Expression of calcium-activated chloride channels TMEM16A and TMEM16B in adult mouse vomeronasal epithelium and during embryonic development of the olfactory epithelium

Olfaction 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 \u201cone receptor one neuron\u201d 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\u2019s 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

The pH sensing properties of RF sputtered RuO2 thin-film prepared using different Ar/O2 flow ratio

The influence of the Ar/O2 gas ratio during radio frequency (RF) sputtering of the RuO2 sensing electrode on the pH sensing performance is investigated. The developed pH sensor consists in an RF sputtered ruthenium oxide thin-film sensing electrode, in conjunction with an electroplated Ag/AgCl reference electrode. The performance and characterization of the developed pH sensors in terms of sensitivity, response time, stability, reversibility, and hysteresis are investigated. Experimental results show that the pH sensor exhibits super-Nernstian slopes in the range of 64.33-73.83 mV/pH for Ar/O2 gas ratio between 10/0-7/3. In particular, the best pH sensing performance, in terms of sensitivity, response time, reversibility and hysteresis, is achieved when the Ar/O2 gas ratio is 8/2, at which a high sensitivity, a low hysteresis and a short response time are attained simultaneously

A Role for STOML3 in Olfactory Sensory Transduction

Stomatin-like protein-3 (STOML3) is an integral membrane protein expressed in the cilia of olfactory sensory neurons, but its functional role in this cell type has never been addressed. STOML3 is also expressed in dorsal root ganglia neurons, where it has been shown to be required for normal touch sensation. Here, we extended previous results indicating that STOML3 is mainly expressed in the knob and proximal cilia of olfactory sensory neurons. We additionally showed that mice lacking STOML3 have a morphologically normal olfactory epithelium. Due to its presence in the cilia, together with known olfactory transduction components, we hypothesized that STOML3 could be involved in modulating odorant responses in olfactory sensory neurons. To investigate the functional role of STOML3, we performed loose patch recordings from wild type and Stoml3 KO olfactory sensory neurons. We found that spontaneous mean firing activity was lower with additional shift in interspike intervals distributions in Stoml3 KOs compared to wild type neurons. Moreover, the firing activity in response to stimuli was reduced both in spike number and duration in neurons lacking STOML3 compared to wildtype neurons. Control experiments suggested that the primary deficit in neurons lacking STOML3 was at the level of transduction and not at the level of action potential generation. We conclude that STOML3 has a physiological role in olfaction, being required for normal sensory encoding by olfactory sensory neurons.Significance Statement Olfactory transduction comprises a series of well-characterized molecular steps that take place in the cilia of olfactory sensory neurons (OSNs) terminating in action potential firing. Here, we introduce a possible new player: stomatin-like protein 3 (STOML3). Indeed, STOML3 is localized in olfactory cilia, and we show that STOML3 plays a role in OSN physiology. First, it allows OSNs to broaden the possible frequency range of their spontaneous activity. Second, STOML3 modulates odorant-evoked action potential firing by regulating both the number of spikes and response duration. These new findings call for a reconsideration of the patterns of the peripheral coding of sensory stimuli

Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The 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. \ua9 2012 Dibattista et al

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

Pheromones 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 \u3bcM 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

A multivesicular body-like organelle mediates stimulus-regulated trafficking of olfactory ciliary transduction proteins

Stimulus transduction in cilia of olfactory sensory neurons is mediated by odorant receptors, Gαolf, adenylate cyclase-3, cyclic nucleotide-gated and chloride ion channels. Mechanisms regulating trafficking and localization of these proteins in the dendrite are unknown. By lectin/immunofluorescence staining and in vivo correlative light-electron microscopy (CLEM), we identify a retinitis pigmentosa-2 (RP2), ESCRT-0 and synaptophysin-containing multivesicular organelle that is not part of generic recycling/degradative/exosome pathways. The organelle's intraluminal vesicles contain the olfactory transduction proteins except for Golf subunits Gγ13 and Gβ1. Instead, Gβ1 colocalizes with RP2 on the organelle’s outer membrane. The organelle accumulates in response to stimulus deprivation, while odor stimuli or adenylate cyclase activation cause outer membrane disintegration, release of intraluminal vesicles, and RP2/Gβ1 translocation to the base of olfactory cilia. Together, these findings reveal the existence of a dendritic organelle that mediates both stimulus-regulated storage of olfactory ciliary transduction proteins and membrane-delimited sorting important for G protein heterotrimerization

Proteome profile of the mature rat olfactory bulb

Olfactory bulbs (OBs) are one of the few brain areas, which show active neurogenesis and neuronal migration processes in adult rats. We constructed a proteome map of the 21 days old rat OBs and identified total 196 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 24 neuronal activity-specific proteins present at high levels, 7 of which are reported for the first time from OBs

Proteome profile of whole cerebellum of the mature rat

Cerebellum is an important brain region involved in motor, cognition, learning and memory functions. Proteome mapping of the 21 days old rat cerebellum identified total 285 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 49 neuronal activity-specific proteins, 7 of which are reported for the first time from the cerebellum in this study. The protein sequence data for 31 proteins reported here have been integrated in the UniProt Knowledgebase

Effect of calcium variation on dielectric response of the sol-gel derived CaCu₃Ti₄O₁₂ thin films

107-110The giant dielectric constant exhibited by CaCu₃Ti₄O₁₂ (CCTO) over a wide temperature and frequency range has generated considerable interest in this material. In this paper, we report the synthesis of sol-gel derived CCTO thin films with varying calcium content and their dielectric characterization. The room temperature XRD-patterns reveal the formation of a single perovskite phase. The dielectric constant, loss tangent and ac conductivity of these films have been investigated as a function of frequency at room temperature. The results show that with increase in the calcium content upto a certain limit the dielectric constant of these films increases