Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50-60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm(3)) surrounding a large dendritic spine, a cylinder (~43 μm(3)) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm(3)) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1).

Synapse clustering facilitates circuit integration, learning, and memory. Long-term potentiation (LTP) of mature neurons produces synapse enlargement balanced by fewer spines, raising the question of how clusters form despite this homeostatic regulation of total synaptic weight. Three-dimensional reconstruction from serial section electron microscopy (3DEM) revealed the shapes and distributions of smooth endoplasmic reticulum (SER) and polyribosomes, subcellular resources important for synapse enlargement and spine outgrowth. Compared to control stimulation, synapses were enlarged two hours after LTP on resource-rich spines containing polyribosomes (4% larger than control) or SER (15% larger). SER in spines shifted from a single tubule to complex spine apparatus after LTP. Negligible synapse enlargement (0.6%) occurred on resource-poor spines lacking SER and polyribosomes. Dendrites were divided into discrete synaptic clusters surrounded by asynaptic segments. Spine density was lowest in clusters having only resource-poor spines, especially following LTP. In contrast, resource-rich spines preserved neighboring resource-poor spines and formed larger clusters with elevated total synaptic weight following LTP. These clusters also had more shaft SER branches, which could sequester cargo locally to support synapse growth and spinogenesis. Thus, resources appear to be redistributed to synaptic clusters with LTP-related synapse enlargement while homeostatic regulation suppressed spine outgrowth in resource-poor synaptic clusters.

Transmission-mode scanning electron microscopy (tSEM) on a field emission SEM platform was developed for efficient and cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizing the resolution and dynamic range necessary for discriminating key subcellular structures, such as small axonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomes which are critical for neuronal function. Individual image fields from the tSEM system were up to 4,295 µm(2) (65.54 µm per side) at 2 nm pixel size, contrasting with image fields from a modern transmission electron microscope (TEM) system, which were only 66.59 µm(2) (8.160 µm per side) at the same pixel size. The tSEM produced outstanding images and had reduced distortion and drift relative to TEM. Automated stage and scan control in tSEM easily provided unattended serial section imaging and montaging. Lens and scan properties on both TEM and SEM platforms revealed no significant nonlinear distortions within a central field of ∼100 µm(2) and produced near-perfect image registration across serial sections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCT™ or Fiji/TrakEM2 software. Axial resolution limits the analysis of small structures contained within a section (∼45 nm). Since this new tSEM is non-destructive, objects within a section can be explored at finer axial resolution in TEM tomography with current methods. Future development of tSEM tomography promises thinner axial resolution producing nearly isotropic voxels and should provide within-section analyses of structures without changing platforms. Brain was the test system given our interest in synaptic connectivity and plasticity; however, the new tSEM system is readily applicable to other biological systems.