On Optical Detection of Densely Labeled Synapses in Neuropil and Mapping Connectivity with Combinatorially Multiplexed Fluorescent Synaptic Markers

Yuriy Mishchenko

doi:10.1371/journal.pone.0008853

Abstract

We propose a new method for mapping neural connectivity optically, by utilizing Cre/Lox system Brainbow to tag synapses of different neurons with random mixtures of different fluorophores, such as GFP, YFP, etc., and then detecting patterns of fluorophores at different synapses using light microscopy (LM). Such patterns will immediately report the pre- and post-synaptic cells at each synaptic connection, without tracing neural projections from individual synapses to corresponding cell bodies. We simulate fluorescence from a population of densely labeled synapses in a block of hippocampal neuropil, completely reconstructed from electron microscopy data, and show that high-end LM is able to detect such patterns with over 95% accuracy. We conclude, therefore, that with the described approach neural connectivity in macroscopically large neural circuits can be mapped with great accuracy, in scalable manner, using fast optical tools, and straightforward image processing. Relying on an electron microscopy dataset, we also derive and explicitly enumerate the conditions that should be met to allow synaptic connectivity studies with high-resolution optical tools.

Highlights

The problem of reconstructing synaptic connectivity in neural circuits has recently attracted much attention [1,2,3,4,5], and a few projects for reconstructing connectivity in different systems, such as C
The process of reconstruction is approached in the following way: tiny synaptic contacts are first located in neuropil using electron microscopy (EM); pre-synaptic axons and post-synaptic dendrites are identified in EM images for each synaptic contact; axonal and dendritic projections are traced to their respective cell bodies using EM images over macroscopically large distances
Theoretical Bounds for Detecting Synapses with light microscopy (LM) We begin this section with a simple calculation involving several basic facts known for mammalian neuropil from neuroanatomy: a) distribution of synapses in neuropil is consistent with a uniform random distribution with the mean density r = 1–2 mm23 [32,33], and b) synapses in mammalian neuropil can be viewed as small disk-shaped objects q = 150–300 nm in diameter [34,35,36,37]

Summary

Introduction

The problem of reconstructing synaptic connectivity in neural circuits has recently attracted much attention [1,2,3,4,5], and a few projects for reconstructing connectivity in different systems, such as C. EM is widely accepted to be the only tool for such reconstructions of neural connectivity with the precision of individual synapses In this paradigm, the process of reconstruction is approached in the following way: tiny synaptic contacts are first located in neuropil using EM; pre-synaptic axons and post-synaptic dendrites are identified in EM images for each synaptic contact; axonal and dendritic projections are traced to their respective cell bodies using EM images over macroscopically large distances (e.g., see [16]). Expected frequency of such errors, is quite high [17]

Methods

Results

Conclusion