Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now the role of microglia in pruning remain under intense investigation.
Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the Purkinje cell layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in–depth histological analysis of the cerebellar cortex.:1 Introduction 6
1.1 Origin of microglia 6
1.2 Synaptic refinement 7
1.3 Fractalkine Receptor CX3CR1 9
1.4 Climbing fiber maturation and PCL development 10
1.5 Aim of the study 12
2 Materials and Methods 13
2.1 Materials 13
2.1.1 General material 13
2.1.1.1.1 Hardware 13
2.1.1.1.2 Consumable supplies 13
2.1.2 Chemicals 14
2.1.3 Solutions 14
2.1.4 Animals 14
2.1.5 Primary Antibodies 15
2.1.6 Secondary Antibodies 15
2.1.7 Software 15
2.2 Methods 15
2.2.1 Genotyping 15
2.2.2 Fixation and cryopreservation 16
2.2.3 Fluorescence Immunohistochemistry 16
2.2.4 Quantification of Microglia in the Cerebellum 16
2.2.5 Assessment of VGluT2 in the cerebellum 18
2.2.6 Statistical Analysis 19
3 Results 20
3.1 Postnatal enrichment of microglia cells in the cerebellar Purkinje cell layer 20
3.2 Microglial proximity to Climbing fibers 22
3.3 Population of the granular und molecular layer of CX3CR1 knock-out mice during early postnatal development 23
3.4 Influence of CX3CR1 knock-out on microglial morphology 25
3.5 Influence of CX3CR1 deletion on the VGluT2 expression during postnatal development 29
4 Discussion 31
4.1 Role of microglia in the developing cerebellum 31
4.2 CX3Cr1 Signaling and influence on microglial motility and morphology 32
4.3 CX3CR1 signaling and synaptic pruning 33
4.4 CX3CR1 signaling and formation of functional synapses 34
4.5 Correlation of immunohistological data with electrophysiological findings 35
5 Summary and conclusion 36
6 Zusammenfassung der Arbeit 38
7 References 41
8 Erklärung über die eigenständige Abfassung der Arbeit 49
9 Publications 50
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:76398 |
| Date | 02 November 2021 |
| Creators | Kaiser, Nicole |
| Contributors | Bechmann, Ingo, Hirrlinger, Johannes, Heppner, Frank, Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.1002/glia.23842 |
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