Isotropic Virtual Planes for Measuring Axonal Length: Population and Single Neuron Approaches in Mice and Human Brain

The isotropic virtual planes (IVP) method is a global spatial sampling approach for length estimation. Thanks to computer-assisted microscopy, IVP can be applied to thick sections oriented in any particular direction, as the stereological probe is randomly rotated inside the section and, therefore, avoids the possible bias that might occur when structures with a given preferred orientation are analyzed. We have applied this stereological tool to estimate axonal length in two different experimental/methodological approaches: (1) axons from populations of projection neurons and (2) axons from single projection neurons, visualized in mice or human tissues. Our results demonstrate that the IVP is an accurate stereological tool that can be used to estimate the axonal length of single or groups of neurons. In the case of single neurons, the IVP allows collecting these quantitative data efficiently, preventing the need to reconstruct the entire cell’s structure manually. The fact that this method is valid to analyze groups of neurons, whose axons provide terminal arborizations that can be either scattered in broad brain regions or restricted to specific brain areas, makes the IVP a suitable tool to be applied in connectomic experiments performed in either small brains, as in rodents, or primates, where the reconstruction of the entire axon is a tedious and time-consuming task. The analysis of the axonal length is of great importance since it is proportional to the number of synapses the axon establishes with its postsynaptic targets, and it is affected by some pathological conditions.