Functional and structural studies investigating macroscopic connectivity in the human cerebral cortex suggest that high-order associative regions exhibit greater connectivity compared to primary ones. However, the synaptic organization of these brain regions remains unexplored. In the present work, we conducted volume electron microscopy to investigate the synaptic organization of the human brain obtained at autopsy. Specifically, we examined layer III of Brodmann areas 17, 3b, and 4, as representative areas of primary visual, somatosensorial, and motor cortex. Additionally, we conducted comparative analyses with our previous datasets of layer III from temporopolar and anterior cingulate associative cortical regions (Brodmann areas 24, 38, and 21). 9,690 synaptic junctions were 3D reconstructed, showing that certain synaptic characteristics are specific to particular regions. The number of synapses per volume, the proportion of the postsynaptic targets, and the synaptic size may distinguish one region from another, regardless of whether they are associative or primary cortex. By contrast, other synaptic characteristics were common to all analyzed regions, such as the proportion of excitatory and inhibitory synapses, their shapes, their spatial distribution, and a higher proportion of synapses located on dendritic spines. The present results provide further insights into the synaptic organization of the human cerebral cortex.

Huntington\'s disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the coding sequence of huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) of the corpus striatum. No effective treatment is still available, thus urging the identification of potential therapeutic targets. While evidence of mitochondrial structural alterations in HD exists, previous studies mainly employed 2D approaches and were performed outside the strictly native brain context. In this study, we adopted a novel multiscale approach to conduct a comprehensive 3D in situ structural analysis of mitochondrial disturbances in a mouse model of HD. We investigated MSSNs within brain tissue under optimal structural conditions utilizing state-of-the-art 3D imaging technologies, specifically FIB/SEM for the complete imaging of neuronal somas and Electron Tomography for detailed morphological examination, and image processing-based quantitative analysis. Our findings suggest a disruption of the mitochondrial network towards fragmentation in HD. The network of interlaced, slim and long mitochondria observed in healthy conditions transforms into isolated, swollen and short entities, with internal cristae disorganization, cavities and abnormally large matrix granules.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.
COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty “ghosts.” To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

Synaptic vesicles can undergo several modes of exocytosis, endocytosis, and trafficking within individual synapses, and their fates may be linked to different vesicular protein compositions. Here, we mapped the intrasynaptic distribution of the synaptic vesicle proteins SV2B and SV2A in glutamatergic synapses of the hippocampus using three-dimensional electron microscopy. SV2B was almost completely absent from docked vesicles and a distinct cluster of vesicles found near the active zone. In contrast, SV2A was found in all domains of the synapse and was slightly enriched near the active zone. SV2B and SV2A were found on the membrane in the peri-active zone, suggesting the recycling from both clusters of vesicles. SV2B knockout mice displayed an increased seizure induction threshold only in a model employing high-frequency stimulation. Our data show that glutamatergic synapses generate molecularly distinct populations of synaptic vesicles and are able to maintain them at steep spatial gradients. The almost complete absence of SV2B from vesicles at the active zone of wildtype mice may explain why SV2A has been found more important for vesicle release.

UNASSIGNED: We previously discovered a pyridazine derivative compound series that can improve cognitive functions in mouse models of Alzheimer\'s disease. One of the advanced compounds from this series, LDN/OSU-0215111-M3, was selected as the preclinical development candidate. This compound activates local protein translation at the perisynaptic astrocytic process (PAP) and enhances synaptic plasticity sequentially. While biochemical evidence supports the hypothesis that the compound enhances the structural plasticity of the tripartite synapse, its direct structural impact has not been investigated.
UNASSIGNED: Volume electron microscopy was used to study the hippocampal tripartite synapse three-dimensional structure in 3-month-old wild-type FVB/NJ mice after LDN/OSU-0215111-M3 treatment.
UNASSIGNED: LDN/OSU-0215111-M3 increased the size of tertiary apical dendrites, the volume of mushroom spines, the proportion of mushroom spines containing spine apparatus, and alterations in the spine distribution across the surface area of tertiary dendrites. Compound also increased the number of the PAP interacting with the mushroom spines as well as the size of the PAP in contact with the spines. Furthermore, proteomic analysis of the isolated synaptic terminals indicated an increase in dendritic and synaptic proteins as well as suggested a possible involvement of the phospholipase D signaling pathway. To further validate that LDN/OSU-0215111-M3 altered synaptic function, electrophysiological studies showed increased long-term potentiation following compound treatment.
UNASSIGNED: This study provides direct evidence that pyridazine derivatives enhance the structural and functional plasticity of the tripartite synapse.

Glaucoma is associated with increased resistance in the conventional aqueous humor (AH) outflow pathway of the eye. The majority of resistance is thought to reside in the juxtacanalicular connective tissue (JCT) region of the trabecular meshwork and is modulated by the inner wall (IW) endothelial cells of Schlemm\'s canal (SC). The IW cells form connections with the underlying JCT cells/matrix, and these connections are thought to modulate outflow resistance. Two ways by which AH crosses the IW endothelium are through: 1) the formation of outpouchings in IW cells called giant vacuoles (GVs) and their intracellular pores (I-pores), and 2) intercellular pores between two adjacent IW cells (B-pores). AH outflow is segmental with areas of high-, low-, and non-flow around the circumference of the eye. To investigate whether changes in cellular connectivity play a role in segmental outflow regulation, we used global imaging, serial block-face scanning electron microscopy (SBF-SEM), and 3D reconstruction to examine individual IW cells from different flow areas of ex vivo perfused normal human donor eyes. Specifically, we investigated the differences in cellular dimensions, connections with JCT cells/matrix, GVs, and pores in SC IW cells between high-, low-, and non-flow areas. Our data showed that: 1) IW cell-JCT cell/matrix connectivity was significantly decreased in the cells in high-flow areas compared to those in low- and non-flow areas; 2) GVs in the cells of high-flow areas had significantly fewer connections beneath them compared to GVs in the cells of low- and non-flow areas; 3) Type IV GVs (with I-pores and basal openings) had significantly fewer connections beneath them compared to Type I GVs (no I-pore or basal opening). Our results suggest that a decreased number of cellular connections between the IW and JCT in high-flow areas is associated with increased numbers of GVs with I-pores and larger Type IV GVs observed in previous studies. Therefore, modulating the number of cellular connections may affect the amount of high-flow area around the eye and thereby modulate AH outflow.

Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron Microscopy (METTEM). This protocol allowed us to image the intracellular distribution of the viral replicase molecules in three-dimensions with METTEM and electron tomography. Our study showed how viral replicase molecules build replication complexes within specialized cell membranes.

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.

In mammalian cochlea, sound-induced vibration is amplified by a three-row lattice of Y-shaped microstructures consisting of electromotile outer hair cell and supporting Deiters cell. This highly organized structure is thought to be essential for hearing of low-level sounds. Prior studies reported differences in geometry and synaptic innervation of the outer hair cells between rows, but how these fine features are achieved at subcellular level still remains unclear. Using serial block-face electron microscopy, we acquired few-hundred-micron-sized cytoarchitecture of mouse organ of Corti at nanometer resolution. Structural quantifications were performed on the Y-shapes as well as afferent and efferent projections to outer hair cells (OHCs). Several new features, which support the previously observed inter-row heterogeneity, are described. Our result provides structural bases for the gradient of mechanical properties and diverse centrifugal regulation of OHC rows.

Increased intraocular pressure (IOP) is the main risk factor for primary open-angle glaucoma and results from impaired drainage of aqueous humor (AH) through the trabecular outflow pathway. AH must pass the inner wall (IW) endothelium of Schlemm\'s canal (SC), which is a monolayer held together by tight junctions, to exit the eye. One route across the IW is through giant vacuoles (GVs) with their basal openings and intracellular pores (I-pores). AH drainage through the trabecular outflow pathway is segmental. Whether more GVs with both basal openings and I-pores are present in the active flow areas and factors that may influence formation of GVs with I-pores have not been fully elucidated due to limitations in imaging methods. In this study, we applied a relatively new technique, serial block-face scanning electron microscopy (SBF-SEM), to investigate morphological factors associated with GVs with I-pores in different flow areas. Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. Six radial wedges of trabecular meshwork including SC (2 each from high-, low-, and non-flow areas) were imaged using SBF-SEM (total: 9802 images). Total GVs, I-pores, basal openings, and four types of GVs were identified. Percentages of GVs with I-pores and basal openings and number of I-pores/GV were determined. Overall, 14.4% (477/3302) of GVs had I-pores. Overall percentage of GVs with both I-pores and basal openings was higher in high- (15.7%), than low- (12.6%) or non-flow (7.3%) areas. Of GVs with I-pores, 83.2% had a single I-pore; 16.8% had multiple I-pores (range: 2-6). Additionally, 180 GVs (90 with I-pores and 90 without I-pores) were randomly selected, manually segmented, and three-dimensionally (3D) reconstructed to determine size, shape, and thickness of the cellular lining. Size of GVs (including median volume, surface area, and maximal cross-sectional area) with I-pores (n = 90) was significantly larger than GVs without I-pores (n = 90) using 3D-reconstructed GVs (P ≤ 0.01). Most I-pores (73.3%; 66/90) were located on or close to GV\'s maximal cross-sectional area with significant thinning of the cellular lining. Our results suggest that larger size and thinner cellular lining of GVs may contribute to formation of GVs with I-pores. More GVs with I-pores and basal openings were observed in high-flow areas, suggesting these GVs do provide a channel through which AH passes into SC and that increasing this type of GV may be a potential strategy to increase aqueous outflow for glaucoma treatment.