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The Ca2+-activated Cl¯ channel TMEM16B carries up to 90% of the transduction current evoked by odorant stimulation in olfactory sensory neurons and control the number of action potential firing and therefore the length of the train of action potentials. A loss of function approach revealed that TMEM16B is required for olfactory-driven behaviors such as tracking unfamiliar odors. Here, we used the electro-olfactogram (EOG) technique to investigate the contribution of TMEM16B to odorant transduction in the whole olfactory epithelium. Surprisingly, we found that EOG responses from Tmem16b knock out mice have a bigger amplitude compared to those of wild type. Moreover, the kinetics of EOG responses is faster in absence of TMEM16B, while the ability to adapt to repeated stimulation is altered in knock out mice. The larger EOG responses in Tmem16b knock out may be the results of the removal of the clamping and/or shunting action of the Ca2+-activated Cl¯ currents leading to the paradox of having smaller transduction current but larger generator potential.

Olfactory marker protein (OMP) was first described as a protein expressed in olfactory receptor neurons (ORNs) in the nasal cavity. In particular, OMP, a small cytoplasmic protein, marks mature ORNs and is also expressed in the neurons of other nasal chemosensory systems: the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. While its expression pattern was more easily established, OMP’s function remained relatively vague. To date, most of the work to understand OMP’s role has been done using mice lacking OMP. This mostly phenomenological work has shown that OMP is involved in sharpening the odorant response profile and in quickening odorant response kinetics of ORNs and that it contributes to targeting of ORN axons to the olfactory bulb to refine the glomerular response map. Increasing evidence shows that OMP acts at the early stages of olfactory transduction by modulating the kinetics of cAMP, the second messenger of olfactory transduction. However, how this occurs at a mechanistic level is not understood, and it might also not be the only mechanism underlying all the changes observed in mice lacking OMP. Recently, OMP has been detected outside the nose, including the brain and other organs. Although no obvious logic has become apparent regarding the underlying commonality between nasal and extranasal expression of OMP, a broader approach to diverse cellular systems might help unravel OMP’s functions and mechanisms of action inside and outside the nose.

This cross-sectional study evaluates self-reported gustatory dysfunction in patients with long-term COVID-19.

Olfactory dysfunction (OD) is one of the most common symptoms of acute and long-COVID-19. QualitativeOD frequently accompanies or follows quantitative olfactory loss . Limited studies to date have combinedboth qualitative and quantitative evaluation of OD . The aim of this study was to estimate the prevalence of qualitative OD and evaluate its functional impact in post-COVID-19 patients by combining a validated questionnaire and a comprehensive olfactory psychophysical evaluation.

Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.

Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, limited (partial) and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and its severe discomfort.

Peripheral sensory cells and the central neuronal circuits that monitor environmental changes to drive behaviors should be adapted to match the behaviorally relevant kinetics of incoming stimuli, be it the detection of sound frequencies, the speed of moving objects or local temperature changes. Detection of odorants begins with the activation of olfactory receptor neurons in the nasal cavity following inhalation of air and airborne odorants carried therein. Thus, olfactory receptor neurons are stimulated in a rhythmic and repeated fashion that is determined by the breathing or sniffing frequency that can be controlled and altered by the animal. This raises the question of how the response kinetics of olfactory receptor neurons are matched to the imposed stimulation frequency and if, vice versa, the kinetics of olfactory receptor neuron responses determine the sniffing frequency. We addressed this question by using a mouse model that lacks the K+-dependent Na+/Ca2+ exchanger 4 (NCKX4), which results in markedly slowed response termination of olfactory receptor neuron responses and hence changes the temporal response kinetics of these neurons. We monitored sniffing behaviors of freely moving wildtype and NCKX4 knockout mice while they performed olfactory Go/NoGo discrimination tasks. Knockout mice performed with similar or, surprisingly, better accuracy compared to wildtype mice, but chose, depending on the task, different odorant sampling durations depending on the behavioral demands of the odorant identification task. Similarly, depending on the demands of the behavioral task, knockout mice displayed a lower basal breathing frequency prior to odorant sampling, a possible mechanism to increase the dynamic range for changes in sniffing frequency during odorant sampling. Overall, changes in sniffing behavior between wildtype and NCKX4 knockout mice were subtle, suggesting that, at least for the particular odorant-driven task we used, slowed response termination of the odorant-induced receptor neuron response either has a limited detrimental effect on odorant-driven behavior or mice are able to compensate via an as yet unknown mechanism.

The purpose of our cohort study was to quantify olfactory deficits in Coronavirus disease 2019 (COVID-19) patients using Sniffin’ Sticks and a pre-post design to evaluate olfactory recovery. Thirty adult patients with laboratory-confirmed mild to moderate forms of COVID-19 underwent a quantitative olfactory test performed with the Sniffin’ Sticks test (SST; Burghardt, Wedel, Germany), considering olfactory threshold (T), odor discrimination (D), and odor identification (I). Results were presented as a composite TDI score (range 1–48) that used to define functional anosmia (TDI ≤ 16.5), hyposmia (16.5 < TDI < 30.5), or functionally normal ability to smell (TDI ≥ 30.5). Patients also self-evaluated their olfactory function by rating their ability to smell on a visual analogue scale (Visual Analog Scale rating) and answering a validated Italian questionnaire (Hyposmia Rating Scale). Patients were tested during hospitalization and about 2 months after symptoms onset. During the hospitalization, the overall TDI score indicated that our cohort had impairments in their olfactory ability (10% was diagnosed with anosmia and more than 50% were hyposmic). Almost all patients showed a significant improvement at around 1 month following the first test and for all the parts of the SST except for odor identification. None of the subjects at 1 month was still diagnosed with anosmia. We also quantified the improvement in the TDI score based on initial diagnosis. Anosmic subjects showed a greater improvement than hyposmic and normosmic subjects. In conclusion, within a month time window and 2 months after symptoms’ onset, in our cohort of patients we observed a substantial improvement in the olfactory abilities.

Olfactory receptor neurons (ORNs) in the nasal cavity detect and transduce odorants into action potentials to be conveyed to the olfactory bulb. Odorants are delivered to ORNs via the inhaled air at breathing frequencies that can vary from 2 to 10 Hz in the mouse. Thus olfactory transduction should occur at sufficient speed such that it can accommodate repetitive and frequent stimulation. Activation of odorant receptors (ORs) leads to adenylyl cyclase III activation, cAMP increase, and opening of cyclic nucleotide-gated channels. This makes the kinetic regulation of cAMP one of the important determinants for the response time course. We addressed the dynamic regulation of cAMP during the odorant response and examined how basal levels of cAMP are controlled. The latter is particularly relevant as basal cAMP depends on the basal activity of the expressed OR and thus varies across ORNs. We found that olfactory marker protein (OMP), a protein expressed in mature ORNs, controls both basal and odorant-induced cAMP levels in an OR-dependent manner. Lack of OMP increases basal cAMP, thus abolishing differences in basal cAMP levels between ORNs expressing different ORs. Moreover, OMP speeds up signal transduction for ORNs to better synchronize their output with high-frequency stimulation and to perceive brief stimuli. Last, OMP also steepens the dose–response relation to improve concentration coding although at the cost of losing responses to weak stimuli. We conclude that OMP plays a key regulatory role in ORN physiology by controlling multiple facets of the odorant response.SIGNIFICANCE STATEMENTOdorant receptors (ORs) form the largest family of G-protein-coupled receptors in mammals and are expressed in olfactory receptor neurons (ORNs). In this paper we show how the olfactory system ensures that monogenic expression of ORs dictates the response profile and the basal noise of ORNs. Olfactory marker protein (OMP), a protein long known to be expressed in mature ORNs, is responsible for controlling appropriate cAMP homeostasis and dynamics to ensure that the expressed OR is the main source of noise. In addition, OMP regulates the dynamic range of ORNs in an OR-dependent way to allow for concentration-dependent odor coding.

TMEM16B is expressed in olfactory sensory neurons, but previous attempts to establish a physiological role in olfaction have been unsuccessful. Pietra et al. find that genetic ablation of TMEM16B results in defects in the olfactory behavior of mice and the cellular physiology of olfactory sensory neurons., The Ca2+-activated Cl− channel TMEM16B is highly expressed in the cilia of olfactory sensory neurons (OSNs). Although a large portion of the odor-evoked transduction current is carried by Ca2+-activated Cl− channels, their role in olfaction is still controversial. A previous report (Billig et al. 2011. Nat. Neurosci. http://dx.doi.org/10.1038/nn.2821) showed that disruption of the TMEM16b/Ano2 gene in mice abolished Ca2+-activated Cl− currents in OSNs but did not produce any major change in olfactory behavior. Here we readdress the role of TMEM16B in olfaction and show that TMEM16B knockout (KO) mice have behavioral deficits in odor-guided food-finding ability. Moreover, as the role of TMEM16B in action potential (AP) firing has not yet been studied, we use electrophysiological recording methods to measure the firing activity of OSNs. Suction electrode recordings from isolated olfactory neurons and on-cell loose-patch recordings from dendritic knobs of neurons in the olfactory epithelium show that randomly selected neurons from TMEM16B KO mice respond to stimulation with increased firing activity than those from wild-type (WT) mice. Because OSNs express different odorant receptors (ORs), we restrict variability by using a mouse line that expresses a GFP-tagged I7 OR, which is known to be activated by heptanal. In response to heptanal, we measure dramatic changes in the firing pattern of I7-expressing neurons from TMEM16B KO mice compared with WT: responses are prolonged and display a higher number of APs. Moreover, lack of TMEM16B causes a markedly reduced basal spiking activity in I7-expressing neurons, together with an alteration of axonal targeting to the olfactory bulb, leading to the appearance of supernumerary I7 glomeruli. Thus, TMEM16B controls AP firing and ensures correct glomerular targeting of OSNs expressing I7. Altogether, these results show that TMEM16B does have a relevant role in normal olfaction.

The first step to perceive molecules in the air as odors is their detection by the olfactory receptors (ORs) present in the cilia of the olfactory sensory neurons (OSNs) in the nasal cavity. The binding of the odorant molecule to the OR triggers a series of biochemical events that lead to the opening of ion channels, creating at first a generator potential that, if the latter reaches threshold, leads to action potential firing. New insights into olfactory transduction introduced new key players and highlighted the necessity to study OSN physiology in an OR-dependent fashion.The necessity of revisiting transduction mechanisms with consideration of the OR that an OSN expresses requires recording methods of odorant responses at single cell levels. A very effective method to do so is the Suction Pipette Technique, which allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. This method can be used in combination with gene targeting and editing techniques to fully address important aspects of the olfactory physiology.

The first step to perceive molecules in the air as odors is their detection by the olfactory receptors (ORs) present in the cilia of the olfactory sensory neurons (OSNs) in the nasal cavity. The binding of the odorant molecule to the OR triggers a series of biochemical events that lead to the opening of ion channels, creating at first a generator potential that, if the latter reaches threshold, leads to action potential firing. New insights into olfactory transduction introduced new key players and highlighted the necessity to study OSN physiology in an OR-dependent fashion.The necessity of revisiting transduction mechanisms with consideration of the OR that an OSN expresses requires recording methods of odorant responses at single cell levels. A very effective method to do so is the Suction Pipette Technique, which allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. This method can be used in combination with gene targeting and editing techniques to fully address important aspects of the olfactory physiology.

Barth syndrome is a rare X-linked disease affecting less than 200 individuals worldwide. Several comorbidities have been associated with the pathology and, among those, cardiac myopathy and neutropenia are the most life threatening. The appropriate nutritive support is important to sustain the everyday life of Barth syndrome patients given the chronic fatigue they experience. Since they often prefer salty and fried food, and avoid vegetables and fruits, their eating habit and food preferences do not always provide the proper amount of vitamins and amino acids. It has been indeed reported that Barth syndrome patients have altered taste sensitivity. As olfaction also contributes to food consumption and flavor perception, we decided to investigate their olfactory abilities using the "Sniffin' sticks' extended test". We found no significant difference in any of the tested olfactory abilities between the group of Barth syndrome patients and the healthy controls. In summary, altered food preference of Barth boys could not be easily explained with an altered olfactory perception.

B-type lamins are major constituents of the nuclear lamina in all metazoan cells, yet have specific roles in the development of certain cell types. Although they are speculated to regulate gene expression in developmental contexts, a direct link between B-type lamins and developmental gene expression in an in vivo system is currently lacking. Here, we identify lamin B1 as a key regulator of gene expression required for the formation of functional olfactory sensory neurons. By using targeted knockout in olfactory epithelial stem cells in adult mice, we show that lamin B1 deficient neurons exhibit attenuated response to odour stimulation. This deficit can be explained by decreased expression of genes involved in mature neuron function, along with increased expression of genes atypical of the olfactory lineage. These results support that the broadly expressed lamin B1 regulates expression of a subset of genes involved in the differentiation of a specific cell type., Emerging evidence suggests that lamins regulate gene expression during cellular differentiation. Gigante et al. show that lamin B1 is necessary for the upregulation of mature neuron-specific genes during olfactory neuron differentiation, and its deficiency leads to attenuated olfactory neuron response to odour in mice.

Ca2+-activated Cl− currents have been implicated in many cellular processes in different cells, but for many years, their molecular identity remained unknown. Particularly intriguing are Ca2+-activated Cl− currents in olfactory transduction, first described in the early 90s. Well characterized electrophysiologically, they carry most of the odorant-induced receptor current in the cilia of olfactory sensory neurons (OSNs). After many attempts to determine their molecular identity, TMEM16B was found to be abundantly expressed in the cilia of OSNs in 2009 and having biophysical properties like those of the native olfactory channel. A TMEM16B knockout mouse confirmed that TMEM16B was indeed the olfactory Cl− channel but also suggested a limited role in olfactory physiology and behavior.

Ca2+-activated Cl- currents have been implicated in many cellular processes in different cells, but for many years, their molecular identity remained unknown. Particularly intriguing are Ca2+-activated Cl- currents in olfactory transduction, first described in the early 90s. Well characterized electrophysiologically, they carry most of the odorant-induced receptor current in the cilia of olfactory sensory neurons (OSNs). After many attempts to determine their molecular identity, TMEM16B was found to be abundantly expressed in the cilia of OSNs in 2009 and having biophysical properties like those of the native olfactory channel. A TMEM16B knockout mouse confirmed that TMEM16B was indeed the olfactory Cl- channel but also suggested a limited role in olfactory physiology and behavior. The question then arises of what the precise role of TMEM16b in olfaction is. Here we review the long story of this channel and its possible roles.

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.

Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception.

A conformational conversion of the cellular prion protein (PrP(C)) is now recognized as the causal event of fatal neurodegenerative disorders, known as prion diseases. In spite of long-lasting efforts, however, the physiological role of PrP(C) remains unclear. It has been reported that PrP(C) is expressed in various areas of the olfactory system, including the olfactory epithelium, but its precise localization in olfactory sensory neurons (OSNs) is still debated. Here, using immunohistochemistry tools, we have reinvestigated the expression and localization of PrP(C) in the olfactory epithelium of adult congenic mice expressing different PrP(C) amounts, that is, wild-type, PrP-knockout, and transgenic PrP(C)-overexpressing animals. We found that PrP(C) was expressed in OSNs, in which, however, it was unevenly distributed, being detectable at low levels in cell bodies, dendrites and apical layer, and more abundantly in axons. We also studied the involvement of PrP(C) in the response of the olfactory epithelium to odorants, by comparing the electro-olfactograms of the 3 mouse lines subjected to different stimulation protocols. We found no significant difference between the 3 PrP genotypes, supporting previous reports that exclude a direct action of PrP(C) in the early signal transduction activity of the olfactory epithelium.

Ca2+-activated Cl− channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca2+-activated Cl− channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca2+-activated Cl− channels. However, a comparison of the functional properties in the whole-cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole-cell voltage-clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca2+ concentration jumps obtained from photorelease of caged Ca2+ and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl− channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co-localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole-cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca2+-activated Cl− current.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca(2+) into the cilia. Ca(2+) activates a Cl(-) current that produces an efflux of Cl(-) ions and amplifies the depolarization. The molecular identity of Ca(2+)-activated Cl(-) channels is still elusive, although some bestrophins have been shown to function as Ca(2+)-activated Cl(-) channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca(2+)-activated Cl(-) channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca(2+)-activated Cl(-) currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca(2+)-activated Cl(-) currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons.

Ca(2+)-activated Cl(-) channels play important physiological roles in various cell types, but their molecular identity is still unclear. Recently, members of the protein family named transmembrane 16 (TMEM16) have been suggested to function as Ca(2+)-activated Cl(-) channels. Here, we report the functional properties of mouse TMEM16B (mTMEM16B) expressed in human embryonic kidney (HEK) 293T cells, measured both in the whole-cell configuration and in inside-out excised patches. In whole cell, a current induced by mTMEM16B was activated by intracellular Ca(2+) diffusing from the patch pipette, released from intracellular stores through activation of a G-protein-coupled receptor, or photoreleased from caged Ca(2+) inside the cell. In inside-out membrane patches, a current was rapidly activated by bath application of controlled Ca(2+) concentrations, indicating that mTMEM16B is directly gated by Ca(2+). Both in the whole-cell and in the inside-out configurations, the Ca(2+)-induced current was anion selective, blocked by the Cl(-) channel blocker niflumic acid, and displayed a Ca(2+)-dependent rectification. In inside-out patches, Ca(2+) concentration for half-maximal current activation decreased from 4.9 microM at -50 mV to 3.3 microM at +50 mV, while the Hill coefficient was >2. In inside-out patches, currents showed a reversible current decrease at -50 mV in the presence of a constant high Ca(2+) concentration and, moreover, an irreversible rundown, not observed in whole-cell recordings, indicating that some unknown modulator was lost upon patch excision. Our results demonstrate that mTMEM16B functions as a Ca(2+)-activated Cl(-) channel when expressed in HEK 293T cells.

The response of olfactory sensory neurons to TNT and RDX as well as to some volatile organic compounds present in the vapors of antipersonnel landmines has been studied both in the pig and in the rat. GC/MS analyses of different plastic components of six different kinds of landmines were performed in order to identify the components of the "perfume" of mines. Studies on rat olfactory mucosa were carried out with electro-olfactogram and calcium imaging techniques, while changes in the cyclic adenosine monophosphate (cAMP) levels following exposure to odorants and explosives were used as a criterion to evaluate the interaction of TNT and RDX with olfactory receptors in a preparation of isolated pig olfactory cilia. These studies indicate that chemical compounds associated with explosives and explosive devices can activate mammalian olfactory receptors.

In recent years, considerable progress has been achieved in the comprehension of the profound effects of pheromones on reproductive physiology and behavior. Pheromones have been classified as molecules released by individuals and responsible for the elicitation of specific behavioral expressions in members of the same species. These signaling molecules, often chemically unrelated, are contained in body fluids like urine, sweat, specialized exocrine glands, and mucous secretions of genitals. The standard view of pheromone sensing was based on the assumption that most mammals have two separated olfactory systems with different functional roles: the main olfactory system for recognizing conventional odorant molecules and the vomeronasal system specifically dedicated to the detection of pheromones. However, recent studies have reexamined this traditional interpretation showing that both the main olfactory and the vomeronasal systems are actively involved in pheromonal communication. The current knowledge on the behavioral, physiological, and molecular aspects of pheromone detection in mammals is discussed in this review.

Hyperpolarization-activated currents ( Ih) are present in several neurons of the central and peripheral nervous system. However, Ihin neurons of the vomeronasal organ (VNO) is not well characterized. We studied the properties of Ihin sensory neurons from acute slices of mouse VNO. In voltage-clamp studies, Ihwas identified by the characteristic kinetics of activation, voltage dependence, and blockage by Cs+or ZD-7288, two blockers of the Ih. Forskolin, an activator of adenylyl cyclase, shifted the activation curve for Ihto less negative potentials. A comparison of Ihproperties in VNO neurons with those of heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, together with RT-PCR experiments in VNO, indicate that Ihis caused by HCN2 and/or HCN4 subunits. In current-clamp recordings, blocking Ihwith ZD-7288 induced a hyperpolarization of 5.1 mV, an increase in input resistance, a decrease in the sensitivity to elicit action potentials in response to small current injections, and did not modify the frequency of action potentials elicited by a large current injection. It has been shown that in VNO neurons some pheromones induce a decrease in cAMP concentration, but the physiological role of cAMP is unknown. After application of blockers of adenylyl cyclase, we measured a hyperpolarization of 5.1 mV in 11 of 14 neurons, suggesting that basal levels of cAMP could modulate the resting potential. In conclusion, these results show that mouse VNO neurons express HCN2 and/or HCN4 subunits and that Ihcontributes to setting the resting membrane potential and to increase excitability at stimulus threshold.

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 microM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 microM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations.

Olfactory receptors (ORs) comprise a large gene family (-1200 proteins encoded by OR genes) and thus are excellent candidates for the fabrication of sensitive biosensors for explosives and other chemicals of interest. Our research project aims to address these two fundamental questions: Which are the volatile components of landmines recognized specifically by animals? How do olfactory neurons located in their nasal cavity respond to the "perfume" of mines? As regards the first question, six different plastic components of six different kinds of landmines have been analyzed. To address the second question, the response of olfactory neurons to these volatile organic compounds present in the vapors of mines, as well as to TNT and RDX, has been studied both in the pig and in the rat. Studies on rat olfactory mucosa have been carried out with electro-olfactogram (EOG) and calcium imaging techniques, while changes in the cAMP levels following exposure to odorants and explosives were used as criterion to evaluate the interaction of TNT and RDX with olfactory receptors in a preparation of isolated pig olfactory cilia. Our studies demonstrate, for the first time, that mammalian olfactory receptors can be activated by explosives and chemical given off from explosive devices and represent a deeper understanding of the molecular mechanisms by which landmine volatiles interact with sensory olfactory neurons and their receptors, necessary for the preparation of a successful biosensor.

In mammalian olfactory receptor neurons (ORNs) signal transduction begins with the binding of an odor molecule to odorant receptors (ORs), which in turn, via a G protein-coupled cascade, activates adenylyl cyclase III (ACIII) and increase intracellular cAMP. cAMP opens the cyclic nucleotide-gated (CNG) channel to initiate odorant-induced depolarization. In the absence of stimulation, basal activity of odorant receptors (ORs) drives basal fluctuations of cAMP, which is observed as baseline current noise that leads to basal action potential firing. Two phosphodiesterases (PDEs) are expressed in ORNs, one located in the cilia (PDE1C) and one restricted to the dendrite and cell body (PDE4A) to lower increased cAMP levels. Perplexingly, neither PDE alone seems to play a significant role in termination of the odorant-induced response, raising the question what their roles might be, not only in signal transduction, but also in cAMP controlled axonal targeting. We investigated the role of PDE1C in ORN transduction by recording from ORNs dissociated from PDE1C knockout mice that had been crossed with odorant receptor-tagged mice (mice expressing GFP in ORNs expressing the highly basally active I7 OR or the mEGOR with low basal activity). We found little difference in peak amplitude and time course in eugenol responses in the mOR-EG ORNs regardless of whether PDE1C was present or not. In contrast, in I7 ORNs the response magnitude to heptanal was greatly reduced and the time for the response to reach its peak magnitude was prolonged once PDE1C was knocked out. Interestingly, PDE1C does not seem to control the overall sensitivity of ORNs since no shift in the dose-response relation was observed in I7 and mOR-EG ORNs. Comparison of I7 and mOR-EG ORN axonal targeting to the bulb in wild-type and PDE1C knockout mice showed that PDE1C did not influence the overall number of main glomeruli innervated by I7 or mOR-EG ORN axons. But in the absence of PDE1C I7 ORNs substantially mis-targeted to a large number of glomeruli neighboring the main glomerulus, which was not observed in mOR-EG ORNs. Together our results suggest that PDE1C is important to retain response magnitude and kinetics across ORNs expressing ORs with different basal activity as well as insuring proper glomerular targeting.