Recombinant Adeno-Associated Virus-mediated rescue of function in a mouse model of Dopamine Transporter Deficiency Syndrome.

Dopamine Transporter Deficiency Syndrome (DTDS) is a rare autosomal recessive disorder caused by loss-of-function mutations in dopamine transporter (DAT) gene, leading to severe neurological disabilities in children and adults. DAT-Knockout (DAT-KO) mouse is currently the best animal model for this syndrome, displaying functional hyperdopaminergia and neurodegenerative phenotype leading to premature death in ~36% of the population. We used DAT-KO mouse as model for DTDS to explore the potential utility of a novel combinatorial adeno-associated viral (AAV) gene therapy by expressing DAT selectively in DA neurons and terminals, resulting in the rescue of aberrant striatal DA dynamics, reversal of characteristic phenotypic and behavioral abnormalities, and prevention of premature death. These data indicate the efficacy of a new combinatorial gene therapy aimed at rescuing DA function and related phenotype in a mouse model that best approximates DAT deficiency found in DTDS.

Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits.

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinson's disease.

Taar1-mediated modulation of presynaptic dopaminergic neurotransmission: role of D2 dopamine autoreceptors.

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

A functional alternative splicing mutation in human tryptophan hydroxylase-2.

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

Implanted reuptake-deficient or wild-type dopaminergic neurons improve ON L-dopa dyskinesias without OFF-dyskinesias in a rat model of Parkinson's disease.

OFF-L-dopa dyskinesias have been a surprising side-effect of intrastriatal foetal ventral mesencephalic transplantation in patients with Parkinson's disease. It has been proposed that excessive and unregulated dopaminergic stimulation of host post-synaptic striatal neurons by the grafts could be responsible for these dyskinesias. To address this issue we transplanted foetal dopaminergic neurons from mice lacking the dopamine transporter (DATKO) or from wild-type mice, into a rat model of Parkinson's disease and L-dopa-induced dyskinesias. Both wild-type and DATKO grafts reinnervated the host striatum to a similar extent, but DATKO grafts produced a greater and more diffuse increase in extra-cellular striatal dopamine levels. Interestingly, grafts containing wild-type dopaminergic neurons improved parkinsonian signs to a similar extent as DATKO grafts, but provided a more complete reduction of L-dopa induced dyskinesias. Neither DATKO nor wild-type grafts induced OFF-L-dopa dyskinesias. Behavioural and receptor autoradiography analyses demonstrated that DATKO grafts induced a greater normalization of striatal dopaminergic receptor supersensitivity than wild-type grafts. Both graft types induced a similar downregulation and normalization of PEnk and fosb/Deltafosb in striatal neurons. In summary, DATKO grafts causing high and diffuse extra-cellular dompamine levels do not per se alter graft-induced recovery or produce OFF-L-dopa dyskinesias. Wild-type dopaminergic neurons appear to be the most effective neuronal type to restore function and reduce L-dopa-induced dyskinesias.

Hyperactivity, elevated dopaminergic transmission, and response to amphetamine in M1 muscarinic acetylcholine receptor-deficient mice.

Acetylcholine serves an important modulatory role in the central nervous system. Pharmacological evidence has suggested that cholinergic activity can modulate central dopaminergic transmission; however, the nature of this interaction and the receptors involved remain undefined. In this study we have generated mice lacking the M1 muscarinic acetylcholine receptor and examined the effects of M1 deletion on dopaminergic transmission and locomotor behavior. We report that M1 deficiency leads to elevated dopaminergic transmission in the striatum and significantly increased locomotor activity. M1-deficient mice also have an increased response to the stimulatory effects of amphetamine. Our results provide direct evidence for regulation of dopaminergic transmission by the M1 receptor and are consistent with the idea that M1 dysfunction could be a contributing factor in psychiatric disorders in which altered dopaminergic transmission has been implicated.

Effects of intrastriatal infusion of D2 and D3 dopamine receptor preferring antagonists on dopamine release in rat dorsal striatum (in vivo microdialysis study).

Dopamine D2-like receptor antagonists haloperidol, spiperone, clozapine, cis -( +)- (1S,2R)-5-methoxy-1-methyl-2-(n -propylamino)tetralin, ( +)-AJ76, cis -( +)- (1S,2R)-5-methoxy-1-methyl-2-(n -di-propylamino)tetralin, ( +)-UH232, and putative D3 dopamine receptor agonist ( +/-)- 7-hydroxy-N,N-di- n -propyl-2-aminotetralin, 7-OH-DPAT, were infused via a transcerebral microdialysis probe into the dorsal striatum of freely moving rats. Local infusion of all the dopamine antagonists studied resulted in concentration-dependent increase of striatal dopamine release in vivo. Subsequent i.p. administration of the drugs did not produce a further rise of dopamine release as compared to the maximal increase elicited by local administration of the same substances. The difference between effects of D2 and D3 dopamine receptor preferring antagonists applied locally was observed only in the degree of dopamine release elevation [the maximal responses were about 160% for haloperidol and spiperone, 190% for clozapine and ( +)-UH232 and 400% for ( +)-AJ76, of basal]. Striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels were elevated only slightly following local infusion of haloperidol, spiperone and clozapine, while systemic administration of the drugs resulted in a marked increase of metabolite extracellular levels. Both ( +)-UH232 and ( +)-AJ76 were found to increase significantly DOPAC and HVA levels during infusion, but the effect was less pronounced in comparison to that produced by systemic drug administration. Infusion of 7-OH-DPAT in the concentration range 5 x 10(-9)to 10(-6) M significantly decreased dopamine release but not metabolite levels down to the values observed following systemic drug administration. The present results give further evidence for the hypothesized leading role of nerve terminal dopamine autoreceptors, presumably of D3 type, in neuroleptic-induced augmentation of dopamine release in rat dorsal striatum.

Effects of a psychostimulant drug sydnocarb on rat brain dopaminergic transmission in vivo.

Transcerebral microdialysis was used to evaluate the effect of a psychostimulant drug, sydnocarb (3-(beta-phenylisopropyl)-N-phenylcarbamoylsydnonimine), on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the dorsal striatum and nucleus accumbens of freely moving rats. Sydnocarb dose dependently (4.4, 8.75 and 17.5 mg/kg, i.p.) induced a relatively modest (up to 350% of control) and long-lasting (up to 6 h) increase in dopamine extracellular level in the rat dorsal striatum. The drug at 8.75 mg/kg, i.p., produced an approximately similar increase in dopamine efflux in the dorsal striatum and in the nucleus accumbens of freely moving rats. Sydnocarb had no effect on DOPAC or HVA extracellular levels in the rat basal ganglia in vivo at any dose studied. It is important that the drug increased the efflux of dopamine in a tetrodotoxin-sensitive and Ca2+-dependent manner. Measurements of behavioral parameters in non-operated rats revealed that sydnocarb markedly increased locomotor activity and induced stereotyped behavior. These data suggest that the stimulant action of sydnocarb is accompanied by a facilitation of central dopaminergic transmission involving an increase in Ca2+-dependent vesicular dopamine efflux.

[The brain dopaminergic systems: receptor heterogeneity, functional role and pharmacological regulation]. / Dofaminergicheskie sistemy mozga: retseptornaia geterogennost', funktsional'naia rol', farmakologicheskaia reguliatsiia.

Recent advances in molecular neurobiology led to a new understanding on mammalian brain dopaminergic system which play a major role in the regulation of motor, cognitive, emotional, neuroendocrine function as well as in the pathogenesis of several pathological conditions, including neurodegenerative diseases, affective disorders, schizophrenia, drug addiction etc. Functional, biochemical and pharmacological heterogeneity of dopamine receptors, which were divided into D1-like (D1 and D5 subtypes) and D2-like (D2, D3 and D4) families of receptors has been postulated. The article reviews the recent advances including author's own results concerning the structure and function of main dopaminergic brain system, i.e. nigrostriatal and mesolimbic. The problem of autoreceptor regulation of dopaminergic neurotransmission, particularly, the processes of dopamine synthesis, release and metabolism has been specially discussed. An involvement of D2 and D3 dopamine autoreceptors in the control of these processes and differences in the mode of action of typical and atypical neuroleptics demonstrating various affinities to D2 and D3 dopamine receptors are analysed in detail. Dopamine and its metabolites have been determined on freely moving rats using brain microdialysis and high performance liquid chromatography. It is hypothesized that dopamine D3 autoreceptor is preferentially involved in the regulation of dopamine release while D2 one is responsible for the control of dopamine synthesis and metabolism in rat basal ganglia in vivo.

In vivo evidence for preferential role of dopamine D3 receptor in the presynaptic regulation of dopamine release but not synthesis.

Brain microdialysis was used to investigate the effects of the putative dopamine D3 receptor agonist (+/-)-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) on dopamine release, metabolism and synthesis in the dorsal striatum and nucleus accumbens of awake rats. The drug administered i.p. dose dependently decreased the release, metabolism and synthesis of dopamine in both brain areas. The potency of 7-OH-DPAT to decrease dopamine release was found to be higher in the nucleus accumbens than in the dorsal striatum (ED50 for nucleus accumbens 0.0096 mg/kg, i.p.; for dorsal striatum 0.068 mg/kg, i.p.). Dopamine metabolism, assessed by measuring 3,4-dihydroxyphenylacetic acid extracellular levels, and dopamine synthesis, determined as 3,4-dihydroxyphenylalanine output following perfusion with the L-aromatic acid decarboxylase inhibitor 3-hydroxybenzylhydrazine (10(-5) M), were decreased at higher dose ranges of 7-OH-DPAT (ED50 for decrease of 3,4-dihydroxyphenylalanine output in nucleus accumbens 0.124 mg/kg, i.p.; in dorsal striatum 0.101 mg/kg, i.p.). The hypomotility of rats induced by 7-OH-DPAT in doses of 0.002-0.25 mg/kg, i.p., was shown to correlate with the decreased dopamine release in the nucleus accumbens. Pretreatment of animals with 7-OH-DPAT at the putative dopamine D3 receptor 'selective' dose of 0.05 mg/kg, i.p., was found to prevent the increase of dopamine release but not the increase in metabolism in the dorsal striatum of freely moving rats induced by (+)-AJ76, cis (+)-(1S,2R)-5-methoxy-1-methyl-1-2-(n-propylamino)tetralin HCI (7 mg/kg, i.p.) and haloperidol (0.1 mg/kg, i.p.). Local application of 7-OH-DPAT by addition into the perfusing medium also resulted in a preferential decrease of dopamine release in the nucleus accumbens as compared with the dorsal striatum (EC50 for nucleus accumbens 1.9 nM; for dorsal striatum 11.3 nM). The present results give further support to the hypothesis that the dopamine D3 autoreceptor is preferentially involved in the presynaptic regulation of dopamine release, while the D2 autoreceptor controls dopamine synthesis.

Simultaneous monitoring of dopamine, its metabolites and trans-isomer of atypical neuroleptic drug carbidine concentrations in striatal dialysates of conscious rats.

1. Transcerebral microdialysis was used to monitor dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and trans-isomer of atypical neuroleptic drug carbidine concentrations in the dialysates from dorsal striatum of freely moving rats following i.p. administration of the drug at doses 0.5, 1,5 and 25 mg/kg. The changes in locomotor activity as well as catalepsy in rats following transcarbidine administration were also evaluated. 2. The microdialysis "point of no net flux" method was used to measure interstitial free concentration (IFC) of trans-carbidine in the dorsal striatum of freely moving rats following i.p. administration of the drug at dose 5 mg/kg. The maximal IFC of trans-carbidine was found to be approximately 1 mu M 20-40 min after injection. 3. The drug at doses up to 1 mg/kg produces elevation of dopamine release not affecting sufficiently its metabolite dialysate levels. IFC of the drug calculated for these doses will not exceed 0.24 pM. At the dose 5 mg/kg, i.p., elevation of both dopamine release and metabolism was observed and dopamine release increased slightly more than DOPAC dialysate levels. 4. Stimulatory action of trans-carbidine on locomotor activity of non-operated rats has been observed at doses 0.2 and 0.5 mg/kg, i.p.. 5. Only the dose 25 mg/kg of trans-carbidine (maximal calculated IFC 4.53 mu M) was found to be cataleptogenic. The drug at this dose failed to increase DA release but induced a marked increase of DOPAC and HVA output. 6. It is concluded that trans-carbidine in in vivo neurochemical and behavioural studies demonstrates the preferential antagonistic action on dopamine release-regulating autoreceptors.

Regulation of dopamine release and metabolism in rat striatum in vivo: effects of dopamine receptor antagonists.

1. The acute effects of some of typical and atypical antipsychotic drugs on the dopamine release and metabolism in the dorsal striatum of freely moving rats were studied using transcerebral microdialysis technique. 2. Classical neuroleptic drugs haloperidol (0.05, 0.1 and 0.2 mg/kg), thioproperazine (0.1, 0.2 and 0.4 mg/kg) and spiperone (0.02, 0.04 and 0.07 mg/kg) administered i.p. induced pronounced elevation of extracellular level of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) up to 250-300% to basal level while producing less increase in that of dopamine (DA) (up to 150-170%). 3. Atypical neuroleptics clozapine and thioridazine (both 2, 5 and 20 mg/kg) increased striatal DA release and DOPAC level approximately at the same degree (maximally up to 200% and 160%, respectively). 4. Dopamine D3 receptor and autoreceptor preferring antagonists (+)-UH232 and (+)-AJ76 (both 4, 7 and 14 mg/kg) more potently increased DA release in comparison with DOPAC dialysate level (+)-AJ76 elevated DA level maximally up to 330%, DOPAC-up to 250%). 5. The features of typical and atypical neuroleptics in preferential action on DA release or DOPAC output were observed in all doses of the drugs studied. 6. The ability of the drugs to affect preferentially DA release or DOPAC extracellular level in rat striatum correlates to their relative affinities at D3 and D2 DA receptors. 7. It is concluded that typical and atypical antipsychotic drugs might be clearly distinguished on the basis of their ability to affect preferentially DA synthesis/metabolism or release in rat dorsal striatum in vivo.

Estimation of the interstitial free concentration of the putative dopamine D3 receptor selective agonist 7-OH-DPAT in the dorsal striatum of freely moving rats.

Using the quantitative microdialysis 'point of no net flux' method, we estimated the interstitial free concentration (IFC) of (+/-)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin (7-OH-DPAT) in the dorsal striatum of freely moving rats after i.p. administration of the drug at the dose of 18.3 mumol/kg. The maximal IFC of 7-OH-DPAT was found to be 1.61 microM 20 min after the injection. Due to the approximately linear relationship between dose and dialysate concentration observed, it may be inferred that the behaviourally active 7-OH-DPAT dose of 0.12 mumol/kg should give an IFC which does not exceed 10 nM. It is concluded that in vivo effects observed following 7-OH-DPAT i.p. administration at doses lower than 0.12 mumol/kg might be considered as mediated by the dopamine D3 receptor.

Dopamine D2 and D3 receptor preferring antagonists differentially affect striatal dopamine release and metabolism in conscious rats.

Classical neuroleptic drugs with high affinity for dopamine D2 receptors in comparison to D3 ones (haloperidol, thioproperazine and spiperone) administered i.p. acutely (0.2, 0.2 and 0.07 mg/kg, respectively) induced a pronounced increase in the extracellular level of 3,4-dihydroxyphenylacetic acid (DOPAC) and only a modest rise in that of dopamine in the dorsal striatum of conscious rats studied by transcerebral microdialysis. Atypical neuroleptics, clozapine and thioridazine (both 20 mg/kg), demonstrating relatively higher affinity for dopamine D3 receptor than typical ones, as well as the dopamine D3 receptor and autoreceptor preferring antagonists, cis-(+)-(1S,2R)-5-methoxy-1-methyl-2-(di-n- propylamino)tetralin HCl ((+)-UH232) and cis-(+)-(1S,2R)-5-methoxy-1-methyl-2-(n-propylamino)tetralin HCl ((+)-AJ76) (both 14 mg/kg), were equally effective or even more potent in increasing dopamine release than DOPAC. It is concluded that the dopamine D2/D3 receptor relative potencies of typical and atypical neuroleptics appear to correspond to their ability to affect preferentially dopamine metabolism or release in rat dorsal striatum in vivo.