Nanoconnectivity: a quantitative approach to examine the structural complexity of the brain

Unraveling the highly complex connectivity at the synaptic level is critical for the understanding of brain function. However, investigating the structure of the brain is time consuming and technically demanding. We have developed and applied a tracing method that allows us to obtain a simplified or skeletonized version of all nerve fibers present within a given brain sample, without the need for full 3D reconstructions. Every tracing or “skeleton” is linked to its corresponding synaptic contacts, so the result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. The tissue sample is obtained by volume electron microscopy (FIB-SEM) and tracing is performed by dedicated software. In addition, the software provides the length of each skeleton as well as the number and positions of its corresponding synapses. With this approach, we have identified clear as well as subtle quantitative differences between three brain regions (the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex). For example, we have found that axons and dendrites are densely packed in the neuropil of these three regions, reaching close to 8 kilometers of nerve fibers per cubic mm in layer 1. In all cases, the predominant fibers were excitatory axons, followed by spiny dendrites, inhibitory axons and smooth dendrites. Other examples include the fact that the excitatory axons in the stratum radiatum establish many more synapses per micron than the axons of the other regions, or the lack of correlation between the number of synapses established on the spines of a given dendrite and the number of synapses established on the dendritic shaft. Using this methodology, we obtain a set of quantitative parameters including the number and proportions of excitatory and inhibitory synapses; the numbers of dendrites and axons; the proportion of excitatory and inhibitory axons; the linear densities of synapses established on dendritic spines and dendritic shafts; the linear densities of synapses established by excitatory and inhibitory axons; and the lengths of the different types of dendrites and axons. Taken together, these data characterize each region, allowing us to compare them on a quantitative basis and to identify subtle traits and differences that would have escaped a qualitative analysis. With this strategy, we can achieve a better understanding of the structure and dynamics of the brain, and we are better equipped to explore how they change during both normal and pathological conditions. ### Competing Interest Statement The authors have declared no competing interest.