Model for photoreceptor synaptic events in the lamina.

<p>A graded depolarization from the soma reaches the axon terminals (1) opening cacophony, which allows the Ca²⁺ influx (2) that triggers exocytosis. Additional mechanisms complement or amplify the Ca²⁺ signal: Ca²⁺ release from the endoplasmic reticulum (ER) (3), PLC activation (4) and Ca²⁺ influx through TRP/TRPL channels (5). Internal Ca²⁺ release could be due to ryanodine receptor (RyR) activation, a mechanism termed Ca²⁺-induced Ca²⁺ release, or mediated by IP₃ receptor (IP₃R) opening as a result of PLC activity. TRP/TRPL working as store-operated channels (SOCs) contribute Ca²⁺ to exocytosis and could also be modulated by PLC-dependent lipid changes. The massive raise of Ca²⁺ from these multiple pathways allows extremely fast exocytosis at the synaptic terminal. In green are displayed the components and pathways shown by us to be involved in vesicle exocytosis. The broken lines denote hypothetical pathways.</p

Intracellular Ca²⁺ stores and PLC contribute to exocytosis in the lamina.

<p>Z-projections of 10 confocal optical sections (Δz = 0.3 mm) showing representative confocal images from slices loaded with FM464. (<b>A</b>) <i>wt</i> slices pre-treated with Thg (10 µM; right) or its vehicle (DMSO; left), and depolarized with high-K⁺. (<b>B</b>) Quantification of the number of synaptic boutons observed for the conditions in A. (<b>C</b>) Bouton labeling induced by depolarization in slices from <i>dserca^TS</i> mutants pre-incubated in chilled Ringer (Control; left) or at 41°C (center) for 2 minutes during depolarization; an example of labeling at 41°C in slices from wt flies is also shown (right). (<b>D</b>) Quantification of the number of synaptic boutons for the conditions in C. (<b>E</b>) Slices from the PLC mutant <i>norpA</i> were depolarized with high-K⁺ (left) and treated with A-23187 in normal Ringer (5 mM K⁺; right). (<b>F</b>) Quantification of the number of synaptic boutons loaded by depolarization in <i>norpA</i> and <i>wt</i> slices. Control: basal loading in <i>norpA</i> in the absence of depolarization. Bars: mean ± SEM, calculated in z-projections of 10 images. Size: x/y/z = 36/36/0.3 µm³. * p<0.05: with respect to <i>wt</i> treated with high-K⁺.</p

Expression of transduction proteins in photoreceptors axons in the lamina.

<p>Z-projections of 10 confocal optical sections (Δz = 0.3 mm) of the lamina showing immunoreaction of antibodies against various proteins in <i>wt</i> brain slices. (<b>A</b>) Phospholipase C (PLC, left); CD8::GFP (center); Merge (right). (<b>B</b>) Protein kinase C (PKC, left); CD8::GFP (center); Merge (right). (<b>C</b>) G_q-Protein subunit (right); CD8::GFP (center); Merge (right). (<b>D</b>) InaD (left); CD8::GFP (center); Merge (right). (<b>E</b>) Cacophony (left); CD8::GFP (center); Merge (right). <i>(F)</i> Rhodopsin (Rh1; left); CD8::GFP (center); Merge (right). (<b>G</b>) Effective colocalization of TRP, TRPL, other transduction proteins and cacophony with photoreceptor axons (CD8::GFP) in the lamina. The segmented signals for the different proteins were randomized within the confined regions of photoreceptor axons as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044182#s4" target="_blank">Methods</a> and illustrated in Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044182#pone.0044182.s002" target="_blank">Figure S2B</a>. A maximum displacement radius of 20 pixels was considered for randomization. n>20 images for all the immunostainings studied.</p

Ca²⁺ signals induced in the lamina by the ‘Ca²⁺ depletion protocol’ are abolished in the <i>trpl;trp</i> mutant.

<p>Slices were bathed with 0-Ca²⁺ solution supplemented with 10 µM Thg (Thg(0Ca²⁺)) during 8 min to produce internal Ca²⁺ stores (ER) depletion. Afterwards the slices were returned to regular Ringer. (<b>A, B</b>) Z-projections of 4 confocal images were obtained from the lamina in slices preloaded with Rhod-2 and treated with the depletion protocol. Rhod-2 fluorescence in <i>wt</i> (A) or <i>trpl;trp</i> (B) slices in regular Ringer (left), during application of Thg(0Ca²⁺) solution (center) and after regular Ringer was restored (right). (<b>C–D</b>) Z-projections of 10 confocal optical sections (Δz = 0.3 mm) showing representative bouton loading of FM4-64 in <i>wt</i> (C) and <i>trpl;trp</i> (D) slices, upon returning to regular Ringer after ER depletion. (<b>E</b>) Quantification of Rhod-2 fluorescence change after restoring Ringer in <i>wt</i> (A) and <i>trpl;trp</i> (B). (<b>F</b>) Quantification of FM4-64 bouton labeling induced by the depletion protocol (C and D, denoted by “0Ca²⁺+Thg”) or upon returning to Ringer after treatment with 0-Ca²⁺ solution without Thg (“0Ca²⁺”). Bars: mean ± SEM calculated in z-projections of 10 images. Size: x/y/z = 36/36/0.3 µm³. * p<0.05 with respect to <i>wt.</i> Pseudocolor scale in arbitrary units.</p

TRP, TRPL and Cacophony Channels Mediate Ca²⁺ Influx and Exocytosis in Photoreceptors Axons in <em>Drosophila</em>

<div><p>In <em>Drosophila</em> photoreceptors Ca²⁺-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel <em>Drosophila</em> brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca²⁺ channel cacophony is also present there. Measurements in the lamina with the Ca²⁺ fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca²⁺ increments mediated by cacophony. Additional Ca²⁺ influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant <em>norpA</em> bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca²⁺ also contributes to exocytosis, since this process was reduced after Ca²⁺-store depletion. Therefore, several Ca²⁺ pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca²⁺ release and the activation of TRP and TRPL coupled to Ca²⁺ depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.</p> </div

Exocytosis in the lamina depends on TRP, TRPL and cacophony.

<p>(<b>A</b>) Z-projections of 10 confocal optical sections (Δz = 0.3 mm) showing the fluorescence of FM4-64-loaded boutons in the lamina from: (<b>a</b>) <i>wt</i> not exposed to high-K⁺ (control); (<b>b</b>) <i>wt</i> after depolarization by high-K⁺; (<b>c</b>) <i>wt</i> depolarized by high-K⁺ in the presence of PLTX-II (100 nM); (<b>d</b>) <i>wt</i> depolarized by high-K⁺ in 0-Ca²⁺ external solution; (<b>e</b>) <i>trp</i> depolarized by high-K⁺; (<b>f</b>) <i>trpl</i> depolarized by high-K⁺; (<b>g</b>) <i>trpl;trp</i> depolarized by high-K⁺. (<b>B</b>) Bouton labeling induced by the Ca²⁺ ionophore, A-23187 (250 nM) in <i>wt</i> (left) and <i>trpl;trp</i> (right) in regular Ringer (5 mM K⁺). (<b>C</b>) Quantification of the number of labeled synaptic boutons for the different conditions shown in A. (<b>D</b>) Bouton labeling induced by high-K⁺ in slices from <i>cac^TS</i> mutants pre-incubated in chilled Ringer (left, Control) or at 37°C (center) for 10 minutes. Right: bouton labeling at 37°C in slices from wt flies. <b>(E)</b> Quantification of the number of synaptic boutons for the conditions shown in D. Bars: mean ± SEM, calculated from z-projections of 10 images. Size: x/y/z = 36/36/0.3 µm³. * p<0.05: respect to <i>wt</i> high-K⁺ labeling.</p

<i>Drosophila</i> visual system and brain slices.

<p>(<b>A</b>) Schematic representation of a section of the fly’s visual system. Photoreceptors somata are arranged in ommatidia in groups of eight (R1–R8). R1–R6 project to the lamina forming a columnar assembly (cartridge) with the axons of the large monopolar neurons (L1–3). (<b>B</b>) Microphotograph of a slice preparation of the visual system. (<b>C</b>) Synaptic boutons fluorescently labeled with FM4-64 in the lamina of a <i>wt</i> fly (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044182#s4" target="_blank">Materials and Methods</a>). The inset shows a detail of the boutons shown in pseudocolor in C. (<b>D</b>) Confocal images displaying fluorescence previous (left) and 60 seconds after light (20 s, white light). (<b>E</b>) Plot of the normalized mean fluorescence measured in the boutons shown by the arrowheads in D (red circles) 60 seconds (t = 60s) after light exposure (t = 0). The control for FM-464 photobleaching (blue circles) was measured in the abdomen, representing a light insensitive region. Normalized mean fluorescence of boutons from slices not exposed to light is also included (black circles). n = 4. Pseudocolor scale in arbitrary units.</p

GCaMP-Ca²⁺ fluorescence from photoreceptor axons in the lamina.

<p>(<b>A–C</b>) Confocal images of the visual system from GMR-Gal4/UAS-G-CaMP transgenic flies showing the expression pattern of the Ca²⁺ indicator protein, G-CaMP, in <i>Drosophila</i> photoreceptors. (<b>D</b>) Pseudocolor fluorescence images illustrating Ca²⁺ increments upon depolarization induced by high-K⁺ (90 mM); effect of the cacophony blocker PLTX-II on G-CaMP/Ca²⁺ fluorescence changes evoked by high-K⁺. (<b>E</b>) Quantification of the G-CaMP/Ca²⁺ fluorescence changes illustrated in (D).</p

DataSheet1_Ontogenesis of the asymmetric parapineal organ in the zebrafish epithalamus.pdf

The parapineal organ is a midline-derived epithalamic structure that in zebrafish adopts a left-sided position at embryonic stages to promote the development of left-right asymmetries in the habenular nuclei. Despite extensive knowledge about its embryonic and larval development, it is still unknown whether the parapineal organ and its profuse larval connectivity with the left habenula are present in the adult brain or whether, as assumed from historical conceptions, this organ degenerates during ontogeny. This paper addresses this question by performing an ontogenetic analysis using an integrative morphological, ultrastructural and neurochemical approach. We find that the parapineal organ is lost as a morphological entity during ontogeny, while parapineal cells are incorporated into the posterior wall of the adult left dorsal habenular nucleus as small clusters or as single cells. Despite this integration, parapineal cells retain their structural, neurochemical and connective features, establishing a reciprocal synaptic connection with the more dorsal habenular neuropil. Furthermore, we describe the ultrastructure of parapineal cells using transmission electron microscopy and report immunoreactivity in parapineal cells with antibodies against substance P, tachykinin, serotonin and the photoreceptor markers arrestin3a and rod opsin. Our findings suggest that parapineal cells form an integral part of a neural circuit associated with the left habenula, possibly acting as local modulators of the circuit. We argue that the incorporation of parapineal cells into the habenula may be part of an evolutionarily relevant developmental mechanism underlying the presence/absence of the parapineal organ in teleosts, and perhaps in a broader sense in vertebrates.</p

Polarization of metastatic cells is dependent on caveolin-1.

<p>(<b>A</b>) Total protein extracts were prepared from both parental MDA-MB-231 and cells stably transduced with a control shRNA targeting luciferase (sh-ctrl) or a shRNA targeting caveolin-1 (shRNA-caveolin-1 #5, sh-cav1). Extracts were separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting with antibodies against caveolin-1 and β-actin. (<b>B</b>) Confluent monolayers of parental, shRNA-control (sh-ctrl) or shRNA-caveolin-1 (sh-cav1) treated MDA-MB-231 cells were wounded with a pipet tip and fixed at 0 (left panel) and 60 minutes (right panel) after monolayer injury. Samples were stained with anti-caveolin-1 (green) and anti-Golgin-97 (red) antibodies, whereas the nuclei were stained with DAPI (blue). The first layer of cells facing the wounded area is distinguished from the rest by a white line. Scale bar, 20 µm. (<b>C</b>) Caveolin-1 localization was analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#pone-0033085-g001" target="_blank">Figure 1C</a>, by measuring the ratio of fluorescence intensity “rear/front”. Data are the mean ± SEM, n = 3. (<b>D</b>) Polarization of both parental (-) and shRNA treated MDA-MB-231 cells with shRNA-luciferase (sh-ctrl) or shRNA-caveolin-1 (sh-cav1) was measured from (C) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>, by analyzing the outer layer of cells facing the wounded area (B). The percentage of polarized cells was evaluated at 0, 60 and 360 minutes after wounding. Data were averaged from three independent experiments (mean ± SEM). *Comparison with control shRNA P<0.05. (<b>E</b>) B16-F10 cells transfected with either pLacIOP (mock) or pLacIOP-caveolin-1 (cav1) were treated or not with 1mM IPTG for 24 hours, grown in monolayers and wounded with a pipet tip to allow migration for 1 hours. Samples were stained with anti-Gigantin-1 polyclonal antibody (blue), phalloidin-Alexa488® (green) and propidium iodide (red). The outer layer of cells facing the wounded area is outlined with a white line. (<b>F</b>) Cell polarization was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>, by analyzing the outer layer of cells facing the wounded area. The percentage of polarized cells was evaluated 0-360 minutes after wounding the monolayer. Data were averaged from three independent experiments (mean ± SEM). Statistically significant differences compared with pLacIOP-transfected B16-F10 (mock) cells are indicated (**, P<0.01 at time 360 min; *, P<0.05 at time 60 min).</p