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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.

Key points In olfactory research it is difficult to deliver stimuli with defined intensity and duration to olfactory sensory neurons. Expression of channelrhodopsin 2 (ChR2) in olfactory sensory neurons provides a means to activate these neurons with light flashes. Appropriate mouse models are available. The present study explores the suitability of an established olfactory marker protein (OMP)/ChR2–yellow fluorescent protein (YFP) mouse model for ex vivo experimentation. Expression of ChR2 in sensory neurons of the main olfactory epithelium, the septal organ and vomeronasal organ is characterized. Expression pattern of ChR2 in olfactory receptor neurons and the properties of light responses indicate that light stimulation does not impact on signal transduction in the chemosensory cilia. Light-induced electro-olfactograms are characterized with light flashes of different intensities, durations and frequencies. The impact of light-induced afferent stimulation on the olfactory bulb is examined with respect to response amplitude, polarity and low-pass filtering. Abstract For the examination of sensory processing, it is helpful to deliver stimuli in precisely defined temporal and spatial patterns with accurate control of stimulus intensity. This is challenging in experiments with the mammalian olfactory system because airborne odorants have to be transported into the intricate sensory structures of the nose and must dissolve in mucus to be detected by sensory neurons. Defined and reproducible activity can be generated in olfactory sensory neurons that express the light-gated ion channel channelrhodopsin 2 (ChR2). The neurons can be stimulated by light flashes in a controlled fashion by this optogenetic approach. Here we examined the application of an olfactory marker protein (OMP)/ChR2–yellow fluorescent protein (YFP) model for ex vivo exploration of the olfactory epithelium and the olfactory bulb of the mouse. We studied the expression patterns of ChR2 in the main olfactory system, the vomeronasal system, and the septal organ, and we found that ChR2 is absent from the sensory cilia of olfactory sensory neurons. In the olfactory epithelium, we characterized light-induced electro-olfactograms with respect to peripheral encoding of stimulus intensity, stimulus duration and stimulus frequency. In acute slices of the olfactory bulb, we identified specific aspects of the ChR2-induced input signal, concerning its dynamic range, its low-pass filter property and its response to prolonged stimulation. Our study describes the performance of the OMP/ChR2–YFP model for ex vivo experimentation on the peripheral olfactory system and documents its versatility and its limitations for olfactory research.

Das Kapitel fuhrt einleitend in die Methoden der Hirnforschung ein und schliest mit Gedanken zu „Geist und Seele – nichts als Chemie und Physik?“ Das hat seine Grunde. Mancher Gelehrter wie Descartes hat sich Gedanken gemacht, warum wir die Welt aufrecht sehen, obwohl die Linse des Auges das Bild auf der Retina verkehrt projiziert. Die Antwort ist: das Auge liefert nur Daten uber eingefangene elektromagnetische Wellen, das Bild entsteht im Gehirn unter dem korrigierenden Einfluss real gewonnener Erfahrung. Wir erfahren, wohin die vom Auge gelieferte Information im Gehirn weitergereicht wird und dass das vom Auge Gelieferte von Station zu Station auf elementare Merkmale analysiert wird. So werden die Winkel von Kanten durch eine Serie von Neuronen erfasst, deren Filter um 10° vom dem Filter des nachsten Neurons abweicht. Place- und grid cells helfen, uns im Raum zu orientieren und uns an bekannte Orte zu erinnern. Wir lesen, dass Eigenschaften von Objekten („Was“) und deren Ort („Wo“) auf getrennten Bahnen reprasentiert sind. Alle erfassten Merkmale werden nach Regeln der Wahrnehmungspsychologie fur die Herstellung und Interpretation eines mentalen Bildes herangezogen, wobei der Gesichtserkennung Vorrang eingeraumt wird. Wir unterliegen bei Bildinterpretationen auch Sinnestauschungen und erfreuen uns an trickreich konstruierten unmoglichen Figuren. Wir wundern uns, dass im Experiment ein Gummiarm als korpereigener wahrgenommen werden und das ICH scheinbar den Korper verlassen kann wie in Nahtod-Erlebnissen und dass es auch unbewusstes Sehen gibt.

Es ist viel von ihnen die Rede, und es gibt sie im Gehirn und uberall im Korper verstreut zu Abermilliarden, doch sie sind winzig, mit Dimensionen unterhalb der Auflosungsgrenze des Lichtmikroskops: Synapsen, die „Erregungs“-ubertragenden Kontaktstrukturen zwischen Sinneszellen und Nervenzellen, zwischen Nervenzellen und Muskelzellen und zwischen Nervenzellen untereinander. Allenfalls motorische Endplatten, die besonders ausgedehnten neuromuskularen Synapsen zwischen der Befehlsleitung einer motorischen Nervenzelle und einer Muskelfaser, konnen noch im Lichtmikroskop lokalisiert werden. Synapsen im lebenden Gewebe zu finden und zu untersuchen, verlangt besondere Kniffe. In der Regel wird mit mehrkanaligen Elektroden der Elektrophysiologie die zuleitende Nervenfaser „gereizt“ und zugleich abgehort, und mit einer zweiten „ableitenden“ Elektrode die Antwort der angesprochenen Zelle registriert. Aus dem Vergleich des Reizmusters in der Zuleitung und der Antwort der Effektorzelle wird indirekt auf das Verhalten der Synapse geschlossen. Technisches soll hier aber nicht weiter zur Sprache kommen.

Alles muss in unserem Korper geregelt und dem Bedarf angepasst werden: Stoffwechsel, Atmung, Herz und Kreislauf, Blutdruck und vieles mehr. Einleitend wird folglich eine Einfuhrung in die Funktionsweise von Regelkreisen gegeben; denn die Regelung „vegetativer“ Funktionen basiert auf biologischen Regelkreisen mit inneren Sinnesorganen als Fuhlern der momentanen Istwerte und zentralen Instanzen im Gehirn als steuernden Kontrolleuren, die im Bedarfsfall den Sollwert verstellen. Im Weiteren wird das periphere vegetative Nervensystem vorgestellt mit Sympathikus und Parasympathikus. Bei der Thermogenese, der biologischen Warmeproduktion, lassen wir das braune Fettgewebe Energie im Leerlauf verpulvern und wir kommen ins Schwitzen.

Die Moglichkeiten der Kommunikation im Tierreich sind auserst vielseitig. Manche Tiere, besonders Meeresbewohner, senden Licht aus (Biolumineszenz), viele Tiere, besonders Insekten, benutzen eine chemische Sprache, die bei Kastenbildung und sozialer Kommunikation im Dienst der Fortpflanzung auch Pheromone umfasst. In diesem Konnex wird diskutiert, ob auch beim Menschen Pheromone geheime Botschaften vermitteln und ob bei der Partnerwahl unser genetischer Personalausweis (MHC=HLA) eine Rolle spielt. Die bewundernswerte Tanzsprache der Bienen ist Beispiel fur eine Symbolsprache, die mehrere Sinnesmodalitaten in Anspruch nimmt. Bienen benutzen sogar seismische, uber das Telefonnetz der Wabe geleitete Signale, mit denen die Entfernung einer ergiebigen Nahrungsquelle angezeigt wird. Seismische Signale und Infraschall sind weitere im Tierreich gebrauchliche Kommunikationsmittel.

Mit der Sinnesphysiologie geraten wir in eine Sphare, die nicht nur aufregend ist dank der erstaunlichen Leistungen menschlicher und tierischer Sinne, sondern auch dank des viel diskutierten, vielleicht nie losbaren Ratsels, wie denn unsere subjektive, mentale Erlebniswelt entsteht. Wir treten ein in den Grenzbereich von „Leib und Seele“.

Gedachtnis beruht auf Systemeigenschaften des Gehirns, die wir primar subjektiv erfahren. Den ersten und wichtigsten Zugang zu diesen fur uns so lebenswichtigen Fahigkeiten eroffnet deshalb zuallererst unsere eigene subjektive Erfahrung. Sie zu analysieren und systematisch zu gliedern ist Aufgabe der Neuropsychologie. In der Praxis beschaftigt sie sich in erster Linie mit den Folgen von Gehirnlasionen sowie in Tierversuchen gewonnenen Erkenntnissen. Aber auch klug ausgedachte Tests mit freiwillig mitarbeitenden Probanden liefern wichtige und subtile Erkenntnisse speziell uber die Fahigkeiten unseres menschlichen Gehirns.

Sprechvermogen und Sprachverstandnis werden aufgrund verletzungsbedingter Aphasien verschiedenen Gehirnregionen zugewiesen, Vermogen zu Sprechen dem Broca-Areal, Verstandnis von Sprache dem Wernicke Areal, doch gibt es mannigfache Querverbinden zwischen diesen Arealen und zu anderen Gehirnregionen. Wie kommt es, dass Sprache nicht nur in mundlicher Form, sondern auch durch Gesten und grafischen Symbolen wie Schrift vermittelt werden kann? Aufsehen erregten Berichte, das bei bestimmten, genetisch bedingten Sprachstorungen defekte Gen FOXP2 habe eine essentielle Funktion bei der Entwicklung des Sprechvermogens. Wie sieht das bei Tieren aus, welche haben das Gen? Oder hat es nur der Mensch?

Unglaubliche Leistungen fernreisender Zugvogel, die im Einzelfall bis zu 80 000 km/Jahr (Kustenseeschwalbe) zurucklegen, hat schon die weltweite Kampagne zur Beringung der Vogel offenbart und das eben gestartete ICARUS-Projekt zur telemetrischen Verfolgung Fernwanderer uber Satelliten als Signalvermittler wird noch viele neue Erkenntnisse bringen. Vor allem das Navigationsvermogen der Fernwanderer lost Erstaunen aus; Wie kann ein fernes Ziel, das nicht mit den Augen, den Ohren oder dem Geruchsinn geortet werden kann, angesteuert werden? Dies ist nicht nur fur Zugvogel ein zu losendes Problem, sondern auch fur fernreisende Langstreckenschwimmer wie Meeresschildkroten, Lachse und Aale, die nach Jahren Aufenthalt in schier unendlicher Ferne zum Laichen zur Statte ihrer Geburt zuruckkehren. Wer diese Orientierungs- und Navigationssysteme erforscht, setzt auf Sonnenkompass, Sternenkompass und auf Navigation mittels des Erdmagnetfeldes. Zu Recht oder blose Hypothesen? Das Kapitel setzt sich mit diesen spannenden Fragen und den technischen Moglichkeiten der Erforschung auseinander.

Sinneszellen und Sinnesorgane, die mechanisch einwirkende Krafte als Quelle von Information nutzen, gibt es in groser Zahl und in vielfaltiger konstruktiver Ausfuhrung. Vielfaltig ist die Struktur der Sensoren, die ihr elektrisches Membranpotential durch Zug- oder Druckkrafte modulieren lassen. Vielfaltig sind auch die Hilfseinrichtungen, welche die Reizflut filtern und selektionieren. Vielfaltig sind schlieslich auch die Empfindungen, die in unserer inneren Erlebniswelt ausgelost werden.

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.

Chemosensation in the mammalian nose comprises detection of odorants, irritants and pheromones. While the traditional view assigned one distinct sub-system to each stimulus type, recent research has produced a more complex picture. Odorants are not only detected by olfactory sensory neurons but also by the trigeminal system. Irritants, in turn, may have a distinct odor, and some pheromones are detected by the olfactory epithelium. Moreover, it is well established that irritants change odor perception and vice versa. A wealth of psychophysical evidence on olfactory-trigeminal interactions in humans contrasts with a paucity of structural insight. In particular, it is unclear whether the two systems communicate just by sharing stimuli, or whether neuronal connections mediate cross-modal signaling. One connection could exist in the olfactory bulb that performs the primary processing of olfactory signals and receives trigeminal innervation. In the present study, neuroanatomical tracing of the mouse ethmoid system illustrates how peptidergic fibers enter the glomerular layer of the olfactory bulb, where local microcircuits process and filter the afferent signal. Biochemical assays reveal release of calcitonin gene-related peptide from olfactory bulb slices and attenuation of cAMP signaling by the neuropeptide. In the non-stimulated tissue, the neuropeptide specifically inhibited the basal activity of calbindin-expressing periglomerular interneurons, but did not affect the basal activity of neurons expressing calretinin, parvalbumin, or tyrosine hydroxylase, nor the activity of astrocytes. This study represents a first step towards understanding trigeminal neuromodulation of olfactory-bulb microcircuits and provides a working hypothesis for trigeminal inhibition of olfactory signal processing. This article is protected by copyright. All rights reserved.

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 receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca2+-permeable channels and Ca2+-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca2+-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca2+-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel.

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.

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.

Wir konzipieren ein ideales, okologisch vertragliches Kraftwerk (Abb. 1.1): Es gehore dem Konzern „Grune Pflanze“ und sei zweigeteilt. Ein erstes Teilkraftwerk, wir nennen es Chloroplast, bezieht Energie von der Sonne, und fuhrt sie in transportfahige und speicherbare Form uber. Dieses erste Teilkraftwerk verbraucht keinen materiellen Kraftstoff, es stellt vielmehr solchen Kraftstoff in Form organischer Molekule her. Das zweite Teilkraftwerk, es heise Mitochondrium, setzt aus dem biologisch synthetisierten Kraftstoff die gespeicherte Energie in arbeitsfahiger Form wieder frei.

Kein Lebewesen kann fur sich allein bestehen. Das Leben aller Organismen ist eingebunden in ein Netzwerk von Beziehungen. Jeder Organismus ist Mitglied einer Fortpflanzungsgemeinschaft und zugleich Mitglied einer Lebensgemeinschaft, die viele Arten umfasst. Koordination zwischen den Mitgliedern einer Fortpflanzungsgemeinschaft verlangt wechselseitige Absprachen. Man muss aufeinander eingehen. In den Staaten der Insekten und in den Verhaltensmustern eines Affenclans wird die Bedeutung der sozialen Kommunikation auch dem aufmerksamen Laien unmittelbar augenfallig.

Auch wenn uns die Vorstellung fremd sein mag, trifft sie doch zu: Wir alle existieren als ein Staat von Zellen. Die Abermilliarden Einzelzellen sind freilich in ihrer Existenz voneinander abhangig und mussen miteinander kooperieren, um den Staat als Ganzes lebensfahig zu halten. Es gibt eine unuberschaubare Fulle von Moglichkeiten, wie Zellen miteinander kommunizieren konnen. Beispielsweise konnen sie auf ihrer Zelloberflache Signalmolekule exponieren, die von Nachbarzellen abgetastet und abgelesen werden konnen. Solche im Nahbereich wirksamen Kommunikationssysteme haben fundamentale Bedeutung in der Embryonalentwicklung. Sie bleiben zeitlebens bedeutsam bei der Kontrolle lokaler Prozesse wie z.B. Entzundungen und Immunreaktionen (s. Kap. 6, Immunologie). Wir betrachten in diesem Kapitel Systeme der Fernkommunikation, besonders die Kommunikation uber Botenstoffe, die unter dem Begriff Hormon (engl.: hormone, von griech.: horman = antreiben, in Gang setzen;) bekannt geworden sind.

So vielgestaltig Lebewesen sind, so mannigfach ihre besonderen Fahigkeiten, die sie in der Evolution erworben haben, um ihre besondere Nische in Okosystemen zu nutzen, so haben sie doch alle auch eine Fulle gleicher Probleme zu bewaltigen. Beispielsweise mussen alle ihre ATP-Akkus nachladen. Zum Gluck fur die Studierenden, die fur Prufungen lernen mussen, benutzen fast alle Lebewesen die gleichen zentralen Stoffwechselwege, um Energie aus Molekulen herauszuholen und in die ATP-Akkus zu leiten.

Wir sprechen noch heute von „vegetativer“ Physiologie, weil die bisher besprochenen Funktionen des Korpers nach Meinung von Gelehrten der Antike (Aristoteles) und ihrer gelehrigen europaischen Schuler bis in die fruhe Neuzeit als „pflanzenhaft“ eingestuft worden sind. Empfindungen und Bewusstsein hatten wir ja bisher allenfalls als weit entfernte schemenhafte Randerscheinungen ins Spiel gebracht, und Geist war, so scheint es, nur zum Verstehen und Lernen des Stoffes notig. Doch das tauscht: Unbewusst war unser Gehirn immer schon im Spiel. Sogar im Schlaf hat sich unser Gehirn mit vegetativen Funktionen befasst.

Es sind nicht nur Minusgrade, die lebensgefahrlich sein konnen. Fallt die Aquarienheizung aus und kuhlt das Wasser von 23°C auf 10°C ab, kippt unser Guppy wie andere Warmwasserfische tot um. Ab 0°C wird es aber fur alle Lebewesen gefahrlich.