Characterization of [³H]Dopamine Uptake Sites and [³H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

Abstract: [³H]Dopamine uptake and [³H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14‐day‐old rat embryos. Specific binding sites for [³H]cocaine and [³H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [³H]Cocaine was not taken up by the cells through an active transport process because [³H]cocaine binding occurred also at 4°C. Moreover, the possibility of [³H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [³H]cocaine from the cells. [³H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [³H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [³H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [³H]dopamine was taken up, [³H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [³H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [³H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [³H]cocaine binding in intact mesencephalic cell cultures was Na⁺ independent. The expression of [³H]dopamine uptake and [³H]cocaine binding sites appeared to be developmentally linked to neuritic outgrowth, supporting the view that cocaine binding sites may be closely associated with the dopamine transporter.