Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy

Recurrent Inhibition to the Medial Nucleus of the Trapezoid Body in the Mongolian Gerbil (<i>Meriones Unguiculatus</i>)

<div><p>Principal neurons in the medial nucleus of the trapezoid body (MNTB) receive strong and temporally precise excitatory input from globular bushy cells in the cochlear nucleus through the calyx of Held. The extremely large synaptic currents produced by the calyx have sometimes led to the view of the MNTB as a simple relay synapse which converts incoming excitation to outgoing inhibition. However, electrophysiological and anatomical studies have shown the additional presence of inhibitory glycinergic currents that are large enough to suppress action potentials in MNTB neurons at least in some cases. The source(s) of glycinergic inhibition to MNTB are not fully understood. One major extrinsic source of glycinergic inhibitory input to MNTB is the ventral nucleus of the trapezoid body. However, it has been suggested that MNTB neurons receive additional inhibitory inputs via intrinsic connections (collaterals of glycinergic projections of MNTB neurons). While several authors have postulated their presence, these collaterals have never been examined in detail. Here we test the hypothesis that collaterals of MNTB principal cells provide glycinergic inhibition to the MNTB. We injected dye into single principal neurons in the MNTB, traced their projections, and immunohistochemically identified their synapses. We found that collaterals terminate within the MNTB and provide an additional source of inhibition to other principal cells, creating an inhibitory microcircuit within the MNTB. Only about a quarter to a third of MNTB neurons receive such collateral inputs. This microcircuit could produce side band inhibition and enhance frequency tuning of MNTB neurons, consistent with physiological observations.</p></div

Schematic summary of the location and projection direction of labelled MNTB principal cells.

<p>A: The two coronal sections of the gerbil MNTB labeled with Nissl, representing the near to rostral and near to caudal end of the MNTB (rostral at– 4.9 mm, caudal at– 5.5 mm relative to Bregma). B: Location of origin and projection directions of all labeled neurons in which the total neurite length was at least 60 μm, shown in the form of arrows in each rostro-caudal section of the MNTB (red outlines top to bottom represent caudal to rostral). The arrows point in the direction of travel of the longest neurite originating from each labeled neuron, and the arrow origin indicates the approximate location of the labeled neuron in the medio-lateral axis of the MNTB normalized to the midline on the right (dashed line). Red arrows indicate neurons that have axons with returning collaterals within the MNTB. Represented are the 16 cases found in coronal preparations out of the total of 17 cases. One additional case (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160241#pone.0160241.g005" target="_blank">Fig 5G</a>) found in a horizontal section is not shown here.</p

Immunohistochemistry in combination with tracer label suggests that MNTB to MNTB collaterals form functional synapses.

<p>A: The compact circular structure labeled in neuron shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160241#pone.0160241.g001" target="_blank">Fig 1B</a> (box). B: Gephyrin staining localized to the cell membrane (arrows) and surrounding extra-nuclear area (arrowheads). C: The overlap of the two channels shown in A and B reveals gephyrin (cyan) juxtaposed to the biocytin labeled axon (magenta, open arrows). D: The presynaptic label SNAP-25 localized at the circumference of the cell soma (not labeled) and E: Gephyrin (arrows and arrowheads as above). F: The overlay of gephyrin (cyan), SNAP-25 (green) and the corresponding axonal ending (magenta) shows very close proximity of neurite with the presynaptic and postsynaptic density (open arrows). G: A third terminal, which appears to form rudimentary fenestrations. H: Gephyrin staining shows a circular shape that suggests a post synaptic cell body. I: The gephyrin label (cyan) seems to be concentrated around the labeled terminal (magenta) that partly encapsulates the putative postsynaptic soma. Scale bars: 10 μm for all panels.</p

Total number of neurons labeled with biocytin juxtacellular electroporation, and patch clamp with dye diffusion in gerbil MNTB brain slices of different ages.

<p>Total number of neurons labeled with biocytin juxtacellular electroporation, and patch clamp with dye diffusion in gerbil MNTB brain slices of different ages.</p

Examples of 3D reconstructions of two biocytin labeled neurons.

<p>The reconstructions are based on intensity thresholds and a contiguous pixel cluster algorithm (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160241#sec002" target="_blank">Methods</a>), and show that the neurites of the labeled neurons projected dorsally (A), or ventrally (B) but remained within the range of a 14 to 30 μm depth within the coronal plane of the slice (z—axis represents the rostro-caudal direction).</p

Tracing collaterals of MNTB principal cells requires sparse labeling of the neurons to avoid overlapping neurites.

<p>A: A neurite originating from the upper cell travels downward into close proximity of the lower neuron’s dendrites. Another neurite originating from the same (upper) cell travels upward and splits, to return and form a terminal in the dashed box. Maximal projection through a depth of 15.2 μm (19 virtual sections of 0.8 μm/slice = 15.2 μm). The dashed square is magnified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160241#pone.0160241.g005" target="_blank">Fig 5D</a>. B: Magnification of the area shown within the solid box in panel A, showing the neurite of the upper cell (arrows) spiraling near the area of the dendritic tree of the lower neuron (arrowhead). Average projection through a depth of 34.8 μm (58 virtual sections of 0.6 μm/slice = 34.8 μm). Scale bar in both panels: 20 μm.</p

Types of axonal endings formed by neurites in the MNTB.

<p>A: Structure with bouton-like swellings formed on the branches which form a circular shape (arrows). B: More classic axonal branches with bouton-like swellings that are thin and spread out (arrows). C: A neuron that formed a terminal (arrows) on its collateral in very close proximity to its originating soma. Scale bars: 10 μm for panels A, B, and 20 μm for panel C.</p

Selective ablation of cochlear hair cells promotes engraftment of human embryonic stem cell-derived progenitors in the mouse organ of Corti

Abstract Background Hearing loss affects 25% of the population at ages 60–69 years. Loss of the hair cells of the inner ear commonly underlies deafness and once lost this cell type cannot spontaneously regenerate in higher vertebrates. As a result, there is a need for the development of regenerative strategies to replace hair cells once lost. Stem cell-based therapies are one such strategy and offer promise for cell replacement in a variety of tissues. A number of investigators have previously demonstrated successful implantation, and certain level of regeneration of hair and supporting cells in both avian and mammalian models using rodent pluripotent stem cells. However, the ability of human stem cells to engraft and generate differentiated cell types in the inner ear is not well understood. Methods We differentiate human pluripotent stem cells to the pre-placodal stage in vitro then transplant them into the mouse cochlea after selective and complete lesioning of the endogenous population of hair cells. Results We demonstrate that hair cell ablation prior to transplantation leads to increased engraftment in the auditory sensory epithelium, the organ of Corti, as well as differentiation of transplanted cells into hair and supporting cell immunophenotypes. Conclusion We have demonstrated the feasibility of human stem cell engraftment into an ablated mouse organ of Corti. Graphical abstrac

Single cell electroporation reveals putative MNTB to MNTB collaterals.

<p>A: brainstem section containing the MNTB with several biocytin-labeled neurons labeled with the single cell electroporation approach. The dashed line delineates borders of the MNTB based on the autofluorescence of the nucleus. Six neurons are labeled, two of them (arrowheads) have distinct neurites that turn within the MNTB. Note that dimmer cells, probably passively labeled by a puff do not have labeled neurites. B: Another example of brainstem section with MNTB and three labeled neurons located further apart, with one neurite travelling across the MNTB between the upper two cells. The red arrow indicates a location where neurites appear to intermingle. The red box indicates an area that is shown magnified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160241#pone.0160241.g006" target="_blank">Fig 6A</a>. Scale bar 100 μm in both panels.</p