Measurement of Dopamine Uptake in Neuronal Cells and Tissues - Drugs of Abuse: Neurological Reviews and Protocols - page 454

Many pharmacological agents exert profound effects on the neuronal uptake
of DA. Certain antidepressant (e.g., clomipramine) and anticholinergic drugs
(e.g., benztropine) are shown to inhibit DA uptake (9-11) . Drugs of abuse (e.g.,
cocaine and amphetamine) are also potent neuronal DA reuptake inhibitors
(12 , 13) . Because the inhibition of neuronal DA uptake by cocaine will enhance
synaptic DA activity, manifest acute behavioral and reinforcing effects, and
signifi cantly contribute to abuse liability, preclinical studies indicate that DA
transporter inhibitors (e.g., GBR 12909, WIN 35065-2) may be potentially
effective in reducing cocaine use (14) . Several DA transporter inhibitors have
been employed as ligands for labeling and quantitating the transporter within
innervated neurons (15) . Nevertheless, the neuronal DA uptake assay is still
considered a very useful experimental approach for determining the action
of neuroactive drugs on neurotransmission. In addition, detecting the active
DA uptake can also serve as a functional indication for neuronal integrity, as
degenerated neurons will lose their ability to take up the neurotransmitter.
A wide variety of tissue preparations have been used in vitro to study
the neuronal uptake transport of DA including cultured cells, tissue slices,
homogenates, crude P 2 synaptosomal fraction, purified synaptosomes, or
synaptic vesicles. The selection of an appropriate preparation for study depends
on the tissue source and availability and time and equipment limitations. To
investigate the effect of a potential new drug on DA uptake, a purifi ed bovine
synaptosomal preparation may be used, as it provides a suffi cient source of
tissue such that a broad range of [ 3 H]DA concentrations can be applied for the
determination of uptake kinetics. On the other hand, to study in vivo effects of
drug treatment on DA uptake in small rodents (such as in the mouse), where
tissue is very limited and performing further purifi cation is not feasible, a
crude homogenate would be the best choice. In this chapter, DA uptake assays
in cultured primary mesencephalic cells and in rat and mouse striatal tissue
preparations are described.
2. Materials
2.1. Solutions for Cellular [ 3 H]DA Uptake
1. Phosphate-buffered saline (PBS): 140 m M NaCl, 2.6 m M KCl, 1 m M KH 2 PO 4 ,
1 m M Na 2 HPO 4 , pH 7.4. Dissolve 8.18 g of NaCl (mol wt 58.44), 0.19 g of KCl
(mol wt 74.56), 0.14 g of Na 2 HPO 4 (mol wt 141.96), and 0.14 g of KH 2 PO 4
(mol wt 136.09) in 900 mL of H 2 O ( see Note 1 ). After adjusting the pH to
7.4 with HCl, the volume of the solution is adjusted to 1000 mL with H 2 O. Store
the solution in a closely capped container in refrigerator.
2. Cell preincubation buffer: PBS supplemented with 0.75 m M MgCl 2 , 0.75 m M
CaCl 2 , and 33 m M D -glucose. To prepare 100 mL of this buffer, dissolve