Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling

Andrea Santuy,Seth G N Grant,Zhen Qiu,José-Rodrigo Rodríguez,Juncal González-Soriano,Laura Tomás-Roca,Javier Defelipe,Angel Merchan-Perez,Fei Zhu

doi:10.1038/s41598-020-70859-5

Abstract

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm3) and the bi-dimensional densities obtained with SDM (puncta/100 µm2). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.

Highlights

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole
PSD95-positive and SAP102-positive synapses were identified as fluorescent puncta using Spinning Disc confocal Microscopy (SDM) (Fig. 2), and FIB-scanning electron microscope (SEM) was used to visualize and reconstruct synaptic junctions in the same regions
When we compared the densities of PSD95 and SAP102 puncta measured with SDM and the densities of AS measured with FIB-SEM, we found that both methods revealed that stratum radiatum (SR) and stratum oriens (SO) had similar densities, while stratum lacunosum-moleculare (SLM) had a lower density (Fig. 5)

Summary

Introduction

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Given that volume electron microscopy cannot be applied brain-wide, we wanted to obtain an estimate of the number of synapses in other regions of the brain where measurements of PSD95 and SAP102 puncta were a vailable[16]. We based this estimate on the quantitative relationship or conversion factor between SDM and FIB-SEM data previously obtained in the hippocampus. Even though this approach has several limitations and underestimates the actual numbers of synapses, it provides valuable information on the minimum number of excitatory synapses that are present in more than a hundred brain regions

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Conclusion