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Chronic Two-Photon Imaging of Neuronal Activity in Alzheimer's Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 Chronic two-photon imaging of neuronal activity in alzheimer’s disease animal models Bhat, Annapoorna Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-72425 Dissertation Published Version Originally published at: Bhat, Annapoorna. Chronic two-photon imaging of neuronal activity in alzheimer’s disease animal mod- els. 2012, University of Zurich, Faculty of Science. Chronic Two-photon Imaging of Neuronal Activity in Alzheimer’s Disease Animal Models Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Annapoorna Bhat aus Indien Promotionskomitee Prof. Dr. Roger M. Nitsch (Vorsitz, Leitung der Dissertation) Prof. Dr. Fritjof Helmchen (Leitung der Dissertation) Prof. Dr. Markus Rudin Prof. Dr. Esther Stöckli Zürich, 2012 1 2 “It is good to have an end to journey toward; but it is the journey that matters, in the end.” ― Ernest Hemingway 3 4 Table of Contents 1. Abbreviations ...........................................................................................................7 2. Summary/Zusammenfassung ...............................................................................11 3. Introduction............................................................................................................15 3.1 Alzheimer’s Disease .....................................................................................................17 3.1.1 Prevalence and risk factors.....................................................................................17 3.1.2 Clinical features......................................................................................................20 3.1.3 Neuropathology ......................................................................................................22 3.1.4 Neuronal dysfunction in AD ..................................................................................24 3.1.5 Mouse models of AD .............................................................................................26 3.1.6 Therapeutic strategies.............................................................................................29 3.1.7 Microglia in AD .....................................................................................................32 3.2 In Vivo Two-photon Imaging in Neocortex ...............................................................35 3.2.1 In vivo two-photon microscopy..............................................................................35 3.2.2 Calcium indicators to study neuronal networks .....................................................39 3.2.3 Rodent barrel cortex ...............................................................................................42 3.4 Two-Photon Microscopy in AD research...................................................................45 3.5 Aim and outline of thesis.............................................................................................48 4. Materials and Methods..........................................................................................51 4.1 Animals .........................................................................................................................53 4.2 Surgical Procedures.....................................................................................................53 4.2.1 Anesthesia ..............................................................................................................53 4.2.2 Post-surgery treatment and monitoring ..................................................................54 4.2.3 Preparation of thinned-skull and craniotomy .........................................................54 4.2.4 Mounting of a head post for head immobilization .................................................56 4.2.5 Preparation of chronic cranial windows.................................................................56 4.2.6 Termination criteria for recovery following surgery..............................................57 4.2.7 Brain dissection, fixation and histological reconstruction .....................................58 4.2.8 Immunohistochemistry...........................................................................................58 4.3 In Vivo Labeling Procedures.......................................................................................59 4.3.1 Viral gene transfer of genetically encoded markers and sensors ...........................59 4.3.2 Labeling of brain beta-amyloid plaques in vivo.....................................................60 4.3.3 Intravenous injection of fluorescent markers for labeling of blood vessels...........61 4.3.4 Topical application of fluorescent markers ............................................................61 4.4 Imaging Procedures.....................................................................................................61 4.4.1 Functional mapping using intrinsic signal imaging ...............................................61 4.4.2 In vivo two-photon microscopy..............................................................................62 4.4.3 Longitudinal imaging using cranial windows over several weeks.........................63 4.4.4 Sensory stimulation and optical recordings............................................................64 4.4.5 Time-lapse imaging of microglia ...........................................................................64 4.5 Immunotherapy ...........................................................................................................64 4.5.1 Intracerebral application of anti-Abeta antibodies .................................................64 4.5.2 Systemic passive immunization .............................................................................65 4.6 Data analysis.................................................................................................................65 5. Results .....................................................................................................................67 5.1 Standardization of chronic imaging protocols ..........................................................69 5.1.1 Use of head post reduces movement artifacts ........................................................69 5 5.1.2 Comparison between imaging through thinned-skull and craniotomy in living mice .........................................................................................................................................70 5.1.3 Improvements to the chronic craniotomy window imaging technique ..................71 5.1.4 Labeling of beta-amyloid plaques in living mice...................................................73 5.2 Various techniques to study microglia-plaque interaction ......................................74 5.2.1 In vivo appearance of typical parenchymal and vascular plaque ...........................74 5.2.2 Plaque targeted antibody application......................................................................75 5.2.3 Application of fluorescently tagged antibody ........................................................77 5.2.4 Time-lapse imaging of microglia after treatment with tagged and untagged antibody ...........................................................................................................................79 5.3 Imaging Cortical Neuronal Activity in Transgenic Models of Brain Amyloidosis 84 5.3.1 Expression of the genetically encoded calcium indicator: YC3.60........................84 5.3.2 Measuring calcium transients.................................................................................85 5.3.3 Spontaneous Activity .............................................................................................86 5.3.4 Evoked Activity in AD and wt control mice..........................................................89 5.3.5 Neuropil activity.....................................................................................................93 5.3.6 Immunohistochemistry...........................................................................................96 6. Discussion .............................................................................................................101 6.1 Standardization of chronic imaging protocols ........................................................103 6.2 Various techniques to study microglia-plaque interaction ....................................104 6.3 Neocortical Neuronal Activity in Alzheimer’s Disease Animal Models................105 6.3.1 Expression of YC3.60 and measurement of calcium tranients.............................105 6.3.2 Spontaneous activity in AD transgenic and wt control mice ...............................106 6.3.3 Evoked Activity in AD transgenic and wt control mice.......................................107 6.3.4 Neuropil Activity in AD transgenic and wt control mice.....................................108 6.3.5 Immunohistochemistry.........................................................................................108 7. Conclusion and Outlook......................................................................................111 8. References.............................................................................................................115 9. Supplementary .....................................................................................................131
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