Presynaptic and Postsynaptic Mesolimbic Dopamine D3 Receptors Play Distinct Roles in Cocaine Versus Opioid Reward in Mice.

BACKGROUND: Past research has illuminated pivotal roles of dopamine D3 receptors (D3R) in the rewarding effects of cocaine and opioids. However, the cellular and neural circuit mechanisms that underlie these actions remain unclear. METHODS: We employed Cre-LoxP techniques to selectively delete D3R from presynaptic dopamine neurons or postsynaptic dopamine D1 receptor (D1R)-expressing neurons in male and female mice. We utilized RNAscope in situ hybridization, immunohistochemistry, real-time polymerase chain reaction, voltammetry, optogenetics, microdialysis, and behavioral assays (n ≥ 8 animals per group) to functionally characterize the roles of presynaptic versus postsynaptic D3R in cocaine and opioid actions. RESULTS: Our results revealed D3R expression in ∼25% of midbrain dopamine neurons and ∼70% of D1R-expressing neurons in the nucleus accumbens. While dopamine D2 receptors (D2R) were expressed in ∼80% dopamine neurons, we found no D2R and D3R colocalization among these cells. Selective deletion of D3R from dopamine neurons increased exploratory behavior in novel environments and enhanced pulse-evoked nucleus accumbens dopamine release. Conversely, deletion of D3R from D1R-expressing neurons attenuated locomotor responses to D1-like and D2-like agonists. Strikingly, deletion of D3R from either cell type reduced oxycodone self-administration and oxycodone-enhanced brain-stimulation reward. In contrast, neither of these D3R deletions impacted cocaine self-administration, cocaine-enhanced brain-stimulation reward, or cocaine-induced hyperlocomotion. Furthermore, D3R knockout in dopamine neurons reduced oxycodone-induced hyperactivity and analgesia, while deletion from D1R-expressing neurons potentiated opioid-induced hyperactivity without affecting analgesia. CONCLUSIONS: We dissected presynaptic versus postsynaptic D3R function in the mesolimbic dopamine system. D2R and D3R are expressed in different populations of midbrain dopamine neurons, regulating dopamine release. Mesolimbic D3R are critically involved in the actions of opioids but not cocaine.

The antidepressant-like effect and proposed mechanism of action of TPN672MA, a novel serotonin-dopamine receptor modulator for the treatment of schizophrenia.

TPN672MA, an innovative antipsychotic drug candidate currently in clinical trials, acts as a dopamine D2/D3 receptor partial agonist, serotonin 5-HT1A receptor agonist, and serotonin 5-HT2A receptor antagonist. Preclinical investigations have demonstrated its potential in treating the core symptoms of schizophrenia. The present study highlights TPN672MA's significant antidepressant-like effects in classical behavioral models, such as the chronic social defeat stress paradigm. The pronounced 5-HT1A receptor agonism and D2/D3 receptor partial agonism of TPN672MA likely contribute to its therapeutic effects in depression. Additionally, TPN672MA's antidepressant-like efficacy may be linked to its ability to enhance the expression levels of brain-derived neurotrophic factor (BDNF) and postsynaptic density protein-95 (PSD95) in the hippocampus. Furthermore, TPN672MA displayed a more rapid onset of antidepressant-like action. In conclusion, TPN672MA represents a promising new drug candidate for the treatment of symptoms of schizophrenia and depression.

IRL790 modulated striatal D1 neurons synaptic plasticity ameliorating levodopa-induced dyskinesia in mouse.

Objective: Levodopa (L-dopa) therapy is the principal pharmacological treatment for Parkinson's disease (PD). Nevertheless, prolonged use of this drug may result in different involuntary movement symptoms caused by the medication, referred to as levodopa-induced dyskinesia (LID). LID is associated with changes in synaptic plasticity of the D1 medium spiny neurons (MSNs) located in the dorsal striatum (dStr). Within the striatum, the amount of Dopamine D3 receptor (D3R) is notably increased in LID, demonstrating colocalization with D1R expression in neurons, and the level of D3R expression is directly related to the intensity of LID. IRL 790, as a D3R antagonist, can ameliorate LID. This study aims to explore if IRL 790 improves LID by regulating the synaptic plasticity of D1+ MSNs in dStr. Methods: The electrophysiology and synaptic spine density of D1+ MSNs in dStr were recorded for sham mice, LID mice, and LID mice treated with IRL 790. The regulation of synaptic plasticity in LID D1+ MSNs by IRL 790 was analyzed. Behavioral tests were conducted to confirm the treatment effect of IRL 790 on LID. Results: In LID D1+ MSNs, there was persistent abnormal LTP, absence of LTD, and an increase in spontaneous excitatory postsynaptic currents (sEPSCs). IRL 790 treatment restored normal LTP, LTD, and sEPSCs. Treatment with IRL 790 also restored the reduced dendritic spine density in D1+ MSNs of LID mice. IRL790 improved dyskinetic manifestations in LID mice. Conclusion: IRL790 ameliorates LID by regulating the synaptic structure and functional plasticity of striatal D1+ MSNs.

Antinociceptive role of the thalamic dopamine D3 receptor in descending modulation of intramuscular formalin-induced muscle nociception in a rat model of Parkinson's disease.

Pain in Parkinson's disease (PD) has been validated as one of the major non-motor dysfunctions affecting the quality of life and subsequent rehabilitation. In the present study, we investigated the role of the dopamine D3 receptor in the thalamic mediodorsal (MD) and ventromedial (VM) nuclei mediated descending control of nociception and intramuscular (i.m.) 2.5% formalin-induced persistent muscle nociception. Paw withdrawal reflexes were measured in naive rats and rats subjected to PD induced by unilateral microinjection of 6 µg 6-OHDA into the rat striatum. Formalin-induced muscle nociception in phase 1, inter-phase, and phase 2 was significantly greater in PD rats compared to naive and vehicle-treated rats (P ˂ 0.001). PD rats exhibited bilaterally mechanical hyperalgesia and heat hypoalgesia in formalin-induced muscle nociception. Microinjection of SK609, a dopamine D3 receptor agonist, at various doses (2.5-7.5 nmol/0.5 µl) into the thalamic VM nucleus dose-dependently prolonged heat-evoked paw withdrawal latencies in both naive and PD rats. Administration of SK609 to either the MD or VM nuclei had no effect on noxious mechanically evoked paw withdrawal reflexes. Pre-treatment of the thalamic MD nucleus with SK609 significantly attenuated formalin-induced nociception, and reversed mechanical hyperalgesia, but not heat hypoalgesia. Pre-treatment of the thalamic VM nucleus with SK609 inhibited formalin-induced nociception in the late phase of phase 2 (30-75 min) and heat hypoalgesia, but not mechanical hyperalgesia (P < 0.05). It is suggested that the dopamine D3 receptors in the thalamus play an antinociceptive role in the descending modulation of nociception. Activation of D3 receptors within the thalamic MD and VM nuclei attenuates descending facilitation and enhances descending inhibition in rats during PD.

D3 Receptor-Targeted Cariprazine: Insights from Lab to Bedside.

Until the late 1800s, drug development was a chance finding based on observations and repeated trials and errors. Today, drug development must go through many iterations and tests to ensure it is safe, potent, and effective. This process is a long and costly endeavor, with many pitfalls and hurdles. The aim of the present review article is to explore what is needed for a molecule to move from the researcher bench to the patients' bedside, presented from an industry perspective through the development program of cariprazine. Cariprazine is a relatively novel antipsychotic medication, approved for the treatment of schizophrenia, bipolar mania, bipolar depression, and major depression as an add-on. It is a D3-preferring D3-D2 partial agonist with the highest binding to the D3 receptors compared to all other antipsychotics. Based on the example of cariprazine, there are several key factors that are needed for a molecule to move from the researcher bench to the patients' bedside, such as targeting an unmet medical need, having a novel mechanism of action, and a smart implementation of development plans.

Involvement of dopamine D3 receptor in impulsive choice decision-making in male rats.

Impulsive decision-making has been linked to impulse control disorders and substance use disorders. However, the neural mechanisms underlying impulsive choice are not fully understood. While previous PET imaging and autoradiography studies have shown involvement of dopamine and D2/3 receptors in impulsive behavior, the roles of distinct D1, D2, and D3 receptors in impulsive decision-making remain unclear. In this study, we used a food reward delay-discounting task (DDT) to identify low- and high-impulsive rats, in which low-impulsive rats exhibited preference for large delayed reward over small immediate rewards, while high-impulsive rats showed the opposite preference. We then examined D1, D2, and D3 receptor gene expression using RNAscope in situ hybridization assays. We found that high-impulsive male rats exhibited lower levels of D2 and D3, and particularly D3, receptor expression in the nucleus accumbens (NAc), with no significant changes in the insular, prelimbic, and infralimbic cortices. Based on these findings, we further explored the role of the D3 receptor in impulsive decision-making. Systemic administration of a selective D3 receptor agonist (FOB02-04) significantly reduced impulsive choices in high-impulsive rats but had no effects in low-impulsive rats. Conversely, a selective D3 receptor antagonist (VK4-116) produced increased both impulsive and omission choices in both groups of rats. These findings suggest that impulsive decision-making is associated with a reduction in D3 receptor expression in the NAc. Selective D3 receptor agonists, but not antagonists, may hold therapeutic potentials for mitigating impulsivity in high-impulsive subjects.

A review on the pharmacology of cariprazine and its role in the treatment of negative symptoms of schizophrenia.

Management of negative symptoms is one of the most challenging and important unmet needs of schizophrenia treatment. Negative symptoms together with positive symptoms result in significant psychosocial impairment and poor quality of life. Existing studies on atypical antipsychotics reported limited treatment adherence due to higher prevalence of treatment-emergent adverse events, such as diabetes, weight gain, hyperlipidemia, hyperprolactinemia and hypertension. A compound with greater affinity for dopamine D2/D3 receptors may improve negative symptoms, mood, and cognitive impairment associated with schizophrenia. In 2015, the US FDA has approved cariprazine, a partial D2/D3 agonist for treatment of schizophrenia, mania or mixed episodes. Midlands and Lancashire Commissioning Support Unit, UK (2019) has particularly suggested cariprazine for the treatment of predominant negative symptoms of schizophrenia. India's Central Drugs Standard Control Organization (CDSCO) has approved cariprazine in 2021 for the treatment of schizophrenia, manic or mixed episodes associated with bipolar I disorder. A ten-fold greater affinity for D3 receptors and partial agonism to serotonin receptors, along with longer half-life make cariprazine distinct when compared with other atypical antipsychotics. Cariprazine is also reported to have fewer incidents of metabolic and hormonal adverse events, and has been shown to provide better relapse prevention. Recent evidence indicates promising effect of cariprazine in ameliorating negative symptoms as well as psychotic symptoms in patients with schizophrenia. In addition, improved adherence to treatment (adjunctive/monotherapy) with cariprazine in patients having inadequate response to an ongoing antipsychotic treatment has also been clinically established. This review presents the evidence-based safety and efficacy of cariprazine for treatment of predominant negative symptoms of schizophrenia.

Aromatic linker variations in novel dopamine D2 and D3 receptor ligands.

Dopamine D2-like receptors, especially D2 and D3 receptor subtypes, are important targets of antipsychotic agents. Many of these antipsychotics share an aliphatic linker element between a protonable amine group and an acyl-like moiety. Here, we have modified this aliphatic linker into phenylmethyl and phenylethyl linkers substituted in different positions. The design, synthesis, and in vitro evaluation of 18 dopamine D2 and D3 receptor ligands were performed in this study. Using a radioligand displacement assay, all ligands were found to have modest nanomolar affinity to D2R and D3R. N-(4-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl}phenyl)acetamide (6c) demonstrates the highest D3R and D2R affinity values (pKi values of 7.83 [D2R] and 8.04 [D3R]), featuring a slight preference to D3R. This derivative can be taken as a reference structure for the development of a new class of D2R and D3R ligands.

In vitro characterization of [125I]HY-3-24, a selective ligand for the dopamine D3 receptor.

Introduction: Dopamine D3 receptor (D3R) ligands have been studied for the possible treatment of neurological and neuropsychiatric disorders. However, selective D3R radioligands for in vitro binding studies have been challenging to identify due to the high structural similarity between the D2R and D3R. In a prior study, we reported a new conformationally-flexible benzamide scaffold having a high affinity for D3R and excellent selectivity vs. D2R. In the current study, we characterized the in vitro binding properties of a new radioiodinated ligand, [125I]HY-3-24. Methods: In vitro binding studies were conducted in cell lines expressing D3 receptors, rat striatal homogenates, and rat and non-human primate (NHP) brain tissues to measure regional brain distribution of this radioligand. Results: HY-3-24 showed high potency at D3R (Ki = 0.67 ± 0.11 nM, IC50 = 1.5 ± 0.58 nM) compared to other D2-like dopamine receptor subtypes (D2R Ki = 86.7 ± 11.9 nM and D4R Ki > 1,000). The Kd (0.34 ± 0.22 nM) and Bmax (38.91 ± 2.39 fmol/mg) values of [125I]HY-3-24 were determined. In vitro binding studies in rat striatal homogenates using selective D2R and D3R antagonists confirmed the D3R selectivity of [125I]HY-3-24. Autoradiography results demonstrated that [125I]HY-3-24 specifically binds to D3Rs in the nucleus accumbens, islands of Calleja, and caudate putamen in rat and NHP brain sections. Conclusion: These results suggest that [125I]HY-3-24 appears to be a novel radioligand that exhibits high affinity binding at D3R, with low binding to other D2-like dopamine receptors. It is anticipated that [125I]HY-3-24 can be used as the specific D3R radioligand.

Microbial bile acid metabolite ameliorates mycophenolate mofetil-induced gastrointestinal toxicity through vitamin D3 receptor.

Mycophenolate mofetil (MMF) is one of the most used immunosuppressive drugs in organ transplantation, but frequent gastrointestinal (GI) side effects through unknown mechanisms limit its clinical use. Gut microbiota and its metabolites were recently reported to play a vital role in MMF-induced GI toxicity, but the specific mechanism of how they interact with the human body is still unclear. Here, we found that secondary bile acids (BAs), as bacterial metabolites, were significantly reduced by MMF administration in the gut of mice. Microbiome data and fecal microbiota transfer model supported a microbiota-dependent effect on the reduction of secondary BAs. Supplementation of the secondary BA lithocholic acid alleviated MMF-induced weight loss, colonic inflammation, and oxidative phosphorylation damage. Genetic deletion of the vitamin D3 receptor (VDR), which serves as a primary colonic BA receptor, in colonic epithelial cells (VDRΔIEC) abolished the therapeutic effect of lithocholic acid on MMF-induced GI toxicity. Impressively, we discovered that paricalcitol, a Food and Drug Administration-approved VDR agonist that has been used in clinics for years, could effectively alleviate MMF-induced GI toxicity. Our study reveals a previously unrecognized mechanism of gut microbiota, BAs, and VDR signaling in MMF-induced GI side effects, offering potential therapeutic strategies for clinics.

Unraveling Activation-Related Rearrangements and Intrinsic Divergence from Ligand-Specific Conformational Changes of the Dopamine D3 and D2 Receptors.

Effective rational drug discovery hinges on understanding the functional states of the target protein and distinguishing it from homologues. However, for the G protein coupled receptors, both activation-related conformational changes (ACCs) and intrinsic divergence among receptors can be misled or obscured by ligand-specific conformational changes (LCCs). Here, we unraveled ACCs and intrinsic divergence from LCCs of the dopamine D3 and D2 receptors (D3R and D2R), by analyzing their experimentally determined structures and the molecular dynamics (MD) simulation results of the receptors bound with various ligands. In addition to the ACCs common to other aminergic receptors, we revealed unique ACCs for these two receptors, including the extracellular portion of TM5 (TM5e) and TM6e shifting away from TM2e and TM3e, with a subtle rotation of TM5e. In identifying intrinsic divergence, we found more outward tilting of TM6e in the D2R compared to the D3R in both the experimental structures and simulations bound with ligands in different scaffolds. However, this difference was drastically reduced in the simulations bound with nonselective agonist quinpirole, suggesting a misleading effect of LCCs. Further, in the quinpirole-bound simulations, TM1 showed a greater disparity between these receptors, indicating that LCCs may also obscure intrinsic divergence. Importantly, our MD simulations revealed divergence in the dynamics of these receptors. Specifically, the D2R exhibited heightened flexibility compared to the D3R in the extracellular loops and TMs 5e, 6e, and 7e, associated with its greater ligand binding site plasticity. Our results lay the groundwork for crafting ligands specifically targeting the D2R and D3R with more precise pharmacological profiles.

DRD3 Predicts Cognitive Impairment and Anxiety in Parkinson's Disease: Susceptibility and Protective Effects.

Background: A possible genetic contribution of dopamine D3 receptor (DRD3) to cognitive impairment in Parkinson's disease (PD) has yet to be investigated. Objective: To explore the effects of rs6280 (Ser9Gly) genotype on PD patients' cognitive performance and to clarify possible interactions with psychopathology. Methods: Two hundred and fifty-three consecutive PD patients underwent neurological and neuropsychological evaluations, which included: Unified Parkinson's Disease Rating Scale (UPDRS), Hoehn & Yahr scale (H&Y), Dementia Rating Scale-2 (DRS-2), and Hospital Anxiety and Depression Scale (HADS). rs6280 polymorphism was genotyped for all PD patients and for 270 ethnically matched healthy volunteers (HC). Non-parametric group comparisons and logistic regressions were used for data analyses. Results: rs6280 genotype did not differ between PD and HC groups. PD patients with rs6280 CC genotype had more impaired cognitive performance (i.e., <1st percentile of demographically adjusted norms) on DRS-2 subscales Initiation/Perseveration and Construction than those with TT genotype. These associations remained statistically significant when other covariates (e.g., demographic features, disease duration, severity of motor symptoms in OFF and ON states, anti-parkinsonian medication, and psychopathology symptoms) were taken into consideration. PD patients with rs6280 TC had less anxiety (i.e., HADS Anxiety≥11) than those with TT (p = 0.012). This association was also independent of other covariates. Conclusions: Study findings suggest that rs6280 CC genotype predisposes to executive dysfunction and visuoconstructional deficits, whereas the heterozygous genotype protects from anxiety in PD. These effects do not appear to be dependent of one another. rs6280 is not a genotypic susceptibility factor for PD.

Modulation of D3R Splicing, Signaling, and Expression by D1R through PKA→PTB Phosphorylation.

The D1R and D3R receptors functionally and synergistically interact in striatonigral neurons. Dopaminergic denervation turns this interaction antagonistic, which is correlated with a decrement in D3nf isoform and an increment in D3R membranal expression. The mechanisms of such changes in D3R are attributed to the dysregulation of the expression of their isoforms. The cause and mechanism of this phenomenon remain unknown. Dopaminergic denervation produces a decrement in D1R and PKA activity; we propose that the lack of phosphorylation of PTB (regulator of alternative splicing) by PKA produces the dysregulation of D3R splicing and changes D3R functionality. By using in silico analysis, we found that D3R mRNA has motifs for PTB binding and, by RIP, co-precipitates with PTB. Moreover, D1R activation via PKA promotes PTB phosphorylation. Acute and 5-day D1R blockade decreases the expression of D3nf mRNA. The 5-day treatment reduces D3R, D3nf, and PTB protein in the cytoplasm and increases D3R in the membrane and PTB in the nucleus. Finally, the blockade of D1R mimics the effect of dopaminergic denervation in D1R and D3R signaling. Thus, our data indicate that through PKA→PTB, D1R modulates D3R splicing, expression, and signaling, which are altered during D1R blockade or the lack of stimulation in dopaminergic denervation.

Dopamine D3 receptor modulates D2 receptor effects on cAMP and GABA release at striatopallidal terminals-Modulation by the Ca2+-Calmodulin-CaMKII system.

Dopamine D2 receptor (D2R) is expressed in striatopallidal neurons and decreases forskolin-stimulated cyclic adenine monophosphate (cAMP) accumulation and gamma-aminobutyric acid (GABA) release. Dopamine D3 receptor (D3R) mRNA is expressed in a population of striatal D2R-expressing neurons. Also, D3R protein and binding have been reported in the neuropil of globus pallidus. We explore whether D2R and D3R colocalize in striatopallidal terminals and whether D3R modulates the D2R effect on forskolin-stimulated [3H]cAMP accumulation in pallidal synaptosomes and high K+ stimulated-[3H]GABA release in pallidal slices. Previous reports in heterologous systems indicate that calmodulin (CaM) and CaMKII modulate D2R and D3R functions; thus, we study whether this system regulates its functional interaction. D2R immunoprecipitates with CaM, and pretreatment with ophiobolin A or depolarization of synaptosomes with 15 mM of K+ decreases it. Both treatments increase the D2R inhibition of forskolin-stimulated [3H]cAMP accumulation when activated with quinpirole, indicating a negative modulation of CaM on D2R function. Quinpirole also activates D3R, potentiating D2R inhibition of cAMP accumulation in the ophiobolin A-treated synaptosomes. D2R and D3R immunoprecipitate in pallidal synaptosomes and decrease after the kainic acid striatal lesion, indicating the striatal origin of the presynaptic receptors. CaM-kinase II alfa (CaMKIIα) immunoprecipitates with D3R and increases after high K+ depolarization. In the presence of KN62, a CaMKIIα blocker, D3R potentiates D2R effects on cAMP accumulation in depolarized synaptosomes and GABA release in pallidal slices, indicating D3R function regulation by CaMKIIα. Our data indicate that D3R potentiates the D2R effect on cAMP accumulation and GABA release at pallidal terminals, an interaction regulated by the CaM-CaMKIIα system.

Dissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)2D3 in A431 squamous cell carcinoma.

BACKGROUND: Human skin is the natural source, place of metabolism, and target for vitamin D3. The classical active form of vitamin D3, 1,25(OH)2D3, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D3 receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)2D3 at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)2D3 resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group "immune response". This may indicate the existence of an alternative, secondary response to 1,25(OH)2D3. In contrast, treatment of A431 ΔRXRA cells with 1,25(OH)2D3 for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)2D3 targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)2D3 treatment. Comparison of transcriptome changes induced by 1,25(OH)2D3 in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)2D3. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)2D3. In summary, VDR or RXRA independent genomic activities of 1,25(OH)2D3 suggest the involvement of alternative factors, opening new challenges in this field.

Carbazole and tetrahydro-carboline derivatives as dopamine D3 receptor antagonists with the multiple antipsychotic-like properties.

Dopamine D3 receptor (D3R) is implicated in multiple psychotic symptoms. Increasing the D3R selectivity over dopamine D2 receptor (D2R) would facilitate the antipsychotic treatments. Herein, novel carbazole and tetrahydro-carboline derivatives were reported as D3R selective ligands. Through a structure-based virtual screen, ZLG-25 (D3R Ki = 685 nmol/L; D2R Ki > 10,000 nmol/L) was identified as a novel D3R selective bitopic ligand with a carbazole scaffold. Scaffolds hopping led to the discovery of novel D3R-selective analogs with tetrahydro-ß-carboline or tetrahydro-γ-carboline core. Further functional studies showed that most derivatives acted as hD3R-selective antagonists. Several lead compounds could dose-dependently inhibit the MK-801-induced hyperactivity. Additional investigation revealed that 23j and 36b could decrease the apomorphine-induced climbing without cataleptic reaction. Furthermore, 36b demonstrated unusual antidepressant-like activity in the forced swimming tests and the tail suspension tests, and alleviated the MK-801-induced disruption of novel object recognition in mice. Additionally, preliminary studies confirmed the favorable PK/PD profiles, no weight gain and limited serum prolactin levels in mice. These results revealed that 36b provided potential opportunities to new antipsychotic drugs with the multiple antipsychotic-like properties.

Effects of Vitamin D3 Supplementation and Aerobic Training on Autophagy Signaling Proteins in a Rat Model Type 2 Diabetes Induced by High-Fat Diet and Streptozotocin.

The aim of this study was to investigate the combined effects of vitamin D3 supplementation and aerobic training on regulating the autophagy process in rats with type 2 diabetic induced by a high-fat diet and streptozotocin. A total of 40 Wistar rats were divided into five groups: normal control (NC), diabetic control (DC), diabetic + aerobic training (DAT), diabetic + vitamin D3 (DVD), and diabetic + aerobic training + vitamin D3 (DVDAT). The rats underwent eight weeks of aerobic training with an intensity of 60% maximum running speed for one hour, along with weekly subcutaneous injections of 10,000 units of vitamin D3. The protein levels of different autophagy markers were assessed in the left ventricular heart tissue. The results showed that the protein levels of AMPK, pAMPK, mTOR, and pmTOR were significantly lower in the DC group compared to the NC group. Conversely, the levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D proteins were significantly higher in the DC group. However, the interventions of aerobic training and vitamin D3 supplementation, either individually or in combination, led to increased levels of AMPK, pAMPK, mTOR, and pmTOR, and decreased levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D (p < 0.05). Additionally, the aerobic capacity in the DAT and DVDAT groups was significantly higher compared to the NC, DC, and DVD groups (p < 0.05). These findings suggest that type 2 diabetes is associated with excessive autophagy in the left ventricle. However, after eight weeks of vitamin D3 supplementation and aerobic training, a significant reduction in excessive autophagy was observed in rats with type 2 diabetes.

Enhancement of memory consolidation by an avoidance conditioned stimulus: Modulation by the D3 receptor.

Conditioned stimuli (CS) paired with foot-shock can enhance memory consolidation. Because the dopamine D3 receptor (D3R) has been implicated in mediating various responses to CSs, the current study explored its potential role in modulation of memory consolidation by an avoidance CS. Male Sprague-Dawley rats trained to avoid foot-shocks in a two-way signalled active avoidance task (8 sessions, 30 trials per session, 0.8 mA foot-shock) were pre-treated with the D3R antagonist NGB-2904 (Vehicle, 0.1 or 5 mg/kg) and exposed to the CS immediately after the sample phase of an object recognition memory task. Discrimination ratios were assessed 72 h later. Immediate, but not delayed (6 h), post-sample exposure to the CS enhanced object recognition memory and this effect was blocked by NGB-2904. Control experiments with the beta-noradrenergic receptor antagonist propranolol (10 or 20 mg/kg) and D2R antagonist pimozide (0.2 or 0.6 mg/kg) indicated that NGB-2904 targeted post-training memory consolidation. Exploring the pharmacological selectivity of the NGB-2904 effect, it was found that: 1) 5 mg/kg NGB-2904 blocked conditioned memory modulation produced by post-sample exposure to a "weak" CS (one day of avoidance training) and concurrent stimulation of catecholamine activity by 10 mg/kg bupropion; and 2) post-sample exposure to a "weak" CS and concurrent administration of the D3R agonist 7-OH-DPAT (1 mg/kg) enhanced consolidation of object memory. Finally, because 5 mg/kg NGB-2904 had no effect on modulation by avoidance training in the presence of foot-shocks, the findings herein support the hypothesis that the D3R plays an important role in modulation of memory consolidation by CSs.

Unveiling the Differences in Signaling and Regulatory Mechanisms between Dopamine D2 and D3 Receptors and Their Impact on Behavioral Sensitization.

Dopamine receptors are classified into five subtypes, with D2R and D3R playing a crucial role in regulating mood, motivation, reward, and movement. Whereas D2R are distributed widely across the brain, including regions responsible for motor functions, D3R are primarily found in specific areas related to cognitive and emotional functions, such as the nucleus accumbens, limbic system, and prefrontal cortex. Despite their high sequence homology and similar signaling pathways, D2R and D3R have distinct regulatory properties involving desensitization, endocytosis, posttranslational modification, and interactions with other cellular components. In vivo, D3R is closely associated with behavioral sensitization, which leads to increased dopaminergic responses. Behavioral sensitization is believed to result from D3R desensitization, which removes the inhibitory effect of D3R on related behaviors. Whereas D2R maintains continuous signal transduction through agonist-induced receptor phosphorylation, arrestin recruitment, and endocytosis, which recycle and resensitize desensitized receptors, D3R rarely undergoes agonist-induced endocytosis and instead is desensitized after repeated agonist exposure. In addition, D3R undergoes more extensive posttranslational modifications, such as glycosylation and palmitoylation, which are needed for its desensitization. Overall, a series of biochemical settings more closely related to D3R could be linked to D3R-mediated behavioral sensitization.

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3R cells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.