Rotating Disk Electrode Voltammetric Measurements of Dopamine Transporter Activity: An Analytical Evaluation

Earles, Cynthia A.; Schenk, James O.

doi:10.1006/abio.1998.2850

Cited by 48 publications

(61 citation statements)

References 20 publications

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“…Recently, additional assay technologies have been explored. These include rotating disk electrophysiology (Burnette et al, 1996;Earles and Schenk, 1998), amperometric detection (Galli et al, 1998;Kawagoe et al, 1991;Khoshbouei et al, 2003), conjugated nanocrystals for detection of cell surface transporters (Rosenthal et al, 2002;Savchenko et al, 2003), and fluorescence-based measurement of monoamine transporter activity (Fowler et al, 2006;Haunsø and Buchanan, 2007;Mason et al, 2005;Schwartz et al, 2003;Wagstaff et al, 2007). The latter technology utilizes the fluorescent substrate ASP + , which was appropriated in recognition of its physical similarity to the neurotoxin N-methyl-4-phenylpyridine, a well-recognized biogenic amine transporter substrate (Javitz et al, 1985).…”

Section: Discussionmentioning

confidence: 98%

Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity

Jørgensen

Nielsen

Peters

et al. 2008

Journal of Neuroscience Methods

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Section: Discussionmentioning

confidence: 98%

Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity

Jørgensen

Nielsen

Peters

et al. 2008

Journal of Neuroscience Methods

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“…Unlike conventional approaches in large beakers, these studies are performed in a small vessel with small volumes (0.5 mL) to decrease mixing time of the whole sample. Constant delivery of solution to the electrode surface and subsequent transfer of electrons from the solution's electroactive substances enable mathematical prediction, even in these low volume chambers, of the detection current using the Levich equation (Earles and Schenk, 1998):

i_{normalL} = 0.62 {italicnFAD}^{2 ∕ 3} {italicCv}^{- 1 ∕ 6} ω^{1 ∕ 2}

where i L is the limiting current in amperes, n the number of electrons transferred per mole of analyte, F the Faraday's constant (96,485 coulombs/mol), A the area of the electrode in cm 2 , D the diffusion coefficient in cm 2 /s, C the analyte concentration in mol/L, ν the kinematic viscosity of the solution in cm 2 /s (0.01 cm 2 /s for an aqueous solution), and ω the angular velocity of the RDE in rad/s (given by ω = 2 πN where N is the number of rotations/s) (Earles and Schenk, 1998; Schenk et al, 1990). By overcoming diffusion limitations, RDEV offers the advantages of sensitive, kinetically resolved, real-time measures of neurotransmitter transporter uptake, release, and efflux.…”

Section: Methodsmentioning

confidence: 99%

Rotating disk electrode voltammetric measurements of serotonin transporter kinetics in synaptosomes

Hagan

Neumaier

Schenk

2010

Journal of Neuroscience Methods

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Altered serotonin (5-HT) signaling is implicated in several neuropsychiatric disorders, including depression, anxiety, obsessive-compulsive disorder, and autism. The 5-HT transporter (SERT) modulates 5-HT neurotransmission strength and duration. This is the first study using rotating disk electrode voltammetry (RDEV) to measure 5-HT clearance. SERT kinetics were measured in whole brain synaptosomes. Uptake kinetics of exogenous 5-HT were measured using glassy carbon electrodes rotated in 500 uL glass chambers containing synaptosomes from SERTknockout (−/−), heterozygous (+/−), or wild-type (+/+) mice. RDEV detected 5-HT concentrations of 5 nM and higher. Initial velocities were kinetically resolved with K m and V max values of 99 ± 35 standard error of regression (SER) nM and 181 ± 11 SER fmol / (s x mg protein), respectively in wild-type synaptosomes. The method enables control over drug and chemical concentrations, facilitating interpretation of results. Results are compared in detail to other techniques used to measure SERT kinetics, including tritium labeled assays, chronoamperometry, and fast scan cyclic voltammetry. RDEV exhibits decreased 5-HT detection limits, decreased vulnerability to 5-HT oxidation products that reduce electrode sensitivity, and also overcomes diffusion limitations via forced convection by providing a continuous, kinetically resolved signal. Finally, RDEV distinguishes functional differences between genotypes, notably, between wild-type and heterozygous mice, an experimental problem with other experimental approaches.

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“…The mPFC was dissected and weighed. The tissue was immediately chopped on an ice-cold glass plate and placed into 500 l of physiological buffer composed of (in mM): 124 NaCl, 1.80 KCl, 1.30 MgSO 4 , 1.24 KH 2 PO 4 , 2.50 CaCl 2 , 26 NaHCO 3 , 10 glucose, saturated with 95% O 2 -5% CO 2 gas mixture) as described (Meiergerd and Schenk, 1995;Earles et al, 1998;Earles and Schenk, 1999) and maintained at 37°C in a temperature-controlled chamber. Composition of the buffer in the low Na ϩ condition consisted of replacement of 124 mM NaCl with choline chloride.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Extracellular Dopamine Clearance in the Medial Prefrontal Cortex: Role of Monoamine Uptake and Monoamine Oxidase Inhibition

Wayment¹,

2001

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In vitro rotating disk electrode (RDE) voltammetry and in vivo microdialysis were used to characterize dopamine clearance in the rat medial prefrontal cortex (mPFC). RDE studies indicate that inhibition by cocaine, specific inhibitors of the dopamine transporter (DAT) and norepinephrine transporter (NET), and low Na ϩ produced a 50-70% decrease in the velocity of dopamine clearance. Addition of the monoamine (MAO) inhibitors, L-deprenyl, clorgyline, pargyline, or in vivo nialamide produced 30-50% inhibition. Combined effects of uptake inhibitors with L-deprenyl on dopamine clearance were additive (up to 99% inhibition), suggesting that at least two mechanisms may contribute to dopamine clearance. Dopamine measured extracellularly 5 min after exogenous dopamine addition to incubation mixtures revealed that most conditions of DAT/NET inhibition did not produce elevated dopamine levels above controls. Inhibition of MAO produced elevated dopamine levels only after long-term, but not short-term, incubation in vitro. Short-term incubation of L-deprenyl combined with DAT and NET uptake inhibitors increased dopamine above control levels, consistent with more than one mechanism of dopamine clearance. Local infusion of pargyline (100 or 300 M) into the mPFC or striatum via microdialysis produced more pronounced and immediate increases in mPFC dopamine levels compared with striatum. Furthermore, dopamine elevation in the mPFC was not accompanied by a decrease in the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, as found in the striatum. These findings may have revealed a unique mechanism of mPFC dopamine clearance and therefore contribute to the understanding of multiple behaviors that involve mPFC dopamine transmission, such as schizophrenia, drug abuse, and working memory function.

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Rotating Disk Electrode Voltammetric Measurements of Dopamine Transporter Activity: An Analytical Evaluation

Cited by 48 publications

References 20 publications

Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity

Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity

Rotating disk electrode voltammetric measurements of serotonin transporter kinetics in synaptosomes

Characterization of Extracellular Dopamine Clearance in the Medial Prefrontal Cortex: Role of Monoamine Uptake and Monoamine Oxidase Inhibition

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