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Development of novel treatments for motivational deficits experienced by individuals with schizophrenia and major depressive disorder requires procedures that reliably assess effort-related behavior in pre-clinical models. High-throughput touchscreen-based testing, that parallels the computerized assessment of human patients, offers a platform for the establishment of tasks with high level of translational validity. Considerable efforts have been made to validate the touchscreen version of tasks that measure the degree of effort an animal is willing to invest for a reward, such as progressive ratio task. While motivational studies primarily focus on reporting alterations of a breakpoint, touchscreen assessment allows to collect multiple measures, especially if additional tasks would be adapted to the touchscreen environment. Classifying these measures to distinct behavioral subdomains is necessary for an evaluation of pre-clinical models. Here we apply data-driven classification techniques to identify behavioral clusters from dataset obtained in progressive ratio task and a novel effort-related choice task that we established and validated in the touchscreen boxes. Moreover, we measure the effect of pharmacological manipulations of the level of dopamine, a key regulator of reward- and effort-related processing, on individual behavioral subdomains that describe effort-related activity, non-specific activity, locomotion, and effort-related choice. Our approach expands the touchscreen-based assessment of pre-clinical models of motivational symptoms, identifies the most relevant behavioral measures in assessing the degree of reward-driven effort and contributes to the understanding of the role of dopamine in mediating distinct aspects of effort-related motivation.

Background Motor complications, characterized by “off” periods – when anti-parkinsonian medications are ineffective – and dyskinesia, are the hallmark of advanced Parkinson's disease (PD). While levodopa is the gold standard PD medication in terms of efficacy, its short duration of effect coupled with progressive loss of dopaminergic neurons leads to motor complications and fails to treat off periods. Purpose of review This review focuses on novel dopaminergic therapies that were recently made clinically available or are currently in development for the treatment of motor complications. First, it will discuss rescue therapies for the treatment of off episodes, including novel apomorphine and levodopa formulations. Second, it will highlight adjunctive dopaminergic medications approved to reduce total daily off time. Third, it will discuss longer-acting levodopa formulations in development and introduce a novel selective dopamine agonist under study. Finally, it will cover novel dopaminergic delivery mechanisms, with specific focus on continuous subcutaneous infusions in development. Summary The breadth of dopaminergic therapies recently approved or in development for motor complications, and specifically off time reduction, evokes cautious optimism. Gains in reducing off time with rescue therapies, adjunctive medications or longer-acting levodopa formulations are modest, and underscore the need for more continuous dopaminergic delivery to address the underlying pathophysiology and translate to clinically meaningful improvement in motor complications.

Impairment in various aspects of cognition is recognized as an important non-motor symptom of Parkinson's disease (PD). Mild cognitive impairment in PD (PD-MCI) is common in non-demented PD patients and is often associated with severity of motor symptoms, disease duration and increasing age. Further, PD-MCI can have a significant negative effect on performance of daily life activities and may be a harbinger of development of PD dementia. Thus, there is significant interest in developing therapeutic strategies to ameliorate cognitive deficits in PD and improve cognitive functioning of PD patients. However, due to significant questions that remain regarding the pathophysiology of cognitive dysfunction in PD, remediation of cognitive dysfunction in PD has proven difficult. In this paper, we will focus on PD-MCI and will review some of the current therapeutic approaches being taken to try to improve cognitive functioning in patients with PD-MCI.

Recent findings from this laboratory demonstrate that ethanol reduces the intrinsic excitability of orbitofrontal cortex (OFC) neurons via activation of strychnine-sensitive glycine receptors. Although the mechanism linking ethanol to the release of glycine is currently unknown, astrocytes are a source of neurotransmitters including glycine and activation of dopamine D1-like receptors has been reported to enhance extracellular levels of glycine via a functional reversal of the astrocytic glycine transporter GlyT1. We recently reported that like ethanol, dopamine or a D1/D5 receptor agonist increases a tonic current in lateral OFC (lOFC) neurons. Therefore, in this study, we used whole-cell patch-clamp electrophysiology to examine whether ethanol inhibition of OFC spiking involves the release of glycine from astrocytes and whether this release is dopamine receptor dependent. Ethanol, applied acutely, decreased spiking of lOFC neurons and this effect was blocked by antagonists of GlyT1, the norepinephrine transporter or D1-like but not D2-like receptors. Ethanol enhanced the tonic current of OFC neurons and occluded the effect of dopamine suggesting that ethanol and dopamine may share a common pathway. Altering astrocyte function by suppressing intracellular astrocytic calcium signaling or blocking the astrocyte-specific Kir4.1 potassium channels reduced but did not completely abolish ethanol inhibition of OFC neuron firing. However, when both astrocytic calcium signaling and Kir4.1 channels were inhibited, ethanol had no effect on firing. Ethanol inhibition was also prevented by inhibitors of phospholipase C and conventional isoforms of protein kinase C (cPKC) previously shown to block D1R-induced GlyT1 reversal and PKC inhibition of Kir4.1 channels. Finally, the membrane potential of OFC astrocytes was depolarized by bath application of a Kir4.1 blocker, a D1 agonist or ethanol and ethanol effect was blocked by a D1 antagonist. Together, these findings suggest that acute ethanol inhibits OFC neuron excitability via a D1 receptor-mediated dysregulation of astrocytic glycine transport.

Abuse of psychostimulants like cocaine that inhibit dopamine (DA) reuptake through the dopamine transporter (DAT) represents a major public health issue, however FDA-approved pharmacotherapies have yet to be developed. Recently a class of ligands termed “atypical DAT inhibitors” has gained attention due to their range of effectiveness in increasing extracellular dopamine (DA) levels without demonstrating significant abuse liability. These compounds not only hold promise as therapeutic agents to treat stimulant use disorders but also as experimental tools to improve our understanding of DAT function. Here we used patch clamp electrophysiology in mouse brain slices to explore the effects of two atypical DAT inhibitors (R-modafinil and JHW 007) on the physiology of single DA neurons in the substantia nigra and ventral tegmental area. Despite their commonalities of being DAT inhibitors that lack cocaine-like behavioral profiles, these compounds exhibited surprisingly divergent cellular effects. Similar to cocaine, R-modafinil slowed DA neuron firing in a D2 receptor-dependent manner and rapidly enhanced the amplitude and duration of D2 receptor-mediated currents in the midbrain. In contrast, JHW 007 exhibited little effect on firing, slow DAT blockade, and an unexpected inhibition of D2 receptor-mediated currents that may be due to direct D2 receptor antagonism. Furthermore, pretreatment with JHW 007 blunted the cellular effects of cocaine, suggesting that it may be valuable to investigate similar DAT inhibitors as potential therapeutic agents. Further exploration of these and other atypical DAT inhibitors may reveal important cellular effects of compounds that will have potential as pharmacotherapies for treating cocaine use disorders.

Dopaminergic signaling and neurodevelopment alterations are associated with several neuropsychiatric disorders. Knockout mice for dopamine transporters (DAT) as well as site-specific knockout mice lacking dopaminergic D2 autoreceptors in dopaminergic neurons (DA-D2RKO) display behavioral alterations such as hyperlocomotion and abnormal prepulse inhibition. However, it is possible that dopaminergic imbalances may have different effects during varied neurodevelopmental windows. In our previous study, we observed that elevated levels of dopamine during the perinatal developmental window increased exploratory behavior of juvenile (4-week-old) Swiss female mice and impaired hedonic behavior in males. In this study, we investigated whether these behavioral alterations persist through young adulthood. In order to do so, we administered daily doses of l-Dopa to mice pups beginning from postnatal day 1 (PD1) to PD5. At the age of 8 weeks, we submitted the young adult males and females to the open field test, elevated plus maze, forced swimming test, and sucrose preference test. We observed that augmentation of dopamine levels during the perinatal developmental window increased locomotor behavior in females, but not males. We also observed an increase in anxiety-behavior in females and anxiolytic-like behavior in males. In addition, we observed stress-coping behavior in males and an increase of hedonic behavior in females. Our results show that dopamine signaling is important for behavioral development and that transient imbalances of dopamine levels can cause permanent behavioral alterations - alterations which are different in males than in females. These data may help in better understanding the spectrum of symptoms associated with different neuropsychiatric disorders.

Selective blockade of serotonin 2A (5-HT

Histone deacetylase 6 (Hdac6), a multifunctional cytoplasmic deacetylase, is abundant in brain. We previously demonstrated that global Hdac6 depletion causes aberrant emotional behaviors in mice. Identification of affected brain systems and its molecular basis will lead to new insights into relations between protein acetylation events and psychiatric disorders. Here we report the dopaminergic abnormalities in Hdac6 KO mice. The dopamine transmission mediated by D1-like and D2-like G protein-coupled dopamine receptors is known to play roles in controlling movement, cognition, and motivational processes, and its dysfunction causes psychiatric disorders. We found that Hdac6 KO mice showed significantly increased locomotor response to novel, but not to habituated environment. In addition, Hdac6 KO mice showed a long-lasting sensitivity to psychostimulants, increased locomotor response to D2-like, but not D1 dopamine receptor agonists, and rapid locomotor response to apomorphine, a direct dopamine agonist, in dopamine-depleted condition. Hdac6 protein was expressed in dopaminergic neurons and their terminals in adult mice brain, and Hdac6-depletion augmented acetylation levels of dopamine-enriched synaptosomal proteins. In Hdac6 KO mice, the striatal content of dopamine and its metabolites was normal in basal condition, but mRNA level of D2 dopamine receptor in the striatum was decreased by 30%. Taken together, our results provide evidence that Hdac6 deficiency leads to aberrant dopamine-dependent behaviors by enhancing postsynaptic dopamine D2 receptor response. This study points out the possibility that Hdac6 and reversible-acetylation events play a regulatory role in D2 dopamine receptor signaling, and thus participate in the pathology of the dopamine-related psychiatric disorders such as schizophrenia.

Recessive mutations in the PTEN-induced putative kinase 1 (PINK1) gene cause early-onset Parkinson's disease (PD). We investigated the interaction between endocannabinoid (eCB) and dopaminergic transmission at corticostriatal synapses in PINK1 deficient mice. Whole-cell patch-clamp and conventional recordings of striatal medium spiny neurons (MSNs) were made from slices of PINK1(-/-), heterozygous PINK1(+/-) mice and wild-type littermates (PINK1(+/+)). In PINK1(+/+) mice, CB1 receptor (CB1R) activation reduced spontaneous excitatory postsynaptic currents (sEPSCs). Likewise, CB1R agonists (ACEA, WIN55,212-3 and HU210) induced a dose-dependent reduction of cortically-evoked excitatory postsynaptic potential (eEPSP) amplitude. While CB1R agonists retained their inhibitory effect in heterozygous PINK1(+/-) mice, conversely, in PINK1(-/-) mice they failed to modulate sEPSC amplitude. Similarly, CB1R activation failed to reduce eEPSP amplitude in PINK1(-/-) mice. Parallel biochemical measurements revealed no significant difference in the levels of the two main eCBs, 2-arachidonoylglycerol (2-AG) and anandamide (AEA) in PINK1(-/-) striata. Similarly, no change was observed in the enzymatic activity of both fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), responsible for eCB hydrolysis. Instead, a significant reduction of binding ability of CB1R agonists was found in PINK1(-/-) mice. Notably, the CB1R-dependent inhibition of synaptic activity was restored either by amphetamine or after chronic treatment with the D2 dopamine receptor agonist quinpirole. Additionally, CB1R binding activity returned to control levels after chronic pretreatment with quinpirole. Consistent with the hypothesis of a close interplay with dopaminergic neurotransmission, our findings show a CB1R dysfunction at corticostriatal synapses in PINK1(-/-), but not in PINK1(+/-) mice, and provide a mechanistic link to the distinct plasticity deficits observed in both genotypes.

© 2020 Elsevier Ltd To date, there are no interventions that impede the inexorable progression of Alzheimer's disease (AD), and currently-available drugs cholinesterase (AChE) inhibitors and the N-Methyl-D-Aspartate receptor antagonist, memantine, offer only modest symptomatic benefit. Moreover, a range of mechanistically-diverse agents (glutamatergic, histaminergic, monoaminergic, cholinergic) have disappointed in clinical trials, alone and/or in association with AChE inhibitors. This includes serotonin (5-HT) receptor-6 antagonists, despite compelling preclinical observations in rodents and primates suggesting a positive influence on cognition. The emphasis has so far been on high selectivity. However, for a multi-factorial disorder like idiopathic AD, 5-HT6 antagonists possessing additional pharmacological actions might be more effective, by analogy to “multi-target” antipsychotics. Based on this notion, drug discovery programmes have coupled 5-HT6 blockade to 5-HT4 agonism and inhibition of AchE. Further, combined 5-HT6/dopamine D3 receptor (D3) antagonists are of especial interest since D3 blockade mirrors 5-HT6 antagonism in exerting broad-based pro-cognitive properties in animals. Moreover, 5-HT6 and dopamine D3 antagonists promote neurocognition and social cognition via both distinctive and convergent actions expressed mainly in frontal cortex, including suppression of mTOR over-activation and reinforcement of cholinergic and glutamatergic transmission. In addition, 5-HT6 blockade affords potential anti-anxiety, anti-depressive and anti-epileptic properties, and antagonising 5-HT6 receptors may be associated with neuroprotective (“disease-modifying”) properties. Finally D3 antagonism may counter psychotic episodes and D3 receptors themselves offer a promising hub for multi-target agents. The present article reviews the status of “R and D” into multi-target 5-HT6 and D3 ligands for improved treatment of AD and other neurodegenerative disorders of aging. This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

Presentation of non-aversive light stimuli for several seconds was found to reliably induce locomotor activation and exploratory-like activity. Light-induced locomotor activity (LIA) can be considered a convenient simple model to study sensory-motor activation. LIA was previously shown to coincide with serotonergic and dopaminergic activation in specific cortical areas in freely moving and anesthetized animals. In the present study we explore the neuropharmacology of LIA using a receptor antagonist/agonist approach in rats. The non-selective 5-HT 2 -receptor antagonist ritanserin (1.5–6 mg/kg, i.p.) dose-dependently reduced LIA. Selective antagonism of either the 5-HT 2A -receptor by MDL 11,939 (0.1–0.4 mg/kg, i.p.), or the 5-HT 2C -receptor by SDZ SER 082 (0.125–0.5 mg/kg, i.p.), alone or in combination, had no significant influence on LIA. Also the selective 5-HT 1A -receptor antagonist, WAY 100635 (0.4 mg/kg, i.p.) did not affect LIA. Neither did the preferential dopamine D2-receptor antagonist, haloperidol (0.025–0.1 mg/kg, i.p.) nor the D2/D3-receptor agonist, quinpirole (0.025–0.5 mg/kg, i.p.) affect the expression of LIA. However, blocking the glutamatergic NMDA-receptor with phencyclidine (PCP, 1.5–6 mg/kg, i.p.) dose-dependently reduced LIA. This effect was also observed with ketamine (10 mg/kg, i.p.). These findings suggest that serotonin and dopamine receptors abundantly expressed in the cortex do not mediate light-stimulus triggered locomotor activity. PCP and ketamine effects, however, suggest an important role of NMDA receptors in LIA.

Others and we have reported that prior methamphetamine (METH) exposure attenuates the persistent striatal dopaminergic deficits caused by a subsequent high-dose "binge" METH exposure. The current study investigated intermediate neurochemical changes that may contribute to, or serve to predict, this resistance. Rats self-administered METH or saline for 7 d. On the following day (specifically, 16 h after the conclusion of the final METH self-administration session), rats received a binge exposure of METH or saline (so as to assess the impact of prior METH self-administration), or were sacrificed without a subsequent METH exposure (i.e., to assess the status of the rats at what would have been the initiation of the binge METH treatment). Results revealed that METH self-administration per se decreased striatal dopamine (DA) transporter (DAT) function and DA content, as assessed 16 h after the last self-administration session. Exposure to a binge METH treatment beginning at this 16-h time point decreased DAT function and DA content as assessed 1 h after the binge METH exposure: this effect on DA content (but not DAT function) was attenuated if rats previously self-administered METH. In contrast, 24 h after the binge METH treatment prior METH self-administration: 1) attenuated deficits in DA content, DAT function and vesicular monoamine transporter-2 function; and 2) prevented increases in glial fibrillary acidic protein and DAT complex immunoreactivity. These data suggest that changes 24 h, but not 1 h, after binge METH exposure are predictive of tolerance against the persistence of neurotoxic changes following binge METH exposures.

Stimulation of D1-like dopamine receptors (D1DRs) or D2-like dopamine receptors (D2DRs) in the nucleus accumbens (NAc) shell reinstates cocaine seeking in rats, an animal model of relapse. D2DRs and D1DRs activate protein kinase C (PKC) and recent studies indicate that activation of PKC in the NAc plays an important role in the reinstatement of drug seeking induced by a systemic cocaine priming injection. In the present study, pharmacological inhibition of PKC in the NAc shell attenuated cocaine seeking induced by intra-accumbens shell microinjection of a D2DR agonist, but not a D1DR agonist. D1DRs and D2DRs are primarily expressed on different accumbens medium spiny (MSN) neurons. Neuronal signaling and activity were assessed in these two populations of NAc neurons with transgenic mice expressing fluorescent labels under the control of D1DR and D2DR promoters. Following the extinction of cocaine self-administration, bath application of a PKC inhibitor produced similar effects on single evoked excitatory and inhibitory post-synaptic currents in D1DR- and D2DR-positive MSNs in the NAc shell. However, inhibition of PKC preferentially improved the ability of excitatory, but not inhibitory, synapses to sustain responding to brief train of stimuli specifically in D2DR-positive MSNs. This effect did not appear to involve modulation of presynaptic release mechanisms. Taken together, these findings indicate that the reinstatement of cocaine seeking is at least partially due to D2DR-dependent increases in PKC signaling in the NAc shell, which reduce excitatory synaptic efficacy in D2DR-expressing MSNs.

The focus of this study was the regulation of the D2-like dopamine autoreceptor (D2 autoreceptor) by protein kinase Cβ, a member of the protein kinase C (PKC) family. Together with the dopamine transporter, the D2 autoreceptor regulates the level of extracellular dopamine and thus dopaminergic signaling. PKC regulates neuronal signaling via several mechanisms, including desensitizing autoreceptors to increase the release of several different neurotransmitters. Here, using both PKCβ(-/-) mice and specific PKCβ inhibitors, we demonstrated that a lack of PKCβ activity enhanced the D2 autoreceptor-stimulated decrease in dopamine release following both chemical and electrical stimulations. Inhibition of PKCβ increased surface localization of D2R in mouse striatal synaptosomes, which could underlie the greater sensitivity to quinpirole following inhibition of PKCβ. PKCβ(-/-) mice displayed greater sensitivity to the quinpirole-induced suppression of locomotor activity, demonstrating that the regulation of the D2 autoreceptor by PKCβ is physiologically significant. Overall, we have found that PKCβ downregulates the D2 autoreceptor, providing an additional layer of regulation for dopaminergic signaling. We propose that in the absence of PKCβ activity, surface D2 autoreceptor localization and thus D2 autoreceptor signaling is increased, leading to less dopamine in the extracellular space and attenuated dopaminergic signaling.

The long held view is cocaine's pharmacological effects are mediated by monoamine reuptake inhibition. However, drugs with rapid brain penetration like sibutramine, bupropion, mazindol and tesofensine, which are equal to or more potent than cocaine as dopamine reuptake inhibitors, produce no discernable subjective effects such as drug "highs" or euphoria in drug-experienced human volunteers. Moreover they are dysphoric and aversive when given at high doses. In vivo experiments in animals demonstrate that cocaine's monoaminergic pharmacology is profoundly different from that of other prescribed monoamine reuptake inhibitors, with the exception of methylphenidate. These findings led us to conclude that the highly unusual stimulant profile of cocaine and related compounds, eg methylphenidate, is not mediated by monoamine reuptake inhibition alone. We describe the experimental findings which suggest cocaine serves as a negative allosteric modulator to alter the function of the dopamine reuptake transporter (DAT) and reverse its direction of transport. This results in a firing-dependent, retro-transport of dopamine into the synaptic cleft. The proposed mechanism of cocaine is, therefore, different from other small molecule negative allostereric modulators of the monoamine reuptake transporters, eg SoRI-6238, which merely reduce the rate of inward transport. Because the physiological role of DAT is to remove dopamine from the synapse and the action of cocaine is the opposite of this, we have postulated that cocaine's effect is analogous to an inverse agonist. If this hypothesis is validated then cocaine is the prototypical compound that exemplifies a new class of monoaminergic drugs; DAT "inverse agonists". This article is part of the Special Issue entitled 'CNS Stimulants'.

Examination of a drug's abuse potential at multiple levels of analysis (molecular/cellular action, whole-organism behavior, epidemiological data) is an essential component to regulating controlled substances under the Controlled Substances Act (CSA). We reviewed studies that examined several central nervous system (CNS) stimulants, focusing on those with primarily dopaminergic actions, in drug self-administration, drug discrimination, and physical dependence. For drug self-administration and drug discrimination, we distinguished between experiments conducted with rats and nonhuman primates (NHP) to highlight the common and unique attributes of each model in the assessment of abuse potential. Our review of drug self-administration studies suggests that this procedure is important in predicting abuse potential of dopaminergic compounds, but there were many false positives. We recommended that tests to determine how reinforcing a drug is relative to a known drug of abuse may be more predictive of abuse potential than tests that yield a binary, yes-or-no classification. Several false positives also occurred with drug discrimination. With this procedure, we recommended that future research follow a standard decision-tree approach that may require examining the drug being tested for abuse potential as the training stimulus. This approach would also allow several known drugs of abuse to be tested for substitution, and this may reduce false positives. Finally, we reviewed evidence of physical dependence with stimulants and discussed the feasibility of modeling these phenomena in nonhuman animals in a rational and practical fashion. This article is part of the Special Issue entitled 'CNS Stimulants'.

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.

SKF-83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine] is reported to be a functionally selective dopamine D1 receptor ligand with high bias for D1-mediated phospholipase C (PLC) versus D1-coupled adenylate cyclase signaling. This signaling bias is proposed to explain behavioral activity in both rat and primate Parkinson’s disease models, and a D1-D2 heterodimer has been proposed as the underlying mechanism. We have conducted an in-depth pharmacological characterization of this compound in dopamine D1 and D2 receptors in both rat brain and heterologous systems expressing human D1 or D2 receptors. Contrary to common assumptions, SKF-83959 is similar to the classical, well-characterized partial agonist SKF38393 in all systems. It is a partial agonist (not an antagonist) at adenylate cyclase in vitro and ex vivo, and is a partial agonist in D1-mediated β-arrestin recruitment. Contrary to earlier reports, it does not have D1-mediated effects on PLC signaling in heterologous systems. Because drug metabolites can also contribute, its 3-N-demethylated analog also was synthesized and tested. As expected from the known structure-activity relationships of the benzazepines, this compound also had high affinity for the D1 receptor and somewhat higher intrinsic activity than the parent ligand, and also might contribute to in vivo effects of SKF-83959. Together, these data demonstrate that SKF-83959 is not a highly-biased functionally selective D1 ligand, and that its reported behavioral data can be explained solely by its partial D1 agonism in canonical signaling pathway(s). Mechanisms that have been proposed based on the purported signaling novelty of SKF-83959 at PLC should be reconsidered.

Dopamine abnormalities underlie a wide variety of psychopathologies, including ADHD and schizophrenia. A new imaging technique, pharmacological magnetic resonance imaging (phMRI), is a promising non-invasive technique to visualize the dopaminergic system in the brain. In this review we explore the clinical potential of phMRI in detecting dopamine dysfunction or neurotoxicity, assess its strengths and weaknesses and identify directions for future research. Preclinically, phMRI is able to detect severe dopaminergic abnormalities quite similar to conventional techniques such as PET and SPECT. phMRI benefits from its high spatial resolution and the possibility to visualize both local and downstream effects of dopaminergic neurotransmission. In addition, it allows for repeated measurements and assessments in vulnerable populations. The major challenge is the complex interpretation of phMRI results. Future studies in patients with dopaminergic abnormalities need to confirm the currently reviewed preclinical findings to validate the technique in a clinical setting. Eventually, based on the current review we expect that phMRI can be of use in a clinical setting involving vulnerable populations (such as children and adolescents) for diagnosis and monitoring treatment efficacy. This article is part of the Special Issue Section entitled ‘Neuroimaging in Neuropharmacology’.

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) expressed in several mammalian brain areas and activated by “trace amines” (TAs). TAs role is unknown; however, discovery of their receptors provided an opportunity to investigate their functions. In vivo evidence has indicated an inhibitory influence of TAAR1 on dopamine (DA) neurotransmission, presumably via modulation of dopamine transporter (DAT) or interaction with the D2 DA receptor and/or activation of inwardly rectifying K + channels. To elucidate the mechanisms of TAAR1-dependent modulation, we used TAAR1 knockout mice (TAAR1-KO), a TAAR1 agonist (RO5166017) and a TAAR1 antagonist (EPPTB) in a set of neurochemical experiments. Analysis of the tissue content of TAAR1-KO revealed increased level of the DA metabolite homovanillic acid (HVA), and in vivo microdialysis showed increased extracellular DA in the nucleus accumbens (NAcc) of TAAR1-KO. In fast scan cyclic voltammetry (FSCV) experiments, the evoked DA release was higher in the TAAR1-KO NAcc. Furthermore, the agonist RO5166017 induced a decrease in the DA release in wild-type that could be prevented by the application of the TAAR1 antagonist EPPTB. No alterations in DA clearance, which are mediated by the DAT, were observed. To evaluate the interaction between TAAR1 and D2 autoreceptors, we tested the autoreceptor-mediated dynamics. Only in wild type mice, the TAAR1 agonist was able to potentiate quinpirole-induced inhibitory effect on DA release. Furthermore, the short-term plasticity of DA release following paired pulses was decreased in TAAR1-KO, indicating less autoinhibition of D2 autoreceptors. These observations suggest a close interaction between TAAR1 and the D2 autoreceptor regulation.

Most socially living species are organized hierarchically, primarily based on individual differences in social dominance. Dominant individuals typically gain privileged access to important resources, such as food, mating partners and territories, whereas submissive conspecifics are often devoid of such benefits. The benefits associated with a high social status provide a strong incentive to become dominant. Importantly, motivational- and reward-related processes are regulated, to a large extent, by the mesolimbic system. Consequently, several studies point to a key role for the mesolimbic system in social hierarchy formation. This review summarizes the growing body of literature that implicates the mesolimbic system, and associated neural circuits, on social hierarchies. In particular, we discuss the neurochemical and pharmacological studies that have highlighted the contributions of the mesolimbic system and associated circuits including dopamine signaling through the D1 or D2 receptors, GABAergic neurotransmission, the androgen receptor system, and mitochondria and bioenergetics. Given that low social status has been linked to the emergence of anxiety- and depressive-like disorders, a greater understanding of the neurochemistry underlying social dominance could be of tremendous benefit for the development of pharmacological treatments to dysfunctions in social behaviors. This article is part of the Special Issue entitled 'The neuropharmacology of social behavior: from bench to bedside'.

Cocaine administration can be both rewarding and aversive. While much effort has gone to investigating the rewarding effect, the mechanisms underlying cocaine-induced aversion remain murky. There is increasing evidence that the lateral habenula (LHb), a small epithalamic structure, plays a critical role in the aversive responses of many addictive drugs including cocaine. However, the effects of cocaine on LHb neurons are not well explored. Here we show that, in acute brain slices from rats, cocaine depolarized LHb neurons and accelerated their spontaneous firing. The AMPA and NMDA glutamate receptor antagonists, 6, 7-dinitroquinoxaline-2, 3-dione, DL-2-amino-5-phosphono-valeric acid, attenuated cocaine-induced acceleration. In addition, cocaine concentration-dependently enhanced glutamatergic excitation: enhanced the amplitude but reduced the paired pulse ratio of EPSCs elicited by electrical stimulations, and increased the frequency of spontaneous EPSCs in the absence and presence of tetrodotoxin. Dopamine and the agonists of dopamine D1 (SKF 38393) and D2 (quinpirole) receptors, as well as the dopamine transporter blocker (GBR12935), mimicked the effects of cocaine. Conversely, both D1 (SKF 83566) and D2 (raclopride) antagonists substantially attenuated cocaine’s effects on EPSCs and firing. Together, our results provide evidence that cocaine may act primarily via an increase in dopamine levels in the LHb that activates both D1 and D2 receptors on the glutamatergic terminals which make synapses on LHb neurons. This leads to an increase in glutamate release and LHb neuron activity, and may contribute to the aversive effect of cocaine observed in vivo.

N-oleoyl-dopamine (OLDA) is an amide of dopamine and oleic acid, synthesized in catecholaminergic neurons. The present study investigates OLDA targets in midbrain dopaminergic (DA) neurons. Substantia Nigra compacta (SNc) DA neurons recorded in brain slices were excited by OLDA in wild type mice. In transient receptor potential vanilloid 1 (TRPV1) knockout (KO) mice, however, SNc DA neurons displayed sustained inhibition of firing. In the presence of the dopamine type 2 receptor (D2R) antagonist sulpiride or the dopamine transporter blocker nomifensine no such inhibition was observed. Under sulpiride OLDA slightly excited SNc DA neurons, an action abolished upon combined application of the cannabinoid1 and 2 receptor antagonists AM251 and AM630. In ventral tegmental area (VTA) DA neurons from TRPV1 KO mice a transient inhibition of firing by OLDA was observed. Thus OLDA modulates the firing of nigrostriatal DA neurons through interactions with TRPV1, cannabinoid receptors and dopamine uptake. These findings suggest further development of OLDA-like tandem molecules for the treatment of movement disorders including Parkinson's disease.

Schizophrenia is a debilitating cognitive disorder. The link between cognitive debilitation and functional outcome in patients with schizophrenia has prompted research to develop procognitive therapies. It is hoped that by improving cognition in these patients, their functional outcome will also improve. Although no established treatments exist as yet, progress has been made toward understanding how to evaluate putative compounds in the clinic. Genetic mouse models and pharmacological rat models of cognitive disruption are being developed that may help to evaluate these putative compounds preclinically. Considering the increased number of genetic mouse models relevant to schizophrenia, there is a need to evaluate pharmacological manipulations on cognition in mice. Here we review the current literature on mouse pharmacological models relevant to schizophrenia. In this review, we discuss where different pharmacological effects between rats and mice on cognitive tasks are observed and assess the validity offered by these models. We conclude that the predictive validity of these models is currently difficult to assess and that much more needs to be done to develop useful mouse pharmacological models of cognitive disruption in schizophrenia.

Antipsychotic drugs for the treatment of schizophrenia arrived in the clinic in the fifties of the previous century and have since been the most effective treatment for patients with this devastating disorder. In spite of the more than half a century of clinical experience, and the introduction of a large number of chemical divers antipsychotic drugs, several recent large, multi-center studies have shown that, although novel (second generation) antipsychotics seem to be tolerated somewhat better (especially in relation to neurological side effects), their therapeutic potential is comparable to that of first generation antipsychotics. Hence there is still an urgent need for better pharmacological tools to treat schizophrenic patients. The current paper reviews the benefits and shortcomings of the currently available drugs, and gives an outlook towards the drugs and targets that are currently being pursued in clinical trials. Given the uncertainty of the drug discovery process and the relatively poor predictive validity of the currently available animal models, it is, at present, impossible to predict which of these drugs will ultimately become available for treating schizophrenic patients.

Electrical activity of ventral tegmental area (VTA) dopamine (DA) neurons is immediately inhibited following in vivo administration of cocaine and other DA-related drugs. While various forms of synaptic modulation were demonstrated in the VTA following exposure to DA-related drugs, comprehensive understanding of their ability to inhibit the activity of DA neurons, however, is still lacking. In this study, using whole-cell patch-clamp recordings from rat brain slices, a novel form of synaptic modulation induced by DA-related drugs was isolated. DA exposure was shown to cause potentiation of γ-amino-butyric acid (GABA) receptor type A (GABA A R)-mediated evoked inhibitory postsynaptic currents (eIPSCs), recorded from VTA DA neurons, under conditions of potassium channels blockade. The potentiation of these eIPSCs lasted for more than twenty minutes, could be mimicked by activation of D2-like but not D1-like DA receptors, and was accompanied by an increase in the frequency of GABA A R-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Furthermore, exposure to inhibitors of DA transporter (DAT) led to potentiation of GABA A currents in a manner similar to the DA-mediated potentiation. Finally, a prolonged presence of l -NAME, an inhibitor of nitric-oxide (NO) signaling was found to conceal the potentiation of GABA A currents induced by the DA-related drugs. Taken together, this study demonstrates a new modulatory form of VTA GABA A neurotransmission mediated by DA-related drugs. These results also suggest better understanding of the initial inhibitory action of DA-related drugs on the activity of DA neurons in the VTA.

Nitric oxide (NO) is associated with dopamine (DA) release. Previously, we demonstrated that rats treated with a non-selective nitric oxide synthase inhibitor, N-omega-nitro-L-arginine (L-NNA) at postnatal days 4-6 (PD4-6) show increased locomotion and disrupt neuronal cytoarchitecture after puberty (PD60). Here, we investigate whether the modulation of NO production in rats at PD4-6 causes long term changes of NO system, its impact on DA innervation, and schizophrenia-like behaviors. NO levels were measured in seven brain areas at PD35, PD60, PD90, and PD120. Autoradiographic studies explored the effect of l-NNA on the expression of D1 and D2 receptors in the caudate-putamen (CPu) and nucleus accumbens (NAcc) at PD60. Locomotor activity was assessed at PD60 using the non-selective DA agonists, amphetamine and apomorphine, and the selective DA receptor agonist [D2, quinpirole; D3, 7-hydroxy-N,N-di-n-propylaminotetralin ((+/-)-7-OH-DPAT)]. L-NNA treatment produced decreases in NO levels in the frontal cortex, striatum, brainstem and cerebellum, while in the occipital cortex changes were observed at PD120. Hippocampus and temporoparietal cortex showed differential levels of NO. Receptor autoradiography revealed increases in D1 receptor levels in the NAcc (shell), while decreases in D2 receptor binding were observed in the CPu and NAcc (core). Amphetamine and quinpirole treatments resulted in increases in locomotion. In contrast, treatment with 7-OH-DPAT produced hypolocomotion at low doses, while increased locomotion was seen at the highest dose. These results show that modulation of NO levels early postnatally (PD4-6) produces long term alteration in NO levels, with possible consequences on DA transmission, and related behaviors relevant to schizophrenia.

Mice lacking the dopamine (DA) transporter (DAT) gene exhibit a phenotype reminiscent of schizophrenia and attention deficit hyperactivity disorder (ADHD), including hyperDAergia, hyperactivity and deficits in cognitive performance, which are alleviated by antipsychotic agents. Numerous studies suggest a dysfunction of nicotinic neurotransmission in schizophrenia and show increased tobacco intake in schizophrenic and ADHD patients, possibly as a self-medication. Thus, we examined the potential alteration of nicotinic neurotransmission in DAT knock-out (KO) mice. We showed that constitutively hyperDAergic DAT KO mice exhibited modifications in nicotinic receptor density in an area- and subtype-dependent manner. In some DAergic areas, the small decrease in the beta2* nicotinic subunit (nAChR) density contrasted with the higher decrease and increase in the alpha6* and alpha7 nAChR densities, respectively. Mutant mice were hypersensitive to the stimulant locomotor effects of nicotine at low doses, probably due to enhanced nicotine-induced extracellular DA level. They also showed hypersensitivity to the hypolocomotion induced by nicotine. In contrast, no hypersensitivity was observed for other nicotine-induced behavioral effects, such as anxiety or motor activity in the elevated plus maze. Co-administration of nicotinic agonists at sub-active doses elicited opposite locomotor effects in wild-type and DAT KO mice, as reported previously for methylphenidate. Interestingly, such a co-administration of nicotinic agonists induced synergistic hypolocomotion in DAT KO mice. These findings show that a targeted increase of DA tone can be responsible for significant adaptations of the cholinergic/nicotinic neurotransmission. This study may provide potential leads for the use of nicotine or combined nicotinic agonists for the therapy of psychiatric disorders.

In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [(35)S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018. The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 μM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003-0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose-response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds.

Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A(2A) receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

Social isolation rearing (SI) is a model of early life stress that results in neurobiological alterations leading to increased anxiety-like behaviors. These animals also exhibit an increased propensity to administer psychostimulants, such as cocaine; however, the mechanisms governing this increased addiction vulnerability remains to be elucidated. Long-term stressors have been shown to produce important alterations in nucleus accumbens core (NAc) function. The NAc regulates motivated and goal-directed behaviors, and individual differences in NAc function have been shown to be predictive of addiction vulnerability. Rats were reared in group (GH; 4/cage) or SI (1/cage) conditions from weaning (PD 28) into early adulthood (PD 77) and dopamine release was assessed using voltammetry in brain slices containing the NAc and dorsomedial striatum. SI rats exhibited enhanced dopamine release and uptake in both regions compared to GH rats. In regard to psychostimulant effects directly at the dopamine transporter (DAT), methylphenidate and amphetamine, but not cocaine, inhibited uptake more in SI than GH rats. The increased potencies were positively correlated with uptake rates, suggesting that increased potencies of amphetamine-like compounds are due to changes in DAT function. Cocaine’s effects on uptake were similar between rearing conditions, however, cocaine enhanced evoked dopamine release greater in SI than GH rats, suggesting that the enhanced cocaine reinforcement in SI animals involves a DAT independent mechanism. Together, the results provide the first evidence that greater psychostimulant effects in SI compared to GH rats are due to effects on dopamine terminals related to uptake dependent and independent mechanisms.

Depression is a common co-morbid condition most often observed in subjects with mild cognitive impairment (MCI) and during the early stages of Alzheimer's disease (AD). Dysfunction of the central noradrenergic nervous system is an important component in depression. In AD, locus coeruleus (LC) noradrenergic neurons are significantly reduced pathologically and the reduction of LC neurons is hypothesized to begin very early in the progression of the disorder; however, it is not known if dysfunction of the noradrenergic system due to early LC neuronal loss is involved in mediating depression in early AD. Therefore, the purpose of this study was to determine in an animal model if a loss of noradrenergic LC neurons results in depressive-like behavior. The LC noradrenergic neuronal population was reduced by the bilateral administration of the neurotoxin 6-hydroxydopamine (6-OHDA) directly into the LC. Forced swim test (FST) was performed three weeks after the administration of 6-OHDA (5, 10 and 14 μg/μl), animals administered the 5 μg/μl of 6-OHDA demonstrated a significant increase in immobility, indicating depressive-like behavior. This increase in immobility at the 5 μg/μl dose was observed with a minimal loss of LC noradrenergic neurons as compared to LC neuronal loss observed at 10 and 14 μg/μl dose. A significant positive correlation between the number of surviving LC neurons after 6-OHDA and FST immobile time was observed, suggesting that in animals with a minimal loss of LC neurons (or a greater number of surviving LC neurons) following 6-OHDA demonstrated depressive-like behavior. As the 6-OHDA-induced loss of LC neurons is increased, the time spent immobile is reduced. Depressive-like behavior was also observed with the 5 μg/μl dose of 6-OHDA with a second behavior test, sucrose consumption. FST increased immobility following 6-OHDA (5 μg/μl) was reversed by the administration of a single dose of L-1-3-4-dihydroxyphenylalanine (DOPA) or l-threo-3,4-dihydroxyphenylserine (DOPS) prior to behavioral assessment. Surviving LC neurons 3 weeks after 6-OHDA (5 μg/μl) demonstrated compensatory changes of increased firing frequency, a more irregular firing pattern, and a higher percentage of cells firing in bursts. These results indicate that depressive-like behavior in mice is observed following the administration of 6-OHDA and the loss of LC noradrenergic neurons; however, the depressive-like behavior correlates positively with the number of surviving LC neurons with 6-OHDA administration. This data suggests the depression observed in MCI subjects and in the early stages of AD may due to the hypothesized early, minimal loss of LC neurons with remaining LC neurons being more active than normal.

Although most commonly associated with actions at cannabinoid CB1 receptors on the extracellular surface of the plasma membrane, the endocannabinoid anandamide (AEA) is also transported into the cell, by the putative anandamide membrane transporter (AMT), and activates the vanilloid receptor 1 (TRPV1) at an intracellular site. AEA is then inactivated by fatty acid amide hydrolase (FAAH). As systemic administration of TRPV1 ligands reduces locomotor activity in normal rodents, we hypothesised that activation of TRPV1 by endocannabinoids could play a role in the control of voluntary movement and that such actions could be regulated by AMT and FAAH. Motor activity was assessed in normal, in reserpine-treated, and in reserpine-treated rats treated with L-DOPA. In normal rats, the TRPV1 agonist capsaicin (1 mg/kg) or the FAAH inhibitor URB597 (10 mg/kg) caused a significant reduction in movement in both the horizontal (locomotion) and vertical (rearing) planes (-45% and -53% respectively with capsaicin; -33% and -37% for URB597). Capsaicin-induced hypolocomotion was attenuated by the TRPV1 antagonist, capsazepine. There was no effect of capsaicin, URB597 or the AMT inhibitor OMDM-2 on motor activity in reserpine-treated rats. L-DOPA treatment of reserpine-treated rats elicited high levels of motor activity in both the horizontal and vertical planes. Horizontal activity was attenuated by capsaicin (1 mg/kg, -60%), but not by URB597 (10 mg/kg) or OMDM-2 (5 mg/kg). Vertical activity was attenuated by capsaicin (1 mg/kg, -61%) and by URB597 (10 mg/kg, -54%), but not by OMDM-2. These data suggest that activation of the TRPV1 system can suppress spontaneous locomotion in normal animals and modulates several L-DOPA-induced behaviours in reserpine-treated rats.

There is growing evidence indicating that oxidative stress is a key contributor to the pathogenesis and progression of Parkinson's disease. The brain, and particularly the basal ganglia, possesses a local rennin-angiotensin system. Angiotensin activates NAD(P)H-dependent oxidases, which are a major intracellular source of superoxide, and angiotensin converting enzyme inhibitors (ACEIs) have shown antioxidant properties. We treated mice with MPTP and the ACEI captopril to study the possible neuroprotective and antioxidant effects of the latter on the dopaminergic system. Pre-treatment with captopril induced a significant reduction in the MPTP-induced loss of dopaminergic neurons in the substantia nigra and a significant reduction in the loss of dopaminergic terminals in the striatum. Furthermore, captopril reduced the MPTP-induced increase in the levels of major oxidative stress indicators (i.e. lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum. Captopril did not reduce striatal MPP(+) levels, MAO-B activity or dopamine transporter activity, which may reduce MPTP neurotoxicity. Our results suggest that angiotensin-converting enzyme inhibitors may be useful for treatment of Parkinson's disease, and that further investigation should focus on the neuroprotective capacity of these compounds.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is involved in neuronal adaptions that underlie cocaine-induced sensitization and reward. mTOR exists in two functionally distinct multi-component complexes known as mTORC1 and mTORC2. In this study, we show that increased mTORC1 activity induced by cocaine is mediated by the dopamine D1 receptor (D1R). Specifically, cocaine treatment increased the phosphorylation on residues Thr2446 and Ser2481 but not on Ser2448 in the nucleus accumbens (NAc) and that this increase in phosphorylated mTOR levels was also apparent when complexed with its binding partner Raptor. Furthermore, the increase in phosphorylated mTOR levels, as well as phosphorylated 4E-BP1 and S6K, downstream targets of mTORC1 were blocked with SCH23390 treatment. Similar results were also observed in the dopamine-transporter knockout mice as the increase in phosphorylated mTOR Thr2446 and Ser2481 was blocked by SCH23390 but not with raclopride. To further validate D1R role in mTORC1 signaling, decrease in phosphorylated mTOR levels were observed in D1R knockout mice, whereas administration of SKF81297 elevated phosphorylated mTOR in the NAc. Lastly deletion of mTOR or Raptor in D1R expressing neurons reduced cocaine-induced locomotor activity. Together, our data supports a mechanism whereby mTORC1 signaling is activated by cocaine administration through the stimulation of D1R.

This study examines the effects of serotonin (5-HT)1A receptor ligands on the in vivo release of 5-HT and dopamine (DA) in the prefrontal cortex of mice. Oral MKC-242 and 8-OH-DPAT, selective 5-HT1A receptor agonists, decreased cortical 5-HT release at low and high doses, while the receptor agonists increased cortical DA release only at a high dose. Local application of the selective 5-HT1A receptor antagonist, WAY100635, via a dialysis probe, antagonized oral MKC-242-induced increase in cortical DA release, but did not affect the decrease in cortical 5-HT release. Local application of 8-OH-DPAT at 100 and 300 nM via a dialysis probe increased cortical DA release, but did not affect cortical 5-HT release. The effects of oral MKC-242 and 8-OH-DPAT on 5-HT release were blocked by low and high doses of WAY100635, while blocking the agonist-induced increase in DA release required a high dose of WAY100635. These results suggest that 5-HT release and DA release in the frontal cortex of mice are regulated by pre- and postsynaptic 5-H1A receptors, respectively, and that the presynaptic 5-HT1A receptor-mediated response is more sensitive to inhibition by WAY100635 than the postsynaptic 5-HT1A receptor-mediated response in mice.

Apomorphine has been introduced in the treatment of late-stage Parkinson's Disease (PD). The disadvantage of a short half-life of apomorphine is now overcome by the use of a continuous subcutaneous (s.c.) self-delivering system. We examined whether continuous s.c. infusion of apomorphine rescues nigro-striatal dopaminergic neurons from toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Apomorphine was continuously infused in mice by means of a s.c. minipump that delivered the drug at a rate of 0.5 or 3.15 mg/kg/day. MPTP induced a >80% reduction in striatal dopamine (DA) after one day. DA levels were still substantially reduced one month following MPTP injection, in spite of a partial recovery. Similarly, striatal immunoreactivity for tyrosine hydroxylase and dopamine transporter was markedly reduced at this time interval. Continuous s.c. infusion of apomorphine starting 40 h following MPTP injection rescued striatal dopaminergic terminals, as assessed by measurements of DA and its metabolites, as well as TH and DAT immunostaining after one month. The neurorescuing effect was more remarkable at a delivery rate of 3.15 mg/kg/day of apomorphine. In contrast, no rescue was observed when apomorphine was administered as a single daily s.c. bolus of 1 or 5 mg/kg starting 40 h following MPTP. We conclude that apomorphine is able to rescue nigro-striatal dopaminergic neurons when continuously delivered at doses that are comparable to those delivered by minipumps in PD patients. These results suggest that continuous s.c. infusion of apomorphine not only relieves the symptoms, but also reduce the ongoing degeneration of nigro-striatal dopaminergic neurons in PD patients.

Methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that MDMA-induced neurotoxicity is mediated by oxidative stress probably due to the inhibition of mitochondrial complex I activity. In this study we investigated the contribution of dopamine (DA) to such effects. For this, we modulated the dopaminergic system of mice at the synthesis, uptake or metabolism levels. Striatal mitochondrial complex I activity was decreased 1 h after MDMA; an effect not observed in the striatum of DA depleted mice or in the hippocampus, a dopamine spare region. The DA precursor, L-dopa, caused a significant reduction of mitochondrial complex I activity by itself and exacerbated the dopaminergic deficits when combined with systemic MDMA. By contrast, no damage was observed when L-dopa was combined with intrastriatal injections of MDMA. On the other hand, dopamine uptake blockade using GBR 12909, inhibited both, the acute inhibition of complex I activity and the long-term dopaminergic toxicity caused by MDMA. Moreover, the inhibition of DA metabolism with the monoamine oxidase (MAO) inhibitor, pargyline, afforded a significant protection against MDMA-induced complex I inhibition and neurotoxicity. Taken together, these findings point to the formation of hydrogen peroxide subsequent to DA metabolism by MAO, rather than a direct DA-mediated mitochondrial complex I inhibition, and the contribution of a peripheral metabolite of MDMA, as the key steps in the chain of biochemical events leading to DA neurotoxicity caused by MDMA in mice.

Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP(+) accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP(+)). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signalling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

Adenosine A(2A) agonists are known to induce catalepsy and inhibit dopamine mediated motor hyperactivity. An antagonistic interaction between adenosine A(2A) and dopamine D(2) receptors is known to regulate GABA-mediated neurotransmission in striatopallidal neurons. Stimulation of adenosine A(2A) and dopamine D(2) receptors has been shown to increase and inhibit GABA release respectively in pallidal GABAergic neurons. However, the role of GABAergic neurotransmission in the motor effects of adenosine A(2A) receptors is not yet known. Therefore in the present study the effect of GABAergic agents on adenosine A(2A) receptor agonist (NECA- or CGS 21680) induced catalepsy and inhibition of amphetamine elicited motor hyperactivity was examined. Pretreatment with GABA, the GABA(A) agonist muscimol or the GABA(B) agonist baclofen potentiated whereas the GABA(A) antagonist bicuculline attenuated NECA- or CGS 21680-induced catalepsy. However, the GABA(B) antagonists phaclophen and delta-aminovaleric acid had no effect. Administration of NECA or CGS 21680 not only reduced spontaneous locomotor activity but also antagonized amphetamine elicited motor hyperactivity. These effects of NECA and CGS 21680 were potentiated by GABA or muscimol and antagonized by bicuculline. These findings provide behavioral evidence for the role of GABA in the motor effects of adenosine A(2A) receptor agonists. Activation of adenosine A(2A) receptors increases GABA release which could reduce dopaminergic tone and induce catalepsy or inhibit amphetamine mediated motor hyperactivity.

Riluzole, has previously been shown to be protective in animal models of Parkinson's disease in vivo. In the present study the effects of riluzole on the intrastriatal formation and accumulation of MPP(+), after i.p. injection of MPTP were tested in mice, using two different experimental protocols. In the first protocol, mice were treated with a single dose (15 mg/kg i.p.) of MPTP and MPP(+) accumulation was measured 30 min, 1 h and 3 h after the injection of the toxin. Riluzole (10 mg/kg p.o.), administered 30 min before MPTP, did not modify the accumulation kinetic of MPP(+). Contrarily to riluzole, a single dose of 50 mg/kg p.o. of 7-nitroindazole (7-NI), a non-selective non hypertensive inhibitor of nitric oxide synthase (NOS), significantly decreased MPP(+) levels. In the second protocol, consisting of 3 injections of MPTP (15 mg/kg i.p.), riluzole, administered 4 times at the dose of 5 mg/kg p.o., had no effect on MPP(+) levels. The protective effect of repeated treatments of riluzole and 7-NI against MPTP-induced depletion of dopamine (DA) is also reported. Our data obtained with 7-NI (in agreement with previous studies reported by others) suggest that a part of the protection observed with this NOS inhibitor is probably due to in vivo inhibition of monoamine oxidase-B (MAO-B). That riluzole does not modify MPP(+) accumulation demonstrates that its protective effect against MPTP toxicity was not due to an in vivo interference with MPTP metabolism.

The expression of cyclooxygenase-2 (COX-2) and striatal dopamine (DA) depletion in BALB/cAnNcrj (BALB/c) mice following a neurotoxic dose of methamphetamine (METH) was investigated. METH-treatment (4 mg/kg x 4, 2 h intervals, s.c.) induced a significant hyperthermia and a persistent depletion of striatal DA levels 72 h after the treatment. COX-2, a marker of the cytotoxic effect of inflammation and oxidative stress and thiobarbituric acid (TBA) were significantly induced in the striatum 72 h after the METH-treatment, but not in the hippocampus. These results suggest that COX-2 may participate in METH-induced neurotoxicity in striatum.

The present study aimed to determine the kinetics of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4 and RBE 4B), to define the type of interaction with 3-O-methyl-L-DOPA (3-OM-L-DOPA), sensitivity to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC), N-(methylamino)-isobutyric acid (MeAIB) and sodium. Non-linear analysis of the saturation curves for L-DOPA and 3-OM-L-DOPA revealed in RBE 4 cells Km values (in microM) of 72 (53, 91) and 40 (25, 57) and in RBE 4B cells Km values (in microM) of 60 (46, 74) and 44 (13, 75), respectively. IC50 values for 3-OM-L-DOPA (RBE 4, 642 [542, 759] microM; RBE 4B, 482 [475, 489] microM) obtained in the presence of a nearly saturating (250 microM) concentration of L-DOPA were greater than the corresponding Ki values (RBE 4, 143 [121, 170] microM; RBE 4B, 93 [92, 95] microM) obtained in the presence of a nearly saturating (250 microM) concentration of 3-OM-L-DOPA; this is compatible with a competitive type of interaction between L-DOPA and 3-OM-L-DOPA. Uptake of both L-DOPA and 3-OM-L-DOPA in RBE 4 and RBE 4B cells was sensitive to BHC with similar IC50 values. MeAIB (up to 2.5 mM) was found not to interfere with the uptake of both L-DOPA and 3-OM-L-DOPA. Uptake of (250 microM) L-DOPA and 3-OM-L-DOPA in the absence of sodium in the incubation medium was similar to that observed in the presence of increasing concentrations of sodium (20-140 mM). Homogenates of both cell lines were endowed with considerable COMT activity. Incubation of RBE 4 and RBE 4B cells with L-DOPA (25 microM) in the presence of a methyl donor (S-adenosyl-L-methionine) resulted in the formation of 3-OM-L-DOPA; this was abolished by 1 microM tolcapone. The fractional outflow of intracellular L-DOPA through the luminal and abluminal cell side was not affected by the presence of intracellular 3-OM-L-DOPA. The fractional outflow of exogenous 3-OM-L-DOPA applied from the luminal cell border was similar to that observed for 3-OM-L-DOPA with origin in L-DOPA. It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which L-DOPA and 3-OM-L-DOPA can be taken up, and 3-OM-L-DOPA behaves as a competitive inhibitor for the uptake of L-DOPA. This, however, only occurs for luminal cell inward movement but not for abluminal cell outward movement of the substrates.

Objective The present study aimed at evaluating the effect of opicapone, a third generation nitrocatechol catechol- O -methyltransferase (COMT) inhibitor, on the systemic and central bioavailability of 3,4-dihydroxy- l -phenylalanine (levodopa) and related metabolites in the cynomolgus monkey. Methods Four monkeys, implanted with guiding cannulas for microdialysis probes, in the substantia nigra , dorsal striatum and prefrontal cortex, were randomized in two groups that received, in a crossover design, vehicle or 100 mg/kg opicapone for 14 days. Twenty-three hours after last administration of vehicle or opicapone, animals were challenged with levodopa/benserazide (12/3 mg/kg). Extracellular dialysate and blood samples were collected over 360 min (at 30 min intervals) for the assays of catecholamine and COMT activity. Results Opicapone increased levodopa systemic exposure by 2-fold not changing C max values and reduced both 3- O -methyldopa (3-OMD) exposure and C max values by 5-fold. These changes were accompanied by ∼76–84% reduction in erythrocyte COMT activity. In dorsal striatum and substantia nigra , opicapone increased levodopa exposure by 1.7- and 1.4-fold, respectively, reducing 3-OMD exposure by 5- and 7-fold respectively. DOPAC exposure was increased by 4-fold in the substantia nigra . In the prefrontal cortex, opicapone increased levodopa exposure and reduced 3-OMD levels by 2.3- and 2.4-fold, respectively. Conclusions Opicapone behaved as long-acting COMT inhibitor that markedly increased systemic and central levodopa bioavailability. Opicapone is a strong candidate to fill the unmet need for COMT inhibitors that lead to more sustained levodopa levels in Parkinson's disease patients.

The selective dopamine D3 receptor antagonist [3H](+)S 14297 ((+)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho(2,3b)dihydro,2,3-furane]), labelled to high specific activity (145 Ci/mmol), bound to cloned human dopamine D3 receptors but displayed negligible binding to cloned human D2 receptors. [3H](+)S 14297 exhibited rapid association and dissociation, high affinity saturable binding (Kd = 7.0 nM) and a competition binding profile highly correlated with that of [125I]iodosulpride (r = 0.98).

A combined pharmacological and genetic approach was undertaken to investigate the contribution of endogenous dopamine to the motor actions of nociceptin/orphanin FQ (N/OFQ) receptor (NOP receptor) ligands. Motor activity was evaluated by a battery of behavioural tests in mice. The involvement of the various DA receptor subtypes in the motor effects of N/OFQ and NOP receptor antagonists was evaluated pharmacologically, using D1/D5 (SCH23390), D2/D3 (raclopride, amisulpride) and D3 (S33084) receptor antagonists, and by using D2 receptor knockout mice. Low doses of N/OFQ and NOP receptor antagonists promoted movement whereas higher doses inhibited it. Motor facilitation was selectively prevented by raclopride while motor inhibition was prevented by amisulpride. Amisulpride also attenuated the hypolocomotion induced by the D2/D3 receptor agonist pramipexole and dopamine precursor l -3,4-dihydroxyphenylalanine, whereas raclopride (and S33084) worsened it. To dissect out the contribution of pre- and postsynaptic D2 receptors, mice lacking the D2 receptor (D2R −/− ) or its long isoform (D2L −/− ) were used. Motor facilitation induced by N/OFQ and NOP receptor antagonists was lost in D2R −/− and D2L −/− mice whereas motor inhibition induced by NOP receptor antagonists (and pramipexole) was lost in D2R −/− but preserved in D2L −/− mice. N/OFQ-induced hypolocomotion was observed in both genotypes. We demonstrate that motor actions of NOP receptor ligands rely on the modulation of endogenous dopamine. Motor facilitation induced by NOP receptor antagonists as well as low dose N/OFQ is mediated through D2L postsynaptic receptors whereas motor inhibition observed with higher doses of N/OFQ occurs by direct inhibition of mesencephalic DA neurons. Motor inhibition seen with high doses of NOP receptor antagonists appears to be mediated through the D2 presynaptic autoreceptors. These data confirm that endogenous N/OFQ is a powerful modulator of dopamine transmission in vivo and that the effects of NOP receptor antagonists on motor function reflect the blockade of this endogenous N/OFQ tone.

Despite evidence suggesting the interaction among glutamate (GLU), dopamine (DA) and ascorbic acid (AA) in the striatum, their actions are often studied separately. Microdialysis was used here to quantify the extracellular interaction among GLU-DA-AA in the striatum of rats, an interaction which was compared with those studied in the substantia nigra (SN). Perfusion of GLU by reverse microdialysis increased DA and decreased 3,4-dihydroxyphenylacetic acid (DOPAC) in the extracellular medium of the striatum, but increased both DA and DOPAC in the SN. The increase of extracellular DA-concentration induced by the local DA-perfusion decreased the extracellular level of GLU and glutamine, an effect that, as suggested by the GLU and glutamine increase observed after the haloperidol administration, probably involves the D2 dopamine receptor. Local administration of AA increased the extracellular DA, decreased DOPAC and had no effect on GLU and glutamine. Present data suggest that, in the striatum, GLU-release inhibits DA-uptake, DA-release inhibits GLU-release, and AA-release prevents DA-oxidation increasing its extracellular diffusion. These effects were different in the SN where GLU probably promoted the DA-release instead of inhibiting the DA-uptake as presumably occurred in the striatum. Present data denote a marked GLU-DA-AA interaction in the striatum, which might be relevant for the pharmacological control of basal ganglia disorders.