Tracing nerve fibers with volume electron microscopy to quantitatively analyze brain connectivity.

The highly complex structure of the brain requires an approach that can unravel its connectivity. Using volume electron microscopy and a dedicated software we can trace and measure all nerve fibers present within different samples of brain tissue. With this software tool, individual dendrites and axons are traced, obtaining a simplified "skeleton" of each fiber, which is linked to its corresponding synaptic contacts. The result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. To test this methodology, we apply it to the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex of the mouse. We find that nerve fibers are densely packed in the neuropil, reaching up to 9 kilometers per cubic mm. We obtain the number of synapses, the number and lengths of dendrites and axons, the linear densities of synapses established by dendrites and axons, and their location on dendritic spines and shafts. The quantitative data obtained through this method enable us to identify subtle traits and differences in the synaptic organization of the samples, which might have been overlooked in a qualitative analysis.

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 µm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 µm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

Glutamatergic Dysfunction and Synaptic Ultrastructural Alterations in Schizophrenia and Autism Spectrum Disorder: Evidence from Human and Rodent Studies.

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.

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

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.

Area-Specific Synapse Structure in Branched Posterior Nucleus Axons Reveals a New Level of Complexity in Thalamocortical Networks.

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.SIGNIFICANCE STATEMENT Many long-distance brain connections depend on neurons whose branched axons target separate regions. Using 3D electron microscopy and single-cell transfection, we investigated the mouse Posterior thalamic nucleus (Po) cell axons that simultaneously innervate motor and sensory areas of the cerebral cortex involved in whisker movement control. We demonstrate significant differences in the size of the boutons made in each area by individual Po axons, as well as in functionally-relevant parameters in the composition of their synapses. In addition, we found similarly large differences between the synapses of Po versus ventral posteromedial thalamic nucleus axons in the whisker sensory cortex. Area-specific synapse structure in individual axons implies a new, unsuspected level of complexity in long-distance brain connections.

Study of the Size and Shape of Synapses in the Juvenile Rat Somatosensory Cortex with 3D Electron Microscopy.

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.

Quantitative 3D Ultrastructure of Thalamocortical Synapses from the "Lemniscal" Ventral Posteromedial Nucleus in Mouse Barrel Cortex.

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.

Haptically assisted connection procedure for the reconstruction of dendritic spines.

Dendritic spines are thin protrusions that cover the dendritic surface of numerous neurons in the brain and whose function seems to play a key role in neural circuits. The correct segmentation of those structures is difficult due to their small size and the resulting spines can appear incomplete. This paper presents a four-step procedure for the complete reconstruction of dendritic spines. The haptically driven procedure is intended to work as an image processing stage before the automatic segmentation step giving the final representation of the dendritic spines. The procedure is designed to allow both the navigation and the volume image editing to be carried out using a haptic device. A use case employing our procedure together with a commercial software package for the segmentation stage is illustrated. Finally, the haptic editing is evaluated in two experiments; the first experiment concerns the benefits of the force feedback and the second checks the suitability of the use of a haptic device as input. In both cases, the results shows that the procedure improves the editing accuracy.