Functional Architecture of the Medial Entorhinal Cortex D i s s e r t a t i o n zur Erlangung des akademischen Grades D o c t o r r e r u m n a t u r a l i u m (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von M.Sc. Saikat Ray Präsident der Humboldt-Universität zu Berlin Prof. Dr. Jan-Hendrik Olbertz Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Richard Lucius Gutachter/innen: 1. Prof. Dr. Michael Brecht 2. Prof. Dr. Imre Vida 3. Dr. James Poulet Tag der mündlichen Prüfung: 22 June, 2016 Table of Contents List of figures ............................................................................................................................ 8 List of tables............................................................................................................................ 11 Statutory Declaration ............................................................................................................ 12 Abstract ................................................................................................................................... 13 Zusammenfassung.................................................................................................................. 14 1. Introduction ........................................................................................................................ 15 Neuronal circuits .......................................................................................................... 16 Memory microcircuit ................................................................................................... 16 Entorhinal cortex .......................................................................................................... 16 Spatial cells in the medial entorhinal cortex ................................................................ 17 Medial entorhinal cortex microcircuit ......................................................................... 18 References .................................................................................................................... 20 2. Grid-layout and theta-modulation of layer 2 pyramidal neurons in medial entorhinal cortex ....................................................................................................................................... 22 References and notes .................................................................................................... 28 Figures.......................................................................................................................... 30 Supporting online material ........................................................................................... 37 Supplementary figures ..................................................................................... 37 Material and methods ....................................................................................... 45 Brain tissue preparation ....................................................................... 45 Histochemistry and immunohistochemistry ........................................ 45 Retrograde neuronal labeling ............................................................... 46 Image acquisition ................................................................................. 47 Cell counts and patch sizes .................................................................. 47 Quantification of axonal orientation and cholinergic boutons ............. 47 Analysis of spatial periodicity ............................................................. 48 Electrophysiology methods .................................................................. 49 2 Data analysis ........................................................................................ 50 Method references ................................................................................ 51 3. Pyramidal and stellate cell specificity of grid and border representations in layer 2 of medial entorhinal cortex ........................................................................................................ 52 Summary ...................................................................................................................... 54 Introduction .................................................................................................................. 55 Results .......................................................................................................................... 56 Discussion .................................................................................................................... 59 Methods summary ........................................................................................................ 60 References .................................................................................................................... 61 Acknowledgments........................................................................................................ 63 Supplementary data ...................................................................................................... 69 Inventory list of supplemental data .................................................................. 70 Supplementary figures ..................................................................................... 71 Supplementary table ......................................................................................... 81 Supplementary note ......................................................................................... 82 Supplemental experimental procedures ........................................................... 88 Freely-moving juxtacellular recordings ............................................... 88 Tetrode recordings ............................................................................... 89 Neurobiotin labeling and calbindin immunohistochemistry ................ 89 Spine density measurement .................................................................. 89 Estimate of the fraction of unintentionally included non-layer 2 cells 89 Analysis of theta locking ..................................................................... 90 Analysis of spatial modulation............................................................. 90 Analysis of spatial information ............................................................ 91 Analysis of gridness ............................................................................. 92 Analysis of border cells ....................................................................... 92 Analysis of head direction ................................................................... 92 Classification of non-identified cells into putative cell types .............. 92 Classification of cells into functional categories ................................. 93 Supplemental references .................................................................................. 94 3 4. Conserved size and periodicity of pyramidal patches in layer 2 of medial/caudal entorhinal cortex .................................................................................................................... 95 Abstract ........................................................................................................................ 97 Introduction .................................................................................................................. 98 Materials and methods ............................................................................................... 100 Animals .......................................................................................................... 100 Human brain tissue ........................................................................................ 100 Tissue preparation .......................................................................................... 100 Histochemistry ............................................................................................... 101 Acetylcholinesterase activity ............................................................. 101 Myelin ................................................................................................ 101 Cytochrome oxidase........................................................................... 101 Synaptic zinc ...................................................................................... 101 NADPH diaphorase ........................................................................... 101 Immunohistochemistry .................................................................................. 101 SMI-32 ............................................................................................... 102 Calbindin ............................................................................................ 102 NeuN .................................................................................................. 102 Cell counts and patch sizes ............................................................................ 103 Analysis of spatial periodicity ....................................................................... 103 Light and fluorescence microscopy ............................................................... 105 Estimate of the neuron number in a human layer four finger-module ........... 105 Results .......................................................................................................................