Extracellular Dopamine Levels Research Articles

Noradrenaline enhances the excitatory effects of dopamine on medial prefrontal cortex pyramidal neurons in rats.

AimOur previous studies showed that exposure to acute restraint stress enhanced cocaine‐induced conditioned place preference (cocaine‐CPP) and suggested the possibility that co‐activation of adrenergic transmission boosts the increase in medial prefrontal cortex (mPFC) neuronal activity by the activation of dopaminergic transmission. To examine this possibility, the effects of the co‐treatment with dopamine (DA) and noradrenaline (NA) on mPFC neurons were compared with those of treatment with DA alone using whole‐cell patch‐clamp recordings.MethodsThe effects of DA alone and a mixture of DA and NA on the membrane potentials and spontaneous excitatory postsynaptic currents (sEPSCs) were examined by electrophysiological recordings of mPFC pyramidal neurons in brain slices of male Sprague Dawley rats. Extracellular DA and NA levels in the mPFC during and after restraint stress exposure were also examined by in vivo microdialysis.ResultsDopamine significantly produced depolarizing effects on mPFC neurons and tended to increase sEPSC frequency. Co‐administration of NA with DA produced stronger depolarizing effects and significantly increased sEPSC frequency. The findings suggest that the additional depolarizing effect of NA on DA‐responsive neurons, rather than the excitation of DA‐nonresponsive neurons by NA, contributes to the stronger effect of co‐treatment of NA with DA.ConclusionThe present study suggests that NA released by restraint stress exposure cooperates with DA to stimulate DA‐responsive neurons in the mPFC, thereby causing the stress‐induced enhancement of cocaine‐CPP.

Absence of methamphetamine-induced conditioned place preference in weaver mutant mice.

AimsG protein‐activated inwardly rectifying potassium (GIRK) channels are related to rewarding effects of addictive drugs. The GIRK2 subunit is thought to play key roles in the reward system. Weaver mutant mice exhibit abnormal GIRK2 function and different behaviors that are caused by several addictive substances compared with wild‐type mice. However, mechanisms of reward‐related alterations in weaver mutant mice remain unclear. The present study investigated changes in the rewarding effects of methamphetamine (METH) in weaver mutant mice.MethodsThe rewarding effects of METH (4.0 mg/kg) were investigated using the conditioned place preference (CPP) paradigm. Extracellular dopamine level in the nucleus accumbens (NAc) was measured by in vivo microdialysis. To identify brain regions that were associated with these changes in rewarding effects, METH‐induced alterations of Fos expression were investigated by immunohistochemical analysis.Results Weaver mutant mice exhibited a significant decrease in METH‐induced CPP and dopamine release in the NAc. Methamphetamine significantly increased Fos expression in the posterior NAc (pNAc) shell in wild‐type but not in weaver mutant mice.ConclusionsMethamphetamine did not induce rewarding effects in weaver mutant mice. The pNAc shell exhibited a significant difference in neuronal activity between wild‐type and weaver mutant mice. The present results suggest that the absence of METH‐induced CPP in weaver mutant mice is probably related to an innate reduction of dopamine and decreased neural activity in the pNAc shell that is partially attributable to the change of GIRK channel function. GIRK channels, especially those containing the GIRK2 subunit, appear to be involved in METH dependence.

An acetylcholine-dopamine interaction in the nucleus accumbens and its involvement in ethanol's dopamine-releasing effect.

Alcohol use disorder is a chronic, relapsing brain disorder causing substantial morbidity and mortality. Cholinergic interneurons (CIN) within the nucleus accumbens (nAc) have been suggested to exert a regulatory impact on dopamine (DA) neurotransmission locally, and defects in CIN have been implied in several psychiatric disorders. The aim of this study was to investigate the role of CIN in regulation of basal extracellular levels of DA and in modulation of nAc DA release following ethanol administration locally within the nAc of male Wistar rats. Using reversed in vivo microdialysis, the acetylcholinesterase inhibitor physostigmine was administered locally in the nAc followed by addition of either the muscarinic acetylcholine (ACh) receptor antagonist scopolamine or the nicotinic ACh receptor antagonist mecamylamine. Further, ethanol was locally perfused in the nAc following pretreatment with scopolamine and/or mecamylamine. Lastly, ethanol was administered locally into the nAc of animals with accumbal CIN‐ablation induced by anticholine acetyl transferase‐saporin. Physostigmine increased accumbal DA levels via activation of muscarinic ACh receptors. Neither scopolamine and/or mecamylamine nor CIN‐ablation altered basal DA levels, suggesting that extracellular DA levels are not tonically controlled by ACh in the nAc. In contrast, ethanol‐induced DA elevation was prevented following coadministration of scopolamine and mecamylamine and blunted in CIN‐ablated animals, suggesting involvement of CIN‐ACh in ethanol‐mediated DA signaling. The data presented in this study suggest that basal extracellular levels of DA within the nAc are not sustained by ACh, whereas accumbal CIN‐ACh is involved in mediating ethanol‐induced DA release.

Xie2-64, a novel CB2 receptor inverse agonist, reduces cocaine abuse-related behaviors in rodents

Cocaine abuse remains a public health threat around the world. There are no pharmacological treatments approved for cocaine use disorder. Cannabis has received growing attention as a treatment for many conditions, including addiction. Most cannabis-based medication development has focused on cannabinoid CB1 receptor (CB1R) antagonists (and also inverse agonists) such as rimonabant, but clinical trials with rimonabant have failed due to its significant side-effects. Here we sought to determine whether a novel and selective CB2R inverse agonist, Xie2-64, has similar therapeutic potential for cocaine use disorder. Computational modeling indicated that Xie2-64 binds to CB2R in a way similar to SR144528, another well-characterized but less selective CB2R antagonist/inverse agonist, suggesting that Xie2-64 may also have CB2R antagonist profiles. Unexpectedly, systemic administration of Xie2-64 or SR144528 dose-dependently inhibited intravenous cocaine self-administration and shifted cocaine dose-response curves downward in rats and wild-type, but not in CB2R-knockout, mice. Xie2-64 also dose-dependently attenuated cocaine-enhanced brain-stimulation reward maintained by optical stimulation of ventral tegmental area dopamine (DA) neurons in DAT-Cre mice, while Xie2-64 or SR144528 alone inhibited optical brain-stimulation reward. In vivo microdialysis revealed that systemic or local administration of Xie2-64 into the nucleus accumbens reduced extracellular dopamine levels in a dose-dependent manner in rats. Together, these results suggest that Xie2-64 has significant anti-cocaine reward effects likely through a dopamine-dependent mechanism, and therefore, deserves further study as a new pharmacotherapy for cocaine use disorder.

Diabetes Causes Dysfunctional Dopamine Neurotransmission Favoring Nigrostriatal Degeneration in Mice.

BackgroundNumerous studies indicate an association between neurodegenerative and metabolic diseases. Although still a matter of debate, growing evidence from epidemiological and animal studies indicate that preexisting diabetes increases the risk to develop Parkinson's disease. However, the mechanisms of such an association are unknown.ObjectivesWe investigated whether diabetes alters striatal dopamine neurotransmission and assessed the vulnerability of nigrostriatal neurons to neurodegeneration.MethodsWe used streptozotocin‐treated and genetically diabetic db/db mice. Expression of oxidative stress and nigrostriatal neuronal markers and levels of dopamine and its metabolites were monitored. Dopamine release and uptake were assessed using fast‐scan cyclic voltammetry. 6‐Hydroxydopamine was unilaterally injected into the striatum using stereotaxic surgery. Motor performance was scored using specific tests.ResultsDiabetes resulted in oxidative stress and decreased levels of dopamine and its metabolites in the striatum. Levels of proteins regulating dopamine release and uptake, including the dopamine transporter, the Girk2 potassium channel, the vesicular monoamine transporter 2, and the presynaptic vesicle protein synaptobrevin‐2, were decreased in diabetic mice. Electrically evoked levels of extracellular dopamine in the striatum were enhanced, and altered dopamine uptake was observed. Striatal microinjections of a subthreshold dose of the neurotoxin 6‐hydroxydopamine in diabetic mice, insufficient to cause motor alterations in nondiabetic animals, resulted in motor impairment, higher loss of striatal dopaminergic axons, and decreased neuronal cell bodies in the substantia nigra.ConclusionsOur results indicate that diabetes promotes striatal oxidative stress, alters dopamine neurotransmission, and increases vulnerability to neurodegenerative damage leading to motor impairment. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Electrophysiological Effects of Transcranial Direct Current Stimulation on Neural Activity in the Rat Motor Cortex.

Transcranial direct current stimulation (tDCS) is a non-invasive technique that modulates the neuronal membrane potential. We have previously documented a sustainable increase in extracellular dopamine levels in the rat striatum of cathodal tDCS, suggesting that cathodal tDCS enhances the neuronal excitability of the cortex. In the present study, we investigated changes in neuronal activity in the cerebral cortex induced by tDCS at the point beneath the stimulus electrode in anesthetized rats in vivo. Multiunit recordings were performed to examine changes in neuronal activity before and after the application of tDCS. In the cathodal tDCS group, multiunit activity (indicating the collective firing rate of recorded neuronal populations) increased in the cerebral cortex. Both anodal and cathodal tDCS increased the firing rate of isolated single units in the cerebral cortex. Significant differences in activity were observed immediately following stimulation and persisted for more than an hour after stimulation. The primary finding of this study was that both anodal and cathodal tDCS increased in vivo neuronal activity in the rat cerebral cortex underneath the stimulus electrode.

Dopaminergic loss of cyclin-dependent kinase-like 5 recapitulates methylphenidate-remediable hyperlocomotion in mouse model of CDKL5 deficiency disorder.

Cyclin-dependent kinase-like 5 (CDKL5), a serine-threonine kinase encoded by an X-linked gene, is highly expressed in the mammalian forebrain. Mutations in this gene cause CDKL5 deficiency disorder, a neurodevelopmental encephalopathy characterized by early-onset seizures, motor dysfunction, and intellectual disability. We previously found that mice lacking CDKL5 exhibit hyperlocomotion and increased impulsivity, resembling the core symptoms in attention-deficit hyperactivity disorder (ADHD). Here, we report the potential neural mechanisms and treatment for hyperlocomotion induced by CDKL5 deficiency. Our results showed that loss of CDKL5 decreases the proportion of phosphorylated dopamine transporter (DAT) in the rostral striatum, leading to increased levels of extracellular dopamine and hyperlocomotion. Administration of methylphenidate (MPH), a DAT inhibitor clinically effective to improve symptoms in ADHD, significantly alleviated the hyperlocomotion phenotype in Cdkl5 null mice. In addition, the improved behavioral effects of MPH were accompanied by a region-specific restoration of phosphorylated dopamine- and cAMP-regulated phosphoprotein Mr 32kDa, a key signaling protein for striatal motor output. Finally, mice carrying a Cdkl5 deletion selectively in DAT-expressing dopaminergic neurons, but not dopamine receptive neurons, recapitulated the hyperlocomotion phenotype found in Cdkl5 null mice. Our findings suggest that CDKL5 is essential to control locomotor behavior by regulating region-specific dopamine content and phosphorylation of dopamine signaling proteins in the striatum. The direct, as well as indirect, target proteins regulated by CDKL5 may play a key role in movement control and the therapeutic development for hyperactivity disorders.

The Role of Dopamine in the Stimulant Characteristics of Novel Psychoactive Substances (NPS)-Neurobiological and Computational Assessment Using the Case of Desoxypipradrol (2-DPMP).

Stimulant drugs, including novel psychoactive substances (NPS, formerly “legal highs”) have addictive potential which their users may not realize. Stimulants increase extracellular dopamine levels in the brain, including the reward and addiction pathways, through interacting with dopamine transporter (DAT). This work aimed to assess the molecular and atomistic mechanisms of stimulant NPS actions at DAT, which translate into biological outcomes such as dopamine release in the brain’s reward pathway. We applied combined in vitro, in vivo, and in silico methods and selected 2-diphenylmethylpiperidine (2-DPMP) as an example of stimulant NPS for this study. We measured in vitro binding of 2-DPMP to rat striatum and accumbens DAT by means of quantitative autoradiography with a selective DAT-radioligand [125I]RTI-121. We evaluated the effects of intravenously administered 2-DPMP on extracellular dopamine in the accumbens-shell and striatum using in vivo microdialysis in freely moving rats. We used dynamic modeling to investigate the interactions of 2-DPMP within DAT, in comparison with cocaine and amphetamine. 2-DPMP potently displaced the radioligand in the accumbens and striatum showing dose-dependence from 0.3 to 30 μM. IC50 values were: 5.65 × 10-7M for accumbens shell and 6.21 × 10-7M for dorsal striatum. Dose-dependent responses were also observed in accumbens-shell and striatum in vivo, with significant increases in extracellular dopamine levels. Molecular dynamics simulations identified contrasting conformational changes of DAT for inhibitors (cocaine) and releasers (amphetamine). 2-DPMP led to molecular rearrangements toward an outward-facing DAT conformation that suggested a cocaine-type effect. The present combination of molecular modeling with experimental neurobiological procedures allows for extensive characterization of the mechanisms of drug actions at DAT as the main molecular target of stimulants, and provides an insight into the role of dopamine in the molecular and neurobiological mechanisms of brain responses to stimulant NPS that have addictive potential. Such knowledge reveals the risk of addiction related to NPS use. The research presented here can be adapted for other psychostimulants that act at their membrane protein targets.

Diverging changes in rat striatal extracellular dopamine and DOPAC levels and in frequency-modulated 50-kHz ultrasonic vocalizations rate during repeated amphetamine treatment

One characteristic feature of addictive drugs is their ability to induce, after a single exposure, a lasting sensitization to the next doses; the underlying neuroplastic changes supposedly involve the brain dopamine system. We aimed at identifying putative relationships between alterations in extracellular dorsal striatal dopamine, HVA and DOPAC levels and in frequency-modulated 50-kHz ultrasonic vocalizations rate response during repeated intraperitoneal amphetamine treatment. Measurements were performed before and after amphetamine doses 1, 2, 7 and 8 (Amph1, Amph2, Amph7 and Amph8; treatment days 1, 7, 12 and 23, respectively). Amphetamine was confirmed to induce sensitization of the vocalization response, but an extended recording time (180 instead of 20 min) revealed that sensitization of this response requires more time to develop than hitherto believed. Baseline extracellular dopamine level increased initially, declined after a series of daily amphetamine doses and showed some tendency for recovery after drug withdrawal. Baseline extracellular DOPAC (but not HVA) showed a continuous decline during the treatment. There was no significant change in the integrated short-term (3-h) extracellular dopamine response, whereas the respective DOPAC collection lowered significantly after repeated drug treatment. Extracellular DOPAC is believed to originate mostly from newly synthesized dopamine, hence the declines in its baseline and post-amphetamine levels suggest falling dopamine synthesis. These results indicate that sensitization of the appetitive vocalization response to amphetamine continues despite reduced dorsal striatal dopamine synthesis and involves no changes in amphetamine-induced dopamine release.

Triple reuptake inhibition of serotonin, norepinephrine, and dopamine increases the tonic activation of α2-adrenoceptors in the rat hippocampus and dopamine levels in the nucleus accumbens

Clinical studies have shown the therapeutic efficacy of an increase in dopamine (DA) transmission in treatment of major depressive disorder (MDD). In the present study, we investigated whether blockade of DA transporters in addition to serotonin (5-HT) and norepinephrine (NE) produced additional adaptations of monoaminergic systems. In vivo electrophysiological recordings were carried out in male anesthetized rats. Vehicle, the 5-HT reuptake inhibitor escitalopram, the NE/DA reuptake blocker nomifensine and their combination (triple reuptake inhibition; TRI) were delivered for 2 or 14 days. Firing activity of NE, 5-HT and DA neurons was assessed. Tonic activation of 5-HT1A receptors and α1- and α2-adrenoceptors was determined in the hippocampus and extracellular DA levels in the nucleus accumbens (NAc). Unlike escitalopram, nomifensine and TRI administration increased the tonic activation of α2-adrenoceptors in the hippocampus despite decreasing NE neuronal firing activity after 2 and 14 days of administration. The firing activity of 5-HT neurons was increased after prolonged nomifensine and TRI regimens, while addition of nomifensine to escitalopram prevented the early 2-day suppression of firing by 5-HT reuptake inhibition. The tonic activation of 5-HT1A receptors was enhanced only with escitalopram. Whereas escitalopram and nomifensine decreased firing activity of DA neurons after a 2-day administration, their combination normalized it to baseline level after 14 days; this was accompanied by a robust increase in extracellular DA levels in the NAc. In summary, these results indicate that TRI increases NE and DA but not 5-HT transmission, suggesting a differential efficacy profile in MDD patients.

Neurophysiological and Neurochemical Effects of the Putative Cognitive Enhancer (S)-CE-123 on Mesocorticolimbic Dopamine System.

Treatments for cognitive impairments associated with neuropsychiatric disorders, such as attention deficit hyperactivity disorder or narcolepsy, aim at modulating extracellular dopamine levels in the brain. CE-123 (5-((benzhydrylsulfinyl)methyl) thiazole) is a novel modafinil analog with improved specificity and efficacy for dopamine transporter inhibition that improves cognitive and motivational processes in experimental animals. We studied the neuropharmacological and behavioral effects of the S-enantiomer of CE-123 ((S)-CE-123) and R-modafinil in cognitive- and reward-related brain areas of adult male rats. In vivo single unit recordings in anesthetized animals showed that (S)-CE-123, but not R-modafinil, dose-dependently (1.25 to 10 mg/kg i.v.) reduced firing of pyramidal neurons in the infralimbic/prelimbic (IL/PrL) cortex. Neither compound the affected firing activity of ventral tegmental area dopamine cells. In freely moving animals, (S)-CE-123 (10 mg/kg i.p.) increased extracellular dopamine levels in the IL/PrL, with different patterns when compared to R-modafinil (10 mg/kg i.p.); in the nucleus accumbens shell, a low and transitory increase of dopamine was observed only after (S)-CE-123. Neither (S)-CE-123 nor R-modafinil initiated the emission of 50-kHz ultrasonic vocalizations, a behavioral marker of positive affect and drug-mediated reward. Our data support previous reports of the procognitive effects of (S)-CE-123, and show a minor impact on reward-related dopaminergic areas.

Deficit in working memory and abnormal behavioral tactics in dopamine transporter knockout rats during training in the 8-arm maze

Understanding the role of the dopamine system in learning and memory processes is very important for uncovering central mechanisms underlying complex behavioral responses that can be impaired in patients with neuropsychiatric disorders caused by dopamine system dysfunction. One of the most useful animal models for dopaminergic dysregulation is the strain of dopamine transporter knockout (DAT-KO) rats that have no dopamine re-uptake and thus elevated extracellular dopamine levels. It is known that dopamine is involved in various cognitive processes such as learning, memory and attention. This investigation was focused on the ability of DAT-KO rats to learn and perform a behavioral task in the 8-arm radial maze test. It was found that DAT-KO rats are able to learn the behavioral task, but the level of task performance did not reach that of WT group. The behavioral tactics used by animals during training significantly differ in mutants. The behavioral tactics used by DAT-KO rats involved perseverations and resulted in worse task fulfillment in comparison to wild-type controls. The data obtained indicate that deficient dopamine reuptake results in an impairment of working memory and perseverative behavioral tactics in DAT-KO rats.

Impaired alcohol-induced dopamine release in the nucleus accumbens in an inflammatory pain model: behavioral implications in male rats.

Recent studies have drawn the attention to the link between alcohol use disorder and the presence of pain. Indeed, the correct management of pain in patients with a previous history of alcohol use disorder has been reported to decrease the risk of relapse in alcohol drinking, suggesting that in this prone population, pain may increase the vulnerability to relapse. Previous data in male rats revealed that inflammatory pain desensitizes mu-opioid receptors in the ventral tegmental area and increases intake of high doses of heroin. Owing to the relevant role of mu-opioid receptors in alcohol effects, we hypothesize that pain may also alter alcohol reinforcing properties and therefore affect alcohol relapse in male rats. Our microdialysis studies show that the presence of inflammatory pain blunted the increase of extracellular dopamine levels in the nucleus accumbens induced by 1.5 g/kg of ethanol (s.c.). Moreover, we also revealed that the administration of 52 nmol of ethanol into the ventral tegmental area failed to induce place preference only in inflammatory pain-suffering animals, and a higher dose (70 nmol) was necessary to reverse this effect. Finally, we evaluated the effect of inflammatory pain on the alcohol deprivation effect in long-term ethanol-experienced male rats. After 4 cycles of free ethanol intake and abstinence periods, inflammatory pain induced alcohol deprivation effect without affecting its magnitude. These intriguing data reveal the impact of pain on neurochemical and behavioral effects after alcohol administration but also underscore the necessity of finding an appropriate paradigm to determine the long-term behavioral consequences.

ErbB4 Null Mice Display Altered Mesocorticolimbic and Nigrostriatal Dopamine Levels as well as Deficits in Cognitive and Motivational Behaviors.

Natural genetic variants of Neuregulin1 (NRG1) and its cognate receptor ErbB4 are associated with a risk for schizophrenia. Whereas most studies on NRG1-ErbB4 signaling have focused on GABAergic interneurons, ErbB4 is also expressed by midbrain dopaminergic neurons where it modulates extracellular dopamine (DA) levels. Here, we report that extracellular steady-state levels of DA are reduced in the medial prefrontal cortex (mPFC; −65%), hippocampus (−53%) and nucleus accumbens (NAc; −35%), but are elevated in the dorsal striatum (+25%) of ErbB4 knock-out mice (ErbB4 KOs) relative to wild-type controls. This pattern of DA imbalance recapitulates the reported prefrontal cortical reduction and striatal increase of DA levels in schizophrenia patients. Next, we report on a battery of behavioral tasks used to evaluate locomotor, cognitive and motivational behaviors in ErbB4 KOs relative to controls. We found that ErbB4 KOs are hyperactive in a novel open field but not in their familiar home cage, are more sensitive to amphetamine, perform poorly in the T-maze and novel object recognition (NOR) tasks, exhibit reduced spatial learning and memory on the Barnes maze, and perform markedly worse in conditioned place preference (CPP) tasks when associating cued-reward palatable food with location. However, we found that the poor performance of ErbB4 KOs in CPP are likely due to deficits in spatial memory, instead of reward seeking, as ErbB4 KOs are more motivated to work for palatable food rewards. Our findings indicate that ErbB4 signaling affects tonic DA levels and modulates a wide array of behavioral deficits relevant to psychiatric disorders, including schizophrenia.

Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study.

Central dopamine plays a key role in sexual behavior. Recently, a Dopamine Transporter knockout (DAT KO) rat has been developed, which displays several behavioral dysfunctions that have been related to increased extracellular dopamine levels and altered dopamine turnover secondary to DAT gene silencing. This prompted us to characterize the sexual behavior of these DAT KO rats and their heterozygote (HET) and wild type (WT) counterparts in classical copulatory tests with a sexually receptive female rat and to verify if and how the acquisition of sexual experience changes along five copulatory tests in these rat lines. Extracellular dopamine and glutamic acid concentrations were also measured in the dialysate obtained by intracerebral microdialysis from the nucleus accumbens (Acb) shell of DAT KO, HET and WT rats, which underwent five copulatory tests, when put in the presence of an inaccessible sexually receptive female rat and when copulation was allowed. Markers of neurotropism (BDNF, trkB), neural activation (Δ-FosB), functional (Arc and PSA-NCAM) and structural synaptic plasticity (synaptophysin, syntaxin-3, PSD-95) were also measured in the ventral tegmental area (VTA), Acb (shell and core) and medial prefrontal cortex (mPFC) by Western Blot assays. The results indicate that the sexual behavior of DAT KO vs. HET and WT rats shows peculiar differences, mainly due to a more rapid acquisition of stable sexual activity levels and to higher levels of sexual motivation and activity. These differences occurred with differential changes in dopamine and glutamic acid concentrations in Acb dialysates during sexual behavior, with lower increases of dopamine and glutamic acid in DAT KO vs. WT and HET rats, and a lower expression of the markers investigated, mainly in the mPFC, in DAT KO vs. WT rats. Together these findings confirm a key role of dopamine in sexual behavior and provide evidence that the permanently high levels of dopamine triggered by DAT gene silencing cause alterations in both the frontocortical glutamatergic neurons projecting to the Acb and VTA and in the mesolimbic dopaminergic neurons, leading to specific brain regional changes in trophic support and neuroplastic processes, which may have a role in the sexual behavior differences found among the three rat genotypes.

Possible synergies between isatin, an endogenous MAO inhibitor, and antiparkinsonian agents on the dopamine release from striatum of freely moving rats.

Isatin is an endogenous indole that inhibits monoamine oxidase (MAO). When exogenously administered, it increases the striatal dopamine and acetylcholine levels and presents neuroprotective effects in the brain. Previous studies show that intrastriatal administration of isatin increased the in vivo dopamine release from striatum in a concentration-dependent form. In the present work, we investigated the effects of combined administration of isatin together with other substances actually used in antiparkinsonian pharmacotherapy on in vivo dopamine overflow. For this, we co-administered isatin with the MAO inhibitors selegiline and clorgyline, l-DOPA, the catechol-o-methyl-transferase (COMT) inhibitors tropolone and dinitrocatechol, with the dopaminergic agonist ropinirole, and with the psychostimulant caffeine, in order to evaluate possible synergies between these substances to increase the dopamine extracellular levels in freely moving rats. Intrastriatal administration of isatin (10 mM, 60 min) significantly increased dopamine release to 1164 ± 152%, compared to the baseline. Co-administration of isatin together with selegiline (1 mM) or clorgyline (1 mM) alone or in combinations showed a similar profile to increase in vivo dopamine release. Intrastriatal infusion of isatin together with antiparkinsonian drugs l-DOPA (25 μM), tropolone (1 mM), dinitrocatechol (100 μM), amantadine (5 mM) and caffeine (5 mM) significantly elevated extracellular dopamine levels more than any single drug, showing a good neurochemical synergy by improving the effect of isatin on the extracellular dopamine levels in the striatum. Infusion of isatin + ropinirole (5 mM) did not change the isatin-induced increase in dopamine overflow. These results could be useful to carry out further investigations with a possible clinical application.

Hispidulin attenuates the social withdrawal in isolated disrupted-in-schizophrenia-1 mutant and chronic phencyclidine-treated mice.

Hispidulin is a flavonoid isolated from Clerodendrum inerme that was found to inhibit intractable motor tics. Previously, we found that hispidulin attenuates hyperlocomotion and the disrupted prepulse inhibition induced by methamphetamine and N-methyl-d-aspartate (NMDA) receptor antagonists, two phenotypes of schizophrenia resembling positive symptoms. Hispidulin can inhibit COMT, a dopamine-metabolizing enzyme in the prefrontal cortex (PFC) that is important for social interaction. Here, we investigated whether hispidulin would affect social withdrawal, one of the negative symptoms of schizophrenia. We examined whether acute administration of hispidulin would attenuate social withdrawal in two mice models, juvenile isolated disrupted-in-schizophrenia-1 mutant (mutDISC1) mice and chronic phencyclidine (PCP)-treated naïve mice. In chronic PCP-treated mice, hispidulin (10 mg·kg-1 , i.p.) attenuated social withdrawal similar to that observed with dopamine D1 receptor antagonist (SCH-23390, 0.02 mg·kg-1 , i.p.) and was mimicked by the selective COMT inhibitor, OR-486 (10 mg·kg-1 , i.p.). Hispidulin increased extracellular dopamine levels in the PFC of chronic PCP-treated mice. In isolated mutDISC1 mice, hispidulin also reversed social withdrawal. In both models, intra-PFC microinjection of a D1 agonist (SKF-81297: 10 nmol/mouse/bilateral) reversed the impairment of Ser897 phosphorylation at the GluN1 subunit of NMDA receptors, suggesting the association between GluN1 Ser897 -phosphorylation and D1 activation in the PFC exits in both models. Hispidulin attenuated social withdrawal by activating D1 receptors indirectly through elevated dopamine levels in the PFC by COMT inhibition. This nature of hispidulin suggests that it a potential novel therapeutic candidate for the treatment of negative symptoms in schizophrenia.

Regional Effects of Monoamine Transporter Blockers on Extracellular Monoamine Levels

Monoamine transporter inhibitors are used in the treatment of a number of psychiatric disorders, including major depression and ADHD, and have high abuse potential. These drugs increase extracellular levels of monoamines by blocking the reuptake of monoamines through their respective transporters. Although selective norepinephrine (NE), dopamine (DA) and serotonin (5‐HT) transporter inhibitors increase NE, DA and 5‐HT, respectively, through blockade of their cognate transporter, there is substantial evidence showing that these selective inhibitors can also block the reuptake of monoamines from non‐cognate transporters. For example, studies have shown that norepinephrine transporter (NET) inhibitors can increase extracellular NE and DA levels in the PFC and hippocampus. Studies have also shown increased extracellular NE, DA and 5‐HT levels in the PFC by administration of a selective serotonin reuptake inhibitor (SSRI). Moreover, studies from dopamine transporter (DAT) and serotonin transporter (SERT) KO mice have also shown that selective NET and SERT blockers can increase extracellular DA levels in both the PFC and striatum. Although these non‐selective effects have been reported, there has not been a systematic investigation of brain‐region specific effects of selective monoamine transporter inhibitors on extracellular monoamine levels. We therefore sought to determine the effects of selective inhibitors of monoamine transporters on extracellular monoamine levels in the prefrontal cortex (PFC), nucleus accumbens (NAcc) and dorsal striatum (dStr). We used an ex vivo neurotransmitter release assay to determine the dose‐dependent effects of a NET inhibitor, desipramine, a DAT inhibitor, GRB 12909 and a SERT inhibitor, fluoxetine, on extracellular NE, DA and 5‐HT from PFC, NAcc, and dStr slices of wild‐type (WT) mice. We also examined the dose‐dependent effects of amphetamine on extracellular monoamine levels in the brain regions of interest of WT mice. The findings from WT mice were subsequently compared to those obtained from DAT and NET knockout mice. The results show that selective blockade of NET, DAT and SERT increase extracellular levels of NE, DA, and 5‐HT respectively, as well as alter the extracellular levels of non‐selective monoamines in a brain‐region and genotype specific manner. These findings will guide future studies exploring how region‐specific changes in monoamine levels affect the activity of neuronal circuits, physiology and behavior.Support or Funding InformationBBRF/NARSAD Young Investigator award to Nikhil Urs

RGS2 Modulates Cocaine Self‐administration by Controlling Dopamine D2 Autoreceptor Activity

IntroductionRepeated exposure to drugs of abuse reduces midbrain dopamine (DA) D2 autoreceptor (D2AR) levels, which is associated with enhanced motivation for drug and escalation of drug intake. Notably, drug self‐administration disrupts the ability of D2ARs to inhibit DA release in the nucleus accumbens (NAc) and abolishes midbrain D2AR‐mediated G protein signaling. However, what regulates midbrain D2AR function remains unclear. The family of regulator of G protein signaling (RGS) proteins negatively modulates GPCR signaling by accelerating GTP hydrolysis and terminating G protein signaling. Among all the subtypes of RGS proteins, we previously showed that RGS2 is expressed in midbrain dopaminergic neurons where D2ARs are located. Importantly, we demonstrated that RGS2 is a negative modulator of D2R‐mediated G protein signaling in neuroblastoma 2a cells. We hypothesize that RGS2 in DA neurons may regulate D2AR activity and drug self‐administration. Thus, this project will determine whether RGS2 regulates cocaine self‐administration by modulating D2AR function in midbrain dopaminergic neurons.MethodsViral microinjectionsMale and female TH‐Cre Long Evans rats (~10 weeks old) were injected into bilateral VTA with an AAV expressing a RGS2 mutant with N‐terminus‐deletion (RGS2ΔN) or empty control vector. Three weeks post‐virus infusion, infection was verified using immunocytochemistry and Western blotting.Behavioral AssaysThree weeks post‐viral infusion, baseline locomotor activity was measured. Intravenous catheters were implanted and rats were trained to self‐administer cocaine. Multiple facets of cocaine self‐administration behaviors were measured including behavioral economic assessment of motivation to take cocaine (threshold protocol), extinction training and cue/cocaine‐primed reinstatement of cocaine seeking. In a separate cohort of rats, cocaine‐induced locomotor activity was measured.Functional CharacterizationIn vivo microdialysis experiments were performed in the NAc to measure extracellular DA levels elicited by cocaine or amphetamine challenge. Further, fast‐scan cyclic voltammetry was used to measure D2AR‐mediated inhibition of DA release and potassium Kv1.2 regulation of DA release in NAc slices of rats virally infused with the RGS2ΔN or empty control virus.ResultsWe found that inhibiting RGS2 activity by overexpressing RGS2ΔN in the VTA of rats significantly reduced motivation for cocaine and cocaine‐primed reinstatement of cocaine seeking. Cocaine‐induced locomotor activity was also reduced by overexpression of RGS2ΔN and was paralleled by blunted extracellular DA levels in the NAc after psychostimulant exposure. Further, RGS2ΔN overexpression significantly increased D2AR‐mediated inhibition of DA release and reduced electrically‐evoked DA release via upregulation of Kv1.2 channels in the NAc. Additionally, RGS2ΔN overexpression reduced D2AR‐mediated inhibition of locomotor activity; however, baseline and D1R‐stimulated locomotor activity were not altered. The present study provides first time evidence that RGS2 plays a critical role in cocaine self‐administration by regulating midbrain D2AR function.Support or Funding InformationThis work is supported by NIH R01DA042862, P50DA006634, F31AA025532

Investigating the Roles of Orexinergic System in Methamphetamine‐induced Addiction

Methamphetamine (METH) is a powerful neurotoxic psychostimulant characterized to affects activity of dopamine transpoters leading to blockage of dopamine (DA) uptake into the synaptic vesicle as well as DA reuptake through the plasma membrane inducing synaptic DA release, which resulted in continuous excess extracellular DA levels in the synaptic cleft. Orexinergic system has been reported to play crucial roles in regulation of arousal, wakefulness, and motivated behaviors for drug abuse. In this study, we have investigated the roles of orexin and/or orexin receptors on METH‐induced addictive behaviors by conducting conditioned place preference (CPP) test using SB334867, an orexin‐1 receptor antagonist. The changes in the dopaminergic system integrity and orexin‐related signaling molecules were examined to elucidate underlying molecular mechanisms. C57BL/6 mice were administered with METH (1 mg/kg, i.p.) alternately on each other day for 8 days, allowed to undergo withdrawal period without injection of METH for additional 8 days, and then challenged again with METH. METH‐administered mice exhibited increased levels of orexin and orexin‐1 receptor in the brain regions of hippocampus, striatum, hypothalamus, and amygdala. The application of SB334867 significantly attenuated the acquisition, expression, and reinstatement of the METH‐induced CPP. These findings suggest that orexinergic system may play a role in the acquisition and relapse of METH‐induced addictive behaviors, thereby providing a novel therapeutic target for the METH addiction.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)