Johannes Reisert - US grants

DESCRIPTION (provided by applicant): Animals live in an environment of constantly changing and complex odorous signals, which are delivered by the inhaled air to olfactory receptor neurons (ORNs) in the nasal cavity. ORNs recognize odorants and convert odorant stimulation into action potentials to be conveyed to the first relay station in the brain, the olfactory bulb. This is achieved by activation of odorant receptors, leading to cAMP generation via a G protein-coupled cascade and the opening of ion channels present on the olfactory cilia and subsequent depolarization. Odorant specificity is provided by the expression of only one type of odorant receptor in a given ORN out of ~1000 different receptors in mice and 350 in humans. Most major components of olfactory signal transduction have been identified. Our goal is to determine what limits and controls the kinetics with which olfactory transduction components interact, how this controls action potential generation and coding and what the behavioral implications are for, in particular, odorant discrimination and initiation of sniffing. Using electrophysiological techniques, we will investigate how mouse ORNs transduce odorant stimulation. Using rapid, repetitive stimulation designed to simulate high-frequency, sniffing-driven odorant delivery, we will establish whether ORNs merely report these rapid changes in odorant concentration or if in fact they themselves actively process this information in a stimulation-frequency-dependent manner. We will determine the functional role in olfactory transduction kinetics of olfactory marker protein (the function of which has not been found since its discovery in 1972) as well as determining the role of different odorant receptors in shaping the time-course of the odorant-induced response. The importance of fast and precise olfactory transduction will be studied using behavioral testing on genetically altered mice to investigate speed-accuracy tradeoff in odorant identification. Monitoring the breathing frequency during active olfactory exploration will allow us to establish the contribution of ORN kinetics and peripheral-versus-central influence on controlling changes in breathing and sniffing rates. PUBLIC HEALTH RELEVANCE: The proposed work will address the importance of both precise timing and fast transduction of odorous signals by G protein-coupled receptors in olfactory receptor neurons from the single-cell to the complex-behavioral levels such as tracking a food source or avoiding a predator. The work has broader implications in that the results will yield fundamental insights into how members of the G protein-coupled receptor family (which comprise a large part of the genome) and neurons that express them, control time-dependent cellular processes ranging from heart beat regulation to conveying hormonal signals.

Summary Our senses convert environmental stimuli into electrical signals that are ultimately interpreted by the brain to guide our behavioral decisions. The conversion of stimuli relies on the ion channels expressed in sensory cells, and their properties thus determine how we perceive our environment. Olfactory receptor neurons (ORNs) in the nasal cavity recognize odorants and, unlike other sensory neurons such as photoreceptors and hair cells, are in direct contact with the external environment, protected only by a thin mucus layer. Olfactory cilia, the cellular compartment that contains the machinery that transduces odorants, must survive in this environment while remaining functional, adding extra demands on membrane integrity and function. The initial event of an odor molecule binding to an odorant receptor in the ciliary membrane leads, via activation of adenylyl cyclase, to opening of the olfactory cyclic-nucleotide gated (CNG) channel that allows Ca2+ influx, which in turn activates an excitatory Ca2+-activated Cl- channel, further depolarizing the neuron. This two-tiered sensory transduction mechanism based on one cationic and one anionic channels, is unique to ORNs and highly conserved across all vertebrates. Both the reason why ORNs use this two-stage ion channel system in general and why a combination of cation and anion conductances in particular is used to perceive odorants are unclear, as is the role of the Ca2+-activated Cl- channel. Only in 2009 was the molecular identity of the olfactory Ca2+-activated Cl- channel determined to be anoctamin 2 (Ano2), and despite a knockout model being available, the roles of Ano2, and therefore also of the CNG channel, remain unclear. We propose to use an Ano2-knockout mouse, electrophysiological and molecular approaches to define how these two ion channels shape the odorant-induced response. We will characterize which specific aspects of the response (adaptation, response reliability, action potential coding, etc.) are determined by a single ion channel or jointly by both. In addition, because the two channels must function in the constraints of the ciliary membrane, we will investigate how the channels rely on membrane constituents for their function and how altered membranes leads to detrimental olfactory function. By examining how the two-tiered sensory transduction mechanism of a cationic and an anionic ion channel operates seamlessly as a dual-component system, we will address fundamental questions in olfaction that have remained unanswered for the past 25 years. The long-term goal of this proposal is to establish how ORNs use their signal transduction in general and their ion channels in particular to reliably encode odorant stimuli, how transduction functions within the constraints of the ciliary membrane, and how this ultimately determines how odorants are perceived.