanted in the LHb and VTA, respectively. To investigate whether optogenetic inhibition of the LHbD1R-VTA circuit could produce antidepressant-like effects, the adeno-associated virus was injected into the bilateral LHb in the D1R-Cre CRS model, and optical fibers were implanted in the bilateral VTA. The D1R-expressing neuronal activity in the LHb was increased in the CRS depression model. Optogenetic activation of the D1R-expressing neurons in LHb induced behavioral despair and anhedonia, which could also be induced by activation of the LHbD1R-VTA axons. Conversely, optogenetic inhibition of the LHbD1R-VTA circuit improved behavioral despair and anhedonia in the CRS depression model. D1R-expressing glutamatergic neurons in the LHb and their projections to the VTA are involved in the occurrence and regulation of depressive-like behavior.

The dopamine D1 receptor positive allosteric modulator, DETQ, improves cognition and social interaction in aged mice and enhances cortical and hippocampal acetylcholine efflux.

Dopamine (DA) D1 and D2 receptors (Rs) are critical for cognitive functioning. D1 positive allosteric modulators (D1PAMs) activate D1Rs without desensitization or an inverted U-shaped dose response curve. DETQ, [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one] is highly selective for the human D1Rs as shown in humanized D1R knock-in (hD1Ki) mice. Here, we have ascertained the efficacy of DETQ in aged [13-23-month-old (mo)] hD1Ki mice and their corresponding age-matched wild-type (WT; C57BL/6NTac) controls. We found that in aged mice, DETQ, given acutely, subchronically, and chronically, rescued both novel object recognition memory and social behaviors, using novel object recognition (NOR) and social interaction (SI) tasks, respectively without any adverse effect on body weight or mortality. We have also shown, using in vivo microdialysis, a significant decrease in basal DA and norepinephrine, increase in glutamate (Glu) and gamma-amino butyric acid (GABA) efflux with no significant changes in acetylcholine (ACh) levels in aged vs young mice. In young and aged hD1Ki mice, DETQ, acutely and subchronically increased ACh in the medial prefrontal cortex and hippocampal regions in aged hD1Ki mice without affecting Glu. These results suggest that the D1PAM mechanism is of interest as potential treatment for cognitive and social behavioral deficits in neuropsychiatric disorders including but not restricted to neurodegenerative disorders, such as Parkinson's disease.

Optogenetic activation of dopamine D1 receptors in island cells of medial entorhinal cortex inhibits temporal association learning.

A critical feature of episodic memory formation is to associate temporally segregated events as an episode, called temporal association learning. The medial entorhinal cortical-hippocampal (EC-HPC) networks is essential for temporal association learning. We have previously demonstrated that pyramidal cells in the medial EC (MEC) layer III project to the hippocampal CA1 pyramidal cells and are necessary for trace fear conditioning (TFC), which is an associative learning between tone and aversive shock with the temporal gap. On the other hand, Island cells in MECII, project to GABAergic neurons in hippocampal CA1, suppress the MECIII input into the CA1 pyramidal cells through the feed-forward inhibition, and inhibit TFC. However, it remains unknown about how Island cells activity is regulated during TFC. In this study, we report that dopamine D1 receptor is preferentially expressed in Island cells in the MEC. Optogenetic activation of dopamine D1 receptors in Island cells facilitate the Island cell activity and inhibited hippocampal CA1 pyramidal cell activity during TFC. The optogenetic activation caused the impairment of TFC memory recall without affecting contextual fear memory recall. These results suggest that dopamine D1 receptor in Island cells have a crucial role for the regulation of temporal association learning.

Anterior and Posterior Basolateral Amygdala Projections of Cell Type-Specific D1-Expressing Neurons From the Medial Prefrontal Cortex Differentially Control Alcohol-Seeking Behavior.

BACKGROUND: Alcohol use disorder is characterized by compulsive alcohol-seeking behavior, which is associated with dysregulation of afferent projections from the medial prefrontal cortex to the basolateral amygdala (BLA). However, the contribution of the cell type-specific mechanism in this neuronal circuit to alcohol-seeking behavior remains unclear. METHODS: Mice were trained with 2-bottle choice and operant alcohol self-administration procedures. Anterograde and retrograde viral methods traced the connection between dopamine type 1 receptor (D1R) neurons and BLA neurons. Electrophysiology and in vivo optogenetic techniques were used to test the function of neural circuits in alcohol-seeking behavior. RESULTS: Chronic alcohol consumption preferentially changed the activity of posterior BLA (pBLA) neurons but not anterior BLA (aBLA) neurons and overexcited D1R neurons in the medial prefrontal cortex. Interestingly, we found that 2 populations of D1R neurons, anterior and posterior (pD1R) neurons, separately targeted the aBLA and pBLA, respectively, and only a few D1R neurons innervated both aBLA and pBLA neurons. Furthermore, pD1R neurons exhibited more excitability than anterior D1R neurons in alcohol-drinking mice. Moreover, we observed enhanced glutamatergic transmission and an increased NMDA/AMPA receptor ratio in the medial prefrontal cortex inputs from pD1R neurons to the pBLA. Optogenetic long-term depression induction of the pD1R-pBLA circuit reduced alcohol-seeking behavior, while optogenetic long-term depression or long-term potentiation induction of the anterior D1R-aBLA circuit produced no change in alcohol intake. CONCLUSIONS: The pD1R-pBLA circuit mediates chronic alcohol consumption, which may suggest a cell type-specific neuronal mechanism underlying reward-seeking behavior in alcohol use disorder.

The Role of Retinal Dopamine D1 Receptors in Ocular Growth and Myopia Development in Mice.

Dopamine is a key neurotransmitter in the signaling cascade controlling ocular refractive development, but the exact role and site of action of dopamine D1 receptors (D1Rs) involved in myopia remains unclear. Here, we determine whether retinal D1Rs exclusively mediate the effects of endogenous dopamine and systemically delivered D1R agonist or antagonist in the mouse form deprivation myopia (FDM) model. Male C57BL/6 mice subjected to unilateral FDM or unobstructed vision were divided into the following four groups: one noninjected and three groups that received intraperitoneal injections of a vehicle, D1R agonist SKF38393 (18 and 59 nmol/g), or D1R antagonist SCH39166 (0.1 and 1 nmol/g). The effects of these drugs on FDM were further assessed in Drd1-knock-out (Drd1-KO), retina-specific conditional Drd1-KO (Drd1-CKO) mice, and corresponding wild-type littermates. In the visually unobstructed group, neither SKF38393 nor SCH39166 affected normal refractive development, whereas myopia development was attenuated by SKF38393 and enhanced by SCH39166 injections. In Drd1-KO or Drd1-CKO mice, however, these drugs had no effect on FDM development, suggesting that activation of retinal D1Rs is pertinent to myopia suppression by the D1R agonist. Interestingly, the development of myopia was unchanged by either Drd1-KO or Drd1-CKO, and neither SKF38393 nor SCH39166 injections, nor Drd1-KO, affected the retinal or vitreal dopamine and the dopamine metabolite DOPAC levels. Effects on axial length were less marked than effects on refraction. Therefore, activation of D1Rs, specifically retinal D1Rs, inhibits myopia development in mice. These results also suggest that multiple dopamine D1R mechanisms play roles in emmetropization and myopia development.SIGNIFICANCE STATEMENT While dopamine is recognized as a "stop" signal that inhibits myopia development (myopization), the location of the dopamine D1 receptors (D1Rs) that mediate this action remains to be addressed. Answers to this key question are critical for understanding how dopaminergic systems regulate ocular growth and refraction. We report here the results of our study showing that D1Rs are essential for controlling ocular growth and myopia development in mice, and for identifying the retina as the site of action for dopaminergic control via D1Rs. These findings highlight the importance of intrinsic retinal dopaminergic mechanisms for the regulation of ocular growth and suggest specific avenues for exploring the retinal mechanisms involved in the dopaminergic control of emmetropization and myopization.

Axo-glial interactions between midbrain dopamine neurons and oligodendrocyte lineage cells in the anterior corpus callosum.

Oligodendrocyte progenitor cells (OPCs) receive synaptic innervation from glutamatergic and GABAergic axons and can be dynamically regulated by neural activity, resulting in activity-dependent changes in patterns of axon myelination. However, it remains unclear to what extent other types of neurons may innervate OPCs. Here, we provide evidence implicating midbrain dopamine neurons in the innervation of oligodendrocyte lineage cells in the anterior corpus callosum and nearby white matter tracts of male and female adult mice. Dopaminergic axon terminals were identified in the corpus callosum of DAT-Cre mice after injection of an eYFP reporter virus into the midbrain. Furthermore, fast-scan cyclic voltammetry revealed monoaminergic transients in the anterior corpus callosum, consistent with the anatomical findings. Using RNAscope, we further demonstrate that ~ 40% of Olig2 + /Pdfgra + cells and ~ 20% of Olig2 + /Pdgfra- cells in the anterior corpus callosum express Drd1 and Drd2 transcripts. These results suggest that oligodendrocyte lineage cells may respond to dopamine released from midbrain dopamine axons, which could affect myelination. Together, this work broadens our understanding of neuron-glia interactions with important implications for myelin plasticity by identifying midbrain dopamine axons as a potential regulator of corpus callosal oligodendrocyte lineage cells.

The Molecular Mechanism of Positive Allosteric Modulation at the Dopamine D1 Receptor.

The dopamine D1 receptor (D1R) is a promising target for treating various psychiatric disorders. While upregulation of D1R activity has shown potential in alleviating motor and cognitive symptoms, orthosteric agonists have limitations, restricting their clinical applications. However, the discovery of several allosteric compounds specifically targeting the D1R, such as LY3154207, has opened new therapeutic avenues. Based on the cryo-EM structures of the D1R, we conducted molecular dynamics simulations to investigate the binding and allosteric mechanisms of LY3154207. Our simulations revealed that LY3154207 preferred the horizontal orientation above intracellular loop 2 (IL2) and stabilized the helical conformation of IL2. Moreover, LY3154207 binding induced subtle yet significant changes in key structural motifs and their neighboring residues. Notably, a cluster of residues centered around the Na+-binding site became more compact, while interactions involving the PIF motif and its neighboring residues were loosened upon LY3154207 binding, consistent with their role in opening the intracellular crevice for receptor activation. Additionally, we identified an allosteric pathway likely responsible for the positive allosteric effect of LY3154207 in enhancing Gs protein coupling. This mechanistic understanding of LY3154207's allosteric action at the D1R paves the way for the rational design of more potent and effective allosteric modulators.

Exercise ameliorates skeletal muscle insulin resistance by modulating GRK4-mediated D1R expression.

Exercise has been recommended as a nonpharmaceutical therapy to treat insulin resistance (IR). Previous studies showed that dopamine D1-like receptor agonists, such as fenoldopam, could improve peripheral insulin sensitivity, while antipsychotics, which are dopamine receptor antagonists, increased susceptibility to Type 2 diabetes mellitus (T2DM). Meanwhile, exercise has been proved to stimulate dopamine receptors. However, whether the dopamine D1 receptor (D1R) is involved in exercise-mediated amelioration of IR remains unclear. We found that the D1-like receptor antagonist, SCH23390, reduced the effect of exercise on lowering blood glucose and insulin in insulin-resistant mice and inhibited the contraction-induced glucose uptake in C2C12 myotubes. Similarly, the opposite was true for the D1-like receptor agonist, fenoldopam. Furthermore, the expression of D1R was decreased in skeletal muscles from streptozotocin (STZ)- and high-fat intake-induced T2DM mice, accompanied by increased D1R phosphorylation, which was reversed by exercise. A screening study showed that G protein-coupled receptor kinase 4 (GRK4) may be the candidate kinase for the regulation of D1R function, because, in addition to the increased GRK4 expression in skeletal muscles of T2DM mice, GRK4 transgenic T2DM mice exhibited lower insulin sensitivity, accompanied by higher D1R phosphorylation than control mice, whereas the AAV9-shGRK4 mice were much more sensitive to insulin than AAV9-null mice. Mechanistically, the up-regulation of GRK4 expression caused by increased reactive oxygen species (ROS) in IR was ascribed to the enhanced expression of c-Myc, a transcriptional factor of GRK4. Taken together, the present study shows that exercise, via regulation of ROS/c-Myc/GRK4 pathway, ameliorates D1R dysfunction and improves insulin sensitivity.

Effects of endogenous dopamine induced by low concentration atropine eye drops on choroidal neovascularization in high myopia mice.

AIM: To evaluate effects of endogenous dopamine induced by low concentration atropine eye drops on choroidal neovascularization (CNV) in high myopia mice. METHODS: The C57BL/6J mice were deprived of the right eye for 4wk, and the high myopia was diagnosed by optometry, the diopter was less than -6.00 D, and CNV was induced by 532 nm laser. The changes of dopamine D1 receptor (DRD1), dopamine D2 receptor (DRD2), and vascular endothelial growth factor A (VEGFA) were detected by Western blot technology at 0.5, 1, 2h, and 7d after 0.01%, 0.05%, and 0.1% atropine eye drops, respectively, the area of CNV was measured. RESULTS: Significant increases were observed on the expression of DRD2 in mouse high myopia model at 0.5, 1, 2h, 7d with 0.05% and 0.1% atropine eye drops (P<0.05). Significant decreases were observed on the expression of DRD1 and VEGFA in mouse high myopia model at 0.5, 1, 2h, 7d with 0.05% and 0.1% atropine eye drops (P<0.05). The area of CNV induced by laser in the drug-treated group was significantly smaller than that in the control group, and the higher the concentration, the more significant the inhibitory effect (P<0.05). CONCLUSION: The 0.01%, 0.05%, 0.1% atropine eye drops can decrease the level of VEGFA and inhibit high myopia CNV indirectly by up-regulating the level of DRD2 and down-regulating the level of DRD1, and the effect of 0.05% and 0.1% atropine eye drops is more significant.

Starburst amacrine cells form gap junctions in the early postnatal stage of the mouse retina.

Although gap junctional coupling in the developing retina is important for the maturation of neuronal networks, its role in the development of individual neurons remains unclear. Therefore, we herein investigated whether gap junctional coupling by starburst amacrine cells (SACs), a key neuron for the formation of direction selectivity, occurs during the developmental stage in the mouse retina. Neurobiotin-injected SACs coupled with many neighboring cells before eye-opening. The majority of tracer-coupled cells were retinal ganglion cells, and tracer coupling was not detected between SACs. The number of tracer-coupled cells significantly decreased after eye-opening and mostly disappeared by postnatal day 28 (P28). Membrane capacitance (Cm), an indicator of the formation of electrical coupling with gap junctions, was larger in SACs before than after eye-opening. The application of meclofenamic acid, a gap junction blocker, reduced the Cm of SACs. Gap junctional coupling by SACs was regulated by dopamine D1 receptors before eye-opening. In contrast, the reduction in gap junctional coupling after eye-opening was not affected by visual experience. At the mRNA level, 4 subtypes of connexins (23, 36, 43, and 45) were detected in SACs before eye-opening. Connexin 43 expression levels significantly decreased after eye-opening. These results indicate that gap junctional coupling by SACs occurs during the developmental period and suggest that the elimination of gap junctions proceeds with the innate system.

Dopamine D1 receptor in orbitofrontal cortex to dorsal striatum pathway modulates methamphetamine addiction.

The orbitofrontal cortex (OFC)-dorsal striatum (DS) is an important neural circuit that contributes to addictive behavior, including compulsive reinforcement, yet the specific types of neurons that play a major role still need to be further elucidated. Here, we used a place conditioning paradigm to measure the conditioned responses to methamphetamine (MA). The results demonstrated that MA increases the expression of c-Fos, synaptic plasticity in OFC and DS. Patch-clamp recording showed that MA activated projection neurons from the OFC to the DS, and chemogenetic manipulation of neuronal activity in OFC-DS projection neurons affects conditioned place preference (CPP) scores. And the combined patch-electrochemical technique was used to detect the DA release in OFC, the data indicated that the DA release was increased in MA group. Additionally, SCH23390, a D1R antagonist, was used to verify the function of D1R projection neurons, showing that SCH23390 reversed MA addiction-like behavior. Collectively, these findings provide evidence for the D1R neuron is sufficient to regulate MA addiction in the OFC-DS pathway, and the study provides new insight into the underlying mechanism of pathological changes in MA addiction.

Computational insights into ligand-induced G protein and ß-arrestin signaling of the dopamine D1 receptor.

The dopamine D1 receptor (D1R), is a class A G protein coupled-receptor (GPCR) which has been a promising drug target for psychiatric and neurological disorders such as Parkinson's disease (PD). Previous studies have suggested that therapeutic effects can be realized by targeting the ß-arrestin signaling pathway of dopamine receptors, while overactivation of the G protein-dependent pathways leads to side effects, such as dyskinesias. Therefore, it is highly desirable to develop a D1R ligand that selectively regulates the ß-arrestin pathway. Currently, most D1R agonists are signaling-balanced and stimulate both G protein and ß-arrestin pathways, with a few reports of G protein biased ligands. However, identification and characterization of ß-arrestin biased D1R agonists has been a challenge thus far. In this study, we implemented Gaussian accelerated molecular dynamics (GaMD) simulations to provide valuable computational insights into the possible underlying molecular mechanism of the different signaling properties of two catechol and two non-catechol D1R agonists that are either G protein biased or signaling-balanced. Dynamic network analysis further identified critical residues in the allosteric signaling network of D1R for each ligand at different conformational or binding states. Some of these residues are crucial for G protein or arrestin signals of GPCRs based on previous studies. Finally, we provided a molecular design strategy which can be utilized by medicinal chemists to develop potential ß-arrestin biased D1R ligands. The proposed hypotheses are experimentally testable and can guide the development of safer and more effective medications for a variety of CNS disorders.