Breathe, Walk and Chew, Volume 187: The Neural Challenge: Part I (Progress in Brain Research)

List of Contributors I. Arsenault, Faculté de médecine dentaire and Groupe de Recherche sur le Système Nerveux Central du FRSQ, Université de Montréal, Succursale Centre-ville, Montreal, Quebec, Canada O. Britz, Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA R.M. Brownstone, Department of Surgery (Neurosurgery) and Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B.R. Brush, Department of Applied Science, McGlothlin-Street Hall, The College of William & Mary, Williamsburg, Virginia, USA T.V. Bui, Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada J.-M. Cabelguen, Neurocentre Magendie, INSERM U 862, Université de Bordeaux, Bordeaux Cedex, France J. Champagnat, C.N.R.S., Centre de Recherche de Gif-sur Yvette—C.N.R.S., FRC 3115, bât. 33, 91198, Gif-sur-Yvette, France F. Clarac, CNRS, P3M, Chemin Joseph Aiguier, Marseille, Cedex, France C.A. Del Negro, Department of Applied Science, McGlothlin-Street Hall, The College of William & Mary, Williamsburg, Virginia, USA R. Dubuc, Département de Kinanthropologie, Université du Québec à Montréal, Montréal, Québec, Canada. Groupe de Recherche sur le Système Nerveux Central, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada. A. El Manira, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden J.L. Feldman, Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA G. Fortin, C.N.R.S., Centre de Recherche de Gif-sur Yvette—C.N.R.S., FRC 3115, bât. 33, 91198, Gif-sur-Yvette, France J.-F. Gariépy, Groupe de Recherche sur le Système Nerveux Central, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada A. Giraudin, Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA M. Goulding, Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA K.S. Grossmann, Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA R.M. Harris-Warrick, Department of Neurobiology and Behavior, Seeley G. Mudd Hall, Cornell University, Ithaca, New York, USA

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J.A. Hayes, Department of Applied Science, McGlothlin-Street Hall, The College of William & Mary, Williamsburg, Virginia, USA A. Ijspeert, Swiss Federal Institute of Technology, Lausanne (EPFL), Lausanne, Switzerland Y. Kang, Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, Yamadaoka, Suita, Osaka, Japan A. Kolta, Faculté de médecine dentaire and Groupe de Recherche sur le Système Nerveux Central du FRSQ, Université de Montréal, Succursale Centre-ville, Montreal, Quebec, Canada A. Kyriakatos, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden S. Lamarque, Neurocentre Magendie, INSERM U 862, Université de Bordeaux, Bordeaux Cedex, France R. Lavoie, Faculté de médecine dentaire and Groupe de Recherche sur le Système Nerveux Central du FRSQ, Université de Montréal, Succursale Centre-ville, Montreal, Quebec, Canada K. Missaghi, Département de Kinanthropologie, Université du Québec à Montréal, Montréal, Québec, Canada. Groupe de Recherche sur le Système Nerveux Central, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada P. Morquette, Faculté de médecine dentaire and Groupe de Recherche sur le Système Nerveux Central du FRSQ, Université de Montréal, Succursale Centre-ville, Montreal, Quebec, Canada J. Morris-Wiman, Department of Orthodontics, University of Florida College of Dentistry, JHMHSC, Gainesville, Florida, USA E. Nanou, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden R.W. Pace, Department of Applied Science, McGlothlin-Street Hall, The College of William & Mary, Williamsburg, Virginia, USA D. Ryczko, Groupe de Recherche sur le Système Nerveux Central, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada L. Saint-Amant, Département de pathologie et biologie cellulaire, Groupe de Recherche sur le Système Nerveux Central, Centre d' Excellence en Neuromique de l'Université de Montréal, Université de Montréal, Montréal, Québec, Canada M. Saito, Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, Yamadaoka, Suita, Osaka, Japan H. Sato, Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, Yamadaoka, Suita, Osaka, Japan R. Teruyama, Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA M. Thoby-Brisson, C.N.R.S., Centre de Recherche de Gif-sur Yvette—C.N.R.S., FRC 3115, bât. 33, 91198, Gif-sur-Yvette, France H. Toyoda, Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, Yamadaoka, Suita, Osaka, Japan D. Verdier, Faculté de médecine dentaire and Groupe de Recherche sur le Système Nerveux Central du FRSQ, Université de Montréal, Succursale Centre-ville, Montreal, Quebec, Canada C.G. Widmer, Department of Orthodontics, University of Florida College of Dentistry, JHMHSC, Gainesville, Florida, USA J. Zhang, Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA

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Subject Index

Active Notch signaling, 23 Amyotrophic lateral sclerosis (ALS), 6 Androgen. See Hormonal influences Asynergia, 7 Atonal homolog 1 (Atoh1/Math 1), 42–43 Axial locomotor networks. See Rhythmogenesis

neuronal/network oscillators, 214 outward currents, 216 pattern variability and sensory modulation, 141 rhythmic motor behaviors, 112–114 rhythm mutants, 55–56 role, in rhythmogenesis, 217 synaptic currents, glutamate lamprey spinal cord, 217–218 respiratory network, 218 Chemoreception hypothesis central chemoreceptors, exercise hyperpnoea, 176 CO2/O2 ratio, 174 during exercise, 175 gas concentrations/pH changes, 176 Kþ ions, 177 peripheral chemoreceptors, exercise hyperpnoea, 176–177 Coupling and peripheral afferents peripheral nervous feedback, 183–184 piston mechanism, 182–183 spinal cord CPGs, 183 supraspinal influences, 183

Brainstem, 181 Calcium channels activated nonspecific cation, 123–128 rhythmogenesis mechanisms, 140–141 Cell ablation, 29–30 Central command hypothesis localization, 178–181 peripheral nervous feedback hypothesis, 181–182 Central congenital hypoventilation syndrome (CCHS), 42 Central pattern generator (CPG) boundaries and components, 138–139 genetic markers identification functional redundancy and complexity, 27–28 modular nature, mammalian locomotor, 25–26 vs. respiratory CPGs, 28 glial cells, 219–220 inspiratory network, 218–219 ionic currents calcium-activated nonselective current/ICAN, 215–216 calcium-dependent potassium current (IK(Ca)), 216–217 NMDA, 216 sodium current/INa(P), 214–215

Defective cell generation, 29–30 Diaphragm central partitioning, motoneurons, 69 hormonal influences, 74–76 neuromuscular compartments, 65–67, 72–73 (see also Neuromuscular compartments) Egr2 (Krox-20) gene, 40–41 Embryonic parafacial (e-pF) oscillator, 40–41 Endocannabinoids, 106 223

224

Excitatory commissural interneurons, 89 Excitatory neurons, rhythmogenesis lamprey, 194–196 salamanders, 198–199 Xenopus tadpoles, 197–198 zebrafish, 196–197

Hormonal influences diaphragm, 74–76 lateral gastrocnemius (LG), 74 masseter muscle, 76 Hox genes, 41 Hyperpnea, 176

Gastrocnemius. See Lateral gastrocnemius (LG) Genetic dissection, rhythmic motor networks caudal neuroaxis development, 20–23 postmitotic transcription factors, 23–25 ventral interneurons generation, 23 cell activity manipulation, 30–31 defective cell generation, 29–30 functional analysis, 32 genetic manipulations, V1 interneurons, 31–32 genetic markers identification functional redundancy and complexity, 27–28 modular nature, mammalian locomotor, 25–26 vs. respiratory CPGs, 28 hindbrain and spinal cord, 20 Glutamatergic interneuron, 48 Group I excitatory interneurons, 87–88 Group-pacemaker mechanism properties, in rhythm generation a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), 119 preBötC neurons, 118 Raphé neurons, 121 RIL application, 120 Riluzole blocks, 117 respiratory rhythmogenesis chloride mediated synaptic inhibition, 114–115 emergent network properties, 128–130 oscillatory bursting properties, neurons, 115–117 rhythmic motor behaviors, 112–114 synaptically triggered burst-generating conductances calcium-activated nonspecific cation, 123–128 PreBötC neurons, 121–123

Ia inhibitory interneurons (IaINs), 85 Inhibitory neurons lamprey, rhythmogenesis, 199 salamanders, rhythmogenesis, 201 Xenopus tadpoles, rhythmogenesis, 200–201 zebrafish, rhythmogenesis, 200 Isometric contraction development, 164 KNa channels, 103–104 Kolmer–Agduhr (KA) neurons, 48 Lamprey locomotor network, 106 rhythmogenesis excitatory neurons, 194–196 inhibitory neurons, 199 locomotor behavior, 190–191 spinal neurons, 192 synaptic currents, glutamate, 217–218 Last-order excitatory interneurons, spines excitatory commissural interneurons, 89 group I excitatory interneurons, 87–88 mediating cutaneous inputs, 88–89 mid-lumbar group II interneurons, 88 spinal modulatory neurons, 89–90 V2a interneurons, 89 V3 interneurons, 89 Last-order inhibitory interneurons, spines in cats, 86 Ia inhibitory interneurons (IaINs), 85 medial lamina V/VI GABAergic neurons, 87 nonreciprocal inhibitory INs, 85–86 Renshaw cells (RCs), 83, 85 V2b interneurons, 87 V0 interneurons, 87 Lateral gastrocnemius (LG) central partitioning, motoneurons, 68 hormonal influences, 74

225

neuromuscular compartments, 64–65, 72 (see also Neuromuscular compartments) Lbx1 gene, 40 Locomotion vs. respiration central command hypothesis localization, 178–181 peripheral nervous feedback hypothesis, 181–182 chemoreception hypothesis central chemoreceptors, exercise hyperpnoea, 176 CO2/O2 ratio, 174 during exercise, 175 gas concentrations/pH changes, 176 Kþ ions, 177 peripheral chemoreceptors, exercise hyperpnoea, 176–177 coupling and peripheral afferents peripheral nervous feedback, 183–184 piston mechanism, 182–183 spinal cord CPGs, 183 supraspinal influences, 183 Locomotor behavior lamprey, rhythmogenesis, 190–191 salamanders, rhythmogenesis, 191–192 Xenopus tadpoles, rhythmogenesis, 191 zebrafish, rhythmogenesis, 191 Locomotor circuitry-ionic mechanisms calcium, 102 embedded modulation, 104–106 KNa channel, 103–104 potassium, 102–103 Locomotor movements, 2 Mandibular movements, in humans, 4–5 Masseter motoneurons Ia-EPSPs, 167 isometric contraction development, 164 jaw-closing motoneurons, 167 orderly recruitment, 164–165 stretch reflex circuit, 165–167 TASK channels, 168–169 Masseter muscle central partitioning, motoneurons, 69–71 hormonal influences, 76

neuromuscular compartments, 67–68, 73–74 (see also Neuromuscular compartments) Mastication bursting properties development, 139–140 putative cellular mechanisms, sensory modulation, 141–144 See also Slow-closing phase Medial lamina V/VI GABAergic neurons, 87 Metabotropic glutamate receptors (mGluRs), 126 Mice genetic dissection, rhythmic motor networks caudal neuroaxis development, 20–23 postmitotic transcription factors, 23–25 ventral interneurons generation, 23 cell activity manipulation, 30–31 defective cell generation, 29–30 functional analysis, 32 genetic manipulations, V1 interneurons, 31–32 genetic markers identification functional redundancy and complexity, 27–28 modular nature, mammalian locomotor, 25–26 vs. respiratory CPGs, 28 hindbrain and spinal cord, 20 Mid-lumbar group II interneurons, 88 Motoneurons, 82. See also Spinal interneurons Motor rhythms development, zebrafish embryos physiology and pharmacology embryonic swimming rhythms, 53–54 evoked rhythms, 52–53 first motor rhythms, 50–52 rhythm mutants, 55–56 spinal cord morphology, 48–50 swimming frequency, in larvae, 54–55 Mouse genetics. See Mice genetic dissection, rhythmic motor networks Muscle compartmentalization. See Neuromuscular compartments Muscle spindle, 164 Neural circuits genetic factors atonal homolog 1 (Atoh1/Math 1), 42–43 embryonic parafacial (e-pF) oscillator, 40–41 inhibitory interneurons insertion, 44 paired-like homeobox 2b (Phox2b) gene, 42 pre-Bötzinger complex, 44–45

226

Neural circuits genetic factors (Continued) rhombomeric patterning, parafacial hindbrain, 41 spinal connection, 43–44 Neuromuscular compartments diaphragm crus region, 65–66 rostral–caudal organization, 72–73 sternocostal region, 65 type IIb MyHC, 67 lateral gastrocnemius (LG) cross-compartmental potentials, 72 evoked electromyographic (EMG) mapping, 64 flexion–withdrawal reflex, 65 masseter muscle layers, in mouse, 73 MyHC type IIa, 74 reflex partitioning, 67–68 Nonreciprocal inhibitory INs, 85–86 Pacemaker properties. See Group-pacemaker mechanism Paired-like homeobox 2b (Phox2b) gene, 42 Parafacial respiratory rhythm generator. See Embryonic parafacial (e-pF) oscillator Periodic depolarizations (PDs), 51–52 Peripheral nervous feedback hypothesis, 181–182 Piston mechanism, 182–183 Plasticity, spinal cord, 106 Pneumogastric nerve role, 4 Potassium channels, 102–103 PreBötC neurons, 121–123 pre-Bötzinger complex, 44–45 Reflex function definition, 8–10 peripheral control, breathing, 10–12 somatic muscular reflexes, 12–13 Renshaw cells (RCs), 83, 85 Respiratory rhythm generation chloride mediated synaptic inhibition, 114–115 emergent network properties, 128–130

oscillatory bursting properties, neurons, 115–117 synaptically triggered burst-generating conductances calcium-activated nonspecific cation, 123–128 PreBötC neurons, 121–123 Rhythmic motor networks, in mice caudal neuroaxis development, 20–23 postmitotic transcription factors, 23–25 ventral interneurons generation, 23 cell activity manipulation, 30–31 defective cell generation, 29–30 functional analysis, 32 genetic manipulations, V1 interneurons, 31–32 genetic markers identification functional redundancy and complexity, 27–28 modular nature, mammalian locomotor, 25–26 vs. respiratory CPGs, 28 hindbrain and spinal cord, 20 Rhythmic movements measurement, 2 Rhythm mutants, 55–56 Rhythmogenesis glial cells, 219–220 inspiratory network, 218–219 ionic currents calcium-activated nonselective current/ICAN, 215–216 calcium-dependent potassium current (IK(Ca)), 216–217 NMDA, 216 outward currents, 216 sodium current/INa(P), 214–215 lamprey excitatory neurons, 194–196 inhibitory neurons, 199 locomotor behavior, 190–191 spinal neurons, 192 neuronal/network oscillators, 214 role, in rhythmogenesis, 217 salamanders excitatory neurons, 198–199 inhibitory neurons, 201 locomotor behavior, 191–192 spinal neurons, 193–194

227

synaptic currents, glutamate lamprey spinal cord, 217–218 respiratory network, 218 Xenopus tadpoles excitatory neurons, 197–198 inhibitory neurons, 200–201 locomotor behavior, 191 spinal neurons, 193 zebrafish excitatory neurons, 196–197 inhibitory neurons, 200 locomotor behavior, 191 spinal neurons, 193 Rhythmogenic properties modulation, 140–141

Renshaw cells (RCs), 83, 85 V2b interneurons, 87 V0 interneurons, 87 Spinal locomotor circuitry, 101–102 Spinal modulatory neurons, 89–90 Spinal neurons lamprey, rhythmogenesis, 192 salamanders, rhythmogenesis, 193–194 Xenopus tadpoles, rhythmogenesis, 193 zebrafish, rhythmogenesis, 193 Swimming frequency, in zebrafish embryos, 54–55 Synaptic transmission. See Locomotor circuitryionic mechanisms

Salamanders locomotion (see Vertebrates locomotion) rhythmogenesis excitatory neurons, 198–199 inhibitory neurons, 201 locomotor behavior, 191–192 spinal neurons, 193–194 Slow-closing phase, 164–166 Spinal cord genetic classification, 21 genetic tools, 29 and hindbrain, rhythmic motor networks, 20 morphology, zebrafish embryos, 48–50 plasticity, endocannabinoids, 106 unit burst generation, 100–101 Spinal interneurons last-order excitatory interneurons excitatory commissural interneurons, 89 group I excitatory interneurons, 87–88 mediating cutaneous inputs, 88–89 mid-lumbar group II interneurons, 88 spinal modulatory neurons, 89–90 V2a interneurons, 89 V3 interneurons, 89 last-order inhibitory interneurons in cat, 86 Ia inhibitory interneurons (IaINs), 85 medial lamina V/VI GABAergic neurons, 87 nonreciprocal inhibitory INs, 85–86

TASK channels, 168–169 V2a interneurons, 89 V1 and V3 interneuron, 24, 31 V2b interneurons, 87 Vertebrates locomotion appendage lower vertebrates, 151–154 mammals, 150–151 axial-based locomotion, 154–156 neural mechanisms adaptive mechanisms, 156–157 CPGs axial locomotor, 156 vs. limb locomotor, 157–158 V0 interneurons, 87 V3 interneurons, 89 Xenopus tadpoles, rhythmogenesis excitatory neurons, 197–198 inhibitory neurons, 200–201 locomotor behavior, 191 spinal neurons, 193 Zebrafish embryos, motor rhythms physiology and pharmacology embryonic swimming rhythms, 53–54 evoked rhythms, 52–53 first motor rhythms, 50–52

228

Zebrafish embryos, motor rhythms (Continued) rhythm mutants, 55–56 spinal cord morphology, 48–50 swimming frequency, in larvae, 54–55 Zebrafish, rhythmogenesis

excitatory neurons, 196–197 inhibitory neurons, 200 locomotor behavior, 191 spinal neurons, 193

SERIES EDITORS

STEPHEN G. WAXMAN Bridget Marie Flaherty Professor of Neurology Neurobiology, and Pharmacology; Director, Center for Neuroscience & Regeneration/Neurorehabilitation Research Yale University School of Medicine New Haven, Connecticut USA

DONALD G. STEIN Asa G. Candler Professor Department of Emergency Medicine Emory University Atlanta, Georgia USA

DICK F. SWAAB Professor of Neurobiology Medical Faculty, University of Amsterdam; Leader Research team Neuropsychiatric Disorders Netherlands Institute for Neuroscience Amsterdam The Netherlands

HOWARD L. FIELDS Professor of Neurology Endowed Chair in Pharmacology of Addiction Director, Wheeler Center for the Neurobiology of Addiction University of California San Francisco, California USA

Preface This book, divided into two volumes of Progress in Brain Research, is largely inspired from presentations made at the 31st International Symposium of Research Group on the Central Nervous System at the University of Montreal held on May 4–5, 2009. The meeting was a special occasion to honor three outstanding neuroscientists, who have been world leaders in their respective fields of motor control, James P. Lund, Serge Rossignol, and Jack L. Feldman. The three honored scientists gave plenary presentations summarizing part of their outstanding accomplishments. In December 2009, James P. Lund prematurely and suddenly deceased. We were stunned and greatly moved by this sad news. We unanimously decided to dedicate this book to his memory. The symposium and this book highlight new findings on the neural control of rhythmic movements with an emphasis on common neuronal mechanisms. The book is divided into four sections from genes and molecules to system physiology. It begins with an historical overview by François Clarac who relates the early research carried out by true pioneers using crude and simple methods to study breathing, walking, and chewing. The first section describes how recent molecular genetics has revolutionized the study of the neuronal networks controlling rhythmic motor behaviors. The second section digs deep into ionic and cellular mechanisms underlying the function of different rhythmic networks. The third section covers broadly the modulation and the plasticity of rhythmic circuits, from chloride homeostasis to spasticity in human subjects. Finally, the 4th and last section, introduced by Dr. Sten Grillner, relates the ideas, contribution, and spirits of the three honorees, Professors Lund, Feldman, and Rossignol. The scope of the knowledge covered here is nothing but breathtaking. From discoveries of the nineteenth century to “in press” material, from zebra fish to humans, with students or senior scientists, rhythmogenesis and its control is explained and discussed, reviewed, and summarized. We thank all the 89 authors who contributed to this book. Jean-Pierre Gossard Réjean Dubuc Arlette Kolta

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Other volumes in PROGRESS IN BRAIN RESEARCH Volume 149: Cortical Function: A View from the Thalamus, by V.A. Casagrande, R.W. Guillery and S.M. Sherman (Eds.) – 2005 ISBN 0-444-51679-4. Volume 150: The Boundaries of Consciousness: Neurobiology and Neuropathology, by Steven Laureys (Ed.) – 2005, ISBN 0-444-51851-7. Volume 151: Neuroanatomy of the Oculomotor System, by J.A. Büttner-Ennever (Ed.) – 2006, ISBN 0-444-51696-4. Volume 152: Autonomic Dysfunction after Spinal Cord Injury, by L.C. Weaver and C. Polosa (Eds.) – 2006, ISBN 0-444-51925-4. Volume 153: Hypothalamic Integration of Energy Metabolism, by A. Kalsbeek, E. Fliers, M.A. Hofman, D.F. Swaab, E.J.W. Van Someren and R.M. Buijs (Eds.) – 2006, ISBN 978-0-444-52261-0. Volume 154: Visual Perception, Part 1, Fundamentals of Vision: Low and Mid-Level Processes in Perception, by S. Martinez-Conde, S.L. Macknik, L.M. Martinez, J.M. Alonso and P.U. Tse (Eds.) – 2006, ISBN 978-0-444-52966-4. Volume 155: Visual Perception, Part 2, Fundamentals of Awareness, Multi-Sensory Integration and High-Order Perception, by S. Martinez-Conde, S.L. Macknik, L.M. Martinez, J.M. Alonso and P.U. Tse (Eds.) – 2006, ISBN 978-0-444-51927-6. Volume 156: Understanding Emotions, by S. Anders, G. Ende, M. Junghofer, J. Kissler and D. Wildgruber (Eds.) – 2006, ISBN 978-0-444-52182-8. Volume 157: Reprogramming of the Brain, by A.R. Mller (Ed.) – 2006, ISBN 978-0-444-51602-2. Volume 158: Functional Genomics and Proteomics in the Clinical Neurosciences, by S.E. Hemby and S. Bahn (Eds.) – 2006, ISBN 978-0-444-51853-8. Volume 159: Event-Related Dynamics of Brain Oscillations, by C. Neuper and W. Klimesch (Eds.) – 2006, ISBN 978-0-444-52183-5. Volume 160: GABA and the Basal Ganglia: From Molecules to Systems, by J.M. Tepper, E.D. Abercrombie and J.P. Bolam (Eds.) – 2007, ISBN 978-0-444-52184-2. Volume 161: Neurotrauma: New Insights into Pathology and Treatment, by J.T. Weber and A.I.R. Maas (Eds.) – 2007, ISBN 978-0-444-53017-2. Volume 162: Neurobiology of Hyperthermia, by H.S. Sharma (Ed.) – 2007, ISBN 978-0-444-51926-9. Volume 163: The Dentate Gyrus: A Comprehensive Guide to Structure, Function, and Clinical Implications, by H.E. Scharfman (Ed.) – 2007, ISBN 978-0-444-53015-8. Volume 164: From Action to Cognition, by C. von Hofsten and K. Rosander (Eds.) – 2007, ISBN 978-0-444-53016-5. Volume 165: Computational Neuroscience: Theoretical Insights into Brain Function, by P. Cisek, T. Drew and J.F. Kalaska (Eds.) – 2007, ISBN 978-0-444-52823-0. Volume 166: Tinnitus: Pathophysiology and Treatment, by B. Langguth, G. Hajak, T. Kleinjung, A. Cacace and A.R. Mller (Eds.) – 2007, ISBN 978-0-444-53167-4. Volume 167: Stress Hormones and Post Traumatic Stress Disorder: Basic Studies and Clinical Perspectives, by E.R. de Kloet, M.S. Oitzl and E. Vermetten (Eds.) – 2008, ISBN 978-0-444-53140-7. Volume 168: Models of Brain and Mind: Physical, Computational and Psychological Approaches, by R. Banerjee and B.K. Chakrabarti (Eds.) – 2008, ISBN 978-0-444-53050-9. Volume 169: Essence of Memory, by W.S. Sossin, J.-C. Lacaille, V.F. Castellucci and S. Belleville (Eds.) – 2008, ISBN 978-0-444-53164-3. Volume 170: Advances in Vasopressin and Oxytocin – From Genes to Behaviour to Disease, by I.D. Neumann and R. Landgraf (Eds.) – 2008, ISBN 978-0-444-53201-5. Volume 171: Using Eye Movements as an Experimental Probe of Brain Function—A Symposium in Honor of Jean BüttnerEnnever, by Christopher Kennard and R. John Leigh (Eds.) – 2008, ISBN 978-0-444-53163-6. Volume 172: Serotonin–Dopamine Interaction: Experimental Evidence and Therapeutic Relevance, by Giuseppe Di Giovanni, Vincenzo Di Matteo and Ennio Esposito (Eds.) – 2008, ISBN 978-0-444-53235-0. Volume 173: Glaucoma: An Open Window to Neurodegeneration and Neuroprotection, by Carlo Nucci, Neville N. Osborne, Giacinto Bagetta and Luciano Cerulli (Eds.) – 2008, ISBN 978-0-444-53256-5. Volume 174: Mind and Motion: The Bidirectional Link Between Thought and Action, by Markus Raab, Joseph G. Johnson and Hauke R. Heekeren (Eds.) – 2009, 978-0-444-53356-2. Volume 175: Neurotherapy: Progress in Restorative Neuroscience and Neurology — Proceedings of the 25th International Summer School of Brain Research, held at the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands, August 25–28, 2008, by J. Verhaagen, E.M. Hol, I. Huitinga, J. Wijnholds, A.A. Bergen, G.J. Boer and D.F. Swaab (Eds.) –2009, ISBN 978-0-12-374511-8. Volume 176: Attention, by Narayanan Srinivasan (Ed.) – 2009, ISBN 978-0-444-53426-2. Volume 177: Coma Science: Clinical and Ethical Implications, by Steven Laureys, Nicholas D. Schiff and Adrian M. Owen (Eds.) – 2009, 978-0-444-53432-3. Volume 178: Cultural Neuroscience: Cultural Influences On Brain Function, by Joan Y. Chiao (Ed.) – 2009, 978-0-444-53361-6. Volume 179: Genetic models of schizophrenia, by Akira Sawa (Ed.) – 2009, 978-0-444-53430-9. Volume 180: Nanoneuroscience and Nanoneuropharmacology, by Hari Shanker Sharma (Ed.) – 2009, 978-0-444-53431-6.

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Other volumes in PROGRESS IN BRAIN RESEARCH

Volume 181: Neuroendocrinology: The Normal Neuroendocrine System, by Luciano Martini, George P. Chrousos, Fernand Labrie, Karel Pacak and Donald W. Pfaff (Eds.) – 2010, 978-0-444-53617-4. Volume 182: Neuroendocrinology: Pathological Situations and Diseases, by Luciano Martini, George P. Chrousos, Fernand Labrie, Karel Pacak and Donald W. Pfaff (Eds.) – 2010, 978-0-444-53616-7. Volume 183: Recent Advances in Parkinson's Disease: Basic Research, by Anders Björklund and M. Angela Cenci (Eds.) – 2010, 978-0-444-53614-3. Volume 184: Recent Advances in Parkinson's Disease: Translational and Clinical Research, by Anders Björklund and M. Angela Cenci (Eds.) – 2010, 978-0-444-53750-8. Volume 185: Human Sleep and Cognition, by Gerard A. Kerkhof and Hans P.A. Van Dongen (Eds.) – 2010, 978-0-444-53702-7. Volume 186: Sex Differences in the Human Brain, their Underpinnings and Implications, by Ivanka Savic (Ed.) – 2010, 978-0-44453630-3.

In Memoriam

James P. Lund (Jim) sadly deceased on December 8, 2009, leaving an enormous hole in the personal and professional lives of many of us. Jim graduated as a dentist in Australia in 1966 from University of Adelaide. With the exceptional intellectual curiosity that characterized him, he gave up private practice after a year to begin a Ph.D. under the supervision of Peter Dellow. The concept of “central pattern generators” (CPGs) was still debated in those years and the “Sherringtonian” view that mastication consisted in a series of alternating jaw opening and jaw closing reflexes prevailed in many laboratories. Jim's pioneering work during his Ph.D. concluded to the existence of a CPG for mastication in the brainstem. After receiving his Ph.D. in 1971, he joined the Medical Research council (MRC) group of the University of Montreal, first as a postdoctoral fellow with Yves Lamarre and later as a faculty member. These were determinant years in Jim's career, and this is when he established himself as an inevitable authority in the field of mastication. His laboratory was recognized as the world's leading center for fundamental studies of orofacial motor function. He asked important questions and touched almost every aspect involved in the neural control of mastication. After studying the cortical influence on the brainstem CPG, he became interested in reflex modulation during movements and showed that reflexes were phasically modulated to avoid perturbation of the movement while maintaining protection of the tissues. With K. Olsson and later K.-G. Westberg, he described the role and connections of brainstem interneurons involved in patterning mastication and modulating jaw reflexes. ix

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It had been proposed that one way to modulate reflexes was by presynaptic inhibition and antidromic discharges evoked in sensory afferents. He investigated this issue in the trigeminal system and with his collaborators subsequently discovered that the propagation of antidromic discharges was controlled by GABAergic synapses along the axonal trunk causing a compartmentalization of the axon. Jim was also interested on how sensory inputs interacted with the CPG. Most of his work on this issue, conducted on animal models, eventually led him to formulate a conceptual model about how sensory inputs from nociceptors altered movements. In collaboration with C. Stohler, he developed the pain adaptation model, which shows how pain itself can cause motor and sensory symptoms. This model has been validated by several clinical research groups throughout the world and is prompting changes in clinical practice. Nociceptive inputs are not the only sensory inputs susceptible to alter movements. In an effort at understanding how other types of sensory inputs affect mastication, he worked in close collaboration with J. Feine to document the effects of loss of inputs from teeth and of patient's treatment on the efficiency of mastication and on the consequences on nutrition. More than a highly prolific and respected scientist, Jim was also a tireless visionary who led many battles to improve research in dental faculties and increase knowledge transfer toward clinical applications. For this reason, he generously accepted to serve as Vice Dean of Research at University of Montreal for 8 years and as Dean of Dentistry at McGill University for 13 years. Throughout those years, he founded research networks and built a world leader pain research center at McGill. Jim never failed to consider issues from different perspectives and to think outside the box. He influenced many institutional and health care decisions, but even more importantly, he left an indelible trace in the life of many scientists that he trained or helped recruiting. He deeply cared for his trainees, collaborators, and colleagues. He always managed to provide his trainees with what great emulation needed to foster their growth. He was an outstanding mentor to people at all levels of career development from undergraduate and postgraduate students to junior academic staff and peers in administrative positions. We will miss Jim deeply for his insights, brightness, contagious energy, and genuine caring for others. Jean-Pierre Gossard Réjean Dubuc Arlette Kolta

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 1

Once upon a time . . . . . . the early concepts of breathing, walking, and chewing François Clarac* CNRS, P3M, Chemin Joseph Aiguier, Marseille, Cedex, France

Abstract: Many important notions relative to breathing, walking, and chewing originated from early concepts developed in the nineteenth century. We will consider successively: – The measurements of the parameters of these behaviors done by great experts on movement recordings. It corresponded to the first elements of biomechanics. – The physiology of central motor activities in locomotion and in respiration completed by the analysis of motor neuropathologies where specific deficits have highlighted the role of some crucial motor structures. – The studies of different reflexes, a predominant concept at that time, that have characterized some particular pathways between the sensory receptors and the different motor output. The goal of this review is to show how true pioneers, often using some crude and simple methods of investigation, have addressed important issues relative to control of these three rhythmic motor behaviors. Keywords: walking; breathing; chewing; history of motor control; biomechanics; reflexes.

Introduction

whereas chewing was only superficially studied. The goal of this review is to retrace how the pioneers of neuroscience investigated these motor behaviors and how they were able to address important issues related to their neural organization. These early pioneers considered movements in general to be of two types; the automatic and the voluntary behaviors: The first, linked with “the machine” as suggested by the philosopher

Once upon a time. . .there was breathing, walking, and chewing. . . these rhythmic activities have been studied scientifically since the nineteenth century. The initial concepts of rhythmic motor control were related to breathing and walking, *Corresponding author. Tel.: þ33-491164139. DOI: 10.1016/S0079-6123(10)87001-3

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René Descartes (1594–1650), originated from a low level of the central nervous system (CNS). By contrast, the second type was linked to the cortex, its psychical functioning and issued from the “soul” (Jeannerod, 2006). Thomas Willis (1621–1675), professor of natural philosophy at Oxford university, dissociated, in the “De motu musculari” (1670), stereotyped involuntary movements initiated by the cerebellum and the brain stem like respiration and circulation and adaptive involuntary movements originating from the striatum, like locomotion. The notion of “soul” discussed with voluntary movements, was progressively abandoned and the problem of the nervous origin of movements became one of the most intriguing debates in the nineteenth century (Brazier, 1988). Considering the three automatic movements, we would like – First, to explain how these rhythmic movements were recorded and measured. During the nineteenth century, the measurement of a biological phenomenon was the first step toward a scientific analysis. Moreover, kinematics and force recordings introduced a first approach to biomechanics. – Second, to summarize the data collected on these automatic functions of the CNS. It was assumed that the cortex, with its different localized areas, was devoted to the conscious actions, whereas the lower part of the CNS was considered the origin of these repetitive activities. – Third, to analyze why the notion of reflex predominated during the second half of the nineteenth century and became the first elaborated mechanism with regard to CNS function.

Measurement of rhythmic movements and first elements of biomechanics These fundamental rhythmic movements, walking and breathing, were introduced by Hippocrates and later mentioned by Galen who described them in relation with their functions.

From Borelli to Weber' brothers The first real work on the mechanical study of locomotion was performed by a Professor of Mathematics in Pisa, Giovanni Borelli (1608–1679). He was the leader of the “Iatrophysicists,” an Italian school that explained animal and human activities with physical, biomechanical, and mathematical laws. He published a fundamental book, “De Motu Animalium” (Borelli, 1680), in which he described in detail the functioning of the human body and he analyzed the different types of biological movements. This book is the first scientific presentation of locomotor movements, in which Borelli described quite well the various forces exerted, and the propulsive action during the stance phase. He mentioned the particular role of the pelvis rotation and of the different joints. He later widened his description to animals like horses and insects. In this field, very little was presented after this Italian study prior to the very extensive work done by the Weber brothers in 1836. Their classical treatise on Die Mechanik Der Menschlichen Gerverkzeuge, established the mechanism of muscular action on a scientific basis. Wilhelm Eduard Weber (1804–1891) and Eduard Friedrich Wilhelm Weber (1806–1871) hypothesized that the body was maintained in an erect position primarily by tension of the ligaments, with little or no muscular exertion. They proposed that during walking or running, the forward motion of the limb could be compared with a passive pendular movement of the leg in relation with gravity (Weber and Weber, 1836). Walking corresponded to a succession of falling forward. They were the first to follow the movements of the center of gravity in chronological order. The Webers analyzed the contraction of individual muscles and explained the role of bones as mechanical levers. Guillaume Duchenne de Boulogne (1806–1875) a French physician, contested the view of a passive pendular movement of the leg during the oscillation phase. He explained that a limb could not oscillate in an extended position. The three

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segments, the thigh, the leg, and the foot, are slightly bent due to the synergic contraction of the flexor muscles (Duchenne de Boulogne, 1867). The study of animal mechanics was expanded by different types of works. Perhaps the most original and talented scientist was the Irishman Samuel Haughton of Dublin (1821–1897) who wrote numerous papers, and in particular analyzed in his laboratory the geometry of skeletal muscle and criticized Darwin's theory of natural selection in Principles of Animal Mechanics (Haughton, 1873). For respiration, after the description of oxygen consumption in 1789, by Antoine Lavoisier (1743–1794), the following studies on this rhythm were mainly on the capacity of lungs and on the respiratory movements. Dr Bourgery in 1843, in Paris and the Dr Hutchinson (1811–1861) in Britain, can be cited for their precise analysis, but it seemed that it was Edmund Goodwyn (1788) who first measured lung content values.

Edweard Muybridge and Etienne Jules Marey These two scientists, one English and the other French, can be considered as great experts on movement recordings. Their lives spanned the same period of time, 1830–1904. Muybridge passed away on May 8th and Marey on the 15th of the same month. Muybridge visited Marey's studio in France in 1881. Muybridge was not really a scientist but rather a professional photographer. He was open to the real life and became a sort of reporter covering different events in various countries. Marey was a physicist and professor at the Collège de France. His analysis in cardiology and aerodynamics as well as his pioneering work in photography and chronophotography are quite well known. Muybridge became in 1867, the official photographer of the U.S. troops in Alaska. Between 1868 and 1873, he visited the entire American west coast and took more than 2000 photos. His

photographs of Yosemite particularly interested Leland Stanford (1824–1893), the wealthy California governor, and also owner of a famous trotting horse named “Occident.” Both Marey and Muybridge thought that during galloping, there was a brief period when all four hooves left the ground. The first experiment took place on June 11, 1878 with the press present. Using a series of 12 stereoscopic cameras and taking pictures at one thousandth of a second, he recorded different horse strides. This confirmed the idea of a short absence of contacts during galloping, although not with the legs fully extended forward and back, as contemporary illustrators tended to imagine. After that, Muybridge became very popular and produced more than 20,000 individual photographs of human and animal locomotion at the University of Pennsylvania from 1883 to 1886 (Haas, 1976). In 1882, he published The horse in motion, and in 1887 wrote his monumental “Animal Locomotion” in 11 volumes (Muybridge, 1887). At the Chicago 1893 World's Columbian Exposition, Muybridge gave a series of lectures on the “Science of Animal Locomotion.” Marey considered himself as a “physicistengineer” or a “life-engineer,” searching throughout his life for the best scientific method to record a given movement. He had an antireductionist attitude, promoting work on intact animals to see very carefully the global output of a function that in general corresponds to a movement (Marey La Machine Animale 1873). He focused first on the circulation of the blood and on the heart beat, and after several trials, he built a “sphygmograph,” the first scientific apparatus that recorded properly the pulse in 1868. He also recorded respiration comparing the trunk and the abdominal movements. Exactly as Muybridge, he considered that it was necessary to make much faster photographs in a sequential order to have the evolution of kinematics and dynamics of movement. He used the discovery of Jules Jansen (1824–1907), a French Astronomer who in 1874 created the “photographic gun,” to record the

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movement of the planet Venus. In a few years, Marey adapted the apparatus and developed in 1882 what he called the chronophotographic gun. This instrument, used mainly for bird flight, was capable of taking, on the same picture, 12 consecutive frames per second. Studying human locomotion, Marey simplified his procedure, recording only the outlines of a movement. He dressed his subject in black and white clothes to limit the data collected to only two or three geometric lines with clear defined angles. The stick diagram representation was thus invented by Marey! A new technical step was reached in 1888, when he was able to use a mobile paper tape and a photosensitive film without perforations and a camera that gave him his technique of “film chronophotography.” He summarized his discoveries, in his book “Le Mouvement” (Marey, 1894a). His work on dog walking, trotting, and galloping was used by Philipson who in 1905 gave a proper analysis of the gait with three extension phases and one flexion phase (see Clarac, 2008). In 1894, Marey analyzed the movements of the mandibule in humans, and explained that it was in fact quite difficult to analyze due to the great motility of the condyles (Marey, 1894b). The movements of this very complex joint are dependent on the physiological activity produced. He said that opening the mouth during chewing is not equal to that of opening the mouth during speaking or singing. To record mandibular movement in humans, he used some dental cement to fix a moving rod with a luminous indicator needed for chronophotography. He described several types of movements: opening and closing, retraction and protraction as well as masticatory movements. With chewing, he dissociated the rhythmic movements around the incisors from those around the molars. In the former case, the lower maxilla goes up and forward and its two branches are more or less in parallel. In the latter molar masticatory movements, the mandible moves around the extremity of its condyles. Marey also described lateral movements of the mandible (see Fig. 1).

However, the three different chewing movements were not studied further, because mastication was considered of minor importance, and was invariably linked with facial movements. Locomotion was viewed as a better test for clinical analysis (see further). Respiration was studied in detail by the physiologists as a fundamental movement as the blood circulation.

The central control of movements During the nineteenth century, the knowledge of the nervous system expanded based on new data about the brain and the role of the different areas of the cortex (Brazier, 1988). However, it was the same period that the spinal cord and the medulla oblongata were described properly. After the great debate between Sir Charles Bell (1774–1842) and François Magendie (1783–1855) on the role of the dorsal and the ventral spinal roots, the anatomy of the spinal cord was finally established by Charles Prosper Ollivier d'Angers (1796–1845) with the publication of his comprehensive work “De la moelle épinière et de ses maladies” (On the spinal cord and its diseases, 1st ed. 1824, 3rd 1837). In the third chapter, he described respiration and the role of the pneumogastric nerve (Grossmann et al., 2006). Jacob Augustus Lockhart Clarke (1817–1880), still known for the column in the spinal cord, which bears his name, worked mainly between 1851 and 1868 and described several nuclei in the medulla oblongata (Clarke, 1869). Sir Astley Paston Cooper (1768–1841), an English surgeon and anatomist specialist of otology, hernia, and vascular surgery, wrote a book on “Illustrations of diseases of the breast” (1829).

Notions of coordination and synergies with posture and locomotion Posture and locomotion have been analyzed by clinicians with respect to a variety of different sorts of pathologies (Clarac et al., 2009). Duchenne who

5 (a)

(b)

(c)

MA

MP

MP

Fig. 1. Mandible movements recorded in human (see Marey, 1894b). (a) Presentation of the moving rod that reproduces the mandible joint angle. (b) Opening and closing the mouth with the luminous indicator for chronophotography. (c) Chewing movements with the incisors (MA) and with the molars (MP).

was a physician originally from “Boulogne sur mer,” a truly self-taught man, clearly defined the notion of muscle coordination, which he called “synergy.” He used an induction coil device to produce faradic current with which he started to treat patients by applying electric current. He quickly realized that it also provided the opportunity to study muscle contractions either alone or in groups. When studying the effects of single muscle faradisation, he stated that “An isolated muscle contraction does not exist in nature” (Duchenne de Boulogne, 1867) and that muscle faradisation must be associated with clinical observations in

patients in order to understand how movements result in the activation of several muscles. Duchenne was also probably the first to use the biopsy procedure to obtain tissue for patients for microscopic examination (Parent, 2005). Duchenne described two pathologies, one altering the motor system and the other, the sensory pathways. The first pathology was described together with Aran (1850), the “progressive muscular atrophy,” with its usual onset at the distal end of the upper limbs, its slowly progressive worsening, with muscular cramp and muscular fascicles. It was the neurosurgeon Jean Cruveilhier (1791–1874) who

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in 1853 explained its origin when he conducted the autopsy of a celebrated acrobat, Lecomte, and found a complete atrophy of the spinal ventral roots. This was later confirmed in 1860 by the neuro-anatomist Jules-Bernard Luys (1828–1897) who found an atrophy of the cells of the ventral horn in the spinal cord. This pathology is at the origin of the amyotrophic lateral sclerosis (ALS) that involves a progressive motor paralysis, leading inevitably to death. Using his signature “anatomo-clinical” method, Jean-Martin Charcot (1825–1893) described ALS in several articles with Alex Joffroy (1844–1908) in 1869. They described the atrophy of the motoneuronal nuclei and also a symmetrical sclerosis of the lateral spinal column (Charcot and Joffroy, 1869). The work was continued with his student Albert Gombault (1844–1904) and documented the lesions of the anterior pyramids and of the brain stem (see Fig. 2; Gombault, 1877).

The second pathology concerned patients who presented troubles of coordination of leg movements during walking and equilibrium disturbance during stance (Duchenne de Boulogne, 1858). Interestingly, the deficits increased in the dark, indicating a compensatory influence of vision. Duchenne showed that these patients had deficient cutaneous sensation on the soles of the feet. Nevertheless, some of these patients were able to move their limbs without sight of the legs. He called this pathology, the “progressive locomotor ataxia,” better known as “tabes dorsalis” described in 1846, by Moritz Heinrich Romberg (1795–1873), a German neurologist of the University of Berlin. He described it with an increased leg and trunk oscillations in the absence of vision, known as the Romberg sign (see Pearce, 2005). This description was identical to the locomotor ataxia of Duchenne, and was associated with a diminished “muscle sense” of central origin

(a)

A A (b)

A

A

A

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(c) B V A

A′

B′

D′ C′ C D

B

B′

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Fig. 2. Nervous lesions in amyotrophic lateral sclerosis (ALS; see Gombault, 1877). (a) Three transverse sections of the spinal cord at three different levels (from left to right: cervical, dorsal, and lumbar). The lateral spinal column is sclerosed mainly at the upper levels. (b) Transverse section of the medulla oblongata with the sclerosis of the anterior pyramids. (c) Transverse section of the medulla oblongata at the level of the hypoglossi nuclei. On the right, the nucleus is intact with about 30 nerve cells. On the left, it is severely degenerated. B and B0 represent the pneumogastric nuclei that are intact.

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(see, Jones, 1972, Schiller, 1995). In fact, the English physician, Robert Bentley Todd (1809–1860) mentioned a lack of coordination of movements linked with the posterior column lesions as early as 1847 in “the physiology of the nervous system.” Jacob Clarke, who in 1868 after a visit from Duchenne, confirmed that locomotor ataxia is associated with the destruction of the posterior roots (Clarke, 1869). Charcot did not himself study human locomotor organization, but he explained it in one of his famous Tuesday lectures, on March 5, 1889 (Gasser, 1995). He dissociated the two levels of locomotor activation; the rhythmic automatic level that he supposed originated in the spinal cord, and the voluntary command that he located within the cortex. He even suggested that the spinal level is the more complicated of the two based on the complexity of the different mechanisms of muscle coordination; the cortex serving only to induce and arrest the behavior. Joseph Babinski (1857–1932) analyzed deficits in motor coordination in cerebellar patients, which he called “asynergia” when a subject was unable to walk. At the onset of locomotion, the failure of the trunk to lean forward prevented the execution of the first step. When a standing patient was asked to look upward by tilting the head and trunk backward, there was a lack of forward displacement of the hip and knee. These two synergies confirmed the absence of equilibrium control during the movement and the anticipatory postural adjustments associated with the movement. His term “asynergia” was not due to muscular weakness but to a lack of coordination during a succession of muscular contractions. Contrary to the innate synergies proposed by Duchenne, the synergies of Babinski were learned, stored in cerebellum, and used to anticipate the perturbation of posture and equilibrium associated with movement performance. During WWI, much of neurology involved analyzing the consequences of different sorts of traumatic injuries to the nervous system. An Irishman, Gordon Holmes (1876–1965), treated many soldiers with traumatic head injuries in battlefield

hospitals in France. This ultimately led to his classical analysis of the symptoms of cerebellar lesions (Holmes, 1917). Similarly, Jean Lhermitte (1877–1959) studied different cases of patients with complete spinal cord sections and described a variety of varying degrees of recovery. He explained that he found some defensive reflexes and some automatic movements analogous to those of locomotion (Roussy and Lhermitte, 1918). This very important discovery was unfortunately subsequently forgotten.

Brain stem and medulla centers of respiration At the onset of the nineteenth century, animal experimentation was a problem. The physiologists who wanted to use dissected preparations had to open the animals. For animals such as the frog, it was possible to keep them for several hours but the problem was for the mammals that they could keep alive for no more than a couple of minutes because of bleeding and respiratory failure. Naturalists then tried to find where respiration was induced. They localized the respiratory centers within the medulla, but for some people, it was very localized, whereas for others, it was largely diffused. Anne Charles de Lorry (1726–1783), a French physician, was able to maintain a dog alive for 15 min, with a normal pulse and a normal breathing, without its cerebrum and cerebellum. A section at the level of the medulla suppressed the vital functions. Jean Legallois (1770–1840) in 1806 worked on decerebrated rabbits that were able to survive for 15 min. He found that after cutting the medulla at the level of the 8th cranial nerve, the respiration stopped immediately: “It is not on the entire brain that the respiration depends, but only on a very circumscribed area of the medulla oblongata situated a short distance from the occipital opening toward the origin of the 8th pair of nerves. (Legallois, 1812 cited in Finger, 1994, p. 27)” In fact, for Legallois, the 8th pair was the pneumogastric nerve. In patients with defects of encephalic development, such as

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absence of the cerebrum and the cerebellum, when the medulla oblongata and the ventral pons were still present at the base of the skull, breathing can continue for a long time. Marie Jean-Pierre Flourens (1794–1867), a French physiologist, examined the behavioral deficits resulting from localized lesions of the brains of rabbits and pigeons. He used two complementary technics: systematic serial sections in the CNS and electrical stimulations of the different areas sectioned. He proposed the concept of a “vital node for respiration” (“Le noeud vital,” Flourens, 1824, 1851). Then, despite Flourens opposition to Franz-Joseph Gall (1758–1828) and his theory of localized brain functions, he himself localized respiration at the apex of the calamus scriptorius (the caudal floor of the fourth ventricle) in a center no larger than the head of a pin. In 1858, he redescribed the node as a bilateral structure, extending on both side of the middle in the caudal part of the medulla (Flourens, 1858). This was not accepted and was successively criticized by several authors. François-Achille Longet (1811–1871) destroyed the restiform bodies and the pyramids, and maintained a form of breathing. In contrast, when he eliminated the reticular formation, breathing stopped (Longet, 1847). Other authors reached similar conclusions. Gad and Marinesco (1892) attempted to reconcile the divergent data by experimenting on cats, dogs, and rabbits destroying different medullary centers with small hot glass rods. They cauterized the vital node, the centers defined by Gierke or by Mislawsky, but never blocked breathing completely. Subsequently, they found that the ventral part of the reticular formation was essential for breathing as confirmed by the postmortem examination of serial sections. More recently, this view has been confirmed but considerably strengthened and extended. Two divergent hypotheses were presented on the mechanisms underlying the rhythm generation of breathing (Pitts, 1946). On the one hand, the rhythm was thought to be produced by the

expression of an inherent autorhythmicity of neurons composing the respiratory centers with a reciprocal innervation between the inspiratory and expiratory groups. It would be said today that respiration is induced by CPG centers. On the other hand, breathing was viewed as a maintained tonic discharge of the medullary respiratory centers, interrupted by two inhibitory mechanisms, the vagal reflex afferent (see later), and the brain stem control through the pneumotaxic center. The two views were often discussed because the data obtained in the cat and the rabbit were different. Marckwald made several transections through the brain stem and the medulla of the rabbit (Marckwald, 1888). When the brain stem was transected through the pons, below the inferior colliculli, respiration persists, but as soon as the vagal nerves were sectioned, the animal inspires deeply. When the section was done between the superior and the inferior colliculi, after the section of the vagal nerves, a very slow rhythm appears due to the “pneumotaxic center.” Lumsden confirmed in cats the presence of this very slow rhythm and proposed in addition an “apneustic center” which stimulated tonically the inspiratory center (Lumsden, 1923). At the beginning of the twentieth century, the idea of several respiratory centers emerged and the problem was to analyze their relationships.

Reflex function and its generalization Reflex is an old notion described first by Descartes and by Willis but mainly detailed by Jiri Prochaska (1749–1820). He was Professor of anatomy and ophthalmology in Prague and finished his career in Vienna. He described the reflex in a small book “De functionibus systematis nervosi commentatio”(see Fearing, 1970, Prochazka et al., 2000) where two important notions are explained. The “vis nervosa” explains its automatic reproducibility and its brief duration, corresponding to an elemental form of energy. The second is the “sensorium commune” that

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corresponds to the nervous system centers where the sensations are integrated before the motor response. It is located in the brain but also in the medulla and in the spinal cord. For him, the spinal cord was the first level of integration, exactly as was defined one century later by Sherrington.

Emergence of the notion of reflexes At the onset of the nineteenth century, reflexes were very well established not only by physiologists but also by psychologists. Even with the fierce controversies he induced, Marshall Hall (1790–1857) established definitively that notion when he came up with the concept of the reflex arc, describing the sensory influences coming into the medulla and reaching the upper centers in addition to the segmental effector responses (Hall, 1833). He explained that the reflex activity that took place at the spinal level could be influenced by “will.” After a removal of the brain, there is an exaggeration of the reflex. More interestingly, speaking about walking, he presupposed a sort of stepping reflex : “In the actions of walking in man, I imagine the reflex function to play a very considerable part, although there are, doubtless facts which demonstrate that the contact of the sole with the ground is not unattended by a certain influence upon action of certain muscles (Hall, 1836 cited in Brazier, 1988)” Hall established blinking, sneezing, and vomiting as reflexes controlled by the CNS in humans. For respiration, in a short book (1855) he published in French in honor of Flourens, he accepted the vital node but considered that this point must be activated by a reflex. A great debate occurred between Wilhelm Pflüger (1829–1910) and Rudolf Hermann Lotze (1817–1881) concerning the role of the spinal cord: According to Pflüger, the spinal cord possessed some psychical properties like higher brain structures. In opposition, Lotze believed that the spinal cord was under the control of the brain

and had no particular properties. For the psychologists, the notion of reflex was described in the context of evolutionary biology. Thomas Laycock (1812–1876) insisted on the continuity in nature of the evolution of mind. Like Griesinger, he made a parallel between the development of the nervous structures and the emergence of the psychical functions in the animal kingdom (Clarke and Jacyna, 1987). Laycock found that Hall's idea of reflex was too narrow, and published an article in 1845 with the following title “On the reflex function of the Brain.” Following these different analyses, the English Psychologist Herbert Spencer (1820–1903) described in detail the evolution of reflex in his book published in 1855, “The principle of psychology.” He followed the evolution of living animals and of the nervous system describing the different steps of increasing complexity. If the lower animals have only rigid automatic reactions, species situated at a higher level in the evolutionary scale have a much weaker organization, but their reactions, even their plasticity, are quite similar. Spencer developed the idea of a unitary living world were mind is at the top but has emerged from the most simple expression of life. His reasoning used the concept of emergence where reflex is not only at the basis but also at the summit. The will was for him a certain form of reflexology, the only difference was that in humans, the sensory information was accumulated, and became conscious and expressed later in life, through different sorts of reactions. “The difference between an involuntary movement of the leg and a voluntary one. . .is, that whereas in the case of the involuntary movement, the psychical states accompanying the impression and the action, are so coherent that the one follows the other instantaneously; in the voluntary they are so imperfectly coherent, that the psychical state accompanying the action does not follow instantaneously. . .thus the cessation of automatic action and the dawn of volition, are one and same thing.” (Spencer, 1855, p. 614). Before the Darwin revolutionary theory, physiologists and psychologists perhaps influenced by Jean Lamarck's ideas (1744–1829), expressed

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the concepts on motor control with a dynamical presentation of the world. The Scottish Alexander Bain (1818–1903) who like Spencer was a member of the Associationist School following Thomas Hobbes (1588–1679), John Locke (1632–1704), John Stuart Mill (1806–1873), and William James (1842–1910), can be considered as part of this empirical lineage that gave a primary role to the sensory-motor organization. In Russia also, the notion of reflex will become essential. Ivan Sechenov (1829–1905), the father of the Russian school of physiology, went first to Germany and later to Paris to work under the influence of Claude Bernard (1813–1878). There, he used a decapitated frog to study the role of the brain-stem on the spinal reflex putting salt crystals at various levels of the transected neuraxis and measuring the timing between the stimulus and the onset of leg withdrawal. He observed that such application delayed the reflex. He interpreted the brain stem stimulation as providing an inhibition of the spinal reflex (Sechenov, 1863). Sechenov conceptualized from his experiments in frog, that all mental processes of man were reflex in nature. He formalized this concept in one of his major publications: “Reflexes of the brain (1863),” in which he considered the mental state in three successive activities, sensory, integrative, and motor. Following the principles of excitations and inhibitions, he proposed that facilitation is associated with the motor phase and linked with emotional excitement. By contrast, inhibition of motor action is present during the second phase or thought. In the nineteenth century, the reflex was only partially understood and we will see the consequences for concepts of breathing and walking.

Peripheral control of breathing In contrast to the experiments done in the medulla oblongata centers, several others were conducted on the nerves. Hall (1836) proposed that breathing was driven by intrapulmonary CO2, and that the respiratory centers needed to

be fed by sensory inputs. Volkmann was the first to stimulate electrically the vagus nerve in 1838, but he did not accept the inhibition of the heart that he observed. It was only in 1845 when Ernst Weber and his brother Eduard observed that inhibitory response in the frog that the phenomenon was really taken into account. Later, Rosenthal (1862) stimulated the vagus and evoked a contraction of the inspiratory muscles. By contrast, Paul Bert (1833–1886), by stimulating the same nerves, inhibited the respiratory rhythm completely (Bert, 1869). Stimulating the superior larynx and the nasal nerves, Bert demonstrated that the larynx was more sensitive to chemical irritants than the nose. Despite contradictory results, it was admitted that different afferent fibers of the vagus, laryngeal, trigeminal, and other nerves could interrupt breathing, which developed into a new story with the famous Hering-Breuer reflex (1868). Ewald Hering (1834–1918) who specialized in color vision (his book “theory of binocular vision” was published in 1868) and was named professor of physiology, first in Vienna and then later in Prague. Hering's greatest achievement was to have attracted two medical students and a novice doctor to work with him, although he performed no experiments himself. Within 5 years, he established the foundation of our knowledge of respiratory reflexes. Joseph Breuer (1842–1925) was a clinical student, who in 1867 was awarded the title of Privat-Dozent. He took a sabbatical to do research in the Military Academy in Vienna with Hering. After his work, he published a single paper on “The self-steering of breathing through the vagus nerve” (Breuer, 1868, see Breuer, 1970). Then, he continued his medical studies and became a psychiatrist. The case study of Anna O., treated by Breuer, has been the source of work, leading to publications such as the famous Studies on Hysteria, in 1895. With this, Breuer can be considered the cofounder with Freud of psychoanalysis. His work on respiration was conducted on various animals, cats, dogs, and rabbits, where he

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used the intravenous application of opium as an anaesthetic. To monitor the ventilation of the lungs, Breuer used a manometer and measured the pressure in the trachea, which decreased during a normal inspiration and increased during the ensuing expiration. He used natural stimuli, inflation, and deflation of the lungs and showed that an expansion of the lungs inhibits inspiration, with the response being closely related to the stimulation. In contrast, a reduction of the lung volume stopped immediately any expiration and could elicit inspiration. This reflex disappeared with the section of the vagi (see Fig. 3a). Florin Kratschmer (1843–1922) also graduated in medicine from the Joseph's Academy in 1869. After a single year on respiration problems, he pursued a career as a military physician. In 1903, he became titular professor of hygiene at the University of Vienna. Kratschmer worked essentially on the upper airways, and used various stimuli like cold air, carbon dioxide. . .etc., which he applied to the different parts of the upper airways. He recorded a variety of responses and established their nervous pathways by sectioning the relevant nerves. He detailed the mechanisms of respiratory and cardiovascular reflexes from the nose and larynx. Kratschmer's work (1870) has been largely ignored, and was only recently translated into English (Kratschmer, 2001). Henry Head (1861–1940) was a medical student in Cambridge, who went to work with Hering for 3 years between 1884 and 1886. Afterward, he came back to England and became a famous neurologist known for his work on sensory afferents. He continued the analysis that Breuer had not finished. Working on rabbits, he used a better hypnotic agent; chloral hydrate. He experimented with a manometer connected to the trachea recording respiration in a much better way (Fig. 3b). Head prepared a slip of diaphragm and used this as an indicator of the degree of activation of the respiratory muscles, which did not interfere with the integrity of the thoracic cavity. His two papers describing the technique of blocking the vagus nerves by cold thus enabling the

(a)

C.VI A

C.VI B

(b)

A

Curve II

Curve X B

Fig. 3. Hering Breuer reflex. (a) Original records from the Breuer's paper (see Widdicombes, 2006). First upper beam: At the top is the intratracheal pressure. The blood pressure is at the bottom. Inflation of the lung increases the pressure inducing a longlasting cessation of respiratory movement. Lower beam: After vagotomy, the reflex is suppressed (the records are going from right to left). (b) Recordings from Head (1889a). Curve II represents a distention of the lung that inhibits inspiration. The tracing at the top is from a control lever attached to the wall of the chest. The dotted line is the tracing of a mercury manometer connected with the trachea. A rise indicates the distention of the lungs. The curve below is that of a lever attached to the diaphragm slips of the rabbit; it represents the movement of the diaphragm as a whole. Last line is a time marker. Curve X is a similar reaction with a short inflation but with pure hydrogen. It shows that the results obtained are not due to a cessation of respiration owing to an increased supply of air.

possibility to repeat the experiment on the same animal came up with two essential points (Head, 1889a,b). One experiment demonstrated that the deflation reflex had a separate pathway from that

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of the inflation reflex. The second described the inspiratory effort due to lung inflation when the vagus nerves were recovering from cold block. At about 10  C, the classical inflation reflex was replaced by a paradoxical one. The modern explanation is that the afferent signals of the two reflexes are conducted in different populations of vagal fibers. At that temperature, the large sensory fibers are still blocked while the smallest ones can transmit the stimulation. J. Widdicombe (2006) summarizing this reflex story concluded with this humoristic sentence “When we digest the papers we find that Breuer offers gourmet food, Head a stodgy suet pudding containing a couple of plums and Kratschmer a dogs dinner; but together they provide a wonderful three-course meal.” If this reflex is essential in animals, it is not the case for most adult humans at rest. However, the reflex may determine breathing rate and depth in newborns.

The somatic muscular reflexes and their use in clinical studies During the second half of the nineteenth century, experiments on reflexes were not limited to the classical frog, other animals were used, mammals in particular, as well as studies conducted routinely on patients. In 1870, Whilhelm Henrich Erb (1840–1921) was the first to recognize the diagnostic utility of the patellar or “knee jerk” reflex and the use of percussion hammer to elicit it. A hammer created by the German clinician Max A. Wintrich (1812–1882) was thought to be a more effective tool than a physician's finger (Pinto, 2003). When Erb wanted to submit his paper about the patellar jerk, he discovered that the editor of the “German Archives of Psychiatry and Neurology” the neurologist and the psychiatrist, Carl Friedrich Otto Westphal (1833–1890) had written a similar paper. They agreed to publish simultaneously (Erb, 1875; Westphal, 1875). However, they had two opposing hypotheses about the very quick response they recorded. If

Erb was right in thinking that the effect was really a reflex involving the spinal cord, Westphal proposed that the response was due to a direct stimulation of the muscle. In the 1880s, the British William Gowers (1845–1915) coined the formulae “Myotatic reflex.” These reflexes were then used as a tool by the majority of clinicians. Their various responses were the basis of modern neurology. Erb started at Heidelberg as assistant to the pathologist Nikolaus Friedreich (1825–1882). His interest in neurology made him a leader in the field. In 1880, he went to the University of Leipzig where he served as a director of the neurology clinic. Like Duchenne he used electricity in the diagnosis and treatment of nervous disorders. Both were associated in a Duchenne–Erb paralysis, a disturbance caused by nerve lesion of plexus brachialis during birth. Among these clinicians, Babinski was perhaps the most impressive in his examination of patients, conducted with “great ceremony” with his assistants (Fulton, 1933). The patient was examined totally naked, with Babinski working in total silence, broken occasionally to request the patient to perform some particular movement or walk about the room to analyze specific contractions of some particular muscles. He used his hammer to induce various types of reflexes, which helped him understand the internal functioning of the nervous system. If, after several hours, he was not satisfied, he would sometimes continue on another day! Babinski was known in particular for the cutaneous reflex of the toes: the “sign” which he described at the meeting of the Société de Biologie in a communication of 28 lines (Babinski, 1896): The pathologic extensor plantar response when the sole is stimulated while in normal situation, the same stimulation gives a general flexion. In its simplicity, Babinski's sign has hardly any equal in medicine. His first publication was nearly ignored. He gave a full account of the reaction and calls it the “signe de l'éventail” in 1903, the fanning of the toes often occurring simultaneously with the extension of the great

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toe with the lesion of the pyramidal tract (Babinski, 1934). Concerning mastication, very little information is available. David Ferrier (1843–1928), the pioneer Scottish neurologist studied the different cortical areas of the monkey in great detail. At the great meeting of neurology of London in August 1881, Ferrier presented the role of the motor cortical area in great detail. He was in complete opposition with Freiderich Leopold Golz (1834–1902) who defended the cortical holist theory. Moreover, Ferrier was able to demonstrate that the high-intensity stimulation of a particular part of the motor cortical areas caused repetitive chewing movements (Ferrier, 1873). Except for that, we can only mention the article of Sherrington where he considered the pinna reflex and the jaw reflexes (Sherrington, 1917). This increasing predominance of reflexology is obvious at the end of the nineteenth century with the discovery of the muscle spindles (Scheerer 1987). Henri Charlton Bastian (1837–1915) gave a quite complete description of that peripheral view: the muscular sense is predominant and the movement initiation results from unconscious kinaesthetic (or visual) images within the kinaesthetic (or visual) centers (Bastian, 1888). It was then that Sherrington with his experiments on the cat spinal cord compared the different peripheral sensory localizations, and defined proprioception in comparison with exteroception and interoception. Sherrington viewed reflexes as the basic units of neural function (Sherrington, 1906). He described with Grundbaum (Grünbaum and Sherrington, 1903) the exact location of the cortical motor center, although he promoted the idea that locomotion was due to a chain of spinal reflexes (Sherrington, 1910). His ideas predominated the first part of the twentieth century, even though it contradicted the central spinal hypothesis developed by Maurice Philipson (1877–1938) in 1905 and by T. GrahamBrown (1882–1965), with his theory of “half centers” in 1914 (see Stuart and Hultborn, 2008).

Conclusion A fairy tale starting with “once upon a time” always finishes with “they married and had many children”. . .We can say that the children of early twentieth century neurology were three giants; two anatomists, Camillo Golgi and Santiago Ramon y Cajal, and the neurophysiologist, Sir Charles Sherrington (Jacobsen, 1995). Their works completely changed our vision of the CNS although they overlooked much of CNS due to the limitations of the physiological methods for exploring brain and spinal organization. For this reason, the functions of the peripheral regions were studied in priority and reflexes were predominantly described. It was necessary to wait about 50 years to see the reemergence of the concept of the central pattern generator (Grillner, 1975). We have today a much more satisfactory approach of the organization of motor control, and in particular with those that will be presenting their data at this 31st International Symposium of the “Groupe de Recherche sur le Système Nerveux Central.”

Acknowledgments We thank Dr A. El Manira and J. C. Viemari for their helpful discussions and valuable comments on an earlier version of the manuscript. We also thank very much the reviewers who made some very precise and useful corrections on the last version of the text. References Aran, F. A. (1850). Recherches sur une maladie non encore décrite du système musculaire -l'atrophie musculaire progressive- [Researches on a pathology not yet describedthe muscular progressive atrophy]. Archives générales de médecine, 3, 172. Babinski, J. (1896). Sur le réflexe cutané plantaire dans certaines affections organiques du système nerveux central. CR. Soc. Biol., 48, 207–208.

14 Babinski, J. (1934). Oeuvre Scientifique, recueil des principaux travaux. In published by J.A. Barre, J. Chaillous and A. Charpentier. Masson Paris. Bastian, H. C. (1888). The “muscular sense”, its nature and cortical localisation. Brain, 10, 1–137. Bert, P. (1869). Des effets de l'excitation du nerf pneumogastrique, du nerf laryngé supérieur et du nerf nasal sur la respiration. Arch. de Physiol. Normale et pathol., 12, 179–196. Borelli, G. (1680). De Motu animalium. Rome: Superiorum Permissu. Brazier, M. A. B. (1988). A History of Neurophysiology in the 19th Century. New York: Raven Press. Breuer, J. (1970). The self-steering of respiration through the nervous vagus. Translated by E. Ullmann. In R. Poter (Ed.), Breathing: Hering-Breuer Centenary Symposium (pp. 357–394). London: Churchill (Ciba fundation symposium). Charcot, J. M., & Joffroy, A. (1869). Deux cas d'atrophie musculaire progressive avec lésions de la substance grise et des faisceaux antéro-latéraux de la moelle épinière. Archiv. Physiol. Neurol. Path., 2, 354–367; pp. 629–650 and pp. 745–760. Clarac, F. (2008). Some historical reflections on the neural control of locomotion. Brain Res. Rev., 57, 13–21. Clarac, F., Massion, J., & Smith, A. M. (2009). Duchenne, Charcot and Babinski, three neurologists of La Salpetrière Hospital, and their contribution to concepts of the central organization of motor synergy. J. Physiol. Paris., 103(6), 361–376. Clarke, J. L. (1869). On locomotor ataxy. Br. Med. J., 2(448), 121–122; and 25 (448) pp. 344–345. Clarke, E., & Jacyna, L. S. (1987). Nineteenth-Century Origins of Neuroscientific Concepts. Berkeley, Los Angeles, London: University of California Press. Duchenne de Boulogne, G. (1858). De l'ataxie locomotrice progressive caractérisée spécialement par des troubles généraux de coordination des mouvements. Arch. Gen. Med., 12, 641–652. Duchenne de Boulogne, G. (1867). Physiologie des mouvements démontrée à l'aide de l'expérimentation électrique et de l'observation clinique et applicable à l'étude des paralysies et des déformations; J. B. Baillière (Ed.), Paris. Erb, W. H. (1875). Uber Sehen reflexe bei gesunden und bei Rückenmarkskranken. Archiv. für Psychiatr., 5, 792–802. Fearing, F. (1970). Reflex Action. Cambridge, Massachussetts, London: MIT Press; 2nd ed. 1970. Ferrier, D. (1873). Experimental researches in cerebral physiology and pathology. West Riding lunatic Asylum Reports., 3, 30–96. Finger, S. (1994). Origins of Neuroscience. Oxford, New-York: Oxford University Press. Flourens, M. J. P. (1824). Recherches expérimentales sur les propriétés et les fonctions du système nerveux dans les animaux Vertébrés. J. B. Baillère (Ed.), Paris.

Flourens, M. J. P. (1851). Note sur le point vital de la moelle allongée. Comptes Rendus A.S. XXXIII, 17, 437–439. Flourens, M. J. P. (1858). Nouveaux détails sur le noeud vital. Compte Rendus A.S. XLVII; pp. 803–806. Fulton, J. F. (1933). Joseph François Felix Babinski. Arch. Neurol. Psy., 29, 168–174. Gad, J., & Marinesco, G. (1892). Recherches expérimentales sur le centre respiratoire bulbaire. Compte Rendus A. S. CXV; pp. 444–447. Gasser, J. (1995). Aux origines du cerveau moderne. In Fayard (Ed.), Paris. 335 pp. Gombault, A. (1877). Contribution à l'étude de la sclérose latérale amyotrophique. Progrès Médical, (Suppl.), 1–86. Grillner, S. (1975). Locomotion in vertebrates. Central mechanisms and reflex interaction. Physiol. Rev., 55, 247–304. Grossmann, S., Maeder, I. M., & Dollfus, P. (2006). Treatise on the spinal marrow and its diseases by Ollivier d'Angers (1794-1845). Spinal Cord, 44(12), 700–707. Grünbaum, A. S. F., & Sherrington, C. (1903). Observation on the physiology of the cerebral cortex of the anthropoid ape. Proc. Roy. Soc., 72, 152–155. Haas, R. B. (1976). Muybridge: Man in Motion. Berkeley: University of California Press. Hall, M. (1833). On the reflex function of the medulla oblongata and medulla spinalis. Phil. Trans.R. Soc., 123, 635–665. Hall, M. (1836). Lectures on the Nervous System and its Diseases. Philadelphia: Carey and Hart. Haughton, S. (1873). Principles of Animal Mechanics. London: Longmans, Green, & Co.. Head, H. (1889a). On the regulation of respiration. Part I. Experimental. J. Phyiol., 10, 1–70. Head, H. (1889b). On the regulation of respiration. Part II. Theoretical. J. Physiol., 10, 279–290. Holmes, G. (1917). The symptoms of acute cerebellar injuries due to gunshot injuries. Brain (IV), 40, 461–535. Jacobsen, M. (1995). Foundations of Neuroscience. New York: Plenum Press. Jeannerod, M. (2006). The origin of voluntary action history of a physiological concept. C.R. Biologies, 329, 354–362. Jones, E. G. (1972). The development of the « muscular sense » concept during the nineteenth century and the work of H. Charlton Bastian. J Hist med. Allied Sci., 27(3), 298–311. Kratschmer, F. (2001). On the reflexes from the nasal mucous membrane on respiration and circulation. translated by E; Ullmann. Respir. Physiol., 127, 93–104. Legallois, J. J.-C. (1812). Expérience sur le principe de la vie, notemment sur celui des mouvements du cur et sur le siège de ce principe. Paris, d' Hautel. (translation by Nancrede NC., Nancrede JG., Experiments on the principle of life. Philadelphia, 1813).

15 Longet, F. A. (1847). Expériences relatives aux effets de l'inhalation de l'ether sulfurique sur le système nerveux des animaux. Arch. Gén. Médic. ser. IV, 13, 374–412. Lumsden, T. (1923). Observations of the respiratory centres in the cat. J. Physiol., 57, 153–160. Marckwald, M. (1888). Movements of Respiration and Their Innervation in the Rabbit. Translated by T. A. Haig, London, Blackie and son. Marey, E. J. (1873). La Machine Animale, Locomotion Terrestre et aérienne (Animal mechanism, terrestrial and air locomotion). Paris: Germer Bailliére. Marey, E. J. (1894a). Le movement [the movement]. In G. Masson (Ed.), Paris. 347 pp. Marey, E. J. (1894b). Les mouvements articulaires étudiés par la photographie. (the joint movements studied by photography). Comptes Rendus A.S., 118, 1019–1025. Muybridge, E. (1887). Animal Locomotion. University of Pennsylvania. The Philadelphia Print Shop. Parent, A. (2005). Duchenne De Boulogne: A Pionneer in Neurology and medical Photography. Can. J. Neurol. Sci., 35, 369–377. Pearce, J. M. S. (2005). Romberg and his sign. Eur. Neurol., 53, 210–213. Pinto, F. (2003). A short history of the reflex hammer. Practical Neurology, 3, 336–371. Pitts, R. F. (1946). Organization of the respiratory center. Physiol. Rev., 26, 609–630. Prochazka, A., Clarac, F., Loeb, G., Rotwell, J., & Wolpaw, J. (2000). What do “voluntary” and “reflex” mean? Modern views on an ancient debate. Exp. Br. Res., 130, 417–432. Roussy, G., & Lhermitte, J. (1918). Les blessures de la moelle et de la queue de cheval. Masson (Ed.) Paris 202 pp.

Scheerer, E. (1987). Muscle sense and innervation feelings : A chapter in the history of perception and action. In H. Heuer & A. F. Sanders (Eds.), Perspectives on Perception and ActionHillsdale, N.J.: Erlbaum. pp. 171–194. Schiller, F. (1995). Staggering gait in Medical History. Ann Neurol, 37, 127–135. Sechenov, I. M. (1863). Reflexes of the Brain. Cambridge, M.A.: M.I.T. Press; Translated from the Russian by S. Belsky, edited by G. Gibbons, with notes by S. Gellerstein, 1965. Sherrington, C. S. (1906). The integrative Action of the Nervous System (5th ed. (1947)). Cambridge: Cambridge University Press. Sherrington, C. S. (1910). Flexion-reflex of the limb, crossed extension reflex, and reflex stepping and standing. J. Physiol. (London), 40, 28–121. Sherrington, C. (1917). Reflexes elicitable in the cat from pinna, vibrissae and jaws. J. Physiol., 51, 404–431. Spencer, H. (1855). The Principle of Psychology. London: Longman, Brown, Green and Longmans; Republished in 1970 by Gregg International Publishers Limited Westmead, Farnborough, Hants Englad, 620pp. Stuart, D. G., & Hultborn, H. (2008). Thomas Graham Brown (1882-1965), Anders Lundberg (1920-), and the neural control of stepping. Br. Res. Rev., 59(1), 74–95. Weber, W., & Weber, E. (1836). Mechanik der Menschlichen Gehwerkzeuge [The Mechanics of Human Locomotion]. Germany: Dieterichschen Buchhandlung Gottingen. Westphal, C. F. O. (1875). Uber einge Bewegunsersqcheinungen an gelähmten Gliedern. Arch. f. Psych., 5, 803–834. Widdicombe, J. (2006). Reflexes from the lungs and Airways : historical perspectives. J. Applied Physiol., 101, 628–634.

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 2

Genetic dissection of rhythmic motor networks in mice Katja S. Grossmann1, Aurore Giraudin1, Olivier Britz, Jingming Zhang and Martyn Goulding* Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA

Abstract: Simple motor behaviors such as locomotion and respiration involve rhythmic and coordinated muscle movements that are generated by central pattern generator (CPG) networks in the spinal cord and hindbrain. These CPG networks produce measurable behavioral outputs and thus represent ideal model systems for studying the operational principles that the nervous system uses to produce specific behaviors. Recent advances in our understanding of the transcriptional code that controls neuronal development have provided an entry point into identifying and targeting distinct neuronal populations that make up locomotor CPG networks in the spinal cord. This has spurred the development of new genetic approaches to dissect and manipulate neuronal networks both in the spinal cord and hindbrain. Here we discuss how the advent of molecular genetics together with anatomical and physiological methods has begun to revolutionize studies of the neuronal networks controlling rhythmic motor behaviors in mice. Keywords: locomotion; respiration; spinal cord; CPG; interneuron; mouse genetics.

Rhythmic motor outputs such as locomotion, respiration, and mastication are, in their simplest forms, highly stereotyped motor behaviors. In fish and tadpoles, locomotion primarily consists of repetitive lateral bending movements of the axis, which are produced by waves of contraction and relaxation that propagate rostrocaudally along the body axis. In contrast, terrestrial vertebrates

propel themselves by flexing and extending their limbs. This more complex mode of locomotion requires extensive coordination between the forelimbs and hindlimbs on each side of the animal and between individual limb joints. The additional demands of land-based locomotion appear to have driven the evolutionary elaboration of the spinal motor circuitry in terrestrial vertebrates, both in terms of numbers and types of neurons, as well as sensory and supraspinal connectivity. In higher vertebrates, the spinal locomotor system has thus

*Corresponding author, 1equal contribution. Tel.: þ1-858-453-4100-1558; Fax: þ1-858-450-2172. DOI: 10.1016/S0079-6123(10)87002-5

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evolved into a highly dynamic network that produces a varied and flexible array of motor outputs in response to sensory feedback pathways and descending influences from rubrospinal, reticulospinal, vestibulospinal, and corticospinal pathways. Rhythmic motor circuits in the hindbrain and spinal cord The core neuronal networks that control rhythmic respiratory and locomotor motor behaviors reside in the hindbrain and spinal cord, respectively. These CPG networks generate simple organized motor rhythms in an autonomous manner. Initial efforts to decipher the general organization of these simple motor CPGs in vertebrates relied heavily on electrophysiological and pharmacological approaches. Such efforts were greatly aided by the development of in vitro hindbrain–spinal cord preparations in neonatal rodents (Kudo and Yamada, 1987; Smith and Feldman, 1987). In addition to enabling investigators to localize two rhythmic CPGs in the medulla that drive respiratory movements, the pre-Bötzinger complex and parafacial respiratory group (Onimaru and Homma, 1987; Smith et al., 1991; this book), this in vitro preparation has been used to map the hindlimb locomotor CPG. For instance, serially sectioning the lumbar spinal cord, both rostrocaudally and dorsoventrally, has defined the minimal area needed to generate a locomotor rhythm. The area that is circumscribed encompasses laminae VII, VIII, and X of the lumbar cord, with lumbar segments L1–L2 being particularly important for rhythm generation (Bracci et al., 1996; Cazalets et al., 1995; Kjaerulff and Kiehn, 1996). Forelimb movements are likewise controlled by networks distributed in the cervical spinal cord (Ballion et al., 2001). Further insights into the organization of the spinal locomotor CPG network have come from in vitro pharmacological studies. From these studies, it appears that locomotor rhythmogenesis is primarily reliant on

glutamatergic ipsilateral neurons (Bracci et al., 1996; Kjaerulff and Kiehn, 1997), whereas the alternating patterns of flexor–extensor and left–right activities are secured by GABAergic and glycinergic pathways (Bracci et al., 1996; Kjaerulff and Kiehn, 1996, 1997). Over the past few years, efforts to delineate the genetic programs that pattern the caudal neuroaxis have provided new avenues for probing the composition of these CPG networks. An important thematic that has emerged has been the role that genetically driven developmental programs play in directing neuronal specification and differentiation in the hindbrain and spinal cord. Significant progress has been made in understanding how such developmental programs determine neuronal identity and how neurons are wired together into functional motor circuits. These studies suggest commonalities in the cellular composition of CPG networks in the hindbrain and spinal cord, in so far that these circuits are built from interneuron cell types that share a similar embryonic heritage. In this review, we will focus on genetic approaches used in mice to identify cells that are components of motor networks and assess their contribution to rhythmic motor behaviors, with an emphasis on locomotion. The developmental program of the caudal neuroaxis The developmental events that pattern the caudal neural tube play a central role in assembling sensorimotor circuits in the hindbrain and spinal cord (Goulding, 2009; Jessell, 2000). The position that a progenitor cell occupies along the dorsoventral (DV) axis confers a specific genetic code to these cells and thus serves as a major determinant of cell identity (Fig. 1a). This DV patterning program segregates newborn neurons into generic populations that are arrayed as longitudinal columns along the antero-posterior axis of the hindbrain and spinal cord. As development proceeds, neurons within these columns migrate

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Fig. 1. Genetic classification, properties, and organization of spinal neurons. (a) Schematic cross-section through the early embryonic mouse spinal cord. Eleven progenitor domains (pd1-pV3) have been identified in the ventricular zone that give rise to distinct postmitotic neuronal populations (dI1-V3). Selected transcription factors expressed within the progenitor domains are shown on the left and those specific to postmitotic populations of neurons born around embryonic day E11 are depicted on the right. Postmitotic neurons that contribute to the locomotor CPG machinery are shown in color, whereas neurons implicated in sensory pathways are shown in gray. (b) Summary of molecular factors as well as cellular and physiological properties that characterize each locomotor neuronal class. (c) Schematic of the motor circuitry in the postnatal ventral spinal cord. The color code used is the same as in (a). The locations of neuron classes and their known subsets, as well as some known connections between these classes, are illustrated. p, progenitor domain. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this chapter.)

22

extensively to form the different laminae seen in the adult cord. Many premotor commissural interneuron cell types, for example, migrate from the intermediate zone to lamina VIII, while ventral interneurons that project ipsilaterally migrate laterally to lamina VII (Fig. 1c). Interestingly, the early DV organization of postmitotic neurons appears to be conserved across widely divergent vertebrate phyla from fish to mammals and it represents the developmental ground plan from which different CPG modules in the spinal cord and hindbrain emerge (Goulding, 2009; Grillner and Jessell, 2009). The molecular programs that control the acquisition of DV positional identity have been explored in some detail. In short, the graded activity of several signaling molecules, sonic hedgehog (Shh) and TGF-like bone morphogenic proteins (BMPs), retinoic acid (RA), and Wnts, initiate and orchestrate the DV patterning of dividing progenitors (Chiang et al., 1996; Liem et al., 1997; Timmer et al., 2002 and reviewed in Jessell, 2000) in part by establishing dorsoventrally restricted domains of patterning factors, including the Pax3/7, Pax6, Nkx6-1, and Nkx2-2 transcription factors, within the ventricular zone of the neural tube. These patterning factors function instructively in a combinatorial manner to subdivide the neural tube into 11 progenitor zones (Goulding, 2009). As noted previously, this developmental program operates in all vertebrate embryos to activate the expression of unique sets of homeodomain (HD) and basic helix-loop-helix (bHLH) transcription factors in the postmitotic neurons that arise from these 11 progenitor domains (Goulding, 2009; Fig. 1a). Six classes of early born interneurons, the dI1–dI6 interneurons, are generated in the dorsal alar plate. Two additional late born populations of dorsal neurons have been identified that are prevalent in birds and mammals, the dILA and dILB (Gross et al., 2002; Mizuguchi et al., 2006; Muller et al., 2002; Wildner et al., 2006). These cells are similar in molecular composition to dI4 and dI5 interneurons, and it is suspected that they represent an evolutionary expansion of

the early born dI4 and dI5 populations in terrestrial vertebrates (Gross et al., 2002; Mizuguchi et al., 2006; Muller et al., 2002; Wildner et al., 2006). Although the majority of dorsal cell types differentiate as sensory interneurons and sensory-relay neurons, some appear to make cellular contributions to the locomotor CPG machinery. However, most of the cell types within the locomotor CPG are derived from six generic neuronal classes that develop from basal plate progenitors: motoneurons and V0, V1, V2a, V2b, and V3 interneurons (Goulding, 2009; Jessell, 2000). Derivatives of these ventral classes of interneurons settle in regions of the spinal cord that are known to contain locomotor CPG networks in quadrupedal mammals (Goulding, 2009; Kiehn, 2006). Postmitotic populations of V0, V1, V2a, V2b, and V3 interneurons are marked by the expression of the transcription factors Evx1/2, En1, Chx10, Gata2/3, and Sim1, respectively (Al-Mosawie et al., 2007; Croneet al., 2008; Karunaratne et al., 2002; Kimura et al., 2006; Lundfald et al., 2007; Moran-Rivard et al., 2001; Peng et al., 2007; Saueressig et al., 1999; Zhang et al., 2008; reviewed in Goulding, 2009; Jessell, 2000; Fig. 1a and b). dI6 neurons that arise more dorsally express Lbx1, Pax2, and Lhx1 and settle in lamina VIII (Gross et al., 2002; Muller et al., 2002). These cells are also thought to contribute to the premotor circuitry (Fig. 1c). The hindbrain displays a similar DV organization of early neuronal cell populations. Of the 14 progenitor domains described in the hindbrain, 10 are equivalent to the spinal progenitor domains and they generate cell types that share many features with their spinal cord counterparts (Gray, 2008). Antero-posterior differentiation events that are orchestrated by the homeobox class of HD transcription factors are of primary importance for the development of hindbrain nuclei that arise from the same embryonic population. Embedded in these nuclei are circuits that control vital autonomic functions such as respiration, as well as mastication, audition, and the supraspinal control of locomotion (Dubreuil et al., 2009;

23

Fujiyama et al., 2009; Pagliardini et al., 2008; Storm et al., 2009). In the next section, we will summarize what is currently known about the early development of the spinal motor circuitry.

Generating ventral interneurons: instructive role of progenitor domain-specific transcription factors As noted previously, each of the early DV progenitor domains expresses a unique combination of transcription factors that play an instructive role in generating V0, V1, V2a, V2b, and V3 interneurons (Fig. 1). Changes in the expression of these factors within each progenitor domain lead to major specification defects in their neuronal progeny. For instance, the HD transcription factor Dbx1 is an essential factor for the specification of V0 interneurons. In Dbx1 mutant mice, neural progenitors that normally give rise to V0 interneurons are respecified to produce V1 and dI6 interneurons (Lanuza et al., 2004; Pierani et al., 2001). Ablation of Nkx6-2, a homeobox transcription factor specific to the V1 progenitor domain, reportedly increases V0 interneuron numbers at the expense of V1 interneurons (Vallstedt et al., 2001). Further ventrally, Nkx22 regulates V3 progenitor identity. In Nkx22 mutants, these cells undergo a ventral-to-dorsal transformation in fate, thereby generating motoneurons at the expense of V3 interneurons (Briscoe et al., 1999). These findings support the idea that transcription factors present in adjacent progenitor domains suppress the differentiation programs that operate in the progenitor domains they abut. They do so by cross-repressive interactions that position the boundaries between adjacent domains. Consequently, loss of one factor often results in the expansion of an adjacent progenitor domain and the cell types that arise from that domain. Other transcription factors are expressed in more than one progenitor domain, where they are required for the generation of several neuron classes. Nkx6-1 has been

shown to be important for the generation of both V2 interneurons and motoneurons (Sander et al., 2000). Inactivation of Pax6, in addition to causing a loss of V1 interneurons, also alters the generation of branchiomotoneurons in the hindbrain (Burrill et al., 1997; Ericson et al., 1997; Sapir et al., 2004). The presence of several transcription factors within each progenitor domain allows further subspecification of domains characterized by the expression of Nkx6-1 or Pax6. The V2 progenitor domain, for example, is defined by coexpression of the forkhead transcription factor Foxn4 and the bHLH transcription factor Ascl1. Interestingly, the V2 progenitor domain, unlike others in the spinal cord, gives rise to two intermingled but molecularly distinct classes, termed V2a and V2b interneurons. Foxn4 and Ascl1 are critical for establishing V2a and V2b cell fate (Del Barrio et al., 2007; Kimura et al., 2008; Li et al., 2005; Peng et al., 2007). In V2 progenitors, Foxn4 induces expression of Ascl1 and the Notch ligand Dll4, which in turn activates Notch signaling in adjacent progenitors. Active Notch signaling in progenitors undergoing a final cell division leads to the generation of V2b interneurons, whereas an absence of Notch signaling in progenitors results in differentiation into V2a interneurons (Del Barrio et al., 2007; Kimura et al., 2008; Li et al., 2005; Peng et al., 2007). A similar mechanism involving Notch signaling has been implicated in the generation of dILA and dILB interneurons in the dorsal spinal cord (Mizuguchi et al., 2006; Wildner et al., 2006).

Generating diversity: postmitotic transcription factors in interneuron differentiation Neurons upon exiting the cell cycle migrate into the mantle zone. During this transition, their transcription factor profile changes and they begin to express factors exclusive to postmitotic neurons. These changes in gene expression mark the acquisition of cell type-specific properties such as cell

24

body position in the cord, neurotransmitter phenotype, axonal projection pattern, and synaptic target selection. Many V0 interneurons are commissural neurons of mixed neurotransmitter phenotypes that extend axons rostrally in the embryonic cord (Moran-Rivard et al., 2001; Pierani et al., 2001). The postmitotic transcription factor Evx1 is specific to V0V neurons that are predominantly glutamatergic but is not expressed in dorsally derived GABA/glycinergic V0D interneurons (G. Lanuza and M. Goulding, unpublished). In Evx1 mutants, a large fraction of V0V interneurons acquire the fate of V1 interneurons and extend axons into the ventrolateral funiculus instead of across the ventral midline. Consequently, Evx1 functions as a postmitotic determinant of V0V interneuron identity (Lanuza et al., 2004; Moran-Rivard et al., 2001). Other postmitotic neuron populations are also known to diverge into smaller subpopulations of neurons. This diversification probably represents the essential developmental and evolutionary changes needed to meet the increased requirements higher vertebrates have with respect to complex locomotor movements and respiration. The further specialization that occurs within these generic populations of interneurons is perhaps best exemplified by the V1 interneuron class. V1 interneurons are marked by their transient expression of the HD protein En1, although En1 has no discernable impact on V1 generic identity or diversification (Sapir et al., 2004; Saueressig et al., 1999). Recently, it has been shown that En1 expressing V1 cells differentiate into Renshaw cells and Ia inhibitory interneurons (Alvarez et al., 2005; Sapir et al., 2004). Renshaw cells receive strong excitatory innervation from motoneuron axon collaterals and in turn inhibit motoneurons, as well as Ia inhibitory interneurons, via synapses that use both glycine and GABA. Remarkably, Renshaw cells and Ia inhibitory interneurons represent less than 25% of all V1-derived cells, demonstrating that there are additional uncharacterized V1-derived cell types. Current efforts to molecularly map these V1 subpopulations have

relied on a combination of expression screens and candidate gene approaches. We have used genetic marking of En1 expressing V1 interneurons to purify these cells from E12.5 spinal cords. Microarray analysis of mRNA isolated from these cells has led to the identification of a number of genes that are enriched in this population (T. Hendricks and M. Goulding, unpublished). Many of them appear to be expressed in subsets of V1 interneurons and may therefore subdivide the V1 population into functional units (Table 1). Several transcription factors exhibit more selective patterns of expression. Pitx2, for example, is differentially expressed by a small subpopulation of ipsilaterally projecting V0 interneurons (V0C interneurons) that form cholinergic synaptic connections on motoneurons (Zagoraiou et al., 2009). In V1 interneurons, MafB is selectively expressed in Renshaw cells but is absent from other V1 cells (T. Hendricks, F. Stam, M. Goulding, unpublished). Glutamatergic V2a and GABA/glycinergic V2b interneuron populations are marked by their differential expression of Chx10 and Gata2/3, respectively (Al-Mosawie et al., 2007; Lundfald et al., 2007). Chx10 and Gata2/3 appear not to govern the neurotransmitter identity or axonal morphology of the V2a and V2b interneurons; however, inactivation of Gata2 in mice suggests this transcription factor has a role in maintaining postmitotic V2 cell identity (Zhou et al., 2000). V3 interneurons that express the PAS-bHLH factor Sim1 can be subdivided into dorsal and ventral populations (Zhang et al., 2008). However, none of Table 1. Genes enriched in V1 and V3 interneuron populations at E12.5

V1 enriched genes V3 enriched genes

Transcription factors

Other

En1, Foxd3, MafB Otx-like, Hoxa3, Cux2 Neurog3, Olig3, Sim1 Nkx2-2, Uncx

Nrxn3, Chmp1b, Nrip3 Kcnk13, Scn2a1 Gtpbp6, Plekhh1, Adam11 Sema5a, Slc5a6

25

the V3-enriched genes that have been identified to date delineate these two populations (Table 1), even though both populations do exhibit differences in their axonal morphology and electrophysiological properties (E. Geiman, Y. Zhang, and M. Goulding, unpublished). Although efforts to map the genetic pathways that establish interneuron diversity in the developing spinal cord are still in their infancy, they are likely to provide a clearer picture of the interneuronal composition of the spinal motor circuitry. This will in turn facilitate further anatomical, physiological, and functional studies of specialized interneuron cell types so as to determine their contributions to motor control. Genetically defined interneuronal populations that shape the locomotor rhythm The identification of genetic markers for different spinal interneuronal populations has laid the foundation for functional studies aimed at assessing the contribution that genetically defined cell types make to the CPG networks controlling locomotor or respiratory rhythmogenesis (Chapter 3 in this book). These efforts have been centered on broad interneuron classes including the V0, V1, V2a, and V3 interneurons (Stepien and Arber, 2008); however, attention is now turning to subtypes within these larger populations, such as the V0C interneurons (Zagoraiou et al., 2009). In characterizing and determining the function of genetically defined interneuron populations in the spinal cord, the following themes have emerged.

Modular nature of the mammalian locomotor CPG Interneurons that make up each of the early generic populations in the embryonic cord typically share a number of common characteristics, for example, morphology of the primary axonal process or neurotransmitter phenotype. Moreover,

this subdivision of embryonic interneurons into discrete anatomical groupings has led to the suggestion that the spinal motor circuitry comprises functional modules that are made up of genetically defined populations of interneurons. This proposition has been confirmed by genetic studies in mice showing that particular interneuron cell types control discrete aspects of the locomotor output. Loss-of-function manipulations that target a particular interneuron population modify specific aspects of the locomotor rhythm and pattern, and leave others unchanged. Some interneuron cell types regulate left–right alternation, while others determine the speed of stepping movements or control the balance between motor outputs on the left and right sides of the cord. In Dbx1 null mutants, where the V0 population is markedly depleted (Lanuza et al., 2004; Pierani et al., 2001), intermittent episodes of left–right synchronous activity are seen in the locomoting cord (Fig. 2c). This deficit is selective, with ipsilateral flexor–extensor alternating activity being maintained and no major changes occurring in the step cycle period. In addition to identifying a neuronal population that is required for proper left–right coordination during locomotion, this study provided evidence that the locomotor CPG has a modular composition with respect to functional control by genetically defined populations of interneurons. More recently, deletion experiments targeting V2a excitatory neurons have revealed an interesting twist to the story, by identifying V2a interneurons as an ipsilateral component of the commissural pathways that secure left–right alternation (Crone et al., 2008; Figs. 1c and 2c). Once again the effects of deleting the V2a interneurons are primarily restricted to left–right stepping behaviors. Moreover, left–right coordination is only altered when mice lacking V2a interneurons step quickly or trot, which suggests that the V2a interneurons are primarily active at higher stepping speeds (Crone et al., 2009). The observation that V2a ipsilateral neurons project onto V0 commissural interneurons confirms that the V2a cells are part of the CPG

26 (a) iFlexor

iExtensor

(b) WT

F-F

cF F-E

iF iE

cFlexor

(c)

V1

F-F

V3

F-F

cF F-E

iF

F-E

iE V0

F-F

V2a

F-F

cF iF

F-E

F-E

iE Fig. 2. Fictive locomotor activities in mice defective in ventral interneurons. (a) Schematic of an isolated spinal cord from a neonate mouse. When bathed with a cocktail of serotonin and N-methyl-D-aspartate, a fictive locomotor rhythm with left–right and flexor–extensor coordinations can be recorded from flexor L2 and extensor L5 lumbar ventral roots. (b) Schematic of locomotorlike activity observed in wild-type spinal cords. Bursts of activity are illustrated by rectangles, polar plots represent coordination between two antagonistic roots. Note the strict left–right and flexor–extensor alternations. (c) Schematic of locomotor activity recorded from spinal cords of different mutant mice. Defects in V1 or V3 interneurons disturb the locomotor step cycle resulting in a slower step cycle period or an unbalanced locomotor rhythm without affecting bilateral and flexor–extensor coordination. The absence, or ablation, of V0 and V2a interneurons results in a partial impairment of bilateral alternation, which is manifested by episodes of left–right cocontraction. c, controlateral; i, ipsilateral; F, flexor; E, extensor; WT, wild type.

module that controls left–right coordination. They most likely provide rhythmic excitatory drive to commissural inhibitory neurons that generate reciprocating left–right flexor and left–right extensor activity. When a second population of excitatory interneurons, the commissural Sim1þ V3 interneurons, were targeted, near-normal patterns of left–right and flexor–extensor alternation were retained, even though there was a marked reduction in the coherency of the locomotor rhythm. Cords in which Sim1þ V3 interneurons were silenced did, however, exhibit a pronounced imbalance in the duration of motor bursts on each side of the cord. This typically involved bursts from one L2 ventral root being prolonged, while those in the other

root were truncated (Zhang et al., 2008 and Fig. 2c). V3 excitatory pathways are therefore predisposed to balancing the locomotor output between both sides of the cord, as opposed to establishing alternating patterns of left–right or flexor–extensor motor activity. Further evidence for the modular organization of the locomotor CPG network has come from experiments analyzing the role of the V1 class of interneurons. When V1 interneurons are depleted in the embryonic spinal cord, marked changes occur in the speed of locomotor rhythm (Gosgnach et al., 2006; Fig. 2). In these mice, left–right stepping and flexor–extensor reciprocation is normal, suggesting the primary role of these cells in the walking CPG is to regulate burst termination and initiation during each step cycle.

27

Functional redundancy and complexity in the locomotor CPG Experiments demonstrating that the deletion of V0 interneurons produces only partial deficits in left–right coordination reveal a degree of redundancy in the commissural pathways that secure left–right alternation. While it seems likely that additional interneuron cell types help coordinating left–right alternation, their identity is not known. The recent demonstration that Netrin-1 mutant cords display a switch from alternating left–right to synchronous left–right locomotor activity supports the idea that multiple commissural interneurons control left–right stepping (Rabe et al., 2009). Moreover, Rabe et al. (2009) also noted that the axons of glutamatergic V3 commissural neurons still cross the ventral midline when netrin-1 is absent, demonstrating that this excitatory commissural pathway cannot secure left–right alternation. We also know that when V3 interneurons are inactivated, left–right alternation is not compromised (Zhang et al., 2008), arguing the contribution this pathway makes to left–right stepping is at best minor. The observation that misspecification of a second class of putative excitatory commissural neurons, the Evx1þ V0V cells, also leaves left–right stepping behaviors intact (Moran-Rivard et al., 2001; Lanuza et al., 2004) leads us to believe that excitatory commissural pathways in the cord do not have essential roles in controlling left–right alternation. In support of this idea, when V3 transmission is abrogated in the Dbx1/ mutant cord, we saw no further degradation of left–right alternation over and above that previously described for the Dbx1/ spinal cord (Lanuza et al., 2004; Y. Zhang and M. Goulding, unpublished). A second example of potential redundancy in the interneuron pathways that control the locomotor output has emerged from efforts to understand how the locomotor CPG produces an alternating pattern of flexor–extensor activity. Multiple manipulations that abrogate ipsilateral inhibitory V1 interneurons function leave

flexor–extensor coordination intact in the isolated CPG or in adult mice (Gosgnach et al., 2006). This was somewhat surprising in so far as an earlier anatomical study had shown that V1 interneurons give rise to cells with the features of Ia disynaptic inhibitory interneurons, a key component of the reciprocal inhibition in the stretch reflex involving flexor–extensor alternation (Alvarez et al., 2005). Experiments undertaken in the lab of Eric Frank examined the integrity of disynaptic inhibitory pathways in the spinal cord of mice lacking V1 interneurons. These analyses revealed that the disynaptic inhibitory pathway between quadriceps sensory afferents and posterior biceps and semitendinosus motor neurons is still operational in Pax6 mutant animals (Wang et al., 2008) and in mice that lack V1 neurotransmission (Z. Wang, J. Zhang, M. Goulding, and E. Frank, unpublished). It now seems that an additional population of inhibitory interneurons act in concert with the V1 population to control this aspect of the motor output (J. Zhang, G. Lanuza, and M. Goulding, unpublished). With the discovery of additional interneuron markers such as those described in Table 1, it is now becoming apparent that the interneuron composition of the spinal cord is quite complex. As noted previously, three-quarters of the V1 interneurons remain unidentified (Alvarez et al., 2005; Sapir et al., 2004), and even less is known about the identity and function of cell types that arise from other embryonic interneuron classes. Using functional criteria to classify spinal interneurons may be problematic, given the likelihood that several interneuron cell types may together regulate a particular motor behavior. Consequently, the loss of one cell population may not lead to pronounced behavioral deficits, especially when one is assaying rudimentary motor patterns such as those produced in the isolated spinal cord or in hindbrain slices. This is borne out by experiments that demonstrate the slowing of the locomotor rhythm caused by V1 depletion is not reproduced when Renshaw cells alone are

28

silenced (Myers et al., 2005). As such, the complex organization of spinal motor circuitry in higher vertebrates may prove problematic for future functional studies due to redundancy and compensation by other spinal interneuron populations that have similar or overlapping functions.

Comparison with respiratory CPGs Many of the embryonic interneuron populations that are present in the spinal cord form longitudinal columns that extend into the hindbrain where they contribute to other motor networks including those controlling respiration. Furthermore, many interneuron cell types that are known to control locomotion appear to have essential roles in respiration, as inactivating them often results in perinatal lethality. For example, inhibitory cell types that are derived from the V1 and V2b interneuron populations also have essential roles in establishing a normal respiratory rhythm (J. M. Zhang, J. Ramirez, M. Goulding, J. Feldman, unpublished). In Lbx1 mutant mice, the altered organization of some hindbrain excitatory nuclei results in an immature respiratory rhythm at birth (Pagliardini et al., 2008). Among these Lbx1-expressing cells are Tlx3þ glutamatergic neurons that are important for respiration (Cheng et al., 2004). Together, these studies give heft to the argument that the respiratory and locomotor CPGs are formed from equivalent neuronal substrates in the hindbrain and spinal cord, respectively. It also highlights the potential insights that can be gained by comparative functional studies between the locomotor and respiratory CPGs with respect to the cellular organization of motor networks in the CNS. Other groups have taken the approach of probing these circuits using genes that are expressed either in the hindbrain rhombomeres from which these CPGs develop or in populations of putative respiratory interneurons (Abbott et al., 2009; Dubreuil et al., 2009; Gray et al., 2001; McKay and Feldman, 2008; Tan et al., 2008; Thoby-Brisson

et al., 2009). For example, studies analyzing Phox2b mutant mice have revealed a key role for parafacial/retrotrapezoid nucleus in chemosensitivity (Dubreuil et al., 2009). Future experiments to genetically dissect these respiratory circuits are likely to use combinatorial methodologies to selectively target particular interneuron populations within a single rhombomere or region of the medulla, some of which are described in the next section. New genetic approaches for studying motor circuits in the spinal cord The elaboration of a genetic classification scheme for the interneuron cell types involved in spinal motor control has opened up new routes for manipulating the spinal motor system and determining how specific motor behaviors are generated. The approaches used so far involve deleting or inactivating broad interneuron classes and assessing how this affects network activity (Table 2). This is usually achieved by generating transgenic animals in which a recombination event such as Cre-mediated excision is used to express an effector molecule in a particular group of neurons (Fig. 3). Class-specific promoters in combination with site-specific recombinases allow ablating, silencing, or activating a population. Most of what we know about the function of genetically-defined spinal interneuron classes comes from in vitro analysis using the isolated spinal cord preparation, in which fictive locomotor activity is determined by extracellular ventral root recordings (Crone et al., 2008; Gosgnach et al., 2006; Lanuza et al., 2004; Zhang et al., 2008). While such studies have provided a broad functional overview of the cellular makeup of the spinal locomotor CPG, the finer aspects of motor control have been largely ignored due to the limitations of these approaches. Nonetheless an arsenal of second-generation tools is being assembled that builds on the lessons learnt from these cruder studies.

29 Table 2. Genetic tools for studying locomotion and respiration Method

Target of action

Effect

Inducer

Caveats

Advantages

Knock out (a–f)

Gene inactivation

Loss of protein function

–



Often perinatal lethal Can affect multiple cell types  Respecification of cells  Adaptation possible

 Many mouse mutants available

DTA (b, g)

Inhibition of RNA translation

Cell death

–

 Presynaptic cells may be affected by loss of their target  Adaptation possible  Not neuron specific

 Highly efficient method to remove one defined population  Ablation is easy to monitor

TeNT (h)

Cleavage of VAMP-2 synaptic protein

Silencing (synaptic transmission)

–

Adaptation possible Silencing difficult to assess

 Targeted cells remain in the circuit  Affects only neuronal cells

AlstR (b, h)

Activation of GIRK channel

Silencing (hyperpolarization)

Alst (peptide)

 Cannot be injected systemically (does not cross BBB)  Desensitization after prolonged exposure

 Inducible/reversible within minutes  Local effect by intrathecal application of Alst  Allows analysis of shortterm effects  Allows analysis in adult mice

ChR2 (i)

Expression of light sensitive cation channel

Activation (depolarization)

Blue light

 High expression levels required  Few transgenic mice successfully generated  Light penetration into deep tissue

 Inducible/reversible within milliseconds  Local excitation possible  Allows analysis of shortterm effects



 

DTA, Diphtheria Toxin A subunit; DTR, Diphtheria Toxin Receptor; TeNT, Tetanus toxin light chain; AlstR, Allatostatin Receptor; Alst, Allatostatin; ChR2, Channelrhodopsin 2; BBB, blood–brain barrier; GIRK, G-protein-coupled inwardly rectifying potassium; VAMP-2, vesicle-associatedmembrane protein 2 (synaptobrevin2). a, Lanuza et al., 2004; b, Gosgnach et al., 2006; c, Pagliardini et al., 2008; d, Rose et al., 2009; e, Dubreuil et al., 2009; f, Wallen-Mackenzie et al., 2006; g, Crone et al., 2008; h, Zhang et al., 2008; i, Hagglund et al., 2010.

Defective cell generation or cell ablation One of the routes that has been successfully used to analyze motor circuits in the hindbrain and spinal cord makes use of mouse mutants in which transcription factors or signaling molecules that

specify neuronal identity and connectivity during development are inactivated (Fig. 1). Although these factors are required for the generation of distinct populations of spinal interneurons, they are often expressed elsewhere in the developing mouse, which usually leads to perinatal lethality

30 On (a)

Promoter 1

Cre Off

Promoter 2 Recombination event Promoter 2

Stop

Gene of interest

On Gene of interest

(b)

Class specific Cre expression molecular turns on gene marker of interest

Cell lineage permanently labeled

Fig. 3. Cre-dependent manipulation of defined neuronal populations. (a) Two different alleles are used in combination to genetically manipulate defined populations of cells. First, the sitespecific Cre recombinase is expressed from a promoter (1) specific to one neuronal class. A second allele is used to express a gene of interest from a promoter (2) that is, in general, constitutive and broadly active but can also be neuronal or cell type specific. Transcriptional STOP sequences flanked by loxP sites (green triangles) are interposed between the promoter and the coding sequence such that Cre-mediated recombination of loxP sites and excision of the STOP cassette is required to express the gene of interest. This strategy can be used to express a reporter gene such as GFP for morphological and axonal characterization, and fate mapping of the genetic lineage or to express an effector gene like TeNT or DTA, resulting in cell silencing or ablation for functional studies. (b) Schematic picture showing genetic lineage labeling in the spinal cord. The promoter (1) of a molecular marker defining a neuronal class (yellow dots) is used to drive Cre expression in the same cells (green dots). Recombination induces expression of the gene of interest (blue circles), which is dependent on promoter (2). At later stages of development recombined cells have migrated to settle in their final location. Although these cells no longer express the molecular marker nor Cre, they are permanently labeled (blue circles). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

when these genes are inactivated. As locomotorlike and respiratory activity can be recorded in neonate mice using the isolated hindbrain–spinal cord preparation, such mutant animals have

proved useful for studying these behaviors (Gosgnach et al., 2006; Lanuza et al., 2004; Myers et al., 2005). An additional issue is the broad expression of many factors within the spinal cord or hindbrain, as removing these factors typically affects large neuronal populations or results in complex cell fate changes that make interpretation of any observed motor deficit difficult. While approaches that selectively ablate a population may avoid some of the complex cell fate changes associated with inactivating developmental control genes, in many instances the effects can still be quite widespread. Expression of the diphtheria toxin A protein (DTA) in molecularly defined cells has been used to selectively eliminate entire classes of interneurons. Examples come from V1 and V2a interneurons, which were successfully ablated using the Rosa-stop-DTA ablator allele and a Chx10-stop-DTA allele, respectively (Crone et al., 2008; Gosgnach et al., 2006). However, use of this strategy is limited as molecular markers of spinal interneurons are often also expressed in non-neuronal tissues, which causes additional phenotypes in mice genetically modified to kill one population. For this reason, cell ablation approaches are often not possible due to collateral tissue damage that results in embryonic lethality. Examples of this are the V1 and V3 interneuron-specific factors En1 and Sim1 that are expressed in muscle cells or the V2b marker Gata3, which is expressed in the placenta and in endothelial cells (Chen and Johnson, 2002; Coumailleau and Duprez, 2009; Ng et al., 1994). Using a neuronal driver such as Eno2-StopDTA allele restricts ablation to neurons, thereby avoiding the effects in nonneural tissue (Kobayakawa et al., 2007).

Manipulating cell activity A different approach to assessing neuronal function involves silencing or blocking neurotransmission in selected neurons and analyzing the effects on particular behaviors. Synaptic transmission can

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be abolished in a particular spinal cell class by expression of the light chain of the tetanus toxin (TeNT; Yamamoto et al., 2003; Zhang et al., 2008). TeNT prevents neurotransmitter release at the synapse, thereby blocking the activation or inhibition of postsynaptic target neurons. This approach has the advantage that the genetically targeted cells are still present. However, expression of TeNT is difficult to assess functionally. While VAMP2 expression can be used as a read out of TeNT activity, electrophysiological studies provide the most compelling validation of TeNT activity. For example, experiments recently undertaken in the cerebellum revealed that TeNT expression in cerebellar granule cells disrupts synaptic transmission to Purkinje cells (Kim et al., 2009). A different strategy for acute silencing involves expressing the insect allatostatin G-coupled protein receptor, AlstR (Gosgnach et al., 2006; Zhang et al., 2008). Upon allatostatin ligand binding, this receptor activates inwardly rectifying Kþ channels (GIRK channels) leading to transient hyperpolarization and decreased excitability of the neurons in which it is expressed (Lechner et al., 2002; Tan et al., 2008). Neuronal signaling can also be altered by inactivating critical components of the neurotransmission machinery. This approach was successfully used by Zagoraiou et al. (2009) since they specifically knockout choline acetyltransferase (ChAT) in Pitx2þ V0C cells thus preventing synaptic transmission from this particular neuronal population (Zagoraiou et al., 2009). Finally, genetic systems that can be used to stimulate or increase the excitability of defined neurons are being developed in mice, and in zebrafish (Armbruster et al., 2007; Wyart et al., 2009). Neurons can be remotely activated by the expression of channelrhodopsin 2 protein (ChR2), a microbial light sensitive monovalent cation channel that allows entrance of Naþ ions in the cell following exposition to blue light (Luo et al., 2008; Nagel et al., 2003). Thus, illumination of ChR2 expressing neurons leads to their depolarization and activation (Boyden et al., 2005). ChR2 expression was

recently targeted to VGlut2 neurons in the hindbrain and the spinal cord and illumination of each region was seen to induce and maintain locomotor-likeactivity in the spinal preparation. This finding confirms that glutamatergic neurons have a key role in elaborating the locomotor rhythm (Hagglund et al., 2010). Approaches using “designer” muscarinic GPCR receptors that can be exclusively activated by synthetic ligands provide additional avenues for selectively stimulating particular interneuron cell types (Armbruster et al., 2007).

Case history: genetic manipulations involving V1 interneurons Many of the aforementioned techniques for genetically manipulating neurons have now been trialed, each of which has advantages and disadvantages. However, by judiciously tailoring the approach used, one can undertake elegant manipulations of these spinal motor networks. Loss of cells is readily quantifiable, whereas silencing activity requires electrophysiological techniques to confirm reduced activity in the targeted neurons. In the Goulding lab, several genetic approaches have been used to remove the V1 class from the spinal circuits. These include developmental deficits in V1 specification (Pax6/), V1-specific cell ablation (En1-DTA), or blocking transmission/silencing (En1-TeNT and En1-AlstR, respectively). All four methods cause a strong slowing of the locomotor-like rhythm while preserving other aspects of the motor output: rhythmogenesis, left–right and flexor–extensor alternation (Fig. 2c). The phenotypes of the Pax6 mutant and En1-DTA mice are very similar, whereas En1-TeNT mice produce a comparatively slower motor rhythm. While cell “silencing” with AlstR produces a slow rhythm when compared to wild-type animals, the rhythm is significantly faster than that observed in En1-TeNT cords. This, in all likelihood, represents partial silencing/inactivation of the V1

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population. The rhythm in the En1-AlstR cords also returns to its normal frequency approximately 15 min after exposure to allatostatin, which probably reflects desensitization of the GIRK channels following prolonged exposure to allatostatin.

Studies in adult behaving mice Functional analysis in vitro using the isolated spinal cord preparation usually only allows identification of gross defects in motor activity. Analyzing complex motor behaviors at adult stages in genetically manipulated mice is often not possible because of neonatal lethality and deficits in more anterior regions of the CNS or in nonneuronal structures. The transcription factor Lbx1 for instance, which is important for specifying dorsal neural fate including the dI6 interneurons, is expressed in the diaphragm (Gross et al., 2000; Storm et al., 2009) and hindbrain, where it is involved in respiratory rhythmogenesis (Pagliardini et al., 2008). In some instances, genetic approaches have been applied to adult animals. Silencing the V3 cells in adult mice by using the AlstR system results in defects in walking (Zhang et al., 2008). Similar to the findings from in vitro experiments, the conditional silencing of these commissural interneurons in adult mice produced less stable and robust locomotor cycles during walking, thus validating the fictive locomotion analysis performed in the neonatal spinal cord. Studying locomotor behaviors in adult mice can also be used to refine observations that have been made using the isolated spinal cord preparation. In vitro studies of V2a interneurons in the neonatal cord (Crone et al., 2008) were extended by adult kinematic analyses showing that V2a interneurons are needed for left–right stepping at faster gait speeds (Crone et al., 2009). Acute silencing of lumbar level V3 interneurons was used to confirm the altered rhythm that occurs in vitro following TeNT blockade of V3

transmission (Zhang et al., 2008). Finer aspects of motor control have also been tested in behaving mice. For example, analysis of the V0C population in adult mice unraveled the specific function of these neurons during particular locomotor behavior. EMG recordings from gastrocnemius and tibialis anterior muscles, respectively ankle extensor and flexor, during walking and swimming tasks revealed that in the absence of V0C neuron activity, the task-dependent modulation of the gastrocnemius muscle was impaired (Zagoraiou et al., 2009). In vitro approaches would not have revealed these kinds of modulatory functions, particularly with respect to small neuronal populations.

Looking forward A next and crucial step for understanding the functional contribution these spinal interneuron classes make to motor control involves acutely eliminating each of them locally from spinal networks in the adult. It is yet not clear whether there are developmental compensatory changes that occur in spinal neuronal networks when a particular neuronal component is removed from the circuit as it is being wired. Loss of cells, especially as circuits are forming, may result in network rearrangements that compensate for the absence of these cells. In particular, little is known about the consequences on growing axons seeking out for synaptic partners when their target cells are absent. Inducing cell ablation after the circuits have wired and fully matured may therefore be critical. An elegant approach is to render mouse cells susceptible to the diphtheria toxin by specific expression of a simian or human diphtheria toxin receptor (DTR). Selective cell death can then be achieved by injecting the diphtheria toxin intraperitoneally at desired time points. Functional compensation has been reported in the circuits controlling feeding and nociception using this strategy (Cavanaugh et al., 2009; Luquet et al., 2005).

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Most molecular markers of interneurons being also expressed in supraspinal neurons or other tissue types, new strategies need to be implemented where genetic targeting is restricted to neuronal classes only in the spinal cord. This would reveal the exclusive role these subpopulations have in shaping motor output during walking, without affecting the functionality of other tissues/organs, or regions of the nervous system. For this purpose, approaches using intersecting combinations of expressed genes are promising. This has successfully been done using Cre driver lines in combination with Flp driver lines (Awatramani et al., 2003). Several dual recombinase-response alleles have now been generated that enable cell labeling or silencing (Farago et al., 2006; Jensen et al., 2008; Kim et al., 2009; Yamamoto et al., 2009). This strategy could allow dissecting the roles of genetically defined cells present in multiple regions in the rostrocaudal axis. For instance, silencing of all neurons of the Atoh1 lineage, including granule cells in the cerebellum and the dI1 class of spinal cells, results in a robust motor coordination defect in behaving mutant mice (Kim et al., 2009). Generating Flp lines specific to either the cerebellum or the spinal cord would allow one to test the contribution each region makes to motor control. The intersectional strategy also represents an attractive method for subdividing the current broad neuronal classes into smaller subsets. Within the V1 interneuron class defined by expression of En1, only the Renshaw cells coexpress the Ca2þ binding protein calbindin (Carr et al., 1998; Sapir et al., 2004). Using these two genes in a combinatorial manner to target this subset of V1 interneurons would thus allow to functionally testing the role of this subpopulation. Double recombinase approaches could also be helpful to unravel the functional contribution of specific cell types related to respiration. Patterning programs in the hindbrain specify interneurons that integrate different functional circuitry according to their antero-posterior localization. Manipulating one interneuronal population in specific rhombomeres would allow one to

unravel the roles different classes of hindbrain interneurons play in respiratory rhythmogenesis. Together with inducible systems for activation and silencing of neurons, the dual recombinase approach represents an elegant way to study specific neuronal populations in adult mice. Nevertheless, in addition to generating novel genetic tools for studying locomotion, more effort needs to be invested in developing robust and precise behavioral tests that are able to detect the subtle locomotor phenotypes when smaller groups of cells are inactivated in adult mice. Conclusion The delineation of the transcriptional code for neuronal cell populations in the ventral spinal cord and the emergence of several genetic approaches to manipulate cells of interest have paved the way for genetically and functionally dissecting the neural circuits controlling locomotion. The embryonic building blocks that make up these motor circuits are shared between the networks controlling respiration and locomotion. Thus, understanding how neuronal cell populations that comprise the hindlimb locomotor CPG are organized should provide important insights into how other CPGs function. It is also hoped that efforts to understand motor behaviors such as locomotion and respiration will provide some of the keys for unlocking more complex neuronal networks. The studies described in this chapter highlight some of the fundamental findings that have shaped our understanding of how interneurons in the ventral spinal cord form neuronal circuits that elaborate coordinated rhythmic motor outputs. However, there is a high degree of complexity in the composition of these circuits, with recent studies revealing the existence of specialized subpopulations within the generic populations of interneurons that emerge from the ventral spinal cord. Although, significant progress has been made in understanding the coarse function of large cell

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groups in controlling particular aspects of locomotor rhythm and pattern generation, many more investigations are waiting to be undertaken which elucidate the role of smaller subpopulations in fine-tuning and shaping locomotor output. Such efforts will depend heavily on the identification of specialized neuronal cell types and the development of new methodologies to genetically target and manipulate them. Acknowledgments K. S. G. is funded by a Feodor Lynen Fellowship from Alexander von Humboldt Foundation. Research in the Goulding lab is supported by grants from the National Institutes of Health (NS031249, NS031978 and NS037075) and the Christopher and Dana Reeve Foundation. We would particularly like to thank Tim Hendricks, Floor Stam, and other members of the Goulding Lab for allowing us to cite their unpublished findings.

Abbreviations Alst AlstR bHLH BMPs ChAT ChR2 CPGs DTA DTR DV EMG GIRK GPCR HD RA Shh TeNT TGF

allatostatin allatostatin receptor basic helix-loop-helix bone morphogenetic proteins choline acetyltransferase channelrhodopsin 2 central pattern generators diphtheria toxin A subunit diphtheria toxin receptor dorsoventral electromyogram G-protein-coupled inwardly rectifying potassium channel G-protein-coupled receptor homeodomain retinoic acid sonic hedgehog light chain of the tetanus toxin transforming growth factor

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glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation. The Journal of Neuroscience, 26(47), 12294–12307. Wang, Z., Li, L., Goulding, M., & Frank, E. (2008). Early postnatal development of reciprocal Ia inhibition in the murine spinal cord. Journal of Neurophysiology, 100(1), 185–196. Wildner, H., Muller, T., Cho, S. H., Brohl, D., Cepko, C. L., Guillemot, F., et al. (2006). dILA neurons in the dorsal spinal cord are the product of terminal and non-terminal asymmetric progenitor cell divisions, and require Mash1 for their development. Development, 133(11), 2105–2113. Wyart, C., Del Bene, F., Warp, E., Scott, E. K., Trauner, D., Baier, H., et al. (2009). Optogenetic dissection of a behavioural module in the vertebrate spinal cord. Nature, 461 (7262), 407–410. Yamamoto, M., Shook, N. A., Kanisicak, O., Yamamoto, S., Wosczyna, M. N., Camp, J. R., et al. (2009). A multifunctional reporter mouse line for Cre- and FLP-dependent lineage analysis. Genesis, 47(2), 107–114. Yamamoto, M., Wada, N., Kitabatake, Y., Watanabe, D., Anzai, M., Yokoyama, M., et al. (2003). Reversible suppression of glutamatergic neurotransmission of cerebellar granule cells in vivo by genetically manipulated expression of tetanus neurotoxin light chain. The Journal of Neuroscience, 23(17), 6759–6767. Zagoraiou, L., Akay, T., Martin, J. F., Brownstone, R. M., Jessell, T. M., & Miles, G. B. (2009). A cluster of cholinergic premotor interneurons modulates mouse locomotor activity. Neuron, 64(5), 645–662. Zhang, Y., Narayan, S., Geiman, E., Lanuza, G. M., Velasquez, T., Shanks, B., et al. (2008). V3 spinal neurons establish a robust and balanced locomotor rhythm during walking. Neuron, 60(1), 84–96. Zhou, Y., Yamamoto, M., & Engel, J. D. (2000). GATA2 is required for the generation of V2 interneurons. Development, 127(17), 3829–3838.

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 3

Genetic factors determining the functional organization of neural circuits controlling rhythmic movements: the murine embryonic parafacial rhythm generator J. Champagnat*, M. Thoby-Brisson, and G. Fortin Neurobiologie et Développement, UPR 3294, C.N.R.S., IFR 144 Neuro-Sud Paris, France C.N.R.S., Inst. Curie, INRA., Univ. Paris-Sud 11, Institut de Neurobiologie Alfred Fessard, FRC 2118, France C.N.R.S., Centre de Recherche de Gif-sur Yvette—C.N.R.S., FRC 3115, bât. 33, 91198, Gif-sur-Yvette, France

Abstract: In mammals, fetal movements governed by central pattern generators are essential for the development of adaptive behaviors such as breathing, walking, and chewing, which are vital after birth. Combining targeted mutations and genetic fate mapping can help to define the molecular determinants that control the development of these central pattern generators. In this chapter, recent results are presented on the embryonic parafacial (e-pF) rhythm generator, one of the two oscillators involved in controlling the breathing behavior and chemosensitive responsiveness. Keywords: parafacial respiratory rhythm generator; pre-Bötzinger complex; Hox genes; Egr2 (Krox-20) gene; Phox2b gene; Atoh1 (Math1) gene; Lbx1 gene; central congenital hypoventilation syndrome.

Introduction

neurobiology of neonatal rodents suggest that breathing rests on two prominent rhythmogenic sites, the parafacial respiratory group and the preBötzinger complex (preBötC, see Feldman et al., 2006, 2009; Bouvier et al., 2010 and references herein). The complex circuitry in which central pattern generators participate, is established during development and thought to rely on spatially and temporally ordered appearance of neurons. The parafacial region is well understood with respect to the

The respiratory rhythm generator is probably one of the best models to understand how genes have been selected and conserved to control development of central pattern generators responsible for adaptive behavior in vertebrates. Recent advances in the

*Corresponding author. Tel.: þ33-1-6982-3406; Fax: þ33-1-6982-4178 DOI: 10.1016/S0079-6123(10)87003-7

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transcription factors that underlie specification of neural progenitors. Hox paralogs and Hox-regulating genes such as Egr2 (Krox-20) govern transient formation of developmental compartments, the rhombomeres, in which rhythmic neuronal networks develop (r1–r7; see Thoby-Brisson et al., 2009 and references herein). Along the dorsoventral axis of the rhombomeres, and in the spinal cord, the different neuronal types to be incorporated in synaptic circuits appear in progenitor domains, such as the “dorsal” domain broadly defined by the expression of Pax7 (dA1dB4), then Lbx1 (dB1–dB4; see Pagliardini et al., 2008) and further subdivided by, inter alia, basic helix-loop-helix (bHLH) proneural genes (see Storm et al., 2009). In addition to these neural-type specific transcription domains, developmental gene expression might also match neuronal populations sharing the same connectivity and function: this is the case of Phox2b expression (three distinct columns (dA3, dB2, V3) in the neural tube along the DV axis, spanning different rhombomeres on the anteroposterior axis) which eventually provides molecular fingerprinting of the entire visceral nervous system from afferent chemosensory function (in carotid bodies, area postrema) to efferent branchiomotor, preganglionic, and autonomic nuclei and ganglia (see Dubreuil et al., 2009a,b). A general view is emerging on the role of developmental transcription factors allowing the coordinated integration of different neuronal types to produce motor rhythmic patterns. The present chapter concentrates on recent findings on the embryonic parafacial (e-pF) rhythm generator in the more general context of respiratory and motor development.

Molecular identification of central oscillators during embryonic development: the e-pF oscillator Evidence for the developmental origin and functional nature of the respiratory rhythm generating circuits involved in fetal and neonatal breathing

has been obtained using mutant mice in which developmental genes encoding transcription factors are inactivated. Inactivation may lead to abnormal breathing behavior as a result of the elimination of crucial neuronal systems. In particular, inactivation of the gene for the zinc finger transcription factor Egr2 that controls the formation of odd rhombomeric segments r3 and r5 results in defective breathing attributed to a reorganization of neuronal circuits within the caudal pons that consequently leads to poor survival at birth (Jacquin et al., 1996). Recently, the candidate cell group deriving from Egr2-positive territories, and explaining the respiratory deficits of Egr2 null mutants, has been identified as a bilateral e-pF population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice (Thoby-Brisson et al., 2009). These cells expressing Phox2b, Lbx1, and Atoh1 derive from Egr2-expressing precursors and their development depends on the integrity of Egr2 (Dubreuil et al., 2009a,b; Pagliardini et al., 2008; Rose et al., 2009; Thoby-Brisson et al., 2009). Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötC), led to an abnormally slow and irregular rhythm, demonstrating that the e-pF controls the respiratory rhythm (Thoby-Brisson et al., 2009). The e-pF oscillator is the only one active at E14.5: it entrains and then couples with the preBötC, which emerges independently at E15.5 in mice (Thoby-Brisson et al., 2005). These data raise the possibility that the e-pF may be influencing the emergence of the preBötC. However, the preBötC oscillator is spared in Egr2 null mutants, in which the e-pF oscillator does not form: therefore, the latter is not required for the emergence of the former. We propose that a two-step developmental process establishes fetal breathing in mice. In the first step, the Egr2dependent e-pF neuronal population clusters at the ventral surface of the hindbrain and functions as an oscillator. In a second step, a second oscillator, the preBötC, emerges independently, and the e-pF couples with it. In that manner, the dual

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organization of the RRG is established at the onset of fetal breathing (Thoby-Brisson et al., 2005, 2009).

Odd rhombomeric (Egr2/Krox20, Hox) patterning of the parafacial hindbrain Comparing the postnatal behavior of several mouse models (reviewed by Champagnat et al., 2009) demonstrated that specific respiratory deficits in vivo are assignable to anteroposterior segments of the brainstem, suggesting that the respiratory neuronal network is organized according to the rhombomeric patterning by Egr2 and Hox expression. Three anteroposterior levels can be distinguished with corresponding deficits. Inactivation of e-pF activity causes life-threatening neonatal apnoeas alternating with respiration at low frequency (see above). At a more caudal (postotic) level, another rhythm abnormality, the complete absence of breathing, is induced when neuronal synchronization fails to develop in the preBötC (Bouvier et al., 2010; Wallén-Mackenzie et al., 2006). A third type of respiratory deficits is induced by modifying the rostral pontine (r1-derived) level, leading to an abnormal inspiratory shaping without affecting respiratory frequency and apnoeas (see Champagnat et al., 2009). These studies also revealed the efficacy of prenatal compensatory mechanisms allowing a normal breathing frequency in conditions that would have otherwise stopped the respiratory rhythm (e.g., the inactivation of acetylcholinesterase, as discussed in Champagnat et al., 2009). Thus, because segmentation is a transient feature, and because a dramatic reconfiguration of neurons and synapses takes place during fetal and postnatal stages, genetic elimination of crucial circuits is not sufficient to demonstrate that the rhombomeric pattern of Hox and Egr2 expression influences directly the establishment of the e-pF in the active brainstem. A study of the Hoxa1(/) mutant (Del Toro et al., 2001) showed that changes in Hox expression patterns did allow the selection of

novel neuronal circuits regulating the breathing behavior. Hoxa1 is the earliest Hox gene expressed (at E8 in mice) in the developing hindbrain and is rapidly downregulated. Analysis of Hoxa1(/) parafacial rhombomeres revealed ectopic neuronal groups with a paratrigeminal phenotype in a parafacial location. These cells establish supernumerary neuronal circuits that do not exist in wild-type controls, that escape apoptosis during development and that are functional postnatally. Facial motoneurons, which normally migrate caudally and ventrally over long distances, adopt a trigeminal like dorsal location when respecified by the Hoxa1 mutation. In this area, which is normally devoid of respiratory-related function, an ectopic group of reticular progenitors adopts a paratrigeminal (r2-like) identity, integrates the respiratory network and controls the preBötC activity at birth. Thus, the selective modification of the expression pattern of a Hox gene may incorporate a novel functional circuit in the respiratory rhythm generator. Because hindbrain neurons control adaptive behaviors, these findings have considerable significance both on developmental and evolutionary grounds. The evolution of neural networks of multirhombomeric origin may be facilitated by the partitioning of the early hindbrain in a number of metameric units initially developing as independent modules. As a result, subsets of neurons with serial homology along the anteroposterior axis, may be developmentally isolated from each other and allowed to evolve independently (see Hurley et al., 2006). Therefore, Hox genes may provide a genetic basis for segment-specific modulation of neuronal development and connectivity. Local changes in the regulation of Hox genes and changes in Hox cisregulatory modules or downstream targets may offer opportunities for the evolution of distinct subsets of neurons (e.g., wild-type parafacial), without affecting the function of others (e.g., wild-type preBötC), eventually resulting in novel functional features (e.g., a para-trigeminal rhythm in Hoxa1 mutants).

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The paired-like homeobox 2b (Phox2b) gene, a visceral cell-type fingerprint in respiratory control: link with central chemosensitivity and the congenital central hyperventilation syndrome There are now several mice models showing that Phox2b is a major actor during the e-pF development (Dubreuil et al., 2009a,b and references herein). Phox2b is the homeodomain transcription factor specifically expressed (since E9) and required in cells that form the phylogenetically ancient visceral reflex circuits controlling digestive, cardiovascular, and respiratory functions, thereby maintaining bodily homeostasis through branchial motor, parasympathetic, sympathetic, and enteric output neurons. Therefore, the Phox2bþ e-pF might be considered as a relay of the visceral nervous system through which the respiratory behavior can also be controlled. On the afferent side of the visceral system, Phox2b is also required for the development of chemoceptive and chemoafferent visceral structures including carotid bodies, their petrosal afferents, and relay neurons in the nucleus tractus solitarius. Within the e-pF area, Phox2b is expressed in chemoresponsive retrotrapezoid neurons (RTN) identified as essential for sensing hypercapnia in the adult rat (Stornetta et al., 2006). The same is true in pre- and neonatal mice, since a low pH stimulation strongly increases the e-pF rhythm at the earliest stages of its development. In addition, lack of response to hypercapnia is a major and consistent abnormality resulting in all cases from the invalidation of Phox2b (Dubreuil et al., 2009a,b) so that the e-pF can be considered a forerunner of the adult RTN. On the whole, evidence from human and mouse genetics as well as neurobiological observations in vivo and in vitro converges to implicate this small population of Phox2b-dependent neurons in the hypercapnic drive of breathing in mammals. Recent studies have also uncovered the selective vulnerability of these e-pF/chemoresponsive neurons to the toxic effect of abnormal Phox2b proteins. Heterozygous Phox2b mutations whereby

a stretch of 20 alanine located 30 to the homeodomain is expanded by 4–13 residues, have been identified as the cause of central congenital hypoventilation syndrome (CCHS), a rare disease defined by an inability to sustain robust breathing during sleep and a marked insensitivity to hypercapnic stimulation (Amiel et al., 2003). Phox2b(27Ala/ þ) mice that bear a frequent CCHS-causing human mutation do not respond to hypercapnia and die shortly after birth from central apnoea. They are therefore a reliable animal model for CCHS. Neurons of the e-pF were found severely depleted in these Phox2b(27Ala/þ) mice, in the absence of notable neuronal loss in other Phox2b expressing structures (see Dubreuil et al., 2009a,b). Selective vulnerability to abnormal Phox2b protein product, together with rhythm generating membrane properties, is an important phenotypic trait that differentiates e-pF from other neurons forming the Phox2bþ chemosensitive visceral nervous system and raises questions about additional factors other than Phox2b and Egr2 that may further specify the e-pF population.

The e-pF expresses the proneural mouse atonal homolog 1 (Atoh1/Math 1) One important class of genes that regulates cellular diversity in the nervous system encodes bHLH transcription factors, which act as generic proneural factors within the Notch pathway to single out neuronal progenitors and promote their differentiation. Subsequent analysis revealed the important roles of bHLH factors and their interaction in the determination of neuronal fates, in relation to the morphogenetic gradients of bone morphogenetic proteins (dorsal) and sonic hedgehog (ventral), along the dorsoventral axis of the spinal and hindbrain neural tube (see references in Dubreuil et al., 2009a,b; Storm et al., 2009). Neurons migrate extensively from dorsal progenitor domains characterized at E10–E12 in mice by the expression of bHLH factors. For example, Atoh1 (also known as Math1) is required to

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determine the fate of the most dorsal progenitors (dA1). Mash1þ progenitors are located further ventrally (dA3) giving rise to (Phox2bþ) neurons of the nucleus of the solitary tract, while Ptf1aþ (dA4) progenitors of the inferior olivary nucleus arise, and so on. Atoh1-dependent (Phox2b negative) progenitors at the dorsal lip (dA1) migrate to populate the lateral reticular, external cuneate, pontine and reticulo-tegmental nuclei. Recently, an additional Atoh1 expressing population of “paratrigeminal” and “parafacial” neurons derived from dB2 progenitors was identified and found to form the e-pF. This population co-expresses Atoh1 with Phox2b and Lbx1 at relatively late stages of the bHLH patterning (from E12.0 in mice) and migrates to the vicinity of facial and trigeminal branchial motor nuclei (Dubreuil et al., 2009a,b; Rose et al., 2009). Imaging rhythmic activity showed that e-pF pacemakers and Atoh1 expression are suppressed after the conditional deletion of Phox2b neurons in the Lbx1- and Egr2-deriving populations, while the e-pF remains active and Atoh1 expressed in other mutants in which the facial branchial motor nucleus is eliminated (Dubreuil et al., 2009b). These results confirm that the e-pF is formed by the caudally migrating population of Egr2þ derivatives, and demonstrate that facial branchiomotor neurons are not required for the e-pF maturation and migration. Two major theoretical implications follow. On the one hand, data now clearly exclude that preBötC and e-pF are serial homologs, since caudal to the e-pF, r7 (and spinal levels) lack both an odd-rhombomeric Egr2 expression and the dB2 (Lbx1þ, Phox2bþ) class of interneurons later expressing Atoh1, migrating and producing the e-pF (see Storm et al., 2009). This view is also largely supported by the large number of mutant mice models now showing that the e-pF can be genetically abolished without eliminating the preBötC (see above) and that the preBötC can be disrupted without eliminating the e-pF (Bouvier et al., 2010). On the other hand, results reveal a serial homology between the paratrigeminal and

parafacial Atoh1þ dB2 populations (Rose et al., 2009), in keeping with the induction of a paratrigeminal respiratory group in Hoxa1 mutants (del Toro et al., 2001, see above) as well as the paratrigeminal location of a respiratory rhythm generator in the lamprey (Martel et al., 2007). On the whole, therefore, the emerging scheme of e-pF formation places emphasis on the evolutionary period at which the odd rhombomeric pattern of Egr2 expression was achieved, shortly after two rounds of whole genome duplication, and during which pharyngeal breathing is shifting from a passive to an active mode (see Hurley et al., 2006; Martel et al., 2007; Murakami et al., 2005).

Perspective: the spinal connection Recent studies shed some light upon cell autonomous genetic network specifying the e-pF neuronal lineage and eventually endowing it with rhythm generating and chemoresponsive cellular properties. As important would be to understand how the e-pF rhythm may influence coordinated breathing movements. In vertebrates, the cell bodies of spinal motor neurons are organized in columns along the rostrocaudal extent of the brainstem and spinal cord. Neurons in a column send axons to a distinct set of muscles during early embryonic development (see Dasen et al., 2008; Rousso et al., 2008). Respiratory muscles are controlled by three different motoneurons subsets, forming the “hypaxial motor column” (HMC): (i) the inspiratory phrenic nucleus, a derivative of the cervical HMC, projects to the diaphragm muscle, (ii) the respiratory thoracic HMC neurons include inspiratory motoneurons innervating the external intercostal muscles and expiratory motoneurons projecting to internal intercostal muscles and, (iii) abdominal HMC motoneurons drives expiratory muscles. The columnar identity of spinal motoneurons is genetically specified. For example, the “lateral motor column,” which projects to limb muscle, is specified by the coexpression of Hox6 (forelimb) or

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Hox10 (hindlimb) paralogs and Foxp1 (Dasen et al., 2008; Rousso et al., 2008). The respiratory HMC identity in mammals might also be predetermined by a currently unknown transcriptional program allowing motor neurons to be connected by their proper rhythm generator and to connect with their specific target muscles. It is a major challenge for the future to uncover this program. It is expected that HMC populations that control air breathing emerged relatively late in evolution. While agnathians such as lampreys do have HMC, its function is to control axial muscles for the swimming behavior. Motor neurons and muscles have evolved to mediate additional functions, in particular air breathing. The recruitment of axial muscles to form an expiratory pump (most probably in amphibians, see Brainerd and Owerkowicz, 2006) has been the first step of the transfer of lung ventilation from head (visceral) to trunk (somatic) regions. Hence, understanding the genetic programs coordinating central (e-pF) and motor (expiratory) sides of the network might shed light on how neuronal developmental programs may switch on functional innovations during evolution.

Perspective: insertion of inhibitory interneurons There is increasing consensus to consider the rodent parafacial group as the controller of active expiration, with neurons active in late expiration (preinspiration; Onimaru et al., 2008) and with preferential action on expiratory (e.g., lumbar) motoneurons (Janczewski and Feldman, 2006). Virtually all e-pF neurons are glutamatergic, while rhythm generation and synchronization within each e-pF cell cluster does not require glutamatergic synapses (in clear contrast with the pre-BötC oscillator, in which both rhythm generation and intercellular synchrony rely on glutamatergic transmission, Thoby-Brisson et al., 2009). Therefore, the e-pF oscillator is optimally designed to play a role in the set up of expiratory circuits through a glutamatergic excitatory control. However, in mammals, the expiratory motor pattern is characterized by phasic

(inspiratory) inhibition of a tonic (chemosensitive) motor background activity (Janczewski and Feldman, 2006, and references herein to the classical works of Tom Sears and coll.). Nothing is known on the inhibitory neuronal circuit, except that this chemoresponsive, phasically inhibited (chloridedependent) pattern is also a characteristic of the gill breathing in fishes and amphibians (see Wilson et al., 2006) and that unbalanced excitation and inhibition in mice mutants (Tlx3 mutants; see Cheng et al., 2004) seriously affect respiratory rhythm generation. Observations in chicken suggest that Egr2 may initiate induction of inhibitory GABAergic circuits in brainstem rhythmic networks. There is in chicken embryos a multifunctional “primordial” activity found in a variety of motor, reticulospinal, and vestibuloocular neurons (Mochida et al., 2009) and eventually forming “episodes” comprising a series of bursts of activity separated by long intervals, maintained during several days of development and driving episodic axial body movements observable in ovo. Fortin and colleagues (Fortin et al., 1999) demonstrated that the parafacial expression of Egr2 in the chicken initiates the induction of episodes by eventually incorporating a GABAergic circuit into the primordial rhythmic network. A noncell autonomous influence of Egr2-expressing progenitors was found sufficient to endow adjacent rhombomeres with the capacity to induce episodes. Induction is robust because it can be reproduced with r2, r4, and r6 and with any hindbrain territory that normally expresses Egr2 (r3, r5) or can be forced to do so (r1, r4; Coutinho et al., 2004). It would therefore be interesting to investigate whether such noncell autonomous induction of synaptic inhibition by Egr2 is conserved in the mouse and contribute to generate the expiratory motor pattern.

Perspective: the pre-Bötzinger complex Because the respiratory rhythm generator includes multiple oscillators in lampreys (Martel et al., 2007), the murine preBötC might be considered

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a conserved homologous of e-pF's partners during gnathostome evolution. This, however, is probably not the case because anteroposterior location and function of postotic rhythm generators seem variable in different vertebrates (see Bass et al., 2008; Kinkead, 2009; Wilson et al., 2006). Isolation and imaging of nonmammal oscillators together with an analysis of Egr2 and Phox2b expression would be required to clarify this issue. It seems, however, more likely that the preBötC correlates with the late emergence of the mammalian diaphragm/ribcage apparatus. Indirect evidence, including late emergence during ontogenesis (Thoby-Brisson et al., 2005, 2009), inspiratory function in rodents (Janczewski and Feldman, 2006), and lack of molecular similarity with the e-pF (Bouvier et al., 2010) and with the evolutionary ancient visceral nervous system (Dubreuil et al., 2009a), suggest that preBötC might have been exapted from nonvisceral postotic progenitors at this occasion (Bouvier et al., 2010). Acknowledgments This work was supported by institutional support from the CNRS (Centre National de la Recherche Scientifique, France) and a grant ANR-07-Neuro007-01 (Agence Nationale de la Recherche, France) to G.F. This work benefited from the facilities and expertise of the Imagif Cell Biology Unit of the CNRS Research Center of Gif-sur-Yvette. References Amiel, J., Laudier, B., Attié-Bitach, T., Trang, H., de Pontual, L., Gener, B., et al. (2003). Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nature Genetics, 33, 459. Bass, A. H., Gilland, E. H., & Baker, R. (2008). Evolutionary origins for social vocalization in a vertebrate hindbrainspinal compartment. Science, 321, 417–421. Bouvier, J., Thoby-Brisson, M., Renier, N., Dubreuil, V., Ericson, J., Champagnat, J., et al. (2010). Hindbrain interneurons and axon guidance signaling critical for breathing. Nature Neuroscience, [13,1066-1074].

Brainerd, E. L., & Owerkowicz, T. (2006). Functional morphology and evolution of aspiration breathing in tetrapods. Respiratory Physiology & Neurobiology, 154, 73–88. Champagnat, J., Morin-Surun, M. P., Fortin, G., & ThobyBrisson, M. (2009). Developmental basis of the rostrocaudal organization of the brainstem respiratory rhythm generator. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 364, 2469–2476. Cheng, L., Arata, A., Mizuguchi, R., Qian, Y., Karunaratne, A., Gray, P. A., et al. (2004). Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nature Neuroscience, 7, 510–517. Coutinho, A. P., Borday, C., Gilthorpe, J., Jungbluth, S., Champagnat, J., Lumsden, A., et al. (2004). Induction of a parafacial rhythm generator by rhombomere 3 in the chick embryo. The Journal of Neuroscience, 24, 9383–9390. Dasen, J. S., De Camilli, A., Wang, B., Tucker, P. W., & Jessell, T. M. (2008). Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell, 134, 304–316. Del Toro, E. D., Borday, V., Davenne, M., Neun, R., Rijli, F. M., & Champagnat, J. (2001). Generation of a novel functional neuronal circuit in Hoxa1 mutant mice. The Journal of Neuroscience, 21, 5637–5642. Dubreuil, V., Barhanin, J., Goridis, C., & Brunet, J.-F. (2009a). Breathing with Phox2b. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 364, 2477–2483. Dubreuil, V., Thoby-Brisson, M., Rallu, M., Persson, K., Pattyn, A., Birchmeier, C., et al. (2009b). Defective respiratory rhythmogenesis and loss of central chemosensitivity in Phox2b mutants targeting retrotrapezoid nucleus neurons. The Journal of Neuroscience, 29, 14836–14846. Feldman, J. L., Kam, K., & Janczewski, W. A. (2009). Practice makes perfect, even for breathing. Nature Neuroscience, 12, 961–963. Feldman, J. L., & Del Negro, C. A. (2006). Looking for inspiration: New perspectives on respiratory rhythm. Nature Reviews. Neuroscience, 7, 232–242. Fortin, G., Jungbluth, S., Lumsden, A., & Champagnat, J. (1999). Segmental specification of GABAergic inhibition during development of hindbrain neural networks. Nature Neuroscience, 2, 873–877. Hurley, I., Hale, M. E., & Prince, V. E. (2006). Duplication events and the evolution of segmental identity. Evolution and Development, 7, 556–567. Jacquin, T. D., Borday, V., Schneider-Maunoury, S., Topilko, P., Ghilini, G., Kato, F., et al. (1996). Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice. Neuron, 17, 747–758. Janczewski, W. A., & Feldman, J. L. (2006). Distinct rhythm generators for inspiration and expiration in the juvenile rat. Journal de Physiologie, 570, 407–420.

46 Kinkead, R. (2009). Phylogenetic trends in respiratory rhythmogenesis: Insights from ectothermic vertebrates. Respiratory Physiology & Neurobiology, 168, 39–48. Martel, B., Guimond, J. C., Gariépy, J. F., Gravel, J., Auclair, F., Kolta, A., et al. (2007). Respiratory rhythms generated in the lamprey rhombencephalon. Neuroscience, 148, 279–293. Moshida, H., Fortin, G., Champagnat, J., & Glover, J. C. (2009). Differential involvement of projection neurons during emergence of spontaneous activity in the developing avian hindbrain. Journal of Neurophysiology, 101, 591–602. Murakami, Y., Uchida, K., Rijli, F. M., & Kuratani, S. (2005). Evolution of the brain developmental plan: Insights from agnathans. Developmental Biology, 280, 249–259. Onimaru, H., Ikeda, K., & Kawakami, K. (2008). CO2sensitive pre-inspiratory neurons of the parafacial respiratory group express Phox2b in trhe neonatal rat. The Journal of Neuroscience, 28, 12845–12850. Pagliardini, S., Ren, J., Gray, P. A., Vandunk, C., Gross, M., Goulding, M., et al. (2008). Central respiratory rhythmogenesis is abnormal in lbx1-deficient mice. The Journal of Neuroscience, 28, 11030–11041. Rose, M. F., Ren, J., Ahmad, K. A., Chao, H.-T., Klisch, T. J., Flora, A., et al. (2009). Math1 is essential for the development of hindbrain neurons critical for perinatal breathing. Neuron, 64, 341–354. Rousso, D. L., Gaber, Z. B., Wellik, D., Morrisey, E. E., & Novitch, B. G. (2008). Coordinated actions of the Forkhead

protein Foxp1 and Hox proteins in the columnar organization of spinal motor neurons. Neuron, 59, 226–240. Storm, R., Colewa-Waclaw, J., Reuter, K., Bröhl, D., Sieber, M., Treier, M., et al. (2009). The bHLH transcription factor Olig3 marks the dorsal neuroepithelium of the hindbrain and is essential for the development of brainstem nuclei. Development, 136, 295–305. Stornetta, R. L., Moreira, T. S., Takakura, A. C., Kang, B. J., Chang, D. A., West, G. H., et al. (2006). Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. The Journal of Neuroscience, 26, 10305–10314. Thoby-Brisson, M., Karlén, M., Wu, N., Charnay, P., Champagnat, J., & Fortin, G. (2009). Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex. Nature Neuroscience, 12, 1028–1035. Thoby-Brisson, M., Trinh, J. B., Champagnat, J., & Fortin, G. (2005). Emergence of the pre-Bötzinger respiratory rhythm generator in the mouse embryo. The Journal of Neuroscience, 25, 4307–4318. Wallén-Mackenzie, A., Gezelius, H., Thoby-Brisson, M., Nygård, A., Enjin, A., Fujiyama, F., et al. (2006). Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation. The Journal of Neuroscience, 26, 12294–12307. Wilson, R. J., Vasilakos, K., & Remmers, J. E. (2006). Phylogeny of vertebrate respiratory rhythm generators: The oscillator homology hypothesis. Respiratory Physiology & Neurobiology, 154, 47–60.

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 4

Development of motor rhythms in zebrafish embryos Louis Saint-Amant* Département de pathologie et biologie cellulaire, Groupe de Recherche sur le Système Nerveux Central, Centre d' Excellence en Neuromique de l'Université de Montréal, Université de Montréal, Montréal, Québec, Canada

Abstract: The nervous system can generate rhythms of various frequencies; on the low-frequency side, we have the circuits regulating circadian rhythms with a 24-h period, while on the high-frequency side we have the motor circuits that underlie flight in a hummingbird. Given the ubiquitous nature of rhythms, it is surprising that we know very little of the cellular and molecular mechanisms that produce them in the embryos and of their potential role during the development of neuronal circuits. Recently, zebrafish has been developed as a vertebrate model to study the genetics of neural development. Zebrafish offer several advantages to the study of nervous system development including optical and electrophysiological analysis of neuronal activity even at the earliest embryonic stages. This unique combination of physiology and genetics in the same animal model has led to insights into the development of neuronal networks. This chapter reviews work on the development of zebrafish motor rhythms and speculates on birth and maturation of the circuits that produce them. Keywords: Spinal cord; mutant; motor; swimming; patch clamp; spontaneous; embryo; larvae. contribution of motor activity generated by the CPG is crucial to the development of motor circuits, as altering activity within the CPG has been shown to affect aspects of circuit formation in the chick (Hanson and Landmesser, 2004), mouse (Myers et al., 2005), and zebrafish (Menelaou et al., 2008). However, the relationship between the networks that generate the early spontaneous motor rhythms and the network involved in more mature behaviors such as locomotion, chewing, or breathing is still unclear. The study of networks that generate swimming rhythms in Xenopus larvae and lamprey has yielded

Introduction Spontaneous motor activity, such as human fetal movements, is common in all vertebrates during development and has been speculated to emanate from a collection of neurons that are defined as a central pattern generator or CPG (Corner, 1978; Hamburger, 1963; Kudo and Nishimaru, 1998; Narayanan et al., 1971; O'Donovan et al., 1998, 2005; Ren and Greer, 2003; Yvert et al., 2004). The *Corresponding author. Tel.: 514-343-7746; Fax: 514-343-5755 DOI: 10.1016/S0079-6123(10)87004-9

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circuit maps of rhythmogenic CPG located in the spinal cord (Grillner, 2003, 2005; Roberts, 1990; Roberts et al., 1998). These maps have provided key insights into how different types of neurons participate in the function of CPGs, but the limited genetic and molecular tools in higher vertebrate models have slowed the progress of research aimed at elucidating the molecular bases of CPG formation and function. The zebrafish has recently emerged as an important model to study the genetics of neural circuit development (Fetcho and Liu, 1998; Kullander, 2005). Electrophysiological and imaging techniques have been developed to study the electrical activity of zebrafish embryos and larvae in vivo, rendering the study of circuit function of these embryos as accessible as most in vitro models of higher vertebrates (Brustein et al., 2003; Buss et al., 2003; Drapeau et al., 1999, 2002; Fetcho and O'Malley, 1997; Higashijima et al., 2003; Masino and Fetcho, 2005; McDearmid and Drapeau, 2006; O'Malley et al., 2003; Ribera and Nusslein-Volhard, 1998; Saint-Amant and Drapeau, 2003; Takahashi et al., 2002). On the molecular biology front, the use of reverse genetic techniques in zebrafish, which are achieved by inactivating genes of interest with antisense morpholino oligos that interfere with translation (Nasevicius and Ekker, 2000), is yielding new data on genes involved in the proper execution of motor behavior, while forward genetic work on motor mutants isolated in mutagenesis screens performed in the past decades is also continuing to reveal genes that play an important role in the development and function of motor circuits. This review focuses on changes in the spinal cord, as previous studies in other swimming vertebrates have shown the importance of the spinal cord in the creation of motor rhythms (Grillner, 2003, 2005; Roberts, 1990; Roberts et al., 1998).

Morphology of the embryonic zebrafish spinal cord The zebrafish spinal cord is very immature when motor rhythms first appear. There are less than 20 neurons with axons per side of each

somite at 24 h postfertilization (hpf) (Bernhardt et al., 1990; Kuwada and Bernhardt, 1990; Kuwada et al., 1990), and their neurotransmitter phenotypes have been worked out (Higashijima et al., 2004b). These neurons and their transmitter phenotypes are represented in Fig. 1a. Starting at the ventral border of the spinal cord, Kolmer–Agduhr (KA) neurons are GABAergic neurons that contact the central canal. GABAergic neurons were not included in the figure as no data was found supporting GABAergic participation in early embryonic behaviors although they may have a role in triggering behaviors in older larvae (Saint-Amant and Drapeau, 2000, 2001; Wyart et al., 2009). There are only two types of glutamatergic interneurons that project descending axons ipsilaterally along several somites as early as 17 hpf. First, the IC interneurons (ipsilateral caudal axon) are a population of early born neurons, with numbers ranging from 1 to 2 per segment that span the hindbrain/spinal cord border but are not present after the sixth somite (Mendelson, 1986a,b). The second ventrally located glutamatergic interneuron with a long descending axon is the ventral lateral descending (VeLD). VeLD cell bodies are located throughout the spinal cord with approximately 2 cells per segment (Bernhardt et al., 1990; Kuwada and Bernhardt, 1990). Before 26 hpf there are only three cholinergic motoneurons per side of each segment (grouped as PMN in Fig. 1a): the CaP (caudal primary), MiP (middle primary), and RoP (rostral primary) motoneurons (Eisen et al., 1986; Myers, 1985; Westerfield et al., 1986). Three interneurons are added in a second wave of neurogenesis before 24 hpf (Bernhardt et al., 1990; Kuwada and Bernhardt, 1990; Kuwada et al., 1990); consequently, they have shorter axons and are presumably slowly added to the existing network. The first late addition is the circumferential descending (CiD) interneuron, which sends an axon first ventrally then caudally. It has been speculated that the CiD and VeLD are part of the same category of neurons in later larval stages and the difference observed here in their length of projections is only

49 ∗

(a)

RB CoPA

VeLD CoB

CoSA

CiA

CiD PMN

IC

∗∗

Spontaneous rhythms

(b)

Evoked rhythms

(c)

Swimming rhythms

(d)

1s

2s

50 ms

PD

PD SB

Left Gap junctions

Glycine + Glutamate + Gap junctions

Gap junctions + Glutamate

Glycine

Glycine

Right

NMDA Cav Nav AMPA

Kca Kv?

Nav Cav?

HB

NMDA Cav Nav AMPA

Glycine Kca Kv

HB

Somites 1–6

Somites 1–6

SC S

–

iE

+

S + –

iE

+ –

iE

cl

–

+

Fig. 1. For legend, see next page.

cE

+

cl

–

–

+

+ +

M

+

SC

cE

+

cl

Kv

HB RS

SC

Glycine Kca

M

–

+ –

M

–

50 Fig. 1 (Continued). Zebrafish embryonic rhythm circuits. (a) Diagram of one segment of the spinal cord. The segment is shown in an open book configuration as if cut along the midline in a sagittal section (modified from Roberts, 2000). Fewer than 20 neurons with axons are present per segment of the spinal cord at this stage and they can be grouped in nine distinct cell types based on morphology (see main text for their description). Glycinergic neurons are shown in black, glutamatergic neurons in white, and cholinergic motoneurons in gray. The asterisks denote points of exit from the spinal cord, * to the skin, ** to the ventral root. (b) Top, schematic representation of the repeating and alternating coiling behavior seen in embryos at 24 hpf. Middle, representation of the electrical activity seen in spinal neurons during spontaneous coiling. Periodic depolarizations (PD) are driven by excitation propagated mostly through a gap junction coupled network of ipsilateral neurons; while synaptic bursts (SB) occur when commissural neurons carry an inhibitory conductance (shunting) during contralateral PDs. The enlarged insets, in this and later figures, show ion channels that have been shown and are speculated to be required for the oscillations. Bottom, hypothetical circuit diagram of the spinal cord during the period of spontaneous coiling (diagram modified from Grillner, 2003; Roberts et al., 1998). The shaded neurons represent the contralateral side of the spinal cord and the dashed boxes represent the rostral to caudal somitic divisions. The zig-zags represent gap junction connections. (c) Top, representation of the coiling contractions in response to touching the left side of the embryo. The first contraction is always contralateral to touch and is usually followed by 1–3 alternating contractions. Middle, representation of the electrical activity during touch-evoked coils. The episode of activity starts with a glycinergic burst on the side ipsilateral to the touch and is quickly followed by a burst of excitatory activity. Bottom, hypothetical circuit diagram of the spinal cord during appearance of touch-evoked coils, where the sensory component of the spinal cord is now connected to the preexisting network of gap junction coupled neurons. The squares represent glutamatergic synapse, while the circles represent glycinergic synapses. (d) Top, representation of the swimming response to touch. The first contraction is always contralateral to touch and is usually followed by a large number of rapidly alternating contractions of low amplitude. Middle, representation of the electrical activity during a swimming episode. Spinal neurons show a sustained plateau of synaptic activity upon which action potentials are seen to alternate from side to side. Bottom, hypothetical circuit diagram of the spinal cord during the appearance of swimming, where the sensory component of the spinal cord is now connected to the hindbrain and the hindbrain excites the spinal cord.

due to a temporary developmental process (Hale et al., 2001; Higashijima et al., 2004b). The second late addition is the circumferential ascending (CiA) interneuron that is glycinergic and projects an axon ventrally to the floor plate then rostrally on the same side of the embryo. The third late addition is the commissural bifurcating (CoB), which projects an axon ventrally, crosses the midline, and then projects axons both rostrally and caudally. Commissural primary ascending (CoPA) interneurons are large, early born glutamatergic interneurons with 1–2 somata per somite that are located dorsally and project axons first ventrally before crossing to the other side of the spinal cord and projecting rostrally in the dorsolateral fasciculus (DLF). Most of commissural secondary ascending (CoSA) interneurons are glycinergic but some are speculated to be glutamatergic (Hale et al., 2001; Higashijima et al., 2004b). CoSAs project their axons like the CoPAs, first ventrally then contralaterally and rostrally, but have smaller somata and extend their growth cones slightly later than

CoPAs. Dorsolateral ascending (DoLA) interneurons are GABAergic neurons that have 1–2 somata per segment, are located slightly more dorsally than CoPAs, and project their axons rostrally in the DLF. At the dorsal most edge of each somite, we find 2–3 Rohon–Beard sensory neurons, which are glutamatergic, project neurites to the skin, and are the first spinal cord neurons to extend axons in the DLF of the spinal cord. For ease of discussion, these spinal neurons are grouped into five broad categories based on their axonal projections and neurotransmitters phenotypes (see Table 1).

Physiology and pharmacology of slow rhythms The first motor rhythms in zebrafish occur at 17 hpf (Kimmel et al., 1974; Saint-Amant and Drapeau, 1998). This immature motor behavior consists of spontaneous repeating, alternating coils of the tail that persist over the course of

51 Table 1. Spinal neurons in five broad categories M

Motoneurons

iE

Ipsilaterally projecting excitatory interneurons Contralaterally projecting excitatory interneurons Contralaterally projecting inhibitory interneurons Sensory neuron

cE cE S

CaP, MiP, RoP VeLD, IC, CiD CoPA, CoSAa CoB, CoSAa RB

a

This population is heterogenous.

several hours (Fig. 1b). The frequency of these coils in embryos removed from their chorions peaks at 1 Hz at 19 hpf and slowly decreases to 0.1 Hz by 26 hpf. The synchronicity of activation provided by this slow rhythm throughout the spinal network may provide a permissive environment for the formation and strengthening of synaptic contacts by promoting coincident preand postsynaptic excitation. In order to understand the role of slow rhythms in development, we need to know how they are generated and how they spread within the network. Spontaneous coils were shown to be neural in origin by abolishing the coils with injections of the nicotinic acetylcholine receptor blockers d-tubocurarine and alpha-bungarotoxin (Grunwald et al., 1988; Saint-Amant and Drapeau, 1998). Lesions showed that only the rostral spinal cord is essential for the rhythm (Pietri et al., 2009; Saint-Amant and Drapeau, 1998). The first pattern in membrane potential observed in spinal neurons prior to 1 day of development consists of sustained voltage increases lasting 300–500 milliseconds (ms) upon which action potentials are often superimposed (Drapeau et al., 1999; Saint-Amant and Drapeau, 2000, 2003). Because of their rhythmic nature, they were named periodic depolarizations (PDs, Fig. 1b). PDs are present in all the categories of neurons outlined above, with the notable exception of neurons that do not project axons ventrally. Indeed, sensory neurons and newly born neurons

with short axons did not show PDs. Experiments with blockers of glutamate receptors revealed that PDs were not dependent on glutamatergic synapses, or any other chemical synapses for transmission (Saint-Amant and Drapeau, 2000). Each side of the spinal cord was shown to contain a gap junction coupled network of neurons which act like members of a large “syncytium,” in which hyperpolarization of one neuron can shut down the whole group (Saint-Amant and Drapeau, 2000, 2001). Central to the creation of smooth alternating rhythm is the need to prevent bilateral contractions. Vertebrates, such as Xenopus, prevent coactivation of both sides of the axial musculature during swimming with mid-cycle inhibition (Soffe et al., 1984). In Xenopus larvae, the glycinergic neurons responsible for the mid-cycle inhibition are the commissural interneurons, which project a commissural axon which bifurcates and extends both rostrally and caudally upon crossing the spinal cord (Li et al., 2001). In the zebrafish, there are only three different types of commissural neurons at this stage: the CoPAs, CoSAs, and CoBs. The CoPA interneurons and a subset of CoSA interneurons were found to be positive for the expression of vesicular glutamate transporter indicating that these neurons are presumably glutamatergic (Higashijima et al., 2004a) and therefore not inhibitory. The CoB interneurons and a second subset of CoSA interneurons, on the other hand, were positive for expression of the neuronal glycine transporter (Higashijima et al., 2004b), suggesting that they are good candidates for the commissural inhibitory interneurons in zebrafish. Contralateral inhibition events in embryonic zebrafish would have to last as long as PDs and occur on the side opposite to the PDs. Indeed, an event fitting this description was observed in embryonic neuron recordings. This event was composed of volleys of synaptic activity and was thus called “synaptic bursts” (SBs, Fig. 1b). SBs are a grouping of glycinergic synaptic events, which occur within a 200–400 ms time period and are also present in

52

all types of neurons with the exception of sensory neurons. As predicted, paired recordings showed that SBs were simultaneous with PDs on the other side of the spinal cord. These data support a model in which PDs spread to all the neurons on the same side of the spinal cord via a ventrally located gap junction network (iE, Fig. 1b), while contralaterally projecting neurons (cI, Fig. 1b) carry glycinergic inputs to the opposite side (Saint-Amant and Drapeau, 2001). The ionic mechanism by which PDs are created in zebrafish is still unknown, although we expect that some neurons in the rostral spinal cord may generate the rhythm through their own pacemaker properties and spread this activity through their descending axons. One of the first types of neuron to extend axons down the length of the spinal cord, the IC interneurons, also have the PDs with the largest amplitude making them an ideal candidate for the pacemaker neurons of the spontaneous rhythm (Saint-Amant and Drapeau, 2001). At the molecular level, experiments with ion channelspecific toxins have yielded some insight into the ionic basis of rhythmogenesis. Tetrodotoxin (sodium channel) blockade reveals that voltagedependent sodium channels are necessary for the generation of PDs, suggesting an important role for sodium influx in either the creation or the propagation of the events (Saint-Amant and Drapeau, 2001). The duration of PDs was shown to increase significantly during application of apamin, a blocker of small conductance calcium activated potassium channels, further suggesting that there is also significant calcium entry during PDs, and that a calcium activated potassium conductance may play a role in PD termination in addition to other potassium conductances (SaintAmant and Drapeau, 2001). The first zebrafish motor behavior is thus produced by a very simple pacemaker-bursting network that oscillates as one unit based on cellular pacemaker properties resulting from a handful of ion channels (Fig. 1b). This would make the early zebrafish rhythmgenerating network akin to CPG in invertebrates. Indeed, the CPG that generates the gastric mill

rhythm in lobsters is crafted by a handful of neurons, some of which are connected by gap junctions and have inherent pacemaker properties (Grillner, 2006; Marder, 1984; Selverston, 2005). It is interesting to note that the stomatogastric ganglion produces events that resemble the PDs we observe in zebrafish. It remains to be determined whether other vertebrates have a similar simple rhythmgenerating mechanism during their early development.

Physiology and pharmacology of evoked rhythms While they still undergo spontaneous coiling, zebrafish embryos acquire the ability to respond to tactile stimuli by 21 hpf. As the coiling frequency wanes, the tactile responses become stronger and easier to isolate from the spontaneous rhythm (Saint-Amant and Drapeau, 1998). At 24 hpf of development, head and tail stimulation produces the same stereotypical response, which typically consists of a strong flexion of the trunk on the side contralateral to touch, followed by one to three alternating contractions (Fig. 1c). Although the touch-evoked coils are similar to the spontaneously occurring ones, touch-evoked coils are stronger and the successive contractions are higher in frequency than the spontaneously occurring coils, suggesting a stronger recruitment of the musculature and faster circuit kinetics (Saint-Amant and Drapeau, 1998). This new rhythmic behavior in response to stimuli suggests a change in the coiling motor network and implies, at the very least, new functional connections from dorsally located sensory neurons to the preexisting ventrally located gap junction network. Past studies have generated mixed results in their efforts to pinpoint the neural substrate for the early touch responses. Some studies had shown that the hindbrain is required for proper tactile responses (Saint-Amant and Drapeau, 1998) while other showed that the hindbrain is not necessary (Downes and Granato, 2006). A recent study reexamined the issue and

53

found that the neural substrate necessary for the full coil of the trunk was located in the most rostral part of the spinal cord as was found previously for the spontaneous coils (Pietri et al., 2009). These results support the concept that the ventrally located motor network is common to both spontaneous and touch-evoked coils, and further suggest that the physiology underlying both behaviors should be similar. Electrophysiological recordings in vivo showed that ipsilateral neurons begin their touch response with a burst of glycinergic events, which is quickly followed by a spiking plateau (Fig. 1c). The faster kinetics of the response may result from changes in the ionic conductances of the participating neurons and their lowered input resistances (Saint-Amant and Drapeau, 2000). Touch responses are dependent on glutamatergic pathways, as they are completely abolished when blockers of glutamatergic transmission are injected into the embryos, whereas spontaneous coiling continues at a normal frequency in the presence of the blockers (Pietri et al., 2009; Saint-Amant and Drapeau, 2000, 2006). Because of limited knowledge of the wiring of the spinal cord in the zebrafish, it is still unclear where the stringent requirement for glutamatergic transmission comes from. The continued propagation of spontaneous PDs through the gap junction network during the glutamatergic blockade suggests that the sensory input never reaches the interconnected premotor network without glutamatergic transmission, otherwise some form of coiling or twitch would still be observed. The presence of an essential synapse between sensory neurons and the premotor network does not, however, preclude a role for glutamatergic synapses in the interneuronal patterning of the touch-evoked motor rhythm. Stronger contractions in response to touch suggest that more synaptic drive is reaching the motoneurons and this is indeed what is observed in recordings (Zhou et al., 2006). The physiological and pharmacological results suggest that touchevoked rhythms result from the addition of glutamatergic and glycinergic chemical synaptic inputs to the preexisting gap junction network resulting

in stronger contractions with faster side-to-side alternation. We know from past experiments that sensory neurons are not electrotonically connected to the gap junction network, suggesting that an intermediate neuron is involved. The identification of the commissural CoPA interneurons as a glutamatergic interneuron puts this neuron at the head of a very short list to carry sensory excitation to the contralateral side of the spinal cord (Higashijima et al., 2004a) (cI, Fig. 1c). Indeed, a very similar type of interneuron in the Xenopus larvae, the dorsolateral commissural sensory interneurons, is a glutamatergic interneuron responsible for relaying and amplifying sensory information from the sensory neurons to motoneurons and premotor interneurons on the opposite side of the spinal cord l (Li et al., 2003; Roberts, 2000). It remains to be demonstrated by electrophysiological techniques whether this neuron is activated during embryonic touch responses and whether it functions downstream of sensory neurons. Collectively, these results are consistent with the hypothesis that this new response is produced when the nascent dorsal sensory component of the spinal cord is connected to the gap junction-mediated ventral motor network of the spinal cord (Fig. 1c).

Physiology and pharmacology of embryonic swimming rhythms Embryonic zebrafish can be induced to swim as early as 28 hpf, long before they naturally hatch during the third day of development. At this stage, embryos can differentiate between head and tail touches and produce different types of initial escape contractions. As with the touch-evoked rhythms, this behavior is very stereotypical and rigid. Swimming embryos perform low-amplitude contractions at a higher frequency than the spontaneous coils. Although a tail stimulus tends to generate a higher percentage of responses with swimming, the frequency of swimming resulting from any stimuli is the same (Saint-Amant and Drapeau, 1998). The absence of flexibility in the

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frequency of the rhythm suggests that before the third day of development, the mechanism that generates the swimming rhythm may represent an all or none switch. The frequency of tail beats in the first swimming bouts is around 20 Hz, a 20-fold increase in frequency when compared to the fastest spontaneous rhythms (Fig. 1d). As development continues, the frequency of tail beats during swimming increases linearly, which may reflect a gradual maturation process of the neurons involved such as their basic membrane properties of input resistance and capacitance. The sudden and dramatic shift in frequency from low-frequency touch responses at 26 hpf to higher frequency swimming at 28 hpf suggests that swimming onset may be the result of a single modification to the preexisting circuit. One key difference between swimming and the previous motor behaviors is that swimming requires the hindbrain in addition to the rostral spinal cord (Saint-Amant and Drapeau, 1998). Interestingly, the interaction between the hindbrain and spinal cord may not be just functional but also developmental as removal of the hindbrain can prevent the normal maturation of the spinal CPG (Chong and Drapeau, 2007). Fictive swimming as revealed by motoneuron recordings in the embryo (Saint-Amant et al., 2008) shows rhythmic bursts of action potentials with high firing frequency occurring on top of a sustained tonic drive (Fig. 1d). Additionally, results from larval fish have shown that spinal neurons receive both glycinergic and glutamatergic inputs during swimming (Buss and Drapeau, 2001; Buss et al., 2003; McDearmid and Drapeau, 2006; McLean et al., 2007, 2008). It is possible to bypass the requirement for the hindbrain by providing an exogenous tonic drive. Indeed, ventral root recordings from spinalized zebrafish have shown that NMDA induces fictive swimming and that only two spinal segments are necessary for generating rhythmic activity, which is consistent with results in other swimming vertebrates (Grillner et al., 1991; Masino and Fetcho, 2005; McDearmid and Drapeau, 2006). As swimming prior to the third day of development occurs in a tight range of

frequencies, the drive from the hindbrain may be relatively homogenous. Taken together, the evidence for a strong glutamatergic component and the need for the hindbrain suggest the simple hypothesis that swimming appears when the hindbrain first contacts the spinal cord and produces a tonic synaptic drive. This new tonic drive induces the spinal CPG to produce a long string of alternating and kinetically faster PDs and SBs (Fig. 1d).

Increasing the frequency of swimming in larvae After 3 days of development, swimming switches to an intermittent mode of swimming, where motor events are composed of a string of a few cycles of high-frequency tail beats punctuated by periods of inactivity (Buss and Drapeau, 2001, 2002; Muller and van Leeuwen, 2004). After 4 days, zebrafish larvae can start choosing from distinct swimming rhythms: slow swims and burst swims. Burst swims are associated with stimulievoked escape responses and as such involve stronger bends of the tail, higher frequencies of tail beats, and faster swimming speeds than the more routine slow swimming (Budick and O'Malley, 2000). After 5 days of development, zebrafish larvae show a even larger repertoire of motor behaviors in terms of frequency and amplitude that includes prey tracking (Mcelligott and O'malley, 2005) and prey capture (Borla et al., 2002). The ability of larval fish to generate a variety of motor rhythms suggests that the spinal cord no longer functions as a single CPG unit. Indeed, researchers have recently shown through careful mapping of neuronal activity at different frequencies of fictive swimming that some types of neurons, with ventrally located cell bodies, are active during slow rhythms and become inhibited at higher frequencies while other types of neurons, with more dorsal cell bodies, show the opposite pattern of recruitment (Fetcho et al., 2008; Liao and Fetcho, 2008; McLean and Fetcho, 2008; McLean et al., 2007). These

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intriguing results clearly show that late larval zebrafish spinal circuitry is not simply shared for all behaviors and suggests that there are functional subdivisions in the zebrafish spinal cord rhythm generator. These findings support a concept where some components may be included or excluded from the spinal CPG. The inclusion or exclusion of a type of spinal neuron in the execution of a behavior likely results from differences how the spinal circuitry is recruited by the higher brain centers such as the hindbrain.

Rhythm mutants Several mutagenesis screens targeted at motor behaviors have been performed in the past 15 years. Studying the genetic roots of motor mutants should yield insights into motor circuit function in normal animals (Bate, 1999; Fetcho and Liu, 1998). Several motility mutants that have been studied to date affect motor activity by perturbing touch sensitivity (Granato et al., 1996; Low et al., 2010a; Nakano et al., 2010; Ribera and Nusslein-Volhard, 1998), the escape response (Burgess et al., 2009; Lorent et al., 2001), neuronal proliferation (Gray et al., 2001; van Eeden et al., 1996), motor axon guidance (Birely et al., 2005; Palaisa and Granato, 2007; Rodino-Klapac and Beattie, 2004; Zeller and Granato, 1999; Zhang et al., 2004), the neuromuscular junction (Behra et al., 2002; Downes and Granato, 2004; Lefebvre et al., 2004; Ono et al., 2001, 2002; Panzer et al., 2005; Saint-Amant et al., 2008; Westerfield et al., 1990; Zhang et al., 2004), or excitation contraction coupling (Gleason et al., 2004; Hirata et al., 2004, 2007; Ono et al., 2001; Schredelseker et al., 2005; Zhou et al., 2006). Although these mutants were very informative in our search to understand motor behavior as a whole, they did not reveal insights into the mechanisms that generate rhythm. Two mutations were shown to prevent the CPG from creating normal motor rhythms. The phenotype of the mutant bandoneon is described

as an accordion because tactile stimulation causes both sides of the mutant to briefly contract simultaneously which reduces the length of the trunk much like an accordion. The spontaneous contractions occur normally in bandoneon mutants, but the responses to touch in the same animals at 24 hpf are clearly of the accordion type (Hirata et al., 2005). When wild-type 24 hpf embryos are placed in the glycine receptor blocker strychnine, they exhibit the same phenotype of normal spontaneous contractions with abnormal bilateral touch responses. This result suggests that bandoneon affects crossed glycinergic synaptic transmission. The bandoneon gene was found to encode for the b2 subunit of the glycine receptor (glrb2), which leads to an absence of glycinergic synaptic signaling (Hirata et al., 2005). The presence of bilateral contraction suggests an important role in cross inhibition while the absence of rhythmic output suggests an additional role in patterning of CPG output. The shocked mutation was first isolated because of a lack of swimming at 3 days (Granato et al., 1996). Further analysis of the mutants revealed that they cease spontaneous contractions abruptly at 21 hpf and fail to respond to tactile stimulation at 24 hpf: by 48 hpf, the larvae respond to touch with a vigorous contralateral contraction but fail to initiate swimming (Cui et al., 2004, 2005). The mutation which causes shocked was found to perturb the function of the CNS glycine transporter (glyt1) (Cui et al., 2005). Glyt1 is expressed extensively by nonneuronal cells in the hindbrain and spinal cord of embryos and larvae. It is thought that the absence of the glycine transporter in shocked mutants leads to an aberrant accumulation of glycine levels in the CNS and the shunting of sustained activity (Cui et al., 2005). Indeed, exposing the animals to strychnine recovers normal spontaneous coils in the embryos and partial swimming at 2 days, while removing glycine from the hindbrain during electrophysiological recordings uncovered a normal fictive swimming rhythm (Cui et al., 2005; Mongeon et al., 2008). These mutants provide evidence for

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the importance of tightly regulating glycinergic signaling in motor networks. Two mutations in the sodium channel gene scn8aa were recently isolated and characterized from the zebrafish nonactive lines (nav) (Low et al., 2010b). These recessive mutants were identified by their lack of sustained motor responses following tactile stimulation. As in mammals, the sodium channel produced from this gene (NaV1.6a) was shown to possess a noninactivating sodium conductance commonly referred to as a persistent sodium current (Crill, 1996), which has been shown to be involved in the creation of sustained patterns of motor output (Low et al., 2010b; Tazerart et al., 2007, 2008; Zhong et al., 2007). It should prove interesting to determine which neurons require this persistent sodium conductance to produce swimming and whether these neurons are an integral part of a core CPG for locomotion in the zebrafish. The mutants described in the last paragraph are some of the few zebrafish mutants which seem to perturb the production of motor rhythms by the CPG. This rarity of identified CPG mutants in zebrafish may be due to either genetic and functional redundancy or the difficulty of establishing the root cause of the defect when it lies deep in the CPG. Developing assays to specifically screen for subtle perturbations in the earliest manifestations of rhythmicity should address the former, while the development of new electrophysiological and imaging techniques should remedy the latter. Zebrafish research is showing that there is still ample room for forward genetic approaches in the study of nervous system development and function in vertebrates.

Future research The molecular mechanisms that lead to the precise synaptic connectivity during development in the embryonic spinal cord are still largely unknown. A variety of genetic tools and techniques are available in the zebrafish such as transgenic lines (Kawakami, 2005; Kuwada, 1995), enhancer lines

(Balciunas et al., 2004; Ellingsen et al., 2005; Parinov et al., 2004; Woolfe et al., 2005), targeted protein knockdown with morpholinos (Nasevicius and Ekker, 2000), and motility mutants (Granato et al., 1996). The combination of these various tools will permit researchers to modify the spinal cord expression patterns of any protein of interest in vivo while assaying the effects on neuron morphology, synaptic junction formation, network activity, and behavior. A promising area of interest to study the mechanisms involved in synaptic specificity is to look at proteins involved in cell–cell interactions such as the cadherin family of proteins (Down et al., 2005; Jontes et al., 2004). Exciting techniques have been developed to permit the stimulation of zebrafish neuronal networks with light which opens the door to define the role of specific types of neurons in creating a variety of motor behaviors (Douglass et al., 2008; Szobota et al., 2007; Wyart et al., 2009) especially when combined to genetic techniques to label specific neuronal types (Scott, 2009; Scott et al., 2007). Identification of these active neurons should be followed by lesion experiments ascribing a behavioral function to each type of neuron and ultimately to a detailed electrophysiological analysis of the synaptic drive mediated by these neurons during motor behaviors. This type of research will lead to a greater understanding of how vertebrate circuits generate rhythmic activity and behavior.

Summary In this review, we present a model for the stepwise development of the zebrafish embryonic motor circuitry that is based on our knowledge of the sequential appearance of behaviors, their physiology, and their pharmacology. Our model proposes that the circuitry begins with a very simple CPG composed of gap junction connected neurons, which produce periodic activity that is dependent on the basic membrane properties dictated by a rudimentary complement of ion channels. This basic rhythmogenic circuit is not

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removed with further development but rather modified by adding layers of complexity. Indeed, the ability to respond to touch may be achieved by adding sensory inputs and chemical neurotransmission, while swimming may be added to the repertoire, by connecting neurons from the hindbrain. Although this model has many components that have not been verified experimentally, it provides a framework to design new experiments and to test new hypotheses. Acknowledgments The author acknowledges Dr Sean Low, Mathieu Lachance, and Joël Ryan for comments on the manuscript. References Balciunas, D., Davidson, A. E., Sivasubbu, S., Hermanson, S. B., Welle, Z., & Ekker, S. C. (2004). Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC Genomics, 5, 62. Bate, M. (1999). Development of motor behaviour. Current Opinion in Neurobiology, 9, 670–675. Behra, M., Cousin, X., Bertrand, C., Vonesch, J. L., Biellmann, D., Chatonnet, A., et al. (2002). Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nature Neuroscience, 5, 111–118. Bernhardt, R. R., Chitnis, A. B., Lindamer, L., & Kuwada, J. Y. (1990). Identification of spinal neurons in the embryonic and larval zebrafish. The Journal of Comparative Neurology, 302, 603–616. Birely, J., Schneider, V. A., Santana, E., Dosch, R., Wagner, D. S., Mullins, M. C., et al. (2005). Genetic screens for genes controlling motor nerve-muscle development and interactions. Developmental Biology, 280, 162–176. Borla, M. A., Palecek, B., Budick, S., & O'malley, D. M. (2002). Prey capture by larval zebrafish: Evidence for fine axial motor control. Brain, Behavior and Evolution, 60, 207–229. Brustein, E., Saint-Amant, L., Buss, R. R., Chong, M., Mcdearmid, J. R., & Drapeau, P. (2003). Steps during the development of the zebrafish locomotor network. Journal of Physiology, Paris, 97, 77–86. Budick, S. A., & O'malley, D. M. (2000). Locomotor repertoire of the larval zebrafish: Swimming, turning and prey capture. The Journal of Experimental Biology, 203, 2565–2579.

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behavioral mutation disrupts acetylcholine receptor localization and motor axon stability. Developmental Neurobiology, 68, 45–61. Schredelseker, J., Di Biase, V., Obermair, G. J., Felder, E. T., Flucher, B. E., Franzini-Armstrong, C., et al. (2005). The beta 1a subunit is essential for the assembly of dihydropyridinereceptor arrays in skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America, 102, 17219–17224. Scott, E. K. (2009). The Gal4/UAS toolbox in zebrafish: New approaches for defining behavioral circuits. Journal of Neurochemistry, 110, 441–456. Scott, E. K., Mason, L., Arrenberg, A. B., Ziv, L., Gosse, N. J., Xiao, T., et al. (2007). Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nature Methods, 4, 323–326. Selverston, A. I. (2005). A neural infrastructure for rhythmic motor patterns. Cellular and Molecular Neurobiology, 25, 223–244. Soffe, S. R., Clarke, J. D., & Roberts, A. (1984). Activity of commissural interneurons in spinal cord of Xenopus embryos. Journal of Neurophysiology, 51, 1257–1267. Szobota, S., Gorostiza, P., Del Bene, F., Wyart, C., Fortin, D. L., Kolstad, K. D., et al. (2007). Remote control of neuronal activity with a light-gated glutamate receptor. Neuron, 54, 535–545. Takahashi, M., Narushima, M., & Oda, Y. (2002). In vivo imaging of functional inhibitory networks on the mauthner cell of larval zebrafish. The Journal of Neuroscience, 22, 3929–3938. Tazerart, S., Viemari, J. C., Darbon, P., Vinay, L., & Brocard, F. (2007). Contribution of persistent sodium current to locomotor pattern generation in neonatal rats. Journal of Neurophysiology, 98, 613–628. Tazerart, S., Vinay, L., & Brocard, F. (2008). The persistent sodium current generates pacemaker activities in the central pattern generator for locomotion and regulates the locomotor rhythm. The Journal of Neuroscience, 28, 8577–8589. Van Eeden, F. J., Granato, M., Schach, U., Brand, M., Furutani-Seiki, M., Haffter, P., et al. (1996). Mutations affecting somite formation and patterning in the zebrafish, Danio rerio. Development, 123, 153–164. Westerfield, M., Liu, D. W., Kimmel, C. B., & Walker, C. (1990). Pathfinding and synapse formation in a zebrafish mutant lacking functional acetylcholine receptors. Neuron, 4, 867–874. Westerfield, M., Mcmurray, J. V., & Eisen, J. S. (1986). Identified motoneurons and their innervation of axial muscles in the zebrafish. The Journal of Neuroscience, 6, 2267–2277. Woolfe, A., Goodson, M., Goode, D. K., Snell, P., Mcewen, G. K., Vavouri, T., et al. (2005). Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biology, 3, e7. Wyart, C., Del Bene, F., Warp, E., Scott, E. K., Trauner, D., Baier, H., et al. (2009). Optogenetic dissection of a behavioural module in the vertebrate spinal cord. Nature, 461, 407–410.

61 Yvert, B., Branchereau, P., & Meyrand, P. (2004). Multiple spontaneous rhythmic activity patterns generated by the embryonic mouse spinal cord occur within a specific developmental time window. Journal of Neurophysiology, 91, 2101–2109. Zeller, J., & Granato, M. (1999). The zebrafish diwanka gene controls an early step of motor growth cone migration. Development, 126, 3461–3472. Zhang, J., Lefebvre, J. L., Zhao, S., & Granato, M. (2004). Zebrafish unplugged reveals a role for muscle-specific kinase

homologs in axonal pathway choice. Nature Neuroscience, 7, 1303–1309. Zhong, G., Masino, M. A., & Harris-Warrick, R. M. (2007). Persistent sodium currents participate in fictive locomotion generation in neonatal mouse spinal cord. The Journal of Neuroscience, 27, 4507–4518. Zhou, W., Saint-Amant, L., Hirata, H., Cui, W. W., Sprague, S. M., & Kuwada, J. Y. (2006). Non-sense mutations in the dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrafish relaxed mutants. Cell Calcium, 39, 227–236.

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 5

Limb, respiratory, and masticatory muscle compartmentalization: Developmental and hormonal considerations C. G. Widmer* and J. Morris-Wiman Department of Orthodontics, University of Florida College of Dentistry, JHMHSC, Gainesville, Florida, USA

Abstract: Neuromuscular compartments are subvolumes of muscle that have unique biomechanical actions and can be activated singly or in groups to perform the necessary task. Besides unique biomechanical actions, other evidence that supports the neuromuscular compartmentalization of muscles includes segmental reflexes that preferentially excite motoneurons from the same compartment, proportions of motor unit types that differ among compartments, and a central partitioning of motoneurons that innervate each compartment. The current knowledge regarding neuromuscular compartments in representative muscles involved in locomotion, respiration, and mastication is presented to compare and contrast these different motor systems. Developmental features of neuromuscular compartment formation in these three motor systems are reviewed to identify when these compartments are formed, their innervation patterns, and the process of refinement to achieve the adult phenotype. Finally, the role of androgen modulation of neuromuscular compartment maturation in representative muscles of these motor systems is reviewed and the impact of testosterone on specific myosin heavy chain fiber types is discussed based on recent data. In summary, neuromuscular compartments are prepatterned output elements in muscle that undergo refinement of compartment boundaries and muscle fiber phenotype during maturation. Further studies are needed to understand how these output elements are selectively controlled during locomotion, respiration, and mastication. Keywords: Masseter; diaphragm; gastrocnemius; development; muscle compartmentalization; androgen. organization of their motor nuclei. This hypothesis was based on mounting evidence that many classically defined muscles, including lateral gastrocnemius (LG) (English and Letbetter, 1982), medial gastrocnemius (Letbetter, 1974), biceps femoris and semitendinosus (English and Weeks, 1989), trapezius (Keane and Richmond, 1981), biventer cervicis (Armstrong et al., 1988), extensor digitorum

Introduction English et al. (1993) proposed the partitioning hypothesis to describe the relationship between compartmentalization of muscles and the spatial *Corresponding author. Tel.: þ1-352-273-5696; Fax: þ1-352-846-0459

DOI: 10.1016/S0079-6123(10)87005-0

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longus (Balice-Gordon and Thompson, 1988), gluteus maximus (English, 1990), biceps brachii (Segal, 1992), and masseter (Herring et al., 1979; Widmer et al., 1997), have a complex anatomical organization composed of multiple, functionally distinct, neuromuscular compartments. Thus, these compartments represent output elements that may be independently activated to generate unique biomechanical actions. It has also been shown that neuromuscular compartments are differentially recruited in specific reflex pathways (Ia afferents, flexion reflex), further supporting the idea that these compartments have functionally distinct roles as unique output elements. Centrally, the motoneurons that innervate these distinct muscle regions have been found to have a spatial organization within the muscle motoneuron pool. Recently, to further characterize these compartments, we, as well as others, have begun to investigate the factors responsible for the developmental patterning of compartments, the spatial organization of motoneuron subpopulations in the motoneuron pool responsible for the innervation of specific neuromuscular compartments, and hormonal influences that might affect both compartment motoneurons centrally and the phenotype of compartment muscle fibers peripherally. Knowledge of the properties, organization, and activation of these output elements as they function as synergists or antagonists in the context of motor control in locomotion, respiration, and mastication is required for refinement of models for these three motor systems. The focus of this chapter is to provide an overview of neuromuscular compartmentalization and to review developmental aspects and hormonal influences that affect compartment formation and functional characteristics in the LG, diaphragm, and masseter muscles, representing muscles of locomotion, respiration, and mastication. Neuromuscular compartmentalization of muscle Neuromuscular compartments are discrete subvolumes of muscle that are innervated by a unique collection of motoneurons (English and

Letbetter, 1982). The original work by Letbetter (1974) on cat medial gastrocnemius laid the foundation for examination of other limb, neck, respiratory, and masticatory muscles that have been shown to be compartmentalized into functional subvolumes. The number of neuromuscular compartments that are contained within any specific muscle varies widely and may be related to the complexity of the biomechanical actions that are performed by the muscle/neuromuscular compartments. Different neuromuscular compartments may also contain muscle fibers with different proportions of particular myosin heavy chain (MyHC) isoforms and, thus, the speed of contraction and fatigue characteristics will vary between neuromuscular compartments.

Lateral gastrocnemius The lateral gastrocnemius (LG) is composed of four neuromuscular compartments. Four regions of the LG muscle in the cat and rat: LG1, LG2, LG3, and LGm (Donahue and English, 1987; English, 1984) are innervated by four primary nerve branches of the LG muscle nerve, each of which contain a unique collection of motor axons. English and Letbetter (1982) used the method of glycogen depletion to determine the innervation territories of the four primary nerve branches and found four distinct and non-overlapping regions of muscle fibers. The compartmentalization of the LG defined by the primary motor nerve branches, was further analyzed using evoked electromyographic (EMG) mapping of stimulated isolated single motor axons (English and Weeks, 1989) in conjunction with glycogen depletion (English and Weeks, 1984). It was found that the motor unit territories were confined to a single compartment. Thus, individual compartments in the LG are independently innervated and have the potential to be activated either singly or as groups of compartments to perform specific tasks. For example, slow walking involves preferential activation of compartment

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LG3 over compartments LG1 and LG2, with the LGm compartment remaining almost totally inactive (English, 1984). Further evidence for the differential activation of LG neuromuscular compartments has been provided through the study of reflex partitioning. Nichols et al. (1993) evaluated the activation of LG compartments by various reflexes (flexion– withdrawal reflex, caudal cutaneous sural nerve reflex, crossed– extension reflex) in the cat. Statistically significant differences in LG compartment recruitment were observed only during the flexion–withdrawal reflex with the LGm compartment having a higher response than the LG1, LG2, or LG3 compartments. This outcome is as one might predict given the dorsal location of this compartment provides a mechanical advantage for LGm in this flexion–withdrawal reflex (Vanden Noven et al., 1986). No differences in activation were found among compartments after the caudal cutaneous sural nerve or crossed–extension reflexes were elicited. Ia afferent sensory partitioning has also been observed in LG after electrical stimulation of LG primary nerve branches. Significantly, greater amplitude monosynaptic EPSPs were recorded from homonomous LG motoneurons innervating the same compartment compared to EPSPs recorded from heteronomous motoneurons innervating the three other compartments after normalization for different motoneuron types in each compartment (Vanden Noven et al., 1986). In addition, statistically greater amplitude heteronomous EPSPs were found in the LG2 branch after stimulation of the soleus nerve compared to the other LG nerve branches. Similar compartment Ia afferent partitioning has been reported for the medial gastrocnemius (Lucas and Binder, 1984). Detailed reviews of monosynaptic Ia EPSP afferent excitation of motoneurons of neuromuscular compartments in various muscles has been published (Stuart et al., 1988; Windhorst et al., 1989). Further evidence for the autonomy of neuromuscular compartments is also provided by the distinctly different biomechanical actions that are elicited by each compartment. Average torque

trajectories and magnitudes differ significantly among LG neuromuscular compartments (Fig. 1a) (Carrasco et al., 1999). The LG1 compartment has a statistically greater plantarflexion torque than the other compartments while LGm has the smallest. Compartments located more laterally (LG1 and LG2) produce higher off-sagittal torques (abduction–adduction, inversion–eversion) about the ankle than those located near the mid-sagittal plane (LG3, LGm). These recordings were conducted with a fixed ankle joint angle (100 ) relative to the tibia and it is possible that further differences in torques produced by the individual LG compartments may be observed as the foot is positioned in functional 3D space.

Diaphragm The mammalian diaphragm is a major inspiratory muscle that is composed of at least two architecturally distinct regions: the sternocostal region and the crus region surrounding the esophagus (Pickering and Jones, 2002). These two regions have functionally distinct actions. During respiration, both the sternocostal region and the crus are activated. However, during emesis, regurgitation, swallowing, or eructation, there is activation of only the sternocostal region with inhibition of the crus to allow the bolus to move in the lower esophagus (Monges et al., 1978). The innervation of the sternocostal diaphragm and crus consists of four branches of the phrenic nerve (SC1, SC2, SC3, and Cr) (Hammond et al., 1989) with potentially additional innervation of costal regions of the sternocostal diaphragm by cervical regions (C4, C5) and innervation of the crus by branches from the vagus nerve and cervical regions (C5, C6) (Young et al., 2010). Unilateral electrical stimulation of the crural branch has been shown to only activate the ipsilateral crus diaphragm while stimulation of the three branches innervating the sternocostal diaphragm elicits responses in distinct but overlapping regions (Duron et al., 1979b; Hammond et al., 1989).

66 Cat lateral gastrocnemius compartment torque plots 0

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Fig. 1. Vector plots of (a) mean lateral gastrocnemius compartment torques about the ankle in the cat (n ¼ 6) (Carrasco et al., 1999) and (b) typical masseter compartment torques about the temporomandibular joint in rabbit (English and Widmer, 2001). Each plot represents pitch versus yaw or pitch versus roll for the two muscles. For the LG compartments, negative pitch is plantarflexion, negative roll is eversion, and negative yaw is adduction. For the masseter compartments, negative pitch is jaw closing, negative yaw is working side rotation, and negative roll is lingual tipping. All compartments were statistically different (ANOVA, Bonferroni, or LSD test, p < 0.05) in at least one torque component (pitch, yaw, roll, or magnitude) for both the LG (n ¼ 6) and masseter (n ¼ 5) muscles.

These findings were verified using glycogen depletion studies that mapped the depleted muscle fiber territories. Differential recruitment of the sternal but not the mid-costal regions during post-inspiratory activity has been shown using EMG recordings in sleeping or anesthetized lambs and is consistent with the observed innervation of these regions by different nerve branches (Henderson-Smart et al., 1982). Thus,

some investigators describe the diaphragm as having three distinct neuromuscular compartments: sternal, costal, and crus. Mechanical actions of the sternocostal and crus regions of the diaphragm have also been reported to be different, further establishing their differential roles. Activation of the sternocostal diaphragm elicits an outward movement of the lower rib cage while activation of the crus had

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no effect on the rib cage (De Troyer et al., 1982). There has, as yet, been no reported assessment of the biomechanical actions of different regions of the sternocostal diaphragm during the stimulation of different nerve branches. Therefore, it is unclear whether there are any mechanical advantages to compartmentalization of the sternocostal portion of the diaphragm. Several studies have reported finding no differences in the proportion of muscle fiber types comprising the different diaphragm compartments. However, we have recently detected significant differences between the expression of type IIb MyHC isoform within the sternocostal and crural diaphragm regions in the mouse (Fig. 2). Type IIb MyHC is not expressed in the lateral costal regions of the sternocostal diaphragm but is readily detected in the sternal regions and in the crus. This differential expression of MyHC mirrors the observed different mechanical actions of these diaphragm regions and provides additional support for the uniqueness of these compartments.

Masseter The masseter muscle is one of four muscles of mastication and has the primary role of closing the jaw in conjunction with two other jaw closing

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muscles, the temporalis and medial pterygoid muscles. The fourth masticatory muscle, the lateral pterygoid, causes jaw protrusion and jaw opening when activated. It has been known for many years that the masseter is composed of superficial, intermediate, and deep layers that have different functions (Hannam et al., 1977; Herring et al., 1979; Schumacher, 1961; Weijs and Dantuma, 1981; Widmer et al., 1997). However, a more thorough evaluation of the rabbit masseter muscle architecture revealed multiple anatomical partitions (Weijs and Dantuma, 1981; Widmer et al., 1997) that could be further subdivided into neuromuscular compartments (English et al., 1999a; Widmer et al., 2003). Unlike the LG where each of four neuromuscular compartments is innervated by a primary muscle nerve branch, the rabbit masseter has been shown to be composed of at least 23 separate compartments that are innervated by unique motor unit axons detected in secondary and tertiary branches of the masseteric motor nerve (Widmer et al., 2003). A similar complexity of the anatomical organization and neural innervation has been reported in the human masseter (Widmer et al., 1996). The complexity of the organization of the masseter muscle output elements makes this muscle an excellent model to investigate

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Fig. 2. Parasagittal sections of mouse diaphragm representing mid-costal (a) and sternocostal (b) regions of an adult male mouse. Fibers containing MyHC type IIa can be observed in both regions of the diaphragm. However, the sternocostal region (b) and the crural region (not shown) have fibers containing type IIb MyHC (asterisks). The discrete localization of MyHC type IIb fibers supports the presence of individual compartments in the mouse diaphragm.

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compartment characteristics and their recruitment during complex movement patterns such as those associated with mastication. Reflex partitioning to specific masseter compartment motoneurons has not yet been examined. However, the biomechanical actions of the individual masseteric compartments have been shown to produce unique torques about the temporomandibular joint (English et al., 1999a; English and Widmer, 2001) (Fig. 1b). The biomechanical actions involve large jaw closing torques as the predominant action, but also produce significant off-sagittal torques to rotate the mandibular condyle about a dorsal–ventral axis to promote medial–lateral movement of the mandible and to rotate the condyle in the rostral–caudal axis to tip the teeth slightly during the power stroke. Comparison of masseter and LG compartment torques reveals larger plantarflexion (pitch) torques and off-sagittal (yaw) torques about the ankle than masseter compartment jaw closing (pitch) torques and off-sagittal torques (yaw) (Fig. 1). However, these differences are more than compensated by the larger number of compartments in the masseter muscle that represent a greater diversity of potential torque production about the temporomandibular joint.

Central partitioning of motoneurons innervating neuromuscular compartments The central organization of motoneurons innervating neuromuscular compartments has been examined for a number of compartmentalized muscles. Several studies suggest a distinct somatotopy of compartment motoneurons in the motor nucleus while some studies of diaphragm, neck, sartorius, and medial gastrocnemius muscles have reported considerable overlap of compartment motoneurons. The differences reported in the observed organization of compartmental motoneurons between different studies may be due in part to differences in the methodology, that is, labeling nerve branches or labeling discrete

regions of the muscle to determine the organization of subpopulations of motoneurons. The functional significance of a somatotopic organization of the compartment motoneurons may be related to an increased success of establishing appropriate synaptic contacts from segmental or descending connections during development.

Lateral gastrocnemius For each of the four neuromuscular compartments of the LG in the cat, a spatially organized group of motoneurons, or compartment nuclei, have been identified (Weeks and English, 1985). A distinct topography of compartment nuclei was reported using HRP labeling of primary nerve branches, with more proximal compartments (innervated by LGm, LG2 nerve branches) generally located more rostrally in the LG motoneuron pool and the more distal compartments (innervated by LG1, LG3 nerve branches) located in the caudal region of the LG motor nucleus. Although an extensive overlap was observed in the distribution of the various compartment nuclei, the central tendencies of each compartment nucleus were significantly different (Weeks and English, 1985). A motoneuron size difference was also identified with a higher proportion of large cells located in the proximal compartment nuclei. This finding is consistent with the prevalent muscle fiber phenotype found in these compartments. The proximal compartments consist of predominantly faster contracting muscle fibers, fast-twitch glycolytic (type FG), that are associated with larger fast-twitch fatigable (FF) motor units. The more distal compartment nuclei have approximately equal numbers of large and small motoneurons, a higher number of muscle spindles, and a higher proportion of slow oxidative (SO) fibers and slow-twitch (type S) motor units (Weeks and English, 1985). The smaller sized motoneurons (< 800 mm2) in these compartments were assumed to be gamma motoneurons.

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Diaphragm The spatial distribution of diaphragm compartment nuclei has been investigated using different combinations of fluorescent tracers or HRP retrograde labeling of phrenic nerve primary branches or the injection of tracers into different regions of the diaphragm (Duron et al., 1979a; Gordon and Richmond, 1990; Laskowski and Sanes, 1987; Rikard-Bell and Bystrzycka, 1980; Tan and Miller, 1986). The results of these studies were generally similar, with the sternal portion of the sternocostal diaphragm innervated by motoneurons located more rostrally in the phrenic motor nucleus while motoneurons innervating the lateral costal portion of the diaphragm were found more caudally. The crural motoneurons were found to have a broad spatial distribution in the middle and caudal regions of the phrenic motoneuron pool when muscle nerve branches were retrogradely labeled (Duron et al., 1979a; Rikard-Bell and Bystrzycka, 1980). However, when labeling was performed by HRP injections into the diaphragm, crural motoneurons were observed to be more extensively distributed throughout the whole phrenic motoneuron pool (Tan and Miller, 1986). Differences in the spatial distribution of motoneurons observed between studies using muscle injection and muscle nerve labeling may be the result of diffusion of the tracer in the muscle (Gordon and Richmond, 1990). Another possible interpretation may be that primary nerve branches innervating the diaphragm contain axons innervating multiple compartments and retrograde labeling of any one primary nerve branch would label multiple compartment nuclei. Since motor unit territories are estimated to be 10–15% the size of the whole diaphragm, it is conceivable that each primary nerve branch could innervate multiple compartments. When discrete regions (potentially single compartments) of the diaphragm were labeled using Nuclear Yellow and Fast Blue, the motoneuron density for each fluorescent label was much more clustered (Laskowski and Sanes, 1987). However,

overlap of each labeled set of motoneurons in the middle of the motor nucleus was still observed. Thus, the spatial distribution of compartment nuclei of the diaphragm (determined by the primary nerve branches of the phrenic nerve) has less distinct somatotopy compared to compartment nuclei of the LG.

Masseter Retrograde labeling of pairs of primary branches innervating different (anterior/posterior) regions of the masseter was investigated using fluorogold and fast blue tracers and no simple spatial patterning of subpopulations of motoneurons could be detected in the rabbit masseteric motoneuron pool (Saad et al., 1997). However, a study using HRP retrograde labeling of motoneurons innervating defined regions of the rabbit masseter muscle found statistically different distributions within the masseteric motor nucleus (Weijs, 1996). The superficial regions of the masseter muscle were innervated by neurons occupying the dorsolateral aspect of the masseteric motoneuron pool while the deep regions were innervated by motoneurons occupying the dorsomedial and central ventromedial aspects. Extensive spatial overlap of motoneurons innervating all masseter regions was found in the rostrocaudal dimension of the masseteric motor nucleus. However, this study was limited by the spread of HRP resulting in the labeling of multiple compartments and the inability to compare retrogradely labeled regions simultaneously using multiple tracers. We have recently conducted retrograde labeling of two discrete regions (anterior and posterior regions of the intermediate layer) of the mouse masseter muscle using injections of cholera toxin B conjugated to two different Alexa fluorochromes. The intermediate layer of the mouse masseter muscle has three distinct regions (at least three compartments) that are each composed of muscle fibers with different proportions of MyHC fiber types (Fig. 3). The anterior third has a high proportion of MyHC IIa

70 Female masseter MyHC type lla

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1 mm

Fig. 3. Representative transverse cryosections of adult male and female mouse masseter muscles immunolabeled for MyHC type IIa (red) or IIb (green). MyHC fiber types are non-uniformly distributed within the masseter in both male and female mice. A higher density of IIa-containing fibers is observed within the anterior region while IIb-containing fibers occupy the posterior region of the muscle. A sexual dimorphic distribution of MyHC IIb is apparent in the posterior region of the masseter. Arrows denote regions in which females are devoid of IIb-containing fibers. In contrast, males have a relatively uniform distribution of MyHC IIb in the posterior masseter. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

fibers; the middle third of the muscle has a high proportion of fibers containing MyHC IIx; while the posterior third is almost exclusively occupied by fibers containing MyHC IIb. Retrograde labeling of the anterior and posterior regions of the intermediate layer allowed the identification of compartment motoneurons to assess their spatial organization within the masseteric motoneuron pool. A discrete somatotopy was observed in the masseteric motor nucleus. The anterior region of the masseter was innervated by motoneurons that occupied the dorsomedial aspect of the masseteric

motor nucleus while motoneurons innervating the posterior masseter were observed in the ventrolateral masseteric motoneuron pool (Fig. 4a). The labeled motoneurons representing each compartment were highly clustered and had no overlap (Fig. 4b). Thus, accurate assessment of the central partitioning of motoneurons innervating compartments in complex muscles such as the masseter muscle appears to depend on the ability to label motor units of individual compartments and these compartments may not be represented by the first order branching of the muscle nerve.

71 (a) MesV

MotV Bregma –5.02 NVsnp

MesV

MotV Bregma –5.08 NVsnp

MesV Bregma –5.14

MotV NVsnp

(b) Labeled motoneurons

R

D

V

R

M

L

C

Centroids of labeled motoneurons

M

V

Transverse R

V

C

L

C

Sagittal

D

D

Frontal R

M

L

C

D

M

L

V

Fig. 4. Spatial organization of motoneurons innervating the anterior and posterior regions of the intermediate masseter layer identified by retrograde labeling using cholera toxin B conjugated to Alexa 555 (anterior region-red) or Alexa 488 (posterior region-green). Tracer (1–3 ml) was injected into the anterior or posterior intermediate masseter of anesthetized mice and allowed to transport for 4–5 days prior to harvesting of the brainstem. Cryosections were mounted on slides and immunostained for NeuN to identify motoneurons. (a) Illustrated are labeled motoneurons (inset magnified) detected within the masseteric motor nucleus (motV) at the brainstem level shown in the linked left panel. (b) A three-dimensional map of the labeled motoneurons for each tracer was constructed and centroids for each group of motoneurons in each section were calculated and plotted. An obvious central partitioning of motoneurons innervating these two masseter intermediate layer compartments of the muscle was observed. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

72

Development of neuromuscular compartments

Lateral gastrocnemius

Recent evidence suggests that cues that may regulate muscle nerve innervation of appropriate muscle targets involve the developmentally regulated expression of homeodomain transcription factors within motoneuron pools (Dalla Torre di Sanguinetto et al., 2008; Marco Garcia and Jessell, 2008). It remains to be determined whether transcription factors that regulate the central organization of motoneurons and the patterning of innervation for whole muscles also regulate the patterning of innervation for neuromuscular compartments. Most of the research examining developmental aspects of neuromuscular compartmentalization has concentrated on the timing and role of nerve–muscle interactions in specifying compartments and on factors involved in the refinement of innervation. Two different mechanisms have been considered for the development of neuromuscular compartments: (1) the muscle is innervated by arbitrary motoneurons in the muscle motoneuron pool, with muscle fibers having polyneuronal innervation, and subsequent refinement of connections are made after birth to form adult neuromuscular compartments by synapse elimination; or (2) compartments are patterned prior to birth with their appropriate motoneuron innervation and the small amount of cross-compartment innervation is refined by selective synapse elimination. Examination of prenatal events in the development of limb and masticatory muscle has provided evidence that nerve–muscle interactions do not play a role in the initial partitioning of muscle groups (Condon et al., 1990; Morris-Wiman and Widmer, 2001, 2003). Additionally, studies examining the neonatal development of LG (Donahue and English, 1987, 1989), extensor digitorum longus (Balice-Gordon and Thompson, 1988), gluteus maximus (English, 1990), tibialis anterior (Iliya and Dum, 1984), and diaphragm (Laskowski and High, 1989) support the conclusion that partitioned innervation to neuromuscular compartments is established at birth.

In studies using EMG recordings to assess responses to electrical stimulation of primary nerve branches in neonatal rat pups, evoked responses were mapped from as many as 60 sites in the four LG neuromuscular compartments (Donahue and English, 1987). Cross-compartmental potentials were rarely observed indicating specificity of the axons in each primary nerve branch. Axon specificity was further tested by electrically stimulating the proximal stump of each severed primary nerve branch and broadly recording from the LG muscle. No EMG potentials were detected after stimulation of each proximal end of the severed primary branch indicating that motor axons did not split prior to the nerve branches and that early postnatally each primary nerve branch contained unique motoneuron axons. To determine if increased synapse elimination in cross-compartment innervation compared to general synapse elimination played a role in the refinement of compartment innervation, unilateral tenotomy of the tendocalcaneus was performed in a group of P0 rat pups to delay synapse elimination and the percentage of polyneuronal innervation in LG was compared on the tenotomy and unoperated sides (Donahue and English, 1989). Cross-compartmental innervation was observed to be eliminated at an earlier time compared to polyneuronal innervation of the compartment. Interestingly, cross-compartmental innervation to LGm was found more frequently in the distal part of the compartment where no physical boundaries are present to separate the compartments (Bennett and Ho, 1988; Donahue and English, 1989). No cross-compartmental innervation was found after postnatal day 8. These findings support the prenatal patterning of compartments in the LG.

Diaphragm The existence in early postnatal rat pups (P0–P3) of a rostral–caudal organization (cervical roots

73

C3–C6) of the innervation of the rostral (sternal and upper costal region) and caudal (mid and lower costal region) diaphragm was demonstrated by recording synaptic potentials or muscle contraction in response to electrical stimulation of cervical ventral roots (Laskowski and High, 1989). Interestingly, the cervical root C3 was found to innervate rostral regions of the diaphragm in the neonate but not in adults. Also, early postnatally, 65% of the diaphragm muscle fibers were innervated by at least two cervical roots and this multiroot innervation was not observed in adults. This postnatal refinement of the C3–C6 rostral–caudal segmental innervation of diaphragm could involve two potential mechanisms (Laskowski and High, 1989): (1) synapse elimination which has been shown to continue through postnatal day 14 (Redfern, 1970); and (2) generation of new muscle fibers during secondary myogenesis which has been observed to continue through postnatal day 21 (Prakash et al., 1993). Although these studies did not investigate the specificity of compartment

Presumptive jaw muscle

innervation by primary nerve branches, nevertheless, the data supported a patterned segmental organization of the innervation of the diaphragm at birth that is subsequently refined early postnatally.

Masseter The three layers of the mouse masseter muscle, superficial, intermediate, and deep, form prenatally from distinct muscle masses (Fig. 5). Each of these anatomical layers contains multiple neuromuscular compartments in the adult mouse; however, no studies to date have examined the boundaries of these compartments or their development. Our lab has investigated the early postnatal development and the influence of the muscle nerve on the patterning of the masseter layers and the phenotype of muscle fibers in the respective layers. Early postnatally, a regionalization of fiber types based on MyHC phenotype can be observed in the mouse that parallels what

gd16 Temp

gd11 Vgang LPt

Vmotor root

Mand

Vgang Segmentation MPt LPt

gd16

Temp Massinter

Vgang Vmotor root

Massinter MPt

Masssup

Tongue Masssup

150 µm

Fig. 5. The developmental process of presumptive jaw muscle mass segmentation begins concomitant with motor root nerve branching at gd11. This mass eventually segments into specific jaw muscles and, in the mouse masseter, into anatomical layers. The right image is a frontal section through the jaw muscles (Temp, temporalis; Masssup, superficial masseter; Massinter, intermediate masseter; LPt, lateral pterygoid; MPt, medial pterygoid) at gd16 that was immunolabeled for desmin to identify the muscle masses. Other structures labeled for orientation include tongue and trigeminal ganglion (V-gang).

74

is observed in the adult masseter with MyHC type IIa distributed in the rostral (anterior) region, type IIb fibers distributed in the caudal (posterior) region, and presumed type IIx fibers located centrally (Widmer et al., 2002). Unilateral injection of b-bungarotoxin into the amnionic sac on gestational day (gd) 12 to eliminate sensory and motor innervation in mouse embryos did not affect the patterning of the masseter layers (Morris-Wiman and Widmer, 2001) and produced no change in the distribution of muscle fiber MyHC content (Morris-Wiman and Widmer, 2003). However, secondary myogenesis appeared to be affected; the volumes of individual masticatory muscle masses as well as the masseter muscle layers were significantly smaller. These data support the premise that neuromuscular compartments, as well as the phenotype of the muscle fibers within each compartment, are prepatterned in the masseter by birth.

Hormonal influences on compartment muscle phenotype Several studies over the past decades have examined the postnatal effects of androgen on muscle at the cellular and molecular levels. Most studies have focused on the effects of supraphysiological levels of testosterone exposure on sexually dimorphic muscles such as the pelvic floor muscles (levator ani), and these studies have provided valuable insight into the target sites of androgen action, the muscle fiber, and the motoneuron. More recent studies have examined anabolic effects of androgens on other muscles and have demonstrated that these effects vary among muscles, muscle fiber types, and experimental conditions. For example: in limb muscle, anabolic effects of androgens on muscle mass are augmented when combined with exercise; the anabolic effects of androgens on diaphragm involve alterations in motoneuron membrane properties, as well as muscle mass; and, in masticatory muscle, the effects of androgen exposure at puberty include alterations in muscle fiber phenotype.

Lateral gastrocnemius Limb muscles such as the LG show very modest responses to androgens either after castration or after supplementation. Castration of male rats or mice has been shown to cause a 10% reduction in gastrocnemius muscle mass after 9 or 11 weeks (Jiao et al., 2009) (Fig. 6). Supplementation of normal physiological levels of testosterone for 3 weeks after prolonged castration caused a modest increase in the muscle wet weight of triceps surae (medial and LG, soleus, and plantaris muscles) but had no effect on tibialis anterior in mice (Fig. 6). It has been suggested that testosterone supplementation must be accompanied by exercise in order to have an anabolic effect on limb muscle. However, endurance exercise combined with testosterone supplementation does not produce significant increases in muscle mass (Borst and Mulligan, 2007; Brown, 2008). Resistance exercise involving eccentric contractions has been shown to produce a modest increase in muscle mass in some studies (Latham et al., 2004; Petrella et al., 2006).

Diaphragm In previous studies, it was observed that after castration diaphragm wet weight of male rats did not change and fiber type proportions were not altered (Prezant et al., 1997). However, supraphysiological levels of testosterone have been shown to produce increases in cross-sectional area of specific fiber types, type IIx and IIb, in the diaphragm (Bisschop et al., 1997; Lewis et al., 2002; Prezant et al., 1997). Additionally, testosterone supplementation has been shown to improve neuromuscular transmission in male rats (Blanco et al., 2001). We have evaluated the effects of 11 weeks of castration and the subsequent addition of physiological levels of testosterone for 3 weeks on the diaphragm in male mice. Diaphragm wet weight increased 10% in response to testosterone after prolonged castration (Fig. 6). However, fiber type proportions within

Tibialis anterior

1.4

Normalized triceps surae wet weight

Normalized tibialis anterior wet weight

75

1.3 1.2 1.1 1.0

Triceps surae

1.4 1.3 1.2 1.1

*

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T + Finasteride

1.0 0.9

0.9 Control

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T + Finasteride

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Experimental group Diaphragm

Masseter

1.4 Normalized masseter wet weight

1.4 Normalized diaphragm wet weight

*

1.3

* 1.2

* 1.1 1.0 0.9

1.3

* *

*

1.2

*

1.1 1.0

0.9 Control

T

T + Finasteride

Experimental group

Control

T

T + Finasteride

Experimental group

Fig. 6. Androgen responsiveness of adult limb, diaphragm, and masseter muscles. CD-1 male mice at 11 weeks post-castration were supplemented for 3 weeks with testosterone (T) or testosterone and finasteride. Controls had no supplementation. Graphs represent wet weights of muscle from the supplementation/control groups normalized to corresponding wet weights from a parallel group of castrated animals. Significant differences in wet weights between experimental groups and castrated condition are indicated by an asterisk (*). The masseter muscle was the only one of the four muscles examined that had a significant reduction in wet weight after castration. Three muscles were found to significantly increase their wet weight after testosterone supplementation (triceps surae, diaphragm, and masseter). Only diaphragm had a significant reduction with testosterone and finasteride. Significant differences between experimental groups are indicated by a { symbol (p < 0.05).

each compartment (sternocostal and crural) were not changed (unpublished data). These data would indicate that testosterone at normal physiological levels does not preferentially affect muscle fibers of a particular phenotype as has been suggested by studies in other muscles. Exposure to finasteride, an inhibitor of the enzyme 5a-reductase responsible for converting testosterone to the more potent DHT, caused a significant decrease

in the testosterone-mediated increase in diaphragm wet weight (Fig. 6). Whereas muscle has little 5a-reductase, many motoneurons are enriched in this enzyme. Studies examining androgen's effects on the sexual dimorphic muscle levator ani have demonstrated that these effects are at least partially mediated through 5a-reductase conversion of testosterone within motoneurons. Our results with finasteride suggest that in diaphragm, the

76

motoneuron, as well as the muscle fiber, may be a target of testosterone action.

Masseter Sexual dimorphism has been reported for both muscle fiber phenotype and motoneuron properties in adult rodent, rabbit, and macaque masseter muscles. In all species examined, a higher proportion of faster MyHC fiber types has been identified in the adult male than in adult female masseter (Eason et al., 2000b; English et al., 1999b; Maxwell et al., 1979; Widmer et al., 2002). In the adult mouse, the male has a higher proportion of MyHC IIb fibers while the female mouse has a higher proportion of IIa fibers (Fig. 3) (Eason et al., 2000b; Widmer et al., 2002). Likewise, in the adult male rabbit masseter, 80% of the muscle fibers contain MyHC type IIa while this same phenotype is found in only 50% of the adult female masseter fibers. Interestingly, the proportion of MyHC type I and IIa fibers and their distribution in the female rabbit masseter are similar to that observed in young adult male rabbits (Eason et al., 2000a). The effect on the rabbit masseter muscle of endogenous androgen during puberty in males or androgen supplementation in castrated young males is to cause a change from a slower to faster MyHC phenotype (Reader et al., 2001). However, castration of adult older male rabbits did not alter the patterning of MyHC phenotype in the masseter, suggesting that this patterning once established is not affected by androgen levels. In male mice, the masseter muscle was observed to decrease by approximately 15% in wet weight 11 weeks after castration when compared to unmanipulated controls (Fig. 6). Testosterone supplementation at physiological levels for 3 weeks resulted in an average increase of 38% in masseter wet weight when compared to masseter from castrated animals. These data indicate that the male masseter response is much more robust than that observed for the triceps surae muscles or the diaphragm and

suggest an enhanced androgen sensitivity of the masseter muscle. The increase in masseter muscle weight was not accompanied by an alteration in the normal patterning of MyHC fiber phenotype, in agreement with what has been observed in adult male rabbit masseter. Median firing rates have been shown to be faster in adult male rabbit masseter motoneurons than in female, and the duration of motor unit activity is shorter in males (English and Widmer, 2003). These results suggest that androgens may mediate changes in masseter muscle fiber phenotype directly by action on the muscle fibers, indirectly by action on motoneurons, or both. The functional significance of sexual dimorphism in the masseter muscle may be related to the requirements of the jaw system for rapid, high force production such as defense or food gathering.

Summary Neuromuscular compartmentalization can be found in limb, respiratory, and masticatory muscles and these compartments serve to subdivide the muscle into functional output elements that can be activated singly or in combination with other compartments/muscles depending on the functional needs. While in lateral gastrocnemius primary nerve branches contain unique motor axons innervating each compartment, the diaphragm and masseter muscles have a more complex arrangement of compartments that are not represented by the primary nerve branches. Ia afferent reflex excitation of motoneurons innervating homonomous compartments over other compartments suggests that motor control mechanisms are specific at the compartment level in limb muscles, but more information is needed to evaluate if respiratory and masticatory muscle compartments have similar Ia afferent controls. Although the spatial organization of compartment motoneurons has substantial overlap with other compartment nuclei and is not as distinct as the muscle motoneuron pool, there is

77

a consistent general somatotopy found for limb, diaphragm, and masseter muscle compartments and this organization may have a role in the targeting of segmental and descending inputs. The formation of neuromuscular compartments in muscles associated with locomotion, respiration, and mastication appears to be based on a patterned developmental program that occurs prior to birth with refinement of innervation to compartments achieved by selective synapse elimination and potentially secondary myogenesis. The specific mechanisms by which appropriate innervation of compartments by their motoneurons is accomplished still remains to be determined, but may be similar to those active in whole muscle development. During puberty, androgens appear to influence the proportion of fibers with a fast MyHC phenotype in some muscles such as masseter, while causing hypertrophy of fast MyHC fiber types in other muscles such as the diaphragm. The preprogrammed formation of neuromuscular compartments involved in locomotion, respiration, and mastication, combined with hormonally induced maturational changes, establishes a repertoire of functional output elements to generate the biomechanical actions necessary to perform the varied tasks of each of these three motor systems. Due to the more complex organization of muscle into neuromuscular compartments, it would seem advantageous to record activity at the compartment level rather than a single muscle site (when appropriate) so that investigations of motor control mechanisms in these three motor systems might have a common anatomical substrate to better understand the selective recruitment of these output elements for specific tasks.

Acknowledgments This study was supported by the National Institutes of Health Grants DE12207, HD055286, and Florida Department of Health 07BB-11.

Abbreviations Cr EMG LG LG1 LG2 LG3 LGm MyHC SC1 SC2 SC3 SO

crural nerve branch of phrenic nerve electromyography lateral gastrocnemius LG compartment 1 LG compartment 2 LG compartment 3 medial LG compartment myosin heavy chain sternocostal nerve branch the phrenic nerve sternocostal nerve branch the phrenic nerve sternocostal nerve branch the phrenic nerve slow oxidative

the

1 of 2 of 3 of

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80 Widmer, C. G., Klugman, D., & English, A. W. (1997). Anatomical partitioning and nerve branching patterns in the adult rabbit masseter. Acta Anatomica (Basel), 159, 222–232. Widmer, C. G., Morris-Wiman, J. A., & Nekula, C. (2002). Spatial distribution of myosin heavy-chain isoforms in mouse masseter. Journal of Dental Research, 81, 33–38.

Windhorst, U., Hamm, T. M., & Stuart, D. G. (1989). On the function of muscle and reflex partitioning. The Behavioral and Brain Sciences, 12, 629–681. Young, R. L., Page, A. J., Cooper, N. J., Frisby, C. L., & Blackshaw, L. A. (2010). Sensory and motor innervation of the crural diaphragm by the vagus nerves. Gastroenterology, 138(3); 1091—1101.

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 6

Spinal interneurons providing input to the final common path during locomotion Robert M. Brownstone{,{,* and Tuan V. Bui{ {

{ Department of Surgery (Neurosurgery), Dalhousie University, Halifax, Nova Scotia, Canada Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada

Abstract: As the nexus between the nervous system and the skeletomuscular system, motoneurons effect all behavior. As such, motoneuron activity must be well regulated so as to generate appropriately timed and graded muscular contractions. Accordingly, motoneurons receive a large number of both excitatory and inhibitory synaptic inputs from various peripheral and central sources. Many of these synaptic contacts arise from spinal interneurons, some of which belong to spinal networks responsible for the generation of locomotor activity. Although the complete definition of these networks remains elusive, it is known that the neural machinery necessary to generate the basic rhythm and pattern of locomotion is contained within the spinal cord. One approach to gaining insights into spinal locomotor networks is to describe those spinal interneurons that directly control the activity of motoneurons, so-called last-order interneurons. In this chapter, we briefly survey the different populations of last-order interneurons that have been identified using anatomical, physiological, and genetic methodologies. We discuss the possible roles of these identified last-order interneurons in generating locomotor activity, and in the process, identify particular criteria that may be useful in identifying putative last-order interneurons belonging to spinal locomotor networks. Keywords: Spinal cord; Central pattern generators; Microcircuits; Motoneurons; Last-order interneurons.

for motor reflexes and muscle contraction (Creed et al., 1932). His work spawned generations of scientists investigating the neural control of movement (Stuart, 2005). The honorees of this symposium are several generations removed from Sherrington; their contributions to our understanding of the circuits underlying rhythmic movement are legion. There is no doubt that our current understanding of breathing, walking, and chewing can be attributed

Introduction A critical question in neuroscience is how neural circuits produce behavior. In the early twentieth century, Sherrington studied neural circuits responsible

* Corresponding author. Tel.: þ1-902-473-6850; Fax: þ1-902-473-6852 DOI: 10.1016/S0079-6123(10)87006-2

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in large measure to the efforts of Jack Feldman, Serge Rossignol, and James Lund. It is almost the centenary of the publication of Thomas Graham Brown's works demonstrating that the mammalian spinal cord has the intrinsic capacity to produce locomotor activity (Brown, 1911, 1914). Our understanding of the spinal circuits responsible for this behavior in terms of connectivity, neuronal intrinsic properties, and modulation has been steadily increasing (for review, see Goulding, 2009; Grillner, 2006; Kiehn, 2006). Although descending and sensory inputs are critical for normal locomotion, the spinal locomotor network, or “central pattern generator” (CPG), regulates the basic rhythm (or speed) and pattern (or coordination) of walking, as well as the degree of motoneuron output (leading to strength of muscle contraction) during locomotion. One strategy in defining the neural network underlying locomotion is to use a bottom-up approach in which the network is described by peeling back the layers starting from the neurons that generate the output of the circuit—motoneurons or “the final common path” (Creed et al., 1932). Hence, in this chapter, we will focus on last-order spinal interneurons—those neurons that project to and synapse directly with motoneurons and thus produce the motor output of locomotion. Many classes of spinal interneurons have been identified based upon anatomical and electrophysiological characteristics in the cat (Jankowska, 2008) and based upon genetic characteristics in the mouse (Goulding, 2009). Given the diversity of spinal interneurons and the difficulty in identifying them, it would be reasonable to ask the question: how would one know if any particular interneuron is directly involved in controlling motoneuron activity during locomotion? To answer this question, a list of criteria could be developed (cf. Brownstone and Wilson, 2008); one could then determine whether or not the criteria are fulfilled for any given neuronal population. Given that motoneurons receive alternating excitatory and inhibitory input during locomotion (Jordan, 1983; Perret, 1983), tentative

criteria lists for both excitatory (Table 1) and inhibitory (Table 2) last-order interneurons can be proposed. It is expected that these lists will be modified as further data regarding locomotor networks is obtained. It should also be recognized that each subset of these last-order interneurons (i.e., excitatory and inhibitory) likely comprises more than one population of neurons. Hence, a single population may not fulfill all criteria (particularly related to their inputs). In addition, it is now recognized that there are also last-order spinal modulatory inputs to motoneurons (Miles et al., 2007; Zagoraiou et al., 2009). These will be considered separately below. From the time of Sherrington to recent years, mammalian locomotor circuits have been explored largely in the cat, in which several populations of last-order neurons have been identified. In recent years, attention has turned to the mouse spinal cord. Advances in molecular biology have generated new tools to dissect, study, and manipulate these circuits, with one aim being to identify interneurons responsible for rhythmic motor output. In particular, these advances have led to the use of fluorescent proteins as markers of gene expression (Chalfie et al., 1994; Zacharias et al., 2000) and tools to activate or silence (Zhang et al., 2007) neurons; these tools will be important in determining whether neurons meet the criteria and are involved in locomotor behavior. Here, we will briefly review some last-order interneuronal populations in mammals that may be involved in the regulation of motoneuron activity during locomotion. It should be noted that even though many of these populations have been found to be rhythmically active during locomotion, none has been found to be critical for the production of motor neuron output during locomotion. The interneurons we will discuss are depicted in Fig. 1. Although the neurons defined in the cat seem to be distinct from those described in rodents, there has been recent progress to relate these functionally and molecularly defined populations. Note that we will not discuss the fundamental work done in invertebrates (see Marder

83 Table 1. Key criteria for last-order excitatory interneurons involved in locomotion Glutamatergic interneurons Located in the thoracolumbar spinal cord Project to and excite limb a-motoneurons At least two distinct populations, one providing excitation primarily to extensor and the other to flexor motoneurons These two populations reciprocally inhibit each other (see Jankowska et al., 1967) Those projecting to extensor motoneurons may receive low threshold primary afferent input (but see Weber et al., 2007) Receive direct reticulospinal input (Noga et al., 2003; Shefchyk and Jordan, 1985) Rhythmically active during locomotion (Kiehn et al., 2008) Their silencing leads to a reduction in output during locomotion Their selective activation may lead to stepping

Table 2. Key criteria for last-order inhibitory interneurons involved in locomotion Glycinergic and/or GABAergic interneurons Located in the thoracolumbar spinal cord Project to and inhibit limb a-motoneurons At least two distinct populations, one providing inhibition primarily to extensor and the other to flexor motoneurons Likely receive input from last-order excitatory interneurons Receive disynaptic reticulospinal input, likely mediated via last-order excitatory interneurons (Noga et al., 2003; Shefchyk and Jordan, 1985) Rhythmically active during locomotion (Kiehn et al., 2008) Their silencing leads to some overlap of flexor and extensor output during locomotion as well as prolongation of motoneuron bursts

and Bucher, 2007 for review) or lower vertebrates (see Grillner et al., 2008 for review), nor will we discuss the experimental approaches to studying these neurons in humans (see Hultborn and Nielsen, 2007).

Last-order inhibitory interneurons Renshaw cells The first two sources of inhibition to motoneurons to be described were Renshaw cells (RCs) (Eccles et al., 1954; Renshaw, 1941, 1946) and Ia

inhibitory interneurons (IaINs) (Jankowska and Roberts, 1972). RCs are responsible for “recurrent inhibition”—they receive inputs from a-motoneuron axon collaterals (Eccles et al., 1954; Lamotte d'Incamps and Ascher, 2008), and in turn monosynaptically inhibit a-motoneurons (as well as gs) through glycinergic/GABAergic synapses (Geiman et al., 2002; Schneider and Fyffe, 1992); for review, see (Alvarez and Fyffe, 2007) and (Hultborn, 2006). Recurrent inhibition is topographically organized, with strongest effects in nearby motoneurons, in both homonymous and synergist motor pools (McCurdy and Hamm, 1994; Trank et al., 1999). They project to other neurons as well, including ventral spinocerebellar tract neurons (Windhorst, 1996) and IaINs (Hultborn et al., 1971) as well as other RCs (Ryall, 1970). There is no evidence of direct reticulospinal input to RCs (Engberg et al., 1968), which receive sparse monoaminergic inputs from brain stem nuclei (Carr et al., 1999). While they receive primary afferent input early in development (Mentis et al., 2006; Naka, 1964), this becomes nonfunctional in the adult (Mentis et al., 2006). The precise functional role of this recurrent inhibition to motoneurons is still not clear. Several studies have raised various suggestions for their role (Alvarez and Fyffe, 2007; Windhorst, 1996), including: they may reverse the size–order recruitment of motoneurons (Friedman et al., 1981); they may change the “gain” of motor pools (Hultborn et al., 2004; Hultborn and PierrotDeseilligny, 1979); they may play a role in decorrelation of motoneuron firing (Maltenfort et al., 1998); and/or they may reduce the amplification of motoneuron inputs mediated by persistent inward currents (Bui et al., 2008; Hultborn et al., 2003; Kuo et al., 2003). Although RCs are rhythmically active during locomotion (McCrea et al., 1980; Nishimaru et al., 2006; Pratt and Jordan, 1987), their role during locomotion is not clear; however, they are not involved in the generation of the rhythm (Pratt and Jordan, 1987). They are believed to receive rhythmic excitatory and inhibitory synaptic input

Mid-lumbar group II inhibitory IN

Vestibulospinal

Reticulospinal

MIDLINE

Corticospinal

Reticulospinal

Rubrospinal

Vestibulospinal

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V2b

V1 EphA4

Group II afferent

Group II excitatory IN

V2a

Lamina V/VI GAD65 Commissural inhibitory IN

Antagonist Group Ia afferent

Ia inhibitory IN Commissural excitatory IN

Group Ia afferent

Motoneuron

Nonreciprocal inhibitory IN

V0 Group Ib afferent Group I excitatory IN

Renshaw cell Cutaneous afferent

Cutaneous excitatory IN

V3 V0C

Fig. 1. Diagram depicting last-order spinal interneurons and some of their known excitatory inputs from descending and primary afferent pathways. It is likely that each population receives inputs from many more types of afferents than those depicted. Filled circles represent classes of neurons identified using molecular biological approaches. Unfilled circles represent classes of neurons identified through anatomical and electrophysiological techniques. These are not mutually exclusive: for example, Renshaw cells and Ia inhibitory interneurons belong to the V1 class of spinal interneurons (indicated by yellow text) and some excitatory interneurons expressing EphA4 belong to the V2a class of interneurons. Note that not all neurons of each class are depicted; for example, some EphA4þ neurons may be last-order inhibitory interneurons and some V0 interneurons are excitatory. Red designates excitatory neurons and boutons, blue designates inhibitory neurons and boutons, and green designates neuromodulatory neurons and boutons. Solid lines represent monosynaptic inputs, and dashed lines represent supraspinal descending inputs that are either mono- or oligosynaptic. The vertical line with large dashes represents the midline. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

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from the CPG (Nishimaru et al. 2010), possibly via commissural interneurons (Nishimaru et al., 2006). Elimination of RC activity by application of the cholinergic antagonist mecamylamine during locomotion leads to an increase in MN firing rate (Noga et al., 1987), suggesting that RCs may play a role in reducing MN firing during locomotion. However, there is no loss of interburst membrane hyperpolarization of motoneurons, nor an increased burst duration in motoneurons (Noga et al., 1987). There is also no evidence of any change in the fidelity of flexor–extensor alternation following RC block. Therefore, while they fulfill several of the criteria that we have listed as belonging to last-order interneurons involved in locomotion, and may play a role in modulating the frequency of MN spike trains, they do not appear to be involved in the termination of rhythmic motoneuron bursts.

Ia inhibitory interneurons IaINs are responsible for “reciprocal inhibition.” That is, they are monosynaptically excited by spindle primary (Ia) afferents and project to and inhibit antagonist motor pools (Eccles and Lundberg, 1958). In addition, they inhibit IaINs that receive Ia afferent inputs from antagonist muscles (Hultborn et al., 1976a). Despite their nomenclature, inputs to IaINs are not restricted to Ia afferents—they are excited, for example, by flexor reflex afferents and cutaneous afferents (Hultborn et al., 1976b) and inhibited by RCs (Hultborn et al., 1971). Descending inputs to IaINs include monosynaptic connections from the ipsilateral vestibulospinal tract and disynaptic inputs from motor cortex and the red nucleus (Hultborn et al., 1976c). A logical role for IaINs would be that they could ensure appropriate alternation of flexor and extensor activity during locomotion. They meet the majority of the criteria laid out in Table 2 wherein they receive primary afferent input, are glycinergic, project to motoneurons, and are

rhythmically active during locomotion (Feldman and Orlovsky, 1975; McCrea et al., 1980; Pratt and Jordan, 1987). In fact, the latter study observed that IaIN activity preceded that of the associated motoneurons, suggesting that they receive an excitatory drive from CPG (these experiments were performed while the cats were paralyzed, thus the excitatory drive could not come from primary afferent inputs). Their ability to ensure proper alternation between flexors and extensors may be enhanced by their ability to reduce motoneuron excitability by inhibiting persistent inward currents (Hyngstrom et al., 2007; Kuo et al., 2003) or by “deselecting” specific motoneurons by reducing their PICs (Heckman et al., 2008; Hyngstrom et al., 2008). Interestingly, systemic administration of strychnine, a glycine receptor antagonist, significantly reduced interburst inhibition in motoneurons but did not seem to extend motoneuron burst duration during fictive locomotion evoked by brainstem stimulation in the decerebrate cat (Pratt and Jordan, 1987). The relationship between flexor and extensor activity was not studied in this preparation, but in young rat and mouse in vitro spinal cord preparations, strychnine typically leads to coactivity in flexor and extensor MNs (Cowley and Schmidt, 1995; Jiang et al., 1999). Taken together, these data suggest that IaINs are involved in rhythmic inhibition of motoneurons during locomotion, but that there may be additional mechanisms (possibly GABA-mediated) involved in terminating motoneuron bursts.

Nonreciprocal inhibitory INs In contrast to reciprocal inhibition, Laporte and Lloyd (1952) reported inhibition of synergist motoneurons when the strength of group I stimulation was increased slightly. This “nonreciprocal inhibition” was felt to be mediated largely by sensory afferents from group Ib fibers which originate in Golgi tendon organs. However, since many interneurons mediating nonreciprocal

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inhibition also receive input from Ia afferents, this reflex pathway is often termed nonreciprocal group I inhibition (Jankowska et al., 1981). Nonreciprocal inhibition is mediated by glycinergic interneurons in laminae V and VI (Brink et al., 1983; Jankowska, 1992; Bannatyne et al., 2009). The organization of Ib inhibition has been shown to be divergent, with the effects of Ib afferent from one muscle being distributed to different muscles through subsets of interneurons for each target motor pool (Jankowska, 1992). Ib inhibitory interneurons have been observed to be mutually inhibitory (Brink et al., 1983). A number of descending inputs to these interneurons include excitatory input from ipsilateral corticospinal and rubrospinal neurons and inhibitory input from reticulospinal inputs (Jankowska, 1992). In a recent study, monosynaptic excitation from the medial longitudinal fascicle (MLF) was demonstrated in ipsilaterally projecting, inhibitory interneurons receiving inputs from group I afferents and located in intermediate laminae (Bannatyne et al., 2009). Given the mixed descending excitatory and inhibitory inputs to these neurons, their contribution, if any, to motoneuron inhibition during locomotion is not clear. However, in keeping with disynaptic group I inhibition being replaced by excitation in extensor motoneurons during fictive locomotion (Gossard et al., 1994; vide infra), recordings from two group I nonreciprocal inhibitory interneurons in the cat revealed that their activity was depressed during fictive locomotion (Angel et al., 2005). Whether the population of these neurons is inhibited during locomotion is not yet clear (see Wilson et al., 2010).

Other last-order inhibitory interneurons identified in the cat Other last-order inhibitory neurons have been identified, including group II interneurons (receiving input from muscle spindle secondaries; Bannatyne et al., 2009), and commissural

inhibitory INs (Arya et al., 1991; Jankowska et al., 2003). Both of these populations receive inputs from reticulospinal pathways (Jankowska et al., 2003; Bannatyne et al., 2009). The possible involvement of inhibitory group II interneurons in locomotion will be treated concurrently with a discussion of excitatory group II interneurons (vide infra, section “Mid-Lumbar Group II Interneurons”).

V1 interneurons Understanding the development of the mouse spinal cord has led to the ability to genetically define populations of spinal interneurons. Interneurons of the ventral spinal cord are largely derived from four progenitor domains (p0–p3), resulting in four classes of postmitotic neurons (V0–V3) (Jessell, 2000). Three of these classes are known to include interneurons that send inhibitory inputs to motoneurons: V0, V1, and V2b. V1 interneurons are specified by the expression of the transcription factor engrailed-1, and include RCs, IaINs, and other yet-to-be-defined inhibitory neurons (Alvarez et al., 2005; Sapir et al., 2004). Recordings of these neurons (other than RCs) in the mouse during locomotor activity have not been reported. Genetic ablation or acute silencing of V1 interneurons reduces locomotor speed (Gosgnach et al., 2006). Perhaps somewhat surprisingly, however, motoneurons still received rhythmic interburst inhibitory input (Gosgnach et al., 2006), suggesting that IaINs (and RCs and the other V1 INs) are not solely responsible for rhythmic inhibition of MNs. Another possible explanation would be that IaINs are not exclusively derived from the V1 population (Wang et al., 2008). This finding illustrates that the physiological and molecular definitions of spinal neurons may not be 100% concordant, and combining data from these different yet complementary paradigms will be helpful in the elucidation of spinal circuits.

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V0 interneurons The homeobox gene Dbx1 controls the specification of a class of commissural interneurons (V0 interneurons), 70% of which are inhibitory and 30% of which are excitatory (Lanuza et al., 2004) (vide infra section “Spinal Modulatory Neurons”). The ventral component of this class (V0V) is composed of commissural interneurons located in lamina VIII. Injection of GFPexpressing pseudorabies virus into hindlimb muscles allowed for mapping of synaptic inputs to motoneurons and demonstrated that 30% of commissural neurons were Dbx1 positive. Therefore, some V0 interneurons project to and synapse directly onto contralateral motoneurons. Postsynaptic inhibition of motoneurons in response to commissural interneuron activation has been demonstrated; whether or not these were V0 interneurons is not known (Quinlan and Kiehn, 2007). V0 neurons have been shown to be rhythmically active during locomotion (Dyck and Gosgnach, 2009). Although ablation of Dbx1 neurons leads to impairments in left–right alternation in neonatal spinal cord preparations (Lanuza et al., 2004), rhythmic activity persists. It is not known whether rhythmic inhibition of motoneurons persists, or whether they simply receive rhythmic excitation in these mutants. Of note, there is no apparent change in the flexor–extensor fidelity or motor burst duration when V0 interneurons are eliminated, suggesting that they are not critical for rhythmic inhibition of motoneurons. Thus, the role of V0 INs in regulating motoneuron output during locomotion remains unknown.

V2b interneurons V2b interneurons are derived from the Lhx3expressing V2 population, and defined by their expression of GATA2/3 (Zhou et al., 2000). It has recently been shown that these neurons express either GABA or glycine, and

project to motoneurons (Al-Mosawie et al., 2007; Joshi et al., 2009; Lundfald et al., 2007). These neurons have not been studied during locomotion.

Medial lamina V/VI GABAergic neurons We have recently described a population of GABAergic neurons in medial laminae V/VI (Wilson et al., 2010). These neurons receive low threshold primary afferent input, and seem to have diverse projections, including to motoneurons. Using 2-photon excitation calcium imaging (Wilson et al., 2007), we demonstrated that they are rhythmically active during locomotion, leading to the suggestion that they may be involved in motoneuronal burst termination. A clearer definition of their role in locomotion awaits future studies in which, for example, these neurons are silenced.

Last-order excitatory interneurons Group I excitatory interneurons Similar to the identification of inhibitory spinal interneurons in the cat, excitatory spinal interneurons have been identified primarily based on their connectivity. The best described source of sensory-derived motoneuron excitation is the direct monosynaptic Ia afferent excitation of motoneurons originating from muscle spindles responsive to muscle stretch. Whether Ia axons, which are rhythmically depolarized during fictive locomotion (Duenas and Rudomin, 1988; Gossard, 1996), contribute to motoneuron excitation is not known. Interestingly, spikes produced by primary afferent depolarization may contribute to rhythmic motoneuron excitation during fictive mastication (Westberg et al., 2000). In the anesthetized cat, group Ib afferents mediate nonreciprocal inhibition of homonymous

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and synergist motoneurons (vide supra). However, during locomotion in acutely spinalized cats, stimulation of Ib afferents leads to excitation rather than inhibition of extensor motoneurons (Gossard et al., 1994). In the presence of active descending systems, this excitation is mediated by a disynaptic excitatory pathway (McCrea et al., 1995). These excitatory responses to Ib stimulation recorded in motoneurons are phase modulated, indicating that the interneurons mediating this reflex pathway are rhythmically active. Indeed, candidate interneurons have been identified (Angel et al., 2005), and are rhythmically active during locomotion. Evidence has been presented demonstrating convergence of mesencephalic locomotor region (MLR)evoked and Ib excitatory postsynaptic potentials in motoneurons (Brownstone et al., 1992b), and group I excitatory interneurons receive reticulospinal input (Bannatyne et al., 2009). Taken together, these data point toward this population of interneurons as a potential source of rhythmic excitation of motoneurons during locomotion (see Table 1).

Mid-lumbar group II interneurons Although other sensory afferents such as group II primary afferents from secondary endings of muscle spindles can contact motoneurons directly (Lundberg et al., 1977; Luscher et al., 1979; Stauffer et al., 1976), their main influence on motoneuron activity is mediated via ipsilaterally projecting interneurons located in laminae VI and VII (Bannatyne et al., 2009; Edgley and Jankowska, 1987; Lundberg et al., 1987; Riddell and Hadian, 2000). These neurons described in the cat can be divided into excitatory and inhibitory populations, are located in mid-lumbar segments, and have descending (Cavallari et al., 1987) and/or ascending (Riddell and Hadian, 2000) projections of several segments. Most midlumbar group II interneurons receive monosynaptic input from descending inputs, including

rubrospinal, vestibulospinal, corticospinal, or reticulospinal neurons (Davies and Edgley, 1994). In addition, they have been shown to receive excitatory inputs from the MLR which are mediated via reticulospinal pathways (Edgley et al., 1988) suggesting a possible role in locomotion. In fact, some mid-lumbar group II interneurons are rhythmically active during fictive locomotion (Shefchyk et al., 1990). However, DOPA administration, which can be used to elicit locomotor activity, depresses these neurons (Edgley et al., 1988), and group II field potentials are depressed during MLR-evoked locomotion (Perreault et al., 1999), suggesting that their role in locomotion may be limited.

Excitatory interneurons mediating cutaneous inputs Excitation from last-order interneurons related to cutaneous sensory afferents has also been described. Activation of hindlimb cutaneous afferents in the cat can generate short-latency EPSPs estimated to be disynaptic in nature (Moschovakis et al., 1991; LaBella et al., 1992). The strength of these EPSPs is modulated in different phases of MLR-evoked locomotion, suggesting that these interneurons receive phasic inputs from locomotor networks during locomotion (Burke et al., 2001, see, however, Gossard et al., 1989 regarding presynaptic inhibition of cutaneous afferents during locomotion). This may translate to gating of behavioral responses to cutaneous stimulation during the step cycle. For example, contact of the paw dorsum with an object can elicit two stereotyped patterns of hindlimb muscle response depending on whether contact was made during the swing or stance phase (Quevedo et al., 2005). There is also evidence that interneurons mediating excitation from cutaneous afferents also receive inputs from muscle afferents, suggesting that some of these interneurons play a multimodal integrative role (Perrier et al., 2000). Whether these interneurons

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are important in mediating rhythmic motoneuron excitation during locomotion remains to be determined.

Excitatory commissural interneurons Excitatory inputs to motoneurons from commissural interneurons have also been demonstrated in the cat. These neurons receive inputs from the reticular formation and vestibular nuclei (Jankowska et al., 2003, 2009; Krutki et al., 2003). Commissural interneurons receiving reticulospinal inputs (their excitatory or inhibitory nature was not identified) have been observed to be active during fictive locomotion in the cat (Matsuyama et al., 2004). In the mouse, last-order commissural interneurons have also been identified (Quinlan and Kiehn, 2007). These neurons may be rhythmically active during locomotion (Zhong et al., 2006). Whether commissural input is a major source of motoneuron excitation during locomotion is questionable given that motoneuron activity is robust in hemisected spinal cords (see Cowley et al., 2009).

V2a interneurons V2a interneurons, which are defined by the expression of the homeobox gene Lhx3 and further specified by the homeodomain proteins chx10 and sox14, send ipsilateral glutamatergic connections to motoneurons (Al-Mosawie et al., 2007; Lundfald et al., 2007). These neurons are located in lamina VII (Al-Mosawie et al., 2007). Ablation of these cells using transgenic methods leads to deficits in right–left coordination of locomotion (Crone et al., 2008). These deficits are apparent at higher locomotor speeds, evidenced by a switch from normal running to an abnormal hopping pattern (Crone et al., 2009). Interestingly, motoneurons are still rhythmically active when these neurons are eliminated, indicating that they are not the sole source of rhythmic excitation of motoneurons during locomotion.

V3 interneurons Another class of ventral interneurons that has been shown to send glutamatergic inputs to motoneurons is the V3 class of interneurons as defined by the expression of Sim1 (Zhang et al., 2008). Unlike the V2a class of interneurons, V3 interneurons project primarily to contralateral motoneurons. Similar to the V2a class, however, ablation of the V3 class compromises the robustness of the locomotor rhythm in the isolated mouse spinal cord (Zhang et al., 2008). It is not known whether these neurons are rhythmically active during locomotion. However, there is no obvious change in the amplitude of motoneuron output, indicating that V3 interneurons do not provide a critical source of rhythmic excitation of motoneurons during locomotion.

Spinal modulatory neurons During locomotion, the postspike after hyperpolarization is modulated in spinal motoneurons (Brownstone et al., 1992a). We have recently demonstrated that this modulation can be produced by activity in neurons supplying the prominent cholinergic C-boutons synapsing on motoneuronal somata (Miles et al., 2007). The neurons supplying these C-boutons are the medial partition neurons (Miles et al., 2007; named by Barber et al., 1984). These neurons, identified by expression of the transcription factor Pitx2, have now been shown to derive from the V0 domain and hence are called V0C (for cholinergic) neurons (Zagoraiou et al., 2009). V0C neurons do not receive direct primary afferent input. They are rhythmically active during locomotion, indicating that they receive input from spinal locomotor networks. Silencing the output of these neurons through elimination of the synthetic enzyme for acetylcholine (choline acetyl transferase) limits the increase in motoneuron output necessary for some locomotor tasks (Zagoraiou et al., 2009). These data indicate that

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the spinal cord not only provides rhythmic excitatory and inhibitory input to motoneurons, but also provides rhythmic modulatory input in a motor task-specific manner.

Concluding remarks In this chapter, we have briefly outlined some last-order interneurons identified in the cat and mouse. While the adult cat preparation has been very useful for the physiological identification of interneurons and the mouse has been useful for molecular identification and manipulation, it is the combination of these approaches, physiological and molecular, that will lead to our understanding of the spinal control of movement. It is likely that there is significant redundancy in these circuits. This is demonstrated by the genetic manipulations which eliminate broad classes of interneurons (V0, V1, V2a, V3). None of these deletions lead to the elimination of rhythmic motoneuron excitation, and it is not clear if any eliminates rhythmic inhibition. That is, there is redundancy (perhaps physiologically similar interneurons derive from different domains; see Wang et al., 2008), and/or the input is derived from dorsal interneurons (e.g., Wilson et al., 2010). However, the spinal neurons responsible for AHP modulation seem to be discretely defined both physiologically and molecularly (Zagoraiou et al., 2009). The understanding of neural circuits in relation to behavior is a key area of investigation in neuroscience today. The contributions of Feldman, Rossignol, and Lund form a solid foundation on which to build this understanding. Using a specific motor output (e.g., breathing, walking, or chewing) as a direct “read-out” of the behavior enables the direct correlation of neural circuit activity and behavior. The combination of new developments in electrophysiology (e.g., adult mouse spinal cord recordings; Manuel et al., 2009), developmental biology (e.g., transcription factors for definition of unique neuronal

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mesencephalic locomotor region. Journal of Neurophysiology, 53, 1345–1355. Shefchyk, S., Mccrea, D., Kriellaars, D., Fortier, P., & Jordan, L. (1990). Activity of interneurons within the L4 spinal segment of the cat during brainstem-evoked fictive locomotion. Experimental Brain Research, 80, 290–295. Stauffer, E. K., Watt, D. G., Taylor, A., Reinking, R. M., & Stuart, D. G. (1976). Analysis of muscle receptor connections by spike-triggered averaging. 2. Spindle group II afferents. Journal of Neurophysiology, 39, 1393–1402. Stuart, D. G. (2005). Integration of posture and movement: Contributions of Sherrington, Hess, and Bernstein. Human Movement Science, 24, 621–643. Trank, T. V., Turkin, V. V., & Hamm, T. M. (1999). Organization of recurrent inhibition and facilitation in motoneuron pools innervating dorsiflexors of the cat hindlimb. Experimental Brain Research, 125, 344–352. Wang, Z., Li, L., Goulding, M., & Frank, E. (2008). Early postnatal development of reciprocal Ia inhibition in the murine spinal cord. Journal of Neurophysiology, 100, 185–196. Weber, I., Puskar, Z., Kozak, N., & Antal, M. (2007). Projections of primary afferent fibers to last-order premotor interneurons in the lumbar spinal cord of rats. Brain Research Bulletin, 71, 337–343. Westberg, K. G., Kolta, A., Clavelou, P., Sandstrom, G., & Lund, J. P. (2000). Evidence for functional compartmentalization of trigeminal muscle spindle afferents during fictive mastication in the rabbit. The European Journal of Neuroscience, 12, 1145–1154. Wilson, J. M., Blagovechtchenski, E., & Brownstone, R. M. (2010). Genetically defined inhibitory neurons in the mouse spinal cord dorsal horn: A possible source of rhythmic inhibition of motoneurons during fictive locomotion. The Journal of Neuroscience, 30, 1137–1148. Wilson, J. M., Dombeck, D. A., Diaz-Rios, M., HarrisWarrick, R. M., & Brownstone, R. M. (2007). Two-photon calcium imaging of network activity in XFP-expressing neurons in the mouse. Journal of Neurophysiology, 97, 3118–3125. Windhorst, U. (1996). On the role of recurrent inhibitory feedback in motor control. Progress in Neurobiology, 49, 517–587. Zacharias, D. A., Baird, G. S., & Tsien, R. Y. (2000). Recent advances in technology for measuring and manipulating cell signals. Current Opinion in Neurobiology, 10, 416–421. Zagoraiou, L., Akay, T., Martin, J. F., Brownstone, R. M., Jessell, T. M., & Miles, G. B. (2009). A cluster of cholinergic premotor interneurons modulates mouse locomotor activity. Neuron, 64, 645–662. Zhang, F., Aravanis, A. M., Adamantidis, A., De Lecea, L., & Deisseroth, K. (2007). Circuit-breakers: Optical technologies for probing neural signals and systems. Nature Reviews. Neuroscience, 8, 577–581.

95 Zhang, Y., Narayan, S., Geiman, E., Lanuza, G. M., Velasquez, T., Shanks, B., et al. (2008). V3 spinal neurons establish a robust and balanced locomotor rhythm during walking. Neuron, 60, 84–96. Zhong, G., Diaz-Rios, M., & Harris-Warrick, R. M. (2006). Intrinsic and functional differences among commissural

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 7

Beyond connectivity of locomotor circuitry—ionic and modulatory mechanisms Abdeljabbar El Manira*, Alexandros Kyriakatos and Evanthia Nanou Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

Abstract: Discrete neural networks in the central nervous system generate the repertoire of motor behavior necessary for animal survival. The final motor output of these networks is the result of the anatomical connectivity between the individual neurons and also their biophysical properties as well as the dynamics of their synaptic transmission. To illustrate how this processing takes place to produce coordinated motor activity, we have summarized some of the results available from the lamprey spinal locomotor network. The detailed knowledge available in this model system on the organization of the network together with the properties of the constituent neurons and the modulatory systems allows us to determine how the impact of specific ion channels and receptors is translated to the global activity of the locomotor circuitry. Understanding the logic of the neuronal and synaptic processing within the locomotor network will provide information about not only their normal operation but also how they react to disruption such as injuries or trauma. Keywords: Modulation; Synaptic transmission; Ion channels; Plasticity; Endocannabinoids; Spinal cord; Motor behavior.

Introduction

at birth such as those controlling respiration (Feldman et al., 2003). Others undergo changes to adapt to the behavioral needs such as those controlling feeding behavior that switches from sucking in early life to chewing later on (Kolta et al., 2007; Lund and Kolta, 2006). And finally those networks controlling locomotion are operational at birth, but in some species their output is not fully shaped/tuned because of the absence of adequate postural control (Clarac et al., 2004; Grillner, 1975, 2003; Kiehn, 2006; Rossignol et al.,

Motor behavior represents the final output of multiple processing stages in the central nervous system that allows the animal to interact with the environment. Discrete neural networks govern specific motor behaviors. Some networks underlie vital functions and are therefore fully functional * Corresponding author. Tel.: þ46-8-524-86911; Fax: þ46-8-349544. DOI: 10.1016/S0079-6123(10)87007-4

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2006; Vinay et al., 2005). All these networks comprise discrete groups of neurons that develop and form appropriate connections in response to specific molecular cues. These molecular cues have begun to be elucidated in the circuits controlling locomotion and respiration (Goulding and Pfaff, 2005; Goulding, 2009; Gray, 2008; Jessell, 2000). The advent of genetic manipulations has opened up a novel era of circuit analysis for motor behaviors since the importance of specific classes of neurons in generating motor behaviors can be assessed using fluorescent markers, optogenetics, and cell-specific interference (El Manira and Grillner, 2008; Fetcho et al., 2008; Gabriel et al., 2008, 2009; Grillner and Jessell, 2009; Kiehn, 2006; McLean and Fetcho, 2008). It also facilitates the comparison of organization principles between different animal models. The basic locomotor functions are governed by local networks residing in the spinal cord that work in close interaction with sensory feedback to produce optimal motor outputs that propel animals in space. Although they can operate autonomously, these networks require inputs from descending centers that provide a “go” signal to initiate locomotor behavior (Drew et al., 2004; Dubuc et al., 2008; Jordan et al., 2008). Exactly how this signal is transformed into rhythmic motor programs is still not entirely clear. A salient feature of locomotor networks is the alternation between phasic excitation and inhibition that ensures the alternating activity of antagonistic muscles. Accessible model systems such as the lamprey and Xenopus tadpole have been instrumental in providing a framework of the organization of locomotor networks because they enabled characterization of some of the key component neurons and their connectivity (Buchanan, 2001; Grillner, 2003; Roberts et al., 2008; Sillar et al., 2008). Genetically accessible systems such as zebrafish and mouse now help to broaden the analysis of spinal networks by taking into account not only the transmitter phenotype of neurons but also their molecular identity (Brownstone and Wilson, 2008; Fetcho et al.,

2008; Goulding, 2009; Grillner and Jessell, 2009; Kiehn, 2006). The locomotor pattern is not simply the result of a hardwired network of neurons with fast ionotropic synaptic transmission, but it is also the consequence of the intrinsic properties of the constituent neurons and the dynamics of their synaptic integration that can lead to short- and long-term plasticity of the global activity of the network (El Manira and Kyriakatos, 2010; El Manira et al., 2008; Nistri et al., 2006; Sillar et al., 2008; Thorn Perez et al., 2009). It is therefore necessary to characterize the biophysical properties of interneurons and motoneurons as well as the synaptic plasticity resulting from repetitive activity or intrinsic modulation. The wealth of information available on the organization of the lamprey locomotor network has allowed us to undertake a detailed characterization of the biophysical properties of specific ion channels and their role in shaping the activity of network neurons. In addition, the role of glutamate as an intrinsic modulator via activation of metabotropic receptors and their interactions with endocannabinoids has also been clarified. In this chapter, we will summarize some of these results and discuss their general impact for the operation of the spinal locomotor networks. Unit burst generation in the spinal cord One of the important questions that have been possible to resolve using the experimental accessibility of the lamprey spinal cord is whether the locomotor network is organized as half-center or as unit burst generators. In other words, is reciprocal inhibition indispensable for the rhythmic activity generated by the spinal network? The answer to this question is that the rhythmicity is inherent to networks in each hemi-part of the spinal cord and that this rhythmicity is maintained after blockade of glycine receptors with strychnine (Cohen and Harris-Warrick, 1984). Furthermore, each hemi-segment is able to generate long-lasting rhythmic activity in response to a

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brief stimulation (Cangiano and Grillner, 2003, 2005). These results led to the conclusion that each hemi-segment represents a unit burst generator consisting of a network of excitatory interneurons with mutual synaptic interactions (Cangiano and Grillner, 2005). Paired recordings from excitatory neurons in the hindbrain of Xenopus tadpoles have demonstrated the existence of mutual excitation that is sufficient to produce rhythmic activity (Li et al., 2006). While the inhibition is dispensable for the generation of the basic rhythm, it plays a pivotal role in producing the alternating pattern between the two sides of the spinal cord. This organization bears some similarities with that of the respiratory network in the brainstem where the inspiratory motor pattern generated in the pre-Bötzinger complex persists in the absence of inhibition and is thus thought to be the result of an excitatory synaptic interaction together with inherent pacemaker properties (Feldman et al., 2003; Ramirez et al., 2004; Smith et al., 2009). Organization of the locomotor network The excitatory drive in the spinal network is mediated by ipsilateral glutamatergic interneurons

while inhibitory glycinergic interneurons ensure the left–right alternation of the motor pattern (Buchanan, 2001; Grillner, 2003). The excitatory interneurons excite each other and drive the activity of ipsilateral motoneurons leading to muscle contraction on the same side of the body (Fig. 1). At the same time, these excitatory interneurons also activate glycinergic inhibitory neurons, which project to the contralateral side and mediate reciprocal inhibition (Fig. 1; Mahmood et al., 2009). This ensures the alternating pattern of activity between the two sides of the body that is characteristic for locomotor behavior. During forward swimming, the locomotor bursts propagate from the rostral to the caudal segments of the spinal cord with a frequency dependent delay to allow the undulation of the body necessary to propel the animal in the water. Finally, in the intact spinal cord stretch receptors, edge cells, located in the lateral margins of the spinal cord are activated by the tension resulting from the contraction of the muscles of the opposite side of the body. These sensory neurons are of two types: one is excitatory, projecting ipsilaterally, and the other is inhibitory, projecting to the contralateral side (Viana di Prisco et al., 1990). Once activated these sensory neurons excite network neurons on the same side and inhibit those on the opposite side. This

Excitatory interneurons Inhibitory commissural interneurons Motoneurons Fig. 1. Organization of the spinal locomotor circuitry. Locomotor activity is generated by segmental networks consisting of ipsilateral glutamatergic interneurons (red) driving the activity of motoneurons (green), which project their axons through ventral roots. The left–right alternation is ensured by commissural glycinergic inhibitory interneurons (blue). The activity is propagated from rostral to caudal segments with a constant phase-lag to produce the waveform underlying swimming in the intact animal. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this chapter.)

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movement-related sensory input provides a negative feedback to the locomotor network and contributes to the transition of the activity from one side of the body to the other. The spinal network organization imposes that the locomotor cycle is composed of an on-cycle excitation alternating with mid-cycle inhibition. The balance between the excitatory and inhibitory phase determines the cycle duration and consequently the frequency of the swimming in the intact animal. Therefore the final locomotor output is not the result only of the connectivity but also of the mechanisms that change the duration and strength of the excitatory and inhibitory phase of the cycle. In the remainder of this chapter, we will briefly summarize some of the biophysical properties of the network neurons and mechanisms of modulation of synaptic transmission that play a role in forming the locomotor behavior.

channels plays a major role in mediating synaptic transmission from presynaptic axons (Fig. 2; Krieger et al., 1999) and activation of KCa channels in postsynaptic neurons (Fig. 2; El Manira et al., 1994; Wikstrom and El Manira, 1998). Blockade of N-type channels during locomotor activity strongly disrupts the rhythm indicating that the cellular and synaptic mechanisms controlled by this type of Ca2þ channels are of importance for the operation of the locomotor network (Buschges et al., 2000). Modulatory systems such as 5-HT, dopamine, and GABA act specifically on the different types of Ca2þ channels to modulate synaptic transmission and firing properties of neurons and thus change the frequency of the locomotor rhythm (Biro et al., 2006; El Manira et al., 1997a, b; Hill et al., 2003; Schwartz et al., 2005, 2007; Zhang et al., 1996).

Calcium—a pivotal current for firing and release of transmitter

Voltage-activated Kþ channels play an important role in shaping the action potential waveform and thus control the amount of Ca2þ flowing into neurons. We have characterized and examined the function of a particular Kþ current in the lamprey locomotor network. We showed that spinal cord neurons possess a fast-activating and fastinactivating Kþ current that activates in a relatively high voltage range (Hess and El Manira, 2001; Toledo-Rodriguez et al., 2005). This transient current has been found in motoneurons and network interneurons, and is specifically sensitive to catechol. The biophysical properties and pharmacological profile of the current are similar to those of Kv3.4 channels. Specific blockade of the transient Kþ current in lamprey spinal cord neurons using catechol revealed the role of this current in controlling the firing properties of neurons, synaptic transmission, and locomotor pattern generation. Blockade of the transient Kþ current in motoneurons and network interneurons does not affect the action potential onset or threshold, but broadens the action potential

Calcium currents are important for the control of the firing of neurons by activating Ca2þdependent Kþ channels and for triggering release of transmitter. Therefore several modulatory systems act on Ca2þ currents to change the operation of the locomotor network. Ca2þ channels exist in many types that are activated at different membrane potentials and can play separate roles in the network as well as be the target for different modulatory systems. It was necessary to define the types of channels present in the lamprey spinal cord neurons. Indeed, locomotor network neurons possess both low- (LVA) and high-voltage-activated (HVA) Ca2þ channels. Several types of HVA Ca2þ channels could be characterized with the largest component of the current being mediated via N-type channels and only a small fraction being mediated by Land P/Q-type channels (El Manira and Bussieres, 1997). Calcium influx through N-type and P/Q-type

Potassium—currents shaping action potentials

103 Presynaptic axon

Ca2+ (P / Q-type)

Ca2– (N-type)

K+

KCa Ca2+ (P/Q-type)

K+

K+

K+

+

+ Na+

Na+ Ca2+

NMDA-R

KNa

Ca2+ (N-type) AMPA-R

Postsynaptic neuron Fig. 2. Mechanisms shaping synaptic transmission in the spinal locomotor circuitry. Synaptic transmission is mediated by Ca2þ influx via N- and P/Q-type channels in presynaptic axon terminals. On the postsynaptic side, these Ca2þ channels are coupled to Ca2þactivated Kþ (KCa) channels that underlie the slow afterhyperpolarization and control the firing properties of neurons. Synaptically induced Naþ influx via AMPA receptors activates Naþ-activated Kþ (KNa) channels that act as a negative feedback mechanism to shape excitatory synaptic transmission.

and decreases the amplitude of the fast afterhyperpolarization. This results in increased inactivation of Naþ channels during the action potential and decreases the deinactivation after the action potential, thereby preventing repetitive firing. The transient Kþ current is also expressed in axons in the lamprey spinal cord and therefore limits transmitter release by keeping the action potential short (Lamotte d'Incamps et al., 2004). During locomotor network activity, the transient Kþ current is involved in controlling the frequency of rhythmic activity. Locomotor activity is significantly altered when the high-threshold transient Kþ channels are blocked. In catechol, the cycle duration is decreased as are the ventral root burst proportion and the number of action potentials fired by motoneurons during each locomotor cycle. These effects can be accounted for by changes in the firing properties of single

neurons and in their synaptic transmission. A decrease in firing of neurons on one side results in a decrease in mid-cycle inhibition. This, combined with the increase in excitatory synaptic transmission, enables neurons on the contralateral side to become activated earlier, resulting in a faster alternation between left and right ventral roots, and thereby increasing the locomotor burst frequency. KNa channels—a built-in feedback system Naþ influx into neurons has been mainly considered as a charge carrier without any major indirect effects. This view has changed; we now know that Naþ can activate specific Kþ channels (Dryer, 1994). Two types of Naþ-activated Kþ channels have been cloned and their roles in

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neuronal and synaptic processes are beginning to emerge (Bhattacharjee and Kaczmarek, 2005). Naþ enters neurons via two routes; one is mediated by voltage-activated channels and the other by ionotropic receptors, mainly glutamate receptors. We will discuss the role of KNa currents activated by Naþ via these two sources, but before we will begin by discussing the evidence for the existence of KNa channels in lamprey spinal cord neurons. In voltage-clamp conditions, depolarization of spinal cord neurons not only activates voltage-activated Kþ and Naþ currents but also reveals an outward Kþ current that is tightly coupled to the Naþ current (Hess et al., 2007). This KNa current consists of two components one transient and the other sustained. The transient KNa is coupled to the Naþ influx underlying the action potential and appears to initiate the early repolarization of the action potential. The sustained current is activated by Naþ influx via leak channels and seems to be activated by Naþ influx during repetitive firing and mediating the part of the slow afterhyperpolarization that is Ca2þ-independent (Wallen et al., 2007). The two KNa currents seem to act as a negative feedback by sensing Naþ influx during action potentials and via leak channels and thus tend to counteract the resulting depolarization. The existence of KNa channels was also shown in spinal cord neurons in Xenopus tadpoles (Dale, 1993). Another major role of KNa channels is to shape synaptic transmission by interaction with AMPA receptors, which provide a major source of Naþ influx into neurons (Fig. 2). We showed using coimmunoprecipitation that KNa channels closely interact with AMPA receptors in lamprey spinal cord neurons (Nanou and El Manira, 2007; Nanou et al., 2008). These channels can be activated by synaptically induced Naþ via AMPA receptors and shape the decay of the AMPAmediated current and the amplitude of the resulting EPSP. The coupling between KNa channels and AMPA receptors by synaptically induced Naþ transients represents an inherent negative feedback that acts as a mechanism to

scale down the magnitude of excitatory synaptic transmission (Fig. 2; Nanou et al., 2008). KNa channels are also targets of modulatory systems such as metabotropic glutamate receptors that are known to affect the activity of the locomotor network (Nanou and El Manira, 2010). Embedded modulation—setting the baseline locomotor frequency The baseline locomotor frequency is set by a combination of activation of ionotropic receptors mediating fast synaptic transmission and a modulatory network involving many G-protein-coupled receptors. The glutamate released from excitatory interneurons does not only provide the on-cycle excitatory drive via activation of ionotropic AMPA and NMDA receptors but also activates mGluRs responsible for an ongoing modulatory tone during locomotor activity (El Manira and Kyriakatos, 2010; El Manira et al., 2002, 2008; LeBeau et al., 2005; Nistri et al., 2006). A significant part of the baseline frequency is dependent on the activation of mGluR1. Blockade of this receptor type decreases the locomotor burst frequency (Krieger et al., 1998). Conversely, pharmacological activation of mGluR1 induces a short- and long-term increase in the locomotor frequency (Kyriakatos and El Manira, 2007). The short-term increase in the locomotor frequency is mediated via inhibition of leak channels and potentiation of NMDA receptors (Fig. 3; Kettunen et al., 2002, 2003). These two effects of mGluR1 are mediated by different intracellular signaling pathways with the modulation of leak channels, but not that of NMDA receptors, requiring Ca2þ release from internal stores and protein kinase C (PKC; Kettunen et al., 2003; Nanou et al., 2009). The fact that the different cellular mechanisms activated by mGluR1 can be separated based on the intracellular pathways allowed us to explore their individual contribution to the overall locomotor network activity. By interfering with the mGluR1-induced

105 NO

CB-R

NOS

+ CB-R

+ PIP2

Endocannabinoids (2-AG)

–

Lipase

–

yR Gl

A NMD

PA AM

PLC

mGluR1

Inhibition

+

Excitation

DAG

CB1-R activation

Control Fig. 3. Short- and long-term plasticity of the spinal locomotor circuitry. Glutamate, endocannabinoids, and NO are released during locomotion and set the baseline burst frequency. The release of endocannabinoids and NO is also triggered by activation of metabotropic glutamate receptor 1 (mGluR1). Endocannabinoid and NO signaling depress inhibitory synaptic transmission and potentiate of the excitatory synaptic drive. The shift in the balance between inhibition and excitation mediates the long-term increase in the locomotor frequency induced by mGluR1. The mGluR1-mediated short-term increase in the locomotor frequency is mediated by potentiation of NMDA receptors and inhibition of leak channels.

modulation of leak channels, we show that this cellular effect, indeed, plays a role in the mGluR1-induced short-term increase of the locomotor frequency (Nanou et al., 2009). Thus, mGluR1 uses separate intracellular pathways to modulate leak channels and NMDA receptors that seem to act synergistically to induce the short-term increase in the locomotor frequency. Endocannabinoids and nitric oxide (NO) also act individually to set the level of activity of the locomotor network (El Manira and Kyriakatos, 2010; Kettunen et al., 2005; Kyriakatos and

El Manira, 2007; Kyriakatos et al., 2009). Endocannabinoids are released from somata and dendrites of network neurons and act as retrograde messengers to activate presynaptic cannabinoid 1 receptors (CB1-R) and modulate inhibitory and excitatory synaptic transmission and as a result increase the locomotor frequency (Fig. 3; El Manira and Kyriakatos, 2010; Kettunen et al., 2005; Kyriakatos and El Manira, 2007). Similarly, nitric oxide is also synthesized within the locomotor network and increases the burst frequency. Interfering with NO synthesis (NOS) by blocking its

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synthesizing enzyme decreases the locomotor burst frequency (Kyriakatos et al., 2009). The NO modulation also induces a shift in the balance between excitation and inhibition in the spinal locomotor network (Fig. 3; Kyriakatos et al., 2009). These two modulatory systems seem to act in a serial manner because the effect of NO can be occluded by blocking cannabinoid receptors. Thus, the modulation within the locomotor network does not only involve classical transmitters but is also dependent on retrograde signaling involving endocannabinoids and NO. Plasticity in the spinal cord—contribution of endocannabinoids and NO In the lamprey spinal cord activation of mGluR1 induces short- and long-term potentiation of the locomotor frequency (Kyriakatos and El Manira, 2007). The long-term potentiation requires activation of CB1 receptors by endocannabinoid release upon activation of mGluR1 and blockade of CB1 receptors completely suppresses the long-term potentiation of the locomotor frequency. This long-term plasticity is mediated by a network of modulatory signaling involving mGluR1 and the release of endocannabinoids and NO (Fig. 3; Kyriakatos and El Manira, 2007; El Manira and Kyriakatos, 2010). Endocannabinoids are released from motoneurons and network interneurons “on demand” during locomotor network operation by activation of mGluR1 by glutamate release from excitatory CPG interneurons. They then act in synergistic manner with NO to induce long-term depression of mid-cycle inhibition and long-term potentiation of on-cycle excitation (Kyriakatos et al., 2009). A similar interaction has been shown between tachykinin receptors activated by substance P and endocannabinoids (Thorn Perez et al., 2009). Previous studies in the Xenopus tadpole spinal locomotor network have shown that NO plays a critical role in modulating the locomotor activity and mid-cycle inhibition (Sillar et al., 2002). In the spinal circuitry endocannabinoids

and NO use similar synaptic mechanisms to regulate the locomotor activity. In addition, they are recruited by activation of mGluR1 to regulate the activity of the locomotor network. It thus appears that endocannabinoids and NO signaling act synergistically to mediate long-term plasticity in the spinal circuitry; the precise mechanisms of this interaction have not yet been clarified. Common mechanisms with other model systems The mechanisms discussed above are by no means exclusive to the lamprey locomotor network; they are shared by many networks controlling motor behaviors. A detailed characterization of the ionic modulatory mechanisms exists in the locomotor system of Xenopus tadpoles where the roles of specific ion channels and receptors have been clarified (Dale and Kuenzi, 1997; Roberts et al., 2010; Sillar et al., 2008). In the rodent spinal cord, the impact of ionic conductances and modulatory transmitters on the activity of single neurons and locomotor activity has also been studied (Heckman et al., 2003; Kwan et al., 2009; Nistri et al., 2006; Tazerart et al., 2007, 2008; Zhong et al., 2007). The existence of pacemaker properties has been shown in the respiratory (Feldman and Del Negro, 2006; Mironov, 2009; Ramirez and Viemari, 2005; Rybak et al., 2007) as well as masticatory networks (Brocard et al., 2006). Although the importance of these properties for the generation of the motor pattern has been debated, they contribute to the production of the final motor output and are the target of many modulatory systems. Conclusion Based on the results discussed above, both in the lamprey spinal cord and other networks underlying motor behavior, it is becoming clear that the identification of the constituent neurons and the mapping of the circuitry is only a first step toward

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revealing the inner function of how these networks generate behavior. The dynamic nature and plasticity of these networks are governed by the particular blend of ion channels and modulatory receptors found on individual neurons, which can shape the activity of the network on shortand long-term scales. Understanding the logic of plasticity of motor circuitries will provide information about not only their normal operation but also how they react to disruption such as injuries or trauma. Acknowledgments We would like to thank Drs J. Ausborn, K. Dougherty, and R. Hill for comments on the chapter. The support from the Swedish Research Council, Söderberg Foundation, The European Union (FP7, Spinal Cord Repair), and Karolinska Institutet is gratefully acknowledged. References Bhattacharjee, A., & Kaczmarek, L. K. (2005). For Kþ channels, Naþ is the new Ca2þ. Trends in Neurosciences, 28, 422–428. Biro, Z., Hill, R. H., & Grillner, S. (2006). 5-HT modulation of identified segmental premotor interneurons in the lamprey spinal cord. Journal of Neurophysiology, 96, 931–935. Brocard, F., Verdier, D., Arsenault, I., Lund, J. P., & Kolta, A. (2006). Emergence of intrinsic bursting in trigeminal sensory neurons parallels the acquisition of mastication in weanling rats. Journal of Neurophysiology, 96, 2410–2424. Brownstone, R. M., & Wilson, J. M. (2008). Strategies for delineating spinal locomotor rhythm-generating networks and the possible role of Hb9 interneurones in rhythmogenesis. Brain Research Reviews, 57, 64–76. Buchanan, J. T. (2001). Contributions of identifiable neurons and neuron classes to lamprey vertebrate neurobiology. Progress in Neurobiology, 63, 441–466. Buschges, A., Wikstrom, M. A., Grillner, S., & El Manira, A. (2000). Roles of high-voltage-activated calcium channel subtypes in a vertebrate spinal locomotor network. Journal of Neurophysiology, 84, 2758–2766. Cangiano, L., & Grillner, S. (2003). Fast and slow locomotor burst generation in the hemispinal cord of the lamprey. Journal of Neurophysiology, 89, 2931–2942.

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 8

Synaptically activated burst-generating conductances may underlie a group-pacemaker mechanism for respiratory rhythm generation in mammals Christopher A. Del Negro{,*, John A. Hayes{, 1, Ryland W. Pace{, Benjamin R. Brush{, Ryoichi Teruyama} and Jack L. Feldman{ { {

Department of Applied Science, McGlothlin-Street Hall, The College of William & Mary, Williamsburg, Virginia, USA Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA } Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA

Abstract: Breathing, chewing, and walking are critical life-sustaining behaviors in mammals that consist essentially of simple rhythmic movements. Breathing movements in particular involve the diaphragm, thorax, and airways but emanate from a network in the lower brain stem. This network can be studied in reduced preparations in vitro and using simplified mathematical models that make testable predictions. An iterative approach that employs both in vitro and in silico models argues against canonical mechanisms for respiratory rhythm in neonatal rodents that involve reciprocal inhibition and pacemaker properties. We present an alternative model in which emergent network properties play a rhythmogenic role. Specifically, we show evidence that synaptically activated burst-generating conductances—which are only available in the context of network activity—engender robust periodic bursts in respiratory neurons. Because the cellular burst-generating mechanism is linked to network synaptic drive we dub this type of system a group pacemaker.

1

Present address: Neurobiologie & Développement, Institut de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique, Gif sur Yvette cedex, France * Corresponding author. Tel.: þ1-757-221-7808; Fax: þ1-757-221-2050 DOI: 10.1016/S0079-6123(10)87008-6

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Keywords: preBötzinger Complex; pre-Bötzinger Complex; central pattern generator (CPG); metabotropic glutamate receptors; calcium-activated nonspecific cation current; mathematical models; emergent network properties; breathing.

Introduction Understanding the neural origins of behaviors like walking, running, swimming, chewing, suckling, and breathing will be tenable via the application of a broad spectrum of techniques from biomechanics to molecular genetics. These crossdisciplinary tools will enable us to cohere data from many levels of analysis spanning intact behaving organisms to intrinsic membrane properties and biochemical signaling pathways studied in vitro. Here we examine breathing behavior in mammals: rhythmic movements of the diaphragm, thorax, and airways to produce ventilation. We seek cellular- and synaptic-level mechanisms that produce the respiratory rhythm. We limit our focus to a specialized region of the lower brain stem (preBötzinger Complex; preBötC), which is essential for breathing in awake intact adult rodents, and is necessary and sufficient for inspiratory motor rhythms in vitro (Feldman and Del Negro, 2006; Rekling and Feldman, 1998; Smith et al., 1991). To focus on the neural origins of the essential underlying inspiratory rhythm, we necessarily set aside other, equally important, issues related to the neural control of breathing, including—but not limited to—the developmental genetics of preBötC circuits, integrated functions of the preBötC within the larger respiratory network of the lower medulla, formation of an appropriate spatiotemporal motor pattern for ventilatory movements, regulation of the rhythm via neuromodulation, sensorimotor integration, for example, mechanosensitive feedback from the lungs, as well as peripheral and central chemosensation related to oxygen, carbon dioxide, and pH. Our analysis of rhythm generation in the preBötC addresses the following two linked questions: (1)

Which neurons? (2) How? The preBötC is a functionally and anatomically defined site in the lower brain stem containing several thousand neurons in rodents, and we are interested in understanding which ones are rhythmogenic. Here, we consider intrinsic membrane and anatomical properties that may underlie rhythmogenicity. Several models—conceptual as well as explicitly mathematical—outline two general paradigms for the mechanism underlying respiratory rhythmogenesis: reciprocal inhibition and/or pacemaker neurons. Here we evaluate some straightforward predictions of these models and conclude that the respiratory rhythm cannot be adequately explained using these paradigms. Instead we present an alternative model: group-pacemaker hypothesis (Feldman and Del Negro, 2006; Rekling and Feldman, 1998; Rekling et al., 1996a; Rubin et al., 2009a). In this model, recurrent synaptic excitation boosts and propagates activity like a conventional network oscillator (Grillner, 2006), yet constituent neurons generate spike bursts with an underlying plateau-like depolarization during the active phase, which is behavior typically associated with intrinsic pacemaker properties (Coombes and Bressloff, 2005).

Rhythmic motor behaviors studied in vitro Central pattern generator (CPG) networks produce neural rhythms for motor behaviors without need of sensory feedback or commands from higher brain centers (Grillner, 2006; Marder, 2001). Invertebrate model systems provide valuable insights into the structure and function of CPGs because these systems comprise a limited number of constituent neurons (100) that can be identified and, more importantly, selectively

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recorded in the context of behavior in vitro. Detailed analyses of CPG neurons and their intrinsic and synaptic properties have led to mathematical models that reconstitute the behaving system in silico. Explicit models make testable predictions to complement experimental data. This engenders an iterative process in which models are created from empirical measurements, and refined by testing their predictions, which ultimately leads to critical experiments (particularly, in vivo) that advance understanding about the neural underpinnings of these behaviors (Fig. 1a). Unraveling CPGs in mammalian systems including spinal locomotor networks as well as hindbrain oral-motor and respiratory oscillators is considerably more difficult. Mammalian CPGs are composed of far more neurons (> 1000), and with some notable exceptions, function, for example, rhythm generation, may be anatomically dispersed. Moreover, basic morphological or even intrinsic features may not be sufficient to classify neurons and impute their function. To discern the identity of a neuron and analyze its function in the CPG, we must consider a host of challenging approaches to measure properties, such as transmitter phenotype, projection patterns, that is, synaptic connectivity, morphology, and intrinsic membrane properties as they relate to influencing burst generation and rhythmic activity. Regarding intrinsic neuronal properties, there is no substitute at present for intracellular (b)

(a)

Dorsal

recordings in the context of fictive behavior in vitro. Molecular genetics has helped define and test the roles of specific classes of spinal interneurons (Goulding, 2009; Goulding and Pfaff, 2005), which can then be isolated and studied in reduced in vitro preparations (Dougherty and Kiehn, 2010; Dyck and Gosgnach, 2009; Gosgnach et al., 2006; Hinckley and Ziskind-Conhaim, 2006; Hinckley et al., 2005; Wilson et al., 2005, 2007; Zhong et al., 2010). However, despite profound breakthroughs in defining and understanding the composition of spinal locomotor circuits that produce left–right alternation and enforce rhythmic stability, finding the core of the CPG—that is, elucidating its rhythmogenic constituents—has remained elusive (Brownstone and Wilson, 2008; Kiehn et al., 2008). The oral-motor CPG has also been isolated and studied in vitro in neonatal rats. Recordings can be obtained from the motor branch of the trigeminal nerve or, in preparations where the mandible is left attached, rhythmic jaw movements can be monitored in situ (Kogo et al., 1996; Tanaka et al., 1999). The rhythmogenic circuits appear to be located within the lateral reticular formation at the level of the trigeminal motor nucleus. An interesting neuronal phenotype that develops intrinsic bursting behavior in response to decreasing levels of extracellular Ca2þ has been identified in the trigeminal sensory nucleus (Brocard et al., 2006; Kolta et al., 2007). Whether these neurons (c) 20 mV

Behavior (in vivo) XII Fictive behavior (in vitro) Reconstituted behavior (in silico)

NA

VM

preBötC XIIn

Ventral

XII

1s

Fig. 1. The neural control of breathing studied at multiple levels. (a) Hierarchy of model systems from breathing studied in vivo to reduced preparations in vitro, which includes en bloc brain stem–spinal cords and slices, and finally to in silico mathematical models. (b) A cartoon of the slice preparation from neonatal rodents in vitro. (c) Whole-cell patch-clamp recording of a preBötC neuron with XII respiratory motor output in a slice preparation.

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function as rhythm-generators for fictive behavior in vitro or during oral-motor behaviors in vivo remains a compelling notion to be tested. The respiratory CPG is particularly advantageous with regard to defining the putative rhythm-generating neurons at the core of a CPG. First, inspiratory rhythmogenic circuits are contained in the preBötC (Gray et al., 1999; Guyenet and Wang, 2001; Guyenet et al., 2002; Smith et al., 1991; Stornetta et al., 2003; Wang et al., 2001). Second, inspiratory rhythms must be fully developed at birth to then function unceasingly in support of life-sustaining breathing movements. The combination of these factors yields a robust system that fortuitously can be reliably reduced to 300–500 mm thick transverse slice preparations from neonatal rodents that isolate the preBötC and can spontaneously generate respiratory-related rhythmic motor nerve output from the hypoglossal (XII) nerve (Fig. 1b and c). Slices provide optimal experimental access to the constituent rhythmogenic neurons for imaging and electrophysiology in the transverse plane, in the context of behaviorally relevant network functions. The slice preparation serves as a reliable and convenient in vitro experimental model widely used since 1991 by numerous laboratories. Discoveries in the slice have consistently motivated new models of rhythm generation (e.g., Butera et al., 1999a,b; Del Negro et al., 2001; Kosmidis et al., 2004; Rubin et al., 2009a; Smith et al., 2007) and lead to novel tests in behaving animals (Gray et al., 2001; McKay et al., 2005; Tan et al., 2008; Wenninger et al., 2004a,b).

Role of pacemaker properties in respiratory rhythmogenesis Early models featured a role for chloridemediated synaptic inhibition Prior to 1989, the dominant models of respiratory rhythm generation incorporated a critical and obligatory role for synaptic inhibition (Bradley

et al., 1975; Feldman and Cowan, 1975). The general structure of these models posited (a minimum of) three interconnected populations of respiratory neurons including inspiratory and expiratory timing circuits, as well as well as a ramp-generator circuit for inspiratory bursts. The interaction of these populations was thought to generate two or three distinct phases of the respiratory cycle in vivo: inspiration and expiration, which some models divided into stage 1 expiration, that is, the postinspiratory phase, and stage 2 expiration. Inhibitory synaptic interactions were postulated to account for the following aspects of the respiratory cycle: (1) regulation of the augmenting ramp of inspiratory activity via recurrent inhibition, which serves as a check against runaway recurrent excitation and linearized inspiration; (2) termination of inspiratory bursts via a transient surge of inhibition; (3) promotion of a phase transition from stage 1 to stage 2 expiration via synaptic inhibition between postinspiratory (post-I) neurons and the augmentingexpiratory (aug-E) neurons that become active in stage 2; (4) assurance of mutually exclusive activity patterns between inspiratory and expiratory neurons through reciprocal inhibition (for a thorough review, see Feldman, 1986). This general model was in many ways the respiratory analog of that originally proposed by T. G. Brown for locomotion, in which two opposing phases of a motor pattern could be generated by reciprocal inhibition of the “half centers” (Brown, 1911, 1914). In the respiratory cycle, however, inhibitory interactions were further postulated to underlie the additional functions. However, when put to the test using the brain stem–spinal cord preparation in vitro, respiratory rhythm continued unabated after the removal of chloride-mediated synaptic inhibition (Feldman and Smith, 1989). This fundamental result has been extensively replicated using a variety of pharmacological and ion substitution methods in a variety of experimental systems including the en bloc brain stem–spinal cord preparation and the slice preparation from neonatal rats and mice,

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in turtle en bloc preparations, and in the working heart–brainstem preparation from adult mice (Brockhaus and Ballanyi, 1998; Büsselberg et al., 2001; Gray et al., 1999; Johnson et al., 2002; Ren and Greer, 2006; Ritter and Zhang, 2000; Shao and Feldman, 1997). Ionotropic synaptic inhibition contributes to normal breathing patterns in vivo (Büsselberg et al., 2003; Richter and Spyer, 2001) and influences the respiratory pattern formed by the brain stem circuits in which the preBötC is embedded (Rubin et al., 2009b; Smith et al., 2007) but it is not necessary for generating the fundamental rhythm in vitro, and thus is not an essential feature of the respiratory CPG in the preBötC (Feldman and Del Negro, 2006).

Neurons with oscillatory bursting properties become integrated into models of rhythmogenesis The original report that proposed the preBötC as a site of respiratory rhythmogenesis (Smith et al., 1991) identified a subset of inspiratory neurons with voltage-dependent pacemaker properties, and suggested that such neurons could be rhythmogenic. Some preBötC inspiratory neurons, when depolarized with bias current, generate ectopic bursts in the interburst interval, that is, between XII motor discharges. After blocking excitatory synaptic interactions, these same neurons continue to burst rhythmically, as long as baseline membrane potential remains sufficiently depolarized. A thorough search using unit and whole-cell recordings in vitro reveals inspiratory (both excitatory and inhibitory; Morgado-Valle et al., 2010), expiratory, and nonrespiratory (tonic) neurons with intrinsic bursting properties after synaptic isolation via low Ca2þ/high Mg2þ Ringer solution. Respiratorymodulated and nonmodulated neurons with bursting properties are widely distributed throughout the ventral medulla, including a region coextensive with the preBötC (Johnson et al., 1994). These results are consistent with the originally speculative notion that intrinsic oscillatory bursting neurons

could be the source of respiratory rhythm (Feldman and Cleland, 1982). We refer to neurons with pacemaker properties rather than pacemaker neurons because bursting in the absence of synaptic transmission has not been established in any way to constitute a distinct classification based on expression of intrinsic conductances, transmitter phenotype, or developmental lineage. The proposal that neurons with voltage-dependent bursting properties are rhythmogenic, that is, the pacemaker hypothesis, became widely accepted without significant tests or proof. In part, the idea of pacemaker-driven CPG was a logical extension of principles well established in invertebrate systems, and the key prediction of the pacemaker hypothesis had been validated, namely that a subset of neurons with bursting-pacemaker properties are found within the preBötC (Johnson et al., 1994; Smith et al., 1991). Butera et al., 1999a,b proposed a concrete mathematical model, which they dubbed a hybrid pacemaker-network (Smith et al., 1995) because it addressed the role of excitatory synaptic interactions in shaping rhythmic activity. Nevertheless, the pacemaker fraction of the network plays an obligatory role in the production of respiratory rhythm, unless the synaptic conductance was raised to unphysiological extremes (Butera et al., 1999b). The constituent neurons were assembled from a limited complement of ionophores, including persistent Naþ current (INaP) and leakage Kþ current (IK-Leak). These two intrinsic properties are distributed heterogeneously; the proper balance of gNaP and gK-Leak endowed voltage-dependent pacemaker properties. Figure 2 plots gNaP versus gK-Leak in the Butera model. Pacemaker behavior is expressed by neurons whose gNaP and gK-Leak values are within the gray wedge in the center of the distribution. Within this wedge, the neurons express enough gNaP to give “burstiness” combined with low enough gK-Leak to ensure that the baseline membrane potential is depolarized within the voltage-dependent activation range for INaP.

116 5 Pacemaker activity Quiescient 4

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Fig. 2. Pacemaker properties in preBötC neurons are a natural byproduct of heterogeneity. The distribution of gNaP versus gK-Leak in the model proposed by Butera et al. (1999a,b) demonstrates that pacemaker properties are expected in 5–25% of constituent neurons. The white area of the plot denotes tonically active neurons; the gray area reflects bursting-pacemaker activity; and finally the black area indicates a quiescent state. Superimposed experimental data points were obtained from whole-cell recordings of preBötC neurons in neonatal rats. The two-tuple (gNaP, gK-Leak) was added to the existing gNaP versus gK-Leak plot from the model, and the points were color-coded based on whether the recorded neurons showed pacemaker activity after synaptic isolation. The data matched the theoretical distribution, indicating that the ratio of gNaP/gK-Leak governs whether intrinsic bursting is possible in any given neuron. This suggests that pacemaker properties are not specialized, but rather a natural byproduct of heterogeneity in a network of cells that all express gNaP and gK-Leak.

Neurons with gNaP and gK-Leak outside this wedge region are either silent (black, lower right) or tonically spiking (white, upper left; Fig. 2). For neurons in the silent or tonic states, applied current could offset the intrinsic gK-Leak and shift the cell behavior into the bursting regime (Butera et al., 1999a). Experimental data have been superimposed on the gNaP–gK-Leak plot in Fig. 2. These points are paired measurements gNaP and gK-Leak obtained from neurons with and without pacemaker properties in the neonatal rat preBötC. This demonstrates that a fraction of neurons with voltage-dependent bursting properties in the absence of synaptic transmission is a natural byproduct of heterogeneity. The role of INaP was originally based on the observation of bursting in low Ca2þ solution

(Johnson et al., 1994), and the role of IK-Leak was a straightforward prediction based on voltage-dependence of burst frequency. These ionic mechanisms for bursting have since been documented repeatedly (Del Negro et al., 2001, 2002a; Koshiya and Smith, 1999, 2008; Koizumi et al., 2010; Rybak et al., 2003; Smith et al., 1991). In 2001, Thoby-Brisson and Ramirez (2001) recognized, however, that bursting activity in synaptically isolated neurons could have another ionic basis, one that was sensitive to blockade by Cd2þ. We now know that this bursting mechanism involves the Ca2þ-activated nonspecific cationic current (ICAN; Del Negro et al., 2005; Pena et al., 2004). Although ICAN is expressed throughout the perinatal period in  96% of neurons in the preBötC, the number

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of neurons with ICAN-mediated bursting properties after synaptic isolation is miniscule prior to P8 (0.6%), but can be as high as 8% after P8 (Del Negro et al., 2005; Pace et al., 2007a; Pena et al., 2004). Throughout postnatal development the fraction of preBötC with voltage-dependent pacemaker properties attributable to gNaP/gKLeak is between 5% and 25% (Del Negro et al., 2005; Pagliardini et al., 2005; Pena et al., 2004).

Evaluating the role of pacemaker properties in rhythm generation

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Direct tests of whether bursting-pacemaker activity is obligatory for rhythm generation came with use of the anticonvulsant drug riluzole (RIL) used, for some years, to treat amyotrophic lateral sclerosis. The therapeutic potential of RIL

seemed attributable to both its ability to depress excitatory transmission (and thus prevent excitotoxicity), as well as its ability to decrease neural excitability through effects on Naþ channels, INaP in particular (Doble, 1996). Applying RIL in the preBötC for the first time, we showed that RIL selectively attenuated INaP with a half-maximal effective concentration of  3 mM, but had little effect on action potentials at concentrations less than 100 mM (Fig. 3). These effects matched those of RIL in neocortex (Urbani and Belluzzi, 2000) and were consistent for all preBötC neurons, not just neurons with bursting properties. RIL also hyperpolarized preBötC neurons by up to 5 mV by blocking the fraction of INaP open at baseline membrane potential, which typically is between  45 and  55 mV in the rhythmic slice preparation (Del Negro et al., 2002b). These effects of RIL on INaP in the preBötC

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and throughout the ventral medulla are now well established (Koizumi and Smith, 2008; Ptak et al., 2005). Most important from the perspective of testing the pacemaker hypothesis, RIL completely precludes voltage-dependent bursting in synaptically isolated preBötC neurons (Fig. 4a). As to its effects on network activity, bath application of RIL at concentrations 1–200 mM has no effect on the frequency of respiratory rhythm in slice preparations from P0 to P4 neonatal rats and mice, in which ICAN pacemaker activity is ostensibly nonexistent (Del Negro et al., 2002b). However, even while the frequency remained stable, longer exposure to RIL diminishes XII motor output, and causes the rhythmic signal in vitro to

(a)

become undetectable (Fig. 5a; Del Negro et al., 2005). The decrease in XII motor output occurs more slowly than the time required to block INaP and can be accounted for by decreases in cell excitability as well as the depression of excitatory transmission (Del Negro et al., 2005; Doble, 1996; Pace et al., 2007b). Nevertheless, to resolve whether the respiratory network could still function after blocking bursting-pacemaker activity, we performed three important follow-up experiments. We repeated the experiment in P0–4 mice using 20-nM tetrodotoxin (TTX), which like RIL, fully blocks INaP. Bath application of 20 nM TTX to rhythmically active slices causes a time-dependent decrease in the XII motor output accompanied by a decrease in respiratory frequency. Prolonged

Low Ca2+/high Mg2+ ACSF

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20 mV

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Fig. 4. Pharmacology of intrinsic bursting in preBötC neurons. (a) Ten micromolars of RIL blocks INaP-dependent bursting in a representative preBötC neuron synaptically isolated using low Ca2þ/high Mg2þ artificial cerebrospinal fluid (ACSF). (b) Ten micromolars of FFA blocks ICAN-dependent bursting in a representative preBötC neuron. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (APV), picrotoxin (PTX), and strychnine hydrochloride (STR) are excitatory synaptic receptor antagonists used to synaptically isolate the neuron in (b). In both (a) and (b) the baseline membrane potential was ramped from 70 to 40 to test (unsuccessfully) for the presence of voltage-dependent bursting activity. Some plotted data were modified with permission from Del Negro et al. (2005).

119 (a) [RIL]

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VM Iapp 250 pA XII 5s Fig. 5. The effects of RIL on respiratory motor rhythm and inspiratory preBötC neurons recorded in vitro. (b) Motor activity recorded from the hypoglossal nerve (XII) in a cumulative dose–response experiment. RIL concentration is displayed above each trace. Raw and integrated traces (XII) are shown. (b) Substance P (SP) at 2 mM restores respiratory rhythm in a mouse slice exposed to 20 nM TTX. This inspiratory neuron hyperpolarized by 7 mV in TTX; therefore þ250 pA was applied (Iapp) to restore baseline VM to 60 mV. After the rhythm stopped, 2 mM SP revived it. The amplitude of the cellular drive potentials and XII amplitude recovered in SP, but spike discharge did not occur with baseline VM at 60 mV. Data have been modified with permission from Del Negro et al. (2002b, 2005).

exposure to 20-nM TTX stops rhythmic activity in slices (Fig. 5b). However, these experiments could not be unambiguously interpreted because 20 nM TTX (unlike RIL) also attenuated action potentials in preBötC neurons (Del Negro et al., 2005). Even though the number of neurons with ICANmediated bursting properties is vanishingly small P0–P4, we repeated the RIL experiments in rhythmically active slices in the presence of 100-mM flufenamic acid (FFA) (Fig. 6). This dose of FFA completely blocks ICAN-mediated pacemaker activity (Fig. 4b) even though it does not block ICAN completely (Del Negro et al., 2005; Pace et al.,

2007a). Interestingly, whether the rhythm stops by 20 nM TTX or the RIL þ FFA cocktail, 0.5–2 mM of the excitatory neuropeptide substance P (SP) or 0.5 mM of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) is sufficient to revive it (Figs. 5b and 6). Neither SP nor AMPA induce a region of negative slope resistance in the steadystate current–voltage curve of preBötC neurons (Gray et al., 1999; Hayes and Del Negro, 2007; Pena and Ramirez, 2004) and thus cannot restore voltagedependent bursting-pacemaker activity to neurons whose pacemaker-like activity has been precluded by RIL or low doses of TTX. The recovery of respiratory network rhythmicity after pharmacological

120 Control

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XII Fig. 6. Respiratory rhythm in the presence of 100 mM FFA and 20 mM RIL. Continuous segments of the experiment showing control, FFA, recovery, and then FFA þ RIL coapplication, and finally FFA þ RIL þ 2 mM SP conditions in a mouse slice. SP rescued the rhythm after its cessation due to FFA þ RIL. Examples of inspiratory bursts and XII output are shown with greater time resolution (lower panels). Data have been modified with permission from Del Negro et al. (2005).

blockade of pacemaker activity strongly suggests RIL and low doses of TTX diminish respiratory motor output by attenuating excitability, rather than blocking pacemaker activity. Finally, we injected RIL directly into the preBötC to selectively affect the rhythmogenic circuitry and avoid effects in other parts of the slice such as premotor and motor neurons, as well as neurons in the Raphé that provide serotonergic and peptidergic input to the preBötC (Ptak et al., 2009). Microinjection pipettes were targeted to the preBötC bilaterally and connected to a pressure source (400–600 mm Hg) gated by TTL pulses. With this system, we apply RIL with very short duration repetitive pulses (8 ms at 3 Hz) while visualizing preBötC neurons under videomicroscopy to ensure that microinjection did not damage the tissue. With both microinjection pipettes properly positioned in the preBötC bilaterally, RIL microinjection for 20–40 min fails to perturb either the amplitude or frequency of respiratory rhythm (Fig. 7, lower trace; Pace et al., 2007b). This fundamental result is the

same when 100 mM FFA is present to preclude ICAN-mediated pacemaker activity. As a control, to ensure that our protocol was effective for drug delivery, we microinjected the GABAA agonist muscimol, which rapidly stops respiratory rhythm (Fig. 7, upper traces). However, when either the left or right microinjection pipette is displaced, the unilateral microinjection of muscimol fails to impact rhythmogenesis (Pace et al., 2007b). Koizumi and Smith (2008) also performed bilateral microinjection of RIL using thin slice preparations from neonatal rats. However, their protocol was slightly different and causes dosedependent attenuation of respiratory frequency. One possible explanation for the disparity in the results is that Koizumi and Smith used thinner slices ( 300 mm), which are more susceptible to decreases in excitability, which invariably accompanies RIL application. Another explanation is that Koizumi and Smith used continuous low-pressure infusion (10–20 mm Hg). This could create a channel in the tissue and direct

121 Washout

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Fig. 7. Microinjection of RIL into the preBötC does not perturb respiratory rhythm. Bilateral microinjection of muscimol is a positive control to ensure the correct placement of the pipettes in the preBötC. After recovery from muscimol, 10 mM RIL microinjection has no effect on XII motor amplitude or frequency for >30 min. Original data are displayed in the figure, but similar experiments, and a more complete set of pharmacological conditions, can be found in Pace et al. (2007b).

the drug to the midline Raphé neurons, which play a critical role by delivering serotonin and SP to maintain excitability in the preBötC (Ptak et al., 2009). In fact, we tested this notion by targeting our microinjection pipettes to the Raphé in rhythmically active mouse slices and found that RIL microinjection to the Raphé bilaterally stops the respiratory rhythm in a few minutes (Fig. 8; Pace et al., 2007b). The experiments recounted above suggest that bursting-pacemaker activity, whether its ionic mechanism depends on voltage-dependent INaP or ICAN, is not obligatory for respiratory rhythm generation.

Role of synaptically triggered burst-generating conductances in respiratory rhythmogenesis PreBötC neurons with early-inspiratory activity and robust bursts may be rhythmogenic If pacemaker properties after synaptic isolation are not necessarily a reliable and accurate phenotype for putative respiratory rhythm-generators, then one should examine how respiratory neurons behave and, what intrinsic properties they utilize in the context of the functioning network, to elucidate the mechanisms of rhythmogenesis. This type of analysis is possible using slice preparations that

122 Microinjection protocol

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Fig. 8. Microinjection of RIL into the midline Raphé region stops respiratory rhythm in vitro. Plotted data have been modified with author permission from Pace et al. (2007b).

Frequency

retain the preBötC in vitro (Fig. 1b and c). An important pioneering work in that regard was the 1996 report by Rekling and colleagues which classified respiratory neurons in the preBötC and adjacent regions of the ventrolateral medulla according to several criteria including inspiratory

drive, or burst, latency, which quantifies the interval during which neurons depolarize and start to discharge spikes prior to the upstroke of XII motor output (see Fig. 9; Rekling et al., 1996a). Neurons with the earliest inspiratory drive latency also had the highest input resistance, showed Depolarization block

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Fig. 9. Intrinsic properties of NK1Rþ preBötC neurons. Inspiratory drive latency is plotted versus whole-cell capacitance (CM) in the lower graph. Histogram (top) shows the frequency of NK1Rþ neurons with CM separated into 10-pF bins. Note that small (i.e., low CM) NK1Rþ neurons predominate in the preBötC. A fast sweep of a typical inspiratory burst is shown at right with a diagram of characteristic measures (inspiratory drive latency and drive amplitude). Depolarization block of intraburst spike frequency is also indicated, which may have functional significance (see Fig. 16). Some data have been modified with permission from Hayes and Del Negro (2007).

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a ramp-like depolarization during the expiratory phase, and consistently achieved inspiratory burst amplitudes of 10–20 mV. In fact, these inspiratory bursts were so strong that intraburst depolarization block of spiking activity was common (see Fig. 9). When these same neurons were hyperpolarized using bias current to preclude spiking altogether, the inspiratory drive potential, denuded of spikes, was substantially identical in amplitude and duration to the envelope of the burst at normal baseline membrane potential with spiking present. This latter point suggested that the drive potential was triggered by synaptic activity and was not primarily attributable to voltagedependent intrinsic currents (e.g., such as INaP). Moreover, whole-cell recordings with the intracellular Naþ channel antagonist QX-314 in the patch solution did not diminish the drive potential amplitude, nor duration, which is further confirmation that inspiratory bursts are not primarily INaP mediated (Pace et al., 2007b). Neurons with the above properties were suggested to be most closely involved in rhythm generation because they were the first subset of respiratory neurons to activate, that is, they showed the earliest burst latency; their inspiratory bursts were robust, strong, and synaptically triggered; and finally their membrane properties were sensitive to modulation by neuropeptides that also modulated the respiratory period (Rekling et al., 1996a,b). Gray and colleagues (Gray et al., 1999) then showed that peptide receptor expression, the neurokinin1 receptor (NK1R) in particular, delineates the borders of the preBötC and that peptidergic modulation of the rhythm is a potential indicator for rhythmogenic neurons. Suzue recognized the modulatory effects of SP as early as 1984, but did not extend his analyses to the cellular, rhythmogenic level (Suzue, 1984). When NK1Rexpressing (NK1Rþ) neurons are destroyed in awake behaving rats using substance P (SP)-conjugated saporin toxin, normal breathing is abolished and the rats are left with an unphysiological ataxic respiratory pathology (Gray et al., 2001; McKay et al., 2005). Based on these studies in vitro and in vivo, the NK1Rþ

subpopulation became an important putative rhythm-generating subset within the preBötC. The NK1Rþ marker is imperfect, however. Some NK1Rþ neurons in the preBötC, particularly in the caudal region are substantially larger neurons with bulbospinal projections suggesting their function is likely of a premotor, not rhythmogenic, nature (Guyenet et al., 2002; Stornetta et al., 2003). When we began to label NK1Rþ neurons using a fluorescent tagging approach in vitro (Hayes and Del Negro, 2007; Pagliardini et al., 2005), we also found that NK1Rþ neurons are divisible according to size, quantified by whole-cell capacitance (CM). The histogram of CM is bimodal (Fig. 9), which suggests that NK1Rþ neurons in the preBötC can be subdivided according to size. Neurons with larger CM ( 85 pF) could represent the bulbospinal premotor neurons, whereas the smaller CM ( 45 pF) neurons are more likely to represent the putative rhythmgenerators. These  45 pF neurons are far more numerous, as shown by the histogram in Fig. 9, and also show significantly longer inspiratory drive latency ( 250 ms), consistent with Rekling's original description ( 300 ms). We dubbed this subset of small neurons early-inspiratory to avoid confusion with the phenotype called preinspiratory by Onimaru and colleagues (Ballanyi et al., 1999), which are more rostrally located in a different respiratory nucleus, not the preBötC (Onimaru et al., 2008, 2009). These  45 pF, early-inspiratory preBötC neurons also show robust inspiratory bursts in which the underlying amplitude of the drive potential is 10–20 mV and depolarization block of intraburst spiking is common (Fig. 9, right), which suggests a strong and synaptically linked mechanism for inspiratory burst generation and was consistent with a rhythmogenic role in preBötC network function.

Calcium-activated nonspecific cation current triggered by excitatory synaptic input We recorded early-inspiratory neurons to dissect the synaptic and intrinsic factors of inspiratory burst generation. Using patch clamp to record in

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the whole-cell configuration, we added drugs to the patch solution that would block signaling pathways or intrinsic properties upon whole-cell dialysis. In that way, we could measure the inspiratory burst prior to drug application via a perforated patch, and then, after rupturing the membrane with suction, apply the drug from the cytoplasmic side to perturb the synaptic-intrinsic burst-generating mechanism, while leaving the entire network, other than the cell we recorded, pharmacologically unperturbed.

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The large magnitude inspiratory bursts with intraburst depolarization block of spiking, which are shown schematically in Fig. 9, share some similarities with synaptically triggered plateau potentials that can have Ca2þ or Ca2þ-dependent ionic mechanisms. Therefore, we applied 30 mM K4-BAPTA in the patch solution, which caused significant attenuation of the inspiratory burst and underlying drive potential (Fig. 10). When we subsequently added 100 mM FFA to the bath, there was no further reduction in the drive

Rupture (0 min)

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VM XII Fig. 10. Role of ICAN in generating inspiratory bursts: chelation of intracellular Ca2þ by BAPTA attenuates inspiratory drive potentials. Patch-electrode filling solution contained 30 mM K4-BAPTA as well as Lucifer yellow. The perforated-patch configuration (left cartoon and photo) is confirmed by the failure of the Lucifer yellow in the patch-pipette solution to dialyze the neuron. The whole-cell configuration (right cartoon and photo) allows Lucifer yellow to fill the neuron. Control conditions in perforated patch are shown at 5 and 35 min. BAPTA is introduced into the cytosol via patch rupture and causes a progressive attenuation of the inspiratory burst. Subsequent bath application of 100 mM FFA has no additional attenuating effects even after 15 min of exposure to the drug. Baseline membrane potential was 60 mV throughout the experiment. Dotted line is drawn to facilitate comparison of the size of the inspiratory drive potential at different time points during the experiment. Data have been modified with author permission from Pace et al. (2007a).

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potential, which suggests that the inspiratory burst is mediated by ICAN (Pace et al., 2007a). We also evoke ICAN directly by adding the photolyzable Ca2þ chelator DM-nitrophen to the patch solution and exposing the recorded soma and proximal dendrites to flashes of ultraviolet light (1–2 s). ICAN reverses at  0 mV, which is expected for a nonspecific cationic current (Fig. 11). Plateau-like bursts can also be evoked with current pulses after whole-cell dialysis with Csþ, which enabled us to evoke ICAN and evaluate its ionic basis using ion substitution (choline for Naþ) and test its Ca2þ activation mechanism by blocking voltagedependent Ca2þ channels with 200 mM Cd2þ (Pace et al., 2007a). Either choline or Cd2þ is able to fully block ICAN-mediated evoked plateaus. However, 100 mM FFA is an incomplete ICAN blocker. In the presence of 100 mM FFA, some ICANmediated plateau response could still be evoked by current pulse, and choline substitution is able to block the remaining response fully (Pace et al., 2007a).

Given the pharmacological limitations of 100 mM FFA, the fact that this dose of the drug did not stop respiratory rhythm in vitro was not a dispositive result (see Fig. 6). However, raising the concentration to 300–350 mM FFA fully blocked ICANmediated evoked plateaus and, furthermore, stopped the respiratory rhythm. Once stopped, the respiratory rhythm could not be restarted using 1–2 mM SP (Fig. 12; Pace et al., 2007a). This experiment strongly suggests that ICAN is an essential cellular mechanism for rhythmogenesis. Nevertheless, concentrations of FFA exceeding 100 mM produce side effects that make interpretation of the results problematic (Pace et al., 2007a; Teulon, 2000). The ion channels that underlie ICAN are not known, but a potentially attractive set of candidates come from the transient receptor potential (TRP) family (Ramsey et al., 2006). In particular, TRPM4 and TRPM5 are unique because they are activated by Ca2þ but selective in their permeability for monovalent cations (Hofmann et al., 2003; Launay et al., 2002). We detected mRNA for TRPM4 and

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Fig. 11. Role of ICAN in preBötC neurons: Ca2þ uncaging by ultraviolet illumination of the photolyzable chelator DM-Nitrophen. ICAN was measured at a range of membrane potentials in voltage clamp. ICAN reversed at 0 mV, consistent with a mixed monovalent cation current. Similar data were obtained in current clamp (not shown).

126 300 mM FFA

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XII 5s Fig. 12. A high dose of FFA (300 mM) blocks inspiratory activity in 70% of slices (top traces). 350-mM FFA blocks the respiratory rhythm in the other 30% (bottom traces). One micromolar of substance P (SP) does not restart the rhythm in 350 mM FFA, but rhythmic activity does recover after >1 h of washout. Baseline membrane potential was 60 mV for traces in the top. Data have been modified with author permission from Pace et al. (2007a).

TRPM5 in preBötC tissues (Fig. 13a; Crowder et al., 2007). The expression of TRPM4 and TRPM5 was tested using commercial antibodies from Santa Cruz Biotechnologies (Santa Cruz, CA) in adult rodent slice preparations (Fig. 13b). This resulted in heavy staining in preBötC and the neighboring nucleus ambiguous (NA). We additionally showed that ICAN-mediated inspiratory drive potentials were labile to modulation of membrane phospholipids, notably phosphatidylinositol 4,5bisphosphate (Crowder et al., 2007), which is characteristic behavior for TRPM4 and TRPM5 (Liu and Liman, 2003; Zhang et al., 2005). However, the molecular identity of ICAN is by no means a solved problem. TRPM4 and TRPM5 are likely candidates, but other TRP channels including those from the canonical TRPC subfamily may be involved as well. The experiments above showed that ICAN played a substantial role in generating inspiratory bursts in the context of network activity. However,

the synaptic activation mechanism was still unclear. It has been well established since the early 1990s that AMPA-type ionotropic glutamate receptors are critical for respiratory rhythm and that Nmethyl-D-aspartate (NMDA) receptors are normally present but not essential for rhythmogenesis (Funk et al., 1993, 1997; Ge and Feldman, 1998; Greer et al., 1991; Morgado-Valle and Feldman, 2007). If NMDA receptors were not the source of Ca2þ influx to activate ICAN then it seemed possible that AMPA receptors could be Ca2þ permeable, despite anatomical evidence that AMPA receptors in the preBötC contain edited GluR2 subunits (Paarmann et al., 2000). We tested this physiologically by recording isolated preBötC neurons in the presence of 1 mM TTX. Short-duration pressure applications, that is, puffs, of 500 nM AMPA caused a transient depolarization as a model of excitatory synaptic drive. Puffs were applied to the dendrite ( 100 mm) to target the region likely to contain synapses and to avoid dislodging the patch-recording pipette at the soma. The AMPA puff response was completely blocked by GYKI 53655 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine), but could not be blocked using the selective antagonist for Ca2þpermeable AMPA receptors IEM-1460 (N,N,N,Trimethyl-5-[(tricyclo[3.3.1.13,7]dec-1-ylmethyl) amino]-1-pentanaminiumbromide hydrobromide). Surprisingly, the response could be reduced by 70–75% by either FFA or Cd2þ, which indicated that the AMPA receptor was coupled to ICAN activation, but not directly (Fig. 14). The coupling relies on local synaptic depolarization by AMPA receptors to evoke voltage-gated Ca2þ channels (Pace and Del Negro, 2008). We also tested the role of metabotropic glutamate receptors (mGluRs) because group I mGluRs are coupled via phospholipase C (PLC) to the synthesis of inositol 1,4,5-trisphosphate (IP3), which evokes intracellular Ca2þ release and could be a source of ICAN activation. Approximately 40% of the inspiratory drive potential can be attributed to mGluR5 (Fig. 15a), one of the two mGluR subtypes that comprise group I. The mGluR5-mediated

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50 mm GADPH Fig. 13. Evidence for TRPM4 and TRPM5 channels in the preBötC. (a) The mRNA coding for TRPM4 and TRPM5 is expressed in the preBötC region. Total RNA was extracted from preBötC and positive control tissues, and then reverse transcribed. Amplified products of the expected sizes were obtained for TRPM4 (301 bp), TRPM5 (483 bp), and GADPH (452 bp). Negative control reactions were performed without reverse transcriptase and amplified nothing. (b) Immunohistochemistry from adult rats. Top panels (positive controls) show TRPM4 and TRPM5 in the compact nucleus ambiguous (cNA) taken from the ventral medulla immediately rostral to preBötC. Lower panels show TRPM4 and TRPM5 labeling in the preBötC at increasing magnification. Data in (a) were replotted with author permission from Crowder et al. (2007).

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component of the inspiratory drive potential was due to intracellular Ca2þ release causing ICAN activation, as confirmed by intracellular blockade of IP3 receptors by xestospongin (Fig. 15b). The other

member, mGluR1, was coupled to transient closure of Kþ channels in preBötC neurons, which also contributed to drive potential generation, but not via ICAN (Pace et al., 2007a,b).

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VM XII Fig. 15. Role of mGluR5 in inspiratory burst generation. (a) Bath application of the selective mGluR5 antagonist MPEP (10 mM) attenuates inspiratory bursts in the context of ongoing network rhythm. (b) Downstream of mGluR5, IP3 receptors cause intracellular Ca2þ release and lead to ICAN activation. Perforated-patch recordings serve as control. The IP3 receptor antagonist xestospongin (1 mM), applied intracellularly by patch rupture, reduces inspiratory bursts. Subsequent application of MPEP causes no further change in the drive potential. Data have been modified with author permission from Pace et al. (2007a).

Group-pacemaker hypothesis of respiratory rhythm generation A new paradigm for respiratory rhythmogenesis based on emergent network properties In the absence of obligatory pacemaker neurons, one alternative explanation for rhythm generation is based on recurrent synaptic excitation. This concept remained purely hypothetical without the identification of specific mechanisms through which it could be implemented. However, we now recognize that ICAN activation is linked to mGluRs and AMPA receptor-mediated recruitment of Ca2þ channels, which is a viable mechanism for the normally latent ICAN to be

evoked synaptically in the lead-up to inspiration (Mironov, 2008; Pace et al., 2007a). Guided by experimental results, we assembled a mathematical model of a network with synaptically activated burst-generating conductances, which was dubbed a group pacemaker by Rekling and colleagues (Rekling and Feldman, 1998; Rekling et al., 1996a). Putatively rhythmogenic early-inspiratory neurons, for example, Fig. 9 right, show ramp-like depolarization and low-rate spiking prior to the inspiratory phase, which is evidence of recurrent excitation (Hartelt et al., 2008; Pace, 2007; Rekling et al., 1996a, 2000). There is significant depolarization block during the inspiratory burst, which produces a scoop-like attenuation of spike amplitude as the burst progresses. A transient period of quiescence follows burst termination, before the next cycle of recurrent excitation begins and synaptic depolarization and low-rate spiking restarts (Del Negro et al., 2009). These characteristic behaviors are visible during on-cell recording, as well as after wholecell dialysis (Fig. 16a), and are reproduced in the model (Fig. 16b; Rubin et al., 2009a). Constituent neurons in the group-pacemaker model are not required to have pacemaker properties; that is, they need not exhibit rhythmic bursting without synaptic interactions. Low-rate spontaneous spiking activity in a subset of neurons engenders positive feedback via recurrent excitation. Glutamate causes postsynaptic depolarization via ionotropic AMPA receptors, and acts at mGluRs, which results in intracellular Ca2þ elevations that evoke ICAN. Postsynaptic ICAN contributes to burst generation by providing the plateau-like envelope of depolarization that makes up the inspiratory drive potential. ICAN also helps terminate the burst. Intraburst spike attenuation, due to ICAN-mediated depolarization block, diminishes recurrent excitation by causing a net reduction in transmitter release. Attenuated spikes in the model decrease excitatory synaptic drive by failing to reach a voltage-dependent threshold for release (Brody and Yue, 2000; Dobrunz et al., 1997; Forsythe et al., 1998), but physiological mechanisms

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Fig. 16. The group-pacemaker hypothesis realized in an explicit mathematical model. (a) On-cell patch recording in the preBötC with inspiratory motor discharge (XII). Below, whole-cell recording of the same cell as above after rupture of the patch, also with XII output. Baseline membrane potential was 60 mV. (b) Data from another preBötC neuron in whole-cell recording is plotted with the model behavior. (c) Fast sweep of a burst in the model from (b) (above) coded with numerals to illustrate the sequence of physiological steps in one model burst in the Naþ–Ca2þ phase plane, which is also displayed. Specific captions are superimposed with the circle-enclosed numbers to encapsulate model dynamics in sequence. See text for more detail. These data were modified with permission from Rubin et al. (2009a).

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include depletion of releasable vesicle pools or retrograde signaling that acts presynaptically to decrease release probability (Best and Regehr, 2008; Gibson et al., 2008; Kettunen et al., 2005). This sets the stage for burst termination by halting Ca2þ accumulation and deactivating ICAN. Also, because Naþ is the predominant charge carrier for inward ICAN, intraburst Naþ accumulation triggers activity-dependent outward currents such as the electrogenic Naþ/Kþ ATPase pump (Ipump; Del Negro et al., 2009), Naþ-dependent Kþ current (IK-Na), and ATP-dependent Kþ current (IK-ATP, which activates as the ATPase pumps consume and thus diminish the available cytosolic ATP). Ipump is the main activity-dependent current featured in detailed analyses of the model, but a wide array of activity-dependent outward currents can accomplish the same basic burst-terminating function (Rubin et al., 2009a). Contrary to conventional wisdom (Hille, 2001), Ca2þ-dependent Kþ currents play no significant role in terminating inspiratory bursts (Crowder et al., 2007; Onimaru et al., 2003; Zavala-Tecuapetla et al., 2008). The Naþdependent outward currents actively hyperpolarize constituent neurons, leading to a transient quiescent state in the network. After a recovery period, sporadic spiking activity in the preBötC rekindles excitatory interconnections and restarts the cycle. Figure 16 illustrates how network activity leads to an increase in intracellular Ca2þ, which subsequently evokes ICAN. In turn, ICAN causes Naþ accumulation to evoke Ipump and other outward currents to cause burst termination. In the model system, this cycle can be visualized in the Naþ–Ca2þ phase plane and its underlying dynamics can be analyzed geometrically (Rinzel and Ermentrout, 1998). Recurrent excitation and activity-dependent outward currents are well-established neurophysiological factors that influence cell and network activity. However, our CPG model depends on glutamatergic signaling to gate postsynaptic burst-generating ion channels. We further propose that short-term synaptic disfacilitation may also contribute to burst termination. This mechanism reflects a new paradigm for central pattern

generation in which the rhythmogenic unit is the ensemble of pre- and postsynaptic features and can be properly considered a group pacemaker (Feldman and Del Negro, 2006; Rekling and Feldman, 1998; Rekling et al., 1996a). This model is undoubtedly oversimplified, in that the individual neuron components are limited to single compartments in which synaptic integration and spike generation both takes place. This may not be an accurate depiction of burst generation in preBötC or in any CPG interneuron where significant processing depends on active dendrites (Stuart et al., 2007). However, the present model has the advantage of simplicity and tractability for mathematical analysis and can be readily assembled in networks.

Conclusions CPGs in vertebrates incorporate thousands of highly interconnected neurons, each of which represents a complex dynamical system with many degrees of freedom (Grillner, 2006; Kozlov et al., 2009). To understand CPGs, we must necessarily focus on the essential rhythmogenic modules. Reciprocal inhibition—where active synaptic inhibition acts as a barrier that must be overcome for phase switching to occur—or specialized pacemaker neurons—which provide a rhythmic template for network activity—are modules that encapsulate rhythmic function in particular synaptic, that is, inhibitory, or cellular properties (i.e., autooscillatory neurons with pacemaker, properties). Both mechanisms find widespread support in real systems. (Grillner, 2006; Marder and Bucher, 2001; Marder and Calabrese, 1996; Stein et al., 1997), but neither mechanism can explain preBötC rhythms (Feldman and Del Negro, 2006; Johnson et al., 2007; Mironov, 2008; Morgado-Valle et al., 2010; Pace et al., 2007a). The next level of module complexity would combine specific synaptic and cellular components, such as the synaptically activated burst-generating conductances that we propose underlie preBötC rhythmicity.

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In CPG systems that depend on an active phase of inhibition or pacemaker properties, we speculate that synaptically activated burst-generating conductances may also be present, which could contribute to enhancing the robustness or duration of the active phase. Such a synaptically triggered conductance would be a convenient target for neuromodulation because its contribution could be modified to regulate the burst-phase without necessarily modifying the frequency or overall pattern of the motor rhythm. Within the preBötC a synaptically activated burst-generating conductance appears to be important in conveying both the frequency and overall pattern of rhythmic activity. Yet there are many aspects of respiratory rhythm generation that are still not well understood. The group-pacemaker framework offers many directions for future tests to answer these questions using in silico models and a robust network with preserved function in vitro. Consequently, the principles brought to light may advance our understanding of the neural control of breathing and provide some insights into the neural origins of other motor behaviors as well.

Acknowledgments The work was supported by NIH HL-40959, NIH NINDS R21, NS070056-01, NIH-NHLBS R01 HL104127-01, and the Undergraduate Biological Sciences Education and Research Training Grant to The College of William & Mary by the Howard Hughes Memorial Institute.

Abbreviations AMPA aug-E BAPTA

a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid augmenting-expiratory 1,2-bis(o-aminophenoxy)ethaneN,N,N0 ,N0 -tetraacetic acid

CPG FFA GABA GYKI 53655 ICAN IEM-1460

IK-ATP IK-Leak IK-Na INaP IP3 Ipump mGluRs NK1R NMDA PLC post-I Pre-BötC QX-314

RIL SP TRP TTX XII

central pattern generator flufenamic acid gamma-aminobutyric acid 1-(4-aminophenyl)-4-methyl-7,8methylenedioxy-5H-2,3benzodiazepine Ca2þ-activated nonspecific cationic current N,N,N,-trimethyl-5-[(tricyclo [3.3.1.13,7]dec-1-ylmethyl) amino]-1-pentanaminiumbromide hydrobromide ATP-dependent Kþ current leakage Kþ current Naþ-dependent Kþ current persistent Naþ current inositol 1,4,5-trisphosphate Naþ/Kþ ATPase pump metabotropic glutamate receptors neurokinin-1 receptor N-methyl-D-aspartate phospholipase C postinspiratory preBötzinger complex N-(2,6dimethylphenylcarbamoylmethyl) triethylammonium bromide riluzole substance P transient receptor potential tetrodotoxin hypoglossal

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 9

Modulation of rhythmogenic properties of trigeminal neurons contributing to the masticatory CPG Arlette Kolta*, Philippe Morquette, Raphaël Lavoie, Isabel Arsenault and Dorly Verdier Faculté de médecine dentaire and Groupe de Recherche sur le Système Nerveux Central du FRSQ, Université de Montréal, Succursale Centre-ville, Montreal, Quebec, Canada

Abstract: Increasing evidence suggests that the dorsal part of the principal sensory nucleus of the trigeminal nerve (NVsnpr) contains a significant core of the central pattern generator (CPG) circuitry required for mastication (Tsuboi et al., 2003). Like many trigeminal brainstem neurons, those of NVsnpr are rhythmically active in phase with fictive mastication in vivo (Tsuboi et al., 2003) and project directly to the trigeminal motoneurons (Kolta et al., 2000), but in contrast with the others, they are the only neurons with intrinsic bursting abilities (Sandler et al., 1998; Brocard et al., 2006) within the minimal area of the brainstem necessary to produce rhythmic activity in trigeminal nerves (Bourque and Kolta, 2001). Development of bursting in NVsnpr neurons closely follows the development of mastication. It is mediated by a persistent Naþ current (INaP) that is expressed only within a certain membrane potential range and that is modulated by the extracellular Ca2þ concentration ([Ca2þ]e), the lower the concentration, the larger the magnitude of INaP. Under physiological [Ca2þ]e, bursting can also be induced in vitro by repetitive electrical stimulation of the trigeminal sensory tract, which projects massively to NVsnpr or by local applications of N-methyl-D-aspartic acid. Both types of stimuli also depolarize glial cells recorded in NVsnpr and increase coupling between them. Glial cells play a determinant role in setting [Ca2þ]e and hence are in a key position to influence NVsnpr neuronal firing pattern. Keywords: Mastication; central pattern generator; rhythmogenesis; INaP; bursting; calcium; sensory modulation.

* Corresponding author. Tel.: þ1-514-343-7112; Fax: +1-514-343-2111 DOI: 10.1016/S0079-6123(10)87009-8

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As everyone knows food intake is essential for life. Ingested food must be broken down in pieces and prepared for digestion. This is done by chewing. Mastication seems like a simple act, but in fact, it requires orchestration of more than 20 oro-facial muscles or muscle compartments and needs to be coordinated with swallowing and respiration. A century ago, (Sherrington, 1917) proposed that mastication was a chain of alternating jaw opening and jaw closing reflexes, even if (Ferrier, 1873) had already shown that it could be induced by stimulating the cerebral cortex in several species of animals. It is only much later, in the early 1970s that Lund and Dellow (1971) showed that the basic pattern of mastication is produced by a brainstem CPG.

complex composed at this level by the trigeminal main sensory nucleus (NVsnpr) and the rostral pole of the spinal nucleus (Fig. 1). Rhythmic activity has been recorded in neurons of all of those nuclei during fictive mastication; that is, mastication elicited by stimulation of the cortical masticatory area in paralyzed and anesthetized animals. Because there is actually no movement and thus no rhythmic sensory feedback, rhythmic firing recorded under these conditions results from activity of rhythmogenic neurons or from inputs received from these rhythmogenic neurons. Rhythmic outputs of CPGs can result from intrinsic properties of individual neurons (“pacemaker neurons”), from interactions within a network (“network pacemaker”), or a combination of the two (see Chapters 8 and 14). We used an

Boundaries and components of the CPG Despite the fact that the brainstem location of the masticatory central pattern generator (CPG) has been identified nearly 40 years ago, its exact boundaries and the elements that form it have not been unequivocally described yet. In the mid1980s, in vivo studies suggested that the essential core of the CPG lies between the rostral poles of the trigeminal (NVmt) and hypoglossal (NXII) motor nuclei. However, it was difficult to ascertain in those studies that the manipulations executed to isolate the masticatory CPG did not also affect the vital and respiratory brainstem centers (Nozaki et al., 1986a,b). Later, transection studies on “en bloc” brainstem preparations showed that rhythmical activity elicited in trigeminal nerves by addition of N-methyl-D-aspartic acid (NMDA) to the bath persists as long as a bloc (gray shadow box in Fig. 1) extending from the rostral pole of NVmt to the rostral pole of the facial nucleus and approximately half a millimeter from the lateral border of the brainstem remains intact (Kogo et al., 1996; Tanaka et al., 1999). This area contains the trigeminal motoneurons embedded in a column of premotor interneurons forming the peritrigeminal area (Peri V) and lateral to which extends the trigeminal sensory

NVmes NPont PeriV NVmt NVsnpr GPC NVII NVspo tractus NXII NVspi NVspc

Fig. 1. Schematic representation of the brainstem nuclei involved in oro-facial movements and their connections (arrows). The gray 3D box defines the boundaries of the masticatory CPG.

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in vitro slice preparation of rat brainstem to examine the intrinsic properties and synaptic inputs of most groups of neurons within the critical brainstem area essential for mastication, and found that most premotor interneurons of the Peri V could not generate rhythmic activity either intrinsically or through the robust reciprocal connections that they shared (Bourque and Kolta, 2001), except for about 5% of those recorded in the area dorsal and slightly lateral to NVmt, called the supratrigeminal area. However, the dorsal part of NVsnpr immediately adjacent to these few bursting neurons was found to contain a large population of rhythmically firing neurons. This was a surprise because this nucleus is generally considered as a sensory relay station that faithfully transmits inputs from primary afferents to the thalamus. Sandler et al. (1998) had shown that about half of NVsnpr neurons recorded in vitro in slices of the gerbil brainstem have plateau properties that allow them to transform a depolarizing input into bursts which was a puzzling property for neurons with this putative function, and there was anecdotal descriptions (including some of our work) in the literature of projections from NVsnpr to motor nuclei that control the muscles responsible for feeding, the Vth, VIIth, and XIIth motor nuclei (Kolta et al., 2000; Li et al., 1993; Pinganaud et al., 1999; Travers and Norgren, 1983; Yoshida et al., 1998), but there had been no attempt to interpret the functional significance of these data. Therefore, we decided to investigate the firing of NVsnpr neurons during fictive mastication. A quarter of the neurons recorded throughout the nucleus changed their firing patterns during fictive mastication induced by repetitive stimulation of the left and right cortical masticatory areas and only about a third of these fired rhythmically in phase with trigeminal motoneurons. The others discharged in bursts at more than twice the frequency of trigeminal motoneurons. Most rhythmic masticatory neurons had inputs from muscle spindle afferents and periodontal receptors and were concentrated in the dorsal part, whereas

nonmasticatory rhythmical neurons had receptive fields on the lips and face and were spread throughout the nucleus but were denser in the ventral part of NVsnpr. The latter may have been related to whisking. Later, using the expression of c-Fos-like protein as a functional marker of activity, we showed that the activity level increases induced by fictive mastication appear only in dorsal NVsnpr (Athanassiadis et al., 2005).

Development of bursting properties in relation to mastication On the basis of this evidence, we decided to further investigate the intrinsic properties of NVsnpr neurons in the in vitro slice preparation. Under standard control conditions, two-thirds of the population fired tonically upon depolarization, while the others had one or several bursts of action potentials upon depolarization. Bursting occurred almost exclusively in the dorsal part of the nucleus and was maintained and even enhanced under conditions that reduce synaptic transmission (removal of Ca2þ from the artificial cerebrospinal fluid [aCSF] used to perfuse the preparation), suggesting that it is an intrinsic property of NVsnpr neurons. One of the first interesting observations that we made was that these intrinsic bursting properties develop in parallel with mastication. In rats, the transition from suckling to chewing occurs gradually over nearly a week. The first masticatory movements appear around postnatal day 12, and the adult pattern is fully established between postnatal day 18 and day 21 (Westneat and Hall, 1992). It is unclear whether mastication and suckling depend on the same CPG network which evolve during that period or on different CPGs. Both movements involve jaw opening and closing, but in one case, chewing, the power stroke occurs during jaw closure, while in the other it occurs during opening with the tongue moving in peristaltic manner. Over the period of transition from suckling to adult mastication, the membrane properties of

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NVsnpr neurons undergo important changes (Brocard et al., 2006); particularly those that determine the ability to generate rhythmic bursting. By postnatal day 10 (P10), some neurons generate one or more bursts at the onset of depolarization ( 5%). The frequency of occurrence increases abruptly after P12 (25%) and by P14, about 45% of dorsal NVsnpr cells discharge bursts during sustained depolarization, and 25% of these do this repetitively. In these, bursting frequency is linearly related to injected current. In Ca2þ-free aCSF, the proportion of cells expressing recurrent bursting increased at all ages, but displayed the same developmental profile (< 15% at P10;  55% at P10–12; > 85% at P16–17). In the same time frame, the proportion of cells showing an afterdepolarization (ADP) after the spike increased in parallel and there was a clear correlation between the presence of ADP and the ability to burst. Similar age-related changes in membrane properties occur in other brainstem nuclei including the more ventral region of NVsnpr containing whisking-related neurons (Lo and Erzurumlu, 2001), the spinal trigeminal nucleus (Guido et al., 1998), and in several other brainstem regions (Bao et al., 1995; Berger et al., 1996; Nguyen et al., 2004; Núñez-Abades et al., 1993; Tanaka et al., 2003; Tsuzuki et al., 1995).

Mechanisms of rhythmogenesis Our evidence (see Brocard et al., 2006 and Kolta et al., 2007) suggests that bursting is primarily mediated by a persistent sodium conductance (INaP), which is also responsible for burst firing in neurons of the pre-Bötzinger complex (Butera et al., 1999; Del Negro et al., 2002, 2005), cultured spinal cord (Darbon et al., 2004), mouse spinal locomotor CPG (Tazerart et al., 2007; Zhong et al., 2007), hippocampus (Azouz et al., 1996; Jinno et al., 2003; Mattia et al., 1997; Su et al., 2001), subthalamic nucleus (Beurrier et al., 2000), neocortex (Brumberg et al., 2000; Franceschetti et al., 1995; Guatteo et al., 1996), and trigeminal primary

afferents of the mesencephalic nucleus (Wu et al., 2005). First, bursting persisted in Ca2þ-free aCSF and in presence of a Ca2þ chelator (1,2-bis (oaminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid [BAPTA]) injected into recorded cells ruling out involvement of Ca2þ-mediated currents. It was not affected by blockers of voltage-activated (tetraethylammonium [TEA]) and Ca2þ-activated (apamine and charybdotoxin) Kþ currents, although TEA substantially increased the duration of bursts and charybdotoxin (blocker of BKCa) increased the amplitude and duration of the ADP facilitating the transition from single spike firing to firing in doublets or triplets. Most importantly, Riluzole, which suppresses INaP predominately (Urbani and Belluzzi, 2000; Wu et al., 2005), and tetrodotoxin (TTX), inhibited bursts and the underlying plateau potentials. The TTX-sensitive plateau potentials and bursts are only seen within a voltage range that corresponds to the activation and steady-state inactivation voltages typical of INaP (Crill, 1996) and both the plateau potential and bursting emerge during the second postnatal week, which corresponds with the developmental profile characteristic of INaP (Alzheimer et al., 1993; Huguenard et al., 1988) and appearance of bursting in other areas of the brain (Franceschetti et al., 1993, 1998; Kriegstein et al., 1987). Finally, lowering the extracellular Ca2þ concentration ([Ca2þ]e) increases the amplitude of the ADP as well as the proportion of neurons that burst and the duration of TTX-sensitive plateau potentials (Brocard et al., 2006). These results indicate that [Ca2þ]e regulates the firing pattern of NVsnpr neurons by modulating the magnitude of INaP. A similar relationship has been found in spinal interneurons thought to be part of the locomotor CPG (Tazerart et al., 2008) and several other areas of the central nervous system (Azouz et al., 1996; Li and Hatton, 1996; Su et al., 2001). To summarize, NVsnpr neurons have intrinsic bursting properties, mediated by INaP, but expression of these properties depends on factors external to the cell (network properties?). We suggest that the Ca2þ-sensitivity of INaP provides a mean for the system to trigger bursting when

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appropriate and to modulate bursting frequency in function of inputs (whether sensory or other). In other words, Ca2þ-dependency may be the mechanism that enables sensory and/or cortical feedback to adjust the output pattern.

Pattern variability and sensory modulation of CPGs By definition, CPGs can generate a rhythmic output in absence of any input. This is not to say that inputs to CPGs do not play an important role. It is well known that sensory feedback is essential to adapt the movement pattern to the prevailing conditions. In absence of cortical or sensory inputs, CPGs produce stereotyped movements that would be identical from cycle to cycle. However, as everyone has experienced before, the form of masticatory movements varies between foods and even within a single masticatory sequence. Cortical input to the masticatory CPG is thought to play an important role in anticipating the pattern of movements required, while sensory inputs provide feedback to adjust the movements to the changes in texture and hardness, error corrections, and to counteract unexpected perturbations. This, of course, is not unique to the masticatory CPG. Many studies in different systems have reported about the importance of sensory feedback in determining different parameters of the movement pattern and phase transition (for review, see Rossignol et al., 1988) but very few have examined the cellular mechanisms at the basis of this interaction between sensory inputs and the rhythm generating neurons. For instance, stretch of pulmonary muscle spindle afferents and diaphragmatic proprioceptors contributes to the termination of inspiration (Speck et al., 1993), while flexing the hip can profoundly alter the locomotor pattern and even block completely the locomotion (Pearson and Rossignol, 1991). In the masticatory system, as the teeth bite into the food, pressure builds within the periodontal ligament and the velocity of closure drops rapidly, causing activation of periodontal receptors (Olsson

et al., 1988) and jaw closing muscle spindle afferents (Goodwin and Luschei, 1975). In both cases, this activation is proportional to the food resistance and is determinant for the duration and amplitude of the electromyogram bursts of the jaw closing muscles (Komuro et al., 2001; Lavigne et al., 1987). Coincidently, these are precisely the inputs received by most dorsal NVsnpr neurons that fired rhythmically in phase with trigeminal motoneurons during fictive mastication (Tsuboi et al., 2003). How signals from these afferents interact with the rhythmogenic properties of NVsnpr is still unknown.

Putative cellular mechanisms underlying sensory modulation of bursting in NVsnpr neurons In in vivo animal experimental models, mastication can be elicited by repetitive stimulation of the cortical masticatory area or of sensory nerves. In both cases, stimulation needs to last hundreds of milliseconds before mastication is triggered despite the fact that both types of inputs reach most brainstem trigeminal neurons at short latencies (< 10 ms). Similarly, mastication continues several hundreds of milliseconds after cessation of stimulation indicating that a slow process is involved in the command. This observation led us to propose the functional model described in Fig. 2. We postulated that this prolonged activity of afferent inputs causes a drop in [Ca2þ]e that in turn triggers activation of INaP. Because of the voltage-dependency of INaP, this would occur only within a defined membrane potential range; between approximately  60 and  50 mV in our cells. Thus, little afferent activity would cause tonic firing in NVsnpr neurons and a slight depolarization, bringing the cell closer to the activation range of INaP (stage 1 in Fig. 2). Further afferent activity would cause a small reduction of [Ca2þ]e, perhaps causing an inactivation of BKCachannels (which have a lower Ca2þ affinity) switching the afterhyperpolarization (AHP) in an ADP and hence promoting firing in doublets (stage 2 in Fig. 2). Additional activity of afferents would

142 Afferent inputs activity

Glial cell activation

[Ca++]e

Membrane potential

Firing pattern

Channels activated INaT IK SKCa BKCa INaP

Depolarized –51

5

4

3

2

1 – 65 Hyperpolarized′

Fig. 2. Functional model of rhythmogenesis in NVsnpr neuron. The model shows the changes in glial cell activation and [Ca2þ]e that could occur as a result of increasing activity in afferent inputs to NVsnpr (going from stages 1 to 5) and the effects that these changes would have on neuronal membrane potential and firing pattern. The right most column shows the ionic channels activated under these different conditions and responsible for the firing pattern represented.

then cause greater Ca2þ depletion extracellularly and stronger intracellular depolarization leading to full activation of INaP and bursting (stage 3 in Fig. 2); bursting frequency being proportional to the depolarization level (stage 4 in Fig. 2). Excessive afferent activity, like would occur if the teeth encountered a hard object for instance, would depolarize the cell above INaP activation range causing burst termination and reversal to tonic firing (stage 5 in Fig. 2). According to this model, sustained activation of inputs that normally trigger mastication should elicit rhythmic bursting in NVsnpr neurons. This bursting depends on the cell membrane potential and on the [Ca2þ]e.

What are the experimental evidences in support of this model? As stated above, in in vitro slice preparations, bursting is readily observed in NVsnpr neurons when [Ca2þ]e is artificially lowered in the perfusing medium, but we showed in a recent study that under physiological [Ca2þ]e (1.6 mM) repetitive stimulation of the sensory tract containing the fiber terminals of sensory afferents (see arrows in Fig. 1), causes bursting in NVsnpr and synchronization of units (Bernier et al., 2010). Interestingly, the optimal stimulation frequency to elicit bursting was found to be between 40 and 60 Hz, a value close to the natural firing frequency (50 and 60 Hz

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during mastication) of the intraoral mechanoreceptors and periodontal receptors which project heavily to dorsal NVsnpr (Appenteng et al., 1982; Trulsson, 2007; Trulsson and Johansson, 2002) and to the optimal stimulation frequency of the cortical masticatory area to elicit mastication in vivo (20–100 Hz) (Huang et al., 1989; Lund and Dellow, 1971). Cortical and sensory fibers projecting to NVsnpr are both glutamatergic, so we used local NMDA applications as a mean of mimicking sustained activation of these fibers. In extracellular recordings, local NMDA applications first increased the firing frequency of the recorded units and triggered rhythmic burst firing after a few seconds, when the excitatory effects were wearing off (see Figure 7 of Bernier et al., 2010). NMDA-induced bursting supported by Ca2þmediated plateau potentials has been described in a number of instances before (Bertrand and Cazalets 2002; Büschges et al., 2000; Gabriel et al. 2008; Hsiao et al. 2002; Zhu et al. 2004). However, the NMDA-induced bursting in NVsnpr neurons probably involves different mechanisms because, in intracellular recordings, it occurs only when hyperpolarizing current is injected into the cell concomitantly to counteract the excessive depolarization produced by NMDA, suggesting a different voltage-dependency from classical NMDA receptors-mediated events. Also, it is insensitive to fluofenic acid (blocker of ICAN) but is readily blocked by Riluzole or TTX. Thus, the above results suggest that sustained activation of NVsnpr neurons by stimulation of their inputs or application of NMDA elicits bursting. Intense, sustained neuronal activity observed under physiological or pathological conditions has been associated to decreases of [Ca2þ]e in a number of instances (Amzica et al., 2002; Nicholson et al., 1978; Pumain and Heinemann, 1985; Pumain et al., 1983; Somjen, 1980); [Ca2þ]e drops to levels as low as 0.8 mM in the cerebellar cortex of the cat during repetitive local stimulation (Nicholson et al., 1978), 0.1 mM during cortical bursting (Somjen, 1980), and 0.08 mM in rat neocortex after local glutamate application (Amzica

et al., 2002). Moreover, bursting in many brain areas is accompanied by drastic drops of [Ca2þ]e (Benninger et al., 1980; Heinnemann et al., 1990; Rusakov and Fine, 2003; for review, see Cohen and Fields, 2004). Variations of [Ca2þ]e need not to be extreme to induce bursting. In fact, in our hands local extracellular applications of BAPTA triggered bursting in all cells tested (n ¼ 5) and this bursting was blocked by Riluzole indicating that it was mediated by INaP (unpublished results). The next question then is: what causes the [Ca2þ]e drops? Several factors can be responsible for the extracellular Ca2þ variations, but implication of glial cells is one of the first that comes to mind given the slow timecourse of the process described above. Glial cells are sensitive to surrounding neuronal activity; they have receptors for many neurotransmitters including glutamate, GABA, acetylcholine, and ATP (for review, see Verkhratsky et al., 1998), and are activated by Kþ released during neuronal firing. Glial processes ensheath synaptic elements (Grosche et al., 1999, 2002; Salpeter, 1987; Spacek, 1985) and evidence is building that glia influences neuronal activity there and at extrasynaptic sites as well by regulating the concentration of several ions (Kþ and Ca2þ among others) in the extracellular space and by releasing a number of factors or transmitters such as glutamate, D-serine, ATP, and so on (Auld and Robitaille, 2003). According to a simulation model, activation of a single calcium channel on a glial perisynaptic process could draw nearly all the Ca2þ in the synaptic cleft (Smith, 1992). Thus, we propose that glia controls the extracellular levels of Ca2þ and by this means plays a role in the onset of bursting. A feature that may be especially important in CPGs is that glial cells form extended networks through which Ca2þ waves propagate rapidly via gap junctions. This could provide a way to activate and perhaps eventually synchronize large assemblies of neurons. Bursts would cease with release of Ca2þ eventually from neurons and/or glial cells coupled to reduced sensory feedback from softened food.

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Such a mechanism would provide an explanation to an observation often made during in vivo experiments where mastication stops even when the cortical stimulation goes on. Preliminary (unpublished) results (Arsenault et al. 2007; Lavoie et al., 2009) indicate that both repetitive stimulation of the sensory fibers and local NMDA applications produce large depolarizations of glial cells recorded in dorsal NVsnpr and dramatically increase coupling between them. Interestingly, glial syncitia formed under these conditions remain confined to dorsal NVsnpr. Finally, addition of carbenoxolone (a blocker of gap junctions) to the bath disrupts NMDA-induced neuronal bursting and we assume that this effect is achieved by an action on glial gap junctions since very little coupling (if any) exists between neurons in this nucleus. Globally, these results point to a possible glial implication.

Conclusion In summary, NVsnpr neurons can be excited by cortical inputs. They also have sensory inputs from muscle spindle, periodontal, and other intraoral mechanoreceptors, which provide the feedback that is necessary for rapid adaptation of the motor pattern. Most importantly, they are capable of generating bursts within the frequency range of natural mastication, when INaP is activated. INaP activation depends on the cell membrane potential and on the [Ca2þ]e. This property enables the network to interact with the intrinsic pacemaker properties of the rhythmogenic neurons to elicit rhythmic bursting only under restricted conditions. Glial cells may contribute to this process by controlling [Ca2þ]e. In his chapter in this book, Harris-Warrick discusses the necessity of multiple and redundant mechanisms for rhythmogenesis in CPGs that are responsible for vital function. This is clearly the case in the respiratory CPG. We cannot rule out that it may also be the case in the masticatory CPG. However, we also propose that redundancy

may not be in the mechanisms as much as in the neuronal population participating to rhythmogenesis. We propose that the rhythmogenic properties reported above are present wherever INaP is present and will appear in groups of neurons that receive the appropriate level of synchronous input and that are encompassed in an organized glial syncitium. Therefore, the foyer of rhythmogenic neurons may be constantly changing (see also chapter 29) according to the inputs. It is difficult to conclude this chapter without acknowledging the tremendous contributions of Jim Lund to much of the work and the ideas described above. He is and will remain greatly missed.

Acknowledgment This work was supported by an operating grant from the CIHR and an infrastructure grant from the FRSQ.

Abbreviations aCSF ADP AHP ATP BAPTA

BKCa [Ca2þ]e CPG GABA ICAN INaP NMDA

artificial cerebrospinal fluid afterdepolarization afterhyperpolarization adenosine triphosphate 1,2-bis (o-aminophenoxy) ethane-N,N,N0 ,N0 -tetraacetic acid big Ca2þ-dependent potassium current extracellular calcium concentration central pattern generator gamma-aminobutyric acid Ca2þ-activated nonspecific cationic current persistent sodium conductance N-methyl-D-aspartic acid

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NPont NVmes NVmt NVsnpr NVspc NVspi NVspo NVII NXII P Peri V SKCa TEA TTX

nucleus pontis (caudalis and oralis) trigeminal mesencephalic nucleus trigeminal motor nucleus principal sensory nucleus of the trigeminal nerve caudalis area of the spinal nucleus of the trigeminal tract interpolaris area of the spinal nucleus of the trigeminal tract oralis area of the spinal nucleus of the trigeminal tract facial nuclei hypoglossal motor nuclei postnatal day peritrigeminal area small Ca2þ-dependent potassium currents tetraethylammonium tetrodotoxin

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 10

Axial dynamics during locomotion in vertebrates: lesson from the salamander Jean-Marie Cabelguen{,*, Auke Ijspeert{, Stéphanie Lamarque{ and Dimitri Ryczko} {

}

Neurocentre Magendie, INSERM U 862, Université de Bordeaux, Bordeaux Cedex, France { Swiss Federal Institute of Technology, Lausanne (EPFL), Lausanne, Switzerland Groupe de Recherche sur le Système Nerveux Central, Département de physiologie, Université de Montréal, Montréal, Québec, Canada

Abstract: Much of what we know about the flexibility of the locomotor networks in vertebrates is derived from studies examining the adaptation of limb movements during stepping in various conditions. However, the body movements play important roles during locomotion: they produce the thrust during undulatory locomotion and they help to increase the stride length during legged locomotion. In this chapter, we review our current knowledge about the flexibility in the neuronal circuits controlling the body musculature during locomotion. We focus especially on salamander because, as an amphibian, this animal is able to display a rich repertoire of aquatic and terrestrial locomotor modes. Keywords: Axial networks; Dynamical systems; Locomotion; Salamander.

Kashin, 1976; Jayne, 1988; Lindsey, 1978). By contrast, in appendage-based modes of locomotion, the animals use primarily their limb muscles to propel themselves (Biewener, 2003; Orlovsky et al., 1999). However, the axial musculature serves two important roles in these animals: the production of trunk bending to increase the stride length and the stabilization of the trunk. Much of what we know about axial muscle functions during locomotion is derived from studies examining animals moving within a single, homogeneous environment, at a constant speed

Introduction The function of the axial musculature during locomotion in vertebrates is of critical importance. In species using axial-based undulatory locomotion, animals (e.g., lampreys, most fish, and snakes) employ axial muscles in the production of lateral undulations of the trunk and tail to propel in aquatic and/or terrestrial medium (Grillner and * Corresponding author. Tel.: þ33-5-57-57-40-52; Fax: þ33-5-57-57-40-51 DOI: 10.1016/S0079-6123(10)87010-4

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(or across a narrow range of speeds), and along a straight track. However, animals must be able to maneuver through complex and changing external environments at varying speeds, and using different modes of locomotion (dynamic conditions). As a result, the functions of the axial musculature during locomotion have remained relatively unexplored. Among vertebrates, salamanders offer a remarkable opportunity to investigate the capacity of the neuronal circuits that control the body axis (“axial networks”) to adapt their output in response to alterations in external conditions, and to switches between locomotor modes. Indeed, adult salamanders are tetrapods capable of displaying a variety of aquatic and terrestrial locomotor modes. In water, they can both walk along the bottom and swim in the water column; while on land they can crawl, walk, or trot. Since aquatic and terrestrial environments pose different challenges for salamanders attempting to move, an analysis of the modifications of the activation patterns of the axial musculature associated with their different forms of locomotor behaviors should provide information about the flexibility of the axial locomotor networks. The goal of this chapter is to review our current understanding of the adaptive processes that occur in axial networks during locomotion in vertebrates, with examples drawn mainly from salamanders. First, we review the current data about the changes in the axial locomotor pattern in response to changes in either external conditions or internal goals. Second, neurobiological data about the neural processes underlying the flexibility of the axial locomotor networks are presented.

Diversity and variability of axial locomotor patterns Several studies have documented the patterns of activation of axial muscles during locomotion, but a few have documented their modification

in response to internal goals or external conditions. This part of the review focuses on such modifications, with examples drawn from appendage-based locomotion and axial-based locomotion.

Appendage-based locomotion Mammals Several studies have addressed the modifications of the axial locomotor patterns in mammals in relation to speed and types of gait. In cats and dogs, the transition from alternate stepping to galloping is associated with a switch from a synchronous bilateral activation of lumbar back muscles twice per cycle during alternate stepping, to once per cycle during galloping (Carlson et al., 1979; English, 1980; Tokuriki, 1974; Zomlefer et al., 1984). Moreover, in intact dogs trotting on a treadmill, the double activation pattern of epaxial muscles is more consistent at higher trotting speed than at lower trotting speed (Ritter et al., 2001). In human, the pattern of lumbar back muscle activity during both walking and running is phasic, with two bursts of activity per stride cycle on each side of the spine (Carlson et al., 1988; Cromwell et al., 2001; De Sèze et al., 2008; Saunders et al., 2005; Thorstensson et al., 1982; Waters and Morris, 1972). The two-burst pattern becomes more prominent during loading of the trunk, as also reported in the trotting dog (Ritter et al., 2001; Thorstensson, 1986). Interestingly, a recent systematic study of the activation pattern of back muscles in humans provided evidence of waves of electromyography (EMG) activity traveling posteriorly along the vertebral column during forward or backward treadmill walking (De Sèze et al., 2008). Traveling waves of lateral bending have also been reported during free overground walking in the domestic ferret (Kafkafi and Golani, 1998). In human, the effects of locomotor mode and those of speed on the pattern of activation of paraspinal muscles can be further studied separately. A transition from walking to running at

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the same speed produces substantial alterations in the pattern of activity of lumbar back muscles (Cappellini et al., 2006; Saunders et al., 2004, 2005; Thorstensson et al., 1982). Conversely, increases in speed during a given locomotor mode (walking or running) are associated with increases in the intensity and relative duration of muscle activations of paraspinal muscles, and only minor changes in their relative timings (Anders et al., 2007; Cappellini et al., 2006; Saunders et al., 2004, 2005; Thorstensson et al., 1982). Interestingly, interindividual variability is larger at low speeds than at high speeds of walking in humans (Ivanenko et al., 2009). The amplitude and the activation pattern of back muscle during stepping in mammals are shaped by the direction of progression (De Sèze et al., 2008; Hase and Stein, 1999), the physical environment (Chevutschi et al., 2007; Masumoto et al., 2004, 2005; Wada et al., 2006a, 2006b), and during voluntary modifications of the locomotor task (Carlson et al., 1988; De Sèze et al., 2008). Previous studies have demonstrated that some hypaxial muscles are phasically activated during locomotion in humans (Anders et al., 2007; Cappellini et al., 2006; Saunders et al., 2004; Waters and Morris, 1972) and dogs (Carrier, 1996; Deban and Carrier, 2002; Fife et al., 2001). However, the role of the hypaxial musculature during stepping in mammals remains poorly understood. The main reason is that this musculature performs simultaneously two functions (locomotion and lung ventilation) that are difficult to differentiate one from the other. However, hypaxial muscles with a pure or a primarily locomotor function (stabilization of the trunk against sagittal shearing torques) have been identified in dogs running on a treadmill (Deban and Carrier, 2002; Fife et al., 2001). Lower vertebrates While in mammals the pattern of activation of epaxial muscles is typically biphasic during alternated forms of stepping, lower legged

vertebrates (salamanders and lizards) show single bursting patterns per locomotor cycle (Delvolvé et al., 1997; Frolich and Biewener, 1992; Ritter, 1995, 1996). However, a comparison between salamanders and lizards reveals differences in epaxial muscle activation patterns during stepping. In salamanders, the activity pattern of the main epaxial muscles (dorsalis trunci) during walking trot conforms to the one expected for the production of lateral bending of the trunk with fixed nodes close to the girdles (“standing wave”; Delvolvé et al., 1997; Frolich and Biewener, 1992; Fig. 1). Indeed, the myomeres located between the forelimb and hindlimb (mid-trunk myomeres) express single synchronous bursts of activity, contralateral to the hindlimb support during each step cycle. Conversely, in trotting lizards, the uniphasic activation of epaxial muscles occurs ipsilateral to the hindlimb support and contributes to the dynamic stabilization, and not to the lateral bending, of the trunk (Ritter, 1995, 1996). Similarly in mammals, the epaxial muscles stabilize the trunk and pelvis against inertial loadings, and the forces applied to it by the hindlimb muscles during stepping on a level surface (Carlson et al., 1979; English, 1980; Ritter et al., 2001; Schilling and Carrier, 2009; Thorstensson et al., 1982; Tokuriki, 1973a, 1973b). It has been suggested that the difference in the activity pattern and function of the epaxial muscles may be associated with differences between vertebrate groups as to the anatomy of the epaxial musculature (i.e., myomeric vs. tripartite), the dorsoventral flexibility of the body axis, the limb position (parasagittal vs. lateral), and the locomotor habit (amphibious vs. fully terrestrial) (Ritter, 1995, 1996). In salamanders, the lateral hypaxial musculature often exhibits a double bursting pattern during stepping, with one burst (main burst) present in every step cycle and one burst (facultative burst) of lower intensity and more variable in occurrence (Bennett et al., 2001). Furthermore, the timing of the bursts within the locomotor cycle supports the view that the hypaxial

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Scapular girdle (0.31 SVL)

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Fig. 1. Comparison between the locomotor patterns during terrestrial and underwater forward stepping in the same salamander. (a) Plots of lateral displacements versus time for 11 midline marker dots during a period of 2.5 s of terrestrial (left) and underwater (right) forward trotting. The locations (drawing at left) and the lateral displacement of the markers dots are expressed in percentage of snout vent length (SVL). In each plot, zero displacement is arbitrary, corresponding to the direction of travel (thin line). Upward deflections indicate movements toward one side and downward deflections movements toward the opposite side. Data are from the same Pleurodeles waltlii (SVL: 9.60 cm). (b) Relative timing of EMG activities of ipsilateral mid-trunk and tail myomeres, and of one protractor muscle (PIFI: pubioischiofemoralis internus) of the contralateral hindlimb during terrestrial (left) and underwater (right) forward stepping cycle. Anatomical positions of the recorded muscles are indicated in drawing at left. i, ipsilateral; co, contralateral. The step cycle (abscissa) is defined as the time interval between two successive onsets of activity in the 0.64 SVL myomere. For each longitudinal location (ordinate), bars illustrate the mean duration of the burst of activity expressed as a percentage of the step cycle. Error bars indicate SEM. Terrestrial stepping: average of 18 cycles. Underwater stepping, average of 55 cycles. Note that during underwater stepping, the EMG bursts have a low amplitude (gray-filled bars), or are absent in some strides (empty bars).

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musculature plays a dual role during terrestrial locomotion in salamanders: (i) resisting torsional forces translated to the trunk by the limbs (main burst) and (ii) bending the trunk (facultative burst) (Bennett et al. 2001; Carrier, 1993; Deban and Schilling, 2009; O'Reilly et al., 2000). In lizards, some lateral hypaxial muscles are primarily involved in lateral bending of the trunk and not in long-axis stabilization of the trunk against torsional loads during terrestrial locomotion (Ritter, 1995, 1996). Several studies have provided evidence of a high degree of variability in the axial locomotor pattern in salamanders, depending on gait and physical environment (e.g., aquatic vs. terrestrial; dry vs. slippery surface). During forward stepping on land, combinations of standing and traveling mechanical waves have been reported in salamanders depending on the speed of locomotion or type of gait (Ashley-Ross, 1994a; Edwards, 1977). It has further been suggested that the more reduced the limbs are, the slower the speed at which the traveling waves appear, that is, salamanders with very reduced limbs rely more on traveling waves on ground, while those with stronger limbs mainly use standing waves (Ashley-Ross, 1994a). Interestingly, salamanders with reduced forelimbs and no hindlimbs (e.g., the aquatic Siren lacertina) combine alternating use of the forelimbs with traveling undulatory waves during aquatic walking (Azizi and Horton, 2004). Similar observations have been previously reported in lizards (Daan and Belterman, 1968; Ritter, 1992, 1996). Salamanders use either standing or head-to-tail traveling waves of axial undulation and epaxial muscle activity during straightforward stepping performed along a submerged trackway (i.e., during reduced gravity and increased viscosity; Fig. 1; Lamarque et al., 2009; see also Deban and Schilling, 2009). Interestingly, the variability in the mid-trunk pattern (Lamarque et al., 2009) and in limb kinematics (Ashley-Ross et al., 2009) is greater during aquatic than during terrestrial stepping. The higher variability of the motor

pattern during aquatic stepping likely results from reduced constraints on the underlying substratum because of buoyancy. Furthermore, the tail EMG activity is absent or very weak during underwater forward stepping in a straight line, although the concomitant tail movements are large (Fig. 1). This suggests that the tail movements during aquatic straightforward stepping mainly result from a passive transmission of the active mid-trunk movements to the tail. This further supports the view that the part of the axial network generating the tail movements can be functionally decoupled from that controlling the mid-trunk movements (Delvolvé et al., 1997). In contrast, during straightforward stepping on a wet surface, tail myomeres typically display an additional burst of activity in phase with every ipsilateral mid-trunk burst (Delvolvé et al., 1997). This double bursting pattern can help the animal to maintain the tail as much as possible aligned to the direction of movement during straightforward stepping (Ashley-Ross, 1994a, 1994b; Roos, 1964). A similar stabilizing function of the head has been attributed to the myomeres located rostrally to the scapular girdle (AshleyRoss, 1994a, 1994b; Roos, 1964). Salamanders can spontaneously exhibit short episodes of backward stepping during maneuvering on land. Thereafter, they switch to a more efficient forward stepping. Much longer episodes of backward stepping can be induced by training the animal to walk backward on a motorized treadmill. In these conditions, backward walking involves a very different and more variable hindlimb motor pattern than that observed during forward walking (Ashley-Ross and Lauder, 1997). Unfortunately, the precise bending pattern of the trunk (i.e., the presence of traveling or standing waves) and the EMG pattern of axial muscles have not been investigated. The flexibility of the spinal pattern-generating networks can also be revealed by comparing the axial motor pattern in restrained and freestepping salamanders. When grasped by the pelvic girdle during ongoing overground stepping,

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salamanders respond with slow, strong rhythmic trunk movements characterized by waves of undulations and epaxial muscle activity traveling from tail to head (Lamarque et al., 2009). This motor behavior is quite similar to that exhibited during struggling behavior in the tadpole (Kahn and Roberts, 1982) and zebrafish (Liao and Fetcho, 2008). In conclusion, the studies of pattern of activity of axial muscles during stepping in various limbed vertebrates have demonstrated some aspects of the dynamics of the stepping networks which allows organisms to adapt to the various demands of a particular situation, such as speed and physical environment.

Axial-based locomotion In lampreys (Davis et al., 1993; Williams, 1986), eels (Gillis, 1998a; Grillner and Kashin, 1976), larval zebrafish (Budick and O'Malley, 2000), Xenopus tadpoles (Kahn et al., 1982; Soffe et al., 1983), and in some snakes (Jayne, 1988), steady undulatory swimming in water is characterized by waves of lateral displacement traveling from head to tail. The waves of lateral bending are produced by epaxial muscle activations which alternate between the two sides of the body, and travels caudally with a constant speed (Gillis, 1998b; Grillner and Kashin, 1976; Hoff and Wasserburg, 2000; Wallén and Williams 1984). The speed of swimming is mainly modulated by the frequency and the amplitude of the body undulations which are controlled by both recruitment of new motor units and increase in their firing frequency (Gillis 1998b; Grillner and Kashin, 1976; McLean et al., 2007). In water, salamanders are capable of rapidly switching from forward stepping to forward swimming. The swimming mode is similar to that of lampreys and eels, with axial undulations being propagated as traveling waves from head to tail (Carrier, 1993; D'Août and Aerts, 1997; Daan and Belterman, 1968; Deban and Schilling, 2009;

Frolich and Biewener, 1992; Gillis, 1997; Roos, 1964). The amplitude of these axial undulations typically increases along the body of the salamander. Such a swimming mode is called anguilliform swimming and does not involve limb movements: limbs are folded backward along the body. As in the lamprey, the average wavelength usually corresponds to the length of the body (i.e., the body produces one complete wave per cycle) and does not vary with the frequency of oscillation (D'Août and Aerts, 1997; Frolich and Biewener, 1992). An interesting difference between the two locomotor modes of the salamander (forward swimming and forward stepping) is that the frequency of the swimming movements is generally two to three times higher than that of the trotting movements (Bennett et al., 2001; Deban and Schilling, 2009; Delvolvé et al., 1997; Frolich and Biewener, 1992). There is generally no overlap of frequencies between the two locomotor modes (i.e., no slow frequency swimming or high-frequency trotting). Previous EMG data are consistent with the hypothesis that the dorsalis trunci muscles and hypaxial muscles act synergistically to produce lateral bending of the body during salamander swimming (Bennett et al., 2001; Carrier, 1993; Deban and Schilling, 2009; Delvolvé et al., 1997; Frolich and Biewener, 1992). The EMG recordings from the dorsalis trunci muscles further revealed that nonuniformities in both the intersegmental coordination pattern and the speed propagation of the EMG traveling waves occur at two specific trunk positions, one located caudal to the pectoral girdle and the other close to the pelvic girdle (Delvolvé et al., 1997; Frolich and Biewener, 1992). These nonuniformities have been related to the presence of the limbs (Delvolvé et al., 1997), since they have not been observed in anguilliform swimmers without paired fins (lamprey) or with reduced pectoral fins and no pelvic fins (eel) (Gillis, 2000; Grillner and Kashin, 1976; Williams et al., 1989). Although the EMG waves have a nonuniform propagation speed along the body, the propagation speed of lateral bending appeared to be constant along the entire length of the salamander

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(D'Août and Aerts, 1997; Frolich and Biewener, 1992). This suggests that the locomotor command is well matched to the mechanical properties of the body and to hydrodynamical forces applied along the body, in order to provide mechanical waves with constant propagation speed during swimming. In salamanders (D'Août et al., 1996; Frolich and Biewener, 1992), lampreys (Williams et al., 1989), and eels (Gillis and Blob, 2001; Grillner and Kashin, 1976), the waves of EMG activity travel down the body faster than the mechanical waves of body curvature. As a consequence, a progressive, posteriorly increasing delay occurs between the timing of muscle activity and lateral bending (or muscle strain). It has been suggested that axial musculature earlier in its muscle strain cycle can provide a mechanism for increasing power production or stiffening the tail for improving transmission of propulsive forces (see, e.g., Altringham and Ellerby, 1999; D'Août et al., 1996; Ellerby et al., 2001; Williams, 1986). Some studies in eels and lampreys have shown that the timing between EMG activity and lateral bending depends on the external environment (aquatic vs. terrestrial). Indeed, similar to swimming, eels use waves of lateral bending traveling from head to tail to produce propulsive thrust during undulatory locomotion on land (crawling) (Gillis, 1998a). However, the epaxial muscles are activated much more strongly and later in their strain cycle during crawling than during swimming (Gillis, 2000; Gillis and Blob, 2001). Furthermore, when a lamprey is taken out of the water and placed on a wet bench, it tends to make undulations which look almost like standing waves because the lateral displacement does not increase along the body but forms quasi-nodes (i.e., points with very little lateral displacements) at specific points along the body (Bowtell and Williams, 1994). These data support the view that the sensory feedback plays a key role in the operating mode of the axial locomotor networks in order to adjust the stiffness of the animal body relative to the

compliance of the environment (Biewener and Gillis, 1999; Gillis and Blob, 2001). Salamanders sometimes use a terrestrial crawling resembling swimming movements performed on the ground (i.e., with limbs folded against the body; Edwards, 1977). However, it is not known whether these “terrestrial swimming movements” are generated by caudally directed waves of EMG activity, as previously reported during terrestrial locomotion in the eel (Gillis and Blob, 2001). The study of swimming in an aquatic medium of increased viscosity and in water stream should also provide further insight on how the environment affects the neuromuscular control of aquatic locomotion in salamanders. Lampreys (Islam et al., 2006; McClellan, 1989) and eels (D'Août and Aerts, 1999) can display brief episodes of backward undulatory swimming (e.g., when encountering an obstacle). By contrast, as in most vertebrates, backward undulatory swimming has never been observed in salamanders. Backward swimming in the lamprey is characterized by waves of epaxial muscle activations which propagate rostrally along the body, with a speed higher than that of the associated kinematic waves (Islam et al., 2006). Therefore, backward swimming seems to result from a functional reversal of the operating mode of the axial locomotor network generating forward swimming (Matsushima and Grillner, 1992). Backward swimming, however, shows important kinematic differences from forward swimming. The snout shows considerable lateral deflections during backward, but not during forward swimming, and the cycle duration during backward swimming is much longer than during forward swimming (D'Août and Aerts, 1999; Islam et al., 2006). These kinematic differences likely reflect the asymmetry of the body mechanics (e.g., heavy head/light tail) which contribute to the translation of the locomotor drive into effective movements. Salamanders can use other specialized forms of aquatic locomotion such as paddling behavior (Ashley-Ross, 1994a, 1994b; Delvolvé et al., 1997; Frolich and Biewener, 1992) and steering

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behavior in three-dimensional space which have received much less attention. It would be interesting to investigate the axial motor patterns underlying these locomotor behaviors since this may help us to get a deeper insight into the flexibility of the axial networks in salamanders.

Neural mechanisms underlying the flexibility of axial networks During locomotion in vertebrates, the rhythmic and coordinated activations of axial and limb muscles are generated by neural networks located in the spinal cord and called central pattern generators (CPGs) (Grillner, 1981). It is convenient to distinguish the network controlling axial motion (axial locomotor CPG) from those controlling limb motions (limb locomotor CPGs; Ijspeert, 2008).

Architecture of the axial locomotor CPG The axial locomotor CPGs have been extensively investigated in limbless, swimming vertebrates. A combination of neurobiological and modeling work in the lamprey (Grillner, 2006) and the Xenopus laevis embryo (Roberts et al., 1997) has demonstrated the segmental structure for the axial CPG for swimming, with a double chain of reciprocally coupled identical oscillators (pools of neurons that exhibit a rhythmic activity) distributed all along the spinal cord, and mutually coupled by local and long distance intersegmental coordinating systems. These data further suggest that each hemisegmental oscillator is a pool of excitatory (glutamatergic) neurons generating recurrent bursting, coupled with a pool of inhibitory (glycinergic) neurons to ensure inhibition of the controlateral hemisegment, and generate left–right alternation (Cangiano and Grillner, 2005; Roberts et al., 2008). The left–right inhibitory connections also lower the burst frequency of the axial network and make the oscillations more robust.

By contrast, there is little information about the organization of the axial locomotor CPGs in limbed vertebrates. Recent experiments on surgically isolated segments or hemisegments from the axial spinal cord of adult salamanders provide direct evidence of a strong similarity of the global architecture, and operating mode of the axial CPG of the salamander with that of the swimming CPG of the lamprey (Ryczko et al., 2010). Our previous modeling studies also support this proposition (Bem et al., 2003; Ijspeert et al., 2007). However, some differences between salamanders and lampreys as to the intrinsic neuronal properties underlying the bursting of spinal segments have been observed. For example, the mechanism for terminating each burst does not involve the Ca2þ-activated Kþ channels in salamanders (Ryczko et al., 2010), while it does in lampreys (El Manira et al., 1994). Taken together, these results support the hypothesis that the basic design of the axial locomotor CPG has been conserved during evolution, albeit with different neural mechanisms for bursting (Falgairolle et al., 2006; Grillner, 2006; Ryczko et al., 2010).

Adaptive mechanisms in the axial locomotor CPG Propagated waves of motor activity along the spinal cord have been observed during chemically induced locomotor-like activity in isolated spinal cords of adult lampreys, adult salamanders, and newborn rodents (Bonnot et al., 2002; Delvolvé et al., 1999; Falgairolle and Cazalets, 2007; Wallén and Williams, 1984; see also Cuellar et al., 2009). A systematic investigation of the different patterns of ventral root activity produced by the in vitro isolated spinal cord of salamander has revealed a high degree of flexibility in the intersegmental coordination pattern, that is, in the operating mode of the axial networks (Ryczko et al., 2009). Intersegmental phase lags range from positive values (i.e., backward propagating waves) to

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negative values (i.e., forward propagating waves), including all the values which can be observed in vivo (Ryczko et al., 2009). A similar flexibility in the operating mode of the axial networks has previously been observed in the lamprey (Matsushima and Grillner, 1992), and to a lesser extent, in the tadpole (Green and Soffe, 1996). The flexibility in the intersegmental phase lag value has been related to the diversity of the locomotor behaviors observed in vivo (Green and Soffe, 1996; Matsushima and Grillner, 1992; Ryczko et al., 2009). Interestingly, during pharmacologically induced fictive locomotion in the high spinal cat, the muscle nerves innervating the lumbar back muscles also display a variety of rhythmic patterns corresponding to the different gaits observed in freely moving animals (Koehler et al., 1984). The neuronal mechanisms underlying the flexibility in the axial locomotor CPG are not fully understood, although several biological and modeling studies suggest that the descending and movement-related sensory inputs play a critical role by regulating the coupling between segmental bursting units (Friesen and Cang, 2001; Grillner and Wallén, 2002).

Coupling of axial and limb locomotor CPGs During locomotion, the axial and limb networks are working together to increase the propulsive efficiency. In vertebrates, the limb locomotor networks are located within the cervical segments for the forelimbs (Ballion et al., 2001) and within the lower thoracic-upper lumbar segments for the hindlimbs (for review see Kiehn, 2006). Spinal sections and pharmacological manipulations further show that these regions can be separated into left and right neural networks, which independently coordinate each limb, and are connected by reciprocal (inhibitory and excitatory) connections (for review, see Butt et al., 2002). The reciprocal excitatory connections have been proposed to be functionally important for in-phase

gaits. In salamander, they might be involved during the synchronous tonic activation of a pair of limbs during swimming or for in-phase paddling. The bursting frequency of the surgically isolated two sides of the spinal cord is higher than that of the intact spinal cord in salamanders (Ryczko et al., 2009), lampreys (Cangiano and Grillner, 2003), and Xenopus embryos (Soffe, 1989). In contrast, the bursting frequency of surgically or pharmacologically uncoupled two halves of the lumbosacral spinal cord is lower than that of the intact spinal cord in neonatal rodents (Bracci et al., 1996; Kjaerulff and Kiehn, 1997; Kudo and Yamada, 1987; Whelan et al., 2000; see however Kremer and Lev-Tov, 1997) and turtles (Samara and Currie, 2007). This difference may reflect a species difference and/or a difference in the left–right coupling mechanisms within axial and limb oscillatory networks. However, more experiments are needed to determine the contribution of developmental changes to the left–right coordinating mechanisms in rodents (Nakayama et al., 2002). Studies on surgically isolated spinal segments in salamander provide evidence that the intrinsic frequency of the segmental networks controlling the limb musculature is lower than that of the segmental network controlling the axial musculature (Ijspeert et al., 2007). The axial and the hindlimb locomotor CPGs can be coupled in different ways and the mode of coupling can be changed by peripheral inputs in high decerebrate cats (Koehler et al., 1984). It is reasonable to postulate a similar flexibility for the coupling between axial and forelimb locomotor CPGs. A flexibility of the coupling of limb CPGs has also been revealed in several vertebrates, depending on descending and peripheral inputs (reviewed in Pearson, 2000). The flexibility of the couplings (axial/limb and limb/limb) not only enables a spinal adaptation of locomotion to different external conditions but may also serve as a basis for the use of these spinal facilities by higher centers for the performance of complex goal-directed movements (Grillner et al., 2008).

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As an example, the mesencephalic locomotor region (MLR) can activate (via reticulospinal neurons), depending on strength, specific spinal networks to produce motor synergies underlying specific locomotor modes (Cabelguen et al., 2003; Steeves et al., 1987). In salamanders, walking occurs during electrical microstimulation of the MLR at low intensity, whereas swimming requires higher intensities (Cabelguen et al., 2003). Our previous numerical simulations (Ijspeert et al., 2007) suggest that the limb oscillators are recruited at low intensity and drive the axial network at a low frequency (walking). At higher intensity, the limb oscillators become silent (saturate), and release the faster swimming rhythms due to the higher intrinsic frequencies of the body oscillators. This is consistent with swimming being the fastest mode of locomotion and also explain why salamander stepping and swimming frequencies do not overlap (Delvolvé et al., 1997; Frolich and Biewener, 1992), but have distinct ranges with a gap between them. The exact mechanism underlying this induced gait transition is still unknown. One possible explanation is that the saturation of the limb oscillator is a spinal mechanism (i.e., it is an intrinsic property of the oscillator), another possible explanation is a gating mechanism in the reticular formation and/or the MLR (i.e., the descending drive is not transmitted to the limb oscillator when it exceeds some threshold).

Conclusion This review provides a brief survey of data from various vertebrates which emphasize the importance of considering locomotion as a complex motor task involving not only the limbs but also the body axis. These data also support the view that the axial locomotor networks are dynamical systems. The dynamics of the axial networks, in combination with that of the limb networks, allows the generation of robust and stable muscle synergies appropriate for locomotion in a continuously varying environment.

One important goal for future research is to investigate the mechanisms that enable flexibility in the axial motor output depending on the locomotor task. This should be evaluated by considering the complex interplay between the central nervous system, the sensory receptors, the body dynamics, and the environment. A promising approach is to combine neurobiological experiments, modeling, and robotics, as we have previously done in the salamander (Ijspeert et al., 2007).

Acknowledgment The support from European Community (LAMPETRA Grant: FP7-ICT-2007-1-216100) and ANR (ImNet Grant: ANR-07-NEURO-015-01) is gratefully acknowledged.

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 11

Recruitment of masseter motoneurons by the presumed spindle Ia inputs Youngnam Kang*, Mitsuru Saito, Hiroki Toyoda and Hajime Sato Department of Neuroscience & Oral Physiology, Osaka University Graduate School of Dentistry, Yamadaoka, Suita, Osaka, Japan

Abstract: The slow-closing phase of the mastication cycle plays a major role in the mastication of foods. However, the neuronal mechanism underlying the slow-closing phase remains unknown. The isometric contraction of jaw-closing muscles is developed through the recruitment of jaw-closing motoneurons during the slow-closing phase. It is well established that motor units are recruited depending on the order of sizes or input resistances (IRs) of motoneurons, which is known as the size principle. Two-pore-domain acid-sensitive Kþ (TASK1/3) channels are recently found to be the molecular correlates of the IR, and also found to be expressed in the masseter motoneurons. Here, we addressed the question whether spindle Ia inputs onto masseter motoneurons can induce the orderly recruitment of motoneurons in slice preparations of the rat brain using voltage-sensitive dye imaging and whole-cell patch-clamp methods. Voltage-sensitive dye imaging revealed the recruitment of many motoneurons in the whole nucleus of masseter in response to repetitive stimulation of the presumed spindle Ia inputs. Dual whole-cell recordings obtained from two adjacent motoneurons revealed the IR-ordered recruitment of motoneurons in response to repetitive stimulation of the presumed spindle Ia inputs. Thus, Ia inputs are likely to play a crucial role in the orderly recruitment of motoneurons of the trigeminal motor nucleus, which would be progressed during the slow-closing phase of the mastication cycle. Possible involvements of TASK channels in the orderly recruitment are discussed. Keywords: slow-closing phase; isometric contraction; orderly recruitment; muscle spindle; masseter motoneuron; TASK channel.

* Corresponding author. Tel.: þ81-66879-2881; Fax: þ81-66879-2885 DOI: 10.1016/S0079-6123(10)87011-6

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Development of isometric contraction during the slow-closing phase The vertical movement of the jaw during rhythmic masticatory movements can be represented as masticatory cycles, each of which consists of three phases: fast- and slow-closing phases and opening phase (Lund and Olsson, 1983; Fig. 1). Jaw-closing masseter motoneurons display membrane depolarization and subsequent firing during the fast-closing phase, and display membrane hyperpolarization during the opening phase. Jaw-opening digastric motoneurons only display membrane depolarization during the opening phase. These membrane potential changes are caused by central pattern generator (CPG; Lund and Olsson, 1983). During the slow-closing phase of the masticatory cycle, the length of the jaw-closing muscles remains almost constant. Therefore, the muscle contraction during the slow-closing phase can be regarded as the isometric contraction. In association with the progress of the slow-closing phase, the isometric contraction

force of jaw-closing muscles increased, as is reflected in the increases in the electromyogram (EMG) activity of these muscles. However, it is not clear whether the synaptic inputs from CPG caused these increases in EMG activity, because the removal of periodontal inputs or spindle Ia afferents decreases the EMG activity during the slow-closing phase (Lavigne et al., 1987; Morimoto et al., 1989). Indeed, during the slow-closing phase, the activity of muscle spindles increased, which in turn caused increases in the firing activity of periodontal mechanoreceptors (Cody et al., 1975; Luschei and Goldberg, 1981; Taylor et al., 1981).

Orderly recruitment of masseter motoneurons during isometric contraction by the activity of g-motoneurons It is well established that during isometric contraction, motor units are recruited depending on the order of sizes or input resistances (IRs) of

Fig. 1. Relationships between mastication phases and relevant neuronal and muscle activities. Development of isometric contraction of masseter muscle seen during the slow-closing phase, in association with the increased activities of periodontal mechanoreceptors and muscle spindle. Mass, masseter muscle; Dig, digastric muscle; RA, rapidly adapting; SA, slowly adapting; Pri, primary ending; Sec, secondary ending (Adapted from Lund and Olsson, 1983).

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motoneurons, which is known as the size principle (Henneman, 1991). It has also been reported that in the human lumbrical muscle, Ia discharge is evoked as soon as the isometric tension is increased ramp-likely, and is maintained constant throughout the contraction (Fig. 2; Vallbo, 1970). This Ia discharge may be caused by the activity of g-motoneurons because the muscle length remained constant. Then, the isometric contraction may be developed by the activity of gmotoneurons. Indeed, it is reported that an activation of stretch reflex pathway caused the rank-ordered recruitment of motor units (Bawa et al., 1984; Calancie and Bawa, 1985). Therefore, it is possible that the rank-ordered recruitment of motor units during isometric contraction is at least partly caused by spindle discharges that are produced by the activity of g-motoneurons (Fig. 3). There may be a common neuronal mechanism underlying the isometric contraction between lumbrical and jaw-closing muscles. In fact, it was reported that Ia afferents show high activities during the isometric contraction due to the coactivation of dynamic g-motoneurons in the trigeminal motor nucleus (TMN) with the a-motoneurons (Larson et al., 1981; Lund et al., 1979). Thus, it is possible that the activity of g-motoneurons is crucial for (a)

the generation of isometric contraction during the slow-closing phase.

Specialized stretch reflex circuit of jaw-closing muscles In contrast to the role in limb movement, the role of g-motoneurons is considered to be very special in jaw-closing movement because of the difference in the stretch reflex circuit between large limb muscle and jaw-closing masseter muscle (Fig. 4). In masseter muscle, the number of intrafusal fibers included in single muscle spindle was found to be extremely large, up to 36 (Fig. 4a; Eriksson et al., 1994), while the number of synapses between Ia afferents and a-motoneurons is much smaller (Fig. 4a; Dessem et al., 1997; Yabuta et al., 1996), comparing with the limb muscle (Fig. 4b; Redman and Walmsley, 1983a, 1983b). Thus, it is likely that in limb muscle, the spatial summation of Ia-excitatory postsynaptic potentials (EPSPs) would easily activate a-motoneurons, while in masseter muscle, the temporal summation of Ia-EPSPs would be required to activate a-motoneurons, as reflected in the difficulty in evoking H-reflex in resting masseter muscles (Fujii and Mitani, 1973). Motor units (b)

(c) DRG

la activity

DRG

(Hz) 30–

Instant. freq. a

0

Tension 2s

g

L

a g

EMG Load B

C

Fig. 2. a–g linkage. (a) During voluntary isometric contraction of human lumbrical muscles, a constant discharge activity of spindle Ia afferent fibers was produced as soon as EMG activity and muscle tension were increased (adapted from Vallbo, 1970). Diagram of a–g linkage during resting state (b) and (c) during isometric contraction, muscle length (L) is kept constant, indicating that g-motoneuron (g) activates Ia sensory endings through the contraction of intrafusal fibers. DRG, dorsal root ganglion.

166 JCMN pool in MotV

MesV

CPG

? a MN

a MN

a MN

small

large

g MN

La fiber (H-reflex arc)

Periodontal Extrafusal mechanoreceptor muscle

Muscle spindle

Masseter Fig. 3. A hypothesized model showing that the isometric contraction during the slow-closing phase is developed by the recruitment of masseter motoneurons through the activation of Ia inputs. MTN, mesencephalic trigeminal nucleus; CPG, central pattern generator.

(a)

of human jaw-closing muscles were orderly recruited during voluntary isometric contraction (Yemm, 1977). Then, it can be hypothesized that the isometric contraction during the slow-closing phase is developed by the recruitment of masseter a-motoneurons that can be caused by the activity of g-motoneuron through Ia inputs, although the driving synaptic inputs to g-motoneuron has not been known yet. We aimed to investigate the neuronal mechanisms underlying the recruitment of masseter a-motoneurons, which would mediate the development of isometric contraction seen during the slow-closing phase of masticatory jaw movements. Whole-cell patch-clamp recording and optical recording were performed in in vitro slice preparation of rat brain, which were cut at 15 tilted rostrally against the coronal plane (Verdier et al., 2003; Fig. 5). As reported by Shigenaga et al. (1990), stem axon from the mesencephalic trigeminal nucleus (MTN) does not directly enter the TMN, but only axon collaterals arising from a single stem axon make synaptic connections onto almost all jaw-closing motoneurons in the dorsolateral area of the TMN. In such transverse slices, the stem axon of an MTN neuron sends the peripheral axon ventrolaterally to, and the central axon ventromedially to, the TMN. Therefore, we placed the monopolar tungsten electrode on the (b) DRG

MTN 10−20

2−3 a

a

g

g −36

2−10

Fig. 4. Differences in the stretch reflex circuit between masseter (a) and limb (b) muscles. A muscle spindle in masseter muscles contains many intrafusal fibers (up to 36, in human), while a spindle in limb muscles contains a few fibers. In contrast, the number of synaptic connections between a single Ia fiber and a a-MN innervating masseter muscles is much smaller than that innervating limb muscles.

167

and Appenteng, 1995), 10 successive stimuli were applied at 100 Hz to the presumed spindle Ia fibers through a tungsten microelectrode. With repetition of stimulation, the number of excited jaw-closing motoneurons or the excited area gradually increased within the TMN. Finally, in response to 10th stimulation, the whole motoneuron pool was excited (unpublished data). This result indicates that the recruitment of jaw-closing motoneurons can be caused through the temporal summation and facilitation of Ia inputs, although the order of recruitment is not clear.

Orderly recruitment of jaw-closing motoneurons by Ia-EPSPs

Fig. 5. Experimental setup in slice preparation. Collaterals of central axons of an MTN neuron invades into the TMN from the dorsomedial border of the nucleus, and makes synaptic connections onto almost all jaw-closing motoneurons. Monopolar tungsten electrode was placed at the dorsomedial border of TMN to stimulate the fibers from MTN.

axon bundle of MTN neurons just dorsomedial to TMN, to stimulate the Ia inputs to jaw-closing motoneurons. To investigate whether the recruitment occurs in response to stimulation of the presumed Ia inputs in brain slices, we performed optical recording using voltage-sensitive dye. Recruitment of jaw-closing motoneurons by temporal summation and facilitation of Ia inputs Since Ia-EPSPs evoked in jaw-closing motoneurons display facilitation in response to 50–200 Hz stimulation of Ia inputs (Grimwood

As schematically shown in Fig. 6, the rank-ordered recruitment of motor units can be seen during muscle stretch. In Fig. 6a, the larger amplitude of impulses represents larger motor units, and they are recruited as the muscle length is increased. The smaller neurons are more easily activated than larger neurons because of their larger IR. Therefore, with an increase in impulse frequencies in Ia afferents, motoneurons are recruited in a manner dependent on their IR. To investigate whether jaw-closing motoneurons are recruited in a rank-order manner, we performed simultaneous recordings from a pair of motoneurons. When simultaneous recordings were obtained from a pair of smaller and larger motoneurons visible under Nomarski optics, the IR was larger in the smaller MN than in the larger MN. The apparent spike threshold was always lower in the smaller MN than in the larger one (unpublished data). In response to 100 Hz stimulation of the presumed afferent axons from MTN, activation of the smaller MN invariably preceded the activation of the larger MN in response to stimulation of Ia inputs, regardless of stimulus intensities (unpublished data). Thus, it is likely that jaw-closing motoneurons were activated or recruited in a manner dependent on their IRs in response to stimulation of Ia inputs.

168 (b) Muscle stretch

(a)

Motor unit 1 2 Tension

3

g activity or muscle stretch

2 1

1 Muscle spindle

2

la I1

3 4

3

R

a

a

R

I3

R

b

b

I2

c

c

5 Ra > Rb > Rc E= IR

∴

Ea

> Eb > Ec

I1

< I2 < I3

Fig. 6. Rank-ordered recruitment of motor units seen not only during voluntary isometric contraction but also during stretch reflex. (a) With an increase in muscle stretch, motoneurons are recruited in a manner dependent on motor unit size. (b) With an increase in muscle stretch or Ia impulses, motoneurons are recruited (filled in gray) in a manner dependent on their input resistance. R, resistance; I, current; E, voltage.

Possible involvements of leak Kþ channels, TASK1/3, in IR-ordered recruitment What is the molecular mechanism critical for rankordered recruitment? It is known that leak Kþ currents play an essential role in determining the IR. The family of two-pore (2P)-domain Kþ channels provides molecular correlates for physiologically identified leak Kþ conductances (Duprat et al., 1997; Goldstein et al., 2001; Patel and Honore, 2001). 2P-domain Kþ channels are subdivided into two groups: 2P-domain weak inward rectifier Kþ channels (TWIK) and TWIK-related channels. Among 2P-domain Kþ channels, TWIKrelated acid-sensitive Kþ (TASK) channels, TASK1 and TASK3, are the most likely candidates for the leak Kþ channels in various central neurons (Goldstein et al., 2001; Kang et al., 2004; Meuth et al., 2003; Millar et al., 2000; Patel and Honore, 2001; Sirois et al., 2000; Talley et al., 2000). Since it is already known that TASK1 and TASK3 channels are strongly expressed in trigeminal motoneurons (Karschin et al., 2001; Talley et al., 2000), it is likely that TASK channels play an important role in rank-ordered

recruitment during the slow-closing phase. Singlechannel conductance of TASK3 channels is twice as large as that of TASK1 channels, and there was a difference in the pH-sensitivity between TASK1 and TASK3 channels (Lesage, 2003). At physiological pH, the open probability of TASK3 channels is almost maximal while that of TASK1 channels is 30–40% of the maximal probability, although the open probability of both TASK1 and TASK3 channels increases with pH increases (Lesage, 2003). Therefore, it is very important to address how differentially TASK1/3 channels are distributed in motoneurons depending on their sizes.

Modulation of orderly recruitment by alteration of TASK channels activity TASK channels are inhibited by many neuromodulators that activate Gq-coupled receptors and by local anesthetics and proton (Bayliss et al., 2003; Lesage, 2003). By contrast, endogenous neuromodulators activating TASK channels in neurons remained unknown, although TASK1

169 (b)

(a) Local anesthetics

General anesthetics

MTN

H+

Nitric oxide (NO)

TASK1/3 channel mGluR1 Endogenous neuromodulator TASK1

Gq a MNs in TMN

Hypoxia Inhibition Activation

Fig. 7. Modulation of leak Kþ (TASK) channels. (a) TASK1 channels are known to be inhibited by many neuromodulators in addition to local anesthetics and Hþ, and activated by volatile anesthetics. We recently found that nitric oxide, one of endogenous neuromodulators, activates TASK1 channels in the basal forebrain cholinergic neurons. (b) TASK1 and TASK3 channels could be modulated by endogenous neuromodulators, affecting the order and extent of recruitment.

channels are activated by general anesthetics such as halothane and sevoflurane (Bayliss et al., 2003; Lesage, 2003). However, we have recently found that TASK1-mediated leak Kþ currents in basal forebrain cholinergic neurons were activated by nitric oxide (NO) signaling through cGMP/ cGMP-dependent protein kinase (PKG) transduction pathway (Kang et al., 2007; Toyoda et al., 2008; Toyoda et al., 2010). Cholinergic neurons located in the pedunculopontine and laterodorsal tegmentum nuclei and the ventromedial medullary reticular formation are known to be a source of nitrergic input to the TMN (Pose et al., 2005; Travers et al., 2005). Therefore, it is possible that NO can modulate TASK channels expressed in the TMN, thereby affecting the order and extent of the recruitment of masseter motoneurons (Fig. 7). This remains to be determined in future experiments. References Bawa, P., Binder, M. D., Ruenzel, P., & Henneman, E. (1984). Recruitment order of motoneurons in stretch reflexes is highly correlated with their axonal conduction velocity. Journal of Neurophysiology, 52(3), 410–420.

Bayliss, D. A., Sirois, J. E., & Talley, E. M. (2003). The TASK family: Two-pore domain background Kþ channels. Molecular Interventions, 3(4), 205–219. Calancie, B., & Bawa, P. (1985). Voluntary and reflexive recruitment of flexor carpi radialis motor units in humans. Journal of Neurophysiology, 53(5), 1194–1200. Cody, F. W., Harrison, L. M., & Taylor, A. (1975). Analysis of activity of muscle spindles of the jaw-closing muscles during normal movements in the cat. The Journal of Physiology, 253 (2), 565–582. Dessem, D., Donga, R., & Luo, P. (1997). Primary- and secondarylike jaw-muscle spindle afferents have characteristic topographic distributions. Journal of Neurophysiology, 77(6), 2925–2944. Duprat, F., Lesage, F., Fink, M., Reyes, R., Heurteaux, C., & Lazdunski, M. (1997). TASK, a human background Kþ channel to sense external pH variations near physiological pH. The EMBO Journal, 16(17), 5464–5471. Eriksson, P. O., Butler-Browne, G. S., & Thornell, L. E. (1994). Immunohistochemical characterization of human masseter muscle spindles. Muscle Nerve, 17(1), 31–41. Fujii, H., & Mitani, H. (1973). Reflex responses of the masseter and temporal muscles in man. Journal of Dental Research, 52(5), 1046–1050. Goldstein, S. A., Bockenhauer, D., O'Kelly, I., & Zilberberg, N. (2001). Potassium leak channels and the KCNK family of two-P-domain subunits. Nature Reviews Neuroscience, 2(3), 175–184. Grimwood, P., & Appenteng, K. (1995). Effects of afferent firing frequency on the amplitude of the monosynaptic EPSP elicited by trigeminal spindle afferents on trigeminal motoneurones. Brain Research, 689(2), 299–303.

170 Henneman, E. (1991). The size principle and its relation to transmission failure in Ia projections to spinal motoneurons. Annals of the New York Academy of Sciences, 627, 165–168. Kang, Y., Dempo, Y., Ohashi, A., Saito, M., Toyoda, H., Sato, H., Koshino, H., Maeda, Y., & Hirai, T. (2007). Nitric oxide activates leak Kþ currents in the presumed cholinergic neuron of basal forebrain. Journal of Neurophysiology, 98 (6), 3397–3410. Kang, D., Han, J., Talley, E. M., Bayliss, D. A., & Kim, D. (2004). Functional expression of TASK-1/TASK-3 heteromers in cerebellar granule cells. The Journal of Physiology, 554(1), 64–77. Karschin, C., Wischmeyer, E., Preisig-Muller, R., Rajan, S., Derst, C., Grzeschik, K. H., Daut, J., & Karschin, A. (2001). Expression pattern in brain of TASK-1, TASK-3, and a tandem pore domain Kþ channel subunit, TASK-5, associated with the central auditory nervous system. Molecular and Cellular Neuroscience, 18(6), 632–648. Larson, C. R., Smith, A., & Luschei, E. S. (1981). Discharge characteristics and stretch sensitivity of jaw muscle afferents in the monkey during controlled isometric bites. Journal of Neurophysiology, 46(1), 130–142. Lavigne, G., Kim, J. S., Valiquette, C., & Lund, J. P. (1987). Evidence that periodontal pressoreceptors provide positive feedback to jaw closing muscles during mastication. Journal of Neurophysiology, 58(2), 342–358. Lesage, F. (2003). Pharmacology of neuronal background potassium channels. Neuropharmacology, 44(1), 1–7. Lund, J. P., & Olsson, K. A. (1983). The importance of reflexes and their control during jaw movement. Trends in Neurosciences, 6, 458–463. Lund, J. P., Smith, A. M., Sessle, B. J., & Murakami, T. (1979). Activity of trigeminal a- and g-motoneurons and muscle afferents during performance of a biting task. Journal of Neurophysiology, 42(3), 710–725. Luschei, E. S., & Goldberg, L. J. (1981). Neural mechanisms of mandibular control: Mastication and voluntary biting. In V. B. Brooks (Ed.), Handbook of physiology, vol. II. Motor control (pp. 1237–1274). Bethesda, ML: American Physiological Society. Meuth, S. G., Budde, T., Kanyshkova, T., Broicher, T., Munsch, T., & Pape, H. C. (2003). Contribution of TWIK-related acid-sensitive Kþ channel 1 (TASK1) and TASK3 channels to the control of activity modes in thalamocortical neurons. The Journal of Neuroscience, 23 (16), 6460–6469. Millar, J. A., Barratt, L., Southan, A. P., Page, K. M., Fyffe, R. E., Robertson, B., & Mathie, A. (2000). A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 3614–3618.

Morimoto, T., Inoue, T., Masuda, Y., & Nagashima, T. (1989). Sensory components facilitating jaw-closing muscle activities in the rabbit. Experimental Brain Research, 76(2), 424–440. Patel, A. J., & Honore, E. (2001). Properties and modulation of mammalian 2P domain Kþ channels. Trends in Neurosciences, 24(6), 339–346. Pose, I., Fung, S., Sampogna, S., Chase, M. H., & Morales, F. R. (2005). Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat. Brain Research, 1041(1), 29–37. Redman, S., & Walmsley, B. (1983a). Amplitude fluctuations in synaptic potentials evoked in cat spinal motoneurones at identified group Ia synapses. The Journal of Physiology, 343, 135–145. Redman, S., & Walmsley, B. (1983b). The time course of synaptic potentials evoked in cat spinal motoneurones at identified group Ia synapses. The Journal of Physiology, 343, 117–133. Shigenaga, Y., Mitsuhiro, Y., Shirana, Y., & Tsuru, H. (1990). Two types of jaw-muscle spindle afferents in the cat as demonstrated by intra-axonal staining with HRP. Brain Research, 514(2), 219–237. Sirois, J. E., Lei, Q., Talley, E. M., Lynch, C., 3rd, & Bayliss, D. A. (2000). The TASK-1 two-pore domain Kþ channel is a molecular substrate for neuronal effects of inhalation anesthetics. The Journal of Neuroscience, 20(17), 6347–6354. Talley, E. M., Lei, Q., Sirois, J. E., & Bayliss, D. A. (2000). TASK-1, a two-pore domain Kþ channel, is modulated by multiple neurotransmitters in motoneurons. Neuron, 25(2), 399–410. Taylor, A., Appenteng, K., & Morimoto, T. (1981). Proprioceptive input from the jaw muscles and its influence on lapping, chewing, and posture. Canadian Journal of Physiology and Pharmacology, 59(7), 636–644. Toyoda, H., Saito, M., Okazawa, M., Hirao, K., Sato, H., Abe, H., Takada, K., Funabiki, K., Takada, M., Kaneko, T., & Kang, Y. (2010). Protein kinase G dynamically modulates TASK1-mediated leak Kþ currents in cholinergic neurons of the basal forebrain. The Journal of Neuroscience, 30(16), 5677–5689. Toyoda, H., Saito, M., Sato, H., Dempo, Y., Ohashi, A., Hirai, T., Maeda, Y., Kaneko, T., & Kang, Y. (2008). cGMP activates a pH-sensitive leak Kþ current in the presumed cholinergic neuron of basal forebrain. Journal of Neurophysiology, 99(5), 2126–2133. Travers, J. B., Yoo, J. E., Chandran, R., Herman, K., & Travers, S. P. (2005). Neurotransmitter phenotypes of intermediate zone reticular formation projections to the motor trigeminal and hypoglossal nuclei in the rat. The Journal of Comparative Neurology, 488(1), 28–47. Vallbo, A. B. (1970). Slowly adapting muscle receptors in man. Acta Physiologica Scandinavica, 78(3), 315–333.

171 Verdier, D., Lund, J. P., & Kolta, A. (2003). GABAergic control of action potential propagation along axonal branches of mammalian sensory neurons. The Journal of Neuroscience, 23(6), 2002–2007. Yabuta, N. H., Yasuda, K., Nagase, Y., Yoshida, A., Fukunishi, Y., & Shigenaga, Y. (1996). Light microscopic observations of the contacts made between two spindle afferent types and

alpha-motoneurons in the cat trigeminal motor nucleus. The Journal of Comparative Neurology, 374(3), 436–450. Yemm, R. (1977). The orderly recruitment of motor units of the masseter and temporal muscles during voluntary isometric contraction in man. The Journal of Physiology, 265(1), 163–174.

Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 12

The interactions between locomotion and respiration Jean-François Gariépy{, Kianoush Missaghi{,{ and Réjean Dubuc{,{,* {

{ Département de Kinanthropologie, Université du Québec à Montréal, Montréal, Québec, Canada Groupe de Recherche sur le Système Nerveux Central, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada

Abstract: Respiration is a vital motor activity requiring fine-tuning to adjust to metabolic changes. For instance, respiration increases in association with exercise. In this chapter, we review the mechanisms underlying respiratory changes during exercise. Three specific hypotheses were proposed. First, the chemoreception hypothesis suggests that chemoreceptors located centrally or peripherally modify breathing by detecting metabolic changes in arterial blood or cerebrospinal fluid. Second, the central command hypothesis stipulates that central neural connections from brain motor areas activate the respiratory centers during exercise. Third, the neural feedback hypothesis stipulates that sensory inputs from the contracting limb muscles modulate the respiratory centers during exercise. We present evidence from the literature supporting possible contributions from these three mechanisms. This review also addresses future research challenges relative to respiratory modulation during exercise. Keywords: locomotion; respiration; brainstem; lamprey; hyperpnea; chemoreception; feedback.

other in order to generate motor output that is consistent with specific biomechanical and physiological constraints. For instance, swallowing is associated with a pause in respiratory output to prevent food from being directed through the respiratory system (Matsuo et al., 2007; McFarland et al., 1994; Miller, 1986). Another example of such an interaction is the way by which respiration increases in frequency and depth to compensate for increased needs for gas

Introduction Mastication, locomotion, and respiration are rhythmical motor activities patterned by neural networks referred to as central pattern generators (CPGs). The CPGs responsible for each of these three rhythmical activities interact with each * Corresponding author. Tel.: 514-343-5729; Fax: 514-343-6611 DOI: 10.1016/S0079-6123(10)87012-8

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exchanges during exercise. In this chapter, we focus on how exercise modulates breathing. Respiration is a rhythmic motor activity that is maintained throughout life to allow gas exchanges between the blood and the environment. Despite its automatic nature, it is strongly influenced by motor and mental activities. Exercise elevates the total demand of oxygen (O2) and increases the production of carbon dioxide (CO2). To compensate for this, the rate and depth of breaths increase during exercise. These changes usually correlate with the intensity of the effort (see Mateika and Duffin, 1995). Moreover, in some animal species running at high speed, there is a 1:1 coupling, that is, there is a respiratory cycle for each locomotor cycle. Physiologists have been intrigued with the question relative to the mechanisms underlying increases in respiration during exercise for a long time. They initially hypothesized that chemical changes in the blood such as an increase of partial pressure of CO2, a decrease of partial pressure of O2, or acidification were the important signals that triggered increased activity of the respiratory centers (Foster, 1866; Haldane and Priestley, 1905; Morat and Doyon, 1900; Volkman, 1841; Winterstein, 1911). Krogh and Lindhard (1913) took a whole different view and proposed that respiratory adjustments during locomotion were due to central neural connections from the motor centers responsible for initiating movements to the respiratory centers in the brainstem. Another view was adopted by others and it was hypothesized that somatic afferent fibers carried excitation to the respiratory centers during exercise (Haouzi et al., 2004b; Morin and Viala, 2002; Viala, 1997; Zuntz and Geppert, 1886). A recent published debate summarizes the arguments in favor of the central command and the peripheral feedback mechanism (Haouzi, 2006; Waldrop and Iwamoto, 2006). Taken together, the hypothesized mechanisms of respiratory modulation during exercise can be regrouped in (1) the chemoreception hypothesis, (2) the central command hypothesis, and (3) the peripheral nervous feedback hypothesis (Fig. 1). It is now generally accepted that each of

these mechanisms might play complementary roles in modulating respiration during exercise. In this chapter, we will review part of the literature supporting these three hypotheses by focusing on debates that have driven scientific works in each of these fields.

The chemoreception hypothesis The influence of CO2 and O2 on breathing has been known since the demonstration that increasing the CO2/O2 ratio in inspired air of human subjects increases their breathing rate (Haldane and Priestley, 1905; Pflüger, 1868). It was originally hypothesized that CO2 or O2 concentrations were directly detected by central respiratory centers. This remained the dominant view during the first quarter of the twentieth century (see Heymans, 1963; Remmers, 2005). However, new findings in the 1920s and 1930s challenged this hypothesis. It was found that respiratory effects induced by O2 deprivation were abolished by cutting the carotid sinus nerve, which innervates the carotid body known today to contain chemoreceptor cells (Heymans and Heymans, 1927). This stressed a contribution of peripheral receptors. Corneille Heymans received the Nobel Prize in Physiology or Medicine in 1938 for this discovery. However, the breathing rate was still increased by CO2 inhalation (Schmidt, 1932). These observations led to the conclusion that the carotid body detected deficits in O2, whereas CO2 was detected centrally. However, long periods of hypoxia also induce a respiratory decrease which is attributed to central effects since it is not associated with a change in the firing of the carotid sinus nerve (Vizek et al., 1987). This was confirmed recently using genetic removal of Task2 potassium channels in the ventral medulla which abolishes this decrease (Gestreau et al., 2010). The original view of chemoreception was further subjected to debate with the proposal of Winterstein (1911, 1949). His reaction theory stated that chemoreceptors detect Hþ ions concentration (pH) rather than gas concentrations. Perfusion of the subarachnoid space with solutions

175

Central effects

Motor centers

CPG

Central chemoreceptors

Periferal effects

Carotid body Activated by Lack of O2 Other stimuli

Limb afferents

CO2 production O2 consumption

Activated by Mechanoreceptors Metabolic changes Temperature

Fig. 1. Graphical representation of the three groups of hypotheses explaining respiratory modulation during exercise. Chemoreceptors located in the carotid body and the central nervous system can detect gas partial pressure changes, acidity changes, or other metabolic changes in the blood or cerebrospinal fluid. The carotid body projects to the brainstem, where respiratory centers are located, through the sinus nerve, a branch of the glossopharyngeal nerve. Central commands are hypothesized to come from various regions of the brain. The premotor and motor cortex, the brainstem locomotor centers, and the spinal cord CPGs have been hypothesized to project directly or indirectly to the respiratory centers. Somatic afferents have also been hypothesized to send projections to the respiratory CPG. These afferents carry signals related to mechanoreception, changes in temperature, and metabolic changes in skeletal muscles. These signals could in turn increase respiration during exercise and could also be responsible for locomotor–respiratory coupling.

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of varying pH and constant CO2 concentration was shown to increase respiration proportionally to the reduction of pH. In contrast, perfusion with solutions of constant pH and increasing CO2 concentrations had no effect (Guyenet et al., 2008; Loeschcke, 1982; Loeschcke et al., 1958). They even produced a small depression of respiration, which supported the crucial role of acidity in modulating breathing. The mechanisms responsible for the detection of gas concentrations/pH changes by the peripheral chemoreceptors are not fully understood. There is evidence that a decrease in potassium leak currents would result in a depolarization of the carotid body's type I cells (chemoreceptors) (Buckler, 1997; Gonzalez et al., 1994; López-Barneo et al., 1988, 2001; Wyatt and Peers, 1995). The disruption of mitochondrial processes can mimic the effect of hypoxia on those currents (Wyatt and Buckler, 2004). Recent hypotheses stipulate that an AMPactivated protein kinase or reactive oxygen species generated in low-oxygen conditions could constitute the hypoxia-detecting mechanisms (Dinger et al., 2007; Gonzalez et al., 2007; Wyatt and Evans, 2007). The mechanisms by which the central chemoreceptors operate are also not fully understood. Some researchers propose that chemoreception is achieved by different types of neurons distributed in the brain; others support a specialized chemoreception theory which stipulates that some specific brain nucleus such as the retrotrapezoid nucleus is responsible for chemoreception (for review, see Guyenet et al., 2008). In accordance with specialized chemoreception, a recent study showed that glial cells of the retrotrapezoid nucleus can detect changes in pH (Gourine et al., 2010). The involvement of central chemoreceptors in exercise hyperpnoea As indicated above, there is strong evidence for the presence of chemoreceptors in the central nervous system. However, their contribution to the respiratory changes during exercise is still not fully

established. Changes in gas concentrations or acidity were examined in the brainstem cerebrospinal fluid near during exercise. Exercise in horses maintained for 9 min neither reduced the pH nor increased the partial pressure of CO2 in cerebrospinal fluid. On the contrary, there was a slight increase in pH and a decrease in the partial pressure of CO2 (Bisgard et al., 1978). These results suggest that possible changes that would be detected by central chemoreceptors do not occur during exercise and as such, these results do not support an important role for central chemoreception in the modulation of respiration during exercise. Reductions in pH or increases of the partial pressure of CO2 in the cerebrospinal fluid can be elicited by other methods such as addition of CO2 to inspired air, occlusion of respiratory pathways, and injection of NaHCO3 in the blood (Eldridge et al., 1984). These methods do increase respiratory activity showing that central chemoreception likely contributes to adjustment of respiration in different physiological states. However, there is no evidence indicating that such control would occur during locomotion. A role of central chemoreceptors for increasing respiration during longer exercise periods or in other animal species cannot be excluded, but it is not supported by currently available data. The involvement of peripheral chemoreceptors in exercise hyperpnoea Duke et al. (1952) showed that the discharge of the carotid body nerve fibers is not modified by breathing CO or CO2 when these gases are mixed with pure O2. Because of this, investigators began using 100% O2 inhalation as a tool to suppress the contribution of peripheral chemoreceptors in the modulation of respiration (for review, see Dejours, 1962). Using this technique, it was found that the peripheral chemoreceptors could be responsible for about 15% of the respiratory drive at rest (Dejours et al., 1957, 1958). This technique was also used to suppress the effect of peripheral chemoreceptors

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during exercise (Jeyaranjan et al., 1987; Wasserman et al., 1979; Whipp et al., 1992). In these studies, respiratory changes are maintained after breathing 100% O2 during exercise, but the breathing rate is reduced by 15–20%. These results were interpreted as an indication that peripheral chemoreceptors contributed to a minor part of the respiratory adjustments during exercise. The contribution of the peripheral chemoreceptors in the carotid body to exercise hyperpnoea was also supported by data obtained from humans with bilateral carotid body resection. Although there was still a very significant respiratory response to exercise in these individuals, it was reduced as compared to that of control subjects (Honda, 1985). Other chemoreceptor mechanisms The lack of strong evidence indicating that CO2, O2, or pH at the peripheral and central level could be responsible for a major part of the respiratory effect during exercise prompted researchers to propose that some other factor, yet unknown, could be responsible for the regulation of respiration (Mitchell et al., 1958; Sinnott, 1961). Kþ ions released by muscles during exercise could modulate respiration (Paterson et al., 1989). Intra-arterial injections of KCl near the carotid body increases ventilation through an action of peripheral chemoreceptors (Band et al., 1985). However, experiments using sinusoidal variations in work rate showed that the concentration of Kþ ions in the blood was not strongly correlated with the variations in ventilation (Casaburi et al., 1995). It was also proposed that release of catecholamines or adenosine as well as changes in blood osmolarity could play a role in the changes in respiratory activity during exercise (for review, see Mateika and Duffin, 1995). Some of these mechanisms were shown to affect respiration in nonphysiological conditions, but there is no direct evidence that they could be responsible for a major part of respiratory increases during active locomotion.

The central command hypothesis Krogh and Lindhard (1913) initially proposed that neural mechanisms might be responsible for at least the early respiratory effect observed during locomotion. They showed that respiratory changes occurred at latencies of less than a second after the onset of locomotion in human subjects. They argued that such abrupt changes in respiration could not be induced by variations in the CO2 content of the blood since blood from the contracting muscles could not reach the respiratory centers in such a short time. Ranson and Magoun (1933) then showed that stimulation of the hypothalamus of decorticated cats generated locomotion associated with increases in respiration. These results were pivotal to indicate that the control of breathing and locomotion might be colocalized in similar regions of the brain. Eldridge et al. (1981, 1985) performed similar experiments in anesthetized cats in which muscular contractions were blocked. In this paralyzed preparation, chemical or electrical stimulation of the hypothalamic locomotor region produces fictive locomotion, that is, the motor nerves normally involved in locomotion show patterns of rhythmic activation, but the muscles do not actually contract. In those conditions, the increase of respiratory activity was similar to that observed during active locomotion. The authors concluded that central commands originating from locomotor regions played a major role in activating respiratory centers during locomotion. The advantage of the experimental setup used by Eldridge et al. (1981, 1985) was the complete isolation of the central nervous component from any peripheral changes that would result from muscular contractions. It was also shown that stimulation of the dorsolateral spinal funiculus, the cervical spinal cord, or the medullary locomotor strip elicited bouts of locomotion that were also associated with similar increases in respiration (Romaniuk et al., 1994). These observations suggested that either a wide variety of locomotion-inducing neural structures project to respiratory centers, or these structures converge to a single locomotor region,

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which in turn projects to respiratory centers. Another important issue relative to the central command hypothesis is the diversity of motor behaviors other than locomotion that influence respiration. For instance, hand gripping (Williamson et al., 2002), cycling (Krogh and Lindhard, 1913), weight lifting (Ratamess et al., 2007), or leg movements (Decety et al., 1993) all influence respiration. It is unlikely that the putative projections from locomotor centers to respiratory centers discussed above will account for all these adjustments of respiration during motor behavior. The central mechanisms involved in respiratory changes during these motor activities will need further investigation. The central command is the only proposed mechanism capable of modifying respiration before any muscular contraction. It has been shown in subjects performing mental simulation of exercise that respiration and heart rate are increased proportionally to the intensity of the mentally simulated effort (Decety et al., 1993). Higher brain areas activated during imaginary efforts have been identified using positron emission tomography (Thornton et al., 2001). Also in humans, respiration is modified before the onset of movement when the subjects are instructed to start moving after a given delay (Tobin et al., 1986). The activation of locomotor regions of cats at a subthreshold intensity for eliciting locomotion could still induce respiratory effects (Eldridge et al., 1981, 1985). These findings support a role for a central command in producing anticipatory adjustments of respiration in association with exercise. However, Eldridge et al. (1981) also showed that the respiratory increase was maintained throughout the fictive locomotor bouts. This suggests that the central command may be responsible not only for anticipatory and early respiratory responses to movement but also for adjusting the respiratory output during the entire movement episode. It was shown by Haouzi et al. (2004a) that the peaks of breathing activity do not occur synchronously with the peaks of locomotor activity in sheeps walking on a treadmill when sinusoidal variations of speed are imposed. Rather, the

peaks of breathing activity were synchronous with the peaks of CO2 output and this led the authors to propose that respiratory changes were, in major part, dissociated from the influence of locomotor centers (Haouzi, 2006). These observations neither obligatorily dismiss the contribution of a central command nor ascribe a major part of the respiratory changes to peripheral mechanisms. There is a delay between the changes in locomotor frequency and respiratory activity, but whether this delay is caused by peripheral or central mechanisms still needs to be determined. Localization of the central command The mechanism proposed to account for the central effects of locomotion on respiration involves connections from supraspinal locomotor centers to respiratory regions (Eldridge et al., 1981; Waldrop and Iwamoto, 2006). An alternative hypothesis is that the locomotor CPGs in the spinal cord would provide inputs to the brainstem respiratory generator during locomotion. This hypothesis was proposed following the demonstration that pharmacological activation of the lumbar spinal cord increased respiration in an in vitro isolated brainstem–spinal cord preparation from rats (Morin and Viala, 2002). This paper addressed mainly the coupling between locomotion and respiration on a cycle to cycle basis. In addition, the authors suggested that the activation of locomotor CPGs could increase the frequency of the respiratory rhythm without coupling it to locomotion. The mechanisms by which the spinal locomotor networks can modulate the respiratory rhythm will need to be investigated in more details. Recent studies on lampreys in our laboratory have provided strong arguments in favor of a supraspinal localization of the connectivity responsible for adjusting respiratory activity to locomotor output (Gravel et al., 2007). The central nervous system of lampreys displays a very similar organization to that of other vertebrate species including mammals. It contains far fewer neurons than that

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of mammals and the neurons are easily accessible to an array of neurobiological techniques, making the lamprey model a very useful one to study the neural control of locomotion and respiration. Respiration in this animal consists in rhythmical contractions of gill muscles, which expels water from the gill baskets through the gill pores. Seven gill pores are present on each side of the body. Despite the considerable differences in the peripheral apparatus responsible for respiration compared to that of mammals, the general organization of the neural networks responsible for controlling both systems present important similarities. Respiratory rhythmogenesis in all vertebrates is accomplished by groups of cells in the hindbrain. In mammals, the rhythmic pattern activates different motoneuron pools including spinal motoneurons innervating the diaphragm,

intercostal, and abdominal muscles (Bianchi and Pásaro, 1997; Giraudin et al., 2008). There are also brainstem motoneurons innervating muscles of the upper respiratory airways which also receive inputs from the respiratory generator. These motoneurons are located in the facial (VII), glossopharyngeal (IX), and vagal (X) nuclei (Bianchi and Pásaro, 1997). In lampreys, the respiratory motoneurons were also identified in the VII, IX, and X motor nuclei (Guimond et al., 2003; Martel et al., 2007; Rovainen, 1985; Thomspon, 1985). The rostrocaudal topography of respiratory motoneurons reflects the rostrocaudal innervations of gill muscles (Guimond et al., 2003). The respiratory rhythm can be recorded in the in vitro isolated brainstem with or without the gills attached (Fig. 2) and with or without the tail attached (Fig. 3). In the 1980s, the

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Fig. 3. Locomotion-induced respiratory increases in a semi-intact lamprey preparation with tail attached. Top: Sketch of the preparation. The tail is left intact and free to swim in a deep part of the bath. EMGs are placed in each side of the tail to record locomotor activity. The brainstem is exposed and an extracellular recording electrode is placed over the right vagal nucleus (Xr) to record respiratory activity. Mechanical stimulation (light touch) of the head induces a bout of swimming in this preparation. (a) Respiratory activity and EMG recordings at rest and during locomotion. Bottom: Statistical analyses on the changes in frequency and area of respiratory bursts in four semi-intact preparations. (b) Respiratory activity after complete lesion of the spinal cord at rest and after applying the same mechanical stimulus used in (a). Bottom: Statistical analyses on the changes in frequency and area of the respiratory bursts in four semi-intact preparations after removal of the spinal cord. (Adapted from Gravel et al. (2007).)

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organization of the respiratory networks was examined in the in vitro isolated brainstem of lampreys. Lesions and intracellular recordings suggested that a region located rostrolaterally to the trigeminal motor nucleus was essential for respiratory rhythmogenesis (Rovainen, 1985; Russell, 1986; Thomspon, 1985). This region is referred to as the paratrigeminal respiratory group (pTRG; Mutolo et al., 2007). It was also suggested that there would be a more caudal respiratory generator in the region corresponding to the medulla oblongata of mammals (Kawasaki, 1979, 1984; Thomspon, 1985). It was recently found using lesions and local drug applications that the pTRG is responsible for the generation of the regular, fast respiratory pattern (see small bursts in Fig. 2), whereas the caudal part of the brainstem generates large bursts that occur more sporadically (Martel et al., 2007; Mutolo et al., 2007, 2010). These large bursts are believed to play a role in emptying the gills from particles (see arrows in Fig. 2). One advantage of lampreys as a model is the ability to use semi-intact preparations, in which the brain and rostral spinal cord are exposed whereas the tail of the lamprey is left intact, capable of swimming freely in a deeper part of the recording chamber (Fig. 3a). Brainstem regions can thus be recorded while the animal displays active swimming. Fictive respiration can be recorded from respiratory motoneurons in the facial (VII), glossopharyngeal (IX), or vagal (X) motor nuclei (Guimond et al., 2003). To elicit bouts of swimming, sensory stimulation was applied by touching skin on the dorsal surface of the head or the tail. This type of stimulation is known to induce locomotion through the activation of the sensory inputs to reticulospinal cells (Antri et al., 2009; Viana di Prisco et al., 1997, 2000). We found that locomotion was associated with marked increases in the frequency and area of respiratory bursts (Fig. 3b). A complete transverse section at the level of the obex was performed to separate the spinal cord from the brainstem. Sensory stimulation did not induce locomotion anymore, but the effects on respiration were preserved (Fig. 3c). These effects were of slightly lesser magnitude than those

observed when muscle contractions occurred (91.5  43.8% increase of respiratory frequency in control vs. 60.1  31.3% after spinal transection for frequency of respiratory bursts; Fig. 3b and c). These results suggest that an important part of the central component of the respiratory changes during locomotion relies on brainstem connections and that active locomotion is not essential to augment respiration. We are currently investigating electrophysiologically and anatomically neuronal projections from locomotor regions to respiratory regions in the brainstem of lampreys. In conclusion, a central command component of respiratory modulation has been identified. The respiratory adjustments are preserved in the absence of chemical changes in the blood or cerebrospinal fluid and afferent activity resulting from muscular contractions. Furthermore, we have shown that connections in the brainstem might play an important role in respiratory modulation during locomotion (Gravel et al., 2007). Despite the strong evidence for the existence of a central command, cellular and network mechanisms responsible for respiratory increases during locomotion still need to be identified. The peripheral nervous feedback hypothesis It was proposed that peripheral nervous feedback from the contracting muscles contributed to the increase of respiratory activity during exercise (Haouzi et al., 2004b; Mateika and Duffin, 1995; Zuntz and Geppert, 1886). This hypothesis is supported by data indicating that stimulation of ventral roots causing contractions of hind limb muscles in anesthetized dogs and cats induces respiratory changes (Comroe and Schmidt, 1943). Cutting the dorsal roots abolished the respiratory response, suggesting that somatic afferents were involved (McCloskey and Mitchell, 1972). A fast respiratory response also occurred when limbs were passively moved both in awake and asleep humans (Ishida et al., 1993). It was proposed that the signal from the exercising limbs was

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carried by type III and IV afferents, which can be activated by movement, accumulation of exercise by-products, local inflammation, and rise in muscle temperature (for review, see Haouzi et al., 2004b). For instance, the receptors located in the skeletal muscles could detect local vascular responses, which would provide an estimation of metabolic changes and thus excite respiratory centers proportionally to CO2 production (Haouzi and Chenuel, 2005). The importance of the peripheral feedback mechanisms during exercise is difficult to ascertain because experimental manipulations aiming at isolating peripheral feedback from other mechanisms do not always do so. For instance, when limbs are passively moved or electrically stimulated, the observed respiratory changes may also be due to changes in awareness, wakefulness, behavioral state, or different cognitive processes. Such interactions between the respiratory effects of passive limb movements and cognitive processes have been described in human subjects. Bell and Duffin (2004) showed that respiratory changes induced by passive limb movements were greatly reduced when the subjects solved a computer puzzle. These observations could be viewed as an indication that cognitive processes can suppress part of the respiratory changes induced by peripheral inputs. It is well documented in other motor systems that central processes gate sensory transmission during movements (Chapman et al., 1988; Clarac et al., 2000; Sillar, 1991; for review, see Rossignol et al., 2006).

Locomotor–respiratory coupling and peripheral afferents Another aspect of respiratory adjustments during locomotion is the coupling that occurs between the two rhythms at high speed of locomotion. Locomotor–respiratory coupling is the synchronization of the phases of the respiratory rhythm and the locomotor rhythm during locomotion. It is

proposed that this coupling is necessary to keep harmonized contractions between muscles responsible for locomotion and respiration and avoid biomechanical conflicts that would result in inefficient muscular contractions or energy losses (Viala, 1997). This coupling was reported by Bannister et al. (1954) in humans running on a treadmill and was also shown to occur during pedaling on a bicycle using cross-correlation analysis (Bechbache and Duffin, 1977). It was originally observed that locomotor cycles were integer-multiples of breathing cycles, leading to locomotor–respiratory coupling ratios of 1:1, 2:1, 3:1, or 4:1. These observations were confirmed by some (Hill et al., 1988; Paterson et al., 1987), whereas others did not find coupling between the two rhythmic motor activities (Kay et al., 1975). Other types of locomotor–respiratory coupling such as 5:2 and 3:2 were also reported, although 2:1 seemed to be the dominant ratio observed in humans (Bramble and Carrier, 1983). Locomotor–respiratory coupling was seen more often during running than during cycling (Bernasconi and Kohl, 1993). Moreover, it was more frequent in experienced than nonexperienced runners (Bramble and Carrier, 1983). However, several studies made on nonexperienced runners have also found a coupling (see Viala, 1997). The coupling between respiratory and locomotor activities was also observed in cats running on a treadmill (Iscoe, 1981). It was predominantly a 1:1 ratio. Other studies on different quadruped species have shown that 1:1 locomotor–respiratory coupling occurs mostly when the animals were galloping or hopping but also, in some occasions, during trotting (Baudinette et al., 1987; Bramble and Carrier, 1983; Kawahara et al., 1989; Young et al., 1992a). The piston mechanism One mechanism proposed to explain locomotor–respiratory coupling is the visceral piston mechanism (Bramble and Carrier, 1983). There would be a passive coupling between

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locomotion and respiration due to the visceral mass rhythmically pushing on lungs during locomotion. This hypothesis was supported by kinematic data in dogs showing that oscillations of the visceral mass were pushing the diaphragm during locomotion, thus driving ventilation independently from muscular contractions (Bramble and Jenkins, 1993). In horses, a similar phenomenon caused by back flexion was observed (Young et al., 1992b). However, it was suggested that this passive biomechanical phenomenon might be less likely to occur in humans due to their upright position during walking (Viala, 1997). As reviewed in the following sections, evidences have now been obtained indicating that although such passive mechanisms could be present in some animals, there are also neural components actively coupling respiratory muscle contractions to the locomotor rhythm. The spinal cord CPGs The locomotor networks of the spinal cord are believed to play at least a partial role in coordinating respiration and locomotion in different coupling ratios. It was reported in different animal preparations that paralysis does not prevent locomotor–respiratory coupling (Corio et al., 1993; Funk et al., 1992b; Perségol et al., 1988; Viala et al., 1987). This indicates that central connections are sufficient to couple the respiratory rhythm to fictive locomotion in these experimental conditions and such mechanisms might also be important to couple respiration to active locomotion. Morin and Viala (2002) showed that pharmacologically activating the lumbar locomotor generator was not sufficient to induce locomotor–respiratory coupling in vitro. This suggests that the activation of the cervical locomotor generator or the activation of both generators might be necessary to entrain respiration. The relative importance of this coupling mechanism compared to the other mechanisms remains open to investigation.

Supraspinal influences Some studies have raised the possibility that cognitive processes could be involved in locomotor– respiratory coupling. For instance, Bechbache and Duffin (1977) showed that subjects pedaling at frequencies determined by a metronome were more likely to develop locomotor–respiratory coupling than subjects who are asked to follow a certain speed using a speedometer. These results suggest that respiratory muscles could be entrained by acoustic stimuli. Lower limb movements are known to be entrained by auditory inputs (Brown et al., 2006; Rossignol and Jones, 1976). Similar mechanisms could thus be responsible for the coupling of respiration to locomotion. Moreover, Bramble and Carrier (1983) reported that locomotor–respiratory coupling occurred earlier during locomotion and was stronger in experienced runners compared to inexperienced runners. This suggests that locomotor–respiratory coupling can be learned or acquired by training. Studies made on decerebrate preparations in which locomotor–respiratory coupling is preserved have provided arguments against a contribution of forebrain structures to the coupling (Corio et al., 1993; Funk et al., 1989; Viala, 1997). These studies showed that locomotor–respiratory coupling can occur without cortical areas and therefore that subcortical and peripheral mechanisms are involved. However, to our knowledge, no attempt was made to determine the relative importance or to completely exclude the contribution of cognitive processes to locomotor–respiratory coupling in humans, in which the locomotor–respiratory coupling patterns are more varied and often consist in subharmonics (2:1, 4:1) rather than the predominantly pure phaselock (1:1) observed in many animal preparations. The peripheral nervous feedback In mammals, repetitive stimulation of muscle and cutaneous afferents entrain respiration in different ratios (Howard et al., 1969; Iscoe and Polosa,

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1976). Single stimulation can also reset the respiratory rhythm when applied during expiration (Kawahara et al., 1988). Passive wing flapping has been shown to entrain respiration in the Canada goose (Funk et al., 1992a). It was shown that low-threshold stimulation of sensory pathways from hindlimb muscles can effectively reset respiration in neonatal rats (Morin and Viala, 2002). These data suggest that there is connectivity between somatic afferents and the respiratory centers and that these connections can entrain the respiratory rhythm. It is thus possible that during locomotion, the rhythmical activation of these afferents entrain respiration in the different locomotor–respiratory coupling ratios that are known. However, as for the other proposed mechanisms, the relative importance of this mechanism compared to others during active locomotion is still unknown.

Conclusions Modulation of respiration during exercise has been the center of a century-old debate opposing supporters of the chemoreception hypothesis, the central command hypothesis and the peripheral nervous feedback hypothesis. Most likely, the respiratory adjustments during locomotion result from a combination of these mechanisms since no isolating paradigm has successfully shown that either of these mechanisms alone could explain all the adjustments that occur during exercise. Respiratory centers are being increasingly well described in terms of connectivity and cellular properties, and animal models in which these properties can be studied have become available, including some where locomotor centers are still present and functional (Gravel et al., 2007; Smith and Feldman, 1987). Using these preparations, it will likely be possible to identify in a near future the neural cellular mechanisms responsible for respiratory adjustments during locomotion.

Acknowledgments We are very grateful to Danielle Veilleux for her technical assistance, to Christian Valiquette for his skilful programming of data analysis software, and to Frédéric Bernard for his help with the figures. This work was supported by grants to R. D. from the Natural Sciences and Engineering Research Council of Canada (NSERC), an individual and group grant from the Canadian Institutes of Health Research (CIHR), and a group grant from the Fonds de la Recherche en Santé du Québec (FRSQ). R. D. receives grants from the Great Lakes Fishery Commission. J.-F. G. received studentships from the FRSQ and CIHR. K. M. received studentships from NSERC. Abbreviations VII IX X pTRG

facial nucleus glossopharyngeal nucleus vagal nucleus paratrigeminal respiratory group

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 13

Rhythmogenesis in axial locomotor networks: an interspecies comparison Dimitri Ryczko{, Réjean Dubuc{,{,* and Jean-Marie Cabelguen} {

Groupe de Recherche sur le Système Nerveux Central, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada { Département de Kinanthropologie, Université du Québec à Montréal, Montréal, Québec, Canada } Neurocentre Magendie, INSERM U 862, Université de Bordeaux, Bordeaux Cedex, France

Abstract: During locomotion, specialized neural networks referred to as “central pattern generators” ensure precise temporal relations between the axial segments, both in limbed and limbless vertebrates. These neural networks are intrinsically capable of generating coordinated patterns of rhythmic activity in the absence of sensory feedback or descending command from higher brain centers. Rhythmogenesis in these neural circuits lies on several mechanisms, both at the cellular and the network levels. In this chapter, we compare the anatomical organization of the axial networks, the role of identified spinal neurons, and their interactions in rhythmogenesis in four species: lamprey, zebrafish, Xenopus tadpole, and salamander. The comparison suggests that several principles in axial network design are phylogenetically conserved among vertebrates. Keywords: Rhythmogenesis; axial network; spinal neurons; lamprey; zebrafish; Xenopus tadpole; salamander.

Introduction

vertebrate motor system the “central pattern generators” (CPGs) concept, originally described in invertebrates (Wilson, 1961). There is now strong evidence that this concept is a general principle for the control of locomotion in several other vertebrate species (e.g., rabbit: Viala and Buser, 1971; dogfish: Grillner, 1974; cat: Perret and Cabelguen, 1976, 1980; lamprey: Cohen and Wallén, 1980; Poon, 1980; larval newt: Soffe et al., 1983; Xenopus embryo: Dale and Roberts, 1984; rat: Kudo and Yamada, 1987; bird: Sholomenko et al., 1991;

In the 1970s, it was shown that the neuronal circuitry localized in the spinal cord could intrinsically generate coordinated, complex patterns of locomotor output (cat: Grillner and Zangger, 1979). This considerable research achievement provided the first direct evidence for extending to the * Corresponding author. Tel.: þ1-514-343-5729; Fax: þ1-514-343-6611 DOI: 10.1016/S0079-6123(10)87013-X

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goldfish: Fetcho and Svoboda, 1993; adult salamander: Delvolvé et al., 1999; turtle: Juranek and Currie, 2000; larval zebrafish: McDearmid and Drapeau, 2006; adult zebrafish: Gabriel et al., 2008; for reviews, see Delcomyn, 1980; Grillner, 1981; Orlovsky et al., 1999). Spinal networks controlling axial movements (axial locomotor CPG) have been studied in great detail at the cellular level utilizing an array of anatomical, pharmacological, neurophysiological, and modeling approaches in limbless swimming vertebrates, including the lamprey (for review, see Grillner, 2006) and the Xenopus tadpole (for review, see Roberts et al., 2010). Recently, imaging and genetic approaches provided new insights into the role of some spinal cell populations during locomotion in the zebrafish (for review, see Fetcho and McLean, 2010; Fetcho et al., 2008; McLean and Fetcho, 2008; see also Saint-Amant, 2010). In contrast, the available data concerning the design of axial locomotor circuits are scarce in limbed vertebrates (Falgairolle and Cazalets, 2007; Ryczko et al., 2010). Some theoretical and experimental studies suggest that the design of the axial locomotor networks is evolutionarily conservative (De Sèze et al., 2008; Fetcho and Reich, 1992; Schilling and Carrier, 2010; for review, see Cohen, 1988; Falgairolle et al., 2006; Fetcho, 1987, 1992; Grillner, 2006; Katz and Harris-Warrick, 1999; Kiehn et al., 1997; ten Donkelaar, 2001; see also Ijspeert et al., 2007). Moreover, interesting new findings in mammals indicate that caudorostrally propagated waves of activity are present in the axial ventral roots of neonatal rat isolated spinal cords during fictive locomotion (Falgairolle and Cazalets, 2007; for review, see Falgairolle et al., 2006). Similarly, a wave of activity propagates rostrocaudally in the spinal cord of cats during fictive scratching (Cuellar et al., 2009). These waves show a similar pattern to those seen during fictive locomotion in lampreys (e.g., Wallén and Williams, 1984), zebrafish (e.g., Gabriel et al., 2008; McDearmid and Drapeau, 2006), Xenopus tadpoles (Kahn and Roberts, 1982a), and salamanders (Delvolvé et al., 1999; Ryczko et al.,

2010). In salamanders, the axial spinal cord comprises rhythm-generating networks that are distributed bilaterally (Ryczko et al., 2010) as observed in lampreys (Cangiano and Grillner, 2003, 2005) and Xenopus embryos (Soffe, 1989). A single hemisegment is capable of producing a rhythmic motor output, both in lampreys (Cangiano and Grillner, 2003) and salamanders (Ryczko et al., 2010). The mechanisms of rhythmogenesis have been recently investigated in isolated axial hemisegments and segments in salamanders (Ryczko et al., 2010), but the detailed organisation of the underlying neural network is unknown. In this context, insight into neuronal circuit design can be gained by comparing closely related species (Katz and Harris-Warrick, 1999). Here, we review the current available information about the cellular, synaptic, and circuit design of axial networks in the lamprey, zebrafish, Xenopus tadpole, and salamander. Common mechanisms underlie rhythmogenesis, and similar neurons play analogous functional roles in different species, suggesting a conservation of the basic building blocks in the axial motor networks.

Locomotor behavior Lamprey The lamprey is an “anguilliform” swimmer: all segments of the body are flexible and participate in the propulsive movement (Williams et al., 1989). The lamprey usually swims forward, but backward swimming also occurs when the animal encounters obstacles. Experimentally, backward swimming can be elicited by tactile stimulation applied on the anterior part of the body (Islam et al., 2006). A left-right alternation pattern is present during both swimming behaviors, but each behavior has a specific intersegmental coordination pattern. Forward swimming is characterized by periodic rostrocaudally traveling waves of excitation of motoneurons (MNs) along each side of the cord, that is, with a positive intersegmental

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“phase lag” (see Grillner, 1974) of þ 1% of cycle duration (Wallén and Williams, 1984). Backward swimming results from caudorostrally traveling waves, that is, with a negative intersegmental phase lag of  1% of cycle duration (Islam et al., 2006). During backward swimming, the cycle duration is longer. The body progression is less efficient and the amplitude of body undulations is larger. Spatial orientation is not maintained during backward swimming (Islam et al., 2006). Lampreys can also crawl in tight places where undulatory movements are not possible (Archambault et al., 2001; Ayers et al., 1983; Zelenin, 2005); they also display slow forward swimming when a tactile stimulation (grasping-like) is performed in the middle part of the body (Ayers et al., 1983; Islam and Zelenin, 2007).

Zebrafish During the first days after fertilization, the repertoire of motor programs expressed by young zebrafishes changes considerably (for review, see Saint-Amant, 2010). Here, we will mostly refer to the data concerning the larval stage (3- to 5-dayold larvae) at which time the following locomotor programs are observed: an escape response, a slow and a fast swimming patterns, and a struggling behavior (Kimmel et al., 1995; Liao and Fetcho, 2008; McLean et al., 2007; Ritter et al., 2001). The escape response is characterized by a brief burst of ventral root activity occurring on the contralateral side at a short latency after the onset of the stimulus (Liao and Fetcho, 2008; Liu and Fetcho, 1999). This is generally followed by swimming. Slow swimming is characterized by weak axial undulations of the body axis coordinated with paddling movements of the pectoral fins; during fast swimming, the fins are held against the body while axial undulations are generated (McLean et al., 2007; Ritter et al., 2001; Thorsen et al., 2004). As in other anguilliform swimmers, both patterns of swimming are associated with rostrocaudally traveling waves of undulation that

become larger as they approach the tail (Müller and van Leeuwen, 2004). During struggling, the cycle duration is longer, the amplitude of the body wave increases compared to swimming, and the wave direction reverses to travel from tail to head (Liao and Fetcho, 2008; see also Sankrithi and O'Malley, 2010).

Xenopus tadpole Xenopus tadpoles generate three axial motor programs: an escape or C-shape flexion response (Boothby and Roberts, 1995), forward swimming (Kahn et al., 1982), and struggling (Kahn and Roberts, 1982b). A gentle tactile stimulation triggers a flexion response, usually followed by a swimming bout. The swimming behavior is anguilliform: the lateral undulations travel rostrocaudally with an increasing amplitude (Kahn and Roberts, 1982a). The myotome activity alternates between sides (Kahn et al., 1982). When restrained, the animal displays a struggling behavior that involves slow, powerful, rhythmic waves of myotome contractions that propagate from the tail to the head. The movement amplitude is larger than for swimming. This behavior is due to rhythmic bursts of activity in myotomal muscles that alternate on either side of a segment, and that are propagated in a caudorostral sequence on each side of the body (Kahn and Roberts, 1982b).

Salamander Salamanders are amphibians displaying terrestrial and aquatic locomotor patterns (for review, see Cabelguen et al., 2010; Chevallier et al., 2008). In water, salamanders typically use an anguilliform forward swimming very similar to that of the lamprey (Delvolvé et al., 1997; Frolich and Biewener, 1992). The swimming pattern of salamanders is based on axial undulations with traveling waves propagated rostrocaudally. The amplitude of the wave increases as it travels to the tail. The limbs

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are folded backwards along the body, as observed for the pectoral fins in zebrafish larvae during the fast swimming pattern (Thorsen et al., 2004). On the ground, salamanders switch to a stepping gait, with movements of the limbs coordinated with the body making S-shaped waves (Delvolvé et al., 1997; Frolich and Biewener, 1992). Salamanders are also capable of backward walking (AshleyRoss and Lauder, 1997), forward underwater stepping (Ashley-Ross et al., 2009; see also Cabelguen et al., 2010), and recent results show that salamanders may also express a struggling behavior when restrained (Lamarque et al., 2009). In both cases, however, further studies are needed to characterize the axial dynamics during these behaviors (see Cabelguen et al., 2010). The embryo of the newt Triturus vulgaris expresses flexion responses, forward swimming, and “slow alternation” behaviors (Soffe et al., 1983). A light touch induces flexures of the body, driven by synchronous bursts of motor root discharge, as observed in Xenopus tadpoles and zebrafish larvae (Soffe et al., 1983). The swimming pattern is similar to that of Xenopus tadpoles. The “slow alternation” behavior is characterized by rostrocaudal waves of electromyographical activity. This pattern is induced by a strong bilateral pressure to the tail and functionally appears similar to the struggling behavior in Xenopus tadpoles, despite the fact that caudorostral waves are observed during struggling in the Xenopus (Kahn and Roberts, 1982b) and zebrafish larvae (Liao and Fetcho, 2008). In the four animal species, there are several similarities in the characteristics of the rostrocaudal mechanical and electromyographical waves involved in forward swimming. Caudorostral waves are also present during struggling or backward swimming in the lamprey. Despite being tetrapods, salamanders also share many similarities in their kinematic and electromyographic patterns with limbless vertebrates. Behavioral similarities could result from a similar design of the axial locomotor networks and we now compare the spinal neurons that are part of the axial networks in the four animal species.

Spinal neurons Lamprey In lampreys, eight classes of spinal neurons have been characterized on the basis of morphological (axonal projection, soma size location) and physiological (excitatory vs. inhibitory) characteristics. The spinal neurons include primary mechanosensory dorsal cells (DCs), second-order sensory giant excitatory interneurons with ascending contralateral axons (GIs), intraspinal stretch-sensitive edge cells (ECs) (Grillner et al., 1982, 1984) that include excitatory (SR-Es) and inhibitory stretch receptors (SR-Is), lateral inhibitory interneurons with an ipsilateral descending axons (LINs) (Rovainen, 1974a), contralaterally and caudally projecting interneurons (CCINs) that include excitatory (CC3 class) and inhibitory neurons (CC1 and CC2 classes) (Buchanan, 1982), excitatory interneurons with ipsilateral axons (EINs) (Buchanan and Grillner, 1987; Buchanan et al., 1989), inhibitory interneurons with ipsilateral axons (IINs) (Buchanan and Grillner, 1988), and MNs (for review, see Buchanan, 2001). The eight cell classes show no significant difference in resting potential, action potential amplitude, and duration (Buchanan, 1993). This contrasts with the observations made in Xenopus tadpoles where some of the spinal neurons (dINs) display action potential shapes clearly distinguishable from that of other interneurons (Li et al., 2007). During fictive swimming, membrane potential oscillations are large in CCINs, ECs, EINs, IINs, LINs, and MNs, but small or even absent in GIs and DCs (Biró et al., 2008; Buchanan, 1982; Buchanan and Cohen, 1982; Buchanan and Grillner, 1988; Buchanan and Kasicki, 1995; Buchanan et al., 1989; El Manira et al., 1996; Kahn, 1982; Russell and Wallén, 1983; Vinay et al., 1996). The excitatory depolarizing phase alternates with a chloridedependent inhibitory repolarizing phase, as shown after injecting chloride into MNs (Russell and Wallén, 1983), LINs and CCINs (Kahn, 1982), EINs (Buchanan et al., 1989), and ECs (Vinay et al., 1996).

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Zebrafish

Xenopus tadpole

In the larval zebrafish (4-day-old larvae), different classes of spinal interneurons have been described on the basis of their morphology (Bernhardt et al., 1990; Hale et al., 2001) including their neurotransmitter phenotype (Higashijima et al., 2004). Rohon-Beard cells (RBs) and dorsal root ganglion (DRG) cells provide the principle source of mechanosensory input to the spinal cord. Glutamatergic interneurons include: ipsilaterally projecting circumferential descending interneurons (CiDs); commissural primary ascending interneurons (CoPAs); multipolar commissural descending interneurons (MCoDs); unipolar commissural descending interneurons (UCoDs); some of the commissural secondary ascending interneurons (CoSAs); and ventral medial interneurons (VeMes) whose descending axonal trajectory has not been clearly revealed (Higashijima et al., 2004). Glycinergic neurons include: ipsilaterally projecting circumferential ascending interneurons (CiAs); commissural bifurcating longitudinal interneurons (CoBLs); some of the CoSAs; commissural longitudinal ascending interneurons (CoLAs); and commissural local interneurons (CoLos). There are also a number of GABAergic interneurons that include Kolmer–Agduhr (K–A) cells and dorsal longitudinal ascending interneurons (DoLAs), as well as the class of ventral serotonergic interneurons (VeSes). There are two classes of MNs, primary and secondary, named for their respective appearance during development (McLean and Fetcho, 2008; Myers et al., 1986). Primary MNs are large, early born, dorsally located, whereas secondary MNs are smaller, ventrally located, and later born (Liu and Westerfield, 1988; Myers, 1985; Westerfield et al., 1986). Among primary MNs, rostral primary (RoP), middle primary (MiP), and caudal primary (CaP) MNs are distinctive based on their location with respect to one another and their regions of axial muscle innervations. Secondary MNs can be divided into classes that innervate either the pectoral fins or the axial musculature (for review, see McLean and Fetcho, 2008).

Ten classes of spinal neurons have been identified in Xenopus tadpoles based on the location of their soma, their dendritic branching pattern, and their axonal projections (Li et al., 2007; Roberts and Clarke, 1982; for review, see Roberts, 2000; Roberts et al., 2010). Excitatory neurons include: sensory RBs; sensory dorsolateral ascending interneurons (dlas); sensory dorsolateral commissural interneurons (dlcs); sensory commissural interneurons (ecINs); descending interneurons with an ipsilateral axon (dINs); descending interneurons with an ipsilateral axon that show repetitive firing (dINrs); and MNs. Inhibitory neurons include: ascending interneurons with an ipsilateral axon (aINs); commissural interneurons (cINs); K–A cells. The dINs can be distinguished from other neurons on the basis of their long spike duration (Li et al., 2006). It seems that there is only one class of MNs, for producing flexion, swimming, and struggling movements (Roberts et al., 1999).

Salamander In salamanders, the morphology of MNs was described (Fetcho, 1986), but there is very little information on interneurons in the axial spinal cord. Most of the data were collected early during development. For instance, a study performed in tadpoles of T. vulgaris and Ambystoma mexicanum (the Axolotl) near the time of hatching (when swimming and struggling are possible) revealed nine types of interneurons using horseradish peroxidase labeling together with GABA and glycine immunohistochemistry (Harper and Roberts, 1993). Six types of non-GABA/glycine neurons were found. These include dorsal Rohon-Beard cells with ascending and descending axons (RBs); giant dorsolateral commissural interneurons with ascending axons (GCs); dorsolateral commissural interneurons with ascending axons; dorsolateral ascending interneurons with ipsilateral axons;

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descending interneurons with ipsilateral axons; and ventrolateral MNs. Three types of inhibitory neurons were found: glycinergic commissural interneurons with ascending and descending axons; GABAergic ascending interneurons with ascending axons; and GABAergic K–A cells with ascending axons. In adult mudpuppies (Necturus maculosus), the morphology of axial MNs is linked to their position in the ventral horn. Large multipolar MNs lay in the lateral tip of the ventral horn. Smaller unipolar MNs are located more medially and are more numerous than lateral MNs. MNs located near the central canal have very few dendritic processes (Fetcho, 1986). There are several similarities between the interneurons in the axial spinal networks of the four vertebrate species. For example, eight neuron classes out of the ten described here for the Xenopus larva correspond very closely to those in the newt, and to a lesser extent, to those in the zebrafish (Harper and Roberts, 1993; Roberts, 2000). A clear difference is the presence of the giant dorsolateral commissural interneuron in newt embryos, absent in Xenopus embryos, but present in lampreys (for review, see Buchanan, 2001). The organization of axial MNs was investigated in the neotenic salamander Necturus maculosus (Fetcho, 1986) and in the terrestrial salamander Ambystoma tigrinum (postmetamorphosis) (Fetcho and Reich, 1992). Epaxial and hypaxial MNs are intermingled in the motor column, as observed in the lamprey (Wallén et al., 1985) and goldfish Carassius auratus (Fetcho, 1986). Intraspinal marginal neurons having morphological similarities to ECs of lampreys (Grillner et al., 1982, 1984) have been found in urodeles (Schroeder and Egar, 1990), but their role as stretch-sensitive sensors remains to be demonstrated (Bem et al., 2003; Ijspeert et al., 2005; for review, see Chevallier et al., 2008). Such similarities between these cells suggest possible homologies in their function. The following section reviews the similarities and the differences in the rhythmogenic role played by putatively homologous neurons in the four species. We will first review the role of excitatory neurons, and then inhibitory neurons.

The role of spinal neurons in rhythm generation Excitatory neurons Lamprey Excitatory interneurons with ipsilateral descending axons (EINs). In lampreys, EINs have been identified as a key component of the rhythmogenic network by Buchanan and Grillner (1987). EINs have ipsilaterally descending projecting axons (Dale, 1986), exciting MNs (Buchanan and Grillner, 1987), EINs (Buchanan et al., 1989; Parker and Grillner, 2000), LINs, and CCINs through glutamatergic synapses (Buchanan and Grillner, 1987; Buchanan et al., 1989). In each hemisegment, the EINs are believed to constitute a sparsely connected recurrent network: excitation would feed back onto any given neuron via several other interposed EINs (Buchanan and Grillner, 1987; Buchanan et al., 1989; Cangiano and Grillner, 2005). EINs have been shown to be monosynaptically connected with other EINs, but reciprocal connections were not identified (Parker and Grillner, 2000). Pharmacological experiments revealed that the rhythmic activity induced by electrical stimulation in a surgically isolated hemicord is dramatically affected by blocking glutamatergic receptors (i.e., AMPA/Kainate and NMDA) (Cangiano and Grillner, 2005). The presence of electrical coupling between EINs and its role in rhythmogenesis remain to be determined. Simulation studies showed that hemisegmental oscillations can be generated by reciprocally connected EINs when a negative feedback mechanism is included as an intrinsic property of EINs (e.g., spike frequency adaptation; Kotaleski et al., 1999; Kozlov et al., 2007). Simulation studies suggest that the negative feedback could result from a slow build up of outward currents hyperpolarizing the neuron through afterhyperpolarization summation and leading to spike frequency adaptation or burst termination. Such a function could be accomplished via calcium-activated potassium channels (KCa) (El Manira et al., 1994; Hill et al., 1992; but see Meer and Buchanan, 1992) and/or

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sodium-activated potassium channels (KNa) that mediate the Ca2þ insensitive component of the slow afterhyperpolarization in lamprey neurons (Cangiano et al., 2002; Hess et al., 2007; Nanou and El Manira, 2007; Nanou et al., 2008; Wallén et al., 2007; see also Huss et al., 2007). In addition, theoretical studies suggest that the strength of the synaptic interactions could determine the strength of the synchrony between EINs (Brunel, 2000; van Vreeswijk and Hansel, 2001; see also Alvarez and Vibert, 2002). The intensity of the background excitation that the network receives would control the frequency of the bursts generated by the network via a modulation of spike frequency adaptation in EINs (Kotaleski et al., 1999; Kozlov et al., 2007). The background excitation of the network is controlled by a descending drive: EINs receive monosynaptic glutamatergic inputs from descending reticulospinal (RS) neurons (Ohta and Grillner, 1989; see also Buchanan and Grillner, 1987; Shaw et al., 2010), which in turn are driven by specific locomotor centers in the brainstem (mesencephalic locomotor region, MLR; Brocard and Dubuc, 2003; Brocard et al., 2010; Sirota et al., 2000; Smetana et al., 2010; for review, see Dubuc et al., 2008; Le Ray et al., 2010) and the forebrain (diencephalic locomotor region, DLR; El Manira et al., 1997; Ménard and Grillner, 2008). The level of RS excitatory drive determines the burst rate of the spinal network. For instance, during MLR-induced locomotion, RS neurons from the middle (MRRN) and the posterior (PRRN) rhombencephalic reticular nuclei show different recruitment pattern (Brocard and Dubuc, 2003). RS neurons in the MRRN are involved in initiation and maintenance of low-frequency swimming, and at higher frequency, RS cells in both the MRRN and the PRRN are recruited. Additionally, the frequency of rhythmic activity recorded from the ventral roots in isolated spinal cord (Brodin et al., 1985) or hemicord (Cangiano and Grillner, 2003) increases with higher concentrations of glutamatergic agonists, and models of the lamprey CPG have been build on the basis of such

observations (Kozlov et al., 2009; Tråvén et al., 1993). Whether different populations of EINs are involved in backward versus forward swimming, or other motor behaviors, is currently unknown. In a recent study, large RS axons in the spinal cord were recorded using multielectrode techniques in freely behaving lampreys. Some RS cells were selectively activated during forward swimming and others during backward swimming (Zelenin et al., 2009; see also Zelenin, 2005). However, neither the RS cells nor their spinal targets have been identified yet. Excitatory contralaterally and caudally projecting interneurons (CC3). CC3 are excitatory (Buchanan, 1982; Buchanan and McPherson, 1995) and may use glutamate as a neurotransmitter (Mahmood et al., 2009). CC3 receive a disynaptic inhibition from the ipsilateral I1 Müller cells and elicit EPSPs in contralateral LINs, myotomal MNs (Buchanan, 1982), and fin MNs (Mentel et al., 2008). This monosynaptic connection to fin MNs is involved in the alternating activity between fin MNs and myotomal MNs located on the same side of the spinal cord during forward symmetrical swimming (Mentel et al., 2006, 2008; Shupliakov et al., 1992). Excitatory stretch receptors (SR-Es). SR-Es have been identified in the lamprey. SR-Es project to MNs and CCINs on the same side (Grillner et al., 1982, 1984; Rovainen, 1974a; Viana Di Prisco et al., 1990; for review, see Buchanan, 2001; Wallén, 1997). The initiation of the burst in the hemisegmental network is facilitated by the activation of ipsilateral SR-Es (Wallén, 1997). In behaving lampreys, when a burst on the right side bends the body segment, the left side of the spinal cord becomes stretched, activating SR-Es of the left side of the cord. This activation leads to excitation of left side neurons that, in turn, contribute to the onset of burst activity on the left side (Wallén, 1997). Hence, if swimming movements are mimicked by experimentally imposing rhythmic lateral movements to an

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in vitro preparation of the spinal cord, the centrally generated rhythm is efficiently entrained to follow the rhythm of the movement (Grillner et al., 1981; Tytell and Cohen, 2008). Zebrafish As in lampreys, glutamatergic interneurons are involved in rhythmogenesis in zebrafish larvae. The synaptic drive to MNs during fictive swimming involves both excitatory and inhibitory inputs (Buss and Drapeau, 2001; McDearmid and Drapeau, 2006). Moreover, fictive locomotor activity is abolished by application of glutamate antagonists CNQX and AP-5 in larvae (Buss and Drapeau, 2001), indicating that glutamatergic inputs are involved. Multipolar commissural descending interneurons and circumferential ipsilateral descending interneurons (MCoDs and CiDs). In recent studies, McLean et al. (2007, 2008) found that ventrally located MCoDs were recruited at slow speed of swimming but inhibited at intermediate speeds, and when more dorsally located, CiDs were recruited. As the swimming speed still further increased, the ventrally located CiDs that were active at intermediate frequencies were switched off by inhibition while more dorsally located CiDs became recruited (McLean et al., 2007; 2008; see also Bhatt et al., 2007; Kimura et al., 2006; Ritter et al., 2001; for review, see Fetcho and McLean, 2010). These results demonstrated that shifts could occur between classes of interneurons as well as within a specific class of interneurons. In both cases, synaptic inhibition determines which excitatory interneurons are active according to the locomotor speed. Ablation experiments revealed that MCoDs, but not CiDs, are essential to produce the rhythmic axial bending of the body (but not the rhythmic fin paddling) during slow swimming movements (McLean et al., 2007). MCoDs excite contralateral small ventral MNs via a

monosynaptic mixed glutamatergic/electrotonic synapse (McLean et al., 2008). Interestingly, anatomical data suggest that MCoDs excite each other through axon collaterals that are in close proximity to contralateral MCoD somata (McLean et al., 2008). Whether recurrent excitation is involved remains to be determined. Experiments examining fast swimming showed that the ablation of CiDs, but not MCoDs, leads to kinematic deficits in the behavior (see McLean et al., 2007). CiDs are excited monosynaptically by the contralateral Mauthner cell and provide a source of excitation that drives locomotor activity through direct connections on ipsilateral MNs (Bhatt et al., 2007; Kimura et al., 2006; Ritter et al., 2001). The presence of recurrent connections between ipsilaterally projecting glutamatergic neurons has not been shown yet (Kimura et al., 2006). They are probable (Fetcho and McLean, 2010) and have been proposed in locomotor network modeling experiments based on identified zebrafish neurons (Knudsen et al., 2006). At the larval stage, the involvement of gap junctions between spinal neurons in rhythmogenesis remains unknown (Buss and Drapeau, 2001). Before the larval stage, there is a gap junction-coupled network that generates a periodic depolarization spreading to all neurons. This is involved in the spontaneous bursting activity of the spinal motor network (Saint-Amant and Drapeau, 2001; for review, see Saint-Amant, 2010). There is a clear recruitment pattern within the populations of MNs according to the locomotor speed. The smallest ventral MNs are rhythmically active during slow swimming. As the swimming speed increases, larger MNs located further dorsally are recruited, and those active at lower speed remain active (McLean et al., 2007). This recruitment pattern follows the classical “size principle” (Henneman et al., 1965). Interestingly, this principle does not appear to apply to premotor interneurons. Indeed, there is no clear correlation between their soma size and their recruitment (McLean et al., 2007, 2008).

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Moreover, as the speed increases, the recruitment of new interneurons is associated with the deactivation of those that are active at lower speed (McLean et al., 2008; for review, see Fetcho and McLean, 2010). Xenopus tadpole Descending excitatory interneurons with an ipsilateral axon (dINs and dINrs). The excitatory interneurons constituting the rhythmogenic core for swimming and struggling show very different properties. Two types of specialized descending excitatory interneurons with an ipsilateral axon (dINs) have been characterized: dINs fire a single action potential per cycle during swimming and are not active during struggling. However, dINrs fire repetitively during struggling and are not active during swimming (Li et al., 2007; for review, see Roberts et al., 2010). They are anatomically similar except that dINs do not have ascending axons. During swimming, dINs are responsible for the rhythmic excitatory drive through a corelease of glutamate and acetylcholine (Li et al., 2004c) to all rhythmically active ipsilateral spinal neurons (dINs, aINs, cINs, MNs; Dale and Roberts, 1985; Li et al., 2006; Soffe et al., 2009). The authors elegantly provided direct evidence for the involvement of recurrent excitation between dINs in rhythmogenesis: the dINs connect to each other and their mutual excitation sums up from cycle to cycle to keep them depolarized (Dale and Roberts, 1985; Li et al., 2006). Such a positive feedback mechanism within the premotor circuitry may explain the long-lasting fictive swimming activity that persists several seconds after a brief cutaneous stimulus (also observed in vivo). Modeling experiments have also supported this idea (Roberts and Tunstall, 1990; for review, see Roberts and Perrins, 1995). An independent rhythmogenic network appears distributed on each side of the spinal cord, as isolated hemicords can generate rhythmic activity (Soffe, 1989). This

is similar to the lamprey (Cangiano and Grillner, 2003, 2005) and the salamander (Ryczko et al., 2009, 2010). Additionally, a rebound firing property intrinsic to dINs is recruited following reciprocal inhibition from the contralateral side (Soffe et al., 2009) and enhances the reliability of their firing, as proposed by modeling (Roberts and Tunstall, 1990; Sautois et al., 2007). Furthermore, dINs are specifically coupled to each other by gap junctions, and experimental data suggest that such coupling could contribute to maintaining swimming as well as synchronized neural activity (Li et al., 2009). Due to a strong adapting property, dINs fire one spike per cycle and are not capable of discharging repetitively (Li et al., 2006). During struggling, another population of excitatory cells is involved in rhythmogenesis. dINrs provide the excitatory drive via monosynaptic glutamatergic inputs to ipsilateral neurons on the same side (aINs, cINs, dINrs, MNs). Contrasting with dINs, they display high-frequency nonadapting, repetitive firing (Li et al., 2007). Excitatory, sensory-relay interneurons (dlas, dlcs, ecINs). Interestingly, swimming and struggling are initiated by qualitatively different sensory stimulations. Whereas a brief sensory stimulation can induce long-lasting bouts of swimming, repetitive sensory stimulation is needed to elicit struggling. The sensory pathway involved in swimming is as follows: RBs relay cutaneous inputs from the trunk through glutamatergic synapses that strongly activate two types of sensory-relay interneurons: dlas and dlcs (Clarke and Roberts, 1984; Clarke et al., 1984; Li et al., 2001, 2003, 2004b). dlas excite neurons of the ipsilateral hemisegmental network (i.e., dINs, aINs, cINs, MNs), whereas dlcs excite the contralateral hemisegmental network mainly through glutamatergic synapses (Li et al., 2003, 2004b; Roberts and Sillar, 1990). The dlas and dlcs participate in reflexes and swimming (Li et al., 2003; Roberts and Sillar, 1990; Sillar and Roberts, 1988). During swimming and struggling, both

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receive early glycinergic inhibition from premotor aINs and do not fire (for review, see Roberts et al., 2010). The pathway that induces struggling is not fully understood. Struggling appears when the skin is repetitively stimulated. This leads to a tonic activation of RBs, which relay this input to cINs, aINs, and MNs (multifunctional cells, i.e., also active during swimming) and to sensory excitatory commissural interneurons (ecINs) and dINrs, which are active only during struggling (specialized cells) (Li et al., 2007; for review, see Roberts et al., 2010). The ecINs receive weak excitation from the ipsilateral RBs that sums slowly. The ecINs fire repetitively, are weakly adapting, and excite contralateral neurons including cINs, ecINs, and MNs through glutamate synapses (Li et al., 2007). When external cutaneous stimulation ends, so does the struggling. In contrast, swimming behavior persists long after cutaneous stimulation has ended. Additionally, in Xenopus tadpoles, there is direct evidence for electrical connections between MNs. Moreover, central nicotinic synapses from MNs to other MNs and to rhythmically active interneurons have been described (Perrins and Roberts, 1995a,b,c; Zhang et al., 2009). Recurrent excitation may help synchronize firing in addition to intrinsic membrane properties such as Kþ conductances. This would limit or “tune” rhythmic firing to the appropriate frequencies for swimming (Roberts et al., 1997). Salamander AMPA/Kainate receptor activation is required for rhythmogenesis both in surgically isolated hemisegments and segments from the mid-trunk spinal cord of the juvenile salamander Pleurodeles waltlii (Ryczko et al., 2010). This suggests that as described in lampreys, zebrafish larvae, and Xenopus tadpoles, a core of glutamatergic interneurons would provide excitatory inputs to MNs in each hemisegment. Interestingly, rhythmic

excitatory inputs have been described in MNs of the larva of the newt Triturus vulgaris (Perrins and Soffe, 1996; Soffe and Perrins, 1997). Interneurons with bi- or multipolar somata, extensive dorsal and ventral dendrites, and a descending ipsilateral axon have been described both in Triturus vulgaris larvae (Harper and Roberts, 1993) and Xenopus embryos (Roberts and Clarke, 1982). Data in the Xenopus suggest that these interneurons would be active during swimming and would provide the excitatory component within the spinal CPG (Dale and Roberts, 1984; Li et al., 2006; Roberts and Alford, 1986). It remains unknown whether these interneurons are present in juvenile and adult salamanders, and if they provide glutamatergic excitatory inputs to MNs. In a recent study, no effect of the gap junction blocker carbenoxolone was detected on the rhythm-generating capabilities of isolated hemisegments and segments from the axial network of juvenile salamanders (Ryczko et al., 2010). This contrasts with results obtained from young Xenopus tadpoles, in which electrical coupling between dINs (Li et al., 2009) and between MNs (Perrins and Roberts, 1995a,b,c; Zhang et al., 2009) was found to contribute to locomotor rhythm maintenance and synchronization, respectively. Whether electrical coupling between spinal interneurons and/or MNs declines as development proceeds in the salamander, as previously reported in the Xenopus (Hinckley and ZiskindConhaim, 2006; Zhang et al. 2009), is unknown. Neurons participating in rhythm generation often exhibit postinhibitory rebound in their membrane potential when injected with a hyperpolarizing current (for review, see Marder and Bucher, 2001). In isolated segments and hemisegments of the axial network of the salamander, pharmacologically blocking the hyperpolarization activated cation current (Ih) does not affect the ability of axial hemisegments/segments to generate rhythmic bursting. However, cycle durations are longer (Ryczko et al., 2010). This suggests that Ih acts as a depolarizing leak current

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contributing to the triggering of a new burst rather than being essential for rhythmogenesis (Kiehn, 2000).

Inhibitory neurons Lamprey Inhibitory contralaterally and caudally projecting interneurons (CC1 and CC2). Among the three classes of CCINs that have been identified in lampreys (Buchanan, 1982), class 1 (CC1) and class 2 (CC2) are inhibitory glycinergic interneurons. The postsynaptic targets of these interneurons include MNs, LINs, and other CCINs on the opposite side of the cord (Buchanan, 1982). These interneurons have been shown indirectly to inhibit contralateral EINs (Buchanan et al., 1989). CC1 receive monosynaptic excitatory inputs from the ipsilateral RS cell B1 and contralateral Mauthner cell. CC2 receive monosynaptic excitatory inputs from the ipsilateral RS cells B2–B4. Immunochemistry experiments suggest that both of these interneurons use glycine, but not GABA as a neurotransmitter (Mahmood et al., 2009). EINs activate inhibitory CCINs that coordinate the EIN networks on both sides so that alternation emerges (Buchanan, 1999). Additionally, CCINs slow down the burst frequency of the segmental network and stabilize the activities of left and right hemisegmental networks (Cangiano and Grillner, 2005; Kozlov et al., 2009). However, CCINs are not essential for rhythmogenesis. Indeed, rhythm generation occurs in a spinal locomotor network surgically deprived of crossed connections (i.e., hemicord) and even in one single hemisegment (Cangiano and Grillner, 2003, 2005; but see Buchanan, 1999; Jackson et al., 2005). Furthermore, pharmacological blockade of glycinergic neurotransmission does not disrupt rhythmogenesis in the spinal cord (Alford and Williams, 1989; Aoki et al., 2001; Cohen and Harris-Warrick, 1984; Hagevik and McClellan, 1994).

Inhibitory ipsilaterally projecting interneurons (IINs). IINs are small cells with ipsilaterally projecting axons that produce IPSPs in nearby CCINs and MNs (Buchanan and Grillner, 1988; for review, see Buchanan, 2001). Due to their low abundance in the spinal cord, little is known about these neurons. Recently, they have been proposed to generate the inhibition that fin MNs receive while ipsilateral myotomal MNs are excited, during forward symmetrical swimming. The inputs to these cells have not been described but may include inputs from ipsilateral EINs (see discussion in Mentel et al., 2008). Lateral interneurons (LINs). LINs are large cells with lateral dendrites. They are mostly present in the rostral spinal cord (Rovainen, 1974a) and have an ipsilateral axon that can project caudally as far as the tail region of the cord (Rovainen, 1974a). LINs inhibit ipsilateral CCINs (Buchanan, 1982) but have been shown in rare cases to inhibit MNs (Rovainen, 1982). These cells are directly activated by ipsilateral EINs (Buchanan and Grillner, 1987), but they also receive monosynaptic inputs from contralateral CCINs (Buchanan, 1982) and polysynaptic inputs from DCs (Rovainen, 1974a). The LINs receive excitatory descending inputs from B2–B4 Müller cells (Rovainen, 1974b). The LINs may be involved in a propriospinal function (Grillner, 2003). Inhibitory stretch receptors (SR-Is). Among the two types of ECs that have been identified in lampreys (Grillner et al., 1982, 1984; Rovainen, 1974a; Viana Di Prisco et al., 1990), SR-Is project contralaterally to inhibit ECs, LINs, and CCINs. SR-Is also receive input from the I2 Müller cell. In behaving lampreys, the termination of the depolarized phase partially results from the activation of contralateral SR-Is. When the myotomes on the right side of the body contract, the left side of the spinal cord becomes stretched. This results in an activation of the SR-Is of the left side and leads to the termination of the ongoing burst on the right side (for review, see Buchanan, 2001; Wallén, 1997).

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Zebrafish As indicated above, the synaptic drive of MNs during fictive swimming consists of excitatory and inhibitory inputs (Buss and Drapeau, 2001; McDearmid and Drapeau, 2006). In the presence of the glycinergic antagonist strychnine, bilateral alternation is lost but a rhythmic activity is maintained (Buss and Drapeau, 2001; McDearmid and Drapeau, 2006). In the zebrafish, glycinergic neurotransmission is thus not essential for rhythmogenesis as shown in lampreys (Alford and Williams, 1989; Cohen and Harris-Warrick, 1984). Ipsilaterally projecting circumferential ascending interneurons and commissural bifurcating longitudinal interneurons (CiAs and CoBLs). Inhibitory inputs select in accordance to locomotor frequency the set of active neurons that are involved in the rhythm-generating network (McLean et al., 2007, 2008). The origin of these inhibitory inputs is not identified. However, some inhibitory cells in the spinal cord, CiAs and CoBLs, are recruited according to the locomotor frequency during swimming (McLean et al., 2007). The pattern of recruitment is reversed as compared to the excitatory neurons (i.e., MCoDs and CiDs). Dorsal neurons (CiAs) are active during slow swimming frequencies, whereas ventral interneurons (CoBLs) are recruited at higher frequencies (McLean et al., 2007). Additionally, when comparing the active spinal cells during swimming and struggling, Liao and Fetcho (2008) found specialized and multifunctional glycinergic interneurons in the axial network of the zebrafish. Specialized glycinergic interneurons (CoLAs and CoLos). CoLAs are active only during struggling. Because these neurons have long ascending projections, they were proposed to provide inhibition to the rostral segments (Liao and Fetcho, 2008). An inhibition of rostral segments would decrease their excitability. As such the “trailing oscillator” hypothesis, described in lampreys (Matsushima and Grillner, 1992; see also Kozlov

et al., 2009), would predict that a caudorostral gradient of excitability would be created along the spinal cord. This would result in a caudorostral propagating wave. A similar role for ascending inhibition during struggling was proposed in the Xenopus (Green and Soffe, 1998; Tunstall and Roberts, 1994). Other specialized inhibitory interneurons are the CoLos that are selectively activated during the escape response (Liao and Fetcho, 2008; Satou et al., 2009). They receive direct electrotonic inputs from the Mauthner cell. CoLos monosynaptically excite contralateral primary MNs and their ablation impairs the escape response (Satou et al., 2009). Multifunctional glycinergic interneurons (CoSAs and CoBLs). CoSAs were shown to discharge during multiple behaviors such as swimming, escape, and struggling (Liao and Fetcho, 2008). However, CoBLs are active only during struggling and swimming but not during escape. During swimming, CoBLs are also recruited in a dorsoventral manner according to speed (McLean et al., 2007). GABAergic K–A cells. Recently, a function for the K–A cells was proposed. Specific photostimulation of these cells induces slow swimming. Furthermore, silencing them reduces the frequency of spontaneous free swimming, indicating that activity of K–A cells provides a necessary excitability tone for spontaneous forward swimming (Wyart et al., 2009). Xenopus tadpole Commissural interneurons (cINs). The cINs are glycinergic and send ascending and descending branches on the contralateral side (Dale, 1985; Soffe et al., 1984). They elicit a strychnine-sensitive inhibition of contralateral rhythmically active MNs and premotor interneurons. Some cINs also have an additional ipsilateral axonal branch, which may be responsible for recurrent on-cycle

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inhibition of other cINs, dINs, and MNs (Roberts et al., 1997). This inhibition from cINs to dINs is crucial to allow the latter cells to fire on rebound and therefore to drive swimming (Li et al., 2007; Roberts et al., 2008). However, this rebound cannot be the only mechanism contributing to rhythm generation, because the surgically isolated hemicord can still generate a rhythm. Moreover, blocking glycinergic inhibition in the hemicord does not abolish rhythmic activity (Soffe, 1989). The cINs are active during both swimming and struggling (i.e., multifunctional cells), but some that fire unreliably during each swimming cycle are recruited during struggling (Li et al., 2007). It is noteworthy that cINs show high-frequency bursting (up to 245 Hz) during struggling as do MNs (Li et al., 2007). The increase in the firing frequency of these inhibitory neurons may be the source of a contextdependent synaptic depression, as shown by experimental and modeling data (Li et al., 2007). As proposed a long time ago by Brown (1911), synaptic depression of reciprocal inhibition could act as a burst termination mechanism. During struggling, the dynamic depression of the inhibitory synapses would allow the contralateral inhibitory neurons to “escape” from the inhibition, start firing, and then terminate the burst. Context-dependent synaptic depression was observed in cINs when they discharge at high (i.e., during struggling), but not at low, frequency (i.e., during swimming; Li et al., 2007; for review, see Roberts et al., 2010). The underlying mechanisms are currently unknown, but could result from a depletion of readily releasable vesicles, as reviewed elsewhere (Schneggenburger et al., 2002). Inhibitory ipsilaterally projecting interneurons (aINs). The aINs have an ipsilateral ascending axon (Li et al., 2001) that provides a recurrent glycinergic inhibition to all ipsilateral neurons, providing a negative feedback to each ipsilateral network (Li et al., 2004a; Roberts and Tunstall, 1990; Soffe, 1989). These cells mainly gate sensory inputs to the spinal cord by inhibition on sensory dlcs (Li et al., 2002) and dlas (Li et al., 2004b). These cells are weakly active during swimming, but strongly active

during struggling (Li et al., 2002, 2004a, 2007). Therefore, strong filtering of dla and dlcs would be needed during struggling (Li et al., 2007). Finally, a physiological characterization of the function of GABAergic K–A cells is still lacking so far in the Xenopus (Dale et al., 1987), although their implication in struggling via ascending inhibition has been proposed (Green and Soffe, 1996). However, a similar role to that characterized recently in the zebrafish (Wyart et al., 2009) may also be hypothesized. Salamander In the juvenile salamander, reciprocal glycinergic connections may ensure alternation between the activities of left and right hemisegments. Indeed blocking glycinergic transmission abolishes the leftright alternation but not rhythmogenesis (Ryczko et al., 2010), as seen in other species presented here. In Triturus vulgaris larvae, axial MNs were shown to receive glycinergic inputs during fictive swimming (Perrins and Soffe, 1996). Indeed, local application of the glycinergic antagonist strychnine blocks midcycle inhibition and significantly increases the peak on-cycle depolarization in MNs during swimming (Perrins and Soffe, 1996). Immunohistochemical data in the same animals suggest that the source of glycine would come from commissural interneurons (Harper and Roberts, 1993). This needs to be confirmed experimentally as well as examined at other developmental stages (juvenile and adult). Interestingly, commissural neurons including glycinergic neurons were found in the brachial segments (i.e., that innervate both the forelimb and the axial muscles) of the adult mudpuppy (Jovanovic and Burke, 2004; Jovanović et al., 1999). Conclusions Altogether, these studies suggest a phylogenetic conservation of basic building blocks of the axial locomotor networks among vertebrates. There are clear common circuit organization, functional

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properties, and cellular and synaptic mechanisms in the different species. One common basic building block of vertebrate spinal locomotor CPG is a hemisegmental core of excitatory interneurons that provides rhythmic, glutamatergic synaptic drive to other premotor interneurons and MNs (for review, see Grillner, 2003; Kiehn, 2006). In accordance with the findings described above in the four vertebrate species, glutamatergic interneurons were recently shown to also be essential for rhythmogenesis in mammals. Selective photoactivation of ventral glutamatergic neurons in the lumbar spinal region of transgenic mice initiated and maintained fictive locomotion (Hägglund et al., 2010). Glutamatergic neurons are also involved in rhythmogenesis in limb locomotor CPGs of salamanders (Lavrov and Cheng, 2004). This is also the case for other rhythmic activities such as the respiratory network in lampreys (Bongianni et al., 1999; Martel et al., 2007) and mammals (Greer et al., 1991; for review, see Feldman and Del Negro, 2006). Excitatory interactions are involved in the generation of the oscillations in the hippocampus and neocortex (for review, see Yuste et al., 2005). Altogether, these observations suggest that a core of excitatory neurons acts as a common building block in oscillatory networks throughout the CNS (for review, see Grillner et al., 2005). Another common feature of axial rhythmogenic networks is the presence of recurrent excitation between its elements. Multiple sources of recurrent excitation can be involved, as demonstrated in the Xenopus tadpole (for reviews, see Roberts and Perrins, 1995; Roberts et al., 2010). Recurrent excitation is a common feature of several structures in the CNS. Such a positive feedback increases the distribution of excitation onto elements of the circuit by amplifying feedforward input (Douglas et al., 1995). This could help to increase the computational power of networks (Maass et al., 2002). Another common feature in axial network design is the presence of reciprocal inhibition between the two sides, in parallel with crossed

excitation. Inhibitory neurons are essential for the coordination of the left and right sides. However, the role of crossed excitatory neurons is less clear. Whether crossed excitatory connections would be involved in the coordination between both sides in accordance with the locomotor speed or gait, as proposed in the zebrafish, remains to be determined (McLean et al., 2007, 2008; for review, see Fetcho and McLean, 2010).

Dynamic reconfiguration of the axial locomotor network Recent studies addressed the involvement of specific spinal neural populations during locomotion. The rhythm-generating network for locomotion may dynamically change with speed and with the locomotor mode both in the zebrafish (Bhatt et al., 2007; Kimura et al., 2006; Liao and Fetcho, 2008; McLean et al., 2007, 2008; Ritter et al., 2001; Satou et al., 2009) and the Xenopus tadpole (swimming vs. struggling: Li et al., 2007). Moreover, the rhythmogenic mechanisms may also change from one behavior to another (Li et al., 2007). Dynamic reconfiguration of the locomotor network was previously proposed to rely on combinations of specialized interneurons and multifunctional interneurons (for review, see Jankowska, 2001). This is strengthened by results not only in Xenopus tadpoles (for review, see Roberts et al., 2010) and in zebrafish larvae (for review, see Fetcho and McLean, 2010) but also in the cat (Frigon and Gossard, 2010; for review, see Frigon, 2009) and in the turtle (Berkowitz, 2002, 2005, 2007, 2008; for review, see Berkowitz et al., 2010). Interestingly, dynamic changes in the composition of the circuits generating motor patterns have also been described in invertebrates (for review, see Kristan et al., 1988; Marder and Calabrese, 1996; Selverston, 2010). Whether dynamic reconfiguration of the rhythmogenic core also occurs in the spinal locomotor network of lampreys or salamanders is unknown.

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In lampreys, significant differences do exist among cell classes in size-related parameters, such as input resistance and rheobase (Buchanan, 1993). Whether such differences could result in a differential recruitment of these cells at different locomotor frequencies (slow vs. fast swimming) or for different swimming behaviors (forward vs. backward) remains unknown. In salamanders, such a reconfiguration of the spinal locomotor network could occur during transitions from walking to swimming (Ijspeert et al., 2007). However, the characterization of the spinal locomotor cells constituting the adult axial network in this species is still lacking. Additionally, little is known about the cellular properties that determine the recruitment of a neuronal type. Interestingly, the involvement of intrinsic properties as a condition for neuronal recruitment is clearly illustrated in the spinal cord of the Xenopus larva (Li et al., 2007): two types of excitatory cells (dINs and dINrs) are selectively involved in two different motor behaviors generated over considerably different time scales (swimming and struggling). In the salamander, swimming and stepping behaviors are performed in nonoverlapping locomotor frequencies (Delvolvé et al., 1997). It is tempting to suggest that a similar principle could determine the recruitment of specialized cells. Therefore, the nervous system could use different solutions to optimize the activity of specialized cells (Prinz et al., 2004). Little is known about the supraspinal neural populations that drive the specialized and multifunctional spinal cells and how they reconfigure the spinal CPG. For instance, increasing MLR stimulation induces an increase in the locomotor frequency via RS neurons (Brocard and Dubuc, 2003; Brocard et al., 2010; Le Ray et al., 2003; Shik and Orlovsky, 1976; Shik et al., 1966; Sirota et al., 2000). It even elicits gait transitions in many animal species (for review, see Le Ray et al., 2010). Indeed, increasing MLR stimulation leads to switching from walking to trotting, and to

galloping in cats (Shik et al., 1966) as well as a transition from stepping to swimming in salamanders (Cabelguen et al., 2003). Whether different RS nuclei are involved during the transition from one locomotor behavior to another is still an open question. In the lamprey, RS cells show a specific recruitment pattern during MLR-induced swimming. Indeed, MRRN RS cells play a prominent role in the initiation and maintenance of low-intensity swimming, whereas both MRRN and PRRN RS cells contribute to higher frequency swimming (Brocard and Dubuc, 2003). The highest swimming frequencies require the activation of a pool of muscarinoceptive cells that in turn project to RS neurons (Smetana et al., 2010). The recruitment of additional RS cell populations in the brainstem in lampreys may lead to the recruitment of distinct rhythmogenic cores in the spinal axial networks. Acknowledgments We are very grateful to Danielle Veilleux, François Auclair, and Jean-François Gariépy for their comments on this chapter. R. D. receives grants from the Canadian Institutes of Health Research (CIHR; individual and group grants), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Fonds de la Recherche en Santé du Québec (FRSQ; group grant), and the Great Lakes Fishery Commission (GLFC). J.-M. C. receives grants from the European Community (LAMPETRA grant: FP7-ICT-2007-1-216100) and ANR (ImNet grant: ANR-07-NEURO-015-01). D. R. receives salary support from the Groupe de Recherche sur le Système Nerveux Central (GRSNC).

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Jean-Pierre Gossard, Réjean Dubuc and Arlette Kolta (Eds.) Progress in Brain Research, Vol. 187 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 14

General Principles of Rhythmogenesis in Central Pattern Generator Networks Ronald M. Harris-Warrick* Department of Neurobiology and Behavior, Seeley G. Mudd Hall, Cornell University, Ithaca, New York, USA

Abstract: The cellular and ionic mechanisms that generate the rhythm in central pattern generator (CPG) networks for simple movements are not well understood. Using vertebrate locomotion, respiration and mastication as exemplars, I describe four main principles of rhythmogenesis: (1) rhythmogenic ionic currents underlie all CPG networks, regardless of whether they are driven by a network pacemaker or an endogenous pacemaker neuron kernel; (2) fast synaptic transmission often evokes slow currents that can affect cycle frequency; (3) there are likely to be multiple and redundant mechanisms for rhythmogenesis in any essential CPG network; and (4) glial cells may participate in CPG network function. The neural basis for rhythmogenesis in simple behaviors has been studied for almost 100 years, yet we cannot identify with certainty the detailed mechanisms by which rhythmic behaviors are generated in any vertebrate system. Early studies focused on whether locomotor rhythms were generated by a chain of coupled reflexes that require sensory feedback, or by a central neural network. By now there is general agreement that for the major rhythmic behaviors (including locomotion, respiration, and mastication, the subjects of this book), there exist CPG networks within the central nervous system that are able to drive the basic rhythmic behavior in the complete absence of sensory feedback. This of course does not eliminate an important role for sensory feedback, which certainly affects cycle frequency and for some behaviors determines the timing of one phase of the behavior (Borgmann et al., 2009; Pearson, 2008). Given the existence of CPGs, the question of rhythmogenesis can be rephrased to ask how these networks determine the timing of the rhythmic behavior. In this chapter, I focus on cellular and molecular mechanisms that could underlie rhythmogenesis in CPG networks, especially those that drive locomotion, respiration, and mastication. Keywords: Central Pattern Generator; rhythmogenesis; bursting; modulation; ion channel; receptor; calcium.

*Corresponding author. Tel.: þ1-607-254-4355; Fax: þ1-607-254-1303 DOI: 10.1016/S0079-6123(10)87014-1

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Neuronal or network oscillators? Two competing mechanisms have been proposed to underlie rhythmogenesis in central pattern generator (CPG) networks, often presented in an either/or manner: either rhythms are driven by endogenously oscillatory neurons which serve as “pacemaker neurons,” or rhythms are generated by the pattern of synaptic connections within the network, forming a “network pacemaker” mechanism. The central point of the “pacemaker neuron” hypothesis is that the CPG network contains neurons that, even when completely isolated from all synaptic input, continue to oscillate and fire rhythmic bursts of action potentials. Further, these pacemaker neuron oscillations provide the rhythmic synaptic drive to the “follower” neurons to generate the rhythm. A corollary is that if the pacemaker neurons were blocked from oscillating, or eliminated, the rhythmic output from the CPG would halt. In contrast, the “network pacemaker” hypothesis has no requirement for oscillatory neurons at all in the network; if they exist, they are not necessary to generate the rhythm (i.e., eliminating them does not block the rhythm), or they are driven by synaptic input to fire in patterns or phases that are not controlled by their intrinsic rhythmicity. Instead, it is the pattern of synaptic connectivity between otherwise tonically active or silent neurons that generates the rhythmic output. The half-center model, for example, generates rhythmic bursting and alternation by a pattern of reciprocal inhibition between two otherwise tonically active neuron pools; one pool fires tonically and inhibits the other pool until it fatigues or the inhibited pool escapes from inhibition, and then the pools reverse their activity. In the CPG networks that are discussed in this book, inhibitory synaptic input plays a critical role in shaping the frequency and phasing of the rhythmic motor pattern, but is not essential for rhythmogenesis: blockade of g-aminobutyric acid (GABA) and glycine inhibition alters but does not abolish the rhythmgenerating ability of the CPG, showing that they are not organized entirely on a simple half-center

model. Alternative models for network pacemakers are based on networks of mutually excitatory interneurons that receive tonic input to initiate a burst, and terminate it by some fatigue mechanism. In my opinion, these models are not as far apart as they seem, and it is unnecessary to place them in an “either/or” dichotomy. Indeed, there are a number of common features that underlie both the “pacemaker neuron” and “network pacemaker” models for rhythmogenesis. These features arise out of new research, in part reported in this book, describing the neuronal mechanisms in CPG networks. Below I describe four general principles of rhythmogenesis that could serve to guide future research in this area.

Principle I: instead of thinking about rhythmic neurons or circuits, consider rhythmogenic ionic currents The research described in this volume describes the ubiquitous activity of a number of ionic currents which can participate in generating rhythmic outputs from both bursting pacemaker-based and network pacemaker-based CPG circuits. These currents have relatively slow kinetics and can help to maintain neurons in a prolonged depolarized or hyperpolarized state. Regardless of the model for rhythmogenesis, these currents appear to be critical components.

Persistent sodium current or INa(P) The persistent sodium current is the slowly or noninactivating component of the fast voltagedependent sodium current. It can be selectively reduced by low concentrations of riluzole, though this drug is less specific at higher concentrations. INa(P) appears to be involved in rhythmogenesis in most of the neural networks discussed in this volume. Blockade of INa(P) has been shown to abolish fictive respiration in the isolated pre-Bötzinger complex (PBC) slice (Del Negro et al., 2002;

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Koizumi and Smith, 2008), although there is argument over whether this effect is directly on the PBC itself (Koizumi and Smith, 2008) or on adjacent nuclei (Pace et al., 2007b). Blockade of this current abolishes bursting in one major class of pacemaker neurons in the PBC (Del Negro et al., 2002; Pena et al., 2004), but also weakens or abolishes repetitive spiking in many neurons, and thus would also weaken any network-based oscillator (Kuo et al., 2006). In summary, it is not clear whether riluzole blocks the respiratory rhythm via its effects on pacemaker neurons or the network, but INa(P) is clearly important. In the mouse spinal locomotor CPG, blockade of INa(P) by low concentrations of riluzole in normal Ringer solution abolishes fictive locomotion evoked either by transmitter application (N-methyl-D-aspartic acid [NMDA] and 5-HT) or by caudal spinal cord stimulation (Tazerart et al., 2007; Zhong et al., 2007). In both of these studies, the cycle frequency was not affected by riluzole; instead, the strength of the motor output became weaker and weaker, at the same cycle frequency, until the pattern was not detectable. This result was explained by riluzole's effect to reduce repetitive firing in both motoneurons and CPG interneurons, which would weaken synaptic drive at each point in the network until it ceases to function. Interestingly, riluzole has no detectable effect on fictive locomotion evoked in the salamander spinal cord by NMDA and d-serine (Chapter 10). As described by Kolta (Chapter 9), the CPG network driving rhythmic mastication is thought to be centered in the trigeminal sensory nucleus. A subset of neurons in this nucleus fire rhythmic bursts of action potentials upon depolarization; such bursting is significantly enhanced in low-calcium saline, which enhances INa(P) (Li and Hatton, 1996; Tazerart et al., 2008). This bursting in low calcium, as well as bursting evoked by NMDA, is blocked by riluzole. Kolta and colleagues suggest that high levels of activity in the nucleus may activate a mechanism to reduce extracellular calcium, thus activating INa(P)-dependent pacemaker neurons which drive the masticatory rhythm. While

this hypothesis has not yet been tested, it raises the very interesting question of whether significant changes in extracellular ion concentrations can arise during normal motor network activation. In mouse spinal cord slices, zero calcium solutions can also evoke riluzole-sensitive bursting in interneurons thought to be part of the locomotor CPG (Tazerart et al., 2008). Tazerart et al. also argue that significant reductions of calcium might arise normally during locomotion, and that the locomotor CPG may be driven by bursting pacemaker neurons whose rhythmicity is activated by a calciumdependent shift in the voltage dependence of INa(P) activation; however, this appears to conflict with experiments in normal calcium Ringer, where the locomotor CPG works well and its cycle frequency is not affected by riluzole (Tazerart et al., 2007; Zhong et al., 2007). These interesting hypotheses will need to be tested by using ion-selective microelectrodes to measure activity-dependent changes in extracellular ion concentrations in normal calcium-containing solutions.

Calcium-activated nonselective current or ICAN ICAN has no intrinsic voltage dependence, but is activated by increases in intracellular Ca2þ with very slow activation and deactivation kinetics. With these properties, it can support prolonged plateau potentials (Zhang et al., 1995) or rhythmic bursting in neurons. In the respiratory CPG, Ramirez and colleagues identified a set of Cd2þ-sensitive bursting pacemaker neurons in mice older than P5 and suggested that these neurons use ICAN to provide some of the rhythmic drive for respiration under normal conditions; in contrast, during gasping evoked by hypoxia, rhythmogenesis is entirely dependent on riluzole-sensitive currents (Pena et al., 2004). An alternative role for ICAN has been proposed by Del Negro and colleagues (2008) (Rubin et al., 2009): in this “group pacemaker model,” a set of mutually excitatory neurons all contain significant densities of ICAN; a small increase in spike activity in a subset of neurons could initiate

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firing in all of them, which in turn activates ICAN, providing the prolonged depolarizing drive for the burst. Thus, in both pacemaker-based and network-based models, some of the drive that holds the neurons depolarized during an inspiratory burst is mediated by ICAN.

Other inward currents In addition to these two important inward currents, several others have been implicated in rhythm generation. NMDA is often used, with other transmitters, to activate fictive motor patterns in rhythmic CPG networks. In the lamprey spinal cord, NMDA-activated inward currents have been hypothesized to participate in swimming rhythm generation, though whether the current's ability to evoke intrinsic bursting is necessary or not may depend on the cycle frequency (see below). The hyperpolarization-activated inward current, Ih, can be activated by the accumulated hyperpolarization separating bursts of action potentials, and provides a depolarizing ramp current to drive the next cycle of activity. Blockade of Ih significantly slows the frequency of fictive locomotion in the salamander spinal cord (Chapter 10), as well as the well-studied pyloric CPG in the lobster stomatogastric ganglion (Peck et al., 2006). In addition, low-threshold, slowly deactivating calcium currents are thought to support repeated firing in many neurons, and these currents could also provide a depolarizing ramp to sustain rhythmic bursting in CPG networks.

Outward currents The other essential component of a rhythmic system is a mechanism to terminate the burst of action potentials. A number of outward currents play roles in both ending bursts and shaping the spike frequency within the burst; modification of these currents often changes the cycle frequency of the rhythm.

Calcium-dependent potassium current, IK(Ca) IK(Ca) is thought to be the most common current to terminate spike bursts. During a burst of action potentials, calcium channels are activated, resulting in an accumulation of intracellular calcium and increasing activation of IK(Ca); this current eventually outweighs the inward currents (synaptic or intrinsic) that maintain the burst, causing the neurons to fall silent. This in turn closes the IK(Ca) channels either through their intrinsic voltage dependence (BK-type) or as a result of the fall in intracellular calcium during the quiet period (SKtype), allowing synaptic or intrinsic ramp currents to reexcite the neurons and initiate the next burst. IK(Ca) is thought to be the burst-terminating current in the swim CPG in the lamprey spinal cord, as the SK channel blocker apamin reduces the neuronal postburst afterhyperpolarization and slows the fictive locomotor pattern by prolonging each burst (El Manira et al., 1994). Similar effects of apamin are seen in the rat spinal locomotor CPG (Cazalets et al., 1999) and in the masticatory system (Del Negro et al., 1999). Interestingly, blockade of IK(Ca) can stabilize a weak locomotor rhythm in the salamander spinal cord, suggesting that too much IK(Ca) can disrupt the rhythm-generating mechanism (Chapter 10). Other currents that have been implicated in regulation of cycle frequency include the hyperpolarizing current generated by the electrogenic sodium–potassium ATPase as a consequence of the accumulation of intracellular sodium during the burst (Rubin et al., 2009), and A-type transient potassium currents, which help set the rate of repolarization after a spike or burst. Hess and Manira (2001) described a catechol-sensitive high-threshold A-type current in lamprey neurons. This current plays an important role in spike termination: during catechol application, motoneuron spikes are significantly broadened. This in turn causes enhanced sodium channel inactivation, and reduced repetitive spike ability. When the A-current is blocked during fictive locomotion, the neurons fire fewer spikes per burst, reducing crossed inhibition and accelerating

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the cycle frequency. The potassium-dominated leak current can also play an important role in regulating cycle frequency. Smith and colleagues have demonstrated both computationally (Smith et al., 2000) and experimentally (Koizumi and Smith, 2008) the importance of the leak in regulating cycle frequency in the inspiratory rhythm generated in the PBC. Reduction of the leak (by application of substance P) causes bursting neuron depolarization and an acceleration in the cycle frequency (Koizumi and Smith, 2008).

Roles of these currents in rhythmogenesis These examples show the varied but ubiquitous functions of intrinsic ionic currents in shaping rhythmogenesis. By evoking nonlinear responses to subthreshold voltage changes, these currents enable prolonged firing that would sustain a burst, and then burst termination, that are essential for rhythmic motor pattern generation. The appropriate balance of currents is critical for rhythmogenesis to occur (Purvis et al., 2007; Rubin et al., 2009; Smith et al., 2000); this might be modified by modulatory inputs that enable or disable network function. Note that the subthreshold currents that activate these rhythmogenic currents could be endogenous (thus from pacemaker neurons) or synaptic (thus from a network-based oscillator); the requirement for nonlinear enhancement of the subthreshold voltages is the same for both models of rhythmogenesis. Blockade of these currents dramatically affects neurons' abilities to remain depolarized and fire prolonged bursts, regardless of whether they are activated by endogenous mechanisms or by synaptic drive. Models such as Del Negro's “group pacemaker” model (Chapter 8; Rubin et al., 2009) are starting to combine the previously separate models by allowing synaptic drive to initiate the burst-generating intrinsic currents for each burst. Ramirez (this volume) has said that the importance of synaptic or intrinsic drive to initiate a burst may vary from burst to burst: “Each breath is a new breath.” The critical

point is that these rhythmogenic currents are present to amplify the subthreshold input into full bursts of neuronal firing.

Principle II: synaptic currents do not simply depolarize of hyperpolarize the cell by rapid synaptic action, but can also evoke nonlinear membrane responses Pure “network-based” models for rhythmogenesis try to generate a motor rhythm based solely on the rapid transmitter actions of the main transmitters (glutamate, GABA, and glycine) and the spikegenerating mechanisms in the neurons. This is very oversimplified because the “standard” fast synaptic transmitters do far more than simply activating ligand-gated ion channels to depolarize or hyperpolarize the cell. The ionic currents that flow through these channels can in turn activate other currents by methods not simply related to membrane potential. In addition, all neurotransmitters except for glycine also activate slow metabotropic receptors that trigger second messenger pathways to modify ionic currents for prolonged times. Thus, synaptic release of glutamate, for example, need not only cause a simple depolarization.

Multiple actions of glutamate in the lamprey spinal cord Excellent examples of this principle have been demonstrated by El Manira and colleagues studying the multiple actions of glutamate in the swimming CPG of the lamprey spinal cord (Chapter 7). Glutamate activates NMDA receptors, which as described above have been implicated in rhythmogenesis. Glutamate also activates a-amino-3-hydroxyl-5-methyl4-isoxazole-propionate (AMPA) receptors, rapidly depolarizing the neurons, via sodium entry into the cell. Nanou et al. (2008) showed that this sodium entry in turn activates a Slack-type sodium-activated potassium current, IK(Na), which generates a prolonged outward current following AMPA receptor

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activation. This secondary and delayed effect of AMPA receptor activation causes an accumulated diminution of synaptic strength through shunting during a burst of excitatory postsynaptic potentials (EPSPs). Wallen et al. (2007) showed that IK(Na) is also activated by sodium entry during a train of action potentials, and contributes to the postburst hyperpolarization amplitude. The hyperpolarization increases with increasing spike frequency, and could participate (with IK(Ca)) in regulation of burst frequency. These experiments clearly show that the glutamate-evoked AMPA current can, by increasing intracellular sodium, evoke prolonged outward currents that outlast the transmitter action. In addition to activating ionotropic receptors, glutamate also activates metabotropic glutamate receptors (mGluR) in the lamprey spinal cord (for review, see LeBeau et al., 2005). mGluR1 activation leads to a variety of effects. Postsynaptically, it excites the neuron by reducing a leak Kþ current, and increasing the current and calcium influx through NMDA receptors. In addition, it activates endocannabinoid synthesis; these molecules diffuse from the cell and bind to presynaptic CB1 receptors on glycine nerve terminals, reducing glycine release. The combined increase in EPSP and decrease in inhibitory postsynaptic potential response reduces mid-cycle inhibition during fictive locomotion, increasing the cycle frequency (El Manira et al., 2008; Kyriakatos and El Manira, 2007).

Glutamate actions in the respiratory network Similar secondary actions of glutamate have been seen in the inspiratory CPG in the PBC. According to the group pacemaker model of Del Negro and colleagues (Chapter 8; Rubin et al., 2009), mutual synaptic excitation between network neurons is thought to initiate each burst of activity, but alone cannot sustain the full inspiratory burst. Instead, glutamate acts through both AMPA receptors and mGluR to activate ICAN, which is provides the sharp rise to the burst. AMPA receptor activation depolarizes the cell,

which in turn activates voltage-gated calcium currents. mGluR5 activates a PLC-based mechanism resulting in release of calcium from IP3sensitive intracellular stores. These two sources of calcium provide the drive to activate ICAN, which then rapidly depolarizes the neuron and increases its spike frequency during the burst. These examples show the general principle that rapid synaptic events cannot be easily separated from slower intrinsic neuronal currents. Intrinsic currents are triggered by the synaptically evoked change in membrane potential or intracellular ion concentration. In addition, metabotropic glutamate and GABA receptors transform the responses to these traditionally rapid receptors into slow changes in neuronal excitability. Katz and Frost (1995) first described the phenomenon of “intrinsic neuromodulation” of neural networks which contain neurons releasing neuromodulators as well as other transmitters; if the expression of metabotropic glutamate and GABA receptors is common in CPG networks, then intrinsic neuromodulation would be a ubiquitous feature to be taken into account in all models of rhythmogenesis.

Principle III: there are multiple mechanisms for rhythm generation in any system, which could vary with age, species, and modulatory state Given the central significance of respiration, locomotion, and mastication, it is very unlikely that a single mechanism accounts for rhythm generation in any of these systems. It is more likely that multiple and redundant rhythmogenic mechanisms cooperate with one predominating under some conditions, while another may predominate at other times. For example, in the lamprey spinal cord, there appear to be two modes of rhythmogenesis. A slow mode, which is seen in the isolated hemicord activated with NMDA, depends on slow bursting sustained by the voltage-dependent activation of NMDA receptors, while a fast mode is seen during application of D-glutamate and does not require NMDA voltage-dependent activation (Cangiano

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and Grillner, 2003). In the inspiratory network of the PBC, the relative importance of different currents varies under many conditions. For example, ICAN-based oscillatory neurons are very rare before P5, though of course younger animals breathe well (Pena et al., 2004). Under normoxia, ICAN is thought to be an important driver for the eupneic rhythm (Pace et al., 2007a; Pena et al., 2004). However, both in vitro (Pena et al., 2004) and in vivo (Paton et al., 2006), hypoxia evokes a much slower gasping motor pattern which is entirely dependent on INaP. Ramirez and colleagues have elegantly shown how neuromodulators can dramatically alter the cycle frequency as well as the regularity and amplitude of the inspiratory rhythm, due to differential effects on INaP and ICAN, as well as other currents (Ramirez, this volume). In the masticatory CPG, there is a parallel between the development of INaP and depolarization-evoked bursting in neurons in the trigeminal main sensory nucleus, and this in turn correlates with the switch from suckling to chewing (Chapter 9). The principle of multiple and redundant mechanisms for rhythmogenesis is clear even in the most well-studied CPG, that driving the pyloric rhythm in the crustacean stomatogastric ganglion. This 14-neuron network is considered the prototype of an oscillatory neuron-driven rhythmic pattern, where a single neuron, the Anterior Burster (AB) is the pacemaker for the rhythm. However, in the earliest experiments to test the essential role of the AB neuron, Miller and Selverston (1982a) photoinactivated the AB neuron; the remaining network was still able to generate a rhythmic motor pattern, upon stimulation of modulatory inputs, though it had different phasing than the normal rhythm. Only when modulatory inputs were removed did the rhythm stop (Miller and Selverston, 1982a; Russell and Hartline, 1978); this has been shown to be due to the loss of rhythmogenic currents in the absence of neuromodulators. Even under these basal nonmodulated conditions, two of the neurons could form a mutually inhibitory halfcenter oscillator provided that one of the neurons

was slightly depolarized to make it active (Miller and Selverston, 1982b). Even for the single AB neuron, there are multiple and redundant ionic mechanisms for generating oscillations. We have shown that dopamine and serotonin can each evoke rhythmic bursting in a synaptically isolated AB neuron, but use different sets of currents to drive the burst (Harris-Warrick and Flamm, 1987). Dopamine-induced bursting is abolished by removal of calcium or block of release of calcium from intracellular stores, and depends on a flufenamic acid (FFA)-sensitive ICAN to drive the oscillations; DA-evoked bursting is insensitive to blockade of INaP by riluzole or tetrodotoxin (TTX). In contrast, serotonin-evoked bursting is insensitive to FFA, but is blocked by low concentrations of either riluzole or TTX, as well as reductions in extracellular sodium (L. Kadiri and R. Harris-Warrick, unpublished). Thus, depending on the modulatory milieu, this neuron can burst by a calcium-based or a sodium-based mechanism; presumably both mechanisms contribute in varying degrees as the modulatory milieu is changed.

Principle IV: it may not be only neurons: the possible role of glial cells Glial cells constitute the majority of the cells in the brain. It has been known for years that glia can express voltage-activated ion channels as well as transmitter receptors and pumps (for review, see Baker, 2002; Sontheimer, 1994), yet these cells are routinely ignored as possible participants in active networks. Kolta (Chapter 9; Kolta et al., 2007) has raised the very interesting hypothesis that glial regulation of extracellular calcium may play a critical role in maintenance of rhythmogenesis in the masticatory system. According to this hypothesis, during chewing, glial mechanisms reduce extracellular calcium and thereby enhance INaP. This allows bursting of neurons in the trigeminal principal sensory nucleus, which drives the masticatory rhythm. Although this hypothesis remains untested, it emphasizes the possible active participation of glial

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cells in setting the conditions under which rhythmogenesis can be initiated or modulated. This could be a fruitful area for research in the future. Conclusion While the neural mechanisms underlying rhythmogenesis have been studied for many years, the chapters in this volume demonstrate how much recent progress has been made in understanding how simple rhythmic behaviors are generated. These mechanisms appear quite varied, but this is not unexpected. Indeed, multiple mechanisms for rhythmogenesis have been demonstrated in simple invertebrate CPGs, and are very likely in vertebrate networks as well. We may be approaching a synthesis of the traditionally competing models of rhythmogenesis by cellular versus network mechanisms: since all the networks described here depend on boosting by rhythmogenic currents such as INaP or ICAN, it may be a small difference (perhaps enough to vary on a cycle-by-cycle basis) between a burst that is initiated by synaptic input and one that is initiated by intrinsic burst currents. Traditional network oscillator models ignore the many nonlinear sequelae of traditional rapid synaptic input, including activation of ion-sensitive currents such as IK(Na) and ICAN as well as the participation of metabotropic receptors at many if not most synapses. With the possible participation of glial mechanisms to regulate neuronal excitability by altering extracellular ion concentrations or other mechanisms, we have a new area of research into the mechanisms of rhythmogenesis. The next decade should be an exciting time for research into the cellular and molecular mechanisms of rhythmogenesis in locomotor, respiratory and masticatory CPGs. Acknowledgments I would like to thank Abdel El Manira, Christopher Del Negro, Arlette Kolta, Jean-Marie Cabelguen, and Jan-Marino Ramirez for very interesting discussions that helped to generate this

chapter. This work was supported by grants from the National Science Foundation, the National Institutes of Health, the Christopher and Dana Reeve Foundation and the New York State Spinal Cord Injury Research Board.

Abbreviations AB CPG FFA PBC PLC TTX

anterior burster neuron central pattern generator flufenamic acid pre-Bötzinger complex phospholipase C tetrodotoxin

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