Dopamine Storage Research Articles

43.4 THE ROLE OF MOLECULAR IMAGING IN GUIDING DRUG DEVELOPMENT

BackgroundMolecular imaging with Positron Emission Tomography (PET) has the unique capability of examining molecules in the brains of human subjects in real time. This has been well exploited in the study of dopaminergic transmission in schizophrenia. The results have on one hand confirmed some predictions, and on the other hand they have brought up unexpected findings, that have the potential to affect drug development in a major way.MethodsPET studies of dopamine release, synthesis, storage, receptors and transporters across labs and different experiments will be reviewed to extract findings that differ from preconceived notions. These have the capability of re-focusing drug development based on new evidence.ResultsExpected findings that PET studies have confirmed are those of striatal dopaminergic excess, measured as enhanced presynaptic release capacity and presynaptic enzymatic synthesis capacity. Also expected was hypodopaminergic transmission in the dorso-lateral prefrontal cortex.The unexpected findings from PET have been as follows: 1) the striatal excess is most predominant in the associative striatum, rather than the limbic striatum. 2) hypodopaminergia is generalized to all extrastriatal areas of the brainThese observations of opposite presynaptic dopaminergic dysfunction between striatal and extrastriatal regions suggest that the striatal excess may not be driven by activity levels of midbrain DA cells but may result from abnormal striatal local regulation of presynaptic DA release, affecting specifically the associative striatum, region which shows largest magnitude of excess DA. Furthermore alterations in dopaminergic signaling in opposing directions may have a dual effect on learning across functional domains by weakening learning from salient or relevant signals and reinforcing stimulus independent signals, thus explaining different symptom domains.DiscussionMore work is needed to understand the cellular mechanisms of dopaminergic dysfunction in schizophrenia. The evidence presented here, while limited, highlights the challenges of drug development in the absence of real in vivo information from patients, and the need for multidisciplinary cross talk to address these challenges.

Dopaminergic dysregulation and impaired associative learning behavior in zebrafish during chronic dietary exposure to selenium

A growing body of evidence indicates that exposure to selenium (Se) can cause neurotoxicity, and this can occur because of its interference with several neurotransmitter systems in humans and animals. Dopamine is a critical modulator of a variety of brain functions and a prime target for environmental neurotoxicants. However, effects of environmentally relevant concentrations of Se on dopaminergic system and its neurobehavioral effects are still largely unknown. For this purpose, we exposed zebrafish, a model organism, to different concentrations of dietary l-selenomethionine (control, 3.5, 11.1, 27.4, and 63.4 μg Se/g dry weight) for a period of 60 days. Cognitive performance of fish was evaluated using a plus maze associative learning paradigm. Oxidative stress, as the main driver of Se neurotoxicity, was assessed by measuring the ratio of reduced to oxidized glutathione (GSH:GSSG), lipid peroxidation (LPO) levels, and mRNA expression of several antioxidant enzymes in the zebrafish brain. Dopamine levels in the brain and the expression of genes involved in dopamine synthesis, storage, reuptake, metabolism, and receptor activation were examined. Moreover, transcription of several synaptic plasticity-related immediate-early and late response genes was determined. Overall, fish fed with the two highest concentrations of dietary Se displayed impaired associative learning. Se exposure also induced oxidative stress in the zebrafish brain, as indicated by a reduction in GSH:GSSG ratio, increased LPO levels, and up-regulation of antioxidant genes in fish treated with the two highest concentrations of Se. An increase in brain dopamine levels associated with altered expression of dopaminergic cell markers was evident in different treatment groups. Moreover, Se exposure led to the down-regulation of immediate-early and late response genes in fish that exhibiting learning impairment. Taken together, the results of this study imply that the induction of oxidative stress and dysregulation of dopaminergic neurotransmission may underlie Se-induced impairment of associative learning in zebrafish.

Valbenazine in tardive dyskinesia: a profile of its use

Valbenazine (Ingrezza®) is an orally bioavailable, selective and reversible vesicular monoamine transporter 2 (VMAT2) inhibitor that is indicated for the once-daily treatment of adults with tardive dyskinesia (TD) in the USA. By inhibiting VMAT2, thereby interfering with the presynaptic uptake and storage of dopamine and other monoamines, valbenazine is thought to counteract the heightened postsynaptic dopaminergic activity that is believed to cause TD. In adults with dopamine-receptor blocking agent-induced TD and underlying schizophrenia, schizoaffective disorder or mood disorder, valbenazine was associated with rapid, significant and sustained on-treatment improvements in TD severity relative to placebo. Somnolence is the most common treatment-emergent adverse event associated with valbenazine and, importantly, depression and extrapyramidal symptoms have not been associated with its use.

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats.

Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.

Metabolism of Dopamine in Nucleus Accumbens Astrocytes Is Preserved in Aged Mice Exposed to MPTP.

Parkinson disease (PD) is prevalent in elderly individuals and is characterized by selective degeneration of nigrostriatal dopamine (NSDA) neurons. Interestingly, not all dopamine (DA) neurons are affected equally by PD and aging, particularly mesolimbic (ML) DA neurons. Here, effects of aging were examined on presynaptic DA synthesis, reuptake, metabolism and neurotoxicant susceptibility of NSDA and mesolimbic dopamine (MLDA) neurons and astrocyte DA metabolism. There were no differences in phenotypic markers of DA synthesis, reuptake or metabolism in NSDA or MLDA neurons in aged mice, but MLDA neurons displayed lower DA stores. Astrocyte metabolism of DA to 3-methoxytyramine (3-MT) in the striatum was decreased in aged mice, but was maintained in the nucleus accumbens. Despite diminished DA vesicular storage capacity in MLDA neurons, susceptibility to acute neurotoxicant exposure was similar in young and aged mice. These results reveal an age- and neurotoxicant-induced impairment of DA metabolic activity in astrocytes surrounding susceptible NSDA neurons as opposed to maintenance of DA metabolism in astrocytes surrounding resistant MLDA neurons, and suggest a possible therapeutic target for PD.

The Design and Evaluation of an l-Dopa-Lazabemide Prodrug for the Treatment of Parkinson's Disease.

l-Dopa, the metabolic precursor of dopamine, is the treatment of choice for the symptomatic relief of the advanced stages of Parkinson’s disease. The oral bioavailability of l-dopa, however, is only about 10% to 30%, and less than 1% of the oral dose is estimated to reach the brain unchanged. l-Dopa’s physicochemical properties are responsible for its poor bioavailability, short half-life and the wide range of inter- and intrapatient variations of plasma levels. An l-dopa–lazabemide prodrug is proposed to overcome the problems associated with l-dopa absorption. Lazabemide is a monoamine oxidase (MAO)-B inhibitor, a class of compounds that slows the depletion of dopamine stores in Parkinson’s disease and elevates dopamine levels produced by exogenously administered l-dopa. l-Dopa was linked at the carboxylate with the primary aminyl functional group of lazabemide via an amide, a strategy which is anticipated to protect l-dopa against peripheral decarboxylation and possibly also enhance the membrane permeability of the prodrug. Selected physicochemical and biochemical properties of the prodrug were determined and included lipophilicity (logD), solubility, passive diffusion permeability, pKa, chemical and metabolic stability as well as cytotoxicity. Although oral and i.p. treatment of mice with the prodrug did not result in enhanced striatal dopamine levels, 3,4-dihydroxyphenylacetic acid (DOPAC) levels were significantly depressed compared to saline, l-dopa and carbidopa/l-dopa treatment. Based on the results, further preclinical evaluation of the l-dopa–lazabemide prodrug should be undertaken with the aim of discovering prodrugs that may be advanced to the clinical stages of development.

Initial steps of the dopamine story

At the end of 1959, it was while preparing a review on monoamine oxidase inhibitors (MAOIs) at the Faculty of Pharmacy, that I discovered the early development of modern neuropharmacology, which started around 1955. Surprised by the sedative properties of chlorpromazine in patients recovering from surgery, Henri Laborit suggested that Jean Delay and Pierre Deniker use the new compound in schizophrenic patients. It was also at this time that the antidepressant properties of MAOIs were observed in patients treated for tuberculosis. The research then into neurotransmission was mainly performed on peripheral cholinergic and noradrenergic neurons, and almost nothing was known of brain neurotransmission. Then, in 1957, Marthe Vogt reported that noradrenaline levels were decreased in the hypothalamus of rats injected with morphine. That same year, Arvid Carlsson and his colleagues described the presence of large amounts of dopamine in the rat striatum and suggested that this catecholamine was not only the precursor of noradrenaline, but could also act as well as a neurotransmitter in some central neurons. Shortly thereafter, these researchers reported that homovanillic acid, the main dopamine metabolite, was markedly increased in the striatum of rats treated with chlopromazine. This led them to propose that this neuroleptic was blocking dopamine receptors, a remarkable hypothesis that was later confirmed. These authors also indicated that dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan, the respective precursors of dopamine and serotonin, were able to reverse the parkinsonism-like syndrome due to reserpine in rabbits. These striking findings of the Swedish researchers, and the elegant metabolic studies of Julius Axelrod and Georg Hertting on peripheral noradrenergic neurons, performed thanks to the use of radioactive noradrenaline, were responsible for my own project when I started my PhD thesis in 1960 at the Pasteur Institute. The goal was to prepare radioactive dopamine from carbon-14-labelled tyrosine and to study its metabolism following its intraventricular injection into the rat brain. My interest in dopamine research was reinforced by the recent studies of Oleh Hornykiewicz and colleagues in Vienna, who observed, on autopsy, that dopamine levels were markedly reduced in the pars compacta, caudate nucleus and putamen of parkinsonian patients. These authors were also responsible for the introduction of L-DOPA treatment for Parkinson's disease, which was rapidly taken up by Andre Barbeau in Quebec. Rapid progress was being made in dopamine synthesis, storage and turnover in the rat striatum from 1963 to 1966, when I joined Axelrod's team at the US National Institutes of Health in Bethesda, Maryland. The field was markedly accelerated by the histochemical report by Kjell Fuxe and Annika Dahlstrom in 1964, which described, for the first time, the mapping of the central dopaminergic neurons as well as other aminergic neurons.

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

SummaryDevelopment of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.

Cocaine sensitization in adult Long-Evans rats perinatally exposed to polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are ubiquitous environmental toxicants known to adversely affect the nervous system and more specifically the dopamine system. Developmental PCB exposure in rats has been shown to produce alterations in dopaminergic signaling that persist into adulthood. The reinforcing properties of psychostimulants are typically modulated via the dopaminergic system, so this project used a behavioral sensitization paradigm to evaluate whether perinatal PCB exposure altered sensitization to the psychostimulant cocaine. Long-Evans rats were perinatally exposed to 0, 3 or 6mg/kg/day of PCBs throughout gestation and lactation. One male and female pup from each litter was retained for behavioral testing. Both horizontal and vertical activity were used to measure cocaine sensitization following repeated injections of 10mg/kg cocaine (IP) on post-natal day (PND) 91–96 and again after a week in the home cage on PND 103. A final locomotor activity session following a challenge injection of 20mg/kg was given on PND 110 to further evaluate the availability of presynaptic dopamine stores. The PCB-exposed rats appeared to be pre-sensitized to cocaine as they exhibited a greater degree of cocaine-induced locomotor activation to the initial injections of cocaine and therefore demonstrated a more rapid onset of cocaine behavioral sensitization compared to non-exposed controls. These results add to the literature detailing how perinatal exposure to dopamine-disrupting contaminants can change the developing brain, thereby producing permanent changes in the neurobehavioral response to psychostimulants later in life.

Dopamine Toxicity Following Chronic Co‐exposure to ‘Bath Salt’ Constituents MDPV, Mephedrone, and Methylone

In recent years, the global abuse of synthetic designer drugs has rapidly increased to become a public health threat. Popularized under the term ‘bath salts,’ synthetic cathinones continue to be one of the most frequently abused classes of designer drugs. According to DEA statistics, bath salts commonly contain one or a mixture of the following synthetic cathinones, 3, 4‐methylenedioxypyrovalerone (MDPV), 4‐methylmethcathinone (mephedrone), and 3, 4‐methylenedioxymethcathinone (methylone). Although these compounds were permanently classified as schedule I controlled substances in the Synthetic Drug Abuse Prevention Act of 2012 (DEA, 2011), they are currently not controlled by international laws and abuse continues to thrive with internet serving as a central marketplace (Karila, et. al. 2015). Pharmacological research regarding synthetic cathinone abuse has primarily focused on the individual mechanisms and associated toxicities of MDPV, mephedrone, and methylone, despite DEA statistics and numerous case reports indicating that many of the frequently abused bath salt ‘brands’ contain various mixtures of these compounds (Araujo, et. al. 2015; DEA, 2011). Given the reported dissimilarities between MDPV, mephedrone, and methylone in regards to selectivity and mechanism of action at the dopamine transporter, it was hypothesized that chronic co‐exposure to the synthetic cathinones would produce significantly altered effects on dopaminergic tone as compared to individual exposure. Our previous work has demonstrated that repeated, combined exposure to MDPV, mephedrone, and methylone, induces significant reductions in dopamine (DA) within the nucleus accumbens (NAc), striatum (STR), substantia nigra pars compacta (SNpc), and ventral tegmental area (VTA) brain regions, a result that was not observed following chronic individual administration. The present study aims to correlate the observed DA depletions following co‐synthetic cathinone exposure with changes in the expression of key proteins involved in DA synthesis, storage, release, and uptake. For this study, adolescent male Swiss‐Webster mice were administered 10 mg/kg intraperitoneal injections of MDPV, mephedrone, or the cathinone cocktail (comprised of MDPV, mephedrone, and methylone) every other day for 14 days (7 injections total). Although the transporter selectivity profile for methylone is similar to mephedrone, releasing assays have shown that it is only half as potent at the dopamine transporter (DAT) and thus was not individually examined in this study (Baumann, et. al. 2012). Brains were removed and microdissections of the NAc, STR, SNpc, and VTA were collected 48 hours after the final exposure. Tissue expression levels of tyrosine hydroxylase (TH), monoamine oxidase B (MAO‐B), catechol‐O‐methyltransferase (COMT), dopamine transporter (DAT‐1) and vesicular monoamine transporter 2 (VMAT2), were determined using Protein simple western blot analysis. Our analyses revealed significantly reduced expression levels of these key proteins involved in DA synthesis, metabolism, and transport in all collected brain regions following repeated dosing of the cathinone cocktail. These data, along with our previous findings, suggest that chronic combined exposure to MDPV, mephedrone, and methylone may be toxic to dopaminergic neurons of the mesolimibic and nigrostriatal brain pathways.

A study of the age-related effects of lactational atrazine exposure

A growing number of reports have demonstrated that the widely-used herbicide Atrazine (ATR) can cause injury to dopamine (DA) neurons, but the exact mechanism remains unclear. In this study, we examined the effects of lactational ATR exposure in Sprague-Dawley rats on dopaminergic neuron health later in life.Compared with control rats, rats exposed to ATR during a critical period of neural development showed decreased striatal DA content and increased rates of DA turnover. The expression of Monoamine oxidase (MAO), which is associated with DA degradation, was up-regulated, and the expression of Vesicular Monoamine Transporter 2 (VMAT2), which is associated with DA transport, was down-regulated. The expression of transcription factor Nuclear Receptor Related Factor 1 (Nurr1), which is associated with DA neuron development, was down-regulated. Increased age (6–12 months old) increased the statistical significance of the differences of the above indicators in the ATR-treated rats compared to the control rats (P<0.05).Taken together, our results indicate that ATR exposure during the critical neural development period causes a down-regulation of Nurr1, which in turn affects Nurr1 target genes, including MAO, VMAT2 and DAT, which are involved in DA degradation and transport. Reduced expression of these genes impairs the capacity for vesicular storage or reuptake of DA, causing decreased levels of striatal DA, which can ultimately lead to DA neuron injury. DA neuron injuries become more severe over time, which suggests that aging can synergistically promote the ATR-associated DA neuron injuries.

Serotonin and dopamine transporter PET changes in the premotor phase of LRRK2 parkinsonism: cross-sectional studies

SummaryBackgroundPatients with Parkinson’s Disease (PD) may exhibit premotor neurochemical changes in dopaminergic (DA) and nondopaminergic systems. Using positron emission tomography (PET), we studied participants with leucine-rich repeat kinase 2 (LRRK2) mutations and with sporadic PD to assess whether DA and serotonin transporter (SERT) changes were similar in LRRK2 PD and sporadic PD, and whether asymptomatic LRRK2 mutation carriers exhibited PET changes in the absence of motor symptoms.MethodsBetween July 1999 and May 2016, we did two cross sectional PET studies at the Pacific Parkinson’s Research Centre (Vancouver, Canada) with LRRK2 mutation carriers with or without manifest PD, patients with sporadic PD, and age-matched healthy controls, all aged 18 years or older. Patients with PD were diagnosed by a neurologist with movement disorder training in accordance with the UK Parkinson’s Disease Society Brain Bank criteria. LRRK2 carrier status was confirmed by bi-directional Sanger sequencing. First, affected and unaffected LRRK2 carriers seen from July 1999 to January 2012 were imaged with PET tracers for the membrane dopamine transporter (DAT) and dopamine synthesis and storage (18F-6-fluoro-L-dopa; FDOPA) and compared with sporadic PD and age-matched healthy controls. Second, distinct groups of LRRK2 mutation carriers, sporadic PD patients, and age-matched healthy controls seen from November 2012 to May 2016 were studied with tracers for the SERT and vesicular monoamine transporter 2 (VMAT2). Striatal DAT binding, DTBZ binding, FDOPA uptake and SERT binding in multiple brain regions were compared using analysis of covariance adjusted for age.FindingsUsing data from 40 LRRK2 mutation carriers, 63 patients with sporadic PD, and 35 controls, we identified significant group differences in striatal DAT binding (all age ranges p<0·0001 in caudate and putamen) and FDOPA uptake (age 50 or lower in caudate, p=0·0002; all other age ranges p<0·0001; in putamen, all age ranges p<0·0001). Affected LRRK2 mutation carriers (n=15) had reduced striatal DAT binding and FDOPA uptake, comparable to sporadic PD of similar duration. Unaffected carriers (n=25) had greater FDOPA uptake and DAT binding than sporadic PD (n=63), with FDOPA uptake comparable to and DAT binding lower than healthy controls. Unaffected LRRK2 carriers (n=9) had significantly elevated SERT binding in hypothalamus (greater than healthy controls, 7 LRRK2 PD and 13 sporadic PD subjects; p<0·0001), striatum (greater than sporadic PD; p=0·02) and brainstem (greater than affected LRRK2 carriers; p=0·01) after adjustment for age. SERT binding in cortex was not significantly different between groups after age adjustment. Striatal DTBZ binding was reduced in all affected patients and asymmetrically reduced in one unaffected carrier.InterpretationDopaminergic and serotonergic changes progress in a similar fashion in LRRK2 PD and sporadic PD, but unaffected LRRK2 mutation carriers exhibit increased SERT binding in striatum, brainstem and hypothalamus, possibly reflecting compensatory changes in serotonergic innervation preceding the motor onset of PD.FundingCanada Research Chairs, Michael J. Fox Foundation, National Institutes of Health, Pacific Alzheimer Research Foundation, Pacific Parkinson’s Research Institute, National Research Council of Canada

Brief Social Isolation in the Adolescent Wistar-Kyoto Rat Model of Endogenous Depression Alters Corticosterone and Regional Monoamine Concentrations.

The Wistar-Kyoto rat (WKY) model has been suggested as a model of adult and adolescent depression though face, predictive and construct validities of the model to depression remain equivocal. The suitability of the WKY as a diathesis model that tests the double-hit hypothesis, particularly during critical periods of brain and behavioural development remains to be established. Here, effects of post-weaning social isolation were assessed during early adolescence (~30pnd) on behavioural despair and learned helplessness in the forced swim test (FST), plasma corticosterone levels and tissue monoamine concentrations in brain areas critically involved in depression, such as prefrontal cortex, nucleus accumbens, striatum and hippocampus. Significantly increased immobility in the FST was observed in socially-isolated, adolescent WKY with a concomitant increase in corticosterone levels over and above the FST-induced stress. WKY also demonstrated a significantly increased release and utilization of dopamine, as manifested by levels of metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid in nucleus accumbens, indicating that the large dopamine storage pool evident during adolescence induces greater dopamine release when stimulated. The serotonin metabolite 5-hydroxy-indoleacetic acid was also significantly increased in nucleus accumbens, indicating increased utilization of serotonin, along with norepinephrine levels which were also signficantly elevated in socially-isolated adolescent WKY. Differences in neurochemistry suggest that social or environmental stimuli during critical periods of brain and behavioural development can determine the developmental trajectories of implicated pathways.

Disrupted iron regulation in the brain and periphery in cocaine addiction

Stimulant drugs acutely increase dopamine neurotransmission in the brain, and chronic use leads to neuroadaptive changes in the mesolimbic dopamine system and morphological changes in basal ganglia structures. Little is known about the mechanisms underlying these changes but preclinical evidence suggests that iron, a coenzyme in dopamine synthesis and storage, may be a candidate mediator. Iron is present in high concentrations in the basal ganglia and stimulant drugs may interfere with iron homeostasis. We hypothesised that morphological brain changes in cocaine addiction relate to abnormal iron regulation in the brain and periphery. We determined iron concentration in the brain, using quantitative susceptibility mapping, and in the periphery, using iron markers in circulating blood, in 44 patients with cocaine addiction and 44 healthy controls. Cocaine-addicted individuals showed excess iron accumulation in the globus pallidus, which strongly correlated with duration of cocaine use, and mild iron deficiency in the periphery, which was associated with low iron levels in the red nucleus. Our findings show that iron dysregulation occurs in cocaine addiction and suggest that it arises consequent to chronic cocaine use. Putamen enlargement in these individuals was unrelated to iron concentrations, suggesting that these are co-occurring morphological changes that may respectively reflect predisposition to, and consequences of cocaine addiction. Understanding the mechanisms by which cocaine affects iron metabolism may reveal novel therapeutic targets, and determine the value of iron levels in the brain and periphery as biomarkers of vulnerability to, as well as progression and response to treatment of cocaine addiction.

Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion

Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Genetic Isolation of Hypothalamic Neurons that Regulate Context-Specific Male Social Behavior.

Nearly all animals engage in a complex assortment of social behaviors that are essential for the survival of the species. In mammals, these behaviors are regulated by sub-nuclei within the hypothalamus, but the specific cell types within these nuclei responsible for coordinating behavior in distinct contexts are only beginning to be resolved. Here, we identify a population of neurons in the ventral premammillary nucleus of the hypothalamus (PMV) that are strongly activated in male intruder mice in response to a larger resident male but that are not responsive to females. Using a combination of molecular and genetic approaches, we demonstrate that these PMV neurons regulate intruder-specific male social behavior and social novelty recognition in a manner dependent on synaptic release of the excitatory neurotransmitter glutamate. These data provide direct evidence for a unique population of neurons that regulate social behaviors in specific contexts.

Evaluation of the effort-related motivational effects of the novel dopamine uptake inhibitor PRX-14040

Psychiatric disorders are often marked by effort-related motivational symptoms such as anergia, fatigue, psychomotor retardation, and alterations in effort-based decision making. Animal studies of effort-related choice behavior are being used to model these symptoms. With these procedures, animals are offered a choice between high effort instrumental actions leading to highly valued reinforcers vs. low effort/low reward options. In the present experiments the motivational effects of a novel dopamine (DA) uptake inhibitor, PRX-14040 (PRX), were assessed using tests of effort-based choice in rats. For the two experiments, rats were tested using the concurrent fixed ratio 5 (FR5)/chow feeding choice task. In the first 2 experiments, the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine (TBZ), which blocks DA storage and depletes DA, was used to produce a shift in effort-related choice, decreasing lever pressing and increasing chow intake. Co-administration of PRX reversed the effects of TBZ, increasing lever pressing and decreasing chow intake in TBZ-treated rats. In experiment 2, PRX was compared with the catecholamine uptake inhibitor and antidepressant bupropion (Wellbutrin), the stimulant drug methylphenidate, and the wakefulness agent modafinil. All four drugs reversed the effects of TBZ, and PRX compared favorably with these compounds. In the final experiment, PRX was assessed for its ability to increase work output in rats responding on a progressive ratio (PROG)/chow feeding choice task in rats that were otherwise untreated. PRX biased animals towards greater exertion of effort, increasing PROG lever pressing output while decreasing chow intake. In summary, PRX was able to reverse the effects of TBZ, and to increase selection of high effort activities. Taken together, these results suggest that PRX could be useful as a treatment for effort-related motivational dysfunction in humans.

Rauwolfia vomitoria inhibits olfaction and modifies olfactory bulb cells.

The rising cost of orthodox medication has endeared so many to the use of herbs for the management of neurological conditions. Rauwolfia vomitoria (RV) one of such herbs is a rainforest shrub whose parts are used locally in the management of psychiatry and other medical issues. Its usefulness though not in doubt is wrapped with adverse reports as its active constituents depletes brain monoamine and dopamine stores. This motivated this research on the effects of the root bark extract on olfaction and the olfactory bulb of adult Wistar rats. Eighteen adult Wistar rats (220g average) were divided into three groups (n=6); control (placebo), 200mg/kg and 400mg/kg RV root bark extract, respectively. The oral administration lasted for seven days and on day 8, test of olfaction was carried out and the animals immediately anaesthetized with ketamine hydrochloride (i.p.) and perfuse-fixed with 10% neutral buffered formalin. All the brains were processed for histology and immunoreactivity. Results showed loss of body weights and olfaction in the 200mg/kg and 400mg/kg RV groups. There was hypertrophy and atrophy of mitral cells respectively, in the 200mg/kg and 400mg/kg RV groups, while there was hyperplasia of cells in the internal granular and plexiform layers of both groups. There was decreased neuron specific enolase (NSE) and neurofilament (NF) expression in the 200mg/kg RV group, while NF and glial fibrillary acidic protein (GFAP) expression was decreased in the 400mg/kg RV group. However, NSE expression was enhanced in the 400mg/kg group, while GFAP expression was enhanced in the 200mg/kg RV group. These results suggest that these doses of RV affect olfaction and appetite, and stimulate adverse cellular changes in the olfactory bulb.

The inherent high vulnerability of dopaminergic neurons toward mitochondrial toxins may contribute to the etiology of Parkinson′s disease

Although the exact mechanism(s) of the degeneration of dopaminergic neurons in Parkinson's disease (PD) is not well understood, mitochondrial dysfunction is proposed to play a central role. This proposal is strongly strengthened by the findings that compromised mitochondrial functions and/or exposure to mitochondrial toxins such as rotenone, paraquat, or MPTP causes degeneration of the midbrain dopaminergic system and manifest symptoms similar to Parkinson's disease in primates and rodents (Goldman, 2014). In fact, the specific dopaminergic toxin MPTP is one of the most commonly used models in the mechanistic studies of environmental factors associated with the etiology of PD, particularly due to the availability of direct and unequivocal clinical and biochemical evidence from human and primate subjects. Several decades of intense studies in many laboratories have led to the proposition of a general mechanism for the specific dopaminergic toxicity of MPTP (Przedborski and Jackson-Lewis, 1998). The salient features of this mechanism are (a) lipophilic pro-toxin, MPTP, freely crosses the blood-brain barrier and enters the brain; (b) in glial cells monoamine oxidase-B converts it to the toxic metabolite MPP+; (c) the polar MPP+ is extruded into the extracellular space through organic cation transporter-3; (d) presynaptic dopamine transporter (DAT) takes it up specifically into dopaminergic neurons; (e) in dopaminergic neurons, MPP+ accumulates in the synaptic vesicles and/or mitochondria; (f) mitochondrial MPP+ inhibits the mitochondrial complex-I of the electron transport chain, leading to cellular ATP depletion and excessive reactive oxygen species (ROS) production causing apoptotic cell death (Lotharius and O’Malley, 2000; Storch et al., 2004). Although this mechanism is generally well accepted, numerous recent studies seriously challenge the central dogma of this proposal that the specific dopaminergic toxicity of MPP+ is primarily due to the specific uptake into dopaminergic neurons through presynaptic DAT.