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Transcript of Interplay between BDNF/TrkB signalling and GABAergic ... between BDNF/TrkB signalling and GABAergic

Interplay between BDNF/TrkB signalling

and GABAergic inhibition in the visual

cortex of mice

Dissertation

Zur Erlangung des Grades

Doktor der Naturwissenschaften

Am Fachbereich Biologie

Der Johannes Gutenberg-Universitt Mainz

vorgelegt von

Daniele Marongiu

geb. am 16.07.1985 in Italien

Mainz, 2013

Tag der mndlichen Prfung: 24.September.2013

Table of Contents

1

Table of Contents

Table of Contents ....................................................................................................................... 1

List of Figures ............................................................................................................................ 4

Abstract ...................................................................................................................................... 6

Zusammenfassung...................................................................................................................... 8

Abbreviations .......................................................................................................................... 10

1. Introduction ........................................................................................................................ 13

1.1. The mouse visual system ............................................................................................ 13

1.1.1. Structure and circuits of the primary visual cortex .......................................... 15

1.2. Plasticity in the visual cortex ...................................................................................... 16

1.3. The GABAergic system .............................................................................................. 18

1.3.1. GABA (-amminobutyric acid) ...................................................................... 18

1.3.2. GABA receptors............................................................................................... 19

1.3.3. The role of Chloride Transporters on GABAergic function ............................ 20

1.4. Brain Derived Neurotrophic Factor (BDNF) ............................................................. 22

1.4.1. BDNF receptors and signaling pathways ......................................................... 23

1.4.2. Effect of BDNF on GABAergic and glutamatergic synaptic transmission ..... 26

1.4.3. BDNF and long term plasticity ........................................................................ 27

1.4.4. TrkB receptor agonists and antagonists ........................................................... 28

1.5. Flavonoids and 7,8-Dihydroxyflavone ....................................................................... 28

1.6. Interplay between BDNF and GABAergic inhibition (in the regulation of visual

cortical plasticity) ...................................................................................................... 30

1.7. Focal laser lesion in the visual cortex ......................................................................... 33

1.8. Objectives of the study ............................................................................................... 34

2. Materials and Methods ....................................................................................................... 35

2.1. Animals ....................................................................................................................... 35

2.2. In-vivo laser lesion in the mouse ................................................................................ 35

2.3. Electrophysiology ....................................................................................................... 36

2.3.1. The patch clamp technique .............................................................................. 36

2.3.2. Acute slices preparation ................................................................................... 39

2.3.3. The patch clamp setup ..................................................................................... 39

2.3.4. Glass micropipettes production........................................................................ 40

2.3.5. Intracellular solutions....................................................................................... 40

2.3.6. Performing a patch clamp recording ................................................................ 40

2.3.7. Data acquisition ............................................................................................... 41

2.3.8. Design of the experiments ............................................................................... 42

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2.3.9. Isolation of excitatory and inhibitory postsynaptic currents ............................ 42

2.3.10. Stimulation protocols ....................................................................................... 43

2.3.11. Analysis of electrophysiological data .............................................................. 43

2.3.12. Drugs ................................................................................................................ 44

2.3.13. 7,8 DHF in vitro and in vivo treatment ............................................................ 44

2.4. Histology..................................................................................................................... 44

2.4.1. Nissl staining .................................................................................................... 44

2.5. BDNF quantification .................................................................................................. 45

2.6. Statistical analysis ....................................................................................................... 45

3. Results ................................................................................................................................ 47

3.1. Modulatory effects of the novel TrkB receptor agonist 7,8-Dihydroxyflavone on

synaptic transmission and intrinsic neuronal excitability in vitro .............................. 47

3.1.1. 7,8-DHF depresses GABAergic transmission ................................................ 47

3.1.2. 7,8-DHF does not affect the glutamatergic transmission................................. 52

3.1.3. 7,8-DHF alters layer 2/3-pyramidal neurons intrinsic properties .................... 56

3.2. Modulatory effects of the novel TrkB receptor agonist 7,8-Dihydroxyflavone on

synaptic transmission and intrinsic neuronal excitability in vivo ............................... 58

3.2.1. Chronic administration of 7,8-DHF reduces GABAA receptors-mediated

mIPSCs frequency ............................................................................................... 58

3.2.2. Chronic administration of 7,8-DHF does not rescue the impairment of

GABAergic transmission in BDNF (+/) mice ................................................... 59

3.3. Lesion-induced alterations of GABAergic transmission in the visual cortex of WT

and BDNF (+/) mice ................................................................................................. 61

3.3.1. Histology of the lesion ..................................................................................... 61

3.3.2. Unaltered intrinsic properties of pyramidal neurons in layers 2/3 post-lesion 62

3.3.3. Lesion-induced effects on basal GABAergic synaptic transmission ............... 64

3.3.4. Increased Paired-Pulse ratio (PPR) post-lesion ............................................... 66

3.3.5. Effects of the lesion on eIPSCs ........................................................................ 68

3.3.6. The effects of the lesion on the GABAergic neurotransmission are mimicked

by an acute block of the TrkB receptor ............................................................... 71

3.3.7. The lesion doe not reduce BDNF protein levels .............................................. 73

4. Discussion .......................................................................................................................... 75

4.1. Characterization of the pharmacological profile of the new TrkB receptor agonist

7,8-Dihydroxyflavone ................................................................................................. 75

4.1.1. In vitro 7,8-DHF effects on synaptic transmission and intrinsic neuronal

excitability ........................................................................................................... 75

4.1.2. In vivo 7,8-DHF effects on GABAergic transmission in WT and BDNF (+/)

mice ..................................................................................................................... 79

4.2. Lesion-induced alterations of the GABAergic transmission in the visual cortex of

WT and BDNF (+/) mice .......................................................................................... 82

4.2.1. Lesion-induced effects on layer 2/3-pyramidal neurons intrinsic properties... 83

Table of Contents

3

4.2.2. Presynaptic functional properties of GABAergic synapses in shamand lesion-

treated WT and BDNF (+/) mice........................................