Dopamine D1 Receptor Signaling Research Articles

D1R/PP2A/p-CaMKIIα signaling in the caudate putamen is involved in acute methamphetamine-induced hyperlocomotion

Drug addiction is underscored by the transition from experimental use to dependent use of addictive drugs. Acute use of methamphetamine (METH) causes a range of clinical symptoms, including hyperlocomotion. Dopamine D1 receptor (D1R)-mediated negative regulation of phosphorylated calcium/calmodulin-dependent protein kinase IIα (p-CaMKIIα, threonine [Thr] 286) is involved in the acute effects induced by single METH administration. Protein phosphatase 2A (PP2A) is a potential bridge that links D1R and p-CaMKIIα (Thr 286) after acute METH administration. However, the mechanisms underlying hyperlocomotion induced by single METH administration remain unclear. In this study, SCH23390 (a D1R inhibitor) and LB100 (a PP2A inhibitor) were administered to examine the involvement of D1R and PP2A signaling in acute METH-induced hyperlocomotion in mice. The protein levels of methylated PP2A-C (m-PP2A-C, leucine [Leu] 309), phosphorylated PP2A-C (p-PP2A-C, tyrosine [Tyr] 307), PP2A-C, p-CaMKIIα (Thr 286), and CaMKIIα in the prefrontal cortex (PFc), nucleus accumbens (NAc), and caudate putamen (CPu) were measured. Administration of 0.5 mg/kg SCH23390 reversed the acute METH-induced increase in protein levels of m-PP2A-C (Leu 309) and the decrease in protein levels of p-PP2A-C (Tyr 307) in the CPu, but not in the PFC and NAc. Moreover, prior administration of 0.1 mg/kg LB100 attenuated hyperlocomotion induced by single METH administration and reversed the decrease in protein levels of p-CaMKII (Thr 286) in the PFC, NAc, and CPu. Collectively, these results indicate that the D1R/PP2A/p-CaMKIIα signaling cascade in the CPu may be involved in hyperlocomotion after a single administration of METH.

Subregion-Specific Regulation of Dopamine D1 Receptor Signaling in the Striatum: Implication for L-DOPA-Induced Dyskinesia.

The striatum is the main structure of the basal ganglia. The striatum receives inputs from various cortical areas, and its subregions play distinct roles in motor and emotional functions. Recently, striatal maps based on corticostriatal connectivity and striosome-matrix compartmentalization were developed, and we were able to subdivide the striatum into seven subregions. Dopaminergic modulation of the excitability of medium spiny neurons (MSNs) is critical for striatal function. In this study, we investigated the functional properties of dopamine signaling in seven subregions of the striatum from male mice. By monitoring the phosphorylation of PKA substrates including DARPP-32 in mouse striatal slices, we identified two subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Low D1 receptor signaling in the two subregions was maintained by phosphodiesterase (PDE)10A and muscarinic M4 receptors. In an animal model of 6-hydroxydopamine (6-OHDA)-induced hemi-parkinsonism, D1 receptor signaling was upregulated in almost all subregions including the DL-IR, but not in the IC. When L-DOPA-induced dyskinesia (LID) was developed, D1 receptor signaling in the IC was upregulated and correlated with the severity of LID. Our results suggest that the function of the striatum is maintained through the subregion-specific regulation of dopamine D1 receptor signaling and that the aberrant activation of D1 receptor signaling in the IC is involved in LID. Future studies focusing on D1 receptor signaling in the IC of the striatum will facilitate the development of novel therapeutics for LID.SIGNIFICANCE STATEMENT Recent progress in striatal mapping based on corticostriatal connectivity and striosome-matrix compartmentalization allowed us to subdivide the striatum into seven subregions. Analyses of D1 receptor signaling in the seven subregions identified two unique subregions with low D1 receptor signaling: the dorsolateral portion of the intermediate/rostral part (DL-IR) and the intermediate/caudal part (IC). Aberrant activation of D1 receptor signaling in the IC is involved in L-DOPA-induced dyskinesia (LID). Previous studies of LID have mainly focused on the DL-IR, but not on the IC of the striatum. Future studies to clarify aberrant D1 receptor signaling in the IC are required to develop novel therapeutics for LID.

Developmental effects of early-life stress on dopamine D2 receptor and proteins involved in noncanonical D2 dopamine receptor signaling pathway in the prefrontal cortex of male rats.

Dopamine neurotransmission is implicated in multiple neuropsychiatric disorders, most strikingly in Parkinson's disease, bipolar disorder, attention-deficit hyperactivity disorder and schizophrenia. In addition to canonical pathway, D2-receptor (D2R) exerts some of its biological actions through regulating the activity of Akt and GSK3, which in turn were found to be altered in several psychiatric illnesses. The present study examined the impacts of maternal separation, an early-life stress model which has been associated with disturbed neurodevelopment and appearance of many psychiatric disorders, on developmental changes in dopamine concentration and the expression of D2Rs, Akt and GSK-3β in the medial prefrontal cortex (PFC; a key target of stress) in adolescent and young adult male rats. Maternal separation was performed 3 h per day from postnatal days 2 to 11. The PFC protein and dopamine contents were determined using western blotting analysis and Eliza, respectively. Results indicated long-term increases in the prefrontal dopamine levels in stressed adolescent and young adult male rats, accompanied by significant downregulation of D2R as well as upregulation of p-Akt and GSK-3β contents in stressed adolescence compared to controls, with all protein levels that returned to control values in stressed adult rats. Our findings suggest that early-life stress differentially modulates prefrontal D2R/Akt/GSK-3β levels during development. Since adolescence period is susceptible to the onset of specific mental illnesses, disruption of noncanonical components of D2R signaling during this critical period may have an important role in programming neurobehavioral phenotypes in adulthood and manipulations influencing Akt/GSK-3β pathway may improve the expression of specific dopamine-related behaviors and the effects of dopaminergic drugs.

ERK1/2 kinases and dopamine D2 receptors participate in the anticonvulsant effects of a new derivative of benzoylpyridine oxime and valproic acid

Inhibition of the activity of extracellular signal-regulated kinases (ERK1/2) induced by the activation of the dopamine D2 receptor signalling cascade may be a promising pharmacological target. The aim of this work was to study the involvement of ERK1/2 and dopamine D2 receptor in the mechanism of the anticonvulsant action of valproic acid (VA) and a new benzoylpyridine oxime derivative (GIZH-298), which showed antiepileptic activity in different models of epilepsy. We showed that subchronic exposure to maximal electroshock seizures (MES) for 5 days reduced the density of dopamine D2 receptors in the striatum of mice. GIZH-298 counteracted the decrease in the number of dopamine D2 receptors associated with MES and increased the number of ligand binding sites of dopamine D2 receptors in mice without MES. The affinity of dopamine D2 receptors to the ligand was not changed by GIZH-298. MES caused an increase in ERK1/2 and synapsin I phosphorylation in the striatum while GIZH-298, similar to VA, reduced the levels of both phospho-ERK1/2 and phosphosynapsin I after MES, which correlated with the decrease in the intensity of seizure in mice. In addition, GIZH-298 suppressed ERK1/2 phosphorylation in SH-SY5Y human neuroblastoma cells at therapeutic concentrations, while VA inhibited ERK1/2 phosphorylation in vivo but not in vitro. The data obtained expand the understanding of the mechanisms of action of VA and GIZH-298, which involve regulating the activity of ERK1/2 kinases, probably by modulating dopamine D2 receptors in limbic structures, as well as (in the case of GIZH-298) directly inhibiting of the ERK1/2 cascade.

CRISPR/Cas9‐mediated knockout of G proteins and β‐arrestins defines their specific but interdependent roles in dopamine D1 receptor signaling

The dopamine D1 receptor (D1R) is a G protein-coupled receptor which regulates key brain functions including voluntary movement, memory, attention, and reward. Selective agonist activation of the D1R may also provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. The D1R canonically activates the heterotrimeric G proteins Gαs, and possibly Gαolf, which activate adenylyl cyclase (AC) to increase cAMP/PKA signaling. The D1R also engages β-arrestin proteins which may transduce β-arrestin dependent signaling by scaffolding and activating kinases. However, the distinct roles of Gαs, Gαolf versus β-arrestins 1 and 2 for D1R signaling remains largely undefined. Here we utilize CRISPR/Cas9 engineered HEK293 cells in which Gαs, Gαolf, β-arrestin1 and β-arrestin2 are stably knocked out (KO) to delineate D1R pharmacology and signaling mechanisms. Western blots confirmed a complete loss of Gαs, Gαolf, β-arrestin1 or β-arrestin2 proteins in KO cells versus parental wildtype cells. Dose responses with the D1R agonist SKF-81297 or β2AR agonist isoproterenol induced robust cAMP production in wildtype cells that was eliminated in Gαs/olf KO cells. Re-expression of Gαs or Gαolf in KO cells rescued D1R-mediated cAMP signaling similar to wildtype cells. Interestingly, the D1R had significantly higher cAMP signaling when coupling via Gαs as compared to Gαolf, while the β2AR only coupled with Gαs to stimulate cAMP signaling. Western blotting indicated Gαs/olf KO completely blocked agonist induced CREB phosphorylation by both D1R and β2AR agonists, indicating a strict dependence on Gαs/olf and cAMP/PKA signaling to activate CREB. In major contrast, β-arrestin1/2 KO significantly increased the intensity and duration of cAMP signaling and CREB phosphorylation by the β2AR. β-arrestin1/2 KO also entirely blocked agonist-induced D1R internalization and impaired D1R desensitization while Gαs/olf KO had no effect on D1R internalization. Key controls of re-expressing Gαs, Gαolf, β-arrestin1 or β-arrestin2 in KO cells completely restored signaling or internalization similar to wildtype cells, indicating CRISPR/Cas9 targeting did not result in non-specific or compensatory effects in the pathways under study. Notably, β-arrestin1/2 KO also potentiated and prolonged D1R or β2AR-induced ERK1/2 phosphorylation. D1R-induced ERK1/2 phosphorylation in β-arrestin1/2 KO cells was eliminated by the SRC inhibitor SU6656 or the MEK inhibitor U0126, suggesting that D1R signaling via a Gαs/SRC/MEK pathway is essential for ERK1/2 activation. Taken together, our studies indicate that Gαs is the dominant transducer for D1R cAMP, CREB and ERK1/2 signaling and that β-arrestins provide distinctly interdependent roles with G proteins in modulating the duration and intensity of D1R and β2AR signaling cascades. Using CRISPR/Cas9 KO model cells, future studies will examine the distinct contributions of β-arrestins 1 and 2 and Gαs/olf for D1R pharmacology, which will be useful in the development of novel D1R biased agonists.

New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D2 receptor regulation and signaling

The dopamine D2 receptor (D2R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.

Proof-of-Mechanism Study of the Phosphodiesterase 10 Inhibitor RG7203 in Patients With Schizophrenia and Negative Symptoms

BackgroundReduced activation of dopamine D1 receptor signaling may be implicated in reward functioning as a potential driver of negative symptoms in schizophrenia. Phosphodiesterase 10A (PDE10A), an enzyme that is highly expressed in the striatum, modulates both dopamine D2- and D1-dependent signaling. MethodsWe assessed whether augmentation of D1 signaling by the PDE10 inhibitor RG7203 enhances imaging and behavioral markers of reward functions in patients with schizophrenia and negative symptoms. In a 3-period, double-blind, crossover study, we investigated the effects of RG7203 (5 mg and 15 mg doses) and placebo as adjunctive treatment to stable background antipsychotic treatment in patients with chronic schizophrenia with moderate levels of negative symptoms. Effects on reward functioning and reward-based effortful behavior were evaluated using the monetary incentive delay task during functional magnetic resonance imaging and the effort-cost-benefit and working memory reinforcement learning tasks. ResultsPatients (N = 33; 30 male, mean age ± SD 36.6 ± 7.0 years; Positive and Negative Syndrome Scale negative symptom factor score 23.0 ± 3.5 at screening) were assessed at three study centers in the United States; 24 patients completed the study. RG7203 at 5 mg significantly increased reward expectation–related activity in the monetary incentive delay task, but in the context of significantly decreased overall activity across all task conditions. ConclusionsIn contrast to our expectations, RG7203 significantly worsened reward-based effortful behavior and indices of reward learning. The results do not support the utility of RG7203 as adjunctive treatment for negative symptoms in patients with schizophrenia.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

The role of orphan receptor GPR139 in neuropsychiatric behavior.

Orphan G protein Coupled Receptors (GPCRs) present attractive targets both for understanding neuropsychiatric diseases and for development of novel therapeutics. GPR139 is an orphan GPCR expressed in select brain circuits involved in controlling movement, motivation and reward. It has been linked to the opioid and dopamine neuromodulatory systems; however, its role in animal behavior and neuropsychiatric processes is poorly understood. Here we present a comprehensive behavioral characterization of a mouse model with a GPR139 null mutation. We show that loss of GPR139 in mice results in delayed onset hyperactivity and prominent neuropsychiatric manifestations including elevated stereotypy, increased anxiety-related traits, delayed acquisition of operant responsiveness, disruption of cued fear conditioning and social interaction deficits. Furthermore, mice lacking GPR139 exhibited complete loss of pre-pulse inhibition and developed spontaneous 'hallucinogenic' head-twitches, altogether suggesting schizophrenia-like symptomatology. Remarkably, a number of these behavioral deficits could be rescued by the administration of μ-opioid and D2 dopamine receptor (D2R) antagonists: naltrexone and haloperidol, respectively, suggesting that loss of neuropsychiatric manifestations in mice lacking GPR139 are driven by opioidergic and dopaminergic hyper-functionality. The inhibitory influence of GPR139 on D2R signaling was confirmed in cell-based functional assays. These observations define the role of GPR139 in controlling behavior and implicate in vivo actions of this receptor in the neuropsychiatric process with schizophrenia-like pathology.

Impaired Acquisition of Nicotine-Induced Conditioned Place Preference in Fatty Acid-Binding Protein 3 Null Mice.

Nicotine causes psychological dependence through its interactions with nicotinic acetylcholine receptors in the brain. We previously demonstrated that fatty acid-binding protein 3 (FABP3) colocalizes with dopamine D2 receptors (D2Rs) in the dorsal striatum, and FABP3 deficiency leads to impaired D2R function. Moreover, D2R null mice do not exhibit increased nicotine-induced conditioned place preference (CPP) following chronic nicotine administration. To investigate the role of FABP3 in nicotine-induced CPP, FABP3 knockout (FABP3-/-) mice were evaluated using a CPP apparatus following consecutive nicotine administration (0.5mg/kg) for 14days. Importantly, nicotine-induced CPP was suppressed in the conditioning, withdrawal, and relapse phases in FABP3-/- mice. To resolve the mechanisms underlying impaired nicotine-induced CPP in these mice, we assessed c-Fos expression and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) signaling in both dopamine D1 receptor (D1R)- and D2R-positive neurons in the nucleus accumbens (NAc). Notably, 64% of dopamine receptor-positive neurons in the mouse NAc expressed both D1R and D2R. Impaired nicotine-induced CPP was correlated with lack of responsiveness of both CaMKII and ERK phosphorylation. The number of D2R-positive neurons was increased in FABP3-/- mice, while the number of D1R-positive neurons and the responsiveness of c-Fos expression to nicotine were decreased. The aberrant c-Fos expression was closely correlated with CaMKII but not ERK phosphorylation levels in the NAc of FABP3-/- mice. Taken together, these results indicate that impaired D2R signaling due to lack of FABP3 may affect D1R and c-Fos signaling and underlie nicotine-induced CPP behaviors.

Corticosterone Attenuates Reward-Seeking Behavior and Increases Anxiety via D2 Receptor Signaling in Ventral Tegmental Area Dopamine Neurons.

Corticosteroids (CORT) have been widely used in anti-inflammatory medication. Chronic CORT treatment can cause mesocorticolimbic system dysfunctions, which are known to play a key role for the development of psychiatric disorders. The VTA is a critical site in the mesocorticolimbic pathway and is responsible for motivation and reward-seeking behaviors. However, the mechanism by which chronic CORT alters VTA dopamine neuronal activity is largely unknown. We treated periadolescent male mice with vehicle, 1 d, or 7 d CORT in the drinking water, examined behavioral impacts with light/dark box, elevated plus maze, operant chamber, and open field tests, measured the effects of CORT on VTA dopamine neuronal activity using patch-clamp electrophysiology and dopamine concentration using fast-scan cyclic voltammetry, and tested the effects of dopamine D2 receptor (D2R) blockade by intra-VTA infusion of a D2R antagonist. CORT treatment induced anxiety-like behavior as well as decreased food-seeking behaviors. We show that chronic CORT treatment decreased excitability and excitatory synaptic transmission onto VTA dopamine neurons. Furthermore, chronic CORT increased somatodendritic dopamine concentration. The D2R antagonist sulpiride restored decreased excitatory transmission and excitability of VTA dopamine neurons. Furthermore, sulpiride decreased anxiety-like behavior and rescued food-seeking behavior in mice with chronic CORT exposure. Together, 7 d CORT treatment induces anxiety-like behavior and impairs food-seeking in a mildly aversive environment. D2R signaling in the VTA might be a potential target to ameliorate chronic CORT-induced anxiety and reward-seeking deficits.SIGNIFICANCE STATEMENT With widespread anti-inflammatory effects throughout the body, corticosteroids (CORT) have been used in a variety of therapeutic conditions. However, long-term CORT treatment causes cognitive impairments and neuropsychiatric disorders. The impact of chronic CORT on the mesolimbic system has not been elucidated. Here, we demonstrate that 7 d CORT treatment increases anxiety-like behavior and attenuates food-seeking behavior in a mildly aversive environment. By elevating local dopamine concentration in the VTA, a region important for driving motivated behavior, CORT treatment suppresses excitability and synaptic transmission onto VTA dopamine neurons. Intriguingly, blockade of D2 receptor signaling in the VTA restores neuronal excitability and food-seeking and alleviates anxiety-like behaviors. Our findings provide a potential therapeutic target for CORT-induced reward deficits.

Shifts in the neurobiological mechanisms motivating cocaine use with the development of an addiction-like phenotype in male rats.

The development of addiction is accompanied by a shift in the mechanisms motivating cocaine use from nucleus accumbens (NAc) dopamine D1 receptor (D1R) signaling to glutamate AMPA-kainate receptor (AMPA-R) signaling. Here, we determined whether similar shifts occur for NAc-D2R signaling and following systemic manipulation of D1R, D2R, and AMPA-R signaling. Male rats were given short-access (20 infusions/day) or extended-access to cocaine (24h/day, 96 infusions/day, 10days). Motivation for cocaine was assessed following 14days of abstinence using a progressive-ratio schedule. Once responding stabilized, the effects of NAc-D2R antagonism (eticlopride; 0-10.0μg/side) and systemic D1R (SCH-23390; 0-1.0mg/kg), D2R (eticlopride; 0-0.1mg/kg), and AMPA-R (CNQX; 0-1.5mg/kg) antagonism, and NAc-dopamine-R gene expression (Drd1/2/3) were examined. Motivation for cocaine was markedly higher in the extended- versus short-access group confirming the development of an addiction-like phenotype in the extended-access group. NAc-infused eticlopride decreased motivation for cocaine in both the short- and extended-access groups although low doses (0.1-0.3μg) were more effective in the short-access group and high doses (3-10μg/side) tended to be more effective in the extended-access group. Systemic administration of eticlopride (0.1mg/kg) was more effective in the extended-access group, and systemic administration of CNQX was effective in the extended- but not short-access group. NAc-Drd2 expression was decreased in both the short- and extended-access groups. These findings indicate that in contrast to NAc-D1R, D2R remain critical for motivating cocaine use with the development of an addiction-like phenotype. These findings also indicate that shifts in the mechanisms motivating cocaine use impact the response to both site-specific and systemic pharmacological treatment.

Development of pyrimidone D1 dopamine receptor positive allosteric modulators

MLS1082 is a structurally novel pyrimidone-based D1-like dopamine receptor positive allosteric modulator. Potentiation of D1 dopamine receptor (D1R) signaling is a therapeutic strategy for treating neurocognitive disorders. Here, we investigate the relationship between D1R potentiation and two prominent structural features of MLS1082, namely the pendant N-aryl and C-alkyl groups on the pyrimidone ring. To this end, we synthesized 24 new analogues and characterized their ability to potentiate dopamine signaling at the D1R and the closely related D5R. We identified structure–activity relationship trends for both aryl and alkyl modifications and our efforts afforded several analogues with improvements in activity. The most effective analogues demonstrated an approximately 8-fold amplification of dopamine-mediated D1R signaling. These findings advance the understanding of structural moieties underlying the activity of pyrimidone-based D1R positive allosteric modulators.

Dopamine D1 Receptor in Cancer.

Simple SummaryCirculating hormones and their specific receptors play a significant role in the development and progression of various cancers. This review aimed to summarize current knowledge about the dopamine D1 receptor’s biological role in different cancers, including breast cancer, central nervous system tumors, lymphoproliferative disorders, and other neoplasms. Treatment with dopamine D1 receptor agonists was proven to exert a major anti-cancer effect in many preclinical models. We highlight this receptor’s potential as a target for the adjunct therapy of tumors and discuss possibilities and necessities for further research in this area.Dopamine is a biologically active compound belonging to catecholamines. It plays its roles in the human body, acting both as a circulating hormone and neurotransmitter. It acts through G-protein-coupled receptors divided into two subgroups: D1-like receptors (D1R and D5R) and D2-like receptors (D2R, D3R, D4R). Physiologically, dopamine receptors are involved in central nervous system functions: motivation or cognition, and peripheral actions such as blood pressure and immune response modulation. Increasing evidence indicates that the dopamine D1 receptor may play a significant role in developing different human neoplasms. This receptor’s value was presented in the context of regulating various signaling pathways important in tumor development, including neoplastic cell proliferation, apoptosis, autophagy, migration, invasiveness, or the enrichment of cancer stem cells population. Recent studies proved that its activation by selective or non-selective agonists is associated with significant tumor growth suppression, metastases prevention, and tumor microvasculature maturation. It may also exert a synergistic anti-cancer effect when combined with tyrosine kinase inhibitors or temozolomide. This review provides a comprehensive insight into the heterogeneity of dopamine D1 receptor molecular roles and signaling pathways in human neoplasm development and discusses possible perspectives of its therapeutic targeting as an adjunct anti-cancer strategy of treatment. We highlight the priorities for further directions in this research area.

Maternal stress programs accelerated aging of the basal ganglia motor system in offspring

Early-life stress involved in the programming of stress-related illnesses can have a toxic influence on the functioning of the nigrostriatal motor system during aging. We examined the effects of perinatal stress (PRS) on the neurochemical, electrophysiological, histological, neuroimaging, and behavioral correlates of striatal motor function in adult (4 months of age) and old (21 months of age) male rats. Adult PRS offspring rats showed reduced dopamine (DA) release in the striatum associated with reductions in tyrosine hydroxylase-positive (TH+) cells and DA transporter (DAT) levels, with no loss of striatal dopaminergic terminals as assessed by positron emission tomography analysis with fluorine-18-l-dihydroxyphenylalanine. Striatal levels of DA and its metabolites were increased in PRS rats. In contrast, D2 DA receptor signaling was reduced and A2A adenosine receptor signaling was increased in the striatum of adult PRS rats. This indicated enhanced activity of the indirect pathway of the basal ganglia motor circuit. Adult PRS rats also showed poorer performance in the grip strength test and motor learning tasks. The aged PRS rats also showed a persistent reduction in striatal DA release and defective motor skills in the pasta matrix and ladder rung walking tests. In addition, the old rats showed large increases in the levels of SNAP-25 and synaptophysin, which are synaptic vesicle-related proteins in the striatum, and in the PRS group only, reductions in Syntaxin-1 and Rab3a protein levels were observed. Our findings indicated that the age-dependent threshold for motor dysfunction was lowered in PRS rats. This area of research is underdeveloped, and our study suggests that early-life stress can contribute to an increased understanding of how aging diseases are programmed in early-life.

Persistently Elevated mTOR Complex 1-S6 Kinase 1 Disrupts DARPP-32–Dependent D1 Dopamine Receptor Signaling and Behaviors

BackgroundThe serine-threonine kinase mTORC1 (mechanistic target of rapamycin complex 1) is essential for normal cell function but is aberrantly activated in the brain in both genetic-developmental and sporadic diseases and is associated with a spectrum of neuropsychiatric symptoms. The underlying molecular mechanisms of cognitive and neuropsychiatric symptoms remain controversial. MethodsThe present study examines behaviors in transgenic models that express Rheb, the most proximal known activator of mTORC1, and profiles striatal phosphoproteomics in a model with persistently elevated mTORC1 signaling. Biochemistry, immunohistochemistry, electrophysiology, and behavior approaches are used to examine the impact of persistently elevated mTORC1 on D1 dopamine receptor (D1R) signaling. The effect of persistently elevated mTORC1 was confirmed using D1-Cre to elevate mTORC1 activity in D1R neurons. ResultsWe report that persistently elevated mTORC1 signaling blocks canonical D1R signaling that is dependent on DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein). The immediate downstream effector of mTORC1, ribosomal S6 kinase 1 (S6K1), phosphorylates and activates DARPP-32. Persistent elevation of mTORC1-S6K1 occludes dynamic D1R signaling downstream of DARPP-32 and blocks multiple D1R responses, including dynamic gene expression, D1R-dependent corticostriatal plasticity, and D1R behavioral responses including sociability. Candidate biomarkers of mTORC1–DARPP-32 occlusion are increased in the brain of human disease subjects in association with elevated mTORC1-S6K1, supporting a role for this mechanism in cognitive disease. ConclusionsThe mTORC1-S6K1 intersection with D1R signaling provides a molecular framework to understand the effects of pathological mTORC1 activation on behavioral symptoms in neuropsychiatric disease.

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson’s disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to l-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with l-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

Characterization of dopamine D2 receptor binding, expression and signaling in different brain regions of control and schizophrenia-model Wisket rats

In previous studies we have shown that a three-hit animal model of schizophrenia (Wisket rat) has several behavioral impairments related to the disorder along with altered mu-opioid (MOP) and cannabinoid (CB1) receptor signaling. As the dopamine hypothesis of schizophrenia is central to research in the field, the goal of the present study was to investigate dopaminergic D2 receptor (D2R) functions (binding capacity, G-protein activation and expression) in several brain regions (hippocampus, prefrontal cortex, striatum, olfactory bulb, cerebellum, brainstem, cortex and diencephalon) of control (Wistar) and Wisket rats.It was found that the D2R mediated maximal activation of G-proteins was substantially higher in hippocampus, striatum and olfactory bulb membranes prepared from the Wisket than in control animals, which was accompanied with lower potency of the D2R-mediated G-protein activation. In contrast, enhanced potency was detected in the prefrontal cortex without changes in the maximal activation. In saturation binding assays the maximal binding capacity of D2Rs was higher in the model animals in cerebral cortex, striatum and lower in the brainstem, while no changes in the dissociation constant values were detected. The D2R mRNA expression showed a trend for greater level in the investigated areas, while the D2R protein expression was significantly higher of Wisket rats compared to Wistar animals in the hippocampus and in the prefrontal cortex but not in the cerebellum.This study proved that the Wisket animals show altered D2 receptor expression and function which might be related to the schizophrenia-like symptoms.

Dopamine D2 receptor signaling on iMSNs is required for initiation and vigor of learned actions

Striatal dopamine D2 receptors (D2Rs) are important for motor output. Selective deletion of D2Rs from indirect pathway-projecting medium spiny neurons (iMSNs) impairs locomotor activities in a task-specific manner. However, the role of D2Rs in the initiation of motor actions in reward seeking and taking is not fully understood, and there is little information about how receptors contribute under different task demands and with different outcome types. The iMSN-D2Rs modulate neuronal activity and synaptic transmission, exerting control on circuit functions that may play distinct roles in action learning and performance. Selective deletion of D2Rs on iMSNs resulted in slower action initiation and response rate in an instrumental conditioning task, but only when performance demand was increased. The iMSN-Drd2KO mice were also slower to initiate swimming in a T-maze procedural learning task but were unimpaired in cognitive function and behavioral flexibility. In contrast, in a Pavlovian discrimination learning task, iMSN-Drd2KO mice exhibited normal acquisition and extinction of rewarded responding. The iMSN-Drd2KO mice showed performance deficits at all phases of rotarod skill learning. These findings reveal that dopamine modulation through iMSN-D2Rs influences the ability to self-initiate actions, as well as the willingness and/or vigor with which these responses are performed. However, these receptors seem to have little influence on simple associative learning or on stimulus-driven responding. The loss of normal D2R roles may contribute to disorders in which impaired dopamine signaling leads to hypokinesia or impaired initiation of specific voluntary actions.

ATP Signaling Controlling Dyskinesia Through P2X7 Receptors.

Dopamine replacement therapy with L-3,4-dihydroxyphenylalanine (L-DOPA) is the only temporary therapy for Parkinson’s disease (PD), but it triggers dyskinesia over time. Since dyskinesia is associated with increased neuronal firing that bolsters purinergic signaling, we now tested whether the selective and blood-brain barrier-permeable P2X7 receptor antagonist Brilliant Blue-G (BBG, 22.5–45 mg/kg ip) attenuated behavioral, neurochemical and biochemical alterations in rats turned hemiparkinsonian upon unilateral striatal injection of 6-hydroxydopamine (6-OHDA) and treated daily with L-DOPA (30 mg/kg by gavage) for 22 days. The blockade of P2X7 receptors decreased L-DOPA-induced dyskinesia and motor incoordination in hemiparkinsonian rats. In parallel, BBG treatment rebalanced the altered dopamine D1 and D2 receptor density and signaling as well as some neuroinflammation-associated parameters in the striatum and substantia nigra. These findings herald a hitherto unrecognized role for purinergic signaling in the etiopathology of dyskinesia and prompt P2X7 receptor antagonists as novel candidate anti-dyskinesia drugs.