Serial block face scanning electron microscopy of a specific gut chemosensory cell.

<p><b>A.</b> Enteroendocrine cell in the ileum of a Pyy-GFP transgenic mouse. Inset shows a reconstruction of a confocal z-stack from the dotted region. <b>B.</b> These cells are rare as shown by flow cytometric analysis. For every 10,000 epithelial cells in the colon or ileum, there are only 5.5 or 11.8 Pyy-GFP cells, respectively. Non-viable cells stained with propidium iodide are indicated in red, GFP negative cells in blue, and the area in the lower right corner contains GFP positive cells. <b>C.</b> The basal process in Pyy-GFP cells weaves in between epithelial cells, making it difficult to analyze by conventional transmission electron microscopy. <b>D.</b> This hurdle can be overcome by serial block face scanning electron microscopy (SBEM). For this, intestinal tissue from a Pyy-GFP mouse was harvested and trimmed with a vibrating blade microtome. The resulting 300 µm wide by 45 µm thick tissue block contained a cell of interest and was imaged with a confocal microscope. E. The block was processed for SBEM. Then, the confocal z-stack was matched to the SBEM image of the entire block face to identify the cell of interest. Bars = 10 µm.</p

Bridging structure to function: neurotrophic factors and the formation of neuropods.

<p><b>A.</b> 3D reconstruction of a confocal z-stack shows glial fibrillary acidic protein (GFAP) immunoreactive enteric glia contacting basal processes in Pyy-GFP cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089881#pone.0089881.s004" target="_blank">Figure S4</a> contains fluorescence data. <b>B.</b> Pyy-GFP enteroendocrine cells express neurotrophic factor receptors trkA, trkB, gfra1, and gfra3. Fold expression is relative to non-GFP intestinal epithelial cells. <b>C.</b> Intestinal organoids were used as a model to test the effects of neurotrophic factors on enteroendocrine cells. Treatments were applied to organoids after 4 days of culture. <b>D.</b> Organoids were cultured from an analogous Cck-GFP mouse model. Cck-GFP organoids had about six GFP positive cells per organoid compared to less than one for every 10 organoids in Pyy-GFP organoids. Representative image of a 4-day old Cck-GFP intestinal organoid. <b>E.</b> Top: Upon the addition of nerve growth factor - β (NGF-β) or artemin, the percentage of enteroendocrine cells with neuropods increased in a dose-dependent manner. Bottom: compared to controls, 10 ng/mL of NGF-β or artemin significantly increased the length of neuropods in enteroendocrine cells. <b>F.</b> Representative images of NGF-β or artemin effects after 24 h of a 10 ng/mL treatment. Bars = 10 µm.</p

An emerging model for the enteroendocrine cell.

<p>Here we used correlative confocal-3D electron microscopy to study the ultrastructure of a single chemosensory cell in the gut. The 3D ultrastructure uncovered unique features of an axon-like neuropod in these cells, including neurofilaments, secretory vesicles, and their relationship to glia. This technical advance can be applied to similar systems in which cells of interest are rare, dispersed, and with convoluted morphology.</p

Trade-Offs in Capacity and Rechargeability in Nonaqueous Li–O 2 Batteries : Solution-Driven Growth versus Nucleophilic Stability

<div><p>The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY – both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia – the cells that nurture enteric neurons. We extended these structural findings into an <i>in vitro</i> intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.</p></div

3D ultrastructure reveals axonal process escorted by enteric glia.

<p><b>A.</b> Enteroendocrine cells compared to other intestinal epithelial cells express neurofilaments light and medium (top panel). Neurofilament proteins light and medium are expressed in 22% and 47% of Pyy-GFP cells, respectively (bottom panel). This quantification was performed using immunohistochemistry with neurofilament-specific antibodies. <b>B.</b> Top panel is a representative image showing that neurofilament heavy is expressed in subepithelial myofibroblasts but not in enteroendocrine cells. Neurofilament light is contained within the Pyy-GFP cell basal process (bottom panel). <b>C.</b> Enteroendocrine cells contain neurofilament medium within the neuropod. Inset shows the position of the cell in the epithelium of the ileum. 3D reconstruction of confocal z-stacks depicts the neurofilament medium contained within the Pyy-GFP cell neuropod. <b>D.</b> The SBEM data also revealed the relationship between the neuropod in the Pyy-GFP cell and enteric glia. Enteric glia trespass the basal lamina and penetrate into the epithelium (inset). SBEM data segmentation revealed that the enteric glia extends a cytoplasmic process into the epithelium that contacts the enteroendocrine cell neuropod. Bars in B and C = 10 µm, in D = 1 µm.</p

Reconstructing the enteroendocrine cell ultrastructure in 3D.

<p><b>A.</b> Data set from serial block face scanning electron microscopy (SBEM) containing a 5 nm/pixel 510 image that spanned a tissue volume of 5,2327 cubic micrometers. <b>B.</b> All nuclei were reconstructed in the block using Imaris software. The block contained 145 epithelial cells, including 129 enterocytes, 11 goblet cells, 4 cells of various types, and 1 enteroendocrine cell. <b>C.</b> The enteroendocrine cell was traced on each slice to reveal its entire ultrastructure. On the left the cell has a tuft of microvilli exposed to the gut lumen, and on the right there is a prominent neuropod that extends towards the basal lamina propria. <b>D.</b> This neuropod is populated by mitochondria, in particular at the tip (blue), secretory vesicles (yellow), and filament-like structures (orange). Top panels show the reconstructions of the cells, and bottom panels show a representative SBEM image of each feature. Structures of interest in the bottom panel have been pseudocolored to facilitate their visualization. Bars = 1 µm.</p

Cuprizone Intoxication Induces Cell Intrinsic Alterations in Oligodendrocyte Metabolism Independent of Copper Chelation

Cuprizone intoxication is a common animal model used to test myelin regenerative therapies for the treatment of diseases such as multiple sclerosis. Mice fed this copper chelator develop reversible, region-specific oligodendrocyte loss and demyelination. While the cellular changes influencing the demyelinating process have been explored in this model, there is no consensus about the biochemical mechanisms of toxicity in oligodendrocytes and about whether this damage arises from the chelation of copper <i>in vivo</i>. Here we have identified an oligodendroglial cell line that displays sensitivity to cuprizone toxicity and performed global metabolomic profiling to determine biochemical pathways altered by this treatment. We link these changes with alterations in brain metabolism in mice fed cuprizone for 2 and 6 weeks. We find that cuprizone induces widespread changes in one-carbon and amino acid metabolism as well as alterations in small molecules that are important for energy generation. We used mass spectrometry to examine chemical interactions that are important for copper chelation and toxicity. Our results indicate that cuprizone induces global perturbations in cellular metabolism that may be independent of its copper chelating ability and potentially related to its interactions with pyridoxal 5′-phosphate, a coenzyme essential for amino acid metabolism