Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

SKF82958, a dopamine D1 receptor agonist, disrupts prepulse inhibition in the medial prefrontal cortex and nucleus accumbens in C57BL/6J mice.

Prepulse inhibition (PPI) is a crucial indicator of sensorimotor gating that is often impaired in neuropsychiatric diseases. Although dopamine D1 receptor agonists have been found to disrupt PPI in mice, the underlying mechanisms are not fully understood. In this study, we aimed to identify the brain regions responsible for the PPI-disruptive effect of the D1 agonist in mice. Results demonstrated that intraperitoneal administration of the selective dopamine D1 receptor agonist SKF82958 dramatically inhibited PPI, while the dopamine D1 receptor antagonist SCH23390 enhanced PPI. Additionally, local infusion of SKF82958 into the nucleus accumbens and medial prefrontal cortex disrupted PPI, but not in the ventral hippocampus. Infusion of SCH23390 into these brain regions also failed to enhance PPI. Overall, the study suggests that the nucleus accumbens and medial prefrontal cortex are responsible for the PPI-disruptive effect of dopamine D1 receptor agonists. These findings provide essential insights into the cellular and neural circuit mechanisms underlying the disruptive effects of dopamine D1 receptor agonists on PPI and may contribute to the development of novel treatments for neuropsychiatric diseases.

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.

Spinal dopaminergic D1 and D5 receptors contribute to reserpine-induced fibromyalgia-like pain in rats.

Fibromyalgia is a complex pain syndrome without a precise etiology. Reduced monoamines levels in serum and cerebrospinal fluid in fibromyalgia patients has been reported and could lead to a dysfunction of descending pain modulatory system producing the painful syndrome. This study evaluated the role of D1-like dopamine receptors in the reserpine-induced fibromyalgia-like pain model in female Wistar rats. Reserpine-treated animals were intrathecally injected with different dopamine receptors agonists and antagonists, and small interfering RNAs (siRNAs) against D1 and D5 receptor subtypes. Withdrawal and muscle pressure thresholds were assessed with von Frey filaments and the Randall-Selitto test, respectively. Expression of D1-like receptors in lumbar spinal cord and dorsal root ganglion was determined using real time polymerase chain reaction (qPCR). Reserpine induced tactile allodynia and muscle hyperalgesia. Intrathecal dopamine and D1-like receptor agonist SKF-38393 induced nociceptive hypersensitivity in naïve rats, whilst this effect was prevented by the D1-like receptor antagonist SCH-23390. Moreover, SCH-23390 induced a sex-dependent antiallodynic effect in reserpine-treated rats. Furthermore, transient silencing of D1 and D5 receptors significantly reduced reserpine-induced hypersensitivity in female rats. Reserpine slightly increased mRNA D5 receptor expression in dorsal spinal cord, but not in DRG. This work provides new insights about the involvement of the spinal dopaminergic D1/D5 receptors in reserpine-induced hypersensitivity in rats.

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

2-(Fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl (UCM-1306), an Orally Bioavailable Positive Allosteric Modulator of the Human Dopamine D1 Receptor for Parkinson's Disease.

Tolerance development caused by dopamine replacement with l-DOPA and therapeutic drawbacks upon activation of dopaminergic receptors with orthosteric agonists reveal a significant unmet need for safe and effective treatment of Parkinson's disease. In search for selective modulators of the D1 receptor, the screening of a chemical library and subsequent medicinal chemistry program around an identified hit resulted in new synthetic compound 26 [UCM-1306, 2-(fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl] that increases the dopamine maximal effect in a dose-dependent manner in human and mouse D1 receptors, is inactive in the absence of dopamine, modulates dopamine affinity for the receptor, exhibits subtype selectivity, and displays low binding competition with orthosteric ligands. The new allosteric modulator potentiates cocaine-induced locomotion and enhances l-DOPA recovery of decreased locomotor activity in reserpinized mice after oral administration. The behavior of compound 26 supports the interest of a positive allosteric modulator of the D1 receptor as a promising therapeutic approach for Parkinson's disease.

Anticancer efficacy of endo- and exogenous potent ligands acting at dopaminergic receptor-expressing cancer cells.

Cancer is one of the most common and dreaded diseases affecting the vastness of society. Unfortunately, still some people die especially when cancer is not diagnosed and thus caught early enough. On the other hand, using available chemo- or radiotherapy may result in serious side effects. Therefore, cancer-specific medications seem to be the most desired and safe therapy. Knowing that some cancers are characterized by overexpression of specific receptors on the cell surface, target-mediated drugs could serve as a unique and effective form of therapy. In line with this, recently dopaminergic receptors were presented important in cancer therapy as several dopaminergic ligands revealed their efficacy in tumor growth reduction as well as in apoptosis mediation. Unfortunately, the indication of whether DA receptor agonists or antagonists are the best choices in cancer treatment is quite difficult, since both of them may exert either pro- or anticancer effects. In this review, we analyze the therapeutic efficacy of compounds, both of exogenous and endogenous origin, targeting dopaminergic receptor-expressing cancers.

Dopamine D1-like receptor blockade and stimulation decreases operant responding for nicotine and food in male and female rats.

Dopamine has been implicated in the reinforcing effects of smoking. However, there remains a need for a better understanding of the effects of dopamine D1-like receptor agonists on nicotine intake and the role of sex differences in the effects of dopaminergic drugs on behavior. This work studied the effects of D1-like receptor stimulation and blockade on operant responding for nicotine and food and locomotor activity in male and female rats. The effects of the D1-like receptor antagonist SCH 23390 (0.003, 0.01, 0.03 mg/kg) and the D1-like receptor agonist A77636 (0.1, 0.3, 1 mg/kg) on responding for nicotine and food, and locomotor activity were investigated. The effects of SCH 23390 were investigated 15 min and 24 h after treatment, and the effects of the long-acting drug A77636 were investigated 15 min, 24 h, and 48 h after treatment. Operant responding for nicotine and food and locomotor activity were decreased immediately after treatment with SCH 23390. Treatment with SCH 23390 did not have any long-term effects. Operant responding for nicotine was still decreased 48 h after treatment with A77636, and food responding was decreased up to 24 h after treatment. Treatment with A77636 only decreased locomotor activity at the 48 h time point. There were no sex differences in the effects of SCH 23390 or A77636. In conclusion, the D1-like receptor antagonist SCH 23390 reduces nicotine intake and causes sedation in rats. Stimulation of D1-like receptors with A77636 decreases nicotine intake at time points that the drug does not cause sedation.

Structure-Functional Selectivity Relationship Studies on A-86929 Analogs and Small Aryl Fragments toward the Discovery of Biased Dopamine D1 Receptor Agonists.

Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through D1- (D1R and D5R) and D2-class (D2R, D3R, and D4R) receptors and activates both G protein- and ß-arrestin-dependent signaling pathways. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinson's disease or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity relationship (SAR) studies have embarked on discovering ß-arrestin-biased dopamine agonists, focused on D2 partial agonists, noncatechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R ß-arrestin-biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold to identify chemical motifs responsible for ß-arrestin-biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein-biased, but none of them were exclusively arrestin-biased. Additionally, various small-fragment molecular probes displayed weak bias toward the ß-arrestin pathway. Continued in-depth SFSR (structure-functional selectivity relationship) studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate the discovery of potent and efficacious arrestin-biased dopamine receptor ligands.

The selective dopamine D1 receptor agonist SKF81297 modulates NMDA receptor currents independently of D1 receptors.

Dopamine D1 receptor (D1R) agonists are frequently used to study the role of D1Rs in neurotransmission and behaviour. They have been repeatedly shown to modulate glutamatergic NMDAR currents in the prefrontal cortex (PFC), giving rise to the idea that D1R activation tunes glutamatergic networks by regulating NMDAR activity. We report that the widely used D1R agonist SKF81297 potentiates NMDAR currents in a dose-dependent manner, independently of D1R activation in mPFC slices, cortical neuron cultures and NMDAR-expressing recombinant HEK293 cells. SKF81297 potentiated NMDAR currents through both GluN2A and GluN2B subtypes in the absence of D1R expression, while inhibiting NMDAR currents through GluN2C and GluN2D subtypes. In contrast, the D1R ligands SKF38393, dopamine and SCH23390 inhibited GluN2A- and GluN2B-containing NMDAR currents. SKF81297 also inhibited GluN2A- and GluN2B-containing NMDAR currents at higher concentrations and when glutamate/glycine levels were high, exhibiting bidirectional modulation. To our knowledge, these findings are the first report of a D1R-independent positive modulatory effect of a D1R ligand on NMDA receptors. Importantly, our results further emphasize the possibility of off-target effects of many D1R ligands, which has significant implications for interpreting the large body of research relying on these compounds to examine dopamine functions.

Activation of dopamine D2 receptor promotes pepsinogen secretion by suppressing somatostatin release from the mouse gastric mucosa.

In vivo administration of dopamine (DA) receptor (DR)-related drugs modulate gastric pepsinogen secretion. However, DRs on gastric pepsinogen-secreting chief cells and DA D2 receptor (D2R) on somatostatin-secreting D cells were subsequently acquired. In this study, we aimed to further investigate the local effect of DA on gastric pepsinogen secretion through DRs expressed on chief cells or potential D2Rs expressed on D cells. To elucidate the modulation of DRs in gastric pepsinogen secretion, immunofluorescence staining, ex vivo incubation of gastric mucosa isolated from normal and D2R-/- mice were conducted, accompanied by measurements of pepsinogen or somatostatin levels using biochemical assays or enzyme-linked immunosorbent assays. D1R, D2R, and D5R-immunoreactivity (IR) were observed on chief cells in mouse gastric mucosa. D2R-IR was widely distributed on D cells from the corpus to the antrum. Ex vivo incubation results showed that DA and the D1-like receptor agonist SKF38393 increased pepsinogen secretion, which was blocked by the D1-like receptor antagonist SCH23390. However, D2-like receptor agonist quinpirole also significantly increased pepsinogen secretion, and D2-like receptor antagonist sulpiride blocked the promotion of DA. Besides, D2-like receptors exerted an inhibitory effect on somatostatin secretion, in contrast to their effect on pepsinogen secretion. Furthermore, D2R-/- mice showed much lower basal pepsinogen secretion but significantly increased somatostatin release and an increased number of D cells in gastric mucosa. Only SKF38393, not quinpirole, increased pepsinogen secretion in D2R-/- mice. DA promotes gastric pepsinogen secretion directly through D1-like receptors on chief cells and indirectly through D2R-mediated suppression of somatostatin release.

A computational model of dopaminergic modulation of hippocampal Schaffer collateral-CA1 long-term plasticity.

Dopamine plays a critical role in modulating the long-term synaptic plasticity of the hippocampal Schaffer collateral-CA1 pyramidal neuron synapses (SC-CA1), a widely accepted cellular model of learning and memory. Limited results from hippocampal slice experiments over the last four decades have shown that the timing of the activation of dopamine D1/D5 receptors relative to a high/low-frequency stimulation (HFS/LFS) in SC-CA1 synapses regulates the modulation of HFS/LFS-induced long-term potentiation/depression (LTP/LTD) in these synapses. However, the existing literature lacks a complete picture of how various concentrations of D1/D5 agonists and the relative timing between the activation of D1/D5 receptors and LTP/LTD induction by HFS/LFS, affect the spatiotemporal modulation of SC-CA1 synaptic dynamics. In this paper, we have developed a computational model, a first of its kind, to make quantitative predictions of the temporal dose-dependent modulation of the HFS/LFS induced LTP/LTD in SC-CA1 synapses by various D1/D5 agonists. Our model combines the biochemical effects with the electrical effects at the electrophysiological level. We have estimated the model parameters from the published electrophysiological data, available from diverse hippocampal CA1 slice experiments, in a Bayesian framework. Our modeling results demonstrate the capability of our model in making quantitative predictions of the available experimental results under diverse HFS/LFS protocols. The predictions from our model show a strong nonlinear dependency of the modulated LTP/LTD by D1/D5 agonists on the relative timing between the activated D1/D5 receptors and the HFS/LFS protocol and the applied concentration of D1/D5 agonists.

Dopamine D1R Receptor Stimulation as a Mechanistic Pro-cognitive Target for Schizophrenia.

Decades of research have highlighted the importance of optimal stimulation of cortical dopaminergic receptors, particularly the D1R receptor (D1R), for prefrontal-mediated cognition. This mechanism is particularly relevant to the cognitive deficits in schizophrenia, given the abnormalities in cortical dopamine (DA) neurotransmission and in the expression of D1R. Despite the critical need for D1R-based therapeutics, many factors have complicated their development and prevented this important therapeutic target from being adequately interrogated. Challenges include determination of the optimal level of D1R stimulation needed to improve cognitive performance, especially when D1R expression levels, affinity states, DA levels, and the resulting D1R occupancy by DA, are not clearly known in schizophrenia, and may display great interindividual and intraindividual variability related to cognitive states and other physiological variables. These directly affect the selection of the level of stimulation necessary to correct the underlying neurobiology. The optimal mechanism for stimulation is also unknown and could include partial or full agonism, biased agonism, or positive allosteric modulation. Furthermore, the development of D1R targeting drugs has been complicated by complexities in extrapolating from in vitro affinity determinations to in vivo use. Prior D1R-targeted drugs have been unsuccessful due to poor bioavailability, pharmacokinetics, and insufficient target engagement at tolerable doses. Newer drugs have recently become available, and these must be tested in the context of carefully designed paradigms that address methodological challenges. In this paper, we discuss how a better understanding of these challenges has shaped our proposed experimental design for testing a new D1R/D5R partial agonist, PF-06412562, renamed CVL-562.

The DR1­CSE/H2S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine­Î³­lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)­induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α­smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1­CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

D1/D5 Dopamine Receptors and mGluR5 Jointly Enable Non-Hebbian Long-Term Potentiation at Sensory Synapses onto Lamina I Spinoparabrachial Neurons.

Highly correlated firing of primary afferent inputs and lamina I projection neurons evokes synaptic long-term potentiation (LTP), a mechanism by which ascending nociceptive transmission can be amplified at the level of the spinal dorsal horn. However, the degree to which neuromodulatory signaling shapes the temporal window governing spike-timing-dependent plasticity (STDP) at sensory synapses onto projection neurons remains unclear. The present study demonstrates that activation of spinal D1/D5 dopamine receptors (D1/D5Rs) creates a highly permissive environment for the production of LTP in male and female adult mouse spinoparabrachial neurons by promoting non-Hebbian plasticity. Bath application of the mixed D1/D5R agonist SKF82958 unmasked LTP at STDP pairing intervals that normally fail to alter synaptic efficacy. Furthermore, during D1/D5R signaling, action potential discharge in projection neurons became dispensable for LTP generation, and primary afferent stimulation alone was sufficient to induce strengthening of sensory synapses. This non-Hebbian LTP was blocked by the D1/D5R antagonist SCH 39166 or genetic deletion of D5R, and required activation of mGluR5 and intracellular Ca2+ release but was independent of NMDAR activation. D1/D5R-enabled non-Hebbian plasticity was observed across multiple neuronal subpopulations in the superficial dorsal horn but was more prevalent in spinoparabrachial neurons than interneurons. Interestingly, the ability of neonatal tissue damage to promote non-Hebbian LTP in adult projection neurons was not observed in D5R knock-out mice. Collectively, these findings suggest that joint spinal D1/D5R and mGluR5 activation can allow unfettered potentiation of sensory synapses onto the output neurons responsible for conveying pain and itch information to the brain.SIGNIFICANCE STATEMENT Synaptic LTP in spinal projection neurons has been implicated in the generation of chronic pain. Under normal conditions, plasticity at sensory synapses onto adult mouse spinoparabrachial neurons follows strict Hebbian learning rules, requiring coincident presynaptic and postsynaptic firing. Here, we demonstrate that the activation of spinal D1/D5Rs promotes a switch from Hebbian to non-Hebbian LTP so that primary afferent stimulation alone is sufficient to evoke LTP in the absence of action potential discharge in projection neurons, which required joint activation of mGluR5 and intracellular Ca2+ release but not NMDARs. These results suggest that D1/D5Rs cooperate with mGluR5 receptors in the spinal dorsal horn to powerfully influence the amplification of ascending nociceptive transmission to the brain.

Decreased dopaminergic inhibition of pyramidal neurons in anterior cingulate cortex maintains chronic neuropathic pain.

Pyramidal neurons in the anterior cingulate cortex (ACC), a prefrontal region involved in processing the affective components of pain, display hyperexcitability in chronic neuropathic pain conditions, and their silencing abolishes hyperalgesia. We show that dopamine, through D1 receptor (D1R) signaling, inhibits pyramidal neurons of mouse ACC by modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Activation of Gs-coupled D1R by dopamine induces the opening of HCN channels at physiological membrane potentials, driving a significant decrease in input resistance and excitability. Systemic L-DOPA in chronic neuropathic mice rescues HCN channel activity, normalizes pyramidal excitability in ACC, and blocks mechanical and thermal allodynia. Moreover, microinjection of a selective D1R agonist in the ACC relieves the aversiveness of ongoing neuropathic pain, while an ACC D1R antagonist blocks gabapentin- and lidocaine-evoked antinociception. We conclude that dopaminergic inhibition via D1R in ACC plays an analgesic role in physiological conditions and is decreased in chronic pain.

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells.

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D1R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H2O2 and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.

Pharmacological perturbation reveals deficits in D2 receptor responses in Thap1 null mice.

The primary dystonia DYT6 is caused by mutations in the transcription factor Thanatos-associated protein 1 (THAP1). To understand THAP1's functions, we generated mice lacking THAP1 in the nervous system. THAP1 loss causes locomotor deficits associated with transcriptional changes. Since many of the genes misregulated involve dopaminergic signaling, we pharmacologically challenged the two striatal canonical dopamine pathways: the direct, regulated by the D1 receptor, and the indirect, regulated by the D2 receptor. We discovered that depleting THAP1 specifically interferes with the D2 receptor responses, pointing to a selective misregulation of the indirect pathway in DYT6 with implications for pathogenesis and treatment.

Salicylate Induced GABAAR Internalization by Dopamine D1-Like Receptors Involving Protein Kinase C (PKC) in Spiral Ganglion Neurons.

BACKGROUND Sodium salicylate (SS) induces excitotoxicity of spiral ganglion neurons (SGNs) by inhibiting the response of γ-aminobutyric acid type A receptors (GABAARs). Our previous studies have shown that SS can increase the internalization of GABAARs on SGNs, which involves dopamine D1-like receptors (D1Rs) and related signaling pathways. In this study, we aimed to explore the role of D1Rs and their downstream molecule protein kinase C (PKC) in the process of SS inhibiting GABAARs. MATERIAL AND METHODS The expression of D1Rs and GABARγ2 on rat cochlear SGNs cultured in vitro was tested by immunofluorescence. Then, the SGNs were exposed to SS, D1R agonist (SKF38393), D1R antagonist (SCH23390), clathrin/dynamin-mediated endocytosis inhibitor (dynasore), and PKC inhibitor (Bisindolylmaleimide I). Western blotting and whole-cell patch clamp technique were used to assess the changes of surface and total protein of GABARγ2 and GABA-activated currents. RESULTS Immunofluorescence showed that D1 receptors (DRD1) were expressed on SGNs. Data from western blotting showed that SS promoted the internalization of cell surface GABAARs, and activating D1Rs had the same result. Inhibiting D1Rs and PKC decreased the internalization of GABAARs. Meanwhile, the phosphorylation level of GABAARγ2 S327 affected by PKC was positively correlated with the degree of internalization of GABAARs. Moreover, whole-cell patch clamp recording showed that inhibition of D1Rs or co-inhibition of D1Rs and PKC attenuated the inhibitory effect of SS on GABA-activated currents. CONCLUSIONS D1Rs mediate the GABAAR internalization induced by SS via a PKC-dependent manner and participate in the excitotoxic process of SGNs.

Dopaminergic mechanisms underlying the expression of antipsychotic-induced dopamine supersensitivity in rats.

Antipsychotic treatment can produce a dopamine-supersensitive state, potentiating the response to dopamine receptor stimulation. In both schizophrenia patients and rats, this is linked to tolerance to ongoing antipsychotic treatment. In rodents, dopamine supersensitivity is often confirmed by an exaggerated psychomotor response to d-amphetamine after discontinuation of antipsychotic exposure. Here we examined in rats the dopaminergic mechanisms mediating this enhanced behavioural response, as this could uncover pathophysiological processes underlying the expression of antipsychotic-evoked dopamine supersensitivity. Rats received 0.5 mg/kg/day haloperidol via osmotic minipump for 2 weeks, before treatment was discontinued. After cessation of antipsychotic treatment, rats showed a supersensitive psychomotor response to the D2 agonist quinpirole, but not to the D1 partial agonist SKF38393 or the dopamine reuptake blocker GBR12783. Furthermore, acute D1 receptor blockade (using SCH39166) decreased the exaggerated psychomotor response to d-amphetamine in haloperidol-pretreated rats, whereas acute D2 receptor blockade (using sulpiride) enhanced it. Thus, after discontinuation of antipsychotic treatment, D1- and D2-mediated transmission differentially modulate the expression of a supersensitive response to d-amphetamine. This supersensitive behavioural response was accompanied by enhanced GSK3ß activity and suppressed ERK1/2 activity in the nucleus accumbens (but not caudate-putamen), suggesting increased mesolimbic D2 transmission. Finally, after discontinuing haloperidol treatment, neither increasing ventral midbrain dopamine impulse flow nor infusing d-amphetamine into the cerebral ventricles triggered the expression of already established dopamine supersensitivity, suggesting that peripheral effects are required. Thus, while dopamine receptor-mediated signalling regulates the expression of antipsychotic-evoked dopamine supersensitivity, a simple increase in central dopamine neurotransmission is insufficient to trigger this supersensitivity.