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-Universität Mainz vorgelegt von Daniele Marongiu geb. am 16.07.1985 in Italien Mainz, 2013 Tag der mündlichen Prüfung: 24.September.2013 Table of Contents 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 (γ-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 1 Table of Contents 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 2 Table of Contents 4.2.2. Presynaptic functional properties of GABAergic synapses in shamand lesion- treated WT and BDNF (+/−) mice....................................................................... 83 4.2.3. Postsynaptic properties of GABAergic in sham and lesion-treated WT and BDNF (+/−) mice ................................................................................................ 85 4.2.4. Implication of the BDNF-TrkB signaling in the lesion-induced alterations of GABAergic transmission ..................................................................................... 89 5. Concluding remarks ..........................................................................................................