Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain

Ling Li,Stephen J Smith,Kristina D Micheva,Liqun Luo,Vsevolod M Ivanov,Maria L Spletter,Bosiljka Tasic,Brian D Mccabe

doi:10.1371/journal.pone.0011503

Ling Li, Stephen J Smith + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0011503

Copy DOI

Journal: PloS one	Publication Date: Jul 9, 2010
Citations: 131	License type: CC BY 4.0

Affiliation: Howard Hughes Medical Institute, Stanford University

Abstract

BackgroundProper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons.Methods and FindingsIn this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs.ConclusionsThe ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits.

Highlights

A fundamental goal of neuroscience is to describe the structure of neural circuits at the levels of single cells and synapses and to understand how this structure determines nervous system function
The ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits
Synapses can be clearly identified by electron microscopy (EM) based on the synaptic vesicles and density of accumulated proteins in the preand post-synaptic regions, and can be visualized by light microscopy through immunostaining for synaptic components or by using transgenic synaptic components tagged with fluorescent molecules

Summary

Methods and Findings

We present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates.

Introduction

A Genetic Strategy to Label Presynaptic Terminals in Single Neurons

Conclusion

Materials and Methods