Search

The crystal structure of the dopamine D3 receptor (D3R) in complex with eticlopride inspired the design of bitopic ligands that explored (1) N-alkylation of the eticlopride's pyrrolidine ring, (2) shifting of the position of the pyrrolidine nitrogen, (3) expansion of the pyrrolidine ring system, and (4) incorporation of O-alkylations at the 4-position. Structure activity relationships (SAR) revealed that moving the N- or expanding the pyrrolidine ring was detrimental to D2R/D3R binding affinities. Small pyrrolidine N-alkyl groups were poorly tolerated, but the addition of a linker and secondary pharmacophore (SP) improved affinities. Moreover, O-alkylated analogues showed higher binding affinities compared to analogously N-alkylated compounds, e.g., O-alkylated 33 (D3R, 0.436 nM and D2R, 1.77 nM) vs the N-alkylated 11 (D3R, 6.97 nM and D2R, 25.3 nM). All lead molecules were functional D2R/D3R antagonists. Molecular models confirmed that 4-position modifications would be well-tolerated for future D2R/D3R bioconjugate tools that require long linkers and or sterically bulky groups.

We have performed theoretical calculations with 70 drugs that have been considered in 231 clinical trials as possible candidates to repurpose drugs for schizophrenia based on their interactions with the dopaminergic system. A hypothesis of shared pharmacophore features was formulated to support our calculations. To do so, we have used the crystal structure of the D2-like dopamine receptor in complex with risperidone, eticlopride, and nemonapride. Linagliptin, citalopram, flunarizine, sildenafil, minocycline, and duloxetine were the drugs that best fit with our model. Molecular docking calculations, molecular dynamics outcomes, blood-brain barrier penetration, and human intestinal absorption were studied and compared with the results. From the six drugs selected in the shared pharmacophore features input, flunarizine showed the best docking score with D2, D3, and D4 dopamine receptors and had high stability during molecular dynamics simulations. Flunarizine is a frequently used medication to treat migraines and vertigo. However, its antipsychotic properties have been previously hypothesized, particularly because of its possible ability to block the D2 dopamine receptors.

Dopamine neurons in the periaqueductal gray (PAG)/dorsal raphe are key modulators of antinociception with known supraspinal targets. However, no study has directly tested whether these neurons contribute to descending pain inhibition. We hypothesized that PAG dopamine neurons contribute to the analgesic effect of D-amphetamine via a mechanism that involves descending modulation via the rostral ventral medulla (RVM). Male C57BL/6 mice showed increased c-FOS expression in PAG dopamine neurons and a significant increase in paw withdrawal latency to thermal stimulation after receiving a systemic injection of D-amphetamine. Targeted microinfusion of D-amphetamine, L-DOPA, or the selective D2 agonist quinpirole into the PAG produced analgesia, while a D1 agonist, chloro APB, had no effect. In addition, inhibition of D2 receptors in the PAG by eticlopride prevented the systemic D-amphetamine analgesic effect. D-amphetamine and PAG D2 receptor-mediated analgesia were inhibited by intra-RVM injection of lidocaine or the GABA(A) receptor agonist muscimol, indicating a PAG-RVM signaling pathway in this model of analgesia. Finally, both systemic D-amphetamine and local PAG microinjection of quinpirole, inhibited inflammatory hyperalgesia induced by carrageenan. This hyperalgesia was transiently restored by intra-PAG injection of eticlopride, as well as RVM microinjection of muscimol. We conclude that D-amphetamine analgesia is partially mediated by descending inhibition and that D2 receptors in the PAG are responsible for this effect via modulating neurons that project to the RVM. These results further our understanding of the antinociceptive effects of dopamine and elucidate a mechanism by which clinically available dopamine modulators produce analgesia.

The rising popularity of alcohol mixed with energy drinks (AmEDs) has become a significant public health concern, with AmED users reporting higher levels of alcohol intake than non-AmED users. One mechanism proposed to explain this heightened level of alcohol intake in AmED users is that the high levels of caffeine found in energy drinks may increase the positive reinforcing properties of alcohol, an effect that may be dependent on interactions between adenosine receptor signaling pathways and the dopamine D2 receptor. Therefore, the purpose of the current study was to confirm whether caffeine does increase the positive reinforcing effects of alcohol using both fixed ratio (FR) and progressive ratio (PR) designs, and to investigate a potential role of the dopamine D2 receptor to caffeine-induced increases in alcohol self-administration. Male Long-Evans rats were trained to self-administer a sweetened alcohol solution (10% v/v alcohol + 2% w/v sucrose) on an FR2 schedule of reinforcement, and the effects of caffeine (0, 5, 10, and 20 mg/kg, i. p. [intraperitoneally]) on the maintenance of alcohol self-administration and alcohol break point were examined. Parallel experiments in rats trained to self-administer sucrose (0.8% w/v) were conducted to determine whether caffeine's reinforcement-enhancing effects extended to a non-drug reinforcer. Caffeine pretreatment (5–10 mg/kg) significantly increased sweetened alcohol self-administration and motivation for a sweetened alcohol reinforcer. However, similar increases in self-administration of a non-drug reinforcer were not observed. Contrary to our hypothesis, the D2 receptor antagonist eticlopride did not block a caffeine-induced increase in sweetened alcohol self-administration, nor did it alter caffeine-induced increases in motivation for a sweetened alcohol reinforcer. Taken together, these results support the hypothesis that caffeine increases the positive reinforcing effects of alcohol, which may explain caffeine-induced increases in alcohol intake. However, the reinforcement-enhancing effects of caffeine appear to be independent of D2 receptor function.

Propofol has shown strong addictive properties in rats and humans. Adenosine A2A receptors (A2AR) in the nucleus accumbens (NAc) modulate dopamine signal and addictive behaviors such as cocaine- and amphetamine-induced self-administration. However, whether A2AR can modulate propofol addiction remains unknown. AAV-shA2AR was intra-NAc injected 3 weeks before the propofol self-administration training to test the impacts of NAc A2AR on establishing the self-administration model with fixed ratio 1 (FR1) schedule. Thereafter, the rats were withdrawal from propofol for 14 days and tested cue-induced reinstatement of propofol seeking behavior on day 15. The propofol withdrawal rats received one of the doses of CGS21680 (A2AR agonist, 2.5–10.0 ng/site), MSX-3 (A2AR antagonist, 5.0–20.0 μg/site) or eticlopride (D2 receptor (D2R) antagonist, 0.75–3.0 μg/site) or vehicle via intra-NAc injection before relapse behavior test. The numbers of active and inactive nose-poke response were recorded. Focal knockdown A2AR by shA2AR did not affect the acquisition of propofol self-administration behavior, but enhance cue-induced reinstatement of propofol self-administration compared with the AAV-shCTRLgroup. Pharmacological activation of the A2AR by CGS21680 (≥ 5.0 ng/site) attenuated cue-induced reinstatement of propofol self-administration behavior. Similarly, pharmacological blockade of D2R by eticlopride (0.75–3.0 μg/site) attenuated propofol seeking behavior. These effects were reversed by the administration of MSX-3 (5.0–20.0 μg/site). The A2AR- and D2R-mediated effects on propofol relapse were not confounded by the learning process, and motor activity as the sucrose self-administration and locomotor activity were not affected by all the treatments. This study provides genetic and pharmacological evidence that NAc A2AR activation suppresses cue-induced propofol relapse in rats, possibly by interacting with D2R.

RATIONALE: The development of addiction is accompanied by a shift in the mechanisms motivating cocaine use from nucleus accumbens (NAc) dopamine D(1) receptor (D(1)R) signaling to glutamate AMPA-kainate receptor (AMPA-R) signaling. OBJECTIVE: Here we determined whether similar shifts occur for NAc-D(2)R signaling and following systemic manipulation of D(1)R, D(2)R, and AMPA-R signaling. METHODS: Male rats were given short-access (20 infusions/day) or extended-access to cocaine (24-hr/day, 96 infusions/day, 10 days). Motivation for cocaine was assessed following 14-days of abstinence using a progressive-ratio schedule. Once responding stabilized, the effects of NAc-D(2)R antagonism (eticlopride; 0-10.0 μg/side) and systemic D(1)R (SCH-23390; 0-1.0 mg/kg), D(2)R (eticlopride; 0-0.1 mg/kg), and AMPA-R (CNQX; 0-1.5 mg/kg) antagonism, and NAc-dopamine-R gene expression (Drd1/2/3) were examined. RESULTS: 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 (0.1-0.3-μg) were more effective in the short-access group and high doses (3-10-μg/side) tended to be more effective in the extended-access group. Systemic administration of eticlopride (0.1 mg/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. CONCLUSION: These findings indicate that in contrast to NAc-D(1)R, D(2)R 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.

Caffeine, the most commonly consumed psychoactive drug in the world, is readily available in dietary sources, including soft drinks, chocolate, tea and coffee. However, little is known about the neural substrates that underlie caffeine's rewarding and aversive properties and what ultimately leads us to seek or avoid caffeine consumption. Using male Wistar rats in a place conditioning procedure, we show that systemic caffeine at a low intraperitoneal dose of 2 mg/kg (or 100 µM injected directly into the rostral, but not caudal, portion of the ventral tegmental area) produced conditioned place preferences. By contrast, high doses of systemic caffeine at 10 and 30 mg/kg produced conditioned place aversions. These aversions were not recapitulated by a caffeine analog restricted to the periphery. Both caffeine reward and aversion were blocked by systemic D1-like receptor antagonism using SCH23390, while systemic D2-like receptor antagonism with eticlopride had smaller effects on caffeine motivation. Most important, we demonstrated that pharmacological blockade of dopamine receptors using α-flupenthixol injected into the nucleus accumbens shell, but not core, blocked caffeine-conditioned place preferences. Conversely, α-flupenthixol injected into the nucleus accumbens core, but not shell, blocked caffeine-conditioned place aversions. Thus, our findings reveal two dopamine-dependent and functionally dissociable mechanisms for processing caffeine motivation, which are segregated between nucleus accumbens subregions. These data provide novel evidence for the roles of the nucleus accumbens subregions in mediating approach and avoidance behaviours for caffeine.

Dopamine is a neurotransmitter of great physiological relevance. Disorders in dopaminergic signal transduction are associated with psychiatric and neurological pathologies such as Parkinson’s disease, schizophrenia and substance abuse. Therefore, a detailed understanding of dopaminergic neurotransmission may provide access to novel therapeutic strategies for the treatment of these diseases. Caged compounds with photoremovable groups represent molecular tools to investigate a biological target with high spatiotemporal resolution. Based on the crystal structure of the D3 receptor in complex with eticlopride, we have developed caged D2/D3 receptor ligands by rational design. We initially found that eticlopride, a widely used D2/D3 receptor antagonist, was photolabile and therefore is not suitable for caging. Subtle structural modification of the pharmacophore led us to the photostable antagonist dechloroeticlopride, which was chemically transformed into caged ligands. Among those, the 2-nitrobenzyl derivative 4 (MG307) showed excellent photochemical stability, pharmacological behavior and decaging properties when interacting with dopamine receptor-expressing cells.

Inner ear hair cells form synapses with afferent terminals and afferent neurons carry signals as action potentials to the central nervous system. Efferent neurons have their origins in the brainstem and some make synaptic contact with afferent dendrites beneath hair cells. Several neurotransmitters have been identified that may be released from efferent terminals to modulate afferent activity. Dopamine is a candidate efferent neurotransmitter in both the vestibular and auditory systems. Within the cochlea, activation of dopamine receptors may reduce excitotoxicity at the inner hair cell synapse via a direct effect of dopamine on afferent terminals. Here we investigated the effect of dopamine on sodium currents in acutely dissociated vestibular afferent calyces to determine if dopaminergic signaling could also modulate vestibular responses. Calyx terminals were isolated along with their accompanying type I hair cells from the cristae of gerbils (P15-33) and whole cell patch clamp recordings performed. Large transient sodium currents were present in all isolated calyces; compared to data from crista slices, resurgent Na+ currents were rare. Perfusion of dopamine (100 μM) in the extracellular solution significantly reduced peak transient Na+ currents by approximately 20% of control. A decrease in Na+ current amplitude was also seen with extracellular application of the D2 dopamine receptor agonist quinpirole, whereas the D2 receptor antagonist eticlopride largely abolished the response to dopamine. Inclusion of the phosphatase inhibitor okadaic acid in the patch electrode solution occluded the response to dopamine. The reduction in calyx sodium current in response to dopamine suggests efferent signaling through D2 dopaminergic receptors may occur via common mechanisms to decrease excitability in inner ear afferents.

The dopaminergic system is involved in a wide range of neuropsychiatric and neurodegenerative disorders. The lack of receptor subtype specificity is related to several pharmacological side effects that are observed during therapy among parkinsonian and schizophrenic patients. It is of paramount importance to search for new compounds that act on dopamine receptors with therapeutic potential, higher clinical effectiveness, and fewer adverse effects. In the present study, we performed a molecular docking study of D2, D3, and D4 receptor interactions with 92 metabolites from Curcuma longa using an in silico approach. We sought to identify compounds for possible drug development. A virtual library of compounds was built using molecules that were identified in the phytochemical characterization of C. longa. Protocols that were validated by redocking were applied to a virtual scan of this library using the Autodock-v4.2.3, Autodock Vina, and Molegro-v6.0 Virtual Docker programs, with four repetitions each. The three-dimensional structures of D2, D3, and D4 receptors in complex with risperidone, eticlopride, and nemonapride were obtained from the Protein Data Bank. Four compounds—stigmasterol, β-sitosterol, cholest-5-en-3-one, and cholestan-3-ol,2-methylene-(3β, 5α)—were the most likely to bind D2, D3, and D4 dopamine receptors, suggesting their potential for possible drug development. El sistema dopaminérgico está involucrado en una amplia gama de trastornos neuropsiquiátricos y neurodegenerativos. La falta de especificidad del subtipo de receptor está relacionada con varios efectos secundarios farmacológicos que se observan durante la terapia entre pacientes parkinsonianos y esquizofrénicos. Es de suma importancia buscar nuevos compuestos que actúen sobre los receptores de dopamina con potencial terapéutico, mayor efectividad clínica y menos efectos adversos. En el presente estudio, realizamos un estudio de acoplamiento molecular de las interacciones de los receptores D2, D3 y D4 con 92 metabolitos de Curcuma longa utilizando un enfoque in silico. Buscamos identificar compuestos para el posible desarrollo de fármacos. Se construyó una biblioteca virtual de compuestos utilizando moléculas que se identificaron en la caracterización fitoquímica de C. longa. Los protocolos que fueron validados por redocking se aplicaron a un escaneo virtual de esta biblioteca usando los programas Autodock-v4.2.3, Autodock Vina y Molegro-v6.0 Virtual Docker, con cuatro repeticiones cada uno. Las estructuras tridimensionales de los receptores D2, D3 y D4 en complejo con risperidona, eticloprida y nemonaprida se obtuvieron del Protein Data Bank. Cuatro compuestos (estigmasterol, β-sitosterol, colest-5-en-3-ona y colestán-3-ol, 2-metilen- (3β, 5α)) fueron los más propensos a unirse a los receptores de dopamina D2, D3 y D4 , lo que sugiere su potencial para el posible desarrollo de fármacos. O sistema dopaminérgico está envolvido em uma ampla gama de doenças neuropsiquiátricas e neurodegenerativas. A falta de especificidade do subtipo de receptor está relacionada a vários efeitos colaterais farmacológicos que são observados durante a terapia entre pacientes parkinsonianos e esquizofrênicos. É de suma importância a busca por novos compostos que atuem nos receptores da dopamina com potencial terapêutico, maior eficácia clínica e menor número de efeitos adversos. No presente estudo, realizamos um estudo de docagem molecular das interações dos receptores D2, D3 e D4 com 92 metabólitos da Curcuma longa usando uma abordagem in silico. Procuramos identificar compostos para possível desenvolvimento de drogas. Uma biblioteca virtual de compostos foi construída a partir de moléculas que foram identificadas na caracterização fitoquímica de C. longa. Os protocolos que foram validados por redocagem e foram aplicados a uma varredura virtual dessa biblioteca usando os programas Autodock-v4.2.3, Autodock Vina e Molegro-v6.0 Virtual Docker, com quatro repetições cada. As estruturas tridimensionais dos receptores D2, D3 e D4 em complexo com risperidona, eticloprida e nemonaprida foram obtidas no Protein Data Bank. Quatro compostos - estigmasterol, β-sitosterol, colest-5-en-3-ona e colestan-3-ol, 2-metileno- (3β, 5α) - foram os mais propensos a se ligar aos receptores de dopamina D2, D3 e D4 , sugerindo seu potencial para possível desenvolvimento de drogas.

The rewarding effect of opiates is mediated through dissociable neural systems in drug naïve and drug-dependent states. Neuroadaptations associated with chronic drug use are similar to those produced by chronic pain, suggesting that opiate reward could also involve distinct mechanisms in chronic pain and pain-naïve states. We tested this hypothesis by examining the effect of dopamine (DA) antagonism on morphine reward in a rat model of neuropathic pain.Neuropathic pain was induced in male Sprague-Dawley rats through chronic constriction (CCI) of the sciatic nerve; reward was assessed in the conditioned place preference (CPP) paradigm in separate groups at early (4-8 days post-surgery) and late (11-15 days post-surgery) phases of neuropathic pain. Minimal effective doses of morphine that produced a CPP in early and late phases of neuropathic pain were 6 mg/kg and 2 mg/kg respectively. The DA D1 receptor antagonist, SCH23390, blocked a morphine CPP in sham, but not CCI, rats at a higher dose (0.5 mg/kg), but had no effect at a lower dose (0.1 mg/kg). The DA D2 receptor antagonist, eticlopride (0.1 and 0.5 mg/kg), had no effect on a morphine CPP in sham or CCI rats, either in early or late phases of neuropathic pain. In the CPP paradigm, morphine reward involves DA D1 mechanisms in pain-naïve but not chronic pain states. This could reflect increased sensitivity to drug effects in pain versus no pain conditions and/or differential mediation of opiate reward in these two states.

D2 receptors (D2Rs) located in both pre- and postsynaptic membranes of medium spiny neurons (MSNs) in the nucleus accumbens (NAc) are involved in the stress response and associated behaviors. The role of D2Rs in chronic unpredictable stress (CUS)-induced depression-like behaviors is not clear. Quinpirole (a D2R agonist) and eticlopride (a D2R antagonist) were stereotactically delivered into the NAc before Sprague Dawley rats underwent CUS. CUS-induced depression-like behaviors were accompanied by a significant decrease in both the dopamine (DA) level and D2R expression in the NAc. Eticlopride reversed CUS-induced depression-like behavior and rescued the DA levels in the NAc, and microinjection of DA into the NAc of CUS individuals had the same effect as eticlopride. By contrast, delivery of quinpirole into the NAc of control animals induced depression-like behaviors accompanied by a decrease in the DA level in the NAc. These results show that DA plays a key role in CUS-induced depression-like behaviors and the D2R exerts a presynaptic negative feedback on DA levels during CUS. Microinjection of quinpirole into the NAc also decreased the level of the kalirin-7 protein in the NAc of both control and stressed animals, while eticlopride increased its level in the NAc of rats. In agreement with these results, intraperitoneal injection of eticlopride in mice also caused an increase in both the kalirin-7 protein level in the NAc and spine density in MSNs, while quinpirole reduced them. These results suggest that regulation of kalirin-7 through D2R in the NAc is a general pathway in rats and mice, and is involved in CUS-induced depression-like behaviors. Kalirin-7 may be directly regulated through the D2R postsynaptic pathway or indirectly through the presynaptic pathway in the NAc. The interaction between D2R and kalirin-7 needs to be investigated further.

Most studies on discriminative stimulus effects of 3,4-methylenedioxymethamphetamine (MDMA) have been conducted using a relatively low dose (1.5 mg/kg), and those studies have invariably implicated serotonergic mechanisms. In contrast, dopaminergic mechanisms mediate the discriminative stimulus effects of amphetamine (AMPH). Some studies have suggested that the discriminative stimulus effects of a higher (3.0 mg/kg) dose of MDMA might rely on both serotonergic and dopaminergic mechanisms. This study aimed to determine effects of selective dopamine (DA) and serotonin (5HT) antagonists on the discriminative stimulus properties of AMPH (0.5 mg/kg) and MDMA (3.0 mg/kg). Separate groups of rats were trained to discriminate AMPH (0.5 mg/kg) or MDMA (3.0 mg/kg) from saline using a food-reinforced drug-discrimination procedure. Effects of DA (SCH 23390: 0.003-0.03 mg/kg and eticlopride: 0.03-0.3 mg/kg) or 5HT (ritanserin: 1.0-10.0 mg/kg, WAY-100635: 0.3-1.0 mg/kg and GR129375: 1.0-3.0 mg/kg) antagonists on the discriminative stimulus effects of both drugs were determined. Both DA antagonists dose-dependently decreased the AMPH but not the MDMA discrimination. None of the 5HT antagonists altered the discriminative stimulus effects of either drug. The MDMA (3.0 mg/kg) stimulus comprises both a DAergic and 5HTergic response, and the results suggest that either one is sufficient, but not required, to maintain the stimulus effects.

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.

l-dopa and dopamine D2 receptor (D2R) agonists are commonly used to relieve the motor deficits of Parkinson's disease. However, long-term treatment with l-dopa or D2R agonists can induce adverse effects such as abnormal involuntary movements (AIMs), which are major limiting factors in achieving long-term control of parkinsonian syndromes. The pathophysiological mechanisms involved in the development of dopaminergic agonist-induced adverse effects are not well understood. Here, we examined the role of two D2R isoforms, D2S and D2L, in l-dopa-induced AIMs using dopamine D2L knockout (D2L KO) mice (expressing purely D2S) and wild-type mice (expressing predominantly D2L). We found that D2L KO mice displayed markedly enhanced AIMs in response to chronic treatment of l-dopa compared to wild-type mice. The l-dopa-induced enhancement of AIMs in D2L KO mice was significantly reduced by the D2R antagonist eticlopride. D2L KO mice also displayed markedly enhanced AIMs in response to chronic treatment with quinpirole, a preferential D2R agonist. These results suggest that D2S contributes more than D2L to dopaminergic agonist-induced AIMs. Our findings may uncover a new factor that contributes to the pathophysiology of dopaminergic drug-induced AIMs, a characteristic manifestation of dyskinesia and also present in psychosis. There is a possibility that the increased ratio of D2S to D2L in the brain plays a significant role in the development of AIM side effects induced by l-dopa or D2R agonists. See Video Abstract, http://links.lww.com/WNR/A622.

Dopamine (DA) is important for the performance of operant behavior as revealed by psychopharmacological studies that manipulate the activity of DA subtype receptors. However, the effects of SKF83959, an atypical DA D1 receptor agonist, on operant behavior and the underlying pharmacological mechanisms remain unclear. The present study sought to determine whether blockade of DA D1- and D2-subtyped receptors would reverse the operant behavior altered by SKF83959. Male rats were trained to respond on either a fixed-interval 30 s (FI30) schedule or a differential reinforcement of low-rate response 10 s (DRL10) schedule, two timing-relevant tasks but with distinct reinforcement contingencies. Pharmacological evaluation was conducted with injection of a selective D1 (or D2) receptor antagonist alone or in combined with SKF83959 (1.0 mg/kg) following a stable baseline of behavioral performance. The results showed that SKF83959 treatment alone significantly disrupted the performance of FI30 and DRL10 behaviors mainly by showing the decreases of the response-related measures, and the distinct profiles of the behavior altered by the drug were manifested by the qualitative analysis of inter-response time data on both tasks. The effects of SKF83959 were not significantly affected/reversed by the pretreatment of either SCH23390 or eticlopride injected at the doses of 0.02 and 0.06 mg/kg; however, a subtle reversal effect was observed in the treatment of low-dose eticlopride. Despite that these results confirm the FI30 and DRL10 behaviors changed by SKF83959, the absence of pharmacological reversal effect by DA receptor antagonist suggests that either D1- or D2-subtyped receptors may not play a critical role in the alteration of timing-relevant operant behavior produced by SKF83959.

The dopamine system is important for incentive salience attribution, where motivational value is assigned to conditioned cues that predict appetitive reinforcers. However, the role of dopamine in this process may change with extended training. We tested the effects of dopamine D1-like and D2-like receptor antagonism on the expression of sign-tracking and goal-tracking conditioned responses following extended Pavlovian conditioned approach (PCA) training. We also tested if amphetamine-induced psychomotor sensitization accelerates the enhanced acquisition of sign-tracking that is observed with extended training. In experiment 1, 24 male Long-Evans rats received 20 PCA sessions in which one lever (CS+, 10 s) predicted 0.2 mL sucrose (10%, w/v) delivery and the other lever (CS−) did not. SCH-23390 (D1-like antagonist) or eticlopride (D2-like antagonist) were administered before non-reinforced behavioural tests at doses of 0, 0.01, and 0.1 mg/kg (s.c.). In experiment 2, rats received vehicle or 2 mg/kg amphetamine (i.p.) for 7 days (n = 12/group). Ten days later, they received 16 PCA training sessions. Both doses of SCH-23390 reduced sign- and goal-tracking, but also reduced locomotor behaviour. A low dose of eticlopride (0.01 mg/kg) selectively reduced goal-tracking, without affecting sign-tracking or locomotor behaviour. Amphetamine produced psychomotor sensitization, and this did not affect the acquisition of sign- or goal-tracking. Following extended PCA training, dopamine D2-like receptor activity is required for the expression of goal-tracking but not sign-tracking. Psychomotor sensitization to amphetamine did not impact incentive salience attribution; however, more selective manipulations of the dopamine system may be needed.

The cerebellum receives dopaminergic innervation and expresses the five types of described dopaminergic receptors. The cerebellar function involves both motor movement and cognition, but the role of cerebellar dopaminergic system on these processes remain unclear. The present study explores the behavioral responses to intracerebellar microinjection of dopaminergic agents in motor and emotional memory. For this, naïve Swiss mice had their cerebellar vermis implanted with a guide canula, received a intravermis microinjection of Dopamine, D1-like antagonist SCH-23390 or D2-like antagonist Eticlopride, and underwent a behavioral analysis of motor learning (by a Rotarod and balance beam learning protocol) or aversive memory acquisition (by the inhibitory avoidance task). The mixed-effects analysis was used to evaluate groups performance, followed by Tukey’s post hoc when appropriated. In this study, Dopamine, SCH-23390 and Eticlopride at the doses used did not affected motor control and motor learning. In addition, the administration of Dopamine and SCH-233390 had no effects on emotional memory acquisition, but the animals that received the highest dose of Eticlopride had an improvement in aversive memory acquisition, shown by a suppression of its innate preference for the dark compartment of the inhibitory avoidance apparatus following an exposure to a foot shock. We propose that cerebellar dopaminergic D2 receptors seem to participate on the modulation of aversive memory processes, without influencing motor performance at the doses used in this study.

Background The release of dopamine (DA) in the posterior ventral tegmental area (pVTA) plays an important role in cue-related learning, reward, and relapse. On the other hand, studies have shown that the use of N-methyl-D-aspartate receptor (NMDAR) antagonist (AP5) inhibits the expression of morphine (5 mg/kg, s. c) conditioned place preference (CPP). In this study, we have tried to show the interaction effect of the DA stimulatory agents through D1-like receptor (D1R) agonist (SKF38393) and D2-like receptor (D2R) antagonist (eticlopride; through disinhibition) with NMDAR antagonist into the pVTA on the expression of morphine CPP. Materials and methods The SKF38393 and eticlopride, individually and simultaneously (in ineffective doses), were injected into the pVTA with the AP5 in rats, and animals were then placed in a CPP apparatus. Results Concomitant administration of D1R agonist (4 μg/rat) with NMDAR antagonist (1 μg/rat) induced the expression of morphine CPP, but the administration of D2R antagonist with NMDAR antagonist was unaffected on the expression of morphine CPP. Furthermore, concomitant administration of ineffective doses of D1R agonist and D2R antagonist with NMDAR antagonist had no effect on the expression of morphine CPP. Conclusions The results showed using higher doses of D1R agonist with NMDAR antagonist could reverse the blocked expression of morphine CPP by NMDAR antagonists, while, the use of D2R antagonist with NMDAR antagonist could not. Therefore, presynaptic receptors such as D1R probably through releasing other stimulatory neurotransmitters can play a vital role in the expression of morphine CPP and cue-related learning.

Schizophrenia is a long term psychosis caused by dysregulation of dopamine D2R. Existing antipsychotic drugs that alleviate positive symptoms of schizophrenia exhibit varied side effects for which designing antipsychotic drugs with reduced side effects remains an important therapeutic goal. This research project has been designed to develop potential amino acid and small molecule-based lead compounds for the reduction of schizophrenic symptoms using in silico drug design tools and methods. Use of LIGDREAM resulted in generation of 100 analogues of Eticlopride. Those with better ADMET properties were subjected to molecular docking against D2R receptor using AutoDock Vina and iGEM dock which yielded two analogues that had more substantial binding affinities for D2R than Eticlopride. NCS-1 is a signalling protein that desensitizes Dopamine D2R. Hence, a protein-based therapeutic compound for schizophrenia was being developed by conserved mutations of two amino acid residues on NCS1 (Phe55 and Leu189) by more hydrophilic and hydrophobic substituent, respectively. Protein-protein docking using the CLUSPRO docking portal and subsequent energy analysis have shown that increased hydrophilicity one had enhanced stabilization. Results obtained from both the modified Eticlopride leads and NCS1 mutations indicate that they can be collectively taken further downstream in anti-schizophrenia drug development.

The rising popularity of alcohol mixed with energy drinks (AmEDs) has become a significant public health concern, with AmED users reporting higher levels of alcohol intake than non-AmED users. One mechanism proposed to explain heightened levels of alcohol intake in AmED users is that the high levels of caffeine found in energy drinks may increase the reinforcing properties of alcohol, an effect which may be dependent on interactions between adenosine signaling pathways and the dopamine D2 receptor. Therefore, the purpose of the current study was to confirm whether caffeine increases the reinforcing efficacy of alcohol using both fixed ratio (FR) and progressive ratio (PR) designs, and to investigate a potential role of the dopamine D2 receptor in caffeine’s reinforcement-enhancing effects. Male Long Evans rats were trained to self-administer a sweetened alcohol or sucrose solution on an FR2 schedule of reinforcement. Pretreatment with caffeine (5-10 mg/kg) significantly increased operant responding for the sweetened alcohol reinforcer, but not sucrose. PR tests of motivation for alcohol or sucrose likewise confirmed a caffeine-dependent increase in motivation for a sweetened alcohol solution, but not sucrose. However, the D2 receptor antagonist eticlopride did not block the reinforcementenhancing effects of caffeine using either an FR or PR schedule of reinforcement. Taken together, these results support the hypothesis that caffeine increases the reinforcing efficacy of alcohol, which may explain caffeine-induced increases in alcohol intake. However, the reinforcement-enhancing effects of caffeine appear to be independent of D2 receptor function.

Context can influence reactions to environmental cues and this elemental process has implications for substance use disorder. Using an animal model, we show that an alcohol-associated context elevates entry into a fluid port triggered by a conditioned stimulus (CS) that predicted alcohol (CS-triggered alcohol-seeking). This effect persists across multiple sessions and, after it diminishes in extinction, the alcohol context retains the capacity to augment reinstatement. Systemically administered eticlopride and chemogenetic inhibition of ventral tegmental area (VTA) dopamine neurons reduce CS-triggered alcohol-seeking. Chemogenetically silencing VTA dopamine terminals in the nucleus accumbens (NAc) core reduces CS-triggered alcohol-seeking, irrespective of context, whereas silencing VTA dopamine terminals in the NAc shell selectively reduces the elevation of CS-triggered alcohol-seeking in an alcohol context. This dissociation reveals new roles for divergent mesolimbic dopamine circuits in the control of responding to a discrete cue for alcohol and in the amplification of this behaviour in an alcohol context., Alcohol craving can be enhanced by alcohol-associated cues and by alcohol-associated contexts. Here the authors investigate the role of the ventral tegmental area (VTA)-to-nucleus accumbens (NAc) core and VTA-to-NAc shell circuits in mediating these distinct aspects of alcohol seeking behaviour in rats.

The high functional plasticity of the glutamatergic synapse of the vestibular epithelium is supported by a delicate balance of excitatory and inhibitory interactions. One of the peptides co-localized with acetylcholine (ACh) in the efferent fibers is dopamine. Using external perfusion of the synaptic zone and multiunit recording of an afferent fibers activity the effect of dopamine antagonists on the background firing rate of the semicircular canal sensory fibers was studied on the isolated frog vestibular. The present research revealed that the dopamine receptor antagonist (D1) SCH-23390 significantly reduced the level of background activity at high concentrations. In contrast, D2 antagonist eticlopride caused positive-negative answer of the background activity of the sensory fibers in dose-depending manner. The data confirm the hypothesis that dopamine, being tonic released from the efferent fibers, realizes neuroprotective inhibitory control over the afferent glutamatergic synapse activity in the vestibular epithelium via D1 and D2 receptors.

By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands., eLife digest Almost a third of prescribed drugs work by acting on a group of proteins known as GPCRs (short for G-protein coupled receptors), which help to transmit messages across the cell’s outer barrier. The neurotransmitter dopamine, for instance, can act in the brain and body by attaching to dopamine receptors, a sub-family of GPCRs. The binding process changes the three-dimensional structure (or conformation) of the receptor from an inactive to active state, triggering a series of molecular events in the cell. However, GPCRs do not have a single ‘on’ or ‘off’ state; they can adopt different active shapes depending on the activating molecule they bind to, and this influences the type of molecular cascade that will take place in the cell. Some evidence also shows that classes of GPCRs can have different inactive structures; whether this is also the case for the dopamine D2 and D3 receptors remained unclear. Mapping out inactive conformations of receptors is important for drug discovery, as compounds called antagonists can bind to inactive receptors and interfere with their activation. Lane et al. proposed that different types of antagonists could prefer specific types of inactive conformations of the dopamine D2 and D3 receptors. Based on the structures of these two receptors, the conformations of D2 bound with the drugs risperidone and eticlopride (two dopamine antagonists) were simulated and compared. The results show that the inactive conformations of D2 were very different when it was bound to eticlopride as opposed to risperidone. In addition D2 and D3 showed a very similar conformation when attached to eticlopride. The two drugs also bound to the inactive receptors in overlapping but different locations. These computational findings, together with experimental validations, suggest that D2 and D3 exist in several inactive states that only allow the binding of specific drugs; these states could also reflect different degrees of inactivation. Overall, the work by Lane et al. contributes to a more refined understanding of the complex conformations of GPCRs, which could be helpful to screen and develop better drugs.

Methylenedioxypyrovalerone (MDPV) is an abused synthetic cathinone, commonly referred to as a “bath salt.” Because the dopamine (DA) transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2) are key regulators of both the abuse and neurotoxic potential of structurally and behaviorally related agents, the impact of MDPV on these transporters was investigated. Results revealed that a single in vivo MDPV administration rapidly (within 1 hour) and reversibly increased both rat striatal DAT and VMAT-2 activity, as assessed via [(3)H]DA uptake in synaptosomes and synaptic vesicles, respectively, prepared from treated rats. There was no evidence of an MDPV-induced increase in plasmalemmal membrane DAT surface expression. Plasma concentrations of MDPV increased dose-dependently as assessed 1 hour after 2.5 and 5.0 mg/kg (s.c.) administration and returned to levels less than 10 ng/ml by 18 hours after 2.5 mg/kg (s.c.). Neither pretreatment with a D1 receptor (SCH23390), a D2 receptor (eticlopride), nor a nicotinic receptor (mecamylamine) antagonist attenuated the MDPV-induced increase in DAT activity. In contrast, eticlopride pretreatment attenuated both the MDPV-induced increase in VMAT-2–mediated DA uptake and an associated increase in cytoplasmic-associated vesicle VMAT-2 immunoreactivity. SCH23390 did not attenuate the MDPV-induced increase in VMAT-2 activity. Repeated MDPV injections did not cause persistent DAergic deficits, as assessed 7 to 8 days later. The impact of MDPV on striatal and hippocampal serotonergic assessments was minimal. Taken together, these data contribute to a growing pharmacological rubric for evaluating the ever-growing list of designer cathinone-related stimulants. The profile of MDPV compared with related psychostimulants is discussed. SIGNIFICANCE STATEMENT: Pharmacological characterization of the synthetic cathinone, 3,4-methylenedioxypyrovalerone (MDPV; commonly referred to as a “bath salt”), is critical for understanding the abuse liability and neurotoxic potential of this and related agents. Accordingly, the impact of MDPV on monoaminergic neurons is described and compared with that of related psychostimulants.

Acetylcholine (ACh) neurotransmission is important for attention, while dopamine (DA) signaling modulates impulsive behavior. Prior studies have established an existing relationship between ACh and DA that mediates dopamine release in the prefrontal cortex of the brain in rats performing the 5-choice serial reaction time task (5-CSRTT). This study is aimed to identify cholinergic and dopaminergic interactions that govern attention and impulsive behavior, using adult Long-Evans rats of both sexes and a 5-CSRTT, with variable short and long cue delays. In Experiment 1, the effects of single cholinergic and dopaminergic drugs were evaluated on 5-CSRTT performance. Drugs like nicotinic ACh receptor (nAChR) agonist nicotine, amphetamine, and GBR12909 that increase the synaptic levels of ACh and DA respectively all increased impulsive behavior. In addition, amphetamine and GBR 12909 decreased attention while nicotine had no effect on attention. The antagonists mecamylamine, a general nAChR antagonist, flupenthixol a DA 1/2 receptor antagonist, and SCH 23390 a DA 1 receptor antagonist, all decreased impulsive behavior, with mixed effects on attention. In contrast, dihydro-β-erythroidine hydrobromide (DHBE), an α4β2 subunit-specific nAChR antagonist, had no significant effects on attention or impulsivity across doses administered. Eticlopride, a DA 2 receptor antagonist, decreased attention at the shortest cue delay but did not affect impulsivity. The acetylcholinesterase inhibitor donepezil decreased both attention and impulsive behavior. Subsequently in Experiment 2, effects of nicotine and amphetamine were determined after pretreatment with SCH 23390 or eticlopride. SCH 23390 attenuated the effects of nicotine and amphetamine to increase impulsivity, while eticlopride only attenuated the effect of nicotine on impulsivity. Minimal effects were seen on attention in the combination trials. This study confirms that dopamine D1 receptor plays an essential role in modulation of impulsive behavior, as measured by the 5-CSRTT. More importantly, it establishes that impulsive behavior is altered by interactions between cholinergic and dopaminergic neurotransmission.

Repeated exposure to psychostimulants during adolescence produces long-lasting changes in behavior that may be mediated by disrupted development of the mesocorticolimbic dopamine system. Here, we tested this hypothesis by assessing the effects of amphetamine (AMPH) and dopamine receptor-selective drugs on behavior and neuron activity in the prelimbic region of the medial prefrontal cortex (PFC). Adolescent male, Sprague-Dawley rats were given saline or 3 mg/kg AMPH between postnatal day (P) 27 and P45. In Experiment 1, locomotor behavior was assessed during adulthood following challenges with a dopamine D1 (SKF 82958) or D2 (quinpirole) receptor-selective agonist. In Experiment 2, pre-exposed rats were challenged during adulthood with AMPH and a D1 (SKF 83566) or D2 (eticlopride) receptor-selective antagonist. In Experiment 3, the activity of putative pyramidal cells in the prelimbic cortex was recorded as rats behaved in an open-field arena before and after challenge injections with AMPH and one of the antagonists. We found that compared to controls, adolescent pre-exposed rats were more sensitive to the stimulant effects of AMPH and the dopamine receptor agonists, as well as to the ability of the antagonists to reverse AMPH-induced stereotypy. Prelimbic neurons from AMPH pre-exposed rats were also more likely to respond to an AMPH challenge in adulthood, primarily by reducing their activity, and the antagonists reversed these effects. Our results suggest that exposure to AMPH during adolescence leads to enduring adaptations in the mesocorticolimbic dopamine system that likely mediate heightened response to the drug during adulthood.

Individuals vary in sensitivity to the behavioral effects of nicotine, resulting in differences in vulnerability to nicotine addiction. The role of rearing environment in determining individual sensitivity to nicotine is unclear. The neuropharmacological mechanisms mediating the effect of rearing environment on the behavioral actions of nicotine are also poorly understood. The contribution of rearing environment in determining the sensitivity to the interoceptive effects of nicotine was determined in rats reared in isolated conditions (IC) or enriched conditions (EC). The role of dopamine receptors and α4β2*-nicotinic acetylcholine (nACh) receptors in mediating the differential effect of IC and EC on the interoceptive action of nicotine was determined. The interoceptive action of nicotine was measured as the discriminative stimulus effect of nicotine. Mecamylamine- and eticlopride-inhibition of the nicotine stimulus were used to examine nACh and dopamine receptors, respectively. α4β2*-nACh receptor expression in the mesolimbic dopamine pathway was determined by quantitative autoradiography of [125I]-epibatidine binding. EC-reared rats are less sensitive than IC-reared rats to the discriminative stimulus effects of nicotine at all but maximally effective doses. Mecamylamine inhibited the nicotine stimulus threefold more potently in EC-reared rats (IC50 = 0.25 mg/kg) compared to IC-reared rats (IC50 = 0.75 mg/kg); eticlopride inhibition was not different. [125I]-epibatidine binding in the ventral tegmental area of EC-reared rats was reduced (2.8 ± 0.3 fmol) compared to that of IC-reared rats (4.0 ± 0.4 fmol); there was no difference in the nucleus accumbens. Rearing environment regulates the sensitivity to the interoceptive effects of nicotine and α4β2*-nACh receptor expression in the mesolimbic dopamine pathway.

Two key transmitters in the medial prefrontal cortex (mPFC), dopamine and acetylcholine, are believed to interact with each other to modulate local glutamatergic transmission, although molecular mechanisms underlying their crosstalk are poorly understood. Here we investigated effects of pharmacological manipulations of dopamine and muscarinic receptors on phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the adult rat mPFC in vivo. We found that an agonist selective for Gαs-coupled dopamine D1 receptors, SKF81297, increased AMPA receptor GluA1 subunit phosphorylation at a protein kinase A-sensitive site (S845), while SKF81297 had no effect on GluA1 phosphorylation at S831. An agonist for Gαi/o-coupled dopamine D2 receptors, quinpirole, also increased S845 but not S831 phosphorylation. When coinjected, the two agonists induced an additive increase in S845 phosphorylation. The D1 receptor antagonist SCH23390 blocked the SKF81297/quinpirole-stimulated S845 phosphorylation. The D2 antagonist eticlopride also partially blocked S845 responses to SKF81297/quinpirole. VU0152100, a positive allosteric modulator selective for Gαi/o-coupled muscarinic M4 receptors, reduced the S845 phosphorylation induced by SKF81297 and quinpirole injected alone or together. In contrast, coinjection of subthreshold doses of tropicamide, an M4 antagonist, and SKF81297 facilitated S845 phosphorylation. Additionally, coadministered SFK81297 and quinpirole increased the abundance of mPFC GluA1 at extrasynaptic sites. These data reveal that both D1 and D2 receptors upregulate GluA1 phosphorylation in mPFC neurons probably via a direct and indirect mechanism, respectively. The indirect mechanism involves M4 receptors which generally counteract the effect of dopamine on GluA1 phosphorylation.