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

Abstract

34We have estimated the densities of synapses per unit volume in the whole mouse brain. To do 35 this, we combined Spinning Disc confocal Microscopy (SDM) that acquires images of synaptic 36 proteins labeled with fluorophores in large brain areas, and FIB-SEM, a three-dimensional 37 electron microscopy technique that provides a resolution beyond that of the confocal 38 microscope, but in relatively small areas. The postsynaptic scaffold proteins PSD95 and SAP102 39were genetically labeled to visualize excitatory synapses with SDM. We calculated the densities 40 of synaptic puncta (with SDM) and the actual number of synapses (with FIB-SEM) in the CA1 41 region of the hippocampus. We then calculated the quantitative relationship between the 42 densities of fluorescent puncta and the number of synapses. Finally, we applied this 43 conversion factor to other regions of the brain where densities of puncta were available. We 44 observed three different groups of brain regions, one with the highest densities of synapses 45(isocortex, olfactory areas, hippocampal formation and cortical subplate), a second group with 46 low densities of synapses (pallidum, hypothalamus, brainstem and cerebellum), and a third 47 group comprising structures which showed a wide range of synapse densities (striatum and 48 thalamus). 49 50 Keywords 51 FIB-SEM, PSD95, SAP102, Hippocampus, CA1 52 53 Significance statement 54 Neurons communicate through synapses. Determining the number of synapses that are 55present in different brain regions is therefore crucial to understand brain connectivity as a 56whole. Here we have developed a technique that allows us to estimate the number of 57 synapses that are present in more than one hundred regions brain-wide. We found that, in 58 general, rostral parts of the brain -including the neocortex-have the highest numbers of 59 synapses, while caudal regions -including the cerebellum and brainstem-have much lower 60 numbers of synapses. Other regions, such as the thalamus, are highly heterogeneous. These 61 quantitative data will help to better understand the structure of brain microcircuits and to 62 build realistic whole-brain models. 63 64 65

Three-dimensional synaptic organization of the human hippocampal CA1 field

“…In the present study, the density of inhibitory synapses was particularly low in most CA1 layers (AS:SS ratio in all layers was around 95:5 except for in SLM, where the ratio was close to 90:10). This data is in line with our study using FIB/SEM to analyze the synaptic density in the mouse (where the proportion of synapses that were inhibitory was 8% in the SLM, and approximately 2% in the case of the SR and SO) (22) and in the rat CA1 field (where 4% of the synapses in SR were inhibitory; Blazquez-Llorca et al, in preparation).…”

“…These values are much higher than the ones found in the present work for the human CA1 (Table 1). Such huge differences in synaptic density between humans and rodents -together with the above-mentioned divergences in the morphology and distribution of pyramidal cells in the SP of CA1 (18), as well as differences in other anatomical, genetic, molecular and physiological features (18,(21)(22)(23)(24)(25)-further support the notion that there are remarkable differences between the human and rodent CA1. These differences clearly need to be taken into consideration when making interpretations in translational studies comparing one species to another.…”

“…However, in recent studies from our group using FIB/SEM to analyze the synaptic density in the rodent CA1 field, the following values were obtained: 2.53 synapses/µm 3 in SO, 2.36 synapses/µm 3 in SR and 1.72 synapses/µm 3 in SLM in the mouse (22), and 2.52 synapses/µm 3 in SR in the rat (Blazquez-Llorca et al, in preparation). These values are much higher than the ones found in the present work for the human CA1 (Table 1).…”

3d Synaptic Organization of the Rat Ca1 and Alterations Induced by Cocaine Self-Administration

“…In the present study, we found that the AS:SS ratio was 96.25:3.75. Preliminary results from our laboratory, using the FIB/SEM methodology, have shown that the AS:SS ratios are 98:2 and 96:4 in the stratum radiatum of the mouse and human CA1, respectively 29,30 . That is, the ratio of AS and SS in the CA1 field is similar in humans and rodents.…”

“…We found a synaptic density of 2.52 synapses / µm 3 . Using FIB/SEM, we have previously shown that the synaptic density in the stratum radiatum of CA1 of the mouse is 2.36 synapses / µm 3 (29) , whereas in the stratum radiatum of CA1 of the human hippocampus, it is 0.67 synapses / µm 3 (30) . Thus, the synaptic density of the rat and mouse is remarkably similar but there are huge differences in synaptic density between humans and rodents.…”