Genetic tools for the analysis of neural networks in flies [Elektronische Ressource] / vorgelegt von Thomas Hendel

Thomas Hendel

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 23 Mo

Extrait

Genetic Tools for the Analysis of Neural
Networks in Flies
Thomas Hendel
Munchen 2008Genetic Tools for the Analysis of Neural
Networks in Flies
Thomas Hendel
Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften
an der Fakult at fur Biologie
der Ludwig{Maximilians{Universit at
Munc hen
vorgelegt von
Thomas Hendel
aus Buchen im Odenwald
Munc hen, den 8.01.2008Erstgutachter: Prof. Alexander Borst
Zweitgutachter: Prof. Rainer Uhl
Tag der mundlic hen Prufung: 11.02.2008Contents
Abstract xiii
1 Introduction 1
1.1 Motion Vision in Flies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Calcium Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.1 Synthetic Calcium Indicators . . . . . . . . . . . . . . . . . . . . . 10
1.2.2 Genetically Encoded Calcium Indicators - GECIs . . . . . . . . . . 10
1.3 Fly Larvae as GECI Testbed - the Neuromuscular Junction . . . . . . . . 20
1.4 2-Photon Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
1.5 Expression Systems for Transgenes in Fruit Flies . . . . . . . . . . . . . . . 26
1.5.1 UAS-Gal4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.5.2 LexA-pLOT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.6 Transgenes for the Inhibition of Neural Activity . . . . . . . . . . . . . . . 29
1.7 Project Outline: Manipulation & Observation of a Neural Circuit . . . . . 29
2 Materials & Methods 31
2.1 Flies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.1 Flycare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.2 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.3 Transfection & Stock Breeding . . . . . . . . . . . . . . . . . . . . . 34
2.2 Physiology & Optical Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.2.1 Neuromuscular Junction Preparation . . . . . . . . . . . . . . . . . 35
2.2.2 2-Photon Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.3 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.3 Quantitative Calcium Measurements with Synthetic Indicators . . . . . . 39
2.3.1 Dye Application by Anterograde Loading of Nerve Fibres . . . . . 39
2.3.2 2-Photon Guided Dye Injection . . . . . . . . . . . . . . . . . . . . 40vi Contents
2.3.3 Calcium Measurement without Wavelength Ratioing . . . . . . . . 41
2.3.4 GECI Performance in Neurons of Living Animals and in the Cuvette 43
2.4 Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.4.1 General DNA Methods . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.4.2 Site Directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . 48
2.4.3 P element Mapping by iPCR . . . . . . . . . . . . . . . . . . . . . 48
2.4.4 Enhancer Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.4.5 The Driver Construct: Enhancer-LexA Fusion . . . . . . . . . . . . 52
2.4.6 The E ector TN-XXL and Yellow Cameleon 3.60 in pLOT 52
2.4.7 GECIs in pUAST . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.5 Recipes, Solutions, Materials . . . . . . . . . . . . . . . . . . . . . . . . . 54
3 Results 61
3.1 Quantitative Calcium Measurements with Synthetic Indicators . . . . . . . 62
3.1.1 Dye Loading into Presynaptic Boutons . . . . . . . . . . . . . . . . 62
3.1.2 Stimulus-Response Relation at the Neuromuscular Junction . . . . 65
3.1.3 Calcium Quanti cation with OGB-1 . . . . . . . . . . . . . . . . . 67
3.2 GECIs at the Neuromuscular Junction . . . . . . . . . . . . . . . . . . . . 71
3.2.1 GECI Responses to Sustained Neural Activity . . . . . . . . . . . . 71
3.2.2 GECI Responses in the Cuvette and in Living Neurons . . . . . . . 77
3.2.3 Transient Calcium Events Reported by OGB-1 and GECIs . . . . . 81
3.3 Directional Selective Calcium Responses in Fruit Fly LPTCs . . . . . . . . 86
3.3.1 DB331 - Yellow Cameleon 3.60 . . . . . . . . . . . . . . . . . . . . 87
3.3.2 DB331 - TN-XXL . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3.4 The Transgene Expression System LexA-pLOT . . . . . . . . . . . . . . . 93
3.4.1 E ector Strains: pLOT-GECI . . . . . . . . . . . . . . . . . . . . . 93
3.4.2 Driver Constructs: Enhancer-LexA . . . . . . . . . . . . . . . . . . 96
4 Discussion 105
4.1 Calcium Quanti cation with Synthetic Indicators . . . . . . . . . . . . . . 105
4.2 GECI Function at the Neuromuscular Junction . . . . . . . . . . . . . . . 107
4.3 Directional Selective Motion Responses in LPTCs . . . . . . . . . . . . . . 115
4.4 Transgene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Contents vii
A Appendix 121
A.1 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
A.2 Matlab Scripts for Data Analysis . . . . . . . . . . . . . . . . . . . . . . . 122
Acknowledgement 145
Curriculum Vitae 147
Versicherung 148viii ContentsList of Figures
1.1 Visual system of the blow y . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Anatomy and preferred direction responses of some LPTCs . . . . . . . . . 4
1.3 Columnar neurons implicated in motion vision in ies . . . . . . . . . . . . 6
1.4 Schematic of proposed motion detection pathways . . . . . . . . . . . . . . 7
1.5 Fluorescence Excitation & Emission . . . . . . . . . . . . . . . . . . . . . . 9
1.6 Schematic structure of the GFP beta barrel . . . . . . . . . . . . . . . . . 11
1.7 Illustration of FRET in Yellow Cameleons . . . . . . . . . . . . . . . . . . 13
1.8 Composition of GECIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.9 Calmodulin amino acid sequence from 3 species . . . . . . . . . . . . . . . 17
1.10 The Larval Neuromuscular Junction . . . . . . . . . . . . . . . . . . . . . . 20
1.11 Neuromuscular junction: preparation & experiments . . . . . . . . . . . . . 22
1.12 Sketch of 2-photon imaging in optic ganglia . . . . . . . . . . . . . . . . . 25
2.1 Schematic of the optical paths in the 2-photon microscope . . . . . . . . . 37
2.2 Anterograde dye loading into motoneuron axons . . . . . . . . . . . . . . . 39
2.3 Dye di usion in injected motoneurons . . . . . . . . . . . . . . . . . . . . . 42
2.4 Schematic of the vector for LexA expression . . . . . . . . . . . . . . . . . 52
2.5 Schematic of the vector for GECI under LexA control . . . . . . 53
3.1 Motoneuron lled with dextrane conjugated OGB-1 . . . . . . . . . . . . . 62
3.2 Larval preparation with injection and stimulation electrodes . . . . . . . . 63
3.3 2-photon guided dye injection . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.4 Calcium measurements using Fluo-4FF . . . . . . . . . . . . . . . . . . . . 66
3.5ts using Magnesium Green . . . . . . . . . . . . . . . 67
3.6 Calcium measurements using OGB-1 . . . . . . . . . . . . . . . . . . . . . 68
3.7 pH e ect on calcium accumulation . . . . . . . . . . . . . . . . . . . . . . 69
3.8 In situ determination of F and F for OGB-1 . . . . . . . . . . . . . 70min max
3.9 Translation of stimulus intensity into calcium concentrations . . . . . . . . 70x List of Figures
3.10 GECI responses to sustained neural activity . . . . . . . . . . . . . . . . . 74
3.11 GECI comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.12 GECI time-constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.13 Response kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.14 In vivo vs. in vitro comparison of GECI responses . . . . . . . . . . . . . . 79
3.15 GECI dissociation constants and hill coe cients in vivo and in vitro . . . . 80
3.16 OGB-1 reports single action potentials . . . . . . . . . . . . . . . . . . . . 81
3.17 GECI responses to calcium transients . . . . . . . . . . . . . . . . . . . . . 82
3.18 Power spectra of OGB-1 line scans . . . . . . . . . . . . . . . . . . . . . . 83
3.19 Gal4 expression pattern of the y line DB331 . . . . . . . . . . . . . . . . 86
3.20 Adult y brain expressing Yellow Cameleon 3.60 in LPTCs . . . . . . . . . 88
3.21 Directional selective calcium responses in VS cells . . . . . . . . . . . . . . 90
3.22e calcium responses in VS cells 2 . . . . . . . . . . . . . 92
3.23 LexA controls expression of Yellow Cameleon 3.60 . . . . . . . . . . . . . . 94
3.24 LexA controls of TN-XXL . . . . . . . . . . . . . . . . . . . . . 95
3.25 Gal4 expression pattern of the y line NP2056 . . . . . . . . . . . . . . . . 97
3.26 Genomic map of the NP2056 insertion . . . . . . . . . . . . . . . . . . . . 98
3.27 Gal4 expression pattern of the y line NP1372 . . . . . . . . . . . . . . . . 99
3.28 Genomic map of the DB331 insertion . . . . . . . . . . . . . . . . . . . . . 99
3.29 Gal4 expression pattern of the y line 3A . . . . . . . . . . . . . . . . . . . 100
3.30 Gal4 expression of the y line 1187 . . . . . . . . . . . . . . . . . . 101
3.31 Genomic map of the 1187 insertion . . . . . . . . . . . . . . . . . . . . . . 102