Manual reconstruction of a bouton its synaptic partner, and all membrane organelles contained.

<p><b>A</b>, <b>B</b>, The FIBSEM image series can be used to segment <i>in vivo</i> imaged structures (<b>A</b>, inset) including all their organelles: axonal bouton – yellow, mitochondria – green, synapse – red, synaptic vesicles – gold, endoplasmic reticulum – blue, dendritic spine – pink, dendritic endoplasmic reticulum – orange. The reconstruction is made from an image volume (6.4 µm×8.0 µm×5.0 µm) (<b>B</b>, inset left) that also includes the synaptically coupled dendritic spine (<b>B</b>, inset right). Scale bar in <b>A</b> is 1 µm, and in <b>A</b> (inset) is 5 µm.</p

Interactive segmentation of neurites.

<p><b>A</b>, The ilastik software allows users to select regions for segmentation (yellow box), and compare the generated 3D model (<b>B</b>) with the <i>in vivo</i> image (<b>C</b>) of the neurite of interest. A GFP dendrite from a layer 5 pyramidal neuron is shown in <b>C</b>. The method can also be used with other fluorescent markers, such as tdTomato (<b>D</b>), here expressed in GABAergic axons and dendrites in a different animal. When the density of neurites (<b>E</b>) is high the neurites can still be distinguished. Panel <b>E</b>, shows the reconstructed axons (red) and dendrites (grey) that are shown in the in vivo image in <b>D</b>. Scale bar in <b>C</b> and <b>D</b> is 5 µm.</p

<i>In vivo</i> imaging, laser branding and tissue preparation.

<p><b>A</b>, Cortical surface showing the vasculature on the surface of the brain. Dotted lines indicate the blood vessels that can also be seen as dark shadows in the 2PLSM (<b>B</b>), with the white square indicating the region imaged at higher magnification (inset). After fixation and sectioning this region (<b>C</b>) was then laser branded, and reimaged using 2PLSM. These branding marks were visible (<b>D</b>) in the resin block (indicated with white arrow heads) without any further enhancement. Their position is also highlighted with laser etching on the surface (black arrows) that can be seen in the FIBSEM (<b>E</b>). This indicates the region to be imaged (<b>F</b>) so that imaging and milling will capture the branded region (white arrow heads). Scale bar in <b>A</b> and <b>B</b> is 100 µm, and 10 µm in (<b>C</b>–<b>F</b>).</p

Mouse 2

Image stack and blender files with the 3D mesh data reconstructed from a 24 months old layer I somatosensory cortex neuropi

Spine and bouton volume correlate closely with synapse size in the aged and adult neuropil.

<p><b>A</b>, Histogram showing the distribution of vesicle density normalized to the synaptic contact surface area shows no difference between aged and adult animals (p = 0.4, Kolmogorov-Smirnov test). <b>B</b>, Correlation between spine head volume and synaptic surface area for adult (N = 207; R² = 0.673) and aged (N = 197; R² = 0.817) mice. The slopes are not significantly different (ANOVA, p = 0.42). <b>C</b>, Correlation between volume of excitatory boutons and synaptic surface area for adult (N = 169; R² = 0.369) and aged (N = 146; R² = 0.526) mice. The slopes are significantly different (ANOVA, p = 0.0045).</p

Somatosensory cortex of aged mice is thinner than adults.

A, Semi-thin coronal sections of mouse somatosensory cortex indicating a reduced cortical thickness in the aged example (left, adult, 4 months old; right, aged, 24 months old). B, Measurements of cortical thickness from 3 aged and 3 adult mice, in the semi-thin sections, showed a reduction of 15.6%, (adult, 1.03 ± 0.047 mm; aged, 0.87 ± 0.003 mm; unpaired t-test, p = 0.0297). C, Measurements of layer I thickness from the same mice, in the semi-thin sections, showed a reduction of 19.4% (adult, 0.103 ± 0.004 mm; aged, 0.083 ± 0.002 mm; N = 10 per each of the six mice; p = 0.0009, unpaired t-test). D, Counts of cell profiles from sections used in B, across the cortical thickness, show no difference between adult and aged animals in any of the five bins positioned from the pial surface to the white matter. Bars indicate the mean ± sem. Differences are not statistically significant; Kolmogorov-Smirnov test, p > 0.9. Scale bar in A is 100 micrometers.</p

Synapses are larger in aged layer 1 neuropil.

<p><b>A</b>, FIBSEM electron micrograph shows two boutons making asymmetric synapses; synapse 1 is made with a single synaptic bouton, 2 and 3 with a multi-synaptic bouton. <b>B</b>, the same synapses were segmented in the TrakEM2 software in FIJI (shown in red: <a href="http://www.fiji.sc/" target="_blank">www.fiji.sc</a>) and reconstructed in 3D. <b>C</b>, There is a strong correlation between the diameter of circles used to annotate 57 synapses and their surface areas measured from their 3D reconstruction (slope of second polynomial regression, y = 0.032–0.09x + 0.95x²; R² = 0.84). <b>D,</b> Frequency distribution of asymmetric synapse sizes, estimated from the maximum diameter measurements shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198131#pone.0198131.g002" target="_blank">Fig 2</a></b> (Inset shows average values of all measurements, N = 2800 adult synapses, 377.8 ± 3.2 nm; N = 2423 aged synapses, 427.7 ± 3.9 nm; p < 0.0001, Kolmogorov-Smirnov test), showed that synapses in layer 1 of aged mice are larger. <b>E</b>, The same data plotted on a logarithmic scale for adult, and <b>F</b>, aged mice. Both show a log-normal distribution (adult, red fitting; center = 287.5, amplitude = 7.27, width = 0.33, R² = 0.96; aged, blue fitting; center = 305.1, amplitude = 4.9, width = 0.47, R² = 0.94). <b>G</b>, Examples of a series of micrographs containing a perforated synaptic density (top) and its segmentation (bottom) by means of the automated detection method, and its 3D rendering on the right. <b>H</b>, Graph showing the percentage of asymmetric synapses that displayed one or more perforation.</p

A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner

Synapse density per type.

<p>Synapse density per type.</p

Measurements on saturated reconstructions.

<p>Measurements on saturated reconstructions.</p