Further studies on the cationic staining of retinal photoreceptors.

In prior communications to this journal, and elsewhere, we have described a series of cytochemical properties of the nerve terminals of the frog (Rana pipiens) photoneceptors which we believe pertain to the internal ionic envinonment of synaptic vesicles. We first showed that the synaptic vesicles of the terminals, in aldehyde-fixed retinas, form electron-dense deposits when incubated in lead ions at bow pH, followed by osmium tetnoxide (2,9,15). We proposed that this property, which we later demonstrated to be shared by synaptic vesicles in many types of neurons (15), signals the presence offixabbe anions or other cation-binding sites within synaptic yesides [see also (6,11)]. In following up our initial findings, we reported in this journal (14) that the “plexiform layers” of the retina, the regions containing most of the intraretinal synapses of retinal neurons, stain with Neutral Red, a weakbase, cationic vital dye. After fixation, the plexiform layers also stain with Alcian Blue at pH 1. Such Alcian Blue staining at bow pH has often been used for demonstrating extracelbulan pobyanions. The staining of the retma, however, differed from that of typical extracebbular materials in that it was seen only if the preparations were first exposed to saponin, a detergent widely used to permeabibize cells without extensive disruption. We made similar observations on the cells of the rat adrenal medulla. This was of particular interest because the medulbary cells store catechobamines, which are cations, whereas the amino acids believed to serve as neurotransmitters for the photoreceptors are anions. Subsequently we found that Acridine Orange, another weak-base vital dye, accumulates extensively in the presynaptic terminals of retinal photoreceptors (13). This accumulation is antagonized by agents that disrupt energy metabolism, and by ammonium chloride, a “weak base” which is widely used to raise intnacompartmental pH in cells [reviewed in (5)]. These last observations suggest that staining of the retinal synapses by weak-base vital dyes is likely to reflect the trapping ofcationic forms ofthe dyes within acidified compartments in the terminals. This is in agreement with present views on staining of other types of structures with vital dyes (5) and � -. . . � ,. � .,,.. . - �: ?‘ � � � � ., . :� - “� ‘ � . - . . ‘ ‘