Acknowledgements Pursuit of a Ph.D degree is an important time in my life and an exciting journey into discovery I would like to first and foremost thank my supervisors Professor Sheu FwuShan for his guidance, encouragement and help Prof Sheu has been exceedingly generous with his time and advice all the years and taught me molecular and cellular neuroscience I also would like to thank my co-supervisor Professor Lim Tit Meng for his support in the most tough 2-year extension to complete my PhD works in DBS My heartfelt thanks also go to Professor Aryeh Routtenberg in Northwestern University for his precious suggestions in all the three projects of my PhD work I would thank Professor Frank Watt in Center for Ion Beam Applications (CIBA), Dept of Physics, NUS for his advice in the project of quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy; Dr Chuang Kai-Hsiang in Singapore Bioimaging Consortium (SBIC) for his professional knowledge in the collaboration of the project using manganese-enhanced magnetic resonance imaging (MEMRI) Thanks also go to Dr Sashi Kesavapany in Dept of Biochemistry, NUS for the generous providing the transgenetic mice from his lab I am also grateful to Dr Ren Minqin in Center for Ion Beam Applications (CIBA), Dept of Physics, NUS for her invaluable suggestions and help in my research I could not complete the work without her knowledge and emotional support I would also acknowledge the financial support of National University of Singapore and spiritual support from my family through this special and important period of my life i Table of Contents Acknowledgements……………………………………………………………………… i Table of Contents………………………………………………………………………….ii Abstract ………………………………………………………………………………… x List of Tables…………………………………………………………………………….xii List of Figures………………………………………………………………………… xii Publications…………………………………………………………………………… xiv Part I Investigations on Learning-Induced Remodeling of Hippocampal Mossy Fibers: The Role of Presynaptic Structural Plasticity in Long-Term Memory ……………………… Chapter Introduction………………………………………….…………………… 1.1 Introduction of hippocampal mossy fibers…… 1.1.1 Anatomy of the hippocampal mossy fibers……………………………… 1.1.1.1 Introduction of the hippocampus……………………………… 1.1.1.2 Introduction of the mossy fibers………………………………… 1.1.2 Histology of Mossy fiber pathways……………………………………… 1.1.3 Anatomic plasticity of the mossy fibers…………………… 1.2 Plasticity of the mossy fibers……………… .9 1.2.1 Short-term plasticity… 10 1.2.2 Mechanisms of short-term plasticity…… 11 1.2.2.1 A1 adenosine receptors………………… .12 ii 1.2.2.2 Metabotropic glutamate receptors………………… 12 1.2.2.3 Kainate receptors………………………………………… 13 1.2.2.4 Intrinsic conductances………………………… 15 1.2.3 Long-term plasticity - long-term potentiation……… 15 1.2.3.1 NMDA receptor independent……………… .16 1.2.3.2 Postsynaptic calcium elevation……………… 17 1.2.3.3 Cyclic AMP ………………………………………… 17 1.2.3.4 Downstream targets of PKA and PKC … 18 1.2.4 Long-term depression………………………………………… 18 1.3 Mossy fibers in learning and memory……………………………………… 19 1.3.1 Role of mossy fibers plasticity in learning and memory 19 1.3.2 Species and strains difference in mossy fibers…… 21 1.4 Significance of studies………………………………………… .23 1.4.1 Missing gap………………………………………… 23 1.4.2 Objectives………………………………………… 25 Chapter Materials and methods…………………………………… …… 27 2.1 Animal model ………………………………………… 27 2.2 Neuroanatomical methods ………………………………………… .27 2.2.1 Brain tissue preparation………………………………………… 28 2.2.1.1 Preparation of unfixed fresh-frozen brain tissue……… 28 2.2.1.1.1 Materials………………………………………… .28 2.2.1.1.2 Protocol………… 28 iii 2.2.1.2 Perfusion fixation………………… .29 2.2.1.2.1 Materials…………………………………… .29 2.2.1.2.2 Protocol……………………………… 30 2.2.2 Cryostat sectioning of frozen brain tissue…………………………… 31 2.2.2.1 Materials………………………………………… 32 2.2.2.2 Protocol……………………………………… 32 2.2.3 Histological study……………………………… .33 2.2.3.1 Nissl stain … 33 2.2.3.2 Timm’s stain .……………………………… 34 2.2.3.2.1 Solution preparation 34 2.2.3.2.2 Protocol 37 2.2.3.3 Immunohistochemistry…………………………… 37 2.2.3.3.1 Protocol for avidin-biotin complex (ABC) method 38 2.3 Behavioral studies – Morris water maze ………… 39 Chapter In vivo analysis of hippocampal mossy fiber remodeling by manganeseenhanced magnetic resonance imaging (MEMRI) … .40 3.1 Introduction ………… .40 3.1.1 Remodeling of the mossy fibers ………… 40 3.1.2 Histological method of detection ………… .40 3.1.3 Introduction of MEMRI ………… 42 3.1.4 Objectives ………… 44 3.2 Materials and methods ………… 45 iv 3.2.1 Subjects ………… 45 3.2.2 Hidden platform water maze task ………… 46 3.2.2.1 Apparatus ………… 46 3.2.2.2 Procedures ………… 46 3.2.3 MEMRI experiments ………… .47 3.2.3.1 Animal preparation ………… 47 3.2.3.2 MRI acquisition ………… 47 3.2.3.3 Data processing ………… 48 3.2.3.3.1 Alignment of 3D MRI Volumes ………… 48 3.2.3.3.2 Manual Segmentation of the Mossy Fibers ……… 51 3.2.3.3.3 Quantification of Mossy Fiber Area .52 3.2.3.3.4 Statistical Analysis ………… .52 3.2.4 Timm’s staining ………… 52 3.2.4.1 Timm’s staining… 52 3.2.4.2 Timm’s analysis… .53 3.2.5 Statistical analysis ………… 55 3.3 Results ………… .55 3.3.1 MEMRI allows imaging hippocampal mossy fiber in vivo and longitudinally .55 3.3.2 LH rats performed better than the Wistar rats in the hidden platform overtraining water maze task .57 3.3.3 Overtraining-induced expansion of mossy fiber terminal fields was observed in Wistar rats not in Lister-Hooded rats .61 v 3.3.4 MEMRI analysis of learning-induced remodeling of the hippocampal mossy fibers in vivo 68 3.4 Discussion 77 Chapter Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy .81 4.1 Introduction .81 4.1.1 Biological functions of Zn 81 4.1.2 Zn in the brain .81 4.1.3 Rationale 83 4.1.4 Aim 85 4.2 Materials and methods .85 4.2.1 Animals and tissue preparations 85 4.2.2 Histology analysis .87 4.2.2.1 Timm’s staining 87 4.2.2.2 Nissl staining 88 4.2.3 Mapping and quantifying Zn: Nuclear microscopy 88 4.2.3.1 Introduction of nuclear microscopy .88 4.2.3.2 Mapping and quantifying Zn .90 4.3 Results 91 4.3.1 Elemental mapping of hippocampus 91 4.3.2 Zn profile by line scan in CA3 93 4.3.3 Quantitative analysis of Zn 98 vi 4.4 Discussion .102 PART II The Role of Cyclin-Dependent Kinase (Cdk5) in Consolidation of Fear Memory 106 Chapter Hippocampus dependent memory is impaired in heterozygous cyclindependent kinase knockout mice (Cdk5+/-) 106 5.1 Introduction .106 5.1.1 Introduction of fear conditioning 106 5.1.2 Introduction of Cdk5 108 5.1.2.1 Cdk5 in the development of the nervous system .109 5.1.2.2 Cdk5 in presynapse 110 5.1.2.3 Cdk5 in postsynapse 110 5.1.3 Pharmacological studies on Cdk5 in learning and memory 112 5.1.4 Rational and objective .113 5.2 Materials and methods .113 5.2.1 Animals .114 5.2.2 Apparatus 114 5.2.3 Experiment procedures .115 5.2.3.1 Fear conditioning training 115 5.2.3.2 Short-term contextual memory and cued memory test 115 5.2.3.3 Retention and extinction of long-term contextual memory and cued memory 116 5.2.4 Genotyping analysis 116 vii 5.2.5 Western blot 118 5.2.6 Immunohistochemistry 119 5.2.7 Optical density measurements 120 5.2.8 Statistical analysis .120 5.3 Results .120 5.3.1 Reduction of Cdk5 protein in the Cdk5+/- mice 120 5.3.2 Training of contextual fear conditioning was not affected in Cdk5+/mice 125 5.3.3 Both short-term and long-term contextual memory was impaired in Cdk5+/mice 125 5.3.4 Cdk5+/- mice showed no impairments in cued fear memory .131 5.3.5 Reduction of Cdk5 facilitates contextual extinction but not cued extinction 131 5.4 Discussions 132 PART III Summary and Future works 136 Chapter Summary and contributions 136 6.1 In vivo analysis of hippocampal mossy fiber remodeling by manganese-enhanced magnetic resonance imaging (MEMRI) 136 6.2 Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy .137 6.3 Hippocampus dependent memory is impaired in heterozygous cyclin-dependent kinase knockout mice (Cdk5+/-) 138 viii Chapter Future works 140 7.1 In vivo analysis of hippocampal mossy fiber remodeling by manganese-enhanced magnetic resonance imaging (MEMRI) 140 7.2 Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy .140 7.3 Hippocampus dependent memory is impaired in heterozygous cyclin-dependent kinase knockout mice (Cdk5+/-) 141 References………………………………………………………………………………142 ix Abstract There are two parts of work which have been done in this thesis to investigate the underlying mechanisms of learning process and long-term memory formation In part I, two projects have been focused on the hippocampal mossy fibers (MFs), aiming at elucidating the role of presynaptic structural plasticity in long-term memory In the first study, the MRI technique, in which manganese-enhanced magnetic resonance imaging (MEMRI) is employed, has been implemented to detect the learning-induced MFs remodeling during the spatial memory formation in vivo Using a quantitative analysis of a series of sections in the rostral dorsal hippocampus, MEMRI showed an increase in the high contrast area CA3a’ area in trained Wistar rats Besides, the hypothesis that remodeling pattern of the MFs is strain-dependent has been confirmed by comparison between two strains of rats: Wistar and Lister-Hooded (LH) rats In contrast to Wistar, no obvious remodeling of MFs was found in trained LH So far as we know, this is the first study to employ MEMRI to visualize the structural redistribution of the hippocampal MFs that is induced by spatial learning In the second study, the laminar-specific Zn levels in the synapses from MFs to CA3 region of the hippocampus have been mapped and quantified in µg/g dry weight level as well as molar concentration in wet weight level, using an alternative approach nuclear microscopy This is the first reported data showing the Zn concentration in the three different strata (stratum lucidum SL, stratum pyramidale SO, and stratum oriens SP) of hippocampal CA3 region (Zhang et al., 2011) As the axon fibers projecting to CA3 pyramidal neurons, the MFs play a critical role in the hippocampal-related learning and x long-term memory procedures Investigations on the learning-induced presynaptic plasticity might shed lights on the complicated mechanisms underlying the hippocampalrecruited long-term memory In part II, a mutant mouse model, heterozygous knockout mouse (Cdk5+/-) has been employed to evaluate the critical role of cyclin-dependent kinase (Cdk5) in fear learning and memory The data here has shown that deficiencies in both short-term (3 hours) and long-term (24 hours) contextual fear memory were observed as the result of reduced Cdk5 protein in Cdk5+/- mice In addition, Cdk5+/- facilitates the extinction of contextual fear conditioning This is the first study to use heterozygous Cdk5 knockout mice to test the hypothesis that reducing levels of Cdk5 would impair performance in contextual fear conditioning This data is complementary to previous pharmacological studies The mutant model Cdk5+/- employed in the study makes possible for further investigations on the different molecular mechanisms involved in amygdala-related cuedor tone-dependent fear conditioning xi List of Tables Table 4.1 Average concentrations and standard errors of Fe Cu and Zn in different layers and background levels, based on seven sections from seven rats 100 List of Figures Figure 1.1 Basic anatomy of the hippocampus formation .……………… Figure 1.2 Hippocampal mossy fiber pathways of a Wistar rat ……… Figure 3.1 Alignment of 3D MRI to bregma in atlas ………………………49 Figure 3.2 Images for the slices chosen for segmentation at -2.52 mm, -2.64 mm, -2.76 mm, -2.88 mm, -3.00 mm, -3.12 mm, -3.24 mm, -3.36 mm, -3.48 mm to bregma …………………50 Figure 3.3 Segmentation of MRI images .…… 51 Figure 3.4 Coronal MEMRI of rat brain at 20 h, days, and days post intravenous infusion of MnCl2 at the dose of 80 mg/kg ………………………………… 57 Figure 3.5 Lister-Hooded rats (LH) show better performance than Wistar rats in the hidden platform overtraining water maze task ……59 Figure 3.6 Overtraining-induced expansion of mossy fiber terminal fields was observed in Wistar rats .…………………………… 64 Figure 3.7 Hidden platform overtraining water maze exposure has no effect on the expansion of the mossy fibers of LH rats ……………66 Figure 3.8 Overtrained Wistar have apparent remodeling of the MFs in anterior dorsal hippocampus, with coordinates from -2.52 mm to -3.48 mm to bregma ……… 70 Figure 3.9 Quantitative analysis of the mossy fiber terminal fields in CA3a’ of Wistar xii rats by MEMRI ……………………………………………… 72 Figure 3.10 Overtraining task has no effect on the MFs distribution of LH rats …………………………………………………………… 74 Figure 3.11 Quantitative MRI analysis of CA3a’ of LH rats ………………………….76 Figure 4.1 Brain atlas at -3.12 mm from bregma considered as the position reference ………… 87 Figure 4.2 The nuclear microscope at the Center for Ion Beam Applications (CIBA), Dept of Physics, National University of Singapore ……………………………………91 Figure 4.3 PIXE energy spectrum of the hippocampus showing characteristic X-ray peaks: Plotted as a log plot …………………………………………… 95 Figure 4.4 Images of the region of interest CA3 …………………………………96 Figure 4.5 Elemental zinc profile across the layers of CA3 … 97 Figure 4.6 ……… 99 Figure 5.1 Heterozygous cyclin-dependent kinase knockout mice (Cdk5+/-) were confirmed by genotyping through polymerase chain reaction (PCR) 117 Figure 5.2 Heterozygous Cdk5 knockouts (Cdk5+/-) have reduced levels of Cdk5 protein but normal levels of tubulin ……………………………….122 Figure 5.3 Cdk5+/- mice have a uniform decrement in Cdk5 protein in limbic regions ………………………123 Figure 5.4 Cdk5+/- mice showed reduced short-term contextual memory, but no impairments of fear conditioning training and short-term cued fear memory .……127 Figure 5.5 Reduction of Cdk5 impairs long-term contextual memory and facilitates contextual extinction .……………………… 129 xiii Publications (* Publications for the thesis’s work) 1.* B Zhang, M.Q Ren, Fwu-Shan Sheu, F Watt, and A Routtenberg Quantitative elemental analysis of zinc in mossy fiber by nuclear microscope, submitted to Journal of Neuroscience Methods 2.* B Zhang, K.H Chuang, Fwu-Shan Sheu, and A Routtenberg In vivo analysis of hippocampal mossy fiber remodeling by manganese-enhanced magnetic resonance imaging (MEMRI), to be submitted 3.* B Zhang, Sashi Kesavapany, Fwu-Shan Sheu, and A Routtenberg Hippocampal dependent memory is impaired in heterozygous cyclin-dependent kinase knockout mice (cdk5+/-), to be submitted S Homhuan, B Zhang, F Sheu A.A Bettiol and F Watt Single cell electroporation using proton beam fabricated biochips, Proceedings of SPIE Vol 7716, 77160V (2010) S K Vashist, B.B Zhang, D Zheng, K Al-Rubeaan, J.H.T Luong, F Sheu, Sulfo-Nhydroxysuccinimide interferes with bicinchoninic acid protein assay, Analytical Biochemistry 417 (2011) 156–158 S Homhuan, B Zhang, F Sheu A.A Bettiol and F Watt, Mouse Neuroblastoma cell electroporation using proton beam fabricated biochips, (Submitted to Biomedical Microdevices) xiv ... 5.2.3.1 Fear conditioning training 115 5.2.3.2 Short-term contextual memory and cued memory test 115 5.2.3.3 Retention and extinction of long-term contextual memory and cued memory ... which have been done in this thesis to investigate the underlying mechanisms of learning process and long-term memory formation In part I, two projects have been focused on the hippocampal mossy... and x long-term memory procedures Investigations on the learning- induced presynaptic plasticity might shed lights on the complicated mechanisms underlying the hippocampalrecruited long-term memory