Tonic and Phasic Amperometric Monitoring of Dopamine Using Microelectrode Arrays in Rat Striatum

Here we report a novel microelectrode array recording approach to measure tonic (resting) and phasic release of dopamine (DA) in DA-rich areas such as the rat striatum and nucleus accumbens. The resulting method is tested in intact central nervous system (CNS) and in animals with extensive loss of the DA pathway using the neurotoxin, 6-hydroxyDA (6-OHDA). The self-referencing amperometric recording method employs Nafion-coated with and without m-phenylenediamine recording sites that through real-time subtraction allow for simultaneous measures of tonic DA levels and transient changes due to depolarization and amphetamine-induced release. The recording method achieves low-level measures of both tonic and phasic DA with decreased recording drift allowing for enhanced sensitivity normally not achieved with electrochemical sensors in vivo

Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD

Dopamine D (2) receptor regulation of the dopamine transporter expressed in Xenopus laevis oocytes is voltage-independent

ABSTRACT . These results suggest that D 2 R-induced up-regulation of DAT activity occurs via a voltage-independent mechanism that depends on G i/o activation and a rapid increase in expression of functional DAT molecules at the cell surface. The dopamine transporter (DAT) belongs to a large family of neurotransmitter and amino acid transporters that are related functionally by their requirement for extracellular Na ϩ and Cl Ϫ Two DA receptor subtypes were originally proposed based on their ability to either stimulate (D 1 R) or inhibit (D 2 R) adenylyl cyclase activit

Cyclin-Dependent Kinase 5 Inhibitors: Inhibition of Dopamine Transporter Activity

Cyclin-dependent kinase (Cdk) 5 reduces the rewarding properties of psychostimulants by dampening postsynaptic dopamine (DA) receptor signaling. Cdk5 is also present in midbrain DA neurons, where the DA transporter (DAT) is localized and limits DA neurotransmission by removing extracellular DA. Here, we tested the hypothesis that Cdk5 could also affect the disposition of DA by regulating DAT activity. Incubation of rat dorsal striatal (dSTR) synaptosomes with the Cdk5 inhibitors roscovitine, olomoucine, and 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(2-pyridyl)benzenesulfonamide (GW8510) or the inactive congener iso-olomoucine resulted in a rapid, concentration-dependent inhibition of specific [3H]DA uptake. However, roscovitine was the only inhibitor that did not also decrease [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN35,428) binding to dSTR DATs. Roscovitine-induced inhibition of dSTR [3H]DA uptake was explained by decreased maximal uptake velocity, without a change in cell-surface DAT levels. Roscovitine did not enhance [3H]DA release mediated by either DAT reverse-transport or Ca2+ channels in dSTR slices. Instead, roscovitine enhanced spontaneous [3H]DA outflow and inhibited DAT-mediated [3H]DA reaccumulation into dSTR slices. To explore the involvement of Cdk5 in roscovitine-induced down-regulation of DAT activity, Cdk5 protein was knocked down via Cdk5-small interfering RNA by as much as 86% in porcine aortic endothelial cells stably expressing human (h)DATs. However, Cdk5 depletion did not alter hDAT activity. Taken together, our results suggest that roscovitine inhibits DAT activity independently of Cdk5; therefore, results obtained with such inhibitors should be interpreted with caution. Our study is the first to demonstrate that Cdk5 inhibitors reduce brain DAT activity via a mechanism that is independent of DAT trafficking and reverse-transport

A relationship between reduced nucleus accumbens shell and enhanced lateral hypothalamic orexin neuronal activation in long-term fructose bingeing behavior.

Fructose accounts for 10% of daily calories in the American diet. Fructose, but not glucose, given intracerebroventricularly stimulates homeostatic feeding mechanisms within the hypothalamus; however, little is known about how fructose affects hedonic feeding centers. Repeated ingestion of sucrose, a disaccharide of fructose and glucose, increases neuronal activity in hedonic centers, the nucleus accumbens (NAc) shell and core, but not the hypothalamus. Rats given glucose in the intermittent access model (IAM) display signatures of hedonic feeding including bingeing and altered DA receptor (R) numbers within the NAc. Here we examined whether substituting fructose for glucose in this IAM produces bingeing behavior, alters DA Rs and activates hedonic and homeostatic feeding centers. Following long-term (21-day) exposure to the IAM, rats given 8-12% fructose solutions displayed fructose bingeing but unaltered DA D1R or D2R number. Fructose bingeing rats, as compared to chow bingeing controls, exhibited reduced NAc shell neuron activation, as determined by c-Fos-immunoreactivity (Fos-IR). This activation was negatively correlated with orexin (Orx) neuron activation in the lateral hypothalamus/perifornical area (LH/PeF), a brain region linking homeostatic to hedonic feeding centers. Following short-term (2-day) access to the IAM, rats exhibited bingeing but unchanged Fos-IR, suggesting only long-term fructose bingeing increases Orx release. In long-term fructose bingeing rats, pretreatment with the Ox1R antagonist SB-334867 (30 mg/kg; i.p.) equally reduced fructose bingeing and chow intake, resulting in a 50% reduction in calories. Similarly, in control rats, SB-334867 reduced chow/caloric intake by 60%. Thus, in the IAM, Ox1Rs appear to regulate feeding based on caloric content rather than palatability. Overall, our results, in combination with the literature, suggest individual monosaccharides activate distinct neuronal circuits to promote feeding behavior. Specifically, long-term fructose bingeing activates a hyperphagic circuit composed in part of NAc shell and LH/PeF Orx neurons

Increased striatal cell surface and total GluA1 after repeated low dose cocaine.

<p>(<b>a</b>) Cell surface GluA1, as determined by Total-Intracellular levels, was increased in dSTR of cocaine-treated rats vs. saline controls 40 min after day 7 of repeated once-daily 10 mg/kg i.p. cocaine or saline injections (group 5, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037673#pone-0037673-t001" target="_blank">Table 1</a>). (<b>b</b>) Total GluA1 in dSTR was increased in cocaine-treated rats vs. saline controls. Mean values ± SEM are shown for Saline: n = 6, Cocaine: n = 15). *p<0.05, Cocaine vs. Saline. Three cocaine rats were removed from analysis (see Methods, Data Analysis).</p

Increased striatal GluA1^Ser-845 phosphorylation after repeated low dose cocaine.

<p>The level of pGluA1^Ser-845 was increased in the dSTR of cocaine-treated rats vs. saline controls 40 min after day 7 of repeated once-daily 10 mg/kg i.p. cocaine or saline injections (group 3, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037673#pone-0037673-t001" target="_blank">Table 1</a>). Representative western blots are shown in the panel above the bars for the ratios of pGluA1^Ser-845 to total GluA1 in dSTR. Mean values ± SEM are shown for Saline: n = 4, Cocaine: n = 16. *p<0.05, Cocaine vs. Saline.</p

Summary of five experimental groups and their 30 min post-injection cocaine-induced locomotor activity.

*<p>Mean ± SEM, NA = not applicable,</p>**<p> = Ratio of HCR/LCR.</p