Rat Striatal Synaptosomes Research Articles

Cocaine Receptor Identified as BASP1

Cocaine exerts its behavioral actions mainly through inhibition of dopamine reuptake. However, cocaine is a weak and non-selective inhibitor of the monoamine transporters. In addition, many dopamine reuptake inhibitors do not produce psychotropic effects in drug-experienced volunteers. On the other hand, previous evidence indicates more potent effects of cocaine. For example, low nanomolar cocaine regulates dopamine D2 receptor signaling. In planarians, low nanomolar levels of cocaine produces environmental place conditioning. These signaling and behavioral actions of cocaine could not be mediated through the inhibition of monoamine transporters. Furthermore, previous a study describes 16 nM cocaine binding in rat striatal synaptosomes suggesting the existence of a high-affinity receptor for cocaine. However, the identity of such receptor is unknown.

Characterization of AN317, a novel selective agonist of α6β2-containing nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6β2-containing (α6β2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson’s disease. This paper presents the preclinical characterization of AN317, a novel α6β2* agonist exhibiting functional selectivity toward other nAChRs, including α4β2, α3β4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood–brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6β2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6β2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson’s disease.

Lack of evidence for synaptic high-affinity γ-hydroxybutyric acid (GHB) transport in rat brain synaptosomes and 11 Na+ -dependent SLC neurotransmitter transporters.

γ-Hydroxybutyric acid (GHB) is an endogenous compound proposed to act as a neurotransmitter. Na+ -dependent, high-affinity GHB transport has long been considered important evidence supporting this hypothesis. However, the molecular identity of such a high-affinity transporter remains unknown. In this study, we sought to identify and characterize GHB synaptic transport through a series of studies using both native and recombinant systems with the ultimate aim of providing evidence to clarify the proposed role of GHB as a neurotransmitter in the mammalian brain. Native [3 H]GHB transport was studied in isolated rat brain synaptosomes and compared to synaptic membranes. As a targeted approach, GHB was also screened against a panel of Na+ -dependent SLC6 neurotransmitter transporters recombinantly expressed in Xenopus laevis oocytes or tsA201 cells. Finally, the low-affinity GHB transporters, MCT1/2 and SMCT1, were probed as GHB transporters in L-[14 C]lactate uptake assays in synaptosomes. We found no evidence of high-affinity [3 H]GHB transport in purified rat brain cortical or striatal synaptosomes or at any of the 11 SLC6 transporters tested. Instead, our results indicate the binding of [3 H]GHB to an unidentified membrane component, distinct from any of the known GHB targets. In accordance with others, we found that GHB and the analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) can, in millimolar concentrations, inhibit L-[14 C]lactate uptake at MCT1 and/or MCT2 and that this also can occur in synaptosomes. In conclusion, through a variety of invitro pharmacological studies, we were unsuccessful in identifying a specific synaptic high-affinity transporter for GHB. Our findings emphasize the need to reevaluate GHB's role as a potential neurotransmitter. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum

In the striatum, histamine H3 receptors (H3Rs) are co-expressed with adenosine A2A receptors (A2ARs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H3Rs and A2ARs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A2AR302-Gαqi4 and wild-type H3Rs in transfected HEK-293T cells. H3R activation with the agonist RAMH resulted in Ca2+ mobilization (pEC50 7.31 ± 0.23; maximal stimulation, Emax 449 ± 25% of basal) indicative of receptor heterodimerization. Functional H3R-A2AR heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, H3R activation decreased A2AR affinity for the agonist CGS-21680 (pKi values 8.10 ± 0.04 and 7.70 ± 0.04). Moreover, H3Rs and A2ARs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H3R-A2AR heteromer with possible physiological implications for the modulation of the intra-striatal transmission.

Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.

Cocaine modulates allosteric D2-σ1 receptor-receptor interactions on dopamine and glutamate nerve terminals from rat striatum

The effects of nanomolar cocaine concentrations, possibly not blocking the dopamine transporter activity, on striatal D2-σ1 heteroreceptor complexes and their inhibitory signaling over Gi/o, have been tested in rat striatal synaptosomes and HEK293T cells. Furthermore, the possible role of σ1 receptors (σ1Rs) in the cocaine-provoked amplification of D2 receptor (D2R)-induced reduction of K+-evoked [3H]-DA and glutamate release from rat striatal synaptosomes, has also been investigated.The dopamine D2-likeR agonist quinpirole (10nM–1μM), concentration-dependently reduced K+-evoked [3H]-DA and glutamate release from rat striatal synaptosomes. The σ1R antagonist BD1063 (100nM), amplified the effects of quinpirole (10 and 100nM) on K+-evoked [3H]-DA, but not glutamate, release.Nanomolar cocaine concentrations significantly enhanced the quinpirole (100nM)-induced decrease of K+-evoked [3H]-DA and glutamate release from rat striatal synaptosomes. In the presence of BD1063 (10nM), cocaine failed to amplify the quinpirole (100nM)-induced effects.In cotransfected σ1R and D2LR HEK293T cells, quinpirole had a reduced potency to inhibit the CREB signal versus D2LR singly transfected cells. In the presence of cocaine (100nM), the potency of quinpirole to inhibit the CREB signal was restored. In D2L singly transfected cells cocaine (100nM and 10μM) exerted no modulatory effects on the inhibitory potency of quinpirole to bring down the CREB signal.These results led us to hypothesize the existence of functional D2-σ1R complexes on the rat striatal DA and glutamate nerve terminals and functional D2-σ1R-DA transporter complexes on the striatal DA terminals. Nanomolar cocaine concentrations appear to alter the allosteric receptor-receptor interactions in such complexes leading to enhancement of Gi/o mediated D2R signaling.

Clobenpropit, a histamine H3 receptor antagonist/inverse agonist, inhibits [3H]-dopamine uptake by human neuroblastoma SH-SY5Y cells and rat brain synaptosomes.

Clobenpropit, a potent antagonist/inverse agonist at the histamine H3 receptor (H3R), reduced the cytotoxic action of 6-hydroxydopamine (6-OHDA) in neuroblastoma SH-SY5Y cells transfected with the human H3R. We therefore set out to study whether this effect involved a receptor-independent action on dopamine transport. The uptake of [3H]-dopamine was assayed in SH-SY5Y cells and rat striatal or cerebro-cortical isolated nerve terminals (synaptosomes). Clobenpropit binding to the human norepinephrine (NET) and dopamine (DAT) transporters was analyzed by molecular modeling. In SH-SY5Y cells, [3H]-dopamine uptake was inhibited by desipramine (selective NET inhibitor), GBR-12909 (selective DAT inhibitor), and fluoxetine (selective inhibitor of the serotonin transporter, SERT) with IC50 values 37, 537, and 2800nM, respectively. The potency rank order indicates that [3H]-dopamine uptake is primarily performed by NET. Clobenpropit inhibited [3H]-dopamine uptake (maximum inhibition 82.7±2.8%, IC50 490nM), and the effect was reproduced by the H3R antagonist/inverse agonist iodophenpropit, but not by the agonists R-α-methylhistamine and immepip or the antagonists/inverse agonists ciproxifan and A-331440. Clobenpropit also inhibited [3H]-dopamine uptake by rat striatal and cerebro-cortical synaptosomes (-54.6±11.3% and -46.3±9.6%, respectively, at 10μM). Modeling of the human NET and DAT obtained by homology from the crystal of Drosophila melanogaster DAT showed that clobenpropit can bind to a site also recognized in both transporters by nisoxetine, a potent NET inhibitor. These data indicate a direct inhibitory effect of clobenpropit on catecholamine transport.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects.

Amphetamines (AMPHs) are globally abused. With no effective treatment for AMPH addiction to date, there is urgent need for the identification of druggable targets that mediate the reinforcing action of this stimulant class. AMPH-stimulated dopamine efflux is modulated by protein kinase C (PKC) activation. Inhibition of PKC reduces AMPH-stimulated dopamine efflux and locomotor activity. The only known CNS-permeant PKC inhibitor is the selective estrogen receptor modulator tamoxifen. In this study, we demonstrate that a tamoxifen analog, 6c, which more potently inhibits PKC than tamoxifen but lacks affinity for the estrogen receptor, reduces AMPH-stimulated increases in extracellular dopamine and reinforcement-related behavior. In rat striatal synaptosomes, 6c was almost fivefold more potent at inhibiting AMPH-stimulated dopamine efflux than [3H]dopamine uptake through the dopamine transporter (DAT). The compound did not compete with [3H]WIN 35,428 binding or affect surface DAT levels. Using microdialysis, direct accumbal administration of 1 μM 6c reduced dopamine overflow in freely moving rats. Using LC-MS, we demonstrate that 6c is CNS-permeant. Systemic treatment of rats with 6 mg/kg 6c either simultaneously or 18 h prior to systemic AMPH administration reduced both AMPH-stimulated dopamine overflow and AMPH-induced locomotor effects. Finally, 18 h pretreatment of rats with 6 mg/kg 6c s.c. reduces AMPH-self administration but not food self-administration. These results demonstrate the utility of tamoxifen analogs in reducing AMPH effects on dopamine and reinforcement-related behaviors and suggest a new avenue of development for therapeutics to reduce AMPH abuse.

Histamine H3 receptor activation stimulates calcium mobilization in a subpopulation of rat striatal neurons in primary culture, but not in synaptosomes

The histamine H3 receptor (H3R) is abundantly expressed in the Central Nervous System where it regulates several functions pre and postsynaptically. H3Rs couple to Gαi/o proteins and trigger or modulate several intracellular signaling pathways, including the cAMP/PKA pathway and the opening of N- and P/Q-type voltage-gated Ca2+ channels. In transfected cells, activation of the human H3R of 445 amino acids (hH3R445) results in phospholipase C (PLC) stimulation and release of Ca2+ from intracellular stores. In this work we have studied whether H3R activation induces Ca2+ mobilization from intracellular stores in native systems, either isolated nerve terminals (synaptosomes) or neurons in primary culture. In rat striatal synaptosomes H3R activation induced inositol 1,4,5-trisphosphate (IP3) formation but failed to increase the intracellular calcium concentration ([Ca2+]i). In striatal primary cultures H3R activation resulted in IP3 formation and increased the [Ca2+]i in 18 out of 70 cells that responded with an elevation in the [Ca2+]i to membrane depolarization with KCl (100 mM) as evaluated by microfluorometry. Confocal microscopy studies corroborated the increase in [Ca2+]i induced by H3R activation in a fraction of those cells that were responsive to membrane depolarization. These results indicate that H3R activation stimulates the PLC/IP3/Ca2+ pathway but only in a subpopulation of striatal neurons.

Novel multifunctional pharmacology of lobinaline, the major alkaloid from Lobelia cardinalis

In screening a library of plant extracts from ~1000 species native to the Southeastern United States, Lobelia cardinalis was identified as containing nicotinic acetylcholine receptor (nicAchR) binding activity which was relatively non-selective for the α4β2- and α7-nicAchR subtypes. This nicAchR binding profile is atypical for plant-derived nicAchR ligands, the majority of which are highly selective for α4β2-nicAchRs. Its potential therapeutic relevance is noteworthy since agonism of α4β2- and α7-nicAchRs is associated with anti-inflammatory and neuroprotective properties. Bioassay-guided fractionation of L. cardinalis extracts led to the identification of lobinaline, a complex binitrogenous alkaloid, as the main source of the unique nicAchR binding profile. Purified lobinaline was a potent free radical scavenger, displayed similar binding affinity at α4β2- and α7-nicAchRs, exhibited agonist activity at nicAchRs in SH-SY5Y cells, and inhibited [3H]-dopamine (DA) uptake in rat striatal synaptosomes. Lobinaline significantly increased fractional [3H] release from superfused rat striatal slices preloaded with [3H]-DA, an effect that was inhibited by the non-selective nicAchR antagonist mecamylamine. In vivo electrochemical studies in urethane-anesthetized rats demonstrated that lobinaline locally applied in the striatum significantly prolonged clearance of exogenous DA by the dopamine transporter (DAT). In contrast, lobeline, the most thoroughly investigated Lobelia alkaloid, is an α4β2-nicAchR antagonist, a poor free radical scavenger, and is a less potent DAT inhibitor. These previously unreported multifunctional effects of lobinaline make it of interest as a lead to develop therapeutics for neuropathological disorders that involve free radical generation, cholinergic, and dopaminergic neurotransmission. These include neurodegenerative conditions, such as Parkinson's disease, and drug abuse.

Proteomics Analysis of Dorsal Striatum Reveals Changes in Synaptosomal Proteins following Methamphetamine Self-Administration in Rats.

Methamphetamine is a widely abused, highly addictive drug. Regulation of synaptic proteins within the brain’s reward pathway modulates addiction behaviours, the progression of drug addiction and long-term changes in brain structure and function that result from drug use. Therefore, using large scale proteomics studies we aim to identify global protein expression changes within the dorsal striatum, a key brain region involved in the modulation of addiction. We performed LC-MS/MS analyses on rat striatal synaptosomes following 30 days of methamphetamine self-administration (2 hours/day) and 14 days abstinence. We identified a total of 84 differentially-expressed proteins with known roles in neuroprotection, neuroplasticity, cell cytoskeleton, energy regulation and synaptic vesicles. We identify significant expression changes in stress-induced phosphoprotein and tubulin polymerisation-promoting protein, which have not previously been associated with addiction. In addition, we confirm the role of amphiphysin and phosphatidylethanolamine binding protein in addiction. This approach has provided new insight into the effects of methamphetamine self-administration on synaptic protein expression in a key brain region associated with addiction, showing a large set of differentially-expressed proteins that persist into abstinence. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD001443.

The Protein Kinase C Inhibitor Tamoxifen Inhibits Neurochemical and Reinforcing Behavioral Effects of Amphetamine

The amphetamine (AMPH) class of drugs exerts its reinforcing effects primarily by reversing the normal function of the dopamine transporter (DAT) and increasing synaptic dopamine in the striatum and nucleus accumbens. We found that inhibition of protein kinase C (PKC) decreases AMPH‐stimulated efflux without affecting normal dopamine uptake by the dopamine transporter. Here, we investigate the estrogen receptor modulator tamoxifen, the only known brain permeable PKC inhibitor, as a potential therapeutic scaffold for the treatment of AMPH abuse. We tested the effect of tamoxifen in vitro on AMPH‐stimulated dopamine efflux in rat striatal synaptosomes and DAT‐containing neuroblastoma (N2A) cells, and in vivo on AMPH‐stimulated locomotor behavior and self‐administration of AMPH in rats. Tamoxifen and its metabolite 4‐hydroxytamoxifen (at 3 μM) decrease AMPH‐stimulated dopamine efflux in vitro in rat striatal synaptosomes by approximately 30% and in hDAT‐N2A cells by 40%. The inhibitory activity of tamoxifen on AMPH‐stimulated dopamine efflux was unaffected by knockdown of estrogen receptor alpha, supporting our hypothesis that tamoxifen inhibits AMPH action independently of its ER modulating effects. Most strikingly, pretreatment of rats with tamoxifen blocks AMPH‐stimulated hyperactivity and dose‐dependently (3‐9 mg/kg) decreases responding for AMPH in a self‐administration paradigm. We conclude that tamoxifen shows potential as a pharmaceutical lead in the development of a therapeutic for the treatment of amphetamine abuse. Supported by DA11697, DA007267, GM07767

Single and Binge Methamphetamine Administrations Have Different Effects on the Levels of Dopamine D2 Autoreceptor and Dopamine Transporter in Rat Striatum

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum. Dopamine D2 receptor antagonists and dopamine transporter (DAT) inhibitors protect against neurotoxicity of the drug by decreasing intracellular dopamine content and, consequently, dopamine autoxidation and production of reactive oxygen species. In vitro, amphetamines regulate D2 receptor and DAT functions via regulation of their intracellular trafficking. No data exists on axonal transport of both proteins and there is limited data on their interactions in vivo. The aim of the present investigation was to examine synaptosomal levels of presynaptic D2 autoreceptor and DAT after two different regimens of METH and to determine whether METH affects the D2 autoreceptor-DAT interaction in the rat striatum. We found that, as compared to saline controls, administration of single high-dose METH decreased D2 autoreceptor immunoreactivity and increased DAT immunoreactivity in rat striatal synaptosomes whereas binge high-dose METH increased immunoreactivity of D2 autoreceptor and had no effect on DAT immunoreactivity. Single METH had no effect on D2 autoreceptor-DAT interaction whereas binge METH increased the interaction between the two proteins in the striatum. Our results suggest that METH can affect axonal transport of both the D2 autoreceptor and DAT in an interaction-dependent and -independent manner.

Post‐phosphorylation control of dopamine transporter by peptidyl prolyl cis‐trans isomerase PIN1 (803.6)

The rat dopamine transporter (DAT) is phosphorylated at the N‐terminus on T53, a site that is followed by proline, making it specific for proline‐directed kinases such as ERK. We found that amphetamine (AMPH) stimulates DAT T53 phosphorylation and mutating this site affects DA transport and AMPH‐stimulated efflux, suggesting a role for T53 phosphorylation in these processes. Phosphorylation of proline‐directed residues strongly impacts protein conformation by reducing the rate of cis‐trans isomerization of the phospho (p)‐S/T‐P peptide bond. This in turn slows the rate of p‐S/T‐P dephosphorylation by preventing actions of phosphatases. The peptidyl prolyl cis‐trans isomerase, PIN1, specifically isomerizes p‐S/T‐P motifs and catalyzes the return of the bond to the trans configuration allowing dephosphorylation to occur. To test for such activities on DAT we have used the PIN1‐specific inhibitor juglone. In both rat striatal synaptosomes and heterologous expression systems juglone treatment rapidly increased the p‐T53 level suggesting that PIN1 acts on DAT and regulates dephosphorylation of T53. In addition, juglone strongly stimulated [3H]DA efflux from rat striatal synaptosomes, supporting a mechanistic link between efflux and p‐T53. These results identify a novel mechanism for regulation of DA efflux and suggest PIN1 as an enzyme that functions in post‐phosphorylation control of DAT function.Grant Funding Source: Supported by NIDA R01 DA 13147

Ethylphenidate: From biomarker to designer drug

The following is a brief background and account of the psychostimulant "designer drug" and curious methylphenidate-ethanol transesterification metabolite, ethylphenidate (EPH; Fig 1). That is ethylphenidate with an "E", not it's well known homolog, methylphenidate (MPH; Ritalin, Concerta, others) – the first-line agent for the treatment of attention-deficit/hyperactivity disorder (ADHD). Note that the only structural difference resides in the ester where EPH has an ethyl group rather than the methyl group in MPH.

Overexpression of parkin in the rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity.

HIV-1 Tat Protein Decreases Dopamine Transporter Cell Surface Expression and Vesicular Monoamine Transporter-2 Function in Rat Striatal Synaptosomes

The dopamine (DA) transporter (DAT) and vesicular monoamine transporter (VMAT2) proteins interact as a biochemical complex to regulate dopaminergic neurotransmission. We have reported that HIV-1Tat(1-86) decreases the specific [(3)H]DA uptake and [(3)H]WIN 35,428 binding sites without a change in total DAT immunoreactivity in rat striatum (Zhu et al., 2009b). The present study determined the effects of Tat on DAT phosphorylation and trafficking, and vesicular [(3)H]DA uptake. Pre-incubation of rat striatal synaptosomes with the protein kinase C (PKC) inhibitor bisindolylmaleimide I (1μM) completely blocked Tat(1-86)-induced reduction of [(3)H]DA uptake, indicating that Tat regulates DAT function through a PKC-dependent mechanism. After exposure of synaptosomes to Tat(1-86) (1μM), DAT immunoreactivity was decreased in plasma membrane enriched fractions (P3) and increased in vesicle-enriched fractions (P4) relative to controls without change in total synaptosomal fractions (P2), suggesting that Tat-induced inhibition of DA uptake is attributable to DAT internalization. Although both DAT and VMAT2 proteins are essential for the regulation of DA disposition in synapse and cytosol, Tat inhibited the specific [(3)H]DA uptake into vesicles (P4) and synaptosomes (P2) by 35% and 26%, respectively, inferring that the inhibitory effect of Tat was more profound in VMAT2 protein than in DAT protein. Taken together, the current study reveals that Tat inhibits DAT function through a PKC and trafficking-dependent mechanism and that Tat impacts the dopaminergic tone by regulating both DAT and VMAT2 proteins. These findings provide new insight into understanding the pharmacological mechanisms of HIV-1 viral protein-induced dysfunction of DA neurotransmission in HIV-infected patients.

Varenicline Is a Potent Partial Agonist at α6β2* Nicotinic Acetylcholine Receptors in Rat and Monkey Striatum

Extensive evidence indicates that varenicline reduces nicotine craving and withdrawal symptoms by modulating dopaminergic function at α4β2* nicotinic acetylcholine receptors (nAChRs) (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex). More recent data suggest that α6β2* nAChRs also regulate dopamine release and mediate nicotine reinforcement. The present experiments were therefore done to test the effect of varenicline on α6β2* nAChRs and their function, because its interaction with this subtype is currently unclear. Receptor competition studies showed that varenicline inhibited α6β2* nAChR binding (K(i) = 0.12 nM) as potently as α4β2* nAChR binding (K(i) = 0.14 nM) in rat striatal sections and with ∼20-fold greater affinity than nicotine. Functionally, varenicline was more potent in stimulating α6β2* versus α4β2* nAChR-mediated [(3)H]dopamine release from rat striatal synaptosomes with EC(50) values of 0.007 and 0.086 μM, respectively. However, it acted as a partial agonist on α6β2* and α4β2* nAChR-mediated [(3)H]dopamine release with maximal efficacies of 49 and 24%, respectively, compared with nicotine. We also evaluated varenicline's action in striatum of monkeys, a useful animal model for comparison with humans. Varenicline again potently inhibited monkey striatal α6β2* (K(i) = 0.13 nM) and α4β2* (K(i) = 0.19 nM) nAChRs in competition studies. Functionally, it potently stimulated both α6β2* (EC(50) = 0.014 μM) and α4β2* (EC(50) = 0.029 μM) nAChR-mediated [(3)H]dopamine release from monkey striatal synaptosomes, again acting as a partial agonist relative to nicotine at both subtypes. These data suggest that the ability of varenicline to interact at α6β2* nAChRs may contribute to its efficacy as a smoking cessation aid.

Endogenous and Psychostimulant Substrates but not Blockers Stimulate Dopamine Transporter Phosphorylation at a Proline‐Directed Site

The dopamine transporter (DAT) clears extraneuronal dopamine (DA) and thus controls the spatio‐temporal dynamics of dopaminergic neurotransmission. DAT is the major target for psychostimulants such as cocaine (COC) and amphetamine (AMPH). DAT has a serine cluster on the distal N‐terminus that is phosphorylated in a PKC‐dependent manner and a proline‐directed site, Thr (T) 53, that is phosphorylated in vitro by the MAP kinases. COC and AMPH impact DAT regulatory properties including uptake activity and surface expression. Although, the mechanism of drug action on DAT is unclear, DAT phosphorylation is associated with altered surface expression and DAT activity. In this study, we examined the effect of DA and several psychostimulant drugs on the phosphorylation of DAT using a newly developed phospho‐specific antibody that detects pT53. Immunoblotting revealed that DAT substrates led to increased DAT T53 phosphorylation. This increase was transport or binding dependent as COC blocked the AMPH effect. In rat striatal synaptosomes METH‐stimulated DAT T53 phosphorylation was rapid, occurring within 60 sec. In contrast, uptake blockers did not influence T53 phosphorylation. Because phosphorylation at proline‐directed sites causes cis‐isomerization of the protein backbone, our findings indicate that translocation or binding of substrates induces major structural rearrangements of the membrane‐proximal N‐terminus. We hypothesize that substrate‐mediated phosphorylation‐dependent structural rearrangement resulting from prolyl cis‐isomerization could thus act as a mechanism affecting DAT function.Supported by NIDA R01 DA 13147‐09