Science, Faculty of
Zoology, Department of
DSpace
UBCV
Barraclough, Bridget Nicole
Doctor of Philosophy - PhD
University of British Columbia
Neural plasticity allows us to form new memories and learn new behaviours. After neurotrauma, it allows adaptation to and, to a limited extent, recovery of lost function. Even following severe injuries such as spinal cord injury (SCI), most of which spare some ascending and descending circuitry, there is a degree of functional restoration that reflects reorganization of remaining neural pathways. Understanding and exploiting mechanisms of spontaneous plasticity is an avenue to generating new approaches to a cure for SCI. To date, most of the experimental work on functional recovery following SCI has focused on descending projections that subserve voluntary movement and locomotion; however, these are initiated and informed by (and are thus dependent upon) sensory input from the body to the brain via “spinofugal” projections. Here I have exploited the developmental expression of Hoxb8, which is restricted to the spinal cord, to genetically label ascending projections to the cerebellum. A detailed anatomical analysis of four reporter lines identified many unexpected Hoxb8-lineage neuronal and non-neuronal cell types in targeted and untargeted transgenic strains, but because of the near-exclusive restriction of reporter expression to spinofugal projections, Hoxb8FlpOᵀᵈᵀ mice were selected as the best model for assessing the distribution of spinocerebellar projections and their possible structural plasticity. I used a cervical (C3) lateral hemisection model to document the natural history of sensorimotor performance over the first month following injury, employing established and novel behavioural measures. These revealed that many behavioural parameters changed spontaneously with time during the post-injury period, while several did not (i.e. were stable deficits), or did not change with injury at all. Since some of the behaviours which recovered (e.g. those which depend on grasp) normally require cerebellar function, I asked whether plasticity of spinocerebellar projections might have contributed. I found that at one month after hemisection, remaining spinocerebellar projections had increased in density (i.e. had sprouted), size, and content of presynaptic machinery, implicating these as possible substrates of functional recovery. I also identified transcriptomic changes in the partially-deafferented cerebellum expected as a consequence of axonal degeneration, and others which suggest how these might lead to spontaneous compensatory plasticity.
HOXB8 LINEAGE TRACING TO MAP SPINOFUGAL PROJECTIONS RELATED TO MOTOR RECOVERY FOLLOWING SPINAL CORD INJURY by Bridget Nicole Barraclough B.Sc., Queen’s University, 2020 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2025 © Bridget Nicole Barraclough, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Hoxb8 lineage tracing to map spinofugal projections related to motor recovery following spinal cord injury submitted by Bridget Nicole Barraclough in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Zoology Examining Committee: Matthew Ramer, Associate Professor, Zoology, UBC Supervisor Michael Gordon, Professor, Zoology, UBC Supervisory Committee Member Shernaz Bamji, Professor, Cellular and Physiological Sciences, UBC University Examiner Lara Boyd, Professor, Faculty of Medicine, UBC University Examiner Roy Sillitoe, Professor, Neuroscience, Baylor College of Medicine External Examiner Additional Supervisory Committee Members: Wolfram Tetzlaff, Professor, Zoology, UBC Supervisory Committee Member Brian MacVicar, Professor, Faculty of Medicine, UBC Supervisory Committee Member iii Abstract Neural plasticity allows us to form new memories and learn new behaviours. After neurotrauma, it allows adaptation to and, to a limited extent, recovery of lost function. Even following severe injuries such as spinal cord injury (SCI), most of which spare some ascending and descending circuitry, there is a degree of functional restoration that reflects reorganization of remaining neural pathways. Understanding and exploiting mechanisms of spontaneous plasticity is an avenue to generating new approaches to a cure for SCI. To date, most of the experimental work on functional recovery following SCI has focused on descending projections that subserve voluntary movement and locomotion; however, these are initiated and informed by (and are thus dependent upon) sensory input from the body to the brain via “spinofugal” projections. Here I have exploited the developmental expression of Hoxb8, which is restricted to the spinal cord, to genetically label ascending projections to the cerebellum. A detailed anatomical analysis of four reporter lines identified many unexpected Hoxb8-lineage neuronal and non-neuronal cell types in targeted and untargeted transgenic strains, but because of the near-exclusive restriction of reporter expression to spinofugal projections, Hoxb8FlpOTdT mice were selected as the best model for assessing the distribution of spinocerebellar projections and their possible structural plasticity. I used a cervical (C3) lateral hemisection model to document the natural history of sensorimotor performance over the first month following injury, employing established and novel behavioural measures. These revealed that many behavioural parameters iv changed spontaneously with time during the post-injury period, while several did not (i.e. were stable deficits), or did not change with injury at all. Since some of the behaviours which recovered (e.g. those which depend on grasp) normally require cerebellar function, I asked whether plasticity of spinocerebellar projections might have contributed. I found that at one month after hemisection, remaining spinocerebellar projections had increased in density (i.e. had sprouted), size, and content of presynaptic machinery, implicating these as possible substrates of functional recovery. I also identified transcriptomic changes in the partially-deafferented cerebellum expected as a consequence of axonal degeneration, and others which suggest how these might lead to spontaneous compensatory plasticity. v Lay Summary The nervous system is “plastic”, enabling us to learn, to form new memories, and to partially recover lost function after spinal cord injury (SCI). Mitigating functional deficits after SCI relies on the sensory nervous system by detecting internal and external stimuli in order to guide behaviour. To better understand this process, I examined spontaneous recovery – or lack thereof - after incomplete SCI in mice using a variety of behavioural tests. I also characterized a mouse model which allows for all sensory fibres originating in the spinal cord and terminating in the brain to be visualized under a microscope. I subjected these mice to an incomplete SCI to determine whether spared fibres had made new connections one month after injury in the cerebellum, a site of integration of sensory and motor signals. These results demonstrated structural changes (fibre sprouting) in the sensory system after SCI that may contribute to functional recovery. vi Preface The work in this dissertation is the original, independent work by the author B. Barraclough. All chapters were written by the author and revised and edited by Dr. Matt Ramer. No Generative Artificial Intelligence tools were used in the development or writing of this thesis. All experiments were performed in accordance with the Canadian Council on Animal Care under approval of the Animal Care Committee at the University of British Columbia under certificate number A22-0001. A version of Chapter 3 has been published [Barraclough, B., Stubbs, T., Bohic, M., Upadhyay, A., Abrairia, V., & Ramer, M. (2024). Direct comparison of hoxb8-driven reporter distribution in the brains of four transgenic mouse lines: towards a spinofugal projection atlas. Frontiers Neuroanatomy, 18. doi: https://doi.org/10.3389/fnana.2024.1400015]. I was the primary author responsible for data curation, investigation, methodology, visualization, and writing (original draft, review, and editing). Ramer, M was the supervisory author on this project involved in conceptualization, funding acquisition, and writing (original draft, review, and editing). A portion of the findings in Chapter 3 are also published [Bohic, M., Upadhyay, A., Eisdorfer, J., Keating, J., Simon, R., Briones, B., Azadegan, C., Nacht, H., Oputa, O., Maritnez, A., Bethell, B., Gradwell, M., Romanienko, P., Ramer, M., Stuber, G., Abraira, V. (2023). A new Hoxb8FlpO mouse line for intersectional approaches to dissect developmentally defined adult sensorimotor circuits. Frontiers, 16 doi: 10.3389/fnmol.2023.1176823]. I conducted the experiments and created the figure for this publication with review from Dr. Matt Ramer. vii Monitoring and supportive care for the experiments in Chapters 2, 3, 4, and 5 were done primarily by the author with assistance from Elford, H. Holland, S. Smaila, B. & Drover, J. Behavioural scoring in Chapter 2 was done by the author and Wheeler, S (BMS). The spinal cord hemisections were performed by the author and Ramer, M. The sequencing data (Chapter 5) was produced in collaboration with the author and the Vancouver Prostate Centre Genomics Core Lab. viii Table of Contents Abstract ...................................................................................................................... iii Lay Summary .............................................................................................................. v Preface ....................................................................................................................... vi Table of Contents .................................................................................................... viii List of Tables ............................................................................................................ xii List of Figures .......................................................................................................... xiii List of Abbreviations ............................................................................................... xvi Acknowledgements ............................................................................................... xxiv Dedication ............................................................................................................... xxv Chapter 1: General Introduction ................................................................................ I 1.1 Ascending and Descending Tracts ................................................................ 2 1.2 The Cerebellum ............................................................................................. 3 1.2.1 Synaptogenesis and Development of the Cerebellum ............................... 3 1.2.2 Cerebellar Circuitry ..................................................................................... 7 1.3 The Spinocerebellar Tract .............................................................................. 9 1.4 Spinal Cord Injuries ..................................................................................... 13 1.5 Recovery after SCI ....................................................................................... 14 1.5.1 Avenues to Recovery: Neuroprotection, Regeneration, and Plasticity ..... 14 1.5.2 Long-Term Potentiation in the Hippocampus ........................................... 15 1.5.3 Plasticity in the Cerebellum ...................................................................... 16 1.5.4 Trauma Induced Plasticity ........................................................................ 17 1.5.5 The Synaptic Active Zone and Plasticity .................................................. 18 1.6 Anatomical Methods to Investigate Plasticity ............................................... 19 1.6.1 The Use of Degeneration Studies to Understand Circuitry ...................... 19 1.6.2 Intrinsically Expressed Molecules ............................................................ 20 1.6.3 Viral and Non-Viral Tracing ...................................................................... 20 1.7 Hoxb8-Driven Recombinase Expression Serves as a Strategy to Trace Spinofugal Projections ............................................................................................ 22 1.7.1 Homeobox Genes .................................................................................... 22 1.7.2 Hoxb8 ....................................................................................................... 23 1.8 Hoxb8 Reporter Mice ................................................................................... 24 1.9 Overall Aims ................................................................................................. 25 Chapter 2: The Natural History of Motor-Related Behavioural Changes Following Unilateral Cervical Hemisection ............................................................ 27 2.1 Introduction ....................................................................................................... 27 ix 2.2 Methods ............................................................................................................ 30 2.2.1 Animals ...................................................................................................... 30 2.2.2 Surgical Procedures ................................................................................... 30 2.2.3 Behavioural Tests ....................................................................................... 31 2.2.4 Behavioural Statistical Analysis ................................................................. 33 2.3 Results .............................................................................................................. 36 2.3.1 Noldus CatWalk ......................................................................................... 36 2.3.2 A Finer-Grained Look at Recovery on the Horizontal Ladder, Grasping, and Noldus CatWalk .................................................................................................. 40 ............................................................................................................................ 42 2.3.3 The Combined CatWalk Index Reveals that the Parameters Obtained by the Noldus CatWalk Are Not a Strong Measure for Spontaneous Recovery ...... 42 2.4 Discussion ........................................................................................................ 47 2.4.1 Functional Recovery Occurred Following Unilateral Cervical Hemisection 47 2.4.2 Limitations with the Noldus CatWalk .......................................................... 48 2.4.3 Several Parameters on the Noldus CatWalk Had Stable Deficits or No Change Following C3 Hemisection ..................................................................... 49 2.4.4 Recovery or Adaptation to a “New Normal”? ............................................. 50 2.4.5 The Battery of Tests May Impact Results ................................................... 51 2.4.6 The Noldus CatWalk and Pain ................................................................... 52 2.4.7 Concluding Remarks .................................................................................. 53 Chapter 3: A Comparison of Hoxb8-Driven Reporter Distribution in the Brains of Four Transgenic Mouse Lines ................................................................................. 54 3.1 Introduction ....................................................................................................... 54 3.2 Methods ............................................................................................................ 58 3.2.1 Animals and Specimen Collection .............................................................. 58 3.2.2 Lateral Spinal Hemisection ........................................................................ 59 3.2.3 Tissue Processing ...................................................................................... 59 3.2.4 Immunohistochemistry ............................................................................... 60 3.2.5 Nissl staining – spinal cords ....................................................................... 61 3.2.6 Image acquisition ....................................................................................... 61 3.2.7 Light Sheet Microscopy .............................................................................. 63 3.3 Results .............................................................................................................. 64 3.3.1 Gross tdtomato expression in Hoxb8 reporter mice ................................... 64 3.3.2 Reporter expression in the spinal cord ...................................................... 66 3.3.3 Spinofugal projections in Hoxb8-reporter mice .......................................... 68 3.3.4 Ectopic recombination in non-targeted Hoxb8 transgenic reporter mice ... 82 3.3.5 Microglia ..................................................................................................... 87 3.4 Discussion ........................................................................................................ 87 x 3.4.2 Regulation of Hox Gene Expression and Implications of its Dysregulation in Lineage Tracing .................................................................................................. 88 3.4.3 Concluding Remarks .................................................................................. 93 Chapter 4: Spinocerebellar Tract Plasticity Following Incomplete Spinal Cord Injury .......................................................................................................................... 94 4.1 Introduction ....................................................................................................... 94 4.2 Methods ............................................................................................................ 98 4.2.1 Animals and Surgical Procedures .............................................................. 98 4.2.2 Specimen Collection and Lightsheet Microscopy ...................................... 98 4.2.3 Short-Term Hemisection ............................................................................ 99 4.2.4 Confocal Microscopy Sample Preparation ................................................. 99 4.2.5 Immunohistochemistry ............................................................................. 100 4.2.6 Confocal Microscopy ................................................................................ 101 4.2.7 Measurements and Statistical Analysis .................................................... 102 4.2.8 VGLUT1 Analysis ..................................................................................... 103 4.2.9 cFOS Immunoreactivity Analysis ............................................................. 104 4.3 Results ............................................................................................................ 107 4.3.1 Unilateral C3 Hemisection Effectively Eliminates Spinofugal Projections from the Ipsilesional Spinal Cord ...................................................................... 107 4.3.2 Spared Mossy Fibre Density Increases with Time Following Hemisection .......................................................................................................................... 108 4.3.3 Increased Reporter and Synaptic Protein Density in Rosettes ................. 113 4.3.4 cFOS Immunoreactivity After Laminectomy and Hemisection .................. 116 4.4 Discussion ....................................................................................................... 118 4.4.1 Technical Considerations for Densitometric Analysis ................................ 118 4.4.2 Anterograde Axonal Transport may be Increased in Spared SCT Fibres . 119 4.4.3 Are New Spinocerebellar Rosettes Functional? ....................................... 120 4.4.4 Plasticity Lateralization in the Cerebellum ............................................... 121 4.4.5 Activity-Dependent Plasticity may be Driven by Sensory and Motor Pathway Convergence ..................................................................................................... 122 4.4.6 Challenges with Determining Functional Synaptogenesis ....................... 124 4.4.7 Laminectomy Can Result in Significant Post-Operative Pain .................. 126 4.4.8 Pre- and Post-Surgical Stress May Influence Pain .................................. 128 4.4.9 Concluding Remarks ................................................................................ 128 Chapter 5: Bulk mRNASeq May Identify Potential Targets for Future Investigation ........................................................................................................... 130 5.1 Introduction ..................................................................................................... 130 5.2 Methods .......................................................................................................... 131 5.2.1 Animals .................................................................................................... 131 xi 5.2.2 Surgical Procedure and Tissue Harvesting .............................................. 131 5.2.3 Bulk mRNAseq Analysis .......................................................................... 132 5.3 Results ............................................................................................................ 132 5.3.1 Bulk mRNAseq Identifies Potential Target for Future Work ..................... 132 5.4 Discussion ...................................................................................................... 139 5.4.1 Astrogliosis after SCI ................................................................................ 140 5.4.2 Microgliosis Occurs after SCI ................................................................... 141 5.4.3 Complement Proteins and Synaptic Stripping ......................................... 143 5.4.4 Synaptic Plasticity – Confounding Findings ............................................. 144 5.4.5 Axon Growth and Regeneration ............................................................... 145 5.4.6 Does Brain Derived Neurotrophic Factor Drive the Plasticity Response? 146 5.4.7 Alternative Approaches ............................................................................ 150 5.4.8 Concluding Remarks ................................................................................ 150 Chapter 6: Discussion ........................................................................................... 152 6.1 Thesis Summary ............................................................................................. 152 6.2 Limitations and Future Directions ................................................................... 154 6.2.1 Transgene Insertion Sites, Sequence Length, and the “Hox Clock” ........ 154 6.2.2 Changes in Motor Behaviours in Sham Operated Animals – Is Another Control Group Warranted? ................................................................................ 155 6.2.3 Does Collaboration Between Corticospinal and Spinocerebellar Plasticity Underpin Spontaneous Functional Improvement? ............................................ 156 6.2.4 Future Experiments Are Warranted Based on Bulk mRNA Sequencing Data .......................................................................................................................... 158 6.3 Final Remarks ................................................................................................. 160 References .............................................................................................................. 162 xii List of Tables Table 2.1. The top 30 Noldus CatWalk Parameters Correlated with Grasping Ability ... 45 Table 2.2. The top 30 Noldus CatWalk Parameters Correlated with the Horizontal Ladder ............................................................................................................................ 46 Table 3.1. Antibodies for immunohistochemistry ........................................................... 61 Table 4.1. Antibodies for Immunohistochemistry ......................................................... 101 Table 5.1. Author-curated list of differentially expressed genes in the cerebellar vermis 14d following C3 Hx ..................................................................................................... 138 xiii List of Figures Figure 1.1. Schematic of cerebellar circuitry. .............................................................. 8 Figure 1.2. Anatomy of the direct spinocerebellar tracts. ........................................ 12 Figure 2.1. Basso mouse scale scoresheet ............................................................... 33 Figure 2.2. Behavioural apparati used in this experiment. ....................................... 35 Figure 2.3. Heatmaps showing changes to behavioural parameters following unilateral hemisection and laminectomy. ................................................................. 39 Figure 2.4. Motor recovery on a variety of behavioural assessments in hemisection and sham mice. ...................................................................................... 42 Figure 2.5. The gait parameters obtained with the Noldus CatWalk do not strongly correlate with grip- and grasp-dependent parameters ............................................. 44 Figure 3.1. Hoxb8 reporter phenotype and genotypes ............................................. 65 Figure 3.2. Reporter distribution in the spinal cords of Hoxb8 reporter mice. ....... 67 Figure 3.3. Medullary reporter fluorescence in Hoxb8 reporter strains. ................. 69 Figure 3.4. Hindbrain reporter fluorescence in Hoxb8 reporter strains. ................. 71 Figure 3.5. Midbrain reporter fluorescence in Hoxb8 reporter strains: inferior colliculus. ..................................................................................................................... 74 Figure 3.6. Midbrain reporter fluorescence in Hoxb8 reporter strains: superior colliculus. ..................................................................................................................... 75 Figure 3.7. Dorsal spinofugal pathways to the midbrain and thalamus. ................. 76 Figure 3.8. Reporter fluorescence in the thalamus of Hoxb8 reporter strains. ...... 77 Figure 3.9. Lateral hemisection reveals origins of spinofugal axons: Hoxb8CreTdt mice ............................................................................................................................... 80 xiv Figure 3.10. Reporter fluorescence at (and rostral to) the level of the anterior commissure (AC) in Hoxb8 reporter strains. ............................................................ 81 Figure 3.11. Ectopic Hoxb8-driven reporter expression in Hoxb8CreTdT mice ....... 84 Figure 3.12. Sparse labeling of retinal ganglion cells in Hoxb8CreTdT mice. .......... 85 Figure 3.13. Ectopic Hoxb8-driven reporter expression in Hoxb8FlpOTdT mice. .... 86 Figure 4.1. Ipsilesional spinofugal projections are eliminated with a left C3 hemisection. ............................................................................................................... 107 Figure 4.2. Rosette density in stripes of the anterior cerebella of intact and hemisected Hoxb8FlpOTdT mice. .............................................................................. 111 Figure 4.3. Rosette density in stripes of the anterior cerebella of intact and hemisected Hoxb8FlpOTdT mice. .............................................................................. 112 Figure 4.4. 28d hemisection animals have significant differences in synaptic marker and reporter expression compared to intact animals. .............................. 115 Figure 4.5. cFOS immunoreactivity in lobule III after rotarod and vibrational exposure in sham and hemisected mice. ................................................................ 117 Figure 5.1. A volcano plot of all genes from bulk mRNAseq. ................................. 133 Figure 5.2. Downstream targets of microtubule associated tau (MAPT) signalling upregulated in the cerebellar vermis 2 weeks after unilateral C3 hemisection. .. 134 Figure 5.3. A ridgeline plot indicating the top 20 gene ontology biological processes identified by gene set enrichment analysis with fold change. ........... 135 Figure 5.4. A ridgeline plot indicating the top 20 gene ontology molecular function categories identified by gene set enrichment analysis with fold change. ........... 136 xv Figure 5.5. A ridgeline plot indicating the top 20 gene ontology cellular component categories identified by gene set enrichment analysis with fold change. ........... 137 Figure 5.6. Downstream targets of brain derived neurotrophic factor (BDNF) signalling upregulated in the cerebellar vermis 2 weeks after unilateral C3 hemisection. ............................................................................................................... 149 Figure 6.1. Hoxb8-lineage microgliosis in the rostral forebrain. ........................... 158 xvi List of Abbreviations AAV: adeno-associated virus ACC: anterior cingulate cortex Aif1: allograft inflammatory factor 1-like AM: anteromedial nucleus of thalamus AMPA: alpha-amino-hydroxy-5-methyl-4-isoxazole propionic acid AOB: accessory olfactory bulb Apoe: Apolipoprotein E ATF3: activating transcription factor 3 Atg12: autophagy related 12 ATP: adenosine triphosphate AV: anteroventral nucleus of thalamus B4U: B4U enhancer BC: basket cell BDA: biotinylated dextran amines BDNF: brain derived neurotrophic factor BMS: basso mouse scale ANOVA: analysis of variance BNST: bed nucleus of the stria terminalis C1qa: complement component 1, q subcomponent, alpha polypeptide C1qb: complement component 1, q subcomponent, beta polypeptide C1qc: complement component 1, q subcomponent, C chain C4b: complement component 4B (Chido blood group) Calm1: calmodulin 1 xvii CCF: common coordinate framework CCI: combined catwalk index Ccl3: chemokine (C-C motif) ligand 3 Ccl6: chemokine (C-C motif) ligand 6 Ccl9: chemokine (C-C motif) ligand 9 CCT: cuneocerebellar tract CF: cephalic flexure CF: climbing fibre CGRP: calcitonin gene-related peptide Cing: cingulum bundle Clec7a: C-type lectin domain family 7, member a CNS: central nervous system CP: choroid plexus CREB: cAMP response element binding protein Crh: corticotropin releasing hormone CSF1R: colony stimulating factor 1 receptor CST: corticospinal tract CTB: cholera toxin subunit b Cu: cuneate ECu: external cuneate Cx3cr1: chemokine (C-X3-C motif) receptor 1 Dbh: dopamine beta hydroxylase DCN: deep cerebellar nuclei xviii DE: dorsal enhancer Dg: deep grey DLG: dorsolateral geniculate nucleus DNA: deoxyribonucleic acid Dock2: dedicator of cyto-kinesis 2 DRG: dorsal root ganglion DSCT: dorsal spinocerebellar tract ECIC: external nucleus of the inferior colliculus eIF: eukaryotic initiation factor ENE: enhancer of nascent expression Fcgr2b: Fc receptor, IgG, low affinity IIb FGF2: fibroblast growth factor 2 FGF8: fibroblast growth factor 8 FlpO: flippase FRT: flippase recognition target GABA: gamma aminobutyric acid GAP43: growth-associated protein 43 Gas1: growth arrest specific 1 GC: granule cell GFAP: glial fibrillary acidic protein GO-BP: gene ontology – biological process GO-CC: gene ontology – cellular compartment GO-MF: gene ontology – molecular function xix GoC: Golgi cell Gr: gracile Grn: granulin GSEA: gene set enrichment analysis Gstp1: glutathione s-transferase pi 1 HRP: horseradish peroxidase Icp: inferior cerebellar peduncle Ifi35: interferon-induced protein 35 Ifi47: interferon gamma inducible protein 47 Ig: intermediate grey IRES: internal ribosome entry site Iw: intermediate white Jph3: junctophilin 3 KCC2: potassium chloride cotransporter 5 Kndc1: kinase non-catalytic C-lobe domain (KIND) containing 1 Lamp5: lysosomal-associated membrane protein family, member 5 Lcn2: Lipocalin 2 LTP: long term potentiation Lyz2: Lysozyme 2 MF: mossy fibre MGM: medial part of the medial geniculate nucleus MGN: medial geniculate nucleus MHB: midbrain-hindbrain boundary xx miRNA: micro ribonucleic acid ML: medial lemniscus mRNA: messenger ribonucleic acid Myh2: myosin, heavy polypeptide 2, skeletal muscle, adult Ndrg2: N-myc downstream regulated gene 2 Nefh: neurofilament, heavy polypeptide NGF: nerve growth factor NM II: non-muscular II NMDA: N-methyl-D-aspartate NPY: neuropeptide y NTS: nucleus of the solitary tract OC: optic chiasm OCT: optimal cutting temperature OML: outer molecular layer PaF: Parafasicular nucleus PAG: periaqueductal grey PB: phosphate buffer PBS: phosphate buffered saline PBSTx: phosphate buffered saline and sodium azide PC: Purkinje cell PF: paraformaldehyde PF: parallel fibre PIL: posterior interlaminar nucleus xxi PKA: protein kinase A PNS: peripheral nervous system PO: posterior complex of the thalamus PoT: posterior triangular complex PTCH1: protein patched homolog 1 Pvalb: parvalbumin RA: retinoic acid RALDH1: retinal dehydrogenase 1 RALDH2 retinal dehydrogenase 2 RARE: retinoic acid response element Rims3: regulating synaptic membrane exocytosis 3 RN: reticular nuclei RNA: ribonucleic acid RSCT: rostral spinocerebellar tract RSPv: retrosplenial cortex Rufy3: RUN and FYVE domain containing 3 SC: satellite cell SCI: spinal cord injury SCT: spinocerebellar tract Serpina3n: serine (or cysteine) peptidase inhibitor, clade A, member 3N Sg: superficial grey Shh: sonic hedgehog Shxt: spinofugal hypothalamic decussating tract xxii Slc10a4: solute carrier family 10 (sodium/bile acid cotransporter family), member 4 Slc8a3: solute carrier family 8 (sodium/calcium exchanger), member 3 SMC: sensory motor cortex Spp1: secreted phosphoprotein 1 SPVC: spinal nucleus of the trigeminal nerve Stmn1: stathmin 1 Str: striatum STT: spinothalamic tract Stx7: syntaxin 7 SUB: subiculum Syt4: synaptotagmin IV t-SNARE: target membrane SNARE TH: thalamus Tlr2: toll-like receptor 2 TMS: transcranial magnetic stimulation Tmsb4x: thymosin, beta 4, X chromosome Trem2: triggering receptor expressed on myeloid cells 2 TrkB: tropomyosin receptor kinase B Tuba1a: tubulin, alpha 1A Tyrobp; TYRO protein tyrosine kinase binding protein UBC: unipolar brush cell UTR: untranslated region v-SNARE: vesicular SNARE xxiii V3: third ventricle VGLUT1: vesicular glutamate transporter 1 VON: vomeronasal nerve VPL: ventroposterolateral nucleus VPM: ventroposeromedial nucleus VSCT: ventral spinocerebellar tract WGA: wheat germ agglutinin ZI: zona incerta xxiv Acknowledgements I would like to begin by offering my deepest gratitude to my supervisor Dr. Matt Ramer, who patiently worked with me, answered my questions, and continuously challenged me to be better. This work would not have been possible without the continued support, motivation, and inspiration that you provided me. I would also like to sincerely thank Drs. Michael Gordon, Brian MacVicar, Wolfram Tetzlaff and Matt Ramer for taking the time to be members of my committee and helping to improve this work with constructive criticism and thought-provoking questions. I am also grateful for the funding support from the Rick Hansen Foundation through the Blusson Integrated Cures Partnership without whom this work would not have been possible. I extend my gratitude to the animal care technicians, vets, and compliance offers who helped to ensure the well-being of the animals. I would also like to thank all members of the International Collaboration on Repair Discoveries (ICORD) for fostering a supportive research environment. I want to thank my lab-mates, Sadie Holland, Brittney Smaila, and Hanna Elford for their friendship, support, and advice throughout this process. Finally, I would like to thank my husband and parents for their encouragement and support, especially through times of stress. xxv Dedication To my parents I Chapter 1: General Introduction Spinal cord injury (SCI) interrupts the connection between the brain and the rest of the body. This disconnection results in the impaired transmission of signals from supraspinal structures to effector organs as well as from the external and internal environments to the brain. In the clinical world, there is an emphasis on reversing paralysis following injury. This has led to an abundance of research focusing on restoring connections to aid in the recovery of movement, which possess both reflexive and voluntary components. However, the importance of sensory feedback in regulating movements has generated far less attention, despite evidence demonstrating that it is crucial (Rossignol et al., 2008; Akay & Murray, 2021). Unlike the peripheral nervous system (PNS), the central nervous system (CNS) is unable to recover completely following trauma such as SCI. However, the CNS is plastic, meaning it adapts to changes in the environment, and these changes can aid functional recovery. Learning and memory are the best studied examples of plasticity in the intact CNS, but recovery following SCI and other traumatic injuries highlights the compensatory plasticity that can occur following an injury. The experiments in this thesis aim to highlight the structural plasticity that occurs in a specific CNS structure, the cerebellum, that might be associated with recovery in locomotion as well as in finer motor behaviours including grasping and changes in gait. A challenge with assessing plasticity of inputs ascending from the spinal cord to the brain (spinofugal projections) is identifying the entirety of their distributions. The traditional means to trace spinofugal projections have limitations associated with them 2 that result in a relatively small number of neurons labeled. This limitation, which is particularly pronounced in the spinal cord, where neurons of interest are widespread, has motivated me to take advantage of a developmentally expressed gene, Hoxb8, to bulk-label ascending projections to the brain. 1.1 Ascending and Descending Tracts Communication between the brain and spinal cord occurs through axons that are organized into specific tracts (Nami et al., 2022). Descending tracts carry information from the brain to the spinal cord to allow us to control specific movements. These include the corticospinal (CST), vestibulospinal, and rubrospinal tracts (Watson et al., 2009). Conversely, ascending tracts carry information from the spinal cord to the brain. One of the main spinofugal projections is the spinothalamic tract (STT) which transmits information pertaining to pain and temperature from the periphery to the thalamus (Steward, 2000). The spinotectal tract is also found within the anterolateral system. This pathway carries information related to spinovisual reflexes from the spinal cord to the superior and inferior colliculi and pretectal nuclei (Watson et al., 2009; Al-Chalabi et al., 2023). Another ascending tract is the spinocerebellar tract (SCT), which transmits unconscious proprioceptive information from muscle spindles to the cerebellum (Steward, 2000). Despite evidence that the cerebellar circuitry adapts greatly in response to SCIs that damage the SCT (Visavadiya & Springer, 2016), there is a dearth of literature surrounding the structural plasticity of these projections. In this thesis, I will investigate the plasticity of the SCT which follows incomplete SCI. 3 1.2 The Cerebellum The cerebellum is an ovoid-shaped structure in the hindbrain that is essential for motor control and coordination (Farini et al., 2021; Beckinghausen & Sillitoe, 2019). It is divided into a central vermis with a region located on each side known as the paravermis. There are three anatomical lobes (anterior, posterior, and flocculonodular) that are further subdivided into transverse zones each of which contain parasagittal stripes comprised of Purkinje cells (Consalez et al., 2021). During development the structure becomes highly foliated and forms 10 lobules (Beckinghausen & Sillitoe, 2019). There are three cellular layers: the superficial molecular layer, the middle Purkinje cell layer, and the deepest granule cell layer (Fig. 1.1) (Voogd & Glickstein, 1998). The deep cerebellar nuclei are the main output of the cerebellum and are surrounded by cerebellar white matter (Fig. 1.1) (Baumel et al., 2009). 1.2.1 Synaptogenesis and Development of the Cerebellum The development of the cerebellum begins in utero for the mouse and other mammals, and in mice is not complete until postnatal day 15 (Chizhikov & Millen, 2003). In general, the central nervous system is derived from the neural plate which then forms the neural tube and is involved in the formation of both the anterior-posterior and dorsal-ventral axes (Chizhikov & Millen, 2003). The neural tube is divided into three main vesicles: the forebrain, midbrain, and hindbrain (Martí-Clua, 2022). The hindbrain portion is further divided into rhombomeres (swellings) from which the anterior most-rhombomere (rhombomere 1) develops into the cerebellum (Martí-Clua, 2022). The anterior limit of the cerebellum is determined by a structure that develops at the midbrain-hindbrain boundary known as the isthmic organizer (Chizhikov & Millen, 2003). 4 The caudal boundary of the cerebellum is set by the transcription factor Hoxa2 which functions to prevent caudal expansion (Chizhikov & Millen, 2003). The isthmic organizer supplies extracellular signals to allow the cerebellar anlage to develop very early on in embryonic development (E8.0-8.5) and is fully developed by E11.5 (Consalez, et al., 2021; Chizhikov & Millen, 2003). Additionally, there are several transcription factors and signalling molecules that work together to create the cerebellum, with sonic hedgehog (Shh) being the main player (Farini et al., 2021). Shh binds to the membrane receptor protein patched homolog 1 (PTCH1). This complex is internalized, and proliferation of local cells and specific precursors begins under the regulation of transcription factors (Farini et al., 2021). This proliferation of nearby cells driven by Shh serves to produce the highly foliated appearance of the cerebellum (Farini et al., 2021). The development of specific cerebellar neurons follows a unique path, however glutamatergic cerebellar neurons are primarily generated from the rhombic lip, while GABAergic cerebellar neurons mainly arise from the ventricular zone (Leto et al., 2016). The main output neurons of the cerebellum are GABAergic Purkinje cells which provide inhibitory input to the deep cerebellar nuclei (Fig. 1.1) (Voogd & Glickstein, 1998). The development of these neurons begins between embryonic days 10 and 13 when precursors migrate along the radial glial process from the ventricular zone to the pial surface (Beckinghausen & Sillitoe, 2019). Granule cells are small glutamatergic neurons that are the most numerous in the cerebellum (Fig 1.1) (Voogd & Glickstein, 1998). Early on in utero, granule cells proliferate and migrate to form the transient outer external granule cell layer wherein they will continue to proliferate for 3 weeks in the mouse before forming postmitotic 5 granule cells (Beckinghausen & Sillitoe, 2019; Consalez et al., 2021). These cells transit the inner external granule layer and move along Bergmann glia, past the previously generated Purkinje cells, to terminate in the maturing granule cell layer. During the migration process, a portion of the axons bifurcate and travel mediolaterally to form parallel fibres (Fig. 1.1) (Consalez et al., 2021; Beckinghausen & Stillitoe, 2019). There are three types of inhibitory interneurons present in the cerebellum, one of which is Golgi cells. Golgi cells are found in the granule cell layer and provide inhibitory input to granule cells (Fig. 1.1) (Voogd & Glickstein, 1998). These neurons begin their development at E13.5 from a common pool of GABAergic precursor neurons in the ventricular zone (Leto et al., 2016; Beckinghausen & Sillitoe, 2019). Stellate and basket cells are also inhibitory interneurons in the cerebellum and begin their development after Golgi cells (E13-P5) from precursors in the prospective white matter (Leto et al., 2016). The prospective white matter is a postnatal region in the cerebellum containing a variety of cells that share a lineage relationship to astrocytes, but differ functionally and molecularly (Leto et al., 2016). These neurons reside in the molecular layer and function to provide inhibitory input to Purkinje cells (Fig. 1.1) (Leto et al., 2016). The patterning of development of these inhibitory interneurons is inside-out, meaning the neurons that develop earlier (Golgi cells) will make up the inner layers and the last to develop (Basket and Stellate cells) comprise the outer layers (Beckinghausen & Stillitoe, 2019). Unipolar brush cells are excitatory interneurons found in the granule cell layer (Fig. 1.1) (Leto et al., 2016). There are two classes of unipolar brush cells (type I and II) that share the same morphological structure (but differ chemically) to form a specialized synapse with a mossy fibre terminal (Leto et al., 2016; Consalez et al., 2021). These 6 neurons develop from the rhombic lip where they migrate dorsally through the white matter to reach the granule layer by post-natal day 10. Type I unipolar brush cells begin their development earlier (E14-17) than type II unipolar brush cells (E16-P0). Two of the cerebellar afferent inputs are climbing fibres and mossy fibres. The development of climbing fibres begins in the inferior olive in the brainstem (Beckinghausen & Sillitoe, 2019). The neurons from which these axons emerge are generated in the caudal rhombic lip at E15. These fibres project to the contralateral cerebellum where they eventually terminate on the dendrites of non-spiny Purkinje cells (Fig. 1.1) (Beckinghausen & Sillitoe, 2019; Voogd & Glickstein, 1998). The development of mossy fibres is less well-defined. The earliest mossy fibres begin their development at embryonic day 12 where they originate from a number of areas in the brain and spinal cord and eventually terminate in a glomerulus which contains the dendrites of granule cells (Fig. 1.1). Collaterals of mossy fibres also provide excitatory input to the deep cerebellar nuclei (Fig. 1.1). Mossy fibres terminate in compartmented, medio-laterally arranged stripes (Beckinghausen & Sillitoe, 2019). Throughout development, granule cells possess specific properties to ensure correct timing of synaptogenesis occurs between them and mossy fibres. When granule cells are in their progenitor stage, they exhibit properties that prevent the outgrowth of mossy fibres, however once in their mature stage granule cells promote growth of mossy fibres and synaptogenesis (Manzini et al., 2006). The deep cerebellar nuclei are the sole output of the cerebellum (Fig. 1.1) and are comprised of three main nuclei: the fastigial (medial), interposed, and dentate (lateral) (Baumel et al., 2009). Within these nuclei, there are 50 – 100 000 neurons including 7 excitatory neurons which project to other brain regions, inhibitory neurons that only project to the inferior olive, and local inhibitory interneurons (Baumel et al., 2009). The glutamatergic neurons that comprise these nuclei begin their development from the rhombic lip at E10.5-12.5 in mice, while the GABAergic neurons develop from the ventricular zone at E13.5-16.5 (Fink et al., 2006). 1.2.2 Cerebellar Circuitry Mossy fibres ascend from the brainstem and spinal cord to synapse with granule cells. The majority of these synapses comprise a structure known as a glomerulus which is inhibited by Golgi cells. There is also a small population of mossy fibres that interact with unipolar brush cells to amplify the signal to the granule cells. Mossy fibre collaterals also provide excitatory input to the deep cerebellar nuclei. Granule cells then ascend to the molecular layer where they give rise to parallel fibres. These parallel fibres bifurcate to relay information to Purkinje cells via excitatory synapses with their dendritic spines. Climbing fibres ascend from the contralateral inferior olive and have an excitatory synapse with Purkinje cells which then transmit inhibitory signals to the deep cerebellar nuclei. Purkinje cells are also inhibited by stellate cells and basket cells in the molecular layer (Consalez et al., 2021; Purves et al., 2001) (Fig. 1.1). 8 Figure 1.1. Schematic of cerebellar circuitry. MF = mossy fibres, UBC = unipolar brush cells, GC = granule cells, GoC = Golgi cells, PF = parallel fibres, SC = stellate cells, BC = basket cells, PC = Purkinje cells, DCN = deep cerebellar nuclei, CF = climbing fibres, + = an excitatory synapse, and - = an inhibitory synapse. The dotted circle indicates a glomerulus. The layers of the cerebellum are indicated: grey represents the brainstem/spinal cord, green is the white matter, yellow is the granule cell layer, pink is the Purkinje cell layer, and blue is the molecular layer (Beckinghausen & Sillitoe, 2019; Consalez et al., 2021). 9 1.3 The Spinocerebellar Tract The SCT is comprised of direct and indirect pathways. The direct pathways are primarily involved in conveying unconscious proprioceptive information (Morrow, 2014). These direct pathways include the dorsal spinocerebellar tract (DSCT), ventral spinocerebellar tract (VSCT), rostral spinocerebellar tract (RSCT) and cuneocerebellar tract (CCT) (Fig. 1.2). When tracing spinocerebellar fibres from the cerebellum to the spinal cord with horseradish peroxidase, Xu and Grant (2005) corrected the longstanding idea that the DSCT enters the cerebellum via the inferior cerebellar peduncle and the VSCT enters via the superior cerebellar peduncle. This newer information indicates that all axons enter the cerebellum via both the superior and inferior peduncles. The DSCT transmits unconscious proprioceptive information relating to fine coordination of the hindlimbs to the cerebellum (Oscarsson, 1965; Watson et al., 2009). In rats and cats, neurons in this pathway originate in Clarke’s column between cervical vertebrae 8 and lumbar vertebrae 2 where they ascend on the ipsilateral side of the spinal cord in the dorsolateral funiculus (Oscarsson, 1965; Morrow, 2014). These axons enter the cerebellum and terminate as mossy fibres on granule cells in the intermediate and vermal zones of the cerebellum (lobules I, II, III, IV, V, VIII) (Oscarsson, 1965; Morrow, 2014). The DSCT is thought to primarily innervate only the ipsilateral cerebellum (Oscarsson, 1965). The CCT is similar to the DSCT, however it carries proprioceptive information about the forelimb (Oscarsson, 1965). The cells that make up this tract originate in lamina 1 of the spinal cord. The axons then ascend ipsilaterally where they enter the cuneate 10 fasciculus and then project to the external and rostral cuneate nuclei. From this location, the axons then enter the cerebellum to terminate primarily in lobules I, II, IV, VI, and IX (Watson et al., 2009). The VSCT carries motor pattern information pertaining to the hindlimbs (Biousse, 2003). The first order neurons from this tract receive their afferents from group 1 muscle fibres and the contralateral flexors in spinal columns in Rexed’s laminae V-VIII which are found more caudal than Clarke’s column (Oscarsson, 1965; Morrow, 2014). These neurons ascend and decussate at the level of the anterior commissural fibres following which they ascend on the contralateral side in the lateral funiculus (Morrow, 2014). Neurons in this tract reach the cerebellum after which they decussate again to terminate on the ipsilateral side of the cerebellum (Oscarsson, 1965). This part of the spinocerebellar tract terminates in longitudinal zones (or stripes) in the medial portion of the intermediate and vermal zones of the anterior lobe. Despite the fact that this tract decussates again once it enters the cerebellum, it has bilateral innervation (Oscarsson, 1965). The termination pattern of mossy fibres is most dense on the side ipsilateral to its origin, however, there is a significant presence on the contralateral side supplied by collaterals (Oscarsson, 1965). The VSCT also has a forelimb equivalent known as the RSCT. The cells that supply this tract originate in laminae 5-7 of cervical vertebrae 5 through 8 (Watson et al., 2009). This pathway has a more complicated path whereby two thirds of it follows the VSCT, while the remaining one-third moves dorsally to join the DSCT (Oscarsson, 1965). These axons terminate almost exclusively in lobules 4 and 5 of the anterior lobe with the majority supplying the ipsilateral side, however there is axonal branching resulting in 11 axons of this tract reaching the contralateral side (Oscasson, 1965; Watson et al., 2009). While outside the scope of this thesis, it is important to note that there are two indirect pathways in the spinocerebellar tract (Morrow, 2014). These indirect pathways include the spino-olivary and spino-lateral reticular nucleus tracts (Pop et al., 2022). Much less is known about these tracts compared with the direct SCT. It is known that the spino-lateral reticular nucleus pathway projects both ipsilaterally and contralaterally to the lateral reticular nucleus in the medulla, while the spino-olivary tract projects contralaterally to the inferior olive in the medulla (Pop et al., 2022). It is speculated that a portion of the indirect SCT only projects locally within the spinal cord (Pop et al., 2022). The lack of knowledge surrounding the anatomy and connectivity of the indirect SCT have driven our focus on the well-defined direct SCT. 12 Figure 1.2. Anatomy of the direct spinocerebellar tracts. The left and right DSCT are in blue (light and dark blue, respectively) and the left and right VSCT/RSCT are in orange (light and dark orange, respectively). 13 1.4 Spinal Cord Injuries SCI results in an interruption of connections between the brain and the spinal cord resulting in a loss of motor, sensory, and autonomic function. Injury to the spinal cord comprises two main phases – primary and secondary (Ahuja et al., 2017). It is the primary phase consisting of the initial traumatic event whereby the severity of the injury is determined. The secondary phase is defined as the events that occur following the primary injury that expand the area of initial damage and further limit neurological recovery (Ahuja et al., 2017). During the secondary phase of SCI, several immune cell types are recruited to the injury site leading to inflammation and cell death (Ahuja et al., 2017). SCIs can be either complete or incomplete, of which the latter is more common. An anatomically complete lesion results in a lack of voluntary motor control or sensory perception being permitted across the lesion (O’Shea et al., 2017). An incomplete injury is formed by one or several smaller lesions (O’Shea et al., 2017). Up until recently, it was thought that axons in the CNS could not regenerate following injury unlike those in the PNS (Steuer & Guertin, 2009). It has since been discovered that under certain conditions these axons can at least partially regenerate (Sofroniew, 2018). However, it is more common for plasticity and axonal sprouting to occur, whereby new axons grow from those spared by the injury. The presence of uninjured axons after incomplete SCI allows for spontaneous synapse reorganization and thus these injuries are more likely to be associated with functional recovery. 14 1.5 Recovery after SCI 1.5.1 Avenues to Recovery: Neuroprotection, Regeneration, and Plasticity Following SCI, therapeutic interventions can be used to aid neuroprotection and regeneration. Neuroprotection can, in some cases, be defined as the weakening of destructive physiological processes that occur after acute injury to minimize secondary damage (Hilton et al., 2017). Many experimental therapeutics for SCI focus on promoting neuroprotection by working to limit prolonged inflammation (O’Shea et al., 2017). These include surgical decompression, in which pressure is removed from the spinal cord and has shown success in animals, as well as pharmacological therapies (Siddiqui et al., 2015). Axonal regeneration (the growth of injured axons) can also be promoted therapeutically after SCI to lead to recovery. Strategies focused on regeneration aim to modulate the glial scar and overcome the inhibitory effect of myelin-derived growth inhibitors (Siddiqui et al., 2015; Zheng & Tuszynski, 2023). Currently in the field of SCI, bioengineering scaffolds are of interest as a means to allow regeneration to occur (Siddiqui et al., 2015). In SCI, regardless of the location or the type of injury, spontaneous plasticity occurs. Plasticity is broadly described as the changes that occur to an altered environment and include axonal sprouting and synaptogenesis (Onifer et al., 2011). This spontaneous plasticity can be linked to functional recovery. The idea of plasticity in the CNS warrants a brief digression on its discovery and function in the hippocampus and the cerebellum. 15 1.5.2 Long-Term Potentiation in the Hippocampus The first form of plasticity to be discovered was long-term potentiation (LTP) in CA1 hippocampal slices. LTP is a form of synaptic plasticity that results in the strengthening of synapses to increase signal transmission between neurons (Leal et al., 2017). Over time when forming a memory, the synapses that are consistently firing together undergo synaptic plasticity in the form of LTP to result in increased transmission between the neurons involved in this memory. N-methyl-D-aspartate (NMDA) and alpha-amino-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are the two main glutamate receptors that are involved in LTP in the hippocampus. LTP is comprised of three primary stages: induction, expression, and maintenance (Hayashi, 2022). Following a high frequency tetanic stimulation, glutamate activates AMPA receptors resulting in a large postsynaptic depolarization. This large depolarization removes the magnesium block on the postsynaptic NMDA receptors resulting in a calcium influx. This influx of calcium triggers a series of calcium-dependent second messengers allowing for the modification of AMPA receptors resulting in maintenance of LTP. LTP can be driven by both presynaptic and postsynaptic changes that strengthen synapses. For example, presynaptically there can be an increase in the amount of neurotransmitter released, while postsynaptically, there can be an increase in receptor density. LTP can be divided into two main phases: early and late. The late phase of LTP is longer-lasting, requires structural changes at the synapse, and involves protein kinases. The induction of late LTP leads to the activation of cAMP-dependent protein kinase (PKA) and MAPK followed by the phosphorylation of cAMP response element 16 binding protein (CREB) and the subsequent transcription of several downstream genes, one of which is brain derived neurotrophic factor (BDNF) (Lu et al., 2008). While there is some evidence to support the role of BDNF in early LTP (Nagappan & Lu, 2005), the majority of work focuses on the role of BDNF in late LTP. Several studies in mice found that knocking out BDNF or tropomyosin receptor kinase B (TrkB) (the receptor for BDNF) significantly impaired late LTP (Lu et al., 2008). Additionally, treating hippocampal slices with antibodies against TrkB also inhibits late LTP (Lu et al., 2008). The role of BDNF in LTP is not unique to the hippocampus, as administration of BDNF in mice enhanced synaptic responses in the anterior cingulate cortex, while bath application of TrkB antagonists blocked this LTP (Miao et al., 2021). 1.5.3 Plasticity in the Cerebellum Learning new motor tasks requires plasticity in the cerebellum at several synapses including between granule cells and mossy fibres and/or projections from deep cerebellar nuclei (D’Angelo, 2014). One of the main afferents of the cerebellum, mossy fibres, transmit information to the dendrites of granule cells in a specialized structure known as a rosette (Ito, 2014). Rosettes are potential sites of plasticity in the cerebellum, which will further be discussed in this thesis. Plasticity in the cerebellum is evident during Pavlovian conditioning of mice to generate an eye-blink in response to a conditioning tone (Boele et al., 2013). Boele et al. (2013) found that in the conditioned group plasticity occurred in the form of significant axon outgrowth and synaptogenesis in the deep cerebellar nuclei. Additionally, there was a specific growth of mossy fibres in the cerebellar nuclei that receive input from Purkinje cells related to the eye-blinking task (Boele et al., 2013). 17 1.5.4 Trauma Induced Plasticity In the hippocampus and cerebellum plasticity occurs as a normal physiological process. However, plasticity also occurs following a traumatic injury that has the potential to aid in functional recovery. A stroke that affects the primary motor cortex hinders movement based on damage to the CST (Dodd et al., 2017). Under normal physiological conditions, the contralateral primary motor cortex inhibits the ipsilateral side during a motor task. After a stroke, there is an interhemispheric imbalance whereby the ipsilesional primary motor cortex no longer inhibits the contralesional hemisphere, and the contralesional hemisphere inhibits the ipsilesional (Dodd et al., 2017). Transcranial magnetic stimulation (TMS) can be used to promote plasticity and thus functional recovery. TMS can be used to excite the ipsilesional hemisphere, allowing it to be active despite inhibition from the contralesional hemisphere. Alternatively, TMS can inhibit the contralesional hemisphere’s usual inhibition of the ipsilesional hemisphere, thereby allowing the ipsilesional hemisphere to be disinhibited. Both of these methods promote neuronal reorganization and circuit formation (Dodd et al., 2017). In a study on thoracic spinal hemisection in rats, Cao et al. (2021), found that spontaneous hindlimb locomotor recovery occurred 5 weeks following injury which was accompanied by sprouting of the contralesional CST below the lesion. Similar findings have been demonstrated following a cervical hemisection that disrupted all descending tracts on the lesioned side (Zörner et al., 2014). In these animals, there was a significant increase retrogradely labelled normalized cells in the gigantocellular reticular nucleus on the contralesional side 4-weeks after injury (compared to the acute state) 18 accompanied by a high degree of locomotor recovery (Zörner et al., 2014). These examples of plasticity in the brain after stroke and SCI primarily focus on the descending tracts. There has been a lack of attention in the literature to ascending (“spinofugal”) projections and their plasticity after SCI. 1.5.5 The Synaptic Active Zone and Plasticity Plasticity in the brain, whether in a naïve state or in response to trauma, can manifest as changes in activation pattern, changes in the strength of existing synapses, or creation of new neuronal connections (Ding et al., 2005). An action potential in the presynaptic neuron induces neurotransmitter release which in turn depolarizes the postsynaptic neuron and, in some cases, leads to an action potential in the postsynaptic neuron. This process occurs through several steps starting with the release of vesicles from the readily releasable pool of vesicles in the presynaptic neuron. These vesicles then dock at the active zone in an adenosine triphosphate (ATP) dependent process before subsequently undergoing fusion triggered by the release of calcium. Neurotransmitters are then released from the vesicles and interact with receptors on the postsynaptic neuron (Kandel et al., 1991). This process is tightly regulated by the SNARE complex and other associated proteins. The vesicular and target membrane are brought together by a series of proteins that belong to the SNARE complex. Vesicular SNARE (v-SNARE), synaptobrevin, and target membrane SNARE (t-SNARE), syntaxin-1 and SNAP-25, interact in a coiled-coil to fuse the membranes together. This interaction remains stable when there is no calcium present (Kandel et al., 1991; Rizo, 2022). Munc13 is another protein involved that interacts with the SNARE complex and is required for vesicle fusion. Rab3 and RIM interact to fuse 19 the synaptic vesicles to the active zone, following which Munc13 interacts with RIM to form the Munc13-RIM-Rab3 complex (Rizo, 2022). Munc13 is an essential protein for neurotransmitter release, membrane bridging, and the opening of t-SNARE syntaxin-1 as demonstrated by the lack of vesicle release in Munc13 knockout mice (Rizo, 2022). The last essential protein in this system is synaptotagmin. Synaptotagmin is only present in neurons and acts as a brake for synaptic transmission. When calcium is not present, synaptotagmin prevents the release of neurotransmitters from vesicles. The presence of calcium removes this inhibition, allowing for vesicular release and action potential transmission through neurotransmitter interaction with the postsynaptic membrane receptors (Kandel et al., 1991). 1.6 Anatomical Methods to Investigate Plasticity There exist several approaches to determining the normal distribution of axons in the spinal cord and their response to trauma. Each has its advantages and disadvantages. 1.6.1 The Use of Degeneration Studies to Understand Circuitry In the 1800s, scientists used the process of Wallerian degeneration to understand neurocircuitry. In the 1940s, the technique of silver-staining was created to visualize the degeneration process. This can allow for the visualization of tracts projecting from the lesion, however there are gaps in the understanding of Wallerian degeneration: the exact spatiotemporal pattern of Wallerian degeneration is poorly understood, and there are conflicting thoughts on the direction of axon degeneration (anterograde, retrograde, both) (Beirowski et al., 2005). 20 1.6.2 Intrinsically Expressed Molecules Another approach makes use of the expression of genes expressed specifically in neuronal subpopulations. For example, primary afferent neurons project to the dorsal horn of the spinal cord and terminate in specific laminae. Individual subpopulations of dorsal root ganglion (DRG) neurons (and their axons) can be distinguished immunohistochemically based on their expression of, for example, neuropeptides such as calcitonin gene-related peptide (CGRP) and Substance P, ion channels such as the ATP receptor P2X3, or vesicular proteins such as vesicular glutamate transporter 1 (VGLUT1). Each of these has been used to study plasticity of primary afferent projections in the dorsal horn following injury to their neighbours via dorsal root injury (Soril et al., 2008; Ramer et al., 2007). When applying therapeutic strategies to enhance or suppress plasticity, however, caution is warranted in interpretation since both injury and treatment can alter gene expression. For example, application of neurotrophic factors intended to stimulate growth of specific sensory neuronal populations changes the expression of the very molecules used to identify those populations (Ramer et al., 2001; Ramer et al., 2003). 1.6.3 Viral and Non-Viral Tracing The most common method to understand the connectivity in the brain is with the use of viral and non-viral tracers. Chemical tracers were developed in the 1960s to allow the labeling of neural connections (Xu et al., 2020). These tracers can either be anterograde or retrograde tracers. Common retrograde tracers include horseradish peroxidase (HRP) and cholera toxin beta-subunit (CTB, which is also an anterograde tracer), while anterograde tracers include biotinylated dextran amines (BDA) and wheat 21 germ agglutinin (WGA) (Xu et al, 2020). Shortly after the development of chemical tracers came the generation of viral tracers (Xu et al., 2020; Qiu et al., 2022). Viral tracers exist in three main forms: anterograde, retrograde, and trans-synaptic. Anterograde viral tracers infect the soma of a neuron, and the viral transgene is transported from the soma (source) to the synapse (terminal) to disclose the output of a neuron. Retrograde viral tracers infect the synapse and are transported to the soma to highlight afferent inputs (Qiu et al., 2022). Adeno-associated viruses (AAV) are the most commonly used anterograde viral tracing tool. The advantages of AAVs include: the inability to self-replicate allowing for restricted expression in the injected neuron, low toxicity, and high stability and expression for a prolonged period of time (Qiu et al., 2022). AAVs are useful for determining neuronal morphology, however they are unable to discern synaptic connections. Transsynaptic viral tracers are ones that have the ability to spread to other neurons via synapses due to the self-replication properties of a virus to label neural circuits (Qiu et al., 2022). Transsynaptic viral tracers can travel in the anterograde or retrograde direction and are usually conjugated to a fluorescent protein (Qiu et al., 2022). While viral tracers are commonly used for their ability to self-replicate, they can also spread locally and non-specifically to neighbouring neurons and glial cells, be toxic to neurons, and generate weak signals (Xu et al., 2020; Li et al., 2021). Assessing structural plasticity requires visualization of axons and terminal arborizations for the tracts of interest. This requirement and the limitations associated with conventional tracing techniques have motivated us to take advantage of a developmentally expressed gene (Hoxb8) to bulk-label spinofugal projections and assess the plasticity of the SCT. 22 1.7 Hoxb8-Driven Recombinase Expression Serves as a Strategy to Trace Spinofugal Projections 1.7.1 Homeobox Genes The development of the nervous system is a highly regulated process that relies heavily on transcription factors, including Hox proteins, to ensure correct neuronal (and non-neuronal) specification, migration, and axonal projection (Philippidou & Dasen, 2013). In most vertebrates, Hox proteins are encoded by 39 genes distributed across four clusters, HoxA through D. These genes have a 60 amino acid region that encodes the deoxyribonucleic acid (DNA)-binding homeodomain (Philippidou & Dasen, 2013). Each of the four clusters exist on a different chromosome. At embryonic day 7, Hox genes begin their activation starting with genes located at the 3’ end of the complex and moving sequentially to the 5’ end, where the most 5’ genes to develop begin to be expressed until E9 (Nolte et al., 2019; Noordermeer et al., 2014). This process is known as temporal collinearity. Hox genes also exhibit spatial collinearity whereby Hox genes are expressed in the body in the same spatial order that they are positioned in their clusters (Noordermeer et al., 2014). Spatial collinearity is regulated in part by retinoic acid (RA) and fibroblast growth factors (FGF2 and FGF8). Hox genes near the 3’ end of the cluster are more responsive to RA, while genes at the 5’ end have increased responsivity to FGF8 (Nolte et al., 2019). FGF8 and FGF2 function to keep cells in an undifferentiated state, reducing their responsiveness to RA. This difference in signalling gradient and responsiveness governs spatial and temporal collinearity of Hox genes (Nolte et al., 2019). 23 Early studies on Drosophila melanogaster revealed that following genetic mutations in the fruit fly, there were homeotic transformations of one body region (or segment) to another (e.g. legs in place of antennae) (Kaufman et al., 1990). Immediately after neural induction in vertebrates, the hindbrain is divided into 7 segment-like swellings, known as rhombomeres (Gonçalves et al., 2020). The importance of Hox genes in developing the anterior-posterior axis of the hindbrain was highlighted in loss-of-function studies. In Hoxa1 mutant mice, the fifth rhombomere is lost leading to fusion between rhombomere 4 and 6 (Carpenter et al., 1993). 1.7.2 Hoxb8 Hox genes are expressed in both the hindbrain and spinal cord (Philippidou & Dasen, 2013). The anterior border of most Hox genes in groups 5-9 are in the cervical region of the spinal cord. Hoxb8 is one of the Hox genes that is expressed mainly in the dorsal or sensory region of the spinal cord (Nolte & Krumlauf, 2013). The anterior borders of the B family Hox genes are found more rostral in the cervical region of the spinal cord than any other group (Nolte & Krumlauf, 2013). Hoxb8 is involved in the differentiation of the noradrenergic system and of myeloid progenitor cells (a known source of microglia) in the hematopoietic system (Huber et al., 2012; Chen et al., 2010). Mouse knock-out studies have revealed a role in the nociceptive system and anxiety-circuits for Hoxb8 related to overgrooming (Holstege et al., 2008; Chen et al., 2010; Tränkner et al., 2019; Nagarajan & Capecchi, 2023). The availability of Hoxb8 transgenic mice and the serendipitous expression pattern in some of these lines has motivated their use as a substitute for injectable anterograde tracers. 24 1.8 Hoxb8 Reporter Mice There are several transgenic mouse models available, of which four take advantage of the temporal and spatial expression of Hoxb8 in order to drive DNA recombinase expression. Two of these use the Cre-Lox system. Cre recombinase is a 38 kDA protein composed of 343 amino acids that recognizes pairs of 34 base pair DNA sequences known as LoxP sites (Sauer, 1987; Sauer, 1998; Song & Palmiter, 2018; Bouabe & Okkenhaug, 2013). When Cre recombinase recognizes the two LoxP sites, it will generate a DNA loop that subsequently excises or inverts the segment of DNA between the two LoxP sites depending on their orientation (Song & Palmiter, 2018). It is common to breed Cre-driver lines with reporter mice whereby the expression of the reporter is activated via deletion of a stop sequence between the two LoxP sites (Song & Palmiter, 2018). The two Hoxb8-Cre lines driving reporter expressed used here are targeted (i.e. knock-in) and non-targeted (i.e. random insertion) transgenics (Witschi et al., 2010; Chen et al., 2010). The two other transgenic lines used in this thesis exploit the FlpO-frt system. In this case Flippase (FlpO) is used in place of Cre-recombinase and flippase recognition target (FRT) is used instead of LoxP sites (Bouabe & Okkenhaug, 2013). As with the Cre lines, one FlpO line is targeted, the other untargeted (Bohic et al., 2023; Zhang et al., 2018). 25 1.9 Overall Aims The importance of environmental feedback on executing behaviours, and the plasticity that is present after SCI in descending tracts has motivated the overall aim of this thesis: to investigate plasticity of spinofugal axons following incomplete SCI. The prevalence of cervical level injuries in humans and the deficits to hand function brought about by these injuries motivated aim 1 of this work. In this aim, I assessed the natural history of motor-related behavioural changes following unilateral cervical hemisection. The spontaneous recovery found in aim 1 of this thesis, in particular related to grip- and grasp-dependent behaviours, likely relies on the sensory system and ascending tracts. The limitations associated with commonly used tracing techniques, the restriction of Hoxb8 to the hindbrain and spinal cord, and the existence of Hoxb8 transgenic mice motivated me to use Hoxb8 lineage tracing to identify and clarify the course of spinofugal axons. In aim 2 of this thesis, I sought to compare Hoxb8-driven reporter distribution in the brain of all four transgenic mouse lines with the primary goal being to select the preferred model for subsequent aims. The bulk of the spontaneous recovery in aim 1 was with grip- and grasp-dependent behaviours. The role of the cerebellum in these motor behaviours, and the significant changes to the cerebellar circuitry in rats after SCI (Visavadiya & Springer, 2016) motivated aim 3 of this work. The goal of aim 3 was to determine the spontaneous plasticity of spinocerebellar tract axons following incomplete SCI. Lastly, I wanted to ascertain if the anatomical plasticity of the SCT was accompanied by synaptic 26 activation of target neurons, and identify transcriptomic changes that occur in response to partial deafferentation. In summary, the overall goal of this thesis is to better understand the potential role of a main ascending tract, the SCT, in spontaneous motor recovery following unilateral cervical hemisection. Understanding beneficial spontaneous changes that occur following SCI will provide impetus for seeking out their underlying mechanisms, and exploit them to enhance recovery in people with SCI. 27 Chapter 2: The Natural History of Motor-Related Behavioural Changes Following Unilateral Cervical Hemisection 2.1 Introduction In humans, 60-70% of SCIs that occur are incomplete and are accompanied by a notable degree of spontaneous functional recovery (Noonan, 2023; Raineteau & Schwab, 2001). Incomplete cervical contusion injuries, specifically at C5, are the most prevalent type of SCI in humans and lead to impaired hand function (Alizadeh et al., 2019; Anderson et al., 2004; Khaing et al., 2012; Weidner et al., 2001). Following SCI, a large portion of patients with severe sensorimotor loss will attain some degree of recovery (Dietz et al., 1998; Fawcett et al., 2007). Work by Steeves et al. (2010) determined that the majority of individuals recovered at least one motor level on the upper extremity motor score assessment one year after traumatic cervical sensorimotor complete injury. Furthermore, after several months of trained treadmill walking, Wernig and Müller (1992) found a significant improvement on the use of paralyzed limbs during locomotion. All of these findings suggest that there is some degree of spontaneous recovery after traumatic SCI which gives promise for possible therapeutics directed at further improvement. Equally, or perhaps even more important, is to understand what behaviours do not improve spontaneously in order to direct therapeutics to these parameters. Animal models, commonly rodents, are used in SCI research with the most clinically relevant injuries being contusions and clip-compressions (Alizadeh et al., 2019). Following thoracic contusion SCI in mice, spontaneous functional recovery is evident in tests including Basso mouse scale (BMS), beam walking, and rotarod 28 (Kakuta et al., 2019; Hill et al., 2009; Gale et al., 1985). Similarly, following incomplete thoracic SCI in rats there is significant locomotor recovery on open field locomotion (the Basso, Beattie and Bresnahan scale) two weeks after injury (Kaegi et al., 2002). Despite their clinical relevance, compression and clip-contusions models do not completely sever specific ascending and descending tracts, meaning that it is difficult to ascribe any potential recovery to specific populations of neurons. To circumvent this limitation and allow for more precise and reproducible SCIs, pyramidotomy and hemisection models can be used. After pyramidotomy (cutting one or both pyramids to remove corticospinal input to the cord), performance in beam walking and forelimb reaching tasks are significantly impaired in cats and monkeys (Lawrence & Kuypers, 1968; Schwartzman, 1978; Armstrong, 1986; Whishaw et al., 1993). In order to assess motor recovery and sprouting of the CST, a unilateral pyramidotomy is preferred because it allows for the assessment of the intact CST from the spared side (Kathe et al., 2014). Following damage to the CST with thoracic hemisection or unilateral pyramidotomy, the spared CST sprouts across the midline and this sprouting is associated with locomotor recovery (Cao et al., 2021; Weidner et al., 2001). While hemisection and pyramidotomy studies have primarily been focused on the functional recovery and plasticity of the CST, there are multiple other ascending and descending pathways that are probably also involved. Most research in spontaneous recovery focuses on thoracic level injury; however cervical injuries are most prevalent in humans and result in deficits to hand function, which is a significant disability for humans, highlighting the importance of expanding mouse models to the cervical level. Earlier work by Anderson et al. (2004) found that 29 following a C5 hemisection in mice, gripping ability is initially completely lost yet recovers fully over time. More recently, Engmann et al. (2020) observed ipsilesional forelimb and hindlimb functional impairment 7-days after C4 hemisection which spontaneously recovered by 4-weeks post-injury. Weidner et al. (2001) found improvement in skilled forelimb reaching tasks in rats following bilateral pyramidotomy that correlated with CST plasticity. The work focused on functional recovery following cervical hemisection is largely restricted to grip assessments; however, these SCIs negatively impact gait and other aspects of movement, and thus other gait-specific behavioural paradigms warrant investigation. Gait parameters obtained via the Noldus CatWalk have largely been used to assess motor improvements after contusion and other more severe SCIs (Hamers et al., 2006), but there is a dearth of literature that applies this assessment to high hemisection models. The goal of this chapter is to compare Noldus CatWalk gait data to BMS and grasp-dependent behaviours (horizontal ladder and wire cage lid-grasping) to determine if any Noldus CatWalk parameters behave in the same way. Currently, in human research, a large number of clinical trials for SCI do not lead to clinical improvement. There may be more success in these clinical trials if one had increased knowledge pertaining to the natural history of functional changes after SCI (Steeves et al., 2010). Therefore, in this chapter I not only address the tests that spontaneously recover and thus suggest innate plasticity, but I also speak to the parameters that remain stable after injury. Here, I demonstrate that following a unilateral C3 hemisection in mice, significant spontaneous recovery occurred in both horizontal ladder and lid-grasping assessments 30 by the 28-day time point, while there was no significant improvement in BMS score. Additionally, there were few Noldus CatWalk measurements that followed a similar recovery pattern as the grip-dependent tests, while the majority resulted in stable deficits or no change following injury. 2.2 Methods 2.2.1 Animals All experiments were carried out in accordance with guidelines of the Canadian Council on Animal Care and the UBC Animal Care Committee. The Hoxb8FlpOTdT and Hoxb8IRESCreTdT mice (described in Chapter 3) were group-housed in a reverse light cycle. The mice used were 8-12 weeks of age and both sexes were assessed. 2.2.2 Surgical Procedures Unilateral C3 hemisection (n=7) and sham (n=7) surgeries were done on Hoxb8FlpOTdT mice (3F, 4M in each group). Immediately prior to surgery, animals received subcutaneous injections of buprenorphine (0.01mg/kg) and lactated ringers (0.1mL). The animals were anesthetized with isoflurane (5% induction, 2-3% maintenance) following which eye lubricant was liberally applied, the upper back was shaved (excess hair removed with Nair), and the skin surface was disinfected with ethanol and chlorhexidine. Both hemisection and sham groups received a midline incision over the cervical spinal cord. A unilateral laminectomy of C3 and C4 was performed with rongeurs. At this point the sham animals were sutured and closed. Hemisections were made by first piercing the dura and cord with a 25-gauge needle just to the right of the dorsal midline vein. One blade of a pair of microscissors was then inserted into the puncture to the bottom of the spinal canal; the other was placed 31 outside the lateral side of the left hemicord and the scissors were closed. The muscle and skin layer were closed with sutures. Both groups of mice survived for 28 days during which they underwent appropriate monitoring, post-surgical care, and behavioural assessments. Both groups of mice received buprenorphine (0.01mg/kg) and lactated ringers (0.1mL) subcutaneously three times daily for the first three days post-surgery. The same hemisection surgery as above was performed on Hoxb8IRESCreTdT mice (n=9, 4F, 5M) to increase the sample size for the Noldus CatWalk, horizontal ladder, wire-lid grasping, and BMS pooled analysis. 2.2.3 Behavioural Tests Acclimation Sham and hemisection Hoxb8FlpOTdT and Hoxb8IRESCreTdT mice were acclimated to the behavioural tests for 5 days prior to surgery. Day 1 consisted of the animals’ cages being placed in the testing room for ~ 1 hour. On day 2, handling began. Day 3 involved the animals exploring the behavioural apparati with their cage mates before exploring them on their own on day 4. Finally, on day 5 the animals went through the exact conditions of the behavioural tests. Following acclimation, baseline testing was done for each behavioural paradigm. Noldus CatWalk The Noldus Catwalk (https://www.noldus.com/catwalk-xt) was used to assess motor recovery (Fig. 2.2 A, A’). In a dark room, the mice walked from one end of the walkway to the other (at which their homecage hut had been placed as motivation). Three compliant trials were required in which the mice had to walk across the recording 32 area in a maximum time of 5 seconds. The testing commenced on experimental day 5 and continued every 4 days until experimental endpoint (4 weeks). Horizontal Ladder A regularly spaced horizontal ladder test was done in which the ladder was elevated 30cm with clear walls on each side and a mirror behind. The mice started from a neutral cage and walked towards their homecage with the hut on top (Fig. 2.2 B). Each trial was filmed with a GoPro to allow for subsequent analysis. The percent success was calculated over three trials in which a step or replacement was a success, and a slip and a miss corresponded to an error. The mice were tested starting 4 days post-injury and assessed every 4 days until the experimental endpoint (4 weeks). Basso Mouse Scale To assess open field locomotion the mice were tested on the BMS (Fig. 2.2 C). The animals were scored by two judges (myself and Sarah Wheeler) on a 9-point scale. If the animals were unable to successfully move or perform sufficient movement after 3 minutes a score of 0 was given (Fig. 2.1). Testing began on experimental day 5 and continued every 4 days until the experimental endpoint (4 weeks). 33 Grasping Ability The mice were placed on a wire-cage lid which was then inverted to suspend the mice upside-down (Fig. 2.2 D). Scores were determined on a semi-quantitative scale in which no grasping ability corresponded to a score of 0, partial grasping ability (mice can hold on to cage lid for a few seconds but paw does not grasp around the wire) was indicated by a score of 1, and full grasping ability resulted in a score of 2. Testing began 4 days post-injury and continued every 4 days until the experimental endpoint (4 weeks, time-point and frequency based on preliminary studies in the Ramer lab and work by Anderson et al., 2004). 2.2.4 Behavioural Statistical Analysis In all cases the earliest post-operative timepoint (day 4 or 5) was excluded from analysis due to excess variability owing to the severity of the surgical procedure and buprenorphine dosing. The behavioural results (horizontal ladder, BMS, grasping ability, and some Noldus CatWalk parameters) were statistically analyzed using GraphPad Prism with a 2-way repeated measures multiple-comparisons analysis of variant (ANOVA) where multiple comparisons were corrected with statistical hypothesis test Figure 2.1. Basso mouse scale scoresheet. The score sheet used for BMS assessment (Basso et al., 2006). 34 using Dunnett’s test. A post-hoc power calculation was done to determine achieved power with G*Power 3.1. The results from all behavioural assessments (Hoxb8FlpOTdT and Hoxb8IRESCreTdT) were pooled. The results from each animal for day 8/9 (horizontal ladder and grasping) and 12/13 (Noldus CatWalk and BMS) were averaged to generate “post-injury early time-points” and data from day 24/25 (horizontal ladder and grasping) and 28/29 (Noldus CatWalk and BMS) were averaged to determine “post-injury late time-points”. In GraphPad Prism a heatmap was created for each behavioural measurement with pre-operative, early and late timepoints. A one-way ANOVA with Dunnett’s multiple comparisons test was performed on each behavioural measurement, with each time point compared to the “early” post-injury time point. The combined catwalk index (CCI) (methods adapted from Crowley et al., 2018) was calculated for the regularly spaced horizontal ladder and wire-cage lid grasping (ipsilesional forepaw). In Microsoft Excel, linear regression data were calculated for each time point and animal in the hemisected mice (Hoxb8FlpOTdT and Hoxb8IRESCreTdT). For each Noldus CatWalk parameter the CCI coefficients were calculated using the slope, intercept, and RSQ functions with grasping or horizontal ladder as the Y data and CatWalk as the X data. Next, the “adjusted CatWalk parameters” were calculated based on the previously calculated CCI coefficient (R2(Slope x CatWalk Data + Intercept). The CCI scores were then calculated by summing the adjusted CatWalk parameters and dividing by the total number of CatWalk parameters used in the analysis (205). Linear regression analysis was performed for the 35 CCI and grasping or horizontal ladder results and a regression plot was created in Microsoft Excel. Figure 2.2. Behavioural apparati used in this experiment. A. The Noldus CatWalk apparatus. A’. Recording of a Hoxb8FlpOTdT mouse on the Noldus CatWalk where LH represent left hindpaw and RF is right forepaw. B. A Hoxb8FlpOTdT mouse on the regularly spaced horizontal ladder. C. A photo of a Hoxb8FlpOTdT mouse undergoing the BMS assessment. D. A photo of a mouse on the lid-grasping test where the arrow represents full grasping ability (score of 2). 36 2.3 Results 2.3.1 Noldus CatWalk To determine if the Noldus CatWalk would be a useful tool for high hemisection studies, I initially created a heatmap of all of the behavioural outcomes from this thesis (Hoxb8FlpOTdT), along with data from a second experiment done with the same behavioural conditions in order to increase the sample size (Hoxb8IRESCreTdT). This heatmap contained an overwhelming amount of data, some of which failed to follow a clear pattern of recovery throughout the 28-day period. Furthermore, Work by Batka et al. (2014) found that most gait parameters vary with speed, which itself varies day-to-day. In an effort to mitigate this confound, I re-assessed the behavioural parameters at three time points: pre-injury, “early” post-injury (averaged days 8/9 and 12/13), and “late” post-injury (averaged days 24/25 and 28/29). I conducted this analysis with the goal of using averaged time points to control for speed variability in order to better detect patterns of recovery or lack thereof (Fig. 2.3 A, B). To allow for a meaningful interpretation of the results in this heatmap, the behavioural parameters were grouped based on the type of change that occurred after injury. These changes include: a change that occurred immediately after the injury and then changed back towards baseline (a change with injury and regression with time), a change that occurred immediately after the injury and progressed with time (a change with injury and progression with time), a change that occurred only at the late timepoint post-injury (a change with time), a change that occurred after injury and remained stable with time (a stable change), and no change. 37 The ability to grip/grasp is impaired after cervical SCI (Anderson et al., 2004). Grip-dependent tests (the regularly spaced horizontal ladder and lid-grasping) are valuable tools to assess recovery following this type of injury. The ipsilesional forepaw had a significantly decreased ability on the horizontal ladder and the lid-grasping test at the early post-injury assessment when compared to pre-injury. These findings then significantly improved by the late time point post-injury (Fig 2.3 A). Only a few of the Noldus CatWalk measurements followed a similar pattern of recovery, one of them being the percentage of time spent standing on lateral paws. This time period increased significantly immediately following injury. While it did show significant improvements by 28-days, it was not at the level of pre-injury measurements, suggesting that the animals initially spent most of their time standing on the spared side, with this behaviour decreasing as the animals recovered (Fig. 2.3 B). Another Noldus CatWalk parameter that had a similar pattern of recovery was the ipsilesional forepaw stand index (Fig. 2.3 A). The paw was lifted from the runway at a significantly decreased speed immediately after injury and this improved by the late time point. The behaviours that demonstrated a stable change (increase or decrease) are important to highlight, as they represent a change that is not improving with time. In the regularly spaced horizontal ladder there was a stable improvement in percent success for the contralesional forepaw after injury (Fig 2.3 A). In terms of the Noldus CatWalk, the ipsilesional forepaw had a significant decrease in measurements pertaining to contact intensity and the amount of time spent standing on the injured paw (max contact max intensity, max intensity, and single stance). On the other hand, the contralesional 38 forepaw had a significantly stable increase in the time spent standing on this paw (stand) and contact area (max contact area) (Fig 2.3 A). The last type of change that occurred in the behavioural testing was a significant change at only the late time point post-injury, which potentially suggested that the animals had formed a new strategy to perform the task as a result of recovery in other areas. This type of change did not occur outside of the Noldus CatWalk parameters and included a significant increase in the percentage of time spent standing on four paws (standing on four %), and a significant decrease in the contralesional forepaw maximum intensity (Fig 2.3 A, B). While some of the Noldus CatWalk parameters had a pattern of recovery, several measurements did not change despite being relevant to the injury model. For example, there was no change in time spent standing on the ipsilesional forepaw (stand), the contact area of the ipsilesional forepaw (max contact area), or the mean intensity of this paw. Additionally, there were several significant changes in the sham animals – some of these might be attributable to the severity of the surgical procedure to expose the cervical spinal cord. 39 Figure 2.3. Heatmaps showing changes to behavioural parameters following unilateral hemisection and laminectomy. A-B. Each row represents a condition and paw. Horizontal ladder, grasping, BMS, and BMS subscore are displayed at the top, and the Noldus CatWalk parameters are below. Within each condition there are data for each timepoint post-injury. Results are shown as a change from baseline where the clear colour and numerical value 0 represent baseline, while dark red and dark green are the furthest point from baseline (numerical value 1.0 and -1.0, respectively). +/- indicate a significant increase or decrease to day 8-12, respectively, calculated with a one-way ANOVA. C. A legend outlining the type of changes that occur after injury. * in B indicate corrections to phase dispersion and coupling to correct the cyclic nature of the measurement. 40 2.3.2 A Finer-Grained Look at Recovery on the Horizontal Ladder, Grasping, and Noldus CatWalk Behavioural assessment was executed for 1 month following hemisection or sham surgeries with the purpose of objectively assessing functional recovery. Figure 2.4 provides a more in-depth look at the behavioural parameters that displayed functional recovery determined through the heatmap analysis (Fig. 2.3). On the regularly spaced horizontal ladder test, the ipsilesional forepaw and hindpaws of hemisected mice had a significant improvement in percent success over the assessment period (Fig. 2.4 A, B, post-hoc power 1.00 and 0.84, respectively). The grasping ability of the ipsilesional forepaw of hemisected mice also improved over the duration of the assessment, with some animals regaining fully grasping ability by the experimental endpoint (Fig. 2.4 E, post-hoc power 0.999). Unsurprisingly, there were no significant changes in any of the paws assessed on the horizontal ladder or grasping tests for sham animals (Fig. 2.4 C, D, F). Motor recovery in BMS and BMS subscore did not show any significant improvements in sham or hemisected animals (data not shown). In the Noldus CatWalk, there was a significant improvement at some time points for the ipsilesional forepaw stand index (Fig. 2.4 G, post-hoc power 0.96), the percentage of time spent standing on lateral paws (Fig. 2.4 H, post-hoc power 1.00), and some phase dispersion and coupling measurements (Fig. 2.4 I, J, post-hoc power 1.00). There were no significant findings in these parameters obtained with the Noldus CatWalk for the sham animals (Fig. 2.4 G-J). 41 42 2.3.3 The Combined CatWalk Index Reveals that the Parameters Obtained by the Noldus CatWalk Are Not a Strong Measure for Spontaneous Recovery Work by Crowley et al., (2018) generated the CCI using a mathematical model to better understand what Noldus CatWalk parameters were important for assessing motor recovery after SCI. They correlated their Noldus CatWalk data with BMS, as it was a strong marker of motor recovery in their experiment. In this work, broad and coarse analysis of spontaneous recovery after C3 hemisection (Fig. 2.3, 2.4) demonstrated that the regularly-spaced horizontal ladder and wire-cage lid grasping assessments were the best parameters to assess spontaneous functional recovery. Therefore, I calculated the CCI using these parameters and plotted it against the corresponding grasping ability or regularly spaced horizontal ladder data (Fig. 2.5, R2=0.462, 0.4079, p <0.0001). These data suggest that the Noldus CatWalk was not a useful tool to measure spontaneous recovery following unilateral C3 hemisection. However, this analysis also ranked all of the gait data obtained on the Noldus CatWalk against grasping or horizontal ladder data (Table 2.1, 2.2). The four strongest correlated gait parameters with the regularly-spaced Figure 2.4. Motor recovery on a variety of behavioural assessments in hemisection and sham mice. A-B. Horizontal ladder percent success for hemisected animals over the 28-day assessment period for both ipsilesional and contralesional forepaws (A) and hindpaws (B). C-D. Horizontal ladder percent success for sham animals over the 28-day assessment period for both ipsilesional and contralesional forepaws (C) and hindpaws (D). E-F. Grasping ability for ipsilesional and contralesional forepaws in both hemisected (E) and sham (F) animals. G. The left front stand index of hemisection (purple) and sham (green) animals over the 28d period. H. The percentage of time spent standing on lateral paws for hemisection (purple) and sham (green) animals over the 28d period. I. The phase dispersion for the right front paw (anchor) to the right hindpaw (target) in hemisection (purple) and sham (green) animals over the 28d period. J. The coupling for the right front paw (anchor) to the right hindpaw (target) in hemisection (purple) and sham (green) animals over the 28d period. * Indicates statistically significant findings and n.s. represents not statistically significant results (Two-way repeated measures ANOVA). 43 horizontal ladder are the same parameters that displayed the same type of change (improvement with time) on the heatmap analysis (Fig. 2.3). While this reveals that a few gait parameters obtained with the Noldus CatWalk spontaneously recovery after unilateral C3 hemisection, the majority do not. In the future, it may be useful to use the CCI to investigate how treatment-induced recovery differs from spontaneous recovery. 44 Figure 2.5. The gait parameters obtained with the Noldus CatWalk do not strongly correlate with grip- and grasp-dependent parameters. A-B. Linear regression plots for ipsilesional forepaw grasping ability (A) and ipsilesional forepaw horizontal ladder (B) and the CCI. 45 Rank CatWalk Parameter Name Slope Intercept RSQ 1 Support_Single_(%) -0.043023 1.352441 0.27002 2 LH_MaxIntensity_Mean 0.011803 -0.403759 0.250629 3 LH_MaxContactMaxIntensity_Mean 0.011868 -0.343658 0.247257 4 LF_MaxContactMaxIntensity_Mean 0.01746 -0.658679 0.23937 5 Couplings_RF->RH_CStat_Mean -0.031216 3.012118 0.238394 6 LH_MeanIntensityOfThe15MostIntensePixels_Mean 0.015781 -0.572738 0.237862 7 LH_MeanIntensity_Mean 0.037629 -1.60401 0.23184 8 RH_MeanIntensity_Mean 0.036934 -1.484006 0.228635 9 LH_DutyCycle_(%)_Mean 0.030005 -0.697924 0.222086 10 LF_MaxIntensity_Mean 0.015631 -0.58907 0.219078 11 LF_PrintWidth_(cm)_Mean 1.371417 0.24644 0.214788 12 Support_Lateral_(%) -0.070822 1.462957 0.212648 13 Couplings_RH->RF_CStat_Mean 0.032122 -0.119448 0.212223 14 PhaseDispersions_RF->RH_CStat_Mean -0.032122 2.996063 0.207894 15 LH_MaxContactMeanIntensity_Mean 0.038445 -1.498917 0.205917 16 LF_MeanIntensityOfThe15MostIntensePixels_Mean 0.020452 -0.761467 0.202599 17 RH_MaxContactMeanIntensity_Mean 0.034442 -1.205004 0.179248 18 StepSequence_RegularityIndex_(%) 0.013956 -0.016302 0.169587 19 Couplings_LF->LH_CStat_R 1.405023 0.124482 0.162663 20 Couplings_LF->RH_CStat_R 1.445635 0.094921 0.160963 21 Couplings_RF->RH_Mean -0.031976 3.005538 0.157851 22 LF_MeanIntensity_Mean 0.034752 -1.263238 0.155361 23 LF_DutyCycle_(%)_Mean 0.030809 -0.466184 0.151844 24 Couplings_LF->RF_CStat_R 1.405354 0.152837 0.146542 25 RH_MaxIntensity_Mean 0.008575 -0.029668 0.145703 26 LH_ManualPrintLength_(cm)_Mean 1.603713 -0.036467 0.145674 27 LF_MaxContactMeanIntensity_Mean 0.037302 -1.284919 0.145247 28 Couplings_RF->LF_Mean -0.044108 3.434893 0.144199 29 RH_MaxContactMaxIntensity_Mean 0.008742 0.006403 0.143915 30 LH_Swing_(s)_Mean -4.62639 1.614289 0.140268 Table 2.1. The top 30 Noldus CatWalk Parameters Correlated with Grasping Ability 46 Rank CatWalk Parameter Name Slope Intercept RSQ 1 Support_Lateral_(%) -0.041409 0.798763 0.289619 2 PhaseDispersions_RF->RH_CStat_Mean -0.01713 1.599463 0.23331 3 Couplings_RF->RH_CStat_Mean -0.014924 1.498776 0.215016 4 LF_StandIndex_Mean 0.053961 0.938593 0.191928 5 Couplings_RF->LF_Mean -0.024219 1.874261 0.173511 6 Couplings_RH->RF_CStat_Mean 0.014374 0.036905 0.16771 7 Couplings_RF->LF_CStat_Mean -0.016753 1.487132 0.155952 8 LF_PrintWidth_(cm)_Mean 0.565923 0.229711 0.145977 9 Support_Single_(%) -0.015672 0.663875 0.142756 10 LF_DutyCycle_(%)_Mean 0.014897 -0.173062 0.142646 11 LF_MaxIntensity_Mean 0.006252 -0.094593 0.139878 12 LF_MaxContactMaxIntensity_Mean 0.006661 -0.091343 0.139044 13 LF_MeanIntensityOfThe15MostIntensePixels_Mean 0.008122 -0.158443 0.127515 14 LH_MeanIntensityOfThe15MostIntensePixels_Mean 0.005759 -0.032048 0.126408 15 LF_ToeSpread_(cm)_Mean 0.524333 0.351703 0.124973 16 PhaseDispersions_LF->RF_CStat_Mean 0.015902 -0.149505 0.124947 17 LH_MaxIntensity_Mean 0.00414 0.049867 0.123043 18 LH_MaxContactMaxIntensity_Mean 0.00417 0.070089 0.121821 19 StepSequence_AA_(%) 0.014012 0.485723 0.111452 20 Couplings_RF->RH_Mean -0.012948 1.351342 0.102136 21 LH_PrintWidth_(cm)_Mean 0.426271 0.265513 0.101043 22 LF_Stand_(s)_Mean 2.555598 0.160351 0.099824 23 LF_PrintArea_(cm²)_Mean 1.122984 0.291126 0.092738 24 Couplings_LF->RF_CStat_Mean 0.012506 -0.003608 0.08582 25 LH_ManualPrintLength_(cm)_Mean 0.602224 0.153131 0.081517 26 LF_ManualPrintLength_(cm)_Mean 0.830491 0.102093 0.077637 27 StepSequence_CA_(%) 0.010076 0.474176 0.074159 28 LH_MaxContactMeanIntensity_Mean 0.011504 -0.204279 0.073585 29 LF_PrintLength_(cm)_Mean 0.467213 0.251431 0.071094 30 LH_ToeSpread_(cm)_Mean 0.492477 0.320289 0.070539 Table 2.2. The top 30 Noldus CatWalk Parameters Correlated with the Horizontal Ladder 47 2.4 Discussion 2.4.1 Functional Recovery Occurred Following Unilateral Cervical Hemisection The goal of this chapter was to characterize the natural history of motor-related behavioural changes following C3 spinal hemisection. Research surrounding cervical SCIs in rodent models is becoming increasingly prevalent due to the high occurrence of cervical SCIs in humans (Khaing et al., 2012). These injuries result in deficits to hand function, and accordingly, impairments in grip/grasp assessments in rodents (Anderson et al., 2004; Khaing et al., 2012). Both coarse and fine-grained analysis of each measurement revealed that the horizontal ladder and lid-grasping assessments had a significant deficit immediately after injury that improved over 28 days (Fig. 2.3, 2.4). A few of the gait parameters obtained with the Noldus CatWalk followed the same pattern of recovery (Fig. 2.3). This demonstrated that there was spontaneous functional recovery following unilateral C3 hemisection, similar to other studies (Anderson et al., 2004; Khaing et al. 2012). There is evidence to suggest that plasticity can be promoted with training or exercise, and that regular behavioural assessment thus might have confounded spontaneous changes (Zou, 2021; Zhang et al., 2019; Rauti et al., 2020). However, Caudle et al. (2011) determined that rats reach maximal behavioural recovery by ambulating in their cage without further training following thoracic injury. This suggests the behavioural assessments performed here were likely insufficient to underlie any improvements. 48 2.4.2 Limitations with the Noldus CatWalk When the Noldus CatWalk was developed, one of its perceived primary benefits was its ability to assess multiple parameters pertaining to each paw individually and simultaneously (Zheng et al., 2021). However, the large amount of information provided by the system has led researchers to “cherry-pick” either the most significant measurement or parameters that are commonly reported in the literature. Neckel et al. (2020) highlight the importance of assessing each parameter obtained with the CatWalk in a coordinated manner due to locomotion being based on the coordinated movement of the whole body, and not individual values. Another important factor to consider is speed. Locomotor tests are characterized under two categories: forced or unforced based on whether speed is imposed on the subject (Batka et al., 2014). In unforced tests, such as the Noldus CatWalk, speed is not regulated with the goal to represent natural locomotion more accurately. In 2014, Batka et al. showed that all CatWalk parameters were uniquely dependent on speed and suggested the CatWalk data should therefore be analyzed using a linear mixed model ANOVA. While outside the scope of this thesis, in the future it would be interesting to determine if analyzing the data using this statistical method would generate similar results. In an effort to circumvent these limitations, I generated a heatmap (Figure 2.3 A, B) to look at the changes in all gait parameters generated with the CatWalk at early and late time points post-injury to determine the pattern of recovery. Dellon et al. (1991) found that the weight gain of rats over the CatWalk assessment period following sciatic nerve injury led to significant increases in print length, toe spread, and intermediate toe spread. Similarly, Zheng et al. (2023) found 49 that changes in weight affected the validity of the paw intensity measurements, and caution that a dramatic loss of weight after injury could affect the interpretation. Intuitively, paw intensity measurements are based on the pressure of the paw on the runway, therefore it stands to reason that a lighter subject could potentially have a decreased intensity measurement based solely on the change in weight. In the results presented here, there was a significant decrease in the left forepaw print width, maximum intensity measurements, and toe spread that occurred after injury and remained stable with time. While it is likely that these changes are attributed to injury, it is possible that the transient weight loss in this experiment affected these results. Additionally, there was a significant increase at the “late” post-injury time point when compared to “early” post-injury of the ipsilesional hindpaw max contact area and print area. The weight change during the 28-day assessment period (d0 mean weight = 23.519 g, SEM = 1.113, n = 16, d14 mean weight = 20.713 g, SEM = 0.612, n= 16, and d28 mean weight = 24.019 g, SEM = 1.113, n = 16) could have impacted the findings. While these results indicate a positive trend for spontaneous recovery, this recovery could have been confounded by the drastic reduction in weight during the “early” assessment post-injury. 2.4.3 Several Parameters on the Noldus CatWalk Had Stable Deficits or No Change Following C3 Hemisection The Noldus CatWalk, BMS, and gait-based assessments proved valuable for determining changes after injury and spontaneous recovery. There were several parameters on the Noldus CatWalk related to the injured forepaw that had stable deficits over the assessment period (print width, maximum intensity, single stance). Additionally, 50 several paw parameters (for all paws), the regularity index, and forepaw base of support did not change significantly over the assessment period. These represent parameters that are unable to change on their own (spontaneously recover), therefore making them key outcome measures to address with future treatment strategies. There are several examples of pharmacological, genetic, and exercise-based treatments that lead to improvement in BMS, grip strength, and parameters on the Noldus CatWalk following SCI (Wang et al., 2011; Goldshmit et al., 2020; Ahuja et al., 2017). Focusing treatment on these stable change parameters could pave the way for novel therapeutics. 2.4.4 Recovery or Adaptation to a “New Normal”? Motor recovery is commonly interpreted by comparison of pre- and post-injury measurements. While animals clearly regain functional skills, it is very rare for the animal to recover to pre-injury levels of mobility (Fouad & Tse, 2008). In this experiment, following the 28 d hemisection, the mice showed quantitative and qualitative improvements on motor performance but did not return to preinjury levels, leading to the question as to whether the mice had adapted to a “new normal”. Work by Ballermann and Fouad (2006), found that rats had changes in muscle activation pattern, postural stance, and kinematics to compensate for damage caused by a T8 SCI. Following a C2 lesion, rats formed alternate limb movements to successfully perform the food pellet reaching task (McKenna & Whishaw, 1999). Furthermore, Grasso et al. (2004) demonstrated that humans with an SCI trained to perform “body weight supported stepping” do so by learning a new motor strategy. Thus, motor recovery following SCI can occur via the development of compensatory motor strategies rather than simply recovering the pre-injury mechanism. 51 In the present work, the mice recovered on the horizontal ladder assessment in the ipsilesional forepaw; however, they never reached pre-injury levels (Fig. 2.4 A). While the BMS did not show statistically significant findings, the ipsilesional hindlimbs did have a stable change over time, suggesting that the mice may have formed a “new normal” motor strategy (Fig. 2.3 A). Neckel et al. (2020) subjected rats to the Noldus CatWalk following a C5 hemisection and found that only the most affected forepaw recovered to pre-injury like behaviour. Interestingly, the less impaired paws took more abnormal steps in an effort to compensate for the impairment. In the Noldus CatWalk data the contralesional forepaw had a stable increase in several parameters including time spent standing on this paw, print area, and max contact area (Fig. 2.3 A). In this experiment, the significant improvement in the mice suggests that a new locomotor strategy was established, which may have been fueled by changes in neural circuitry governing the uninjured paws. 2.4.5 The Battery of Tests May Impact Results Mice were subjected to four behavioural tests with each mouse completing the Noldus CatWalk and BMS on one day, and the regularly spaced horizontal ladder and grasping ability the following day. Subjecting the same mice to a battery of tests rather than having groups assessed on each parameter allows one to use fewer mice, compare outcomes between tests, and have more confidence interpreting the motor recovery results (MclIwain et al., 2001). The primary disadvantage to this model is that mice subjected to numerous tests may perform differently than those trained and tested solely on one test (McIlwain et al., 2001). It is important to be cognisant of these potential drawbacks when interpreting the results. 52 2.4.6 The Noldus CatWalk and Pain The Noldus CatWalk has been used in a variety of different studies to look at neuropathic, inflammatory, and arthritic pain. To use the Noldus CatWalk to assess pain, researchers focus on paw intensity parameters (max contact area, print area, intensity, and print length) and coordination (regularity index, swing, and duty cycle) (Ferreira-Gomes et al., 2008; Möller et al., 2008; Sakuma et al., 2013; Xu et al., 2019). Möller et al. (2008) assessed the effectiveness of certain drugs in arthritic pain in rats and found that rats with arthritic pain had a reduction in print area, stance duration, and mean intensity of the injured paw. Following SCI in humans, 60-80% experience pain, of which one-third of these individuals report the pain to be severe (Masri & Keller, 2012). This chronic pain results in decreased physical and psychosocial function (Hunt et al., 2021). The STT, an ascending pathway that originates in the dorsal horn of the spinal cord and decussates at-level, carries nociceptive- and temperature-related information to the thalamus (Al-Chalabi et al., 2023). A unilateral C3 hemisection severs the contralateral STT leading to a loss of pain and temperature sensation on the spared side of the body, while leaving the ipsilesional STT intact. Activity of STT neurons is inhibited, directly or indirectly by descending monoaminergic systems (Millan, 2002). It is therefore possible that some of the Noldus CatWalk results are related to the pain the mice are experiencing following hemisection. There was a significantly stable decrease in several paw intensity measurements following hemisection in the ipsilesional forepaw (max contact max intensity, max intensity, and single stance) (Fig. 2.3 A). These changes were not consistent with the 53 sham animals (a change that regressed with time for the intensity measurements and no change for single stance) (Fig 2.3 A). Additionally, there was also a significantly stable decrease in the duty cycle (the percentage in stance during a single-step cycle) in the ipsilesional forepaw following injury, while there was no change in this parameter for the sham animals (Fig. 2.3 A). Based on these findings, it is reasonable to speculate that the hemisected animals are experiencing pain that is unrelated to post-surgical pain. Furthermore, it is possible that the pain present in the hemisected mice was masked in the horizontal ladder and lid-grasping tests due to the motivation not to slip or fall off of the apparatus. 2.4.7 Concluding Remarks After unilateral C3 hemisection, it is evident that the natural course of recovery (or lack thereof) involves a wide variety of behavioural changes (i.e. transient change, stable change, and no change). The grasp-dependent behaviours clearly demonstrated spontaneous recovery, as did some of the gait parameters from the Noldus CatWalk, while BMS and other Noldus CatWalk measurements provided evidence of a “new normal”, as well as parameters that may benefit from further treatment. The findings presented in this chapter highlight the importance of assessing a wide range of behavioural outcomes following SCI. In doing so, they emphasize the integral role of these tests in evaluating treatments that might benefit humans with SCI beyond that afforded by limited spontaneous functional recovery. 54 Chapter 3: A Comparison of Hoxb8-Driven Reporter Distribution in the Brains of Four Transgenic Mouse Lines 3.1 Introduction Animal behaviour relies on the ability to detect and respond to external and internal stimuli. In the somatosensory system, primary afferent neurons of the DRG are the first to be activated upon stimulation. This information is transmitted to the spinal cord and brainstem where it is modulated by local and descending influences. Long-distance projection neurons then carry this processed information to supraspinal structures where it can be integrated and acted upon. The positioning of ascending tracts in the mouse spinal cord has been well-established (Watson & Harrison, 2012), but their routes to supraspinal termination zones – particularly the midbrain and thalamus – is less well-understood, despite a relatively deep knowledge of their cells of origin in the spinal cord (Kayalioglu, 2009). Historically, the primary method to label axons ascending from the spinal cord to the brain (“spinofugal” projections) – a prerequisite for investigating the circuitry responsible for relaying sensory information – has employed degeneration studies and anterograde and retrograde tracing techniques. As highlighted in Chapter 1, there are several limitations associated with these approaches (Cliffer et al., 1991; Saleeba et al., 2019). The most pertinent when it comes to the spinal cord is the relatively small number of neurons that anterograde tracers can reliably label, especially when the neurons of interest are widely distributed, as they are along the length of the cord. This prompted us to ask whether genes expressed specifically at the level of the spinal cord 55 could be exploited as drivers for reporter expression in order to permanently label spinofugal projections. Given their restricted expression to the caudal neuraxis (brainstem and spinal cord), Hox genes represent promising candidates. Rostral expression boundaries of the different Hox proteins are correlated with their position in their respective clusters (i.e. 3’-positioned Hox genes expressed more rostrally, 5’-positioned Hox genes expressed more caudally). One family of Hox genes (Hoxb) shows a marked restriction in expression to the dorsal (alar) plate of the developing spinal cord (Graham et al., 1991), implicating a role in the establishment of spinofugal pathways. Indeed, in mice lacking Hoxb8, fewer neurons of the dorsal spinal cord are generated (or more are lost to apoptosis), their usual laminar positioning is disturbed, and their afferent input is disorganized; these are accompanied by sensory deficits including hyposensitivity to heat and abnormal itch leading to overgrooming (Holstege et al., 2008), although the latter phenomenon has been attributed to the absence of Hoxb8-lineage microglia (Chen et al., 2010). These findings point to Hoxb8 as a useful tool to drive expression of reporter molecules to label spinofugal axons. In this chapter I evaluate the utility of lineage tracing in four transgenic mouse lines (Hoxb8Cre, Hoxb8FlpO, Hoxb8T2A and Hoxb8IRESCre) for the bulk-labeling of all spinofugal projections, with a view to selecting the most appropriate one for assessing their morphological plasticity following incomplete SCI (Chapter 4). The earliest efforts to generate Hoxb8 reporter constructs were carried out by the Deschamps group, who aimed to identify the regulatory elements responsible for setting the anterior expression boundary of Hoxb8 in the brainstem (Charité et al., 1995). An 56 11kb segment (located upstream of the Hoxb8 gene and including part of the adjacent Hoxb9 sequence) was used to generate one line of LacZ reporter mice, and partially mimicked endogenous Hoxb8 expression but with a more caudal expression boundary (the fourth cervical spinal segment rather than the brainstem). Two of the transgenic mouse lines used in this thesis were generated with this DNA sequence fused to either a Cre (Witschi et al., 2010) or a FlpO recombinase cassette (Zhang et al., 2018). Crossing these transgenic mice with appropriate reporter mice should allow identification of axons ascending from the spinal cord to the brain. The utility of random insertions of DNA fragments such as these is affected (either positively or negatively depending on the question being asked) by the absence of regulatory elements controlling endogenous expression. In the case of the construct used to generate Hoxb8-Cre and Hoxb8-FlpO mice, the transgenes are no longer in the vicinity of three important retinoic-acid response elements (RARE), positioned between the nearby Hoxb4 and Hoxb5 genes (Valarché et al., 2003; Oosterveen et al., 2003; Afzal et al., 2023), and responsible for setting the rostral Hoxb8 boundary. These mice also lack a micro-ribonucleic acid (miRNA) target sequence (specific for miR-196a) in the 3’ untranslated region (UTR) of the transgene, required for restricting endogenous Hoxb8 expression (Yekta et al., 2004, Hornstein et al., 2005). Other uncertainties when using this transgenic strategy are the possible disruption of coding sequences at the insertion site, the influence of regulatory elements not normally associated with (Hoxb8) transcription, and the timing of changes in chromatin accessibility as development proceeds (Noordermeer et al., 2014; Neijts et al., 2017). This is of particular importance 57 for the Hox genes which are transcribed sequentially (a processes referred to as the “Hox clock”) (Kmita & Duboule, 2003; Soshnikova & Duboule, 2009). To circumvent some of these confounds, a targeted insertion (knock-in) approach can be used. In this study I investigated two such models. The first is a knock-in that uses an internal ribosome entry site (IRES) upstream of Cre and under the control of endogenous Hoxb8 regulators (Chen et al., 2010). The second Hoxb8 knock-in drives FlpO recombinase expression. In this case a sequence encoding the self-cleaving peptide T2A was inserted before the Hoxb8 stop codon in exon 2 and was fused to FlpO before the 3’ UTR (Bohic et al., 2023). Neither of these targeted models is expected to interfere with Hoxb8 function, and while Cre and FlpO have equivalent excision efficiencies (Birling et al., 2012), subtle differences in transcriptional (Cre) or translational (FlpO) recombinase activity might be expected. Whereas the IRES system results in less efficient transcription of the second gene in the sequence (20-50% of the first, Mizuguchi et al., 2000), the near 100% efficiency of T2A self-cleavage results in a 1:1 ratio of Hoxb8 and FlpO recombinase protein expression. Here, I anatomically characterize these four different Hoxb8 reporter lines using confocal, superresolution, and light-sheet microscopy. I confirm that all models bulk-label ascending projections to the brain, but with important differences given rostral expression boundaries in targeted and non-targeted transgenic mice. The routes taken by these axons through the pons, midbrain and thalamus have also been clarified, and I described a new decussating tract of spinofugal axons ventral to the hypothalamus. Finally, I was able to successfully determine the preferred model for Aim 3 – Hoxb8FlpOTdT mice. 58 3.2 Methods 3.2.1 Animals and Specimen Collection All experiments were carried out in accordance with guidelines of the Canadian Council on Animal Care and the UBC Animal Care Committee. Hoxb8IRESCre (RRID:IMSR_JAX:035978), Hoxb8Cre (obtained from the lab of Hanns Zeilhofer, ETH Zurich, Switzerland), Hoxb8FlpO (C57BL/6-Tg(Hoxb8-Flpo)11Oki/Kctt, sperm obtained from the European Mutant Mouse Archive) mice were crossed with appropriate TdTomato (tdt) reporter mice (Ai14, 007914, RRID:IMSR_JAX:007914; Ai65, 021875, RRDI: IMSR_JAX:021875) (Fig. 3.1). Mice were kept on a standard diet and light cycle. One male and one female (n=2) of the Hoxb8IRESCreTdT strain, two males and one female (n=3) of the Hoxb8CreTdT strain, and three males and one female (n=4) of the Hoxb8FlpOTdT strain 8-12 weeks of age were deeply anesthetized with a 1500mg/kg intraperitoneal injection of urethane and subsequently perfused with heparinized saline followed by 4% paraformaldehyde (PF) in 0.1M phosphate buffer (PB). The brains and rostral portion of the spinal cords were removed and fixed overnight in 4% PF in 0.1M PB, after which they were equilibrated in 20% sucrose in 0.1M PB. Four percent PF-fixed brains and cervical spinal cords of Hoxb8T2AFlpOTdT (n=2, 1M, 1F) mice were provided by Dr. Victoria Abraria (Rutgers University) (Bohic et al., 2023). Photos of each specimen were obtained using two UV lights, one on each side to visualize the rostral boundary of reporter expression in an intact CNS. The brains were then frozen in Optical Cutting Temperature (OCT) compound and stored at -80°C. One neonatal Hoxb8FlpOTdT mouse was used (2 days of age). The mouse was deeply anesthetized using isoflurane following which it was decapitated. The head of 59 the mouse was fixed for 48 hours in 4% PF in 0.1M PB, after which the brain was removed. The brain was then fixed and equilibrated as described above. 3.2.2 Lateral Spinal Hemisection Four additional Hoxb8CreTdTmice underwent lateral hemisection at C3 in order to injure all reporter-positive spinofugal axons on one side of the animal. Immediately prior to surgery, animals received subcutaneous injections of buprenorphine (0.01 mg/kg) and lactated ringers (0.1mL). The animals were anesthetized with isoflurane (5% induction, 2-3% maintenance) following which eye lubricant was liberally applied, the upper back was shaved (excess hair removed with depilatory cream), and the skin surface was disinfected with ethanol and chlorhexidine. Mice received a midline incision over the upper cervical spinal cord, and a C3-C4 laminectomy was performed. Hemisections were made by first piercing the dura and cord with a 25-gauge needle just to the right of the dorsal midline vein. One blade of a pair of micro scissors was then inserted to puncture to the bottom of the spinal canal; the other was placed outside the lateral side of the left hemicord and the scissors were closed. The muscle and skin layers were closed with sutures. Mice survived for 6 days (n=1), 2 weeks (n=2) or 4 weeks (n=1) during which they underwent appropriate monitoring and post-surgical care. Mice received buprenorphine (0.01mg/kg) and lactated ringers (0.1mL) subcutaneously three times daily for the first 3 days post-surgery. 3.2.3 Tissue Processing Eight brains from intact mice (two adults of each genotype) were serially-sectioned in the coronal plane at 100 microns using a cryostat (Micron). One male Hoxb8CreTdT and one male Hoxb8FlpOTdT brain were also sectioned parasagittally at 60 100 microns. An additional Hoxb8FlpOTdT mouse was sectioned coronally at 50-micron sections and thaw mounted to Superfrost Plus slides. The 2d old-mouse pup brain was also sectioned coronally at 100 microns. Four (~101 µm) rectangular adhesive vinyl frames custom made on a Cricut (Explore Air) were applied to Superfrost Plus slides before thaw-mounting sections to prevent tissue compression and damage during coverslipping. The slides were air dried for a minimum of 2 hours and washed with phosphate-buffered saline (PBS, 2x, 10 min each). Slides were then delipidated overnight with Cubic-L (TCI, Cat# T3740) to which NaCl was added (to 0.45%) to prevent tissue swelling and detachment from the slides. The following day, the refractive index (RI=1.520) was matched to glass using Cubic-R+(M) (TCI, Cat# T3741) in 2x20 minute applications. Finally, the slides were coverslipped using 24x50mm coverslips and Cubic mounting medium (TCI, Cat# M3294) or ProLong Glass mountant (ThermoFisher Cat# P36984). 3.2.4 Immunohistochemistry Sections were permeabilized and blocked with 10% normal donkey serum and 0.2% Triton X-100 plus 0.02% sodium azide in PBS (PBSTx azide) for one hour before the primary antibodies were applied (Table 3.1) for ~48 hours. Prior to application of the secondary antibodies (Table 3.1), the slides were washed three times (5 minutes each) with PBS. Secondary antibodies were applied overnight at room temperature and subsequently washed 3 times for 5 min each in PBS. The refractive index was matched to glass using Cubic-R+(M) (TCI, Cat#T3741) in one 20-minute application. Slides were coverslipped using 24x50mm coverslips and the cubic mounting media (TCI, Cat# M3294) before being sealed with nail varnish. Slides with 50 micron-thick sections were 61 not cleared with Cubic reagents, and instead coverslipped using ProLong Glass anti-fade mounting media (Invitrogen, P36984). Table 3.1. Antibodies for immunohistochemistry Antigen Source Conjugate/Dilution Sox9 R and D Systems Cat# AF3075, RRID:AB_2194160 NA/1:200 NeuN (Fox3) Millipore Cat# MAB377, RRID:AB_2298772 NA/1:1000 p73 Abcam Cat# ab40658, RRID:AB_776999 NA/1:500 Iba1 FUJIFILM Wako Shibayagi Cat#019-19,741, RRID:AB_839504 NA/1:1000 Iba1 Cell Signaling Technology, Cat#17198S, RRID: AB_2820254 Alexa 488/1:250 PDGFRa R and D Systems Cat# AF1062, RRID:AB_2236897 NA/1:200 Goat IgG Jackson ImmunoResearch, Cat#705-165-147, RRID: AB_2307351 Alexa 488/1:500 Mouse IgG Jackson ImmunoResearch, Cat#715-546-151, RRID: AB_2340850 Alexa 488/1:500 Mouse IgG Jackson ImmunoResearch, Cat#715-605-150, RRID: AB_2340862 Alexa647/1:500 Rabbit IgG Abcam, Cat#ab96894 Dy 650/1:500 3.2.5 Nissl staining – spinal cords The spinal cords (cervical to lumbar) were harvested from an additional five PF-perfused mice (one of each genotype and a second Hoxb8CreTdT) to examine neuronal recombination in the spinal cord. In this case, 50-micron frozen sections were thaw-mounted onto glass slides and counterstained with NeuroTraceTM 640/660 Deep-Red Fluorescent Nissl Stain (1:200 for 2h, ThermoFisher Catalog number: N21483) 3.2.6 Image acquisition Images of coronally-sectioned brains were taken using a 5x objective on a Zeiss LSM800 scanning confocal microscope and Zen Blue software. Each brain section was 62 imaged in all dimensions using tiling and z-stacking (4-6 z-slices with an optical section thickness of 40-50 µm after orthogonal projection). The images were stitched during acquisition, orthogonally projected, and a single pixel filter was applied to remove digital gain artifacts. To begin registration to the Allen Mouse Atlas coronal average template, any rotation of the microscope images in the rostro-caudal plane was determined using QuPath. The Allen Mouse Atlas average coronal template was then digitally sectioned to account for this rotation (which was always less than ±2.5°). Fiducials were selected that included areas of high TdTomato reporter intensity, major surface structures, and internal landmarks (ex: inner surfaces of the ventricles). In FIJI, 8-12 fiducials were selected on each Allen Mouse Atlas section and then the same landmarks were chosen on the corresponding microscope image (FIJI > region of interest). The thickness of the microscope images and the Allen Mouse Atlas slices were not identical, therefore adjustments were made by skipping an atlas slice or doubling up (i.e., registering multiple microscope images to the same Allen Mouse Atlas slice) to minimize divergence. In general, an effort was made to keep the fiducials in the same location and only make gradual changes as I moved through the Allen Mouse Atlas along the z-axis. The images were registered to the Allen Mouse Atlas coronal average template using FIJI (moving least squares with similarity method in the registration plugin) and stacked (in tools, stack sorter). It should be noted that there is slight rotation along the rostro-caudal axis of our sections (and registration to a slightly rotated average coronal template). Images of sections for immunohistochemistry were taken using a 20x or 63x objecting using a Zeiss LSM800 scanning confocal microscope and Zen Blue software 63 for the above sections. Super resolution microscopy (63x) was used to capture images of p73-labeled cells (Airyscan). 3.2.7 Light Sheet Microscopy One intact Hoxb8IRESCreTdT mouse and one Hoxb8CreTdT mouse, hemisected 28 days earlier, were deeply anesthetized with a 1500 mg/kg intraperitoneal injection of urethane and perfused with heparinized saline followed by 4% PF in 0.1M PB. Their brains were removed and fixed overnight in 4% PF in 0.1M PB. Brains were washed for 6h (3 x 2h) in PBS on a room temperature shaker protected from light. Brains were delipidated for 7 days with Cubic-L (TCI, Cat# T3740) to which NaCl was added (to 0.45%) to prevent tissue swelling in a 37° incubated shaker protected from light. The cubic-L solution was changed daily for the first 4 days, after which it was changed every second day. The refractive index (RI = 1.520) was matched to glass using Cubic-R+(N) (TCI, Cat# T3983) for 2 days on a room temperature shaker protected from light. The cleared cerebella were embedded in 2.5% agarose in Cubic-R+(N) (TCI, Cat#T3983). Images of the cerebella were acquired using a Zeiss Z.1 Light sheet microscope with Zen Black software with tiling and z-stacking. 64 3.3 Results 3.3.1 Gross tdtomato expression in Hoxb8 reporter mice Transgenic Hoxb8-driven reporter expression was easily identified with UV illumination (Fig. 3.1A). Fluorescence in knock-in strains extended from the tail to the nape of the neck, and included the forelimb; this was not the case in non-targeted strains, in which the forelimb was spared, and reporter expression was limited to skin below mid-torso (Fig. 3.1A). Developmental Hoxb8 mRNA expression extends from the caudal end of the spinal cord to the brainstem (Witschi et al., 2010). This is reflected in the extent of reporter expression in the CNS of Hoxb8IRESCreTdT and Hoxb8T2AFlpOTdT knock-in strains (Bohic et al., 2023, Fig. 3.1B-E), as demonstrated under UV illumination. In the transgenic reporter models (Hoxb8CreTdT and Hoxb8FlpOTdT), the rostral expression boundary was close to C4 (Fig. 3.1D, E), as expected based on previously demonstrated expression of the transgenic constructs in mouse embryos and adult reporters (Charité et al., 1995; Witschi et al., 2010). In 3 of the mouse lines maintained in our facility, recombination occasionally occurred throughout the body. This was not the result of stop codon excision in reporter-positive male gametes: while Hoxb8 is expressed, at least in the human, in spermatogonia/spermatocytes (The Human Protein Atlas), only Sertoli cells, and no cells of the spermatocyte lineage had undergone recombinase in mice (data not shown). Such mice were excluded from analyses. 65 Figure 3.1. Hoxb8 reporter phenotype and genotypes. A. tdtomato fluorescence of mouse pups and adult mice under ultraviolet illumination. Note more caudal reporter boundary (sparing forelimbs) in non-targeted transgenic pups (Hoxb8-CreTdT). Non-fluorescent mice in each panel are littermates from crosses between mice hemizygous for the recombinase and homozygous for tdtomato. “X” indicates exons. B-E. Hoxb8 driven recombinase constructs, reporter strains, and ultraviolet-illuminated brains for targeted (B, C) and non-targeted (D, E) transgenic mice. 66 3.3.2 Reporter expression in the spinal cord Almost all spinal cord neurons caudal to Hoxb8 expression boundaries underwent recombination in all reporter lines (Fig. 3.2). As expected, tdtomato intensity was greatest in the dorsal grey matter given the high neuronal density. In non-targeted transgenic strains, only axon and their terminals were detected in the cord above C4 (Fig. 3.2E). In the thoracic and lumbar cords (but not the cervical cord), vascular-associated cells were apparent, in line with the mesodermal distribution of Hoxb8 expression during development (Deschamps et al., 1999). Therefore, I am able to conclude that all spinofugal projections are tdtomato-positive (but only up to C4 in non-targeted reporter strains). 67 Figure 3.2. Reporter distribution in the spinal cords of Hoxb8 reporter mice. A. Spinal cord cross sections from cervical (below C4), thoracic and lumbar cords of all reporter strains. B. Cross section and equatorial horizontal sections at the Hoxb8 transition zone (C4) from non-targeted transgenic strains. C. Higher-power images of Nissl-stained sections of the intermedial lumbar grey matter illustrating near-complete recombination in neurons. A rare example of a reporter negative neuron is indicated in the bottom panel (arrow). bv, blood vessel. D. As in (C) but at the Hoxb8 transition zone; recombined neurons are indicated with arrows. E. Horizontal sections from the ventral cord of Hoxb8CreTdT mice spanning the hoxb8-transition zone. Recombined motoneurons are indicated by arrows. WM, white matter; GM, grey matter. 68 3.3.3 Spinofugal projections in Hoxb8-reporter mice Spinofugal projections were effectively labeled in all four Hoxb8 reporter lines, but with slight differences between strains depending on the brain region (Fig. 3.3-3.10). Primary afferent terminals (and possible post-synaptic dorsal column axon terminals) were densely labeled in the gracile (Gr), cuneate (Cu) and external cuneate nuclei (ECu) in all strains (Fig. 3.3B, C), as were a few neuronal somata in the gracile nucleus (but not the other dorsal column nuclei) in targeted transgenic models (Fig. 3.3D) (Bohic et al., 2023). Other axons extended from ventral ascending tracts into the reticular nuclei (RN). There were a few unilaterally apparent neurons in the caudal spinal nucleus of the trigeminal nerve (SPVC) in Hoxb8IRESCreTdT mice due to a slightly yawed section (Fig. 3.3B and data not shown). Labeling was also more pronounced in the spinal tract of the trigeminal nerve in the knock-in models, but these were not trigeminal in origin, given the lack of terminal labeling in the SPVC. Additionally, there were some neurons apparent in the nucleus of the solitary tract (NTS) in knock-in strains (NTS, Hoxb8IRESCreTdT > Hoxb8T2AFlpOTdT); these were not present in the untargeted transgenic reporter mice (Fig. 3.3B, C). 69 Figure 3.3. Medullary reporter fluorescence in Hoxb8 reporter strains. A. Schematic indicating plane of section (inset) and the equivalent image from the YSK Unified Anatomical Atlas (https://kimlab.io/brain-map/atlas/). B. Overview of reporter fluorescence from each strain. C. Detail of the dorsal medulla. Reporter-positive neurons are present within the nucleus of the solitary tract (NTS) of targeted strains, and absent from others. D. Reporter-positive neurons (arrows) in the gracile nucleus of targeted transgenic reporter strains. 70 Reporter-positive axons and mossy fibre terminals were present as parasagittal bands – stripes – in the cerebellar granular layer in lobules in the anterior and posterior cerebellum with no obvious difference in density between strains (lobules II, III, IV, VIII and IX) (Fig. 3.4 B, C). In Hoxb8IRESCreTdT mice, but in no other strain, a population of tdtomato-positive spinofugal (or possibly bulbofugal) axons was present in the core of the facial nucleus (Fig. 3.4 C). 71 Figure 3.4. Hindbrain reporter fluorescence in Hoxb8 reporter strains. A. Schematic indicating plane of section (inset) and the equivalent image from the YSK Unified Anatomical Atlas (https://kimlab.io/brain-map/atlas/).B. Overview of reporter fluorescence from each strain (III and IV indicate cerebellar lobules; VII indicates the facial motor nucleus). C. Details of the cerebellum (lobule III) and ventral hindbrain. 72 At the level of the midbrain (Fig. 3.5), axon terminals of the periaqueductal grey (PAG) received a dense projection of reporter-positive axons, which extended well into the thalamus. In targeted transgenic strains (Hoxb8IRESCreTdT and Hoxb8T2AFlpOTdT), many reporter-positive axon terminals were present in the external nucleus of the inferior colliculus (ECIC), a region that receives input from the spinal cord and brainstem (Aitkin et al., 1978; Davidson et al., 2010). Their absence in non-targeted strains revealed thick axons that continued rostrally toward the thalamus (Fig. 3.7). In all mice, tdtomato-positive axons projecting to the intermediate grey (ig) of the superior colliculus formed a meshwork. The midbrain-hindbrain boundary (MHB) is an important signaling centre in development that is responsible for determining cell fates anteriorly in the midbrain and posteriorly in the cerebellum (Gibbs et al., 2017). It represents a major point of divergence of anterolateral ascending tracts, where spinotectal and spinothalamic axons leave the inferior cerebellar peduncle, some of which projected to the PAG and others to the ig layer of the superior colliculus (Fig. 3.7). The finest axons also projected to two vertically-oriented bands extending from the ig dorsomedially to the aqueduct ventromedially, and to the lateral border of the PAG ventrolaterally. In the intermediate white layer (iw) of the superior colliculus, the middle calibre axons decussate and connect the left and right deep grey of the superior colliculus, which itself is continuous with the ig (Fig. 3.7). The thickest axons to leave the MHB traveled dorsally in the midbrain, and curved rostrally toward the diencephalon (Fig. 3.7) to approach the caudal end of the ventral posterior lateral nucleus (VPLN) of the thalamus and the posterior intralaminar nucleus (PIL) (Fig. 3.7D). 73 These large-calibre axons travel rostrally toward the posterior complex of the thalamus (PO) where they cluster at the dorso-medial border of the medial geniculate nucleus (MGN) and more rostrally at the dorso-medial border of the ventral posterior medial nucleus of the thalamus (VPM) (Fig. 3.8). From here, these axons traversed the MGN and VPM, and arborized in the posterior intralaminar nucleus (PIL) and ventral posterior lateral nucleus (VPL), respectively (Fig. 3.8). The medial part of the MGN (multimodal part) was heavily labeled in all strains. Axons of the STT are reporter-positive in all strains, however they are notably more apparent in the knock-in models, likely attributable to the majority of spinothalamic axons originating in the spinal cord above C3 (Davidson et al., 2010) (Fig. 3.8). 74 Figure 3.5. Midbrain reporter fluorescence in Hoxb8 reporter strains: inferior colliculus. A. Schematic indicating plane of section (inset) and the equivalent image from the YSK Unified Anatomical Atlas (https://kimlab.io/brain-map/atlas/). B. Reporter fluorescence from each strain. Arrows indicate the external cortex of the inferior colliculus (ECIC), absent from non-targeted transgenic strains. C. Higher-power view of decussating axons in the intermediate white matter of the superior colliculus (iw). Aq, cerebral aqueduct. 75 Figure 3.6. Midbrain reporter fluorescence in Hoxb8 reporter strains: superior colliculus. A. Schematic indicating plane of section (inset) and the equivalent image from the YSK Unified Anatomical Atlas (https://kimlab.io/brain-map/atlas/). B. Overviews of reporter fluorescence from each strain. C. Details of the dorsal midbrain. Ig, intermediate grey layer of the superior colliculus; iw, intermediate white layer of the superior colliculus; PAG, periaqueductal grey; ECIC, external nucleus of the inferior colliculus; stt, spinothalamic tract. 76 Figure 3.7. Dorsal spinofugal pathways to the midbrain and thalamus. A-C. Collicular transition from inferior (A) to superior in Hoxb8T2AFlpOTdT mouse. D,E. Parasagittal sections from a Hoxb8CreTdT mouse illustrating the origins of the dorsally-coursing spinocerebellar tract from the inferior cerebellar peduncle (icp) at the midbrain-hindbrain boundary (MHB) (D) and the continuity of projections from the deep grey (dg) to the intermediate grey (ig) (E). F. Schematic of four separate projections to the midbrain and thalamus emerging from the icp. cb, cerebellum; cbx, cerebellar commissure, dg, deep grey matter of the superior colliculus; ECIC, external nucleus of the inferior colliculus; ig, intermediate grey of the superior colliculus; iw, intermediate white matter of the superior colliculus; PAG, periaqueductal grey; stt, spinothalamic tract; TH, thalamus. 77 Figure 3.8. Reporter fluorescence in the thalamus of Hoxb8 reporter strains. A. Schematic indicating plane of section (inset) and the equivalent image from the YSK Unified Anatomical Atlas (https://kimlab.io/brain-map/atlas/). B. Overviews of reporter fluorescence from each strain. C, D. Higher-power schematic (reference atlas image numbers indicated) and photomicrographs of the posterior thalamus at 400-micron intervals. Magenta arrows indicate stt axons; cyan arrows indicate the medial lemniscus (ml); yellow arrows indicate the spinofugal hypothalamic decussating tract (shxt). E. Higher power view of the shx, shxt and a possible termination zone in the zona incerta (ZI) in four Hoxb8 reporter lines. F. Parasagittal sections (medial to lateral) from a Hoxb8T2AFlpOTdT mouse of the shxt. Cing, cingulum bundle; cp, choroid plexus; DG, dentate gyrus; MGM, medial part of the medial geniculate nucleus; MGN, medial geniculate nucleus; PaF, parafasicular nucleus; PO, posterior complex of the thalamus; PoT, posterior triangular complex; RSPv, retrosplenial cortex; V3, third ventricle; VPM, ventral posterior medial nucleus of the thalamus; VPL, ventral posterior nucleus of the thalamus. 78 This lineage-tracing model revealed a previously undescribed tract which I call the spinofugal hypothalamic decussating tract (shx). I believe that the axons of this tract emerge from the caudal end of the VPL or possibly the rostral end of the PIL, travel ventrally between the internal capsule and optic tract, and decussate just posterior to the optic chiasm (oc) in the ventral-most part of the thalamus (Fig. 3.8 E, F) with the zona incerta (ZI) the likely site of termination (double yellow arrows in Fig. 3.5A). This tract was more prominent in targeted knock-in strains, indicating a contribution from spinal neurons above C4 (Fig. 3.6E). While reporter-positive axons were apparent in all strains in the VPL of the thalamus, the lateral PO, and the ZI, labeling was slightly less extensive in non-targeted Hoxb8-reporters when compared to knock-in strains (Fig. 3.8). Tdtomato-positive axons were also apparent more dorsally in the medial part of the PO and in the parafasicular nucleus (PaF) (Fig. 3.8). In an effort to better understand the path of the shxt, four Hoxb8CreTdT mice were subjected to a unilateral spinal hemisection at C3 (Fig. 3.9) to eliminate all reporter positive axonal input ascending from the ipsilesional spinal cord to the brain. Gross inspection of tdtomato fluorescence confirmed loss of reporter-positive terminals ipsilaterally (and to a lesser extent contralaterally) in the cervical cord (Fig. 3.9A), pons and midbrain (Fig. 3.9B), and caudal thalamus including the ZI (Fig. 3.9C). Importantly, while these findings indicate an ipsilaterally ascending path for neurons from the uninjured side of the cord, a path from the ipsilateral thalamus to the contralateral ZI could not be traced. However, there was a surprising preservation of axons in the anterior VPL nucleus (Fig. 3.7D) at two (and even four) weeks post-hemisection (Fig. 79 3.9 E, F), raising the possibility of recurrent projections from the ipsilateral anterior VPL to the more posterior contralateral ZI. At the level of the anterior commissures, there are reporter-positive axons in the fusiform nucleus of the bed nucleus of the stria terminalis (BNST) in both knock-in strains which likely arise from neurons in the NTS, based on previous tracing studies in the rat (Riche et al., 1990; Lebow & Chen, 2016), and the absence of NTS recombination in non-targeted strains. At the rostral-most end of the brain, but only in Hoxb8CreTdT mice, axons of the vomeronasal nerve (VON) and their terminations in glomeruli of the accessory olfactory bulb were reporter positive (Fig. 3.10D). Interestingly, these were more prominent in female than male specimens. 80 Figure 3.9. Lateral hemisection reveals origins of spinofugal axons: Hoxb8CreTdt mice. A. Gross view of the site of C3 hemisection (left panel, arrow), a horizontal section through the injury site middle panel, and a cross-sectional view demonstrating ipsilateral terminal degeneration. B. Loss of spinotectal and spinopontine axons ipsilaterally. C. Thalamic projections (Zona incerta, ZI, highlighted) are largely preserved contralateral to injury, implying an ipsilateral pathway decussating in the diencephalon. D. The anterior VPL nucleus is spared contralaterally, and only partly diminished ipsilaterally. E. Light-sheet imaging demonstrates anterior VPL nucleus sparing at 4 weeks-post hemisection (Hx). F. Schematic demonstrating orientation of perspective in E (3D viewer from www.connectivity.brain-map.org). Cyan arrows indicate the medial lemniscus (ml); yellow indicates the spinofugal hypothalamic decussating tract (shxt). 81 Figure 3.10. Reporter fluorescence at (and rostral to) the level of the anterior commissure (AC) in Hoxb8 reporter strains. A. Schematic indicating plane of section (inset) and the equivalent image from the YSK Unified Anatomical Atlas (https://kimlab.io/brain-map/atlas/). B. Overviews of reporter fluorescence from each strain. C. Details of the region surrounding the AC. D. Projections of the vomeronasal nerve (VON) to the accessory olfactory bulb (AOB) in female and male mice. E, F. Details of (D). 82 3.3.4 Ectopic recombination in non-targeted Hoxb8 transgenic reporter mice While Hoxb8-driven recombinase mouse lines are often touted as “brain sparing” in terms of neuronal expression (Witschi et al., 2010; Chen et al., 2010), this was not true for the non-targeted transgenic models investigated. In Hoxb8CreTdT mice, scattered neurons in the cortex, hippocampus and cerebellum were reporter-positive, but in no consistent way; a cerebellar Purkinje cell (PC) is shown in the bottom right panel of Fig. 3.11A. As previously reported (Szabo et al., 2015), a population of Hoxb8-lineage neurons was present in the anteroventral (AV) and anteromedial (AM) nuclei of the thalamus in Hoxb8CreTdT mice (Fig. 3.11A). It was possible to follow the course of their axons, at least from the AV nucleus, laterally (Fig. 3.11B) and anteriorly (Fig. 3.11C) through the dorsal part of the striatum, after which they converged in the cingulum bundle (cing) (Fig. 3.11D). Caudally, at the level of the midbrain, these radiated toward the retrosplenial cortex (RSPv) (Fig. 3.11A, E, G) and additionally terminated in the subiculum (SUB) (Fig. 3.9G). More anteriorly, termination zones of cing axons could be found in the anterior cingulate cortex (ACC) (Fig. 3.11F). These results were unchanged following spinal hemisection (data not shown) and agree with projections of the AVN and AMN demonstrated in the Allen Mouse Brain Connectivity Atlas. Additionally, in Hoxb8CreTdT mice there were reporter-positive astrocytes (verified with Sox9 immunohistochemistry) (Sun et al., 2017) forming a distinct border between the ventral midbrain and thalamus at the cephalic flexure (CF) (Fig. 3.11A, H-J). Consistent with other scattered cells, a few recombined retinal ganglion cells were apparent in the optic tract (Fig. 3.12A), and optic chiasm (Fig. 3.12B, Inset). These 83 followed the optic tract dorsally and terminated in the dorsal lateral geniculate nucleus of the thalamus, as well as in the superficial grey of the superior colliculus in discrete tufts (Fig. 3.12B) – these were also unaffected by C3 hemisection (data not shown). In the other non-targeted strain, Hoxb8FlpOTdT, an unexpected population of reporter-positive cells was present in the outer molecular layer (OML) of the dentate gyrus. Morphological characterization and p73 immunoreactivity (and a lack of NeuN staining) revealed these to be late-surviving Cajal-Retzius cells (Fig. 3.13A-D) (Anstötz & Maccaferri, 2020). Cajal-Retzius cells not of the Hoxb8-lineage are present in the cortex and dentate gyrus of neonatal mice only and die within the first two weeks. Lastly, the choroid plexus of Hoxb8FlpOTdT mice contained a dense population of tdtomato-positive PDGFRa-positive mesenchymal cells (fibroblasts) (Fig. 3.13E, F). This contrasted with the other mouse lines, in which reporter-positive choroid plexus cells were of the monocytic lineage, expressing Iba1 (Fig. 3.13F) and f4/80 (data not shown). 84 Figure 3.11. Ectopic Hoxb8-driven reporter expression in Hoxb8CreTdT mice. A. Overviews of coronal and sagittal/parasagittal brain sections. Occasional Purkinje cells underwent recombination (PC, inset). B. Detail of the rostral thalamus indicating the anteroventral (AV) and anteromedial (AM) nuclei and the lateral course of axonal projections (green arrows). C. The rostral course of AV and AM-originating axons through the striatum (Str). D. AV and AM axons converge in the cingulum bundle (cing). E. AV and AM-originating axons radiate from the cingulum bundle towards the RSPv. F,G. Coronal sections from the level of the anterior thalamus (G) and the rostral midbrain (H, I) showing AV and AM projections to the anterior cingulate cortex (AC), the RSPv, and the subiculum (SUB). H, I. Detail from A showing reporter-positive cells ventral to the cephalic flexure (CF). J. Sox9 immunohistochemistry revealed these to be astrocytes. ml. medial lemniscus; stt, spinothalamic tract 85 Figure 3.12. Sparse labeling of retinal ganglion cells in Hoxb8CreTdT mice. A. Thalamic section demonstrating axons in the optic tract (opt) and termination in the dorsal lateral geniculate nucleus (DLG), just lateral to the rostro-caudal interface between the medial geniculate nucleus (MGN) and ventral posterior medial nucleus (VPN). B. Sparsely-labeled axons decussate in the optic chiasm (inset). Opt axons also terminate as tufts in the superficial grey layer (sg) of the superior colliculus. Ig, intermediate grey layer of the superior colliculus; ml, medial lemniscus; PIL, posterior intralaminar nucleus; PoT, posterior triangular complex of the thalamus; shxt, spinofugal hypothalamic decussating tract; stt, spinothalamic tract; ZI, zona incerta. 86 Figure 3.13. Ectopic Hoxb8-driven reporter expression in Hoxb8FlpOTdT mice. A. Coronal section at the level of the caudal thalamus indicating the spinothalamic tract (stt) and medial lemniscus (ml), and the dentate gyrus. B. High-power images of reporter-expressing dentate gyrus cells, and their expression of Cajal-Retzius-specific p73. C. Cajal-Retzius cells are NeuN-negative. D. In the P2 dentate gyrus, but not the P2 cortex, about 50% of Cajal-Retzius cells are also reporter-positive – reporter-positive cortical Cajal-Retzius cells are rare at this age. E. Heavy reporter expression in the choroid plexus (cp) of Hoxb8FlpOTdT mice. Occasional neurons in the lateral septum of Hoxb8T2AFlpOTdT mice had undergone recombination (inset). F. In Hoxb8IRESCreTdT (and other) mice, reporter-expressing cells of the choroid plexus are Iba1-positive macrophages, whereas in Hoxb8FlpOTdT mice, they are Iba1-negative, PDGFRa-positive mesenchymal fibroblasts. 87 3.3.5 Microglia Previous studies using Hoxb8 reporter mouse lines have highlighted the presence of Hoxb8-lineage microglia, thought to comprise one-third of all adult microglia in the mouse brain (Chen et al., 2010; Szabo et al., 2015; Bohic et al., 2023). Microglia were present in all Hoxb8 reporters assessed here, but differed markedly in abundance. Hoxb8-lineage microglia were most numerous in the cortex of Hoxb8IRESCreTdT mice followed by Hoxb8CreTdT mice (most readily apparent in Fig. 3.8D, 3.10B, C). Interestingly, even fewer microglia were present in the other knock-in model (Hoxb8T2AFlpOTdT) (Bohic et al., 2023). There were almost no Hoxb8-lineage microglia in Hoxb8FlpOTdT mice. 3.4 Discussion The overarching aim of this chapter was to determine the feasibility of using Hoxb8 reporter mice to study the distal anatomy of spinofugal projections, with a longer-term goal of understanding their plasticity in the wake of neurotrauma such as spinal cord injury. Specifically, I wanted to determine which of the four Hoxb8-driven recombinase lines, if any, would be suitable for the study of spinofugal projections and their plasticity following incomplete SCI. I have used the YSK Unified Anatomical Atlas https://kimlab.io/brain-map/atlas/) (Chon et al., 2019) to map axons of spinal origin onto specific regions throughout the brain. This atlas was made in an effort to resolve discrepancies between the Allen Common Coordinate Framework (CCF) (Wang et al., 2020) and the Franklin-Paxinos mouse brain atlas (Paxinos & Franklin, 2019). Another issue is the relationship between axonal projections and cytoarchitectonically-defined nuclei: spinofugal projections are 88 not necessarily overlaid onto the entirety of particular nuclei. The cerebellum is an example of this: subdivisions of spinofugal input are not apparent from gross granule cell cytoarchitecture, and therefore not included in standard atlases (Ozol and Hawkes, 1997; Gebre et al., 2012). Additionally, in more laminated structures such as the superior colliculus, dendrites (which receive the bulk of synaptic input) extend into distant layers. These findings highlight the importance for development of a spinofugal projection atlas; this work represents an important step in that direction. The absence of Hoxb8-lineage microglia and neurons (with the exception of Cajal-Retzius cells in the dentate gyrus) in Hoxb8FlpOTdT mice allows for specific visualization of axons. An additional convenience in these mice is that a lesion can be made above the fourth cervical segment (i.e. rostral to the Hoxb8 transgene expression boundary) in order to interrupt all reporter-positive axons ascending on one side of the spinal cord. While the overall goal of this chapter has been met, the differences between Hoxb8-recombinase strains raises intriguing questions about Hoxb8 regulation under normal circumstances, and perturbations that might be brought about by transgene insertion in native and non-native loci. Without knowledge of the transgene insertion site, or the effects of chromatin lengthening in the knock-in models, much of what follows is speculative. Nevertheless, some discussion of Hox gene regulation under normal circumstances is required to begin to address these questions. 3.4.2 Regulation of Hox Gene Expression and Implications of its Dysregulation in Lineage Tracing Many of the differences between targeted and non-targeted reporter lines were expected based on the differing rostral Hoxb8 expression boundaries (C4 in both non- 89 targeted strains). Obvious examples include the relatively lower density of labeled axons projecting to the thalamus via the medial lemniscus in Hoxb8CreTdT and Hoxb8FlpOTdT mice, and the absence of a projection from the NTS to the BNST. These, as well as more notable occurrences of ectopic recombination in Hoxb8CreTdT mice (anterior thalamic nuclei, astrocytes beneath the cephalic flexure, retinal ganglion and vomeronasal axons) and Hoxb8FlpOTdT mice (Cajal-Retzius cells in the dentate gyrus, choroid plexus fibroblasts) are most easily explained by the absence of important negative regulatory elements in the transgene constructs and resulting sensitivity to nearby sources of diffusible signaling molecules during development (more on this below). The differences that exist between mice in which recombinase was targeted to the native Hoxb8 locus are more difficult to explain. For example, there was an obvious difference in reporter-positive microglial density between Hoxb8IRESCreTdT and Hoxb8T2AFlpOTdT mice. The presence of tdtomato-positive axons in the core of the facial motor nucleus in Hoxb8IRESCreTdT mice only, may also speak to artefactually elevated regulation of Hoxb8 protein expression. As such, some discussion of Hox gene regulation under normal circumstances is warranted. First, let us consider the non-targeted strains in which the extent of reporter expression is both absent from tissues normally expressing Hoxb8 (e.g. the upper cervical cord), and expressed ectopically in more rostral structures. In Hoxb8FlpO and Hoxb8Cre mice, the transgene is randomly inserted into the genome and thus no longer near the distal element, which is responsible for extension of Hoxb8 expression from C4 to the obex (Oosterveen et al., 2003; Valarché et al., 2003; Afzal et al., 2023). 90 On the other hand, ectopic recombination could be attributed to the absence of suppressive regulatory sequences, insertion of the transgene in the vicinity of non-canonical promoters or enhancers, or temporally-altered chromatin accessibility. The responsible mechanism, or mechanisms, requires a stimulus for the activation of Hoxb8 expression. The dependence of Hoxb8 transcription on retinoic acid, makes developmental exposure to this diffusible signalling molecule a likely cause (Charité et al., 1995; Bel-Vialar et al., 2002; Forlani et al., 2003; Afzal et al., 2023). Indeed, ectopic recombination occurred close to tissues expressing retinoic synthesizing enzymes (RALDH1 and RALDH2) (olfactory epithelium, retina, craniofacial mesenchyme below the cephalic flexure, choroid plexus (Ruberte et al., 1993; Yamamoto et al., 1998; Luo et al., 2004) and atlas.brain-map.org), and/or in brain regions rendered sensitive to endogenous retinoic acid using a RARE-driven LacZ reporter (anterior thalamic nuclei, dentate gyrus) (Luo et al., 2004). If RA is the responsible driver of ectopic recombination, then timing of transgene RARE accessibility is most likely to underpin differences between Hoxb8Cre and Hoxb8FlpO expression. RA-dependent Hox gene regulation has many layers, and relief from Hoxb8 suppression must contribute to ectopic expression. Recently, Afzal et al. (2023), used single molecule in situ hybridization on mouse embryos in conjunction with CRISPR/Cas9 to generate mutations in three RAREs (DE, B4U and ENE) thought to be involved in the regulation of Hoxb genes. In single mutant studies of the DE and ENE, there was increased transcription of both Hoxb9 and Hoxb1, with a greater increase in the ENE mutant embryos (Afzal et a., 2023). The B4U mutation did not result in changes from wildtype mice in Hoxb9 or Hoxb1 transcription, however Hoxb4 transcription was increased. This 91 suggests that the DE and ENE are important in the regulation of all Hoxb genes, while B4U only acts on nearby or adjacent genes in the Hoxb cluster (Afzal et al., 2023). Given that loss of control by ENE led to an increase in Hoxb9 transcription, its absence may similarly affect the Hoxb8 transgene leading to its ectopic expression due to the importance of the “Hox clock” on Hox gene regulation. The regulation of Hoxb8 is also dictated by miRNAs through their role in posttranslational regulation through interaction with sequences in the 3’UTR (Humphreys et al., 2005). Translation comprises three main phases: initiation, termination, and elongation. There exists a debate as to which phase of translation with which miRNAs interfere, but Humphreys et al. (2005), suggest that miRNAs affect translational initiation. In the naïve state, translational initiation requires several eukaryotic initiation factors (eIF) and a poly(A) tail to recognize the start codon in the 3’UTR (Humphreys et al., 2005). This interaction results in either cleavage and degradation of mRNAs or the inhibition of translation. The former mechanism is less common and only occurs if there is near perfect complementarity between the miRNA and the target sequence in the 3’UTR (Humphreys et al., 2005). Non-targeted reporter mice also lack a miRNA target sequence (specific for miR196a) in the 3’UTR of the transgene, required for restricting anterior Hoxb8 expression (Yekta et al,. 2004; Hornstein et al., 2005). Without it, and under the influence of appropriate Hoxb8-inducing signals, one might expect transgene expression rostral to the brainstem. Neither of the knock-in recombinase models is expected to interfere with Hoxb8 function, and while Cre and FlpO have equivalent excision efficiencies (Birling et al., 92 2012), subtle differences in transcriptional or translational control might be anticipated: whereas the IRES system results in less efficient transcription of the second gene in the sequence (20-50% of the first, (Mizuguchi et al., 2000)), the near 100% efficiency of T2A self-cleavage should result in a 1:1 ratio of Hoxb8 and FlpO recombinase protein. The IRES has been reported to protect flanking sequences from miRNA-mediated inhibition of translation (Humphreys et al., 2005; Mathonnet et al., 2007; Thermann & Hentze, 2007). If this is the case for Hoxb8IRESCre mice, then it may be considered a Hoxb8 over-expressor, potentially explaining the abundance of Hoxb8-lineage microglia and the unique presence of spinofugal projections to the facial nucleus’ core (Fig. 3.3). The differences between the four reporter strains described here raise questions about the fidelity of permanent reporter expression to endogenous Hoxb8 expression. The two targeted transgenic models are a case in point: there was an obvious difference in the number of reporter-positive microglia (Hoxb8IRESCreTdT >> Hoxb8T2AFlpOTdT), and lateral septal neurons were only apparent in Hoxb8T2AFlpO mice (Bohic et al., 2023). One hypothesis is that this is a result of the relationship between differing lengths of the knocked-in constructs and the timing of Hox gene expression (i.e. temporal collinearity or the “Hox clock”). Increased genomic distance between the Hoxb8 sequence and the miR196a target in the 3’UTR may result in a prolonged expression of Hoxb8 and evasion of miRNA-mediated mRNA degradation. An approach to determining the true distribution of Hoxb8 expression might be to identify a sequence specifically transcribed in response to Hoxb8, and construct a reporter driven by its promoter. A first step towards this goal is identification of genes upregulated by Hoxb8 overexpression and has recently been accomplished in the chick (Wilmerding et al., 2021). 93 3.4.3 Concluding Remarks Overall, Hoxb8-driven reporter expression is a viable approach to map axonal projections from the spinal cord to the brain. It offers superb visualization of trajectories otherwise inferred (spinothalamic and spinotectal tracts) or previously unknown (spinofugal hypothalamic decussating tract). Differences in transgenic constructs also provide some information on somatotopy (i.e. projections from the whole cord or from that up to C4 only). Models of choice for defining ascending pathways are the FlpO lines, given the relative paucity of Hoxb8-lineage microglia in both strains, and spatial separation of ascending pathways from the dentate gyrus in the Hoxb8-FlpO line. 94 Chapter 4: Spinocerebellar Tract Plasticity Following Incomplete Spinal Cord Injury 4.1 Introduction The CNS has the capacity, albeit limited, to compensate for functional deficits brought about by neurotrauma. Pioneering studies by Kaas and Darian-Smith groups have shown that the loss of sensory input from the body to the CNS via amputation, peripheral nerve injury, dorsal root section, or spinal cord injury, results in large-scale reorganization of somatosensory circuitry, from the spinal cord to the cortex (Florence & Kaas, 1995; Kaas, 2000; Fisher et al., 2020). When the loss of afferent input is small, there are immediate adjustments in the sensitivity of remaining circuitry that mitigate functional deficits. When more extensive, however, structural remodelling (the growth of new axons and formation of new synapses) is thought to contribute to functional recovery. This is known to occur at the level of the first afferent synapse in the spinal cord or dorsal column nuclei (Wu & Kaas, 2002; Fisher et al., 2020). It also occurs in the spinal cord of rats and non-human primates, where both dorsal root and spinal cord injury lead to sprouting of axons originating in the primary sensory cortex (Darian-Smith et al., 2013; Darian-Smith et al., 2014; McCann et al., 2020). Evidence for sprouting of intermediate-order sensory neurons (e.g. spinothalamic, spinocerebellar, spinotectal and other “spinofugal” projections) after injury to their neighbours is scant. Clinically, the majority of spinal cord injuries (~70%) are incomplete in that the cord retains some lesion-spanning connections (Kirke-Rogers & Todd, 2016). In these instances, some functional recovery usually occurs (Kirke-Rogers & Todd, 2016), and this is often ascribed to compensation or plasticity subserved by spared circuitry. 95 Humans who suffer a SCI are often primarily concerned with regaining hand function, however until 2004, there has been a paucity of behavioural assessments designed for mice to assess forelimb function (Anderson et al., 2004). Work by the Steward group showed that following cervical hemisection in mice, gripping disability recovered over time and was accompanied by structural changes in the sensorimotor cortex (SMC) (Anderson et al., 2004; Blanco et al., 2007). These changes to the SMC included reorganization such that gripping was controlled by both the ipsilesional and contralesional SMC (Blanco et al., 2007). Grasping and gripping are not governed by the cortex alone: studies in patients with cerebellar disease have demonstrated significant reaching and grasping dysfunction whereby task performance deviates from internal models of what form specific movements “should” take (Nowak et al., 2013). This is very likely to be a result of deficiencies in detection or interpretation of proprioceptive input arising from the spinal cord via the SCT which carries unconscious proprioceptive and motor information to the cerebellum (Koh & Markovich, 2022). SCT axons originate in Clarke’s Column of the thoracic and upper lumbar spinal cord (dorsal spinocerebellar tract), laminae 7-9 in the lumbosacral cord (ventral spinocerebellar tract), laminae 5-7 of C5-C8 (rostral spinocerebellar tract), laminae 1 and 5-7 above C8 and the external cuneate nucleus (cuneocerebellar tract). The axons of all of these neurons terminate primarily in the anterior lobe of the cerebellum (lobules I-V), but with additional innervation of posterior lobules VIII and IX (Watson et al., 2009), and are arranged as medio-lateral compartments or “stripes” in the granule cell layer. Therein they form “rosettes” – stereotypically-shaped pre-synaptic boutons contacting 96 multiple post-synaptic granule cells (Gebre et al., 2012). Rosettes in these stripes contain VGLUT2, and alternate with others containing VGLUT1 and arise from dorsal column nuclei mossy fibres (Gebre et al., 2012). One aim of this chapter was to determine if there were structural changes to spinocerebellar projections following unilateral cervical hemisection that accompanied the motor recovery described in Chapter 2. This aim was motivated in part by findings made by Visavadiya and Springer (2016) in rats. Following a thoracic contusion injury, there were changes in the cerebellar circuitry of rats including limited granule cell activation, and a loss of parallel fibre influence on Purkinje cell dendrites (Visavadiya & Springer, 2016). The apparent changes in cerebellar circuitry following SCI warranted further investigation into the plasticity of SCT axons. Work done by Matson et al. (2022) provides further rationale for this aim: these authors found changes in gene expression towards a regenerative state in neurons in the lumbar spinal cord following a thoracic level injury. The spatial expression of regeneration-associated genes, the presence of excitatory neurotransmitters, and the fact that the SCT neurons are lost following axotomy (Feringa et al., 1985; Feringa et al., 1987), suggested that these are remaining (uninjured) spinocerebellar neurons. These changes in gene expression were accompanied by some evidence for collateral axon growth caudal to the injury (Matson et al., 2022). Importantly, the authors reported no change in mossy fibre density in the cerebellum, although the viral tracing method employed was inadequate to draw such conclusions, in part because of the limited number of neurons labeled (see Chapter 3), the inability to distinguish surviving axons 97 from those undergoing slow CNS-type Wallerian degeneration, and the focus on axons rather than rosettes. Another aim in this chapter was to determine if anatomical plasticity of SCT fibres is accompanied by synaptic activation of target neurons. Excitation-transcription coupling can be a valuable tool to probe functional synaptogenesis, and is initiated by intense post-synaptic activity elevating [Ca2+]i resulting in CREB phosphorylation and induction of cFOS expression (Kida, 2012). We have used this method previously to demonstrate functional re-connection of injured dorsal root axons with neurons in the spinal dorsal horn (Scott & Ramer, 2010), and injured vagal sensory axons with targets in the brainstem (Ramer, 2003). Previous work in rats has demonstrated cFOS activation in the granular layer of the cerebellum with walking on a rotarod (Jasmin et al., 1994), and so here I attempt to determine whether cFOS immunoreactivity is similarly induced in mice, and whether changes with time following unilateral C3 hemisection indicate a return of post-synaptic activation (i.e cFOS induction) of granule cells. Because the rotarod relies on voluntary locomotion which may be more difficult to achieve at earlier post-operative timepoints, I also use a more passive whole-body vibratory stimulus (100 Hz). Here, I show that 28-days after hemisection, there is structural plasticity of mossy fibre terminals at the level of the cerebellum as exemplified by changes in synaptic density, size, and the content of synaptic machinery. The magnitude of the increase in synaptic density was greater on the contralesional side of the cerebellum, despite roughly equivalent contributions of the SCT to the distal portions of each stripe in uninjured animals. These results identify a novel site of plasticity that is well-positioned 98 to mediate (or fine-tune) the functional recovery which also occurs following incomplete SCI (Chapter 2). The contralateral bias of plasticity may indicate differential responses of the various neurons of origin of the SCT in the spinal cord, the behavioural compensation that hemisected animals must adapt after injury, and/or contributions of corollary motor circuitry. Lastly, I was unable to determine the role of cerebellar plasticity in spontaneous motor recovery due to significant cFOS immunoreactivity in the sham group. 4.2 Methods 4.2.1 Animals and Surgical Procedures The hemisected Hoxb8FlpOTdT mice (n=7, 3F, 4M) from Chapter 2 were used following acclimation, surgery, and behavioural assessment. The additional mice used in this chapter (uninjured and 5d hemisection) were also Hoxb8FlpOTdT mice. 4.2.2 Specimen Collection and Lightsheet Microscopy One month following hemisection, injured mice (n=7) and uninjured mice (n=4) were deeply anesthetized with a 1500mg/kg intraperitoneal injection of urethane and perfused with heparinized saline followed by 4% PF in 0.1M PB. The brains were removed and fixed overnight in 4% PF in 0.1M PB, after which they were equilibrated in 20% sucrose in 0.1M PB. The cerebella were cut from the rest of the brain and washed for 6 hours (3 x 2 hours) with PBS on a room temperature shaker protected from light. The cerebella were delipidated for 7 days with Cubic-L (TCI, Cat #T3740) to which NaCl was added (to 0.45%) to prevent tissue swelling in a 37°C incubated shaker protected from light. The Cubic-L solution was changed daily for the first four days, after which it was changed every second day. The refractive index (RI=1.520) was matched to glass 99 using Cubic-R+(N) (TCI, Cat# T3983) for two days on a room temperature shaker protected from light. The cleared cerebella were embedded in 2% agarose in Cubic-R+(N) (TCI, Cat#T3983). Images of the cerebella were acquired using a Zeiss Z.1 Light sheet microscope with Zen Black software with tiling and z-stacking (optical section thickness ~20 µm). 4.2.3 Short-Term Hemisection Five Hoxb8FlpOTdT mice (2F, 3M) over 12 weeks of age received a unilateral C3 hemisection with a 5-day survival period as described in Chapter 2 following which the mice were perfused, dissected, and cleared using the Cubic-protocol (Chapter 4. 4.2.2). All clearing parameters were consistent with those previously described, however Cubic-R+(M) (TCI, Cat# T3741) was used in place of R+(N). 4.2.4 Confocal Microscopy Sample Preparation A portion of the 28-day hemisection cerebella used for lightsheet imaging (n=3 injured, n=3 intact) was washed 3 times for 2 hours each time with PBS and equilibrated in 20% sucrose in 0.1M PB overnight. The cerebella were then frozen in OCT compound and stored at -80°C. All of the cerebella were sectioned in the coronal plane at 100 µm using a cryostat (Micron). Four (~101 mm) circular vinyl adhesive stickers custom made on a Cricut (Explore Air) were applied to Superfrost Plus slides before thaw-mounting sections to prevent tissue compression during coverslipping. A subset of the slides was air dried for 1 hour and subsequently washed with PBS (3 x 5 min). The remaining slides were stored at -80°C. The RI was matched to glass using Cubic-R+(M) (TCI, Cat# T3741) in one 20-minute application. The slides were coverslipped with 100 24x50mm coverslips and mounted with ProLong Glass anti-fade mounting media (Invitrogen, P36984). 4.2.5 Immunohistochemistry A subset of thaw-mounted sections was used for post synaptic density protein 95 (PSD95) and Munc13 immunohistochemistry (n=6, 3Hx, 3 intact) and another subset was used for VGLUT2 immunohistochemistry (n=6, 3Hx, 3 intact). The sections were prepared as previously described (Chapter 3 immunohistochemistry) with the specific antibodies listed in Table 4.1. Following primary and secondary antibody incubation, the slides were coverslipped with 24x50mm coverslips using ProLong Glass mounting media (Invitrogen, P36984). 101 Table 4.1. Antibodies for Immunohistochemistry 4.2.6 Confocal Microscopy Images were taken using a 63x oil-immersion objective lens (plan APOCHROMAT, NA 1.4) on a Zeiss LSM800 scanning confocal microscope and Zen Blue software for non-immunohistochemically prepared slides (4.24). Stripes 3I, 1, and 3C in lobule III were imaged. Three images of stripe 3I, 1, and 3C in lobule III were acquired for each animal (n=6, 3Hx, 3 intact). Immunohistochemically prepared slides (Section 4.25) were imaged using a Zeiss LSM800 scanning confocal microscope with super resolution AiryScan module. Antigen Host Species and Clonality IgG Subclass Source Catalog No. Dilution Munc13 Rabbit polyclonal IgG Synaptic Systems 126 103 1:500 PSD95 Mouse monoclonal IgG2a Novus NB300-556 1:500 VGLUT2 Guinea pig monoclonal IgG2 Synaptic Systems 135 418 1:2000 VGLUT1 Guinea pig monoclonal IgG2 Synaptic Systems 135 304 1:1000 cFOS Rabbit polyclonal IgG Santa Cruz Biotech Inc F1715 1:500 Donkey anti-guinea pig IgG Alexa 647 Donkey polyclonal IgG(H+L) Jackson Immuno 706-605-148 1:500 Donkey anti-mouse IgG Alexa 647 Donkey polyclonal IgG(H+L) Jackson Immuno 715-605-150 & 706-606-148 1:500 Donkey anti-rabbit IgG Alexa 488 Donkey polyclonal IgG(H+L) Invitrogen A21206 1:500 Donkey anti-guinea pig IgG Dy405 Donkey polyclonal IgG(H+L) Jackson Immuno 705-476-148 1:500 102 One hundred images of individual rosettes from each animal (n=600, 300Hx, 300 intact) were acquired with AiryScanning with no z-stacking. 4.2.7 Measurements and Statistical Analysis The density of the distal “spray” of each stripe (3C, 2C, 1, 2I, 3I) in lobules II, III, and IV were calculated using z-stacked images in the sagittal plane (z=20) for intact, 5-day hemisection, and 28-day hemisection animals using FIJI. This region was chosen to minimize the potential contribution of any remaining axonal debris, which may persist in the fascicles that run up the centre of each stripe. In FIJI, the noise was removed (Despeckle), background was subtracted (50 pixels, rolling ball), and a threshold was set for each image. A fractional area calculation for each stripe was made and graphed in Prism. The average density across all stripes was also calculated for each group and statistically analyzed using a Welch’s ANOVA in GraphPad Prism. The size of the rosettes in high power images (63x objective, NA 1.4) of the cerebellum was assessed by a scorer who was blinded to the samples (hemisected vs. uninjured was unknown). Confocal image z-stacks (20-40 z-slices with optical section thickness) were first processed in Zen (Zeiss) to equalize histograms (Stack correction). After subsequent smoothing (Gauss) and background subtraction (50 pixels, rolling ball) this allowed rosette profiles to be identifiable with a threshold in FIJI (Yen). The difference in area frequency distribution between intact and hemisection stripes in each lobule was statistically assessed using Kologorov-Smirnov tests. The fractional area of Munc13, PSD95, VGLUT2, and TdTomato was determined for each rosette using FIJI after removing noise, subtracting background (50 pixels, rolling ball), and setting a threshold for each image. In GraphPad Prism outliers were 103 identified (method > ROUT) and removed. For Munc13 and PSD95 the colocalization was also assessed in FIJI (Process > Image Calculator > AND) to generate an image in which only pixels that exist in both PSD95 and Munc13 images were present in the combined image. To assess colocalization density, the fractional area was calculated in the new image (measurement: fractional area). To determine whether changes in overlap was not simply a result of changes in density of both proteins, the colocalization density was divided by the sum of the Munc13 and PSD95 densities. With GraphPad Prism, a histogram of each measurement was created and statistically analyzed using a Kolmogorov-Smirnov test. 4.2.8 VGLUT1 Analysis Twelve Hoxb8FlpOTdT mice 12 weeks of age or older received a unilateral C3 hemisection (as described above) and survived for 5d (n=6) or 28d (n=6). The injured and uninjured (n=4) mice were perfused and dissected as described above. The brains were removed and fixed overnight in 4% PF in 0.1M PB. The cerebella were cut from the rest of the brain and cleared with CUBIC protocol as described in section 4.2.2. Once clear, the cerebella were frozen in optimal cutting temperature compound and stored at -80°C. All of the cerebella were sectioned in the coronal plane at 50 µm using a Cryostat (Micron) and collected as floating sections in 12-well plates (8 sections/well) with 0.1M PB with 0.01% sodium azide. The floating sections were prepared as previously described (Chapter 2 immunohistochemistry) with the specific antibodies listed in Table 4.1. Following primary and secondary antibody incubation, the slides were coverslipped with 24x50mm coverslips using ProLong Glass mounting media (Invitrogen, P36984). 104 Images of the coronally-sectioned cerebella were taken on a Zeiss LSM800 scanning confocal microscope and Zen Blue software using a 20x objective. Three images of each stripe (3C, 2C, 1, 2I, 3I) in lobule III were taken for each animal (n=16) with bidirectional scanning, 2x averaging and Gauss processing. The density of each stripe was determined by a scorer who was blinded to the conditions (injured vs. uninjured). In FIJI, the noise was removed (Despeckle), background was subtracted (50 pixels, rolling ball), and a threshold was set for each image. A fractional area calculation for each stripe was made and graphed in Prism. The average density across all stripes was also calculated for each group and statistically analyzed using a Welch’s ANOVA in GraphPad Prism. 4.2.9 cFOS Immunoreactivity Analysis 4.2.9.1 Animals All experiments were carried out in accordance with guidelines of the Canadian Council on Animal Care and the UBC Animal Care Committee. Male C57BL/6 mice (Charles River) 8-12 weeks of age were group-housed in a standard light cycle. 4.2.9.2 cFOS Surgical Procedures The same hemisection (n=28, M) and sham (n=30, M) surgery described in chapter 2 was performed on C57BL/6 mice. A small group of mice (n=9, M) received the sham surgery as described previously; however, the 25-gauge needle was inserted to puncture the cord immediately left of the dorsal midline vein. The mice survived for 7d (n=13 Hx, n=16 sham) or 28d (n=15 Hx, n=23 sham). The same monitoring and post-surgical care was provided as previously described. 105 4.2.9.3 cFOS Stimulation To ensure successful stimulation, the mice were acclimated to the rotarod for four days prior to stimulation. Intact (n=4), 7d sham (n=8), 7d hemisection (n=6), 28d sham (n=9), and 28d hemisection (n=7) mice were placed on the rotarod for 60 minutes at the lowest speed. If the animal fell off the rotarod, it was immediately placed back on. For vibrational stimulation, intact (n=6), 7d sham (n=8), 7d hemisection (n=7), 28d sham (n=7), and 28d hemisection (n=8) were placed on a subwoofer and covered by a beaker to ensure they remained in place while a 100Hz stimulation was played using the Tone Gen app for 5 minutes. The amplitude was adjusted to be just audible to the experimenter’s ear (the dial at the 10 o’clock position). 4.2.9.4 Specimen Collection and Confocal Microscopy Sample Preparation One hour after the end of vibrational or rotarod stimulation, mice were deeply anesthetized with a 1500mg/kg intraperitoneal injection of urethane and perfused with heparinized saline followed by 4% PF in 0.1M PB. The brains were removed and fixed overnight in 4% PF in 0.1M PB, after which they were equilibrated in 20% sucrose in 0.1M PB. The cerebella were frozen in OCT compound and stored at -80°C. All of the cerebella were sectioned in the coronal plane at 50 µm using a Cryostat (Micron) and collected as floating sections in 12-well plates (8 sections/well) with 0.1M PB with 0.01% sodium azide. A group of intact unstimulated cerebella (n=5) and 28d sham unstimulated cerebella (n=7) were collected and prepared as described above. 4.2.9.5 Immunohistochemistry The floating sections were prepared as described above with specific primary and secondary antibodies listed in Table 4.1. The sections were then mounted on to 106 Superfrost Plus slides and coverslipped using 24x50mm coverslips and the Prolong Glass mounting media (Invitrogen, P36984). 4.2.9.6 Image Acquisition Images of the coronally-sectioned cerebella were taken on a Zeiss LSM800 scanning confocal microscope and Zen Blue software. The rotarod stimulated cerebella were imaged using a 20x objective, and the vibrationally stimulated cerebella were imaged using a 63x oil-immersion objective. Three images of each stripe 3I, 1, and 3C in lobule III were acquired for each animal (n=34 rotarod, n=48 vibration) with bidirectional scanning, 2x averaging and Gauss processing. 4.2.9.7 Measurements and Statistical Analysis The fractional area of cFOS was determined by a scorer who was blinded to the samples (hemisection, sham, intact, stimulation was unknown). In FIJI, confocal images were smoothed (Gaussian blur), underwent background subtraction (50 pixels, rolling ball), and set to a threshold (Triangle, minimum 10, maximum 255, dark background). The fractional area (analyze > measure) was then calculated for cFOS. The fractional area of cFOS was normalized to the 7d sham group in each condition (rotarod and vibration). The data were graphed and statistically analyzed in Prism with a one-way ANVOA with Brown-Forsythe and Welch ANOVA where multiple comparisons were corrected with statistical hypothesis test using Dunnett. 107 4.3 Results 4.3.1 Unilateral C3 Hemisection Effectively Eliminates Spinofugal Projections from the Ipsilesional Spinal Cord A C3 hemisection (Fig 4.1 A, B) effectively eliminated all tdtomato-positive synapses in the ipsilateral midbrain (superior colliculus and periaqueductal grey matter (Fig. 4.1 C)), although some reporter-positive axonal debris persisted in white matter tracts. Figure 4.1. Ipsilesional spinofugal projections are eliminated with a left C3 hemisection. A. The site of the hemisection (marked by arrow) under UV light. B. Hemisected spinal cord acquired with confocal microscopy with the location of the injury indicated by the arrow and the ependymal cells marked by the arrow head showing the rostral extent of Hoxb8 transgene-driven reporter expression. C. Lightsheet images of Hoxb8FlpOTdT hemisected mice of the superior and inferior colliculi (top and bottom, respectively) and PAG. D. Illustration of the SCT pathways and the effect of left C3 hemisection (scissors) on these spinofugal tracts. Circles in the cerebellar vermis represent locations of rosettes. 108 4.3.2 Spared Mossy Fibre Density Increases with Time Following Hemisection The SCT terminates as mossy fibres on granule cells in five medio-laterally arranged stripes (annotated as 3I, 2I, 1, 2C, 3C in relation to the injured or contralateral sides) in the lobules of the cerebellum (Gebre et al., 2012), but the contribution of the SCT on each side of the cord to individual stripes has not been well-defined. To shed light on this uncertainty, and to establish a baseline density measurement specific to the spared SCT, a small number (n=5) of mice survived for 5d following a C3 hemisection. Rosette density in these mice fell to slightly less than 50% of that in intact mice (Fig. 4.2 A-G), a slightly larger drop than anticipated (50%), probably due to gliosis associated with terminal degeneration. With the possible exception of lobule III, there was no obvious differential reduction in density across stripes, indicating a roughly equal distribution of the contralateral SCT to each. By 28d following hemisection, rosette density had increased significantly in each lobule (Fig. 4.2 E’-G’), most notably in lobules II and III, in which overall density approached that of uninjured mice. Analysis of individual stripes (Fig. 4.2 E-G) revealed an obvious bias toward increased density on the contralesional side. To determine if this change in density was specific to VGLUT2-positive axons, the density analysis was also done at the same timepoints with VGLUT1-positive axons. There were no significant differences in rosette density in VGLUT1-positive axons with time after injury (Fig.4.3 A, B), suggesting that the changes in spared mossy fibre density are specific to VGLUT2-positive axons. An increased rosette density can be attributed to a change in quantity, size, or both. Measurement of individual rosettes in stripes 3I, 1 and 3C of lobule III revealed a 109 small but significant rightward shift in area-frequency distribution in the central and contralateral (but not ipsilateral) stripes (Fig. 4.2 J-L), indicating some degree of enlargement. The mean rosette area was similar between the two groups (lobule III 3C Hx mean: 25.9000 microns2, SEM: 0.2215, N: 4080 and intact mean: 23.92 microns2, SEM: 0.1990, N: 3701), indicating a minimal contribution of changes in size to overall density measurements. 110 111 Figure 4.2. Rosette density in stripes of the anterior cerebella of intact and hemisected Hoxb8FlpOTdT mice. A, B, C. Orthogonal projections of lightsheet microscopy images of the cerebella and a 3D reconstruction of intact (A), 5d hemisection (B), and 28d hemisection (C) mice. D. Lightsheet microscopy images of all stripes in lobules II, III and IV (left to right) of intact, 5d hemisection, and 28d hemisection animals. Stripe 1 represents the middle stripes, 2I and 2C are the stripes immediately lateral to stripe 1 on the ipsilateral and contralateral side, respectively, and 3I and 3C are the stripes located most laterally on the ipsilesional and contralesional side, respectively. E-G. Density of rosettes in each stripe across lobules II (E), III (F) and IV (G) in intact, 5d hemisection, and 28d hemisection animals. E’-G’. The density of intact, 5d hemisection, and 28d hemisection animals across all stripes in lobules II (E’), III (F’) and IV (G’). Statistically significant p-values are shown, n.s indicate non-statistically significant findings (Welch’s ANOVA). H-I. High magnification confocal microscopy images of stripes 3C in lobule III of the cerebellum in intact (H) and 28d hemisection (I) mice. J-L. Graphs show the area distribution of rosettes in stripes 3I (J), 1 (K), and 3C (L) in intact and 28d hemisection mice. The area of the rosettes is on the x-axis, and the proportional frequency is on the y-axis. Significant p-values shown and n.s. represents non-statistically significant findings (Kologorov-Smirnov tests). 112 Figure 4.3. Rosette density in stripes of the anterior cerebella of intact and hemisected Hoxb8FlpOTdT mice. A. Confocal microscopy images of lobule III of the cerebella in an uninjured animal with TdTomato in red and VGLUT1 in blue. B. Density of rosettes in each stripe across lobule III of VGLUT1-postivie axons in uninjured, 5d hemisection, and 28d hemisection animals (left). The density of intact, 5d hemisection, and 28d hemisection animals across all stipes in lobule III is on the right. P-values are shown (Welch’s ANOVA). 113 4.3.3 Increased Reporter and Synaptic Protein Density in Rosettes Post-hoc examination of images from light sheet microscopy images of intact and 28d-hemisected mice gave the impression that not only were rosettes more numerous, but also qualitatively brighter (Fig. 4.2 H, I) as though their tdtomato content had increased. I therefore asked whether the change in rosette density and size was accompanied by parallel changes in reporter and synaptic protein content of individual rosettes. I measured the density distribution of TdTomato using superresolution microscopy and found a significantly higher tdtomato density in hemisected animals than in intact mice (Fig. 4.4 C, p<0.0001). Interestingly, in the hemisected mice, there was a prominent peak at approximately 50% area fraction suggesting a possible maximum possible tdtomato content (i.e. a ceiling effect). VGLUT2 specifically labels mossy fibres arising from the spinal cord (Gebre et al., 2012). There was a significant increase in the density distribution of VGLUT2 in rosettes of 28d hemisection animals over intact animals in the contralesional hemisphere (Fig. 4.4 D, p<0.0001). As a first step toward determining whether structural plasticity and increased VGLUT2 content are functionally relevant, I investigated two key components of synaptic machinery. Munc13 is a presynaptic protein that is involved in synaptic vesicle release due to its critical role in the process of vesicle priming (Chen et al., 2013). PSD95 is one of the most numerous proteins found in the postsynaptic density of excitatory synapses, and thus serves as a postsynaptic marker (Béïque & Andrade, 2003). Analysis of these synaptic markers using superresolution microscopy revealed a 114 significant difference in the density distribution between intact and 28d hemisection animals (Hx>Intact, Fig. 4.4 H, I, p<0.0001). Furthermore, there was a significant difference in colocalization density distribution of PSD95 and Munc13 between the 28d hemisection animals and the intact animals (Hemisection > Intact, Fig. 4.4 J, p<0.0001). 115 Figure 4.4. 28d hemisection animals have significant differences in synaptic marker and reporter expression compared to intact animals. A, B. Superresolution microscopy images of rosettes in intact (A) and 28d hemisection (B) mice. C, D. Density distributions of TdTomato (C) and VGLUT2 (D) in 28d hemisection and intact mice. E, F. Superresolution microscope images of intact (E) and 28d hemisection (F) mice. G. Superresolution image of a rosette with TdTomato, PSD95, and Munc13 with the colocalization of PSD95 and Munc13 in the inset indicated by arrows. H-J. Density distributions of Munc13 (H), PSD95 (I), and colocalization (J) in intact and 28d hx mice. 116 4.3.4 cFOS Immunoreactivity After Laminectomy and Hemisection Seven days after unilateral cervical hemisection, there was limited spontaneous functional recovery that had significantly improved by day 28 (Fig 4.5). To determine if functional cerebellar plasticity is involved in spontaneous functional recovery, I aimed to specifically drive the synaptic activity of the SCT. One means to stimulate the SCT was with rotarod exposure, motivated by previous work in rats (Jasmin et al., 1994) and the role of the SCT in balance and coordination of movements (Sengul & Watson, 2015). The SCT conveys unconscious proprioceptive information from the spinal cord to the cerebellum, and thus a proprioceptive stimulus in the form of vibration was also used to specifically drive the synaptic activity of this tract. Rotarod and vibrational stimulation were able to induce cFOS activity in intact mice (Fig. 4.5 A-J). After vibrational and rotarod stimulation, there was a significant increase in cFOS density 28d after injury when compared to 7d (Fig. 4.5 K, L). However, there was also a significant increase in cFOS density 28d later in sham-operated mice when compared to 7d (Fig. 4.5 K, L). The significant changes in the sham group leave me unable to determine whether activation of cFOS in the cerebellum is mediated by SCT neurons, or if there is some other pathway activated (or an inhibitory pathway suppressed) by the surgical procedure. 117 Figure 4.5. cFOS immunoreactivity in lobule III after rotarod and vibrational exposure in sham and hemisected mice. A-B. cFOS activity in intact mice after rotarod exposure (A, A’) and vibrational exposure (B, B’). C-F. Confocal microscopy images of 7d sham (C), 7d hemisection (D), 28d sham (E), and 28d hemisection (F) mice. G-J. Confocal microscopy images of 7d sham (G), 7d hemisection (H), 28d sham (I), and 28d hemisection (J) mice. Graphs showing normalized density of cFOS after vibration (K) or rotarod (L) exposure at various time points and injury conditions. Asterisks represent statistically significant findings, n.s. indicate non-statistically significant findings (Welch’s ANOVA). 50μm 50μm 50μm 50μm 50μm 50μm 50μm 50μm 118 4.4 Discussion In Chapter two, I showed that lateral spinal hemisection, as with other incomplete spinal cord injuries, is followed by variable functional behavioural recovery. This implicates plasticity of spared circuitry. Here, I demonstrated that there was structural plasticity of mossy fibre terminals at the level of the cerebellum characterized by changes in synaptic density and size, and increases in the presynaptic content of synaptic markers 28d following hemisection. Such changes were particularly dramatic in the contralesional hemicerebellum. However, I was unable to find a correlation of SCT plasticity and functional recovery following unilateral cervical hemisection. 4.4.1 Technical Considerations for Densitometric Analysis A crucial requirement of detecting morphological plasticity following SCI, particularly with pre-labeled axons as is the case in our reporter mice, is the ability to distinguish between intact structures and the persistent debris products resulting from protracted Wallerian degeneration in the CNS. Many axonal fragments in white matter tracts endured for the 28d survival period following C3 hemisection, but terminals in grey matter did not, at least in the spinal grey matter, superior colliculus, and periaqueductal grey (Fig. 4.1). Superresolution images of rosettes among granule cells also bore no traits of degeneration (fragmentation or attachment to disintegrating axons), indicating that they were indeed spared. The short-term (5d) hemisections also resulted in a reduction of rosette density in the cerebellum consistent with a complete loss of terminals from the injured hemicord, or approximately 50% of spinocerebellar input. It should be noted that I carefully restricted density measurements to the distal grey matter of the cerebellar granular layer (the distal “spray”) so as to avoid 119 contamination by debris persisting in axon fascicles ascending from the commissural part of the arbor vitae. Fragmentation of injured axons also has the effect of concentrating, and thus increasing the intensity of, fluorescent reporter molecules, and so their inclusion in regions of interest can artificially increase density measurements. This, along with a lack of focus on rosettes, appears to have been the case in the only other examination of spinocerebellar mossy fibre plasticity (to my knowledge) after spinal cord injury (Matson et al., 2022), in which pre-labeled (and clearly fragmented) axons, were not excluded from the analysis, and continued to degenerate between 3 and 6 weeks following spinal cord injury. Fractional area measurements on their own cannot distinguish between size and number of objects being measured. Parsing their relative contributions requires quantification of rosette size-frequency distributions. Although I found a small enlargement of rosette profiles, this was not enough to account for the overall rise in density, indicating an increase in rosette abundance with time following injury. 4.4.2 Anterograde Axonal Transport may be Increased in Spared SCT Fibres I found that there was an increased density of the synaptic proteins VGLUT2 and Munc13 in spared rosettes. While this could be a reflection of their increased transcription, no such change in expression was identified in a previous nuclear RNA sequencing analysis of any lumbar neurons following spinal cord injury (Matson et al., 2022). Together with an increased content of tdtomato in spared rosettes, greater VGLUT2 and Munc13 density suggests a possible increase in speed and/or volume of material axonally transported from the soma to terminal arborizations. This response is a well-studied consequence of axotomy in regeneration-competent peripheral neurons 120 (reviewed in Petrova & Eva, 2018), and for sensory neurons is not restricted to the axotomized peripheral branch but is also present in uninjured dorsal root axons (Mar et al., 2014). There is also precedent for its occurrence in spared axons after CNS injury: after T10 dorsolateral spinal transection in rats and injection of BDA tracer, Brock et al. (2018) found that BDA travelled along spared ventral CST axons more rapidly in injured than in intact animals, returning to normal after 2 weeks. In spared mossy fibre terminals, increased axonal transport following hemisection, if not perfectly balanced by similar alterations in retrograde transport, may also have accounted for the subtle but significant increase in rosette size. 4.4.3 Are New Spinocerebellar Rosettes Functional? I found that there was a significant rightward shift in the Munc13/PSD95 colocalization density distribution in the 28d hemisection animals compared to intact animals (Fig. 4.4 J), and this increase was greater than would be expected based on simple increases in the density of each protein. Enhanced synaptic coupling, a Munc13-dependent process through which inactive synapses become active, may have occurred alongside increased colocalization (Chen et al., 2018). It is important to note that the increased density of PSD95 in the 28d hemisection animals was unexpected, but may have been due either to global increases in material undergoing axonal transport (see above), or to inadvertently including granule cells in the analysis. Rosettes are complex, convoluted structures meaning that it is difficult to separate them from granule cell dendrites (even with superresolution microscopy). They do receive axo-axonic input from Golgi cells (Chapter 1), but this input is inhibitory and thus does not involve PSD95 (Sheng & Kim, 2011). These results raise the possibility 121 that new rosettes are indeed functional, but also that synaptic transmission is enhanced at each rosette/granule cell glomerulus. These notions remain to be investigated by more direct means such as assessing the proclivity for long-term potentiation at the mossy fibre synapse (Hansel et al., 2001). 4.4.4 Plasticity Lateralization in the Cerebellum The striking difference in mossy fibre density between cerebellar hemispheres ipsilateral and contralateral to hemisection requires consideration, particularly since the contribution of the spinocerebellar tracts in one hemicord to granule cell innervation across the medio-lateral extent of the cerebellum has not been well-documented. The clinical picture of spinocerebellar tract anatomy is one of different populations either not crossing the midline (e.g. the DSCT), or crossing at the level of the cord and re-crossing in the cerebellum (e.g. the VSCT). Since C3 lateral hemisection interrupts axons arriving at the cerebellum from ipsilateral (DSCT) neurons and contralateral (VSCT) neurons, one would expect an overall bilateral reduction in mossy fibre terminals. Whether said decrease is actually equivalent for the ipsilateral and contralateral hemispheres would then depend on the relative numbers of DSCT and VSCT neurons caudal to the injury. This textbook description is complicated by the fact that single mossy fibre collaterals (of multiple origins) project to multiple stripes and multiple lobules (Pop et al., 2022, Xu & Grant, 1988; Shinoda et al., 2000). Unilateral injection of fluorochrome-conjugated WGA has provided some clarification to this conundrum: this technique, which should label both DSCT- and VSCT-projecting somata, results in uniformly bilateral mossy fibre labeling in the cerebellum, at least in the anterior lobe upon which I have focused (Reeber et al., 122 2011). Our analysis of rosette density after short-term (5d) hemisection is in alignment with these observations, bringing us back to the question as to why rosette density increases most dramatically in the contralateral cerebellum. In the early days following hemisection the burden of movement is carried out by the contralateral limbs. The associated proprioceptive input can arrive at the cerebellum via the spared DSCT, but not by the neurons of origin of the contralateral VSCT whose axons are severed after their first decussation. One might therefore postulate activity-dependent growth of DSCT axons, but not of VSCT fibres. That said, there is significant motor recovery ipsilateral to hemisection (Chapter 2), which could be expected to engage proprioceptors connected to VSCT neurons on the injured side and strengthen new connections. Spasticity ipsilateral to spinal hemisection may also develop with time (Hsieh et al., 2010; Hultborn & Malmsten, 1983), but how this relates to VSCT activation of cerebellar circuitry is unknown, especially with respect to movement planning and execution as per internal models. 4.4.5 Activity-Dependent Plasticity may be Driven by Sensory and Motor Pathway Convergence In the CNS, increased neuronal activity consolidates new and existing connections, and therefore plays a critical role in reorganization and plasticity (Fouad & Tse, 2008). While proprioceptive input from contralateral limbs is undoubtedly greater than that from the ipsilateral side early on following hemisection, it is unlikely that this alone drives formation of new synapses: descending (motor) output from the ipsilateral sensorimotor cortex to the contralateral cerebellum and/or spinal cord probably also contributes. 123 Cerebellar granule cells constitute the most numerous neuronal cell type in the brain, and about 40% of these receive proprioceptive information from both the sensory cuneocerebellar tract and cortically-driven pontine motor nuclei (Huang et al., 2013). What is more is that the motor input to these “multimodal” granule cells is the same that is transmitted to cord motor circuitry, and corresponds somatotopically to proprioceptive input from the cervical/upper thoracic cord and external cuneate nucleus. Such an arrangement could provide a substrate for Hebbian LTP of granule cell responses to sensory and motor mossy fibre input. So far, this corollary circuit has been demonstrated only for VGLUT1-expressing pontocerebellar and cuneocerebellar mossy fibres, but it is significant that in the cerebellar vermis, VGLUT1-expressing pontocerebellar and cuneocerebellar terminals broadly segregate into complementary stripes (Huang et al., 2013), as do cuneocerebellar and spinocerebellar mossy fibres (Gebre et al., 2012). The pontine grey matter expresses both VGLUT1 and VGLUT2 mRNA (in situ hybridization data from the Allen mouse atlas), and many rosettes in stripes innervated by the SCT also stain positively for VGLUT1 (Gebre et al., 2012), suggesting that they may also arise from the pontine grey. The upshot of these findings is that a convergence of pontine and spinocerebellar circuitry (similar to pontine and cuneocerebellar circuitry) probably exists, and that ipsilateral cortical activity cooperates with contralateral spinofugal input to the cerebellum to preferentially cement new contralateral connections. In this work sprouting only occurred for tdtomato SCT axons and not co-mingling VGLUT1 axons. Another corollary circuit exists in the spinal cord where Clarke’s column (DSCT) neurons receive input from proprioceptive primary afferents and corticospinal axons 124 (either direct or indirect via GABAergic interneurons) (Hantman and Jessell, 2010). Here again, there exists a platform upon which descending cortical input and sensory information about limb position, both more active in the cord contralateral to hemisection, may underpin activity-dependent plasticity in the cerebellum. Ipsilateral to injury, corticospinal axons sprout into the intermediate grey matter from the contralateral side (Ghosh et al., 2009; Aoki et al., 1986; Cao et al., 2021), a phenomenon that can be enhanced, along with functional recovery, by several pharmacological interventions aimed at neutralizing glial scar and myelin-derived inhibitory factors (Lee et al., 2008; Wang et al., 2011). Additionally, electrical stimulation of the spared motor cortex promotes significant axonal sprouting within its usual territory to the intermediate grey matter (Carmel & Martin, 2014). While the post-synaptic targets of these sprouts have not been defined, any excitatory connection with at-level-crossing VSCT neurons might, alongside ipsilateral primary afferent activity, contribute to enhanced proprioceptive input from the ipsilateral cord, and thus activity-dependent structural plasticity and functional recovery. 4.4.6 Challenges with Determining Functional Synaptogenesis To determine if the spontaneous functional recovery that occurred after unilateral cervical hemisection was related to increased activity of the SCT, the immediate early gene cFOS was used as a marker of neuronal activity (Cole & Josselyn, 2008). Following an intense synaptic activity, many second messenger pathways are activated by an influx of calcium. One downstream effect is the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) at Ser133. Once phosphorylated, CREB activates downstream immediate early genes, including cFOS 125 (Kida, 2012). There was a significant increase in cFOS immunoreactivity with time after hemisection (Fig. 4.5 K, L), suggesting an increase in neuronal activity in the cerebellum. The understanding of these results was complicated by the significant increase in cFOS immunoreactivity in sham-operated animals, which has several explanations. Firstly, after rotarod stimulation there was a similar amount of cFOS immunoreactivity in 7d sham and 7d hemisection animals. These findings may be explained by the difficulty of this motor task. Regardless of the injury severity, staying on the rotarod for 60 minutes is difficult for the mice and this possibly led to an increase in neuronal activity. When using a more passive stimulus - vibration - the 7d sham animals had significantly less cFOS density than 7d hemisection animals, possibly explained by the more purely sensory nature of the task. However, this theory still fails to explain the increase in cFOS density that occurs 28d after laminectomy in both conditions. Following thoracic spinal contusion and laminectomy in rats, cFOS immunoreactivity was detected in the spinal cord and brainstem. There was little cFOS immunoreactivity in intact animals (Del-Bel et al., 2000). Spinal laminectomy may result in microtraumas that are not detected behaviourally, however may be sufficient to induce cFOS immunoreactivity (Del-Bel et al., 2000). Therefore, it is possible that the cervical laminectomy damaged peripheral nerves and musculature enough to affect cFOS immunoreactivity without leading to behavioural impairments. The changes to the cerebellar circuitry brought about by the surgical approach used in these experiments makes it difficult to use cFOS immunoreactivity to determine the role of SCT axon plasticity in functional recovery. Activating transcription factor 3 126 (ATF3) is a transcription factor that is upregulated after axonal damage in affected neurons, promotes axonal sprouting, regrowth, and regeneration (Heinrich et al., 2024; Katz et al., 2022). A genetic model in which ATF3 is specifically knocked-out from SCT axons (e.g. VGLUT2-ATF3-/-) could be used to determine if the plasticity and sprouting of the SCT correlates with functional recovery. Not only would this model prevent the sprouting response of the SCT while retaining the spared innervation, but it would also not be affected by “microtraumas” induced by cervical laminectomy. 4.4.7 Laminectomy Can Result in Significant Post-Operative Pain To perform a unilateral cervical laminectomy, a midline incision (~1cm long) was made over the cervical spinal cord, the muscle layer was separated with scissors, the tissue was retracted, and the lamina at C3-C4 (the bony arch on the back of a vertebra) was removed with rongeurs. While this surgery does not result in paralysis, it is fairly invasive and did lead to notable changes in animal behaviour during post-operative care, including signs of pain, decreased alertness, and weight loss. In the literature, there is a lack of standardization when it comes to laminectomy and sham surgeries. Kosta et al. (2012), performed a minimal laminectomy (only L6) in rats, an extensive laminectomy (L5-S1), and a laminectomy combined with ganglionectomy to determine the difference in nociceptive behaviour in the groups. The more severe laminectomy (regardless of ganglionectomy) led to a significant increase in nociceptive behaviour that peaked at 15 days post-injury. Furthermore, the extensive laminectomy group also presented with muscular imbalance and spinal deformity (Kosta et al., 2012). Not only can laminectomy lead to significant pain, but the increase in pain is similar to what is seen in SCI animals. An operant mechanical conflict test can be 127 used to assess neuropathic pain in rodents. This involves the animals crossing sharp probes to escape an area of bright light (Odem et al., 2020). After T10 laminectomy, rats had an enhanced pain-like avoidance when compared to uninjured controls, which was comparable to SCI animals (Odem et al., 2020). In addition to the pain associated with laminectomy, this procedure also involves a large amount of tissue retraction and injury to the skin and muscle. Work in rats found that tissue retraction is associated with postoperative pain (Flatters et al., 2010). Additionally, significant skin and muscle injury in rodents leads to tissue hypoxemia, acidemia, the release of inflammatory mediators, and reactive oxygen species that result in spontaneous activity, reduced threshold, and enlarged receptive fields of nociceptors (Eisenach & Brennan, 2019). Overall, it is likely that the significant damage caused by surgical exposure of the spinal cord, muscle, and skin results in increased nociception in sham and hemisection animals. It is evident that laminectomy (and hemisection) results in significant post-operative pain. While the cerebellum is typically thought to be involved in the unconscious control of movements, nociceptive stimulation leads to the activation of pathways and neurons in the cerebellum (Saab & Willis, 2003). Early findings in cats revealed that nociceptive information transmitted by c-fibres traveled via mossy fibres to affect the discharge of cerebellar Purkinje cells (Jie & Pei-Xi, 1992). Additionally, in rats neurons in the lateral reticular nucleus involved in the modulation of visceral nociception have direct axonal projections to the cerebellar vermis (Ness et al., 1998). The direct effect of nociception on the cerebellum may have impacted the activation of granule cells in a manner that is unrelated to proprioceptive signaling, thus confounding the outcome of the cFOS immunoreactivity experiments. 128 4.4.8 Pre- and Post-Surgical Stress May Influence Pain Prior to surgery and rotarod stimulation, all mice were acclimated to the rotarod with frequent handling and exposure to the apparatus. Additionally, after surgery animals were handled frequently for daily weighing, monitoring and received subcutaneous injections 3 times daily for the first 3 days. Frequent injections, handling, and behaviour testing can be stressful for the animals and may impact the results of the experiment. Cao et al. (2015), exposed rats to pre- and post-operative short-term stress with immobilization and forced-swimming tests to determine the effect of stress on post-operative pain. Rats that were exposed to short-term stress (pre- or post-operatively) had delayed postoperative pain recovery (Cao et al., 2015). While steps were taken to mitigate stress in these experiments (gradually increasing exposure to me, gentle handling, etc), it is likely that the mice still experienced significant pre- and post-operative stress. This stress may have led to worsened post-operative pain recovery, and thus may have impacted the results in the sham-operated mice. 4.4.9 Concluding Remarks Understanding the natural history of plastic changes in the CNS following spinal cord injury will allow exploration of underlying mechanisms that might be exploited to improve outcomes. I have shown that following unilateral C3 hemisection, there was significant structural plasticity of the spared SCT at the level of the cerebellum, particularly in the contralesional hemisphere, despite the cerebellum normally receiving roughly equivalent amounts of SCT innervation to both sides. These findings extend the known catalogue of sites at which plasticity can occur, however more work is needed to 129 understand if this plasticity drives improvements of motor function that also occur spontaneously following incomplete SCI. 130 Chapter 5: Bulk mRNASeq May Identify Potential Targets for Future Investigation 5.1 Introduction The work in this thesis has found spontaneous functional recovery (Chapter 2) following unilateral cervical hemisection, and sprouting of SCT axons alongside an increase in rosettes’ content of synaptically-important proteins (Chapter 4). An unknown remains as to how sprouting of SCT axons in the cerebellum is induced at the molecular level. There are a number of possibilities, one of which being a change in the transcriptome of spared SCT neurons in the spinal cord. This has been explored by Matson et al. (2022), who found that several regeneration-associated genes were upregulated in response to thoracic SCI including activating transcription factor 3 (ATF3), growth associated protein 43 (GAP43), BDNF, and Sox11. Still, the stimulus responsible for inducing these changes remains unknown. A potential mechanism involves changes in expression or availability of growth-promoting signals in the partially-deafferented cerebellum. Classical work by Levi-Montalcini and colleagues demonstrated that limiting amounts of target-derived nerve growth factor (NGF) are required for survival and axonal outgrowth of sensory and sympathetic neurons of the peripheral nervous system (the “Neurotrophic Factor Hypothesis”). It is also well-known that excess NGF promotes collateral sprouting of peripheral axons (Zhou et al., 2003), and that it can be inhibited by NGF-blocking manipulations (Zhou et al., 2003). This principle has been repeatedly validated for numerous developmentally-important morphogens and axonal growth factors (Thoenen et al., 1988; Schörnig & Rohrer, 1993). A similar mechanism might be operational in the cerebellum: removal of synaptic input to granule cells, or processes involved in 131 Wallerian degeneration of severed axons might stimulate new growth of SCT axons and subsequent synaptogenesis. Motivated in part by this and findings in Chapters 2 and 4, I used bulk RNA sequencing of the cerebellar vermis as a “first pass” to determine if there were changes in gene expression after hemisection that reflected glial or neuronal responses to partial denervation which might be related to the sprouting response of SCT axons. Here, I use bulk mRNA sequencing of the anterior vermis from mice two weeks following a C3 hemisection or sham surgery to identify several candidate genes that may participate in the plasticity of spared SCT axons in the partially deafferented cerebellum. 5.2 Methods 5.2.1 Animals All experiments were carried out in accordance with guidelines of the Canadian Council on Animal Care and the UBC Animal Care Committee. Male C57BL/6 mice (Charles River) 8-12 weeks of age were group-housed in a standard light cycle. 5.2.2 Surgical Procedure and Tissue Harvesting The same hemisection (n=6) and sham (n=5) surgery described in chapter 2 was performed on C57BL/6 mice. The animals survived for 14d, and the previously described post-surgical care was provided. The mice were deeply anesthetized with a 1500mg/kg intraperitoneal injection of urethane and hand-perfused with ice-cold sterile PBS (30mL) after which the cerebellar vermis was quickly dissected and immediately flash frozen in liquid nitrogen. Sample preparation, RNA extraction, library preparation and sequencing was done by the Vancouver Prostate Centre Genomics Core Lab. 132 5.2.3 Bulk mRNAseq Analysis The data provided by the Vancouver Prostate Centre Genomics Core Lab was analyzed with ExpressAnalyst (https://www.expressanalyst.ca/). In ExpressAnalyst a single expression table analysis was done (specify organism > mouse, analysis type > differential expression, data type > counts bulk RNAseq, ID type > Ensembl Gene ID). To perform differential analysis, DESeq2 was selected (over Limma and EdgeR) as it is the preferred analysis for RNAseq data with smaller sample sizes. All data were compared against a common group – sham operated animals. To determine the differentially expressed genes, I used unadjusted p-values, a log2fold change of zero to account for both upregulated and downregulated genes, and a p-value of 0.05. For bioinformatics analysis (ridgeline plots), gene set analysis (gene ontology, GO – biological processes (GO-BP), molecular function (GO-MF), cellular component (GO-CC)) over pathway analysis (KEGG) was performed to allow for a broader understanding of the genes of interest. Lastly, gene set enrichment analysis (GSEA) (over ORA) was done to detect more subtle changes in gene expression, which is likely the case in this model. 5.3 Results 5.3.1 Bulk mRNAseq Identifies Potential Target for Future Work Using the unadjusted p-values, 576 differentially-expressed genes were found of which 393 were upregulated and 183 were downregulated in hemisected when compared to sham-operated animals (Fig. 5.1). I looked at the differentially expressed genes and identified genes that are expected to change following partial deafferentation of the cerebellum. Past research, and previous findings in this thesis (Chapter 4), 133 suggest that following unilateral cervical hemisection glial cells become activated and increase in number around injured axons, changes occur to synaptic activity, and axonal sprouting/plasticity occurs. I used these expected biological processes that occur after SCI and deafferentation to identify markers that were expected to change after unilateral C3 hemisection (Table 5.1). Figure 5.1. A volcano plot of all genes from bulk mRNAseq. The differentially expressed genes (576) are shown in blue and red, where blue are downregulated genes (183) and red represent upregulated genes (393). Developed with www.expressanalyst.ca 134 Figure 5.2. Downstream targets of microtubule associated tau (MAPT) signalling upregulated in the cerebellar vermis 2 weeks after unilateral C3 hemisection. Darker shades of red indicate greater upregulation. Generated with QIAGEN IPA Interpret 135 Bioinformaticcs analysis with GSEA and GO-BP/MF/CC revealed ontogenetic categories that differed between sham and hemisected animals (Fig. 5.3, 5.4, 5.5). Several of the genes that I expected to change after hemisection (Table 5.1) were found in these categories. For example, in GSEA-GO-BP analysis, genes involved in regulation of immune system process include Clc3, Clec7a, C1qa, and C1qc. In molecular function analysis, many of the expected genes were found in pattern recognition receptor activity (Clec7a, Tlr2) and peptide receptor activity (Cx3cr1, NPY). Overall, in this bulk mRNAseq analysis, I have identified several genes that are expected to change after hemisection based on known biological changes that happen after partial deafferentation of other regions of the nervous system. Figure 5.3. A ridgeline plot indicating the top 20 gene ontology biological processes identified by gene set enrichment analysis with fold change. Developed with www.expressanalyst.ca 136 Figure 5.4. A ridgeline plot indicating the top 20 gene ontology molecular function categories identified by gene set enrichment analysis with fold change. Developed with www.expressnaalyst.ca 137 Figure 5.5. A ridgeline plot indicating the top 20 gene ontology cellular component categories identified by gene set enrichment analysis with fold change. Developed with www.expressnaalyst.ca 138 Table 5.1. Author-curated list of differentially expressed genes in the cerebellar vermis 14d following C3 Hx 139 The next step in this analysis was to identify which of these expected gene changes may be involved in the plasticity and sprouting of spared SCT axons after unilateral cervical hemisection. It is likely that some of the genes in “axon growth/regeneration” and “synaptic plasticity” processes are involved in this response. For example, a reasonable hypothesis is that the release of Tau protein by degenerating SCT axons triggers protection and an incomplete repair response. The release of microtubule-associated protein tau (MAPT) may drive this plasticity response, and lead to an upregulation of “axon growth/regeneration” genes (e.g. Tuba1a, BDNF, Stmn1) and/or “synaptic plasticity genes” (e.g. Nefh) (Fig. 5.2). In chapter 4, I determined that the sprouting of SCT axons likely occurred seen by a dramatic increase in rosette density that is not accounted for by an increase in rosette size, and thus attributed to more rosettes. It is known that axon growth and guidance is driven by movement of a growth cone which requires specific cytoskeletal machinery (Hur et al., 2013). Work in chapter 4 also found that not only were there more rosettes, but there was an increase in synaptic machinery markers and a greater colocalization of these markers after hemisection. This suggests that hemisected mice may have more synapses, and/or more active synapses. It is reasonable to assume that gene changes in the key synaptic machinery proteins (synaptotagmin, calmodulin, RIM) may reflect this plasticity response. 5.4 Discussion Bulk mRNA sequencing of the anterior cerebellar vermis 14d after unilateral C3 hemisection or sham-surgery was successfully able to identify several genes that were expected to change after SCI. Further analysis of these genes provided insight into 140 potential drivers of plasticity of spared SCT axons in the partially deafferented cerebellum, which will be discussed here. 5.4.1 Astrogliosis after SCI Following damage to the CNS, including SCI, astrocytes transition to a reactive state and proliferate – a process known as astrogliosis (Li et al., 2014). This serves to limit swelling, isolate the damaged areas, and contribute to an immune response (Li et al., 2014). Reactive astrocytes undergo hypertrophy, have lengthened protrusions, enlarged nuclei, produce more glial fibrillary acid protein (GFAP), and help to initiate an immune response by releasing cytokines and chemokines (Li et al., 2014). Fourteen days after injury, hemisected mice had a greater amount of GFAP expression (Table 5.1) when compared to sham animals. This is an expected finding and is consistent with data from chapter 4 where 5d after injury the rosette density fell to slightly more than the expected 50% of that of intact mice likely due to gliosis associated with terminal degeneration. One of the proteins identified with bulk mRNAseq that was upregulated in hemisected when compared to sham mice (Table 5.1) was chemokine ligand 3 (Ccl3). After SCI, proinflammatory chemokines help to drive the secondary damage that occurs through maintaining heightened immune and inflammatory responses (Pelisch et al., 2020). Work by Pelisch et al. (2020) determined that after a thoracic SCI, Ccl3-/- mice had improved locomotor recovery, a smaller lesion, and less neuronal damage when compared to wildtype mice. Therefore, Ccl3 has a detrimental role on SCI recovery. Herein, astrogliosis is expected after SCI and occurs in regions distant from the injury, such as the cerebellum, as a result of Wallerian degeneration. The presence of 141 reactive astrocytes during Wallerian degeneration creates a negative environment for sprouting and regeneration (Wang et al., 2009), and thus these genes likely do not represent promising candidates to reflect the plasticity response. 5.4.2 Microgliosis Occurs after SCI Another form of gliosis that occurs in the partially deafferented cerebellum 14d after injury is microgliosis. In the uninjured state, microglia have a ramified morphology with long cytoplasmic protrusions that continuously scan the environment to detect abnormalities (Porro et al., 2021). After injury (or disease), microglia undergo a morphological change that involves an increase in cell body size and a decrease in protrusion length (Porro et al., 2021). Myosin heavy chain 2 (Myh2) is upregulated in hemisected when compared to sham mice 14d after injury, and has the largest fold change of the potential targets selected (Table 5.1). There is evidence to suggest that Myh2 may be associated with microglial activation. Non-muscle myosin II (NM II) is expressed in neuronal and non-neuronal cells and is involved in shifting microglia to a pro-inflammatory state, protrusion, and migration (Porro et al., 2021). Another gene with greater expression in hemisected when compared to sham mice 14d after injury is triggering receptor expression on myeloid cells 2 (Trem2) (Table 5.1). Trem2 is found on the surface of microglia in the resting state and is upregulated when microglia transition to an activated state (Xu et al., 2022). Interestingly, Trem2 is also involved in neuropathic pain, and is found in higher levels in regions, such as the cerebellum, which are involved in pain processing (Vijayan et al., 2025; Saab & Willis, 2003; Jie & Pei-Xi, 1992). While the literature on the role of microglia in recovery after SCI, or other traumatic injuries, is conflicting, there is sufficient evidence to suggest its 142 negative effects. Work by Li et al. (2020), depleted microglia in mice using a CSF1R-antagonist and found improved locomotor recovery, decreased inflammation, and a reduction in cytokine and chemokine release. This is not unique to SCI, as after ischemic stroke, inhibition of Clec7a (a pattern recognition receptor, upregulated in the cerebella of hemisected mice, that triggers phagocytosis and cytokine production) prevents synaptic loss and improves functional outcome after stroke (Wan et al., 2024). Lastly, after microglia are activated and accumulate in the damaged area, phagocytosis occurs. This is triggered, in part, by the release of cytokines and chemokines from astrocytes, and involves microglia engulfing and digesting cellular debris, pathogens, and other cells (Porro et al,, 2021; Xu et al., 2022). Two weeks after injury, there may be more debris in the deafferented cerebellum due to remaining axonal debris from long-lasting Wallerian degeneration. The increased debris could explain the significant increase in cytokine and chemokine gene expression (Clec7a and Ccl6, Table 5.1) in hemisected when compared to sham mice. Similar to astrogliosis, many genes involved in microgliosis are upregulated after hemisection. While microgliosis can have determinantal effect in spontaneous recovery, there is also evidence to suggest a positive role of microglia in collateral sprouting. Early work found that denervation of the dentate gyrus resulted in microglial activation and collateral sprouting in spared intact axons (Guthrie et al., 1995; Woods et al., 1998). It is possible that some genes involved in microgliosis are upregulated in response to plasticity. 143 5.4.3 Complement Proteins and Synaptic Stripping I found that the complement proteins C1q(a,b,c) and C4 (Table 5.1) were significantly increased in hemisected when compared to sham animals after unilateral C3 hemisection. After deafferentation or axotomy, synapses are removed to allow the brain to reorganize and adapt (Kettenmann et al., 2013). Part of this process involves synaptic stripping. There is ample evidence to suggest that synaptic stripping requires complement proteins, as a loss of the complement protein C1q is associated with an increase in the number of synaptic boutons and additional excitatory synapses in the cortex (Chu et al., 2010). Following a contusion SCI in rats, complement proteins C1q and C4 were strongly activated 1 day after injury, and this activation persisted 6 weeks after injury, and at areas rostral to the injury site (Anderson et al., 2004). While complement proteins play an integral role in synaptic stripping by identifying the neurons that should be eliminated, microglia are essential in eliminating those synapses tagged by complement proteins (Stephan et al., 2012; Presumey et al., 2017; Perry & O’Connor, 2010). The process of synaptic stripping was initially described in 1968 (Blinzinger & Kreutzberg). Work by Salvany et al., (2021), furthered the understanding of how microglia ingest and destroy synaptic terminals, in that after sciatic nerve injury, injured spinal cord motorneurons are internalized by microglia. While the majority of synaptic stripping seems to be detrimental to axonal regeneration, it is essential to remove synapses before forming new circuits. Work by Peterson et al. (2015), used C1q knockout mice to assess changes in CST axon sprouting and midline crossing after SCI. In these knockout mice there were no effects on midline crossing or overall sprouting length, however the SCI-induced sprouting response of CST axons into 144 lamina X did not occur in the knockout mice. These findings highlight a potential beneficial role for the complement protein C1q in axonal sprouting and plasticity after SCI. It would be interesting to see if the upregulation of complement proteins in hemisected mice decreases by 28d after hemisection – by which point some recovery has occurred (Chapters 2 and 4) and inactive synapses removed. 5.4.4 Synaptic Plasticity – Confounding Findings In chapter 4, I found that there was an increase in presynaptic marker Munc13, postsynaptic marker PSD95, and the colocalization of these two markers in hemisected when compared to uninjured mice 28d after injury. These data suggest that hemisected animals have more active synapses, and possibly a greater number of synapses. The bulk mRNAseq data found a protein that is involved with the active zone of a synapse – regulating synaptic membrane exocytosis 3 (Rims3) (Table 5.1). Regulating synaptic membrane exocytosis proteins are scaffold proteins that are found in the presynaptic active zone and are involved in docking, priming, and fusion of synaptic vesicles, as well as the release of neurotransmitters (Wu et al., 2023). Junctophilin 3 (Jph3) is another target involved in the regulation of neuronal synaptic plasticity and associated with locomotor learning that was found in bulk mRNAseq data (NCBI, expressanalyst database). The increased synaptic activity in hemisected mice, and the locomotor recovery led me to believe that Rims3 and Jph3 would be upregulated in hemisected when compared to sham animals. However, both of these genes were slightly downregulated in hemisected animals, as seen with a negative fold change (Table 5.1). This could be explained by the fact that the sham animals are also learning and 145 undergoing synaptic plasticity, or more likely that there have not yet been any long-lasting changes made to neuronal circuits 14d after injury. Again, it may be worth investigating how these synaptic markers change 28d after injury. One of the synaptic plasticity markers that was upregulated 14d after injury in hemisected mice was calmodulin 1 (Calm1, Table 5.1). Calcium is essential for synaptic function due to its role in neurotransmitter release and synaptic plasticity. When calcium binds to calmodulin several downstream signalling events are triggered that in turn affect synaptic function and plasticity (Xia & Storm, 2005). 5.4.5 Axon Growth and Regeneration During development, a series of events occurs to ensure efficient and proper wiring of the nervous system. Neuronal growth cones are located on the tips of growing axons, and they reach their target due to the activity of guidance cues in the extracellular environments (O’Donnell et al., 2016). Axon growth is driven by the polymerization and depolymerization of actin filaments, microtubules and cytoskeletal components (Dent et al., 2011). In order for spared SCT axons to sprout in response to deafferentation, which I demonstrated in Chapter 4, proteins involved in axon growth need to be present. Stathmin 1 is a phosphoprotein involved in regulating microtubules dynamics, and it was upregulated in hemisected mice 14d after injury (Table 5.1). In an effort to determine candidates for CST regeneration after SCI, Fuller et al. (2015) found that Stathmin 1 was enriched in CST axons during development in rats, and that when disrupted, axon outgrowth was significantly impaired in culture. Liedtke et al. (2003), performed in vivo experiments in mice and found that mice without stathmin 1 had 146 axonal pathologies of the CNS and PNS and increased axonal degeneration. Another gene involved in axon growth and regeneration is alpha 1a tubulin isoform 1a (Tuba1a), and this gene was also upregulated after hemisection (Tetzlaff et al., 1991) (Table 5.1). When Tuba1a is knocked-down in Zebrafish, the nervous system formation is impaired, and the normally occurring regeneration of retinal ganglion cells following optic nerve injury does not occur (Veldman et al., 2010). The need for axon growth and regeneration machinery in synaptic sprouting in conjunction with the upregulation of these markers after hemisection suggests that some of these may be a reflection of the plasticity of SCT axons. 5.4.6 Does Brain Derived Neurotrophic Factor Drive the Plasticity Response? BDNF is a member of the neurotrophin family with a well-established role in synaptic plasticity, LTP, learning, memory, and regeneration after injury (Pelisch et al., 2020). The higher affinity BDNF receptor is TrkB. When BDNF binds to TrkB the intracellular tyrosine autophosphorylation and a variety of second messenger pathways occur. One outcome is effects on synaptic plasticity via phospholipase-C which then activates IP3 and causes and increase in Ca2+. Alternatively, BDNF can promote axon growth via the disinhibition of cAMP through extracellular signal-regulated kinases. In addition to plasticity and axon growth, BDNF stimulates the expression of regeneration associated genes, including GAP43 and Tuba1a, which may improve recovery after SCI (Pelisch et al., 2020). BDNF was upregulated in hemisected when compared to sham mice 14d after injury (Table 5.1). The previous findings in this thesis found that 28d after hemisection, mice have improved locomotion and sprouting of SCT axons. It is reasonable to assume that BDNF may be involved in this spontaneous recovery. Work 147 by Jakeman et al. (1998), found significantly improved hindlimb activity, stepping, and growth of cholinergic fibres in rats with treatment of BDNF for 28d after thoracic spinal contusion or transection injury when compared to vehicle-treated animals. There is also evidence to suggest that administering BDNF to SCI animals can promote collateral sprouting (Vavrek et al., 2006). Furthermore, there is significant work that highlights the sprouting of serotonergic and dopaminergic neurons after injury, attributed in part to the presence of TrkB receptors allowing these neurons to respond to the increase in BDNF after injury (Scott et al., 2005). The increase in monoaminergic markers (Dbh, Slc6a2 and Slc10a4, Table 5.1) in hemisected when compared to intact mice provides further support for sprouting in hemisected mice. A less well-discussed role of BDNF is on neuronal network excitability (Ferrini & De Koninck, 2013). When microglia are in the resting state, purinoceptor 4 (P2X4R) is expressed at negligible levels. Following the release of cytokines and chemokines, microglia transition to a reactive state and P2X4R is significantly upregulated. This purinoceptor responds to changes in ATP, which leads to the synthesis and release of BDNF from reactive microglia. BDNF then interacts with the TrkB receptor leading to the downregulation of the potassium chloride cotransporter 2 (KCC2), disruption of chloride homeostasis, and eventually the strength of GABAA-glycine receptor-mediated inhibition is affected leading to disinhibition (Ferrini & De Koninck, 2013). This phenomenon may have significant effects in the cerebellum, as Purkinje neurons have GABAA receptors, and thus there may be significant changes in synaptic strength due to this disinhibition caused by microglia-released BDNF (Huang et al., 2013). Lastly, there is also evidence to suggest that neuropathic pain depends on these microglia-BDNF induced changes, 148 as transgenic mice with BDNF ablated only in microglia were not susceptible to morphine induced hyperalgesia (Ferrini & De Koninck, 2013). The increased pain that hemisected mice experience after injury, when compared to sham-operated animals, may be explained in part by the increase in microglial-released BDNF. Overall, the role of BDNF in plasticity, network excitability, and nociception lead me to believe that BDNF is upregulated by a specific pathway (potentially MAPT) after unilateral cervical hemisection, and then drives the plasticity of SCT axons (Fig. 5.6). 149 Figure 5.6. Downstream targets of brain derived neurotrophic factor (BDNF) signalling upregulated in the cerebellar vermis 2 weeks after unilateral C3 hemisection. Darker shades of red indicate greater upregulation. Generated with QIAGEN IPA Interpret. 150 5.4.7 Alternative Approaches The findings presented here provide a strong starting point for generating future hypotheses in the plasticity response after unilateral cervical hemisection. However, in bulk RNA sequencing, the results are reported in terms of average gene expression across the whole sample (cerebellar vermis), and therefore small differences between cell types are undetectable. This means that small differences in gene expression between individual granule cells, or an increase in transcriptomic markers in neurons and a decrease in glial cells may cancel out and present as a lack of significant findings. Single cell RNA sequencing allows for the analysis of individual cells and can reveal heterogeneity and unique gene expression at the level of a single cell (Haque et al., 2017). Alternatively, it is also plausible that the transcriptomic changes driving the sprouting response originate below the level of the cerebellum driven by activity-dependent plasticity. Clarke’s column (DSCT) neurons receive input from proprioceptive primary afferents and descending corticospinal tract axons (Hantman & Jessel, 2010, explained in Chapter 4). These axons are more active in the contralesional spinal cord after injury, and the increased activity may lead to changes in expression of genes associated with sprouting. 5.4.8 Concluding Remarks Understanding both the expected and surprising transcriptomic changes after unilateral C3 hemisection with bulk mRNA sequencing allows a deeper understanding of the spinofugal tracts after SCI. The timepoint used (14d) provides a snapshot of evolving and overlapping processes including the clearance of injured axons and the 151 sprouting of new ones. In the future, transcriptomic analysis at earlier and later timepoints will help to parse out these processes and determine the true target driving the sprouting response of SCT axons. 152 Chapter 6: Discussion 6.1 Thesis Summary Motivated by the importance of spinofugal projections in providing the environmental feedback required to successfully elicit behaviour, the overall aim of this thesis was to investigate plasticity of said projections to the cerebellum following unilateral cervical hemisection. Completion of this goal was reached by breaking down the requirements into 3 separate aims. This research has been carried out with the long term aim of improving outcomes for humans after cervical SCI (the most prevalent type) and as such it is important to understand the changes that occur during convalescence to help direct future treatments. In Aim 1 of this thesis (Chapter 2), I described the natural course of behavioural recovery following unilateral C3 hemisection using a variety of behavioural assessments. Several measures changed spontaneously over time suggesting innate plasticity, while several had stable changes or did not change at all, rendering them useful for evaluation of experimental treatment strategies. The limitations associated with conventional anterograde tracing techniques (Chapter 1) and the importance of ascending projections in SCI recovery motivated Aim 2 of this thesis, in which I determined that Hoxb8FlpOTdt mice were an effective tool for the bulk-labeling of all ascending projections. While not the primary aim of this chapter, observations made during characterization of four Hoxb8 reporter lines highlighted implications of random transgene insertion into the genome, transgene length (knock-in models), and possibly the repercussions on the “Hox clock” on Hoxb8 reporter-positive cells. They also provide a potential new tool to genetically manipulate specific 153 populations of CNS or CNS-projecting neural cells such as vomeronasal neurons, anterior thalamic nuclei, and cephalic flexure astrocytes (Hoxb8Cre mice), late-surviving Cajal-Retzius cells of the dentate gyrus (Hoxb8FlpO mice), and brain microglia (Hoxb8Cre and Hoxb8IRESCre mice – though not shown in this thesis, work in our lab has found that spinal cord microglia are not of the Hoxb8 lineage). For neuronal populations, intersectional genetics can be used to ablate cells or modulate their activity (Bohic et al., 2023). Recombinase activity (either FlpO or Cre) can be induced in Hoxb8-lineage neurons expressing a GFP reporter via injection of retrograde AAV vectors into their terminal fields (e.g. the retrosplenial cortex for thalamic neurons, the BNST for NTS neurons, the accessory olfactory bulb for vomeronasal neurons) encoding N- and C-terminal fragments of Cre or FlpO, which combine to form the functional recombinase in the presence of GFP (“Cre-DOG” or “FlpO-DOG”, Tang et al., 2015). Crossing these mice with flox-stop-flox or frt-stop-frt diphtheria toxin mice, flox/ftt-stop-flox/frt DREADD mice, or flox/frt-stop-flox/frt channelrhodopsin mice offers unique opportunities ask questions about the functions of these neuronal phenotypes, and a myriad of others, given the number of floxed and frted alleles currently available. In the final aim of this thesis (Chapter 4) I found that there was significant structural and molecular plasticity of the terminations of the spared SCT following unilateral C3 hemisection. While no causal relationship has been established, such plasticity is well-suited to be a substrate for spontaneous improvements in motor performance. Furthermore, I was unable to determine if the synaptogenesis of the SCT was functionally relevant, likely attributed to the significant impact of surgical exposure of the 154 spinal cord. However, bulk mRNAseq analysis allowed identification of candidate genes that may be involved in the plasticity of SCT axons some of which should be subject to further experiments in the future. 6.2 Limitations and Future Directions 6.2.1 Transgene Insertion Sites, Sequence Length, and the “Hox Clock” In the non-targeted Hoxb8 reporter strains (Hoxb8CreTdT and Hoxb8FlpOTdT mice), ectopic reporter expression is most likely due to the absence of the miR196a binding site in their 3’ UTRs, and/or their insertion near one or more new regulatory elements. This may render them as novel “sensors” of some developmentally-important diffusible signalling molecule – most likely RA, given the presence of a RARE in the Hoxb8 promoter sequence (Oosterveen et al., 2003). Circumstantial evidence, in the form of RA-responsiveness of transgenic reporter (Luo et al., 2004; Wilde et al., 2017), suggests this might be the case for thalamic neurons and cephalic flexure-associated astrocytes in Hoxb8CreTdT mice, and Cajal-Retzius cells in Hoxb8FlpOTdT mice (Chapter 3). However, a more complete picture would be painted by determining the insertion sites of the transgenes and identification of nearby promoters activated by other diffusible signalling molecules. The differences between the four reporter strains described here also raise questions about the fidelity of permanent reporter expression to endogenous Hoxb8 expression. The two targeted transgenic (knock-in) models are a case in point: there was an obvious difference in the number of reporter-positive microglia (Hoxb8IRESCre >> Hoxb8T2AFlpO), which may be a result of protection of sequence flanking the IRES from miRNA-196-mediated translational inhibition (Mathonnet et al., 2007; Thermann & 155 Hentze, 2007; Humphreys et al., 2005). An alternate hypothesis is that this is a result of the relationship between differing lengths of the knocked-in constructs and the timing of Hox gene expression (i.e. temporal collinearity or the “Hox clock”). I have speculated that increased genomic distance between the Hoxb8 sequence and the miR196a target in the 3’UTR results in a prolonged expression of Hoxb8 prior to microRNA-mediated mRNA degradation. This notion depends on the sufficiency of miR196a allele in the unaltered Hoxb cluster to interfere with Hoxb8T2AFlpO or Hoxb8IRESCre translation. A simple test of this hypothesis would be to examine reporter expression in mice homozygous for the targeted constructs – delayed Hoxb8 expression would result in more widespread reporter expression. Another approach to determining the true distribution of Hoxb8 expression might be to identify a sequence specifically transcribed in response to Hoxb8, and construct a reporter driven by its promoter. A first step towards this goal is identification of genes upregulated by Hoxb8 overexpression. This has been accomplished in the chick (Wilmerding et al., 2021), but a remaining issue is how specific the upregulated genes are to Hoxb8. 6.2.2 Changes in Motor Behaviours in Sham Operated Animals – Is Another Control Group Warranted? The behavioural testing in chapter 2 may have served as “training” or “exercise” for the mice. While not conventionally viewed as training, the mice were subjected to behavioural assessment for the four tests every four days beginning on day four and five (Chapter 2). Work by Zou (2021) demonstrated that reorganization of the cortex occurred following SCI which allowed the hindlimb motor area to be recruited to control 156 forelimb extensor motor function. However, this reorganization did not occur without continued training; this suggested functional guidance was required (Zou, 2021). Zhang et al. (2019) found that fine-motor control was rescued in CST mutant mice with voluntary wheel running training for 8-12 weeks. This recovery of function was accompanied by an increased in PSD95 expression and brain derived neurotrophic factor (BDNF) in the hippocampus. Hippocampal slices exposed to BDNF for 72 hours led to an elevation in synapse number, spine density, and post-injury changes of the synaptic network (Rauti et al., 2020). As a result, one could postulate that the behavioural assessments employed in Chapter 2 of this thesis served as training for the mice, and that this training led to compensatory rewiring of the SCT. While this is unlikely, it should be excluded experimentally. This could involve a control group of mice that received the same unilateral cervical hemisection without behavioural testing, to determine if the amount of cerebellar plasticity that occurred was consistent with the behavioural group. 6.2.3 Does Collaboration Between Corticospinal and Spinocerebellar Plasticity Underpin Spontaneous Functional Improvement? In Chapter 4, I highlighted several points of convergence between descending cortical output and spinocerebellar afferent circuitry that may cooperate after injury to elicit functional improvement. C3 spinal hemisection removes contralateral corticospinal input from the ipsilateral hemicord, leaving the lion’s share of responsibility for motor behaviour to the ipsilateral hemisphere, and an increase in activity (and thus structural plasticity in the contralateral hemicord, Carmel & Martin, 2014, possibly involving SCT neurons, Hantman & Jessell, 2010) can be inferred. 157 An unexpected observation, made in preliminary experiments using Hoxb8CreTdT mice, was a striking increase in the number of reporter-positive microglia in the unaffected forebrain (Fig. 6.1). What is more is that the brain regions involved (primary motor area, caudoputamen, nucleus accumbens) are established neural substrates for motor learning and execution, cognition, motivation, and reward, and are thus well-situated to underpin plasticity of the spared hemisphere responsible for spontaneous recovery. In the adult, microglia-synapse interaction enhances synaptic activity and network synchrony (Akiyoshi et al., 2018), raising the exciting possibility that these serve as an intermediary between adaptive changes in corticospinal and spinocerebellar circuitry. However, many questions remain: Is this response specific to Hoxb8-lineage microglia? Is endogenous Hoxb8 upregulated or are these cells infiltrating the brain from the circulation (Van Deren et al., 2022)? Is the transgene acting as a sensor of neuronal activity or diffusible signals upregulated in the spared hemisphere? What is the temporal sequence of this microglial response with respect to CST and SCT plasticity and spontaneous recovery? Clearly Hoxb8 reporter mice have more to tell us about the natural history of SCI. 158 6.2.4 Future Experiments Are Warranted Based on Bulk mRNA Sequencing Data The data in Chapter 5 guide two future experiments that will help one to understand the changes that occur in the partially deafferented cerebellum. First, it is well understood that when an axon is severed from its cell body, as is the case in unilateral C3 hemisection, changes occur to the distal axon (Geden & Deshmukh, 2017). From ~72 hours after injury, rapid breakdown of the distal axons occurs resulting in a glial response to remove axonal debris (a process known as gliosis) (Geden & Deshmukh, 2017). While this process has been well-characterized, a question that remains from the results of the bulk mRNA sequencing data is: what is the mechanism through which gliosis is initiated? Several of the transcriptomic changes that occurred in the cerebellar vermis 14d after unilateral C3 hemisection involved genes associated with gliosis (astrogliosis or microgliosis). The IPA analysis found several downstream targets of Tau signaling upregulated in the cerebellar vermis after C3 hemisection, many of which are associated with gliosis (e.g. Tyrobp, Clec7a, Tlr2, Apoe, Ccl3, Fig. 5.2, Table 5.1). Under normal uninjured conditions, Tau is found mainly in neurons, specifically in axons, and is involved in microtubule stabilization, cellular signaling, and neuronal development (Ke et Figure 6.1. Hoxb8-lineage microgliosis in the rostral forebrain. There is a clear microgliosis ipsilateral to a C3 hemisection in the motor cortex, caudoputamen, and nucleus accumbens. 159 al., 2012; Denk et al., 2010). Following damage to the axons at C3, the distal portions of the axons in the cerebellum lose support from the cell body, undergo Wallerian degeneration, break apart, and release several molecules including Tau. At 14d post-injury, it is expected that the axons in the cerebellum are either still undergoing degeneration, or that the changes caused by degeneration are still occurring. The molecules downstream of Tau make it reasonable to postulate that Tau is responsible for gliosis. However, to confirm these findings a future experiment is needed where bulk mRNA sequencing will be done 1 week and 3 weeks post-injury. It is expected that the gliosis-associated genes will be highly expressed at 1 week post-injury. Three weeks after injury, it is expected that there will either be a decrease in gliosis-related genes as axons are cleared, or that these gliosis-associated genes remain highly expressed, and the neuronal plasticity-genes are upregulated at this later timepoint. The other main question that remains as a result of the bulk mRNA sequencing data is: what might be leading to collateral sprouting and other forms of plasticity? The upregulation of BDNF 14d after unilateral cervical hemisection may be the result of Tau being released from degenerating axons, as BDNF upregulation is a consequence of Tau exposure. There is ample evidence for the role of BDNF in axonal sprouting after deafferentation in the PNS, and the CST after SCI (Benítez-Temiño et al., 2023; Sasaki et al., 2009). Additionally, SCT neurons in the spinal cord produce BDNF (Matson et al., 2022), which may be anterogradely transported and released in the cerebellum by spared SCT axons (Fawcett et al., 2000; Lever et al., 2001). The upregulation of BDNF caused by Tau after injury, and the presence of BDNF in spared axons make it a promising candidate to drive the plasticity response. 160 To answer this question and confirm this role a future experiment should be done. Following unilateral cervical hemisection, mice would be assessed behaviourally (Chapter 2) and plasticity would be assessed (Chapter 4) with administration of BDNF or a BDNF antagonist – TrkB-Fc (Soril et al., 2008; Ramer et al., 2007). Prior work on BDNF and TrkB-Fc after dorsal root injury suggests both beneficial and detrimental roles of BDNF on axonal plasticity. Scott and Ramer (2010) found that after dorsal root injury, exogenous BDNF was able to enhance sprouting, and this sprouting response was decreased with TrkB-Fc. Conversely, after dorsal root injury in rats, endogenous BDNF had detrimental effects on the plasticity of mechanosensory primary afferents, seen with a significant improvement in mechanosensation after dorsal root injury with TrkB-Fc administration (Ramer et al., 2007). Based on past findings, it is expected that exogenous BDNF will enhance the sprouting response of SCT axons and lead to improved behavioural recovery. Antagonism of BDNF with TrkB-Fc will inhibit the sprouting of SCT axons, and thus it is expected that these mice will have worse behavioural recovery (as it is hypothesized that the SCT plasticity drives functional recovery). However, since SCT axons are mechanosensory, it will be interesting to see if TrkB-Fc administration will lead to increased plasticity of SCT axons. 6.3 Final Remarks Animal behaviour is choreographed through the integration of sensory input with motor output. Under circumstances in which the flow of information in either direction is interrupted, as it is after spinal cord injury, the primary goal is to re-establish connectivity of ascending and descending systems. In the ideal scenario, this would be accomplished by regeneration of injured axons and reconnection with their original 161 targets. As this is not yet possible, a workaround is to harness the plasticity of spared systems in order to either restore lost function or adapt to a new normal. There are at present several circuits in which such plasticity has been demonstrated, mainly within the damaged cord. One aim of this thesis was to determine whether something similar could happen at distant or intermediate structures such as the cerebellum. This is indeed the case. 162 References Afzal, Z., Lange, J. J., Nolte, C., McKinney, S., Wood, C., Paulson, A., De Kumar, B., Unruh, J., Slaughter, B. D., & Krumlauf, R. (2023). Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube. Development (Cambridge, England), 150(10). https://doi.org/10.1242/DEV.201259 Ahuja, C. S., Nori, S., Tetreault, L., Wilson, J., Kwon, B., Harrop, J., Choi, D., & Fehlings, M. G. (2017). Traumatic Spinal Cord Injury—Repair and Regeneration. Clinical Neurosurgery, 80(3), S22–S90. https://doi.org/10.1093/NEUROS/NYW080 Aitkin, L. M., Dickhaus, H., Schult, W., & Zimmermann, M. (1978). External nucleus of inferior colliculus: auditory and spinal somatosensory afferents and their interactions. Journal of Neurophysiology, 41(4), 837–847. https://doi.org/10.1152/JN.1978.41.4.837 Akay, T., & Murray, A. J. (2021). Relative Contribution of Proprioceptive and Vestibular Sensory Systems to Locomotion: Opportunities for Discovery in the Age of Molecular Science. International Journal of Molecular Sciences 2021, Vol. 22, Page 1467, 22(3), 1467. https://doi.org/10.3390/IJMS22031467 Akiyoshi, R., Wake, H., Kato, D., Horiuchi, H., Ono, R., Ikegami, A., Haruwaka, K., Omori, T., Tachibana, Y., Moorhouse, A. J., & Nabekura, J. (2018). Microglia Enhance Synapse Activity to Promote Local Network Synchronization. ENeuro, 5(5). https://doi.org/10.1523/ENEURO.0088-18.2018 Al-Chalabi, M., Reddy, V., & Gupta, S. (2023). Neuroanatomy, Spinothalamic Tract. StatPearls. https://www.ncbi.nlm.nih.gov/books/NBK507824/ 163 Alizadeh, A., Dyck, S. M., & Karimi-Abdolrezaee, S. (2019). Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. Frontiers in Neurology, 10. https://doi.org/10.3389/FNEUR.2019.00282 Anderson, K. D., Abdul, M., & Steward, O. (2004). Quantitative assessment of deficits and recovery of forelimb motor function after cervical spinal cord injury in mice. Experimental Neurology, 190(1), 184–191. https://doi.org/10.1016/J.EXPNEUROL.2004.06.029 Anderson, A., Robert, S., Huang, W., Young, W., & Cotman, C. (2004). Activation of complement pathways after contusion-induced spinal cord injury. Journal of neurotrauma, 21(12), 1831-1846. https://doi.org/10.1089/neu.2004.21.1831 Anstötz, M., & Maccaferri, G. (2020). A Toolbox of Criteria for Distinguishing Cajal–Retzius Cells from Other Neuronal Types in the Postnatal Mouse Hippocampus. ENeuro, 7(1). https://doi.org/10.1523/ENEURO.0516-19.2019 Aoki, M., Fujito, Y., Satomi, H., Kurosawa, Y., & Kasaba, T. (1986). The possible role of collateral sprouting in the functional restitution of corticospinal connections after spinal hemisection. Neuroscience Research, 3(6), 617–627. https://doi.org/10.1016/0168-0102(86)90058-1 Armstrong, D. M. (1986). Supraspinal contributions to the initiation and control of locomotion in the cat. Progress in Neurobiology, 26(4), 273–361. https://doi.org/10.1016/0301-0082(86)90021-3 Babcock, A. A., Wirenfeldt, M., Holm, T., Nielsen, H. H., Dissing-Olesen, L., Toft-Hansen, H., Millward, J. M., Landmann, R., Rivest, S., Finsen, B., & Owens, T. (2006). Toll-like receptor 2 signaling in response to brain injury: an innate bridge to neuroinflammation. 164 The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 26(49), 12826–12837. https://doi.org/10.1523/JNEUROSCI.4937-05.2006 Ballermann, M., & Fouad, K. (2006). Spontaneous locomotor recovery in spinal cord injured rats is accompanied by anatomical plasticity of reticulospinal fibers. European Journal of Neuroscience, 23(8), 1988–1996. https://doi.org/10.1111/J.1460-9568.2006.04726.X Barber, C. F., Jorquera, R. A., Melom, J. E., & Littleton, J. T. (2009). Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains. The Journal of Cell Biology, 187(2), 295–310. https://doi.org/10.1083/jcb.200903098 Batka, R. J., Brown, T. J., Mcmillan, K. P., Meadows, R. M., Jones, K. J., & Haulcomb, M. M. (2014). The need for speed in rodent locomotion analyses. Anatomical Record (Hoboken, N.J. : 2007), 297(10), 1839–1864. https://doi.org/10.1002/AR.22955 Baumel, Y., Jacobson, G. A., & Cohen, D. (2009). Implications of functional anatomy on information processing in the deep cerebellar nuclei. Frontiers in Cellular Neuroscience, 3(NOV), 795. https://doi.org/10.3389/NEURO.03.014.2009/BIBTEX Bautista, E., Zarco, N., Aguirre-Pineda, N., Lara-Lozano, M., Vergara, P., González-Barrios, J. A., Aguilar-Roblero, R., & Segovia, J. (2018). Expression of Gas1 in Mouse Brain: Release and Role in Neuronal Differentiation. Cellular and Molecular Neurobiology, 38(4), 841–859. https://doi.org/10.1007/s10571-017-0559-0 Beckinghausen, J., & Sillitoe, R. V. (2019). Insights into cerebellar development and connectivity. Neuroscience Letters, 688, 2. https://doi.org/10.1016/J.NEULET.2018.05.013 165 Béïque, J. C., & Andrade, R. (2003). PSD-95 regulates synaptic transmission and plasticity in rat cerebral cortex. The Journal of Physiology, 546(Pt 3), 859. https://doi.org/10.1113/JPHYSIOL.2002.031369 Beirowski, B., Adalbert, R., Wagner, D., Grumme, D. S., Addicks, K., Ribchester, R. R., & Coleman, M. P. (2005). The progressive nature of Wallerian degeneration in wild-type and slow Wallerian degeneration (WldS) nerves. BMC Neuroscience, 6(1), 1–27. https://doi.org/10.1186/1471-2202-6-6/FIGURES/14 Bel-Vialar, S., Itasaki, N., & Krumlauf, R. (2002). Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development, 129(22), 5103–5115. https://doi.org/10.1242/dev.129.22.5103 Benítez-Temiño, B., Hernández, R. G., de la Cruz, R. R., & Pastor, A. M. (2023). BDNF Influence on Adult Terminal Axon Sprouting after Partial Deafferentation. International Journal of Molecular Sciences, 24(13). https://doi.org/10.3390/ijms241310660 Birling, M. C., Dierich, A., Jacquot, S., Hérault, Y., & Pavlovic, G. (2012). Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background. Genesis (New York, N.Y. : 2000), 50(6), 482–489. https://doi.org/10.1002/DVG.20826 Blanco, J. E., Anderson, K. D., & Steward, O. (2007). Recovery of forepaw gripping ability and reorganization of cortical motor control following cervical spinal cord injuries in mice. Experimental Neurology, 203(2), 333–348. https://doi.org/10.1016/J.EXPNEUROL.2006.08.017 166 Blinzinger, K., & Kreutzberg, G. (1968). Displacement of synaptic terminals from regenerating motorneurons by microglial cells. Zeitschrift, 85(2), 145-157. https://doi.org/10.1007/BF00325030 Boele, H. J., Koekkoek, S. K. E., De Zeeuw, C. I., & Ruigrok, T. J. H. (2013). Axonal Sprouting and Formation of Terminals in the Adult Cerebellum during Associative Motor Learning. Journal of Neuroscience, 33(45), 17897–17907. https://doi.org/10.1523/JNEUROSCI.0511-13.2013 Bohic, M., Upadhyay, A., Eisdorfer, J. T., Keating, J., Simon, R. C., Briones, B. A., Azadegan, C., Nacht, H. D., Oputa, O., Martinez, A. M., Bethell, B. N., Gradwell, M. A., Romanienko, P., Ramer, M. S., Stuber, G. D., & Abraira, V. E. (2023). A new Hoxb8FlpO mouse line for intersectional approaches to dissect developmentally defined adult sensorimotor circuits. Frontiers in Molecular Neuroscience, 16, 1176823. https://doi.org/10.3389/FNMOL.2023.1176823/BIBTEX Bouabe, H., & Okkenhaug, K. (2013). Gene Targeting in Mice: a Review. Methods in Molecular Biology (Clifton, N.J.), 1064, 315. https://doi.org/10.1007/978-1-62703-601-6_23 Brock, J. H., Rosenzweig, E. S., Yang, H., & Tuszynski, M. H. (2018). Enhanced axonal transport: A novel form of “plasticity” after primate and rodent spinal cord injury. Experimental Neurology, 301(Pt A), 59. https://doi.org/10.1016/J.EXPNEUROL.2017.12.009 Caillard, O., Moreno, H., Schwaller, B., Llano, I., Celio, M. R., & Marty, A. (2000). Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity. Proceedings of the 167 National Academy of Sciences, 97(24), 13372–13377. https://doi.org/10.1073/pnas.230362997 Cao, J., Wang, P.-K., Tiwari, V., Liang, L., Lutz, B. M., Shieh, K.-R., Zang, W.-D., Kaufman, A. G., Bekker, A., Gao, X.-Q., & Tao, Y.-X. (2015). Short-term pre- and post-operative stress prolongs incision-induced pain hypersensitivity without changing basal pain perception. Molecular Pain, 11, 73. https://doi.org/10.1186/s12990-015-0077-3 Cao, Y., Shi, Y., Xiao, Z., Chen, X., Chen, B., Yang, B., Shu, M., Yin, Y., Wu, S., Yin, W., Fu, X., Tan, J., Zhou, Q., Wu, Z., Jiang, X., & Dai, J. (2021). Contralateral Axon Sprouting but Not Ipsilateral Regeneration Is Responsible for Spontaneous Locomotor Recovery Post Spinal Cord Hemisection. Frontiers in Cellular Neuroscience, 15, 730348. https://doi.org/10.3389/FNCEL.2021.730348/BIBTEX Carmel, J. B., & Martin, J. H. (2014). Motor cortex electrical stimulation augments sprouting of the corticospinal tract and promotes recovery of motor function. Frontiers in Integrative Neuroscience, 8(JUNE). https://doi.org/10.3389/FNINT.2014.00051 Carpenter, E. M., Goddard, J. M., Chisaka, O., Manley, N. R., & Capecchi, M. R. (1993). Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain. Development, 118(4), 1063–1075. https://doi.org/10.1242/DEV.118.4.1063 Caudle, K. L., Brown, E. H., Shum-Siu, A., Burke, D. A., Magnuson, T. S. G., Voor, M. J., & Magnuson, D. S. K. (2011). Hindlimb Immobilization in a Wheelchair Alters Functional Recovery Following Contusive Spinal Cord Injury in the Adult Rat. Neurorehabilitation and Neural Repair, 25(8), 729. https://doi.org/10.1177/1545968311407519 168 Charité, J., De Graaff, W., Vogels, R., Meijlink, F., & Deschamps, J. (1995). Regulation of the Hoxb-8 gene: synergism between multimerized cis-acting elements increases responsiveness to positional information. Developmental Biology, 171, 294–305. Chen, H., Tang, A. H., & Blanpied, T. A. (2018). Subsynaptic spatial organization as a regulator of synaptic strength and plasticity. Current Opinion in Neurobiology, 51, 147–153. https://doi.org/10.1016/J.CONB.2018.05.004 Chen, S. K., Tvrdik, P., Peden, E., Cho, S., Wu, S., Spangrude, G., & Capecchi, M. R. (2010). Hematopoietic origin of pathological grooming in Hoxb8 mutant mice. Cell, 141(5), 775–785. https://doi.org/10.1016/j.cell.2010.03.055 Chen, Z., Cooper, B., Kalla, S., Varoqueaux, F., & Young, S. M. (2013). The Munc13 Proteins Differentially Regulate Readily Releasable Pool Dynamics and Calcium-Dependent Recovery at a Central Synapse. The Journal of Neuroscience, 33(19), 8336. https://doi.org/10.1523/JNEUROSCI.5128-12.2013 Chizhikov, V., & Millen, K. J. (2003). Development and malformations of the cerebellum in mice. Molecular Genetics and Metabolism, 80(1–2), 54–65. https://doi.org/10.1016/J.YMGME.2003.08.019 Chleilat, E., Pethe, A., Pfeifer, D., Krieglstein, K., & Roussa, E. (2020). TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons. International Journal of Molecular Sciences, 21(8). https://doi.org/10.3390/ijms21082735 Chu, Y., Jin, X., Parada, I., Pesic, A., Stevens, B., Barres, B., Prince, D. (2010). Enhanced synaptic connectivity and epilepsy in C1q knockout mice. Proceedings of the National 169 Academy of Sciences of the Unites States of America, 107(17), 7975-7980. https://doi.org/10.1073/pnas.0913449107 Cimino, P. J., Sokal, I., Leverenz, J., Fukui, Y., & Montine, T. J. (2009). DOCK2 is a microglial specific regulator of central nervous system innate immunity found in normal and Alzheimer’s disease brain. The American Journal of Pathology, 175(4), 1622–1630. https://doi.org/10.2353/ajpath.2009.090443 Cliffer, K. D., Burstein, R., & Giesler, G. J. (1991). Distributions of Spinothalamic, Spinohypothalamic, and Spinotelencephalic Fibers Revealed by Anterograde Transport of PHA-L in Rats. The Journal of Neuroscience, 71(3), 652–666. Cole, C. J., & Josselyn, S. A. (2008). Transcription regulation of memory: CREB, CaMKIV, Fos/Jun, CBP, and SRF. Consalez, G. G., Goldowitz, D., Casoni, F., & Hawkes, R. (2021). Origins, Development, and Compartmentation of the Granule Cells of the Cerebellum. Frontiers in Neural Circuits, 14. https://doi.org/10.3389/FNCIR.2020.611841 Crowley, S. T., Kataoka, K., & Itaka, K. (2018). Combined CatWalk Index: an improved method to measure mouse motor function using the automated gait analysis system. BMC Research Notes, 11(1). https://doi.org/10.1186/S13104-018-3374-X D’Angelo, E. (2014). The Organization of Plasticity in the Cerebellar Cortex: From Synapses to Control. Progress in Brain Research, 210, 31–58. https://doi.org/10.1016/B978-0-444-63356-9.00002-9 170 Darian-Smith, C., Lilak, A., & Alarcón, C. (2013). Corticospinal sprouting occurs selectively following dorsal rhizotomy in the macaque monkey. The Journal of Comparative Neurology, 521(10), 2359–2372. https://doi.org/10.1002/CNE.23289 Darian-Smith, C., Lilak, A., Garner, J., & Irvine, K. A. (2014). Corticospinal sprouting differs according to spinal injury location and cortical origin in macaque monkeys. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 34(37), 12267–12279. https://doi.org/10.1523/JNEUROSCI.1593-14.2014 Davidson, S., Truong, H., & Giesler, G. J. (2010). A quantitative analysis of spinothalamic tract neurons in adult and developing mouse. The Journal of Comparative Neurology, 518(16), 3193. https://doi.org/10.1002/CNE.22392 De Schepper, S., Ge, J. Z., Crowley, G., Ferreira, L. S. S., Garceau, D., Toomey, C. E., Sokolova, D., Rueda-Carrasco, J., Shin, S.-H., Kim, J.-S., Childs, T., Lashley, T., Burden, J. J., Sasner, M., Sala Frigerio, C., Jung, S., & Hong, S. (2023). Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer’s disease. Nature Neuroscience, 26(3), 406–415. https://doi.org/10.1038/s41593-023-01257-z Del-Bel, E. A., Borges, C. A. G., Defino, H. L. A., & Guimarães, F. S. (2000). Induction of Fos protein immunoreactivity by spinal cord contusion. Brazilian Journal of Medical and Biological Research, 33(5), 521–528. https://doi.org/10.1590/S0100-879X2000000500006 171 Dellon, E. S., & Dellon, A. L. (1991). Functional assessment of neurologic impairment: track analysis in diabetic and compression neuropathies. Plastic and Reconstructive Surgery, 88(4), 686–694. https://doi.org/10.1097/00006534-199110000-00020 Denk, F., & Wade-Martins, R. (2009). Knock-out and transgenic mouse models of tauopathies. Neurobiology of Aging, 30(1), 1–13. https://doi.org/10.1016/j.neurobiolaging.2007.05.010 Dent, E. W., Gupton, S. L., & Gertler, F. B. (2011). The Growth Cone Cytoskeleton in Axon Outgrowth and Guidance. Cold Spring Harbor Perspectives in Biology, 3(3), a001800–a001800. https://doi.org/10.1101/cshperspect.a001800 Deschamps, J., Van Den Akker, E., Forlani, S., De Graaff, W., Oosterveen, T., Roelen, B., & Roelfsema, J. (1999). Initiation, establishment and maintenance of Hox gene expression patterns in the mouse. In Int. J. Dev. Biol (Vol. 43). www.ehu.es/ijdb Dias, D., Portugal, C. C., Relvas, J., & Socodato, R. (2025). From Genetics to Neuroinflammation: The Impact of ApoE4 on Microglial Function in Alzheimer’s Disease. Cells, 14(4). https://doi.org/10.3390/cells14040243 Dietz, V., Wirz, M., Curt, A., & Colombo, G. (1998). Locomotor pattern in paraplegic patients: training effects and recovery of spinal cord function. Spinal Cord, 36(6), 380–390. https://doi.org/10.1038/SJ.SC.3100590 Dodd, K. C., Nair, V. A., & Prabhakaran, V. (2017). Role of the contralesional vs. Ipsilesional hemisphere in stroke recovery. Frontiers in Human Neuroscience, 11, 279403. https://doi.org/10.3389/FNHUM.2017.00469/BIBTEX 172 Eisenach, J. C., & Brennan, T. J. (2018). Pain after surgery. Pain, 159(6), 1010–1011. https://doi.org/10.1097/j.pain.0000000000001223 Eng, L. F., & Ghirnikar, R. S. (1994). GFAP and Astrogliosis. Brain Pathology, 4(3), 229–237. https://doi.org/10.1111/j.1750-3639.1994.tb00838.x Engmann, A. K., Bizzozzero, F., Schneider, M. P., Pfyffer, D., Imobersteg, S., Schneider, R., Hofer, A. S., Wieckhorst, M., & Schwab, M. E. (2020). The Gigantocellular Reticular Nucleus Plays a Significant Role in Locomotor Recovery after Incomplete Spinal Cord Injury. The Journal of Neuroscience, 40(43), 8292. https://doi.org/10.1523/JNEUROSCI.0474-20.2020 Eskandari-Sedighi, G., & Blurton-Jones, M. (2023). Microglial APOE4: more is less and less is more. Molecular Neurodegeneration, 18(1), 99. https://doi.org/10.1186/s13024-023-00693-6 Farini, D., Marazziti, D., Geloso, M. C., & Sette, C. (2021). Transcriptome programs involved in the development and structure of the cerebellum. Cellular and Molecular Life Sciences, 78(19–20), 6431. https://doi.org/10.1007/S00018-021-03911-W Fawcett, J. P., Alonso-Vanegas, M. A., Morris, S. J., Miller, F. D., Sadikot, A. F., & Murphy, R. A. (2000). Evidence that Brain-Derived Neurotrophic Factor from Presynaptic Nerve Terminals Regulates the Phenotype of Calbindin-Containing Neurons in the Lateral Septum. The Journal of Neuroscience, 20(1), 274–282. https://doi.org/10.1523/JNEUROSCI.20-01-00274.2000 Fawcett, J. W., Curt, A., Steeves, J. D., Coleman, W. P., Tuszynski, M. H., Lammertse, D., Bartlett, P. F., Blight, A. R., Dietz, V., Ditunno, J., Dobkin, B. H., Havton, L. A., Ellaway, P. 173 H., Fehlings, M. G., Privat, A., Grossman, R., Guest, J. D., Kleitman, N., Nakamura, M., … Short, D. (2007). Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials. Spinal Cord, 45(3), 190–205. https://doi.org/10.1038/SJ.SC.3102007 Feringa, E. R., Lee, G. W., & Vahlsing, H. L. (1985). Cell death in Clarke’s column after spinal cord transection. Journal of Neuropathology and Experimental Neurology, 44(2), 156–164. https://doi.org/10.1097/00005072-198503000-00004 Feringa, E. R., Pruitt 2D, J. N., McBride, R. L., & Vahlsing, H. L. (1987). Changes in number and size of Clarke’s column neurons after cord transection. Journal of Neuropathology and Experimental Neurology, 46(6), 695–702. https://doi.org/10.1097/00005072-198711000-00008 Ferreira-Gomes, J., Adães, S., & Castro-Lopes, J. M. (2008). Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study. The Journal of Pain, 9(10), 945–954. https://doi.org/10.1016/J.JPAIN.2008.05.012 Ferrini, F., & De Koninck, Y. (2013). Microglia Control Neuronal Network Excitability via BDNF Signalling. Neural Plasticity, 2013, 1–11. https://doi.org/10.1155/2013/429815 Fink, A. J., Englund, C., Daza, R. A. M., Pham, D., Lau, C., Nivison, M., Kowalczyk, T., & Hevner, R. F. (2006). Development of the Deep Cerebellar Nuclei: Transcription Factors and Cell Migration from the Rhombic Lip. Journal of Neuroscience, 26(11), 3066–3076. https://doi.org/10.1523/JNEUROSCI.5203-05.2006 174 Fisher, K. M., Garner, J. P., & Darian-Smith, X. C. (2020). Reorganization of the Primate Dorsal Horn in Response to a Deafferentation Lesion Affecting Hand Function. Journal of Neuroscience, 40(8), 1625–1639. https://doi.org/10.1523/JNEUROSCI.2330-19.2020 Flatters, S. J. L. (2010). Effect of analgesic standards on persistent postoperative pain evoked by skin/muscle incision and retraction (SMIR). Neuroscience Letters, 477(1), 43–47. https://doi.org/10.1016/j.neulet.2010.04.033 Florence, S. L., & Kaas, J. H. (1995). Large-scale reorganization at multiple levels of the somatosensory pathway follows therapeutic amputation of the hand in monkeys. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 15(12), 8083–8095. https://doi.org/10.1523/JNEUROSCI.15-12-08083.1995 Forlani, S., Lawson, K. A., & Deschamps, J. (2003). Acquisition of Hox codes during gastrulation and axial elongation in the mouse embryo. Development, 130(16), 3807–3819. https://doi.org/10.1242/dev.00573 Fouad, K., & Tse, A. (2008). Adaptive changes in the injured spinal cord and their role in promoting functional recovery. Neurological Research, 30(1), 17–27. https://doi.org/10.1179/016164107X251781 Fuller, H. R., Slade, R., Jovanov-Milošević, N., Babić, M., Sedmak, G., Šimić, G., Fuszard, M. A., Shirran, S. L., Botting, C. H., & Gates, M. A. (2015). Stathmin is enriched in the developing corticospinal tract. Molecular and Cellular Neuroscience, 69, 12–21. https://doi.org/10.1016/j.mcn.2015.09.003 Gagliardi, D., Pagliari, E., Meneri, M., Melzi, V., Rizzo, F., Comi, G. Pietro, Corti, S., Taiana, M., & Nizzardo, M. (2022). Stathmins and Motor Neuron Diseases: Pathophysiology and 175 Therapeutic Targets. Biomedicines, 10(3). https://doi.org/10.3390/biomedicines10030711 Gale, K., Kerasidis, H., & Wrathall, J. R. (1985). Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment. Experimental Neurology, 88(1), 123–134. https://doi.org/10.1016/0014-4886(85)90118-9 Gebre, S. A., Reeber, S. L., & Sillitoe, R. V. (2012). Parasagittal compartmentation of cerebellar mossy fibers as revealed by the patterned expression of vesicular glutamate transporters VGLUT1 and VGLUT2. Brain Structure and Function, 217(2), 165–180. https://doi.org/10.1007/S00429-011-0339-4/FIGURES/10 Geden, M. J., & Deshmukh, M. (2016). Axon degeneration: context defines distinct pathways. Current Opinion in Neurobiology, 39, 108–115. https://doi.org/10.1016/j.conb.2016.05.002 Ghosh, A., Sydekum, E., Haiss, F., Peduzzi, S., Zörner, B., Schneider, R., Baltes, C., Rudin, M., Weber, B., & Schwab, M. E. (2009). Functional and Anatomical Reorganization of the Sensory-Motor Cortex after Incomplete Spinal Cord Injury in Adult Rats. The Journal of Neuroscience, 29(39), 12210. https://doi.org/10.1523/JNEUROSCI.1828-09.2009 Gibbs, H. C., Chang-Gonzalez, A., Hwang, W., Yeh, A. T., & Lekven, A. C. (2017). Midbrain-hindbrain boundary morphogenesis: At the intersection of wnt and Fgf signaling. In Frontiers in Neuroanatomy (Vol. 11). Frontiers Media S.A. https://doi.org/10.3389/fnana.2017.00064 Gloster, A., Wu, W., Speelman, A., Weiss, S., Causing, C., Pozniak, C., Reynolds, B., Chang, E., Toma, J. G., & Miller, F. D. (1994). The T alpha 1 alpha-tubulin promoter specifies 176 gene expression as a function of neuronal growth and regeneration in transgenic mice. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 14(12), 7319–7330. https://doi.org/10.1523/JNEUROSCI.14-12-07319.1994 Goldshmit, Y., Banyas, E., Bens, N., Yakovchuk, A., & Ruban, A. (2020). Blood glutamate scavengers and exercises as an effective neuroprotective treatment in mice with spinal cord injury. Journal of Neurosurgery: Spine, 33(5), 692–704. https://doi.org/10.3171/2020.4.SPINE20302 Gonçalves, C. S., Le Boiteux, E., Arnaud, P., & Costa, B. M. (2020). HOX gene cluster (de)regulation in brain: from neurodevelopment to malignant glial tumours. Cellular and Molecular Life Sciences, 77(19), 3797–3821. https://doi.org/10.1007/S00018-020-03508-9/FIGURES/4 Graham, A., Maden, M., & Krumlauf, R. (1991). The murine Hox-2 genes display dynamic dorsoventral patterns of expression during central nervous system development. Development, 112(1), 255–264. Grasso, R., Ivanenko, Y. P., Zago, M., Molinari, M., Scivoletto, G., Castellano, V., Macellari, V., & Lacquaniti, F. (2004). Distributed plasticity of locomotor pattern generators in spinal cord injured patients. Brain, 127(5), 1019–1034. https://doi.org/10.1093/BRAIN/AWH115 Grenningloh, G., Soehrman, S., Bondallaz, P., Ruchti, E., & Cadas, H. (2004). Role of the microtubule destabilizing proteins SCG10 and stathmin in neuronal growth. Journal of Neurobiology, 58(1), 60–69. https://doi.org/10.1002/neu.10279 177 Guthrie, K. M., Nguyen, T., & Gall, C. M. (1995). Insulin-like growth factor-1 mRNA is increased in deafferented hippocampus: Spatiotemporal correspondence of a trophic event with axon sprouting. Journal of Comparative Neurology, 352(1), 147–160. https://doi.org/10.1002/cne.903520111 Hagemeyer, N., & Prinz, M. (2014). Burning down the house: IRF7 makes the difference for microglia. The EMBO Journal, 33(24), 2885–2886. https://doi.org/10.15252/embj.201490345 Hamers, F. P. T., Koopmans, G. C., & Joosten, E. A. J. (2006). CatWalk-assisted gait analysis in the assessment of spinal cord injury. Journal of Neurotrauma, 23(3–4), 537–548. https://doi.org/10.1089/NEU.2006.23.537 Hansel, C., Linden, D. J., & D’Angelo, E. (2001). Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum. Nature Neuroscience, 4(5), 467–475. https://doi.org/10.1038/87419 Hantman, A. W., & Jessell, T. M. (2010). Clarke’s Column Neurons as the Focus of a Corticospinal Corollary Circuit. Nature Neuroscience, 13(10), 1233. https://doi.org/10.1038/NN.2637 Hayashi, Y. (2022). Molecular mechanism of hippocampal long-term potentiation – Towards multiscale understanding of learning and memory. Neuroscience Research, 175, 3–15. https://doi.org/10.1016/J.NEURES.2021.08.001 Heinrich, R., Aronheim, A., Cheng, Y.-C., & Perlman, I. (2024). Editorial: ATF3: a crucial stress-responsive gene of glia and neurons in CNS. Frontiers in Molecular Neuroscience, 17. https://doi.org/10.3389/fnmol.2024.1484487 178 Higuchi, K., Uyeda, A., Quan, L., Tanabe, S., Kato, Y., Kawahara, Y., & Muramatsu, R. (2024). Synaptotagmin 4 Supports Spontaneous Axon Sprouting after Spinal Cord Injury. The Journal of Neuroscience, 44(43), e1593232024. https://doi.org/10.1523/JNEUROSCI.1593-23.2024 Hill, R. L., Zhang, Y. P., Burke, D. A., Devries, W. H., Zhang, Y., Magnuson, D. S. K., Whittemore, S. R., & Shields, C. B. (2009). Anatomical and Functional Outcomes following a Precise, Graded, Dorsal Laceration Spinal Cord Injury in C57BL/6 Mice. Journal of Neurotrauma, 26(1), 1. https://doi.org/10.1089/NEU.2008.0543 Hilton, B. J., Moulson, A. J., & Tetzlaff, W. (2017). Neuroprotection and secondary damage following spinal cord injury: concepts and methods. Neuroscience Letters, 652, 3–10. https://doi.org/10.1016/J.NEULET.2016.12.004 Hökfelt, T., Stanic, D., Sanford, S. D., Gatlin, J. C., Nilsson, I., Paratcha, G., Ledda, F., Fetissov, S., Lindfors, C., Herzog, H., Johansen, J. E., Ubink, R., & Pfenninger, K. H. (2008). NPY and its involvement in axon guidance, neurogenesis, and feeding. Nutrition, 24(9), 860–868. https://doi.org/10.1016/j.nut.2008.06.010 Holstege, J. C., De Graaff, W., Hossaini, M., Cano, S. C., Jaarsma, D., Van Akker, E. Den, & Deschamps, J. (2008). Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice. Proceedings of the National Academy of Sciences of the United States of America, 105(17), 6338. https://doi.org/10.1073/PNAS.0802176105 Honda, A., Usui, H., Sakimura, K., & Igarashi, M. (2017). Rufy3 is an adapter protein for small GTPases that activates a Rac guanine nucleotide exchange factor to control neuronal 179 polarity. Journal of Biological Chemistry, 292(51), 20936–20946. https://doi.org/10.1074/jbc.M117.809541 Hornstein, E., Mansfield, J. H., Yekta, S., Hu, J. K. H., Harfe, B. D., McManus, M. T., Baskerville, S., Bartel, D. P., & Tabin, C. J. (2005). The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature, 438(7068), 671–674. https://doi.org/10.1038/NATURE04138 Hsieh, T. H., Tsai, J. Y., Wu, Y. N., Hwang, I. S., Chen, T. I., & Chen, J. J. J. (2010). Time course quantification of spastic hypertonia following spinal hemisection in rats. Neuroscience, 167(1), 185–198. https://doi.org/10.1016/J.NEUROSCIENCE.2010.01.064 Huang, C. C., Sugino, K., Shima, Y., Guo, C., Bai, S., Mensh, B. D., Nelson, S. B., & Hantman, A. W. (2013). Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells. ELife, 2(2), 400. https://doi.org/10.7554/ELIFE.00400 Huber, L., Ferdin, M., Holzmann, J., Stubbusch, J., & Rohrer, H. (2012). HoxB8 in noradrenergic specification and differentiation of the autonomic nervous system. Developmental Biology, 363(1), 219–233. https://doi.org/10.1016/J.YDBIO.2011.12.026 HULTBORN, H., & MALMSTEN, J. (1983). Changes in segmental reflexes following chronic spinal cord hemisection in the cat. I. Increased monosynaptic and polysynaptic ventral root discharges. Acta Physiologica Scandinavica, 119(4), 405–422. https://doi.org/10.1111/J.1748-1716.1983.TB07357.X Humphreys, D. T., Westman, B. J., Martin, D. I. K., & Preiss, T. (2005). MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail 180 function. Proceedings of the National Academy of Sciences of the United States of America, 102(47), 16961. https://doi.org/10.1073/PNAS.0506482102 Hunt, C., Moman, R., Peterson, A., Wilson, R., Covington, S., Mustafa, R., Murad, M. H., & Hooten, W. M. (2021). Prevalence of chronic pain after spinal cord injury: a systematic review and meta-analysis. Regional Anesthesia & Pain Medicine, 46(4), 328–336. https://doi.org/10.1136/RAPM-2020-101960 Hur, E.-M., Saijilafu, & Zhou, F.-Q. (2012). Growing the growth cone: remodeling the cytoskeleton to promote axon regeneration. Trends in Neurosciences, 35(3), 164–174. https://doi.org/10.1016/j.tins.2011.11.002 Jakeman, L. B., Wei, P., Guan, Z., & Stokes, B. T. (1998). Brain-Derived Neurotrophic Factor Stimulates Hindlimb Stepping and Sprouting of Cholinergic Fibers after Spinal Cord Injury. Experimental Neurology, 154(1), 170–184. https://doi.org/10.1006/exnr.1998.6924 Jang, E., Lee, S., Kim, J., Kim, J., Seo, J., Lee, W., Mori, K., Nakao, K., & Suk, K. (2013). Secreted protein lipocalin-2 promotes microglial M1 polarization. The FASEB Journal, 27(3), 1176–1190. https://doi.org/10.1096/fj.12-222257 Jasmin, L., Gogas, K. R., Ahlgren, S. C., Levine, J. D., & Basbaum, A. I. (1994). Walking evokes a distinctive pattern of Fos-like immunoreactivity in the caudal brainstem and spinal cord of the rat. Neuroscience, 58(2), 275–286. https://doi.org/10.1016/0306-4522(94)90034-5 181 Ji, L., Xu, S., Luo, H., & Zeng, F. (2022). Insights from DOCK2 in cell function and pathophysiology. Frontiers in Molecular Biosciences, 9. https://doi.org/10.3389/fmolb.2022.997659 Jiang, Q., Xue, S., Pan, X., Yu, T., Wei, X., Li, L., Qi, C., Shi, W., Ren, Z., Hu, D., & Fu, H. (2025). Differential changes in the microglial transcriptome between neonatal and adult mice after spinal cord injury. Scientific Reports, 15(1), 13708. https://doi.org/10.1038/s41598-025-98429-7 Jie, W., & Pei-Xi, C. (1992). Discharge response of cerebellar Purkinje cells to stimulation of C-fiber in cat saphenous nerve. Brain Research, 581(2), 269–272. https://doi.org/10.1016/0006-8993(92)90717-N Junge, H. J., Rhee, J.-S., Jahn, O., Varoqueaux, F., Spiess, J., Waxham, M. N., Rosenmund, C., & Brose, N. (2004). Calmodulin and Munc13 Form a Ca2+ Sensor/Effector Complex that Controls Short-Term Synaptic Plasticity. Cell, 118(3), 389–401. https://doi.org/10.1016/j.cell.2004.06.029 Kaas, J. H. (2000). The reorganization of somatosensory and motor cortex after peripheral nerve or spinal cord injury in primates. Progress in Brain Research, 128, 173–179. https://doi.org/10.1016/S0079-6123(00)28015-1 Kaegi, S., Schwab, M. E., Dietz, V., & Fouad, K. (2002). Electromyographic activity associated with spontaneous functional recovery after spinal cord injury in rats. The European Journal of Neuroscience, 16(2), 249–258. https://doi.org/10.1046/J.1460-9568.2002.02076.X 182 Kakizawa, S., Moriguchi, S., Ikeda, A., Iino, M., & Takeshima, H. (2008). Functional Crosstalk Between Cell-Surface and Intracellular Channels Mediated by Junctophilins Essential for Neuronal Functions. The Cerebellum, 7(3), 385–391. https://doi.org/10.1007/s12311-008-0040-1 Kakuta, Y., Adachi, A., Yokohama, M., Horii, T., Mieda, T., Iizuka, Y., Takagishi, K., Chikuda, H., Iizuka, H., & Nakamura, K. (2019). Spontaneous functional full recovery from motor and sensory deficits in adult mice after mild spinal cord injury. Heliyon, 5(6). https://doi.org/10.1016/J.HELIYON.2019.E01847 Kandel, E., Schwartz, J., & Jessell, T. (1991). Principles of Neural Science (H. Lebowitz & J. Butler, Eds.; 4th ed., Vol. 2). McGraw-Hill. Kann, O., Almouhanna, F., & Chausse, B. (2022). Interferon γ: a master cytokine in microglia-mediated neural network dysfunction and neurodegeneration. Trends in Neurosciences, 45(12), 913–927. https://doi.org/10.1016/j.tins.2022.10.007 Kanno, M., Suzuki, S., Fujiwara, T., Yokoyama, A., Sakamoto, A., Takahashi, H., Imai, Y., & Tanaka, J. (2005). Functional expression of CCL6 by rat microglia: A possible role of CCL6 in cell–cell communication. Journal of Neuroimmunology, 167(1–2), 72–80. https://doi.org/10.1016/j.jneuroim.2005.06.028 Kano, S.-I., Choi, E. Y., Dohi, E., Agarwal, S., Chang, D. J., Wilson, A. M., Lo, B. D., Rose, I. V. L., Gonzalez, S., Imai, T., & Sawa, A. (2019). Glutathione S-transferases promote proinflammatory astrocyte-microglia communication during brain inflammation. Science Signaling, 12(569). https://doi.org/10.1126/scisignal.aar2124 183 Kathe, C., Hutson, T. H., Chen, Q., Shine, H. D., McMahon, S. B., & Moon, L. D. F. (2014). Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies. Journal of Visualized Experiments : JoVE, 94(94), 51843. https://doi.org/10.3791/51843 Katz, H. R., Arcese, A. A., Bloom, O., & Morgan, J. R. (2022). Activating Transcription Factor 3 (ATF3) is a Highly Conserved Pro-regenerative Transcription Factor in the Vertebrate Nervous System. Frontiers in Cell and Developmental Biology, 10. https://doi.org/10.3389/fcell.2022.824036 Kaufman, T. C., Seeger, M. A., & Olsen, G. (1990). Molecular and Genetic Organization of The Antennapedia Gene Complex of Drosophila melanogaster. Advances in Genetics, 27(C), 309–362. https://doi.org/10.1016/S0065-2660(08)60029-2 Kayalioglu, G. (2009). Projections from the Spinal Cord to the Brain. In The Spinal Cord (pp. 148–167). Elsevier Ltd. https://doi.org/10.1016/B978-0-12-374247-6.50014-6 Ke, Y. D., Suchowerska, A. K., van der Hoven, J., De Silva, D. M., Wu, C. W., van Eersel, J., Ittner, A., & Ittner, L. M. (2012). Lessons from tau-deficient mice. International Journal of Alzheimer’s Disease, 2012, 873270. https://doi.org/10.1155/2012/873270 Kettenmann, H., Kirchhoff, F., & Verkhratsky, A. (2013). Microglia: New Roles for the Synaptic Stripper. Neuron, 77(1), 10–18. https://doi.org/10.1016/j.neuron.2012.12.023 Khaing, Z. Z., Geissler, S. A., Jiang, S., Milman, B. D., Aguilar, S. V., Schmidt, C. E., & Schallert, T. (2012). Assessing forelimb function after unilateral cervical spinal cord injury: novel forelimb tasks predict lesion severity and recovery. Journal of Neurotrauma, 29(3), 488–498. https://doi.org/10.1089/NEU.2011.2106 184 Kida, S. (2012). A Functional Role for CREB as a Positive Regulator of Memory Formation and LTP. Experimental Neurobiology, 21(4), 136–140. https://doi.org/10.5607/en.2012.21.4.136 Kirke Rogers, W., & Todd, M. (2016). Acute spinal cord injury. Best Practice and Research: Clinical Anaesthesiology, 30(1), 27–39. https://doi.org/10.1016/J.BPA.2015.11.003 Kmita, M., & Duboule, D. (2003). Organizing axes in time and space; 25 years of colinear tinkering. Science (New York, N.Y.), 301(5631), 331–333. https://doi.org/10.1126/SCIENCE.1085753 Koebis, M., Urata, S., Shinoda, Y., Okabe, S., Yamasoba, T., Nakao, K., Aiba, A., & Furuichi, T. (2019). LAMP5 in presynaptic inhibitory terminals in the hindbrain and spinal cord: a role in startle response and auditory processing. Molecular Brain, 12(1), 20. https://doi.org/10.1186/s13041-019-0437-4 Koh, M., & Markovich, B. (2022). Neuroanatomy, Spinocerebellar Dorsal Tract. StatPearls. https://www.ncbi.nlm.nih.gov/books/NBK556013/ Košta, V., Kojundžić, S. L., Sapunar, L. C., & Sapunar, D. (2009). The extent of laminectomy affects pain-related behavior in a rat model of neuropathic pain. European Journal of Pain, 13(3), 243–248. https://doi.org/10.1016/j.ejpain.2008.04.012 Kritas, S. K., Saggini, A., Cerulli, G., Caraffa, A., Antinolfi, P., Pantalone, A., Rosati, M., Tei, M., Speziali, A., Saggini, R., & Conti, P. (2014). Corticotropin-Releasing Hormone, Microglia and Mental Disorders. International Journal of Immunopathology and Pharmacology, 27(2), 163–167. https://doi.org/10.1177/039463201402700203 185 Larhammar, M., Patra, K., Blunder, M., Emilsson, L., Peuckert, C., Arvidsson, E., Rönnlund, D., Preobraschenski, J., Birgner, C., Limbach, C., Widengren, J., Blom, H., Jahn, R., Wallén-Mackenzie, Å., & Kullander, K. (2015). SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis. Biological Psychiatry, 77(6), 526–536. https://doi.org/10.1016/j.biopsych.2014.07.017 Lawrence, D. G., & Kuypers, H. G. J. M. (1968). The functional organization of the motor system in the monkey. I. The effects of bilateral pyramidal lesions. Brain : A Journal of Neurology, 91(1), 1–14. https://doi.org/10.1093/BRAIN/91.1.1 Leal, G., Bramham, C. R., & Duarte, C. B. (2017). BDNF and Hippocampal Synaptic Plasticity. Vitamins and Hormones, 104, 153–195. https://doi.org/10.1016/BS.VH.2016.10.004 Lebow, M. A., & Chen, A. (2016). Overshadowed by the amygdala: the bed nucleus of the stria terminalis emerges as key to psychiatric disorders. Molecular Psychiatry, 21(4), 450–463. https://doi.org/10.1038/mp.2016.1 Lee, H., Raiker, S. J., Venkatesh, K., Geary, R., Robak, L. A., Zhang, Y., Yeh, H. H., Shrager, P., & Giger, R. J. (2008). Synaptic Function for the Nogo-66 Receptor NgR1: Regulation of Dendritic Spine Morphology and Activity-Dependent Synaptic Strength. The Journal of Neuroscience, 28(11), 2753. https://doi.org/10.1523/JNEUROSCI.5586-07.2008 Lehnart, S. E., & Wehrens, X. H. T. (2022). The role of junctophilin proteins in cellular function. Physiological Reviews, 102(3), 1211–1261. https://doi.org/10.1152/physrev.00024.2021 Leto, K., Arancillo, M., Becker, E. B. E., Buffo, A., Chiang, C., Ding, B., Dobyns, W. B., Dusart, I., Haldipur, P., Hatten, M. E., Hoshino, M., Joyner, A. L., Kano, M., Kilpatrick, D. L., Koibuchi, N., Marino, S., Martinez, S., Millen, K. J., Millner, T. O., … Hawkes, R. (2016). 186 Consensus Paper: Cerebellar Development. Cerebellum (London, England), 15(6), 789–828. https://doi.org/10.1007/S12311-015-0724-2 Lever, I. J., Bradbury, E. J., Cunningham, J. R., Adelson, D. W., Jones, M. G., McMahon, S. B., Marvizón, J. C. G., & Malcangio, M. (2001). Brain-Derived Neurotrophic Factor Is Released in the Dorsal Horn by Distinctive Patterns of Afferent Fiber Stimulation. The Journal of Neuroscience, 21(12), 4469–4477. https://doi.org/10.1523/JNEUROSCI.21-12-04469.2001 Li, E., Guo, J., Oh, S. J., Luo, Y., Oliveros, H. C., Du, W., Arano, R., Kim, Y., Chen, Y. T., Eitson, J., Lin, D. T., Li, Y., Roberts, T., Schoggins, J. W., & Xu, W. (2021). Anterograde Transneuronal Tracing and Genetic Control with Engineered Yellow Fever Vaccine YFV-17D. Nature Methods, 18(12), 1542. https://doi.org/10.1038/S41592-021-01319-9 Li, Y., Ritzel, R. M., Khan, N., Cao, T., He, J., Lei, Z., Matyas, J. J., Sabirzhanov, B., Liu, S., Li, H., Stoica, B. A., Loane, D. J., Faden, A. I., & Wu, J. (2020). Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice. Theranostics, 10(25), 11376–11403. https://doi.org/10.7150/thno.49199 Li, Z.-W., Li, J.-J., Wang, L., Zhang, J.-P., Wu, J.-J., Mao, X.-Q., Shi, G.-F., Wang, Q., Wang, F., & Zou, J. (2014). Epidermal growth factor receptor inhibitor ameliorates excessive astrogliosis and improves the regeneration microenvironment and functional recovery in adult rats following spinal cord injury. Journal of Neuroinflammation, 11(1), 71. https://doi.org/10.1186/1742-2094-11-71 187 Liedtke, W., Leman, E. E., Fyffe, R. E. W., Raine, C. S., & Schubart, U. K. (2002). Stathmin-Deficient Mice Develop an Age-Dependent Axonopathy of the Central and Peripheral Nervous Systems. The American Journal of Pathology, 160(2), 469–480. https://doi.org/10.1016/S0002-9440(10)64866-3 Lindsay, R. (1988). Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. The Journal of Neuroscience, 8(7), 2394–2405. https://doi.org/10.1523/JNEUROSCI.08-07-02394.1988 Lu, Y., Christian, K., & Lu, B. (2008). BDNF: a key regulator for protein synthesis-dependent LTP and long-term memory? Neurobiology of learning and memory, 89(3), 312-323. https://doi.org/10.1016/j.nlm.2007.08.018 Luo, T., Wagner, E., Grün, F., & Dräger, U. C. (2004). Retinoic acid signaling in the brain marks formation of optic projections, maturation of the dorsal telencephalon, and function of limbic sites. Journal of Comparative Neurology, 470(3), 297–316. https://doi.org/10.1002/CNE.20013 Ma, W., Huang, G., Wang, Z., Wang, L., & Gao, Q. (2023). IRF7: role and regulation in immunity and autoimmunity. Frontiers in Immunology, 14. https://doi.org/10.3389/fimmu.2023.1236923 Manzini, M. C., Ward, M. S., Zhang, Q., Lieberman, M. D., & Mason, C. A. (2006). The Stop Signal Revised: Immature Cerebellar Granule Neurons in the External Germinal Layer Arrest Pontine Mossy Fiber Growth. Journal of Neuroscience, 26(22), 6040–6051. https://doi.org/10.1523/JNEUROSCI.4815-05.2006 188 Mar, F. M., Simões, A. R., Leite, S., Morgado, M. M., Santos, T. E., Rodrigo, I. S., Teixeira, C. A., Misgeld, T., & Sousa, M. M. (2014). Brief Communications CNS Axons Globally Increase Axonal Transport after Peripheral Conditioning. https://doi.org/10.1523/JNEUROSCI.4680-13.2014 Martí-Clua, J. (2022). Times of neuron origin and neurogenetic gradients in mice Purkinje cells and deep cerebellar nuclei neurons during the development of the cerebellum. A review. Tissue and Cell, 78, 101897. https://doi.org/10.1016/J.TICE.2022.101897 Masri, R., & Keller, A. (2012). CHRONIC PAIN FOLLOWING SPINAL CORD INJURY. Advances in Experimental Medicine and Biology, 760, 74. https://doi.org/10.1007/978-1-4614-4090-1_5 Masuda, T., Sankowski, R., Staszewski, O., & Prinz, M. (2020). Microglia Heterogeneity in the Single-Cell Era. Cell Reports, 30(5), 1271–1281. https://doi.org/10.1016/j.celrep.2020.01.010 Mathonnet, G., Fabian, M. R., Svitkin, Y. V., Parsyan, A., Huck, L., Murata, T., Biffo, S., Merrick, W. C., Darzynkiewicz, E., Pillai, R. S., Filipowicz, W., Duchaine, T. F., & Sonenberg, N. (2007). MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science (New York, N.Y.), 317(5845), 1764–1767. https://doi.org/10.1126/SCIENCE.1146067 Matson, K. J. E., Russ, D. E., Kathe, C., Hua, I., Maric, D., Ding, Y., Krynitsky, J., Pursley, R., Sathyamurthy, A., Squair, J. W., Levi, B. P., Courtine, G., & Levine, A. J. (2022). Single cell atlas of spinal cord injury in mice reveals a pro-regenerative signature in 189 spinocerebellar neurons. Nature Communications 2022 13:1, 13(1), 1–16. https://doi.org/10.1038/s41467-022-33184-1 Matsuda, T., Takuma, K., & Baba, A. (1997). Na+-Ca2+ Exchanger: Physiology and Pharmacology. The Japanese Journal of Pharmacology, 74(1), 1–20. https://doi.org/10.1254/jjp.74.1 McCann, M. M., Fisher, K. M., Ahloy-Dallaire, J., & Darian-Smith, C. (2020). Somatosensory corticospinal tract axons sprout within the cervical cord following a dorsal root/dorsal column spinal injury in the rat. The Journal of Comparative Neurology, 528(8), 1293–1306. https://doi.org/10.1002/CNE.24826 McIlwain, K. L., Merriweather, M. Y., Yuva-Paylor, L. A., & Paylor, R. (2001). The use of behavioral test batteries: effects of training history. Physiology & Behavior, 73(5), 705–717. https://doi.org/10.1016/S0031-9384(01)00528-5 McKenna, J. E., & Whishaw, I. Q. (1999). Complete Compensation in Skilled Reaching Success with Associated Impairments in Limb Synergies, after Dorsal Column Lesion in the Rat. Journal of Neuroscience, 19(5), 1885–1894. https://doi.org/10.1523/JNEUROSCI.19-05-01885.1999 Miao, H., Miao, Z., Pan, J., Li, X., & Zhuo, M. (2021). Brain-derived neurotrophic factor produced long-term synaptic enhancement in the anterior cingulate cortex of adult mice. Molecular brain, 14(1), 140. https://doi.org/10.1186/s13041-021-00853-z Millan, M. J. (2002). Descending control of pain. Progress in Neurobiology, 66(6), 355–474. https://doi.org/10.1016/S0301-0082(02)00009-6 190 Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E., & Hayakawa, T. (2000). IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Molecular Therapy : The Journal of the American Society of Gene Therapy, 1(4), 376–382. https://doi.org/10.1006/MTHE.2000.0050 Möller, K. Ä., Berge, O. G., & Hamers, F. P. T. (2008). Using the CatWalk method to assess weight-bearing and pain behaviour in walking rats with ankle joint monoarthritis induced by carrageenan: effects of morphine and rofecoxib. Journal of Neuroscience Methods, 174(1), 1–9. https://doi.org/10.1016/J.JNEUMETH.2008.06.017 Morrow, M. J. (2014). Encyclopedia of the Neurological Sciences (Second Edition). Encyclopedia of the Neurological Sciences (Second Edition), 892–893. http://www.sciencedirect.com:5070/referencework/9780123851581/encyclopedia-of-the-neurological-sciences Nagappan, G., & Lu, B. (2005). Activity-dependent modulation of the BDNF receptor TrkB: mechanisms and implications. Trends in Neurosciences, 28(9), 464-471. https://doi.org/10.1016/j.tins.2005.07.003 Nagarajan, N., & Capecchi, M. R. (2023). Optogenetic stimulation of mouse Hoxb8 microglia in specific regions of the brain induces anxiety, grooming, or both. Molecular Psychiatry 2023, 1–15. https://doi.org/10.1038/s41380-023-02019-w Nami, H., Perone, C. S., & Cohen-Adad, J. (2022). Histology-informed automatic parcellation of white matter tracts in the rat spinal cord. Frontiers in Neuroanatomy, 16. https://doi.org/10.3389/FNANA.2022.960475/FULL 191 Naphade, S. B., Kigerl, K. A., Jakeman, L. B., Kostyk, S. K., Popovich, P. G., & Kuret, J. (2010). Progranulin expression is upregulated after spinal contusion in mice. Acta Neuropathologica, 119(1), 123–133. https://doi.org/10.1007/s00401-009-0616-y Neckel, N. D., Dai, H., & Burns, M. P. (2020). A Novel Multi-Dimensional Analysis of Rodent Gait Reveals the Compensation Strategies Used during Spontaneous Recovery from Spinal Cord and Traumatic Brain Injury. Journal of Neurotrauma, 37(3), 517. https://doi.org/10.1089/NEU.2018.5959 Neijts, R., Amin, S., van Rooijen, C., & Deschamps, J. (2017). Cdx is crucial for the timing mechanism driving colinear Hox activation and defines a trunk segment in the Hox cluster topology. Developmental Biology, 422(2), 146–154. https://doi.org/10.1016/J.YDBIO.2016.12.024 Ness, T. J., Follett, K. A., Piper, J., & Dirks, B. A. (1998). Characterization of neurons in the area of the medullary lateral reticular nucleus responsive to noxious visceral and cutaneous stimuli. Brain Research, 802(1–2), 163–174. https://doi.org/10.1016/S0006-8993(98)00608-8 Nimmerjahn, F., & Ravetch, J. V. (2008). Fcγ receptors as regulators of immune responses. Nature Reviews Immunology, 8(1), 34–47. https://doi.org/10.1038/nri2206 Nolte, C., De Kumar, B., & Krumlauf, R. (2019). Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis. Genesis, 57(7–8), e23306. https://doi.org/10.1002/DVG.23306 Nolte, C., & Krumlauf, R. (2013). Expression of Hox Genes in the Nervous System of Vertebrates. https://www.ncbi.nlm.nih.gov/books/NBK6519/ 192 Noonan, V. (2023). A look at spinal cord injury in Canada: Rick Hansen spinal cord injury registry (RHSCIR) – 2021 SCI data summary. Topics in spinal cord injury rehabilitation, 2023, 29. https://doi.org/10.46292/sci23-00031S Noordermeer, D., Leleu, M., Schorderet, P., Joye, E., Chabaud, F., & Duboule, D. (2014). Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci. ELife, 2014(3). https://doi.org/10.7554/ELIFE.02557 Nowak, D. A., Timmann, D., & Hermsdörfer, J. (2013). Deficits of grasping in cerebellar disorders. Handbook of the Cerebellum and Cerebellar Disorders, 1657–1668. https://doi.org/10.1007/978-94-007-1333-8_73/FIGURES/00735 Odem, M. A., Lacagnina, M. J., Katzen, S. L., Li, J., Spence, E. A., Grace, P. M., & Walters, E. T. (2019). Sham surgeries for central and peripheral neural injuries persistently enhance pain-avoidance behavior as revealed by an operant conflict test. Pain, 160(11), 2440–2455. https://doi.org/10.1097/j.pain.0000000000001642 O’Donnell, M., Chance, R. K., & Bashaw, G. J. (2009). Axon Growth and Guidance: Receptor Regulation and Signal Transduction. Annual Review of Neuroscience, 32(1), 383–412. https://doi.org/10.1146/annurev.neuro.051508.135614 Okabe, N., Wei, X., Abumeri, F., Batac, J., Hovanesyan, M., Dai, W., Azarapetian, S., Campagna, J., Pilati, N., Marasco, A., Alvaro, G., Gunthorpe, M. J., Varghese, J., Cramer, S. C., Mody, I., & Carmichael, S. T. (2025). Parvalbumin interneurons regulate rehabilitation-induced functional recovery after stroke and identify a rehabilitation drug. Nature Communications, 16(1), 2556. https://doi.org/10.1038/s41467-025-57860-0 193 Onifer, S. M., Smith, G. M., & Fouad, K. (2011). Plasticity After Spinal Cord Injury: Relevance to Recovery and Approaches to Facilitate It. Neurotherapeutics, 8(2), 283. https://doi.org/10.1007/S13311-011-0034-4 Oosterveen, T., Niederreither, K., Dollé, P., Chambon, P., Meijlink, F., & Deschamps, J. (2003). Retinoids regulate the anterior expression boundaries of 5’ Hoxb genes in posterior hindbrain. The EMBO Journal, 22(2), 262–269. https://doi.org/10.1093/EMBOJ/CDG029 OSCARSSON, O. (1965). Functional Organization of the Spino- and Cuneocerebellar Tracts. Https://Doi.Org/10.1152/Physrev.1965.45.3.495, 45, 495–522. https://doi.org/10.1152/PHYSREV.1965.45.3.495 O’Shea, T. M., Burda, J. E., & Sofroniew, M. V. (2017). Cell biology of spinal cord injury and repair. The Journal of Clinical Investigation, 127(9), 3259. https://doi.org/10.1172/JCI90608 Ozol, K. O., & Hawkes, R. (1997). Compartmentation of the granular layer of the cerebellum. Histology Histopathology, 12, 171–184. Paxinos, G., & Franklink, K. B. J. (2019). Paxinos and Franklin’s the mouse brain in stereotaxic coordinates. London: Academic Press. Pelisch, N., Rosas Almanza, J., Stehlik, K. E., Aperi, B. V., & Kroner, A. (2020). CCL3 contributes to secondary damage after spinal cord injury. Journal of Neuroinflammation, 17(1), 362. https://doi.org/10.1186/s12974-020-02037-3 194 Perry, V. H., & O’Connor, V. (2010). The Role of Microglia in Synaptic Stripping and Synaptic Degeneration: A Revised Perspective. ASN Neuro, 2(5). https://doi.org/10.1042/AN20100024 Peterson, S. L., Nguyen, H. X., Mendez, O. A., & Anderson, A. J. (2015). Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo. The Journal of Neuroscience, 35(10), 4332–4349. https://doi.org/10.1523/JNEUROSCI.4473-12.2015 Petrova, V., & Eva, R. (2018). The Virtuous Cycle of Axon Growth: Axonal Transport of Growth-Promoting Machinery as an Intrinsic Determinant of Axon Regeneration. Developmental Neurobiology, 78(10), 898–925. https://doi.org/10.1002/DNEU.22608 Philippidou, P., & Dasen, J. S. (2013). Hox Genes: Choreographers in Neural Development, Architects of Circuit Organization. Neuron, 80(1), 12–34. https://doi.org/10.1016/J.NEURON.2013.09.020 Pop, I. V., Espinosa, F., Blevins, C. J., Okafor, P. C., Ogujiofor, O. W., Goyal, M., Mona, B., Landy, M. A., Dean, K. M., Gurumurthy, C. B., & Lai, H. C. (2022). Structure of Long-Range Direct and Indirect Spinocerebellar Pathways as Well as Local Spinal Circuits Mediating Proprioception. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 42(4), 581–600. https://doi.org/10.1523/JNEUROSCI.2157-20.2021 Porro, C., Pennella, A., Panaro, M. A., & Trotta, T. (2021). Functional Role of Non-Muscle Myosin II in Microglia: An Updated Review. International Journal of Molecular Sciences, 22(13), 6687. https://doi.org/10.3390/ijms22136687 195 Presumey, J., Bialas, A. R., & Carroll, M. C. (2017). Complement System in Neural Synapse Elimination in Development and Disease (pp. 53–79). https://doi.org/10.1016/bs.ai.2017.06.004 Purves, D., Augustine, G. J., Fitzpatrick, D., Katz, L. C., LaMantia, A.-S., McNamara, J. O., & Williams, S. M. (2001). Circuits within the Cerebellum. https://www.ncbi.nlm.nih.gov/books/NBK10865/ Qiu, L., Zhang, B., & Gao, Z. (2022). Lighting Up Neural Circuits by Viral Tracing. Neuroscience Bulletin, 38(11), 1383–1396. https://doi.org/10.1007/S12264-022-00860-7/FIGURES/4 Raineteau, O., & Schwab, M. E. (2001). Plasticity of motor systems after incomplete spinal cord injury. Nature Reviews Neuroscience 2001 2:4, 2(4), 263–273. https://doi.org/10.1038/35067570 Ramachandran, R., Reifler, A., Parent, J. M., & Goldman, D. (2010). Conditional gene expression and lineage tracing of tuba1a expressing cells during zebrafish development and retina regeneration. Journal of Comparative Neurology, 518(20), 4196–4212. https://doi.org/10.1002/cne.22448 Ramer, L. M., McPhail, L. T., Borisoff, J. F., Soril, L. J. J., Kaan, T. K. Y., Lee, J. H. T., Saunders, J. W. T., Hwi, L. P. R., & Ramer, M. S. (2007). Endogenous TrkB Ligands Suppress Functional Mechanosensory Plasticity in the Deafferented Spinal Cord. The Journal of Neuroscience, 27(21), 5812–5822. https://doi.org/10.1523/JNEUROSCI.0491-07.2007 196 Ramer, M. S. (2003). Spontaneous Functional Viscerosensory Regeneration into the Adult Brainstem. The Journal of Neuroscience, 23(30), 9770–9775. https://doi.org/10.1523/JNEUROSCI.23-30-09770.2003 Ramer, M. S., Bradbury, E. J., & McMahon, S. B. (2001). Nerve growth factor induces P2X3 expression in sensory neurons. Journal of Neurochemistry, 77(3), 864–875. https://doi.org/10.1046/J.1471-4159.2001.00288.X Ramer, M. S., Bradbury, E. J., Michael, G. J., Lever, I. J., & McMahon, S. B. (2003). Glial cell line-derived neurotrophic factor increases calcitonin gene-related peptide immunoreactivity in sensory and motoneurons in vivo. The European Journal of Neuroscience, 18(10), 2713–2721. https://doi.org/10.1111/J.1460-9568.2003.03012.X Rauti, R., Cellot, G., D’Andrea, P., Colliva, A., Scaini, D., Tongiorgi, E., & Ballerini, L. (2020). BDNF impact on synaptic dynamics: extra or intracellular long-term release differently regulates cultured hippocampal synapses. Molecular Brain 2020 13:1, 13(1), 1–16. https://doi.org/10.1186/S13041-020-00582-9 Reeber, S. L., Gebre, S. A., Filatova, N., & Sillitoe, R. V. (2011). Revealing neural circuit topography in multi-color. Journal of Visualized Experiments : JoVE, 57. https://doi.org/10.3791/3371 Reis, D. J., & Ross, R. A. (1973). Dynamic changes in brain dopamine-β-hydroxylase activity during anterograde and retrograde reactions to injury of central noradrenergic axons. Brain Research, 57(2), 307–326. https://doi.org/10.1016/0006-8993(73)90139-X 197 Riche, D., De Pommery, J., & Menetrey, D. (1990). Neuropeptides and catecholamines in efferent projections of the nuclei of the solitary tract in the rat. Journal of Comparative Neurology, 293(3), 399–424. https://doi.org/10.1002/cne.902930306 Rizo, J. (2022). Molecular Mechanisms Underlying Neurotransmitter Release. Https://Doi.Org/10.1146/Annurev-Biophys-111821-104732, 51, 377–408. https://doi.org/10.1146/ANNUREV-BIOPHYS-111821-104732 Rossignol, S., Barrière, G., Frigon, A., Barthélemy, D., Bouyer, L., Provencher, J., Leblond, H., & Bernard, G. (2008). Plasticity of locomotor sensorimotor interactions after peripheral and/or spinal lesions. Brain Research Reviews, 57(1), 228–240. https://doi.org/10.1016/J.BRAINRESREV.2007.06.019 Ruberte, E., Friederich, V., Chambon, P., & Morriss-Kay, G. (1993). Retinoic acid receptors and cellular retinoid binding proteins III. Their differential transcript distribution during mouse nervous system development. Development, 118(1), 267–282. https://doi.org/10.1242/dev.118.1.267 Saab, C. Y., & Willis, W. D. (2003). The cerebellum: organization, functions and its role in nociception. Brain Research Reviews, 42(1), 85–95. https://doi.org/10.1016/S0165-0173(03)00151-6 Sauer, B. (1987). Functional expression of the cre-lox site-specific recombination system in the yeast saccharomyces cerevisiae . Molecular and cellular biology, 7(6), 2087-2096. https://doi.org/10.1128/mcb.7.6.2087-2096.1987 Sauer, B. (1998). Inducible gene targeting in mice using the cre/lox system. Methods, 14(4), 381-392. https://doi.org/10.1006/meth.1998.0593 198 Sakuma, T., Kamoda, H., Miyagi, M., Ishikawa, T., Arai, G., Eguchi, Y., Suzuki, M., Oikawa, Y., Sakuma, Y., Kubota, G., Inage, K., Saino, T., Orita, S., Yamauchi, K., Inoue, G., Takahashi, K., & Ohtori, S. (2013). Comparison of catwalk analysis and von frey testing for pain assessment in a rat model of nerve crush plus inflammation. Spine, 38(15). https://doi.org/10.1097/BRS.0B013E318297BFB6 Saleeba, C., Dempsey, B., Le, S., Goodchild, A., & McMullan, S. (2019). A student’s guide to neural circuit tracing. In Frontiers in Neuroscience (Vol. 13, Issue AUG). Frontiers Media S.A. https://doi.org/10.3389/fnins.2019.00897 Salvany, S., Casanovas, A., Piedrafita, L., Tarabal, O., Hernández, S., Calderó, J., & Esquerda, J. E. (2021). Microglial recruitment and mechanisms involved in the disruption of afferent synaptic terminals on spinal cord motor neurons after acute peripheral nerve injury. Glia, 69(5), 1216–1240. https://doi.org/10.1002/glia.23959 Sanders, M. C., Goldstein, A. L., & Wang, Y. L. (1992). Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells. Proceedings of the National Academy of Sciences of the United States of America, 89(10), 4678–4682. https://doi.org/10.1073/pnas.89.10.4678 Sasaki, M., Radtke, C., Tan, A. M., Zhao, P., Hamada, H., Houkin, K., Honmou, O., & Kocsis, J. D. (2009). BDNF-hypersecreting human mesenchymal stem cells promote functional recovery, axonal sprouting, and protection of corticospinal neurons after spinal cord injury. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 29(47), 14932–14941. https://doi.org/10.1523/JNEUROSCI.2769-09.2009 199 Schafer, D. P., Lehrman, E. K., Kautzman, A. G., Koyama, R., Mardinly, A. R., Yamasaki, R., Ransohoff, R. M., Greenberg, M. E., Barres, B. A., & Stevens, B. (2012). Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron, 74(4), 691–705. https://doi.org/10.1016/j.neuron.2012.03.026 Schörnig, M., Heumann, R., & Rohrer, H. (1993). Synthesis of nerve growth factor mRNA in cultures of developing mouse whisker pad, a peripheral target tissue of sensory trigeminal neurons. The Journal of Cell Biology, 120(6), 1471–1479. https://doi.org/10.1083/jcb.120.6.1471 Schwab, J. M., Frei, E., Klusman, I., Schnell, L., Schwab, M. E., & Schluesener, H. J. (2001). AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats. Journal of Neuroimmunology, 119(2), 214–222. https://doi.org/10.1016/S0165-5728(01)00375-7 Schwartzman, R. J. (1978). A behavioral analysis of complete unilateral section of the pyramidal tract at the medullary level in Macaca mulatta. Annals of Neurology, 4(3), 234–244. https://doi.org/10.1002/ANA.410040308 Scott, A. L. M., Borisoff, J. F., & Ramer, M. S. (2005). Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. European Journal of Neuroscience, 21(1), 81–92. https://doi.org/10.1111/j.1460-9568.2004.03838.x Scott, A. L. M., & Ramer, M. S. (2010a). Differential regulation of dendritic plasticity by neurotrophins following deafferentation of the adult spinal cord is independent of p75NTR. Brain Research, 1323, 48–58. https://doi.org/10.1016/j.brainres.2010.02.004 200 Scott, A. L. M., & Ramer, M. S. (2010b). Schwann cell p75NTR prevents spontaneous sensory reinnervation of the adult spinal cord. Brain, 133(2), 421–432. https://doi.org/10.1093/brain/awp316 Sengul, G., & Watson, C. (2015). Ascending and Descending Pathways in the Spinal Cord. In The Rat Nervous System (pp. 115–130). Elsevier. https://doi.org/10.1016/B978-0-12-374245-2.00008-5 Sheng, M., & Kim, E. (2011). The postsynaptic organization of synapses. Cold Spring Harbor Perspectives in Biology, 3(12). https://doi.org/10.1101/CSHPERSPECT.A005678 Shinoda, Y., Sugihara, I., Wu, H. S., & Sugiuchi, Y. (2000). The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex. Progress in Brain Research, 124, 173–186. https://doi.org/10.1016/s0079-6123(00)24015-6 Siddiqui, A. M., Khazaei, M., & Fehlings, M. G. (2015). Translating mechanisms of neuroprotection, regeneration, and repair to treatment of spinal cord injury. Progress in Brain Research, 218, 15–54. https://doi.org/10.1016/BS.PBR.2014.12.007 Sofroniew, M. V. (2018). Dissecting spinal cord regeneration. Nature, 557(7705), 343–350. https://doi.org/10.1038/S41586-018-0068-4 Song, A. J., & Palmiter, R. D. (2018). Detecting and Avoiding Problems When Using the Cre/lox System. Trends in Genetics : TIG, 34(5), 333. https://doi.org/10.1016/J.TIG.2017.12.008 201 Song, Z., Han, A., & Hu, B. (2024). Thymosin β4 promotes zebrafish Mauthner axon regeneration by facilitating actin polymerization through binding to G-actin. BMC Biology, 22(1), 244. https://doi.org/10.1186/s12915-024-02045-2 Soril, L. J. J., Ramer, L. M., McPhail, L. T., Kaan, T. K. Y., & Ramer, M. S. (2008). Spinal brain-derived neurotrophic factor governs neuroplasticity and recovery from cold-hypersensitivity following dorsal rhizotomy. Pain, 138(1), 98–110. https://doi.org/10.1016/j.pain.2007.11.014 Soshnikova, N., & Duboule, D. (2009). Epigenetic temporal control of mouse Hox genes in vivo. Science (New York, N.Y.), 324(5932), 1321–1323. https://doi.org/10.1126/SCIENCE.1171468 Steeves, J. D., Kramer, J. K., Fawcett, J. W., Cragg, J., Lammertse, D. P., Blight, A. R., Marino, R. J., Ditunno, J. F., Coleman, W. P., Geisler, F. H., Guest, J., Jones, L., Burns, S., Schubert, M., Van Hedel, H. J. A., & Curt, A. (2010). Extent of spontaneous motor recovery after traumatic cervical sensorimotor complete spinal cord injury. Spinal Cord 2011 49:2, 49(2), 257–265. https://doi.org/10.1038/sc.2010.99 Stephan, A. H., Barres, B. A., & Stevens, B. (2012). The Complement System: An Unexpected Role in Synaptic Pruning During Development and Disease. Annual Review of Neuroscience, 35(1), 369–389. https://doi.org/10.1146/annurev-neuro-061010-113810 Steuer, I., & Guertin, P. A. (2009). Spinal Cord Injury Research in Mice: 2008 Review. 9, 490–498. https://doi.org/10.1100/tsw.2009.63 Steward, O. (2000). Somatic Sensation II. Functional Neuroscience, 175–193. https://doi.org/10.1007/978-1-4612-1198-3_10 202 Sun, W., Cornwell, A., Li, J., Peng, S., Joana Osorio, M., Aalling, N., Wang, S., Benraiss, A., Lou, N., Goldman, S. A., & Nedergaard, M. (2017). SOX9 Is an Astrocyte-Specific Nuclear Marker in the Adult Brain Outside the Neurogenic Regions. The Journal of Neuroscience, 37(17), 4493. https://doi.org/10.1523/JNEUROSCI.3199-16.2017 Szabo, N. E., Da Silva, R. V., Sotocinal, S. G., Zeilhofer, H. U., Mogil, J. S., & Kania, A. (2015). Hoxb8 intersection defines a role for Lmx1b in excitatory dorsal horn neuron development, spinofugal connectivity, and nociception. Journal of Neuroscience, 35(13), 5233–5246. https://doi.org/10.1523/JNEUROSCI.4690-14.2015 Takeichi, T., Takarada-Iemata, M., Hashida, K., Sudo, H., Okuda, T., Kokame, K., Hatano, T., Takanashi, M., Funabe, S., Hattori, N., Kitamura, O., Kitao, Y., & Hori, O. (2011). The effect of Ndrg2 expression on astroglial activation. Neurochemistry International, 59(1), 21–27. https://doi.org/10.1016/j.neuint.2011.03.019 Tetzlaff, W., Alexander, S., Miller, F., & Bisby, M. (1991). Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43. The journal of neuroscience, 11(8), 2528-2544. https://doi.org/10.1523/JNEUROSCI.11-08-02528.1991 Thermann, R., & Hentze, M. W. (2007). Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 2007 447:7146, 447(7146), 875–878. https://doi.org/10.1038/nature05878 Thoenen, H., Bandtlow, C., Heumann, R., Lindholm, D., Meyer, M., & Rohrer, H. (1988). Nerve growth factor: Cellular localization and regulation of synthesis. Cellular and Molecular Neurobiology, 8(1), 35–40. https://doi.org/10.1007/BF00712909 203 Timofeeva, Y., & Volynski, K. E. (2015). Calmodulin as a major calcium buffer shaping vesicular release and short-term synaptic plasticity: facilitation through buffer dislocation. Frontiers in Cellular Neuroscience, 9. https://doi.org/10.3389/fncel.2015.00239 Tiveron, M.-C., Beurrier, C., Céni, C., Andriambao, N., Combes, A., Koehl, M., Maurice, N., Gatti, E., Abrous, D. N., Kerkerian-Le Goff, L., Pierre, P., & Cremer, H. (2016). LAMP5 Fine-Tunes GABAergic Synaptic Transmission in Defined Circuits of the Mouse Brain. PloS One, 11(6), e0157052. https://doi.org/10.1371/journal.pone.0157052 Tränkner, D., Boulet, A., Peden, E., Focht, R., Van Deren, D., & Capecchi, M. (2019). A Microglia Sublineage Protects from Sex-Linked Anxiety Symptoms and Obsessive Compulsion. Cell Reports, 29(4), 791-799.e3. https://doi.org/10.1016/J.CELREP.2019.09.045 Trifilo, M. J., Bergmann, C. C., Kuziel, W. A., & Lane, T. E. (2003). CC Chemokine Ligand 3 (CCL3) Regulates CD8 + -T-Cell Effector Function and Migration following Viral Infection. Journal of Virology, 77(7), 4004–4014. https://doi.org/10.1128/JVI.77.7.4004-4014.2003 Valarché, I., De Graaff, W., & Deschamps, J. (2003). A 3’ remote control region is a candidate to modulate Hoxb-8 expression boundaries. International Journal of Developmental Biology, 41(5), 705–714. https://doi.org/10.1387/IJDB.9415490 van Asperen, J. V., Kotaich, F., Caillol, D., & Bomont, P. (2024). Neurofilaments: Novel findings and future challenges. Current Opinion in Cell Biology, 87, 102326. https://doi.org/10.1016/j.ceb.2024.102326 204 Van Deren, D. A., De, S., Xu, B., Eschenbacher, K. M., Zhang, S., & Capecchi, M. R. (2022). Defining the Hoxb8 cell lineage during murine definitive hematopoiesis. Development (Cambridge, England), 149(8). https://doi.org/10.1242/DEV.200200 Vavrek, R., Girgis, J., Teztlaff, W., Hibert, G., & Fouad, K. (2006). BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats. Brain, 129(6), 1534–1545. https://doi.org/10.1093/brain/awl087 Veldman, M. B., Bemben, M. A., & Goldman, D. (2010). Tuba1a gene expression is regulated by KLF6/7 and is necessary for CNS development and regeneration in zebrafish. Molecular and Cellular Neuroscience, 43(4), 370–383. https://doi.org/10.1016/j.mcn.2010.01.004 Vijayan, N., Thanikachalam, P. V., & Patel, S. (2025). Decoding of the Role of TREM2 in Neuropathic Pain: Molecular Pathway and Neuroinflammatory Mechanism. Drug Development Research, 86(4). https://doi.org/10.1002/ddr.70111 Visavadiya, N. P., & Springer, J. E. (2016). Altered Cerebellar Circuitry following Thoracic Spinal Cord Injury in Adult Rats. Neural Plasticity, 2016. https://doi.org/10.1155/2016/8181393 Voogd, J., & Glickstein, M. (1998). The anatomy of the cerebellum. Trends in Neurosciences, 21(9), 370–375. https://doi.org/10.1016/S0166-2236(98)01318-6 Wan, H., He, M., Cheng, C., Yang, K., Wu, H., Cong, P., Huang, X., Zhang, Q., Shi, Y., Hu, J., Tian, L., & Xiong, L. (2024). Clec7a Worsens Long-Term Outcomes after Ischemic Stroke by Aggravating Microglia-Mediated Synapse Elimination. Advanced Science. https://doi.org/10.1002/advs.202403064 205 Wang, L., Hu, B., Wong, W. M., Lu, P., Wu, W., & Xu, X. (2009). Glial and axonal responses in areas of Wallerian degeneration of the corticospinal and dorsal ascending tracts after spinal cord dorsal funiculotomy. Neuropathology, 29(3), 230–241. https://doi.org/10.1111/j.1440-1789.2008.00969.x Wang, Q., Ding, S.-L., Li, Y., Royall, J., Feng, D., Lesnar, P., Graddis, N., Naeemi, M., Facer, B., Ho, A., Dolbeare, T., Blanchard, B., Dee, N., Wakeman, W., Hirokawa, K. E., Szafer, A., Sunkin, S. M., Oh, S. W., Bernard, A., … Ng, L. (2020). The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas. Cell, 181(4), 936-953.e20. https://doi.org/10.1016/j.cell.2020.04.007 Wang, Q., Li, T., Fang, C., & Zhang, B. (2021). Bioinformatics analysis of the wheel treadmill test on motor function recovery after spinal cord injury. Ibrain, 7(4), 265–277. https://doi.org/10.1002/ibra.12006 Wang, T., Molliver, D. C., Jing, X., Schwartz, E. S., Yang, F. C., Samad, O. A., Ma, Q., & Davis, B. M. (2011). Phenotypic switching of nonpeptidergic cutaneous sensory neurons following peripheral nerve injury. PloS One, 6(12). https://doi.org/10.1371/JOURNAL.PONE.0028908 Wang, X., Li, W., Zhang, J., Li, J., Zhang, X., Wang, M., Wei, Z., & Feng, S. (2023). Discovery of therapeutic targets for spinal cord injury based on molecular mechanisms of axon regeneration after conditioning lesion. Journal of Translational Medicine, 21(1), 511. https://doi.org/10.1186/s12967-023-04375-1 Watson, C., Paxinos, G., & Kayalioglu, G. (2009). The Spinal Cord. The Spinal Cord. https://doi.org/10.1016/C2009-0-01677-8 206 Wei, Z., Sun, M., Liu, X., Zhang, J., & Jin, Y. (2014). Rufy3, a protein specifically expressed in neurons, interacts with actin-bundling protein Fascin to control the growth of axons. Journal of Neurochemistry, 130(5), 678–692. https://doi.org/10.1111/jnc.12740 Weidner, N., Ner, A., Salimi, N., & Tuszynski, M. H. (2001). Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury. Proceedings of the National Academy of Sciences of the United States of America, 98(6), 3513. https://doi.org/10.1073/PNAS.051626798 Weishaupt, N., Blesch, A., & Fouad, K. (2012). BDNF: The career of a multifaceted neurotrophin in spinal cord injury. Experimental Neurology, 238(2), 254–264. https://doi.org/10.1016/j.expneurol.2012.09.001 Wernig, A., & Müller, S. (1992). Laufband locomotion with body weight support improved walking in persons with severe spinal cord injuries. Paraplegia, 30(4), 229–238. https://doi.org/10.1038/SC.1992.61 Whishaw, I. Q., Pellis, S. M., Gorny, B., Kolb, B., & Tetzlaff, W. (1993). Proximal and distal impairments in rat forelimb use in reaching follow unilateral pyramidal tract lesions. Behavioural Brain Research, 56(1), 59–76. https://doi.org/10.1016/0166-4328(93)90022-I Wilde, J. J., Siegenthaler, J. A., Dent, S. Y. R., & Niswander, L. A. (2017). Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 37(10), 2565–2579. https://doi.org/10.1523/JNEUROSCI.2121-16.2017 207 Willment, J. A., & Brown, G. D. (2018). CLEC7A. In Encyclopedia of Signaling Molecules (pp. 1154–1161). Springer International Publishing. https://doi.org/10.1007/978-3-319-67199-4_584 Wilmerding, A., Rinaldi, L., Caruso, N., Re, L. Lo, Bonzom, E., Saurin, A. J., Graba, Y., & Delfini, M. C. (2021). HoxB genes regulate neuronal delamination in the trunk neural tube by controlling the expression of Lzts1. Development (Cambridge, England), 148(4). https://doi.org/10.1242/DEV.195404 Witschi, R., Johansson, T., Morscher, G., Scheurer, L., Deschamps, J., & Zeilhofer, H. U. (2010). Hoxb8-Cre Mice: a Tool for Brain-Sparing Conditional Gene Deletion. Genesis (New York, N.Y. : 2000), 48(10), 596. https://doi.org/10.1002/DVG.20656 Woods, A., Guthrie, K., Kurlawalla, M., & Gall, C. (1998). Deafferentation-induced increases in hippocampal insulin-like growth factor-1 messenger RNA expression are severely attenuated in middle aged and aged rats. Neuroscience, 83(3), 663–668. https://doi.org/10.1016/S0306-4522(97)00539-3 Wu, A., Yang, G., Liu, G., & Zhang, J. (2025). SGK1 upregulation in GFAP+ neurons in the frontal association cortex protects against neuronal apoptosis after spinal cord injury. Cell Death & Disease, 16(1), 237. https://doi.org/10.1038/s41419-025-07542-y Wu, C. W. H., & Kaas, J. H. (2002). The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord. Somatosensory & Motor Research, 19(2), 153–163. https://doi.org/10.1080/08990220220133261 208 Wu, S., Fan, J., Tang, F., Chen, L., Zhang, X., Xiao, D., & Li, X. (2023). The role of RIM in neurotransmitter release: promotion of synaptic vesicle docking, priming, and fusion. Frontiers in Neuroscience, 17. https://doi.org/10.3389/fnins.2023.1123561 Xia, Z., & Storm, D. R. (2005). The role of calmodulin as a signal integrator for synaptic plasticity. Nature Reviews Neuroscience, 6(4), 267–276. https://doi.org/10.1038/nrn1647 Xu, L., Wang, J., Ding, Y., Wang, L., & Zhu, Y.-J. (2022). Current Knowledge of Microglia in Traumatic Spinal Cord Injury. Frontiers in Neurology, 12. https://doi.org/10.3389/fneur.2021.796704 Xu, Q., & Grant, G. (1988). Collateral projections of neurons from the lower part of the spinal cord to anterior and posterior cerebellar termination areas. A retrograde fluorescent double labeling study in the cat. Experimental Brain Research, 72(3), 562–576. https://doi.org/10.1007/BF00250601 Xu, Q., & Grant, G. (2005). Course of spinocerebellar axons in the ventral and lateral funiculi of the spinal cord with projections to the posterior cerebellar termination area: an experimental anatomical study in the cat, using a retrograde tracing technique. Experimental Brain Research, 162(2), 250-256. https://doi.org/10.1007/s00221-004-2132-6 Xu, X., Holmes, T. C., Luo, M. H., Beier, K. T., Horwitz, G. D., Zhao, F., Zeng, W., Hui, M., Semler, B. L., & Sandri-Goldin, R. M. (2020). Viral vectors for neural circuit mapping and recent advances in trans-synaptic anterograde tracers. Neuron, 107(6), 1029. https://doi.org/10.1016/J.NEURON.2020.07.010 209 Xu, Y., Tian, N. X., Bai, Q. Y., Chen, Q., Sun, X. H., & Wang, Y. (2019). Gait Assessment of Pain and Analgesics: Comparison of the DigiGaitTM and CatWalkTM Gait Imaging Systems. Neuroscience Bulletin, 35(3), 401. https://doi.org/10.1007/S12264-018-00331-Y Yamamoto, M., Dräger, U. C., Ong, D. E., & McCaffery, P. (1998). Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation. European Journal of Biochemistry, 257(2), 344–350. https://doi.org/10.1046/j.1432-1327.1998.2570344.x Yekta, S., Shih, I. H., & Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science (New York, N.Y.), 304(5670), 594–596. https://doi.org/10.1126/SCIENCE.1097434 Yousefpour, N., Tansley, S. N., Locke, S., Sharif, B., Parisien, M., Bourojeni, F. B., Deamond, H., Mathur, V., Arana, N. R.-K., Austin, J. S., Bourassa, V., Wang, C., Cabana, V. C., Wong, C., Lister, K. C., Rodrigues, R., St-Louis, M., Paquet, M.-E., Carroll, M. C., … Ribeiro-da-Silva, A. (2025). Targeting C1q prevents microglia-mediated synaptic removal in neuropathic pain. Nature Communications, 16(1), 4590. https://doi.org/10.1038/s41467-025-59849-1 Yuan, A., Sershen, H., Veeranna, Basavarajappa, B. S., Kumar, A., Hashim, A., Berg, M., Lee, J.-H., Sato, Y., Rao, M. V, Mohan, P. S., Dyakin, V., Julien, J.-P., Lee, V. M.-Y., & Nixon, R. A. (2015). Neurofilament subunits are integral components of synapses and modulate neurotransmission and behavior in vivo. Molecular Psychiatry, 20(8), 986–994. https://doi.org/10.1038/mp.2015.45 210 Yuan, H., Xu, S., Wang, Y., Xu, H., Wang, C., Zhu, Q., Yang, R.-K., Chen, X., Yang, P.-C., & Shi, X. (2010). Corticotrophin-releasing hormone (CRH) facilitates axon outgrowth. Spinal Cord, 48(12), 850–856. https://doi.org/10.1038/sc.2010.47 Zamanian, J. L., Xu, L., Foo, L. C., Nouri, N., Zhou, L., Giffard, R. G., & Barres, B. A. (2012). Genomic Analysis of Reactive Astrogliosis. The Journal of Neuroscience, 32(18), 6391–6410. https://doi.org/10.1523/JNEUROSCI.6221-11.2012 Zhang, M. D., Su, J., Adori, C., Cinquina, V., Malenczyk, K., & Girach, F. (2018). Ca2+−binding protein NECAB2 facilitates inflammatory pain hypersensitivity. Clinical Investigations, 128, 3757–3768. Zhang, Q., Shi, R., Hao, M., Feng, D., Wu, R., & Shi, M. (2023). NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats. Frontiers in Molecular Neuroscience, 16. https://doi.org/10.3389/fnmol.2023.1149683 Zhang, W., Yang, B., Weng, H., Liu, T., Shi, L., Yu, P., So, K. F., Qu, Y., & Zhou, L. (2019). Wheel running improves motor function and spinal cord plasticity in mice with genetic absence of the corticospinal tract. Frontiers in Cellular Neuroscience, 13. https://doi.org/10.3389/FNCEL.2019.00106/FULL Zhao, T., Di, J., Kang, Y., Zhang, H., Yao, S., Liu, B., & Rong, L. (2025). <scp>TREM2</scp> Impedes Recovery After Spinal Cord Injury by Regulating Microglial Lysosomal Membrane Permeabilization-Mediated Autophagy. Cell Proliferation. https://doi.org/10.1111/cpr.70047 211 Zheng, B., & Tuszynski, M. H. (2023). Regulation of axonal regeneration after mammalian spinal cord injury. Nature Reviews Molecular Cell Biology 2023 24:6, 24(6), 396–413. https://doi.org/10.1038/s41580-022-00562-y Zheng, G., Younsi, A., Scherer, M., Riemann, L., Walter, J., Skutella, T., Unterberg, A., & Zweckberger, K. (2021). The CatWalk XT® Gait Analysis Is Closely Correlated with Tissue Damage after Cervical Spinal Cord Injury in Rats. Applied Sciences 2021, Vol. 11, Page 4097, 11(9), 4097. https://doi.org/10.3390/APP11094097 Zhou, L., Baumgartner, B. J., Hill-Felberg, S. J., McGowen, L. R., & Shine, H. D. (2003). Neurotrophin-3 Expressed In Situ Induces Axonal Plasticity in the Adult Injured Spinal Cord. The Journal of Neuroscience, 23(4), 1424–1431. https://doi.org/10.1523/JNEUROSCI.23-04-01424.2003 Zörner, B., Bachmann, L. C., Filli, L., Kapitza, S., Gullo, M., Bolliger, M., Starkey, M. L., Röthlisberger, M., Gonzenbach, R. R., & Schwab, M. E. (2014). Chasing central nervous system plasticity: the brainstem’s contribution to locomotor recovery in rats with spinal cord injury. Brain, 137(6), 1716–1732. https://doi.org/10.1093/BRAIN/AWU078 Zou, Y. (2021). Targeting axon guidance cues for neural circuit repair after spinal cord injury. Journal of Cerebral Blood Flow & Metabolism, 41(2), 197. https://doi.org/10.1177/0271678X20961852
Thesis/Dissertation
eng
Zoology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ramer, Matthew
Hoxb8 lineage tracing to map spinofugal projections related to motor recovery following spinal cord injury
Text