京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20806号 / 医博第4306号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.

Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.

Much of our understanding of the fascinating complexity of neuronal circuits comes from anatomical tracing studies that use dyes or fluorescent markers to highlight pathways that run through the brain and spinal cord. Viral vectors have been utilized by many previous groups as tools to highlight pathways or deliver transgenes to neuronal populations to stimulate growth after injury. In a series of studies, we explore anterograde and retrograde tracing with viral vectors to trace spinal pathways and explore their contribution to behavior in a rodent model. In a separate study, we explore the effect of stimulating intrinsic growth programs on regrowth of corticospinal tract (CST) axons after contusive injury. In the first study, we use self-complimentary adeno associated viral (scAAV) vectors to trace long descending tracts in the spinal cord. We demonstrate clear and bright labeling of cortico-, rubro- and reticulospinal pathways without the need for IH, and show that scAAV vectors transduce more efficiently than single stranded AAV (ssAAV) in neurons of both injured and uninjured animals. This study demonstrates the usefulness of these tracers in highlighting pathways descending from the brain. Retrograde tracing is also a key facet of neuroanatomical studies involving long distance projection neurons. In the next study, we highlight a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating MN pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. In a related study, we use a modified version of HiRet as part of a multi-vector system that synaptically silences neurons to explore the contribution of the rubrospinal tract (RST) and CST to forelimb motor behavior in an intact rat. This system employs Tetanus toxin at the neuronal synapse to prevent release of neurotransmitter via cleavage of vesicle docking proteins, effectively preventing the propagation of action potentials in those neurons. We find that shutdown of the RST has no effect on gross forelimb motor function in the intact state, and that shutdown of a small population of CST neurons in the FMC has a modest effect on grip strength. These studies demonstrate that the HiRet lentivirus is a unique tool for examining neuronal circuitry and its contribution to function. In the final study, we explore stimulation of the Phosphoinositide 3-kinase/Rac-alpha serine/threonine Protein Kinase (PI3K/AKT) growth pathway by antagonizing phosphatase and tensin homolog (PTEN), a major inhibitor, to encourage growth of CST axons after a contusive injury. We use systemic infusions of four distinct PTEN antagonist peptides (PAPs) targeted at different sites of the PTEN protein. We find robust axonal growth and sprouting caudal to a contusion in a subset of animals infused with PAPs targeted to the PTEN enzymatic pocket, including typical morphology of growing axons. Serotonergic fiber growth was unaffected by peptide infusion and did not correlate with CST fiber density. Though some variability was seen in the amount of growth within our animal groups, we find these PTEN antagonist peptides a promising and clinically relevant tool to encourage CST sprouting, and a potentially useful addition to therapies using combinatory strategies to enhance growth. These studies demonstrate that viral tracing is a powerful tool for mapping spinal pathways and elucidating their ability to reform spinal circuits after injury. Viral vectors can be used in both anterograde and retrograde tracing studies to highlight intricacies of neuronal cell bodies, axons and dendritic arbors with a high degree of fidelity. In the injured state, these tools can help identify pathways that contribute to spontaneous recovery of function by highlighting those that reform circuits past an injury site. In the uninjured state, these vectors can contain neuronal silencing methods that help define the contribution of specific pathways to behavior. / Neuroscience

Le système vestibulaire et le cortex moteur participent au contrôle de la posture, mais la nature de leurs interactions est peu documentée. Afin de caractériser les interactions vestibulo-corticales qui sous-tendent le contrôle de l’équilibre en position debout, l’activité électromyographique (EMG) du soléaire (SOL), du tibial antérieur (TA) et du péronier long (PERL) de la jambe droite a été enregistrée chez 14 sujets sains. La stimulation galvanique vestibulaire (GVS) a été appliquée avec la cathode derrière l’oreille droite ou gauche à différents intervalles inter-stimulus (ISIs) avant ou après la stimulation magnétique transcrânienne induisant des potentiels moteurs évoqués (MEPs) au niveau des muscles enregistrés. Lorsque que la cathode était à droite, une inhibition des MEPs a été observée au niveau du SOL à un ISI de 40 et 130 ms et une facilitation des MEPS a été observée au niveau TA à un ISI de 110 ms. Lorsque la cathode était à gauche, une facilitation des MEPs a été observée au niveau du SOL, du TA et du PERL à un ISI de 50, -10 et 0 ms respectivement. L’emplacement de ces interactions sur l’axe neural a été estimé en fonction des ISIs et en comparant l’effet de la GVS sur les MEPs à son effet sur l’EMG de base et sur le réflexe-H. Selon ces analyses, les modulations observées peuvent avoir lieu au niveau spinal ou au niveau supraspinal. Ces résultats suggèrent que les commandes de la voie corticospinale peuvent être modulées par le système vestibulaire à différents niveaux de l’axe neuronal. / The vestibular system and the motor cortex are involved in the control of posture but the nature of their interactions is poorly documented. To characterize vestibulo-cortical interactions underlying the control of balance during quiet standing, the electromyographic activity (EMG) of the soleus (SOL), tibialis anterior (TA) and peroneus longus (PERL) of the right leg was recorded in 14 healthy subjects. Bipolar galvanic vestibular stimulation (GVS) was applied with the cathode behind the right or left ear at various inter-stimulus intervals (ISI) before and after transcranial magnetic stimulation eliciting motor evoked potentials (MEP) in the muscles recorded. When the cathode was on the right, MEP in the SOL were inhibited at 40 and 130 ms while MEP were facilitated in TA at 110 ms. When the cathode was on the left, MEP were facilitated in the SOL at 50 ms, in TA at -10 ms and in PERL at 0 ms. The localization of these interactions along the neural axis was estimated according to the ISI and by comparing the effect of the GVS on the MEP to its effect on the background EMG and on the SOL H-reflex. Based on these analyses, the observed modulations of MEP observed could have occurred at spinal or supraspinal level. These results suggest that the corticospinal output may be modulated by the vestibular system at different levels of the neural axis.

Cortical representations are plastic and are allocated based on the proportional use or disuse of a pathway. A steady stream of sensory input maintains the integrity of cortical networks; while in contrast, alterations in afferent activation promote sensorimotor reorganization. After an incomplete spinal cord injury (SCI), damage to the ascending and/or descending pathways induces widespread modifications to the sensorimotor system. Strengthening these spared sensorimotor pathways may be therapeutic by promoting functional recovery after injury. Using a technique called transcranial magnetic stimulation (TMS), we show that the leg motor cortex is facilitated by peripheral sensory inputs via disinhibition and potentiation of excitatory intracortical circuits. Hence, in addition to its crucial role in sensory perception, excitation from peripheral sensory afferents can reinforce muscle activity by engaging, and possibly shaping, the activity of the human motor cortex. After SCI, the amount of excitation produced by afferent stimulation reaching the motor cortex is expectantly reduced and delayed. This reduction of sensory inflow to the motor cortex may contribute to our findings that cortical inhibition is down-regulated after SCI, and this compensation may aid in the recruitment of excitatory networks in the motor cortex as a result of the damage to its output neurons. By repeatedly pairing sensory inputs from a peripheral nerve in the leg with direct cortical activation by TMS, in an intervention called paired associative stimulation, we show that the motor system can be potentiated in both uninjured individuals and after SCI. In the uninjured subjects, we show that in order to produce associative facilitation, the time window required for coincident activation of the motor cortex by TMS and peripheral sensory inputs is not as narrow as previously thought (~100 vs. ~20 ms), likely due to the persistent activation of cortical neurons following activation by TMS. The potential to condition the nervous system with convergent afferent and cortical inputs suggests that paired associative stimulation may serve as a priming tool for motor plasticity and rehabilitation following SCI.

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The purpose of the present work was to study hand impairments and manual ability in children with cerebral palsy (CP) as well as to clarify their relationship. Appraising the degree of hand impairments requires normative data to differentiate the real dysfunctions of CP children from the normal difficulties according to their age, sex, or handedness. As there is no normative data for gross manual and fine finger dexterity, a first experiment focused on the normal development of manipulative functions was conducted to establish these norms. A second experiment was carried out to develop and validate through the Rasch model a measure of manual ability in children with CP since such a measure was not yet available. The invariance of the ABILHAND-Kids questionnaire was also tested across relevant demographic and clinical subgroups of CP children. Finally, a third experiment was performed to quantify the hand impairments in children with CP and to investigate their relationship with manual ability as measured with the ABILHAND-Kids questionnaire. Hand motor impairments, markedly more prevalent than hand sensory impairments, were moderately correlated with manual ability measures and predicted 58% of their variance. Consequently, manual ability cannot simply be inferred from hand impairments and should be measured and treated per se. / L'objectif de ce travail était d'étudier les déficiences de la main et l'habileté manuelle chez les enfants infirmes moteurs d'origine cérébrale (IMOC) ainsi que de clarifier leur relation. Apprécier la gravité des déficiences de la main nécessite des données normatives afin de différencier les dysfonctionnements réels des enfants IMOC des difficultés normales compte tenu de leur âge, sexe, ou latéralité. Etant donné l'absence de normes quant à la dextérité manuelle grossière et la dextérité digitale fine, une première étude a été réalisée afin d'examiner le développement normal de ces deux types de dextérité. Des normes sur les dextérités manuelle grossière et digitale fine ont ainsi pu être établies. Une deuxième étude a été effectuée afin de développer et valider à travers le modèle de Rasch une mesure de l'habileté manuelle chez les enfants IMOC. L'invariance du questionnaire ABILHAND-Kids a également testée à travers différents sous-groupes démographiques et cliniques d'enfants IMOC. Enfin, une troisième étude a été réalisée afin de quantifier les déficiences de la main chez les enfants IMOC et d'investiguer leur relation avec l'habileté manuelle. Les déficiences motrices de la main, plus prévalentes que les déficiences sensitives, étaient modérément corrélées avec les mesures d'habileté manuelle et prédisaient 58% de leur variance. En conséquence, l'habileté manuelle ne peut être simplement inférée à partir des déficiences de la main et devrait donc être mesurée et traitée pour elle-même.

The purpose of the present work was to study hand impairments and manual ability in children with cerebral palsy (CP) as well as to clarify their relationship. Appraising the degree of hand impairments requires normative data to differentiate the real dysfunctions of CP children from the normal difficulties according to their age, sex, or handedness. As there is no normative data for gross manual and fine finger dexterity, a first experiment focused on the normal development of manipulative functions was conducted to establish these norms. A second experiment was carried out to develop and validate through the Rasch model a measure of manual ability in children with CP since such a measure was not yet available. The invariance of the ABILHAND-Kids questionnaire was also tested across relevant demographic and clinical subgroups of CP children. Finally, a third experiment was performed to quantify the hand impairments in children with CP and to investigate their relationship with manual ability as measured with the ABILHAND-Kids questionnaire. Hand motor impairments, markedly more prevalent than hand sensory impairments, were moderately correlated with manual ability measures and predicted 58% of their variance. Consequently, manual ability cannot simply be inferred from hand impairments and should be measured and treated per se. / L'objectif de ce travail était d'étudier les déficiences de la main et l'habileté manuelle chez les enfants infirmes moteurs d'origine cérébrale (IMOC) ainsi que de clarifier leur relation. Apprécier la gravité des déficiences de la main nécessite des données normatives afin de différencier les dysfonctionnements réels des enfants IMOC des difficultés normales compte tenu de leur âge, sexe, ou latéralité. Etant donné l'absence de normes quant à la dextérité manuelle grossière et la dextérité digitale fine, une première étude a été réalisée afin d'examiner le développement normal de ces deux types de dextérité. Des normes sur les dextérités manuelle grossière et digitale fine ont ainsi pu être établies. Une deuxième étude a été effectuée afin de développer et valider à travers le modèle de Rasch une mesure de l'habileté manuelle chez les enfants IMOC. L'invariance du questionnaire ABILHAND-Kids a également testée à travers différents sous-groupes démographiques et cliniques d'enfants IMOC. Enfin, une troisième étude a été réalisée afin de quantifier les déficiences de la main chez les enfants IMOC et d'investiguer leur relation avec l'habileté manuelle. Les déficiences motrices de la main, plus prévalentes que les déficiences sensitives, étaient modérément corrélées avec les mesures d'habileté manuelle et prédisaient 58% de leur variance. En conséquence, l'habileté manuelle ne peut être simplement inférée à partir des déficiences de la main et devrait donc être mesurée et traitée pour elle-même.