The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture

Abstract

As a first step toward elucidating mechanisms involved in the sorting of synaptic vesicle proteins in neurons, we have used immunofluorescence microscopy to determine the distribution of two synaptic vesicle proteins, synapsin I and synaptophysin, in hippocampal neurons developing in culture. In mature cultures, synapsin I and synaptophysin immunoreactivity was concentrated in puncta that were restricted to sites where axons contacted neuronal cell bodies or dendrites. Electron-microscopic immunocytochemistry demonstrated that these puncta corresponded to vesicle-filled axonal varicosities that were exclusively presynaptic. At early stages of development, before cell-cell contact, both synapsin I and synaptophysin were preferentially localized in axons, where they were particularly concentrated in the distal axon and growth cone. In axons that did not contact other cells, immunostaining for these two proteins had a granular appearance, which persisted for at least 7 d, but focal accumulations of vesicles comparable to those seen at sites of synaptic contact were not observed. When neurons contacted one another, numerous puncta of synapsin I and synaptophysin formed within the first week in culture. Double-label immunofluorescence demonstrated that the two vesicle antigens were closely codistributed throughout these stages of development. These observations demonstrate that synaptic vesicle proteins assume a polarized distribution within nerve cells beginning early in development, as soon as the axon can be identified. In contrast, differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective sorting of some materials in nerve cells, first appear at a subsequent stage of development. The selective distribution of synaptic vesicle proteins to the axon occurs in isolated cells, independent of interactions with other cells. In contrast, the formation of large clusters of vesicles typical of presynaptic specializations requires contact with an appropriate postsynaptic target. Thus, in cultured hippocampal neurons, the localization of synaptic vesicles in presynaptic specializations is the result of sorting mechanisms intrinsic to individual neurons as well as to mechanisms mediated by cell-cell contact.

Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons

“…Syp is expressed at high levels during synaptogenesis and is one of the earliest synaptic proteins to accumulate at developing synapses in culture (16). Such properties of syp expression are compatible with its role in regulating synapse formation as found in this study.…”

Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks

“…1). The localization of presynaptic proteins in the soma and neurites was also previously demonstrated for synapsin 1 and synaptophysin (Fletcher et al, 1991;Benson and Cohen, 1996). In the present study, the VGAT positivity was considered to reflect axonal transport of the VGAT protein from the Golgi apparatus (see Fig.…”

The neurochip: a new multielectrode device for stimulating and recording from cultured neurons

“…Hippocampal neurons from E18 rats in culture form large numbers (on the order of 1000) of functional synapses, beginning at around 3 days and maturing after approximately 2 weeks (Bartlett and Banker, 1984;Fletcher et al, 1991;Vicario-Abejon et al, 1998). Thus, we expect that the neurochip will be a useful device for studying networks of these neurons.…”

“…We have chosen to work with rat hippocampal neurons for the following reasons. (1) Similar to most CNS neurons, these cells spontaneously form densely interconnected networks in vitro (Bartlett and Banker, 1984;Fletcher et al, 1991). (2) A vast literature base exists regarding their cell culture and physiology.…”