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Neurons communicate through excitatory and inhibitory synapses. Both lines of communication are adjustable and allow the fine tuning of signal exchange required for learning processes in neural networks. Several distinct modes of plasticity modulate glutamatergic and GABAergic synapses in Purkinje cells of the cerebellar cortex to promote motor control and learning. In the present paper, we present evidence for a role of short-term ionic plasticity in the cerebellar circuit activity. This type of plasticity results from altered chloride driving forces at the synapses that molecular layer interneurons form on Purkinje cell dendrites. Previous studies have provided evidence for transiently diminished chloride gradients at these GABAergic synapses following climbing fiber activity. Electrical stimulation of climbing fibers in acute slices caused a decline of inhibitory postsynaptic currents recorded from Purkinje cells. Dendritic calcium-gated chloride channels of the type anoctamin 2 (ANO2) were proposed to mediate this short-term modulation of inhibition, but the significance of this process for motor control has not been established yet. Here, we report results of behavioral studies obtained from Ano2 −/− mice, a mouse line that was previously shown to lack this particular mode of ionic plasticity. The animals display motor coordination deficits that constitute a condition of mild ataxia. Moreover, motor learning is severely impaired in Ano2 −/− mice, suggesting cerebellar dysfunction. This reduced motor performance of Ano2 −/− mice highlights the significance of inhibitory control for cerebellar function and introduces calcium-dependent short-term ionic plasticity as an efficient control mechanism for neural inhibition.

Olfactory and trigeminal chemosensory systems reside in parallel within the mammalian nose. Psychophysical studies in people indicate that these two systems interact at a perceptual level. Trigeminal sensations of pungency mask odour perception, while olfactory stimuli can influence trigeminal signal processing tasks such as odour localization. While imaging studies indicate overlap in limbic and cortical somatosensory areas activated by nasal trigeminal and olfactory stimuli, there is also potential cross-talk at the level of the olfactory epithelium, the olfactory bulb and trigeminal brainstem. Here we explored the influence of olfactory and trigeminal signaling in the nasal cavity. A forced choice water consumption paradigm was used to ascertain whether trigeminal and olfactory stimuli could influence behaviour in mice. Mice avoided water sources surrounded by both volatile TRPV1 (cyclohexanone) and TRPA1 (allyl isothiocyanate) irritants and the aversion to cyclohexanone was mitigated when combined with a pure odorant (rose fragrance, phenylethyl alcohol, PEA). To determine whether olfactory-trigeminal interactions within the nose could potentially account for this behavioural effect we recorded from single trigeminal sensory axons innervating the nasal respiratory and olfactory epithelium using an isolated in vitro preparation. To circumvent non-specific effects of chemical stimuli, optical stimulation was used to excite olfactory sensory neurons in mice expressing channel-rhodopsin (ChR2) under the olfactory marker protein (OMP) promoter. Photoactivation of olfactory sensory neurons produced no modulation of axonal action potential conduction in individual trigeminal axons. Similarly, no evidence was found for collateral branching of trigeminal axon that might serve as a conduit for cross-talk between the olfactory and respiratory epithelium and olfactory dura mater. Using direct assessment of action potential activity in trigeminal axons we observed neither paracrine nor axon reflex mediated cross-talk between olfactory and trigeminal sensory systems in the rodent nasal cavity. Our current results suggest that olfactory sensory neurons exert minimal influence on trigeminal signals within the nasal cavity.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

Physiological processes of vital importance are often safeguarded by compensatory systems that substitute for primary processes in case these are damaged by gene mutation. Ca2+-dependent Cl- secretion in airway epithelial cells may provide such a compensatory mechanism for impaired Cl- secretion via cystic fibrosis transmembrane conductance regulator (CFTR) channels in cystic fibrosis (CF). Anoctamin 1 (ANO1) Ca2+-gated Cl- channels are known to contribute to calcium-dependent Cl- secretion in tracheal and bronchial epithelia. In the present study, two mouse models of CF were examined to assess a potential protective function of Ca2+-dependent Cl- secretion, a CFTR deletion model (cftr-/-), and a CF pathology model that overexpresses the epithelial Na+ channel β-subunit (βENaC), which is encoded by the Scnn1b gene, specifically in airway epithelia (Scnn1b-Tg). The expression levels of ANO1 were examined by mRNA and protein content, and the channel protein distribution between ciliated and non-ciliated epithelial cells was analyzed. Moreover, Ussing chamber experiments were conducted to compare Ca2+-dependent Cl- secretion between wild-type animals and the two mouse models. Our results demonstrate that CFTR and ANO1 channels were co-expressed with ENaC in non-ciliated cells of mouse tracheal and bronchial epithelia. Ciliated cells did not express these proteins. Despite co-localization of CFTR and ANO1 in the same cell type, cells in cftr-/- mice displayed no altered expression of ANO1. Similarly, ANO1 expression was unaffected by βENaC overexpression in the Scnn1b-Tg line. These results suggest that the CF-related environment in the two mouse models did not induce ANO1 overexpression as a compensatory system.

Many animals follow odor trails to find food, nesting sites, or mates, and they require only faint olfactory cues to do so. The performance of a tracking dog, for instance, poses the question on how the animal is able to distinguish a target odor from the complex chemical background around the trail. Current concepts of odor perception suggest that animals memorize each odor as an olfactory object, a percept that enables fast recognition of the odor and the interpretation of its valence. An open question still is how this learning process operates efficiently at the low odor concentrations that typically prevail when animals inspect an odor trail. To understand olfactory processing under these conditions, we studied the role of an amplification mechanism that boosts signal transduction at low stimulus intensities, a process mediated by calcium‐gated anoctamin 2 chloride channels. Genetically altered Ano2 −/− mice, which lack these channels, display an impaired cue‐tracking behavior at low odor concentrations when challenged with an unfamiliar, but not with a familiar, odor. Moreover, recordings from the olfactory epithelium revealed that odor coding lacks sensitivity and temporal resolution in anoctamin 2‐deficient mice. Our results demonstrate that the detection of an unfamiliar, weak odor, as well as its memorization as an olfactory object, require signal amplification in olfactory receptor neurons. This process may contribute to the phenomenal tracking abilities of animals that follow odor trails.

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca(2+). Two family members,ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose.We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.

The mammalian olfactory system detects an unlimited variety of odorants with a limited set of odorant receptors. To cope with the complexity of the odor world, each odorant receptor must detect many different odorants. The demand for low odor selectivity creates problems for the transduction process: the initial transduction step, the synthesis of the second messenger cAMP, operates with low efficiency, mainly because odorants bind only briefly to their receptors. Sensory cilia of olfactory receptor neurons have developed an unusual solution to this problem. They accumulate chloride ions at rest and discharge a chloride current upon odor detection. This chloride current amplifies the receptor potential and promotes electrical excitation. We have studied this amplification process by examining identity, subcellular localization, and regulation of its molecular components. We found that the Na + /K + /2Cl − cotransporter NKCC1 is expressed in the ciliary membrane, where it mediates chloride accumulation into the ciliary lumen. Gene silencing experiments revealed that the activity of this transporter depends on the kinases SPAK and OSR1, which are enriched in the cilia together with their own activating kinases, WNK1 and WNK4. A second Cl − transporter, the Cl − /HCO 3 − exchanger SLC4A1, is expressed in the cilia and may support Cl − accumulation. The calcium-dependent chloride channel TMEM16B (ANO2) provides a ciliary pathway for the excitatory chloride current. These findings describe a specific set of ciliary proteins involved in anion-based signal amplification. They provide a molecular concept for the unique strategy that allows olfactory sensory neurons to operate as efficient transducers of weak sensory stimuli.

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca(2+) for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC-ESI-MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.

Background: Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl− accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl− currents. The intracellular Cl− concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl− currents to signal generation differs between individual afferent neurons, and whether the specific Cl− levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl− homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl− concentrations and on the expression levels of Cl− transporters in rat DRG neurons. Results: We developed an in vitro assay for testing how inflammatory mediators influence Cl− concentration and the expression of Cl− transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE2 for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl−-sensitive dye MQAE revealed an increase of the intracellular Cl− concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na+-K+−2Cl− cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl− levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl− importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl− homeostasis toward a state of augmented Cl− accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators. Conclusion: Our findings indicate that inflammatory mediators impact on Cl− homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl− levels and, hence, the driving force for depolarizing Cl− efflux. These findings corroborate current concepts for the role of Cl− regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl− concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl− currents in the presynaptic terminals. Moreover, excitatory Cl− currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Recent research into the generation of hyperalgesia has revealed that both the excitability of peripheral nociceptors and the transmission of their afferent signals in the spinal cord are subject to modulation by Cl- currents. The underlying Cl- homeostasis of nociceptive neurons, in particular its postnatal maturation, is, however, poorly understood. Here we measure the intracellular Cl- concentration, [Cl-](i), of somatosensory neurons in intact dorsal root ganglia of mice. Using two-photon fluorescence-lifetime imaging microscopy, we determined [Cl-](i) in newborn and adult animals. We found that the somatosensory neurons undergo a transition of Cl- homeostasis during the first three postnatal weeks that leads to a decline of [Cl-](i) in most neurons. Immunohistochemistry showed that a major fraction of neurons in the dorsal root ganglia express the cation-chloride co-transporters NKCC1 and KCC2, indicating that the molecular equipment for Cl- accumulation and extrusion is present. RT-PCR analysis showed that the transcription pattern of electroneutral Cl- co-transporters does not change during the maturation process. These findings demonstrate that dorsal root ganglion neurons undergo a developmental transition of chloride homeostasis during the first three postnatal weeks. This process parallels the developmental "chloride switch" in the central nervous system. However, while most CNS neurons achieve homogeneously low [Cl-](i) levels - which is the basis of GABAergic and glycinergic inhibition - somatosensory neurons maintain a heterogeneous pattern of [Cl-](i) values. This suggests that Cl- currents are excitatory in some somatosensory neurons, but inhibitory in others. Our results are consistent with the hypothesis that Cl- homeostasis in somatosensory neurons is regulated through posttranslational modification of cation-chloride co-transporters. (C) 2007 ISDN. Published by Elsevier Ltd. All rights reserved.

The vanilloid capsaicin, N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide, is the pungent ingredient of chili peppers and is used in pain research as an activating ligand of heat-sensitive transduction channels in nociceptive neurons. Here we describe the synthesis and application of two capsaicin derivatives modified at the hydroxy function of the vanillyl motif: alpha-carboxy-4,5-dimethoxy-2-nitrobenzyl-caged (CDMNB-caged) capsaicin and {7-[bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl-caged (BCMACMOC-caged) capsaicin. These compounds show dramatically reduced pungency, but release active capsaicin upon irradiation with UV light. CDMNB-caged capsaicin can be used to perform concentration-jump experiments, while BCMACMOC-caged capsaicin is membrane-impermeant and can be applied selectively to the intracellular or extracellular sides of a plasma membrane. Both compounds can serve as valuable research tools in pain physiology.

In sensory neurons of the peripheral nervous system, receptor potentials can be amplified by depolarizing Cl currents. In mammalian olfactory sensory neurons (OSNs), this anion-based signal amplification results from the sequential activation of two distinct types of transduction channels: cAMP-gated Ca channels and Ca-activated Cl channels. The Cl current increases the initial receptor current about 10-fold and leads to the excitation of the neuron. Here we examine the activation mechanism of the Ca-dependent Cl channel. We focus on calmodulin, which is known to mediate Ca effects on various ion channels. We show that the cell line Odora, which is derived from OSN precursor cells in the rat olfactory epithelium, expresses Ca-activated Cl channels. Single-channel conductance, ion selectivity, voltage dependence, sensitivity to niflumic acid, and Ca sensitivity match between Odora channels and OSN channels. Transfection of Odora cells with CaM mutants reduces the Ca sensitivity of the Cl channels. This result points to the participation of calmodulin in the gating process of Ca-ativated Cl channels, and helps to understand how signal amplification works in the olfactory sensory cilia. Calmodulin was previously shown to mediate feedback inhibition of cAMP-synthesis and of the cAMP-gated Ca channels in OSNs. Our results suggest that calmodulin may also be instrumental in the generation of the excitatory Cl current. It appears to play a pivotal role in the peripheral signal processing of olfactory sensory information. Moreover, recent results from other peripheral neurons, as well as from smooth muscle cells, indicate that the calmodulin-controlled, anion-based signal amplification operates in various cell types where it converts Ca signals into membrane depolarization.

The fragile X mental retardation syndrome in humans is caused by a mutational loss of function of the fragile X mental retardation gene 1 (FMR1). FMR1 is an RNA-binding protein, involved in the development and function of the nervous system. Despite of its medical significance, the evolutionary origin of FMR1 has been unclear. Here, we report the molecular characterization of HyFMR1, an FMR1 orthologue, from the cnidarian hydroid Hydractinia echinata. Cnidarians are the most basal metazoans possessing neurons. HyFMR1 is expressed throughout the life cycle of Hydractinia. Its expression pattern correlates to the position of neurons and their precursor stem cells in the animal. Our data indicate that the origin of the fraxile X related (FXR) protein family dates back at least to the common ancestor of cnidarians and bilaterians. The lack of FXR proteins in other invertebrates may have been due to gene loss in particular lineages.

Chitinases are enzymes that degrade chitin, the second most abundant polymer in nature. They are ubiquitous among living organisms where they play a role in development, food-digestion and innate immunity. We have cloned and characterized the first cnidarian chitinase cDNA from the hydroid Hydractinia. The Hydractinia chitinase exhibits a typical secreted family 18 hydrolases primary structure. In situ hybridization and RT-PCR experiments showed that it is exclusively expressed in ectodermal tissues of the animal, only following metamorphosis while undetectable in embryonic and larval stages. Most prominent expression was observed in the stolonal compartment of colonies, structures that are covered by a chitinous periderm. Chitinase mRNA was detected in new branching points along stolons and in hyperplastic stolons indicating a role of the enzyme in pattern formation and allorecognition. It was also expressed in polyps where it was mostly restricted to their basal portion. This expression pattern suggests that HyChit1 also fulfills a role in host defense, probably against fungal and nematode pathogens. Endodermal expression of HyChit1 has never been observed, suggesting that the enzyme does not participate in food-digestion.

In a mutant strain of Hydractinia (Cnidaria: Hydrozoa), the polyps develop ectopic supernumerary tentacles and heads (hypostomes) after an initial phase of wild-type growth. In order to elucidate the molecular mechanisms implicated in the development of aberrant phenotypes, we tried to enhance or suppress the expressivity of this hypomorphic mutation by exposing subclones to factors supposedly influencing pattern formation. Upon iterated treatment with alsterpaullone, an inhibitor of GSK-3, the formation of additional, ectopic head structures and the budding of new polyps were dramatically accelerated and enhanced. The endogenous stolon-inducing factor (SIF) had opposite effects by reducing head forming potential while increasing stolon-forming potential. SIF could be used to rescue extremely aberrant phenotypes. In these mutant colonies, long polyps with multiple heads eventually detach from stolons and lose the ability to regenerate stolons. Upon exposure to SIF, such free-floating multi-headed polyps resumed production of stolons and acquired wild-type morphology. We conclude that a canonical WNT signaling cascade is involved in patterning the body axis of polyps and in the initiation of budding, and that SIF counteracts this signaling system.

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.

The mammalian olfactory epithelium contains olfactory receptor neurons and trigeminal sensory endings. The former mediate odor detection, the latter the detection of irritants. The two apparently parallel chemosensory systems are in reality interdependent in various well-documented ways. Psychophysical studies have shown that virtually all odorants can act as irritants, and that most irritants have an odor. Thus, the sensory perception of odorants and irritants is based on simultaneous input from the two systems. Moreover, functional interactions between the olfactory system and the trigeminal system exist on both peripheral and central levels. Here we examine the impact of trigeminal stimulation on the odor response of olfactory receptor neurons. Using an odorant with low trigeminal potency (phenylethyl alcohol) and a non-odorous irritant (CO(2) ), we have explored this interaction in psychophysical experiments with human subjects and in electroolfactogram (EOG) recordings from rats. We have demonstrated that simultaneous activation of the trigeminal system attenuates the perception of odor intensity and distorts the EOG response. On the molecular level, we have identified a route for this cross-modal interaction. The neuropeptide calcitonin-gene related peptide (CGRP), which is released from trigeminal sensory fibres upon irritant stimulation, inhibits the odor response of olfactory receptor neurons. CGRP receptors expressed by these neurons mediate this neuromodulatory effect. This study demonstrates a site of trigeminal-olfactory interaction in the periphery. It reveals a pathway for trigeminal impact on olfactory signal processing that influences odor perception.

The chemosensory neuroepithelia of the vertebrate olfactory system share a life-long ability to regenerate. Novel neurons proliferate from basal stem cells that continuously replace old or damaged sensory neurons. The sensory neurons of the mouse and rat olfactory system specifically express bestrophin 2, a member of the bestrophin family of calcium-activated chloride channels. This channel was recently proposed to operate as a transduction channel in olfactory sensory cilia. We raised a polyclonal antibody against bestrophin 2 and characterized the expression pattern of this protein in the mouse main olfactory epithelium, septal organ of Masera, and vomeronasal organ. Comparison with the maturation markers growth-associated protein 43 and olfactory marker protein revealed that bestrophin 2 was expressed in developing sensory neurons of all chemosensory neuroepithelia, but was restricted to proximal cilia in mature sensory neurons. Our results suggest that bestrophin 2 plays a critical role during differentiation and growth of axons and cilia. In mature olfactory receptor neurons, it appears to support growth and function of sensory cilia. J. Comp. Neurol. 515:585–599, 2009. © 2009 Wiley-Liss, Inc.

The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research.

The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca 2+ signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca 2+ -dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca 2+ /calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca 2+ signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca 2+ -regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography–electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca 2+ -signaling networks in the olfactory system of the rat.

Astacin-like metalloproteases are ubiquitous in the animal kingdom but their phylogenetic relationships and ancient functions within the Metazoa are unclear. We have cloned and characterized four astacin-like cDNAs from the marine hydroid Hydractinia echinata and performed a database search for related genes in the draft genome sequence of the sea anemone Nematostella vectensis. These sequences and those of higher animals' astacins were subjected to phylogenetic analysis revealing five clusters within the Eumetazoa. The bone morphogenetic protein-1/tolloid-like astacins were represented in all eumetazoan phyla studied. The meprins were only found in vertebrates and cnidarians. Two clusters were taxon-specific, and one cluster represented astacins, which probably evolved after the split of the Cnidaria. Interestingly, grouping of astacins according to the protease catalytic domain alone resulted in clusters of proteins with similar overall domain architecture. The Hydractinia astacins were expressed in distinct cells during metamorphosis and some also during wound healing. Previously characterized cnidarian astacins also act during development. Based on our phylogeny, however, we propose that the developmental function of most of them is not homologous to the developmental function assigned to higher animals' astacins.

Wnt/Frizzled/ß-catenin-based signaling systems play diverse roles in metazoan development, being involved not only in the establishment of body axes in embryogenesis but also in regulating stem cell fate in mammalian post-embryonic development. We have studied the role the canonical Wnt cascade plays in stem cell fate determination in Hydractinia, a member of the ancient metazoan phylum Cnidaria, by analyzing two key molecules in this pathway, frizzled and ß-catenin, and blocking GSK-3. Generally, frizzled was expressed in cells able to divide but absent in post-mitotic, terminally differentiated cells such as nerve cells and nematocytes. Transcripts of frizzled were identified in all embryonic stages beginning with maternal transcripts in the oocyte. Following gastrulation and in the planula larva, frizzled expression concentrated in the central endodermal mass from which the first interstitial stem cells and their derivatives arise. In post-metamorphic development, high levels of frizzled transcripts were detected in interstitial stem cells. Activating downstream events of the Wnt-cascade in the post-metamorphic life phase by blocking GSK-3 with paullones induced recruitment of nematocytes and nerve cells from the pool of interstitial stem cells. Terminal differentiation was preceded by an initial burst of proliferation of frizzled-positive i-cells. In activated i-cells, ß-catenin appeared in the cytoplasm, later in the nucleus. It was subsequently again observed in the cytoplasm and eventually faded out during terminal differentiation. Our results suggest an ancient role of Wnt signaling in stem cell fate determination.

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., Stocker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys.337, 300–307]. To localize and follow the processing of pro-astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro-astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach. We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position −1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-specific cleavage would be possible.

Purpose In the vertebrate retina, calcium-activated chloride channels are expressed in photoreceptor synaptic terminals. These channels are involved in the control of transmitter release in the dark. The search for their molecular identity has recently lead to the localization of the protein anoctamin 2 (also TMEM16B) in the outer plexiform layer of the rodent retina. Since both rod and cone photoreceptors have their terminals in this layer, it was not clear which of these express anoctamin 2. Here, we examine rod spherules and cone pedicles for expression of anoctamin 2. Methods Expression of anoctamin genes was studied in the rat eye using RT-PCR. Immunohistochemical experiments were used to localize anoctamins and chloride transporters with their regulatory kinases. Photoreceptor synaptic proteins, as well as the lectins Peanut agglutinin and Griffonia simplicifolia agglutinin, were used to distinguish retinal structures. Results Anoctamin 1, 2, and 10 were found to be expressed in the eye. Anoctamin 2 was expressed as a splice variant that includes exon 15 of the genomic structure. The protein is exclusively expressed in rod terminals and is not present in cone pedicles. Expression is not clustered at the ribbon complex, but spread across the presynaptic membrane where it colocalizes with the plasma membrane calcium pump. The electroneutral chloride transporter NKCC1 is expressed in photoreceptor terminals, together with its regulatory kinases SPAK and OSR1. Conclusions Rod photoreceptor terminals possess the molecular machinery for chloride accumulation and for the generation of calcium-dependent chloride currents conducted through anoctamin 2 channels. We discuss this finding in the framework of the established hypothesis that calcium-activated chloride channels are part of a feedback inhibition mechanism that limits transmitter release in the dark.

Background Astacins are a large family of zinc metalloproteases found in bacteria and animals. They have diverse roles ranging from digestion of food to processing of extracellular matrix components. The C. elegans genome contains an unusually large number of astacins, of which the majority have not been functionally characterized yet. Results We analyzed the expression pattern of previously uncharacterized members of the astacin family to try and obtain clues to potential functions. Prominent sites of expression for many members of this family are the hypodermis, the alimentary system and several specialized cells including sensory sheath and sockets cells, which are located at openings in the body wall. We isolated mutants affecting representative members of the various subfamilies. Mutants in nas-5, nas-21 and nas-39 (the BMP-1/Tolloid homologue) are viable and have no apparent phenotypic defects. Mutants in nas-6 and nas-6; nas-7 double mutants are slow growing and have defects in the grinder of the pharynx, a cuticular structure important for food processing. Conclusions Expression data and phenotypic characterization of selected family members suggest a diversity of functions for members of the astacin family in nematodes. In part this might be due to extracellular structures unique to nematodes.