Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue

Is axonal degeneration a key early event in Parkinson’s disease?

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here by permission of IOS Press for personal use, not for redistribution. The definitive version was published in Journal of Parkinson's Disease 6 (2016): 703-707, doi:10.3233/JPD-160881.Recent research suggests that in Parkinson’s disease the long, thin and unmyelinated axons of dopaminergic neurons degenerate early in the disease process. We organized a workshop entitled ‘Axonal Pathology in Parkinson’s disease’, on March 23rd, 2016, in Cleveland, Ohio with the goals of summarizing the state-of-the-art and defining key gaps in knowledge. A group of eight research leaders discussed new developments in clinical pathology, functional imaging, animal models, and mechanisms of degeneration including neuroinflammation, autophagy and axonal transport deficits. While the workshop focused on PD, comparisons were made to other neurological conditions where axonal degeneration is well recognized

Effect of umbilical cord matrix stem cells on Parkinson’s disease model rats

Doctor of PhilosophyDepartment of Anatomy and PhysiologyMark L. WeissUmbilical cord matrix or Wharton’s Jelly is a mucous connective tissue ensheathing the cord blood vessels and contains mesenchymal-like stem cells. Previously, we have shown that pig umbilical cord matrix stem (pUCMS) cells transplanted into normal rat brain were recovered up to 6 weeks post-transplantation, where a sub-population of pUCMS cells exhibited neuronal morphology and expressed a variety of neuronal markers. Here, approximately 150 pUCMS cells were transplanted into non-immunesuppressed rats that previously received a brain lesion by neurotoxin, 6-hydroxydopamine (6-OHDA), which specifically affects midbrain dopaminergic neurons, leading to pathologic findings similar to that of Parkinson’s disease (PD). The pUCMS cells proliferated up to 8 weeks post-transplantation and there was a significant increase in the percentage and number of pUCMS cells expressing tyrosine hydroxylase (TH), which is a marker for dopaminergic cells. We conclude that 1. Xenotransplants of pig UCMS cells are not rejected by rats at least up to 8 weeks after transplantation and 2. The pig UCMS cells proliferate and differentiate after transplantation into PD model rats. The surface antigen and gene expression profile of human umbilical cord matrix stem (hUCMS) cells resemble that of mesenchymal stem cells. Apomorphine-induced rotatory behavior was used to analyze the motor deficits of the PD model rats. In different experiments 1000, 2500 and 25000 hUCMS cells were transplanted into the brain of non-immunesuppressed PD model rats. There was a dose-dependent decrease in apomorphine-induced rotations; the maximum benefit was found in the rats that received 1000 hUCMS cells. The graft cells were recovered at 2 days and 1 week, but not at 6, 10 or 12 weeks post-transplantation. Quantitative assessment of host TH-positive midbrain dopaminergic neurons revealed a positive correlation between the behavioral improvement and TH-positive cell number in the low-density (1000 cells) transplant group, showing that the hUCMS cells may play a role in rescuing damaged host dopaminergic neurons and promote improvement of motor deficits in PD-model rats. In summary, hUCMS cells appear to be mesenchymal stem cells that can be harvested in great numbers from a non-controversial, inexhaustible source. Human UCMS cells show therapeutic benefit in PD model rats, but the mechanism by which they promote improvement is presently unknown

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue

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

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

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

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