The pH‐Sensitive Dye Acridine Orange as a Tool to MonitorExocytosis/Endocytosis in Synaptosomes

Abstract

Abstract:We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca 2ϩ . The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acidloaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca 2ϩ removal with EGTA evidences endocytosis: Its T 1/2 was 12 Ϯ 0.6 s. The K A ϩ , channel inhibitors 4-aminopyridine (100 M) and ␣-dendrotoxin (10 -100 nM) are known to induce glutamate release by inducing the firing of Na ϩ channels; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca 2ϩ -dependent AO release, which is prevented by the Na ϩ channel inhibitor tetrodotoxin and potentiated by 4␤-phorbol 12-myristate 13-acetate (PMA). With ␣-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd 2ϩ or tetrodotoxin. The T 1/2 of endocytosis, which was unaffected by PMA, was 12 Ϯ 0.4 s with EGTA and Cd 2ϩ and 9.5 Ϯ 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover. Key Words: Acridine orangeExocytosis-Endocytosis-Synaptosome-Rat cerebral cortex-Acidic neurotransmitter-containing vesicles. J. Neurochem. 72, 625-633 (1999).Synaptosomes retain all the machinery for the uptake, storage, and exocytosis of neurotransmitters. In situ the terminal functions largely autonomously from the relatively distant cell body, requiring only the electrical signal from the axonal action potential to trigger release, and replenishment of materials via axonal transport mechanisms for long-term survival. The cerebral cortical preparation contains a high proportion of glutamatergic terminals (Nicholls, 1993), thus making nonspecific systems to monitor exo/endocytosis likely representations of the turnover of glutamate-releasing synapses.Although much information is available on exocytosis, only limited information is presently available on the physiology and biochemistry of endocytosis and on vesicle turnover. Available methods are based on measurements of membrane capacitance (Alvarez de Toledo et al., 1993;Thomas et al., 1994) that are not readily applicable to typical fast synapses or on the distribution of fluorescent optical tracers as N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]py...

The effect of nitric oxide on synaptic vesicle proton gradient and mitochondrial potential of brain nerve terminals

“…Acidifica tion of synaptic vesicles was assessed using acridine orange (AO), a pH-sensitive fluorescent probe that is accumulated in the acidic cell compartments pro portionally to the pH gradient [18,19]. As a result, AO fluorescence is quenched because of protona tion of the dye.…”

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

“…In contrast to carboxyfluorescein, acridine orange can be taken up into cells and has been widely used to monitor pH inside animal (Wieczorek et al, 1991;Zoccarato et al, 1999;Malnic & Geibel, 2000) and plant cells (Pope & Leigh, 1988;DuPont, 1989). Carboxyfluorescein is a large double-negative charged anion that can permeate the plasma membrane only in its non-fluorescing diacetate form (Babcock, 1983;Graber et al, 1986).…”

Vesicular Zinc Regulates the Ca2+Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals

“…We compared the effect of EGTA-AM on depolarization-induced and glutamate-induced glutamate release from hippocampus MFSs. We added 25 mM KCl to induce depolarization (Zoccarato et al, 1999) and 20 pM glutamate to activate presynaptic receptors on MF terminals (Schmitz et al, 2001;Rebola et al, 2008; but see Kwon and Castillo, 2008). Addition of KCl to the synaptosomes lead to large glutamate release, whereas 20 pM glutamate evokes significantly smaller responses.…”