VISUAL DETECTION OF TRANSPORT-P IN PEPTIDERGIC NEURONS

1 Hypothalamic peptidergic neurones possess an uptake process for amin es (transport-P), for which prazosin is a substrate. It is characteriz ed by a paradoxical increase in the accumulation of [H-3]-prazosin whe n the concentration of unlabelled prazosin is increased above 10(-7) M . This increase is due to activation of a proton-dependent, vacuolar-t ype ATPase-linked pump that is blocked by tricyclic antidepressants. T his study utilized a fluorescence method to detect amine uptake in ind ividual cells. 2 Prazosin is fluorescent but most of its emission spec trum is in the ultraviolet range. We therefore used an analogue of pra zosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green par t of the visible spectrum. 3 BODIPY FL prazosin accumulated in immorta lised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL pr azosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for p razosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL praz osin were accumulated by the same uptake process. 4 BODIPY FL prazosin accumulated in a granular distribution, which is compatible with stor age in intracellular vesicles. 5 Hypothalamic cells from foetal rats i n primary culture also accumulated BODIPY FL prazosin by a desipramine -sensitive process. Uptake was predominantly in neurones and glial cel ls did not accumulate the amine. 6 Fluorescent detection provides visu al evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain.