3D reconstruction of synaptic and nuclear corticosteroid receptors distribution density in the amygdala a feasibility study

3D RECONSTRUCTION OF SYNAPTIC AND NUCLEAR CORTICOSTEROID RECEPTORS DISTRIBUTION DENSITY IN THE AMYGDALA: A FEASIBILITY STUDY Stephanie Koo BA Social Science (Psychology) (Honours) Submitted in fulfilment of the requirements for the degree of Masters of Applied Science (Research) HL84 Translational Research Institute (TRI) and Institute of Health and Biomedical Innovation (IHBI) School of Psychology and Counselling Queensland University of Technology (QUT) 2017 Keywords Adrenal Glands, Amygdala, Brain, Cytosol, Dendrite, Fear, Glucocorticoids, Membrane, Mineralocorticoids, Neuron, Nucleus, Post Synaptic Density, Spine, Stress, Synapse 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study i Abstract Disruptions to neuronal populations of corticosteroid receptors (glucocorticoid receptors; GR and mineralocorticoid receptors; MR) have been implicated in a range of stress-related pathologies; referred to as the Receptor Balance Hypothesis Traditionally, however, the receptor balance hypothesis only focuses on genomic populations of corticosteroid receptors, and does not account for membrane-associated corticosteroid receptors In this thesis, we tested the feasibility of using novel methods of reconstructing subcellular structures in order to characterise the distribution densities of GR and MR within the nucleus, and at excitatory post-synaptic terminals in the rat amygdala We used triple-label immunofluorescence in conjunction with confocal imaging to characterise the labelling of corticosteroid receptors Using Imaris™ software, we found that we could three-dimensionally reconstruct corticosteroid receptors, and perform objectbased colocalisation analysis, in order to quantify the populations of corticosteroid receptors located at excitatory post-synaptic sites This provides a novel method of quantifying corticosteroid receptors in amygdala tissue The adaptability of the method suggests that it could be applicable to a range of applications in stress research ii 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study Table of Contents Keywords i Abstract ii Table of Contents iii List of Figures vii List of Tables xiii List of Abbreviations xiv Statement of Original Authorship xv Acknowledgements xvi Chapter 1: Introduction Functional Role of Corticosteroids The Amygdala and Corticosteroids Corticosteroid Receptors 10 A Rationale for Quantifying Corticosteroid Receptor Subpopulations 13 Fluorescent Imaging and Reconstruction of Corticosteroid Receptor Subtypes… 14 Thesis Objectives and Outline 18 Chapter 2: Corticosteroid Receptors 21 Dosage Effects of Corticosteroids on Corticosteroid Receptors 21 Corticosteroid Receptors in the Amygdala 22 Temporal Effects of Corticosteroid Receptors 24 Receptor Balance Hypothesis 30 Summary and Implications 33 Chapter 3: General Method 37 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study iii Subjects 37 Antibodies 38 Primary Antibodies 39 Fluorescent Labels 41 Procedure 43 Tissue Preparation 43 Immunohistochemistry 44 Confocal Imaging 45 Image Processing 46 Design 47 Controls 47 Operationalisation of Variables 49 Ethics and Limitations 53 Ethics and Handling 53 The applicability of Animal Research to Humans in Stress 53 Chapter 4: Protocol Validation 55 Method 55 Subjects and Procedure 55 Design 57 Results and Discussion 59 Reagent optimisation 59 Labelling Specificity 63 Characterisation of Triple Labelling 68 Summary 76 iv 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study Chapter 5: Corticosteroid Receptor Densities in the Amygdala 79 Method 80 Subjects and Procedure 80 Design 83 Results 83 Controls 83 Mosaic Images 84 Deconvolution of Images 86 Nuclear Surfaces 90 Creation of Genomic GR and MR 92 Creation of Extra-nuclear GR and MR and Post-synaptic Terminals 94 Colocalisation of Corticosteroid Receptors at Post-synaptic Terminals 95 Corticosterone levels 98 Analysis 98 Descriptive Statistics 98 Genomic Corticosteroid Receptors vs Corticosteroid Receptors at Post-Synaptic Terminals 104 Proportion of Synapses that contain Corticosteroid Receptors 106 Chapter 6: Discussion 111 Applicability of 3D Reconstruction for Characterising Corticosteroid Receptors112 GR and MR labelling can be Reconstructed as Spots 112 Nuclei can be reconstructed as Surfaces to identify gGR and gMR populations 115 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study v Distribution Densities of GR and MR 116 Distribution of gGR and gMR in Amygdala Subnuclei 116 Distribution of Genomic and Colocalised Corticosteroid Receptors 118 Proportion of Excitatory Post-synaptic Terminals Containing Corticosteroid Receptors 119 Chapter 7: Conclusions 123 Reagent and Protocol Validation 123 3D reconstruction of Corticosteroid Receptors 124 References 129 vi 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study List of Figures Figure The release of corticosteroids (Cort) via the Hypothalamic Pituitary Adrenal (HPA) Axis Figure Depiction of the organisation of subnuclei (labelled for GR) in a coronal section of the rat amygdala, under wide-field epifluorescence Overlay adapted from Figure 31 of Stereotaxic Coordinates (Paxinos & Watson, 1997) 4-point axis refers to the orientation of the section: D, dorsal; V, ventral; M, medial; L, lateral LA, lateral amygdala; BA, basal amygdala; CeA, central amygdala Figure The amygdala receives excitatory inputs from the hippocampus, thalamus and mPFC during stress – these circuits underlie Pavlovian conditioning and drive activation of the HPA axis from the CeA Excitatory intra-amygdaloid circuits are also activated during stress by corticosteroids Figure Factors that interact with MR and GR to affect Cognition and Behaviour 12 Figure Distribution of Genomic and Synaptic GR and MR within a neuron Genomic GR and MR are located within the cytoplasm, and translocate to the nucleus when bound Synaptic GR and MR are located near or within the membrane at synapses; when activated, these receptors can affect neurotransmission 25 Figure Corticosteroid receptors in the BLA-complex mediate neuronal excitability differently to the hippocampus Corticosteroids increase neuronal excitation in the BLA-complex, through mMR This excitability is maintained through gGR Further application of corticosterone depressed neuronal excitability through mGR 3D Reconstruction of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study vii Adapted from research by Karst et al (2010), Groeneweg et al (2011), and Sarabdjitsingh and Jӧels (2014) 29 Figure Excitation and Emission Spectrum for DAPI, Alexa Fluor 488 and Alexa Fluor 594 Adapted from Life Technologies (2015a) 42 Figure The LA, BA and CeA in a coronal section, regions sampled in grey Sections were taken -2.04mm to -3.36mm from the bregma according to the rat brain atlas (Paxinos & Watson, 2007) Adapted from “The Rat Brain in Stereotaxic Coordinates 6th edition,” by G Paxinos and C Watson, 2007, p.56 50 Figure Groups involved in MR titration of antibodies 58 Figure 10 Representative image of fluorescent labelling of PSD-95-like immunoreactivity at concentrations of 1:500, 1:750 and 1:1000 (epifluorescence), in rat brain tissue A non-linear contrast was applied in Photoshop using the curves function Transformation was applied uniformly to all three images to improve the contrast Images were taken with a 60x (1.25 NA) oil objective Scale bar: 10µm 60 Figure 11 a), b), c), and d) show tissue sections incubated with rMR-1D5 at a dilution of 1:200; where the top two images are sections incubated 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Goat Antirabbit IgG H&L (Alexa Fluor 594) Abcam VIC, Australia AB150084 - 1.96mg/ml 4`,6diamidino-2phenylindole, dihydrochlorid e (DAPI) Life Technologies VIC, Australia D1306 1711782 158 Appendices Appendix B Acquisition Parameters Table Acquisition Parameters of Images with Olympus FV1200 Solid State Laser LD 405nm (50nw) LD 473nm (15mw) LD 559nm (15mw) Laser Power 0.9% 3.0% (GR)/5.0% (MR) 3.0% HV 855 760 (GR)/810 (MR) 800 Offset 1 Gain 37 46 (GR)/40 (MR) 37 Appendices 159 Appendix C Nyquist Sampling Parameters Table Nyquist Sampling Parameters from Scientific Volume Imaging (n.d.) Microscopy Parameter Value Microscope Type Confocal Numerical Aperture 1.35 Excitation Wavelength 488 Emission Wavelength 520 Number of Excited Photons 1.0 Lens Immersion Refractive Index Oil, 1.515 Scientific Volume Imaging (n.d.) Microscopy Nyquist rate and PSF calculator Retrieved from: https://svi.nl/NyquistCalculator 160 Appendices Appendix D Ethics Appendices 161 Appendix E Supplementary Videos Table Links to Supplementary Videos of 3D Objects in Imaris (Bitplane) Video URL Video 1, Process of 3D Reconstruction https://youtu.be/9kgq69dt1ls Video 2, High Magnification view of 3D objects in Imaris https://youtu.be/cs3e4UxgAdY 162 Appendices ... Amygdala: A Feasibility Study xiii List of Abbreviations ACTH Adrenocorticotrophic Releasing Hormone BLA Basolateral Amygdala BA Basal Nuclei of the Amygdala CeA Central Nuclei of the Amygdala CRH... of Synaptic and Nuclear Corticosteroid Receptors Distribution Density in the Amygdala: A Feasibility Study Overlay in white displays the boundaries of the different subregions of the amygdala. .. The amygdala can be broadly divided into three subregions; the basolateral amygdala complex (BLA), the central amygdala (CeA) and the intercalated cells (ITC) (Giustino & Maren, 2015; Pape & Pare,