Abstract
Projection neurons are the commonest neuronal type in the mammalian forebrain and their individual characterization is a crucial step to understand how neural circuitry operates. These cells have an axon whose arborizations extend over long distances, branching in complex patterns and/or in multiple brain regions. Axon length is a principal estimate of the functional impact of the neuron, as it directly correlates with the number of synapses formed by the axon in its target regions; however, its measurement by direct 3D axonal tracing is a slow and labor-intensive method. On the contrary, axon length estimations have been recently proposed as an effective and accessible alternative, allowing a fast approach to the functional significance of the single neuron. Here, we analyze the accuracy and efficiency of the most used length estimation tools—design-based stereology by virtual planes or spheres, and mathematical correction of the 2D projected-axon length—in contrast with direct measurement, to quantify individual axon length. To this end, we computationally simulated each tool, applied them over a dataset of 951 3D-reconstructed axons (from NeuroMorpho.org), and compared the generated length values with their 3D reconstruction counterparts. The evaluated reliability of each axon length estimation method was then balanced with the required human effort, experience and know-how, and economic affordability. Subsequently, computational results were contrasted with measurements performed on actual brain tissue sections. We show that the plane-based stereological method balances acceptable errors (~5%) with robustness to biases, whereas the projection-based method, despite its accuracy, is prone to inherent biases when implemented in the laboratory. This work, therefore, aims to provide a constructive benchmark to help guide the selection of the most efficient method for measuring specific axonal morphologies according to the particular circumstances of the conducted research.
Highlights
The highly integrated functioning of the brain relies on axons that directly connect distant regions
In this work we report a computational benchmarking between three experimental tools used for axon length estimation on brain tissue sections
The neurons contained in those files had been uploaded to NeuroMorpho.org by a number of different laboratories, and were produced either through direct reconstruction on a series of sections from a sparsely-labeled brain that spanned the total length of the arbor or, in the case of those belonging to the MouseLight project, through semiautomatic tracing of single cells from a densely labeled brain digitally scanned by means of single two-photon tomography (STPT) [4]
Summary
The highly integrated functioning of the brain relies on axons that directly connect distant regions. Unlike dendrites, which integrate input signals [10,11,12], the axon is a transmission compartment for all-or-none fast action potentials. The functional impact of signals travelling down an axon depends critically on the wiring of the axon, as it constrains the number and distribution of its synapses. The number and distribution of synapses can be reliably derived from the axon length within its target structures [8,9,16]. Accurate measurement of axon length, is key for the functional modelling of brain-wide circuits as it estimates the functional impact of the single neuron [17,18]
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