Characterization of the molecular and cellular mechanisms in vomeronasal signal transduction in mice [Elektronische Ressource] / vorgelegt von Silke Hagendorf

Silke Hey

Description

Characterization of the molecular and cellular mechanisms in vomeronasal signal transduction 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 Silke Hagendorf aus Münster (Westf.) Berichter: Universitätsprofessor Dr. Marc Spehr Universitätsprofessor Dr. Hermann Wagner Tag der mündlichen Prüfung: 4.12.2009 Die Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Contents I. Contents 1. Introduction 1 1.1 The main olfactory system 2 1.2 The accessory olfactory system 5 1.3 Gain control and input-output relationship in chemosensory signaling 10 1.3.1 Adaptation mechanism 10 1.3.2 Homeostatic plasticity 12 1.4 Voltage-gated potassium channels 12 1.4.1 The ether-à-go-go-related gene (ERG) channel 13 1.5 Aims 15 2. Materials and Methods 18 2.1 Equipment 18 2.2 Consumables 19 2.3 Enzymes and Kits2.4 Antibodies2.5 Primer 20 2.6 Chemicals and Inhibitors 21 2.7 Software2.8 Solutions 22 2.9 Animals 23 2.10 VNO preparation2.11 Expression profiling 24 2.12 Immunocytochemistry 25 2.13 Confocal imaging 26 2.14 Immunoblotting 28 2.15 Electrophysiology 29 2.16 Data analysis 33 2.17 RNA extraction and reverse transcription-PCR2.18 3-D reconstruction 34 3. Results 35 3.

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 117
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

Characterization of the molecular and cellular

mechanisms in vomeronasal signal

transduction in mice

thematik, Informatik und Von der Fakultät für Ma

Naturwissenschaften der RWTH Aachen University zur

ades einer Doktorin der Erlangung des akademischen Gr

Dissertation eNaturwissenschaften genehmigt

vorgelegt von

Master of Science

Silke Hagendorf

aus Münster (Westf.)

ofessor Dr. Marc Spehr prsBerichter: Universität

professor Dr. Hermann Wagner sUniversität

üfung: 4.12.2009 hen PrTag der mündlic

Die Dissertation ist auf den Internetseiten der

Hochschulbibliothek online verfügbar.

I. Contents 1. Introduction 2 1.1 The main olfactory system 5 1.2 The accessory olfactory system 1.3 Gain control and input-output relationship in chemosensory signaling 10
1.3.1 Adaptation mechanism
1.3.2 Homeostatic plasticity
12 els1.4 Voltage-gated potassium chann 1.4.1 The ether-à-go-go-related gene (ERG) channel
15 Aims 1.5 Materials and Methods 2. 18 2.1 Equipment 19 2.2 Consumables 19 s and Kits2.3 Enzyme 19 2.4 Antibodies 20 2.5 Primer 21 2.6 Chemicals and Inhibitors 21 2.7 Software 22 s2.8 Solution 23 2.9 Animals 23 2.10 VNO preparation 24 g2.11 Expression profilin 25 cytochemistryo2.12 Immun 26 2.13 Confocal imaging 28 oblotting2.14 Immun 29 physiology2.15 Electro 33 2.16 Data analysis 33 PCRtraction and reverse transcription-x2.17 RNA e 34 2.18 3-D reconstruction 3. Results sal neurons:of sensory output in basal vomeronal 3.1 Homeostatic contro her-à-go-go related activity-dependent expression of et 35 gene potassium channels3.1.1 Activity-dependent expression of voltage-gated
K+ channel genes in the VNO
3.1.2 Coexpression of ERG1, ERG3 and MiRP2 in basal VSNs

Contents 

1 1012 13 18

35 35 39

I 

3.1.3 Biophysical fingerprint of vomeronasal ERG channels:
current activation3.1.4 Biophysical fingerprint of ERG channels:
current availability and deactivation
3.1.5 The ERG1 channel subunit is a major contributor to
vomeronasal IK(ERG)
3.1.6 IK(ERG) is selectively expressed in basal VSNs
ctively translated3.1.7 Regulated transcription is effe yunctionalitd in protein finto change3.1.8 IK(ERG) contributes to the late phase of action potential
n in VSNsrepolarizatio3.1.9 IK(ERG) extends the dynamic range of the vomeronasal
ulus-response functionstim3.2 Ca2+-calmodulin feedback mediates sensory adaptation and inhibits
pheromone-sensitive ion channels in the vomeronasal organ 61
3.2.1 Ca2+-CaM modulation of diacylglycerol-sensitive cation channels
3.2.2 CaM localization to VSN microvilli
65CaM-mediated channel inhibitionendence of 3.2.3 Concentration dep tion of CaM signaling3.2.4 Disrup3.2.5 VNO pheromone response undergo Ca2+-CaM-dependent
68 ptationsensory ada3.2.6 Ca2+-CaM-dependent adaptation modulates pheromone
70al VSNsindividuresponsivity of 3.3 Persistent activity in the mouse vomeronasal organ 73
74 etersmpara3.3.1 Firing yd activit3.3.2 Thapsigargin-induced sustaine3.3.3 Correlation of action potential firing and Ca2+-oscillations 78
2+ 80 -currentsin Ca3.3.4 Thapsigargin induces shift 81yained activit3.3.5 Inhibition of urine-induced sust 83yd activit3.3.6 Inhibition of thapsigargin-induced sustaine 3.3.7 Sustained activity in TRPM4/TRPM5 knock-out animals 84
4. Discussion sal neurons:of sensory output in basal vomeronal 4.1 Homeostatic contro her-à-go-go related activity-dependent expression of et 87 gene potassium channels4.1.1 ERG expression provides basal VSNs with
87naturel sigphysicaa distinct bio

Contents  43 4851  52 54 55 57 61 63 66 77 87

II 

Contents 

4.1.2 Fast ERG-mediated K+ currents contribute to
90 AP repolarization in basal VSNs4.1.3 Regulated ERG expression provides a mechanism for
homeostatic control of the VSN input-output relationship 92
2+ y adaptation and-calmodulin feedback mediates sensor4.2 Cainhibits pheromone-sensitive ion channels in the vomeronasal organ 94
4.2.1Modulation of diacylglyerol-sensitive cation channels by Ca2+-CaM 95
4.2.2 Time-dependent adaptation of sensory responses in VSNs 95
4.2.3 Comparison with adaptation in OSNs 96
97 d activity in the mouse vomeronasal organ4.3 Sustaine 97 tion in VSNsy activance of persistent sensor4.3.1 Existe4.3.2 Identification of pathway molecules 100 
1025. Summary 1046. Abbreviations 107 7. References 1208. Acknowledgement 1219. Appendix 121 9.1 Curriculum vitae 122 9.2 Publication list  

  102 104 107120 121

III 

Introduction 

on i1. Introduct ental chemical cues and translate this information into The ability to detect environmd perhaps the most oldest anlogenetically the phymeaningful cellular responses isthe outside world. During organism to interact with important form of a cell and/or subsets of sensory systemsherefore developed different evolution, organisms have tthat detect and identifcomprise olfaction and gustation which ry chemical stimuli from their envirepresent the dominant sensory modalities foronment. The chemical senses
ense in onsidered a 'lower' sn is often cthe majority of animals. Although olfactiohumans, without this sensory ability we would not be able to enjoy a delicious meal,
would missspoiled or poisonous food. Mo out on many emotions and reover, most mammals that lomemories, and would not be warned ofse the ability to smell are
mate, or even escape their enemies. nonviable. They are not able to suckle,imuli and is, thus, oluble, nonvolatile stry system mainly detects water-soThe gustatresponsible for the evaluation of food. The sense of smell, instead, detects relatively
and plays an important , 1994; Farbman, 1994) et al.hydrophobic substances (Doty role in social communication. The exchange of olfactory stimuli is essential for mate
mine an individuals d deter status of conspecifics, anchoice, to discern the socialgender, genetic compatibility, and health. In addition, olfaction is essential for
marking territories, spatial orientation as well as sexual communication between
potential mating partners. Even emotions such as anxiety or stress can be detected
via the olfactory system. The intraspecific chemical information exchange involves an enigmatic class of
. Despite their crucial role in pheromones instructive semiochemicals  intrinsicallychanisms of pheromone rs, the principle me social, and sexual behavioinstinctive,detection remain largely mysterious.

Figure 1.1: Schematic diagram showing the
heteronose intogen cheeomus ooserngasory subsynization of the rodestems with thent
helium (MOE),ctory epitmain olfa(SO), avomeronandsal Gru organene (Vberg ganNO), glionseptal (GGorga).n
a. , 2006et al. Spehr Modified from

1  

Introduction 

the mammalian nose ischemical stimuli, To meet the bewildering complexity of et (see: Zufall and Munger, 2001; Breer organized into chemosensory subsystems al., 2006). On the anatomical level, at least four distinct chemosensory tissues can be
discriminatthe septal organ (SO), and ed - the main olfactory epithelium (MOE), the the Grueneberg ganglion (GG) vomeronasal organ (VNO), (Fig. 1.1). The most
fundamental division is currently drawn between the main and accessory olfactory
system (Munger et al., 2009; Ma 2007; Breer et al., 2006).

systemyThe main olfactor1.1

a specialized neuronal tissue, the main The primary events in odor detection occur interior part of the nasal septum as well olfactory epithelium (MOE), which lines the posurbinates. The past years have seen an rface of the endotuas the dorsolateral sogy of this chemosensory subsystem. ion of studies on the neurophysiolexplos SNs) reside in the MOE and expressClassical olfactory sensory neurons (O(Buck and Axel, 1991). As a eptor (OR) gene superfamilymembers of the odorant rec ently expresses onlyolfactory neuron appar chemosensory systems, each hallmark ofusive manner lonoallelic and mutually excone member of the OR gene family in a m one neuron hypothesis, however, , 2004; 2005). This one receptor et al.(Serizawa r and Leinders-Zufall, 2005).h, 2004; Speis still far from being proven (MombaertsFor example, in Drosophila melanogaster, the OR83b gene is ubiquitously
, 1999; et al. in almost all OSNs (Vosshall ide a conventional ORcoexpressed alongs2000; Krieger et al., 2003). In addition, members of the V2R2 subfamily of
ssed with conventional V2R receptors e coexpre ar genesvomeronasal receptor(Martini et al., 2001), and also functional interference of taste receptors was shown
(Scott, 2004). CRs) that G protein-coupled receptors (GPORs are members of the superfamily of nus and extracellular amino termi-helical transmembrane domains, an αshare seven an GPCRs are divided into three main an intracellular carboxy terminus. Mammali: the rhodopsin receptor-like family (A), classes based on protein sequence similarity glutamate receptor c (B) and the metabotropithe secretin receptor-like receptor family t Sadee, 2001; Joosfsson, 1999; Graul andfamily (C) (Bockaert and Pin, 1999; Joseon, these receptors change their and Methner, 2002). Upon ligand interacti

2  

brane domain 3 and 6 and, thconformation in transmemheterotrimeric G-proteins.

Introduction 

ereby, bind and activate

Figure 1.2: O