Changes in the size of the synaptic junction are thought to have significant functional consequences. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the rat somatosensory cortex. We have segmented in 3D a large number of synapses (n = 6891) to analyze the size and shape of excitatory (asymmetric) and inhibitory (symmetric) synapses, using dedicated software. This study provided three main findings. Firstly, the mean synaptic sizes were smaller for asymmetric than for symmetric synapses in all cortical layers. In all cases, synaptic junction sizes followed a log-normal distribution. Secondly, most cortical synapses had disc-shaped postsynaptic densities (PSDs; 93%). A few were perforated (4.5%), while a smaller proportion (2.5%) showed a tortuous horseshoe-shaped perimeter. Thirdly, the curvature was larger for symmetric than for asymmetric synapses in all layers. However, there was no correlation between synaptic area and curvature.
Knowing the proportions of asymmetric (excitatory) and symmetric (inhibitory) synapses in the neuropil is critical for understanding the design of cortical circuits. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the juvenile rat (postnatal day 14) somatosensory cortex (hindlimb representation). We segmented in three-dimensions 6184 synaptic junctions and determined whether they were established on dendritic spines or dendritic shafts. Of all these synapses, 87–94% were asymmetric and 6–13% were symmetric. Asymmetric synapses were preferentially located on dendritic spines in all layers (80–91%) while symmetric synapses were mainly located on dendritic shafts (62–86%). Furthermore, we found that less than 6% of the dendritic spines establish more than one synapse. The vast majority of axospinous synapses were established on the spine head. Synapses on the spine neck were scarce, although they were more common when the dendritic spine established multiple synapses. This study provides a new large quantitative dataset that may contribute not only to the knowledge of the ultrastructure of the cortex, but also towards defining the connectivity patterns through all cortical layers.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-017-1470-7) contains supplementary material, which is available to authorized users.
Thalamocortical synapses from "lemniscal" neurons of the dorsomedial portion of the rodent ventral posteromedial nucleus (VPMdm) are able to induce with remarkable efficacy, despite their relative low numbers, the firing of primary somatosensory cortex (S1) layer 4 (L4) neurons. To which extent this high efficacy depends on structural synaptic features remains unclear. Using both serial transmission (TEM) and focused ion beam milling scanning electron microscopy (FIB/SEM), we 3D-reconstructed and quantitatively analyzed anterogradely labeled VPMdm axons in L4 of adult mouse S1. All VPMdm synapses are asymmetric. Virtually all are established by axonal boutons, 53% of which contact multiple (2-4) elements (overall synapse/bouton ratio = 1.6). Most boutons are large (mean 0.47 μm3), and contain 1-3 mitochondria. Vesicle pools and postsynaptic density (PSD) surface areas are large compared to others in rodent cortex. Most PSDs are complex. Most synapses (83%) are established on dendritic spine heads. Furthermore, 15% of the postsynaptic spines receive a second, symmetric synapse. In addition, 13% of the spine heads have a large protrusion inserted into a membrane pouch of the VPMdm bouton. The unusual combination of structural features in VPMdm synapses is likely to contribute significantly to the high efficacy, strength, and plasticity of these thalamocortical synapses.
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/µm 3) and the bi-dimensional densities obtained with SDM (puncta/100 µm 2). 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. Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Synapses can be identified with several methods, including genetic labeling of synaptic scaffolding proteins and electron microscopy (EM). Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that participate in the regulation of cell polarity, cell adhesion and synaptic signal transduction 1-3. PSD95 and SAP102 belong to the MAGUK family and are expressed in the postsynaptic density (PSD) of excitatory glutamatergic synapses 4-12 , where they contribute to the recruitment and retention of glutamate receptors 13-15. Genetic labeling of the endogenous PSD95 and SAP102 postsynaptic proteins and imaging using Spinning Disk confocal Microsocpy (SDM) have been proven to be useful for the characterization of synapse diversity in all brain regions of the mouse. SDM is a rapid method that allows the imaging of entire brain sections, so the simultaneous visualization of millions of synapses is made possible, obtaining bi-dimensional densities of fluorescent puncta per surface area (puncta/100 µm 2) 16. Previous attempts have been made to calculate the density of synapses in the brain using EM. This technique allows the identification of individual synapses, although it is restricted to much smaller fields of view. Furthermore, most of these EM studies apply stereological techniques to a limited number of EM sections. Although stereology is a proven valuable method for object counting, the total number of synapses is an estimation which is subject to several technical limitations [see 17 for a review]. In the present study, we use Focused Ion Beam
Thalamocortical posterior nucleus (Po) axons innervating the vibrissal somatosensory (S1) and motor (MC) cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile snout vibrissae. Here, using fine-scale high-end 3D electron microscopy, we demonstrate in adult male C57BL/6 wild-type mice marked differences between MC versus S1 Po synapses in (1) bouton and active zone size, (2) neurotransmitter vesicle pool size, (3) distribution of mitochondria around synapses, and (4) proportion of synapses established on dendritic spines and dendritic shafts. These differences are as large, or even more pronounced, than those between Po and ventro-posterior thalamic nucleus synapses in S1. Moreover, using single-axon transfection labeling, we demonstrate that the above differences actually occur on the MC versus the S1 branches of individual Po cell axons that innervate both areas. Along with recently-discovered divergences in efficacy and plasticity, the synaptic structure differences reported here thus reveal a new subcellular level of complexity. This is a finding that upends current models of thalamocortical circuitry, and that might as well illuminate the functional logic of other branched projection axon systems.
Mitochondria play a key role in energy production and calcium buffering, among many other functions. They provide most of the energy required by neurons, and they are transported along axons and dendrites to the regions of higher energy demands. We have used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the somatosensory cortex of the juvenile rat. We have estimated the volume fraction occupied by mitochondria and their distribution between dendritic, axonal, and nonsynaptic processes. The volume fraction of mitochondria increased from layer I (4.59%) to reach its maximum in layer IV (7.74%) and decreased to its minimum in layer VI (4.03%). On average, 44% of mitochondrial volume was located in dendrites, 15% in axons and 41% in nonsynaptic elements. Given that dendrites, axons, and nonsynaptic elements occupied 38%, 23%, and 39% of the neuropil, respectively, it can be concluded that dendrites are proportionally richer in mitochondria with respect to axons, supporting the notion that most energy consumption takes place at the postsynaptic side. We also found a positive correlation between the volume fraction of mitochondria located in neuronal processes and the density of synapses.
The correlation between dysfunction in the glutamatergic system and neuropsychiatric disorders, including schizophrenia and autism spectrum disorder, is undisputed. Both disorders are associated with molecular and ultrastructural alterations that affect synaptic plasticity and thus the molecular and physiological basis of learning and memory. Altered synaptic plasticity, accompanied by changes in protein synthesis and trafficking of postsynaptic proteins, as well as structural modifications of excitatory synapses, are critically involved in the postnatal development of the mammalian nervous system. In this review, we summarize glutamatergic alterations and ultrastructural changes in synapses in schizophrenia and autism spectrum disorder of genetic or drug-related origin, and briefly comment on the possible reversibility of these neuropsychiatric disorders in the light of findings in regular synaptic physiology.
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
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