Complete reconstructions of vertebrate neuronal circuits on the synaptic level require new approaches. Here, serial section transmission electron microscopy was automated to densely reconstruct four volumes, totaling 670μm3, from the rat hippocampus as proving grounds to determine when axo-dendritic proximities predict synapses. First, in contrast with Peters’ rule, the density of axons within reach of dendritic spines did not predict synaptic density along dendrites because the fraction of axons making synapses was variable. Second, an axo-dendritic touch did not predict a synapse; nevertheless, the density of synapses along a hippocampal dendrite appeared to be a universal fraction, 0.2, of the density of touches. Finally, the largest touch between an axonal bouton and spine indicated the site of actual synapses with about 80% precision, but would miss about half of all synapses. Thus, it will be difficult to predict synaptic connectivity using data sets missing ultrastructural details that distinguish between axo-dendritic touches and bona fide synapses.

SummaryA morphological description of the differentiation of the outer integument of the Arabidopsis thaliana seed is presented. The period covered starts at about the octant embryo stage, extends to the mature seed, and concludes beyond that at the initial stages of seed imbibition. During this period the two-celllayered outer integument goes through a dramatic differentiation process. The outer cell layer secretes mucilage in a ring between the plasma membrane and the outer cell wall at the corners of the cell. This secretion forces the cytoplasm into a columnar shape in the center of the cell. Before and during this process, starch granules are produced, initially at the center of the outer wall and later within the column. Late in differentiation, the starch granules are degraded as the cell produces a highly reinforced wall surrounding the columnar protoplast and at the radial walls between adjacent cells. This results in a cell containing large amounts of mucilage surrounding and completely outside of a highly reinforced columella. The mucilage and outer wall then dehydrate to leave the columella and radial walls visible as the epidermal plateau and reticulations visible on the mature seed. The inner cell layer of the outer integument also produces and degrades starch granules concomitantly with the outer layer but produces no mucilage. In the mature dry seed the collapsed outer wall remains connected to the top of the columella and the radial walls, but these connections are rapidly broken as the mucilage fully hydrates.

A suite of epidermal characters in Arabidopsis is under the transcriptional control of a combinatorial complex containing WD repeat, bHLH and MYB proteins. Many genetic, molecular and biochemical means have been employed to identify and characterize a complete minimal set of complex members required for the trichome initiation, root hair spacing, anthocyanin production and seed coat tannin production pathways. In addition, the WD and bHLH proteins required for outer seed coat differentiation have been identified. However, until now the MYB complex member(s) required for this last WD-bHLH-MYB complex-dependent character have remained elusive. Here we identify two MYBs, AtMYB5 and TT2, as partially redundant in regulating this outer seed coat developmental process with MYB5 having the major role. MYB5 and TT2 are shown to be expressed in this outer seed coat domain. We also show that MYB5 has weak pleiotropic control over trichome development and tannin production and is also expressed in the appropriate places for these functions. TT8 and the downstream GL2 and TTG2 regulators of seed coat development are found to be downregulated in the MYB mutants. Although the TTG1-dependent R2R3 MYBs are considered to be highly pathway specific, identification of MYBs responsible for outer seed coat development allowed for the elucidation of previously undetected novel developmental pleiotropy among these elements.

Previous studies identified various mechanisms of light scattering reduction in tissue induced by chemical agents. Our results suggest that dehydration is an important mechanism of optical clearing in collagenous and cellular tissue. Photographic and optical coherence tomography images indicate that air-immersed skin and tendon specimens become similarly transparent to glycerol-immersed specimens. Transmission electron microscopy images reveal that dehydration causes individual scattering particles such as collagen fibrils and organelles to become more densely packed, but does not significantly alter size. A heuristic particle-interaction model predicts that the scattering particle volume fraction increase can contribute substantially to optical clearing in collagenous and cellular tissue.

Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long-term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta-burst stimulation and comparisons were made to control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post-induction and to perfusion-fixed hippocampus in vivo. Nascent zones were present at the edges of ~35% of synapses in perfusion-fixed hippocampus and as many as ~50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased without significant change in synapse area, suggesting that presynaptic vesicles were recruited to pre-existing nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed that glutamate receptors can be found in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170±5 nm in perfusion-fixed hippocampus to 251±4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that falloff in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP.