Elevated Cytosolic Cl<sup>−</sup>Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

Untiet, Verena; Moeller, Lisa M.; Ibarra-Soria, Ximena; Sánchez-Andrade, Gabriela; Stricker, M.; Neuhaus, Eva M.; Logan, Darren W.; Gensch, Thomas; Spehr, Marc

doi:10.1093/chemse/bjw077

Cited by 14 publications

(16 citation statements)

References 76 publications

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“…Although we found Ano2 B⌬4 transcripts in the VNO, we also detected mRNA that included exon 4 and thus encodes functional Ano2 channels. We found no evidence for the recently proposed scenario (15) in which functional isoforms of Ano2 are only expressed in supporting cells because we did not detect Ano2 in those cells. Because we cannot exclude that Ano2 mediates minor chloride currents in VSNs, we investigated the physiological role of CaCCs in the VNO using mice lacking both Ano1 and Ano2 in VSNs (⌬(Ano1 olf /Ano2) mice).…”

Section: Ca 2؉ -Activated CL ؊ Currents In the Vomeronasal Organcontrasting

confidence: 99%

“…These data do not rule out an additional expression in supporting sustentacular cells of the VNE as suggested previously (15). However, such an expression is excluded for Ano1 because neuronal-specific OMP-Cre-mediated Ano1 knockout completely abolished Ano1 immunoreactivity of VNO microvilli.…”

Section: Ano1 and Ano2 Co-localization In Microvilli Of Vsnscontrasting

confidence: 61%

“…Diacylglycerol opens Na ϩ -and Ca 2ϩ -permeable transient receptor potential canonical 2 (TRPC2) channels and thereby depolarize VSNs (13,14). Several lines of evidence using Cl Ϫ -sensitive dyes and perforated patch clamp suggest a high Cl Ϫ concentration inside VSN dendrites (15)(16)(17). Ca 2ϩ entering through TRPC2 opens Ca 2ϩ -activated Cl Ϫ channels (CaCCs) (18), resulting in an efflux of chloride that further depolarizes the cell and may amplify the initial TRPC2-mediated VSN response (17,19).…”

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confidence: 99%

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Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression

Münch

Billig

Hübner

et al. 2018

Journal of Biological Chemistry

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Ca-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, Ca-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 Ca-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 Ca-activated Cl currents. Although these currents persisted in the VNOs of mice lacking , they were undetectable in olfactory neuron-specific knockout mice irrespective of the presence of The loss of Ca-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 / double knockout mice. We conclude that Ano1 mediates the bulk of Ca-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.

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Section: Ca 2؉ -Activated CL ؊ Currents In the Vomeronasal Organcontrasting

confidence: 99%

Section: Ano1 and Ano2 Co-localization In Microvilli Of Vsnscontrasting

confidence: 61%

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confidence: 99%

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Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression

Münch

Billig

Hübner

et al. 2018

Journal of Biological Chemistry

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“…Among others, Ca 2+ -FLIM revealed globally increased calcium concentrations in astrocytes close to amyloid plaques in mouse models of Alzheimer's disease (Kuchibhotla et al, 2009) or demonstrated different resting calcium levels along dendrites of hippocampal neurons in rat brain tissue (Zheng et al, 2015). FLIM of ion indicators has also been employed for determination of intracellular chloride concentrations, e.g., in olfactory epithelia (Kaneko et al, 2004), dorsal root ganglia of rodents (Gilbert et al, 2007), cockroach salivary acinar cells (Lahn et al, 2011), cells of the mouse vomeronasal organ (Untiet et al, 2016), or acute slices of mouse brain (Untiet et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Quantitative determination of cellular [Na+] by fluorescence lifetime imaging with CoroNaGreen

Meyer

Untiet

Fahlke

et al. 2019

Journal of General Physiology

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Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na+]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na+]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na+], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na+] between 0 and 150 mM, exhibiting an apparent Kd of ∼80 mM. Based on these calibrations, a [Na+] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na+] of 13.0 mM, whereas [Na+] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na+/K+-ATPase by removal of extracellular K+ caused significant [Na+] increases in all cellular subcompartments. Using an alternative approach for data analysis (“Ratio FLIM”) increased the temporal resolution and revealed a sequential response to K+ removal, with cytosolic [Na+] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na+]. This approach thus represents a valuable tool for quantitative determination of [Na+] and changes thereof in different subcellular compartments.

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“…So far, glial [Cl − ] int has been determined in cultured astrocytes with results varying between 29–46 mM (Bekar and Walz, ; Bevensee et al, ; Kettenmann et al, ; Kimelberg, ; Smith et al, ; Walz and Mukerji, ). Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride‐sensitive dye MQAE (Gensch et al, ; Kovalchuk and Garaschuk, ; Marandi et al, ; Untiet et al, ) in acute cerebellar slices to determine the [Cl − ] int in Bergmann glial cells at different stages of postnatal development and to study pathways involved in its regulation. We found that anion channels associated with two glial glutamate transporters, excitatory amino acid transporter 1 and 2 (EAAT1 and EAAT2), modify the resting [Cl − ] int in adult wild type (WT) glia and dynamically modulate these values in response to cerebellar neuronal activity.…”

Section: Introductionmentioning

confidence: 99%

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Untiet

Kovermann

Gerkau

et al. 2016

Glia

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Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl ] to be controlled by two opposing transport processes: chloride is actively accumulated by the Na -K -2Cl cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl ] to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl ] in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl ] . Other tested chloride channels or chloride transporters do not contribute to [Cl ] under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K -Cl cotransporter change resting Bergmann glial [Cl ] in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

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Elevated Cytosolic Cl⁻Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

Cited by 14 publications

References 76 publications

Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression

Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression

Quantitative determination of cellular [Na+] by fluorescence lifetime imaging with CoroNaGreen

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

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Elevated Cytosolic Cl−Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

Cited by 14 publications

References 76 publications

Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression

Ca2+-activated Cl− currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male–male aggression

Quantitative determination of cellular [Na+] by fluorescence lifetime imaging with CoroNaGreen

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

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Elevated Cytosolic Cl⁻Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons