La posture, composante statique du contrôle moteur permettant une position érigée du corps, repose sur une décharge tonique des motoneurones innervant nos muscles antigravitaires. La décharge prend la forme de « potentiel de plateau » au niveau de motoneurones matures chez de nombreux vertébrés. Pour déterminer une éventuelle concordance entre l'émergence des propriétés de plateau et le développement postural, notre travail a eu pour but d'étudier la maturation et la nature ionique des potentiels de plateau des motoneurones innervant le muscle triceps surae (extenseur de la cheville) chez le rat nouveau-né.La réalisation de ces travaux de thèse nous a permis de dégager un mécanisme fondamental dans la genèse des propriétés de plateau des motoneurones lombaires. Ce mécanisme dont le fondement repose sur l'activation d'un « ménage à trois » jouerait un rôle majeur dans le développement moteur chez le rat. Dans la mesure où les potentiels de plateau des motoneurones sont fortement perturbés à la suite d'une lésion médullaire, cette avancée scientifique permettra éventuellement de mieux comprendre l'origine de certains déficits sensori-moteurs (spasticité, hyperalgésie...) et le développement de nouvelles stratégies thérapeutiques. / Posture allowing an erect posture of the body relies on spiking activity of motoneurons innervating antigravitary muscle. Discharge could take the form of plateau potential on mature motoneurons of numerous vertebrates. To determine a possible concordance between the emergence of plateau potential and postural control development, we studied the maturation and ionic nature of plateau potential of motoneurons innervating triceps surae muscle of neonatal rat.The conclusion of our work allows us to propose a fundamental mechanism in the genesis of plateau potential on lumbar motoneurons. This mechanism based on a "ménage a trois" seems to play an important role in the neonatal motor development. This scientific advance could eventually lead to a better understanding of the origin of some sensori-motor impairments (spasticity, hyperalgesia...) and development of therapeutic strategies.

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The present study examined the role of group I metabotropic glutamate receptors (mGluRs) in mammalian spinal motor networks and investigated the potential role of mGluRs in the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Group I mGluR activation was found to modulate locomotor-related activity recorded from ventral roots of in vitro mouse spinal cord preparations. Activation of group I mGluRs led to an increase in the frequency of locomotor-related bursts and a decrease in their amplitude. The cellular mechanisms underlying group I mGluR-mediated modulation were investigated using whole-cell patch-clamp recordings from spinal neurons. Recordings from motoneurons revealed a wide range of effects, some of which were expected to increase motoneuron excitability, such as membrane depolarisation and hyperpolarisation of action potential thresholds. However, the net modulatory effect of group I mGluR activation was a reduction in motoneuron excitability, likely reflecting a reduction in the density of fast inactivating Na+ currents. The activation of group I mGluRs also reduced excitatory synaptic input to motoneurons, suggesting that modulation of motoneuron properties and synaptic transmission both contribute to group I mGluR-mediated reductions in locomotor motoneuron output. Recordings from spinal interneurons revealed a smaller range of modulatory effects for group I mGluRs. The clearest effect on interneurons, membrane depolarisation, may underlie group I mGluR-mediated increases in the frequency of locomotor activity. Finally, the potential role of group I mGluRs in the pathogenesis of ALS was investigated using a mouse model of the disease. Although no major perturbations in group I mGluR-mediated modulation were demonstrated in ALS affected spinal cords, there appeared to be a difference in the intrinsic excitability of spinal interneurons between wild type and ALS affected animals. Together these data highlight group I mGluRs as important sources of neuromodulation within the spinal cord and potential targets for the treatment of ALS.

Qualquer tarefa motora ativa se dá pela ativação de uma população de unidades motoras. Porém, devido a diversas dificuldades, tanto técnicas quanto éticas, não é possível medir a entrada sináptica dos motoneurônios em humanos. Por essas razões, o uso de modelos computacionais realistas de um núcleo de motoneurônios e as suas respectivas fibras musculares tem um importante papel no estudo do controle humano dos músculos. Entretanto, tais modelos são complexos e uma análise matemática é difícil. Neste texto é apresentada uma abordagem baseada em identificação de sistemas de um modelo realista de um núcleo de unidades motoras, com o objetivo de obter um modelo mais simples capaz de representar a transdução das entradas do núcleo de unidades motoras na força do músculo associado ao núcleo. A identificação de sistemas foi baseada em um algoritmo de mínimos quadrados ortogonal para achar um modelo NARMAX, sendo que a entrada considerada foi a condutância sináptica excitatória dendrítica total dos motoneurônios e a saída foi a força dos músculos produzida pelo núcleo de unidades motoras. O modelo identificado reproduziu o comportamento médio da saída do modelo computacional realista, mesmo para pares de sinal de entrada-saída não usados durante o processo de identificação do modelo, como sinais de força muscular modulados senoidalmente. Funções de resposta em frequência generalizada do núcleo de motoneurônios foram obtidas do modelo NARMAX, e levaram a que se inferisse que oscilações corticais na banda-beta (20 Hz) podem influenciar no controle da geração de força pela medula espinhal, comportamento do núcleo de motoneurônios até então desconhecido. / Any active motor task is accomplished by the activation of a motor unit population. However, due to many ethical and technical difficulties the synaptic input to the motoneurons cannot be measured in humans. For these reasons realistic computational models of a motoneuron nucleus and the innervated muscle fibers have an important role in the study of the human control of muscles. However such models are complex and their mathematical analysis is difficult. In this text a system identification approach of a realistic motor unit nucleus model is presented with the objective of obtaining a simpler model capable of representing the transduction of the motor unit nucleus inputs into the muscle force signal associated to that nucleus. The system identification was based on an orthogonal least squares algorithm to find a NARMAX model, the input being the net dendritic excitatory synaptic conductance of the motoneurons and the output being the muscle force signal produced by the motor unit nucleus. The identified model output reproduced the mean behavior of the output from the realistic computational model even for input-output signal pairs not used during the identification process, such as sinusoidally modulated output muscle force signals. Generalized frequency response functions of the motoneuron nucleus were obtained from the identified NARMAX model, and led to an inference that cortical oscillations in the beta band (20 Hz) can affect force control by the spinal cord, an unknown motoneuron nucleus behavior until now.

Qualquer tarefa motora ativa se dá pela ativação de uma população de unidades motoras. Porém, devido a diversas dificuldades, tanto técnicas quanto éticas, não é possível medir a entrada sináptica dos motoneurônios em humanos. Por essas razões, o uso de modelos computacionais realistas de um núcleo de motoneurônios e as suas respectivas fibras musculares tem um importante papel no estudo do controle humano dos músculos. Entretanto, tais modelos são complexos e uma análise matemática é difícil. Neste texto é apresentada uma abordagem baseada em identificação de sistemas de um modelo realista de um núcleo de unidades motoras, com o objetivo de obter um modelo mais simples capaz de representar a transdução das entradas do núcleo de unidades motoras na força do músculo associado ao núcleo. A identificação de sistemas foi baseada em um algoritmo de mínimos quadrados ortogonal para achar um modelo NARMAX, sendo que a entrada considerada foi a condutância sináptica excitatória dendrítica total dos motoneurônios e a saída foi a força dos músculos produzida pelo núcleo de unidades motoras. O modelo identificado reproduziu o comportamento médio da saída do modelo computacional realista, mesmo para pares de sinal de entrada-saída não usados durante o processo de identificação do modelo, como sinais de força muscular modulados senoidalmente. Funções de resposta em frequência generalizada do núcleo de motoneurônios foram obtidas do modelo NARMAX, e levaram a que se inferisse que oscilações corticais na banda-beta (20 Hz) podem influenciar no controle da geração de força pela medula espinhal, comportamento do núcleo de motoneurônios até então desconhecido. / Any active motor task is accomplished by the activation of a motor unit population. However, due to many ethical and technical difficulties the synaptic input to the motoneurons cannot be measured in humans. For these reasons realistic computational models of a motoneuron nucleus and the innervated muscle fibers have an important role in the study of the human control of muscles. However such models are complex and their mathematical analysis is difficult. In this text a system identification approach of a realistic motor unit nucleus model is presented with the objective of obtaining a simpler model capable of representing the transduction of the motor unit nucleus inputs into the muscle force signal associated to that nucleus. The system identification was based on an orthogonal least squares algorithm to find a NARMAX model, the input being the net dendritic excitatory synaptic conductance of the motoneurons and the output being the muscle force signal produced by the motor unit nucleus. The identified model output reproduced the mean behavior of the output from the realistic computational model even for input-output signal pairs not used during the identification process, such as sinusoidally modulated output muscle force signals. Generalized frequency response functions of the motoneuron nucleus were obtained from the identified NARMAX model, and led to an inference that cortical oscillations in the beta band (20 Hz) can affect force control by the spinal cord, an unknown motoneuron nucleus behavior until now.

Cholinergic input to, and cholinergic mechanisms within the lower lumbar (L6) and upper sacral (S1) spinal cord of rat may influence neuronal excitability and afferent transmission (Thor et al, 2000) and may provide the environment necessary for appropriate central nervous system control of bladder and bowel function. It is unclear, however, if cholinergic terminals and receptors are present in the L6 & S1 spinal segments of rat and when this may develop. Cholinergic mechanisms have been shown to alter sensory afferent transmission, enhance motoneuron excitability, induce plateau potentials via non-linear membrane properties in motoneurons and reveal oscillations in locomotor-related interneurons. The enhanced activity of sphincter motoneurons was attributed to non-linear properties during the continence phase of distention-evoked voiding in the decerebrate cat (Paroschy & Shefchyk, 2000). Candidate neurotransmitters inducing non-linear properties in cat sphincter motoneurons are 5-HT (Paroschy & Shefchyk, 2000) and acetylcholine via motoneuron axon collaterals (Sasaki, 1994) and other spinal sources. We have established using the antibody to the vesicular acetylcholine transporter (VAChT) that cholinergic terminals are present on ventrolateral Onuf (VLO), dorsomedial Onuf (DMO) motoneurons and parasympathetic preganglionic motoneurons (PGN) in the L6 and S1 rat spinal cord segments. Muscarinic receptor (M2), nicotinic-α4 and α7 receptor subunit immunoreactivity was also present on Onuf motoneurons and in regions dorsal to the PGN. One source of the cholinergic puncta on Onuf motoneurons may be from motoneuron axon collaterals which we observed on a postnatal day 15 VLO motoneuron. Cholinergic terminals were observed on vasoactive intestinal polypeptide-immunoreactive (VIP) afferents, interneurons in the intermediolateral (IML) region and perhaps on other afferents in the lateral and medial collateral pathway of L6 and S1 spinal segments. In the ventral horn, the cholinergic puncta and receptors appear to have a mature distribution around two weeks postnatal and the cholinergic terminals appeared to have a mature distribution in the IML region by three weeks postnatal. Using whole cell patch clamp recording techniques and thick slices of the L6 and S1 rat spinal cord, we observed excitatory responses of ventral horn neurons and motoneurons to carbachol (10-50 μM), a non-specific cholinergic agonist. Ventral horn neurons (postnatal day 8- 16) exhibited prolonged firing and prolonged depolarizations (plateau potentials) beyond the duration of the applied excitatory input from cholinergic (n=6/33) and other (n= 4/37) neurotransmitter systems. In a selection of the neurons with plateau potentials, the L-type calcium current played a role in the plateau production (n=5/5) and low frequency oscillations (n=2/2) as revealed by nifedipine. Postnatally, the voiding reflex changes from a perineal-evoked reflex, to the adult bladder-bladder reflex. Cholinergic input may be responsible in part for the bursting activity of the external urethral sphincter and the activation of the bladder, which is required for complete voiding reflexes in the adult rat. Plateau potentials and enhanced excitability due to cholinergic mechanisms could render inessential a constant excitatory drive that is required in the perineal-evoked voiding reflex in the neonatal rat and may underlie changes in the voiding reflexes that occur during postnatal development. / February 2006

L’Esclerosi Lateral Amiotròfica (ELA o ALS) és una malaltia neuromuscular caracteritzada per la degeneració selectiva de les motoneurones (MN) superiors e inferiors del còrtex motor, el tronc de l’encèfal i la medul•la espinal, que resulta en una debilitat, espasticitat i atròfia progressives de la musculatura. Menys del 10% dels casos corresponen a la forma familiar de la malaltia, i un 20% d’aquests estan relacionats a mutacions en el gen de l’enzim superòxid dismutasa 1 (mSOD1). La resta de casos corresponen a la forma esporàdica. Les causes implicades en la degeneració selectiva de les MN en la ELA són encara desconegudes. La seva patogènesi s’ha atribuït a diversos mecanismes com serien l’estrès oxidatiu, l’agregació proteica anormal, la disfunció mitocondrial, el transport axonal aberrant, la neuroinflamació, l’autoimmunitat o l’excitotoxicitat per glutamat. En el present estudi hem treballat amb dues d’aquestes hipòtesis en avaluar l’efecte dels sèrums de pacients amb ELA i altres malalties de la MN sobre l’activitat del receptor ionotròpic de glutamat de tipus N-metil-D-Aspartat (NMDAR), expressat en el model d’oòcit de Xenopus laevis. Mitjançant assaigs de ELISA hem analitzat la presència d’autoanticossos associats a ELA en el sèrum de pacients. L’acció dels sèrums control i patològics en els oòcits de Xenopus produïa la generació de corrents oscil•latoris de clorur (Cl-). Aquests corrents havien estat prèviament descrits en aquestes cèl•lules i són deguts a l’activació dels canals de Cl- dependents de calci (Ca2+), endògens en els oòcits de Xenopus, a causa de la mobilització de Ca2+ intracel•lular. L’alliberació de Ca2+ dels compartiments intracel•lulars es activada per l’acció d’un factor sèric, anomenat àcid lisofosfatídic o lisofosfatidat (LPA), sobre el seu receptor, present en la membrana dels oòcits, i a través d’una via de senyalització de segons missatgers. Així doncs, en aquest model, la generació de corrents oscil•latoris de Cl- és una mesura indirecta de la mobilització intracel•lular de Ca2+. En presència del NMDAR, les respostes generades pel sèrum ELA eren significativament superiors a les activades pel sèrum d’individus sans i d’altres malalties de la MN. La resposta generada pel sèrum ELA presentava una dependència respecte de la presència de les dues subunitats del NMDAR i era sensible al bloqueig del receptor amb MK-801, un antagonista no competitiu. Vàrem reproduir els experiments amb sèrums del model de rata transgènica mSOD1 G93A, considerat un model de la forma familiar de la malaltia. Les mostres de sèrum mSOD1 G93A generaven, en presència del NMDAR, respostes significativament superiors a les activades pel sèrum de rata WT. En analitzar l’acció de la fracció de IgG purificada dels sèrums control i patològics en el model d’oòcit de Xenopus, es generaven corrents transitoris d’entrada de tipus no oscil•latori, els quals diferien dels generats en el cas del sèrum complet. La resposta activada per IgG de pacients amb ELA en presència del NMDAR era també significativament superior a la generada per les IgG d’individus sans. En la segona part d’aquest estudi s’ha comprovat la presència d’anticossos contra la proteïna Semaforina 3A (Sema3A) en alguns sèrums de ELA i Lower Motor Neuron Disease (LMND), una altra forma comuna de malaltia de la MN. La Sema3A és una molècula quimiotàctica de guia axonal recentment relacionada amb la patologia de la ELA en detectar-se una sobreexpressió d’aquesta proteïna en cèl•lules de Schwann terminals del model de ratolí mSOD1 G93A. Tot i descartar-se que els anticossos contra Sema3A siguin un marcador específic de la ELA, al no detectar-se en tots el sèrums de pacients, i alhora, al estar presents també en algunes mostres LMND, aquests autoanticossos podrien tenir un efecte defensiu contra les senyals nocives exercides per Sema3A sobre els axons de les MN. / Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease, characterized by the selective degeneration of the superior motor neurons in the motor cortex and of the inferior motor neurons in the brain-stem and spinal cord. The familial form of the illness is associated with the mutation of the superoxide dismutase enzyme (SOD-1). This and other mutations accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form. In this study we tested the effect of sera from sporadic ALS patients and from mutated human SOD-1 (mSOD1 G93A) transgenic rats on N-methyl-D-aspartate receptors (NMDAR). We hypothesize that an endogenous excitotoxic factor is implicated in neuronal death in ALS, mediated by the activation of NMDAR noncanonical signalling pathways. Sera from ALS patients or healthy subjects were pretreated to inactivate complement pathways and dialysed to remove glutamate. Sera from mSOD1 G93A rats were obtained at different stages of the neurodegenerative progression. Sera from transgenic rats were also pretreated to eliminate complement system and glutamate. Immunoglobulins G (IgGs) from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and glutamate-induced currents were recorded using the two electrode voltage clamp technique. We observed that sera from sporadic ALS patients induced transient oscillatory currents in Xenopus oocytes expressing NMDAR with a total electric charge significantly higher than the electric charge carried by currents induced by sera from healthy subjects. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. Results of sera from mSOD1 G93A transgenic rats were similar to those of sera from ALS patients; samples from patients with another type of neuromuscular disease did not exert this effect. IgG from ALS patients have a significant effect on NMDAR-injected oocytes and that response was doubled respect to the observed in the case of IgG from healthy subjects. Our data agree with the view that ALS patients sera contain some soluble factors that activates NMDAR, not opening directly the ionic conductance, but activating a non-canonical pathway.