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

Abstract

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.

Volume Electron Microscopy Analysis of Synapses in Associative and Primary Regions of the Human Cerebral Cortex

“…We found that the majority of synapses had a macular shape (79-87%) and were the smallest synapses. These findings are in line with previous reports in other brain regions and species (Geinisman et al, 1987;Jones et al, 1991;Neuman et al, 2016;Hsu et al, 2017;Calì et al, 2018;Santuy et al, 2018a;Domínguez-Álvaro et al, 2019Domínguez-Álvaro et al, , 2021aMontero-Crespo et al, 2020;Cano-Astorga et al, 2021, 2023Turégano-López, 2022;Alonso-Nanclares et al, 2023). It has been reported that complex-shaped synapses possess more AMPA and NMDA receptors than macular synapses, characterizing them as a potentially 'powerful' population associated with more enduring memory-related functionality than macular synapses (Geinisman et al, 1987(Geinisman et al, , 1991(Geinisman et al, , 1992a(Geinisman et al, , 1992b(Geinisman et al, , 1993Lüscher et al, 2000;Toni et al, 2001;Ganeshina et al, 2004aGaneshina et al, , 2004bSpruston 2008).…”

“…However, a frequent misunderstanding regarding this characteristic is that it suggests that synapses on dendritic shafts are predominantly SS. In reality, quantitative analyses of synapses in the neuropil reveal that the majority of synapses are AS established on spines, followed by AS on dendritic shafts, and then SS on dendritic shafts (Beaulieu et al, 1992;Peters et al, 2008;Hsu et al, 2017;Calì et al, 2018;Santuy et al, 2018b;Yakoubi et al, 2019;Montero-Crespo et al, 2020, 2021Domínguez-Álvaro et al, 2019Domínguez-Álvaro et al, , 2021aDomínguez-Álvaro et al, , 2021bCano-Astorga et al, 2021, 2023Schmuhl-Giesen et al, 2022;Turégano-López, 2022). Thus, the prevalence of axospinous AS, followed by axodendritic AS and axodendritic SS, can be considered an additional characteristic among the general rules governing human synapses.…”