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• W2804039430 abstract "Ca2+-activated Cl− currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca2+-activated Cl− channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl− efflux. Here, we confirmed the co-localization of the Ca2+-activated Cl− channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca2+-activated Cl− currents. Although these currents persisted in the VNOs of mice lacking Ano2, they were undetectable in olfactory neuron-specific Ano1 knockout mice irrespective of the presence of Ano2. The loss of Ca2+-activated Cl− currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male–male territorial aggression in olfactory Ano1/Ano2 double knockout mice. We conclude that Ano1 mediates the bulk of Ca2+-activated Cl− currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice. Ca2+-activated Cl− currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca2+-activated Cl− channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl− efflux. Here, we confirmed the co-localization of the Ca2+-activated Cl− channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca2+-activated Cl− currents. Although these currents persisted in the VNOs of mice lacking Ano2, they were undetectable in olfactory neuron-specific Ano1 knockout mice irrespective of the presence of Ano2. The loss of Ca2+-activated Cl− currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male–male territorial aggression in olfactory Ano1/Ano2 double knockout mice. We conclude that Ano1 mediates the bulk of Ca2+-activated Cl− currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice. In some animals, the vomeronasal organ (VNO) 3The abbreviations used are: VNOvomeronasal organCaCCCa2+-activated Cl− channelVSNvomeronasal sensory neuronVNEvomeronasal sensory epitheliumTRPCtransient receptor potential canonicalMOEmain olfactory epitheliumOMPolfactory marker proteinPSTHpoststimulus time histogramBESN,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid. plays an important role in social behaviors such as mating, flight, or aggression. Its sensory cells, the vomeronasal sensory neurons (VSNs), detect pheromones that are released by individuals of the same species or kairomones released by predators and that prompt animals to either attract or avoid each other (1Munger S.D. Leinders-Zufall T. Zufall F. Subsystem organization of the mammalian sense of smell.Annu. Rev. Physiol. 2009; 71 (18808328): 115-14010.1146/annurev.physiol.70.113006.100608Crossref PubMed Scopus (231) Google Scholar, 2Chamero P. Leinders-Zufall T. Zufall F. From genes to social communication: molecular sensing by the vomeronasal organ.Trends Neurosci. 2012; 35 (22658923): 597-60610.1016/j.tins.2012.04.011Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Extensive research on the murine VNO has shown that it is crucial for various innate social actions. For example, male–male aggression in mice is almost completely abolished when the VNO is ablated (3Bean N.J. Modulation of agonistic behavior by the dual olfactory system in male mice.Physiol. Behav. 1982; 29 (6891074): 433-43710.1016/0031-9384(82)90262-1Crossref PubMed Scopus (57) Google Scholar, 4Clancy A.N. Coquelin A. Macrides F. Gorski R.A. Noble E.P. Sexual behavior and aggression in male mice: involvement of the vomeronasal system.J. Neurosci. 1984; 4 (6541245): 2222-222910.1523/JNEUROSCI.04-09-02222.1984Crossref PubMed Google Scholar, 5Maruniak J.A. Wysocki C.J. Taylor J.A. Mediation of male mouse urine marking and aggression by the vomeronasal organ.Physiol. Behav. 1986; 37 (3749330): 655-65710.1016/0031-9384(86)90300-8Crossref PubMed Scopus (57) Google Scholar) or when its key signaling components are genetically disrupted (6Leypold B.G. Yu C.R. Leinders-Zufall T. Kim M.M. Zufall F. Axel R. Altered sexual and social behaviors in trp2 mutant mice.Proc. Natl. Acad. Sci. U.S.A. 2002; 99 (11972034): 6376-638110.1073/pnas.082127599Crossref PubMed Scopus (451) Google Scholar, 7Stowers L. Holy T.E. Meister M. Dulac C. Koentges G. Loss of sex discrimination and male–male aggression in mice deficient for TRP2.Science. 2002; 295 (11823606): 1493-150010.1126/science.1069259Crossref PubMed Scopus (662) Google Scholar, 8Chamero P. Katsoulidou V. Hendrix P. Bufe B. Roberts R. Matsunami H. Abramowitz J. Birnbaumer L. Zufall F. Leinders-Zufall T. G protein Gαo is essential for vomeronasal function and aggressive behavior in mice.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21768373): 12898-1290310.1073/pnas.1107770108Crossref PubMed Scopus (125) Google Scholar, 9Norlin E.M. Gussing F. Berghard A. Vomeronasal phenotype and behavioral alterations in Gαi2 mutant mice.Curr. Biol. 2003; 13 (12867032): 1214-121910.1016/S0960-9822(03)00452-4Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Moreover, the Bruce effect, an innate pregnancy block when pregnant females encounter a male different from the one they mated with, is diminished in mice lacking the VNO (10Bruce H.M. An exteroceptive block to pregnancy in the mouse.Nature. 1959; 184 (105, 13805128): 10510.1038/184105a0Crossref PubMed Scopus (487) Google Scholar, 11Bellringer J.F. Pratt H.P. Keverne E.B. Involvement of the vomeronasal organ and prolactin in pheromonal induction of delayed implantation in mice.J. Reprod. Fertil. 1980; 59 (7401039): 223-22810.1530/jrf.0.0590223Crossref PubMed Scopus (134) Google Scholar), yet some pheromone-induced social behaviors do not require a functional VNO (12Tirindelli R. Dibattista M. Pifferi S. Menini A. From pheromones to behavior.Physiol. Rev. 2009; 89 (19584317): 921-95610.1152/physrev.00037.2008Crossref PubMed Scopus (265) Google Scholar), possibly because also other olfactory subsystems may be able to detect pheromones (1Munger S.D. Leinders-Zufall T. Zufall F. Subsystem organization of the mammalian sense of smell.Annu. Rev. Physiol. 2009; 71 (18808328): 115-14010.1146/annurev.physiol.70.113006.100608Crossref PubMed Scopus (231) Google Scholar). vomeronasal organ Ca2+-activated Cl− channel vomeronasal sensory neuron vomeronasal sensory epithelium transient receptor potential canonical main olfactory epithelium olfactory marker protein poststimulus time histogram N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid. VSNs are bipolar neurons that possess one unbranched dendrite that protrudes apically into the vomeronasal lumen. At their apical ends, they feature a dendritic knob from which mucus-embedded microvilli emerge. Microvilli and dendritic knobs are the sites where the VNO receptor potential is generated. Whereas dendritic knobs of all VSNs are located at the apical surface of the vomeronasal sensory epithelium (VNE), their cell bodies are organized in an apical and a basal layer. Single VSNs express receptors from two large G protein–coupled receptor families. Cell bodies of vomeronasal receptor type I–expressing VSNs reside within the apical layer and express the G protein subunit Gαi2, whereas vomeronasal receptors type II–positive VSNs are found in the basal layer of the sensory epithelium and express the G protein subunit Gαo (1Munger S.D. Leinders-Zufall T. Zufall F. Subsystem organization of the mammalian sense of smell.Annu. Rev. Physiol. 2009; 71 (18808328): 115-14010.1146/annurev.physiol.70.113006.100608Crossref PubMed Scopus (231) Google Scholar). Pheromones bind to microvilli of VSNs and thereby trigger the dissociation of the respective G protein complex. Their β- and γ-subunits then activate phospholipase C, which converts phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-triphosphate. Diacylglycerol opens Na+- and Ca2+-permeable transient receptor potential canonical 2 (TRPC2) channels and thereby depolarize VSNs (13Lucas P. Ukhanov K. Leinders-Zufall T. Zufall F. A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: mechanism of pheromone transduction.Neuron. 2003; 40 (14642279): 551-56110.1016/S0896-6273(03)00675-5Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 14Leinders-Zufall T. Storch U. Bleymehl K. Mederos Y. Schnitzler M. Frank J.A. Konrad D.B. Trauner D. Gudermann T. Zufall F. PhoDAGs enable optical control of diacylglycerol-sensitive transient receptor potential channels.Cell Chem. Biol. 2018; 25 (29276045): 215-22310.1016/j.chembiol.2017.11.008Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Several lines of evidence using Cl−-sensitive dyes and perforated patch clamp suggest a high Cl− concentration inside VSN dendrites (15Untiet V. Moeller L.M. Ibarra-Soria X. Sánchez-Andrade G. Stricker M. Neuhaus E.M. Logan D.W. Gensch T. Spehr M. Elevated cytosolic Cl− concentrations in dendritic knobs of mouse vomeronasal sensory neurons.Chem. Senses. 2016; 41 (27377750): 669-67610.1093/chemse/bjw077Crossref PubMed Scopus (8) Google Scholar, 16Kim S. Ma L. Unruh J. McKinney S. Yu C.R. Intracellular chloride concentration of the mouse vomeronasal neuron.BMC Neurosci. 2015; 16 (90, 26667019): 9010.1186/s12868-015-0230-yCrossref PubMed Scopus (11) Google Scholar, 17Yang C. Delay R.J. Calcium-activated chloride current amplifies the response to urine in mouse vomeronasal sensory neurons.J. Gen. Physiol. 2010; 135 (20038523): 3-1310.1085/jgp.200910265Crossref PubMed Scopus (43) Google Scholar). Ca2+ entering through TRPC2 opens Ca2+-activated Cl− channels (CaCCs) (18Zufall F. Ukhanov K. Lucas P. Liman E.R. Leinders-Zufall T. Neurobiology of TRPC2: from gene to behavior.Pflügers Arch. 2005; 451 (15971083): 61-71Crossref PubMed Scopus (71) Google Scholar), resulting in an efflux of chloride that further depolarizes the cell and may amplify the initial TRPC2–mediated VSN response (17Yang C. Delay R.J. Calcium-activated chloride current amplifies the response to urine in mouse vomeronasal sensory neurons.J. Gen. Physiol. 2010; 135 (20038523): 3-1310.1085/jgp.200910265Crossref PubMed Scopus (43) Google Scholar, 19Kim S. Ma L. Yu C.R. Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons.Nat. Commun. 2011; 2 (21694713): 36510.1038/ncomms1368Crossref PubMed Scopus (46) Google Scholar). The incomplete disruption of VNO function observed in TRPC2 knockout mice suggests an alternative activation pathway that bypasses TRPC2. It was postulated that it involves inositol 1,4,5-triphosphate–mediated Ca2+ release from intracellular stores and downstream activation of CaCCs, but this explanation was questioned recently (20Chamero P. Weiss J. Alonso M.T. Rodríguez-Prados M. Hisatsune C. Mikoshiba K. Leinders-Zufall T. Zufall F. Type 3 inositol 1,4,5-trisphosphate receptor is dispensable for sensory activation of the mammalian vomeronasal organ.Sci. Rep. 2017; 7 (28860523)1026010.1038/s41598-017-09638-8Crossref PubMed Scopus (10) Google Scholar). We and others have shown previously that the CaCCs anoctamin 1 and 2 (Ano1 and Ano2, or TMEM16A and TMEM16B) are co-expressed in the apical border of the vomeronasal sensory epithelium (21Dibattista M. Amjad A. Maurya D.K. Sagheddu C. Montani G. Tirindelli R. Menini A. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.J. Gen. Physiol. 2012; 140 (22732308): 3-1510.1085/jgp.201210780Crossref PubMed Scopus (40) Google Scholar, 22Billig G.M. Pál B. Fidzinski P. Jentsch T.J. Ca2+-activated Cl− currents are dispensable for olfaction.Nat. Neurosci. 2011; 14 (21516098): 763-76910.1038/nn.2821Crossref PubMed Scopus (157) Google Scholar). Although a constitutive knockout of Ano1 is postnatally lethal in mice, likely because of a malformation of the trachea (23Rock J.R. Futtner C.R. Harfe B.D. The transmembrane protein TMEM16A is required for normal development of the murine trachea.Dev. Biol. 2008; 321 (18585372): 141-14910.1016/j.ydbio.2008.06.009Crossref PubMed Scopus (177) Google Scholar), Ano2−/− mice did not show a severe phenotype (22Billig G.M. Pál B. Fidzinski P. Jentsch T.J. Ca2+-activated Cl− currents are dispensable for olfaction.Nat. Neurosci. 2011; 14 (21516098): 763-76910.1038/nn.2821Crossref PubMed Scopus (157) Google Scholar). Here we confirm the co-expression and co-localization of Ano1 and Ano2 in the microvilli of murine VSNs. Surprisingly, Ano1 carries the bulk of Ca2+-activated Cl− currents (ICl(Ca)) in VSNs, whereas Ano2 apparently plays only a minor role. Disruption of both channels in mouse VSNs resulted in diminished spontaneous and pheromone-evoked action potential firing. The loss of CaCCs, however, did not result in altered pheromone-induced territorial aggressiveness in male mice. Immunolabeling of paraffin sections of the mouse nose (Fig. 1A) showed that Ano1 and Ano2 co-localize close to the dendritic knob of VSNs and line the apical side of the sensory epithelium (Fig. 1B), in agreement with previous results (21Dibattista M. Amjad A. Maurya D.K. Sagheddu C. Montani G. Tirindelli R. Menini A. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.J. Gen. Physiol. 2012; 140 (22732308): 3-1510.1085/jgp.201210780Crossref PubMed Scopus (40) Google Scholar, 22Billig G.M. Pál B. Fidzinski P. Jentsch T.J. Ca2+-activated Cl− currents are dispensable for olfaction.Nat. Neurosci. 2011; 14 (21516098): 763-76910.1038/nn.2821Crossref PubMed Scopus (157) Google Scholar). Immunostaining of isolated VSNs consistently showed co-expression of both proteins. No cells expressing only one of these channels were detected (Fig. 1C). Co-staining for ezrin, a protein expressed on microvilli of VNO-supporting cells (24Dauner K. Lissmann J. Jeridi S. Frings S. Möhrlen F. Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose.Cell Tissue Res. 2012; 347 (22314846): 327-34110.1007/s00441-012-1324-9Crossref PubMed Scopus (38) Google Scholar), showed a dotted intermittent expression pattern at the apical border of the sensory epithelium that neither co-localized with Ano1 nor with Ano2 (Fig. S1, A and B). Conversely, villin, which is also present in microvilli of VSNs, co-localized with Ano1 (not shown) and Ano2 (Fig. S1C). Hence, the expression of either channel is restricted to microvilli of VSNs. Because in VSNs the subcellular localization of either channel is almost completely restricted to microvilli, immunohistochemistry is ill suited to examine whether these channels are differentially expressed in apical or basal VSNs. We therefore addressed this issue by in situ hybridization that detects Ano1 and Ano2 transcripts in the cell bodies of VSNs. Hybridization of coronal VNO cryosections showed an even distribution of Ano1 and Ano2 mRNAs across both layers of the VNO sensory epithelium (Fig. 1D). Ano1-positive cells were also detected in the nonsensory epithelium, which agrees with the localization of Ano1-expressing mucus-secreting glandular cells and vascular smooth muscle cells in the VNO (1Munger S.D. Leinders-Zufall T. Zufall F. Subsystem organization of the mammalian sense of smell.Annu. Rev. Physiol. 2009; 71 (18808328): 115-14010.1146/annurev.physiol.70.113006.100608Crossref PubMed Scopus (231) Google Scholar). Negative controls using sense RNA probes for Ano1 or Ano2 gave no staining (Fig. 1D). Both Ano1 and Ano2 display various splice variants that differ in their biophysical properties (25Ponissery Saidu S. Stephan A.B. Talaga A.K. Zhao H. Reisert J. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel anoctamin 2.J. Gen. Physiol. 2013; 141 (23669718): 691-70310.1085/jgp.201210937Crossref PubMed Scopus (31) Google Scholar, 26Stöhr H. Heisig J.B. Benz P.M. Schöberl S. Milenkovic V.M. Strauss O. Aartsen W.M. Wijnholds J. Weber B.H. Schulz H.L. TMEM16B, a novel protein with calcium-dependent chloride channel activity, associates with a presynaptic protein complex in photoreceptor terminals.J. Neurosci. 2009; 29 (19474308): 6809-681810.1523/JNEUROSCI.5546-08.2009Crossref PubMed Scopus (171) Google Scholar, 27Ferrera L. Caputo A. Ubby I. Bussani E. Zegarra-Moran O. Ravazzolo R. Pagani F. Galietta L.J. Regulation of TMEM16A chloride channel properties by alternative splicing.J. Biol. Chem. 2009; 284 (19819874): 33360-3336810.1074/jbc.M109.046607Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 28Hwang S.J. Blair P.J. Britton F.C. O'Driscoll K.E. Hennig G. Bayguinov Y.R. Rock J.R. Harfe B.D. Sanders K.M. Ward S.M. Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles.J. Physiol. 2009; 587 (19687122): 4887-490410.1113/jphysiol.2009.176198Crossref PubMed Scopus (310) Google Scholar, 29Davis A.J. Forrest A.S. Jepps T.A. Valencik M.L. Wiwchar M. Singer C.A. Sones W.R. Greenwood I.A. Leblanc N. Expression profile and protein translation of TMEM16A in murine smooth muscle.Am. J. Physiol. Cell Physiol. 2010; 299 (20686072): C948-C95910.1152/ajpcell.00018.2010Crossref PubMed Scopus (103) Google Scholar, 30Ferrera L. Scudieri P. Sondo E. Caputo A. Caci E. Zegarra-Moran O. Ravazzolo R. Galietta L.J. A minimal isoform of the TMEM16A protein associated with chloride channel activity.Biochim. Biophys. Acta. 2011; 1808 (21645494): 2214-222310.1016/j.bbamem.2011.05.017Crossref PubMed Scopus (33) Google Scholar, 31Ubby I. Bussani E. Colonna A. Stacul G. Locatelli M. Scudieri P. Galietta L. Pagani F. TMEM16A alternative splicing coordination in breast cancer.Mol. Cancer. 2013; 12 (75, 23866066): 7510.1186/1476-4598-12-75Crossref PubMed Scopus (33) Google Scholar, 32Caputo A. Caci E. Ferrera L. Pedemonte N. Barsanti C. Sondo E. Pfeffer U. Ravazzolo R. Zegarra-Moran O. Galietta L.J. TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity.Science. 2008; 322 (18772398): 590-59410.1126/science.1163518Crossref PubMed Scopus (996) Google Scholar, 33Tian Y. Schreiber R. Kunzelmann K. Anoctamins are a family of Ca2+-activated Cl− channels.J. Cell Sci. 2012; 125 (22946059): 4991-499810.1242/jcs.109553Crossref PubMed Scopus (138) Google Scholar, 34Sondo E. Scudieri P. Tomati V. Caci E. Mazzone A. Farrugia G. Ravazzolo R. Galietta L.J. Non-canonical translation start sites in the TMEM16A chloride channel.Biochim. Biophys. Acta. 2014; 1838 (23994600): 89-9710.1016/j.bbamem.2013.08.010Crossref PubMed Scopus (24) Google Scholar, 35Xiao Q. Yu K. Perez-Cornejo P. Cui Y. Arreola J. Hartzell H.C. Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21555582): 8891-889610.1073/pnas.1102147108Crossref PubMed Scopus (166) Google Scholar, 36Stephan A.B. Shum E.Y. Hirsh S. Cygnar K.D. Reisert J. Zhao H. ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19561302): 11776-1178110.1073/pnas.0903304106Crossref PubMed Scopus (258) Google Scholar). To determine which splice variants are present in the VNO, we performed RT-PCR on VNO tissue using primers that span exon boundaries and therefore hybridize only to spliced-together exons (see exon labels in Fig. 2). This analysis revealed that vomeronasal transcripts of Ano1 included exon 14 (Fig. 2A). Murine Ano2 mRNA was previously analyzed in tissue from the eye and the main olfactory epithelium (MOE) (26Stöhr H. Heisig J.B. Benz P.M. Schöberl S. Milenkovic V.M. Strauss O. Aartsen W.M. Wijnholds J. Weber B.H. Schulz H.L. TMEM16B, a novel protein with calcium-dependent chloride channel activity, associates with a presynaptic protein complex in photoreceptor terminals.J. Neurosci. 2009; 29 (19474308): 6809-681810.1523/JNEUROSCI.5546-08.2009Crossref PubMed Scopus (171) Google Scholar, 36Stephan A.B. Shum E.Y. Hirsh S. Cygnar K.D. Reisert J. Zhao H. ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19561302): 11776-1178110.1073/pnas.0903304106Crossref PubMed Scopus (258) Google Scholar). Although an Ano2 isoform including exon 1a and exon 2 was found in the retina (later termed isoform A), in the MOE Ano2 is predominantly transcribed using an alternative start site at exon 1b while skipping exon 1a-2 (termed isoform B). A nonfunctional variant of isoform B that lacks exon 4 (termed isoform BΔ4; Fig. 2B) is also expressed in the MOE (25Ponissery Saidu S. Stephan A.B. Talaga A.K. Zhao H. Reisert J. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel anoctamin 2.J. Gen. Physiol. 2013; 141 (23669718): 691-70310.1085/jgp.201210937Crossref PubMed Scopus (31) Google Scholar). While our RT-PCR experiments revealed no expression of the retinal Ano2 isoform A in the VNO, Ano2 isoform B was present with and without exon 4. No Ano2 isoform containing exon 14 could be detected (Fig. 2B). Control experiments showed the expression of Ano2 isoform A in the eye and a lack of Ano2 in the liver (Fig. S2A). Sequencing of cDNAs from the VNO confirmed the existence of an exon 4-containing Ano2 mRNA. Additionally, a sequence overlap in the chromatogram revealed the co-expression of a variant lacking this alternatively spliced exon (Fig. S2B). To investigate the functional roles of CaCCs in the VNO, we generated mouse lines in which Ano1, Ano2, or both are absent from VSNs. Because constitutive deletion of Ano1 in mice is postnatally lethal, likely because of malformation of the trachea (23Rock J.R. Futtner C.R. Harfe B.D. The transmembrane protein TMEM16A is required for normal development of the murine trachea.Dev. Biol. 2008; 321 (18585372): 141-14910.1016/j.ydbio.2008.06.009Crossref PubMed Scopus (177) Google Scholar), we disrupted Ano1 specifically in mature olfactory neurons of the various olfactory subsystems. Floxed Ano1 mice (38Heinze C. Seniuk A. Sokolov M.V. Huebner A.K. Klementowicz A.E. Szijartó I.A. Schleifenbaum J. Vitzthum H. Gollasch M. Ehmke H. Schroeder B.C. Hübner C.A. Disruption of vascular Ca2+-activated chloride currents lowers blood pressure.J. Clin. Invest. 2014; 124 (24401273): 675-68610.1172/JCI70025Crossref PubMed Scopus (104) Google Scholar) were crossed to mice expressing Cre recombinase under the promotor of the olfactory marker protein (OMP) (39Li J. Ishii T. Feinstein P. Mombaerts P. Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons.Nature. 2004; 428 (15042081): 393-39910.1038/nature02433Crossref PubMed Scopus (214) Google Scholar) to yield olfactory-specific Ano1 knockout mice (subsequently called ΔAno1olf). As expected, Ano1 immunoreactivity was absent from the apical part of the sensory epithelium in the VNO of ΔAno1olf mice, whereas Ano2 expression was not affected (Fig. 3A). Western blots of N-deglycosylated VNO lysates confirmed that Ano2 expression levels were unchanged in the VNO of ΔAno1olf (Fig. 3B). Ano1/2 olfactory double knockout mice (Δ(Ano1olf/Ano2)) were obtained by crossing ΔAno1olf mice to constitutive Ano2−/− (ΔAno2) mice. We have described ΔAno2 mice previously (22Billig G.M. Pál B. Fidzinski P. Jentsch T.J. Ca2+-activated Cl− currents are dispensable for olfaction.Nat. Neurosci. 2011; 14 (21516098): 763-76910.1038/nn.2821Crossref PubMed Scopus (157) Google Scholar). VNO tissue lysates from WT, ΔAno1olf, ΔAno2, or Δ(Ano1olf/Ano2) animals were analyzed by Western blotting. A sharp band corresponding to a nonglycosylated shortened Ano1 protein (lacking the ∼6 kDa large part encoded by the floxed exon (38Heinze C. Seniuk A. Sokolov M.V. Huebner A.K. Klementowicz A.E. Szijartó I.A. Schleifenbaum J. Vitzthum H. Gollasch M. Ehmke H. Schroeder B.C. Hübner C.A. Disruption of vascular Ca2+-activated chloride currents lowers blood pressure.J. Clin. Invest. 2014; 124 (24401273): 675-68610.1172/JCI70025Crossref PubMed Scopus (104) Google Scholar)) appeared in VNO lysates from ΔAno1olf and Δ(Ano1olf/Ano2) mice, but not in those from WT and ΔAno2 animals (Fig. 3C). The remaining upper Ano1 band in ΔAno1olf lysates likely stems from nonneuronal glandular or vascular smooth muscle cells which do not express OMP. A broad band corresponding to glycosylated Ano2 was detected in lysates from WT and ΔAno1olf animals but was absent in ΔAno2 and Δ(Ano1olf/Ano2) lysates (Fig. 3C). Absence of the respective proteins was also seen when staining coronal paraffin sections of the nose from WT and Δ(Ano1olf/Ano2) mice. Whereas Ano1 and Ano2 immunoreactivity was detected in WT mice and co-localized with villin at the microvillar endings of VSNs, it was absent in Δ(Ano1olf/Ano2) animals (Fig. 3D). The antibody for Ano1 showed some unspecific staining in VSN cell bodies that persisted in the ΔAno1olf. Expression and localization of villin was not changed in Δ(Ano1olf/Ano2) VNOs, indicating that microvilli of VSN dendritic knobs are intact. Moreover, gross morphological differences concerning the size, shape, and integrity of the VNO layers, as well as changed expression of VNO marker proteins such as ezrin, TRPC2, Gαo, and phosphodiesterase type 4A (PDE4A) could not be observed in Δ(Ano1olf/Ano2) animals (Fig. 3D and Fig. S3C). Ca2+-activated Cl− currents of VSNs were examined in acute slices of the VNO by patching somata in the whole-cell voltage clamp configuration. Recordings with 1.5 μm free [Ca2+] in the pipette revealed robust outwardly rectifying steady-state ICl(Ca) (with 94.8 ± 15.9 pA/pF at +120 mV (mean ± S.E.)) that exhibited slow voltage-dependent activation at positive potentials in most cells (Fig. 4, A and B). As expected, these currents were absent in Δ(Ano1olf/Ano2) VSNs (Fig. 4A), even when pipette [Ca2+] was increased to 13 μm (Fig. 4B). Surprisingly, also VSNs deficient in only Ano1 lacked ICl(Ca) (Fig. 4, A and C), whereas ΔAno2 VSNs showed currents with amplitude, rectification and kinetics comparable with those of WT cells (Fig. 4, A and D, and Fig. S4). Similar results were found when analyzing ICl(Ca) tail currents (Fig. S4). These results indicate that Ano1 carries the bulk of ICl(Ca) in sensory neurons of the VNO. To assess the hypothesis that CaCCs amplify the initial TRPC2-mediated inward current (2Chamero P. Leinders-Zufall T. Zufall F. From genes to social communication: molecular sensing by the vomeronasal organ.Trends Neurosci. 2012; 35 (22658923): 597-60610.1016/j.tins.2012.04.011Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Yang C. Delay R.J. Calcium-activated chloride current amplifies the response to urine in mouse vomeronasal sensory neurons.J. Gen. Physiol. 2010; 135 (20038523): 3-1310.1085/jgp.200910265Crossref PubMed Scopus (43) Google Scholar, 19Kim S. Ma L. Yu C.R. Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons.Nat. Commun. 2011; 2 (21694713): 36510.1038/ncomms1368Crossref PubMed Scopus (46) Google Scholar), we compared spontaneous and stimulus-evoked spiking in VSNs of WT and Δ(Ano1olf/Ano2) animals in the loose-patch configuration. In WT mice 35 of 123 cells (28.5%, 13 mice) showed spontaneous spiking within the baseline observation period of 35 s. However, only 15 of 106 VSNs lacking Ano1 and Ano2 (14.2%, 10 mice) showed spontaneous action potential firing (Fig. 5A, left panel). Next, we examined stimulus-evoked spiking by applying diluted urine of male mice (pooled from 7 male mice, 1:200 in bath solution) to the microvilli of some of the measured VSNs. In WT animals, 20 of 82 VSNs (24.4%) showed an increase in the action potential firing rate upon urine application. In contrast, only 7 of 90 Δ(Ano1olf/Ano2) VSNs (7.8%) responded to the stimulus (Fig. 5A, right panel). Representative original spike registrations of stimulated WT and Δ(Ano1olf/Ano2) VSNs that displayed at least one action potential are presented in a raster plot over time in Fig. 5B (upper panels). Poststimulus time histograms (PSTHs) of all stimulated cells show a mild but clear increase in spiking upon urine stimulation in WT, but not in CaCC-deficient VSNs (Fig. 5B, lower panels). Firing frequencies of all measured cells before and during the stimulation pe" @default.
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• W2804039430 title "Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression" @default.
• W2804039430 cites W1544860128 @default.
• W2804039430 cites W1649056280 @default.
• W2804039430 cites W1940693565 @default.
• W2804039430 cites W1942764539 @default.
• W2804039430 cites W1963546312 @default.
• W2804039430 cites W1981193117 @default.
• W2804039430 cites W1984372033 @default.
• W2804039430 cites W1993169531 @default.
• W2804039430 cites W1998447762 @default.
• W2804039430 cites W1999633796 @default.
• W2804039430 cites W1999832198 @default.
• W2804039430 cites W2005402925 @default.
• W2804039430 cites W2016329811 @default.
• W2804039430 cites W2018710614 @default.
• W2804039430 cites W2032434028 @default.
• W2804039430 cites W2032864009 @default.
• W2804039430 cites W2047200582 @default.
• W2804039430 cites W2058797597 @default.
• W2804039430 cites W2067128597 @default.
• W2804039430 cites W2071900171 @default.
• W2804039430 cites W2078571865 @default.
• W2804039430 cites W2080541137 @default.
• W2804039430 cites W2081786742 @default.
• W2804039430 cites W2082855639 @default.
• W2804039430 cites W2083092470 @default.
• W2804039430 cites W2091580061 @default.
• W2804039430 cites W2092290968 @default.
• W2804039430 cites W2092868876 @default.
• W2804039430 cites W2093272113 @default.
• W2804039430 cites W2095421754 @default.
• W2804039430 cites W2096683144 @default.
• W2804039430 cites W2100316885 @default.
• W2804039430 cites W2113045541 @default.
• W2804039430 cites W2117090406 @default.
• W2804039430 cites W2124674257 @default.
• W2804039430 cites W2130973558 @default.
• W2804039430 cites W2135316980 @default.
• W2804039430 cites W2136425662 @default.
• W2804039430 cites W2139333308 @default.
• W2804039430 cites W2146556180 @default.
• W2804039430 cites W2147771024 @default.
• W2804039430 cites W2148770506 @default.
• W2804039430 cites W2149093695 @default.
• W2804039430 cites W2152827122 @default.
• W2804039430 cites W2153041644 @default.
• W2804039430 cites W2153668688 @default.
• W2804039430 cites W2160801132 @default.
• W2804039430 cites W2162336775 @default.
• W2804039430 cites W2165044121 @default.
• W2804039430 cites W2165495909 @default.
• W2804039430 cites W2225840870 @default.
• W2804039430 cites W2255523442 @default.
• W2804039430 cites W2262749401 @default.
• W2804039430 cites W2412057024 @default.
• W2804039430 cites W2471887092 @default.
• W2804039430 cites W2519590824 @default.
• W2804039430 cites W2563885276 @default.
• W2804039430 cites W2596920990 @default.
• W2804039430 cites W2743082498 @default.
• W2804039430 cites W2749476993 @default.
• W2804039430 cites W2776364703 @default.
• W2804039430 cites W4246649400 @default.
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