Anatomical substrates of central nervous system plasticity induced by spinal cord reflex conditioning and sensorimotor cortex stimulation

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During development and throughout adult life, inputs from the brain combine with inputs from the periphery to induce activity-dependent plasticity in the spinal cord. This activity-dependent plasticity shapes spinal circuitry and helps in acquisition and maintenance of normal motor function. The neural pathways and processes responsible for induction and maintenance of plasticity in the spinal cord remain unclear. Understanding the mechanisms responsible for activity-dependent plasticity in the spinal cord is essential for developing therapies for spinal cord injury. Operant conditioning of the H-reflex, the electrical analog of the spinal stretch reflex (SSR), provides a simple experimental model to study activity-dependent plasticity in the spinal cord. In response to an operant conditioning protocol, monkeys, humans, rats, and mice can gradually increase or decrease the SSR or the H-reflex. Operant-conditioning induces plasticity at multiple sites in the CNS including the spinal cord. Furthermore, conditioning appears to be dependent only on descending influence originating from the contralateral sensorimotor cortex via the corticospinal tract (CST). In addition, a recent study indicated that, like operant conditioning, direct electrical stimulation of the SMC also modulates H-reflex by inducing plasticity in the cortex and the spinal cord. The anatomical basis of spinal cord plasticity responsible for operant conditioning and SMC stimulation-induced modulation in the H-reflex remains to be elucidated. The central goal of this study was to determine if the change in the soleus H-reflex subsequent to operant conditioning and SMC stimulation is associated with changes in the GABAergic terminals on soleus motoneurons. In accord with the central goal, the main hypotheses were that: (1) operant down-conditioning of the H-reflex is associated with an increase in the GABAergic terminals on soleus motoneurons; (2) operant up-conditioning of the H-reflex is associated with a decrease in the GABAergic terminals on soleus motoneurons; and (3) long-term SMC stimulation-induced increase in the H-reflex is associated with a decrease in the GABAergic terminals on soleus motoneurons. These hypotheses were tested by identifying GABAergic terminals based on their immunoreactivity to glutamic acid decarboxylase 67 (GAD67), the main isoform of the enzyme present in terminals on motoneurons. With regard to the first hypothesis, results from operant conditioning studies indicate that successful down-conditioning was associated with an increase in the number, size, and GAD density of GABAergic terminals on motoneurons. These changes probably reflect the CST influence responsible for the decrease in the H-reflex. With regard to the second hypothesis, successful up-conditioning did not change the GABAergic terminal number, although there was an increase in the terminal diameter. Successful up-conditioning did not differ from unsuccessful up-conditioning in any of the measures. Therefore, the terminal changes could reflect non-specific effects of up-conditioning. Together, the results from these two studies support evidence from previous studies indicating that up- and down-conditioning are not mirror images of each other but rather have different mechanisms. With regard to the third hypothesis, results indicate that long-term SMC stimulation-induced increase in the H-reflex is associated with an increase in the GABAergic terminals on the soleus motoneurons. In addition, there was also a decrease in the GABA-B receptor expression on motoneurons. These changes probably reflect compensatory plasticity in response to the primary plasticity responsible for the SMC stimulation-induced increase in the H-reflex. Overall, these results provide valuable insights about the anatomical substrates of plasticity responsible for operant conditioning and SMC stimulation-induced change in the H-reflex. Operant conditioning and SMC stimulation-induced modulation in the H-reflex helps in improvement of locomotor function after certain types of spinal cord injury and these results indicate that activity-dependent plasticity in the spinal GABAergic neural networks plays an important role in mediating this functional recovery.
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Anatomical Substrates of Central Nervous System Plasticity Induced by Spinal Cord Reflex Conditioning and Sensorimotor Cortex Stimulation Shreejith D. Pillai A Dissertation Submitted to the University of Albany, State University of New York in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy School of Public Health Department of Biomedical Sciences 2009 UMI Number: 3355168 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ______________________________________________________________ UMI Microform 3355168 Copyright 2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. _______________________________________________________________ ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106-1346 ACKNOWLEDGEMENTS I would like to acknowledge my mentors, Drs. Xiang Yang Chen and Jonathan Wolpaw for their guidance and encouragement. Thanks especially to Dr. Xiang Yang Chen. I thoroughly appreciate your time and efforts in teaching and guiding me. Without your relentless support, I would not have been able to make it along this journey and successfully complete this project. Thanks to Dr. Jonathan Wolpaw, for your scientific insights, guidance and support. I am indeed grateful to you for having me as part of your lab. I would also like to express my gratitude to Dr. Yu Wang, whose guidance and support helped me immensely in completion of my thesis. I would also like to thank Dr. Jonathan Carp for guiding and helping me during the writing of my dissertation. Thanks to Lu for her help with surgical work and teaching me how to use a vibratome, Dr. Ann Tennissen, for commenting on my manuscripts; Yi Chen, for help with figures and his valuable suggestions; Rongliang Liu, for assistance in animal care. I would also like to express my gratitude to the many wonderful people I have had the opportunity to work with, for making this research possible and for providing me with an enjoyable work environment: Gerwin Schalk, Elizabeth Wolpaw, Marilyn Birmingham, Aiko Thompson, Ren Diao, Tammy Coleman, and Hesham Sheikh. I would also like to thank my dissertation committee members for their excellent guidance and encouragement: William Shain, Xinxin Ding, and David Martin. Also, thanks to Abigail Snyder-Keller, Chris Bjornsson, Karen Smith, and Daniel LeBlanc-Goodspeed for their guidance and assistance. I would also like to express my gratitude to the staff at the Advanced Light Microscopy Core: Rich Cole and Don Swarovski. I would also like to thank the Library personnel, Wadsworth Center Animal Care staff and the Immunology core. I would also ii like to thank Caitlin Reid and Judith Duckor of the School of Public Health for their assistance throughout my graduate career here. I would like to thank all of the friends I have made during my time here at the Wadsworth Center and in Albany for their support and for providing me with a lifetime of great memories. Most importantly, I would like to dedicate this dissertation to my family: my mother, father, sister, and brother-in-law. Much of my success is due to their unwavering love, support, and sacrifice. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv LIST OF FIGURES viii LIST OF TABLES xi LIST OF ABBREVIATIONS xii ABSTRACT xiv CHAPTER 1: INTRODUCTION 1 1A. Background review 1 1B. Activity-dependent plasticity in the spinal cord in development and in adult life 2 1B1. Developmental plasticity 3 1B2. Plasticity with skill acquisition during adult life 4 1B3. Adaptive plasticity after injury 6 1C. Neurochemical basis of spinal cord plasticity 7 1C1. Neurochemical networks in an intact spinal cord 7 1C2. Spinal cord injury (SCI) alters neurochemical mileu of the spinal cord 8 1C2a. Changes in excitatory networks after SCI 9 1C2b. Changes in inhibitory networks after SCI 10 1C2c. Clinical significance of neurochemical basis of plasticity 12 1D. Operant conditioning of the H-reflex 12 1D1. Operant conditioning of the H-reflex as a model to study activity-dependent plasticity in the spinal cord 12 1D2. H-reflex conditioning depends upon supraspinal influence 19 1D3. Operant conditioning of the H-reflex is associated with multi-site plasticity 22 iv 1E. Use of SMC stimulation to modulate H-reflex 26 1F. Clinical application of H-reflex modulation for improvement of function after SCI 27 1G. Specific aims 31 CHAPTER 2: GENERAL METHODOLOGY 34 2A. Operant conditioning of the H-reflex 34 2A1. Subjects 34 2A2. Electrode Implantation 34 2A3. H-reflex conditioning and data collection 35 2B. GABAergic terminal labeling and quantification 36 2B1. Retrograde labeling of soleus motoneurons 36 2B2. Immunohistochemical labeling of GAD67 terminals 37 2B3. Image analysis 39 2B4. Quantification of GABAergic terminals on soleus motoneurons 39 2B5. Data analysis 39 2C. Sensorimotor cortex (SMC) stimulation 43 2C1. Implantation of chronic recording and stimulating electrodes 43 2C2. SMC stimulation 44 2C3. Data analysis 48 2C4. Immunohistochemistry evaluation for SMC 49 2C5. GAD67 terminal and GABA-B receptor labeling in SMC stimulated rats51 CHAPTER 3: EFFECTS OF H-REFLEX DOWN-CONDITIONING ON GABAERGIC TERMINALS ON SOLEUS MOTONEURONS 54 3A. Introduction 54 3B. Experimental Design 54 3C. Results 57 3C1. Soleus motoneurons 57 3C2. GABAergic terminals 58 v 3D. Discussion 65 3D1. GAD-IR comparisions 66 3D2. Effects of successful down-conditioning on GABAergic terminals 67 3D3. Origin of the GABAergic terminals in DS rats 68 3D4. Mechanism of decrease of H-reflex by the modified GABAergic Terminals 70 CHAPTER 4: H-REFLEX UP-CONDITIONING CHANGES GABAERGIC TERMINALS ON IDENTIFIED SOLEUS MOTONEURONS 73 4A. Introduction 74 4B. Experimental design 68 4C. Results 77 4D. Discussion 81 CHAPTER 5: LONG-TERM SENSORIMOTOR CORTEX (SMC) STIMULATION INDUCES SPINAL AND SUPRASPINAL PLASTICITY 85 5A. Introduction 85 5B. Experimental design 85 5C. Results 86 5C1. Effects of SMC stimulation on H-reflex 87 5C2. Effect of the SMC stimulation on the SMC stimulus amplitude 88 5C3. Effect of SMC stimulation on the cortex 93 5D. Discussion 99 5D1. Plasticity responsible for the SMC stimulation-induced H-reflex 99 5D2. Supraspinal plasticity associated with SMC stimulation induced H-reflex increase 102 5D3. Possible therapeutic uses of SMC stimulation 106 vi CHAPTER 6: LONG-TERM SMC STIMULATION-INDUCED INCREASE IN THE H-REFLEX IS ASSOCIATED WITH CHANGES IN GABAERGIC TERMINALS AND RECEPTORS ON MOTONEURONS 108 6A. Introduction 108 6B. Experimental design 109 6C. Results 110 6C1. Effects of SMC stimulation on GABAergic terminals 110 6C2. Effects of SMC stimulation on GABA-B receptors 111 6D. Discussion 122 6D1. Stimulation increases GABAergic terminals on soleus motoneurons 122 6D2. Role of modified GABAergic terminals in SMC stimulation-induced increase in the H-reflex 125 6D3. Origin of the modified GABAergic terminals 125 6D4. SMC stimulation induces plasticity of GABA-B receptors 126 6D5. Role of GABA-B receptors in SMC stimulation-induced H-reflex increase 128 6D6. Role of GABA in activity-dependent plasticity in the CNS 130 CHAPTER 7: CONCLUSIONS 132 7A. Operant down-conditioning alters GABAergic terminals on soleus motoneurons 135 7B. Effects of operant up-conditioning on GABAergic terminals on soleus motoneurons 138 7C. Effects of long-term SMC stimulation on cortex and the spinal cord 141 7D. Future work 145 7D1. Glycinergic terminals and receptors 145 7D2. Cholinergic terminals and receptors 146 7D3. GABA receptors 146 7E. Summary 149 REFERENCES 151 vii LIST OF FIGURES PAGE # Fig 1-1: Spinal stretch reflex (SSR) and its electrical analog, the Hoffman reflex (H-reflex) 14 Fig 1-2: (A) The operant conditioning protocol (B) The top graph shows average daily H-reflexes (±SEM) from rats during control-mode exposure and for the subsequent HRup or HRdown exposure (C) Average results for up-conditioning and down-conditioning of the H-reflex of the triceps surae in monkeys (left), the spinal stretch reflex of the biceps brachii in monkeys (middle), and the SSR of the biceps brachii in humans (right) 15 Fig 1-3: Spinal cord plasticity associated with H-reflex conditioning. Left: Triceps surae motoneurons on the conditioned side of HRdown monkeys had more positive firing thresholds and slightly smaller Ia EPSPs. Right: Conditioning changes the contacts of idealized average F terminals and their active zones on the cell bodies of triceps surae motoneurons on the conditioned sides of HRup and HRdown animals 17 Fig 1-4: H-reflex conditioning depends upon supraspinal influence 21 Fig 1-5: H-reflex conditioning induced plasticity at multiple sites in the CNS 25 Fig 1-6: The CNS circuitry involved in operant conditioning of the H-reflex 30 Fig 2-1: Assessment of glutamic acid decarboxylase-67 immunoreactivity (GAD-IR) 42 Fig 2-2: Sensorimotor cortex (SMC) stimulation protocol 47 Fig 3-1: Timeline for implantation, down-conditioning, dye injection and Perfusion 55 Fig 3-2: Successful down-conditioning increases the GABAergic terminal number on soleus motoneurons 59 Fig 3-3: Average (± SEM) values for: down-conditioning successful (DS), down conditioning failed (DF) and naive control (NC) rat groups for measures that differed significantly among the groups. (Fig A) Motoneuron perimeter glutamic acid decarboxylase immunoreactivity (GAD-IR) intensity. (Fig B) Number of GABAergic terminals per motoneuron (MN). (Fig C) Feret’s diameter of GABAergic terminals. (Fig D) Terminal GAD density (i.e. GAD-IR ⁄ diameter, as described in the text). (Fig E) GABAergic terminal coverage of soma (expressed as percent of perimeter, see text). (Fig F) GAD density (GAD-IR/Terminal viii diameter 61 Fig 4-1: Timeline for implantation, up-conditioning, dye injection and perfusion 76 Fig 4-2: Average (±SE) values for up-conditioning successful (US) and up-conditioning failed (UF) rats of the present study. (A) GAD-IR intensity of motoneuron perimeter. (B) Number of GABAergic terminals/motoneuron. (C) Feret’s diameter of GABAergic terminals. (D) Terminal GAD density (i.e., GAD-IR/diameter, as described in the text). (E) GABAergic terminal coverage of soma (expressed as percent of perimeter, see text) 79 Fig. 5-1: Average data for all rats exposed to the 20-day-on/20-day-off /20-day-on (20/20/ 20) stimulation protocol, for the final 10 control days before and for 60 days after the beginning of SMC stimulation. (A) average daily contralateral and ipsilateral soleus H-reflex, background EMG, and M response in percentage of average value for the final 10 control days. (B) average daily SMC stimulus amplitude in percentage of its value for the first day of stimulation. (C) average daily cord volley in percentage of its value for the first day of stimulation 90 Fig. 5-2: Average values of contralateral and ipsilateral soleus H-reflex, background EMG, and M response for each 10-day period (in percentage of average value for the final 10 control days) for the final 10 control days and the subsequent 60 days of the 20/20/20 SMC stimulation protocol 92 Fig 5-3: Confocal images of sections of left (stimulated) and right (unstimulated) SMC areas from a rat exposed to the 20/20/20 left SMC stimulation protocol. Top to bottom: Nissl staining (showing neurons), GFAP (glial fibrillary acidic protein) labeling (reflecting astrocytes), CyQuant staining (showing nuclei), and merged image 95 Fig 5-4: Effect of the SMC stimulation on neurons: Average (± SEM) values of number of neurons in SMC regions for the stimulated and unstimulated SMCs 96 Fig 5-5: Effect of the SMC stimulation on astrocytes: Average (± SEM) values of number of atrocytes in the superficial (left) and deep (right) SMC for the stimulated and unstimulated SMCs 97 Fig 5-6: Confocal merged images of slices from stimulated (top) and unstimulated (bottom) SMC areas from rats exposed to the 20/20/20 left ix SMC stimulation protocol and perfused 3 days (left panel) and 30 days after (right panel) the end of SMC stimulation 98 Fig 5-7: Comparision of the average number of astrocytes in the superficial (top) and deep (bottom) regions in rats 3 days and 30 days after the end of Stimulation 99 Fig 6-1: Timeline for implantation, SMC stimulation, dye injection and perfusion 109 Fig 6-2: Average (± SEM) values for: stimulated side (contralateral), unstimulated side (ipsilateral) and naive control (NC) rat groups for measures that differed significantly among the groups- (A): GAD67 immunoreactivity on motoneuron perimeter (GAD-IR) intensity (B): Number of GABAergic terminals/motoneuron (MN) (C): Terminal area (D): Feret’s diameter of GABAergic termininals (E): GABAergic terminal coverage of soma (expressed as percent of perimeter, see text). (F): Terminal GAD density (i.e. GAD-IR ⁄ diameter) 114 Fig 6-3: Representative photomicrographs showing GABA-B immuno- reactivity on spinal motoneurons from a control rat (left) and from a stimulated rat: contralateral (right, top) and ipsilateral (right, bottom) sides after the end of SMC stimulation 118 Fig 6-4: (A) Average values (± SE) of the number of GABA-B receptor labeled motoneurons/section from the contralateral and the ipsilateral sides of SMC stimulated rats compared with control rats. (B) Average values (± SE) for intensity of GABA-B receptor labeling from the contralateral and the ipsilateral sides of SMC stimulated rats compared with control rats 120 Fig 7-1: Summary of the effects of successful down-conditioning on GABAergic terminals on soleus motoneurons 131 Fig 7-2: Summary of the effects of successful up-conditioning on GABAergic terminals on soleus motoneurons 141 Fig 7-3: Summary of the effects of SMC stimulation on GABAergic terminals and receptors on soleus motoneurons 144 x LIST OF TABLES Table 3-1: Motoneuron and GABAergic terminal numbers and properties for each experimental group 60 Table 4-1: Motoneuron and GABAergic terminal numbers and properties for up-conditioned successful (US) rats, up-conditioned unsuccessful (UF) rats, and naïve control (NC) rats 78 Table 6-1: Motoneuron and GABAergic terminal numbers and properties for contralateral and ipsilateral sides of SMC stimulated rats and naïve control rats 113 Table 6-2: Effects of SMC stimulation on GABA-B receptor expression on spinal cord motoneurons (MN) 119 xi ABBREVIATIONS ABC avidin biotin complex b.i.d. bis in die CPG central pattern generator CNS central nervous system CST corticospinal tract CTb cholera toxin subunit-b DA dorsal ascending tract DAB diaminobenzidine DC dorsal column DF down-conditioning unsuccessful DS down-conditioning successful DRG dorsal root ganglia EMG electromyograph EPSP excitatory post-synaptic potential GAD67 glutamic acid decarboxylase-67 GAD65 glutamic acid decarboxylase-65 GABA gamma-aminobutyric acid GABA-A gamma-aminobutyric acid-A receptor GABA-B gamma-aminobutyric acid-B receptor GFAP glial fibrillary Acidic Protein GlyT2 glycine transporter xii HBHS hepes buffered hank’s saline HRup H-reflex up-conditioning HRdown H-reflex down-conditioning 5-HT 5-hydroxytryptamine i.m. intramuscular i.p. intraperitoneal LC lateral column LLR long-latency polysynaptic response MEP motor evoked potentials NC naïve Control p.o. per os q.o.d. every other day rTMS repetitive transcranial magnetic stimulation s.q. subcutaneous SCI spinal cord injury SD standard deviation SE standard error SSR spinal stretch reflex TS triceps surae US up-conditioning unsuccessful UF up-conditioning successful VC ventral column VChaT vesicular cholinergic transporter xiii ABSTRACT During development and throughout adult life, inputs from the brain combine with inputs from the periphery to induce activity-dependent plasticity in the spinal cord. This activity-dependent plasticity shapes spinal circuitry and helps in acquisition and maintenance of normal motor function. The neural pathways and processes responsible for induction and maintenance of plasticity in the spinal cord remain unclear. Understanding the mechanisms responsible for activity-dependent plasticity in the spinal cord is essential for developing therapies for spinal cord injury. Operant conditioning of the H-reflex, the electrical analog of the spinal stretch reflex (SSR), provides a simple experimental model to study activity-dependent plasticity in the spinal cord. In response to an operant conditioning protocol, monkeys, humans, rats, and mice can gradually increase or decrease the SSR or the H-reflex. Operant- conditioning induces plasticity at multiple sites in the CNS including the spinal cord. Furthermore, conditioning appears to be dependent only on descending influence originating from the contralateral sensorimotor cortex via the corticospinal tract (CST). In addition, a recent study indicated that, like operant conditioning, direct electrical stimulation of the SMC also modulates H-reflex by inducing plasticity in the cortex and the spinal cord. The anatomical basis of spinal cord plasticity responsible for operant conditioning and SMC stimulation-induced modulation in the H-reflex remains to be elucidated. The central goal of this study was to determine if the change in the soleus H- reflex subsequent to operant conditioning and SMC stimulation is associated with xiv changes in the GABAergic terminals on soleus motoneurons. In accord with the central goal, the main hypotheses were that: (1) operant down-conditioning of the H-reflex is associated with an increase in the GABAergic terminals on soleus motoneurons; (2) operant up-conditioning of the H-reflex is associated with a decrease in the GABAergic terminals on soleus motoneurons; and (3) long-term SMC stimulation-induced increase in the H-reflex is associated with a decrease in the GABAergic terminals on soleus motoneurons. These hypotheses were tested by identifying GABAergic terminals based on their immunoreactivity to glutamic acid decarboxylase 67 (GAD67), the main isoform of the enzyme present in terminals on motoneurons. With regard to the first hypothesis, results from operant conditioning studies indicate that successful down-conditioning was associated with an increase in the number, size, and GAD density of GABAergic terminals on motoneurons. These changes probably reflect the CST influence responsible for the decrease in the H-reflex. With regard to the second hypothesis, successful up-conditioning did not change the GABAergic terminal number, although there was an increase in the terminal diameter. Successful up-conditioning did not differ from unsuccessful up-conditioning in any of the measures. Therefore, the terminal changes could reflect non-specific effects of up- conditioning. Together, the results from these two studies support evidence from previous studies indicating that up- and down-conditioning are not mirror images of each other but rather have different mechanisms. With regard to the third hypothesis, results indicate that long-term SMC stimulation-induced increase in the H-reflex is associated with an increase in the GABAergic terminals on the soleus motoneurons. In addition, there was also a decrease in the GABA-B receptor expression on motoneurons. These changes xv probably reflect compensatory plasticity in response to the primary plasticity responsible for the SMC stimulation-induced increase in the H-reflex. Overall, these results provide valuable insights about the anatomical substrates of plasticity responsible for operant conditioning and SMC stimulation-induced
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