AB2231 Sigma-AldrichAnti-Dopamine Transporter; 100 µg Antibody

The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKIIα, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates.

The development of diet-induced obesity (DIO) can potently alter multiple aspects of dopamine signaling, including dopamine transporter (DAT) expression and dopamine reuptake. However, the time-course of diet-induced changes in DAT expression and function and whether such changes are dependent upon the development of DIO remains unresolved. Here, we fed rats a high (HFD) or low (LFD) fat diet for 2 or 6 weeks. Following diet exposure, rats were anesthetized with urethane and striatal DAT function was assessed by electrically stimulating the dopamine cell bodies in the ventral tegmental area (VTA) and recording resultant changes in dopamine concentration in the ventral striatum using fast-scan cyclic voltammetry. We also quantified the effect of HFD on membrane associated DAT in striatal cell fractions from a separate group of rats following exposure to the same diet protocol. Notably, none of our treatment groups differed in body weight. We found a deficit in the rate of dopamine reuptake in HFD rats relative to LFD rats after 6 but not 2 weeks of diet exposure. Additionally, the increase in evoked dopamine following a pharmacological challenge of cocaine was significantly attenuated in HFD relative to LFD rats. Western blot analysis revealed that there was no effect of diet on total DAT protein. However, 6 weeks of HFD exposure significantly reduced the 50 kDa DAT isoform in a synaptosomal membrane-associated fraction, but not in a fraction associated with recycling endosomes. Our data provide further evidence for diet-induced alterations in dopamine reuptake independent of changes in DAT production and demonstrates that such changes can manifest without the development of DIO.

Chronic cocaine administration regulates the expression of several proteins related to dopaminergic signaling and synaptic function in the mesocorticolimbic pathway, including the prefrontal cortex. Functional abnormalities in the prefrontal cortex are hypothesized to be due in part to the expression of proteins involved in dopamine signaling and plasticity. Adult male rhesus monkeys self-administered cocaine (i.v.) under limited (n = 4) and extended access conditions (n = 6). The abundance of surrogate markers of dopamine signaling and plasticity in the dorsolateral prefrontal cortex (DLPFC), orbitofrontal cortex (OFC), and anterior cingulate cortex (ACC) were examined: glycosylated and non-glycosylated forms of the dopamine transporter (efficiency of dopamine transport), tyrosine hydroxylase (TH; marker of dopamine synthesis) and phosphorylated TH at Serine 30 and 40 (markers of enzyme activity), extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK 2), and phosphorylated ERK1 and ERK2 (phosphorylates TH Serine 31; markers of synaptic plasticity), and markers of synaptic integrity, spinophilin and post-synaptic density protein 95 (roles in dopamine signaling and response to cocaine). Extended cocaine access increased non-glycosylated and glycosylated DAT in DLPFC and OFC. While no differences in TH expression were observed between groups for any of the regions, extended access induced significant elevations in pTH(Ser31) in all regions. In addition, a slight but significant reduction in phosphorylated pTH(Ser40) was found in the DLPFC. Phosphorylated ERK2 was increased in all regions; however, pERK1 was decreased in ACC and OFC but increased in DLPFC. PSD-95 was increased in the OFC but not in DLPFC or ACC. Furthermore, extended cocaine self-administration elicited significant increases in spinophilin protein expression in all regions. Results from the study provide insight into the biochemical alterations occurring in primate prefrontal cortex.

Mitochondria play a pivotal role in the development of pathology associated with Parkinson's disease (PD), traumatic brain injury (TBI), and following exposure to the environmental toxin trichloroethylene (TCE). Evidence from humans indicates that both TBI and TCE can play a role in the development of PD and that each of these insults result in significant mitochondrial dysfunction. In the current studies we hypothesized that exposure to both TCE and TBI would result in increased pathology associated with PD. To test this hypothesis, 16 week old male Fischer 344 rats were administered TCE for either one or two weeks by oral gavage. Following exposure to TCE, rats were subjected to either a sham, mild (1.0mm), or moderate (2.0mm) controlled cortical impact TBI. Given the strong connection between mitochondrial function and PD, TBI, and TCE, tissue from the striatum and substantia nigra were analyzed 6h after the TBI. Neither TCE exposure, TBI, nor the combination of the two insults resulted in mitochondrial deficits at 6h post-TBI in the substantia nigra. Unlike the substantia nigra, the striatum exhibited significant mitochondrial dysfunction. Exposure to TCE alone for two weeks resulted in approximately a 75% reduction in mitochondrial function (pless than 0.05) in the striatum whereas TBI alone resulted in approximately a 30% reduction in striatal mitochondrial function. Following 1 week exposure to TCE followed by TBI, there was a significant reduction (50%) in mitochondrial function (pless than 0.05) which required the presence of both insults. Beginning 12 days after the injury significant motor impairment was observed with Rotarod testing. Animals exposed to TCE and a moderate TBI exhibited performance which was approximately 50% of controls (pless than 0.01). Cylinder testing revealed that at 30 days post-injury animals exposed to TCE and a moderate TBI also had about a 34% reduction in the usage of the contralateral fore paw and this impairment was significantly worse than both control animals and animals exposed to TCE and a mild TBI (pless than 0.05). At 30 days post-injury there was a 13-17% reduction in the number of tyrosine hydroxylase (TH) positive neurons in the substantia nigra (pless than 0.05), which was the result of protein loss and not cell death. Loss of TH positive neurons did not result in changes in striatal TH fiber density or levels of the dopamine transporter or type-2 dopamine receptor. Additionally, exposure to TCE prior to the TBI did not increase the loss of cortical tissue, indicating regional specificity for TCE induced deficits. These studies provide further evidence for the connection between TCE, TBI, and PD and lend support to the concept that PD develops from a multifactorial injury scenario.

Dopaminergic neurotransmission is terminated by the action of the presynaptic dopamine transporter (DAT). It mediates Na(+)/Cl(-) -dependent re-uptake of extracellular dopamine (DA) into the cell, and is regarded as a major regulatory mechanism for synaptic transmission. Previous works have documented that protein kinase C (PKC) activator or inhibitor alters DA uptake by DAT, suggesting that PKC phosphorylation plays an important regulatory mechanism in DAT function. Based on the existence of consensus amino acid sequences for PKC phosphorylation, it has been postulated that PKC regulation of DAT is mediated by the direct phosphorylation of DAT protein. In this study, we try to discover whether the functional regulation of DAT by PKC is due to direct phosphorylation of DAT. The PKC null mutant hDAT, where all putative PKC phosphorylation sites are eliminated, has been constructed by the replacement of serine/threonine residues with glycines. The mutation itself showed no effect on the functional activities of DAT. The DA uptake activity of PKC null mutant was equivalent to those of wild-type hDAT (80-110% of wild-type). Phorbol ester activation of PKC inhibited DA uptake of wild-type hDAT by 35%, and staurosphorine blocked the effect of phorbol ester on DA uptake. The same phenomena was observed in PKC null mutant DAT, although no significant phosphorylation was observed by PKC activation. Confocal microscopic analysis using EGFP-fused DAT revealed that the activation of PKC by phorbol ester elicited fluorescent DAT to be internalized into the intracellular space both in wild-type and PKC null mutant DAT in a similar way. These results suggest that PKC-mediated regulation of DAT function is achieved in an indirect manner, such as phosphorylation of a mediator protein or activation of a clathrin-mediated pathway.

There is evidence both for and against Na(+)- and Cl(-)-dependent neurotransmitter transporters forming oligomers. We found that cross-linking the human dopamine transporter (DAT), which is heterologously expressed in human embryonic kidney 293 cells, either with copper phenanthroline (CuP) or the bifunctional reagent bis-(2-methanethiosulfonatoethyl)amine hydrochloride (bis-EA) increased the apparent molecular mass determined with nonreducing SDS/PAGE from approximately 85 to approximately 195 kDa. After cross-linking, but not before, coexpressed, differentially epitope-tagged DAT molecules, solubilized in Triton X-100, were coimmunoprecipitated. Thus, the 195-kDa complex was a homodimer. Cross-linking of DAT did not affect tyramine uptake. Replacement of Cys-306 with Ala prevented cross-linking. Replacement of all of the non-disulfide-bonded cysteines in the extracellular and membrane domains, except for Cys-306, did not prevent cross-linking. We conclude that the cross-link is between Cys-306 at the extracellular end of TM6 in each of the two DATs. The motif GVXXGVXXA occurs at the intracellular end of TM6 in DAT and is found in a number of other neurotransmitter transporters. This sequence was originally found at the dimerization interface in glycophorin A, and it promotes dimerization in model systems. Mutation of either glycine disrupted DAT expression and function. The intracellular end of TM6, like the extracellular end, is likely to be part of the dimerization interface.

Here, we describe the generation of viable and dopamine-producing neurons derived from pluripotent mouse embryonic stem cells. Neurotrophic factors in combination with survival-promoting factors, such as interleukin-1beta, glial cell line-derived neurotrophic factor, neurturin, transforming growth factor-beta(3) and dibutyryl-cyclic AMP, significantly enhanced Nurr1 and tyrosine hydroxylase (TH) mRNA levels, whereas En-1, mash-1 and dopamine-2-receptor mRNA levels were not upregulated. In parallel, mRNA levels of the anti-apoptotic gene bcl-2 were found to be upregulated at terminal stages. Double immunofluorescence analysis revealed increased numbers of TH- and dopamine transporter-, but not gamma-aminobutyric acid- and serotonin-positive neurons in relation to synaptophysin-labeled cells by survival-promoting factors. Moreover, high-performance liquid chromatography analysis showed detectable levels of intracellular dopamine. We conclude that survival-promoting factors enhance differentiation, survival and maintenance of dopaminergic neurons derived from embryonic stem cells.