Aus der Anatomischen Anstalt der Ludwig-Maximilians-Universität München Lehrstuhl I - Vegetative Anatomie Vorstand: Prof. Dr. med. Jens Waschke The localization and complete extent of the newly defined preganglionic Edinger-Westphal nucleus (EWpg) in human Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Laura Ioana Valeanu aus Sibiu, Rumänien Jahr 2020 1 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. med. Jens Waschke Mitberichterstatter: Prof. Dr. Stefan Glasauer PD Dr. Christoph Trumm Mitbetreuung durch die promovierte Mitarbeiterin: Prof. Dr. rer. nat. Anja Horn-Bochtler Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 19.05.2020 2 Table of contents ABSTRACT ........................................................................................................................................... 8 ZUSAMMENFASSUNG ...................................................................................................................... 9 I. INTRODUCTION ............................................................................................................................. 11 I.1. Anatomy and function of the inner eye muscles .......................................................... 11 I.1.1. The iris sphincter muscle ................................................................................................. 11 I.1.2. The ciliary muscle ............................................................................................................. 11 I.2. Innervation of the inner eye muscles ............................................................................... 13 I.3. Motoneurons of extraocular muscles .............................................................................. 15 I.4. Oculomotor nucleus complex ........................................................................................... 16 I.5. Aim of the study ..................................................................................................................... 18 II. MATERIAL AND METHODS ....................................................................................................... 19 II.1. Cases ....................................................................................................................................... 19 II.2. Preparing the sections ........................................................................................................ 20 II.3. Staining ................................................................................................................................... 20 II.3.1. Nissl-staining .................................................................................................................... 20 II.3.2. Immunostaining ................................................................................................................ 20 II.3.2.1. Antibodies ...................................................................................................................... 21 Cholineacetyltransferase (ChAT) ..................................................................................... 21 Urocortin (UCN)..................................................................................................................... 21 Non-phosphorylated neurofilaments (NP-NF) .............................................................. 21 II.4. Immunoperoxidase procedure .......................................................................................... 21 II.5. Analysis of stained sections ............................................................................................. 23 III. RESULTS ....................................................................................................................................... 24 III.1. Cytoarchitecture of the oculomotor nucleus complex in human ........................... 24 III.2. Delineation of EWpg and EWcp by ChAT- and UCN-immunostaining .................. 26 III.3. Delineation of EWpg from MIF motoneurons by NP-NF-immunostaining ............ 28 III.4. Complete population of the preganglionic neurons of EWpg in human .............. 35 IV. DISCUSSION ................................................................................................................................ 39 IV.1. Edinger-Westphal nucleus ............................................................................................... 39 IV.2. Complete extent of the EWpg-population .................................................................... 40 IV.3. EWpg and EWcp different cell populations with common inputs? ....................... 41 IV.4. EWpg, EWcp and neurodegenerative diseases .......................................................... 43 3 V. CONCLUSION ............................................................................................................................... 43 VI. REFERENCES .............................................................................................................................. 45 Attachment to materials and methods........................................................................................ 51 Eidesstattliche Versicherung/Affidavit ....................................................................................... 59 Acknowledgements ......................................................................................................................... 60 4 Table of figures Figure 1. Schematic drawing of a sagittal section through the eyeball showing the anatomical structures relevant for this study .............................................................. 12 Figure 2. Anatomical scheme of a detailed view of Figure 1 (rectangle).................................................................................................................. 12 Figure 3. Schematic drawing of a sagittal view of the human brainstem showing the cutting plane for Figure 4…………………………..……………………......................... 14 Figure 4. Transverse Nissl-stained section of the oculomotor nucleus complex to demonstrate the cytoarchitecture of functional cell groups of the Edinger-Westphal nucleus (EW)……...…………………………….…………………………….…………… 15 Figure 5. Series of Nissl-stained transverse sections through the oculomotor nucleus complex from caudal to rostral………...……………………...………………………….. 25 Figure 6. Detailed views of the morphology and immunostaining of the Edinger- Westphal nucleus (EW) ………………………………………..…………...…………….. 27 Figure 7. Photographs of neighbouring sections at a rostral level of the oculomotor nucleus (nIII)……………………………………………………………………………….. 29 Figure 8. Detailed views of EWpg dorsal group and ventral group from Figure 7C and D (rectangles), showing double labelled neurons for ChAT und NP-NF in adjacent sections……..…………………….……….…………….………………………...…..…… 30 Figure 9. Photographs of neighbouring sections farther rostral to the sections in Figure 7………….………………………...…………………………...………………..…………. 31 Figure 10. Photograph of a transverse section through the oculomotor nucleus complex at rostral level of nIII, immunostained for NP-NF and counterstained for Nissl to illustrate the dorsal and the ventral EWpg group.………………...………………..… 32 Figure 11. Photographs of neighbouring sections farther rostral then nIII, at the anteromedian nucleus (AM) level, stained for Nissl, UCN, ChAT and NP-NF to show the cytoarchitecture and the exact content of the AM……………….…..……………... 33 5 Figure 12. Detailed views of AM from Figure 11C and D (rectangles), showing double labelled neurons for ChAT und NP-NF in adjacent sections………………………….. 34 Figure 13. and 14. Schematic drawings of sagittal views of the oculomotor nucleus complex, showing transverse cutting planes from caudal to rostral with the distribution of UCN- and ChAT-positive EW neurons………..……………………………..…… 37-38 6 Abbreviations AM: anteromedian nucleus BSA: bovine serum albumin CCN: central caudal nucleus CG: ciliary ganglion ChAT: choline acetyltransferase cMRF: central mesencephalic reticular formation EW: Edinger-Westphal nucleus EWcp: central projecting part of EW EWpg: preganglionic neurons of EW INC: interstitial nucleus of Cajal IO: inferior oblique muscle IR: inferior rectus muscle LR: lateral rectus muscle MIF: multiply-innervated muscle fibre MR: medial rectus muscle NII: optic nerve NIII: oculomotor nerve nIII: oculomotor nucleus nIV: trochlear nucleus nVI: abducens nucleus NP-NF: non-phosphorylated neurofilaments PBS: phosphate buffer solution RN: red nucleus RT: room temperature SIF: singly-innervated muscle fibre SO: superior oblique muscle SR: superior rectus muscle TBS: tris buffered saline UCN: urocortin 7 ABSTRACT Traditionally the Edinger-Westphal nucleus (EW) was considered as the location of preganglionic neurons of the ciliary ganglion (CG) to mediate pupillary constriction and lens accommodation, but was recently shown to refer to two different functional cell groups. Accordingly, a new nomenclature was introduced: In human the cytoarchitecturally defined EW consists of centrally projecting urocortin-positive neurons, which are involved in stress related function, regulation of alcohol and food consumption, is now termed EWcp. In contrast, the preganglionic neurons of the CG are found as an inconspicuous group of cholinergic neurons dorsally, which is now termed EWpg (Kozicz et al. 2011). In this study the complete population and distribution of cholinergic preganglionic
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