RE-EDUCATING THE INJURED SPINAL CORD BY OPERANT CONDITIONING OF A REFLEX PATHWAY DISSERTATION. the Degree Doctor of Philosophy in the Graduate

Transcription

1 RE-EDUCATING THE INJURED SPINAL CORD BY OPERANT CONDITIONING OF A REFLEX PATHWAY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yi Chen, M.S. * * * * * The Ohio State University 2006 Dissertation Committee: Professor Lyn B. Jakeman, Adviser Professor Bradford T. Stokes Approved by Professor John A. Buford Graduate Program in Professor Jonathan Wolpaw Physiology and Cell Biology

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3 ABSTRACT During development and throughout life, input from the brain and the periphery induces activity-dependent plasticity and thus shapes and maintains the spinal circuitry. This activity-dependent plasticity is the foundation for normal spinal function. When spinal cord injury interrupts the pathways of the spinal circuitry, normal input is altered, and pathological plasticity and abnormal reflexes develop. Thus, it becomes necessary to re-connect the interrupted spinal cord reflex pathways, and also to reshape or re-educate these pathways, to restore even partial function. Operant conditioning of the spinal stretch reflex (SSR) or its electrical analog, the H-reflex, provides a new method to induce long-term plasticity within 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. The conditioning changes the spinal cord, since evidence of it remains even after all supraspinal control is removed. Furthermore, the conditioning appears to be dependent only on descending influence in the corticospinal tract (CST), as transection of CST abolishes the acquisition of conditioning while transection of other descending pathways does not. ii

4 The central goal of this study was to determine whether H-reflex conditioning could help to modulate and guide normal functional recovery after spinal cord injury. The central hypotheses, supported by previous studies and preliminary data, were: (1) that H-reflex conditioning affects reflex magnitude during locomotion since up-conditioning will increase it and down-conditioning will decrease it; and (2) that H-reflex conditioning can reduce asymmetry in locomotion after spinal cord injury and (3) that appropriate reflex conditioning can improve locomotion and restore function. These hypotheses were tested by studying soleus muscle activity during locomotion in normal rats and in rats with a lateral column (LC) transection before and after H-reflex conditioning. Results indicate that: 1) in normal rats in which the soleus H-reflex had been decreased by down-conditioning, the H-reflexes elicited during locomotion were also smaller. Similarly, in rats in which the soleus H-reflex had been increased by up-conditioning, the H-reflexes elicited during locomotion were also larger. However, the conditioninginduced changes did not affect the duration, length, or right/left symmetry of the step cycle, even though the conditioned change in the stance H-reflex was positively correlated with change in the amplitude of the soleus locomotor burst. 2) in rats with LC transection in which a persistent asymmetry in treadmill locomotion was evident, up-conditioning increased the H-reflex and ameliorated the iii

5 locomotor asymmetry. This improvement was accompanied by a significant increase in right soleus burst amplitude. In contrast, in LC rats without up-conditioning exposure, the locomotor asymmetry persisted and no other significant changes occurred. These results suggest that operant conditioning of H-reflexes or other spinal reflexes may be able to reduce the functional deficits associated with spinal cord injury or other disorders of motor control. They also suggest that appropriate reflex conditioning may improve function in people with partial spinal cord injuries. In combination with other new therapeutic methods (such as those that promote regeneration), reflex conditioning may be able to maximize restoration of function after spinal cord injury. iv

6 Dedicated to my Mother and Father, and my family v

7 ACKNOWLEDGMENTS My graduate work was initiated from a collaboratory project between the Laboratory of Spinal Cord Injury Research in the Department of Physiology and Cell Biology at the Ohio State University and the Laboratory of Nervous System Disorders at the Wadsworth Center, Albany, New York. I consider myself extremely lucky to have had the distinct honor and privilege to experience my graduate career in these two prestigious labs. I wish to thank the faculty and staff of both labs. Without their help, both scientific and intellectual, this work would not have been possible. Many thanks to Ping Wei, Zhen Guan, FengQin Yin, Patricia Walters, Ming Wang, Todd Lash and Ying Chen for sharing your histological and surgical expertise as well as knowledge and application of the Electromechanical Spinal Cord Injury Device with me. My appreciation extends to Dr. Dana McTigue, Dr. Manhong Ma, and Dr. Daniel Ankeny, for your advice and help. Your kindness and openness cannot be overstated. Thanks to Lu Chen, Ronliang Liu, and Gerwin Schalk. Lu, your help with surgical and histological work, among others, was greatly appreciated. Ronliang, your assistance in treadmill locomotion and animal care was very important to my experiments. Gerwin, vi

8 your programs and ideas greatly facilitated my data processing and analysis. I would also like to express my gratitude to Dr. Elizabeth Winter Wolpaw, Dr. Jonathan Carp, and Dr. Ann Tennissen, for commenting on my manuscripts and dissertation. Your insights, suggestions, and editorial efforts were invaluable. I also extend my thanks to Dr. Dennis McFarland, Dr. Yu Wang, Dr. Aiko Thompson, and Shreejith Pillai, for their suggestions and comments. Thanks especially to Dr. XiangYang Chen. I thoroughly appreciate your time and efforts in teaching and guiding me. Your guidance and advice for my studies were irreplaceable. Without your relentless help and 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. Your enthusiasm for science and ability to progress in several distinct projects simultaneously always intrigue and inspire me. I greatly benefitted from your encouragement and the challenges you presented to me. I am indeed grateful to you for having me as part of your lab. I would like to thank Dr. John Buford for serving on my committee. Your advice and comments were very valuable and I learned from you the importance of positioning my specific scientific questions into a much bigger picture. This point of view, I believe, is important not only for my graduate studies, but also for my career thereafter. vii

9 I would like to express my gratitude to Dr. Brad Stokes. Dr. Stokes, as my first advisor, your guidance and patience will always be appreciated and remembered. Your sense of humor, your laughter and kindness are greatly enjoyed and admired by everyone in the lab. I fully appreciate your open mind to questions and new ideas. Special thanks to Dr. Lyn Jakeman for so many things. Your encouragement, patience, and guidance have always been there with me throughout this research and my graduate career. You challenged me to think like an independent researcher and to grow into a scientist. Aside from this, it is also hard for me to forget your holding my hands and calming me down with Don t worry, you will be doing just fine at my very first seminar years ago. And I guess I did. Thanks for everything! Finally, I would like to thank my parents and thank the people who cared about me both back in China and here in U.S. Mom and Dad, thank you for always being there for me and for supporting and encouraging me. I am deeply indebted to your unconditional love and am proud of you! viii

10 VITA June 27, Born - Suzhou, China B.S. Medicine, The Nanjing Medical University M.S. Physiology and Cell Biology, The Ohio State University present... Graduate Research Associate, The Ohio State University PUBLICATIONS 1. Chen Y, Chen XY, Jakeman LB, Schalk G, Stokes BT, Wolpaw JR. The interaction of a new motor skill and an old one: H-reflex conditioning and locomotion in rats. Journal of Neuroscience 25: , Chen Y, Chen XY, Jakeman LB, Stokes BT, Liu RL, Wolpaw JR. Reeducating the injured spinal cord by operant conditioning of a reflex pathway. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. 3. Chen Y, Jakeman LB, Stokes BT, Chen XY, Liu RL, Wolpaw JR. H-reflex conditioning may modify locomotion after lateral column transection in rats. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. ix

11 4. Chen L, Chen Y, Liu RL, Chen XY, Wolpaw JR. Bilateral globus pallidus ablation in rats prevents down-conditioning of H-reflex. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. 5. Chen XY, Jakeman LB, Stokes BT, Chen Y, Liu RL, Wolpaw JR. H-reflex conditioning may modify locomotion after lateral column transection in rats. BMS Conference, Chen Y, Chen L, Jakeman LB, Stokes BT, Chen XY, Wolpaw JR. The effect of H-reflex up-conditioning on soleus function during locomotion in rats with right lateral column transections. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. 7. Chen XY, Chen L, Chen Y, Liu RL, Schalk G, Jakeman LB, Stokes BT, Wolpaw JR. Down-conditioning of soleus H-reflex reduces the H-reflex and affects soleus activity during locomotion. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. 8. Chen L, Chen Y, Liu RL, Chen XY, Wolpaw JR. Up-conditioning of H-reflex appears to affect soleus reflex function during locomotion in rats. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. 9. Chen Y, Chen L, Liu RL, Schalk G, Jakeman LB, Stokes BT, Chen XY, Wolpaw JR. The effect of H-reflex conditioning on soleus reflex function and EMG activity during locomotion in normal rats and in rats with right lateral column transection. BMS Conference, Chen XY, Jakeman LB, Chen Y, Wolpaw JR, Stokes BT. H-reflex conditioning in spinal cord-injured rats after NT-3 treatment. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. 11. Tennissen AM, Chen XY, Chen L, Schalk G, Wolpaw JR, Jakeman LB, Chen Y, Stokes BT. H-reflex conditioning effects on reflexes during locomotion in normal x

12 and spinal cord-injured rats. Program No Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online. FIELDS OF STUDY Major Field: Physiology and Cell Biology xi

13 TABLE OF CONTENTS Page Abstract... ii Dedication... v Acknowledgments... vi Vita... ix List of Tables... xv List of Figures... xvi List of Abbreviations... xxiv Chapters: 1. General Introduction... 1 (A) Background Review... 1 (1) Activity-Dependent Spinal Cord Plasticity... 1 (2) Operant Conditioning of the H-reflex, the Electrical Analog of the Spinal Stretch Reflex (SSR)... 5 (3) Relevance of H-Reflex Conditioning to Real Life... 7 (4) The Spinal Cord Plasticity Associated with H-Reflex Conditioning (5) Spinal Cord Plasticity Underlying H-Reflex Conditioning Depends on Supraspinal Descending Influence (6) Spinal Cord Pathways Essential for H-reflex Conditioning (7) Locomotion and its Motor Control (8) Reflex Conditioning Could Help to Maximize Restoration of Function after Spinal Cord Injury (9) Summary (B) Preliminary Studies (1) Recording EMG and H-reflexes during Treadmill Locomotion xii

14 (2) Effects of a Unilateral Spinal Cord Injury (i.e., LC Transection) on Locomotion (3) Evidence that H-reflex Conditioning Affects Function during Locomotion (C) Aims of this Project General Methodology (A) Chronic Operant Conditioning of the H-Reflex (B) Right Lateral Column (LC) Transection and Post-lesion Animal Care (C) Assessment of Treadmill Locomotion (D) Spinal Cord Histology Effects of H-reflex Conditioning on Reflex Function During Locomotion in Normal Rats (A) Introduction (B) Methods (1) Animal Preparation and Environment (2) The H-Reflex Conditioning Protocol (3) Treadmill Locomotion and the Locomotor H-Reflexes (4) Perfusion and Postmortem Examination (5) Analysis of H-Reflexes During Locomotion (6) Analysis of Locomotion (C) Results (1) Effects of Conditioning on the Conditioning H-Reflex (2) Effects of Conditioning on the Locomotor H-Reflexes (3) Effects of Conditioning on Locomotion (D) Discussion (1) Effects of Locomotion on H-Reflex Conditioning xiii

15 (2) Effects of H-Reflex Conditioning on Locomotion (3) Etiology of Conditioning Failure (4) Analysis of Skill Interactions (5) Possible Applications of H-Reflex Conditioning H-Reflex Conditioning Reduces Asymmetry in Locomotion Produced by Lateral Column Transection (A) Introduction (B) Methods (1) Electrode Implantation (2) Lateral Column Transection (3) Treadmill Locomotion and the Locomotor H-Reflexes (4) The H-Reflex Conditioning Protocol (5) Perfusion, Postmortem Examination, and Lesion Verification (C) Results (1) Histology (2) Effects of LC transection (3) Effects of Up-conditioning on Locomotor H-Reflexes and the Soleus Locomotor Bursts (4) Effects of Up-conditioning on the Step Cycle (D) Discussion (1) The Locomotor Asymmetry and the Effect of Up-conditioning (2) Possible Therapeutic Applications (3) Future Directions Summary and Conclusion List of References xiv

16 LIST OF TABLES Table Page 1 Effects of conditioning on locomotion. Average (±SE) right and left soleus burst amplitudes and durations and step-cycle duration and right/left symmetry after conditioning for successful HRdown and HRup rats (expressed in terms of their control values), and the correlations between changes in them and the change in stance H-reflex (with the H-reflex change expressed in terms of the control value of soleus burst amplitude). (NS: No significant correlation (P>0.05)) xv

17 LIST OF FIGURES Figure Page 1 Main pathway of the spinal stretch reflex (SSR) and its electrical analog, the Hoffman reflex (H-reflex). The pathway consists of the Ia afferent neuron from the muscle spindle, its synapse on the alpha motoneuron, and the motoneuron itself. Ia afferent excitation excites motoneurons innervating the same muscle and its synergists. If the afferent is excited normally, i.e., by muscle stretch, the muscle's response is the SSR. If it is excited by nerve stimulation, i.e., via an electrical stimulation cuff, the response is the H-reflex. The SSR and the H-reflex are typically measured by EMG, as shown by the inset. The first response after nerve stimulation, M response, results from excitation of the efferent fibers. It is followed by Ia afferent excitation of the H-reflex. While their pathway is wholly spinal, both SSR and H-reflex are affected by descending influence (dashed lines) on the Ia terminal (exerted presynaptically) and on the motoneuron, and the SSR is also influenced by descending control of muscle spindle sensitivity (Modified from Chen and Wolpaw, 1999; Wolpaw, 1987) The H-reflex conditioning protocol and its result (Adapted from Wolpaw, 1997, with permission from Elsevier). A: Soleus background EMG is continuously monitored in a rat with chronically implanted EMG electrodes and a tibial nerve cuff. When background EMG stays within a defined range for a randomly varying s period, nerve stimulation just above M-response threshold elicits an M response (the direct muscle response) and an H-reflex. Under the control mode, no reward occurs after the nerve stimulation, the H-reflex is simply measured to determine its control size. Under the up- or down-conditioning mode, reward (a food pellet) occurs 200 ms after the nerve stimulation if the H-reflex is above (up-conditioning) or below (down-conditioning) a given value. Each rat is first exposed to the control mode for 10 days, and then to the up- or down-conditioning xvi

18 mode for 50 days (Chen and Wolpaw, 1995, 1996). B Top: average daily H-reflex for 9 up-conditioned animals (up triangles) and 12 down-conditioned animals (down triangles) during the control mode (i.e., days -10 to 0) and during subsequent exposure to the up- or down-conditioning mode (in percent of control-mode value). H-reflex size rises (up-conditioned animals) or falls (down-conditioned animals) over several weeks. B Bottom: average post-stimulus EMG response to the nerve cuff stimulus from an up-conditioned rat (left) and a down-conditioned rat (right) for a day before (solid line) and a day after (dashed line) prolonged up- or down-conditioning exposure. The H-reflex is much larger after up-conditioning and much smaller after down-conditioning, while the background EMG (shown here by EMG at zero time) and the M response do not change (Chen and Wolpaw, 1995, 1996). C: Average results for up-conditioning (up triangles) and down-conditioning (down triangles) of triceps surae H-reflex in monkeys (left), biceps brachii SSR in monkeys (middle), and biceps brachii SSR in humans (right). The courses and final magnitudes of change are similar to those found in rats (Wolf et al., 1995; Wolpaw et al., 1994; Wolpaw and O'Keefe, 1984) Changes in the spinal stretch reflex (SSR) pathway during development and during the acquisition of motor skill (Adapted from Wolpaw, 1997, with permission from Elsevier). A: EMG responses of soleus (solid lines) and tibialis anterior (dotted lines) muscles to sudden foot dorsiflexion, which stretches the soleus and shortens its antagonist, the tibialis anterior. In a normal infant, this stimulus produces SSRs in both muscles. In a normal adult, an SSR occurs only in the stretched muscle, that is, the soleus; little or no response occurs in the tibialis anterior. In contrast, in an adult with cerebral palsy, in whom perinatal supraspinal injury has impaired the descending influence responsible for the development of normal adult SSRs, the infantile pattern persists: SSRs occur in both soleus and tibialis anterior (The short latency and unusual form of the tibialis anterior response also suggest the presence of central or peripheral abnormalities, or both.) (Myklebust BM, Gottlieb GL (unpublished data comparable to those in Myklebust et al., 1986 and 1982)). B: Working for reward, monkeys performed an elbow flexion-extension task on which brief perturbations (that is, 10-ms torque pulses) were randomly superimposed. Shown are biceps EMG and elbow angle (flexion is upward) for an unperturbed trial (dotted line), a perturbed trial early in training (solid line), and a perturbed trial in training (broken line). xvii

19 Early in training, perturbation elicits both an SSR and a long-latency polysynaptic response (LLR). After intermittent training over several years, the SSR is much larger and the LLR has disappeared. Over several years, the SSR has gradually taken over the role of opposing the pertubation. This is accompanied by improvement in performance: the deflection superimposed by the torque pulse on the smooth course of elbow flexion is smaller and briefer (Meyer-Lohmann et al., 1986). C: Soleus H-reflexes are much smaller in professional ballet dancers than in other well-trained athletes (for example, runners, swimmers, cyclists). (H-reflexes of sedentary subjects are intermediate.) (Nielsen et al., 1993) Spinal plasticity associated with H-reflex conditioning (Adapted from Wolpaw, 1997, with permission from Elsevier). A: H-reflex conditioning affects motoneuron firing threshold, primary afferent EPSP size, and conduction velocity. Top: After down-conditioning of the H-reflex, motoneurons have more positive firing thresholds and smaller Ia EPSPs. These changes can explain why the motoneurons are less likely to fire in response to nerve stimulation (Carp and Wolpaw, 1994). Bottom: Rat soleus motoneuron axonal conduction velocities from control (solid) and down-conditioned (dashed) rats. Conduction velocity is significantly lower after down conditioning in both rats and monkeys (Carp and Wolpaw 1994; Carp et al. 2001). B: Illustration of average F terminals and their active zones on the cell bodies of the motoneurons of monkey triceps surae muscles after H-reflex conditioning. The size of the terminal is smaller in up-conditioned animals than in down-conditioned animals. In addition, the number of active zone (represented by the filled circles and hemi-circle) is less after up-conditioning than down-conditioning, while there is no significant change in the size of the active zone (Feng-Chen and Wolpaw 1996). C: Results of immunohistochemistry study of GAD 67 -labeled terminals on the soleus motoneuron after H-reflex conditioning. The number, diameter and coverage of GAD 67 -labeled terminals increase in the successful down-conditioning group compared with failed or non-conditioned control group. DS: Down-conditioning successful; DF: Down-conditioning failed; NC: Non-conditioned control (Wang et al., 2006) Contusion injury of the spinal cord impairs H-reflex conditioning. A: Final H-reflex amplitude in % of the control. While in the normal rat group, the change in final H-reflex size was more than 20% and xviii

20 conditioning was successful (represented by open triangles; the direction of the triangles indicates up- or down- conditioning); in the rat group with contusion injury (SCI Rats), conditioning was unsuccessful (represented by filled triangles). This indicates that contusion injury impairs H-reflex conditioning. B: The impairment is significantly correlated with the severity of injury, as assessed by % of white matter remaining. In up-conditioning, those rats with more white matter spared after injury had greater increase and in down-conditioning, they had greater decrease in HR size (Modified from Chen et al., J. Neurotrauma, 1996, 1999) Effect of pathway transection on H-reflex down-conditioning and upconditioning. A: Protocol for the down-conditioning study. Different pathway transections were performed before the rats were exposed to downconditioning. B: Final H-reflex size (in % of initial values) for normal, LC, DC, CST, and DA rats.!: successful rat (change in the correct direction $20%); ": unsuccessful rat. LC and DA rats achieved decrease like those of normal rats. DC and CST rats did not decrease the H-reflex (Modified from Chen and Wolpaw, 1997). C: Protocol for the up-conditioning study. Different pathway transections were performed before up-conditioning. D: Final H-reflex size (in % of initial values) for normal, LC, DC, CST, and DA rats.!: successful rat (change in the correct direction $20%); ": unsuccessful rat. LC and DA rats achieved increase like those of normal rats. DC and CST rats did not increase the H-reflex (Modified from Chen et al., 2002). E: Camera lucida drawings (top) and photomicrographs (bottom) of transverse sections of T8-9 spinal cord from a normal rat (with lateral (LC), dorsal (DC), and ventral (VC) columns labeled), an LC rat, a DC rat, a CST rat, and a DA rat. In lesioned rats, the section shown is at the transection epicenter. In the drawings, hatching is gray matter, stippled areas are the CST, and black areas are necrotic debris, cystic cavities, or fibrous septa (Modified from Chen et al., 2001) General framework of locomotor control in the cat. The EMGs of the inset at the bottom center are taken from Semitendinosus (St, a knee flexor/ hip extensor), Sartorius (Srt, a hip flexor), Vastus Lateralis (VL, a knee extensor), Gastrocnemius Medialis (GM, an ankle extensor) and Triceps brachialis (Tri, an elbow extensor); L and R refer respectively to left and right. DRG, dorsal root ganglion; CPG, central pattern generator; xix

21 E, extensor motoneurons; F, flexor motoneurons; VLF, ventrolateral funiculus; DLF, dorsolateral funiculi, PS, propriospinal interneurons; L3-S1 refer loosely to various lumbar and sacral spinal segments; NE, norepinephrine; 5-HT, serotonine, DA, dopamine, Glu, glutamate (From Rossignol et al., 2002 with permission from Elsevier) A: Right (R; top trace) and left (L; bottom trace) soleus EMG recorded while a normal rat walked on the treadmill. B: Relationship between the right soleus EMG and the right stance phase of locomotion (indicated by horizontal lines). As the vertical dashed lines indicate, the onset of the stance phase is closely related to the onset of the soleus burst. As indicated in Materials and Methods, stance was measured as the period during which the foot was within 1 mm of the treadmill surface, which slightly outlasts the period during which the soleus produces force EMG activity during treadmill locomotion from a normal rat (A and B) and from a rat with right lateral column (LC) transection (C and D). A : Rectified right (top) and left (bottom) soleus EMG activity from the normal rat. The filled circles mark the onsets of the right soleus bursts and the open circles mark those of the left soleus bursts. The gait of this normal rat is symmetrical, characterized by the illustration that the time from the onset of the right burst to that of the left burst is about the same as the time from the onset of the left burst to that of the right burst. B: Average of the rectified EMG activity from both soleus muscles during locomotion from this rat. The right (dashed line) and the left (solid line) soleus EMG bursts are symmetrical in this normal rat. C: Rectified right (top) and left (bottom) soleus EMG activity from the LC rat. The filled circles mark the onsets of the right soleus bursts and the open circles mark those of the left soleus bursts. The gait of this right LC rat is asymmetrical, i.e., the time from the onset of the right burst to that of the left burst is clearly shorter than the time from the onset of the left burst to that of the right burst. D: Average of the rectified EMG activity from both soleus muscles during locomotion of the LC rat. The soleus EMG bursts on the right (dashed line, i.e., the lesioned) side appear to be briefer than those on the left (solid line, i.e., the uninjured) side The effects of H-reflex conditioning on reflex function during locomotion in the preliminary study. A: H-reflex elicited during treadmill locomotion before (solid) and after (dashed) up-conditioning (left) and down-conditioning (right). B: Left: H-reflex elicited during locomotion before (solid) and after (dashed) up-conditioning from a rat with right lateral column transection. xx

22 Right: Superimposed average rectified soleus EMG bursts before (solid line) and after (dotted line) up-conditioning from this rat. C: Left: H-reflex elicited during locomotion before (solid) and after (dashed) down-conditioning from a rat with a 0.7-mm contusion injury. Right: Superimposed average rectified soleus EMG bursts before (solid) and after (dotted) down-conditioning in this contusion injured rat Study protocol. After learning to walk on the treadmill, each rat was implanted with soleus EMG electrodes and nerve-cuff stimulating electrodes. At least 3 weeks later, it was exposed to the control mode for 20 days and then to the HRdown or HRup conditioning mode for 50 days. Locomotion and H-reflexes during locomotion were assessed on the treadmill during the control period and near the end of the conditioning period Locomotor H-reflexes: elicitation of the right soleus H-reflex during the stance and swing phases of the step cycle. A: Left, Stimulus (arrow) during the right soleus burst elicits the stance H-reflex. Right, Average absolute value of right soleus EMG after stimulation (at 0 ms) during stance. The dotted line indicates the background EMG level at the time of stimulation, and the M response and H-reflex are shaded. B: Left, Stimulus (arrow) when right soleus EMG is low elicits the swing H-reflex. Right, Average absolute value of right soleus EMG after stimulation during swing. Note that M-response size (and thus effective stimulus strength) is similar in the stance and swing phases, whereas the H-reflex is much larger during the stance phase when background EMG is much higher. absol val: Absolute value Effects of conditioning on the conditioning H-reflexes and the locomotor H-reflexes. The average (±SE) final values of conditioning, stance, and swing H-reflexes from successful HRdown and HRup rats are shown. The conditioning and locomotor H-reflexes are similarly decreased in the HRdown rats and similarly increased in the HRup rats Conditioning and locomotor H-reflexes before (solid) and after (dotted) conditioning from an HRdown and an HRup rat. The conditioning H-reflexes are each the average of a single day (at least 4000 trials), and the locomotor H-reflexes are each the average of trials obtained during the treadmill session. (In several traces, stimulus artifacts are present in the first millisecond after the stimulus.) After conditioning, the conditioning and locomotor H-reflexes are smaller in the HRdown rat and larger in the HRup rat. absol val: Absolute value xxi

23 15 Conditioning and locomotor H-reflexes before (solid) and after (dotted) conditioning from an unsuccessful HRdown rat. The conditioning H-reflexes are each the average of a single day (at least 4000 trials), and the locomotor H-reflexes are each the average of trials obtained during the treadmill session. Although the conditioning H-reflex is unchanged after conditioning, the stance and swing H-reflexes are markedly increased. absol val: Absolute value Stance H-reflexes and right soleus locomotor bursts before (solid) and after (dotted) conditioning from an HRdown and an HRup rat. The stance H-reflexes are each the average of trials, and the stance bursts are each the average of bursts obtained during the treadmill session. After conditioning, both the stance H-reflex and the soleus burst are smaller in the HRdown rat and larger in the HRup rat. absol val: Absolute value A: Experimental design. After learning to walk on the treadmill, each rat was implanted with soleus EMG electrodes and nerve-cuff stimulating electrodes. Several weeks later it was subjected to an LC transection. At least 16 days after the transection when locomotion was grossly normal (i.e., BBB in both legs), the rat was exposed to the control mode for at least 10 days and then to the HRup conditioning mode for 50 days (HRup group) or was not exposed to the HRup conditioning protocol (Control group). Background EMG amplitude and M response size were stable throughout. Locomotion and H-reflexes during locomotion were assessed on the treadmill during the control period and near the end of the conditioning period. B: Camera lucida drawings of transverse sections of T8-9 spinal cord from a normal rat (with lateral column (LC), dorsal column corticospinal tract (CST), dorsal column ascending tract (DA), and ventral column (VC) labeled) and from an LC-transected rat. In the LC rat, the section shown is at the lesion epicenter. Hatching indicates gray matter and stippled areas are the main corticospinal tract. Scale bar indicates 1 mm Effects of up-conditioning on locomotor H-reflexes and soleus locomotor bursts. Average (±SE) locomotor H-reflexes (A) and left and right soleus burst amplitudes (B) and durations (C) for Control rats (open bars) and HRup rats (solid bars) for the second treadmill session in percent of their values for the first (i.e., initial) session. Asterixes indicate significant changes from the first to the second session (*: P<0.05; **: P<0.01). Locomotor H-reflexes and right soleus burst amplitudes are increased in the HRup rats. The Control rats show no significant change xxii

24 19 Illustration of locomotor H-reflexes and soleus locomotor bursts from a control rat (A) and from a up-conditioned one (B). The main traces are average right soleus bursts and the inset small traces are average stance H-reflexes from a Control rat and an HRup rat for the first (solid) and second (dotted) treadmill sessions. (For the H-reflexes shown, average background EMG and M response size were the same for the first and second sessions (Chen et al., 2005b for method). The horizontal scale bars are 2 ms. for both the Control and HRup rats, while the vertical scale bars are 50 and 200 :V, respectively.) In the second session, the stance H-reflex and the right soleus burst amplitude (and duration) are increased in the HRup rat. The Control rat shows no change in the H-reflex or in the burst Effects of up-conditioning on the step-cycle. A: Average (±SE) step-cycle durations, and B: RBO-LBO (right soleus burst onset to left soleus burst onset) duration of the Control rats (open bars) and of the HRup rats (solid bars) for the second treadmill session as percent of their values for the first (i.e., initial) session. ** indicates a significant change from the first to the second session (P<0.01). Step-cycle duration is unchanged in both Control and HRup rats. However, RBO-LBO duration (which was lower than normal in the first treadmill session) is increased in HRup rats only Right and left soleus bursts (rectified EMG) from an HRup rat for the first (i.e., before up-conditioning; A) treadmill session and the second (i.e., after up-conditioning; B) session. (The horizontal scale bar indicates 0.5 second and the vertical scale is 100 and 150 :V for the right and left bursts, respectively.) Each RBO ( ) and LBO ( ) is marked. The short vertical dashed lines mark the midpoints between RBOs (i.e., midpoints of step-cycles), which is the time when LBOs should occur (as in normal rats). Prior to up-conditioning, LBO occurs too early; after up-conditioning, it occurs on time xxiii

25 LIST OF ABBREVIATIONS b.i.d. CPG CNS CST DA DC DRG EMG EPSP GAD 67 GABA HRup HRdown i.m. LBO LC bis in die central pattern generator central nervous system corticospinal tract dorsal ascending tract dorsal column dorsal root ganglia electromyograph excitatory post-synaptic potential glutamic acid decarboxylase gamma-aminobutyric acid H-reflex up-conditioning H-reflex down-conditioning intramuscular left (soleus) burst onset lateral column xxiv

26 LLR p.o. q.o.d. RBO s.q. SCI SD SE SSR TS VC long-latency polysynaptic response per os every other day right (soleus) burst onset subcutaneous spinal cord injury standard deviation standard error spinal stretch reflex triceps surae ventral column xxv

27 CHAPTER 1 GENERAL INTRODUCTION (A) BACKGROUND REVIEW (1) Activity-Dependent Spinal Cord Plasticity Studies of activity-dependent plasticity have traditionally focused on the cortex (e.g., Van der Loos and Woolsey, 1973; Simons and Land, 1987; Sur and Rubenstein, 2005; Feldman and Brecht, 2005) and other brain areas (i.e., hippocampus, cerebellum, etc.) (Bliss and Lomo, 1973; Andersen et al., 1977; Malenka and Nicoll, 1999; Malenka, 2003; Doyon and Benali, 2005), and ignored the spinal cord. As a result, the mechanisms by which activity-dependent plasticity in the spinal cord modifies spinal cord function remains largely unknown. The spinal cord has traditionally been viewed as little more than a conduit between the brain and the periphery, despite its central role as the final common pathway for behavior. Evidence accumulated over the past 100 years, and focusing on those revealed in recent years, indicates that this traditional view is not correct. A number of clinical and laboratory studies show that the spinal cord is not merely the location of hardwired reflexes, but that, like the rest of the CNS, it undergoes 1

28 activity-dependent plasticity: input from the periphery or from the brain can cause lasting changes in the spinal cord that affect its output for long time periods after alteration (Wolpaw and Tennissen, 2001; Edgerton et al., 2004). Peripheral input to the isolated spinal cord has also been shown to cause sensitization, long-term potentiation, and related phenomena that contribute to chronic pain syndromes (Mendell, 1984; Randic, 1996; Baranauskas and Nistri, 1998; Alvares and Fitzgerald, 1999; Woolf and Costigan, 1999; Dubner and Gold, 1999; Herrero et al., 2000; Willis, 2002; Ji et al., 2003). Classical and operant conditioning protocols and training regimens have been shown to induce changes in spinal cord output (Patterson, 1976; Kandel, 1977; Durkovic, 1985, 1986; Hodgson et al., 1994; Barbeau and Rossignol, 1987; Dietz et al., 1994, 1995; Belanger et al., 1996; Harkema et al., 1997; de Leon et al., 1998, 1999; Dobkin, 1998, 1999; Field-Fote, 2000; Bouyer and Rossignol, 2001; Thompson, 2001; Drew et al., 2002; Cohen and Hallett, 2003; Schneider and Capaday, 2003). During development and during skill acquisition, as well as in spinal cord injury and other disorders, descending input from the brain combines with peripheral input to change the spinal cord (Goode and Van Hoven, 1982; Myklebust et al., 1982, 1986; Casabona et al., 1990; Koceja et al., 1991; O'Sullivan et al., 1991; Nielsen et al., 1993; Levinsson et al., 1999; Rossignol et al., 2002; Cohen and Hallett, 2003; Edgerton et al., 2004; Fernando et al., 2004; Cauraugh and Summers, 2005; Doyon and Benali, 2005). The role of activity-dependent plasticity in the acquisition and maintenance of behaviors in normal states and after CNS injury has begun to be recognized in the spinal 2

29 cord. This new interest is the result of two major findings. The first is the recent developments in spinal cord injury research (Bregman et al., 1997, 2002; Bregman, 1998; Fawcett, 1998; Amar and Levy, 1999; Tuszynski and Kordower, 1999; McTigue et al., 2000; Bradbury et al., 2002; Edgerton and Roy, 2002; Rossignol et al., 2002; Stokes and Jakeman, 2002; Edgerton et al., 2004; Ying et al., 2005) that recognize the possibilities for CNS regeneration and inevitably raise the issue of how regenerated neuronal tissue can become useful and how it will be able to provide normal, or at least acceptable, function (Jakeman et al., 1998; Merkler et al., 2001; Edgerton and Roy, 2002; Dietz and Harkema, 2004; Ma et al., 2004; Ying et al., 2005). Since a normally functioning adult spinal cord is the product of appropriate activity-dependent plasticity during early development and throughout subsequent life, newly regenerated spinal cord will probably not be properly or even acceptably configured for effective use, and is likely to display diffuse infantile reflexes or other disordered and dysfunctional outputs (Muir and Steeves, 1997; Wolpaw and Tennissen, 2001). As the methods for inducing spinal cord regeneration develop, it will be essential to find and develop methods for re-educating the newly regenerated spinal cord. This anticipated need compels attention to activity-dependent spinal cord plasticity and to the processes by which spinal cord neurons and synapses are shaped to serve important and fundamental life functions as diverse as locomotion and urination, as well as non-fundamental functions such as writing or painting. Furthermore, additional incentive for exploring activity-dependent plasticity in the spinal cord comes from recent appreciation of the latent capacities for 3

30 plasticity of the injured but unregenerated spinal cord. In spinal animals, activity-dependent plasticity of spinal neuronal circuits modifies the sensori-motor function of the adult mammalian lumbosacral spinal cord (Barbeau and Rossignol, 1987; de Leon et al., 1998, 1999; Barbeau et al., 1999). Recent evidence that exercise, training, and enriched environments can improve functional recovery after spinal cord injury have provided a compelling basis for understanding the mechanisms by which activity and/or descending control shape neural output (Bouyer and Rossignol, 2001; Lankhorst et al., 2001; Barbeau et al., 2002; Rossignol et al., 2002, 2006; Cauraugh and Summers, 2005; Doyon and Benali, 2005; Ying et al., 2005). Understanding this plasticity is essential for understanding both the changes that occur after injury and the processes that can be accessed and guided so as to restore useful function (Nelson and Mendell, 1979; Cope et al., 1986; Thompson et al., 1992; Calancie et al., 1994; Shefner et al., 1992; Dietz et al., 1994, 1995; McTigue et al., 2000; Wolpaw and Tennissen, 2001 for discussion). The second reason for the new interest in spinal cord plasticity is the growing recognition, based on data from a variety of laboratory models, that the acquisition and maintenance of both normal motor performances and the abnormal behaviors accompanying disease involve activity-dependent plasticity at multiple sites throughout the CNS, including the spinal cord (Lieb and Frost, 1997; Wolpaw, 1997; Lisberger, 1998; Garcia et al., 1999; Chen and Wolpaw, 1999; Pearson, 2000; Wolpaw and Tennissen, 2001; Chen and Wolpaw, 2005; Wolpaw and Chen, 2006). The peripheral and descending inputs that are modulated during practice or with trauma or disease can 4

31 modify the spinal cord, and these changes combine with others elsewhere to modify behavior. Thus, knowledge of the mechanisms of spinal cord plasticity and its interactions with plasticity elsewhere in the CNS is important for understanding normal behaviors as well as the complex disabilities caused by disorders like spinal cord injury. (2) Operant Conditioning of the H-Reflex, the Electrical Analog of the Spinal Stretch Reflex (SSR) As indicated above, until recently, activity-dependent plasticity in the spinal cord has been little explored. Nevertheless, the spinal cord is more technically accessible than the rest of the CNS, and because its inputs and outputs are well defined and controllable, the spinal cord provides a good model to study the mechanisms of activity-dependent plasticity in the central nervous system. Over the past 25 years, Dr. Jonathan Wolpaw s laboratory at the Wadsworth Center, New York State Department of Health has used a simple model to explore the mechanisms of activity-dependent spinal cord plasticity and its role in normal and abnormal motor function. This model, first established in monkeys (Wolpaw et al., 1983; Wolpaw, 1987) and then in humans (Evatts et al., 1989), in rats (Chen and Wolpaw, 1995), and mice (Carp et al., 2005), is based on operant conditioning of the spinal stretch reflex (SSR) pathway (reviewed in Wolpaw, 1997, 2001; Chen and Wolpaw, 1999; Wolpaw and Tennissen, 2001). As shown in Figure 1, the SSR (or tendon jerk), and its electrical analog, the H-reflex, are mediated largely by a two-neuron, monosynaptic 5

32 pathway consisting of the Ia afferent fiber, its synapse on the alpha motoneuron, and the motoneuron itself (Magladery et al., 1951; Matthews, 1972; Baldissera et al., 1981; Henneman and Mendell, 1981; Brown, 1984). Because it is influenced by descending activity from the brain, this pathway can be operantly conditioned. Monkeys, humans, rats (including humans and rats with spinal cord injuries) and mice can gradually increase or decrease the SSR or its electrical analog, the H-reflex, in response to an operant conditioning protocol (Wolpaw et al., 1983; Wolpaw, 1987; Evatts et al., 1989; Chen and Wolpaw, 1995; Carp et al., 2005; and reviewed in Wolpaw, 1997, 2001, and Wolpaw and Tennissen, 2001). Since evidence of learning persists even after all descending activity is abolished (Wolpaw and Lee 1989), the learning involves plasticity in the spinal cord itself. Figure 2 presents the conditioning protocol and its results. The rat H-reflex protocol is shown. The mouse, monkey and human H-reflex and SSR protocols are equivalent to it and provide comparable results (Wolpaw et al., 1983; Wolpaw, 1987; Evatts et al., 1989; Chen and Wolpaw, 1995; Carp et al., 2005). Reward is given if reflex size is above (up-conditioning) or below (down-conditioning) a criterion. A key feature of the design is that the time of reflex elicitation is unpredictable. Thus, because this reflex is the earliest possible CNS response, the subject can change its size only by maintaining continual appropriate descending influence over the spinal arc of the reflex. By linking reward to reflex size, the protocol operantly conditions the subject to maintain such influence. This influence changes reflex size appropriately over days and weeks. As Figure 2B illustrates, the 6

33 H-reflex in rats nearly doubles under the up-conditioning mode, or falls by almost half under the down-conditioning mode. This reflex change occurs without change in background EMG, M response, daily number or distribution of trials, or muscle weight (Chen and Wolpaw, 1995; reviewed in Wolpaw and Carp, 1990 and Chen and Wolpaw, 1999). This change is an adaptive (i.e., mode-appropriate) response to the reward contingency. This adaptive change occurs in two phases, a small rapid phase I in the first few hours or days, and a much slower phase II that continues for weeks (Wolpaw and O'Keefe, 1984; Wolpaw et al., 1994; Chen et al., 2001a). Phase I appears to reflect rapid mode-appropriate change in descending influence over the spinal arc of the reflex, while phase II reflects gradual spinal cord plasticity produced by the chronic continuation of the descending input responsible for phase I. The continuation of this descending influence over the period of conditioning changes the spinal cord (Wolpaw and Lee, 1989) and increases (up-conditioning mode) or decreases (down-conditioning mode) the H-reflex. (3) Relevance of H-Reflex Conditioning to Real Life Probably because the monosynaptic pathway of the SSR and H-reflex is the best-defined spinal cord reflex pathway, this simple behavior is frequently used in the laboratory and in clinical protocols. However, in normal life it is also a part of much more complex behaviors. This reflex pathway participates in posture and locomotion, in sophisticated athletic and technical skills, and in the abnormal motor control associated 7

34 with spinal cord injuries and other neurological disorders (reviewed in Wolpaw and Tennissen, 2001). Much evidence suggests that plasticity like that seen in the laboratory model contributes to motor development in childhood, learning of motor skills later in life, deficits caused by trauma and disease, and the functional effects of therapeutic procedures (Riddoch, 1917; Kuhn, 1950; Shurrager and Dykman, 1951; Myklebust et al., 1982, 1986; Lovely et al., 1986; Meyer-Lohmann et al., 1986; Barbeau and Rossignol, 1987; Koceja et al., 1991; O'Sullivan et al., 1991; Nielsen et al., 1993; Wernig et al., 1995, 1998; Whalen and Pearson, 1997a, 1997b; de Leon, 1998, 1999; O'Sullivan et al., 1998; Ronthal, 1998; Barbeau et al., 1999, 2002; Hiersemenzel et al., 2000; Edgerton and Roy, 2002; Rossignol et al., 2002; Cohen and Hallett, 2003; Dietz and Harkema, 2004; Edgerton et al., 2004; Ying et al., 2005; Wolpaw and Tennissen, 2001 for review). In the newborn infant, SSRs exist in both agonist and antagonist muscles during muscle stretch (Myklebust et al., 1986; O'Sullivan et al., 1991). Antagonist SSRs gradually disappear during childhood (Myklebust et al., 1986). However, in patients with perinatal supraspinal damage (e.g., cerebral palsy) that impairs descending influence, antagonist SSRs still remain (Myklebust et al., 1982; O'Sullivan et al., 1998). As Figure 3A shows, in the absence of normal descending influence, antagonist SSRs may persist into adulthood, and contribute to motor dysfunction. In normal adults, SSRs and H-reflexes are affected by the nature, intensity, and duration of motor training. When monkeys trained to make smooth movements are exposed to random perturbations, the SSRs elicited by the perturbations gradually increase, and the increase accounts for 8

35 improvement in performance (Meyer-Lohmann et al., 1986) (Figure 3B). In professional ballet dancers, H-reflexes and SSRs in the legs (and Group Ia reciprocal inhibition as well) are much smaller than in other highly trained athletes (Koceja et al., 1991, 2004; Nielsen et al., 1993) (Figure 3C). The decreased direct peripheral influence on motoneurons indicated by the smaller reflexes may effectively increase cortical control and allow more precise movement. In cats, partial denervation of hindlimb extensor muscles gradually produces both spinal and supraspinal plasticity that changes the strength of remaining group I inputs during locomotion, and this change helps compensate for the functional deficits produced by denervation (Whalen and Pearson, 1997a, 1997b; reviewed in Wolpaw and Tennissen, 2001). In spinalized animals, treadmill training causes activity-dependent plasticity that improves locomotor function (Shurrager and Dykman, 1951; Lovely et al., 1986; Barbeau and Rossignol, 1987; de Leon, 1998, 1999; Rossignol et al., 2002). In humans, disorders that disrupt descending activity, such as spinal cord injury or stroke, also produce gradual long-term plasticity in spinal cord reflexes, and these changes contribute to spasticity and other disabling problems (Riddoch, 1917; Kuhn, 1950; Ronthal, 1998; Hiersemenzel et al., 2000; Chen et al., 2002a). Treadmill training in humans with spinal cord injuries can improve locomotion and reduce the need for assistive devices (Wernig et al., 1995, 1998; Barbeau et al., 2002; Edgerton and Roy, 2002; Dietz and Harkema, 2004; Cauraugh and Summers, 2005; Ying et al., 2005). As these examples illustrate, H-reflex conditioning represents a phenomenon that plays an integral part in the acquisition and maintenance of motor skills 9

36 throughout life, in the functional deficits that occur with trauma or disease, and in the mechanisms that compensate for these deficits (Wolpaw and Tennissen, 2001 for review). They also indicate that changes in H-reflex or SSR activity do not occur independently of other alterations in spinal cord function. (4) The Spinal Cord Plasticity Associated with H-Reflex Conditioning Since the H-reflex is produced primarily by the Ia afferent synapse on the "- motoneuron and by the "-motoneuron itself, the size of the H-reflex depends on the state of these two CNS elements. Both are influenced by descending pathways from supraspinal regions (Burke and Rudomin, 1978; Baldissera et al., 1981; Chen and Wolpaw, 1997, 2002; Chen et al., 2002b). Exposure to the up- or down-conditioning mode is thought to cause operantly conditioned, mode-appropriate change in this supraspinal influence. As noted above, since the H-reflex is elicited at an unpredictable time and occurs before any other CNS response, the animal can only increase reward frequency by being prepared ahead of time, i.e., by continuously controlling the descending activity so as to keep the spinal reflex arc in the proper state. As a result, mode-appropriate supraspinal influence is present for lengthy periods of each day over the many days of up- or down-conditioning. This long-term supraspinal influence over the spinal arc produces activitydependent plasticity in the spinal cord. This is first shown by the fact that the reflex asymmetry created by the conditioning persists after the removal of descending input: it 10

37 survives for at least several days after the spinal cord is isolated from the brain (Wolpaw and Lee, 1989). Intracellular studies in monkeys and rats indicate that down-conditioning is accompanied by a positive shift in firing threshold, a modest decrease in Ia EPSP amplitude, and by a decrease in axonal conduction velocity (Carp and Wolpaw, 1994; Carp et al., 2001) (Figure 4A). Both the threshold shift and the change in conduction velocity are best explained by a positive shift in sodium channel activation voltage (Halter et al., 1995). The change in threshold can largely account for the H-reflex decrease (Carp and Wolpaw, 1994; Carp et al., 2001). Results also suggest that downconditioning and up-conditioning are not mirror images of each other, but rather have different mechanisms (Carp and Wolpaw, 1994, 1995). Down-conditioning appears to be due mainly to change in the motoneuron itself (Carp and Wolpaw, 1994; Halter et al., 1995). In contrast, H-reflex up-conditioning seems, on the basis of physiological and anatomical data, to be the result of changes in interneurons that convey group I homonymous (and synergist) input to the motoneuron (Carp and Wolpaw, 1994; Halter et al., 1995). Anatomical studies indicate that H-reflex conditioning also includes changes in synaptic terminals on the motoneurons (Feng-Chen and Wolpaw, 1996, 1997; Maniccia et al., 1996, 1997; Pillai et al., 2004; Wang et al., 2004, 2006). Figure 4B illustrates that the average size on F terminal, a presumably inhibitory terminal on the motoneuron cell body, is smaller, and that the number of active zones in this terminal decreases after upconditioning in comparison to down-conditioning. In addition, there have been recent 11

38 studies showing that in rats after successful H-reflex down-conditioning, there is an increase in size and density of the GABAergic terminals on the motoneurons (Figure 4C, adapted from Wang et al., 2004, 2006). Thus, the physiological and anatomical studies to date indicate that H-reflex conditioning produces a complex pattern of spinal cord plasticity. It causes plasticity at multiple sites in the spinal cord (reviewed in Wolpaw, 1997, 2001; Wolpaw and Tennissen, 2001). (5) Spinal Cord Plasticity Underlying H-Reflex Conditioning Depends on Supraspinal Descending Influence Descending pathways from supraspinal areas presumably convey the modeappropriate influence over the H-reflex arc that eventually produces spinal cord plasticity and changes H-reflex size. This assumption was confirmed by studies of the effects of spinal cord lesions on H-reflex conditioning. In rats, contusion injuries of the thoracic spinal cord impaired H-reflex conditioning, and the degree of impairment was correlated with the amount of white matter destroyed (Chen et al., 1996, 1999). As shown in Figure 5, the amount of H-reflex decrease (under the down-conditioning mode) or increase (under the up-conditioning mode) is inversely correlated with the severity of the injury as assessed histologically (or by time to return of bladder function) (Chen et al., 1996). Upconditioning and down-conditioning were similarly sensitive to injury (Chen et al., 1999). The results confirm the importance of descending control from supraspinal structures in mediating the operantly conditioned change in H-reflex size. 12

39 These studies also indicate that, after thoracic spinal cord contusion that reduces the spinal cord crosssection at the lesion by 60-80%, some rats retain the capacity for H- reflex conditioning (Chen et al., 1996, 1999). Thus, in some rats, mode-appropriate change in activity in surviving descending fibers was able to support conditioning, indicating that operant conditioning could still modify function after injury. These data are consistent with data from humans with partial spinal cord injuries which showed that more than 60% of individuals with cervical cord injuries were able to reduce the amplitude of the biceps brachii SSR (Segal and Wolf, 1994). (6) Spinal Cord Pathways Essential for H-Reflex Conditioning To identify spinal cord pathways essential for H-reflex conditioning, studies have been performed to investigate the effects of specific pathway transections at T8-9 on the H-reflex and on its down- and up-conditioning (Figure 6A-D; modified from Chen and Wolpaw, 1997, 2002; Chen et al., 2002b, 2003). Figure 6E shows the four transections performed in these studies. They consisted of: 1) transection of the entire right lateral column (LC) containing the rubrospinal, reticulospinal, and vestibulospinal tracts (which are largely or totally ipsilateral at this level) (Kuypers, 1981; Holstege and Kuypers, 1987; Kennedy, 1990; Tracey, 1995); 2) transection of the dorsal column (DC) containing the main corticospinal tract (CST) and the dorsal ascending tract (DA) (Kuypers, 1981; Holstege and Kuypers, 1987; Kennedy, 1990; Tracey, 1995); 3) transection of the CST alone; and 4) transection of the DA alone. 13

40 In control rats without conditioning, transection of LC, DC, CST, or DA produced an immediate (i.e., first-day) temporary increase in the size of the H-reflex (Chen et al., 2001b). Most important, for all of the transections, the size of the H-reflex and the M response, as well as the background EMG, returned to or very close to pre-transection values within two weeks (Chen et al., 2001b). This excluded the possibility of the effect of lesion itself on the H-reflex at the end of pathway transection studies. In the experimental groups (with one of the four transections or no transection as shown in Figure 6E), the rats were exposed to the control mode for days and then to the down- or up-conditioning mode for 50 days (see Figure 6A and 6C for the protocol for these studies) to investigate the effects of LC, DC, CST, or DA transection on the ability to decrease or increase the H-reflex in response to the down- or up-conditioning protocol (Chen and Wolpaw, 1997, 2002; Chen et al., 2002b). Figure 6B shows the final H-reflex for LC, DC, CST and DA rats after down-conditioning as percent of initial H- reflex size and as compared with data from down-conditioned normal rats (Chen and Wolpaw, 1997, 2002). The five groups differed significantly from one another and this difference was due to the DC and CST rats. Down-conditioning was similarly effective in normal, LC, and DA rats: for each of these groups, the H-reflex decrease was significant, and the final values for LC, DA and normal rats were similar. In contrast, DC and CST rats did not decrease the H-reflex under the down-conditioning mode and the final H-reflex sizes were significantly different from the final sizes for normal, LC, or DA rats. As indicated in the preceding section, CST or DC transection has no lasting effect on the 14

41 H-reflex itself. Thus, the implications of Figure 6B are that the CST is essential for acquisition of H-reflex decrease and that the rubrospinal, reticulospinal, and vestibulospinal tracts (located in the LC (Kuypers, 1981; Holstege and Kuypers, 1987; Kennedy, 1990; Tracey, 1995)) and the DA tract are not essential (Chen and Wolpaw, 1997, 2002). Figure 6D shows the final H-reflex for LC, DC, CST, and DA rats after upconditioning as percent of initial H-reflex size and as compared with data from upconditioned normal rats (Chen et al., 2002b). As in down-conditioning, up-conditioning results for the DA and LC groups were comparable to those in the normal group. For each of these groups, the H-reflex increase was significant, and the final values for LC and DA rats did not differ from those of normal rats. In contrast, the DC and CST rats did not increase the H-reflex after exposure to the up-conditioning mode, and final H-reflex sizes were significantly different from those for normal rats. Over the entire period of data collection in all rats, background EMG and M response amplitude remained stable. The results indicate that the main CST is essential for acquisition of H-reflex increase, and that the LC (which contains rubrospinal, reticulospinal, and vestibulospinal tracts) and the DA tract are not essential. Thus, the results for acquisition are similar for up- and down-conditioning. These results are consistent with human studies indicating that people with spinal cord injuries can be conditioned to increase or decrease the SSR while those with strokes involving sensorimotor cortex (the main origin of the CST) cannot (Segal and Wolf, 1994; Segal, 1997). 15

42 (7) Locomotion and its Motor Control Locomotion is active, rhythmic movement with the goal of transition of the body in space (Grillner, 1981). The characteristic of rhythmicity and alternation simplifies the neural systems that control locomotion. On the other hand, considerable flexibility of locomotion is required for animals to adapt to and survive various environmental conditions (Grillner, 1981; Marder and Calabrese, 1996). A neural network within the spinal cord, i.e., the central pattern generator (CPG), is responsible for coordinated rhythmic locomotor activities (Shik et al., 1969). It contributes to alternating flexor and extensor muscles for rhythmic stepping (Calancie, 1994; Edgerton et al., 1976; Marder and Calabrese, 1996). In the normal condition, the CPG is under the control and modulation of descending input from supraspinal structures, intersegmental (propriospinal) influences, and segmental sensory afferent inputs from the periphery. It has been demonstrated that in animals, the CPG can generate locomotor-like activity even when the cord is isolated from the brain (Shik et al., 1969). In normal animals, the CPG is also involved in the integration of the supraspinal descending inputs and movement-dependent feedbacks for various forms of walking (Buford et al., 1990; Buford and Smith, 1990, 1993). The basic pattern produced by a CPG is usually modified by sensory inputs from peripheral receptors and signals from supraspinal descending pathways and from other regions of the central nervous system. Input from proprioceptive receptors is involved in automatically regulating the timing and amplitude of the stepping pattern (Pearson, 1993, 16

43 1995). The afferent signals from flexor and extensor muscles as well as the Golgi tendon organs contribute to the transition of locomotion from stance phase to swing phase. Stimulation of afferent fibers from the extensor muscles prolongs the stance phase and often delays the onset of swing phase until the stimulation has terminated (Whelan et al., 1995). Therefore, the swing phase is not initiated unless the limb is unloaded (near the end of leg extension when the animals weight is being borne by the other legs and the extensor muscles are shortened and thus unable to produce optimal forces) and the forces exerted by extensor muscles are low, which is signaled by a decrease in activity from Golgi tendon organs. Proprioceptive feedback from muscle spindles and Golgi tendon organs also play a significant role in the generation of burst activity in extensor motor neurons. In humans, spindles of extensor muscles account for up to 30% of the excitatory input to the ankle extensor motor neurons (Yang et al., 1991). In decerebrate cats, static and dynamic gamma motor activities during locomotion show distinctive patterns (Taylor et al., 2000). They play roles in regulating sensitivity of muscle spindle during posture and locomotion, although they are largely independent of EMG activity (Bennett et al., 1996) and vary in different muscles during locomotion (Murphy et al., 2002). We now have quite a good understanding of the neuronal mechanisms generating the motor pattern for walking in animals. The CPG for each hind leg is distributed within the lumbar region of the spinal cord, with the more anterior segments (L1 to L2 in the rat, or L2 to L4 in the cat) appearing to contain the primary elements in the network (Shik and 17

44 Orlovsky, 1976; Grillner, 1981; Armstrong, 1986; Cazalets et al., 1995; Rossignol, 1996; Kremer and Lev-Tov, 1997; Kiehn and Kjaerulff, 1998; Rossignol et al., 2002). Figure 7 summarizes our current understanding of locomotor generation and control in the animal (Rossignol, 1996; Rossignol et al., 2002). The locomotor pattern involves a sequential and bilateral activation of muscles of different joints. The CPG is capable of activating motoneurones in an appropriate sequence. It also sets the excitability of interneurons involved in transmitting information from descending pathways and sensory afferents through which corrections are continuously occurring during various phases of the locomotor cycle. It is thought that the CPG can be activated through different descending pathways through propriospinal interneurons. In most animals, there is also a segmental locomotor-generating mechanism, and rostral and caudal segments may play different roles. Excitatory and inhibitory neurotransmitters can also activate, block or modulate the activity of the CPG. Peripheral afferent inputs do not appear to be essential for the generation of the basic rhythm of the CPG although they are crucial for the appropriate and adaptive expression of the locomotor pattern (Rossignol, 1996; Rossignol et al., 2002). Walking in humans appears to have its own unique characteristics. Its pattern and motor control may not simply be summarized as alternating flexor and extensor muscles for rhythmic stepping. By comparing selected studies on the neural control of human walking with similar studies in reduced animal preparations, Capaday (2002) found that the switch mechanism of walking in the animal preparations differs from that in humans. 18

45 In human walking, group-i afferent feedback from ankle extensors contributes little to the stance-to-swing transition. Capaday proposed that the human spinal CPG, if it exists, is much less robust than those in other mammals (such as rats and cats), and suggested that the neural control of human walking may need to be understood in its own terms (Capaday 2002). There are three types of excitatory pathways involved in the process of conveying information from the extensor sensory fibers to extensor motor neurons: a mono-synaptic pathway from Ia fibers; a disynaptic pathway from Ia and Ib fibers; and a polysynaptic pathway from Ia and Ib fibers, which controls the stance duration as well as the level of extensor activity. Thus, the spinal cord takes charge of the generation and regulation of the basic motor pattern for stepping. On the basis of this locomotion pattern, the supraspinal structures generate and fine-tune more complex voluntary movements. Control from the supraspinal region initiates and controls the speed of walking through descending inputs. While the alternating pattern of hindlimbs during stepping is due to the reciprocal activation of the inhibitory interneuron system of extensor and flexor burst within the spinal cord, the descending control from the brain can affect this spinal circuit. Therefore, the supraspinal region and sensory feedback from the periphery are responsible for the adaption to the environment (Pearson, 1993; Nielsen, 2004). Since reflex conditioning induces plasticity in supraspinal structures and descending influence on the spinal cord, and since it modifies the spinal cord (Wolpaw and Lee, 1989; Carp and Wolpaw, 1994, 1995; Feng-Chen and Wolpaw, 1996; Chen et al., 2004a, 2004b, 2005a; 19

46 Chen and Wolpaw, 2005; Wang et al., 2006; Wolpaw and Chen, 2006), it could provide a means for individuals with spinal cord injury to induce appropriate changes in the brain, in descending influence on the spinal cord, and in the spinal cord itself, including the sensory feedback from the periphery to improve locomotor function. (8) Reflex Conditioning Could Help to Maximize Restoration of Function after Spinal Cord Injury Spinal cord injury triggers a cascade of biological events and leads to motor and reflex deficits and/or dysfunction, sensory loss, chronic pain, and dysfunction in the control of automatic systems (e.g., respiratory impairment, bowel dysfunction, bladder dysfunction, sexual dysfunction, etc.) (McDonald, 1999, 2002; McTigue et al., 2000; Beattie et al., 2002; Sadowsky et al., 2002; Schwartz and Fehlings, 2002). Because spinal cord locomotor function is normally under the control of supraspinal descending influence and input from the periphery, when spinal cord injury disrupts neural circuits in the spinal cord and the supraspinal descending control, pathological spinal cord plasticity occurs and abnormal reflexes, spasticity, and other motor abnormalities appear (Fujimori et al., 1966; Ashby et al., 1974; Ashby and Verrier, 1975; Davis, 1975; Dimitrijevic et al., 1988; Little and Halar, 1988; Burney et al., 1992; Shefner et al., 1992; Doyle et al., 1993; St. George, 1993; Stein et al., 1993; Calancie et al., 1994). Impairment in locomotor function greatly reduces the quality of life of thousands of individuals with spinal cord injury. According to the National Spinal Cord 20

47 Injury Statistical Center (NSCISC), each year in the United States alone, there are over 11,000 new cases of spinal cord injury reported, and a total of approximately one quarter of a million Americans are currently living with a spinal cord injury (DeVivo, 1997). Most of them have an incomplete injury, with some preservation or recovery of sensory and/or motor function caudal to the level of lesion (Go et al., 1995). They are suffering from deficits and disorders brought by the injury. Current actual or potential strategies for treatment of spinal cord injury generally include, in the acute stage (i.e., shortly after the injury): minimizing the lesion: preventing initial tissue loss; inhibiting inflammation; applying neuroprotection; minimizing scar tissue formation; and reducing factors that lead to secondary injury (Bregman, 1998; McDonald, 1999, 2002; Aldskogius and Kozlova, 2002; Gimenez y Ribotta et al., 2002; Schwab, 2002). In the chronic stage, potential treatments include: promoting axonal regeneration and establishing proper connections: replacing destroyed cells; and training or re-educating spared and/or regenerated spinal cord neural circuitry (Olson, 1997; McDonald, 1999, 2002; McTigue et al., 2000; Barbeau et al., 2002; Edgerton and Roy, 2002; Reier et al., 2002; Gimenez y Ribotta et al., 2002; Schwab, 2002). Spinal cord injury affects patients with changes in the central nervous system including loss of the descending inputs that initiate and control locomotion, with changes in the musculoskeletal system (including problems of muscle activation, muscle weakness, hyperactive spinal reflexes, increased stiffness of the ankle joint, inability to bear weight and maintain balance, and increased fatigability of the motor unit), and with changes in 21

48 the electromyographic patterns (including increased tibialis anterior muscle activation during swing and reduced triceps surae activity (Dietz et al., 1981)). Thus, to treat individuals with spinal cord injury, it is not only necessary to re-connect the interrupted spinal cord reflex pathways, but also to train regenerated spinal cord neural circuitry to establish proper functional connections. Aimed at improving the quality of life of spinal cord injured individuals, some treatment strategies can enhance the recovery of motor function following injury. Thus, pharmacological agents and exercise regimens (such as locomotor training, assisted (i.e., weight-supported) walking, computer-assisted gait training, and functional electrical stimulation assisted walking) have been explored in individuals with spinal cord injury to improve their mobility (Merletti et al., 1978; Kralj and Bajd, 1989; Granat et al., 1992; Nakamura et al., 1992; Quintern, 1998; Remy-Neris et al., 1999; Barbeau et al., 2002; Dietz and Harkema, 2004). Individuals with spinal cord injuries are benefitting from these strategies and showing some improvement in their walking ability. As reviewed above, activity-dependent plasticity can still occur after spinal cord injury. Therefore, mobility of patients with spinal cord injury could be improved by taking advantage of the plasticity of the central nervous system. Reflex conditioning can induce activity-dependent plasticity which affects supraspinal structures, descending inputs, and the spinal cord, as well as sensory feedback from the periphery. Therefore, reflex conditioning could provide a new therapeutic strategy for individuals with spinal cord injury for improving locomotor function. Reflex conditioning has a number of 22

49 advantages: it is safe, non-invasive (in humans with surface electrodes), and easy to conduct (Evatts et al., 1989; Segal and Wolf, 1994; Segal, 1997); it can be readily automated so as to minimize the need for the continuing involvement of therapists and other personnel; and it can be used alone or in conjunction with other rehabilitation strategies. Once techniques for achieving significant spinal cord regeneration become available, reflex conditioning could be valuable, perhaps even essential, for re-educating a restored spinal cord to function effectively. (9) Summary The plasticity in spinal cord reflex pathways that occurs during development and continues throughout life is an essential foundation for normal motor function. Spinal cord injury can lead to pathological spinal cord plasticity that alters the strengths and specificities of intraspinal connections, producing abnormal reflexes, spasticity, and other motor abnormalities. Operant conditioning of the spinal stretch reflex (SSR) or its electrical analog, the H-reflex, provides a new method to induce long-term plasticity within the central nervous system including 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. Recent physiological, anatomical, and immunohistochemical studies in conditioned animals have shown that this reflex conditioning produces plasticity at multiple sites in the spinal cord. 23

50 Studies of the effects of spinal cord lesions on H-reflex conditioning indicate that contusion injuries of the thoracic spinal cord in rats impair H-reflex conditioning and that the degree of impairment is correlated with the amount of white matter destroyed. These results confirm the importance of descending control from supraspinal structures in mediating operantly conditioned changes in H-reflex size. Further studies with specific pathway transections indicate that the corticospinal tract (CST) is essential for acquisition of both down- and up-conditioning whereas the rubrospinal, reticulospinal, and vestibulospinal tracts (located in the lateral column) and the dorsal column ascending tract are not. These results are consistent with human studies indicating that people with spinal cord injuries can be conditioned to increase or decrease the SSR while those with strokes involving sensorimotor cortex (the main origin of the CST) cannot. These results have significant implications for rehabilitation of spinal-cord injured people and animals: that as long as the CST remains functional (or can be restored in the future by methods that produce regeneration), animals or humans with extensive spinal cord injuries should still be able to increase or decrease the H-reflex by means of an operant-conditioning protocol. Although studies have demonstrated that reflex conditioning can produce changes in the spinal cord, and suggested that it may provide a means for modulating and guiding functional recovery in individuals with spinal cord-injury, these studies have not explored whether and how reflex function during locomotion will be affected by reflex conditioning. Before reflex conditioning can be used as a new strategy for helping 24

51 individuals with spinal cord injury, it is necessary to understand whether reflex conditioning can affect normal locomotion, and whether it can improve abnormal locomotion such as that observed after spinal cord injury so that it can be used to correct abnormal gait conditions. (B) PRELIMINARY STUDIES (1) Recording EMG and H-Reflexes during Treadmill Locomotion In order to explore the possibility that reflex conditioning might be used to improve locomotion after spinal cord injury, we have developed techniques for recording EMG activity and eliciting H-reflexes during treadmill locomotion. Figure 8A shows EMG recorded from right and left soleus muscles during locomotion from a normal rat. In order to establish the relationship between the soleus locomotor burst and the stance phase of locomotion, a video camera recorded (at 60 frames/sec) treadmill locomotion from the right side while EMG data were collected. A custom offline analysis program used the recorded video to determine for each step cycle the time during which part or all of the right hind foot was within 1 mm of the treadmill surface. This time was defined as the right stance period, and its timing was compared to that of the EMG burst from the right soleus. Figure 8B illustrates the typically close relationship between the onsets of the soleus EMG burst and the stance phase of locomotion. 25

52 (2) Effects of a Unilateral Spinal Cord Injury (i.e., LC Transection) on Locomotion To determine whether H-reflex conditioning could improve locomotion after spinal cord injury, a well-defined spinal cord injury that produced a well-defined locomotor deficit is needed. As spinal cord pathway transection studies indicate, destruction of the entire ipsilateral LC does not affect the ability to operantly condition the H-reflex. In an initial study, we found that this LC lesion produces a clear locomotor asymmetry. In normal rats, the right and left EMG activities are symmetrical: Figure 9 (left) shows soleus EMG activity during treadmill locomotion from a normal rat and demonstrates symmetrical right and left soleus EMG bursts. In addition, the gait appears to be symmetrical: the time interval from the onset of the right burst to that of the left burst is about the same as the time interval from the onset of the left burst to that of the right burst. In contrast, rats with right lateral column transection show asymmetrical EMG activity. Figure 9 (right) shows soleus EMG activity during treadmill locomotion from a rat with a transection of the right LC. The time from onset of the right soleus burst to onset of the left soleus burst is substantially shorter than the time from the onset of the left soleus burst to the onset of the right soleus burst. This finding is consistent with other earlier studies (Gorska et al., 1993; Zmyslowski et al., 1993). It implies that the right stance phase of locomotion is defective: it ends more quickly than it should. This asymmetric locomotion appears to be permanent as it persists for at least three months following the injury. Thus, we showed that right LC transection produces a well-defined 26

53 locomotor abnormality (defective right stance) and provided an abnormality that we could attempt to reduce through H-reflex conditioning. (3) Evidence that H-Reflex Conditioning Affects Function during Locomotion Preliminary data indicated that H-reflex conditioning affects reflex function during locomotion. Figure 10A shows the H-reflex amplitude elicited during locomotion from an up-conditioned rat (left) and from a down-conditioned rat (right) before (solid line) and after (dashed line) successful up- or down-conditioning. The H-reflex elicited during treadmill walking became much larger in the up-conditioned rat and became much smaller in the down-conditioned rat after conditioning. The background EMG and M response before and after the conditioning in both rats were comparable. These initial results suggest that the change in motoneuron response to group I input caused by H-reflex conditioning is not limited to the situation of the conditioning protocol, but is also expressed under the dynamic state of locomotion. Figure 10B (left) shows the soleus H-reflex elicited during treadmill locomotion from a right LC transected rat before (solid line) and after (dashed) up-conditioning. Figure 10B (right) shows the superimposed average rectified soleus EMG bursts from this rat before (solid line) and after (dashed) up-conditioning. The soleus H-reflex elicited during locomotion increased dramatically and the EMG bursts became larger and lasted longer after up-conditioning. These preliminary results suggest that by increasing soleus 27

54 response to agonist group I input, up-conditioning of the right soleus H-reflex might reduce the gait asymmetry produced by the LC transection. Figure 10C shows the soleus H-reflex (left) and the average rectified soleus EMG (right) during locomotion before (solid line) and after (dashed line) down-conditioning from a rat with a 0.7-mm contusion injury. The H-reflex was smaller and the soleus EMG burst was shorter after the down-conditioning. These preliminary data suggest that it may be possible to use H-reflex conditioning to change reflex function during locomotion after spinal cord injury if only the CST or its altered replacement remains functional (or can be restored in the future by methods that produce regeneration). These data also suggest that conditioning of H-reflex or other spinal cord reflexes could help to induce and guide functional recovery after spinal cord injury. (C) AIMS OF THIS PROJECT The central goal of this project was to determine whether chronic conditioning of the H-reflex can help to modulate and guide functional recovery after spinal cord injury. The central hypotheses, supported by previous studies and preliminary data, are: (1) that H-reflex conditioning affects reflex function during locomotion, that up-conditioning will increase reflex function and that down-conditioning will decrease it; and (2) that H-reflex conditioning can reduce asymmetry in locomotion after spinal cord injury and that appropriate reflex conditioning can improve locomotion and restore some function. These hypotheses were tested by first exploring in normal rats the interactions of H-reflex 28

55 conditioning with locomotion. In the first study (Section IV), three questions will be asked: first, whether the H-reflex change created by conditioning is affected by locomotion; second, whether H-reflex conditioning affects locomotion, as assessed by the duration and symmetry of the step cycle; and third, whether H-reflex conditioning affects the CNS activity responsible for locomotion. In the next study (Section V), we wished to determine whether H-reflex conditioning could correct a locomotor abnormality caused by a well-defined spinal cord lesion in rats. The results of this project should establish H-reflex conditioning as a new method for inducing and guiding spinal cord plasticity after injury. In combination with other new therapeutic methods (such as those that promote axonal regeneration), chronic reflex conditioning may be able to maximize restoration of function after spinal cord injury. This study may also contribute to development of more effective therapies for individuals with spinal cord injury. 29

56 Figure 1. Main pathway of the spinal stretch reflex (SSR) and its electrical analog, the Hoffman reflex (H-reflex). The pathway consists of the Ia afferent neuron from the muscle spindle, its synapse on the alpha motoneuron, and the motoneuron itself. Ia afferent excitation excites motoneurons innervating the same muscle and its synergists. If the afferent is excited normally, i.e., by muscle stretch, the muscle s response is the SSR. If it is excited by nerve stimulation, i.e., via an electrical stimulation cuff, the response is the H-reflex. The SSR and the H-reflex are typically measured by EMG, as shown by the inset. The first response after nerve stimulation, M response, results from excitation of the efferent fibers. It is followed by Ia afferent excitation of the H-reflex. While their pathway is wholly spinal, both SSR and H-reflex are affected by descending influence (dashed lines) on the Ia terminal (exerted presynaptically) and on the motoneuron, and the SSR is also influenced by descending control of muscle spindle sensitivity (Modified from Chen and Wolpaw, 1999; Wolpaw, 1987). 30

57 Figure 1. Main pathway of the spinal stretch reflex (SSR) and its electrical analog, the Hoffman reflex (H-reflex). 31

58 Figure 2. The H-reflex conditioning protocol and its result (Adapted from Wolpaw, 1997, with permission from Elsevier). A: Soleus background EMG is continuously monitored in a rat with chronically implanted EMG electrodes and a tibial nerve cuff. When background EMG stays within a defined range for a randomly varying s period, nerve stimulation just above M- response threshold elicits an M response (the direct muscle response) and an H-reflex. Under the control mode, no reward occurs after the nerve stimulation, the H-reflex is simply measured to determine its control size. Under the up- or down-conditioning mode, reward (a food pellet) occurs 200 ms after the nerve stimulation if the H-reflex is above (up-conditioning) or below (down-conditioning) a given value. Each rat is first exposed to the control mode for 10 days, and then to the up- or down-conditioning mode for 50 days (Chen and Wolpaw, 1995,1996). B Top: average daily H-reflex for 9 up-conditioned animals (up triangles) and 12 downconditioned animals (down triangles) during the control mode (i.e., days -10 to 0) and during subsequent exposure to the up- or down-conditioning mode (in percent of controlmode value). H-reflex size rises (up-conditioned animals) or falls (down-conditioned animals) over several weeks. B Bottom: average post-stimulus EMG response to the nerve cuff stimulus from an up-conditioned rat (left) and a down-conditioned rat (right) for a day before (solid line) and a day after (dashed line) prolonged up- or downconditioning exposure. The H-reflex is much larger after up-conditioning and much smaller after down-conditioning, while the background EMG (shown here by EMG at zero time) and the M response do not change (Chen and Wolpaw, 1995, 1996). C: Average results for up-conditioning (up triangles) and down-conditioning (down triangles) of triceps surae H-reflex in monkeys (left), biceps brachii SSR in monkeys (middle), and biceps brachii SSR in humans (right). The courses and final magnitudes of change are similar to those found in rats (Wolf et al., 1995; Wolpaw et al., 1994; Wolpaw and O'Keefe, 1984). 32

59 A Stimulus B AMPLITUDE (% of initial value) Rat H-reflex TIME (days) EMG M H Reward EMG (mv) M H M H C AMPLITUDE (% of initial value) TIME (ms) 200 Monkey H-reflex DAY Monkey SSR DAY TIME (ms) Human SSR SESSION Figure 2. The H-reflex conditioning protocol and its result. 33

60 Figure 3. Changes in the spinal stretch reflex (SSR) pathway during development and during the acquisition of motor skill (Adapted from Wolpaw, 1997, with permission from Elsevier). A: EMG responses of soleus (solid lines) and tibialis anterior (dotted lines) muscles to sudden foot dorsiflexion, which stretches the soleus and shortens its antagonist, the tibialis anterior. In a normal infant, this stimulus produces SSRs in both muscles. In a normal adult, an SSR occurs only in the stretched muscle, that is, the soleus; little or no response occurs in the tibialis anterior. In contrast, in an adult with cerebral palsy, in whom perinatal supraspinal injury has impaired the descending influence responsible for the development of normal adult SSRs, the infantile pattern persists: SSRs occur in both soleus and tibialis anterior (The short latency and unusual form of the tibialis anterior response also suggest the presence of central or peripheral abnormalities, or both.) (Myklebust BM, Gottlieb GL (unpublished data comparable to those in Myklebust et al., 1986 and 1982)). B: Working for reward, monkeys performed an elbow flexion-extension task on which brief perturbations (that is, 10-ms torque pulses) were randomly superimposed. Shown are biceps EMG and elbow angle (flexion is upward) for an unperturbed trial (dotted line), a perturbed trial early in training (solid line), and a perturbed trial in training (broken line). Early in training, perturbation elicits both an SSR and a long-latency polysynaptic response (LLR). After intermittent training over several years, the SSR is much larger and the LLR has disapperaed. Over several years, the SSR has gradually taken over the role of opposing the pertubation. This is accompanied by improvement in performance: the deflection superimposed by the torque pulse on the smooth course of elbow flexion is smaller and briefer (Meyer-Lohmann et al., 1986). C: Soleus H-reflexes are much smaller in professional ballet dancers than in other welltrained athletes (for example, runners, swimmers, cyclists). (H-reflexes of sedentary subjects are intermediate.) (Nielsen et al., 1993). 34

61 A B C Figure 3. Changes in the spinal stretch reflex (SSR) pathway during development and during the acquisition of motor skill. 35

62 Figure 4. Spinal plasticity associated with H-reflex conditioning (Adapted from Wolpaw, 1997, with permission from Elsevier). A:H-reflex conditioning affects motoneuron firing threshold, primary afferent EPSP size, and conduction velocity. Top: After down-conditioning of the H-reflex, motoneurons have more positive firing thresholds and smaller Ia EPSPs. These changes can explain why the motoneurons are less likely to fire in response to nerve stimulation (Carp and Wolpaw, 1994). Bottom: Rat soleus motoneuron axonal conduction velocities from control (solid) and down-conditioned (dashed) rats. Conduction velocity is significantly lower after down conditioning in both rats and monkeys (Carp and Wolpaw 1994; Carp et al. 2001). B: Illustration of average F terminals and their active zones on the cell bodies of the motoneurons of monkey triceps surae muscles after H-reflex conditioning. The size of the terminal is smaller in up-conditioned animals than in down-conditioned animals. In addition, the number of active zone (represented by the filled circles and hemi-circle) is less after up-conditioning than down-conditioning, while there is no significant change in the size of the active zone (Feng-Chen and Wolpaw 1996). C: Results of an immunohistochemistry study of GAD 67 -labeled terminals on the soleus motoneuron after H-reflex conditioning. The number, diameter and coverage of GAD 67 -labeled terminals increase in the successful down-conditioning group compared with failed or non-conditioned control group. DS: Down-conditioning successful; DF: Down-conditioning failed; NC: Non-conditioned control (Wang et al., 2006). 36

63 A C B Figure 4. Spinal plasticity associated with H-reflex conditioning. 37

64 Figure 5. Contusion injury of the spinal cord impairs H-reflex conditioning. A: Final H-reflex amplitude in % of the control. While in the normal rat group, the change in final H-reflex size was more than 20% and conditioning was successful (represented by open triangles; the direction of the triangles indicates up- or downconditioning); in the rat group with contusion injury (SCI Rats), conditioning was unsuccessful (represented by filled triangles). This indicates that contusion injury impairs H-reflex conditioning. B: The impairment is significantly correlated with the severity of injury, as assessed by % of white matter remaining. In up-conditioning, those rats with more white matter spared after injury had greater increase and in down-conditioning, they had greater decrease in HR size (Modified from Chen et al., J. Neurotrauma, 1996, 1999). 38

65 FINAL H-REFLEX AMPLITUDE (% of control) A Normal Rats SCI Rats B (R=0.60, P=0.02) (R=-0.86, P=0.001) HRup Rats HRdown Rats WHITE MATTER REMAINING (%) Figure 5. Contusion injury of the spinal cord impairs H-reflex conditioning. 39

66 Figure 6. Effect of pathway transection on H-reflex down-conditioning and upconditioning. A: Protocol for the down-conditioning study. Different pathway transections were performed before the rats were exposed to down-conditioning. B: Final H-reflex size (in % of initial values) for normal, LC, DC, CST, and DA rats.!: successful rat (change in the correct direction $20%); ": unsuccessful rat. LC and DA rats achieved decrease like those of normal rats. DC and CST rats did not decrease the H-reflex (Modified from Chen and Wolpaw, 1997). C: Protocol for the up-conditioning study. Different pathway transections were performed before up-conditioning. D: Final H-reflex size (in % of initial values) for normal, LC, DC, CST, and DA rats.!: successful rat (change in the correct direction $20%); ": unsuccessful rat. LC and DA rats achieved increase like those of normal rats. DC and CST rats did not increase the H-reflex (Modified from Chen et al., 2002). E: Camera lucida drawings (top) and photomicrographs (bottom) of transverse sections of T8-9 spinal cord from a normal rat (with lateral (LC), dorsal (DC), and ventral (VC) columns labeled), an LC rat, a DC rat, a CST rat, and a DA rat. In lesioned rats, the section shown is at the transection epicenter. In the drawings, hatching is gray matter, stippled areas are the CST, and black areas are necrotic debris, cystic cavities, or fibrous septa (Modified from Chen et al., 2001). 40

67 A Pathway Transection Down-conditioning begins Control mode Down-conditioning mode FINAL H-REFLEX SIZE (% of control) B C DAYS None Pathway Transection LC DC CST DA TRANSECTION Conditioning Begins Control Mode HRup Conditioning Mode D FINAL H-REFLEX (% of control) None LC DC CST DA TRANSECTION E Figure 6. Effect of pathway transection on H-reflex down-conditioning and upconditioning. 41

68 Figure 7. General framework of locomotor control in the cat. The EMGs of the inset at the bottom center are taken from Semitendinosus (St, a knee flexor/ hip extensor), Sartorius (Srt, a hip flexor), Vastus Lateralis (VL, a knee extensor), Gastrocnemius Medialis (GM, an ankle extensor) and Triceps brachialis (Tri, an elbow extensor); L and R refer respectively to left and right. DRG, dorsal root ganglion; CPG, central pattern generator; E, extensor motoneurones; F, flexor motoneurones; VLF, ventrolateral funiculus; DLF, dorsolateral funiculi, PS, propriospinal interneurones; L3-S1 refer loosely to various lumbar and sacral spinal segments; NE, norepinephrine; 5-HT, serotonine, DA, dopamine, Glu, glutamate (Adapted from Rossignol et al., 2002 with permission from Elsevier). 42

69 Figure 7. General framework of locomotor control in the cat. 43

70 Figure 8. A: Right (R; top trace) and left (L; bottom trace) soleus EMG recorded while a normal rat walked on the treadmill. B: Relationship between the right soleus EMG and the right stance phase of locomotion (indicated by horizontal lines). As the vertical dashed lines indicate, the onset of the stance phase is closely related to the onset of the soleus burst. As indicated in Materials and Methods, stance was measured as the period during which the foot was within 1 mm of the treadmill surface, which slightly outlasts the period during which the soleus produces force. 44

71 Figure 8. A: Right and left soleus EMG recorded while a normal rat walked on the treadmill. B: Relationship between the right soleus EMG and the right stance phase of locomotion. 45

72 Figure 9. EMG activity during treadmill locomotion from a normal rat (A and B) and from a rat with right lateral column (LC) transection (C and D). A : Rectified right (top) and left (bottom) soleus EMG activity from the normal rat. The filled circles mark the onsets of the right soleus bursts and the open circles mark those of the left soleus bursts. The gait of this normal rat is symmetrical, characterized by the illustration that the time from the onset of the right burst to that of the left burst is about the same as the time from the onset of the left burst to that of the right burst. B: Average of the rectified EMG activity from both soleus muscles during locomotion from this rat. The right (dashed line) and the left (solid line) soleus EMG bursts are symmetrical in this normal rat. C: Rectified right (top) and left (bottom) soleus EMG activity from the LC rat. The filled circles mark the onsets of the right soleus bursts and the open circles mark those of the left soleus bursts. The gait of this right LC rat is asymmetrical, i.e., the time from the onset of the right burst to that of the left burst is clearly shorter than the time from the onset of the left burst to that of the right burst. D: Average of the rectified EMG activity from both soleus muscles during locomotion of the LC rat. The soleus EMG bursts on the right (dashed line, i.e., the lesioned) side appear to be briefer than those on the left (solid line, i.e., the uninjured) side. 46

73 Figure 9. EMG activity during treadmill locomotion from a normal rat (A and B) and from a rat with right lateral column (LC) transection (C and D). 47

74 Figure 10. The effects of H-reflex conditioning on reflex function during locomotion in the preliminary study. A: H-reflex elicited during treadmill locomotion before (solid) and after (dashed) up-conditioning (left) and down-conditioning (right). B: Left: H-reflex elicited during locomotion before (solid) and after (dashed) up-conditioning from a rat with right lateral column transection. Right: Superimposed average rectified soleus EMG bursts before (solid line) and after (dotted line) up-conditioning from this rat. C: Left: H-reflex elicited during locomotion before (solid) and after (dashed) down-conditioning from a rat with a 0.7-mm contusion injury. Right: Superimposed average rectified soleus EMG bursts before (solid) and after (dotted) down-conditioning in this contusion injured rat. 48

75 A EMG (μv) EMG ( ) μv B M H TIME (ms) M H TIME (ms) M H TIME (ms) C EMG (μv) M H Figure 10. The effects of H-reflex conditioning on reflex function during locomotion in the preliminary study. 49

76 CHAPTER 2 GENERAL METHODOLOGY (A) Chronic Operant Conditioning of the H-Reflex Subjects were Sprague-Dawley rats ( g). All animal procedures were in accord with the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (National Academy Press, Washington, DC 1996). They have been reviewed and approved by the Institutional Animal Care and Use Committee of the Wadsworth Center and are regularly re-reviewed and reapproved. The H-reflex conditioning protocol, described in Chen and Wolpaw (1995, 1997, 2002) and in Wolpaw and Herchenroder (1990), is summarized here. To prepare a rat for conditioning, chronic stimulating and recording electrodes were implanted under ketamine and xylazine anesthesia (ketamine HCl (80 mg/kg) and xylazine (10 mg/kg), both intraperitoneal). To elicit the soleus H-reflex, a silicon rubber cuff containing a pair of stainless steel fine-wire electrodes was placed on the posterior tibial nerve just proximal to the TS branches. Fine-wire EMG electrodes with their final 0.5-cm segments stripped and separated by 3-5 mm were inserted in the soleus. The right 50

77 leg (and sometimes the left as well) was implanted, and up to 6 EMG channels were monitored simultaneously (e.g., soleus and lateral and medial gastrocnemii muscles of right leg or soleus of both legs). The wires from all electrodes passed subcutaneously to a small plug mounted on the skull. The Teflon-coated wires from the nerve cuff and the muscle passed subcutaneously to a connector plug mounted on the skull with stainless steel screws and dental cement. Beginning at least 20 days after surgery, data were gathered from each rat for at least 70 days. Throughout this period, the animal lived in a standard rat cage. A flexible 40-cm cable attached to the plug mounted on the skull carried the wires to an electrical swivel above the cage and thence to EMG amplifiers (1000 gain, 100-5,000 Hz bandpass) and nerve-cuff stimulation units. The cable, which allowed the animal to move freely about its cage, remained in place 24 h/day. Rats had full access to water and standard rat chow, except that during H-reflex conditioning they received food mainly as described below. Animal well-being was carefully monitored, and body weight was measured weekly to ensure that rats continued to gain weight normally. Lab lights were dimmed from each day. A computer system interfaced with up to 18 rats simultaneously and continuously. For each animal, it monitored EMG and delivered the nerve cuff stimuli and (during conditioning) a food-pellet reward. If the absolute value (equivalent to the full-wave rectified value) of background (i.e., ongoing) soleus EMG remained within a specified range (usually 1-2% of maximum possible EMG, which was assessed as maximum 51

78 M-response (direct muscle response), see Chen and Wolpaw 1995) for a randomly varying s period, a stimulus pulse (typically 0.5 ms in duration) was delivered to the tibial nerve cuff and elicited the H-reflex. The computer kept pulse amplitude just above M-response threshold (Chen and Wolpaw, 1995; Wolpaw and Herchenroder, 1990). Thus, background EMG and M-response amplitude remained stable throughout data collection. The M response began about 1.5 ms after nerve stimulation and lasted about 3 ms. The H-reflex started at about 5.5 ms and lasted about 4 ms. Under the control mode, the computer simply digitized the soleus EMG and stored the absolute value for 50 ms following the stimulus. Under the HRup (i.e., to increase the reflex amplitude) or HRdown (i.e., to decrease the reflex) conditioning mode, it also delivered a food pellet reward 200 ms after nerve stimulation if the absolute value of soleus EMG during the H-reflex interval (typically ms after stimulus onset) was more (HRup mode) or less (HRdown mode) than a criterion value. The criterion value was selected on the basis of the control-mode data so that reward occurred on 30-40% of the trials. As H-reflex size changed appropriately over days and weeks and reward probability rose, the criterion values were repeatedly changed to keep reward probability around 30%. It is important to note that, as described above, background EMG and M response amplitudes were kept stable throughout study. During its daily activity, each animal usually satisfied the background EMG criterion, and thus received nerve cuff stimulation, 3,000-4,000 times/ day. Trials occurred throughout the 24 hours of the day, but tended to be less frequent in the afternoon (Chen and Wolpaw, 1994). The 52

79 computer provided a daily summary, including average background EMG amplitude and average post-stimulus course of EMG amplitude for each muscle. (As noted above, all online EMG measurements were absolute value, though raw EMG was stored for later analysis.) Daily H-reflex size was defined as average EMG amplitude in the H-reflex interval minus average background EMG amplitude, and was calculated in units of average background EMG amplitude. The computer control of background EMG and stimulus strength ensured that background EMG and M response were stable over the months of data collection. Data were collected from each rat under the control mode for 20 days. These data defined the animal's initial H-reflex size. Then, for the next 50 days, data were collected under the HRup or HRdown conditioning mode protocol, as described above. To determine the final effect of HRup or HRdown mode exposure (or continued control-mode exposure), final H-reflex size was determined by averaging daily H-reflex for the final 10 days, and expressing the result as percent of average H-reflex for the final 10 days in control-mode period. Thus, a value of 100% indicates no change in the leg's H-reflex. As with operant conditioning in the monkey (Wolpaw et al. 1993) and mouse (Carp et al., 2005), a change of 20% in the correct direction was considered evidence of successful conditioning. In addition, each animal's average daily H-reflex sizes for the final 10 days were compared to those of its initial control-mode period by t-test. (As a rule, a change of 20% in the average value is associated with a significant difference by t-test.) 53

80 Rats remained healthy and active throughout data collection. Body weight increased as expected (Chen and Wolpaw, 1995, 1997, 2002; Chen et al., 1999, 2002b, 2003). At the end, all rats were sacrificed by overdose of pentababital and perfused, as described below. Muscle and nerve condition and electrode placements were assessed, implanted muscles were weighed, and the locations and extent of spinal cord lesions were determined as described below. (B) Right Lateral Column (LC) Transection and Post-lesion Animal Care LC transection was performed by electrocautery (small vessel cauterizer, Fine Science Tools) as described in Chen and Wolpaw (1997, 2002) and Chen et al. (2001b, 2002b). Each rat was anesthetized with an intraperitoneal injection of ketamine HCl (80 mg/kg) and xylazine (10 mg/kg). A one-vertebra dorsal laminectomy was performed at T8-T9 with minimal disturbance of the dural envelope, and the rat was positioned securely in a stereotaxic frame with ear bars and vertebral clamps. The cord was visualized under a dissection microscope. The cauterizer was mounted in a micromanipulator, and during transection, was activated in brief pulses to minimize thermal damage to adjacent tissue. The right lateral column transection was produced by lesioning the lateral half of the right side of the spinal cord (i.e., the lateral 0.9 mm of the cord on the right side). After the lesioning, the site was rinsed with saline and covered with Durafilm to minimize connective tissue adhesions to the dura, and the muscle and skin were sutured in layers. 54

81 Immediately after lesioning, the rat was placed under a heating lamp and given analgesia (Demerol, 0.2 mg, i.m.) before it recovered from the anesthesia. Once awake, it was given a second dose of Demerol, returned to its cage, and allowed to eat and drink freely. For the next 7-10 days, it received special care as follows. The bladder was expressed at least three times daily until spontaneous voiding returns (a rat was considered to have bladder control if the bladder was small or empty 5 times within the previous 2-3 days or was empty all day on the most recent day). If the lower abdomen was wet with urine, the area was washed and dried to prevent skin irritation. Antibiotics (Gentocin (Gentamicin Sulfate; 0.25 mg, b.i.d., i.m.) and Flo-Cillin (Sterile Penicillin G Benzathine and Penicillin G Procaine; 15,000 units, q.o.d., i.m.)) and lactated Ringer's solution (5 ml, b.i.d., s.q.) were given until the return of bladder function. In addition, a vitamin C supplement (2 mg/day) and a piece of apple (>10 g) were given from before the lesion until the return of bladder function. Body weight was measured daily. For the first five days after the lesion, each animal was given a soft mash of water-soaked rat chow. A high-calorie dietary supplement (Nutri-Cal; 2-4 ml/day, p.o.) was given until body weight recovered to its pre-lesion level. (C) Assessment of Treadmill Locomotion Each rat was first trained in 2-4 pre-operative sessions to walk on a treadmill (Eco 3/6 treadmill, Columbus Instruments). The pre-operative training was necessary so that rats were able to perform treadmill walking immediately upon initiation of the locomotor 55

82 EMG data-collection period. For each rat, locomotion EMG data were collected twice with a constant treadmill speed ( m/sec): a first time before conditioning (during days -10 to 0) and a second time after conditioning (during days 41-50) (Figure 11 and 17A). During data collection, the rat was transferred from its cage to the treadmill. The flexible cable was connected to an electronic swivel above the treadmill which allowed the animal to walk freely on the treadmill. The computer monitored the EMG activity from the soleus muscle. When the EMG activity reached at a specific point of a step cycle (e.g., near the end of the stance phase), the computer delivered a stimulus to the tibial nerve through the stimulating cuff which elicited the H-reflex. The stimulus amplitude was adjusted by the computer to maintain a stable M response. For each rat, five min EMG data of treadmill walking were collected for each of the three different conditions: (1) stimulus delivered at a point near the end of the stance phase of the step cycle; (2) stimulus delivered at a point near the end of the swing phase of the step cycle; and (3) no stimulus delivered, the walking EMG was simply recorded. EMG data were processed with programs to calculate: the step cycle; the stance and swing phase duration (Figure 8B); the duty cycle (the stance duration as percentage of the step cycle); the time interval from the onset of the right soleus burst to that of the left burst, the time interval from the left onset to the right onset; and the average amplitude of soleus bursts. These data were used to define normal or control EMG and to quantify changes caused by conditioning or spinal cord injury. 56

83 (D) Spinal Cord Histology At the end of data collection, each rat was given an overdose of sodium pentobarbital intraperitoneally and perfused through the heart with saline followed by 4% paraformaldehyde (or 3% paraformaldehyde and 1% glutaraldehyde) in 0.1 M phosphate buffer (ph 7.3). The placement of the EMG electrodes and the nerve cuff and the integrity of the tibial nerve were verified, and the soleus muscles of both sides were removed and weighed. The spinal cord was removed and stored in 10% sucrose in 0.1 M phosphate buffer overnight. The blocks encompassing the transection were embedded in paraffin. Transverse 20-:m-thick serial sections were cut from the paraffin-embedded blocks and stained with Luxol fast blue (for myelinated fibers) and 0.1% cresyl violet (for Nissl substance). Sections encompassing the transection were assessed to determine the location and size of the transection. Camera lucida drawings were made at 50 x magnification. Remaining white matter was identified at 200 x magnification by the presence of normal Luxol fast blue staining. The tracings were enlarged and then digitized (Summagraphics Corp. digitizing pad and Jandel Scientific Sigmascan program), and the tissue remaining at the epicenter of the transection was measured according to the method of Olby and Blakemore (1996). The extent of the lesion was determined as the area of the remaining LC as a percent of the contralateral LC (Chen and Wolpaw, 1997; 2002; Chen et al., 1996, 1999, 2001b, 2002b, 2003). Thus, for example, a value of 20% indicates that at the transection epicenter, 80% of the LC was damaged or 57

84 absent. The border between LC and the ventral column was defined according to Paxinos and Watson (1986). (The following Chapter 3 and 4 are the published/submitted papers (Chen et al., 2005b, 2006 submitted) that resulted from my study.) 58

85 CHAPTER 3 EFFECTS OF H-REFLEX CONDITIONING ON REFLEX FUNCTION DURING LOCOMOTION IN NORMAL RATS (A) Introduction The nervous system maintains a broad repertoire of adaptive behaviors acquired through practice, commonly referred to as skills (Compact OED, 1993). New skills may interfere, or interact in other ways, with old ones. These interactions are often addressed in terms of unitary concepts of memory consolidation, reactivation, and interference (e.g., Shadmehr and Holcomb, 1997; Krakauer et al., 1999; Goedert and Willingham, 2002; Wigmore et al., 2002; Walker et al., 2003; Caithness et al., 2004). However, it is now clear that even the simplest skills involve complex distributed patterns of activitydependent plasticity (Wolpaw and Lee, 1989; Carrier et al., 1997; Cohen et al., 1997; Lieb and Frost, 1997; Thompson et al., 1997; Whalen and Pearson, 1997b; Lisberger, 1998; Garcia et al., 1999; Medina et al., 2000, 2002; Hansel et al., 2001; King et al., 2001; Wolpaw and Tennissen, 2001; Carey and Lisberger, 2002; van Alphen and De zeeuw, 2002; Wolpaw, 2002; Blazquez et al., 2003). A new skill may involve different kinds of plasticity that occur at different sites at different rates and that differently affect 59

86 (and are differently affected by) the similarly complex plasticity associated with an old skill. Thus, the study of interactions between skills can benefit from simple learning models that allow these interactions to be explored on the level of their neuronal and synaptic mechanisms. The spinal cord provides a unique opportunity for such explorations. As the final common pathway for neuromuscular behaviors, its motoneurons, interneurons, and their connections serve the entire behavioral repertoire, and, like the rest of the CNS, undergo activity-dependent plasticity throughout life (Wolpaw and Tennissen, 2001 for review). During early development and during skill acquisition later on, descending input from the brain combines with peripheral input to change the spinal cord so as to ensure satisfactory performance of all the many behaviors that issue from it. At the same time, the relative simplicity and experimental accessibility of the spinal cord and the tracts that connect it to the brain facilitate localization and exploration of the spinal and supraspinal plasticity associated with the acquisition of a new skill. These advantages are best exemplified by the SSR and the H-reflex, which are the simplest motor behaviors. They are mediated primarily by a two-neuron, monosynaptic pathway consisting of the primary afferent (i.e., group Ia or large group II) fiber, its synapse on the motoneuron, and the motoneuron itself (Magladery et al., 1951; Matthews, 1972; Baldissera et al., 1981; Henneman and Mendell, 1981; Brown, 1984). Because it is influenced by descending activity from the brain, this pathway can be operantly conditioned. In response to an operant conditioning protocol, monkeys (Wolpaw et al., 60

87 1983a; Wolpaw, 1987), humans (Evatts et al., 1989), rats (Chen and Wolpaw, 1995), and mice (Carp et al., 2005) can gradually decrease (i.e., down-conditioning mode) or increase (i.e., up-conditioning mode) the SSR or the H-reflex. Acquisition of these simple motor skills (i.e., a smaller or larger H-reflex), which occurs over days and weeks of practice (i.e., of exposure to the conditioning protocol), is associated with complex patterns of activity-dependent spinal and supraspinal plasticity that include changes in motoneuron firing threshold and conduction velocity, in several different synaptic terminal populations on the motoneuron, and probably in spinal interneurons and supraspinal regions as well (Wolpaw, 2001; Wolpaw and Tennissen, 2001). Because spinal motoneurons and interneurons mediate all motor behaviors, the plasticity directly responsible for H-reflex change is likely to affect behaviors other than the H-reflex. For example, the change in motoneuron response to primary afferent input caused by H-reflex conditioning could affect locomotion; and the change in motoneuron firing threshold could affect essentially every behavior. This study explores in normal rats the interactions of H-reflex conditioning with locomotion, an important, well-characterized, and quantifiable skill in which the primary afferent excitation responsible for the H-reflex plays a major role (Grillner, 1981; Yang et al., 1991; Pearson, 1993; Bennett et al., 1996; Stein et al., 2000). It asks: (1) whether the H-reflex change created by conditioning is affected by locomotion; (2) whether H-reflex conditioning affects locomotion, as assessed by the duration and symmetry of the step cycle; and (3) whether H-reflex conditioning affects the CNS activity responsible for 61

88 locomotion, as assessed by the EMG burst during the stance phase. The results illustrate and clarify the interactions of new and old motor skills. (B) Methods Subjects were 18 Sprague-Dawley rats (14 male, 4 female; g at the beginning of the study). (Evaluation of the data from previous studies of 135 normal rats (82 males and 53 females) (Chen and Wolpaw, ; Chen et al., ; unpublished data) indicated that none of the physiological variables involved in this study (e.g., background EMG level, H-reflex size, M response size) differs significantly between males and females.) All procedures satisfied the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (National Academy Press, Washington, D.C., 1996), and had been reviewed and approved by the Institutional Animal Care and Use Committee of the Wadsworth Center. (1) Animal Preparation and Environment Under general anesthesia (ketamine HCl (80 mg/kg) and xylazine (10 mg/kg), both intraperitoneal), each rat was implanted with chronic stimulating and recording electrodes. To elicit the H-reflex, a silicone rubber nerve cuff containing a pair of stainless steel multi-stranded fine-wire electrodes was placed on the right posterior tibial nerve just above the triceps surae branches. To record soleus EMG activity, pairs of fine- 62

89 wire electrodes with the final 0.5-cm stripped were placed in the right and left soleus muscles (only right soleus in four rats). The Teflon-coated wires from the nerve cuff and the muscles passed subcutaneously to a connector plug mounted on the skull with stainless steel screws and dental cement. Immediately after surgery, the rat was placed under a heating lamp and given an analgesic (Demerol, 0.2 mg, intramuscular). Once awake, it received a second dose of analgesic and was returned to its cage and allowed to eat and drink freely. Throughout the study, the rat lived in a standard rat cage with a 40-cm flexible cable attached to the skull plug. The cable, which allowed the animal to move freely in the cage, carried the wires from the electrodes to a commutator above the cage which connected to EMG amplifiers and a nerve-cuff stimulation unit. The rat had free access to water throughout. During H-reflex conditioning, it received most of its food by performing the task described below. Animal well-being was carefully checked several times each day, and body weight was measured weekly. Laboratory lights were dimmed from 2100 to 0600 daily. (2) The H-Reflex Conditioning Protocol Data collection began at least 20 days after the implantation surgery. A computer system continuously monitored (24 h/day) the right soleus EMG and controlled two outputs: the nerve-cuff stimulus and the reward (i.e., a 20-mg food pellet). If the absolute value of background (i.e., ongoing) EMG (i.e., equivalent to the full-wave rectified value) 63

90 remained within a specified range for a randomly varying sec period, a stimulus pulse (typically 0.5 ms in duration) was delivered by the nerve cuff. Pulse amplitude was initially set just above M-response (i.e., the direct muscle response to nerve stimulation) threshold and then continuously and automatically adjusted by the computer in order to maintain M-response size unchanged throughout the whole period of data collection (which ensured that the effective strength of the nerve-cuff stimulus did not change). Under the control mode, the computer simply measured the absolute value of soleus EMG for 50 ms following the stimulus. Under the HRdown or HRup conditioning mode, a food reward was dispensed 200 ms after nerve stimulation if EMG amplitude in the H- reflex interval (e.g., ms after stimulation) was below (HRdown mode) or above (HRup mode) a criterion value. In the course of its normal activity, the animal usually satisfied the background EMG requirement, and thus received nerve-cuff stimulation, 2,500-8,000 times per day. H-reflex size was calculated as average EMG amplitude in the H-reflex interval minus average background EMG amplitude at the time of stimulation, and was expressed in units of average background EMG amplitude. This H-reflex elicited in the conditioning protocol is designated the conditioning H-reflex to distinguish it from the H-reflexes elicited during the stance and swing phases of locomotion, which are designated the locomotor H-reflexes. Figure 11 summarizes the study design. Data were collected under the control mode for 20 days to determine the initial size of the animal s conditioning H-reflex. It was then exposed to the HRdown or HRup mode for 50 days. To determine the final 64

91 effect on conditioning H-reflex size of HRdown or HRup mode exposure, average H- reflex size for the final 10 days of the exposure was calculated as percent of initial (i.e., average of final 10 control-mode days) H-reflex size. As in the past, successful conditioning was defined as a change of $20% in the correct direction (Wolpaw et al., 1993; Chen and Wolpaw, 1995). (3) Treadmill Locomotion and the Locomotor H-Reflexes As indicated in Figure 11, prior to the implantation surgery each rat learned in 1-2 sessions (one per day, min walking per session) to walk quadrupedally on a motordriven treadmill (Eco 3/6 Treadmill, Columbus Instruments, Columbus, OH) at a speed of 9-16 m/min (Burghardt et al., 2004). During this training, the rat was motivated primarily by food reward (bread or cereal). In a few rats, this was supplemented early in training by a weak (0.69-mA, 0.2-s) electric stimulus from a metal grid just behind the posterior end of the treadmill. This minimal aversive stimulus caused no vocalization or other evidence of significant distress and was administered only once or twice per rat. The preimplantation training was effective: when they were placed on the treadmill for actual locomotor data collection later on, rats typically walked immediately. Because rat H- reflex size displays a diurnal variation (opposite in phase to that found in monkeys) (Chen et al., 2002c), locomotor data from each rat were always collected at the same time of day. 65

92 Locomotor data were collected in one treadmill session during the 20 controlmode days and in another treadmill session during the final 10 days of exposure to the HRdown or HRup mode (i.e., Figure 11). For these sessions, the rat was transferred from its cage to the treadmill, and the head-mounted cable was connected to a commutator above the treadmill. For each rat, treadmill speed was the same before and after conditioning. During locomotion, EMG was continuously recorded ( khz bandpass) from right and left (14 rats) (or only right (4 rats)) soleus muscles, and digitized (4.0 khz) and stored by computer, as shown in Figure 8A. In each treadmill session for each rat, data were collected under three different conditions. In Conditions A and B, whenever right soleus EMG satisfied defined criteria (see below) a stimulus pulse (typically 0.5 ms in duration, and kept just above M- response threshold as described above) was delivered by the nerve cuff to elicit the right soleus H-reflex. In Condition A (Figure 12A), the stimulus was delivered when the right soleus EMG remained in a specified high range (and the left soleus EMG remained in a specified low range) for 200 ms. These criteria placed the stimulus past the middle of the right soleus locomotor burst, and thus in the later part of the right stance phase of the step cycle. In Condition B (Figure 12B), the stimulus was delivered when the right soleus EMG remained in a specified low range (and the left soleus EMG remained in a specified high range) for 200 ms. These criteria placed the stimulus late in the right swing phase of the step cycle. Condition C had the same EMG criteria as Condition A. The difference was that no stimulus was actually delivered to the nerve cuff, even though the computer 66

93 continued to generate and record the stimulus trigger. Conditions A and B were used to study the locomotor H-reflexes, that is, the H-reflexes during the stance and swing phases of locomotion, respectively. Condition C examined right and left soleus EMG during undisturbed locomotion. About 5 min of data were collected under each condition (beginning with Condition C) from each rat in each session and were stored for later analysis. (4) Perfusion and Postmortem Examination After data collection was completed for this study, each rat was used in one of several other studies of the long-term effects of H-reflex conditioning, following which it received an overdose of sodium pentobarbital (intraperitoneal) and was perfused through the heart with saline followed by 4% paraformaldehyde solution. The nerve cuff, the EMG electrodes, and the tibial nerve were examined and the soleus muscles of both sides were removed and weighed. (5) Analysis of H-Reflexes During Locomotion The EMG recorded under Conditions A and B was rectified and used to measure H-reflexes during the stance and swing phases of locomotion, respectively, as illustrated in Figure 12. In these offline analyses, the computer triggered on the stimulus and digitized the EMG for up to 500 ms before and after stimulation. It then averaged those trials for which background EMG amplitude (i.e., EMG amplitude for the 20 ms 67

94 immediately prior to the stimulus) and M-response size satisfied specified criteria. These criteria ensured that the H-reflexes before and after conditioning were measured at the same background EMG amplitude and M-response size, and thereby allowed us to assess the effect of conditioning on the H-reflexes. That is, the criteria applied to the stance H- reflex data ensured that the background EMG amplitude and M-response size for the stance H-reflex measured before conditioning were the same as for the stance H-reflex measured after conditioning; and the criteria applied to the swing H-reflex data ensured that the background EMG amplitude and M-response size for the swing H-reflex measured before conditioning were the same as for the swing H-reflex measured after conditioning. (Soleus burst amplitudes typically varied considerably across step cycles. Thus, even when H-reflex conditioning changed the average burst amplitude, there was enough overlap between burst amplitudes before and after conditioning so that these criteria were able to ensure that locomotor H-reflex measurements before and after conditioning were derived only from step-cycles for which soleus burst amplitudes were comparable.) Just as for the conditioning H-reflexes, locomotor H-reflex sizes were calculated as average EMG amplitude in the H-reflex interval minus average background EMG amplitude at the time of stimulation, and were expressed in units of average background EMG amplitude. The impacts of down- and up-conditioning on these reflexes were assessed by comparing the reflexes before and after conditioning by paired t-test. 68

95 (6) Analysis of Locomotion To assess locomotion, the EMG recorded under Condition C (i.e., no nerve-cuff stimulus) was rectified and low-pass filtered by a 50-ms running average. It was then used to assess the right and left soleus locomotor bursts, and these bursts were in turn used to assess step-cycle duration, length, and symmetry. Automated analysis identified the bursts by detecting the points at which soleus EMG reached (i.e., burst onset) and fell below (i.e., end of burst) 10% of its maximum value, and then calculated for each session of each rat the average: step-cycle duration (time between right burst onsets in sec), stepcycle length (treadmill speed in cm/sec times step-cycle duration in sec), right and left burst durations (time from burst onset to offset); right and left burst amplitudes (total EMG area between burst onset and offset divided by burst duration); and step-cycle symmetry. Step-cycle symmetry was defined as the time from right burst onset to left burst onset divided by the time from right burst onset to the next right burst onset (i.e., the time of a full step cycle). Thus, a value of 0.5 indicates that the right/left timing of the step cycle, as assessed by the soleus bursts, was symmetrical. The impact of conditioning on these locomotor measures was assessed by the correlations (determined by linear regression) between changes in them and change in the stance H-reflex. The stance H- reflex was examined closely because it directly reflects the impact of H-reflex conditioning on the soleus response to primary afferent input during the soleus contribution to locomotion (i.e., during the soleus burst). 69

96 (C) Results All rats remained healthy and active and continued to gain weight throughout the study. Body weight increased from 359(±69SD) g at the time of implantation surgery to 515(±109) g at the time of perfusion. At the end of study, soleus muscle weights (measured as percent of body weight) were symmetrical and did not differ significantly between down-conditioned (HRdown) and up-conditioned (HRup) rats. (1) Effects of Conditioning on the Conditioning H-Reflex In magnitude of conditioning H-reflex change and rate of success (i.e., H-reflex change $20% in the correct direction (Wolpaw et al., 1993; Chen and Wolpaw, 1995)), the results of the present study were similar to those of previous studies (Chen and Wolpaw, 1995, 1996; Chen et al., 1999, 2001a, 2002b; Carp et al., 2001). Conditioning was successful in 13 of the 18 rats (8 of 12 HRdown rats (5 of the 8 males, and 3 of the 4 females) and in 5 of 6 HRup rats (all male)). (As noted in Methods, males and females do not differ in any of the physiological variables assessed here.) In the successful HRdown rats, the conditioning H-reflex size fell to 50% (±2%SE); and in the successful HRup rats it rose to 232% (±56%SE). In the remaining 4 HRdown rats and one HRup rat, the conditioning H-reflex remained within 20% of its control size. In all rats, background EMG and M-response size during measurement of the conditioning H-reflex remained stable throughout data collection. 70

97 (2) Effects of Conditioning on the Locomotor H-Reflexes Successful HRdown or HRup conditioning produced comparable changes in the locomotor H-reflexes elicited during the stance and swing phases of locomotion. In the successful HRdown rats, H-reflex size in the stance phase fell to 39% (±8%SE) of its control value (p= by paired t-test), and H-reflex size in the swing phase fell to 59% (±11%SE) (p=0.007). In the successful HRup rats, H-reflex size in the stance phase rose to 252% (±46%SE) (p=0.03), and H-reflex size in the swing phase rose to 229% (±87%SE) (p=0.2). Figure 13 summarizes these results. Figure 14 shows protocol, stancephase, and swing-phase H-reflexes before and after conditioning from one HRdown rat and one HRup rat. After conditioning, the conditioning and locomotor H-reflexes are all smaller in the HRdown rat and larger in the HRup rat. While M response size differed across animals and across the conditioning, stance, and swing reflexes of individual animals (e.g., Figure 14), these differences (which reflect inter-animal differences in M- response recruitment curves and other factors) did not alter the impact of H-reflex conditioning on H-reflex size. In an ancillary effort to assess the likely impact of soleus H-reflex conditioning on synergist muscles during locomotion, gastrocnemius data gathered in earlier studies of soleus H-reflex conditioning have been evaluated (Chen and Wolpaw, ; Chen et al., ; and unpublished data). In 17 rats in which the soleus H-reflex was decreased (9 HRdown rats) or increased (8 HRup rats) by conditioning, the gastrocnemius H-reflex underwent a similar but lesser change. In the HRdown rats, it decreased 24% 71

98 (±17%SE) as much as the soleus H-reflex; and in the HRup rats, it increased 18% (±8%) as much. These results are consistent with primate data on the muscular specificity of conditioning (Wolpaw et al., 1983b), and imply that the gastrocnemius locomotor H- reflexes were affected by conditioning in the same way as the soleus locomotor H- reflexes, but to a lesser degree. The results from the unsuccessful HRdown rats were surprising. As noted above, in these four rats the conditioning H-reflexes after conditioning were within 20% of their control values, and they averaged 99% (±6%SE). In two of the four, the stance and swing H-reflexes after conditioning were also within 20% of their control values. However, in the other two unsuccessful HRdown rats, the stance and swing H-reflexes were markedly larger after conditioning, rising to 358% and 126% in one rat and to 396% and 183% in the other. For one of these two rats, Figure 15 shows conditioning, stance, and swing H- reflexes before and after conditioning. After attempted down-conditioning, the conditioning H-reflex is nearly the same as its control, while the locomotor H-reflexes are much larger than their controls. (3) Effects of Conditioning on Locomotion Table 1 summarizes the impact of conditioning on locomotion. It shows, for successful HRdown and HRup rats, average (±SE) right and left soleus burst amplitudes and durations and average step-cycle duration and right/left symmetry after conditioning, expressed in terms of their control (i.e., pre-conditioning) values. It also shows the 72

99 correlation of the change in each measure with the conditioned change in the stance H- reflex. 73

100 Right Soleus Burst Left Soleus Burst Step Cycle Amplitude Duration Amplitude Duration Duration Symmetry HRdown Rats 0.88 (0.06) 1.08 (0.04) 0.83 (0.14) 1.10 (0.04) 1.05 (0.02) 1.05 (0.03) HRup Rats 1.29 (0.18) 1.09 (0.04) 0.93 (0.21) 1.03 (0.01) 0.99 (0.03) 1.03 (0.01) Correlation p=0.004 NS NS NS NS NS Table 1. Effects of conditioning on locomotion. Average (±SE) right and left soleus burst amplitudes and durations and step-cycle duration and right/left symmetry after conditioning for successful HRdown and HRup rats (expressed in terms of their control values), and the correlations between changes in them and the change in stance H-reflex (with the H-reflex change expressed in terms of the control value of soleus burst amplitude). (NS: No significant correlation (P>0.05)). 74

101 H-reflex conditioning affected the amplitude of the right soleus burst. This amplitude displayed a strong positive correlation with the conditioned change in the stance H-reflex (P=0.004, R=+0.74, slope=+0.40, intercept=1.05). The regression indicates that, on the average, a given percent change in the stance H-reflex was accompanied by a percent change 0.4 times as large in soleus burst amplitude. Figure 16 shows stance H-reflexes and right soleus bursts before and after conditioning from an HRdown rat and an HRup rat. After conditioning, both the stance H-reflex and the soleus burst are smaller in the HRdown rat and larger in the HRup rat. In contrast, H-reflex conditioning did not significantly affect the durations of the right and left soleus bursts, nor the amplitude of the left soleus burst. Furthermore, conditioning did not appear to affect step-cycle duration or right/left symmetry. Since treadmill speed for each rat was the same before and after conditioning, these results also indicate that conditioning did not affect step-cycle length. In the two unsuccessful HRdown rats that showed large increases in their stance H-reflexes (e.g., Figure 15), right soleus burst amplitudes did not change (i.e., 103% and 104% of control after conditioning, respectively). (D) Discussion This study assessed the interactions between a new motor skill - an operantly conditioned decrease or increase in the right soleus H-reflex - and an old motor skill - locomotion in the normal rat. Both of these skills depend on soleus motoneurons and their 75

102 response to primary afferent input. The difference is that the H-reflex depends almost entirely on soleus motoneurons and their primary afferent response, while locomotion depends also on many other motoneuron populations and their responses to many different kinds of input. (1) The Effects of Locomotion on H-Reflex Conditioning The data summarized in Figure 13 and illustrated in Figure 14 show that, in the normal rat, successful H-reflex conditioning was still evident during locomotion. In both HRdown and HRup rats, the locomotor H-reflexes during stance and swing exhibited changes comparable to those of the conditioning H-reflexes. The presence of these changes did not depend on the level of background EMG or on M response size. As Figure 14 illustrates, the H-reflex changes were evident with higher and lower levels of background EMG and with larger and smaller M responses. This persistence contrasts with the H-reflex changes that occur with a switch from standing to walking or from walking to running (Capaday and Stein, 1986, 1987; Stein, 1995; Faist et al., 1996). Unlike these situations, in which the H-reflex changes markedly when the concurrent task changes, the effects of conditioning on the H-reflex did not disappear when the rat began to walk. This persistence of the effects of conditioning on the H-reflex is consistent with the slow time-course of H-reflex conditioning (both during its initial development and during its reversal by exposure to the opposite mode (Wolpaw et al., 1986; Chen and 76

103 Wolpaw, 1996)). The time course implies that the motoneuron plasticity and other spinal cord plasticity that underlies the H-reflex change develops gradually over days and weeks; and thus would not be expected to simply disappear when the rat begins to walk and reappear when it stops. As reviewed in detail elsewhere (Wolpaw, 2001; Wolpaw and Tennissen, 2001), H-reflex change is associated with changes in motoneuron firing threshold and conduction velocity, in F and C terminal populations on the motoneuron, and probably in spinal interneurons and supraspinal regions as well. For example, the changes in motoneuron threshold and conduction velocity may be best explained by a positive shift in the activation voltage of sodium channels throughout the motoneuron, possibly mediated by protein kinase C (Carp and Wolpaw, 1994; Halter et al., 1995). This change would be unlikely to appear or disappear as the rat shifted from one behavior to another. On the other hand, it was theoretically possible that the effect of this plasticity on soleus response to primary afferent input would be blocked during locomotion by a compensatory change in presynaptic inhibition (i.e., a decrease in presynaptic inhibition during locomotion in HRdown rats and an increase in HRup rats), and thus that the conditioned change in the H-reflex would disappear. However, the data summarized in Figure 13 indicate that this did not happen, rather the H-reflex change remained evident during locomotion. 77

104 (2) The Effects of H-Reflex Conditioning on Locomotion While the conditioned change in the H-reflex was not affected by locomotion, it was quite possible that locomotion would be affected by the conditioned change in the H- reflex. Primary afferent input contributes substantially to the locomotor burst that supports stance. It is estimated to be responsible for 35% of the ankle-extensor force in decerebrate cats (Stein et al., 2000), 23% of the triceps surae force in spinalized cats given clonidine (Bennett et al., 1996), and 30-60% of the soleus burst in normal humans (Yang et al., 1991). Thus, the change in the response to this input caused by H-reflex conditioning might be expected to change, or even impair, locomotion. Right-leg stance might collapse in HRdown rats, while in HRup rats it might be characterized by increased plantarflexion of the ankle. Right-leg stance might become briefer in HRdown rats and longer in HRup rats, leading to asymmetries in the step cycle. (In addition, locomotor adjustments to perturbations such as an unexpected obstacle or a sudden change in the slope of the walking surface might be compromised.) However, the present data provide no evidence for gross disturbances in normal locomotion due to H-reflex conditioning. As Table 1 summarizes, neither HRdown nor HRup conditioning appeared to affect stepcycle or soleus burst durations or right/left symmetry. Nevertheless, H-reflex conditioning did have the expected effects on the strength of the right soleus burst. As shown in Table 1 and illustrated in Figure 16, conditioned change in H-reflex size was strongly correlated with change in burst amplitude. Furthermore, the magnitude of the burst change (i.e., 40% of the magnitude of the H- 78

105 reflex change) is consistent with data on the contribution of primary afferent input to the burst (Yang et al., 1991; Bennett et al., 1996; Stein et al., 2000). This finding indicates that the impact of H-reflex conditioning on locomotion was not prevented by compensatory change in muscle spindle sensitivity (i.e., increased in HRdown rats and decreased in HRup rats), or compensatory changes in other synaptic inputs to the motoneuron (i.e., increased excitation in HRdown rats and decreased excitation in HRup rats). H-reflex conditioning had no significant effect on the left soleus burst, which was unchanged or somewhat smaller in both HRdown and HRup rats. This is consistent with primate data indicating that unilateral H-reflex conditioning has little effect on the contralateral H-reflex of the awake animal (Wolpaw et al., 1993) (although it does change the contralateral spinal cord (Wolpaw and Lee, 1989)). In sum, the plasticity produced by H-reflex conditioning did affect CNS function during locomotion, and this effect was not counteracted by changes in muscle spindle sensitivity, presynaptic inhibition, or other synaptic inputs. Conditioning of the right soleus H-reflex changed the muscle s response to primary afferent input during locomotion and also changed the amplitude of soleus activation during stance. Nevertheless, these unilateral changes did not appear to affect step-cycle duration or symmetry. The absence of such effects has several possible explanations. First, since the soleus is only one, and not the largest, of the muscles providing plantarflexion, the change in the soleus burst caused by H-reflex conditioning might not have been sufficient to 79

106 produce a detectable effect on the step cycle. While this is certainly possible, the data on the effects of soleus H-reflex conditioning on the gastrocnemius H-reflex suggest that the gastrocnemius locomotor burst was similarly affected to some extent. This makes it less likely that the effects of conditioning on the locomotor burst were simply too small to affect the step cycle. (Furthermore, as noted below, preliminary data suggest that soleus H-reflex conditioning affects locomotion in spinal cord-injured rats, and thus imply that it has significant kinematic impact.) Second, the change in the soleus (and probably gastrocnemius) locomotor bursts might have induced automatic changes in the activity of other muscles. For example, in HRdown rats, the increase in ankle dorsiflexion caused by the reduced soleus burst might have produced proprioceptive input that increased activity in right quadriceps muscles and thereby increased knee extension during stance so as to balance out the increased ankle dorsiflexion and maintain body height. Such automatic compensatory changes would be constantly present as the rat moved about the cage, and would therefore be expected to be accompanied by additional activity-dependent plasticity. This possibility is consistent with evidence that changes in muscle activation produced by nerve transections induce compensatory changes in other muscles (Whelan and Pearson, 1997b; Pearson et al., 1999; Bouyer et al., 2001). In the present case, the additional compensatory plasticity would ensure preservation of normal locomotion, despite the fact that the pattern of muscle activations underlying locomotion was different from the pattern before H-reflex conditioning. 80

107 Such compensatory plasticity may help account for the multi-site spinal and supraspinal plasticity associated with H-reflex conditioning, particularly those aspects of it that do not seem to underlie the operantly conditioned change in H-reflex size. For example, H-reflex conditioning in monkeys is accompanied by a large decrease in the amplitude of heteronymous (but not homonymous) primary afferent EPSPs that has no obvious relationship to H-reflex change (Carp and Wolpaw, 1994, 1995). Furthermore, while down-conditioning in monkeys does not change the contralateral H-reflex of the awake animal, it greatly increases the contralateral motoneuron response to primary afferent input when the spinal cord is isolated from supraspinal influence (Wolpaw and Lee, 1989). In a perhaps related fashion, transection of the corticospinal tract in successful HRdown rats leads over 10 days to an H-reflex that is significantly larger than the control H-reflex prior to down-conditioning (Chen and Wolpaw, 2002). Such plasticity, which is inexplicable when viewed simply in terms of H-reflex conditioning, may ensure that the change in muscle response to primary afferent input caused by H- reflex conditioning does not change step duration, length, or symmetry, or impair responses to obstacles, grade changes, or other perturbations. (3) The Etiology of Conditioning Failure Four HRdown rats failed to change the conditioning H-reflex (i.e., it remained within 20% of its control value). In two of these four, the locomotor H-reflexes were also not changed by conditioning. However, in the other two, the locomotor H-reflexes were 81

108 much larger after conditioning, despite the fact that neither the conditioning H-reflexes nor the soleus locomotor bursts were changed. As a wholly unexpected finding, this remarkable increase is comparable to the increased contralateral response seen in the isolated spinal cord of HRdown monkeys (Wolpaw and Lee, 1989), and to the largerthan-control H-reflexes that corticospinal tract transection produces in successful HRdown rats (Chen and Wolpaw, 2002) or that the down-conditioning protocol produces in rats with sensorimotor cortex ablations (Chen et al., 2004b). Taken together, these unexpected and apparently inexplicable results imply that the final effect of H-reflex conditioning reflects the combination of several activitydependent processes. In successful rats, the combined effect is an appropriate H-reflex change, while in unsuccessful rats, the combined effect may be no change, or even an inappropriate change. Furthermore, the combined effect may differ depending on the situation in which it is assessed. For example, in the unsuccessful HRdown rat of Figure 15, the H-reflex elicited in the context of the steady-state muscle activity of the conditioning protocol is little changed, while the H-reflex elicited during or between the soleus locomotor bursts is greatly increased. (4) Analysis of Skill Interactions The results suggest that it is not sufficient to address the interactions of new and old motor skills in terms of overarching concepts such as memory consolidation, which are not readily interfaced with the complexity of the neuronal and synaptic mechanisms 82

109 that contribute to each skill. Locomotion is a skill that comprises activity-dependent plasticity at multiple sites. Any new skill that affects one or more of these sites is likely to affect locomotion in some way. The nature of this effect and its ultimate impact on both skills are issues that will yield only to detailed mechanistic investigations, and these are facilitated by simple models such as H-reflex conditioning. It is from such studies that more adequate general concepts are likely to emerge. For example, by indicating that soleus H-reflex conditioning affects how locomotion is produced, the present results suggest that locomotion never undergoes consolidation, but rather is continually maintained by activity-dependent adaptive processes comparable to those responsible for its original acquisition. (5) Possible Applications of H-Reflex Conditioning As noted above, the preservation of locomotion, despite the change in soleus activation and responsiveness produced by H-reflex conditioning, is likely to reflect automatic adjustments by other muscles and possibly additional adaptive activitydependent plasticity, that is, plasticity that is triggered by disturbances that result from the soleus changes and serves to ensure continued normal locomotion. However, when locomotion is already abnormal, due to spinal cord injury or another chronic disorder of supraspinal control, the same automatic adjustments and the same impetus for adaptation to eliminate the impact of H-reflex conditioning may be impaired or absent. In such pathological situations, H-reflex conditioning might be used 83

110 to substantially modify locomotion, or even to restore more effective locomotion. For example, when stance during locomotion is inadequate or inconsistent, the increase in motoneuron response to primary afferent input caused by H-reflex up-conditioning might help to restore more effective or consistent stance. Spinal reflex conditioning is possible in people with partial spinal cord injuries (Segal and Wolf, 1994), and preliminary studies in spinal cord-injured rats suggest that such conditioning can improve locomotion (Chen et al., 2005b). Activity-dependent plasticity in the spinal cord can be induced by sensory input from the periphery or by descending input from the brain (Wolpaw and Tennissen, 2001 for review). The sensory input created by assisted treadmill locomotion can induce spinal cord plasticity and thereby improve locomotion (e.g., Shurrager and Dykman, 1951; Lovely et al., 1986; Barbeau and Rossignol, 1987; de Leon et al., 2002; Dietz and Harkema, 2004). The descending input created by reflex conditioning might also be used to change the spinal cord so as to improve function. Furthermore, as methods for inducing spinal cord regeneration after injury are developed (Schwab and Bartholdi, 1996; McTigue et al., 2000; Selzer, 2003), H-reflex conditioning and conditioning of other simple reflexes (e.g., Chen et al., 2005a) might provide flexible and precise methods for re-educating the regenerated spinal cord so as to maximize recovery of function. 84

111 Figure 11. Study protocol. After learning to walk on the treadmill, each rat was implanted with soleus EMG electrodes and nerve-cuff stimulating electrodes. At least 3 weeks later, it was exposed to the control mode for 20 days and then to the HRdown or HRup conditioning mode for 50 days. Locomotion and H-reflexes during locomotion were assessed on the treadmill during the control period and near the end of the conditioning period. 85

112 Figure 12. Locomotor H-reflexes: elicitation of the right soleus H-reflex during the stance and swing phases of the step cycle. A: Left, Stimulus (arrow) during the right soleus burst elicits the stance H-reflex. Right, Average absolute value of right soleus EMG after stimulation (at 0 ms) during stance. The dotted line indicates the background EMG level at the time of stimulation, and the M response and H-reflex are shaded. B: Left, Stimulus (arrow) when right soleus EMG is low elicits the swing H-reflex. Right, Average absolute value of right soleus EMG after stimulation during swing. Note that M-response size (and thus effective stimulus strength) is similar in the stance and swing phases, whereas the H- reflex is much larger during the stance phase when background EMG is much higher. absol val: Absolute value. 86

113 Figure 12. Locomotor H-reflexes: elicitation of the right soleus H-reflex during the stance and swing phases of the step cycle. 87

114 Figure 13. Effects of conditioning on the conditioning H-reflexes and the locomotor H-reflexes. The average (±SE) final values of conditioning, stance, and swing H-reflexes from successful HRdown and HRup rats are shown. The conditioning and locomotor H- reflexes are similarly decreased in the HRdown rats and similarly increased in the HRup rats. 88

115 Figure 13. Effects of conditioning on the conditioning H-reflexes and the locomotor H-reflexes. 89

116 Figure 14. Conditioning and locomotor H-reflexes before (solid) and after (dotted) conditioning from an HRdown and an HRup rat. The conditioning H-reflexes are each the average of a single day (at least 4000 trials), and the locomotor H-reflexes are each the average of trials obtained during the treadmill session. (In several traces, stimulus artifacts are present in the first millisecond after the stimulus.) After conditioning, the conditioning and locomotor H-reflexes are smaller in the HRdown rat and larger in the HRup rat. absol val: Absolute value. 90

117 Figure 14. Conditioning and locomotor H-reflexes before (solid) and after (dotted) conditioning from an HRdown and an HRup rat. 91

118 Figure 15. Conditioning and locomotor H-reflexes before (solid) and after (dotted) conditioning from an unsuccessful HRdown rat. The conditioning H-reflexes are each the average of a single day (at least 4000 trials), and the locomotor H-reflexes are each the average of trials obtained during the treadmill session. Although the conditioning H-reflex is unchanged after conditioning, the stance and swing H-reflexes are markedly increased. absol val: Absolute value. 92

119 Figure 16. Stance H-reflexes and right soleus locomotor bursts before (solid) and after (dotted) conditioning from an HRdown and an HRup rat. The stance H-reflexes are each the average of trials, and the stance bursts are each the average of bursts obtained during the treadmill session. After conditioning, both the stance H-reflex and the soleus burst are smaller in the HRdown rat and larger in the HRup rat. absol val: Absolute value. 93

120 Figure 16. Stance H-reflexes and right soleus locomotor bursts before (solid) and after (dotted) conditioning from an HRdown and an HRup rat. 94

121 CHAPTER 4 H-REFLEX CONDITIONING REDUCES ASYMMETRY IN LOCOMOTION PRODUCED BY LATERAL COLUMN TRANSECTION (A) Introduction The spinal cord, like the rest of the CNS, undergoes activity-dependent plasticity in development, during skill acquisition, and in response to trauma and disease (Goode and Van Hoven, 1982; Myklebust et al., 1982, 1986; Casabona et al., 1990; Koceja et al., 1991; O'Sullivan et al., 1991; Nielsen et al., 1993; Straka and Dieringer, 1995; Levinsson et al., 1999; Wolpaw and Tennissen, 2001 for review). The activity that induces spinal cord plasticity comes from peripheral sensory receptors via dorsal root input pathways and from the brain via descending pathways. After spinal cord injury, appropriate sensory input can modify the spinal cord so as to improve motor function (Dietz et al., 1995; Bregman et al., 1997; de Leon et al., 1998, 1999; Dobkin, 1998; Wernig et al., 1998; Behrman and Harkema, 2000; Field-Fote, 2000). However, the possibility that appropriate descending input might also be able to improve the function of the injured spinal cord remains largely unexplored. In uninjured animals and humans, operant conditioning of spinal reflexes can modify descending activity and induce spinal cord 95

122 plasticity (Segal, 1997; Chen et al., 2005b). If such conditioning can also induce plasticity in the injured spinal cord, it might be used to improve motor function. The simplest spinal cord reflex pathways are the largely monosynaptic pathway of the SSR and the H-reflex, and the disynaptic pathway of reciprocal inhibition (Magladery et al., 1951; Matthews, 1972; Baldissera et al., 1981; Henneman and Mendell, 1981; Brown, 1984; Chen et al., 2005a). Because these pathways are influenced by descending activity from the brain, they can be operantly conditioned. Monkeys, humans, rats and mice can gradually decrease (i.e., down-conditioning mode) or increase (i.e., upconditioning mode) the SSR, the H-reflex, or reciprocal inhibition (Wolpaw et al., 1983a; Wolpaw, 1987; Evatts et al., 1989; Chen and Wolpaw, 1995; Carp et al., 2005; Chen et al., 2005a). Conditioning depends on the corticospinal tract, and is not affected by transection of other major descending pathways (Chen and Wolpaw, 1997, 2002; Chen et al., 2002b). The reflex changes are associated with complex patterns of plasticity that include modifications in motoneuron firing threshold and conduction velocity, in several different synaptic terminal populations on the motoneuron, and probably in spinal interneurons and supraspinal regions as well (Carp and Wolpaw, 1994, 1995; Feng-Chen and Wolpaw, 1996; Pillai et al., 2004; Wang et al., 2004; Wolpaw, 2001, and Wolpaw and Tennissen, 2001 for review). Furthermore, recent studies indicate that this plasticity can affect locomotion (Chen et al., 2005b). The change in response to primary afferent input caused by H-reflex conditioning affects the muscle s contribution to the step cycle. 96

123 The present study set out to determine whether H-reflex conditioning could correct a locomotor abnormality caused by a well-defined spinal cord lesion in rats. The results suggest that reflex conditioning protocols could provide a precise and practical new method for improving motor function in people with spinal cord injuries or other chronic CNS disorders. (B) Methods Subjects were 13 Sprague-Dawley rats (female; g at the beginning of the study). All rats received right lateral column transection. Eight were then exposed to the H-reflex up-conditioning protocol. The other five were not exposed to conditioning and served as a control group. Figure 17A summarizes the experimental design. All procedures satisfied the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (National Academy Press, Washington, D.C., 1996), and had been reviewed and approved by the Institutional Animal Care and Use Committee of the Wadsworth Center. Throughout the study, all rats were individually housed in standard rat cages. Laboratory lights were dimmed from 2100 to 0600 daily. (1) Electrode Implantation Under anesthesia (ketamine HCl (80 mg/kg) and xylazine (10 mg/kg), intraperitoneal (i.p.)), each rat was implanted with chronic stimulating and recording 97

124 electrodes. To elicit the H-reflex, a nerve cuff was placed on the right posterior tibial nerve just above the triceps surae branches. To record soleus EMG activity, pairs of finewire electrodes were placed in the right and left soleus muscles. The wires from the nerve cuff and the muscles were led subcutaneously to a connector plug mounted on the skull. After surgery, the rat was kept warm and given an analgesic (Demerol, 0.2 mg, intramuscular (i.m.), twice), and was returned to its cage and allowed to eat and drink freely. (2) Lateral Column (LC) Transection Twenty to 41 days after electrode implantation, each rat was anesthetized (as described above) for LC transection. A one-vertebrate dorsal laminectomy was performed at T8-T9, the cord was visualized with a dissection microscope, and the entire right lateral column (i.e., the lateral half of the right side of the cord) was transected by electrocautery. This procedure destroyed the rubrospinal, vestibulospinal, and reticulospinal tracts (Tracey, 2004), while it left intact the main corticospinal tract (CST), which is located at the base of the dorsal column in the rat (Chung et al., 1987; Smith and Bennett, 1987; Cliffer and Giesler, 1989; Tracey, 2004) and is the only major descending path known to be essential for H-reflex conditioning (Chen and Wolpaw, 1997, 2002; Chen et al., 2002b). The site was rinsed and covered with Durafilm to minimize dural adhesions, and the muscle and skin were sutured in layers. The rat was kept warm and received analgesia (see above). Our standard post-injury care protocol (i.e., bladder management, antibiotics, 98

125 food and vitamin supplements) has been described in detail previously (Chen and Wolpaw, 1997, 2002; Chen et al., 1996, 1999, 2002a, 2003) and in Chapter 2 (B). Bladder function returned within 5 days, and rats resumed gaining weight within 22 days. All rats regained their pre-transection weights by 29 days after LC transection, and remained healthy and active and continued to gain weight until the end of study. Body weight increased from 306(±24SD) g just prior to LC transection to 373(±83) g at the time of perfusion. Locomotion, which was markedly impaired immediately after injury (BBB score (Basso et al., 1995) on Day 1: 1(±2SD) on right, 12(±2SD) on left) improved rapidly in subsequent days. By Day 18, locomotion was grossly normal in all rats (BBB in both legs). Nevertheless, assessment of treadmill locomotion (see below) revealed a persistent locomotor asymmetry as described in the Results. (3) Treadmill Locomotion and the Locomotor H-Reflexes Prior to implantation surgery, each rat learned to walk quadrupedally on a motordriven treadmill at 9-10 m/min (Burghardt et al., 2004; Chen et al., 2005b). Locomotor data were collected in one treadmill session days after LC transection (i.e., at least 4 days after the BBB scale had returned to 20-21) and in a second session days later. For HRup rats, the first session occurred prior to up-conditioning and the second occurred in the final 10 days of up-conditioning. In each rat, treadmill speed was the same for both sessions. During locomotion, EMG was continuously recorded ( khz bandpass) from right and left soleus muscles, digitized (4.0 khz), and stored. 99

126 In each treadmill session for each rat, right and left soleus EMG during undisturbed locomotion was recorded. The EMG was rectified and used to calculate (as described in Chapter 2 (C) and 3 (B) (6): step cycle duration (time between right burst onsets in sec); step cycle length (treadmill speed in cm/sec times step-cycle duration in sec); right and left burst durations (time from burst onset to offset); right and left burst amplitudes (EMG area between burst onset and offset); and step-cycle symmetry. Stepcycle symmetry was defined as the time from right burst onset (RBO) to left burst onset (LBO) divided by the time from RBO to the next RBO (i.e., the time of a full step cycle). Thus, a value of 0.5 indicates that the right/left timing of the step cycle, as assessed by the soleus bursts, was symmetrical. In each session, right soleus H-reflexes during the right stance and swing phases of the step cycle was also elicited as described in Chapter 2 (C) and 3 (B) (3). From the rectified EMG, these locomotor H-reflexes were measured as average EMG amplitude in the H-reflex interval minus average background EMG amplitude at the time of stimulation, and were expressed in units of average background EMG amplitude (Chen and Wolpaw, 1995, 1996, 1997, 2002; Chen et al., 2005b). (4) The H-Reflex Conditioning Protocol During the days between the first and second treadmill sessions (50-70 days for rats exposed to the H-reflex up-conditioning protocol (HRup rats), days for those not exposed), each HRup rat had a 40-cm flexible cable attached to the skull plug continuously (i.e., 24 h/d). The cable, which allowed the animal to move freely in the 100

127 cage, carried the wires from the electrodes to a commutator above the cage which connected to EMG amplifiers and a nerve-cuff stimulation unit. The rat had free access to water throughout. During H-reflex conditioning, it obtained most of its food by performing the task described below. Animal well-being was carefully checked several times each day, and body weight was measured weekly. A computer system continuously monitored (24 h/day) right soleus EMG and controlled the nerve cuff stimulus and a reward (i.e., a 20-mg food pellet). If the absolute value of background (i.e., ongoing) EMG (i.e., equivalent to the full-wave rectified value) remained within a specified range for a randomly varying sec period, a stimulus pulse (typically 0.5 ms in duration) was delivered by the nerve cuff. Pulse amplitude was initially set just above M-response (i.e., the direct muscle response to nerve stimulation) threshold and then continuously adjusted by the computer to maintain constant M- response size for the whole period of data collection. Under the control mode, the computer simply measured the absolute value of soleus EMG for 50 ms following the stimulus. Under the up-conditioning (HRup) mode, a reward was dispensed 200 ms after nerve stimulation if EMG in the H-reflex interval (e.g., ms after stimulation) exceeded a criterion value. In the course of its normal activity, the animal usually satisfied the background EMG requirement, and thus received nerve-cuff stimulation 3,000-9,000 times per day. H-reflex size was calculated as average EMG amplitude in the H-reflex interval minus average background EMG amplitude at the time of stimulation, and was expressed in units of average background EMG amplitude. This H-reflex elicited 101

128 in the conditioning protocol is called the conditioning H-reflex to distinguish it from the locomotor H-reflexes elicited during the stance and swing phases of locomotion. Data were collected under the control mode for at least 10 days to determine the initial size of the rat s conditioning H-reflex. It was then exposed to the HRup mode for 50 days. To determine the final effect on conditioning H-reflex size of HRup mode exposure, average H-reflex size for the final 10 days of the exposure was calculated as percent of initial (i.e., average of final 10 control-mode days) H-reflex size. As in the past, successful conditioning was defined as a change of $20% in the correct direction (Wolpaw et al., 1993; Chen and Wolpaw, 1995). H-reflex up-conditioning was successful (i.e., increase $20% from control H- reflex size (Wolpaw et al., 1993; Chen and Wolpaw, 1995)) in 7 of the 8 rats (i.e., success rate of 87.5%). In these rats, the size of the conditioning H-reflex rose to 198(±45SE)% of control. (In the one unsuccessful rat, final conditioning H-reflex size was 98% of control.) The success rate and average final increase were comparable to previous values for normal rats and LC rats (Chen and Wolpaw 1995,1997, 2002; Chen et al., 1996,1999, 2001a, 2002a,b; Carp et al., 2001, and unpublished data), and thus further confirmed that LC transection does not impair up-conditioning. In all rats, background EMG and M- response size during measurement of the conditioning H-reflex remained stable throughout data collection. The seven successful rats constituted the experimental group of HRup rats for evaluation of the effects of up-conditioning on treadmill locomotion. The five rats not exposed to up-conditioning constituted the Control group. 102

129 (5) Perfusion, Postmortem Examination, and Lesion Verification After data collection was completed, each rat received an overdose of sodium pentobarbital (intraperitoneal) and was perfused through the heart (Chen and Wolpaw, 1997, 2002; Chen et al., 2002b). Nerve cuff, EMG electrodes, and tibial nerve were examined, and soleus muscles of both sides were removed and weighed. Soleus muscle weights (measured as percent of body weight) were symmetrical and did not differ significantly between HRup and Control groups, nor did they differ from soleus muscle weights of normal rats (Chen and Wolpaw, 1995, 1996, 1997, 2002; Chen et al., 2002b, 2005b) The spinal cord was removed and blocks encompassing the transection were embedded in paraffin. Transverse 20-:m-thick serial sections from the paraffinembedded blocks were processed and used to determine the location and size of the lesions as previously described (Chen and Wolpaw, 1997, 2002; Chen et al., 2002b). (C) Results (1) Histology In the 13 LC rats, 28(±18SD)% of the right and all the left LC, 93(±25SD)% of the right and 96(±13SD)% of the left dorsal column corticospinal tract (CST), 76(±28)% of the right and 97(±7)% of the left dorsal column ascending tract (DA), and 91(±22)% of the right and all the left ventral column (VC) remained. Lesion size was comparable in HRup and Control rats. In the 8 HRup rats, 29(±19SD)% of the right and all the left LC, 103

130 all the right and left CST, 78(±23)% of the right and 98(±4SD)% of the left DA, and 75(±39)% of the right and all the left VC remained. In the 5 Control rats, 27(±19SD)% of the right and all the left LC, 81(±43)% of the right and 90(±22)% of the left CST, 83(±30)% of the right and all the left DA, and 95(±10)% of the right and all the left VC remained. There is no significant difference between these two groups (P>0.2 for all comparisons by t-test). Figure 17B shows camera lucida drawings of T8-T9 transverse sections through the lesion epicenter for one of the 13 rats. (2) Effects of LC transection In the first treadmill session, prior to up-conditioning of the HRup group, locomotor H-reflexes and burst amplitudes and durations were not significantly different from those found in normal rats. However, the rats displayed a clear asymmetry in the onset times of the right and left soleus bursts. The time from right soleus burst onset (RBO) to left soleus burst onset (LBO) was less than the time from LBO to RBO. It averaged 0.43(±0.02SE) of the step cycle, different (P=0.015, paired t test) from the 0.50 expected. (In the 11 normal rats of an earlier study (Chen et al., 2005b), the average value was 0.50(±0.03SE).) Thus, in the LC rats of this study, the time from RBO to LBO averaged only 77% of the time from LBO to RBO, implying that the right stance phase of locomotion was shortened. 104

131 (3) Effects of up-conditioning on locomotor H-reflexes and the soleus locomotor bursts To determine the effects of H-reflex up-conditioning, the data from the first and second treadmill sessions were compared for the Control and HRup groups. Figure 18 summarizes the effects on the sizes of the right soleus locomotor H-reflexes (A) and on the amplitudes (B) and durations (C) of the right and left soleus locomotor bursts. For the Control group, the results for the second session are very similar to those of the first session; there are no significant differences. In contrast, for the HRup group, the locomotor H-reflexes and the right soleus burst amplitude are significantly larger in the second session (P=0.003 and P=0.04 by paired t-test, respectively). As in normal rats (Chapter 3), the increase in the stance H- reflex was comparable to that in the conditioning H-reflex (i.e., increases to 280(±38SE)% and 212(±49)%, respectively). The increase in the right burst amplitude, to 144(±15SE)%, indicates that, on the average, an increase in the stance H-reflex was accompanied by an increase 0.32 times as large in soleus burst amplitude. This relationship is consistent with previous results from normal rats (Chapter 3) and with other data on the contribution of primary afferent input to the locomotor burst (Yang et al., 1991; Bennett et al., 1996; Stein et al., 2000). Furthermore, the data from normal rats showing that H-reflex up- and down-conditioning have the expected effects on soleus burst amplitude (i.e., increase and decrease, respectively) indicate that the present 105

132 increase is not a non-specific effect of simply eliciting the H-reflex over the conditioning period, but rather is a specific effect of the up-conditioning protocol. Although no significant effects are present on left burst amplitude or right or left burst durations, the data suggest that up-conditioning was accompanied by an increase in left burst amplitude, as well as by a decrease in left burst duration and an increase in right burst duration. Figure 19B illustrates the effects of up-conditioning on the stance H-reflex and the right burst amplitude and duration. In the HRup rat, the H-reflex and the burst are much larger, and the burst lasts longer, in the second session. In the control rat (Figure 19A), the H-reflex and the burst do not change. (4) Effects of up-conditioning on the step cycle Figure 20 summarizes the effects of up-conditioning on step-cycle duration (A), and on the right/left asymmetry in the step-cycle (B) present in the first session. Stepcycle duration did not change in either group. Since for each rat treadmill speed was the same in the two sessions, this result also indicates that the length of the step-cycle did not change. In the Control group, the asymmetry remained in the second session: the portion of the step-cycle between RBO and LBO remained shorter than the portion between LBO and RBO. However, the asymmetry was gone in the HRup group. The contrast between the groups is particularly striking because in the first session the asymmetry was slightly 106

133 (though not significantly) greater in the HRup group than in the Control group. The RBO- LBO duration of the Control group averaged 0.45(±0.02SE) of the step-cycle in the first session and 0.46(±0.01) in the second; while the HRup group averaged 0.42(±0.04) in the first session and increased to a normal value of 0.52(±0.04) in the second. Figure 21 illustrates the elimination of the asymmetry in an HRup rat. It shows right and left soleus bursts before and after up-conditioning with each RBO (!) and LBO (") marked. The short vertical dashed lines mark the midpoints between RBOs, which is the time when LBOs should occur (as they do in normal rats). Prior to up-conditioning, LBO occurs too early; after up-conditioning, it occurs on time. (D) Discussion (1) The locomotor asymmetry and the effect of up-conditioning Right LC transection created a clear locomotor abnormality that was evident in both HRup and Control LC rats in the treadmill session prior to up-conditioning and persisted in the Control LC rats in the session after up-conditioning. The time from right soleus burst onset (RBO) to left burst onset (LBO) was substantially longer than the time from LBO to RBO. In the light of evidence for the close relationship between the onset of the soleus burst and the onset of the stance phase of locomotion (Chapter 1 (B) (1)), this timing asymmetry implies that right LC rats did not sustain right stance as well as normal rats. This failure could reflect a tendency of the right burst to rise more quickly and 107

134 decline earlier in LC rats than in normal rats (e.g., Figure 9B). It could also reflect injuryinduced abnormalities in motoneuron recruitment and firing that impair the translation of muscle activation (reflected in EMG) to muscle force (reflected in stance maintenance) (Chen et al., 2005b). Up-conditioning of the H-reflex restores right-left symmetry: after conditioning, the time from RBO to LBO equals that from LBO to RBO. Previous evidence of the correspondence between soleus and gastrocnemius activity and evidence that soleus conditioning has a similar though smaller effect on the gastrocnemius H-reflex (Wolpaw et al., 1983b; Chen et al., 2005b), implies that this restored symmetry occurs also in stance onset. The probable origin is the marked increase in the strength of the locomotor burst (i.e., Figure 19). As noted above, this increase is consistent with the expected increased contribution of primary afferent input to the burst (Yang et al., 1991; Bennett et al., 1996; Stein et al., 2000; Chen et al., 2005b). It is also possible that conditioning improves the translation of motor unit activation to actual force production. (2) Possible therapeutic applications The results suggest that H-reflex conditioning could be useful for restoring more normal function to people with partial spinal cord injuries who retain corticospinal tract (CST) function. The present study and previous studies of the effects of pathway specific transections in rats (Chen and Wolpaw, 1997, 2002; Chen et al., 2002b) indicate that the only major descending tract needed for conditioning is the CST; and this is supported by 108

135 evidence that conditioning of the biceps spinal stretch reflex is possible in people with partial spinal cord injuries and is not possible in people with strokes involving sensorimotor cortex, the origin of the CST (Segal and Wolf, 1994). At the same time, the long-term persistence of the effects of conditioning remains uncertain. Available evidence from monkeys indicates that, when exposure to the protocol is stopped, up-conditioning declines with a half-life of about 17 days, while down-conditioning persists essentially unchanged for at least four weeks (Wolpaw et al., 1986); and studies in humans with partial spinal cord injuries indicate that the effects of down-conditioning are still evident four months later (Segal and Wolf, 1994). It is possible that the functional effects of H- reflex conditioning might require periodic reinforcement by re-exposure to the conditioning protocol. On the other hand, in individuals with partial spinal cord injuries in which conditioning had improved locomotion, the improved locomotion itself might be sufficient to ensure the persistence of the adaptive reflex change. The two sources of activity-dependent spinal cord plasticity are sensory inputs from the periphery and descending inputs from the brain. Recent studies in animals and humans indicate that the patterned sensory inputs induced by locomotor training regimens can induce spinal cord plasticity and improve function after spinal cord injury (Dietz et al., 1995; Bregman et al. 1997; de Leon et al., 1998, 1999; Dobkin, 1998; Wernig et al., 1998; Field-Fote, 2000). The present study shows that an operant conditioning protocol can produce descending input that also induces spinal cord plasticity and improves function. These two new therapeutic approaches might complement each other very 109

136 effectively. While locomotor training regimens create complex stereotyped patterns of sensory inputs aimed at restoring complex behaviors, operant conditioning protocols can focus on changing specific spinal cord pathways, that is, the individual elements that underlie and support complex behaviors. Conditioning protocols could be selected to address the particular deficits of individual patients. H-reflex conditioning offers several possibilities (e.g., up- or down-conditioning of right or left soleus/gastrocnemius or tibialis anterior muscles), and reciprocal inhibition can also be conditioned (Chen et al., 2005a). Conditioning protocols could be largely automated so as to require minimal therapist involvement. They might also be used to address other functional abnormalities such as spasticity (St George, 1993; Boorman et al., 1996; Hiersemenzel et al., 2000) or even disorders of autonomic function (Colachis, 1992; Weaver et al., 2002). The ability to target specific reflex pathways and deficits could be particularly important once significant regeneration is possible (Kuffler, 2000; Schwab and Bartholdi, 1996; McTigue et al., 2000; Selzer, 2003). A partially or even wholly regenerated spinal cord will probably not provide normal or perhaps even effective function immediately. Methods for reeducating it, for inducing adaptive plasticity comparable to that occurring during normal development, will be essential. In this context, H-reflex conditioning and comparable conditioning of other simple reflexes could help to shape spinal cord function so as to maximize the recovery of motor control. 110

137 (3) Future directions The present study shows that spinal reflex conditioning could be a clinically practical method for inducing and guiding spinal cord plasticity to help restore function after spinal cord injury. However, before it can be used as an important new method for restoring function, further development of this new therapeutic approach and demonstration of its clinical value in humans with spinal cord injuries are needed. Future studies should aim to define the capabilities and characteristics of this new treatment method. It would be necessary to determine whether properly chosen reflex conditioning can improve a variety of different walking problems associated with spinal cord injuries. This can be done by evaluating, in animals with a range of different spinal cord injuries, the effects on walking of carefully selected reflex conditioning. Future studies should also seek to determine the practical clinical value of this new treatment approach, i.e., demonstrate in humans with spinal cord injuries that reflex conditioning can improve walking. Therefore, it would be necessary to test in humans whether reflex conditioning can improve walking in patients with partial spinal cord injuries and whether these improvements persist. This should probably be done in a individual-specific manner, i.e., to evaluate each person's motor deficits and then design a reflex training program to reduce those deficits and thereby improve motor function. Future studies should also explore whether reflex conditioning can be useful for problems other than abnormal walking, for example, treatment of spasticity developed after spinal cord injury. We know that spinal cord injury in humans is associated with 111

138 increased stretch reflexes and flexor reflex afferent reflexes (Barbeau and Norman, 2003; Dietz, 2000; Roby-Brami and Bussel, 1987; Schmit et al., 2002), and with decreases in Ia-reciprocal inhibition, recurrent inhibition, and presynaptic inhibition (Boorman et al., 1991, 1996; Morita et al., 2001; Mazzocchio and Rossi, 1997; Faist et al., 1994; Hiersemenzel et al., 2000; Dietz, 2000). These reflex abnormalities are thought to contribute to spasticity. As spasticity is more pronounced in patients with incomplete spinal cord injuries than in those with complete injuries (Little et al., 1989; Young 1994), using reflex conditioning to ameliorate spastic symptoms would be particularly promising. H-reflex down-conditioning might be able to decrease hyperactive stretch reflexes and thus to relief muscle spasm. In addition, conditioning of other reflex pathways such as reciprocal inhibition might be used to restore a more normal level of Iareciprocal inhibition (Chen et al., 2005a). Thus conditioning of reciprocal inhibition might provide with an additional effective reflex conditioning method for treatment of spasticity. 112

139 Figure 17. A: Experimental design. After learning to walk on the treadmill, each rat was implanted with soleus EMG electrodes and nerve-cuff stimulating electrodes. Several weeks later it was subjected to an LC transection. At least 16 days after the transection when locomotion was grossly normal (i.e., BBB in both legs), the rat was exposed to the control mode for at least 10 days and then to the HRup conditioning mode for 50 days (HRup group) or was not exposed to the HRup conditioning protocol (Control group). Background EMG amplitude and M response size were stable throughout. Locomotion and H-reflexes during locomotion were assessed on the treadmill during the control period and near the end of the conditioning period. B: Camera lucida drawings of transverse sections of T8-9 spinal cord from a normal rat (with lateral column (LC), dorsal column corticospinal tract (CST), dorsal column ascending tract (DA), and ventral column (VC) labeled) and from an LC-transected rat. In the LC rat, the section shown is at the lesion epicenter. Hatching indicates gray matter and stippled areas are the main corticospinal tract. Scale bar indicates 1 mm. 113

140 Figure 17. A: Experimental design. B: Camera lucida drawings of transverse sections of T8-9 spinal cord from a normal rat and from an LC-transected rat. 114

141 Figure 18. Effects of up-conditioning on locomotor H-reflexes and soleus locomotor bursts. Average (±SE) locomotor H-reflexes (A) and left and right soleus burst amplitudes (B) and durations (C) for Control rats (open bars) and HRup rats (solid bars) for the second treadmill session in percent of their values for the first (i.e., initial) session. Asterixes indicate significant changes from the first to the second session (*: P<0.05; **: P<0.01). Locomotor H-reflexes and right soleus burst amplitudes are increased in the HRup rats. The Control rats show no significant change. 115

142 A ** 300 Locomotor H-Reflexes Percent of Initial * 0 B200 Stance Swing Burst Amplitude * Percent of Initial 100 C100 Percent of Initial 0 50 Left Right Burst Duration 0 Left Right Figure 18. Effects of up-conditioning on locomotor H-reflexes and soleus locomotor bursts. 116

143 Figure 19. Illustration of locomotor H-reflexes and soleus locomotor bursts from a control rat (A) and from a up-conditioned one (B). The main traces are average right soleus bursts and the inset small traces are average stance H-reflexes from a Control rat and an HRup rat for the first (solid) and second (dotted) treadmill sessions. (For the H-reflexes shown, average background EMG and M response size were the same for the first and second sessions (Chen et al., 2005b for method). The horizontal scale bars are 2 ms. for both the Control and HRup rats, while the vertical scale bars are 50 and 200 :V, respectively.) In the second session, the stance H-reflex and the right soleus burst amplitude (and duration) are increased in the HRup rat. The Control rat shows no change in the H-reflex or in the burst. 117

144 A200 Control Rat EMG (μv) TIME (ms) B HRup Rat EMG (μv) TIME (ms) Figure 19. Illustration of locomotor H-reflexes and soleus locomotor bursts from a control rat (A) and from a up-conditioned one (B). 118

145 Figure 20. Effects of up-conditioning on the step-cycle. A: Average (±SE) step-cycle durations, and B: RBO-LBO (right soleus burst onset to left soleus burst onset) duration of the Control rats (open bars) and of the HRup rats (solid bars) for the second treadmill session as percent of their values for the first (i.e., initial) session. ** indicates a significant change from the first to the second session (P<0.01). Step-cycle duration is unchanged in both Control and HRup rats. However, RBO-LBO duration (which was lower than normal in the first treadmill session) is increased in HRup rats only. 119