Unidirectional photoreceptor-to-Müller glia coupling and unique K+ channel expression in Caiman retina.

Müller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Müller cells abundantly express Kir4.1 inwardly rectifying potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and function of potassium channels in Müller cells from the retina of crocodiles remain, hitherto, unknown.We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus), endowed with both diurnal and nocturnal vision, by (i) immunohistochemistry, (ii) whole-cell voltage-clamp, and (iii) fluorescent dye tracing to investigate K+ channel distribution and glia-to-neuron communications.Immunohistochemistry revealed that caiman Müller cells, similarly to other vertebrates, express vimentin, GFAP, S100β, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Müller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Müller cells. Electrophysiological properties of enzymatically dissociated Müller cells without photoreceptors and isolated Müller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Müller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Müller cells thus revealing a uni-directional dye coupling.Our data indicate that caiman Müller glial cells are unique among vertebrates studied so far by predominantly expressing TASK-1 rather than Kir4.1 K+ channels and by bi-directional ion and uni-directional dye coupling to photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type of K+ buffering

A woolly mammoth (Mammuthus primigenius) carcass from Maly Lyakhovsky Island (New Siberian Islands, Russian Federation)

A partial carcass of an adult woolly mammoth (Mammuthus primigenius) found in 2012 on Maly Lyakhovsky Island presents a new opportunity to retrieve associated anatomical, morphological, and life history data on this important component of Pleistocene biotas. In addition, we address hematological, histological, and microbiological issues that relate directly to quality of preservation. Recovered by staff from North-Eastern Federal University in Yakutsk, this individual is a relatively old female preserving soft tissue of the anteroventral portion of the head, most of both fore-quarters, and the ventral aspect of much of the rest of the body. Both tusks were recovered and subjected to computed tomographic analysis in which annual dentin increments were revealed as cycles of variation in X-ray attenuation. Measurements of annual increment areas (in longitudinal section) display a pulsed pattern of tusk growth showing cycles of growth rate variation over periods of 3-5 years. These intervals are interpreted as calving cycles reflecting regular shifts in calcium and phosphate demand for tusk growth vs. fetal ossification and lactation. Brown liquid associated with the frozen carcass turned out to include remains of hemolyzed blood, and blood samples examined microscopically included white blood cells with preserved nuclei. Muscle tissue from the trunk was unusually well preserved, even at the histological level. Intestinal contents and tissue samples were investigated microbiologically, and several strains of lacticacid bacteria (e.g., Enterococcus faecium, Enterococcus hirae) that are widely distributed as commensal organisms in the intestines of herbivores were isolated. (C) 2017 Elsevier Ltd and INQUA. All rights reserved

Unidirectional flow of sulforhodamine-B dye from cone to caiman Müller cell.

<p>(<b>A</b>) Upper panel: whole-cell patch clamp technique using micropipette (MP) filled with sulforhodamine-B penetrating the inner segment of a cone attached to a Müller cell. The dye filled the cone and the Müller cell: tracing of the photoreceptor cell body and inner segment (IS) and spreading throughout the whole Müller cell from the soma (MS) to the endfeet. Lower panel: combined (DIC and fluorescent) image showing that sulforhodamine-B is not permeable to neighboring photoreceptors, but filled only the cone which was patched. Insert shows an enlarged image of fine attachments of three cones to a single glial cell where two cones are not showing fluorescent dye. (<b>B</b>) Upper panel: whole-cell patch clamp of a Müller cell resulted in the dye-tracing of the cell body and endfeet, while no spreading of the dye occurred to the photoreceptors attached to this Müller cell. Lower panel: patch of the Müller cell soma only traced the Müller cell; the dye did not spread to the attached photoreceptors.</p

Expression of retinal glial and photoreceptor markers in the caiman retina.

<p>Immunostaining for glial fibrillary acidic protein (GFAP), glial specific calcium binding protein (S100β), glutamate-to-glutamine converting enzyme glutamine synthetase (GS), and DAPI (blue) which shows nuclei of retinal cells. (<b>A</b>) Confocal (left) and Nomarski (right) images showing the expression of GFAP, (<b>B</b>) S100β, and (<b>C</b>) glutamine synthetase. The markers delineated the overall structure of Müller cells: white arrowheads point to the inner limiting membranes (ILM) in the vitreal endfeet areas. White arrows point to stalks of Müller cells. Blue arrowheads show nuclei of pigment epithelium cells. Scale bar  = 20 µm. ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, ONL-outer nuclear layer, OPL-outer plexiform layer, IS-inner segments of photoreceptors, OS-outer segments of photoreceptors, RPL-retinal pigment epithelium layer.</p

Differential localization of potassium channels and glial markers in the caiman retina.

<p>(<b>A</b>) Inwardly rectifying potassium channels Kir4.1 (green) are localized in the area of the photoreceptors and in outer nuclear layer (red arrowhead), but not in Müller cell processes such as stalks or endfeet. This is different from most of vertebrates where Kir4.1 channels were found in Müller cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097155#pone.0097155-Bringmann1" target="_blank">[3]</a>. (<b>B</b>) Glial Müller cell specific marker vimentin, V (green); photoreceptor specific alpha-1 subunit of transducin, Gαt1 (red); nucleus-specific marker, DAPI (blue). Vimentin staining is observed in endfeet, somata, stalks and in distal processes (yellow arrowhead points somata, white arrow shows stalk). DAPI staining (blue) shows nuclei of retinal cells. Alpha-1 subunit of transducin (Gαt1, red) is a marker of the second messenger G-protein cascade and it is found mostly in photoreceptors. (<b>C</b>) ATP-dependent K⁺ channel, Kir6.1 (black), is localized in stalks (white arrow) and in endfeet, while (<b>D</b>) two pore domain acid sensitive K⁺ channels, TASK-1 (black) are found in whole Müller cell compartments: in endfeet (black arrowhead), stalks (white arrow), somata (yellow arrowhead) and in distal processes (white arrowhead). Scale bar  = 20 µm. ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, ONL-outer nuclear layer, IS- inner segments of photoreceptors.</p

Localization of connexin 43 (Cx43) in the caiman retina.

<p>(<b>A</b>) Immunolocalization of the Müller cell-specific protein glutamine synthetase (GS, red) and staining of the nuclei by Hoechst 33258 (blue). The micrograph shows the overlay of the fluorescence image and the transmitted light with visible retinal layers. (<b>B</b>) Immunostaining of Cx43 (green), yellow arrowheads point to Müller cell-like structures. (<b>C</b>)-(<b>E</b>) Overlay of Cx43 and GS staining clearly demonstrates co-localization in Müller cell processes (yellow arrowheads). Moreover, cone outer segments and cone pedicles are stained by the lectin peanut agglutinin (PNA, purple). Localization of Cx43 in cone pedicles is demonstrated at higher magnification in (<b>E</b>) (white arrowheads). ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, OPL-outer plexiform layer, ONL-outer nuclear layer, OLM-outer limiting membrane, IS-inner segments of photoreceptors, OS-outer segments of photoreceptors.</p

Cellular tandem between Müller cells and photoreceptors: involvement of 2P domain K⁺ channels.

<p>(<b>A</b>) Müller cell soma (MS) with attached cones approached by a patch pipette (MP). In <b>A</b>, <b>B</b>, and <b>C</b>, white arrowheads and white dotted lines show the area of contact between the inner segment (IS) of photoreceptors and MS. S points to the soma of cones. (<b>B</b>) Rods with outer segments (black circle), inner segments (black diamond) and somata (open black square) attached to MS. In <b>B</b> and <b>C</b>, thick white arrow shows MS. (<b>C</b>) Isolated Müller cell without photoreceptors. Curved arrows show endfeet. Thin white arrow shows stalk. (<b>D</b>) Average currents recorded from MS of isolated Müller cells (solid line, n = 5) and recorded from MS of isolated Müller cells with photoreceptors attached (dotted line, n = 5). The I/V-curves were obtained in response to a linear voltage ramp from −150 mV to +150 mV in control ECS K⁺ = 2.5 mM. Müller cells have linear outward currents near K⁺-equilibrium potential. (<b>E</b>) Effect of 2-P domain channel blocker bupivacaine (BUPI 1 mM) on isolated Müller cells with (dotted line) and without photoreceptors attached (solid line). After BUPI, residual currents are reduced ∼15 times. (<b>F</b>) Adding the Kir channel blocker barium (Ba²⁺, 100 µM) to BUPI, caused a near complete block of residual current (from −100 mV to +50 mV). (<b>G</b>) Membrane potentials of cells in 2.5 and 10 mM K⁺ ECS. [K⁺]_o = 10 mM induces depolarization. (<b>H</b>) Application of BUPI 1 mM and BUPI 1 mM with barium 100 µM were used to test for 2P-domain and Kir channels respectively. Single Müller cells (solid black column, n = 8) and cells with photoreceptors attached (grey column, n = 10) show different sensitivity to BUPI. Addition of Ba²⁺ further depressed membrane potentials in both cells, but with no significant difference. Error bars represent standard errors of the mean (SEM), where p<0.05 was considered significant (*).</p

Müller cells in retinal wholemounts.

<p>(<b>A</b>) Recordings from vitreal endfeet of Müller cells. A patch pipette (MP) at the vitreal surface penetrating a single endfoot while many round- shaped endfeet with different diameters are visible. The patch clamp technique was used for recording the electrical currents and voltages as well as for fluorescent dye injection. The insert shows the differential interference contrast (DIC) image of a retinal axial section with a patch pipette positioned at the endfoot membrane. (<b>B</b>) Representative currents from caiman Müller cell endfeet in response to a voltage ramp of 80 ms duration from −150 mV to +150 mV (I/V-curves). The curves show the I/V relationship in different extracellular [K⁺], 2.5 mM and 10 mM, and the washout in 2.5 mM demonstrating classical glial sensitivity to potassium. (<b>C</b>) and (<b>D</b>) Two representative examples for Lucifer Yellow diffusion from injected endfoot (white arrowhead) to neighbor endfoot (white arrow) under control conditions. (<b>E</b>) Lucifer Yellow injection into a single endfoot in a retinal wholemount perfused with the gap junction blocker carbenoxolone (200 µM). The dye is not propagating to the neighboring cells.</p