Expression of ANO1 in neurons of the cerebellar cortex.

<p><b>(A)</b> Schematic representation of the main cell types that constitute the microcircuits of the cerebellar cortex. Inhibitory interneurons <i>(red)</i>: basket cells <i>(BC)</i>, Golgi cells <i>(GC)</i> and stellate cells (SC); excitatory input to Purkinje cells <i>(PC) (green)</i>: climbing fibers <i>(CF)</i>, granule cells <i>(GrC)</i>, mossy fibers <i>(MF)</i> and parallel fibers <i>(PF)</i>. <b>(B)</b> Diaminobenzidine-labelled ANO1_in antiserum produced a continuous staining pattern in the Purkinje cell layer <i>(PCL)</i> as well as a scattered stain in the granule cell layer <i>(GL)</i> and the molecular layer <i>(ML)</i> of the mouse cerebellum. <b>(C)</b> Low-magnification image showing ANO1 immunofluorescence <i>(green)</i> and DAPI nuclear stain <i>(blue)</i>. Only few ANO1-positive cells are located among the many cells of the <i>GL</i>, most ANO-1 cells can be seen in the <i>PCL</i> and <i>ML</i>. <b>(D)</b> Detail of the <i>GL</i>, demonstrating co-localization of ANO1 and GAD^Cre, a marker for GABAergic neurons. <b>(E)</b> Co-localization of ANO1 with GAD^Cre cells in the <i>ML</i>. <b>(F)</b> Purkinje cell somata stained with the ANO1_ex antiserum and <i>NeuroTrace</i>^® to illustrate that all Purkinje cells are ANO1-positive. <b>(G)</b> Small, ANO1-negative granule-cell nuclei <i>(blue)</i> and somewhat larger ANO1-positive Golgi cells <i>(GoC)</i> in the <i>GL</i>. <b>(H)</b> In the molecular layer, ANO-1 positive cells are <i>SC</i> and <i>BC</i> inhibitory interneurons. <b>(I)</b> Preadsorption control for the ANO1_ex antiserum on cerebellar cortex. Scale bars: <i>B</i>, <i>C</i>: 200 μm, <i>D</i>,<i>E</i>: 50 μm, <i>F</i>: 20 μm, <i>G</i>: 50 μm, <i>H</i>: 20 μm, <i>I</i>: 50 μM. Blue represents DAPI nuclear stain.</p

PCR primer pairs used to characterize cerebellar ANO1 and ANO2.

<p>PCR primer pairs used to characterize cerebellar ANO1 and ANO2.</p

Expression of ANO2 protein in Purkinje cells.

<p><b>(A)</b> ANO2 immunosignals from the cerebellar cortex are discernible in the dendrites of Purkinje cells. The signals are weak but stronger than the background signals emanating from the granule cell layer <i>(GL)</i>. <b>(B)</b> In the Purkinje cell, the ANO2 immunosignal <i>(green)</i> is visible in dendrites and the perinuclear region, but only weakly in the plasma membrane of the cell body. In contrast, ANO1 signals <i>(red)</i> label the entire Purkinje cell soma, but are not detectable in dendrites. <b>(C)</b> The ANO2 antiserum does not stain the cerebellar cortex of the Ano2^-/- mouse. <b>(D)</b> ANO2 immunosignals in the glomeruli of the olfactory bulb serving as positive control for ANO2 in brain tissue; <i>gl</i>: glomerular layer, <i>opl</i>: outer plexiform layer. <b>(E)</b> Absence of ANO2 immunosignals from the olfactory bulb of the Ano2^-/- mouse. Blue in <i>B-E</i> represents DAPI nuclear stain. All calibrations bars: 20 μm.</p

Involvement of ANO2 in depolarization-induced depression of inhibition.

<p><b>(A)</b> A cerebellar Purkinje cell loaded with the fluorescent dye Alexa Fluor 568. Scale bar: 10 μm. <b>(B)</b> Spontaneous postsynaptic currents in a Purkinje cell with E_Cl near 0 mV and V_hold = -69 mV. Overlay of 764 current traces showing similar time courses but differing amplitudes, probably reflecting distinct positions of GABAergic synapses on the Purkinje cell dendritic tree. <b>(C)</b> Postsynaptic currents were completely blocked by 50 μM picrotoxin, an inhibitor of GABA_A-receptor chloride channels. <b>(D)</b> Protocol for activation of climbing fibers: Ten 0.1-ms current pulses were applied to the area near the proximal dendrite of a Purkinje cell while recording the whole-cell current of that cell at -70 mV. CF-activation produced characteristic complex spikes, as shown in the inset. <b>(E)</b><i>Upper traces</i>: GABAergic inhibitory postsynaptic currents recorded from a Purkinje cell at V_hold = -48 mV and with 5 mM Cl^- in the pipette solution. The positive polarity of IPSCs indicates Cl^- influx. <i>Lower traces</i>: postsynaptic currents, recorded immediately after the climbing-fiber stimulation, displayed decreased amplitudes. <b>(F)</b> IPSCs recorded from a Purkinje cell of an Ano2^-/—mouse before <i>(upper traces)</i> and immediately after <i>(lower traces)</i> CF-activation. <b>(G)</b> The number of detectable IPSC signals decreased by ~47% through climbing-fiber stimulation (before CF: 30.7 ± 6.5 min^-1; after CF: 14.6 ± 3.4 min^-1; 8 cells; <i>ctrl</i>). In slices from Ano2^-/- mice, more IPSCs were detected (54.5 ± 18.5 min^-1; 4 cells), and the activation of climbing fibers had no effect (52.5 ± 16.2 min^-1; 4 cells).</p

ANO1 and ANO2 expression levels in the cerebellum.

<p><b>(A)</b> Membrane topology model for anoctamin Ca²⁺-activated Cl^- channels based on the X-ray structure of a fungal TMEM16 protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref042" target="_blank">42</a>]. The transmembrane domains 5 and 6 are thought to provide the pore-lining region in the homodimeric channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref095" target="_blank">95</a>]. Five negatively charged amino-acid residues <i>(E</i>, <i>D)</i> and an asparagine residue <i>(N)</i> in transmembrane domains 6–8 serve as Ca²⁺-binding sites involved in channel gating [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref039" target="_blank">39</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref041" target="_blank">41</a>]. Four alternatively spliced segments <i>(a—d)</i> determine the apparent Ca²⁺-sensitivity of the ANO1 channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref005" target="_blank">5</a>]. ANO2 has two isoforms <i>A</i> and <i>B</i> and a regulatory motif at a position homologous to segment <i>c</i> in ANO1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref006" target="_blank">6</a>]. <b>(B)</b> RT-PCR analysis from mouse olfactory epithelium <i>(OE)</i> and mouse cerebellum <i>(CB)</i> yield similarly strong ANO1 signals in cerebellum but weaker signals for ANO2. <b>(C)</b> Immunoblots obtained from lysates of cerebellum <i>(CB)</i> and main olfactory epithelium <i>(OE)</i> from wild-type and Ano2^-/- mice show an ANO1-specific signal at ~120 kDa with the ANO1_in antiserum. <b>(D)</b> Rabbit anti-ANO2_ex serum stains ANO2-specific bands <i>(asterisks)</i> in immunoblots obtained from lysates of main olfactory epithelium <i>(OE)</i> and eye, as well as in membrane-protein preparations of main olfactory bulb <i>(OB)</i> and cerebellum <i>(CB)</i>. ANO2 bands are not present in immunoblots from Ano2^-/- mice.</p

Reversal of GABAergic postsynaptic currents by the ANO2 inhibitor.

<p><b>(A)</b> At a Cl^- concentration of 12 mM in the recording pipette, GABAergic postsynaptic currents were negative (Cl^- efflux), indicating that postsynaptic E_Cl is less negative than V_hold. <b>(B)</b> Shortly after applying 5 μM ANO2 inhibitor, positive currents appear <i>(circles)</i> as some synapses experience a decline of postsynaptic [Cl^-]_i, while others still have high Cl^-<i>(asterisks)</i>. <b>(C)</b> During the continued presence of the ANO2 inhibitor, virtually all postsynaptic currents reverse to positive polarity (Cl^- influx) indicating that GABAergic synapses experience an E_Cl more negative than V_hold. <b>(D)</b> The collected data from 12 Purkinje cells at [Cl^-]_i = 12 mM and V_hold = -60 mV demonstrate the polarity reversal of postsynaptic currents (PSCs) actuated by the ANO2 inhibitor. <b>(E)</b> Schematic representation of an hypothesis for the 12 mM [Cl^-]_i experiment. In the absence of the ANO2 inhibitor <i>(upper scheme)</i>, the basal activity of ANO2 channels <i>(green)</i> provides a Cl^- conductance in the dendritic membrane. ANO2 contributes to the Cl^—transport machinery, whose various pathways are represented by the K⁺/Cl^—cotransporter KCC2 <i>(blue)</i>. Together the Cl^- pathways stabilize a slightly elevated level of [Cl^-]_i which results in a negative driving force (V_m—E_Cl < 0) for Cl^- currents through GABA_A receptors in GABAergic synapses <i>(red)</i>. In this situation, Cl^- currents are outwardly directed and cause negative postsynaptic currents. Application of the ANO2 inhibitor <i>(lower scheme)</i> reduces the Cl^- conductance. This causes a polarity reversal of the Cl^- driving force, as the balance shifts towards Cl^- extrusion, causing local [Cl^-]_i to decrease. This hypothesis provides a qualitative concept for the role of ANO2 channels in the inversion of postsynaptic currents that is depicted in panels A to C. The proximity of Cl^—transport pathways and GABAergic synapses, as well as the occurrence of local Cl^-.gradients within dendritic segments, are inspired by the model for GABA_A-receptor-mediated Cl^- gradients in extended dendritic trees proposed by Jedlicka et al. (2011) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142160#pone.0142160.ref088" target="_blank">88</a>].</p

Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons-4

Mmunfluorescence of calcitonin gene-related peptide (CGRP) and substance P (SP) was evaluated at the indicated times after start of the treatment. The relative number of neuropeptide-expressing neurons was determined in test DRGs and in contralateral control DRGs, and the percent change of positive cells, Δ NP = (NP-NP)/NP·100 [%], is given for each time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**). (B) Cryosections of a control DRG and the treated contralateral test DRG stained with NKCC1 antibodies. The red circles illustrate the area from which the mean fluorescence intensity was obtained. Background subtraction removed most of the pericellular fluorescence as well as the fluorescence originating from the nucleus. All cell sections with intact perimeter were evaluated. (C) Distribution of fluorescence intensities recorded from 3 control DRGs and 3 contralateral test DRGs illustrate a shift to higher intensities after a 3-hr treatment with inflammatory mediators. Solid lines are Gaussian fits to the data.<p><b>Copyright information:</b></p><p>Taken from "Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons"</p><p>http://www.molecularpain.com/content/4/1/32</p><p>Molecular Pain 2008;4():32-32.</p><p>Published online 12 Aug 2008</p><p>PMCID:PMC2526990.</p><p></p

(A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with the chloride indicator dye MQAE

The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Clconcentration. Warm colours represent high intracellular Clconcentration (small τ values). (B) Quantitative evaluation of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after the start of treament with inflammatory mediators. Mean LT values were determined in control DRGs (LT) and in contralateral test DRGs (LT), and the percent increase of LT is depicted as ΔLT = (LT-LT)/LT·100 [%] for each treatment time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**).<p><b>Copyright information:</b></p><p>Taken from "Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons"</p><p>http://www.molecularpain.com/content/4/1/32</p><p>Molecular Pain 2008;4():32-32.</p><p>Published online 12 Aug 2008</p><p>PMCID:PMC2526990.</p><p></p

Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons-0

Mmunfluorescence of calcitonin gene-related peptide (CGRP) and substance P (SP) was evaluated at the indicated times after start of the treatment. The relative number of neuropeptide-expressing neurons was determined in test DRGs and in contralateral control DRGs, and the percent change of positive cells, Δ NP = (NP-NP)/NP·100 [%], is given for each time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**). (B) Cryosections of a control DRG and the treated contralateral test DRG stained with NKCC1 antibodies. The red circles illustrate the area from which the mean fluorescence intensity was obtained. Background subtraction removed most of the pericellular fluorescence as well as the fluorescence originating from the nucleus. All cell sections with intact perimeter were evaluated. (C) Distribution of fluorescence intensities recorded from 3 control DRGs and 3 contralateral test DRGs illustrate a shift to higher intensities after a 3-hr treatment with inflammatory mediators. Solid lines are Gaussian fits to the data.<p><b>Copyright information:</b></p><p>Taken from "Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons"</p><p>http://www.molecularpain.com/content/4/1/32</p><p>Molecular Pain 2008;4():32-32.</p><p>Published online 12 Aug 2008</p><p>PMCID:PMC2526990.</p><p></p

Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons-2

RG (FI) is related to that of its contralateral control DRG (FI). The change of fluorescence intensity ΔFI = (FI-FI)/FI·100 [%] is depicted 1 hr, 2 hr, and 3 hr of treatment, respectively. The data show that 3 hr after the start of the treatment the expression of the Na, K, 2Clcotransporter NKCC1 is enhanced while that of the K, Clcotransporter KCC2 is reduced. (B) A similar result was obtained in experiments where control DRGs and test DRGs were not obtained from the same vertebral segment, but were randomly combined. Indicated significance levels are p ≤ 0.01 (**) and p ≤ 0.001 (***). Numbers in parentheses indicate cells evaluated.<p><b>Copyright information:</b></p><p>Taken from "Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons"</p><p>http://www.molecularpain.com/content/4/1/32</p><p>Molecular Pain 2008;4():32-32.</p><p>Published online 12 Aug 2008</p><p>PMCID:PMC2526990.</p><p></p