A comparison of manual neuronal reconstruction from biocytin histology or 2-photon imaging: morphometry and computer modeling.

Arne V Blackman,P Jesper Sjã¶Strã¶M,Stefan Grabuschnig,Robert Legenstein

doi:10.3389/fnana.2014.00065

Abstract

Accurate 3D reconstruction of neurons is vital for applications linking anatomy and physiology. Reconstructions are typically created using Neurolucida after biocytin histology (BH). An alternative inexpensive and fast method is to use freeware such as Neuromantic to reconstruct from fluorescence imaging (FI) stacks acquired using 2-photon laser-scanning microscopy during physiological recording. We compare these two methods with respect to morphometry, cell classification, and multicompartmental modeling in the NEURON simulation environment. Quantitative morphological analysis of the same cells reconstructed using both methods reveals that whilst biocytin reconstructions facilitate tracing of more distal collaterals, both methods are comparable in representing the overall morphology: automated clustering of reconstructions from both methods successfully separates neocortical basket cells from pyramidal cells but not BH from FI reconstructions. BH reconstructions suffer more from tissue shrinkage and compression artifacts than FI reconstructions do. FI reconstructions, on the other hand, consistently have larger process diameters. Consequently, significant differences in NEURON modeling of excitatory post-synaptic potential (EPSP) forward propagation are seen between the two methods, with FI reconstructions exhibiting smaller depolarizations. Simulated action potential backpropagation (bAP), however, is indistinguishable between reconstructions obtained with the two methods. In our hands, BH reconstructions are necessary for NEURON modeling and detailed morphological tracing, and thus remain state of the art, although they are more labor intensive, more expensive, and suffer from a higher failure rate due to the occasional poor outcome of histological processing. However, for a subset of anatomical applications such as cell type identification, FI reconstructions are superior, because of indistinguishable classification performance with greater ease of use, essentially 100% success rate, and lower cost.

Highlights

Investigations of neuronal morphology have been a key feature of neuroscience since the studies of Ramón y Cajal and before (Ramón y Cajal, 1911; Senft, 2011)
pyramidal cell (PC) were identified by their characteristic apical dendrite, and their axons were largely confined to layer 5 (L5) with the occasional ascending process
Within the two basket cells (BCs) and PC clusters, reconstructions from biocytin histology (BH) or fluorescence imaging (FI) did not further segregate into distinct sub-clusters. These results suggest that both reconstruction methods produce enough detail to reliably classify different neuronal types, while at the same being so similar in terms of outcome that the choice of method does not impact cell classification appreciably

Summary

Introduction

Investigations of neuronal morphology have been a key feature of neuroscience since the studies of Ramón y Cajal and before (Ramón y Cajal, 1911; Senft, 2011). Increases in the number and accessibility of reconstructed neurons promise new approaches; for example, resources such as NeuroMorpho.Org allow researchers access to a large pool of reconstructions from published studies, which can be mined for further data (Ascoli et al, 2007). Use of such interlinked datasets of 3D reconstructions may be key in “big science” initiatives such as the Human Brain Project, and for any project wishing to simulate the brain (Markram, 2013)

Results

Discussion

Conclusion