Synaptic Vesicle Proteins and Early Endosomes in Cultured Hippocampal Neurons : Differential Effects of Brefeldin A in Axon and Dendrites

genesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal-dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagrain, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidaseor Lucifer yellowlabeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery. S YNAPTIC vesicles (SVs) 1 a r e highly specialized secretory organelles of neurons which store and secrete non-peptide neurotransmitters. In mature neurons, SVs are concentrated in nerve terminals where they undergo exocytosis at specialized regions of the presynaptic plasmalemma, the so-called active zones (see Kelly, 1988; for reviews see De Camilli and Jahn, 1990; Stidhof and Jahn, 1991; Trimble et al., 1991). Their exocytosis occurs asynchronously and at very low rates in resting nerve terminals and is greatly stimulated, with minimal temporal delay (fractions of a millisecond), by nerve terminal depolarization (Katz and Miledi, 1968). After exocytosis, SV membranes are rapidly reinternalized and reused for the assembly of new, neurotransmitter-filled SVs (Heuser and Reese, 1973; Ceccarelli et al., 1973; Sulzer and Holtzman, 1989). It rePlease address all correspondence to Dr. Pietro De Camilli, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06510. 1. Abbreviau'o~ used in this paper: BFA, Brefeldin A; SVs, synaptic vesicles; Sy~-A, lumenal domain of rat Syt I; Trf-R, transferrin receptor. mains to be elucidated how SVs are first assembled from newly synthesized components and subsequently regenerated at each cycle of exo-endocytosis. It is still unclear whether SV recycling involves clathrin-coated vesicles and early endosomes (as in the receptor-mediated recycling pathway), or whether endocytosis follows another more specialized pathway (Miller and Heuser, 1984; Pfeffer and Kelly, 1985; Torri-Tarelli et al., 1987; Maycox et al., 1992). Microvesicles that are closely related to neuronal SVs in biochemical and functional properties (referred to as synaptic-like microvesicles) were identified in a variety of endocrine cells and endocrine cell lines (Navone et al., 1986; Wiedenrnann et al., 1988; Baumert et al., 1990; Reetz et al., 1991). These vesicles have been used to study aspects of SV membrane traffic which cannot be easily investigated in neurons (Johnston et al., 1989; Cameron et al., 1991; Linstedt and Kelly, 1991; R6gnier-Vigouroux et al., 1991). Most of these studies have focused primarily on synaptophysin as a model SV protein. It was found that a large fraction of synaptophysin in these cells is colocalized with the transferrin receptor (Trf-R), a well established marker of the receptormediated recycling pathway (Dautry-Varsat et al., 1983), and it was suggested that synaptic-like microvesicles are regener© The Rockefeller University Press, 0021-9525/93/09/1207/15 $2.00 The Journal of Cell Biology, Volume 122, Number 6, September 1993 1207-1221 1207 on A ril 0, 2017 D ow nladed fom Published September 15, 1993