Aus dem Institut für Integrative Neuroanatomie der Medizinischen Fakultät Charité – Universitätsmedizin Berlin DISSERTATION Characterization of inhibitory and projection specific neurons of the presubiculum zur Erlangung des akademischen Grades Doctor medicinae (Dr. med.) vorgelegt der Medizinischen Fakultät Charité – Universitätsmedizin Berlin von Roxanne Lofredi Aus Frankfurt am Main Datum der Promotion: 10.03.2017.................................. TABLE OF CONTENTS DATUM DER PROMOTION: .................................. ................................................................. 1 ABSTRACT (ENGLISH) ............................................................................................................. 4 ABSTRACT (DEUTSCH) ............................................................................................................ 5 LIST OF ABBREVIATIONS ....................................................................................................... 6 LIST OF FIGURES ...................................................................................................................... 8 1. INTRODUCTION ................................................................................................................. 9 1.1 ANATOMY OF THE PRESUBICULUM ....................................................................................... 9 1.2 FUNCTION OF THE PRESUBICULUM ..................................................................................... 14 1.2.1 The sense of orientation ............................................................................................ 14 1.2.2 The head direction signal .......................................................................................... 15 1.3 INFORMATION PROCESSING IN THE PRESUBICULUM ............................................................ 19 1.3.1 Excitatory microcircuit .............................................................................................. 20 1.3.2 Inhibitory microcircuit .............................................................................................. 23 1.4 MAIN QUESTIONS OF THE PRESENT STUDY .......................................................................... 27 2. METHODS AND MATERIALS ........................................................................................ 28 2.1 ANIMALS ............................................................................................................................ 28 2.2 IMMUNOHISTOCHEMISTRY ................................................................................................. 28 2.3 ANALYSIS AND QUANTIFICATION OF NEURONAL DENSITY .................................................. 30 2.4 ANALYSIS AND QUANTIFICATION OF LABELED INTERNEURONS .......................................... 32 2.5 RETROGRADE TRACING ...................................................................................................... 33 2.6 STEREOTACTIC SURGERY ................................................................................................... 34 2.7 VERIFICATION OF INJECTION SITE ....................................................................................... 34 2.8 ELECTROPHYSIOLOGICAL RECORDINGS .............................................................................. 35 2.9 ELECTROPHYSIOLOGICAL ANALYSIS .................................................................................. 36 2.10 3D RECONSTRUCTION OF RECORDED NEURONS .............................................................. 37 2.11 STATISTICS ......................................................................................................................... 38 3. RESULTS ............................................................................................................................. 39 3.1 STRUCTURES AND BOUNDARIES OF THE PRESUBICULUM .................................................... 39 3.2 INTERNEURONS OF THE PRESUBICULUM ............................................................................. 41 3.2.1 Layer distribution of presubicular GABAergic and non-GABAergic neurons ......... 41 3.2.2 Labeling of GABAergic neurons in the presubiculum using molecular markers ..... 45 3.2.3 GABAergic neurons of the presubiculum express different molecular markers ...... 47 3.2.4 Double-labeling of GABAergic neurons ................................................................... 52 3.3 PROJECTION SPECIFIC NEURONAL SUBPOPULATIONS IN THE PRESUBICULUM ...................... 54 3.3.1 Animals ..................................................................................................................... 54 3.3.2 Laminar distribution of LMN and ADN projecting neurons .................................... 54 3.3.3 LMN projecting neurons ........................................................................................... 56 3.3.4 ADN projecting neurons ........................................................................................... 58 4. DISCUSSION ...................................................................................................................... 61 4.1 INTERNEURONS OF THE PRESUBICULUM ............................................................................. 61 4.1.1 Evaluation of chosen methods – Cell counting ......................................................... 62 4.1.2 Neuronal density in the presubiculum ....................................................................... 64 4.1.3 Specific distribution of interneurons in the presubiculum ........................................ 65 2 4.1.4 Colocalization of GABA and marker proteins in the presubiculum ......................... 66 4.1.5 Molecular subtypes of presubicular interneurons ..................................................... 67 4.1.6 Double labeling reveals additional subtypes ............................................................. 70 4.1.7 Functional relevance of the findings ......................................................................... 71 4.1.8 Prospects .................................................................................................................... 74 4.2 PROJECTION- SPECIFIC NEURONAL SUBPOPULATIONS IN THE PRESUBICULUM ..................... 75 4.2.1 Evaluation of chosen methods ................................................................................... 75 4.2.2 Functional role of presubicular projections to subcortical nuclei ............................. 76 4.2.3 Prospects .................................................................................................................... 77 REFERENCES ............................................................................................................................ 79 EIDESSTATTLICHE VERSICHERUNG ............................................................................... 89 PUBLIKATIONSLISTE ............................................................................................................ 90 LEBENSLAUF ............................................................................................................................ 91 DANKSAGUNG .......................................................................................................................... 93 3 ABSTRACT (English) Presubiculum (PrS) is a transitional cortical area of the parahippocampal formation in the temporal lobe, close to hippocampus and entorhinal cortex. PrS is involved in spatial navigation processing by encoding an animal’s head direction (HD). The HD signal is most likely generated in lateral mammillary nucleus (LMN) and relayed in anterodorsal thalamus (ADN) before being processed in the PrS. PrS is thought to be crucial for updating the HD signal with visual landmark information as it receives direct projections of visual cortex and projects back to downstream ADN and LMN. The aim of my work has been twofold. First, I examined the GABAergic neurons of the PrS. GABAergic interneurons are the source of inhibitory activity that is essential for local signal generation. I quantified the total number of presubicular interneurons in the GAD67-GFP transgenic mouse and investigated morphological properties and layer specific distribution of interneuron subtypes. The proportion of interneurons in the PrS was 11% of all neurons. To identify neurochemical subpopulations, I performed double immunolabeling with combinations of Parvalbumin (PV), Calretinin (CR), Calbindin (CB), Somatostatin (SOM), Vasointestinal Peptide (VIP) and Neuropeptide Y (NPY). Largest population of presubicular interneurons was PV+ (36%). CR, CB and SOM interneurons contributed evenly to the population (~18%), while there were less VIP+ interneurons (9%). Double labeling experiments revealed a small subpopulation of presubicular interneurons positive for PV and SOM, a co-expression pattern seen in the hippocampal formation but usually absent in neocortical regions. Indeed, the PrS displays a unique expression pattern of inhibitory cells, and their functional role within the presubicular microcircuit will have to be investigated in future studies. The second part of my work focused on projection-specific neurons of the PrS targeting LMN and ADN. To reveal their morphological and electrophysiological identity, I injected retrogradely transported fluorescent beads into LMN or ADN which allowed assessing the laminar origin of projection neurons. While LMN projecting neurons were exclusively seen in layer IV, ADN projecting neurons were restricted to deep layers. Patch