Immunogold labeling of synaptic vesicle proteins in developing hippocampal neurons

Abstract

Synaptic vesicles (SV) contain high concentrations of specific proteins. How these proteins are transported from soma to synapses, and how they become concentrated at SV clusters at presynaptic terminals were examined by immunogold electron microscopy in dissociated rat hippocampal neurons at 3-6 days in culture, a developmental stage when axonal transport of SV proteins is robust. In neuronal somas, labels for the SV integral membrane proteins (synaptophysin, SV2, VAMP/synaptobrevin, and synaptotagmin) were localized at Golgi complexes and other membranous structures that were dispersed in the cytoplasm as individual vesicle/vacuoles. These vesicles/vacuoles became aggregated in axons, with the size of aggregates ranging from 0.2 to 2 μm in length. Pleomorphic vesicle/vacuoles within the aggregate were typically larger (50-300 nm) than SVs, which were uniform in size at ~ 40 nm. These pleomorphic vesicles/vacuoles are probably transport cargos carrying SV integral membrane proteins from the soma, and then are preferentially sorted into axons at early developmental stages. Serial thin sections of young axons indicated that many labeled aggregates were not synaptic, and in fact, some of these axons were without dendritic contacts. In contrast, labels for two SV-associated proteins, synapsin I and α-synuclein, were not localized at the Golgi complexes or associated with membranous structures in the soma, but were dispersed in the cytoplasm. However, these SV-associated proteins became highly concentrated on clusters of SV-like vesicles in axons, and such clusters were already distinctive in axons as early as 3 days in culture. These clusters consisted of ~ 4-30 vesicles in single thin sections, and the vesicles were of a uniform size (~ 40 nm). Serial sectioning analysis showed that these clusters could be part of nascent synapses or exist in axons without any dendritic contact. Importantly, the vesicles were intensely labeled for SV integral membrane proteins as well as SV-associated proteins. Thus, these EM observations reveal that the two groups of proteins, SV integral membrane and SV-associated, proceed through different routes of biosynthesis and axon transport, and are only sorted into the same final compartment, SV clusters, when they are in the axons.

Keywords: Active zone cytomatrix; Axon transport; Electron microscopy; SV2; Synapsin; Synaptophysin.

Fig. 1

Immunogold labeling of synaptophysin in dissociated hippocampal neuronal culture at 4 (a, b) and 5 (c, d) DIV. In neuronal soma (a), label is localized at the Golgi complex and on membranous structures (arrows in a). In axons (b-d), label is concentrated on aggregates of vesicles/vacuoles (arrow in b). However, not all vesicles/vacuoles are labeled (boxed area in b). The labeled aggregates consist of tubular (arrow in c) and vesicular structures. Clusters of SV-like vesicles of uniform size (~ 40 nm) are also intensely labeled (d). Scale bars = 100 nm, c & d shared the same scale bar

Fig. 2

Label for SV2 is localized at the Golgi complex and membranous structures (arrows in a) in soma, and at individual and aggreges (arrow in b) of vesicles /vacuoles in axons. Enlarged micrograph in c shows concentrated label on a cluster of SV-like vesicle (circled area) and on a nearby tubular structure (arrow). Boxed area shows other nearby tubular-vesicular structures without any label. Samples were 4 DIV. Scale bars = 100 nm

Fig. 3

Clusters of SV-like vesicles are labeled for synaptophysin (a) and SV2 (b). Samples were 4 DIV, and (c) was fixed with glutaraldehyde for better structural preservation. Arrows in (c) points to coated vesicles interpreted as clathrin (enlarged in inset). Scale bar = 100 nm

Fig. 4

Serial sections (1–17 in center column) through two axons (yellow and blue) containing synaptophysin-labeled aggregates (marked as a, b & c) of vesicles/vacuoles at 5 DIV. Insets on both sides of the serial sections show enlargements of these aggregates at various sections. The axon segments containing aggregates a & c do not have contact with dendrites, whereas aggregate d in sections 16–17 is in contact with a dendrite (pink), perhaps forming a nascent synapse. Thick arrows in insets a5 & b5 point to clathrin-coated vesicles

Fig. 5

Label for synapsin I is not associated with the Golgi complex, but dispersed in the cytoplasm in the neuronal soma (a), and concentrated on clusters of SV-like vesicles (arrow in b) in axons. Open arrows point to multivesicular bodies (MVB) in b. Samples were 4 DIV. Scale bars = 100 nm

Fig. 6

Label for α-synuclein is not associated with the Golgi complex, but dispersed in the cytoplasm in the neuronal soma (a), and concentrated on clusters of SV-like vesicles (arrow in b) in axons. Samples were 6 DIV. Scale bars = 100 nm

Fig. 7

Label for SNAP-25 is localized at the Golgi complex in the soma at 4 DIV (a), and preferentially at axolemma (b, c), but not at clusters of SV-like vesicles (c) in axons. Scale bars = 100 nm, a & b share the same bar

Fig. 8

Nascent synapses are formed as early as 3 (a), 4 (b) and 5 (c) DIV, with a few synaptic vesicles and some larger sized vesicles/vacuoles. These vesicles label for SV proteins, SV2 (a), SV-associated protein, synapsin I (b), and AZ cytomatrix, Piccolo (c). Scale bar = 100 nm

Fig. 9

Dense core vesicles (DCV, arrows) sometimes exist in groups in young axons at 3 (c), 4 (a, b) and 6 (d) DIV. The vesicular membranes of DCV label for SV2 (a) and synaptotagmin (b), but not for synaptophysin (c). Label for Piccolo, an AZ cytomatrix protein is located on the outside of the DCV (d). Scale bar = 100 nm