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Expression of calcium-activated chloride channels TMEM16A and TMEM16B in adult mouse vomeronasal epithelium and during embryonic development of the olfactory epithelium
Full text linkOlfaction enables animals to be familiar with the surrounding environmental changes. Exchange of odor molecules between animals is a way to communicate with each other and is necessary for various physiological processes, like reproduction, food preferences, prey detection, etc. The olfactory epithelium is always in contact with the inhaled air that is accompanied by odor molecules. Olfactory sensory neurons are the primary neurons of the olfactory epithelium. These neurons follow the “one receptor one neuron” rule, i.e. each individual olfactory sensory neuron expresses only one type of olfactory receptor out of ~1300 types in mouse. These neurons are specialized to convert chemical interaction, between odor molecules and olfactory receptor, into electrical signals by specific transduction mechanisms, which occur in the cilia of these neurons. The ciliary membrane contains cyclic nucleotide-gated channels and calcium-activated chloride channels. It is well documented that calcium-activated chloride channels are used to enhance signal to noise ratio in olfactory sensory neurons but we do not know about their involvement in the development of olfactory epithelium. TMEM16A and TMEM16B, the members of transmembrane proteins 16 (TMEM16) family, are responsible for the calcium-activated chloride current in various cells. In present work, I studied expression of TMEM16A and TMEM16B proteins during embryonic development of mouse and tried to find their role in olfactory epithelium development.
I found expression of TMEM16A and TMEM16B in the developing olfactory epithelium at different embryonic ages. At embryonic day 12.5 (E12.5), TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium. Olfactory sensory neurons are devoid of TMEM16A but this channel is expressed in the apical organelle free region and microvilli of supporting cells. Nasal septal glands and lateral nasal glands also express TMEM16A at the luminal surface of glands. In contrast, TMEM16B immunoreactivity was observed at E14.5 at the apical surface of the olfactory epithelium. Its expression was observed only in mature olfactory sensory neurons. With the maturation of olfactory sensory neurons and elongation of cilia TMEM16B expression is increased along with ACIII, CNGA2 and acetylated-tubulin. Interestingly, olfactory sensory neurons express only TMEM16B, but I found expression of TMEM16A as well as of TMEM16B in microvilli of vomeronasal sensory neurons. These findings indicate different physiological roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory and vomeronasal epithelia. Taking into account the previous evidences, I hypothesized that the presence of TMEM16A at the apical part and in microvilli of the supporting cells as well as in nasal glands is involved (1) in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium and/or (2) in proliferation and development during embryonic development. By comparing immunohistochemistry experiments on TMEM16A-/- and TMEM16A+/+ littermate mice I excluded the hypothesis that TMEM16A is involved in proliferation or development of the olfactory epithelium. So, either TMEM16A does not play a central role in the development of the olfactory epithelium or its genetic ablation does not affect olfactory development. Supporting cells, Bowman’s and nasal glands morphology remained unchanged in TMEM16A-/- mice, but at present we do not know whether the mucus composition is same as in TMEM16A+/+ littermate mice. Localization of TMEM16B to the cilia of mature olfactory sensory neurons and in microvilli of vomeronasal sensory neurons is consistent with a role in sensory signal transduction mechanism.
In conclusion, the present work explored the dynamic expression pattern of TMEM16A and TMEM16B. It might be possible that different physiological roles of these proteins depend on the intracellular and extracellular factors expressed in the corresponding cells
Developmental expression of the calcium-activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium
No full textCalcium-activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium-activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction
Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.
Full text linkPheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca2+concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca2+in isolated mouse vomeronasal sensory neurons in the absence of intracellular K+. In whole-cell recordings, the average current in 1.5 μM Ca2+and symmetrical Cl-was -382 pA at -100 mV. Ion substitution experiments and partial blockade by commonly used Cl-channel blockers indicated that Ca2+activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca2+-activated Cl-channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca2+-activated Cl-channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca2+-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca2+-activated Cl-currents in mouse vomeronasal sensory neurons
Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons
Full text linkThe rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Activity-dependent regulation of translation in olfactory sensory neurons of naris- occluded mice
No full textVariations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Development of the Olfactory Epithelium and Nasal Glands in TMEM16A-/- and TMEM16A+/+ Mice.
Full text linkTMEM16A/ANO1 is a calcium-activated chloride channel expressed in several types of epithelia and involved in various physiological processes, including proliferation and development. During mouse embryonic development, the expression of TMEM16A in the olfactory epithelium is dynamic. TMEM16A is expressed at the apical surface of the entire olfactory epithelium at embryonic day E12.5 while from E16.5 its expression is restricted to a region near the transition zone with the respiratory epithelium. To investigate whether TMEM16A plays a role in the development of the mouse olfactory epithelium, we obtained the first immunohistochemistry study comparing the morphological properties of the olfactory epithelium and nasal glands in TMEM16A-/- and TMEM16A+/+ littermate mice. A comparison between the expression of the olfactory marker protein and adenylyl cyclase III shows that genetic ablation of TMEM16A did not seem to affect the maturation of olfactory sensory neurons and their ciliary layer. As TMEM16A is expressed at the apical part of supporting cells and in their microvilli, we used ezrin and cytokeratin 8 as markers of microvilli and cell body of supporting cells, respectively, and found that morphology and development of supporting cells were similar in TMEM16A-/- and TMEM16A+/+ littermate mice. The average number of supporting cells, olfactory sensory neurons, horizontal and globose basal cells were not significantly different in the two types of mice. Moreover, we also observed that the morphology of Bowman's glands, nasal septal glands and lateral nasal glands did not change in the absence of TMEM16A. Our results indicate that the development of mouse olfactory epithelium and nasal glands does not seem to be affected by the genetic ablation of TMEM16A
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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