“Physiological characterization of chemosensory mechanisms in mice” Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science Lisa Marie Moeller aus Schwelm Berichter: Universitätsprofessor Dr. rer. nat. Marc Spehr Universitätsprofessor Dr. rer. nat. Frank Müller Tag der mündlichen Prüfung: 03.11.2014 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. I Content 1. Introduction 1 1.1 The main olfactory system 1 1.1.1 Anatomical structure and cells of the main olfactory epithelium 2 1.1.1.1 Olfactory sensory neurons (OSNs) 3 1.1.1.2 Sustentacular cells (SCs) 5 1.1.2 Physiological properties of OSNs 7 1.1.2.1 Signaltransduction in OSNs 7 1.1.2.2 Adaptation mechanisms 8 1.1.2.3 The physiological role of Ca2+ 9 1.2 The accessory olfactory system 10 1.2.1 Anatomy of the AOS 10 1.2.2 Chemoreceptors in the VNO 11 1.2.3 Physiological properties of VSNs 13 1.2.3.1 Signaltransduction in VSNs 13 1.2.3.2 Physiological role of Cl- 14 1.2.4 Pheromones and behavior 15 1.3 Mitochondria 17 1.4 Voltage-gated ion channels 19 Aims 23 2. Materials and Methods 24 2.1 Chemicals 24 2.2 Solutions 25 2.3 Consumables 27 2.4 Equipment 28 2.5 Software 30 2.6 Mouse strains 30 Methods 31 2.7 Animal and tissue preparation 31 2.7.1 Acute coronal MOE slices 31 2.7.2 Acute coronal VNO slices 31 2.7.3 En face preparation of the VNO sensory epithelium 32 2.7.4 Dissociation of olfactory sensory neurons 32 2.8 Electrophysiology 32 I 2.8.1 Patch-clamp technique 32 2.8.2 Electrophysiological recordings from OSNs in acute MOE tissue slices 34 2.8.3 Electrophysiological recordings from sustentacular cells in acute MOE tissue slices 36 2.8.4 Electrophysiological recordings from VSNs in acute VNO tissue slices 36 2.9 Confocal imaging 37 2.9.1 Ca2+ imaging in VNO slices via confocal imaging 38 2.10 Single-cell electroporation 38 2.10.1 Electroporation in VNO slices 38 2.11 Two-Photon fluorescence lifetime imaging microscopy (2P-FLIM) 39 2.11.1 Fluorescence lifetime and collisional quenching using Cl--sensitive MQAE 40 2.11.2 En face Cl- imaging of the VNO sensory epithelium via 2P-FLIM 42 2.11.3 Calibration of fluorescence lifetime in cells of the VNO sensory epithelium 42 2.12 Fluorescence recovery after photobleaching (FRAP) 43 2.12.1 FRAP in OSNs from MOE-slices and dissociated OSNs 43 2.13 Data Analysis 44 3. Results 45 3.1 Mitochondrial Ca2+ sequestration plays a key role in olfactory signaling in mice 45 3.1.1 Mitochondrial Ca2+ uptake shapes the odor-mediated primary receptor current in OSNs ensuring a broad dynamic range 45 3.1.2 Basic response kinetics are not significantly changed in stimulated OSNs under control conditions versus mitochondrial Ca2+ uptake inhibition. 48 3.1.3 Investigation of passive membrane and basic response properties in OSNs 50 3.1.4 Mitochondrial Ca2+ mobilization regulates the action potential output in OSNs 52 3.1.5 Odor-dependent mitochondrial distribution in OSN 55 3.2 Electrophysiological characterization of sustentacular cells in the MOE of mice 58 3.2.1 Analysis of basic passive membrane properties of SCs in MOE slices 58 3.2.2 Investigation of voltage-gated Na+ channels 60 3.2.3 Voltage-activated K+ currents 63 3.2.4 Hyperpolarization-activated cyclic nucleotide-gated channels 66 3.2.5 Sustentacular cells do not show odor-evoked activation 68 3.3 The juvenile mouse pheromone ESP22 activates vomeronasal sensory neurons 70 3.3.1 VSNs show dose-dependent activation upon ESP22 stimulation. 70 3.3.2 VSNs responding to ESP22 are also activated by the natural source of ESP22: juvenile tear fluid. 71 3.3.3 ESP22-induced cytosolic Ca2+ transients in VSNs of VNO slices 73 3.4 Excursus: Single-cell electroporation as an imaging approach in VNO slices 75 3.5 Cl- imaging in the mouse VNO using 2P-FLIM technology 79 II - 3.5.1 The VNO en face preparation allows 2P-FLIM measurements of intracellular Cl concentrations in an intact epithelial environment 79 - 3.5.2 Quantitative calibration of Cl concentrations in 2P-FLIM measurements 82 - - 3.5.3 Cl 2P-FLIM measurements revealed stimulus- and time-dependent [Cl ]i changes in VSN knobs and SCs 83 4. Discussion 87 4.1 Mitochondrial Ca2+ sequestration plays a key role in olfactory signaling in mice 87 4.2 Electrophysiological characterization of sustentacular cells in the MOE of mice 91 4.2.1 Basic passive membrane properties of SCs in MOE slices 92 4.2.2 Voltage-dependent conductances 93 4.2.2.1 Investigation of voltage-gated sodium channels 93 4.2.2.2 Voltage-activated potassium currents 95 4.2.2.3 Hyperpolarization-activated cyclic nucleotide-gated channels 96 4.2.2.4 Voltage-gated calcium currents 97 4.2.3 Sustentacular cells do not show odor-evoked activation 98 4.3 The juvenile mouse pheromone ESP22 activates vomeronasal sensory neurons 98 4.4 Excursus: Single-cell electroporation as an imaging approach in VNO slices 100 4.5 Cl- imaging in the mouse VNO using 2P-FLIM 101 5. Summary 105 6. Abbreviations 106 7. Literature 110 8. Acknowledgement 137 9. Curriculum vitae 138 III 1. Introduction In animals, environmental chemical cues play a crucial role not only for the survival of an individual, but for the whole species. Chemodetection is essential for finding food (including quality control), predator recognition (triggering escape behavior), mate choice and, accordingly, reproduction. Chemical senses comprise the sense of smell (olfactory system) and the sense of taste (gustatory system). Together, these are responsible for the detection of a vast range of molecular signals. The gustatory system mainly detects water-soluble, non- volatile molecules that each elicit either of five distinct perceptual qualities: sweet, bitter, sour, salty and umami (Zhang et al., 2003; Mombaerts, 2004a; Chandrashekar et al., 2006; Yarmolinsky et al., 2009). In contrast, the olfactory system detects an enormous structural complexity of mainly volatile substances. Intraspecific chemical communication is mediated by pheromones, providing information about social hierarchy, sex and age as well as the health and endocrine state of an individual. Moreover, pheromones can trigger stereotyped behaviors and alter endocrine state, e.g. by accelerating puberty or synchronizing estrus in females (Novotny et al., 1999; Brennan & Zufall, 2006). Several anatomically distinct olfactory subsystems evolved to accomplish these diverse olfactory tasks in rodents: the main olfactory epithelium (MOE), the vomeronasal organ (VNO), the Grueneberg ganglion (GG) and the septal organ of Masera (SO) (Tian & Ma, 2004; Breer et al., 2006; Ma, 2007; Brechbühl et al., 2008) (Fig.1.1). Using these dedicated tissues, the olfactory subsystems detect in part overlapping sets of chemosignals (Spehr et al., 2006b). Fig.1.1: Schematic viewing of the mouse olfactory system. Location of the distinct olfactory subsystems composed of the main olfactory epithelium (MOE), vomeronasal organ (VNO), Grueneberg ganglion (GG) and septal organ (SO) in the periphery, and the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) as a part of the central nervous system. Modified from Spehr et al., 2006. 1.1 The main olfactory system The main olfactory system consists of the main olfactory epithelium (MOE) in the periphery and the main olfactory bulb (MOB), where initial central processing takes place (Fig 1.2). The MOE is located in the posterior-dorsal nasal cavity, where it lines both several endoturbinates and the nasal septum. Bipolar canonical olfactory sensory neurons (OSNs) reside in the MOE, extending their cilia into the olfactory mucus. For odor detection, members of the odorant 1 receptor (OR) superfamily are expressed in the ciliary membranes (Buck & Axel, 1991). OSNs project an unmyelinated axons trough the basal lamina and cribriform plate to the MOB. Neurons expressing the same OR send their axons into one or a few spherical structures, called glomeruli, in each hemisphere of the MOB (Vassar et al., 1994; Ressler et al., 1994; Mombaerts et al., 1996) (Fig. 1.2). In those structures, OSN axon terminals synapse with MOB projection neurons, mitral cells, which project to higher brain regions such as the piriform cortex (Stettler & Axel, 2009). The axonal expression of ORs is required for convergence of axons and to target a specific glomerulus forming a topographic map in the MOB (Wang et al., 1998). Fig 1.2: Schematic diagram of organization and location of the MOE components in the rodent nose. The OSNs are situated in the MOE, from where they project their axons through the cribriform plate into glomerular structures in the MOB. In those neuropil structures, OSN axons form synapses with the OB projection neurons, i.e. mitral cells, which, in turn, target higher brain regions. CP, cribriform plate; GL, glomerulus; MC, mitral cell; MOB, main olfactory bulb; MOE, main olfactory epithelium; OSN, olfactory sensory neuron. Modified from Spehr et al., 2006. 1.1.1 Anatomical structure and cells of the main olfactory epithelium The MOE mainly comprises three different cell types: OSNs, responsible for odor detection, sustentacular cells, also called supporting cells (SCs), ensure metabolic support for OSNs and tissue stability, and basal cells (BCs), serving as progenitor cells for newborn OSNs (Fig. 1.3). Additionally, microvillous cells have been described a MOE cell type with yet unclear functional properties. These microvillous cells express IP3 receptors as well as TRPM5 channels (Lin et al., 2008; Pfister et al., 2012).