The Ca2+-activated Cl- channel TMEM16B shapes the response time course of olfactory sensory neurons.

Mammalian olfactory sensory neurons (OSNs) generate an odorant-induced response by sequentially activating two ion channels, which are in their ciliary membranes. First, a cationic, Ca2+-permeable cyclic nucleotide-gated channel is opened following odorant stimulation via a G protein-coupled transduction cascade and an ensuing rise in cAMP. Second, the increase in ciliary Ca2+ opens the excitatory Ca2+-activated Cl- channel TMEM16B, which carries most of the odorant-induced receptor current. While the role of TMEM16B in amplifying the response has been well established, it is less understood how this secondary ion channel contributes to response kinetics and action potential generation during single as well as repeated stimulation and, on the other hand, which response properties the cyclic nucleotide-gated (CNG) channel determines. We first demonstrate that basic membrane properties such as input resistance, resting potential and voltage-gated currents remained unchanged in OSNs that lack TMEM16B. The CNG channel predominantly determines the response delay and adaptation during odorant exposure, while the absence of the Cl- channels shortens both the time the response requires to reach its maximum and the time to terminate after odorant stimulation. This faster response termination in Tmem16b knockout OSNs allows them, somewhat counterintuitively despite the large reduction in receptor current, to fire action potentials more reliably when stimulated repeatedly in rapid succession, a phenomenon that occurs both in isolated OSNs and in OSNs within epithelial slices. Thus, while the two olfactory ion channels act in concert to generate the overall response, each one controls specific aspects of the odorant-induced response. KEY POINTS: Mammalian olfactory sensory neurons (OSNs) generate odorant-induced responses by activating two ion channels sequentially in their ciliary membranes: a Na+, Ca2⁺-permeable cyclic nucleotide-gated (CNG) channel and the Ca2⁺-activated Cl⁻ channel TMEM16B. The CNG channel controls response delay and adaptation during odorant exposure, while TMEM16B amplifies the response and influences the time required for the response to reach its peak and terminate. OSNs lacking TMEM16B display faster response termination, allowing them to fire action potentials more reliably during rapid repeated stimulation. The CNG and TMEM16B channels have distinct and complementary roles in shaping the kinetics and reliability of odorant-induced responses in OSNs.

Paradoxical electro-olfactogram responses in TMEM16B knock-out mice.

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.

Ca2+-Activated Chloride Channels and Phospholipid Scramblases.

The functional characterization of the TMEM16 protein family unexpectedly brought together two different research fields in membrane biology: anion channel and membrane lipid organization [...].

Functional expression of TMEM16A in taste bud cells.

KEY POINTS: Taste transduction occurs in taste buds in the tongue epithelium. The Ca2+ -activated Cl- channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems. Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds. Large Ca2+ -activated Cl- currents blocked by Ani-9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells. ATP indirectly activates Ca2+ -activated Cl- currents in type I cells through TMEM16A channels. These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. ABSTRACT: The Ca2+ -activated Cl- channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl- homeostasis. Here, we examined the presence of Ca2+ -activated Cl- channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co-localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole-cell patch-clamp recordings in isolated cells from taste buds, we measured an average current of -1083 pA at -100 mV in 1.5 µm Ca2+ and symmetrical Cl- in type I cells. Ion substitution experiments and blockage by Ani-9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+ -activated Cl- currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto-ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+ -activated Cl- currents blocked by Ani-9, indicating a possible role of TMEM16A in ATP-mediated signalling. These results provide a definitive demonstration that TMEM16A-mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often-neglected taste cells.

The cyclic AMP signaling pathway in the rodent main olfactory system.

Odor perception begins with the detection of odorant molecules by the main olfactory epithelium located in the nasal cavity. Odorant molecules bind to and activate a large family of G-protein-coupled odorant receptors and trigger a cAMP-mediated transduction cascade that converts the chemical stimulus into an electrical signal transmitted to the brain. Morever, odorant receptors and cAMP signaling plays a relevant role in olfactory sensory neuron development and axonal targeting to the olfactory bulb. This review will first explore the physiological response of olfactory sensory neurons to odorants and then analyze the different components of cAMP signaling and their different roles in odorant detection and olfactory sensory neuron development.

Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel.

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.

TrkB signaling directs the incorporation of newly generated periglomerular cells in the adult olfactory bulb.

In the adult rodent brain, the olfactory bulb (OB) is continuously supplied with new neurons which survival critically depends on their successful integration into pre-existing networks. Yet, the extracellular signals that determine the selection which neurons will be ultimately incorporated into these circuits are largely unknown. Here, we show that immature neurons express the catalytic form of the brain-derived neurotrophic factor receptor TrkB [full-length TrkB (TrkB-FL)] only after their arrival in the OB, at the time when integration commences. To unravel the role of TrkB signaling in newborn neurons, we conditionally ablated TrkB-FL in mice via Cre expression in adult neural stem and progenitor cells. TrkB-deficient neurons displayed a marked impairment in dendritic arborization and spine growth. By selectively manipulating the signaling pathways initiated by TrkB in vivo, we identified the transducers Shc/PI3K to be required for dendritic growth, whereas the activation of phospholipase C-γ was found to be responsible for spine formation. Furthermore, long-term genetic fate mapping revealed that TrkB deletion severely compromised the survival of new dopaminergic neurons, leading to a substantial reduction in the overall number of adult-generated periglomerular cells (PGCs), but not of granule cells (GCs). Surprisingly, this loss of dopaminergic PGCs was mirrored by a corresponding increase in the number of calretinin+ PGCs, suggesting that distinct subsets of adult-born PGCs may respond differentially to common extracellular signals. Thus, our results identify TrkB signaling to be essential for balancing the incorporation of defined classes of adult-born PGCs and not GCs, reflecting their different mode of integration in the OB.

Transplanted human adipose tissue-derived stem cells engraft and induce regeneration in mice olfactory neuroepithelium in response to dichlobenil subministration.

We used immunodeficient mice, whose dorsomedial olfactory region was permanently damaged by dichlobenil inoculation, to test the neuroregenerative properties of transplanted human adipose tissue-derived stem cells after 30 and 60 days. Analysis of polymerase chain reaction bands revealed that stem cells preferentially engrafted in the lesioned olfactory epithelium compared with undamaged mucosa of untreated transplanted mice. Although basal cell proliferation in untransplanted lesioned mice did not give rise to neuronal cells in the olfactory mucosa, we observed clusters of differentiating olfactory cells in transplanted mice. After 30 days, and even more at 60 days, epithelial thickness was partially recovered to normal values, as also the immunohistochemical properties. Functional reactivity to odorant stimulation was also confirmed through electro-olfactogram recording in the dorsomedial epithelium. Furthermore, we demonstrated that engrafted stem cells fused with mouse cells in the olfactory organ, even if heterokaryons detected were too rare to hypothesize they directly repopulated the lesioned epithelium. The data reported prove that the migrating transplanted stem cells were able to induce a neuroregenerative process in a specific lesioned sensory area, enforcing the perspective that they could become an available tool for stem cell therapy.

The physiological roles of anoctamin2/TMEM16B and anoctamin1/TMEM16A in chemical senses.

Chemical senses allow animals to detect and discriminate a vast array of molecules. The olfactory system is responsible of the detection of small volatile molecules, while water dissolved molecules are detected by taste buds in the oral cavity. Moreover, many animals respond to signaling molecules such as pheromones and other semiochemicals through the vomeronasal organ. The peripheral organs dedicated to chemical detection convert chemical signals into perceivable information through the employment of diverse receptor types and the activation of multiple ion channels. Two ion channels, TMEM16B, also known as anoctamin2 (ANO2) and TMEM16A, or anoctamin1 (ANO1), encoding for Ca2+-activated Cl¯ channels, have been recently described playing critical roles in various cell types. This review aims to discuss the main properties of TMEM16A and TMEM16B-mediated currents and their physiological roles in chemical senses. In olfactory sensory neurons, TMEM16B contributes to amplify the odorant response, to modulate firing, response kinetics and adaptation. TMEM16A and TMEM16B shape the pattern of action potentials in vomeronasal sensory neurons increasing the interspike interval. In type I taste bud cells, TMEM16A is activated during paracrine signaling mediated by ATP. This review aims to shed light on the regulation of diverse signaling mechanisms and neuronal excitability mediated by Ca-activated Cl¯ channels, hinting at potential new roles for TMEM16A and TMEM16B in the chemical senses.

Noradrenaline modulates sensory information in mouse vomeronasal sensory neurons.

During the induction of the body's alert state, the sympathetic system modulates sensory modalities and fine-tunes peripheral organs for improved stimulus detection. We explored noradrenaline (NA)'s role in modulating signaling in vomeronasal sensory neurons (VSNs), responsible for detecting pheromones and other semiochemicals. In current-clamp recordings, NA increased the firing frequency in response to natural stimuli of responsive VSNs and induced spiking activity in previously unresponsive neurons. Current injections into VSNs showed an increase in firing frequency during NA application. Combining transcriptomic analysis, electrophysiology, Ca2+ imaging, and a pharmacological approach, we identified alpha 1 adrenergic receptors as crucial for NA-induced firing frequency increases in VSNs. Immunohistochemistry revealed catecholaminergic fibers in the vomeronasal sensory epithelium, suggesting localized NA release near VSNs. This study unveils NA as a key regulator of VSN signaling, shedding light on the intricate interplay between the sympathetic nervous system and chemosensory processing, advancing our understanding of sensory modulation.

Structural heterogeneity of the ion and lipid channel TMEM16F.

Transmembrane protein 16 F (TMEM16F) is a Ca2+-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca2+-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca2+ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions.

Anoctamin 2/TMEM16B: a calcium-activated chloride channel in olfactory transduction.

In vertebrate olfactory transduction, a Ca(2+)-dependent Cl(-) efflux greatly amplifies the odorant response. 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. The Ca(2+) activates a Cl(-) current that, in the presence of a maintained elevated intracellular Cl(-) concentration, produces an efflux of Cl(-) ions and amplifies the depolarization. In this review, we summarize evidence supporting the hypothesis that anoctamin 2/TMEM16B is the main, or perhaps the only, constituent of the Ca(2+)-activated Cl(-) channels involved in olfactory transduction. Indeed, studies from several laboratories have shown that anoctamin 2/TMEM16B is expressed in the ciliary layer of the olfactory epithelium, that there are remarkable functional similarities between currents in olfactory sensory neurons and in HEK 293 cells transfected with anoctamin 2/TMEM16B, and that knockout mice for anoctamin 2/TMEM16B did not show any detectable Ca(2+)-activated Cl(-) current. Finally, we discuss the involvement of Ca(2+)-activated Cl(-) channels in the transduction process of vomeronasal sensory neurons and the physiological role of these channels in olfaction.

Slow Inactivation of Sodium Channels Contributes to Short-Term Adaptation in Vomeronasal Sensory Neurons.

Adaptation plays an important role in sensory systems as it dynamically modifies sensitivity to allow the detection of stimulus changes. The vomeronasal system controls many social behaviors in most mammals by detecting pheromones released by conspecifics. Stimuli activate a transduction cascade in vomeronasal neurons that leads to spiking activity. Whether and how these neurons adapt to stimuli is still debated and largely unknown. Here, we measured short-term adaptation performing current-clamp whole-cell recordings by using diluted urine as a stimulus, as it contains many pheromones. We measured spike frequency adaptation in response to repeated identical stimuli of 2-10 s duration that was dependent on the time interval between stimuli. Responses to paired current steps, bypassing the signal transduction cascade, also showed spike frequency adaptation. We found that voltage-gated Na+ channels in VSNs undergo slow inactivation processes. Furthermore, recovery from slow inactivation of voltage-gated Na+ channels occurs in several seconds, a time scale similar to that measured during paired-pulse adaptation protocols, suggesting that it partially contributes to short-term spike frequency adaptation. We conclude that vomeronasal neurons do exhibit a time-dependent short-term spike frequency adaptation to repeated natural stimuli and that slow inactivation of Na+ channels contributes to this form of adaptation. These findings not only increase our knowledge about adaptation in the vomeronasal system, but also raise the question of whether slow inactivation of Na+ channels may play a role in other sensory systems.

Expression pattern of Stomatin-domain proteins in the peripheral olfactory system.

Recent data show that Stomatin-like protein 3 (STOML3), a member of the stomatin-domain family, is expressed in the olfactory sensory neurons (OSNs) where it modulates both spontaneous and evoked action potential firing. The protein family is constituted by other 4 members (besides STOML3): STOM, STOML1, STOML2 and podocin. Interestingly, STOML3 with STOM and STOML1 are expressed in other peripheral sensory neurons: dorsal root ganglia. In here, they functionally interact and modulate the activity of the mechanosensitive Piezo channels and members of the ASIC family. Therefore, we investigated whether STOM and STOML1 are expressed together with STOML3 in the OSNs and whether they could interact. We found that all three are indeed expressed in ONSs, although STOML1 at very low level. STOM and STOML3 share a similar expression pattern and STOML3 is necessary for STOM to properly localize to OSN cilia. In addition, we extended our investigation to podocin and STOML2, and while the former is not expressed in the olfactory system, the latter showed a peculiar expression pattern in multiple cell types. In summary, we provided a first complete description of stomatin-domain protein family in the olfactory system, highlighting the precise compartmentalization, possible interactions and, finally, their functional implications.

2D MXene interfaces preserve the basal electrophysiology of targeted neural circuits.

Neural interfaces enable the monitoring of the state of the brain and its composite cell networks, as well as stimulate them to treat nervous disorders. In addition to their highly efficient charge transduction and stability during operation, the neural electrodes should avoid altering the physiological properties of targeted neuronal tissues. Two-dimensional (2D) MXene materials integrate the advantages of metallic conductivity, high specific-surface area and surface functionality in aqueous dispersions, showing promising potential in neural interface applications. Here, we apply uncoated Ti3C2Tx MXene to interface neuronal development. The impacts of the uncoated Ti3C2Tx MXene interface on neuronal development and neuronal microcircuit activity were tested for the first time. Compared to the standard neuronal culture with a poly-L-ornithine coated coverslip, uncoated Ti3C2Tx MXene surfaces did not affect the cell morphology, density, neuron ratios, maturation or the compositions of the neuronal network. Moreover, calcium imaging, spontaneous postsynaptic currents (sPSCs) and also miniature postsynaptic currents (mPSCs) were recorded to demonstrate that Ti3C2Tx MXene interfaces preserved the basal physiology of neuronal activity. The ability to interface neuronal circuit development without altering neuronal signaling properties enables the construction of MXene-based neural prosthetic devices for neuroscience research, diagnosis, and therapies.

The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony.

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.

Paving the way for designing drugs targeting TMEM16A.

The calcium-activated chloride channel TMEM16A is involved in several physiological processes and is an important pharmacological target. Dinsdale and colleagues recently unveiled several residues in the outer pore region that constitute a critical site for the design of drugs that modulate TMEM16A channels.

TMEM16A and TMEM16B Modulate Pheromone-Evoked Action Potential Firing in Mouse Vomeronasal Sensory Neurons.

The mouse vomeronasal system controls several social behaviors. Pheromones and other social cues are detected by sensory neurons in the vomeronasal organ (VNO). Stimuli activate a transduction cascade that leads to membrane potential depolarization, increase in cytosolic Ca2+ level, and increased firing. The Ca2+-activated chloride channels TMEM16A and TMEM16B are co-expressed within microvilli of vomeronasal neurons, but their physiological role remains elusive. Here, we investigate the contribution of each of these channels to vomeronasal neuron firing activity by comparing wild-type (WT) and knock-out (KO) mice. Performing loose-patch recordings from neurons in acute VNO slices, we show that spontaneous activity is modified by Tmem16a KO, indicating that TMEM16A, but not TMEM16B, is active under basal conditions. Upon exposure to diluted urine, a rich source of mouse pheromones, we observe significant changes in activity. Vomeronasal sensory neurons (VSNs) from Tmem16a cKO and Tmem16b KO mice show shorter interspike intervals (ISIs) compared with WT mice, indicating that both TMEM16A and TMEM16B modulate the firing pattern of pheromone-evoked activity in VSNs.

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 (OSNs), 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 OSNs. We additionally showed that mice lacking STOML3 have a morphologically normal olfactory epithelium. Because of its presence in the cilia, together with known olfactory transduction components, we hypothesized that STOML3 could be involved in modulating odorant responses in OSNs. To investigate the functional role of STOML3, we performed loose patch recordings from wild-type (WT) and Stoml3 knock-out (KO) OSNs. We found that spontaneous mean firing activity was lower with additional shift in interspike intervals (ISIs) distributions in Stoml3 KOs compared with WT neurons. Moreover, the firing activity in response to stimuli was reduced both in spike number and duration in neurons lacking STOML3 compared with WT 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 OSNs.

Human cord blood CD133+ stem cells transplanted to nod-scid mice provide conditions for regeneration of olfactory neuroepithelium after permanent damage induced by dichlobenil.

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133(+) stem cells (HSC) injected intravenously to nod-scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA-DQalpha1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil-treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro-olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil-treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss.