Keywords: Motor cortex, Human, spinal cord transcranial direct current stimulation (tDCS), extensor carpi radialis

chez l'Homme : altération et récupération fonctionnelle

Monsieur le Professeur François Mauguière et

Monsieur le Docteur Laurent Vinay m'ont fait l'honneur d'être rapporteurs à mon jury de thèse. Je les remercie vivement pour leurs judicieux conseils et leurs critiques constructives.

Madame le Docteur Emmanuelle Apartis-

Bourdieu m'a fait l'honneur de présider ma thèse ; qu'elle en soit ici remerciée.

Monsieur le Professeur Alberto Priori m'a fait l'honneur d'être examinateur de ma thèse. Je l'en remercie vivement.

Madame le Professeur Pascale Pradat-Diehl, m'a accueillie dans le service de Médecine Physique et

Réadaptation de la Pitié-Salpêtrière. Je la remercie de m'avoir permis de réaliser mon travail auprès du Docteur Rose Katz au cours du mon stage de M2, ce qui m'a, par la suite, donné la possibilité de poursuivre par une thèse d'université dans ce laboratoire.

Monsieur le Professeur Emmanuel Pierrot-Deseilligny, fondateur du laboratoire, par ses travails et ses écrits, m'a permis de découvrir et d'apprendre la Neurophysiologie. Qu'il trouve ici mes plus vifs remerciements.

Madame le Docteur Alexandra Lackmy-Vallée, pour sa gentillesse, son aide, sa grande générosité ainsi que pour ses précieux conseils, qu'elle trouve ici le témoignage de ma gratitude.

Madame le Docteur Véronique Marchand-Pauvert, merci d'avoir toujours pris le temps de répondre à mes questions avec patience et gaité.

Monsieur le Docteur Nicolas Roche, sans qui la sélection et l'enregistrement des patients à l'hôpital

Raymond-Poincaré n'auraient pas été possibles.

Madame le Docteur Valérie Achache, pour ses encouragements, sa gaîté et sa gentillesse, qu'elle trouve ici mes plus vifs remerciements.

Monsieur le Docteur Jean-Charles Lamy, merci pour l'aide apportée au cours des expériences, ainsi que pour ses précieux conseils.

Madame Geneviève Bard, je la remercie pour son soutien administratif, ses précieux conseils, son aide et surtout pour son amitié.

Monsieur le Docteur Nachiket Nadkarni, je tiens à le remercier pour avoir toujours pris de son temps pour corriger mes divers écrits en anglais.

Louis-Solal Giboin, Berthe

Introduction

Motor function (i.e. muscle tone, posture and movement) depends on the firing of the motoneurons (MNs) "the final common pathway" (Sherrington, 1906). Features of MN firing (number of MNs activated, frequency of firing and pattern of activation) reflect an integration of the upstream activity in different structures of the central nervous system (CNS) (motor cortex, cerebellum, basal ganglia, vestibular system, and spinal cord circuitry).

Most available data concerning the knowledge of neural pathways involved in motor control come from experiments performed in animal preparations (anaesthetised, decerebrated or spinalised) using invasive methods that cannot be used in humans. Due to phylogenetic evolution, data obtained in animals cannot be always transposed in man without caution for several reasons, for example:

INTRODUCTION

My research projects fit into these frameworks and focus on two aspects:

1. The study of changes in spinal inhibitory network induced by furosemide in healthy subjects and patients with spinal cord injury (SCI).

Recent studies in animals have shown that down-regulation of potassium chloride co-transporter KCC2 after spinal cord transection reverses the effect of gamma aminobutyric acid (GABA) and glycine back to their immature (excitatory) state (Vinay & Jean-Xavier, 2008) and thus results in a reduction of inhibitory synaptic efficiency (Boulenguez et al. 2010). If this phenomenon also exists in humans following SCI, it may contribute to the reflex hyper-excitability exhibited in SCI patients. To address this question, we demonstrated effect of a diuretic (furosemide), a cation-chloride cotransporter antagonist, on spinal network excitability in healthy subjects and SCI patients.

Ipsi-and contralateral corticospinal control on reciprocal inhibition between

forearm muscles in healthy subjects and hemiplegic stroke patients as demonstrated by transcranial direct current stimulation.

Transcranial direct current stimulation (tDCS) has emerged as a method for exploring cortex excitability (Priori et al., 1998;Nitsche & Paulus, 2000), and has also begun to be used as a tool to enhance recovery from hemiplegia. Recent studies in healthy subjects have shown that anodal tDCS over the contralateral hand motor cortex can induce changes in spinal network excitability (i.e. reciprocal inhibition) (Roche et al., 2009(Roche et al., , 2010[START_REF] Roche | Effects of anodal tDCS on lumbar propriospinal system in healthy subjects[END_REF]. It is unknown whether motor activity from the unaffected cerebral hemisphere could be employed after semi-brain damage in patients with hemiplegia. Moreover, little is known about the non-affected limb if it always functions like 'normal' after unilateral stroke. To address this question, the ipsi-and contralateral corticospinal controls on reciprocal inhibition between forearm muscles were explored using anodal tDCS applied over the motor cortex of non-lesioned hemisphere of stroke patients and then compared to healthy subjects.

The common objective of these projects is to disclose pathophysiology of motor impairment following CNS lesions so as to develop new therapeutics treatments (i.e. new rehabilitation programs relying on neurophysiological bases, pharmacological treatments). This thesis is organized into four chapters. The first chapter is dedicated to basic knowledge of physiology and functioning of spinal networks studied in this thesis. The second chapter firstly presents the basic electrophysiological method used in common by the two studies together with its advantages and limitations, followed by two sections separating the methods used in each study. The third chapter is also divided into two sections: i) the first section reports results obtained in the study observing changes in spinal inhibitory network induced by a diuretic (furosemide) in

Chapter 1 Background of physiology CHAPTER 1 BACKGROUND OF PHYSIOLOGY

Presynaptic inhibition of Ia fibres

It has been shown that the synaptic efficacy of the primary sensory afferent (Ia fiber) -α-motoneuron synapse can be regulated by presynaptic inhibition (PSI) at the spinal cord. The sensory information can be thereby modified before reaching the target neuron to optimise motor performance and sensory discrimination.

In 1957, Frank and Fuortes, have shown in the cat that stimulation of sensory afferent fibres leads to a reduction in excitatory postsynaptic potentials (EPSPs) generated in motoneurons in the spinal cord. This decrease occurs without any accompanying change in the postsynaptic membrane. Reproduced from Frank & Fuortes, 1957.

The presynaptic inhibition is mediated by interneurons acting on primary afferent terminals through axo-axonic synapses (reviewed in Eccles, 1964). This results in a depolarization of primary afferents, termed primary afferent depolarization (s) which leads to a reduction in transmitter release at synaptic junction, thus inducing a decrease in excitatory postsynaptic potentials (EPSPs). Pharmacological studies have shown that presynaptic inhibition is reduced by the GABA A antagonists [START_REF] Eccles | Pharmacological studies on presynaptic inhibition[END_REF][START_REF] Curtis | Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat[END_REF]. The existence of GABAergic axo-axonic synapses on the terminal of Ia afferent fibres is proposed as a mechanism underlying presynaptic inhibition (reviewed in [START_REF] Rudomin | Selectivity of presynaptic inhibition: a mechanism for independent control of information flow through individual collaterals of single muscle spindle afferents[END_REF]. Activation of GABA A receptors increases the efflux of CI -ions and thus inducing depolarisation of the terminal of afferent fibres. This induces a reduction of the amplitude of propagated action potential that blocks or reduces Ca 2+ influx, thereby inhibiting the release of neurotransmitter from Ia afferent axons (reviewed in Rudomin & Schmidt, 1999). It has been shown in the cat that the latency of presynaptic inhibition was 1.5-1.8 ms and at least two interneurons (first-order PAD interneuron and last-order PAD interneuron) were basically required in this control (reviewed in Jankowska, 1992). The long-duration presynaptic inhibition has been suggested to be due to sustained activity of PAD interneurons (Eccles et al., 1962b) or the slow dynamic of GABA release and/or uptake (reviewed in [START_REF] Rudomin | Presynaptic Inhibition and Neural Control[END_REF].

CHAPTER 1 BACKGROUND OF PHYSIOLOGY

Electrophysiological studies in animals showed that PAD interneurons can be activated by repetitive electrical stimulation, vibration or stretching of the muscle tendon (Eccles , 1964;[START_REF] Devanandan | Muscle stretch and the presynaptic inhibition of the group Ia pathway to motoneurones[END_REF], 1966[START_REF] Gillies | Presynaptic inhibition of the monosynaptic reflex by vibration[END_REF].

CHAPTER 1 BACKGROUND OF PHYSIOLOGY

1.1

Inputs to PAD interneurons

Peripheral inputs to PAD interneuron

Interneurons mediating presynaptic inhibitions are facilitated by group I afferents. In humans, there is no direct evidence showing that Ib afferents also contribute to the activation of the PAD interneurons inhibition as demonstrated in animals. However, the fact that the threshold of inhibition "D1" of the soleus motoneurons induced by conditioning stimuli of the common peroneal nerve (0.9-1 × motor theshold) (Mizuno et al., 1971;[START_REF] Iles | Evidence for cutaneous and corticospinal modulation of presynaptic inhibition of Ia afferents from the human lower limb[END_REF] is higher than that of disynaptic reciprocal Ia inhibition (0.6-0.7 × motor theshold) would be compatible with the involvement of Ib afferents. Studies in animals and humans showed that low-threshold cutaneous afferent volleys induce a decrease of presynaptic inhibition of Ia fibres (Iles & Robert, 1987;[START_REF] Nakashima | Cutaneous effects on presynaptic inhibition of flexor Ia afferents in the human forearm[END_REF]Meunier & Pierrot Desseilligny, 1998;Rudomin et al., 1983). Anaesthesia of the hand in human subjects increased the amount of presynaptic inhibition. This confirms that cutaneous input can modulate transmission in presynaptic inhibitory pathways in humans [START_REF] Nakashima | Cutaneous effects on presynaptic inhibition of flexor Ia afferents in the human forearm[END_REF]. At least in the cat, articular afferents also induce a decrease on presynaptic inhibition and both cutaneous and articular afferents depress transmission in PAD pathways at the level of the first-order PAD interneurons [START_REF] Lund | Inhibitory action from the flexor reflex afferents on transmission to Ia afferents[END_REF]Rudomin et al., 1983)

Descending inputs to PAD interneuron

Inhibitory descending control

The main descending control influencing PAD interneurons mediating presynaptic inhibition of Ia fibres is depressive. Corticospinal fibres and cutaneous afferents converge onto inhibitory interneurons and suppress the activity of first-order PAD interneurons (reviewed in Lundberg & Vycklicky, 1963;Rudomin et al., 1983). Last-order PAD interneurons are tonically depressed via different reticulospinal pathways (reviewed in Rudomin & Schmidt, 1999). The interruption of this strong tonic inhibitory control results in net increase in the excitability of PAD interneurons presynaptic inhibition after spinalization in decerebrate animals. The brainstem structures responsible for tonic depression of presynaptic inhibition of Ia fibers are controled by inhibition effect from the higher centers. Therefore, presynaptic inhibition is diminished in decerebrate animals. In humans, presynaptic inhibition is consistently decreased in the lower limb after spinal cord lesions [START_REF] Faist | A quantitative assessment of presynaptic inhibition of Ia afferents in spastics. Differences in hemiplegics and paraplegics[END_REF][START_REF] Nielsen | Changes in transmission across synapses of Ia afferents in spastic patients[END_REF]Pierrot-Desilligny, 1990). Study in humans showed that motor cortex stimulation reduces D1 inhibition of the soleus H-reflex [START_REF] Meunier | Cortical control of presynaptic inhibition of Ia afferents in humans[END_REF] confirming corticospinal depression of PAD interneuron of presynaptic inhibition of soleus Ia terminals.

CHAPTER 1 BACKGROUND OF PHYSIOLOGY Facilitatory descending control

Facilitatory descending control on PAD interneuron of presynaptic inhibition probably exists, but it is generally less dominant than inhibitory effects (reviewed in [START_REF] Lundberg | To what extent are brain commands for movements mediated by spinal interneurons[END_REF]. The first-order PAD interneurons could be excited via vestibular nuclei [START_REF] Carpenter | Primary afferent depolarization evoked from the sensorimotor cortex[END_REF].

Postsynaptic inhibition induced by cutaneous stimulation

Postsynaptic inhibition induced by cutaneous stimulation plays a role in withdrawal reflex/flexor reflex. This is a spinal reflex, causing a contraction of limb flexors in response to nociceptive stimulus to protect the body from damage. Flexor reflex was firstly introduced by Sherrington in 1906. In decerebrate animals, noxious skin stimuli on the plantar of foot induce contraction of flexor muscles of hip and knee and dorxiflexor of the foot while antagonist extensor muscles are inhibited (Sherrington 1906[START_REF] Sherrington | Flexion-reflex of the limb, crossed extension-reflex, and reflex stepping and standing[END_REF]. Sherrington also states that the flexion movement at this joint is according to the skin-point stimulated, introducing the concept of 'local sign'. The connection between sensory neuron (impulses from nociceptors) with motoneurons in the flexion reflex circuit is shown to be polysynaptic involving several interneurons (reviewed in [START_REF] Hunt | Spinal reflex mechanisms concerned with skeletal muscle[END_REF]. The flexion reflex was confirmed in humans by [START_REF] Hagbarth | Spinal withdrawal reflexes in the human lower limbs[END_REF]. Noxious stimuli applied to the plantar of foot evoke reflexes, which closely resemble with the flexion reflex of Sherrington. It was suggested by the author that flexor reflex can be elicited in all lower limb muscles when the adequate receptive field is stimulated. The latencies of these reflexes were relatively short which indicate that they are probably mediated by spinal reflex arc (reviewed in [START_REF] Kugelberg | Demonstration of A and C fibre components in the Babinski plantar response and the pathological flexion reflex[END_REF][START_REF] Pedersen | Studies on the central pathway of the flexion reflex in man and animal[END_REF].

Influence of cutaneous volley on the modulation of soleus monosynaptic reflex was demonstrated by [START_REF] Hugon | Exteroceptive reflexes to stimulation of the sural nerve in normal man[END_REF]. After electrical stimulation of the sural nerve (cutaneous foot afferents), reflex response appear at a latency of about 80 ms in different muscles of both legs and also induces an inhibition of the monosynaptic soleus H-reflex. The effects of single cutaneous stimuli on soleus motoneuron excitability depend on the site of stimulation and the inhibition recorded from ankle tendon jerk is greater when cutaneous stimuli applied over the lateral dorsum or the plantar surface of the foot than over the mid-dorsum of the foot. Burke et al. (1991) and Nielsen et al. (1997) presented the evidence that the cutaneous effect induced in the lower limb with latency shorter than 70 ms, is spinal in origin. Their results are also supported by the fact that the cutaneous stimuli induced spinal motoneuron inhibition exists in spinal animals and complete spinal cord lesions in humans (Longchampt & Chanelet, 1968;Logigian et al, 1999).

The descending control seems to have a net facilitatory effect on cutaneous inhibition. A study in patients with spinal cord lesion demonstrated that soleus motoneurons could be inhibited by high-intensity cutaneous stimuli of sural nerve over the same ISI range as normal subject, but the degree of inhibition is less than in normal subjects (Logigian et al, 1999).

CHAPTER 1 BACKGROUND OF PHYSIOLOGY

No direct evidence demonstrates neurotransmitter involved in postsynaptic inhibition induced in soleus motoneuron by cutaneous afferents. Few pharmacological studies have shown that strychnine, an antagonist of spinal postsynaptic inhibition, blocked the effects of glycine on spinal motoneurons and interneurons [START_REF] Curtis | The pharmacology of spinal postsynaptic inhibition[END_REF] and inhibition of motoneuron involved in flexor reflex has been shown to be mediated by glycine [START_REF] Dhawan | Selective inhibition by glycine of some somatic reflexes in the cat[END_REF].

Post-activation depression (synaptic transmission between Ia fibre and motoneuron)

It has been shown in the cat that the amplitude of monosynaptic reflex decreases during repetitive stimulation of afferent fibres [START_REF] Eccles | Effects induced in a monosynaptic reflex path by its activation[END_REF]. A frequency related depression of this reflex has been described at stimulus intervals as long as 10-20 s [START_REF] Lloyd | Reflex depression in rhythmically active monosynaptic reflex pathways[END_REF]. Using intracellular recordings of motoneurons in the cat, [START_REF] Curtis | Synaptic action during and after repetitive stimulation[END_REF] have demonstrated changes in the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve (Figure 4). A) Repetitive monosynaptic EPSP's recorded from gastrocnemius motoneuron in a cat with amplifier and the frequency of the maximum group I volley from posterior tibial nerve of each record B) Repetitive monosynaptic EPSP's at the indicated frequencies (Hz) when the EPSP had attainted a steady state. C) Repetitive monosynaptic EPSP's plotted against the stimulus frequencies. Above the frequency scale, the corresponding stimulus intervals are shown in msec. Reproduced from [START_REF] Curtis | Synaptic action during and after repetitive stimulation[END_REF].

CHAPTER 1 BACKGROUND OF PHYSIOLOGY

The recording reveals changes in amplitude of EPSP following changes in frequencies. There is no change in the EPSP's size until the frequency is in excess of 0.4/sec. There is a progressive depression of EPSP as the frequency is raised to 5-10/sec, whereas, at higher rates, EPSPs increase and reach a maximum at approximately 50/sec. It has been suggested that the depression results from reduced release of neurotransmitter from Ia afferent fibers to motoneurons [START_REF] Kuno | Mechanism of facilitation and depression of the excitatory synaptic potential in spinal motoneurones[END_REF]. Repetitive activation of the Ia afferents can be expected to induce a reduction of neurotransmitter stores in the Ia afferent terminal. If this reduction becomes pronounced, the neurotransmitter release would be insufficient for subsequent activation. The postsynaptic depression appears to depend on the intrinsic neuronal properties of the motoneuron. Facilitation effect is dominant for high-threshold fast motoneuron, while depression is dominant for low-threshold slow motoneuron [START_REF] Honig | Alpha-motoneuron EPSPs exhibit different frequency sensitivities to single Ia-afferent fiber stimulation[END_REF].

It has been also shown in humans that the amplitude of H-reflex decreases during repetitive stimulation (Magladery & McDougal, 1951;Paillard, 1955;[START_REF] Rothwell | Habituation and conditioning of the human long latency stretch reflex[END_REF]Hultborn & Nielsen, 1998;[START_REF] Hultborn | On the mechanism of the post-activation depression of the H-reflex in human subjects[END_REF]Aymard et al., 2000;[START_REF] Lamy | Post-activation depression in various group I spinal pathways in humans[END_REF]. This depression can be attributed to the previous activation of Ia afferent because it is observed after a conditioning stimulus that is subthreshold for the H-reflex or tendon jerk [START_REF] Katz | Conditioning of H reflex by a preceding subthreshold tendon reflex stimulus[END_REF]Hultborn & Nielsen, 1998). A long lasting inhibition of the soleus H-reflex can also be evoked by a preceding soleus H-reflex, by a brief of voluntary ankle flexor or extensor muscle contraction or by a tap applied to Achilles tendon. The time course of this long lasting inhibition was similar in all cases suggesting that the mechanism responsible for this depression is the same. At stimulus intervals between the preceding conditioning stimulus and the soleus H-reflex test longer than 12 s, the size of the H-reflex did not increase, whereas a slight decrease of the H-reflex is observed at a conditioning-test interval of 10 s and the depression increases rapidly when the frequency of stimulation below 10 s (Crone & Nielsen, 1989) (see Figure 5).

In addition, post-activation depression is reported to be decreased in spastic patients compared to healthy subjects [START_REF] Nielsen | Regulated properties of motoneurons and primary afferents: new aspects on possible spinal mechanisms underlying spasticity[END_REF][START_REF] Nielsen | Changes in transmission across synapses of Ia afferents in spastic patients[END_REF], Hultborn & Nielsen, 1998). The amount of post-activation depression of the flexor carpi radialis (FCR) and soleus H-reflex is significantly reduced on the affected side of hemiplegic patients, whereas no changes were observed on the unaffected side and also in normal subjects (Aymard et al., 2000[START_REF] Lamy | Impaired efficacy of spinal presynaptic mechanisms in spastic stroke patients[END_REF]. A) A subthreshold tendon tap applied to the Achilles tendon B) A brief voluntary contraction of tibialis anterior C) A brief voluntary contraction of soleus D) Sustained contraction of soleus. In A-D the test reflex was elicited every 14s and the conditioning stimulus/movement was elicited at varying intervals (1-12 s) before the test reflex. The size of conditioned H-reflex was expressed as a percentage of its control value measured at a stimulus frequency of 1 stimulus per 14 s. Reproduced from Crone & Nielsen, 1989.

Reciprocal inhibition

The concept of reciprocal inhibition was firstly introduced by Sherrington in 1897. Sherrington's law of reciprocal innervation states that: When a muscle contracts, its direct antagonist relaxes to an equal extent allowing smooth movement. Pathway of reciprocal Ia inhibition was firstly investigated in the cat lumbar motoneurons using monosynaptic test reflexes, suggesting that Ia afferents had 'direct effect' on antagonistic motoneuron, introducing 'direct inhibition' (Lloyd, 1946). However, the hypothesis of direct inhibition was failed since intracellular recording subsequently showed that an interneuron is interpolated in the Ia inhibitory pathway [START_REF] Eccles | The central pathway for the direct inhibitory action of impulses in the largest afferent nerve fibers to muscle[END_REF] and activity in this pathway can inhibit the monosynaptic reflex [START_REF] Araki | Correlation of the inhibitory post-synaptic potential of motoneurones with the latency and time course of inhibition of monosynaptic reflexes[END_REF]. The interneuron has been called 'Ia interneuron' [START_REF] Eccles | Types of neurone in and around the intermediate nucleus of the lumbosacral cord[END_REF]. The Ia interneuron was introduced as a mechanism changing the excitatory action of Ia afferents into inhibitory action, determining as a simple 'integrative centre' (Eccles & Lundberg, 1958). Individual Ia inhibitory interneuron inhibits approximately up to 20% of motoneuron of a given nucleus. However, each interneuron induces only simply small inhibitory postsynaptic potentiels (IPSIs) in motoneurons, and so at least 70 interneurons are required to inhibit individual motoneurons (Jankowska & Robert, 1972).

In general, Ia interneuron directly inhibits antagonist motoneuron. However, this rule cannot be applied in some cases. Intracellular recordings showed that reciprocal Ia inhibition is not found between adductors and abductors (Eccles & Lundberg, 1958) or expiratory and inspiratory intercostal muscles of the same segment [START_REF] Sears | Investigations on respiratory motoneurones of the thoracic spinal cord[END_REF]. In contrast, there is an inhibition from knee extensor to motoneuron of the hip flexor (Eccles & Lundberg, 1958). It was shown in decerebrated cat that in the hindlimb that maximal Ia IPSPs are much larger in flexor than in extensor motoneuron (Eccles & Lundberg, 1958). Besides its connection to antagonist motoneuron, Ia inhibitory interneuron also projects to opposite Ia interneuron [START_REF] Hultborn | Convergence on interneurones mediating the reciprocal Ia inhibition of motoneurones. II. Effects from segmental flexor reflex pathways[END_REF]. For example, Ia interneuron activated from flexor Ia afferents inhibits Ia interneuron activated from extensor Ia afferents.

It was shown in human ankle (Mizuno et al., 1971), wrist (Day et al., 1984), and elbow [START_REF] Katz | Reciprocal Ia inhibition between elbow flexors and extensors in the human[END_REF], that group I afferent fibres are involved in reciprocal inhibition. As stressed in the cat by [START_REF] Hultborn | Recurrent inhibition from motor axon collaterals of transmission in the Ia inhibitory pathway to motoneurones[END_REF], Ia inhibitory interneurons can be inhibited by Renshaw cell. Except from Renshaw cells themselves, Ia interneurons are the only spinal interneurons that receive recurrent inhibition. This specific characteristic allows to distinct between reciprocal Ia and non-reciprocal group I inhibition (see Pierrot-Deseilligny & Burke, 2012). In humans, reciprocal inhibition at ankle and elbow levels have been considered as 'true' reciprocal Ia inhibition since i) the CHAPTER 1 BACKGROUND OF PHYSIOLOGY muscles are 'real' antagonists ii) the inhibition can be evoked by a pure Ia volley, and iii) the inhibition is depressed by recurrent inhibition [START_REF] Katz | Reciprocal Ia inhibition between elbow flexors and extensors in the human[END_REF][START_REF] Baret | Evidence for recurrent inhibition of reciprocal inhibition from soleus to tibialis anterior in man[END_REF]. Unlike ankle and elbow levels, the reciprocal inhibition between wrist flexors and extensors is not inhibited by Renshaw cells activation and there is no evidence that the inhibition can be evoked by pure Ia volley (Aymard et al., 1995). Indeed, flexor carpi radialis (FCR) and extensor carpi radialis (ECR) are not real antagonists since they operate as synergistic muscles in wrist abduction movement. The inhibition of ECR motoneurons by activation of Ia afferent fibres from the median nerve is not only inhibition between antagonistic muscles since the median nerve also innerves finger flexors muscles that are synergists of wrist extensor in clenching and grasping. Moreover, the fact that the interneurons mediating reciprocal inhibition between wrist flexors and extensors also receive excitation from group I afferent of the other nerve, including homonymous nerve, suggesting that this inhibition is likely non-reciprocal group I inhibition (reviewed in Pierrot-Deseilligny & Burke, 2012). Further investigation supported the evidence that Ib afferents probably contribute to the activation of interneuron mediating the reciprocal inhibition from ECR to FCR.

Inputs to non-reciprocal interneurons

Segmental inputs a) Group I afferent fibres

Convergence from group I elbow muscle afferents and antagonistic group I afferents onto interneurons mediating reciprocal inhibition from ECR to FCR was revealed in post-stimulus time histogram (PSTH) of single voluntarily activated motor units (Aymard et al., 1995).

Selectively raising the electrical threshold of Ia fibres in the conditioning volley during the prolonged vibration has been shown to increase the electrical threshold of reciprocal Ia inhibition between elbow muscles [START_REF] Katz | Reciprocal Ia inhibition between elbow flexors and extensors in the human[END_REF]. However, such prolonged vibration did not alter the electrical threshold of the inhibition at the wrist level suggesting that the dominant input to the relevant interneurons is Ia in origin at the elbow level but Ib in origin at the wrist level (Wargon et al., 2006). These results could be likely explained by the convergence of Ia and Ib afferents onto the relevant nonreciprocal 'Ib' interneurons, described in cats [START_REF] Harrison | Sources of input to interneurones mediating group I non-reciprocal inhibition of motoneurones in the cat[END_REF].

b) Mutual inhibition

In animal studies, mutual inhibition between opposite-side interneurons has been identified as a general central nervous system mechanism. Baldissera et al. (1987) CHAPTER 1 BACKGROUND OF PHYSIOLOGY also demonstrated that mutual inhibition exists in humans at the wrist level. Stimulating of group I afferents in the radial nerve results in an inhibition of the FCR H-reflex. Similarly, stimulating of group I afferents in the median nerve results in an inhibition of the ECR H-reflex c) Recurrent inhibition Ia interneurons are only spinal interneurons that can be blocked by recurrent inhibition. Contrary to what was observed both in the cat hindlimb and in human elbow muscles, the interneuron mediating reciprocal inhibition between wrist flexors and extensors is not inhibited by Renshaw cells (Aymard et al., 1995). Activation of Renshaw cells by intravenous administration of levo-acetylcarnitine blocked Ia reciprocal inhibition from triceps to biceps muscles but not disynaptic inhibition from ECR to FCR [START_REF] Rossi | Group Ia non-reciprocal inhibition from wrist extensor to flexor motoneurones in humans[END_REF].

Descending inputs

Studies in animals showed that non-reciprocal group I inhibitory interneurons (Ib) receives excitatory supraspinal control from corticospinal and rubrospinal tracts and that they are inhibited by the dorsal reticulospinal and the noradrenergic reticulospinal system (Jankowska & Lundberg, 1981). Studies in humans show that TMS does not increase the reciprocal inhibition from ECR to FCR and sometimes even decreases. It was suggested that the corticospinal excitatory inputs to inhibitory interneurons mediating reciprocal in the human wrist are weak [START_REF] Mercuri | Effects of transcranial electrical and magnetic stimulation on reciprocal inhibition in the human arm[END_REF]Munier & Pierrot-Deseilligny, 1989;[START_REF] Kato | Effects of transcranial magnetic stimulation to the reciprocal Ia inhibitory interneurones in the human wrist[END_REF].

Ionic theory

Conductance increase during an impulse

Important insights into the mechanism of action potential were obtained by [START_REF] Cole | Electric impedance of the squid giant axon during activity[END_REF]. Using an electrode inserted along the squid giant axon, these authors showed that the membrane resistance dramatically decreases when the potential action occurs. A resistance of about 1000 Ω/cm 2 at rest falls to a minimum value of 25 Ω/cm 2 during the action potential, implying an increase of conductance of nerve impulse. This experiment partly confirmed Bernstein's hypothesis (1902) that a nerve impulse represents a temporary "breakdown" of the membrane resistance.

5.2

Reversal in membrane polarity "Na hypothesis"

As a conductance is a measure of the ion permeable aspect of the membrane, it has been believed that the depolarisation of a nerve fibre during excitation involves an increase of permeability to ions. The importance of ionic movements in excitable tissue has been shown by a number of animal experiments. Overton's work in 1902 demonstrated the irritability of frog muscles in relation to the sodium ion (Na + ). The frog muscles quickly lost their power to respond to direct stimulation during they were immersed in a solution containing 10% less of sodium concentration than normally found in the external medium. Hodgkin & Katz (1949) have proposed that the net entry of Na + in one impulse is probably large enough to charge the membrane capacity to the full extent of the action potential, the entry of Na + is the direct cause of the change in membrane potential. It has also been proposed that the rate of rise and amplitude of the action potential are determined by the ratio of concentration of Na + inside and outside of the axon (Hodgkin & Katz, 1949;Huxley & Stämpfil, 1951). These experiments are consistent with the theory that nervous conduction depends on a specific increase in permeability which allows Na + to move from outside of nerve fibre where its region of concentration is high to its region of low concentration inside it. Hodgkin (1977) stated that "In all of these theoretical action potentials the reversed potential difference at the crest of the spike depended on a selective increase in Na permeability and a low internal concentration of Na ions". Moreover, it was also considered that a selective increase in the sodium conductance would induce not only for the depolarization but also for the reversal of the membrane potential.

CHAPTER 1 BACKGROUND OF PHYSIOLOGY

5.3

Resting Membrane Potential and Ionic Distribution during neuron activity Bernstein (1902) suggested that resting membranes are highly permeable to potassium ions (K + ). Hodgkin & Katz (1949) has confirmed Bernstein's hypothesis demonstrating that there is much more K + inside than outside the cell, and the potassium-selective channels are open in resting neuron implying the resting membrane is more permeable to K + than to the other ions. The flow of ions through membrane channels is dependent on ion gradients across the membrane. For example, Na + is actively pumped out of cells while K + is pumped in (Na + /K + -ATPase). The concentrations of chloride ions (Cl -) and calcium ions (Ca 2+ ) are also maintained at greater levels outside the cell. The ionic composition inside the cell is substantially different from that of extracellular fluids, leaving negative charges within the cell.

Using measurement of electric potential across the membrane and radioactive tracers, [START_REF] Keynes | The sodium and potassium content of cephalopod nerve fibres[END_REF] showed that sodium and potassium are important to have reliable values for the intracellular ionic concentrations since a net entry of sodium during nervous activity approximately balances the loss of potassium. The diffusion of Na + into the cell during depolarisation reduces the resting membrane potential creating an excitatory postsynaptic potential (EPSP). Until reaching the threshold, the reduced voltage now opens up a great amount of the sodium channels in the membrane resulting in an increase of Na + influx into the cell; the action potential is then generated.

An outflow of potassium ions has been proposed to be related with repolarisation during the falling phase and this phase may be accelerated by a process which increases the potassium permeability after the action potential has reached its crest (Hodgkin et al., 1949). Chloride conductance has also been suggested to be activated by hyperpolarisation in Aplysia neurons. Increasing the intracellular CIconcentration markedly increased the steady stage conductance [START_REF] Chesnoy-Marchais | Characterization of a chloride conductance activated by hyperpolarization in Aplysia neurones[END_REF]. Hyperpolarisation (inhibitory postsynaptic potential (IPSP)) is thus thought to be caused by an efflux of K + or influx of CI-.

Chloride channel

CI-has been reported to be both distributed passively and actively transported through membrane channel. In resting skeletal muscle and erythrocytes, CIpermeability is extremely high, CI-distributes passively due to the membrane potential, and thereby the intracellular CI-concentration is close to electrochemical equilibrium causing the active transports of CI-hardly work to counteract this high permeability [START_REF] Hodgkin | The influence of potassium and chloride ions on the membrane potential of single muscle fibres[END_REF]Hutter & Nobel, 1960;[START_REF] Adrian | Potassium chloride movement and the membrane potential of frog muscle[END_REF][START_REF] Adrian | Internal chloride concentration and chloride efflux of frog muscle[END_REF]. While, in squid axon, it was shown that the intracellular CI-concentration was 2-3 times higher than equilibrium [START_REF] Keynes | Chloride in the squid giant axon[END_REF]. Studies in neuron cell reveal GABA hyperpolarised neuron response by opening ligand-gated CI-channel showed that the chloride equilibrium potential (ECI) was not equal to resting membrane potential [START_REF] Kuffler | Mechanism of gamma aminobutyric acid (GABA) action and its relation to synaptic inhibition[END_REF][START_REF] Boistel | Membrane permeability change during inhibitory transmitter action in crustacean muscle[END_REF], suggesting that active CI-transport would exist. Active CI-transport mechanisms have consequently been suggested. In skeletal muscle and red blood cell, CI-is in equilibrium since CI-permeability is high that masks active CI-transport. In immature neuron, CI-intruder such as the Na+-K+-2CI-cotransporter (NKCC) establish a higher intracellular CI-concentration than electrochemical equilibrium, whereas CI-extruder such as the Na+-CI-cotransporter (KCC) induces a lower intracellular CI-concentration than electrochemical equilibrium in mature neuron (For reference see [START_REF] Alvarez-Leefmans | Physiology and Pathology of chloride transporters and channels in the nervous system: From molecules to diseases[END_REF].

Cation-chloride cotransporter

General characteristics

During the recent years, there has been an increase in the studies focusing on the role of cation chloride cotransporters (CCCs). The CCCs are secondary active transporters that mediate the couple movement of Na+ and/or K+ to that of CI-across cell membrane, known as Na+-K+-2CI-cotransporter (NKCCs) and Na+-CIcotransporter (KCCs). They move CI-across membrane against their concentration/or electrochemical gradient using the potential energy stored in the electrochemical transmembrane gradient of Na + (generated by the Na+ pump). Two isoforms of NKCC (NKCC1 and NKCC2) and four isoforms of KCC (KCC1-4) have been identified. Chloride exporters are the KCCs, while the NKCCs act as chloride importers.

Neuronal function

The role of CCCs in neurons is currently intensively investigated. It was shown in animal studies that CCCs are determining in the control of the intracellular CI - concentration (Payne et al., 2003). Chloride homeostasis is important for a range of neurophysiological process in mammals. It is known that gamma aminobutyric acid (GABA) and glycine, common inhibitory neurotransmitters in the adult CNS, activate chloride-permeable ionotropic glycine and GABA A receptors (GlyRs and GABA A Rs) [START_REF] Legendre | The glycinergic inhibitory synapse[END_REF][START_REF] Fritschy | Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications[END_REF]. During development of neurons, the expression of CCCs plays an important role in changes of GABA and glycine actions. In embryonic and early postnatal life of the rat, neurons in the brain show strong expression of NKCC1, whereas expression of KCC2 is low [START_REF] Plotkin | Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain[END_REF][START_REF] Clayton | Ontogeny of cation-Clcotransporter expression in rat neocortex[END_REF][START_REF] Wang | Developmental changes in KCC1, KCC2, and REFERENCE NKCC1 mRNA expressions in the rat brain[END_REF], resulting in high intracellular Cl -levels. This forces GABA A receptors to open, inducing an efflux of Cl -through their channel, thus depolarizing of GABA A /Glycine responses. As development progresses, the expression of NKCC1 decreases while the expression of chloride exporters, like KCC2 increases. This results in a decrease of the intracellular chloride concentration, inducing an influx of Cl -, thereby hyperpolarizing of GABA A /Glycine response (reviewed in Ben-Ari, 2002;[START_REF] Blaesse | Cation-chloride cotransporters and neuronal function[END_REF]. A negative shift in the GABA reversal potential (E GABA ) is paralleled by a strong up-regulation of KCC2 near the end of the second postnatal week in the rat hippocampal and neocortical pyramidal neuron [START_REF] Rivera | The K+/Cl-co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation[END_REF]. It has been demonstrated in humans that KCC2 expression in the neocortex begins to increase at 40 weeks after conception [START_REF] Dzhala | NKCC1 transporter facilitates seizures in the developing brain[END_REF]. From these evidences, the developmental shift in GABA and glycine actions from excitatory to inhibitory is likely due to strong expression of co-transporters KCC2 and down-regulation of co-CHAPTER 1 BACKGROUND OF PHYSIOLOGY transporters NKCC1. In addition, it was shown that after neonatal complete spinal cord transection in the rat, thereby removing the influence of the brain on the lumbar spinal cord, prevents or at least delays the switch from depolarizing to hyperpolarizing IPSPs that normally occurs during postnatal development (Jean-Xavier et al., 2006). This reversal appears related to down-regulation of KCC2, thus decreases in intracellular chloride concentration. Ben-Ari, 2002;Jean-Xavier et al, 2006. CHAPTER 1 BACKGROUND OF PHYSIOLOGY

7.3

Physiopathology in the brain

Animal studies

Two members of the CCCs family, KCC2 and NKCC1 have been much interest in studies on GABArergic signaling under pathophysiological conditions, such as seizures, and stroke (reviewed in Kahle al., 2008;[START_REF] Löscher | Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments[END_REF]. The contribution of altered NKCC1 and KCC2 activity to epileptogenesis has been extensively explored. As the NKCC1 expression is high during the neonatal period, elevated intracellular Cl -level induces the excitatory action of GABA, this has been proposed to contribute to increased efficacy of neonatal seizure [START_REF] Dzhala | Excitatory actions of endogenously released GABA contribute to initiation of ictal epileptiform activity in the developing hippocampus[END_REF][START_REF] Khazipov | Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus[END_REF]. Hypersensitivity of GABA response in the hippocampus is also observed after KCC2 knockdown, inducing generalized seizure in the adult mice (Hübner et al., 2001;Woo et al., 2002). A study in the rat revealed a sustained downregulation of KCC2 after focal cerebral ischemia (Jaenisch et al., 2009). It was proposed that this down-regulation of KCC2 would lead to positive shift of the chloride equilibrium potential, which is expected to reduce functional GABAergic inhibition. As epileptic seizure occasionally occur after stroke, the post ischemic KCC2 reduction might be involved in the pathogenesis of epilepsy after brain lesion. However, it is not unclear whether this reduction of KCC2 is a result or the cause of epilepsy. Another aspect is also proposed, the down-regulations of KCC2 after stroke that looks similar with those in the development process, might initiate reorganization of neuronal networks (see Di [START_REF] Filippo | Plasticity and repair in the post-ischemic brain[END_REF].

7.3.2

Human studies [START_REF] Cohen | On the origin of interictal activity in human temporal lobe epilepsy in vitro[END_REF] have demonstrated in vivo in patients with temporal lobe epilepsy that perturbed neuronal chloride transport induces excitatory GABAergic signaling and that might result in epileptogenesis. The excitatory GABAergic signaling was later found to be related to a down-regulation of KCC2 [START_REF] Huberfeld | Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy[END_REF]. A dysfunction in GABA system has been proposed in autism. Using bumetanide, a high specific NKCC1 antagonist, reduces intracellular chloride and thus reinforcing GABAergic inhibition. Bumetanide significantly reduced the score of Childhood Autism Rating Scale in sixty children with autism or Asperger syndrome [START_REF] Lemonnier | A randomised controlled trial of bumetanide in the treatment of autism in children[END_REF].

CHAPTER 1 BACKGROUND OF PHYSIOLOGY

Physiopathology in the spinal cord

In the animal spinal cord, the lack of KCC2 expression induces a rise of intracellular CI-concentration, thereby depolarizing of GABA and glycine (Vinay & Jean-Xavier, 2008), and also results in reduction of inhibitory synaptic strength (Boulenguez et al. 2010). In KCC2 knockout animals, sciatic nerve recordings reveal abnormal spontaneous electrical activity and altered reflex responses to peripheral electrical stimuli (Hübner et al. 2001). KCC2 was also reported to be involved in pain perception through its effects on postsynaptic inhibition in spinal cord neurons. Several studies demonstrated that decreased KCC2 expression in the spinal cord is associated with increased nociception [START_REF] Coull | Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain[END_REF][START_REF] Mantyh | Setting the tone: superficial dorsal horn projection neurons regulate pain sensitivity[END_REF]. Inhibition of KCC2 in the mouse spinal cords led to decreased heat-evoked withdrawal latency, whereas inhibition of NKCC1 resulted in increased response latencies to heat stimuli [START_REF] Austin | Inhibition of KCC2 in mouse spinal cord neurons leads to hypersensitivity to thermal stimulation[END_REF].

Physiopathology in the peripheral nerve

After sciatic serve crush in animals, reduction of KCC2 is observed at the injured side of the spinal cord (lumbar spinal motoneurons) compared to intact animals whose spinal cord shows well defined line surrounding motoneurons where is KCC2 located in non-injured condition (Modol et al., 2014).

The monosynaptic reflex

The stretch reflex, also known as myotatic reflex or tendon reflex is a muscle contraction in response to stretching the muscle. The stretch reflex was firstly defined by [START_REF] Liddell | Reflexesin response to stretch (myotatic reflexes)[END_REF] in the decerebrate cats. This spinal reflex provides automatic regulation of skeletal muscle length. When a muscle is stretched, sensory fibres (Ia fibres) from the muscle spindles excites monosynaptically the α-motoneurons that are located in the ventral horn of the spinal cord [START_REF] Lloyd | Conduction and synaptic transmission of the reflex response to stretch in spinal cats[END_REF](Lloyd, , 1946 a,b ; a,b ;[START_REF] Granit | Reflex self-regulation of muscle contraction and autogenetic inhibition[END_REF][START_REF] Hunt | Monosynaptic reflex response of spinal motoneurones to graded afferent stimulation[END_REF]. The α-motoneuron sends the motor output back to the muscle fibres to contract the muscle. In animal studies, the method of the monosynaptic reflex was introduced in the 1940s as a tool for investigating the excitability changes in the motoneuron pools [START_REF] Renshaw | Activity in the Simplest Spinal Reflex Pathways[END_REF][START_REF] Lloyd | Conduction and synaptic transmission of the reflex response to stretch in spinal cats[END_REF]. Since then, the technique of the monosynaptic reflex has used to reveal various properties of the spinal circuitry that is necessary for the integration of peripheral afferents and descending pathways. The monosynaptic reflex from Ia afferents to motoneurons is probably the best known reflexes. The technique of the monosynaptic reflex constitutes one of the simplest tools available to investigate spinal pathways in both animals and humans. Ia sensory fibre from muscle spindle primary ending has monosynaptic projections to alpha motoneuron innerving the corresponding muscle.

CHAPTER 2 MATERIALS AND METHODS

The H-reflex

Principles of the method

The Hoffmann reflex or H-reflex is the electrical equivalent of the monosynaptic stretcH-reflex in animal studies and has become the main tool in many motor control investigations in humans (for a review, see Pierrot -Deseilligny & Burke, 2012). [START_REF] Sherrington | Flexion-reflex of the limb, crossed extension-reflex, and reflex stepping and standing[END_REF] showed in the cats that the monosynaptic reflex is predominant in the antigravity muscles like soleus, quadriceps and also flexor carpi radialis muscle (FCR) in the forelimb. Similar results were also found in humans. In most healthy subjects at rest, the H-reflex can usually be recorded from the soleus (Hoffman, 1918), quadriceps [START_REF] Gassel | A study of femoral nerve conduction time[END_REF], in the lower limb and from FCR in the upper limb [START_REF] Deschuytere | Monosynaptic reflexes in the superficial forearm flexors in man and their clinical significance[END_REF]. The soleus H-reflex was firstly introduced as a monosynaptic response that is the most reliably elicited in the lower limb in humans by electrical stimulation of the posterior tibial nerve in the popliteal fossa (Hoffman, 1918). Electromyographic recording from the soleus muscle via an electrode percutaneously placed over the muscle shows a direct motor (M) response that is followed by the Hreflex. The latency of the M response is about 5-10 msec, while the soleus H-reflex appears later, with a latency of 25-35 msec (Figure 11). In the upper limb, the FCR reflex is the most reliably elicited. It appears with a latency of 16-22 msec (Figure 12). The latency of the H-reflex is different amongst individuals also depending upon on the height of subjects. The neural circuitry responsible for the H-reflex is characterized by the monosynaptic projection of the group Ia afferents onto the α motoneurons. Using intrathecal recordings Magladery et al. (1951) demonstrated in humans that activation of Ia afferent fibres induced by percutaneous electrical stimulation sends the signal to the spinal cord with a conduction time from the dorsal root to the ventral root of only 1.5 ms. The authors proposed that this delay is too short for interneuronal transmission, determining a monosynaptic connection between the Ia fibre and motoneuron. The soleus H-reflex is elicited by selectively stimulating the Ia fibres projecting onto the homonymous motoneuron, while the direct motor response (M) is due to direct activation of the axon motor. The latency of M response is about 5-10 msec, while the soleus H-reflex appears with a latency of 25-35 msec. The FCR H-reflex is elicited by selectively stimulating the Ia fibres projecting onto the homonymous motoneuron, while the direct motor response (M) is due to direct activation of axon motor. The FCR H-reflex appears with a latency of 15-22 msec.

CHAPTER 2 MATERIALS AND METHODS

H-reflex and M-wave recruitment curves

The recruitement curve

The relationship between the intensity of stimulation and the size of the H-reflex and M response has been studied in both animals and humans. It was shown that the modulations of these two responses depend on the intensity of stimulation. At low stimulus intensity, the H-reflex appears at a lower threshold than the motor response, since Ia afferents (larger diameter) corresponding to the H-reflex are more excitable than the motor axons. Progressive increase of the intensity of stimulation induces an increase of the H-reflex until reaching its maximum (Hmax) and then decreases. Meanwhile, the M response appears at a stimulus intensity corresponding to the motor threshold, which is above the threshold of the H-reflex since the axons of the αmotoneuron are smaller than those of the Ia afferents. Further increase in the intensity of stimulation leads to M response increasing until reaching its maximum response (Mmax); the H-reflex is completely suppressed. This results from the antidromic motor volley set up in motor neurons colliding with and eliminating the H-reflex (Hoffman, 1922). The amplitude of the motor response (Δ) and the H-reflex response expressed as a percentage of the M max (Ο) is plotted against the intensity of the electrical stimulation of the posterior tibial nerve (expressed in multiples of motor threshold). Reproduced from [START_REF] Pierrot-Deseilligny | The monosynaptic reflex: a tool to investigate motor control in humans. Interest and limits[END_REF] The M response represents the direct activation of the axon motor by the electrical stimulation. The maximum M response indicates the recruitment of all motor CHAPTER 2 MATERIALS AND METHODS axons. It provides an estimate of the response given by the entire motoneuron pool tested by the monosynaptic reflex. Expressing the H-reflex as a percentage of M max allows us to compare the results between individual subjects.

2.3

The orderly recruitment of α-motoneurons in the monosynaptic reflex

In general, motoneurons are recruited in an orderly sequence from the smallest to the largest size according to the size principle [START_REF] Henneman | Functional significance of cell size in spinal motoneurons[END_REF][START_REF] Henneman | Functionalorganization of motoneuron pool and its inputs[END_REF]. These authors demonstrated that the smallest motoneurons represented by the smallest impulse amplitude have lower thresholds and thus are recruited first, while larger motor neurons have higher thresholds and are recruited last. The facilitation input will begin to affect motoneurons that just failed to be discharged in the test reflex, while inhibition will affect those that had just been recruited in the test reflex.

A) Test reflex alone B) Test reflex conditioned by an excitatory input CHAPTER 2 MATERIALS AND METHODS

C) Test reflex conditioned by an inhibitory input

Use of the H-reflex

The H-reflex technique is used to test the changes in the excitability of motoneurons in response to a conditioning stimulus. Modulations of the H-reflex amplitude depend on the number of motoneurons involved in the test H-reflex under conditioning stimulation, reflecting changes in the excitability of the motoneuron population. If a conditioning stimulus induces an inhibitory effect, the Ia afferent test volley recruits fewer motoneurons than in the control situation, thus the amplitude of the H-reflex is decreased. In contrast, if motoneurons are facilitated by a conditioning stimulus, those that have not been recruited in the control H-reflex will be fired. This increases the size of the H-reflex as a result of temporal summation of the conditioning and test EPSP.

CHAPTER 2 MATERIALS AND METHODS

2.5

Advantages and disadvantages of the method 2.5.1

Advantages of the method

The monosynaptic reflex is a simple and non-invasive method, and can be used to estimate the excitability of spinal motoneurons at rest and during movement. This method is widely used to study spinal circuits in both animals and humans, and also in patients with CNS lesions. However, it presents some limitations.

2.5.2

Limitation of the method a) Mechanisms controlling the Ia afferent volleys H-reflex is mediated by Ia afferents so the mechanisms controlling the Ia afferent volleys such as alteration of the excitability of Ia afferents, presynaptic inhibition, post-activation depression, curtailment of the compounded EPSP by disynaptic IPSI have to be taken in consideration. Some of these limitations can be overcome: for example post-activation at the fibre Ia -motoneurons synapse rely on the H-reflex frequency (see physiological background). However, keeping the frequency identical for conditioned and unconditioned H-reflex allow discarding its effect.

Possible changes in the amount of presynaptic Ia inhibition, alteration of Ia excitability fibre are much more important during voluntary movement that at rest. However, our experiments were performed at rest. Furthermore, previous experiments have demonstrated that presynaptic Ia inhibition is not modified by tDCS (Roche et al., 2009) b) Limitation due to the property of the motoneuron pool and in homogenous distribution of afferents within the motoneuron pool.

Sensitivity of different sizes of H-reflex

A conditioning stimulation causes facilitatory or inhibitory effects on the Hreflex. These effects vary with the intensity of the conditioning stimulus, and also depend on the size of the test H-reflex itself (Crone et al., 1990;[START_REF] Meinck | Facilitation and inhibition of the human H reflex as a function of the amplitude of the control reflex[END_REF]. These different sensitivities of the reflexes with respect to their different sizes reflects a nonlinear input-output relationship within the motoneuron pool. It was shown that small and large sizes of monosynaptic reflex have a lower sensitivity to facilitatory and inhibitory input than reflexes of intermediate size (Crone et al., 1990). The sensitivity of the monosynaptic reflex to facilitation (upper part) or inhibition (lower part). Reproduced from Crone et al., 1990 When the conditioning stimulus is strong (solid line), the number of additionally recruited (facilitation) or derecruited (inhibition) motoneurons will increase with the size of the test H-reflex, and then decrease. Meanwhile, when the conditioning stimulus is weak (dashed line), a plateau region appears between phases of increase and decrease. Given this, it is therefore necessary to adjust the intensity of the stimulation test to maintain and adjust the size of the H-reflex to where it is sensitive to response to facilitatory or inhibitory effects (20-30% of Mmax, see figure above) and keep a similar size throughout the experiment.

CHAPTER 2 MATERIALS AND METHODS

Plateau potential

It has been shown in animals that α-motoneurons could develop the plateau potential due to persistent inwards currents (for reference see [START_REF] Hultborn | Variable amplification of synaptic input to cat spinal motoneurones by dendritic persistent inward current[END_REF][START_REF] Heckman | Motoneuron excitability: the importance of neuromodulatory inputs[END_REF]. They amplify the response of synaptic inputs leading to a sustained increase of motoneuron activity. This phenomenon may influence the relationship between excitatory synaptic inputs and discharge frequency of motoneurons [START_REF] Hultborn | Variable amplification of synaptic input to cat spinal motoneurones by dendritic persistent inward current[END_REF]. The existence of plateau potential might be considered to affect the results obtained with the technique of the H-reflex. Studies in humans using a stimulus applied directly to the calf muscle suggested that a stimulation frequency of at least 10 Hz is required to disclose the plateau potentials [START_REF] Collins | Large involuntary forces consistent with plateau-like behavior of human motoneurons[END_REF][START_REF] Collins | Sustained contractions produced by plateau-like behaviour in human motoneurones[END_REF]. However, there is no direct evidence proving in humans that the stimulation evoked H-reflex is sufficient to generate plateau potentials, and the monosynaptic reflexes studied in the present study was likely not contaminated with plateau potential since the maximum stimulation frequency used was only 1 Hz.

To sum up, the H-reflex technique is nevertheless the most valuable method to investigate changes in motoneurons excitability in healthy subjects and patients with CHAPTER 2 MATERIALS AND METHODS CNS lesions but the following rules have to be taken into account: i) the amplitude of the unconditioned reflex must be within the ascending part of the recruitment curve (Figure 15) ii) the H-reflex amplitude has to be expressed of the percentage of M max iii) the unconditioned H-reflex amplitude has to be the same in all situations iv) conditioned and unconditioned H-reflex have to randomly alternated.

CHAPTER 2 MATERIALS AND METHODS

Methods used in the project 1

3.1 Electrophysiological recordings

Stimulation test: Soleus H-reflex

Spinal inhibitory circuits were studied using non-invasive electrophysiological techniques based on the H-reflex. The H-reflex was evoked in the soleus muscle by percutaneous stimulation of the posterior tibial nerve (PTN) at the popliteal fossa with a rectangular 1 ms duration stimulus, delivered at 0.33 Hz to the PTN. The H-reflex was recorded from the EMG signal of the soleus muscle using bipolar surface electrodes positioned on the skin parallel to the muscle belly.

The maximum motor response (M max ) and maximum H-reflex response (H max ) were recorded at the beginning of each experiment. The stimulus intensity was then adjusted to elicit an H-reflex amplitude of 20-35% of M max , which corresponded to ~ 50%, of H max . The unconditioned H-reflexes were set to have similar size across subjects and kept constant throughout the experiment, since the sensitivity of the Hreflex to facilitation or inhibition conditioning effects depends on its non-conditioned size (Crone et al. 1990).

3.1.2

Inhibitory conditioning stimulations.

a) Presynaptic inhibition

Several non-invasive methods have been developed to study presynaptic inhibition in humans. The first study on presynaptic inhibition has begun with a phenomenon called the "vibration paradox." Effects of prolonged tendon vibration result in two phenomena i) a discharge in the homonymous Ia afferent fibres acting on the motoneuron which causes the tonic vibration reflex (TVR) (reviewed in [START_REF] Gail | Differential effects on tonic and phasic reflex mechanisms produced by vibration of muscles in man[END_REF][START_REF] Hagbarth | Tonic vibration reflexes (TVR) in spasticity[END_REF] and ii) an inhibition of the H-reflex of that muscle [START_REF] Gail | Differential effects on tonic and phasic reflex mechanisms produced by vibration of muscles in man[END_REF][START_REF] Delwaide | Tendon hyperreflexia in neurologic clinical practice[END_REF][START_REF] Delwaide | Human monosynaptic reflexes and presynaptic inhibition[END_REF]. The coexistence of these two phenomena has led to attribute to presynaptic inhibition of Ia terminals with PAD [START_REF] Delwaide | Human monosynaptic reflexes and presynaptic inhibition[END_REF]). However, application of vibration to the homonymous tendon actives other mechanisms such as homosynaptic depression (see Background of physiology). Another method was subsequently developed to eliminate the lack of specificity is the prolonged vibration of the homonymous tendon. Application of vibration to the tendon of a heteronymous muscle (tibialis anterior or bicep femoris) induces a clear inhibition of the soleus Hreflex [START_REF] Morin | Evidence for presynaptic inhibition of muscle spindle Ia afferents in man[END_REF], Hultborn et al., 1987;[START_REF] Nielsen | Is presynaptic inhibition distributed to corticospinal fibres in man?[END_REF]Nakashima et al., 1989).

CHAPTER 2 MATERIALS AND METHODS

Presynaptic inhibition of Ia fibres may also be evoked by a conditioning stimulus to the nerve innervating the antagonist muscle. This induces an inhibition of the H-reflex in the agonist muscle called D1/D2 inhibition. A conditioning stimulus applied to the common peroneal nerve at 1-1.4 × motor threshold (MT) induces an inhibition of the H-reflex of the soleus. The inhibition is long lasting and composed of two phases, called 'D1' at 5-30 ms, and 'D2' at 70-200 ms (Mizuno et al., 1971). It has been shown that the same conditioning volley that inhibition the H-reflex does not modify the motor evoked potential (MEP) amplitude evoked by cortical stimulation in the soleus at an interstimulus interval (ISI) corresponding to D1 (21 ms) (Faist et al., 1996). This suggests that at this ISI, common peroneal nerve induced H-reflex inhibition is presynaptic in origin. The amplitude of soleus H-reflex expressed as a percentage of its control value is plotted against the interstimulus interval. Reproduced from [START_REF] Morita | Sensitivity of Hreflexes and stretch reflexes to presynaptic inhibition in humans[END_REF] Another method called heteronymous facilitation was proposed to measure the presynaptic inhibition of Ia fibres. A facilitation of the soleus H-reflex induced by conditioning stimulus to the nerve innervating the quadriceps muscle is purely monosynaptic within the first 0.6 ms [START_REF] Hultborn | Assessing changes in presynaptic inhibition of I a fibres: a study in man and the cat[END_REF]. A constant conditioning stimulus elicits a constant EPSP in motoneurons, and thus produces a constant reflex facilitation, unless the amount of presynaptic inhibition of Ia fibres mediating the conditioning volley changes. Thus, modification of motoneurons excitability can result in changes in the reflexes H sizes, which reflects a change in the level of presynaptic inhibition. The more that the facilitation reflex is important, the more that presynaptic CHAPTER 2 MATERIALS AND METHODS inhibition is low. However, this method presents some limitations: i) it requires a fairly high level of heteronymous facilitation to highlight changes in presynaptic inhibition, and ii) to avoid risk of contamination with non-monosynaptic effects, the ISI used to record the monosynaptic facilitation of the H-reflex ought to be chosen between 0.2-0.6 ms. This implies an accurate definition of the ISI that maybe time consuming.

To conclude, as D1 inhibition is the easiest method, it was chosen to investigate presynaptic inhibition of Ia fibres in healthy and patient subjects in the present study. The soleus H-reflex was conditioned with a preceding stimulation of the common peroneal nerve (CPN) through hemispherical bipolar electrodes placed 2 cm below the neck of the fibula. The stimulus consisted of a train of 3 rectangular pulses, each of 1 ms duration, an interpulse interval of 3 ms, with an intensity 1.2 times the tibialis anterior (TA) motor threshold (MT), delivered 21 ms before the test stimulation. The motor threshold was determined by tendon palpation and by the oscilloscope EMG display.

CHAPTER 2 MATERIALS AND METHODS b) Postsynaptic inhibition

Postsynaptic inhibition elicited in soleus motoneurones by electrical stimulation of sural cutaneous afferents is one of the most robust reflexes in animals and humans. It has been shown that stimulation of the sural nerve (a purely cutaneous nerve) induces an inhibition of the soleus H-reflex [START_REF] Hugon | Influence of stimulation of the sural nerve on voluntary motor activity[END_REF]Hugon & Bathien, 1967). The inhibition of the soleus H-reflex appears when cutaneous stimuli applied over the lateral dorsum or the plantar surface of the foot. The average distance from the point of stimulation on the distal of foot to the mid-popliteal region is 50 cm (reviewed in [START_REF] Gassel | Local sign and late effects on motoneuron excitability of cutaneous stimulation in man[END_REF]. The amplitude of soleus H-reflex expressed as a percentage of its control value is plotted against the interstimulus interval Adapted from Pierrot-Deseilligny et al., 1973. In humans, the amount of this inhibition is marked (around 40 % of the unconditioned reflex) (Pierrot-Deseilligny et al., 1973). The soleus H-reflex was conditioned by electrical stimuli applied to cutaneous afferents of the sural nerve through bipolar adhesive electrodes placed on the lateral side of the fifth toe. The stimuli consisted of trains of 17 pulses, each of duration 1 ms, with an interpulse interval of 3 ms, delivered 50 ms before the test stimulus. In such conditions, the induced inhibition is likely caused by postsynaptic inhibition of soleus motoneurons (for reference see Pierrot-Deseilligny & Burke, 2012). The intensity of stimulation was based on the subject's perception threshold (PT) defined as the lowest stimulus intensity at which the subject reported the sensation of the stimulation using a single pulse. As it was shown in our laboratory that this postsynaptic inhibition is significant when the CHAPTER 2 MATERIALS AND METHODS intensity of cutaneous stimulus is greater than 3 times the threshold of perception, the intensity of the stimulus train was slowly increased to 3-5 times the perceptual threshold in order to induce a clear-cut inhibition of the soleus H-reflex. To reproduce the same conditions of stimulation in SCI patients, we have demonstrated an intensity curve of postsynaptic inhibition induced by cutaneous stimulation in six healthy subjects to define the minimum intensity inducing a significant inhibition with the stimulator (D7SA Digitimer Ltd, Welwyn Garden City, UK) used for this protocol. The inhibition is significant in this group of subjects when the intensity of stimulation is greater than 6.5 mA, then reinforces for the currents between 6.5-8.5 mA and remains constant for the currents between 8.5-10 mA reflecting a saturation of inhibition. The intensity of the conditioning stimuli inducing postsynaptic inhibition was therefore set in all SCI patients at approximately 8.5 mA.

CHAPTER 2 MATERIALS AND METHODS

3.1.3

Excitatory conditioning stimulation.

Post-activation depression

Post-activation depression was chosen in the present study to test the possible effect of furosemide on excitatory synapses since it reflects the efficiency of the monosynaptic excitatory transmission between Ia fibre and motoneuron ans is a pure excitatory effect. Alternative possibilities to test pure excitatory effects should be either homonymous or heteronymous monosynaptic facilitation of the soleus H-reflex. However, homonymous monosynaptic facilitation cannot be used in humans to assess purely excitatory effects, due to several factors, including the mixed excitatory and inhibitory actions and duration of after-hyperpolarisation. Heteronymous monosynaptic Ia facilitation from the quadriceps to the soleus requires an accurate determination of the time interval between the conditioning and test stimulus to ensure its monosynaptic origin [START_REF] Hultborn | Assessing changes in presynaptic inhibition of I a fibres: a study in man and the cat[END_REF]. For other excitatory pathways, for example group II disynaptic excitatory pathways, the indirect method we used cannot eliminate possible contamination by monosynaptic afferent projections or by inhibitory projections involved in the pathway. Moreover, testing group II excitatory pathways at rest relying on the use of the quadriceps H-reflex is not as easy as evoking the H-reflex in the soleus muscle.

It has been shown in both animals (Eccles, 1964) and humans (Magladery et al., 1951;Paillard, 1955;Crone & Nielsen, 1989;Hultborn & Nielsen, 1998) that postactivation depression at the Ia fibre-motoneuron synapse reflects the efficiency of the monosynaptic excitatory transmission between Ia fibre and motoneuron and is thus a simple and reliable tool to assess the functioning of excitatory synaptic transmission without contamination of other synaptic mechanisms (Meunier et al., 2007). Postactivation depression can be demonstrated by the depressive effect of increasing the stimulus rate on the size of H-reflexes. The H-reflex is dramatically depressed at short intervals (1 Hz) with gradual recovery as the interval increases to over 10 s (Crone & Nielsen, 1989). In the present study, the H-reflex evoked every 3 s (0.33 Hz) was initially adjusted at H max /2. The soleus H-reflex evoked at low frequency (every 6 s or 0.16 Hz) and high frequency (every 1s or 1 Hz) was measured in each case (see Figure 20). Twenty soleus H-reflexes were evoked for each stimulus rate (1 Hz and 0.16 Hz). The amount of post-activation depression of the H-reflex was evaluated as the size of the H-reflex elicited at 1 Hz (high stimulus rate) expressed as a percentage of its value when elicited at 0.16 Hz (low stimulus rate). This is referred to as the 1/0.16 ratio. This ratio was used to determine the amount of post-activation depression of the H-reflex (Aymard et al., 2000).

CHAPTER 2 MATERIALS AND METHODS

Furosemide

The loop diuretics, furosemide and bumetanide, are thought to modulate inhibitory synapses through their antagonist effect on cation-chloride co-transporters. It has been suggested that furosemide antagonises the KCC2 co-transporter but also the NKCC1 co-transporter. Nevertheless, its effect on the NKCC1 co-transporter is less potent than bumetanide (Gillen et al. 1996;Russell, 2000). To date, no substance has been identified that selectively inhibits KCC2 (Kahle et al. 2008;Löscher & Schmidt, 2011), and some of the KCC2 blockers used in animal studies require very high concentrations (Payne, 1997) that are likely to be toxic in humans.

Furosemide was reported to reduce GABA A inhibition in adult neurons by reducing the neuronal transmembrance chloride gradient [START_REF] Thompson | Residues in Transmembrane Domains I and II Determine γ-Aminobutyric Acid Type AA Receptor Subtype-Selective Antagonism by Furosemide[END_REF]. Nicoll (1978) demonstrated using intracellular recordings from the frog spinal cord that furosemide selectively and reversibly blocked inhibitory postsynaptic potentials (IPSPs) mediated by GABA and glycine without affecting excitatory postsynaptic potentials (EPSPs) mediated by glutamate. It has also been shown in both animals and humans that furosemide has clinically relevant antiepileptic actions. Studies on animal hippocampal slices revealed that furosemide potently blocks epileptiform activity without suppressing neuronal excitability [START_REF] Hochman | Dissociation of synchronization and excitability in furosemide blockade of epileptiform activity[END_REF][START_REF] Hochman | Extracellular chloride and the maintenance of spontaneous epileptiform activity in rat hippocampal slices[END_REF][START_REF] Hochman | Chloride-cotransport blockade desynchronizes neuronal discharge in the "epileptic" hippocampal slice[END_REF]. It was proposed that the action of furosemide is mediated through nonsynaptic mechanisms modulating the size of extracellular space since modulation of electric field interactions via the extracellular space (ECS) might also contribute to neuronal hypersynchrony and epileptogenicity. In humans, a small clinical trial showed that oral furosemide (120 mg daily during 4 months) significantly decreased the frequency of seizures in patients with severe focal epilepsy [START_REF] Ahmad | Controlled trial of frusemide as an antiepileptic drug in focal epilepsy[END_REF]. Recently, it was shown that a single intravenous injection of 20 mg furosemide crosses the blood-brain barrier and completely blocks epileptic activity in the brain (Huglund & Hochman, 2005). Together with its effects described above and given that furosemide is a widely used diuretic which has been employed with a good safety record for many years in humans, we have chosen furosemide to investigate the functioning of inhibitory synapses in the present study.

Furosemide used in the present study

The usual starting oral dose of furosemide in the clinic in adults is 20-80 mg per day. In the present study, the smallest dose, furosemide 20 mg was firstly tested on the spinal inhibitory networks in a small sample of subjects. Orally-administered furosemide 20 mg induced no significant changes in spinal inhibitory networks (see Results), so furosemide 40 mg was subsequently tested using the same experimental design. For the subjects convenience, due to its diuretic effect, the amount inhibition was monitored through repetitive measurements over a time period limited to 70 minutes after administration of the drug since furosemide pharmacokinetics and pharmacodynamics in healthy subjects show that furosemide (40-80 mg) appears in the serum within 10 minutes and peaks from 60 to 90 minutes after oral administration (Branch et al. 1976[START_REF] Branch | Determinants of response to frusemide in normal subjects[END_REF]Tilstone & Fine, 1978).

Recording chain

The test stimulus evoking the soleus H-reflex and the conditioning stimuli were delivered using constant-current stimulators (D7SA Digitimer Ltd., Welwyn Garden City, UK). The stimulations were computer-triggered using a customised script (Signal 4.20, Cambridge Electronic Design, Cambridge, UK). The H-reflex was recorded from the EMG signal of the soleus muscle using bipolar surface electrodes (Delsys Inc., Boston, USA, Ag electrodes DE-2.1) positioned on the skin parallel to the muscle belly. The EMG signals were amplified (×1000), band pass filtered at 20-450 Hz (Delsys, Inc., Boston, MA, US), digitised at 1 kHz (Power 1401 A/D board, Cambridge Electronic Design, Cambridge, UK) and then transmitted to an oscilloscope for visualization and to a computer for storage and offline analysis (Figure 21). The soleus H-reflex is evoked by a stimulus test applied over the posterior tibial nerve at the popliteal fossa using stimulator 1. To evoke presynaptic inhibition, the soleus H-reflex was conditioned with a preceding stimulation of the common peroneal nerve through electrodes placed below the neck of the fibula using stimulator 2. To evoke postsynaptic inhibition, the soleus H-reflex was conditioned by electrical stimuli applied to cutaneous afferents of the sural nerve through electrodes placed on the lateral side of the fifth toe using stimulator 3. A computer with the Signal® system triggers the stimulators in a random sequence. The H-reflex was recorded from the EMG signal of the soleus muscle using bipolar surface electrodes positioned on the skin parallel to the muscle belly. The EMG signal is amplified and then transmitted to an oscilloscope for visualization and to a computer for storage and offline analysis.

CHAPTER 2 MATERIALS AND METHODS

Statistical methods

Data processing

To evaluate the amounts of pre-and postsynaptic inhibition, peak-to-peak amplitudes of unconditioned H-reflexes were compared to those of conditioned Hreflexes. The inhibitory effects were quantified by calculation of the inhibition value as 100 -((conditioned H-reflex/unconditioned H-reflex) × 100). Each inhibition value was then normalized to the percentage of its baseline value using the equation: [%((Inhibition at t x -baseline inhibition)/baseline inhibition)] +100.

To evaluate the effects of post-activation depression, the 1/0.16 ratio was normalized to the percentage of its baseline value using the equation: [% ((1/0.16 ratio at t x -1/0.16 ratio at baseline)/ 1/0.16 ratio at baseline)] +100.

Statistical analysis

The amounts of pre-/postsynaptic inhibition and post-activation depression were averaged among subjects for each time epoch (0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, 51-60 min, and 61-70 min). For healthy subject experiments, a twoway repeated-measures ANOVA (control vs. furosemide as the within-subject factor) was used to examine the effects of furosemide on pre-/postsynaptic inhibitions or postactivation depression. To compare the effects of furosemide in healthy subjects and paraplegic patients, a two-way ANOVA was employed. A post-hoc pairwise comparison was performed using Tukey's HSD test.

A one way repeated-measure ANOVA on time epoch (0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, 51-60 min, and 61-70 min) was also used to determine the impact of time in the control experiment.

For dose-dependent experiments, an a priori power analysis was first performed to determine the minimal sample size. A posteriori, all our data sets were, as assumed in our power analysis, normally distributed and successfully passed the equality of variance test so parametric tests (one way repeated measures ANOVA) were employed.

Significance was taken at P < 0.05. Mean data are provided ± standard error of the mean (SEM). Electromyographic (EMG) activity was recorded from FCR muscle using bipolar surface electrodes positioned on the muscle belly. EMG activity was displayed in a wide analysis window beginning 100 ms before the test stimulus and lasting 300 ms. EMG activity was sampled at 2 kHz, amplified (x 5000 -10000) and band-pass filtered (250 Hz-3 kHz) using a Digitimer D360 amplifier (DIGITIMER LTD, WELWYN GARDEN CITY, Herts, UK). The FCR H-reflex was evoked by electrical stimuli applied to the median nerve. The maximum direct motor response (Mmax) was first determined and the unconditioned H-reflex was adjusted to 10% -20% of Mmax in each subject. The amplitude of the unconditioned FCR H-reflex was adjusted to 50% of the maximum amplitude of H-reflex in the majority of cases and it was kept constant throughout the experiment.

CHAPTER 2 MATERIALS AND METHODS

Methods used in

Conditioning stimulation a) Reciprocal inhibition

It has been shown that a conditioning stimulus applied to the radial nerve induces an early inhibition of the FCR H-reflex [START_REF] Day | Transmission in the spinal reciprocal Ia inhibitory pathway preceding willed movements of the human wrist[END_REF](Day et al., , 1984)). The FCR Hreflex was evoked by electrical stimuli applied to the median nerve near the cubital fossa. It was conditioned with conditioning stimulation of the radial nerve through bipolar electrodes placed at the spiral groove. The conditioning intensity was adjusted below the ECR motor threshold (MT) to prevent recurrent inhibition (Aymard et al., 1995) and was between 0.7-0.9 MT depending on the subject. The motor threshold was determined by tendon palpation and by the EMG displaying on the oscilloscope's screen. The ISI was the time interval between test and conditioning stimuli at which the early radial-induced inhibition of the FCR H-reflex was greatest. The ISI was determined using 0.5 ms steps in the range -3 ms/+ 1 ms (Day et al. 1984).

CHAPTER 2 MATERIALS AND METHODS

Transcranial direct current stimulation

Transcranial direct current stimulation or tDCS is a simple non-invasive cortical modulation technique in which a device sends constant low direct current (DC) delivered to the area of interest through the electrodes. The basic idea design of tDCS, using DC has been used for the treatment of mental and neurologic disorders since Aldini's work in the beginning of 19th century (see [START_REF] Parent | Giovanni Aldini: from animal electricity to human brain stimulation[END_REF]. Since the 1960s, brain polarization has been reintroduced in animals (Creutzfeld et al., 1962;[START_REF] Bindman | The action of brief polarizing currents on the Cerebral cortex of the rat (1) during current flow and (2) in the production of long-lasting after-effects[END_REF]Purpura & McMurtry, 1965) and later in humans (Priori et al., 1998;Nitche & Paulus 2000). There are a number of studies investigating tDCS effects on the central nervous system. Today, transcranial direct current stimulation is beginning to be used more frequently as a new brain stimulation technique and in therapeutic applications in humans.

Animal studies

There was a rise in interest in transcranial direct current stimulation in the 1960s as demonstrated by different researchers (Creutzfeld et al., 1962;[START_REF] Bindman | The action of brief polarizing currents on the Cerebral cortex of the rat (1) during current flow and (2) in the production of long-lasting after-effects[END_REF]Purpura & McMurtry, 1965). They showed that stimulation through the scalp could affect brain function by changing the cortical excitability. This change is likely due to the modulation of resting membrane potential of cortical cells. A tDCS device has an anodal electrode (positively charged electrode) and a cathodal electrode (negatively charged electrode). One of the electrodes is placed over the area of interest and the other one (reference electrode) is placed in another area (e.g. the supraorbital region) to complete the circuit. They also demonstrated that positive and negative stimulation had different effects on cortical excitability. Anodal tDCS causes a depolarization of the resting membrane potential that leads to increases in neuronal excitability of the area being stimulated and more spontaneous cell firing, whereas cathodal tDCS causes a hyperpolarization of the resting membrane potential, leading to decreases in neuronal excitability of the area being stimulated and decreased spontaneous cell firing. Purpura & McMutry (1965) have also demonstrated that tDCS effects on the motor cortex were not only based on what type of stimulation was being used but also on the intensity of simulation as well as the type of cortical cells being stimulated. The effects of tDCS induced with a given polarity can thus be different between pyramidal cells and nonpyramidal cells. Effects of tDCS beyond the end of stimulation were also reported and these are usually related to the duration and intensity of stimulation. The post-effect of tDCS was also observed and proposed to be due to the plasticity concepts of long-term potentiation (LTP) and long-term depression (LTD) [START_REF] Bindman | The action of brief polarizing currents on the Cerebral cortex of the rat (1) during current flow and (2) in the production of long-lasting after-effects[END_REF][START_REF] Weiss | Quenching revisited: low level direct current inhibits amygdala-kindled seizures[END_REF].

CHAPTER 2 MATERIALS AND METHODS

Human studies a) Effects on the brain

The tDCS experiment in humans was first introduced by Priori and colleagues in 1998. They investigated whether direct current could reach and influence the human brain as demonstrated in animal studies. Testing the effect of tDCS on the human motor cortex by assessing the changes in motor potentials evoked by transcranial magnetic brain stimulation (TMS) shows significant modulations in the excitability of the motor cortex. This provides direct evidence that very small electric fields can cross the skull and influence human brain activity. Subsequent experiments by Nitche and Paulus in 2000 also confirmed the effects of tDCS on the motor cortex with significant changes in the motor evoked potential (MEP) size. They also demonstrated the differentiation effects of anodal and cathodal tDCS on the human brain: anodal increases cortical excitability while cathodal decreases cortical excitability. The authors proposed that these effects are probably due to shifts in neuronal resting membrane potential. The tDCS effects observed in their study also outlasted the duration of stimulation but were reversible. Amplitude and endurance beyond the end of stimulation were reported to be current-intensity and stimulation-duration dependent.

The cortical excitability changes induced by tDCS in humans have been reported to be caused by various mechanisms. In 2003, Nitche and colleagues proposed that the cortical excitability modulations elicited during tDCS depend on membrane polarization as seen in animals (Purpura & McMurtry, 1965). Blocking sodium and calcium channels eliminates or reduces the excitability enhancement induced by anodal stimulation during and after tDCS, whereas the reduction in excitability caused by cathodal tDCS is not modulated by ion channel blockade. These results suggest that anodal tDCS caused neuronal depolarization, while cathodal tDCS generated neuronal hyperpolarization since neuronal hyperpolarization would inactive the sodium and calcium channels. Stagg et al (2009) demonstrated using Magnetic Resonance (MR) spectroscopy that the increase of cortical excitability induced by anodal tDCS is due to reduction of cortical inhibitory neurotransmitter (GABA) concentrations while reduction of cortical excitatory neurotransmitter (Glutamate) concentrations (without any modifications of GABA concentration level) causes the decrease of cortical excitability induced by cathodal tDCS.

However, it appears that the mechanisms underlying immediate effects and after-effects of tDCS are different. Mechanisms underlying the post effects induced by tDCS appear to be related to modulations of cerebral plasticity. The long-lasting aftereffects of tDCS on the brain are thought to be mediated at the synaptic level by NMDA receptors [START_REF] Liebetanz | Pharmacological approach to the mechanisms of transcranial DC-stimulation-induced after-effects of human motor cortex excitability[END_REF][START_REF] Nitsche | Pharmacological Modulation of Cortical Excitability Shifts Induced by Transcranial Direct Current Stimulation in Humans[END_REF][START_REF] Siebner | Preconditioning of low-frequency repetitive transcranial magnetic stimulation with transcranial direct current stimulation: evidence for homeostatic plasticity in the human motor cortex[END_REF]. The NMDA

CHAPTER 2 MATERIALS AND METHODS

receptor is known to be involved in cortical neuroplastic changes such as long-term potentiation [START_REF] Bennett | Update on the neurophysiology of pain transmission and modulation: focus on the NMDA-receptor[END_REF]. However, it was subsequently reported that the aftereffects of tDCS do not arise from NMDA receptor synaptic involvement alone. [START_REF] Ardolino | Non-synaptic mechanisms underlie the after-effects of cathodal transcutaneous direct current stimulation of the human brain[END_REF] proposed that the after-effects of cathodal tDCS in the human brain arising through non-synaptic mechanisms possibly involve alterations in transmembrane proteins and changes in pH. Nitche et al., (2009) demonstrated in healthy subjects that under placebo medication, anodal tDCS enhanced, and cathodal tDCS reduced, excitability of the motor cortex for about 60-120 min, whereas oral administration of citalopram (a selective serotonin reuptake inhibitor) enhanced and prolonged the facilitation induced by anodal tDCS and turned cathodal tDCS-induced inhibition into facilitation which was prolonged over 120 minutes after tDCS. Serotonin has a prominent impact on tDCS-induced plasticity in humans since the modulation of the serotonergic system affects long-term potentiation (LTP) and long-term depression (LTD).

In addition, it has been shown that the electrode position and the orientation of the electric field could also contribute to the results in the human brain. Current distribution under electrodes over the chin is less tangential to the cerebral cortex than one under electrodes over the frontal bone. Currents with comparable intensities but with different directions can have dramatically different effects on the excitability of the cortex [START_REF] Priori | Brain polarization in humans: a reappraisal of an old tool for prolonged non-invasive modulation of brain excitability[END_REF].

CHAPTER 2 MATERIALS AND METHODS b) Effects of tDCS applied over the cerebral cortex on the spinal networks

In 2009, Roche and colleagues have demonstrated the effects of tDCS over the contralateral primary motor cortex area (M1) on spinal networks excitability at the cervical level in humans suggesting that the effects of tDCS should not be considered only on cortical neurons but also on spinal circuits. Anodal tDCS applied continuously for 20 minutes over the contralateral hand motor area increased reciprocal inhibition directed from ECR to FCR motoneurons during the application with no modification of presynaptic inhibition directed to FCR Ia fibres and FCR H-reflex recruitment curves. These authors suggested that anodal tDCS probably modifies the rate of spontaneous discharge of M1 and induces an increase in the efficiency of the descending control acting on spinal neurons. The absence of change in presynaptic inhibition of FCR Ia terminals indicates that tDCS likely mainly increases disynaptic inhibitory interneuron excitability. It was also demonstrated in the same study that cathodal tDCS induces no change in both presynaptic Ia inhibition and reciprocal inhibition. A subsequent study by the same authors showed that anodal tDCS also has effects on spinal networks excitability at the lumbar level: anodal tDCS decreased reciprocal Ia inhibition directed from the tibialis anterior muscle to the soleus motoneuron, increased homonymous soleus recurrent inhibition, and induced no change in both presynaptic inhibition of soleus Ia fibres and soleus H-reflex recruitment curves. The authors proposed that anodal tDCS preferentially increases the spontaneous corticospinal cell firing rate projecting to reciprocal inhibition and Renshaw cells [START_REF] Roche | Impact of precision grip tasks on cervical spinal network excitability in humans[END_REF]). It appears that effects of anodal tDCS on reciprocal inhibition between agonist and antagonist muscles are different at cervical spinal and lumbar spinal levels as anodal tDCS over the hand motor cortex increases disynaptic inhibition directed from ECR to FCR while it diminishes reciprocal Ia inhibition directed from the tibialis anterior muscle to the soleus motoneuron. It was proposed that these opposite results may be due to i) the difference in nature of interneurons mediating disynaptic inhibition at wrist and ankle levels, and ii) the differences in cortical structures stimulated by tDCS since tDCS over the leg motor cortex generated an electrical field perpendicular to the target motor cortex, while tDCS over the hand motor cortex generated an electrical field parallel to the stimulated motor cortex. In these series of experiments, tDCS has not been reported induce any after-effects on the spinal networks studied. In a subsequent study in 2012, Roche et al. demonstrated post-effects on the lumbar propriospinal system for at least 20 minutes after the end of anodal tDCS during 20 minutes over the motor cortex [START_REF] Roche | Effects of anodal tDCS on lumbar propriospinal system in healthy subjects[END_REF].

CHAPTER 2 MATERIALS AND METHODS

Physical parameters of tDCS a) Size of electrodes and focality

tDCS is less focal than other brain stimulation methods such as transcranial magenetic stimuation (TMS) and transcranial electrical stimulation (TES) given its electrode size. Some studies in humans showed that the size of active and reference (cathode) electrodes might complicate tDCS results. A smaller active electrode applied over the M1 showed better increased the corticospinal excitability of the extensor carpi radialis muscle than the larger ones [START_REF] Bastani | a-tDCS differential modulation of corticospinal excitability: the effects of electrode size[END_REF]. Anodal tDCS over the supplementary motor area and a reference electrode placed over the contralateral prefrontal cortex impaired complex whole body motor skill learning whereas using a larger reference electrode for the same protocol did not affect the outcome of learning compared to the sham [START_REF] Kaminski | Effect of transcranial direct current stimulation (tDCS) during complex whole body motor skill learning[END_REF].

A study in animals using functional magnetic resonance imaging (fMRI) to measure the effectiveness of tDCS showed that tDCS over the frontal lobe induced neuronal activation in the target cortex and also in its connected brain region (Takano et al., 2011). The cortical stimulation effect of tDCS on the left primary motor cortex (M1) demonstrated by fMRI in humans show that besides its effects on the left M1, there were also activations in the left supplementary motor cortex (SMA) and the right posterior parietal cortex (Kwon et al., 2008).

b) Current intensity

The efficacy of tDCS to induce modification of membrane polarity also depends on current density that determines the induced electrical field strength (Purpura et al., 1965). Nistche & Paulus (2000) showed the dependency of the size of prolonged motor cortex excitability change after anodal tDCS on different current intensities (0.2-1.0 mA) with larger current densities inducing stronger effects. At 2 mA, anodal or cathodal tDCS over the prefrontal cortex in healthy subjects improved or decreased more verbal fluency compared to the result observed with 1 mA [START_REF] Iyer | Safety and cognitive effect of frontal DC brain polarization in healthy individuals[END_REF].

c) Duration

With constant current intensity, duration is an important parameter that influences the occurrence and duration of tDCS post-effects. Nitche & Paulus (2001) demonstrated in humans the long-lasting excitability of modulations after the end of tDCS. After 5-7 minutes of anodal tDCS over the motor cortical representational field of the abductor minimi muscle (ADM), the MEP amplitudes (measured by TMS) return to baseline within the first few minutes of stimulation, whereas 9 -13 minutes of stimulation results in after-effects remaining for a maximum duration of 1.5 hours after

CHAPTER 2 MATERIALS AND METHODS

the end of stimulation. The same results were also found for the cathode. Cathodal tDCS over the hand motor area of the ADM induced prolonged excitability reduction in humans with duration dependency. 5 and 7 min of stimulation resulted in motor cortical excitability reductions, which lasted for minutes after the end of stimulation, while 9 minutes of stimulation induced after-effects for up to an hour after the end of stimulation (Nitche et al., 2003).

d) Electrode position and polarity

The anode is defined as the positively charged electrode, whereas the cathode is the negatively charged one. Most authors place the anode above the motor cortical representational field of the target muscle, and the cathode above the contralateral orbital to increase cortical excitability, while for decreasing the cortical excitability: the cathode is positioned above the representation of target muscles in the motor cortex, while the anode is placed above the contralateral orbital (Nitche & Paulus 2000, 2001;Nitche et al., 2003).

e) Safety

tDCS is generally regarded as a safe method of brain stimulation. However, it is not advised to use tDCS in people susceptible to seizures and/or people who have metallic implants near the electrodes. In addition, possible side effects of tDCS such as headaches, dizziness, nausea, itching sensations as well as transient skin irritation (i.e. burning) under the electrodes should be made known to subjects [START_REF] Poreisz | Safety aspects of transcranial direct current stimulation concerning healthy subjects and patients[END_REF][START_REF] Palm | Skin lesions after treatment with transcranial direct current stimulation (tDCS)[END_REF].

f) Comparison to other brain stimulation technique: rTMS

Recently, repetitive transcranial magnetic stimulation (rTMS) and tDCS have come to be used more often as painless and non-invasive brain stimulation techniques in humans. Regarding their effects on the cortex, tDCS greatly differs from rTMS, since rTMS induces action potentials of axons in of the cortex that then release neurotransmitters at their terminal synapses, while tDCS does not cause action potentials in cortical neurons. In general, high-frequency rTMS and anodal tDCS enhance, and low-frequency rTMS and cathodal tDCS decrease, the excitability of neurons of the targeted cortical areas. Both rTMS and tDCS have also been reported to induce post-effects that outlast the period of stimulation. TMS coils come in a variety of different sizes and configurations and it appears that TMS is able to limit stimulation to an area of about 25 mm 2 , while the current flow of tDCS throughout the brain occurs over a wide area. tDCS intensities indicate the current flow between the electrodes with no indication of how much the current enters the brain and polarizes neurons. However,

CHAPTER 2 MATERIALS AND METHODS

the relationship between stimulation intensity and clinical effects has not yet been clearly reported in both rTMS and tDCS. tDCS is easy to sham and relatively simple to apply compared to rTMS. It was reported that tDCS at an intensity below 1.5 mA is generally not perceived by subjects. If low current intensity is used with large area stimulating electrodes and low impedance, subjects would not distinguish between active and sham stimulation. Both rTMS and tDCS are generally considered as safe methods. However, tDCS induces a small or no risk of a seizure compared to rTMS as it does not induce action potentials in axons. tDCS applied for minutes does not increase the markers of neuronal damage such neuron specific enolase or brain Nacetyl-aspartate. However, whether tDCS is safe for long stimulation duration or high intensities is still unknown (Reviewed in Priori et al., 2009).

To conclude, convincing experiments now show that tDCS can pass through the scalp and influence brain and spinal cord activities during their application and sometimes induce post-effects in motor cortex and some spinal circuits (Priori et al., 1998(Priori et al., , 2009;;Nistche & Paulus, 2001;[START_REF] Priori | Brain polarization in humans: a reappraisal of an old tool for prolonged non-invasive modulation of brain excitability[END_REF]Roche et al., 2009[START_REF] Roche | Impact of precision grip tasks on cervical spinal network excitability in humans[END_REF][START_REF] Roche | Effects of anodal tDCS on lumbar propriospinal system in healthy subjects[END_REF]. Together with its advantages such as non-invasiveness, painlessness, and inducing less chance of causing seizure, we therefore chose tDCS to investigate changes in the excitability of spinal network in hemiplegic patients in the present study.

Application of tDCS in the present study

The experimental procedure was identical to that used by Roche et al. (2009). Application of tDCS used a NEUROCONN DC-STIMULATOR (NEUROCONNGMBH COMPANY, Ilmenau, Germany) with two conductive rubber electrodes placed in saline-soaked sponges (5x7 cm). The cathode was placed over the contralateral supra-orbital region. The effects of tDCS were tested in active and sham conditions. In both conditions, current intensity was fixed at 1.75 mA. Current flowed continuously during 20 minutes for active conditions, and only 120 seconds for the sham condition since Nitsche and Paulus (2000) had previously reported that a duration of at least 3 minutes of tDCS was necessary to induce after-effects. The current was ramped up or down over the first and last 8 seconds of stimulation. The minimum delay between the two recording sessions (active and sham tDCS) was about one week. The active and sham conditions were randomly alternated.

Recording Chain

The test stimulus evoking FCR H-reflex and the conditioning stimulus were delivered using constant-current stimulators (D7SA Digitimer Ltd., Welwyn Garden City, UK). The stimulations were computer-triggered using a customised script (Signal 4.20, Cambridge Electronic Design, Cambridge, UK). The H-reflex was recorded from the EMG signal of the FCR muscle using bipolar adhesive electrodes (Euro ECG electrodes, 32x36 mm, Vicchio, Florence, Italy) positioned on the skin parallel to the muscle belly. The EMG activity was sampled at 2 kHz, amplified (x 5000 -10000) and band-pass filtered (250 Hz-3 kHz) using a Digitimer D360 amplifier (DIGITIMER LTD, WELWYN GARDEN CITY, Herts, UK) and then transmitted to an oscilloscope for visualization and to a computer for storage and offline analysis. Anodal tDCS (sham/active) was applied over the motor cortex during 20 minutes. The recording chain in this present study was similar to that in the project 1 (see Figure 21).

CHAPTER 2 MATERIALS AND METHODS

Statistical methods

Data processing

The H-reflex size was determined by averaging peak-to-peak amplitudes expressed as a percentage of the maximum motor response (M max ). The amount of reciprocal inhibition was defined as 100 -((conditioned H-reflex/unconditioned Hreflex) × 100). The mean amount of reciprocal inhibition from each period was normalised as a percentage of its baseline inhibition measured over 10 minutes before tDCS (t 0 ) according to the equation: [%((Inhibition at t x -inhibition at t 0 )/ inhibition at t 0 )] +100. Therefore, positive value refers to a larger inhibition than baseline inhibition and vice versa for negative value.

Statistical analysis

For individual data, a two-way ANOVA was performed with time period as the first factor (baseline, period 1 tDCS, period 2 tDCS, and post tDCS) and tDCS condition (sham and active ipsilateral tDCS) as the second factor.

A two-way ANOVA was performed on unconditioned H-reflexes (with time period and condition as factors) to ensure that the modulation of reciprocal inhibition resulted from tDCS not from the variation of the H-reflex over time. Grouped data were analysed using two-way repeated-measures ANOVAs to determine effects of tDCS and time period on reciprocal inhibition. When the F value was significant, post hoc pairwise comparisons were performed using the Newman-Keuls test. Significance was taken at P < 0.05. Mean data are provided as mean ± 1 Standard Error of the Mean (SEM). Statistical analysis was performed using the SigmaPlot software 11.0.

Project 1 1. Subjects

The experiments described in this study conformed to the guidelines issued by the World Medical Association (Declaration of Helsinki) on the ethics of experimentation in humans and the approval of the local ethical committee of the CPP Île-de-France VI -Pitié-Salpêtrière. Written informed consent was obtained from all subjects before participation.

Healthy subjects included in the present study had no history of drug allergies. Patients were selected based on the following criteria : i) the presence of paraplegia/tetraplegia after spinal cord injury (complete or incompelete) ii) the presence of a sizeable H-reflex in soleus muscle iii) the absence of medical history of drug allergies. This randomized controled study was performed on 19 healthy volunteer subjects (9 females) aged 22-64 years old (mean±SEM 35.1±2.5 years) and 10 SCI patients (1 female) aged 20-67 years old (mean±SEM 36.4±4.8 years). The patients were selected from patients undergoing rehabilitation at Physical Medicine and Rehabilitation department, the Hôpital Raymond Poincaré (Raymond Poincaré Hospital). The average duration after SCI was 6.3±1.5 months. Nine patients present complete cord injuries.

For the recruitment, some patients were excluded from the study because of the absence of soleus H-reflex. Another constraint presented here was that pre-and postsynaptic inhibition could not be always evoked in all included patients; presynaptic inhibition could be demonstrated in 9 of 10 patients and postsynaptic inhibition could be demonstrated in only 4 of 10 patients (see individual inhibition values Table 2). This constraint especially for postsynaptic inhibition has limited our recordings and also the interpretation of the results.

CHAPTER 3 RESULTS : PROJECT 1

Study design

All of healthy subjects participated in two different sessions to investigate the time course of changes in spinal network excitability for a period of 70 minutes (i) after furosemide intake (furosemide experiment), and (ii) without furosemide (control experiment). The two different experiments (furosemide and control) were performed on different days. Only furosemide experiments were performed in SCI patients.

Experimental procedure

Medical examinations (blood testing for electrolytes, creatinine, glucose and blood pressure) were performed before the experiment to verify that subjects had no contraindications to furosemide.

All experiments were performed at rest. Subjects sat comfortably with the head, neck, back and arms supported in a slightly reclined armchair. The dominant leg was examined and remained in a stationary supported position with hip semi-flexion (120°), slight knee flexion (160°) and plantar flexion (110°). Once inhibition values are evaluated for baseline period, furosemide is then orally taken. The recording is performed after furosemide intake for 70 minutes. In control experiments, the time period of recording is the same as furosemide experiments.

CHAPTER 3 RESULTS : PROJECT 1

The effect of furosemide was also tested on monosynaptic excitatory transmission (synaptic transmission between muscle afferent terminals and soleus motoneurons/post-activation depression of the soleus H-reflex) to explore if furosemide has specific effect on functioning of inhibitory synapses. This experiment was performed in 15 healthy subjects the same way as described previously for pre-and postsynaptic inhibition.

Water intake was recommended to subjects after the experiment. However, apart from the diuretic effect, some side effects of furosemide such as headaches and dizziness were reported from some subjects that participated in the present study.

CHAPTER 3 RESULTS : PROJECT 1

Results from healthy subjects

4.1

Amplitude of H-reflex (as % of M max )

The maximum motor response (M max ) and maximum H-reflex response (H max ) were recorded at the beginning of the each experiment. The stimulus intensity was then adjusted to elicit H-reflex amplitude of 20-35% of M max , which corresponded to ~ 50%, of H max. The mean values of H-reflex amplitude are 24.85 ± 2.8 % M max in control experiments, and 23.17 ± 2.2 % M max in furosemide experiments.

Effects of time on two spinal inhibitory networks

Presynaptic inhibition of Ia fibres.

Presynaptic inhibition was recorded over 70 minutes in 19 healthy subjects in the control experiments (Figure 24). The presynaptic inhibition is not modified over time. A one-way repeatedmeasures ANOVA failed to detect a significant effect of time on presynaptic inhibition in control experiments (P > 0.05).

CHAPTER 3 RESULTS : PROJECT 1

Postsynaptic inhibition induced by cutaneous stimulation.

The experiments were performed in the all of subjects but this type of spinal synaptic inhibition could not be demonstrated in 4 of them since conditioning stimulus intensity required to evoke postsynaptic inhibition was above that evoking an unbearable painful sensation or triggered the flexor reflex in these subjects. The time course of postsynaptic inhibition was observed in 15 healthy subjects over 70 minutes, illustrating in the Figure 25. The amount of presynaptic inhibition in control experiments is constantly at the baseline level over time, while there is a gradual decrease of presynaptic inhibition immediately after furosemide intake until reaching its nadir at 41-50 minutes (38.16±2.36 % of its baseline value) and being continuously at this level up to 90 minutes.

CHAPTER 3 RESULTS : PROJECT 1 b) Furosemide vs. control : Group results

The comparison between variations of presynaptic inhibition in control and furosemide experiments in 19 subjects is shown in Figure 28. There is a significantly decrease of postsynaptic inhibition after drug administration in furosemide experiments (F 14,98 = 17.67, P < 0.001, one way repeatedmeasure ANOVA). The amounts of postsynaptic inhibition is decreased immediately after furosemide intake and progressively decreased until reaching its nadir at 51-60 minutes (21.88 % of its baseline value). The decrease is statistically significant from beginning up to 61-70 minutes (baseline vs. 0-10 minutes, baseline vs. 11-20 minutes, baseline vs. 21-30 minutes, baseline vs. 31-40 minutes, baseline vs. 41-50 minutes, baseline vs. 51-60 minutes, and baseline vs. 61-70 minutes (P<0.01, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001 and P<0.001 respectively, Tukey test)).

CHAPTER 3 RESULTS : PROJECT 1 a) Furosemide vs. control : a representative individual result

Postsynaptic inhibition recorded over 70 minutes in control and furosemide experiments in a representative subject is illustrated in Figure 30. The amount of postsynaptic inhibition in control experiments is gradually decreased over time reaching its nadir at 51-60 minutes (30.55±5.89 % of its baseline value). In furosemide experiment, the amount of postsynaptic inhibition is dramatically decreased after furosemide administration until the absence of postsynaptic inhibition at 41-50 minutes and 51-60 minutes (-18.39±10.94 and -36.73±5.89 % of its baseline value, respectively). The inhibition value is calculated using the following equation: 100 -((conditioned H value/unconditioned H value) × 100), the values in negative means conditioned H value > unconditioned H value, thereby facilitation (the inhibition is not found).

CHAPTER 3 RESULTS : PROJECT 1 b) Furosemide vs. control : Group results

The comparison between variations of postsynaptic inhibition in control and furosemide experiments is presented in Figure 31 (15 subjects). The amounts of postsynaptic inhibition in the furosemide experiments begin to be significantly different from those observed in control experiments at 21-30 minutes after drug administration. Two-way repeated-measures ANOVA detected a significant interaction of condition (control vs. furosemide), time epochs, and condition × time epochs (F 14,84 = 3.43, P condition = 0.034, P time epochs <0.001, P condition × time epochs = 0.004). Post-hoc analysis showed that furosemide significantly reduced postsynaptic inhibition at 21-30 minutes, 51-60 minutes and 61-70 minutes (P<0.01, P<0.001, and P<0.05 respectively, Tukey test).

Effects of two different doses of furosemide on the two spinal inhibitory spinal networks

For dose-dependent experiments, an a priori power analysis was performed to determine the minimal sample size. Given that postsynaptic inhibition decreased over time (see Figure 29), the power analysis was performed on presynaptic inhibition only. A sample size of 6 subjects, each having 0, 20 or 40 mg of furosemide provided 80% power to detect a 40% change in presynaptic inhibition (one way repeated measures ANOVA, two-tailed 5%). This calculation was made assuming that raw data are approximately normally distributed with a standard deviation of 10%. One way repeated measures ANOVA was then employed.

Presynaptic inhibition

The effects of two different doses of furosemide were studied on presynaptic inhibition in 6 subjects. Furosemide effects were evaluated during the 40-70 minutes where effects were mostly observed (Figure 32). Furosemide 0 and 20 mg induce no changes in presynaptic inhibition (97.53±10.69% and, 96.39±10.79% of its baseline value, respectively), while presynaptic inhibition is decreased up to 34.61±9.22% of its baseline value following CHAPTER 3 RESULTS : PROJECT 1 administration of furosemide 40 mg. A one-way repeated-measures ANOVA detected a significant dose-effect (F 5,10 = 13.67, P = 0.001). A Post-hoc Turkey test revealed significant difference between 0 mg vs. 40 mg, and 20 mg vs. 40 mg (P<0.01 and P<0.01, respectively).

Postsynaptic inhibition

The effects of two different doses of furosemide administration were studied on postsynaptic inhibition in 6 subjects. The amounts of postsynaptic inhibition recorded at 40-70 min after drug administration are illustrated in Figure 33. As a percentage of its baseline value, the mean levels of inhibition are: furosemide 0 mg (control) = 92.98±10.60%, 20 mg = 77.12±27.23%, and 40 mg = 38.13±14.11%. There is a trend for a dose-effect in postsynaptic inhibition as observed in presynaptic inhibition. However, a one-way repeated-measures ANOVA failed to detect a significant dose-effect on postsynaptic inhibition: P > 0.05. Since time has an influence on postsynaptic inhibition, the effect of furosemide evaluated at 40-70 minutes could be confounded with this time effect.

CHAPTER 3 RESULTS : PROJECT 1

4.5

Furosemide effects on monosynaptic excitatory transmission.

Post-activation depression

This experiment was performed after the study of pre-and postsynaptic inhibitions was completed. Fifteen subjects from the previous recordings participated in this session (see Table 1). The comparison between variations of post-activation depression determined using 1/0.16 ratio observed from 15 healthy subjects in control and furosemide experiments is shown in Figure 34. Post-activation depression is not modified over 70 minutes of recording in both control and furosemide conditions. A two-way repeated-measure ANOVA failed to detect a significant interaction of condition × time epochs: P > 0.05. There is no significant difference between two groups (control vs. furosemide).

CHAPTER 3 RESULTS : PROJECT 1

Individual raw data of healthy subjects

Individual values (not-normalised) of pre-and postsynaptic inhibition reported as a percentage of amplitude of unconditioned H-reflex, and values of post activation depression (not-normalised) reported as 1/0.16 ratio recorded at baseline and during the 40-70 minutes (grouped all the three subpools data (41-50 minutes, 51-60 minutes, and 61-70 minutes) into one) where effects were mostly observed, are shown in Table 1.

Table 1 : Individual values (not-normalised) of pre-and postsynaptic inhibition, and post-activation depression in healthy subjects.

The inhibition value was calculated using the following equation: 100

-((H conditioned/H unconditioned) × 100).
The values in negative means H conditioned > H unconditioned, thereby facilitation (the inhibition is not found). The sign "-" means not recorded.

CHAPTER 3 RESULTS : PROJECT 1

Results from SCI patients

5.1 Amplitude of H-reflex (as % of M max )

The maximum motor response (M max ) and maximum H-reflex response (H max ) were recorded at the beginning of the each experiment. The stimulus intensity was then adjusted to elicit H-reflex amplitude of 20-35% of M max , which corresponded to ~ 50%, of H max. The mean values of H-reflex amplitude in SCI patients is 27.27 ± 4.7 % M max in furosemide experiments (normal subjects = 23.17 ± 2.2 % M max ).

5.2

Degree of pre-and postsynaptic inhibition

Presynaptic inhibition

The degree of presynaptic inhibition evaluated before drug administration (baseline) in all healthy subjects and SCI patients is illustrated in Figure 35. The degree of presynaptic inhibition in SCI patients is smaller than those observed from healthy subjects. The degree of presynaptic inhibition at the beginning of recording (baseline) is 28.00 ± 1.7 in 19 healthy subjects, and 11.88 ± 3.7 in 10 patients.

CHAPTER 3 RESULTS : PROJECT 1

A t-test reveals a statistically difference between the degree of inhibition in healthy subjects and in SCI patients (p<0.001).

Postsynaptic inhibition

The degree of postsynaptic inhibition evaluated before drug administration (baseline) in all healthy subjects and SCI patients is illustrated in Figure 36. The results show that the degree of postsynaptic inhibition is on average abolished in SCI patients. Some of them present facilitation instead of inhibition (see individual data in table X). The inhibition value is calculated using the following equation: 100 -((conditioned H value/unconditioned H value) × 100), the values in negative means conditioned H value > unconditioned H value, thereby facilitation (the inhibition is not found). The degree of postsynaptic inhibition at baseline is 23.54 ± 2.3 in 15 healthy subjects, and -2.01 ± 4.0 in 10 patients with SCI. A t-test reveals a statistically reliable difference between the degree of inhibition in healthy subjects and in SCI patients (p<0.001).

CHAPTER 3 RESULTS : PROJECT 1

5.3

Effects of furosemide on the two spinal inhibitory networks.

Presynaptic inhibition of Ia fibres.

Presynaptic inhibition recorded over 70 minutes after furosemide administration in 9 SCI patients is illustrated in Figure 37. The presynaptic inhibition following furosemide administration is highly fluctuated during 0-40 minutes after furosemide intake, and subsequently become stable at 41-70 minutes where the inhibition values are above the baseline. A one-way repeated-measures ANOVA failed to detect a significant effect of time on presynaptic inhibition in control experiments (P > 0.05).

CHAPTER 3 RESULTS : PROJECT 1 a) SCI patients vs. healthy subjects

The comparison between variations of presynaptic inhibition in 19 healthy subjects and 9 SCI patients is presented in Figure 38. The amount of presynaptic inhibition is gradually reduced after furosemide administration up to 70 minutes in healthy subject, while after furosemide intake in SCI patients; presynaptic inhibition is fluctuated at the beginning and stays over baseline value at 41-50 minutes until the end of recording. Two-way repeated-measures ANOVA detected a significant interaction of subjects (healthy subjects vs. SCI patient), and subjects × time epochs (F 6,182 = 5.19, P subject = 0.003, P subject × time epochs <0.001). Post-hoc analysis showed that presynaptic inhibition of healthy subjects vs. presynaptic inhibition of SCI patients significantly different at, 41-50 minutes, 51-60 minutes, and 61-70 minutes (P<0.01, P<0.01 and P<0.001 respectively, Tukey test). At these time epochs, furosemide does not decrease presynaptic inhibition contrastively to what observed in healthy subjects.

CHAPTER 3 RESULTS : PROJECT 1

Postsynaptic inhibition induced by cutaneous stimulation

This type of inhibition could be detected only in 4 of 10 patients. The rest of subjects showed a tendency to facilitation instead of inhibition (see individual raw data in Table 2). However, the amounts of postsynaptic inhibition recorded before furosemide administration (baseline) in the 4 patients is very small. The data are not normally distributed, the variances are heterogeneous and the sample size is too small for statistical analysis. However, the results observed from the 4 patients suggest that there no tendency of decrease of the postsynaptic over recording as observed in healthy subjects.

The comparison between postsynaptic inhibition in healthy subjects and SCI patients is not presented here because the sample of patients is very small and the patient's data have too high variation.

Individual raw data of SCI patients

Individual values (not normalised to baseline) of pre-and postsynaptic inhibition and of post-activation depression recorded at baseline and during the 40-70 minutes period are shown in Table 2. Note that three subpools at the intervals with the most prominent effects (41-50 minutes, 51-60 minutes, and 61-70 minutes) were pooled. The inhibition value was calculated using the following equation: 100 -((H conditioned/H unconditioned) × 100). The values in negative means H conditioned > H unconditioned, thereby facilitation (the inhibition is not found, indicated in red). These values were excluded from the calculation. The sign "-" means not record.

Subjects

Healthy volunteer subjects included in the present study had no history of intracranial metal implantation, cochlea implant, or cardiac pacemaker. Patients were selected based on the following criteria: i) the presence of hemiparesis after unilateral stroke ii) the absence of pain and severe clonus following the elctrical stimulation in the tested limb iii) the presence of a sizable H-reflex in flexor carpi radialis (FCR) muscle; iv) the absence of medical history of intracranial metal implantation, cochlea implant, or cardiac pacemaker; v) the ability to sit upright in a wheelchair or armchair during at least one hour; and vi) the ability to understand simple explanation of the study. A randomised, sham-controlled tDCS study was performed on 33 healthy volunteer subjects (20 females, 8-left handeds) aged 20-59 years old (mean±SEM 30.7±1.8 years), 9 hemiplegic patients (4 females) aged 32-69 years old (mean±SEM 53.6±4.0 years). The patients were selected from patients undergoing rehabilitation at Physical Medicine and Rehabilitation service, the Hôpital de la Pitié-Salpêtière (Pitié-Salpêtière Hospital). The average duration after stroke was 4.7±1.7 months.

Study design

Contra-and ipsilateral anodal tDCS effects on reciprocal inhibition between forearm muscles were tested in healthy subjects and hemiplegic stroke patients in the present study.

Contralateral study: anodal tDCS is applied over the hand motor cortex of the non-dominant right hemisphere, and reciprocal inhibition is recorded in the nondominant upper limb in right-handed healthy subjects. This result is then compared to the previous results in the dominant upper limb observed by Roche et al., 2009. In stroke patients, anodal tDCS is applied over the unaffected hemisphere, and reciprocal inhibition is recorded in the non-paretic upper limb.

Ipsilateral study: study in healthy subjects is performed in right-handers and left-handers. Anodal tDCS is applied over the hand motor cortex of the hemisphere that is ipsilateral to their dominant limbs, thus reciprocal inhibition is recorded on the right side in right-handed subjects and on the left side in left-handed subjects. In stroke patient, anodal tDCS is applied over the unaffected hemisphere, and reciprocal inhibition is recorded in the paretic upper limb.

CHAPTER 3 RESULTS : PROJECT 2

The subjects were blind to the conditions of tDCS. The active and sham tDCS were randomly alternated. Each condition was performed on the subjects on different days to avoid carry-over effects.

Experimental procedure

Subjects were seated in a comfortable reclining armchair with the shoulder slightly abducted at about 60°, elbow semi-flexed and slight pronation of the forearm. The distal part of the upper limb was supported by an armrest in order to exclude any active maintenance of wrist posture, and the subjects were asked to keep the recording arm at rest. Once all parameters (unconditioned H reflex amplitude, conditioning stimulation intensity and ISI) were set the baseline inhibition (without tDCS) was determined over the first 10 minutes, being defined as the baseline period. The tDCS electrodes were attached. The anode was placed over the hand motor cortex for 20 minutes in both active and sham conditions. These 20 minutes were divided into two "10 minute" periods: period 1 and period 2. The tDCS electrodes were removed immediately after the end of the stimulation. The amount of reciprocal inhibition was also measured for 10 minutes after the end of stimulation to evaluate post-effects. In each period (baseline, period 1, period 2 and post), the amount of inhibition was assessed with three series of 40 H reflexes (20 conditioned H reflexes and 20 unconditioned H reflexes). Conditioned and unconditioned H-reflexes were evoked every 3 s and randomly alternated. Before application of tDCS, the amount of reciprocal inhibition is recorded during 10 minutes, corresponding to baseline period. tDCS period of 20 minutes is divided into 2 session of 10 minutes; period 1 and period 2. The amount of reciprocal inhibition is recorded during 10 minutes after application of tDCS, corresponding to post-tDCS period. Results obtained from active tDCS resemble with those from sham condition. Both of results from active and sham conditions observed during and after tDCS are very close to baseline value. A two-way measure ANOVA failed to detect a significant interaction of condition × time period: P > 0.05. There was no significant difference between two groups (sham vs. active tDCS). Reciprocal inhibition recorded in the dominant upper limb is increased during tDCS especially at period 2 and restored to its baseline value after tDCS, while, in the non-dominant limb, reciprocal inhibition remained at the baseline level throughout the recording. Two-way measures ANOVA detected a significant different between two groups (dominant vs. non-dominant) (F 1,88 =5.90, P= 0.017). Results from an individual subject show that reciprocal inhibition recorded in the dominant limb is rapidly increased during tDCS especially at the period 2, while in the non-dominant limb, reciprocal inhibition is initially increased at the period 1 but restores to the baseline level during the period 2 and remained at this level until post tDCS.

CHAPTER 3 RESULTS : PROJECT 2

Ipsilateral study

Effects of anodal tDCS over ipsilateral motor area on reciprocal inhibition were firstly demonstrated in right-handed subjects. The experiments were performed in active tDCS and sham conditions. Secondly, results from right-hander were compared to those observed from left-hander, only in active condition.

Right-handed subjects

Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb is observed in healthy right-handed subjects. The reciprocal inhibition values are recorded in both active and sham conditions in 19 healthy right-handed subjects, illustrating in Figure 44. Results obtained from active tDCS experiment strongly resemble with those from sham experiments. Both of results from active and sham conditions observed during and after tDCS are very close to baseline value. A two-way repeated-measure ANOVA failed to detect a significant interaction of condition × time period: P > 0.05. There was no significant difference between two groups (sham vs. active tDCS).

CHAPTER 3 RESULTS : PROJECT 2

Right-handed subjects vs. left-handed subjects

As it has shown previously that sham tDCS induced no changes in reciprocal inhibition (see result above, Figure 44), examining the effect of ipsilateral tDCS in right-handed and left-handed subjects was only focused in active tDCS condition. Figure 45 represents the results obtained in 24 right-handed subjects and 8 left-handed subjects. Reciprocal inhibition is not modified in right-handed subjects, while, little decreased of the inhibition is observed in left-handed subjects. However, a two-way measure ANOVA failed to detect a significant interaction of condition × time period: P > 0.05. There was no significant difference between two groups (right handers vs. left handers).

Individual raw data of healthy subjects

Individual values (not-normalised) of reciprocal inhibition at wrist level reported as a percentage of amplitude of unconditioned H-reflex, recorded at baseline, period 1 tDCS, period 2 tDCS and post-tDCs in non-dominant and/or dominant upper limbs of 33 healthy subjects, are shown in Table 6. The inhibition value was calculated using the following equation: 100 -((H conditioned value/H unconditioned value) × 100). The values in negative means H conditioned > H unconditioned, thereby facilitation (the inhibition is not found). The sign "-" means not record.

Results from stroke patients

Degree of reciprocal inhibition in stroke patients

The levels of reciprocal inhibitions between wrist muscles were evaluated before application of tDCS (baseline) and recorded in 32 dominant limbs and 12 nondominant limbs of healthy subjects, and 7 paretic limbs and 5 non-paretic limbs of hemiplegic patients, illustrating in Figure 46. . The degree of reciprocal inhibition observed in the dominant and the nondominant upper limbs in healthy subjects and in the non-paretic limb in hemiplegic patients are similar, while the levels of inhibition in paretic upper limb is relatively small compared to the others. The levels of reciprocal inhibition are 28.10 ± 1.6 in the dominant limbs, 30.62 ± 3.6 in the non-dominant limbs, 19.93 ± 3.6 in the paretic limbs, and 28.34 ± 4.9 in the non-paretic limbs. A t-test detected a significant different between the dominant limb vs. the paretic limb (P = 0.032) and the non-dominant limb vs. the paretic limb (P = 0.049).

CHAPTER 3 RESULTS : PROJECT 2

5.2

Ipsilateral study

Stroke patients

Effects of anodal tDCS of the unaffected motor cortex on reciprocal inhibition in the paretic upper limb were observed in 7 stroke patients (active n=6, sham n=6), illustrating in Figure 47. Reciprocal inhibition is slightly increased during tDCS in active and sham condition. Nevertheless, the data have a high variation. A two-way measure ANOVA failed to detect a significant interaction of condition × time period: P > 0.05. There was no significant difference between conditions (sham vs. active tDCS).

CHAPTER 3 RESULTS : PROJECT 2

Stroke patients vs. Healthy subjects

The comparison between variations of reciprocal inhibition demonstrated by ipsilateral tDCS in healthy subjects and hemiplegic patients are presented in Figure 48. The reciprocal inhibitions are recorded in 32 healthy subjects and 6 hemiplegic patients in active tDCS condition. The reciprocal inhibition is not modified during ipsilateral tDCS in healthy subjects and in stroke patients. The amount of reciprocal inhibition is close to baseline value throughout the experiment in the both groups. A two-way measure ANOVA failed to detect a significant interaction of condition × time period: P > 0.05. There was no significant difference between the two groups (healthy subjects vs. stroke patients).

CHAPTER 3 RESULTS : PROJECT 2

5.3

Contralateral study

Stroke patients

Effects of contralaetral anodal tDCS of the unaffected motor cortex on reciprocal inhibition in the non-paretic upper limb were recorded in 5 stroke patients (active n=5, sham n=4), illustrating in Figure 49. There is a slightly decreased of reciprocal inhibition in sham condition while, in active condition, the inhibition is extremely decreased during tDCS especially during period 2 and remain reduced after tDCS. However, the sample of subjects is too small for statistical analysis. In healthy subjects, reciprocal inhibition recorded in the dominant limb is increased during contralateral tDCS especially at period 2 and restored to its baseline value after tDCS, while, in the non-dominant limb, reciprocal inhibition remained at the baseline level throughout the recording. In hemiplegic patients, the inhibition is extremely decreased during tDCS, even after tDCS the inhibition is remain reduced. However, statistical analysis is not employed to compare the results in the two groups since the sample of patients is very small. CHAPTER 3 RESULTS : PROJECT 2

Individual raw data of stroke patients

Individual values (not-normalised) of reciprocal inhibition at wrist level reported as a percentage of amplitude of unconditioned H-reflex, recorded at different periods (baseline, period 1 tDCS, period 2 tDCS and post-tDCS) in paretic and non-paretic upper limbs of nine stroke patients, are shown in Table x. The inhibition value was calculated using the following equation: 100 -((H conditioned value/H unconditioned value) × 100). The values in negative means H conditioned > H unconditioned, thereby facilitation (the inhibition is not found). The values in red indicate a very small value of inhibition that were excluded from the calculation. The sign "-" means not record.

Chapter 4 Discussion

Project 1

The main finding of the present study is that oral administration of furosemide at the usual clinical dose (40 mg) decreases the function of inhibitory synapses in the spinal cord without affecting monosynaptic excitatory transmission in healthy subjects. The presynaptic inhibition is not modified over 70 minutes of recording without furosemide while there is a gradual decrease of inhibition after furosemide orallyadministered until the end of recording in the same participants. For postsynaptic inhibition, the amount of inhibition is decreased over time in control experiments (without furosemide). However, after furosemide intake, the amount of postsynaptic inhibition is also decreased and the decrease is more marked than that without furosemide. The reduction of inhibition is also dose-dependent. To verify if furosemide has specific effects on the functioning of inhibitory synapses, effects of furosemide were also tested on post-activation depression at the Ia fibre-motoneuron synapse (an excitatory synaptic transmission). The results reveal no changes of excitatory synaptic transmission observed in both control and furosemide experiments.

In SCI patients, the effect of furosemide was also tested on pre-and postsynaptic inhibitions. First, the levels of pre-and postsynaptic inhibition were evaluated before drug administration (baseline) revealing that presynaptic inhibition is decreased in patients with spinal cord lesion compared to healthy subjects and postsynaptic inhibition induced by cutaneous stimulation is extremely decreased or completely abolished in these patients. The results obtained in SCI patients reveal that the action of furosemide on presynaptic inhibition is less important than in healthy subjects, and the same results likely occur for postsynaptic inhibition but the data had a very high variation and the sample size of patients is too small to conclude.

Methodological considerations

In humans, available methods to study spinal cord networks are indirect and rely on EMG recordings that operate as a window on motoneuron and interneuron excitability (Pierrot-Deseilligny & Burke, 2012). Since the pioneering experiments in humans by Magladery et al. (1951) and Paillard (1955), electrophysiological noninvasive but selective methods have been developed to study the excitability of spinal networks. Recent results obtained in animals show that lesion of the spinal cord induces a down-regulation of KCC2 co-transporter, which reverses the inhibitory pattern of GABAergic and glycinergic neurons back towards their immature and excitatory state (Jean-Xavier et al. 2006) and also results in reduction of inhibitory synaptic strength (Boulenguez et al. 2010). These findings raise the possibility of setting up a technique, which would allow us to determine if such a reversal also exists in patients with spinal cord injury (SCI). Furosemide, a loop diuretic, has been shown to selectively and reversibly block inhibitory postsynaptic potentials (IPSPs) without affecting excitatory postsynaptic potentials (EPSPs) in animal spinal motoneurons (Nicoll, 1978) and has been used as a cation-chloride co-transporter blocker (reviewed in Kahle et al. 2008). Here, we used furosemide to demonstrate changes in spinal inhibitory networks in healthy subjects and patients with spinal cord lesion. The effect of furosemide was also tested on an example of spinal excitatory synapses in healthy subjects to verify whether furosemide affects both inhibitory and excitatory synapses. These spinal circuits were studied using non-invasive electrophysiological techniques based on the H-reflex evoked in soleus muscle at rest. The specific effects of furosemide on spinal inhibitory synapses as demonstrated here provide a window on the intrinsic functioning of inhibitory synapses mediated through cation-chloride cotransporters.

The choice of furosemide

Experiments relying on motoneuron intracellular recordings in the frog demonstrated that furosemide selectively and reversibly blocks IPSPs mediated by GABA and glycine without alteration of EPSPs (Nicoll, 1978). Furosemide has been used as a KCC2 blocker in animal experiments (Gillen et al. 1996;Russell, 2000;[START_REF] Thompson | Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl-in hippocampal CA3 neurons[END_REF]. However, since furosemide is also capable of inhibiting NKCC1, the question may arise as to how we can interpret the results from this nonspecific KCC2 blocker. NKCC1 is known to establish an increase of [Cl -] i that underlies the excitatory effect of GABAergic and glycinergic neurons in the immature state (Ben Ari, 2002;[START_REF] Dzhala | NKCC1 transporter facilitates seizures in the developing brain[END_REF]Yamada et al., 2004), while KCC2 activity is crucial for synaptic inhibition via GABA and glycine (Hübner et al., 2001). If, in our study, furosemide dominantly blocks NKCC1 or equally antagonises NKCC1 and KCC2, this CHAPTER 4 DISCUSSION : PROJECT 1 would lead to an increase or unchanged in inhibitory synapses excitability in healthy subjects instead of reduction. Moreover, Boulenguez et al. (2010) demonstated that the level of NKCC1 is not modified after spinal cord transection in the rat. If this is the case in SCI patients, even though furosemide blocks both NKCC1 and KCC2 cotransporters, its effects on NKCC1 would likely be the same in normal subjects and in SCI patients. The action of GABA and glycine is due to the distribution of Cl-across the membrane regulated by cation-chloride co-transporters. Exploring the effects of a NCCK1 antagonist (i.e. bumetanide) on the inhibitory synaptic activity would be interesting to test especially in SCI patients. However, given that strength of synaptic inhibition is known to be regulated by KCC2 (Boulenguez et al.,2010), and that the aim of our study is to test the excitability of inhibitory synpases, furosemide, which is a safe and widely used diuretic was chosen in the study.

Results in healthy subjects

Changes in inhibitory synapses over time

Habituation is a gradual decrease in responsiveness following repetitive stimulation and is a well-known phenomenon in the nervous system (Harris, 1943;Hernández-Peón et al. 1956). Most studies report habituation as a reduction in excitatory responses. Spontaneously active spinal neurons are progressively inhibited (inhibitory build-up) by repeated stimulation (Haber & Wagam, 1974). The characteristics of inhibitory build-up regarding stimulus intensities are similar to the frequency effects on inhibitory synapses observed in Aplysia [START_REF] Waziri | Organization of inhibition in abdominal ganglion of Aplysia. II. Posttetanic potentiation, heterosynaptic depression, and increments in frequency of inhibitory postsynaptic potentials[END_REF] and in the mammalian spinal cord [START_REF] Kuno | Quantal components of the inhibitory synaptic potential in spinal mononeurones of the cat[END_REF]. Habituation of the flexor reflex has been reported in isolated frog spinal cord showing that habituation is due to a reduction in the efficacy of synapses within the pathway (Farel et al., 1973). It has been shown in decerebrate animals that prolonged stimulation of the sural nerve at 0.5 Hz or higher produced a complete habituation or failure of the C-fibre evoked response in a dorsal horn neuron (Woolf, 1983). However, habituation has also been referred to as a reduction in inhibitory responses. In the decerebrate cat, intracellular recordings of motoneurons participating in the flexion reflex revealed that there was a marked and significant decrease of PSP amplitude during habituation which contains not only the EPSP's habituate, but also decrements of IPSP components. The IPSPs of polysynaptic responses decrease rather than increase during habituation suggesting that the amount of postsynaptic inhibition on the motoneuron is also "habituating" (i.e., decreasing). Besides, the decrease in response during habituation was proposed as a result of reduced input to motoneurons occurring in interneurons (Brazier, 1961). Habituation CHAPTER 4 DISCUSSION : PROJECT 1 has also been observed with the flexor reflex in normal human subjects and patients with complete spinal cord lesion. Regular repeated stimuli delivered to the skin on the medial side of the plantar surface of the foot induce a significant decrease of flexor reflex response in both normal subjects and patients. As habituation can be observed in patients with complete spinal cord injury, it was suggested that this phenomenon may occur partly or totally at the spinal cord level (Dimitrijevic & Nathan, 1970;Dimitrijevic et al., 1972). Recently, studies in normal subjects reported the degree of habituation of the nociceptive flexion reflex evoked by stimulation of the sural nerve is dependent on both interstimulus interval (ISI) and stimulus intensities. Lower stimulus intensities and a shorter ISI lead to strong habituation. The authors proposed that the mechanisms underlying habituation in humans are still unclear; however, psychological adaptation of subjects and also the effects of sensory saturation could be taken into account (see Dincklage et al., 2013).

The experiments performed in this study lasted up to 70 minutes and the same electrical stimuli were repeated at 0.33 Hz throughout. It could thus be argued that changes in inhibitory spinal network excitability were due to a habituation effect, not furosemide. To exclude this hypothesis, we performed control experiments without furosemide in the same healthy subjects. No changes were observed over time for presynaptic inhibition of Ia fibres without furosemide. This finding demonstrates that changes in presynaptic inhibition following furosemide intake in healthy subjects are most likely due to the effects of furosemide. In the case of postsynaptic inhibition, there was a gradual decrease of inhibition over time in control experiments revealing that habituation may involve the function of cutaneous receptors or synapses interposed between afferent fibres and soleus motoneurons. The diminution of postsynaptic inhibition after furosemide administration is more pronounced compared with controls, which is likely caused by furosemide even though the difference was not statistically significant for all epochs. Put together, our results in healthy subjects suggest that furosemide is at least partly responsible for the decrease of the efficacy of pre-and postsynaptic inhibition of soleus motoneurons. This point of view is also supported by the significant dose effect relationship for presynaptic inhibition and by the trend of the dose effect relationship for postsynaptic inhibition.

CHAPTER 4 DISCUSSION : PROJECT 1

Effects of furosemide on spinal inhibitory networks in healthy subjects

Absence of furosemide effect on monosynaptic excitatory transmission

Although furosemide is known from animal experiments to block cationchloride co-transporters, its effects on the human spinal network have never been tested before. The question could be asked as to whether the effects demonstrated for inhibitory synapses are specific to GABAergic and glycinergic synapses, or if they are non-specific, affecting both inhibitory and excitatory synapses. Our results revealed no significant difference observed in post-activation depression at the Ia fibre-motoneuron synapse, a pure excitatory synaptic transmission, between control and furosemide experiments in healthy subjects. These results allow one to exclude the hypothesis that furosemide also acts on excitatory synapses.

Specific effect of furosemide on pre-and postsynaptic inhibitions in healthy subjects

Orally-administered furosemide, at doses commonly used in the clinic (40 mg), significantly reduced spinal inhibitory activity (pre-and postsynaptic inhibitions) for at least 70 minutes from intake compared to control experiments in the same healthy subjects. Although furosemide decreases both pre-and postsynaptic inhibitions in healthy subjects, the effect of furosemide seems to be more important for postsynaptic than presynaptic inhibition. The most likely explanation for the difference between the time courses of pre-and postsynaptic inhibition is that in the control condition (without furosemide), there was no systematic change over time for presynaptic inhibition while the amount of postsynaptic inhibition is progressively decreased over time. This effect of time by itself on postsynaptic inhibition is interpreted above with respect to habituation. Thus, for postsynaptic inhibition, the inhibition following furosemide intake is the sum of the habituation effect and the effect of furosemide itself, while the changes in presynaptic inhibition following furosemide intake are only due to the furosemide effect. Another explanation may be that the kinematics of pre-and postsynaptic inhibitions are different; however, to our knowledge, there is no evidence to support this hypothesis. Altogether with the absence of furosemide action on excitatory synapses, these observations strongly suggest that furosemide selectively decreases the functioning of spinal inhibitory synapses and thus has potential to detect changes in the intrinsic functioning of inhibitory synapses in humans.

CHAPTER 4 DISCUSSION : PROJECT 1

2.2.3

Does furosemide affects all the structure of the CNS?

Our results in healthy subjects show that furosemide induces changes in spinal inhibitory circuits. Since the neurons that compose these networks are controlled by descending (supraspinal) input as well, any change induced by furosemide via changing the efficacy of supraspinal input onto the neurons inhibiting soleus motorneurons preor postsynaptically must also be considered. It has been shown in animals that furosemide decreases IPSPs without modifying EPSPs (Nicoll, 1978), and we also demonstrated here that furosemide decreases the excitability of inhibitory synapses without affecting excitatory synapses. The main supraspinal control input acting on presynaptic inhibition is inhibitory (for references see Pierrot-Deseilligny & Burke, 2012). Thus, at its level, the effect of furosemide is likely, leading to a decrease in the supraspinal inhibitory input acting on presynaptic (PAD) interneurons. This disinhibition of PAD interneurons would likely result in an increase of the amount of presynaptic inhibition. However, we observed here in healthy subjects a reduction of presynaptic inhibition after furosemide intake; this cannot be explained by the act of furosemide on supraspinal control. Logigian et al. (1999) have presented data obtained in human experiments suggesting that the descending control acting on interneurons fed by cutaneous afferents is facilitatory. Since furosemide has no effect on excitatory synapses, the reduction of postsynaptic inhibition following furosemide administration in healthy subjects would not be due to changes in supraspinal control acting on cutaneous interneurons. CHAPTER 4 DISCUSSION : PROJECT 1

Results in SCI patients

3.1

Limitations of the study

Difficulty of recruitment

To be compatible with aninal data, we initially planned to conduct our research only in patients with complete cord injury since down-regulation of KCC2 was reported after complete spinal cord transection in the rat (Boulenguez et al., 2010). However, it was difficult to find patients with complete spinal cord injury. Given that animal experiments showed afterwards that this phenomenon is also present in the case of incomplete spinal cord lesion, we have subsequently requested a research authorization to perform our experiments in both patients with complete and incomplete spinal cord injuries. However, since we received the authorization at the end of 2013, only one patient with incomplete spinal cord injury was recruited in this study.

For the recruitment, 4 of 14 SCI patients were excluded from our study since the amplitude of their soleus H-reflex was too small or disappeared. Modifications of Hreflex amplitude after SCI are still under debate. The H-reflex has been reported to be absent or markedly decreased below the lesion in early human spinal shock and the degree and duration of H-reflex suppression reflects the severity of the injury [START_REF] Leis | Behavior of the Hreflex in humans following mechanical perturbation or injury to rostral spinal cord[END_REF]. This loss of reflex is proposed to be due to reduced excitability of motoneurons caused by the sudden loss of supraspinal inputs. However, the H-reflex starts to rapidly recover after SCI shock [START_REF] Hiersemenzel | From spinal shock to spasticity: Neuronal adaptations to a spinal cord injury[END_REF]. An increase of the soleus H-reflex amplitude in patients with chronic complete cord lesion suggests an increase in central synaptic excitability that may contribute to the appearance of hyperreflexia after SCI [START_REF] Hiersemenzel | From spinal shock to spasticity: Neuronal adaptations to a spinal cord injury[END_REF][START_REF] Little | H-reflex changes following spinal cord injury[END_REF]. However, a study has reported an unchanged of soleus H-reflex threshold, gain and amplitude in chronically complete SCI patients compared to normal subjects [START_REF] Schindler-Ivens | Soleus H-reflex recruitment is not altered in persons with chronic spinal cord injury[END_REF]. Soleus H/M ratio is less than 0.7 in normal subjects [START_REF] Delwaide | Cutaneous nerve stimulation and motoneuronal excitability. II: Evidence for non-segmental influences[END_REF]. In our study, the average soleus H/M ratio found in SCI patients was 0.56±0.08. CHAPTER 4 DISCUSSION : PROJECT 1

Decrease or absence of presynaptic inhibition of Ia fibres and postsynaptic inhibition induced by cutaneous stimulation in SCI patients

Another constraint present in the present study was that pre-and postsynaptic inhibition could not be always be evoked in all included patients. Presynaptic inhibition could be demonstrated in 9 of 10 patients; however, the degree of inhibition observed in these SCI patients is considerably reduced compared to normal subjects, but still detectable. Postsynaptic inhibition induced by cutaneous stimulation could be detected in only 4 of 10 patients. The modifications of postsynaptic inhibition in soleus motoneurons induced by cutaneous stimulation after spinal cord lesion have not been well documented. In our study, we found that this inhibition is extremely reduced after SCI. This unexpected constraint has limited our recordings and the interpretation of results since the sample size of participants is small and the degree of cutaneous inhibition found in this small sample is also small. A recent study has also reported a decrease of flexor reflex response in soleus motoneurons in paraplegic patients during the spastic state following tibial nerve stimulation at the malleolus medialis level. This decrease was stable even with an increase of stimulation intensity [START_REF] Hiersemenzel | From spinal shock to spasticity: Neuronal adaptations to a spinal cord injury[END_REF]. Similarly as we observed here, even with an increase of the conditioning sural stimulus intensity until triggering movement of flexors of the foot, the degree of postsynaptic inhibition in patients was no longer increased. In addition, as this cutaneous inhibition could not easily be demonstrated in SCI patients, another type of postsynaptic inhibition, crossed inhibitory responses in soleus motoneurons [START_REF] Aggelopoulos | Characterization of a descending system that enables crossed group II inhibitory reflex pathways in the cat spinal cord[END_REF][START_REF] Stubbs | Short-latency crossed inhibitory responses in the human soleus muscle[END_REF] was tested in the present study. However, we failed to evoke this crossed inhibition in SCI patients. Alternative possibilities to test postsynaptic inhibition should be either reciprocal inhibition or recurrent inhibition. Ia-reciprocal inhibition has been shown to be reduced [START_REF] Boorman | Impaired "natural reciprocal inhibition" in patients with spasticity due to incomplete spinal cord injury[END_REF] or replaced by reciprocal excitation of antagonistic muscle after SCI [START_REF] Crone | Appearance of reciprocal facilitation of ankle extensors from ankle flexors in patients with stroke or spinal cord injury[END_REF]. Abnormal of recurrent inhibition was reported in SCI patients [START_REF] Shefner | Recurrent inhibition is increased in patients with spinal cord injury[END_REF] and the method used to assess recurrent inhibition in humans is difficult to perform [START_REF] Katz | Recurrent inhibition in humans[END_REF]. Together with these constraints, the study of the effects of furosemide in SCI patients is mainly considered on presynaptic inhibition. CHAPTER 4 DISCUSSION : PROJECT 1

Effects of furosemide on spinal inhibitory networks in SCI patients

The action of furosemide is likely reduced in SCI patients. Changes in presynaptic inhibition of Ia fibres following furosemide were significantly different between healthy subjects and SCI patients. In healthy subjects, the amount of presynaptic inhibition is significantly gradually decreased from baseline at every time epoch after 21-30 minutes following furosemide administration (see Figure 26). In contrast, no significant difference is found for the time course of presynaptic inhibition with furosemide in SCI patients. At the intervals (41-70 minutes) where the most prominent effects were found in normal subjects, the amount of presynaptic inhibition is stable around the baseline in SCI patients (see Figure 37). For postsynaptic inhibition, as the sample size of patients is too small, it is very difficult to interpret the results. However, considering the individual raw data of postsynaptic inhibition, there is a marked decrease of inhibition at 41-70 minutes following furosemide in all individual healthy subjects (see Table 3.), while non-significant changes of postsynaptic inhibition were observed from the 4 patients at this time interval (see Table 4).

To sum up, even if the sample of patients is relatively small, the comparison reveals clearly differences of furosemide action between normal subjects and SCI patients. These differences suggest that the effect of furosemide is likely reduced in SCI patients. However, a larger number of SCI patients is still required to confirm the results.

Cation-chloride co-transporters are regulators of the chloride gradient in neurons. Activation of co-transporter KCC2 (a chloride extruder) during development reduces [CI -] i thus resulting in hyperpolarizing effects of GABA and glycine (Ben Ari, 2002;Takahashi, 1984;Wu et al. 1992;Ziskind-Conhaim, 1998). Several studies in animals have shown that loss or reduction of co-transporter KCC2 function results in the development of central nervous system (CNS) hyper-excitability (Woo et al. 2002;Zhu et al. 2008). In the presence of furosemide, the functioning of inhibitory synapses is markedly decreased in healthy subjects, while there is likely no tendency of decrease in spinal inhibitory synapses in SCI patients. These results lead us to hypothesise that the expression of KCC2 is probably decreased after SCI in humans. This hypothesis is consistent with the data obtained in animals showing that KCC2 co-transporter is downregulated in motoneurons after spinal cord transection [START_REF] Lu | Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with down-regulation of the chloride transporter KCC2 in rat[END_REF]Boulenguez et al., 2010;Modol et al., 2014). A down-regulation of KCC2 induces an accumulation of [Cl -] i that reverses GABA/glycine response polarity to excitation [START_REF] Nabekura | Reduction of KCC2 expression and GABAA receptormediated excitation after in vivo axonal injury[END_REF]Vinay & Jean-Xavier, 2008) and thus results in reduction of inhibitory synaptic efficiency (Boulenguez et al., 2010). To sum up, the preliminary results observed in this study suggest that the intrinsic functioning of inhibitory synapses is reduced in SCI patients.

CHAPTER 4 DISCUSSION : PROJECT 1

Applications of physiopathology related to results obtained in SCI patients

Presynaptic inhibition of Ia fibres is decreased in lower limbs in patients with spinal cord lesions, whatever the nature of the lesion [START_REF] Faist | A quantitative assessment of presynaptic inhibition of Ia afferents in spastics. Differences in hemiplegics and paraplegics[END_REF][START_REF] Nielsen | Changes in transmission across synapses of Ia afferents in spastic patients[END_REF][START_REF] Pierrot-Deseilligny | Electrophysiological assessment of the spinal mechanisms underlying spasticity[END_REF]. As the main descending controls on PAD interneurons mediating presynaptic inhibition of Ia fibres is depressive, the interruption of the corticospinal tract could not explain the reduction of the presynaptic inhibition of Ia fibres in the lower limb in patients with spinal cord lesions (if anything, it would produce an increase of presynaptic inhibition). Thus, the interruption of other descending pathways that normally maintain a tonic level of presynaptic inhibition of Ia fibres under resting conditions in normal subjects has been proposed. The inhibitory action of the cutaneous and articular inputs on first-order PAD interneurons is subjected to a powerful tonic inhibition (see pathway (1) in the Figure 51) via a brainstem pathway that disappears after spinal cord transection in the cat (Rudomin & Schmidt, 1999). Interruption of this strong tonic inhibitory control from the brainstem would consequently liberate the cutaneous and articular inputs to fully exercise their inhibitory actions. This explanation relies on the hypothesis that cutaneous and articular inputs are still generating inhibition in PAD interneurons after spinal cord lesion. However, as observed in our study, cutaneous inhibition to motoneurons is strongly reduced or abolished, and it can be hypothesised that the immobilization of a limb following complete paraplegia reduces the effects of proprioceptive fibres [START_REF] Lundbye-Jensen | Immobilization induces changes in presynaptic control of group Ia afferents in healthy humans[END_REF]. Therefore, if a decrease in efficacy of cutaneous and proprioceptive fibres is also acting at the PAD interneuron level, one can wonder if the interruption of the supraspinal tonic inhibition really reduces presynaptic inhibition. In such condition, the reduction of intrinsic functioning of inhibitory synapses found here would be a possible explanation for the decrease of presynaptic inhibition in SCI patients. This hypothesis is supported by animal studies suggesting that loss or decrease of KCC2 expression would be a cause of reduced inhibitory neuronal activity after SCI which might also contributes to spasticity (Boulenguez et al., 2010). Spasticity is a frequent complication found in patients with spinal cord lesions. [START_REF] Lance | The control of muscle tone, reflexes, and movement: Robert Wartenberg Lecture[END_REF] has stated that "spasticity is a motor disorder characterized by a velocitydependent increase in tonic stretch reflexes (muscle tone) with exaggerated tendon jerk, resulting from hyperexcitability of the stretch reflex, as one component of the upper motoneuron syndrome". Transmission is altered in all studied spinal pathways that have been investigated in patients with spinal cord lesions, always in the direction that would exaggerate the stretch reflex (for reference see Pierrot-Deseilligny & Burke, 2012). Our results are in accordance with this evidence since pre-and postsynaptic inhibition is decreased in SCI patients and this decrease likely contributes to the exaggeration of the stretch reflex. The most general mechanism that have been proposed underlying spasticity is that supraspinal inhibitory and excitatory inputs that exert a balanced control on spinal reflex activity in normal condition are interrupted after CNS lesion (Reviewed in [START_REF] Adams | Spasticity after spinal cord injury[END_REF][START_REF] Sheean | The pathophysiology of spasticity[END_REF]. However, to date, the indirect methods used in humans do not allow distinguishing of between changes in supraspinal control acting on neuron and changes in the intrinsic functioning of the synapse itself.

A previous study showed that soleus motoneurons in patients with spinal cord lesion were inhibited by high-intensity cutaneous stimuli of the sural nerve over the same ISI range as normal subjects, but the degree of inhibition is less than in normal subjects (Logigian et al, 1999). This suggests that there is likely a descending CHAPTER 4 DISCUSSION : PROJECT 1 supraspinal control that has a net facilitory effect on this inhibitory effect of cutaneous stimulation on motoneuron excitability. The interruption of descending control would consequently result in net reduction of the cutaneous inhibition in SCI patients. In addition, the reduction of the intrinsic functioning of the inhibitory synapse suggested in our study would likely also contribute to the reduction of postsynaptic inhibition induced by cutaneous stimulation in SCI patients.

Clinical implication

Furosemide decreases spinal inhibitory synapse excitability in healthy subjects. The question may arise whether prolonged intake of furosemide would affect daily life or physical activity of people. In patients who have spasticity (i.e. SCI patients) the prolonged treatment of furosemide probably requires careful observation since furosemide could reduce the functioning of inhibitory synapses which may results in an increment of spasticity. However, exaggerated reflexes or increase in muscle tone have never been reported as a side effect of furosemide. No systematic study of exaggerated reflexes and muscle tone has been performed during prolonged furosemide treatment. However, none of the healthy volunteers who participated in our experiments reported exaggerated reflexes and changes in muscle tone following a single dose of furosemide administration. We may thus assume that in people with normal functioning of inhibitory synapses, the effects of furosemide have no clinical consequences. However, it would be interesting to further explore the possible muscle tone changes following prolonged furosemide administration, especially in patients with spasticity.

CHAPTER 4 DISCUSSION : PROJECT 1

Therapeutic implication

Our observations provided indirect evidence in humans that the KCC2 is probably down-regulated in SCI patients as demonstrated in the spinal rat. Given that KCC2 plays an important role in regulating the strength of inhibitory synapses in SCI (Boulenguez et al., 2010), it is interesting for the therapy if increases of KCC2 expression would help SCI patients to reduce spasticity. A recent study has demonstrated that restoring chloride homeostasis by activation of the serotonin receptor in spinal motoneurons after SCI (hyperpolarizing the reversal potential of inhibitory postsynaptic potentials (IPSPs), E IPSP ) induces up-regulation of KCC2. This results in the restoration endogenous inhibition in the neonatal spinal rat [START_REF] Bos | Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2[END_REF]. The authors have proposed that up-regulation of KCC2 function has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. This would be a novel therapeutic strategy for spasticity in SCI patients.

Conclusion

Our results suggest that furosemide, a safe and widely used diuretic, at doses commonly used in the clinic, has the potential to test the intrinsic functioning of inhibitory synapses in humans. The difference in furosemide effects on spinal inhibitory synapse excitability in healthy subjects and SCI patients favours the hypothesis of a decrease in inhibitory neuronal activity induced by down-regulation of KCC2 after SCI in humans that likely contributes to spasticity.

CHAPTER 4 DISCUSSION : PROJECT 2

Project 2

The main finding of the present study is that contralateral anodal tDCS induces no change in reciprocal inhibition at the wrist level in the non-dominant upper limb of healthy subjects, while an increase of reciprocal inhibition was reported in their dominant-limb (Roche et al., 2009). Ipsilateral anodal tDCS induces no change in reciprocal inhibition in the dominant upper limb of right-handed healthy subjects. Even though there is a small decrease of inhibition in left-handed subjects, no significant difference between the two groups was observed. Preliminary results in hemiplegic patients show no changes in reciprocal inhibition of the paretic upper limb during ipsilateral anodal tDCS of non-lesioned hemisphere in both active and sham conditions. In contralateral study, anodal tDCS of the unaffected motor cortex dramatically decreases reciprocal inhibition in the non-paretic upper limb.

In addition, the degree of reciprocal inhibition at baseline (before tDCS) is reduced in the paretic upper limb in stroke patients compared to healthy subjects. In the non-paretic upper limb, the degree of reciprocal inhibition is not different from healthy subjects. However, statistical analysis did not reveal any difference between the paretic and the non-paretic upper limbs. In normal subjects, there is no difference of the degree of reciprocal inhibition between the dominant and the non-dominant upper limbs.

Methodological considerations Transcranial direct current stimulation

Repetitive TMS and tDCS are two tools currently available to non-invasively modify excitability of the motor cortex (Priori et al., 2009). Apart from its simplicity, the major argument in favour of tDCS over rTMS is that rTMS produces effects on cortical excitability that may occur not only at the stimulation site but also at distant connected sites. For example, Wassermann et al. (1998) showed that 1 Hz rTMS over the primary motor cortex (M1) changes interhemispheric and corticospinal excitability in the contralateral homologous M1 as the authors found a small decrease in the MEPs indicating a reduced cortical excitability in the contralateral homologous cortex. By contrast, tDCS of the left M1 can facilitate or suppress MEPs triggered from the same hemisphere depending on the direction of current flow but has no effect on MEPs evoked from the opposite M1 (Lang et al., 2004). Thus, despite the fact that tDCS is applied through large electrodes and thus, is less focal, we assume that it is more suitable for the comparison of the effects of ipsi-and contralateral tDCS. However, to distinguish or selectively activate the muscle area target is not possible since the size of the active electrode applied over the motor cortex is large (35 cm 2 ), the cortical areas of agonist muscle (FCR) and antagonist muscle (ECR) were thus simultaneously stimulated. The second argument in favour of tDCS is related to sham stimulation. TMS produces a loud click when each stimulus is delivered and electric current is induced in the scalp as well as in the brain, leading to local activation of sensory nerves and muscles which is readily perceived by subjects. Moreover, even if the click is indistinguishable between real and sham TMS, the lack of induced electric current means that there is no accompanying scalp sensation. In contrast with tDCS, subjects only feel the current intensity variation, so they can be entirely unaware of the difference between real and sham stimulation (Priori et al., 2009), even though here, for simplicity's sake the experimenter was not blind to the intervention.

Since Roche and colleagues ( 2009) have demonstrated the effect of contralateral anodal tDCS applied over the motor cortex on reciprocal inhibition in healthy subjects, this opens up the possibility of using tDCS as a tool to investigate changes in supraspinal descending control on spinal circuitry in humans. Nowadays, tDCS has begun to be used as a rehabilitation tool in patients with CNS lesions. However, knowledge of tDCS effects on spinal networks in normal subjects and patients with CNS lesions is still required.

CHAPTER 4 DISCUSSION : PROJECT 2

Effects of anodal tDCS in healthy subjects

2.1

Contralateral study Roche et al. (2009) previously demonstrated in our laboratory that anodal tDCS over the contralateral cortex induced an increase of reciprocal inhibition between forearm muscles in the dominant upper limb of healthy subjects. The authors proposed that tDCS acts by modifying the rate of spontaneous discharge of M1 neurons and thus induces an increase in the efficiency of the descending control on spinal neurons involved in reciprocal inhibition. Our present findings, using the same protocol as Roche's experiments, show that anodal tDCS induces no change in reciprocal inhibition of non-dominant upper limb. The results from an individual healthy subject who participated in the study of Roche et al., (2009) and in the present study also confirm the difference of reciprocal inhibition between dominant and non-dominant upper limbs during tDCS application.

Interhemispheric transcallosal interactions between both homologous cortical areas have been studied in humans using paired-pulse TMS [START_REF] Ferbert | Interhemispheric inhibition of the human motor cortex[END_REF][START_REF] Hanajima | Interhemispheric facilitation of the hand motor area in humans[END_REF]. These studies show that stimulation of one hemisphere can induce facilitatory, but mostly inhibitory effects in the opposite hemisphere known as inter-hemispheric inhibition (IHI) [START_REF] Ferbert | Interhemispheric inhibition of the human motor cortex[END_REF]. Recording of MEPs of the first dorsal interosseous muscle elicited by TMS in right-handed subjects showed that the IHI originating in the "dominant" left hemisphere is more marked than after stimulation of the 'non-dominant" right hemisphere. In contrast, left-handed subjects show inhomogeneous findings with either right-or left-sided predominant IHI. It was proposed that not handedness, but hemispheric dominance contributes to the laterality of inhibition between the two hemispheres [START_REF] Netz | Hemispheric asymmetry of transcallosal inhibition in man[END_REF]. Other studies have also reported similarly that the IHI between motor cortices is asymmetric, with prominent IHI projections originating in the "dominant" left hemisphere (Baümer et al., 2007;[START_REF] Gorsler | Interhemispheric effects of high and low frequency rTMS in healthy humans[END_REF][START_REF] Giovannelli | Modulation of interhemispheric inhibition by volitional motor activity: an ipsilateral silent period study[END_REF]Wassermann et al., 1998;Ziemaan & Hallett, 2001). As in primates, interhemispheric interaction in human has been considered to be mediated by transcallosal connections [START_REF] Boroojerdi | Transcallosal inhibition in cortical and subcortical cerebral vascular lesions[END_REF][START_REF] Boroojerdi | Interhemispheric inhibition in patients with multiple sclerosis[END_REF][START_REF] Meyer | Inhibitory and excitatory interhemispheric transfers between motor cortical areas in normal humans and patients with abnormalities of the corpus callosum[END_REF]. There is some evidence from developmental studies showing that the transcallosal connection of the hand muscle area plays a role in execution of unilateral movement. The mirror movements of the hand are common in children, who have an immature transcallosal connection, but these movements are rarely seen after transocallosal fibres are completely myelinated (Connonlly & Stratton, 1968;[START_REF] Danek | Cortically evoked motor responses in patients with Xp22.3-linked Kallmann's syndrome and in female gene carriers[END_REF][START_REF] Stern | In Towards a More Refined Analysis of the "Overflow" or "Associated Movement[END_REF]. This transcallosal connection enables healthy adults to perform strictly unilateral tasks even though some mirror movements can be observed in the non-dominant hand if the task is complex (Ziemaan & Hallett, 2008). It was CHAPTER 4 DISCUSSION : PROJECT 2 proposed that the increase in IHI originating from the active M1 to the M1 of the opposite hemisphere is responsible for the inhibition of undesired mirror movement [START_REF] Hübers | Interhemispheric motor inhibition: its role in controlling electromyographic mirror activity[END_REF]. Transcallosal connections between motor cortices have been shown to contribute to control of the corticospinal output from the M1 on forearm muscles (i.e. flexor carpi radialis) [START_REF] Perez | Mechanisms underlying functional changes in the primary motor cortex ipsilateral to an active hand[END_REF]. Note that all subjects that participated in our contralateral study and in study of Roche et al., (2009) were righthanded subjects. Together with this evidence, it can probably be assumed that asymmetrical IHI with stronger inhibition from "dominant" left to "non-dominant" right hemisphere plays a role in the difference of reciprocal inhibition in dominant and nondominant upper limbs in right-handed subjects. Another explanation would be that the sensitivity of the two hemispheres to tDCS is not similar. However, the motor threshold for induction of contralateral MEPs in hand muscles was reported to be the same in both hemispheres [START_REF] Kobayashi | Ipsilateral motor cortex activation on functional magnetic resonance imaging during unilateral hand movements is related to interhemispheric interactions[END_REF]. The last possible explanation would be the excitability of corticospinal control on reciprocal inhibition is different between nondominant and dominant limbs. If this is the case, the degree of inhibition should be different in the both sides. However, the degree of reciprocal inhibition observed at baseline (before tDCS) in this study is similar between dominant and non-dominant upper limbs (see Figure 46). Overall, the hypothesis of asymmetrical IHI seems to mostly favour our results of asymmetrical control on reciprocal inhibition in righthanded subjects.

Ipsilateral study

The objective of an ipsilateral study in healthy subjects is to compare the results with stroke patients. Our results revealed no changes of reciprocal inhibition in the dominant upper limb of right-handed subjects during ipsilateral anodal tDCS over the motor cortex. An anatomical study suggests that ipsilateral control from hand primary motor cortex may be different in left-handed and right-handed subjects (Hervé et al. 2005). The volume of transcallosal in left-handers was found to be larger compared to right-handers [START_REF] Clarke | Anatomical-behavioral relationships: corpus callosum morphometry and hemispheric specialization[END_REF][START_REF] Luders | Relationships between sulcal asymmetries and corpus callosum size: gender and handedness effects[END_REF][START_REF] Preuss | Handedness and corpus callosum morphology[END_REF] suggesting an association between handedness and inter-hemispheric connectivity. To our knowledge, there is no evidence for the difference of ipsilateral corticospinal control between left-handers and right-handers. We thus explored the ipsilateral effects of tDCS on reciprocal inhibition in left-handed subjects. The comparison between the two different groups of handedness revealed no significant difference of results. If anything, it seems likely that there is a small decrease of reciprocal inhibition in left-handed subjects compared to right-handed subjects. However, the sample of left-handed subjects was small compared to right-handers (8 left-handers vs. 24 right-handers).

CHAPTER 4 DISCUSSION : PROJECT 2

The existence of uncrossed corticospinal tracts from the motor cortex to spinal motoneurons has been established in healthy humans and higher primates. It contains only 8-10% of the pyramidal tract [START_REF] Kuypers | Anatomy of the descending pathways[END_REF], and the majority of them finally cross at the segmental spinal cord level [START_REF] Porter | Corticospinal function and voluntary movement[END_REF]. However, such ipsilateral corticospinal fibres preferentially reach proximal rather than distal muscles [START_REF] Colebatch | The distribution of muscular weakness in upper motor neuron lesions affecting the arm[END_REF]. The ipsilateral motor projection to distal upper limb muscles in normal subjects is still under debate. At rest, ipsilateral MEPs evoked by TMS in distal muscles (i.e. the forearm and hand muscles) are rarely found in healthy subjects (Bawa et al., 2004;[START_REF] Carr | Evidence for bilateral innervation of certain homologous motoneurone pools in man[END_REF]Wassermann et al. 1991). A few studies have reported ipsilateral responses in distal muscles in healthy subjects, but under strong voluntary contractions and high intensity TMS (Wassermann et al. 1991;Ziemann et al. 1999). However, the ipsilateral responses in distal muscles are usually small and inconstant (Alagona et al., 2001). In functional magnetic resonance imaging (fMRI) studies, ipsilateral activation of the primary motor area can be observed during unilateral motor tasks in some healthy subjects, but not all of them [START_REF] Allison | Functional MRI cerebral activation and deactivation during finger movement[END_REF][START_REF] Cramer | Activation of distinct motor cortex regions during ipsilateral and contralateral finger movements[END_REF][START_REF] Rao | Functional magnetic resonance imaging of complex human movements[END_REF][START_REF] Singh | Functional MR imaging of cortical activation of the cerebral hemispheres during motor tasks[END_REF]. Such ipsilateral activation is more frequently observed with the non-dominant hand [START_REF] Kobayashi | Ipsilateral motor cortex activation on functional magnetic resonance imaging during unilateral hand movements is related to interhemispheric interactions[END_REF]. Increasing the excitability of the ipsilateral motor cortex has no effect on reciprocal inhibition particular right-handed subjects. This can probably be explained by the few ipsilateral motor projections to spinal motoneurons or interneurons of distal muscles in the upper limbs.

Effects of anodal tDCS in stroke patients

Degree of reciprocal inhibition in stroke patients (before tDCS)

The degree of reciprocal inhibition of the paretic upper limb evaluated in 9 stroke patients is significantly reduced compared to the dominant and non-dominant upper limb of normal subjects (see Figure 45). These results are consistent with studies of Artieda et al., 1999;Nakashima et al., 1989 and[START_REF] Raoul | Etude de la cartographie des circuits reflexes médullaire à point de départ proprioceptif au repos et au cours de contractions volontaires chez les sujets sains et les malades[END_REF]. The decrease in reciprocal inhibition is frequently found in patients with spasticity, but not in patients with muscle tone or flaccic hemiplegia (Nakashima et al., 1989). The most likely explanation of this phenomenon is that the excitability of inhibitory interneurons is reduced as a result of cortical lesion. Reduced activity of reciprocal inhibition interneurons could be partly responsible for motor deficits including abnormalities of contraction of antagonist muscles in these patients. The hypothesis is supported by the fact that in normal subjects anodal tDCS does not modify presynaptic inhibition of Ia fibres (Roche et al., 2009).

CHAPTER 4 DISCUSSION : PROJECT 2

Ipsilateral study

Ipsilateral corticospinal control on reciprocal inhibition at the wrist level was tested by anodal tDCS of unaffected motor cortex in 6 patients with unilateral stroke. No significant difference is found between tDCS and sham groups in these patients as observed in normal subjects.

Some TMS studies report that MEPs in paretic hand muscles of stroke patients can be obtained more frequently than in healthy subjects by TMS of the ipsilateral, unaffected motor cortex [START_REF] Netz | Reorganization of motor output in the nonaffected hemisphere after stroke[END_REF][START_REF] Trompetto | Motor recovery following stroke: a transcranial magnetic stimulation study[END_REF], with some characteristics that differ from those observed in normal subjects (i.e. shorter latency) (Alagona et al., 2001). However, it has been reported that ipsilateral MEPs in the paretic hand are inconsistently found [START_REF] Bastings | Hand Motor Recovery after Stroke: A Transcranial Magnetic Stimulation Mapping Study of Motor Output Areas and Their Relation to Functional Status[END_REF][START_REF] Caramia | Ipsilateral activation of the un affected motor cortex in patients with hemiparetic stroke[END_REF], and that they were more often observed in patients with poor recovery than with good recovery [START_REF] Netz | Reorganization of motor output in the nonaffected hemisphere after stroke[END_REF][START_REF] Turton | Contralateral and ipsilateral EMG responses to transcranial magnetic stimulation during recovery of arm and hand function after stroke[END_REF]. Here, we observed no changes in reciprocal inhibition in paretic limb during ipsilateral tDCS of unaffected motor cortex. A possible hypothesis would be that the unilateral stoke does not induce much changes in the ipsilateral projections to the distal muscles in stroke patients. However, our sample of patients is small. A larger number of patients is required to confirm the results.

Contralateral study

Anodal tDCS of the unaffected motor cortex induces a strong decrease in reciprocal inhibition in the non-paretic upper limb. All hemiparetic patients (n=5) participating in this contralateral study are right-handed subjects (3 right hemiplegia, 2 left hemiplegia). The amounts of reciprocal inhibition in the unaffected side of patients are decreased during contralateral anodal tDCS in all cases (dominant and nondominant side). In all hemiparetic subjects, the clinical disability was presented only in affected side. Nevertheless, a marked depression of reciprocal inhibition during contralateral anodal tDCS was seen in the unaffected side in 4 of 5 patients.

After unilateral stroke, clinical symptoms are visible in the affected side. However, abnormal transmission in some spinal pathways in the upper limb has been described in the unaffected side. For example, presynaptic inhibition of Ia fibres in the upper limb is reduced in the non-paretic upper limb, though to a less extent than on the affected side (Aymard et al., 2000;[START_REF] Lamy | Impaired efficacy of spinal presynaptic mechanisms in spastic stroke patients[END_REF]. It was shown in the paretic side of hemiparetic patients that the response to stretch in biceps muscle is increased and that in triceps muscle is diminished compared to normal subjects. In contrast, the opposite results are found in the unaffected side, decreased response in biceps muscle and increase the response in triceps muscle that not seen in normal subjects [START_REF] Thilmann | Pathological stretch reflexes on the "good" side of hemiparetic patients[END_REF]. The muscle strength is also reduced on the unaffected side compared to normal CHAPTER 4 DISCUSSION : PROJECT 2 subjects; 21% decrease of the muscle strength was reported for the upper limb muscles (reviewed in [START_REF] Gandevia | Strength Changes in Hemiparesis: Measurements and Mechanisms[END_REF]. The preliminary results observed here are likely in accordance with these findings indicating that the non-affected upper limb of stroke patients is not similar to the normal upper limbs of healthy subjects.

To discuss the results in terms of reciprocal inhibition response to contralateral tDCS in normal subjects would be unsuitable to clarify our findings since contralateral anodal tDCS induces either increase of reciprocal inhibition in the dominant limb or no change in the non-dominant upper limb, while the inhibition in the unaffected side is decreased in the both cases (dominant and non-dominant). Moreover, the degree of reciprocal inhibition in the non-paretic upper limb before tDCS is not different from normal subjects. The changes that may occur at cortical level would also be unlikely to explain the results. Indeed, it was reported that the excitability of unaffected hemisphere is increased after unilateral stroke (Leipert et al., 2000;[START_REF] Shimizu | Motor cortical disinhibition in the unaffected hemisphere after unilateral cortical stroke[END_REF]. Anodal tDCS effect is known to increase cortical excitability. If anything, anodal tDCS of the unaffected motor area would produce a strong, even stronger increase of reciprocal inhibition compared to the results in normal subjects.

To date, there is no evident explanation for this finding and this result is still required to be confirmed with a larger number of patients.

Clinical implication

Contralateral anodal tDCS can induce an increase of reciprocal inhibition in the dominant upper limb, but not in the non-dominant upper limb in healthy subjects. As it is known, and we also found in the present study that the reciprocal inhibition between forearm muscles is reduced in paretic hand of stroke patients. Anodal tDCS could probably be used as an intervention to enhance reciprocal inhibition in the paretic upper limb. However, ipsilateral tDCS of the non-lesioned hemisphere seems to have no influence on reciprocal inhibition in the paretic upper limb. If we use contralateral tDCS of the lesioned hemisphere, it should be noted that this would probably work only when the paretic side is the dominant limb.

Studies in stoke patients have reported an increase in the excitability of unaffected hemisphere (Leipert et al., 2000;[START_REF] Shimizu | Motor cortical disinhibition in the unaffected hemisphere after unilateral cortical stroke[END_REF] and an abnormally high IHI drive from intact to lesioned hemisphere during execution of voluntary movement by the paretic hand [START_REF] Murase | Influence of interhemispheric interactions on motor function in chronic stroke[END_REF]. To reduce the inhibition exerted by the unaffected hemisphere on the affected hemisphere and restore the normal balance of IHI, dual-hemisphere tDCS (upregulated excitability in M1 of affected hemisphere by anodal and downregulated excitability in the intact hemisphere M1 by cathodal) has CHAPTER 4 DISCUSSION : PROJECT 2 been described to enhance motor learning in healthy subjects [START_REF] Vines | Dual-hemisphere tDCS facilitates greater improvements for healthy subjects' non-dominant hand compared to unihemisphere stimulation[END_REF] and to facilitate motor recovery in stroke patients (Bolognini et al., 2011;[START_REF] Vandermeeren | Could dual-hemisphere transcranial direct current stimulation (tDCS) reduce spasticity after stroke?[END_REF][START_REF] Lefebvre | Single session of dual-tDCS transiently improves precision grip and dexterity of the paretic hand after stroke[END_REF].Our results suggest that during increased cortical excitability by anodal tDCS of the unaffected hemisphere, a decrease of reciprocal inhibition in the unaffected upper limb is observed. The question may arise that what cathodal tDCS of the unaffected hemisphere would do on reciprocal inhibition in the non-paretic upper limb? However, no study has reported changes in non-paretic limb following dual or unilateral cathodal tDCS of the non-affected motor cortex (reviewed in Ayache et al., 2012). Nevertheless, evaluating changes in the non-paretic upper limb following the intervention would be needed.

Conclusion

It appears that interhemisphere inhibition between motor areas exerted via transcallosal connections plays an important role in the lateralization of neural activity during execution of unimanual movement in healthy subjects [START_REF] Hübers | Interhemispheric motor inhibition: its role in controlling electromyographic mirror activity[END_REF]Ziemaan & Hallett, 2001). Several studies reveal stronger drive of IHI from the dominant to non-dominant hemisphere in normal subjects (especially in right-handed subjects: Baümer et al., 2007;[START_REF] Gorsler | Interhemispheric effects of high and low frequency rTMS in healthy humans[END_REF]Giovanneli et al., 2009;[START_REF] Netz | Hemispheric asymmetry of transcallosal inhibition in man[END_REF]Wassermann et al., 1998). Asymmetrical reciprocal inhibition control on the both sides of upper limb observed in right-handed subjects is likely related to asymmetrical IHI drive. The constraint-induced movement therapy (CIMT) is an intervention often used to improve affected upper limb function in patients with unilateral hemiparesis under the concept of decreasing activity of one hemisphere to release activity of the opposite hemisphere. Recently, it was suggested in healthy subjects that unilateral motor training of the non-dominant hand and contralateral dominant hand restraint (CIMT's concept) induce a decrease of cortical excitability in the dominant hemisphere and decrease in IHI from dominant to non-dominant hemisphere, thus improving performance in the non-dominant hand [START_REF] Williams | Interhemispheric modulation induced by cortical stimulation and motor training[END_REF]. Reciprocal inhibition is important in the process of movement (i.e. stabilizing joint, allowing agonist and antagonist muscles to work in synchrony for smooth movement). Using this type of training to increase reciprocal inhibition in the non-dominant upper limb would be of benefit for performing movements of the non-dominant upper limb in normal subjects.

The results of ipsilateral tDCS in left-handed subjects suggest that they might be different from those observed in right-handers. It would be interesting to confirm this result by increasing the numbers of subjects. This would provide a better understanding of the role of corticospinal control in handedness.

It is still a matter of debate in TMS studies whether ipsilateral activation exists in stroke patients. To our knowledge, the effects of ipsilateral anodal tDCS on spinal networks in stroke patients have not yet been well documented (see Ayache et al., 2012). An increase in the number of stoke patients in our ipsilateral study is still required for further investigation. Changing some parameters of tDCS in the study in patients (i.e. increased intensity, reduced electrode size) would also be interesting for further research in motor recovery.

Finally, our results suggest that the unaffected upper limb does not response to contralateral anodal tDCS with the 'normal' pattern. A larger number of patients is still required to confirm our results and since there is only little knowledge about unaffected side, more research on unaffected side of patients with hemiparesis should be required too. However, the hypothesis that there are also some changes on the 'unaffected side' after unilateral stroke is also supported by our study. Figure 24 : Time course of presynaptic inhibition without furosemide in healthy subjects.

Figure 25 : Time course of postsynaptic inhibition without furosemide in healthy subjects.

Figure 26 : Time course of presynaptic inhibition with furosemide in healthy subjects.

Figure 27 : Time course of presynaptic inhibition with and without furosemide in a healthy individual subject.

Figure 28 : Histogram of time course of presynaptic inhibition with and without furosemide in all healthy subjects.

Figure 29 : Time course of postsynaptic inhibition with furosemide in healthy subjects.

Figure 30 : Time course of postsynaptic inhibition with and without furosemide in a healthy individual subject.

Figure 31 : Histogram of time course of postsynaptic inhibition with and without furosemide in all healthy subjects.

Figure 32 : Effects of two different doses of furosemide on presynaptic inhibition in healthy subjects.

Figure 33 : Effects of two different doses of furosemide on postsynaptic inhibition in healthy subjects.

Figure 34 : Histogram of time course of monosynaptic excitatory transmission (post-activation depression) with and without furosemide in all healthy subjects.

Figure 35 : The average degree of presynaptic inhibition evaluated before drug administration (baseline) in healthy subjects and SCI patients.

Figure 36 : The average degree of postsynaptic inhibition evaluated before drug administration (baseline) in healthy subjects and SCI patients.

Figure 37 : Time course of presynaptic inhibition with furosemide in SCI patients.

Figure 38 : Histogram of time course of presynaptic inhibition with and without furosemide in all healthy subjects and SCI patients. Figure 46 : The degree of reciprocal inhibition in healthy subjects and stroke patients.

Figure 47 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.

Figure 48 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.

Figure 49 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.

Figure 50 : Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.

Figure 51 Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

List of tables

VS vestibulospinal pathway

Ma thèse est consacrée à l'étude des réseaux neuronaux spinaux impliqués dans la motricité chez l'Homme est comprend deux chapitres.

1. Modifications du fonctionnement des réseaux neuronaux inhibiteurs de la moelle épinière, induites par un diurétique (furosémide) chez l'Homme Des travaux récents effectués sur la moelle épinière du rat ont mis en évidence qu'au cours du développement chez les mammifères, les synapses GABAergiques et glycinergiques sont tout d'abord excitatrices avant de devenir inhibitrices et qu'une section de la moelle épinière ne permet pas cette transformation. Cette transition développementale semble due à l'action d'un transporteur transmembranaire (KCC2) au cours de développement qui diminue après section de la moelle épinière. La diminution de l'expression du KCC2 dépolarise l'action du GABA et de la glycine, ce qui conduit donc à une réduction de l'efficacité de synapse inhibitrice. Le but de ce projet est d'explorer si chez l'Homme une section traumatique de la moelle épinière qui prive les neurones inhibiteurs de leur contrôle suprasegmentaire a pour conséquence de modifier leur comportement synaptique, voire de les ramener à un fonctionnement « immature », c'est-à-dire de transformer des synapses inhibitrices en synapses facilitatrices. Pour tester cette hypothèse, nous avons étudié l'effet sur des synapses inhibitrices de la moelle épinière d'une prise per os de furosémide, un antagoniste de KCC2, et comparé ses effets chez des sujets sains et chez des patients porteurs d'une section de la moelle épinière. L'étude sur les sujets sains suggère que le furosémide (40 mg) a pour effet une réduction du fonctionnement des synapses inhibitrices. Cet effet du furosémide sur les synapses inhibitrices semble être réduit chez des patients. Les résultats obtenus chez les sujets sains indiquent que furosémide administré per os à des dose largement utilisé en clinique humain modifie sélectivement le fonctionnement des synapses inhibitrices et permet donc de disposer d'un mesure non-invasive de fonctionnement intrinsèque de la synapse inhibitrice. Les résultats préliminaires obtenus chez les patients porteurs d'une section de la moelle épinière suggèrent une réduction de l'efficacité de synapses inhibitrices qui probablement contribue à la spasticité.

Contrôle ipsi et contralatéral de l'inhibition réciproque au niveau du poignet chez des

sujets sains et des patients atteints d'accident vasculaire cérébral (AVC) démontré par la stimulation transcrânienne de courant continu La stimulation électrique transcrânienne de courant continu encore appelée « transcranial direct current stimulation (tDCS) » par les anglo-saxons, a connu un essor considérable et constitue aujourd'hui une technique de référence pour moduler l'excitabilité du cortex chez l'Homme. En 2009, Roche et al. ont montré dans notre laboratoire, que la tDCS anodale appliquée sur l'hémisphère contralateral pouvait également modifier l'excitabilité des réseaux neuronaux spinaux (i.e. l'inhibition réciproque au niveau du poignet) enregistrée sur le côté dominant chez les sujets sains. L'existence de projection corticale ipsilatérale sur les réseaux neuronaux spinaux de la moelle épinière et leurs éventuelles modifications après lésion cortico-sous-corticale reste très controversée. Dans ce projet, nous avons testé les effets de la tDCS ipsi-et contralarérale du cortex non-lésé sur l'inhibition réciproque chez des patients AVC. La tDCS ipsilatérale n'induit pas de modifications de l'inhibition réciproque chez les sujets sains. Des résultats similaires enregistrés sur le membre supérieur lésé ont été observés chez des patients AVC, mais ces résultats mériteraient d'être confortés avec un plus grand nombre de sujets. La tDCS contralatérale chez les sujets sains n'induit pas de modifications de l'inhibition réciproque enregistrées sur le membre supérieur nondominant. Ce résultat est différent de celui observé sur le membre supérieur dominant par Roche et al. (2009). Ce contrôle asymétrique sur l'inhibition réciproque est argument en faveur de l'hypothèse que l'inhibition inter-hémisphérique (IHI) entre les deux cortex moteurs est asymétrique. L'IHI à partir de l'hémisphère «dominant» est probablement plus importante. Les effets de la tDCS contralatérale sur l'inhibition réciproque du membre supérieur non-lésé est différent des résultats observés chez des sujets sains, indiquant que la commande motrice contralatérale sur le membre supérieur non-lésé n'est pas indemne. Néanmoins, un plus grand nombre de patients est encore nécessaire pour confirmer le résultat.

Mots clés : réseaux neuronaux spinaux, synapses inhibitrices, furosémide, tDCS, sujets sains, patients blessé médullaire, patient hémiplégique.

My thesis is devoted to the study of the spinal circuitry involved in motor functions using non-invasive electrophysiological methods in humans. It comprises two research projects.

Changes in spinal inhibitory networks induced by a diuretic (furosemide) in humans

Studies in animals have shown that during neural development, GABAergic and glycinergic neurons are first excitatory, and then become inhibitory during maturation. This developmental transition is mainly due to the activation of co-transporter KCC2 at the mature state. A downregulation of KCC2 was reported after spinal cord transection in the rat that leads to the depolarising (excitatory) action of GABA and glycine and thus results in a reduction of inhibitory synaptic efficiency. The aim of this project was to explore if spinal cord injury (SCI) in human reverses the pattern of GABAergic and glycinergic neurons back towards the immature state (primarily excitatory). To test this hypothesis, we studied the effects of furosemide (a KCC2 antagonist) on spinal inhibitory synaptic function, and compared the results obtained in healthy subjects and SCI patients. Results in healthy subjects suggest that furosemide (40 mg, orally-administrated) induces a reduction of inhibitory synapse functions. This effect of furosemide on inhibitory synapses seems to be reduced in SCI patients. Our results suggest that furosemide has the potential to test functions of inhibitory synapses in humans. The difference of furosemide effects on spinal inhibitory synapse excitability in healthy subjects and SCI patients favours the hypothesis of a decrease in inhibitory neuronal activity induced by down-regulation of KCC2 after SCI in humans that likely contributes to spasticity.

Ipsi-and contralateral corticospinal control on reciprocal inhibition between forearm

muscles in healthy subjects and hemiplegic stroke patients as demonstrated by transcranial direct current stimulation.

Transcranial direct current stimulation (tDCS) has emerged as a method for exploring cortex excitability in humans. Roche et al. (2009) have shown in our laboratory that using anodal tDCS over contralateral motor cortex can also induce changes in spinal network excitability (i.e. reciprocal inhibition between forearm muscles) in the dominant limb in healthy subjects. It is unknown whether motor activity from the unaffected cerebral hemisphere could be employed after semi-brain damage in patients with hemiplegia. Moreover, little is known about the non-affected limb if it always functions like 'normal' after unilateral stroke. In this project, the ipsi-and contralateral corticospinal controls on reciprocal inhibition between forearm muscles were explored using anodal tDCS applied over the unaffected motor cortex of stroke patients and then compared to the results obtained in healthy subjects. Ipsilateral tDCS induces no change in reciprocal inhibition in healthy subjects. Similar results recorded on the affected upper limb are observed in stoke patients. However a larger number of patients is required to confirm the results. Contralateral anodal tDCS in healthy subjects shows no changes of reciprocal inhibition recorded in the non-dominant upper limb. This result is different from that observed in the dominant upper limb by Roche et al. (2009). This asymmetrical control on reciprocal inhibition would favour the hypothesis that the inter-hemispheric inhibition (IHI) between both motor cortices is asymmetric, with prominent IHI projections originating in the "dominant" left hemisphere. Contralateral anodal tDCS of the unaffected motor cortex induces a strong decrease in reciprocal inhibition in non-affected upper limb in stoke patients. This is different from that observed in both dominant and non-dominant upper limb in healthy subjects suggesting that the pathophysiological changes after unilateral stroke would probably not occur only on the hemiparesis side, but may also the non-affected side. A larger number of patients is still required to confirm the results. Key words: spinal network, inhibitory synapses, furosemide, tDCS, healthy subjects, SCI patients, stroke patients.

peroneal nerve; MT, motor threshold; PT, perception threshold; PAD, primary afferent depolarization; SCI, spinal cord injury; TA, tibialis anterior; SEM, Standard error of the mean.

Introduction

GABA and glycine are depolarising and excitatory in the immature spinal cord, becoming inhibitory in the mature state (Takahashi, 1984;Wu et al. 1992;Ziskind-Conhaim, 1998, Ben Ari, 2002). This developmental transition is generated by the differential expression of cationchloride transporters, the chloride inward co-transporter (NKCC1) being highly expressed in immature neurons whereas the chloride outward co-transporter (KCC2) is dominant in mature neurons. The abundant KCC2 expression on mature neurons lowers the intracellular chloride ion concentrations. When GABA and glycine neurotransmitters contact the neuron, there will subsequently be an inward flux of chloride ions, thus lowering the membrane potential (i.e. inhibition).

In the animal spinal cord, the lack of KCC2 expression leads to depolarising action of GABA and glycine (Vinay & Jean-Xavier, 2008), and also results in reduction of inhibitory synaptic efficiency (Boulenguez et al. 2010). In KCC2 knockout animals, sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered lumbar motoneurons responses to sciatic nerve stimulation (Hübner et al. 2001). Several studies in animals have shown that loss or reduction of co-transporter KCC2 function results in the development of central nervous system (CNS) hyper-excitability (Woo et al. 2002;Zhu et al. 2008).

The loop diuretics, furosemide and bumetanide, are thought to modulate inhibitory synapses through their antagonist effect on cation-chloride co-transporters (Gillen et al. 1996;Payne et al. 2003;Yamada et al. 2004). It has been suggested that furosemide antagonises the KCC2 co-transporter but it is also used as a NKCC1 blocker. Nevertheless, its effect on the NKCC1 co-transporter is less potent than bumetanide (Gillen et al. 1996;Russell, 2000). To date, no substance has been identified that selectively inhibits KCC2 (Kahle et al. 2008;Löscher & Schmidt, 2011), and some of the KCC2 blockers used in animal studies require very high concentrations (Payne, 1997) that are likely to be toxic in humans. Nicoll (1978) demonstrated in the frog that furosemide selectively and reversibly blocks inhibitory postsynaptic potentials (IPSPs) elicited in spinal motoneurons by GABA and glycine without affecting excitatory postsynaptic potentials (EPSPs) elicited in spinal motoneurons by glutamate. Since furosemide has been shown to cross the blood-brain barrier after intravenous injection in humans (Haglund & Hochman, 2005) and is a widely used diuretic which has been employed with a good safety record for many years in humans, we have chosen furosemide to investigate the functioning of inhibitory synapses in this study.

The aim of the present study was to investigate the possibility that orally-administered furosemide at normal clinical doses could modify the functioning of spinal inhibitory synapses in healthy human subjects. We studied two types of inhibitory spinal networks: i) presynaptic inhibition of Ia fibres which is likely mediated via GABAergic synapses, and ii) postsynaptic inhibition produced by cutaneous stimulation which is probably mediated via glycinergic synapses (Jankowska, 1992). In addition, we explored the effects of furosemide on the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons by studying post-activation depression of the H-reflex. The results obtained in healthy subjects reveal that orally-administered furosemide (40 mg) reduces the efficacy of spinal inhibitory interneurons without affecting the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons.

Materials and Methods

Ethical approval.

The experiments described in this study conformed to the guidelines issued by the World Medical Association (Declaration of Helsinki) on the ethics of experimentation in humans and the approval of the local ethical committee of the CPP Île-de-France VI -Pitié-Salpêtrière.

Written informed consent was obtained from all subjects before participation.

Study design.

This randomized controled study was performed on 19 healthy subjects (9 females) aged 22-64 years old (mean±SEM 35.1±2.5 years). All subjects participated in two different sessions to investigate the time course of changes in spinal network excitability for a period of 70 minutes (i) after furosemide intake (furosemide experiment) and, (ii) without furosemide (control experiment). The two different experiments (furosemide and control) were performed on different days.

Experimental procedure

Medical examinations (blood testing for electrolytes, creatinine, glucose and blood pressure) were performed before the experiment to verify that subjects had no contraindications to furosemide.

All experiments were performed at rest. Subjects sat comfortably with the head, neck, back and arms supported in a slightly reclined armchair. The dominant leg was examined and remained in a stationary supported position with hip semi-flexion (120°), slight knee flexion (160°) and plantar flexion (110°).

Each experiment comprised seven time epochs (0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, 51-60 min, and 61-70 min). The amount of pre-/postsynaptic inhibition evaluated at the beginning of recording (during 10 minutes before drug administration in the furosemide experiment, or 10 minutes before the test start time in the control experiment) corresponds to baseline inhibition. After the amount of inhibition was determined for the baseline period, 40 mg furosemide was orally administered. For the subject's convenience, due to its diuretic effect, the amount inhibition was monitored through repetitive measurement over a time period limited to 70 minutes after administration of drug. Furosemide pharmacokinetics and pharmacodynamics in healthy subjects show that furosemide (40-80 mg) appears in the serum within 10 minutes and peaks from 60 to 90 minutes after oral administration (Branch et al. 1976[START_REF] Branch | Determinants of response to frusemide in normal subjects[END_REF]Tilstone & Fine, 1978).

The effects of varying doses of furosemide (0 mg, 20 mg and 40 mg) were also investigated on pre-/postsynaptic inhibition in 6 subjects using the same experiment design.

The experiments testing the effects of furosemide on the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons (post-activation depression of the H-reflex) were performed in 15 subjects the same way as described previously for pre-and postsynaptic inhibition.

Water intake was recommended to subjects after the experiment. However, apart from the diuretic effect, some side effects of furosemide such as headaches and dizziness were reported by some of the subjects that participated in the present study.

Electrophysiological recordings

Method of assessing spinal network excitability

Soleus H-reflex. Spinal inhibitory circuits were studied using non-invasive electrophysiological techniques based on the H-reflex. The H-reflex was evoked in the soleus muscle by percutaneous stimulation of the posterior tibial nerve (PTN) at the popliteal fossa using a constant-current stimulator (D7SA Digitimer Ltd., Welwyn Garden City, UK). A rectangular 1 ms duration stimulus, delivered at 0.33 Hz to the PTN, was computer-triggered using a customised script (Signal 4.20, Cambridge Electronic Design, Cambridge, UK). The H-reflex was recorded from the EMG signal of the soleus muscle using bipolar surface electrodes (Delsys Inc., Boston, USA, Ag electrodes DE-2.1) positioned on the skin parallel to the muscle belly. The EMG signals were amplified (×1000), band pass filtered at 20-450 Hz (Delsys, Inc., Boston, MA, US), digitised at 1 kHz (Power 1401 A/D board, Cambridge Electronic Design, Cambridge, UK) and then stored on computer for offline analysis.

The maximum motor response (M max ) and maximum H-reflex response (H max ) were recorded at the beginning of the each experiment. The stimulus intensity was then adjusted to elicit Hreflex amplitude of 20-35% of M max , which corresponded to ~ 50%, of H max [mean value of H-reflex amplitude 24.85 ± 2.8 % M max (control experiments); 23.17 ± 2.2 % M max (furosemide experiments)]. The unconditioned H-reflexes were set to have similar size across subjects and kept constant throughout the experiment, since the sensitivity of the H-reflex to facilitation or inhibition conditioning effects depends on its non-conditioned size (Crone et al. 1990).

Inhibitory conditioning stimulations. (see Figure 1)

Presynaptic inhibition of Ia fibres. The D1 inhibition of the H-reflex was used to assess presynaptic inhibition of Ia fibres (Mizuno et al. 1971). The soleus H-reflex was conditioned with preceding stimulation of the common peroneal nerve (CPN) through hemispherical bipolar electrodes placed 2 cm below the neck of the fibula. The stimulus consisted of a train of 3 rectangular pulses, each of 1 ms duration, with an interpulse interval of 3 ms, with an intensity 1.2 times the tibialis anterior (TA) motor threshold (MT), delivered 21 ms before the test stimulation (Faist et al. 1996). The motor threshold was determined by tendon palpation and by the oscilloscope EMG display [MT mean value 15.00 ± 0.31 mA (control experiment); 11.60 ± 0.13 mA (furosemide experiment)]. At this inter-stimulus interval (21 ms), the CPN conditioning volley evokes an inhibition of the soleus H-reflex that is probably caused by presynaptic inhibition of soleus Ia afferents (Faist et al. 1996).

Postsynaptic inhibition produced by cutaneous stimulation. It has been shown that stimulation of the sural nerve (a purely cutaneous nerve) induces an inhibition of the soleus H-reflex (Hugon & Bathien, 1967). The soleus H-reflex was conditioned by electrical stimuli applied to cutaneous afferents of the sural nerve through bipolar adhesive electrodes placed on the lateral side of the fifth toe. The stimuli consisted of trains of 17 pulses, each of duration 1 ms, with an interpulse interval of 3 ms, delivered 50 ms before the test stimulus. In such conditions, the induced inhibition is probably caused by postsynaptic inhibition of soleus motorneurons (Pierrot-Deseilligny & Burke, 2012). The intensity of stimulation was based on the subject's perception threshold (PT) defined as the lowest stimulus intensity at which the subject reported sensation of the stimulation using a single pulse [PT mean value 1. Post-activation depression. To test the possible effect of furosemide on spinal excitatory synapses, we used post-activation depression of soleus H-reflexes. It has been shown in both animals (Eccles, 1964) and humans (Magladery et al. 1951;Paillard, 1955;Crone & Nielsen, 1989;Hultborn & Nielsen, 1998) that post-activation depression at the Ia fibre-motorneuron synapse reflects the efficiency of the monosynaptic excitatory transmission between Ia fibre and motoneuron and is thus a simple and reliable tool to assess the functioning of excitatory synaptic transmission without contamination of other synaptic mechanisms (Meunier et al. 2007). Post-activation depression can be demonstrated by the depressive effect of increasing the stimulus rate on the size of H-reflexes. H-reflex is dramatically depressed when the interval between the two consecutive stimuli is 1-2 s with gradual recovery as the interval increase over 10 s (Crone & Nielsen, 1989). In the present study, the H-reflex evoked every 3 s (0.33 Hz) was initially adjusted at H max /2. The soleus H-reflex evoked at low frequency (every 6s or 0.16 Hz) and high frequency (every 1s or 1 Hz) was measured in each case (see Figure 4B). Twenty soleus H-reflexes were evoked for each stimulus rate (1 Hz and 0.16 Hz).

The amount of post-activation depression of the H-reflex was evaluated as the size of the Hreflex elicited every 1 Hz (high stimulus rate) expressed as a percentage of its value when elicited every 0.16 Hz (low stimulus rate). This is referred to as the 1/0.16 ratio. This ratio was used to determine the amount of post-activation depression of the H-reflex (Aymard et al. 2000).

Statistical methods

Data processing

To evaluate the amounts of pre-and postsynaptic inhibition, peak-to-peak amplitudes of unconditioned H-reflexes were compared to those of conditioned H-reflexes. The inhibitory effects were quantified by calculation of the inhibition value as 100 -((conditioned H value/ unconditioned H value) × 100). Each inhibition value was then normalized to the percentage of its baseline value using the equation: [%((Inhibition at t x -baseline inhibition)/baseline inhibition)] +100.

To evaluate the effects of post-activation depression, the 1/0.16 ratio value was normalized to the percentage of its baseline value using the equation: [% ((1/0.16 ratio at t x -1/0.16 ratio at baseline)/ 1/0.16 ratio at baseline)] +100.

Statistical analysis

The amounts of pre-/postsynaptic inhibition and post-activation depression were averaged among subjects for each time epoch (0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, 51-60 min, and 61-70 min). A two-way repeated-measures ANOVA (control vs. furosemide as the within-subject factor) was used to examine the effects of furosemide on pre-/postsynaptic inhibitions or post-activation depression. A post-hoc pairwise comparison was performed using Tukey's HSD test.

A one way repeated-measure ANOVA on time epoch (0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, 51-60 min, and 61-70 min) was also used to determine the impact of time in the control experiment.

For dose-dependent experiments, an a priori power analysis was first performed to determine the minimal sample size. A posteriori, all our data sets were, as assumed in our power analysis, normally distributed and successfully passed the equality of variance test so parametric tests (one way repeated measures ANOVA) was employed.

Significance was taken at P < 0.05. Mean data are provided ± standard error of the mean (SEM).

Results

Effects of time on two examples of spinal inhibitory networks.

Presynaptic inhibition of Ia fibres.

The amount of presynaptic inhibition in the control experiments (without furosemide) observed over 70 minutes is shown in Figure 2A (19 subjects). A one-way repeated-measures ANOVA failed to detect a significant effect of time on presynaptic inhibition in control experiments (P > 0.05).

Postsynaptic inhibition induced by cutaneous stimulation.

The experiments were performed in all 19 subjects but this type of spinal synaptic inhibition could not be demonstrated in four of them since the conditioning stimulus intensity required to evoke postsynaptic inhibition was above that evoking an unbearable painful sensation and/or triggered the flexor reflex in these subjects. The results from postsynaptic inhibition were thus observed in only 15 subjects. The amount of postsynaptic inhibition in the control experiments observed over 70 minutes is shown in Figure 2B. There was a gradual decrease of inhibition over time in control experiments (F 14,98 = 8.56, P < 0.001, one way repeatedmeasure ANOVA). The amounts of postsynaptic inhibition decreased progressively after the onset of the recordings and the decrease is statistically significant from 21-30 minutes up to

Furosemide effects on the two spinal inhibitory networks.

Presynaptic inhibition

The comparison between variations of the amount of presynaptic inhibition (expressed as a percentage of its baseline value) in control (□) and furosemide (■) experiments is shown in Figure 3A (19 subjects). The amounts of presynaptic inhibition in the furosemide experiments start to be significantly different from that observed in control situation at 31-40 minutes after drug administration. The amount of presynaptic inhibition following furosemide decreased progressively and reached its nadir at 61-70 minutes (60.66 % of its baseline value). Two-way repeated-measures ANOVA detected a significant interaction of condition (control vs. furosemide), time epochs, and condition × time epochs (F 18,108 = 2.40, P condition = 0.037, P time epochs <0.001, P condition × time epochs = 0.032). Post-hoc analysis showed that furosemide significantly reduced presynaptic inhibition at 31-40 minutes, 41-50 minutes, 51-60 minutes, and 61-70 minutes (P<0.05, P<0.05, P<0.01 and P<0.01 respectively, Tukey test).

Postsynaptic inhibition

The comparison between variations of postsynaptic inhibition (expressed as a percentage of its baseline value) in control and furosemide experiments are presented in Figure 3B (15 subjects). The strength of postsynaptic inhibition decreased progressively after furosemide administration. The amounts of postsynaptic inhibition in the furosemide experiments began to be significantly different from those observed in control experiments 21-30 minutes after drug administration. Then amount of postsynaptic inhibition decreased continuously and reached its nadir at 51-60 minutes (21.88 % of its baseline value). Two-way repeatedmeasures ANOVA detected a significant interaction of condition (control vs. furosemide), time epochs, and condition × time epochs (F 14,84 = 3.43, P condition = 0.034, P time epochs <0.001, P condition × time epochs = 0.004). Post-hoc analysis showed that furosemide significantly reduced postsynaptic inhibition at 21-30 minutes, 51-60 minutes and 61-70 minutes (P<0.01, P<0.001, and P<0.05 respectively, Tukey test).

Furosemide effects on monosynaptic excitatory transmission.

Post-activation depression.

This supplementary experiment was performed after the study of pre-and postsynaptic inhibitions was completed. Fifteen subjects of the previous recording participated in this session (see Table 1). The comparison between variations of post-activation depression (expressed as a percentage of its baseline value) in control and furosemide experiments, observed in 15 subjects is shown in Figure 4A. A two-way repeated-measure ANOVA failed to detect a significant interaction of condition × time epochs: P > 0.05. There was no significant difference between two groups (control vs. furosemide).

Individual values (not normalised to baseline) of pre-and postsynaptic inhibition and of postactivation depression recorded at baseline and during the 40-70 minutes period are shown in Table 1. Note that three subpools at the intervals with the most prominent effects (41-50 minutes, 51-60 minutes, and 61-70 minutes) were pooled in Table 1.

Effects of two different doses of furosemide

For dose-dependent experiments, an a priori power analysis was performed to determine the minimal sample size. Given that postsynaptic inhibition decreased over time (i.e., habituation, see above), the power analysis was performed on presynaptic inhibition only. A sample size of 6 subjects, each having 0, 20 or 40 mg of furosemide provided 80% power to detect a 40% change in presynaptic inhibition (one way repeated-measures ANOVA, two-tailed 5%). This calculation was made assuming that raw data are approximately normally distributed with a standard deviation of 10%. One way repeated measures ANOVA was then employed.

Presynaptic inhibition

The effects of two different doses of furosemide were studied on presynaptic inhibition in six subjects (Figure 5A). The histogram shows the amount of presynaptic inhibition at different doses observed over a period of 40-70 minutes after furosemide administration where the effects were mostly observed (see Figure 3A). A one-way repeated-measures ANOVA detected a significant dose-effect (F 5,10 = 13.67, P = 0.001). A Post-hoc Tukey test revealed significant difference between 0 mg vs. 40 mg, and 20 mg vs. 40 mg (P<0.01 and P<0.01, respectively).

Postsynaptic inhibition

The effects of two different doses of furosemide administration were studied on postsynaptic inhibition in six subjects (Figure 5B). The histogram shows the amount of postsynaptic inhibition at different doses observed over 40-70 minutes after furosemide administration.

There was a trend for a dose-effect in postsynaptic inhibition as observed in presynaptic inhibition. However, a one-way repeated-measures ANOVA failed to detect a significant dose-effect on postsynaptic inhibition: P > 0.05. Since time has an influence on postsynaptic inhibition, the effect of furosemide evaluated at 40-70 minutes could be confounded with this time effect.

Discussion

The main finding of the present study is that oral administration of furosemide at the usual clinical dose (40 mg) decreases the function of inhibitory synapses in the spinal cord without affecting muscle afferent-evoked excitation of motoneurons.

Spinal networks involved in the present study

We have chosen to assess presynaptic inhibition directed to soleus Ia fibres using D1 inhibition. Presynaptic inhibition of Ia fibres was first described by Frank & Fuortes (1957) since a depression of a monosynaptic EPSP occurs without any change in motoneuron membrane potential. It was then extensively studied by Eccles and Colleagues (1964) and Rudomin and Colleagues (for references, see Rudomin & Schmidt, 1999). There is a general agreement that the pathways mediating presynaptic Ia inhibition involve two interneurons, where the last order interneuron is GABAergic. The D1 inhibition was first described in humans by Mizuno et al. (1971). [START_REF] Berardelli | Evidence favouring presynaptic inhibition between antagonist muscle afferents in the human forearm[END_REF] and Faist et al. (1996) subsequently showed in both upper and lower limbs that the antagonistic conditioning volley eliciting the D1 inhibition was not depressing the motor evoked potential elicited in the target muscle by cortical stimulation. It is therefore likely that the D1 inhibition is presynaptic in origin since postsynaptic inhibition should depress the monosynaptic H-reflex and also the motor evoked potential. The time interval between conditioning and test stimuli used to evoke the D1 inhibition was proposed by Faist et al. (1996) who demonstrated that at this specific time interval (21 ms), the inhibition of the soleus H reflex is only due to presynaptic inhibition acting on soleus Ia afferents.

Among the postsynaptic effects induced in the soleus motoneurons, the inhibition elicited by electrical stimulation of sural cutaneous afferents, which is a part of the withdrawal reflex (for reference see, Pierrot-Deseilligny& Burke, 2012), is one of the most robust reflexes in animals and humans. In humans, the amount of this inhibition is marked (around 40 % of the unconditioned reflex ( Pierrot-Deseilligny et al., 1973). Neurotransmitter involvement in this type of spinal inhibitory network has never been directly demonstrated; however, postsynaptic inhibition has been shown to be mediated by glycine in the cat spinal motoneurons (Curtis et al., 1973).

To study postsynaptic inhibition induced by cutaneous stimulation, we have used the protocol originally described by Hugon & Bathien (1967). The conditioning stimulus was a train of 17 shocks, since it has been shown that cutaneous stimulation induced responses require temporal summation. Later, Burke et al. (1991) and Nielsen et al. (1997) presented the evidence that the cutaneous effect induced in the lower limb with latency shorter than 70 ms, is spinal in origin. Their results are also supported by the fact that the cutaneous effect exists in spinal animals and complete spinal cord lesions in humans (Longchampt & Chanelet, 1968;Logigian et al, 1999). Put together, although the exact pattern of the spinal reflexes fed by cutaneous afferents is not completely known, we favour the hypothesis that the effects are spinal in origin. Post-activation depression was chosen to test the possible effect of furosemide on excitatory effects since it has been thought to reflect the efficiency of the monosynaptic excitatory transmission between Ia fibre and motoneuron that is an example of pure excitatory effect. Alternative possibilities to test pure excitatory effects should be either homonymous or heteronymous monosynaptic facilitation of the soleus H-reflex. However, homonymous monosynaptic facilitation cannot be used in humans to assess purely excitatory effects, due to several factors, for example the mixed excitatory and inhibitory actions and duration of after-hyperpolarisation. Heteronymous monosynaptic Ia facilitation from the quadriceps to the soleus requires an accurate determination of the time interval between the conditioning and test stimulus to ensure its monosynaptic origin (for references, see Pierrot-Deseilligny & Burke, 2012). For other excitatory pathways, for example group II disynaptic excitatory pathways, the indirect method we used cannot eliminate possible contamination by monosynaptic afferent projections or by inhibitory projections involved in the pathway.

Moreover, testing group II excitatory pathways at rest relying on the use of the quadriceps Hreflex is not as easy as evoking the H-reflex in the soleus muscle and group II excitatory effects are much larger in the standing than in the sitting position.

Changes in inhibitory synapses over time

Habituation is a gradual decrease in responsiveness following repetitive stimulation and is a well-known phenomenon in the nervous system (Harris, 1943;Hernández-Peón et al. 1956).

Most studies report habituation as a reduction in excitatory response, for example a decrease in the flexor reflex (Farel et al., 1973) or failure of the C-fibre evoked response in a dorsal horn neuron following prolonged stimulation (Woolf, 1983). However, it has also been referred to as a reduction in inhibition response. In the decerebrate cat, intracellular recording of motoneurons participating in the flexion reflex revealed that there was a marked and significant decrease of PSP amplitude during habituation which contains not only the EPSP's habituate, but also decrements of IPSP components. The IPSPs of polysynaptic responses decrease rather than increase during habituation suggesting that the amount of postsynaptic inhibition on the motoneuron is also "habituating" (i.e., decreasing). Besides, the decrease in response during habituation was proposed as a result of reduced input to motoneurons occurring in interneurons (Brazier, 1961). Habituation can be observed in patients with complete spinal cord injury, suggesting that this phenomenon may occur partly or totally at the spinal cord level (Dimitrijevic & Nathan, 1970;Dimitrijevic et al., 1972). Mechanisms underlying habituation in humans are still unclear; however, psychological adaptation of subjects, and also the effects of sensory saturation, could be taken into account (see Dincklage et al., 2013). The experiments performed in this study lasted up to 70 minutes and the same electrical stimuli were repeated at 0.33 Hz throughout. It could thus be argued that changes in inhibitory spinal network excitability were due to a habituation effect, not furosemide. To exclude this hypothesis, we performed control experiments without furosemide in the same participants. No changes were observed over time for presynaptic inhibition of Ia fibres without furosemide (see Figure 2A). This finding demonstrates that changes in presynaptic inhibition following furosemide intake is most likely due to the effects of furosemide. In the case of postsynaptic inhibition, there was a gradual decrease of inhibition over time in control experiments revealing that habituation may involve the function of cutaneous receptors or synapses interposed between afferent fibres and soleus motoneurons. The diminution of postsynaptic inhibition after furosemide administration is more pronounced compared with control (see Figure 3B), which is likely caused by furosemide even though the difference is not statistically significant for all epochs.

Put together, our results suggest that furosemide is at least partly responsible for the decrease of the efficacy of pre-and postsynaptic inhibition of soleus motoneurons. This point of view is also supported by the significant dose effect relationship for presynaptic inhibition and by the tendency of dose related changes in the postsynaptic inhibition (Figure 5).

Specific effects of furosemide onto spinal inhibitory synapses

Although furosemide is known from animal experiments to block cation-chloride cotransporters, its effects on the human spinal network have never been tested before. The question could be asked as to whether the effects demonstrated for inhibitory synapses are specific to GABAergic and glycinergic synapses, or if they are non-specific, affecting both inhibitory and excitatory synapses. The results obtained from the experiments testing the effects of furosemide on post-activation depression at the Ia fibre-motoneuron (an excitatory synaptic transmission) allow one to exclude the hypothesis that furosemide also acts on the Ia afferent-motoneuron synapse. There was no significant difference observed in post-activation depression between control and furosemide experiments. Altogether, these observations strongly suggest that furosemide decreases the functioning of spinal inhibitory synapses.

Although furosemide decreases both pre-and postsynaptic inhibition, the effect of furosemide seems to be more important for postsynaptic than presynaptic inhibition. The most likely explanation for the difference between the time courses of pre-and postsynaptic inhibition is that in the control condition (without furosemide), there was no systematic change over time for presynaptic inhibition (Figure 2A) while the amount of postsynaptic inhibition is progressively decreased over time (Figure 2B). This effect of time by itself on postsynaptic inhibition is interpreted above with respect to habituation. Thus, for postsynaptic inhibition, the inhibition following furosemide intake is the sum of habituation effect and the effect of furosemide itself, while the changes of presynaptic inhibition following furosemide intake is only due to furosemide effect. Another explanation may be that the kinematics of pre-and postsynaptic inhibition are different; however, to our knowledge, there is no evidence to support this hypothesis.

Obviously, the possible effects of furosemide are not restricted to the spinal cord, but may affect all the structures of the CNS, thereby the functioning of networks projecting on interneurons mediating pre-and postsynaptic inhibition. However, since it has been shown in animals that furosemide decreases IPSPs without modifying EPSPs (Nicoll, 1978), the possible effect of furosemide on these networks would be a decrease in inhibitory controls. A decrease in inhibitory controls acting on pre-and postsynaptic inhibition would result in an increase in the efficiency of these inhibitory pathways and thus cannot be in all likelihood responsible for the decrease of inhibition we have observed.

Furosemide is known to inhibit in a non-competitive fashion GABA and glycine receptor channels mainly through antagonism of the cation-chloride cotransport system, but this drug also modulates the extracellular space involving, for example, electrical resistance of nervous tissue, extracellular ionic concentration or local ephaptic coupling [START_REF] Hochman | Normal and aberrant structure of neurons and glia: The extracellular space and epileptic activity in the adult brain: Explaining the antiepileptic effects of furosemide and bumetanide[END_REF]. Thus, if furosemide modulates the extracellular space, the possibility exists that it would change the distribution of current applied by the transcutaneous electrical conditioning stimuli applied to the peripheral nerves. Therefore, we have verified that the direct motor response evoked in the TA following the stimulation of the CPN was not modified during the experiments (data not shown) suggesting that there is likely no change in the distribution of current induced by furosemide. Obviously, the site of electrical stimuli are far from the spinal cord but the absence of changes in the TA motor response was the only available data in our experiments to test this hypothesis.

Methodological significance and clinical implications

In humans, available methods to study spinal cord networks are indirect and rely on EMG recordings that operate as a window on motoneuron and interneuron excitability (Pierrot-Deseilligny & Burke, 2012). Since the pioneering experiments in humans by Magladery et al. (1951) and Paillard (1955), electrophysiological non-invasive but selective methods have been developed to study the excitability of spinal networks. Recent results obtained in animals show that lesion of the spinal cord induces a down-regulation of KCC2, which reverses the inhibitory pattern of GABAergic and glycinergic neurons back towards their immature and excitatory state (Jean-Xavier et al. 2006). This finding raises the possibility of setting up a technique, which would allow us to determine if such a reversal also exists in patients with spinal cord injury (SCI). The specific effects of furosemide on spinal inhibitory synapses demonstrated here provide a window on the intrinsic functioning of inhibitory synapses mediated through cation-chloride cotransporters. If SCI in humans shows a downregulation of KCC2 as observed in animal studies, the comparison of furosemide effects on spinal inhibitory synapse excitability in healthy subjects and SCI patients would allow us to study these effects.

Furosemide is observed to decrease presynaptic inhibition directed to soleus Ia fibres and postsynaptic inhibition directed to soleus motoneurons in the present study. The question may arise whether prolonged intake of furosemide would affect daily life or physical activity of people. Exaggerated reflexes or increase in muscle tone have never been reported as a side effect of furosemide. Even though, to our knowledge, no systematic study of exaggerated reflexes or muscle tone has been performed during prolonged furosemide treatment. None of the healthy volunteers who participated in our experiments reported changes in reflexes or muscle tone following a single dose of furosemide administration. We may thus assume that in people with normal functioning of inhibitory synapses, the effects of furosemide have no clinical consequences. However, this may be different in patients with modifications of the functioning of inhibitory synapses. If the down-regulation of KCC2 in spinal rats, (Boulenguez et al. 2010) is also present in patients with SCI, it would be interesting to explore the possible muscle tone changes following prolonged furosemide treatment.

In conclusion, furosemide, which is a safe and widely used diuretic, has an effect on spinal inhibitory synapses in healthy subjects. The use of this loop diuretic would be a very useful tool to explore the possibility as to whether the reduced inhibitory interneuronal activity which likely contributes to spasticity, also exists in humans with spinal cord injury. and critical revision of the manuscript. D.J.: conception and design of electrophysiology experiments. J.L.: conception and design of experiments, interpretation of data, critical revision of the manuscript. N.R.: conception and design of experiments, interpretation of data, critical revision of the manuscript. All authors approved the final version of the manuscript, all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Asymmetrical control of reciprocal inhibition at wrist level

Alexandra LACKMY-VALLEE 1 , Wanalee KLOMJAI 1-2 , Bernard BUSSEL 5 , Rose KATZ 1-4 , and Nicolas ROCHE 3-5 1 Sorbonne Universités UPMC Univ Paris 06, ER 6, F-75005, Paris, France

Introduction

The control of neural transmission in pathways mediating reciprocal inhibition in antagonist muscles continues to raise questions ever since Sherrington (1906), who first introduced the concept of reciprocal innervation. Intracellular recordings in cat spinal cord reveal a striking similarity in the descending and segmental convergence on agonist α-motoneurones and Ia inhibitory interneurones of the antagonist α-motoneurones [START_REF] Lundberg | The excitatory control of the Ia inhibitory pathway[END_REF][START_REF] Hultborn | Transmission in the pathway of reciprocal Ia inhibition to motoneurones and its control during the tonic reflex[END_REF]. [START_REF] Lundberg | The excitatory control of the Ia inhibitory pathway[END_REF] proposed that these connections from the brain to corresponding αmotoneurones and Ia interneurones were also used in parallel during voluntary movement in order to achieve a coordinated contraction and relaxation of the antagonist muscles.

Consistent with animal data, it has been shown in humans that pathways mediating reciprocal inhibition in flexor and extensor motoneurones are disynaptic, and that motoneurones and inhibitory interneurones have received similar segmental and descending control (Day et al. 1984[START_REF] Cavallari | Changes in reciprocal Ia inhibition from wrist extensors to wrist flexors during voluntary movement in man[END_REF], Shindo et al. 1984, Rothwell et al. 1984[START_REF] Cowan | The effect of percutaneous motor cortex stimulation on H reflexes in muscles of the arm and leg in intact man[END_REF]). At the level of the elbow and ankle, extensor and flexor muscles operate as real antagonists, so interneurones relaying reciprocal inhibition to flexor and to extensor motoneurones are identified as Ia inhibitory interneurones. At the wrist level, extensor carpis radialis (ECR) and flexor carpis radialis (FCR) not only operate as antagonists but also as agonists in wrist abduction so interneurons mediating reciprocal inhibition to -motoneurons innervating extensor and flexor muscles differ from those mediating Ia inhibition at the ankle and elbow levels. The interneurones mediating reciprocal inhibition between FCR and ECR are not inhibited by Renshaw cells (Aymard et al. 1995) but receive input from both Ia and Ib afferent fibres (Wargon et al. 2006). It has been hypothesised that interneurones mediating reciprocal inhibition between wrist flexors and extensors, probably share more characteristics with Ib than Ia inhibitory interneurones. Regarding their descending control, Ib inhibitory interneurones receive a powerful controlling input from the reticulospinal tract, and thus, are influenced by both contra-and ipsi-lateral descending control [START_REF] Crosby | Correlative Anatomy of the Nervous System[END_REF]. Moreover, [START_REF] Illert | Integration in descending motor pathways controlling the forelimb in the cat. 7. Effects from the reticular formation on C3-C4 propriospinal neurones[END_REF] have shown that forelimb muscle motoneurones receive a concomitant descending control from both cortices. In humans, the role of the ipsilateral motor cortex is still under debate although recent studies [START_REF] Bradnam | Cathodal transcranial direct current stimulation suppresses ipsilateral projections to presumed propriospinal neurons of the proximal upper limb[END_REF][START_REF] Mccambridge | Cathodal transcranial direct current stimulation of the primary motor cortex improves selective muscle activation in the ipsilateral arm[END_REF] suggest that ipsilateral tDCS influences arm motor control. In humans, reciprocal inhibition directed from ECR to FCR and its descending control from the contralateral hand motor cortex have been studied following changes in excitability of contralateral hand motor cortex induced by Transcranial Magnetic Stimulation (TMS) [START_REF] Rothwell | Effects of motor cortex stimulation on spinal interneurones in intact man[END_REF][START_REF] Cowan | The effect of percutaneous motor cortex stimulation on H reflexes in muscles of the arm and leg in intact man[END_REF]) and by Transcranial Direct Current Stimulation (tDCS) (Roche et al. 2009). To our knowledge, the control of transmission of the reciprocal inhibition from FCR to ECR has not yet been described in humans. This is mainly due to the difficulty of evoking an H reflex in extensor muscles in humans. Previous results suggest that corticospinal control is asymmetrical between flexor and extensor ankle motoneurones in humans [START_REF] Crone | Reciprocal Ia inhibition between ankle flexors and extensors in man[END_REF].

Although the presence of an ECR H reflex is rarely seen, we were able to recruit a sample of healthy subjects exhibiting an H reflex in ECR at rest with sizeable amplitude allowing the study of reciprocal inhibition directed to ECR motoneurones. The aims of this study were therefore primarily to explore the effects of contralateral anodal tDCS on the reciprocal inhibition directed from FCR to ECR, in order to compare corticospinal control acting on interneurones mediating reciprocal inhibition from flexors to extensors and vice versa and secondly to explore the possible effects of anodal tDCS applied over the ipsilateral motor cortex on pathways that mediate reciprocal inhibition in flexor and extensor wrist muscles. Materials and Methods

Subjects

Thirty-two healthy subjects ranging from 22 to 60 years (mean 31.1 ± 9.9 years), 20 females and 12 males (9 left-handed and 23 right-handed), were included in this study. Note all subjects participated in every experiment. Among them only 9 subjects had an H reflex in ECR stable enough at rest to explore reciprocal inhibition directed from ECR to FCR. All subjects gave written informed consent before participating in the experiments. This study was performed according to the Code of Ethics of the World Medical Association (Declaration of Helsinki), and was approved by the local ethical committee (CPP Ile de France 6-Pitié-Salpêtrière).

Transcranial direct current stimulation of the motor cortex tDCS was applied using a NEUROCONN DC-STIMULATOR (NEUROCONNGMBH COMPANY, Ilmenau, Germany) via two conductive rubber electrodes placed in salinesoaked sponges (5x7 cm). Transcranial magnetic stimulation (TMS) elicited by MAGSTIM 200 (MAGSTIM DYFED, UK), was used to determine the position of the anode positioned over the hand motor cortex. The cathode was placed over the supra-orbital region. Three conditions of tDCS were tested: i) the active contralateral condition: with the anode placed over the contralateral hand motor cortex and cathode positioned over the ipsilateral supraorbital area; ii) the active ipsilateral condition; anode over the ipsilateral hand motor cortex and cathode over the contralateral supra-orbital area; iii) the sham condition: the electrodes were placed in the same position as in active conditions but, current was activated for only 120 seconds at the beginning and 30 seconds at the end of sham stimulation in order to mimic the sensations of ramp up and ramp down current perceived in the active conditions. In the experiments involving with the ECR H reflex, due to the difficulty of evoking a test H reflex in ECR, the sham condition was performed in only one configuration with anodal tDCS electrode placed over the contralateral motor cortex. We were careful to respect a minimum delay of 48 hours between the different tDCS conditions and on average the delay between two recording sessions was about one week. Moreover, in the present study, active and sham contralateral tDCS was tested only on reciprocal inhibition directed from FCR to ECR.

Effects induced by contralateral tDCS on reciprocal inhibition directed from ECR to FCR were obtained from our previous study (Roche et al. 2009).

In all conditions, current intensity was fixed at 1.75 mA. Current flowed continuously for 20 minutes for both active conditions, and for 150 seconds in the sham condition, since Nitsche and Paulus (2000) had previously reported that a duration of at least 3 minutes of tDCS was necessary to induce after-effects. The current was ramped up or down over the first and last 8 seconds of stimulation. The experimental procedure was identical to that used by Roche et al. (2009).

Measurement of spinal network excitability

Electromyogram recordings

Subjects were seated in a comfortable reclining armchair with the shoulder slightly abducted at about 60 degrees, the elbow semi-flexed and with slight pronation of the forearm. The distal part of the upper limb was supported by an armrest in order to exclude any active maintenance of wrist posture, and the subjects were asked to relax the arm. Electromyographic (EMG) activity was recorded from FCR and ECR muscles using bipolar surface electrodes positioned on the muscle belly. EMG activity was displayed in a wide analysis window beginning 100 ms before, ending 200 ms after the test stimulus. To ensure that subjects relaxed their wrist muscles, background EMG activity was measured a posteriori, in the 100 ms pre-trigger windows with rectified EMG activity. EMG activity was sampled at 2 kHz, amplified (x 5000 -10000) and band-pass filtered (250 Hz-3 kHz) using a Digitimer D360 amplifier (DIGITIMER LTD, WELWYN GARDEN CITY, Herts, UK. In all subjects, experiments were performed on the dominant upper limb based on patient selfreports.

Electrical stimuli

Transcutaneous electrical stimulation (rectangular pulses of 1 ms duration every 3 s) were applied to radial and median nerves, via 3 cm diameter hemi-spherical bipolar electrodes. The electrodes were placed near the cubital fossa to stimulate the median nerve and at the spiral groove to stimulate the radial nerve.

Test stimuli

FCR H reflex

The FCR H reflex was evoked by electrical stimuli applied to the median nerve. First, the maximum direct motor response (Mmax) was first determined and the unconditioned H reflex was adjusted to 10% -20% of Mmax in each subject (mean value 14.17 ± 2.5% Mmax). The amplitude of the unconditioned FCR H reflex corresponded to 50% of the maximum amplitude of H reflex in the majority of cases. It was kept constant throughout the experiment, since the H reflex sensitivity to facilitation or inhibition can vary with its unconditioned amplitude (Crone et al. 1990).

ECR H reflex

The ECR H reflex was evoked by radial nerve stimulation. As mentioned previously, it is difficult to elicit the H reflex in ECR (Pierrot-Deseilligny and Burke 2012). Thirty-two subjects participated in this study, but only 9 subjects had an ECR H reflex large enough at rest to show changes in reciprocal inhibition. As above, the Mmax response was determined first and the unconditioned ECR H reflex was adjusted to 5%-15 % of Mmax in each subject and was kept constant throughout the experiment (mean value 6.62 ± 1.4% Mmax).

Conditioning stimuli

Conditioning stimuli were applied to the nerve innervating the antagonist muscle, i.e. that counteracting the muscle in which the test H reflex was evoked. The intensity of the conditioning stimuli was adjusted to activate group I fibres but was below the activation threshold of Renshaw cells in order to prevent recurrent inhibition [START_REF] Aymard | Recurrent inhibition between motor nuclei innervating opposing wrist muscles in the human upper limb[END_REF]. This implies that the conditioning stimuli did not evoke either a direct motor response (which may have an influence by antidromic activation of Renshaw cells) or an H reflex (which may have influence by orthodromic activation of Renshaw cells). The motor threshold was determined by the oscilloscope display of EMG. The average intensity of conditioning stimuli was 0.88 ± 0.02 motor threshold (MT) for the radial nerve, and was 0.66 ± 0.02 MT for the median nerve (Table 1). The interstimulus interval (ISI) was the time interval between test and conditioning stimuli for which the level of reciprocal inhibition was maximal. By convention, a positive ISI corresponds to a conditioning stimulus preceding the test stimulus. The ISI was determined using 0.5 ms steps in the range -3 ms/+ 1 ms for reciprocal inhibition from ECR to FCR (Day et al. 1984) and -1 ms/+ 3 ms for reciprocal inhibition from FCR to ECR (Wargon et al. 2006). The mean ISI for reciprocal inhibition from ECR to FCR was 0.12 ± 0.6 ms and for reciprocal inhibition from FCR to ECR was 0.60 ± 0.8 ms. The ISI was kept constant throughout the experiments.

Experimental procedure

A randomised, sham-controlled tDCS study was performed. During the experiments, the subjects were blind to the conditions of tDCS. The active contralateral condition, the active ipsilateral condition, and the sham condition were randomly alternated. Each condition was performed on the subjects on different days to avoid carry-over effects.

Once all parameters (unconditioned H reflex amplitude, conditioning stimulation intensity and ISI) were set, the baseline inhibition (without tDCS) was determined over the first 10 minutes, being defined as the baseline period. Next, the tDCS electrodes were attached. The anode was placed over the hand motor cortex for 20 minutes for both active (ipsi and contralateral conditions) and sham tDCS. These 20 minutes were divided into two "10 minute" periods: period 1 and period 2. The tDCS electrodes were removed immediately after the end of the stimulation. The amount of reciprocal inhibition was also measured for 10 minutes following the end of stimulation in order to evaluate post-effects (see diagram on Fig. 1). In each period (baseline, period 1 tDCS, period 2 tDCS and post tDCS), the amount of inhibition was assessed with three series of 40 H reflexes (20 conditioned H reflexes and 20 unconditioned H reflexes). Conditioned and unconditioned H reflexes were evoked every 3 s and randomly alternated.

Analysis

Parameter definitions

The background EMG activity was evaluated by calculating the pre-trigger root mean square (rms) of rectified EMG activity in the 100 ms window preceding the test stimulation. The average H reflex size was determined from peak-to-peak amplitudes expressed as a percentage of the maximum motor response (Mmax). The amount of reciprocal inhibition was defined as ((unconditioned H value -conditioned H value)/unconditioned H value) x 100. The mean amount of reciprocal inhibition from each period was normalised as a percentage of the baseline inhibition measured over the baseline period (t 0 ) according to the equation: ((inhibition t -inhibition t0 )/ inhibition t0 ) x 100. Therefore when amount of reciprocal inhibition was normalised to the baseline, a positive value means that inhibition was larger than the baseline and a negative value means the inhibition was weaker than the baseline .

The normalised amounts of reciprocal inhibition were only used in the graphics representing group data. Statistical analyses were performed on raw data, i.e with amount of reciprocal inhibition expressed as a percentage of unconditioned H value.

Statistical analysis

For individual data, a two-way ANOVA was performed with time period as the first factor (baseline, period 1 tDCS, period 2 tDCS, and post tDCS) and tDCS condition (sham, active contralateral tDCS and active ipsilateral tDCS) as the second factor. When reciprocal inhibition from FCR to ECR was investigated, we compared effects induced during sham, active contralateral tDCS and active ipsilateral tDCS as subjects were examined in all 3 conditions. To evaluate effects induced by active ipsilateral tDCS on reciprocal inhibition from ECR to FCR, we compared the modulations observed from sham and active ipsilateral tDCS conditions, only since active contralateral tDCS had already been tested on another group of subjects in our previous study (Roche et al. 2009).

A two-way ANOVA was performed on unconditioned H reflexes (with time period and condition as factors) to ensure that the modulations of reciprocal inhibition resulted from tDCS not from the variation of the H reflex over time. Moreover i) as amplitude of unconditioned H reflex may affect the conditioned H reflex (Crone et al. 1990) and ii) as the mean amplitude of unconditioned H reflex in ECR was smaller than that in FCR (mean value of unconditioned ECR H reflex was 6.62 ± 1.4% Mmax, compared to mean value of unconditioned FCR H reflex was 14.17 ± 2.5%, cf Table 1) a linear regression between the ratio ECR unconditioned H reflex/FCR unconditioned H reflex and the ratio of ECR conditioned H reflex/FCR conditioned H reflex was calculated in order to ensure that modulations induced by tDCS, were not affected by unconditioned H reflex amplitude .

Group data were analysed using two-way repeated-measures ANOVAs to determine effects of tDCS and time period on reciprocal inhibition. Moreover, to determine if modulations of reciprocal inhibition from ECR to FCR observed in left-handed subjects during active ipsilateral tDCS, were different to that observed in right-handed subjects, a two-way ANOVA was performed with period (baseline, period 1 tDCS, period 2 tDCS, post tDCS) as the first factor and handedness (left-handed subjects, right-handed subjects) as the second factor. Note both two way ANOVAs were performed on raw data using amount of reciprocal inhibition expressed as a percentage of unconditioned H reflex rather than on normalised data (reciprocal inhibition expressed as a percentage of baseline inhibition). To ensure that background EMG activity was constant all over the time, and whatever the tDCS condition a two-way ANOVA (testing tDCS condition and time period as factors) was performed on rms.

When the F value was significant, post hoc pairwise comparisons were performed using the Newman-Keuls test. Degrees of freedom calculated by statistical analyses are indicated in brackets after the F value. Significance was taken at P < 0.05. Mean data are shown as mean ± 1 Standard Error of the Mean (SEM). Statistical analysis was performed using the SigmaPlot software 11.0.

Results

Fig. 2 illustrates a representative example of unconditioned and conditioned reflexes obtained over the baseline period in the same subject. Note that reciprocal inhibition induced in the FCR H reflex (upper traces) is larger than that in the ECR H reflex lower traces. Table 1 summarises the mean unconditioned ECR and FCR H reflex amplitudes: for each tDCS condition (sham, contralateral, ipsilateral) and period (baseline, during tDCS, after tDCS), no significant change of unconditioned test reflex amplitude was observed.

Background EMG activity

To ensure that all experiments were conducted at rest we assessed the background EMG activity by calculating the rms of rectified EMG activity in the 100 ms pre-trigger window.

The mean rms was calculated for experiments testing reciprocal inhibition directed from FCR to ECR (mean rms = 0.103 ± 0.025mV) and for experiments testing reciprocal inhibition directed from ECR to FCR (mean rms = 0.109 ± 0.0143 mV). Statistical analysis with tDCS condition as first factor (sham, active ipsilateral and active contralateral) and time period as second factors attested that background EMG activity was constant over time and that it was not affected by tDCS: experiments exploring reciprocal inhibition directed from ECR to FCR (F(2) = 1.868 P condition = 0.177, F(3)= 0.853 P period = 0.471, F(6)= 0.0198 P condition x period = 0.996); experiments testing reciprocal inhibition directed from ECR to FCR : (F(1) = 0.00561 P condition = 0.960, F(3)= 0.350 P period = 0.789, F(6)= 0.00369 P condition x period = 1.000).

Sham conditions

Fig. 3A shows individual data obtained in 9 subjects in whom it was possible to evoke a stable ECR H reflex at rest (5 right-handers, 4 left-handers). The amount of reciprocal inhibition directed from FCR to ECR was plotted against the four time periods (baseline, period 1 tDCS, period 2 tDCS and post tDCS). The amount of reciprocal inhibition did not change significantly over time. The mean level of inhibition as a percentage of unconditioned H reflex in ECR for each time period was: 17.80% ± 2.9 in baseline period; 15.32% ± 2.6 in period 1 tDCS; 14.43% ± 2.3 in period 2 tDCS and 14.16% ± 3.7 in post tDCS. Fig. 3C shows individual data obtained in 20 subjects (17 right-handers, 3 left-handers). The amount of reciprocal inhibition directed from ECR to FCR did not change significantly over time: the mean level of inhibition as a percentage of unconditioned FCR H reflex for each time period was 34.63 % ± 2.4 in baseline period; 35.44% ± 3.4 in period 1 tDCS; 37.59% ± 3.2 in period 2 tDCS and 34.63% ± 2.3 in post tDCS. These results confirm that whichever reciprocal inhibition pathway explored (from FCR directed to ECR -Fig. 3A or from ECR directed to FCR -Fig. 3C), sham tDCS never induced a significant change of reciprocal inhibition.

Active tDCS applied over the contralateral hand motor cortex

Reciprocal inhibition from FCR to ECR Fig. 3B shows individual data obtained from 9 subjects (5 right-handers, 4 left-handers). The amount of inhibition of the ECR H reflex is plotted against the four time periods. In 9 subjects, the level of inhibition was lower in both periods 1 and 2, compared to baseline.

During the post tDCS period, the level of inhibition increased in 7 subjects compared to period 2. The average reciprocal inhibition expressed as a percentage of unconditioned H reflex in the 9 subjects was: 16.55% ± 1.7 in baseline period; 7.23% ± 3.1 in period 1 tDCS; 3.91% ± 3.1 in period 2 tDCS and 10.92% ± 2.4 in post tDCS Fig. 4A shows group data with the amount of reciprocal inhibition from FCR to ECR normalised to a percentage of its baseline value obtained from sham and active contralateral tDCS conditions. The two-way repeated measures ANOVA showed an effect of time period (baseline, period 1 tDCS, period 2 tDCS, post tDCS) and a significant interaction between time period and tDCS condition (active contralateral, active ipsilateral and sham): F(3) = 2.951 P period = 0.05, F(2) = 0.993 P condition = 0.395, F(2,3) = 3.412 P condition X period = 0.009. These results confirm that the mean values of reciprocal inhibition from the active contralateral tDCS condition were dramatically lower in both periods 1 and 2 compared to those obtained in the baseline period (before tDCS application) and from the sham condition (Newman and Keuls analysis, baseline vs period 1 tDCS P = 0.009, baseline vs period 2 tdCS P < 0.001, baseline vs post tDCS P = 0.01). At the end of stimulation the mean level of reciprocal inhibition was restored to its baseline value (Newman and Keuls analysis; baseline vs post tDCS P = 0.223 post tDCS vs period 1 tDCS P = 0.07, post tDCS vs period 2 tDCS P = 0.004).

Note that the mean levels of reciprocal inhibition recorded during the baseline period were similar in sham and active tDCS conditions. Mean inhibition as a percentage of H unconditioned reflex was 17.80% ± 3.6 in the sham condition compared to 16.55% ± 2.6 with contralateral stimulation. (Newman and Keuls analysis P = 0.879 (Table 1)). Moreover the mean values of unconditioned H reflex amplitudes were not affected by condition or time period (F(2) = 0.799 P condition = 0.412, F(3) = 1.518 P period = 0.250, F(2,3) = 1.55 P condition X period = 0.359). Mean values of unconditioned H reflex amplitude were similar during the baseline period (7.08 ± 1.5% Mmax in the sham condition compared to 7.46 ± 1.3% Mmax with contralateral stimulation) (Table 1).

Reciprocal inhibition from ECR to FCR

The effects of active contralateral tDCS on reciprocal inhibition directed from ECR to FCR were described in our previous study (Roche et al. 2009). The data presented in Fig. 4B were extracted from our previous findings for comparison with the results obtained from the present study of reciprocal inhibition directed from FCR to ECR (Fig. 4A). Reciprocal inhibition was enhanced in periods 1 and 2 compared to that observed in the baseline period and sham condition (Fig. 4B) (two-way repeated measures ANOVA (4 periods (baseline, period 1 tDCS, period 2 tDCS, post tDCS) and 2 conditions (sham, active contralateral tDCS), F(3) = 2.062 P period = 0.03; F(1) = 2.085 P condition = 0.174, F(3,1) = 6.180 P period X condition < 0.001). Reciprocal inhibition was restored to near baseline levels after the end of stimulation for the active tDCS condition: Newman and Keuls analysis; baseline vs period 1 tDCS P = 0.04, baseline vs period 2 tDCS P < 0.001, baseline vs post tDCS P = 0.38.

Influence of the unconditioned H reflex size on the amount of reciprocal inhibition.

It can be seen that the size of the unconditioned ECR H reflex was lower than that of the unconditioned FCR H reflex (cf Table 1). Therefore, to ensure that the size of the unconditioned H reflex did not impact on the results, two ratios; unconditioned ECR H reflex/unconditioned FCR H reflex and conditioned ECR reflex/conditioned FCR H reflex were calculated. A linear regression was performed to determine whether the difference between ECR and FCR reflexes altered the amount of reciprocal inhibition (figure not shown). The analysis revealed a significant linear correlation (P < 0.001; R2 = 0.957) indicating that the level of reciprocal inhibition in ECR and FCR muscles would be equal if unconditioned H reflex evoked in ECR and FCR were similar.

Active tDCS applied over the ipsilateral motor cortex.

Reciprocal inhibition from FCR to ECR

Reciprocal inhibition directed from FCR to ECR was tested in 8 subjects as it was not possible to perform the experiments with active ipsilateral tDCS in one subject. The mean levels of inhibition, expressed as a percentage of unconditioned H test reflex were baseline: 17.46% ± 3.9 in baseline period; 15.65% ± 4.8 in period 1 tDCS; 14.28% ± 5.1 in period 2 tDCS and = 14.13% ± 5.3 in post tDCS. The level of inhibition for the group data is illustrated in Fig. 5A which shows that tDCS applied over the ipsilateral motor cortex did not modify reciprocal inhibition directed from FCR to ECR. The results obtained in sham and active tDCS conditions are strikingly similar since the two curves can be superimposed. This was confirmed by the post-hoc test indicating that the time period had no effect on the amount of inhibition in sham and in active ipsilateral tDCS conditions (P period > 0.05 during active ipsilateral tDCS or during sham tDCS).

Reciprocal inhibition from ECR to FCR First, reciprocal inhibition directed from ECR to FCR was compared during sham (Fig. 3C) and anodal ipsilateral stimulation (Fig. 3D) in 20 subjects. As a percentage of unconditioned test H reflex, the mean levels of inhibition were: 27.74 ± 2.7 in baseline period; 27.9 ± 3.1 in period 1 tDCS; 29.82 ± 3.4 in period 2 tDCS; 28.08 ± 3.0 in post tDCS. The level of inhibition for group data is illustrated in Fig. 5B which shows that ipsilateral tDCS did not induce any statistical significant modification. Results obtained with anodal stimulation strongly resemble those obtained with sham stimulation. This was confirmed by statistical analysis indicating that the time period had no effect on the amount of inhibition in sham and in active ipsilateral tDCS conditions (two-way ANOVA (2 conditions (sham, active ipsilateral tDCS) X 4 time periods (baseline, period 1, period 2, post tDCS)), F(3) = 1.829 P period = 0.152; F(1) = 2.492 P condition = 0.071; F(3,1) = 0.0868; P period x condition = 0.967). Moreover the mean values of unconditioned H reflex amplitudes were not affected by condition or time period (F(1) = 2.904 P condition = 0.104, F(3) = 2.492 P period = 0.071, F(1,3) = 0.119 P condition X period = 0.948). Mean values of unconditioned H reflex amplitude were similar during the baseline period (15.13± 2.3% Mmax in the sham condition compared to 13.00 ± 1.9% Mmax with ipsilateral stimulation) (Table 1).

Subsequently, the number of subjects was increased in order to compare results obtained in right-handed and left-handed subjects. Thirty-one subjects were included, of which 8 were left-handed (individuals data are not shown). In the 23 right-handed subjects we found that the level of inhibition was increased compared to baseline in 9 subjects and decreased in 14 subjects in period 1. In period 2, the values were increased compared to baseline in 15 subjects and decreased in 8 subjects. Mean levels of inhibition as a percentage of unconditioned test H reflex were 24.02 % ± 2.4 in baseline period 22.19 % ± 2.7 in period 1 tDCS; 23.94 % ± 2.7 in period 2 tDCS and 23.62 % ± 3.2 in post tDCS. Moreover for the 8 left-handed subjects included in this series of complementary experiments, we found that in 7 subjects, the values were decreased compared to baseline in period 1 and in 4 subjects in period 2. Mean level of inhibition as a percentage of unconditioned test H reflex was 26.77 % ± 4.0 in baseline period, 22.01 % ± 5.4 in period 1 tDCS, 25.41 ± 6.6 in period 2 tDCS and 25.14 ± 4.9 in post tDCS. Group data are shown in Fig. 5C. Whatever the handedness, no significant modification was seen during or after anodal ipsilateral tDCS (two-way ANOVA F(3) = 0.252, P period = 0.860; F(1) = 0.243, P handedness = 0.623; F(3,1) = 0.004, P period x handedness = 0.987).

Study limitations tDCS methodological considerations

Repetitive TMS and tDCS are two tools currently available to non-invasively modify excitability of the motor cortex but the two techniques differ mainly with regard to their mechanisms and their spatial resolution (Priori et al. 2009). rTMS triggers action potentials in cortical axons and subcortical white matter whereas tDCS leads to polarisation of neurons, but does not induce axonal action potentials. Apart from its simplicity, the major argument in favour of tDCS over rTMS is that rTMS produces effects on cortical excitability that may occur not only at the stimulation site but also at distant connected sites. For example, Wassermann et al. (1998) showed that 1 Hz rTMS applied over the primary motor cortex (M1) changes interhemispheric and corticospinal excitability in the contrateral homologous M1 as the authors found a small decrease in the MEP recruitment curve indicating a reduced cortical excitability in the contralateral homologous cortex. By contrast, it has been shown that tDCS applied to the left primary motor cortex can facilitate or suppress MEPs triggered from the same hemisphere depending on the direction of current flow but has no effect on MEPs evoked from the right primary motor cortex (Lang et al. 2004). Thus, despite the fact that tDCS is applied through large electrodes and thus, less focal, we assume that it is more suitable for the comparison of the effects of ipsi-and contralateral tDCS. The second argument in favour of tDCS is related to of sham stimulation. TMS produces a loud click when each stimulus is delivered and electric current induced in the scalp as well as in the brain, leads to local activation of sensory nerves and muscle which is readily perceived by subjects. However, even if the click is indistinguishable between real and sham TMS, the lack of induced electric current means that there is no accompanying scalp sensation. In contrast with tDCS, subjects only feel the current intensity variation, so they can be entirely unaware of the difference between real and sham stimulation (Priori et al. 2009), even though here, for simplicity's sake the experimenter was not blind to the intervention.

Since the tDCS electrodes applied over the hand motor cortex were always placed at the same position to study the two types of reciprocal inhibition (directed from FCR to ECR and directed from ECR to FCR), it could be argued that cortical structures activated by tDCS were not required for both types of inhibition. However, imaging data have shown that representative map areas of the FCR and ECR are very close or even intermingled [START_REF] Wassermann | Cortical motor representation of the ipsilateral hand and arm[END_REF][START_REF] Devanne | The comparable size and overlapping nature of upper limb distal and proximal muscle representations in the human motor cortex[END_REF]. As the size of the active electrode applied over the motor cortex is large (35 cm 2 ), we assume that the cortical areas of FCR and ECR were simultaneously stimulated during tDCS. Thus, the hypothesis that tDCS did not stimulate a relevant cortical structure can be ruled out.

The main purpose of this study was to test the descending control from the motor cortex to spinal interneurones, so we focussed on effects induced by tDCS on the reciprocal inhibition pathways using only the anodal polarity, which increases cortical activity (Purpura and McMurtry 1965). Moreover, in our previous study (Roche et al. 2009) we showed that cathodal tDCS had no effect on reciprocal inhibition from ECR to FCR.

ECR and FCR H reflex methodological considerations

Thirty-two healthy subjects were enrolled in this study. Among them, 32 had an FCR H reflex and only 9 of them had an ECR H reflex. This finding is in accordance with previous results (Day et al. 1984;Aymard et al. 1995;Wargon et al. 2006). Furthermore, the amplitude of unconditioned reflexes is usually smaller in ECR than in FCR H. The conditioning stimulus, when applied to the radial nerve, does not usually evoke an H reflex in the ECR, while the conditioning stimulus applied to the median nerve, usually evokes an FCR H reflex. To avoid evoking FCR H reflexes which may induce activation of Renshaw cells between FCR and ECR motoneurones (Aymard et al. 1995), a lower average intensity conditioning stimulus was applied to the median nerve than that applied to the radial nerve (cf. Table 1). This lower median nerve conditioning stimulus intensity probably contributes to the lower baseline level of reciprocal inhibition in ECR compared to FCR motoneurones. However, in 1995, Aymard et al. were able to find a subject in which the conditioning stimuli applied to the radial nerve and to the median nerve were identical. In this condition, the amounts of reciprocal inhibition were similar in both ECR and FCR reflexes (Aymard et al. 1995;Fig. 1). However, Crone et al. (1990) emphasised that the amount of inhibition also depends on the unconditioned H reflex amplitude, and the size of the unconditioned ECR H reflex was lower than the FCR H reflex. It could be argued that the differential effects of tDCS on reciprocal inhibition directed from ECR to FCR and from FCR to ECR could be at least partly due to differences in FCR and ECR unconditioned reflex amplitude. However, we found a significant linear correlation between the ratio of unconditioned ECR H reflex/unconditioned FCR H reflex and the ratio of conditioned ECR H reflex/conditioned FCR H reflex indicating that the level of reciprocal inhibition in ECR and FCR would be equal if unconditioned H reflexes evoked in ECR and FCR were similar. This confirms that the effects of anodal tDCS applied over the contralateral hand motor area cannot be explained by the difference in size of conditioned reflexes. Moreover, differences between unconditioned reflex amplitudes may impact on quantitative comparisons but would not influence the qualitative differences (facilitation vs inhibition). Therefore, it is more likely that the reciprocal inhibition behaviour following tDCS strongly reflects differences in the excitability of interneurones.

Among the 32 subjects having an FCR H reflex, 23 were right-handed and 9 left-handed, whereas among the 9 subjects having an ECR H reflex, only 4 were left-handed. Therefore, the comparison between left-handed and right-handed subjects, were restricted to reciprocal inhibition directed from ECR to FCR.

Discussion

The main finding of the present study is that anodal tDCS applied over the contralateral hand motor cortex increases reciprocal inhibition directed from wrist extensors to wrist flexors but decreases reciprocal inhibition from wrist flexors to wrist extensors.

Opposite effects of active tDCS applied to the contralateral hand motor cortex

Since the position and stimulation characteristics of the active tDCS electrode were identical, when we tested reciprocal inhibition from ECR to FCR and from FCR to ECR, hand motor cortex excitability was the same for both directions of reciprocal inhibitions. The most striking finding of the present series of experiments is that increasing the excitability of the same motor cortex area, induces an increase of reciprocal inhibition from ECR to FCR but a decrease in reciprocal inhibition from FCR to ECR. Evidence from both animal and human studies (Purpura and McMurtry 1965;Priori et al. 1998), shows that anodal tDCS applied over the motor cortex decreases the membrane resting potential of cortical cells, which are spontaneously active in M1 [START_REF] Evarts | Motor Control, part 2[END_REF]. In our previous study (Roche et al. 2009), we proposed that the enhancement of the reciprocal inhibition from ECR to FCR, following tDCS, was due to an increase in efficiency of the descending volley reaching the interneurones that mediate reciprocal inhibition from ECR to FCR. To understand the opposing effects of interneurones that mediate reciprocal inhibition from ECR to FCR and vice-versa, following the same changes in motor cortex excitability, we propose to take into account the mutual inhibition between opposite-side interneurones that mediate reciprocal inhibition, for example an FCR interneurone which mediates reciprocal inhibition from FCR to ECR also inhibits the interneurone which mediates reciprocal inhibition from ECR to FCR as depicted in Fig. 6. In animal studies, mutual inhibition between opposite-side interneurones has been identified as a general central nervous system mechanism. Baldissera et al. (1987) also demonstrated that mutual inhibition exists in humans at the wrist level. Thus, we could propose two hypothetical mechanisms: i) that the descending control acting on interneurones that mediate reciprocal inhibition is asymmetrical and is concentrated on interneurones that mediate reciprocal inhibition from ECR to FCR (Fig. 6A), ii) the reciprocal inhibition at spinal level between ECR and FCR is asymmetrical (Fig. 6B).

The first hypothesis is related to a study by Marteens de Noordhout et al. (1999), suggesting that the cortico-motoneuronal synaptic connections are stronger on wrist and finger extensor motoneurones than on flexor motoneurones. Lundberg and collaborators have shown in the cat that there is a corticospinal parallel control of motoneurones and of the corresponding reciprocal Ia interneurones, i.e. the interneurones that mediate reciprocal inhibition to antagonistic motoneurones [START_REF] Lundberg | The excitatory control of the Ia inhibitory pathway[END_REF]. Although the interneurone which mediates reciprocal inhibition at the wrist level exhibits characteristics different from that of Ia interneurones, it might thus be hypothesised that there is parallel descending control of ECR motoneurones and of interneurones that mediate reciprocal inhibition from ECR to FCR (and similarly from FCR motoneurones and interneurones that mediate inhibition from FCR to ECR). If the descending control acting on ECR and interneurones that mediate reciprocal inhibition from ECR to FCR, is stronger than the descending control acting on FCR, the excitability of the interneurones that mediate inhibition from ECR to FCR (the ECR interneurone in Fig. 6) is increased more than that of the interneurones that mediate reciprocal inhibition from FCR to ECR (the FCR interneurone in Fig 6). As a consequence, mutual inhibition between the two opposite-side interneurones is more strongly directed to the FCR interneurone than ECR interneurone. Thus, the net effect of the descending control and mutual inhibition is an increase in the excitability of the ECR interneurone. For the FCR interneurone, its descending control is weaker and leads to the reverse situation: the weaker facilitatory descending control is counteracted by greater mutual inhibition. Hence, this results in a decrease in FCR interneurone excitability.

The second hypothesis is shown in Fig. 6B. The descending control from the hand motor cortex projecting to the ECR and FCR interneurones is similar, but the reciprocal inhibition and the mutual inhibition at the spinal level are asymmetric. The reciprocal inhibition is more strongly directed from ECR interneurones to FCR motoneurones than in the reverse situation. This hypothesis is supported by results observed at the lumbar level indicating that reciprocal inhibition is asymmetric: the reciprocal Ia inhibition from extensors to flexors is more powerful than the reciprocal Ia inhibition from flexors to extensors (R. Eccles and Lundberg 1958;[START_REF] Crone | Reciprocal Ia inhibition between ankle flexors and extensors in man[END_REF]. Moreover, the activity of spinal circuits is often asymmetric with regard to target motoneurones, for example, the monosynaptic reflex in FCR is stronger than that in ECR. In this case, when the mutual inhibition is more powerful on FCR interneurones than on ECR interneurones, the net effect on FCR interneurones is inhibitory whereas the effect on ECR interneurones is facilitatory.

However, the indirect methods we used do not allow us to choose between these two hypotheses. Furthermore both asymmetrical descending control and asymmetrical reciprocal inhibition may coexist.

Effects of tDCS applied to the ipsilateral hand motor cortex

The possible effects of tDCS applied to ipsilateral hand motor cortex on spinal networks may originate i) from ipsilateral connections to spinal neurones; or ii) hemispheric connections from ipsilateral to contralateral homologous motor cortex areas.

Although the existence of uncrossed corticospinal tracts from motor cortex to spinal motoneurones has been established in healthy humans and higher primates [START_REF] Kuypers | Anatomy of the descending pathways[END_REF], the effects of activation of the ipsilateral cortex on spinal motoneurones are still under debate.

A few papers have reported ipsilateral responses in distal muscles during strong voluntary contractions and high intensity TMS (Wassermann et al. 1991;Ziemann et al. 1999). Bawa et al. (2004) concluded that, in general, forearm and hand muscles in healthy subjects, did not show any ipsilateral motor evoked potentials. However, in post-stroke patients, ipsilateral responses have been recorded (Alagona et al. 2001).

Interhemispheric connections between homologous cortical areas are well known and generally considered to be inhibitory although the presence of excitatory interhemispheric connections have also been proposed [START_REF] Bloom | The role of the corpus callosum in interhemispheric transfer of information: excitation or inhibition?[END_REF]. Furthermore, several studies (for example, Hervé et al. 2005) suggest that ipsilateral control may be different in lefthanded and right-handed subjects. In the present study, we were unable to find significant effects with ipsilateral anodal tDCS. If anything, it seems that a small decrease of reciprocal inhibition from ECR to FCR may be occurring in the period 1, more marked in left-handed than in right-handed subjects. Even if this decrease is inconsistent or a small decrease, this effect may have therapeutic consequences. Indeed, it might be expected that coupling anodal contralateral tDCS and cathodal ipsilateral tDCS might facilitate the effects of contralateral anodal tDCS by excluding possible inhibitory effects from ipsilateral cortex.

Functional significance and possible therapeutic applications

Our findings have shown that an increase of excitability of contralateral hand motor cortex results in an increase of reciprocal inhibition to FCR motoneurones, and in contrast a decrease of the reciprocal inhibition to ECR motoneurones. In other words, it seems likely that the function of this descending control is to facilitate ECR motoneurone excitability by reducing inhibitory influences and to depress the excitability of FCR motoneurones by enhancing inhibitory influences. Fine motor hand movements in humans (grasping, writing, typing, etc.) require ECR contraction to stabilise the wrist joint. The descending control acting on reciprocal inhibition is likely to contribute to the facilitation of ECR contraction. In this case, anodal tDCS may be useful for the treatment of patients with cortical lesions who show wrist extensor deficit. It might also be used to facilitate ECR contraction and promote control of hand movements. This therapeutic approach is based on papers published since 2008 (for review see Ayache et al. 2012), that indicate that anodal tDCS applied to the lesioned hemisphere may improve motor performance.

Recent studies performed in stroke patients suggested that cathodal or dual tDCS may reduce spasticity in upper limb muscles [START_REF] Vandermeeren | Could dual-hemisphere transcranial direct current stimulation (tDCS) reduce spasticity after stroke?[END_REF][START_REF] Wu | Effects on decreasing upperlimb poststroke muscle tone using transcranial direct current stimulation: a randomized sham-controlled study[END_REF]. Cathodal tDCS was not tested in the present study but our findings that reveal an increase of reciprocal inhibition directed from ECR to FCR, suggest that contralateral anodal tDCS may be used to reduce flexor spasticity in stroke patients. Indeed, Nakashima et al. (1989) showed that reciprocal inhibition between wrist muscles is decreased in spastic patients. Therefore, it may be hypothesised that a reinforcement of reciprocal inhibition directed to flexor motoneurones may limit the hyper-excitability of the FCR stretch reflex.

Moreover, even though it is unconsistent or small decrease, the ipsilateral effect may also have some therapeutic consequences. Indeed, it could be expected that coupling anodal contralateral tDCS and cathodal ipsilateral tDCS may facilitate the effects of contralateral anodal tDCS by excluding possible inhibitory effects from ipsilateral cortex. Experiments lasted about 40 minutes divided in 4 periods of 10 minutes: 10 minutes before the beginning of tDCS baseline period, period 1 tDCS, period 2 tDCS and 10 minutes after the end of tDCS (post tDCS). Current flowed continuously for 20 minutes for both active conditions divided into two 10 minute periods (period 1 tDCS and period 2 tDCS), and for 120 s at the beginning of period 1 tDCS and 30 sec at the end of period 2 tDCS in the sham condition. Conditioned and unconditioned H reflexes were recorded continuously during each period of 10 minutes. Indeed a sequence of 40 H reflexes (20 conditioned H reflexes and 20 unconditionned H reflexes randomly alternated) lasted 3 minutes so 3 sequences of 40 H reflexes were done in each period (Pperiod = 0.152, Pcondition = 0.071, Pperiod X condition = 0.967) Table 1 Two ways repeated measures ANOVA were performed to compare effects of time period (baseline, period 1, period 2 and post tDCS) and effects of tDCS condition (sham, active contralateral and active ipsilateral) on amount of reciprocal inhibition and unconditioned H reflex amplitudes.

  Hanna-Boutros et Sina Sangari. Merci pour leur soutien et l'ambiance sympathique qu'ils apportent au laboratoire. Toutes les personnes qui ont été les sujets des expériences. Patients, membres du laboratoire, stagiaires, collègues Rimoises : Emmanuelle Girard et Rozenn Derrien, collègues thaïlandais : Ornwijit Chupetch, Pattarawan Sriwatcharin, Somphop Srisitthipaiboon et Alexandre-Pattana Toussaint. Je les remercie vivement pour le temps et la confiance qu'ils m'ont accordés. A ma mère, pour son amour indéfectible, son soutien permanent, et la confiance qu'elle a mise en moi, ขอบคุ ณแม่ ที ่ คอยให้ ก ำลั งใจและสนั บสนุ นลู กสำวคนนี ้ มำตลอด รั กแม่ ค่ ะ A ma famille, à mes amis de Thaïlande et de France, et à Arruck Merci de votre soutien régulier au cours des ces années passées en France ; votre présence morale m'a beaucoup aidée pour atteindre mes objectifsTable of ContentsIntroduction ......................................................................................................................... Chapter 1 ............................................................................................................................. Background of physiology .................................................................................................. 1. Presynaptic inhibition of Ia fibres ............................................................................ 1.1 Inputs to PAD interneurons 1.1.1 Peripheral inputs to PAD interneuron 1.1.2 Descending inputs to PAD interneuron 2. Postsynaptic inhibition induced by cutaneous stimulation ...................................... 3. Post-activation depression ..................................................................................... 4. Reciprocal inhibition .............................................................................................. 4.1 Inputs to non-reciprocal interneurons 4theory .............................................................................................................

Figure 1 :

 1 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition.Reproduced fromFrank & Fuortes, 1957.

Figure 2 :

 2 Figure 2 : Sketch of presynaptic inhibition of Ia fibre.

Figure 3 :

 3 Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. First-order PAD INs receive excitation from Ia and Ib afferents and the VS pathway (2). They receive inhibition through the same inhibitory INs from cutaneous afferents and the CS pathway (however, there is also facilitation effect from corticospinal pathway on first-order PAD INs, indicated by the thin line). Inhibitory INs inhibiting firstorder PAD INs receive descending tonic inhibition (dotted line (1)). Last-order PAD INs receive inhibition from the RS pathways, themselves inhibited from higher centres (3). Adapted from Pierrot-Deseilligny & Burke, 2012. VS = vestibulospinal pathway, CS = corticospinal pathway, RS = reticulospinal pathways

Figure 4 :

 4 Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.

Figure 5 :

 5 Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.

Figure 6 :

 6 Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level. FCR-coupled group I inhibitory interneurons receive their excitatory group I input from FCR and ECR Ia and Ib afferents (projections from ECR Ib afferents have been omitted), are not inhibited from Renshaw cells, and project to ECR motoneurons and ECR-coupled group I inhibitory interneurons (inhibitory projections of ECR-coupled group I inhibitory interneurons to FCR-coupled group I interneurons have been omitted). Renshaw cells activated by recurrent collaterals from FCR motoneurons project to both FCR and ECR motoneurons. Reproduced from Wargon et al., 2006. Ia = Ia fibre afferents, Ib = Ib fibre afferents, FCR = flexor carpi radialis, ECR= extensor carpi radialis, IN = interneuron, CS = Corticospinal control

Figure 7 :

 7 Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon. Reproduced from Hodgkin and Katz's work in 1949.

Figure 8 :

 8 Figure 8 : Control of cytosolic CI-in different cell type. Reproduced from Alvarez-Leefmans & Delpire, 2009.

Figure 9 :

 9 Figure 9 : The expression of NKCC1 and KCC2 in neurons. (a) Early expression of NKCC1 and (b) Late expression of KCC2 determine the change in chloride intracellular concentration in response to GABA and glycine during development. Glycinergic and GABAergic synapses are first excitatory before become inhibitory during develoment while a transection of the spinal cord does not allow this transformation. Adapted fromBen-Ari, 2002; Jean-Xavier et al, 2006.

Figure 10 :

 10 Figure 10 : Sketch of pathway of the monosynaptic reflex.

Figure 11 :

 11 Figure 11 : Example of M response and H-reflex in the soleus muscle.

Figure 12 :

 12 Figure 12 : Example of M response and H-reflex in FCR muscle.

Figure 13 :

 13 Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 14 :

 14 Figure 14 : Orderly recruitment of motoneuron (MNs).A) Given Ia input, the size of monosynaptic Ia EPSP decreases while motoneuron size increases. The dotted line represents the threshold for discharge of the MNs. Only the two smallest motoneurons (black) are fired by the test Ia volley. B) When an excitatory input is applied, there is a summation of the conditioning EPSP (thin line) and test (thick line) EPSP. As a result, MNs which had failed to discharge in the control reflex are raised to firing threshold and the size of the reflex is increased. C) When an inhibitory input is applied, there is a summation of the conditioning IPSP (thin line) and test (thick line) EPSP. As a result, MNs which had been recruited in the control reflex cannot be discharged, thus the size of the reflex in reduced. Reproduced from Pierrot -Deseilligny &Burke, 2012

Figure 15 :

 15 Figure 15 : Schematic of Sensitivity of the different size of the H-reflex.

Figure 16 :

 16 Figure 16 : Time course of presynaptic inhibition induced by conditioning stimulus of common peroneal nerve.

Figure 17 :

 17 Figure 17 : Schematic diagram of the presynaptic inhibition of soleus Ia fibres and example of waveforms of the soleus unconditioned H-reflex and the soleus conditioned H-reflex induced by conditioning stimulus of common peroneal nerve. (A) The conditioning stimulus induces the afferent volley in the tibialis anterior (TA) Ia afferent fibres and activates primary afferent depolarization (PAD) interneurons responsible for the presynaptic inhibition of Ia soleus fibres before the synapse with the soleus motoneuron. Adapted from Pierrot-Deseilligny & Burke, 2012. (B) Examples of waveforms of the soleus unconditioned H-reflex (left), and the soleus conditioned H-reflex (right) when preceded by the TA conditioning stimulus. The arrow indicates the trigger of the test stimulation. The conditioning stimulus consists of a train of 3 rectangular pulses, each of 1 ms duration, with an intensity 1.2 times the TA motor threshold (MT), delivered 21 ms before the test stimulation (see the beginning of the trace).

Figure 18 :

 18 Figure 18 : Time course of postsynaptic inhibition induced by cutaneous stimulation applied at the lateral side the fifth toe.

Figure 19 :

 19 Figure 19 : Schematic diagram of the postsynaptic inhibition induced by cutaneous stimulation and example of waveforms of the soleus unconditioned H-reflex, and the soleus conditioned H-reflex induced by conditioning stimulus of cutaneous afferents of sural nerve. (A) A conditioning stimulus induces the afferent volley in the cutaneous/nociceptive fibres (dotted line) in the sural nerve from the skin of the lateral side of the fifth toe activating spinal interneurons (INs) projecting onto the soleus motoneuron. Adapted from Pierrot-Deseilligny & Burke, 2012. (B) Examples of waveforms of the soleus unconditioned H-reflex, and the soleus conditioned H-reflex by electrical stimuli applied to cutaneous afferents of the sural nerve evoking postsynaptic inhibition. The conditioning stimulus consists of trains of 17 pulses, each of duration 1 ms, with an interpulse interval of 3 ms, delivered 50 ms before the test stimulus. The arrow indicates the triggering of the test stimulus.

Figure 20 :

 20 Figure 20 : Examples of waveforms of the soleus H-reflex preceded by another soleus H-reflex at 1 Hz and at 0.16 Hz

Figure 21 :

 21 Figure 21 : Example of a recording chain to study the pre-and postsynaptic inhibition in the lower limb with a soleus H-reflex.

Figure 22 :

 22 Figure 22 : Schematic diagram of the reciprocal inhibition at the wrist level and example of waveforms of the unconditioned FCR H-reflex, and the FCR conditioned H-reflex induced by conditioning stimulus of radial nerve. (A) A conditioning stimulus applied to the radial nerve induces the afferent volley in extensor carpi radialis (ECR) (dotted line) activating interneurons (IN) of reciprocal inhibition projecting on flexor carpi radilais (FCR) motoneurons. Adapted from Pierrot-Deseilligny & Burke, 2012. (B) Examples of waveforms of the unconditioned Hreflex, and the conditioned H-reflex by electrical stimuli applied to the radial nerve evoking reciprocal inhibition using a rectangular pulse with a duration of 1 ms, with an intensity below the ECR motor threshold (MT).

  Each experiment comprised seven time epochs(0-10 min, 11-20 min, 21-30 min, 31-40 min, 41-50 min, 51-60 min, and 61-70 min). The amount of pre-/postsynaptic inhibition evaluated at the beginning of recording (during 10 minutes before drug administration in the furosemide experiment, or 10 minutes before the test start time in the control experiment) corresponds to baseline inhibition. After the amount of inhibition was determined for the baseline period, 40 mg furosemide was orally administered.

Figure 23 :

 23 Figure 23 : Schematic diagram of experimental procedure of the control or furosemide experiments.

Figure 24 :

 24 Figure 24 : Time course of presynaptic inhibition without furosemide in healthy subjects. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 25 :

 25 Figure 25 : Time course of postsynaptic inhibition without furosemide in healthy subjects. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 26 :

 26 Figure 26 : Time course of presynaptic inhibition with furosemide in healthy subjects. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 27 :

 27 Figure 27 : Time course of presynaptic inhibition with and without furosemide in a healthy individual subject.The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time.The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 28 :

 28 Figure 28 : Histogram of time course of presynaptic inhibition with and without furosemide in all healthy subjects. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 29 :

 29 Figure 29 : Time course of postsynaptic inhibition with furosemide in healthy subjects. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 30 :

 30 Figure 30 : Time course of postsynaptic inhibition with and without furosemide in a healthy individual subject. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 31 :

 31 Figure 31 : Histogram of time course of postsynaptic inhibition with and without furosemide in all healthy subjects. The histogram shows the amounts of postsynaptic inhibition observed from control (□) and furosemide (■) experiment during 70 minutes. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 32 :

 32 Figure 32 : Effects of two different doses of furosemide on presynaptic inhibition in healthy subjects. The histogram shows the amount of presynaptic inhibition at different doses (0, 20, and 40 mg) in six healthy subjects, observed over a period of 40-70 minutes after furosemide administration where the effects were mostly observed. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 33 :

 33 Figure 33 : Effects of two different doses of furosemide on postsynaptic inhibition in healthy subjects. The histogram shows the amount of postsynaptic inhibition at different doses (0, 20, and 40 mg) in six healthy subjects, observed over a period of 40-70 minutes after furosemide administration where the effects were mostly observed. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 34 :

 34 Figure 34 : Histogram of time course of monosynaptic excitatory transmission (post-activation depression) with and without furosemide in all healthy subjects.The histogram shows post-activation depression value determined using 1/0.16 ratio observed from control (□) and furosemide (■) experiment during 70 minutes. The post-activation deprression value was normalized to the percentage of its baseline value and plotted against the recording time. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 35 :

 35 Figure 35 : The average degree of presynaptic inhibition evaluated before drug administration (baseline) in healthy subjects and SCI patients. The histogram shows the degree of presynaptic inhibition observed at the beginning of recording in 19 healthy subjects and 10 patients with SCI. The presynaptic inhibition values presented in the histogram are defined using a following equation: 100 -((conditioned H reflex/unconditioned H reflex) × 100). Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 36 :

 36 Figure 36 : The average degree of postsynaptic inhibition evaluated before drug administration (baseline) in healthy subjects and SCI patients. The histogram shows the degree of postsynaptic inhibition observed at the beginning of recording (baseline) in 15 healthy subjects and 10 patients with SCI. Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 37 :

 37 Figure 37 : Time course of presynaptic inhibition with furosemide in SCI patients. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 38 :

 38 Figure 38 : Histogram of time course of presynaptic inhibition with and without furosemide in all healthy subjects and SCI patients. The presynaptic inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

  Figure x. represents the postsynaptic inhibition recorded over 70 minutes after furosemide administration in the 4 patients.

Figure 39 :

 39 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. The inhibition value was normalized to the percentage of its baseline value and plotted against the recording time. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 40 :

 40 Figure 40 : Diagram of recording of tDCS study.

  Effect of anodal tDCS of the non-dominant right hemisphere on reciprocal inhibition in the non-dominant upper limb is observed in right-handed healthy subjects. Figure41represents the results obtained from 11 right-handed subjects in active condition, and 7 right-handed subjects in sham condition.

Figure 41 :

 41 Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

  Roche et al., (2009) have shown in healthy right-handed subjects that tDCS of the dominant left motor cortex induced net increase of reciprocal inhibition of the dominant upper limb. In the present study, we demonstrated effect of tDCS of the nondominant right motor cortex on reciprocal inhibition recorded in the non-dominant upper limb in 11 healthy right-handed subjects. The present observations are compared with the results from Roche et al., 2009 performed in 13 healthy right-handed subjects (Figure 42).

Figure 42 :

 42 Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).The figure in the right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

  represents results of an individual subject who participated in the study ofRoche et al., 2009 and in our present study.

Figure 43 :

 43 Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM).

Figure 44 :

 44 Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the upper right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

Figure 45 :

 45 Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the upper right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

Figure 46 :

 46 Figure 46 : The degree of reciprocal inhibition in healthy subjects and stroke patients. The reciprocal inhibition values presented in the histogram are defined using a following equation: 100 -((conditioned H reflex/unconditioned H reflex) × 100). Vertical bars represent the standard error of the mean (± 1 SEM). Asterisks () represent significant values (* P < 0.05, ** P <0.01, *** P<0.001).

Figure 47 :

 47 Figure 47 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the upper right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

Figure 48 :

 48 Figure 48 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the upper right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

Figure 49 :

 49 Figure 49 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the upper right shows the side (larger one), which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

  The comparison between variations of reciprocal inhibition demonstrated by contralateral tDCS in healthy subjects and hemiplegic patients are presented in Figure50. The reciprocal inhibitions recorded in the dominant limb in 13 healthy subjects (results fromRoche et al., 2009), the non-dominant limb in 11 healthy subjects and the non-paretic limbs in 5 stroke patients (the dominant upper limb n=2, the non-dominant upper limb n=3).

Figure 50 :

 50 Figure 50 : Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the nondominant upper limb in healthy subjects and in the non-paretic upper limb in stroke patients. The inhibition value was normalized as a percentage of its baseline value and plotted against the time period. The dotted line represents the level of baseline value. Vertical bars represent the standard error of the mean (± 1 SEM). The figure in the upper right shows the side, which the reciprocal inhibition was recorded and the hemisphere that was stimulated (red point).

Figure 51

 51 Figure 51 Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. MN = motoneuron, VS = the vestibulospinal tract, CS = the corticospinal tract, RS = reticulospinal tract. Pathway (1) = tonic control from the brainstem. Adapted from Pierrot-Deseilligny & Bruke, 2012.

Figure 1 :

 1 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 2 :

 2 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 3 :

 3 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 4 :

 4 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 5 :

 5 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 6 :

 6 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 7 :

 7 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 8 :

 8 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 9 :

 9 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 10 :

 10 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 11 :

 11 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 12 :

 12 Figure 1 : Response in motoneurons in the spinal cord corresponds with presynaptic inhibition. .Figure 2 : Sketch of presynaptic inhibition of Ia fibre.Figure 3 : Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat.Figure 4 : Intracellular recording of a cat motoneuron the EPSP generated monosynaptically in gastrocnemius motoneuron following repetitive activation of Ia afferent of tibial nerve.Figure 5 : Size of the soleus H-reflex preceded by difference conditioning stimuli activating soleus Ia fibres.Figure 6 : Pathways of non-reciprocal group I inhibition at wrist level.Figure 7 : The relationship of the height of the spike and the ratio of the concentration of Na inside and outside the axon.Figure 8 : Control of cytosolic CI-in different cell type. .Figure 9 : The expression of NKCC1 and KCC2 in neurons.Figure 10 : Sketch of pathway of the monosynaptic reflex.Figure 11 : Example of M response and H-reflex in the soleus muscle.Figure 12 : Example of M response and H-reflex in FCR muscle.Figure 13 : H-reflex and M-wave recruitment curve. Maximal M wave (Mmax)

Figure 15 :

 15 Figure 14 : Orderly recruitment of motoneuron (MNs).Figure 15 : Schematic of Sensitivity of the different size of the H-reflex.Figure 16 : Time course of presynaptic inhibition induced by conditioning stimulus of common peroneal nerve.Figure 17 : Schematic diagram of the presynaptic inhibition of soleus Ia fibres and example of waveforms of the soleus unconditioned H-reflex and the soleus conditioned H-reflex induced by conditioning stimulus of common peroneal nerve.Figure18: Time course of postsynaptic inhibition induced by cutaneous stimulation applied at the lateral side the fifth toe.Figure19: Schematic diagram of the postsynaptic inhibition induced by cutaneous stimulation and example of waveforms of the soleus unconditioned H-reflex,

Figure 17 :

 17 Figure 14 : Orderly recruitment of motoneuron (MNs).Figure 15 : Schematic of Sensitivity of the different size of the H-reflex.Figure 16 : Time course of presynaptic inhibition induced by conditioning stimulus of common peroneal nerve.Figure 17 : Schematic diagram of the presynaptic inhibition of soleus Ia fibres and example of waveforms of the soleus unconditioned H-reflex and the soleus conditioned H-reflex induced by conditioning stimulus of common peroneal nerve.Figure18: Time course of postsynaptic inhibition induced by cutaneous stimulation applied at the lateral side the fifth toe.Figure19: Schematic diagram of the postsynaptic inhibition induced by cutaneous stimulation and example of waveforms of the soleus unconditioned H-reflex,

Figure 18 :

 18 Figure 14 : Orderly recruitment of motoneuron (MNs).Figure 15 : Schematic of Sensitivity of the different size of the H-reflex.Figure 16 : Time course of presynaptic inhibition induced by conditioning stimulus of common peroneal nerve.Figure 17 : Schematic diagram of the presynaptic inhibition of soleus Ia fibres and example of waveforms of the soleus unconditioned H-reflex and the soleus conditioned H-reflex induced by conditioning stimulus of common peroneal nerve.Figure18: Time course of postsynaptic inhibition induced by cutaneous stimulation applied at the lateral side the fifth toe.Figure19: Schematic diagram of the postsynaptic inhibition induced by cutaneous stimulation and example of waveforms of the soleus unconditioned H-reflex,

Figure 19 :

 19 Figure 14 : Orderly recruitment of motoneuron (MNs).Figure 15 : Schematic of Sensitivity of the different size of the H-reflex.Figure 16 : Time course of presynaptic inhibition induced by conditioning stimulus of common peroneal nerve.Figure 17 : Schematic diagram of the presynaptic inhibition of soleus Ia fibres and example of waveforms of the soleus unconditioned H-reflex and the soleus conditioned H-reflex induced by conditioning stimulus of common peroneal nerve.Figure18: Time course of postsynaptic inhibition induced by cutaneous stimulation applied at the lateral side the fifth toe.Figure19: Schematic diagram of the postsynaptic inhibition induced by cutaneous stimulation and example of waveforms of the soleus unconditioned H-reflex,

Figure 20 :

 20 Examples of waveforms of the soleus H-reflex preceded by another soleus H-reflex at 1 Hz and at 0.16 Hz Figure 21 : Example of a recording chain to study the pre-and postsynaptic inhibition in the lower limb with a soleus H-reflex.

Figure 22 :

 22 Schematic diagram of the reciprocal inhibition at the wrist level and example of waveforms of the unconditioned FCR H-reflex, and the FCR conditioned Hreflex induced by conditioning stimulus of radial nerve.

Figure 23 :

 23 Schematic diagram of experimental procedure of the control or furosemide experiments.

Figure 39 :

 39 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. Figure 40 : Diagram of recording of tDCS study.Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects.Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects.Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject.Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects.Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.Figure46: The degree of reciprocal inhibition in healthy subjects and stroke patients.Figure47: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.Figure48: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.Figure49: Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.Figure50: Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.Figure51Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

Figure 41 :

 41 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. Figure 40 : Diagram of recording of tDCS study.Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects.Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects.Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject.Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects.Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.Figure46: The degree of reciprocal inhibition in healthy subjects and stroke patients.Figure47: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.Figure48: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.Figure49: Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.Figure50: Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.Figure51Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

Figure 42 :

 42 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. Figure 40 : Diagram of recording of tDCS study.Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects.Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects.Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject.Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects.Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.Figure46: The degree of reciprocal inhibition in healthy subjects and stroke patients.Figure47: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.Figure48: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.Figure49: Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.Figure50: Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.Figure51Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

Figure 43 :

 43 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. Figure 40 : Diagram of recording of tDCS study.Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects.Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects.Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject.Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects.Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.Figure46: The degree of reciprocal inhibition in healthy subjects and stroke patients.Figure47: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.Figure48: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.Figure49: Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.Figure50: Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.Figure51Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

Figure 44 :

 44 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. Figure 40 : Diagram of recording of tDCS study.Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects.Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects.Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject.Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects.Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.Figure46: The degree of reciprocal inhibition in healthy subjects and stroke patients.Figure47: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.Figure48: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.Figure49: Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.Figure50: Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.Figure51Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

Figure 45 :

 45 Figure 39 : Time course of postsynaptic inhibition with furosemide in SCI patients. Figure 40 : Diagram of recording of tDCS study.Figure 41 : Effect of contralateral anodal tDCS on reciprocal inhibition in the non-dominant upper limbs of right-handed subjects.Figure 42 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in right-handed subjects.Figure 43 : Effect of contralaetral anodal tDCS on reciprocal inhibition in the dominant and the non-dominant upper limbs in an individual subject.Figure 44 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed subjects.Figure 45 : Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limbs in right-handed and left-handed subjects.Figure46: The degree of reciprocal inhibition in healthy subjects and stroke patients.Figure47: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the paretic upper limbs in stroke patients.Figure48: Effect of ipsilateral anodal tDCS on reciprocal inhibition in the dominant upper limb in healthy subjects and in the paretic upper limbs in stroke patients.Figure49: Effect of contralaetral anodal tDCS on reciprocal inhibition in the non-paretic upper limb in stroke patients.Figure50: Effect of contralaetral anodal tDCS on reciprocal inhibition observed in the dominant and the non-dominant upper limb in healthy subjects and in the nonparetic upper limb in stroke patients.Figure51Pathways of presynaptic inhibition with primary afferent depolarisation (PAD) of Ia terminals in the cat. .

  73 ± 0.02 mA (control experiment); 1.61 ± 0.1 mA (furosemide experiment)]. The intensity of the stimulus train was slowly increased to 3-5 times the perceptual threshold in order to induce a clear-cut inhibition of the soleus H-reflex [intensity mean value 4.33 ± 0.3 PT (control experiment); 3.79 ± 0.3 PT (furosemide experiment)]. Each stimulation sequence comprised three different conditions of 20 stimuli each: 1) test stimulus alone, 2) paired stimulation (test stimulus + conditioning stimulus to CPN evoking presynaptic inhibition), 3) paired stimulation (test stimulus + conditioning stimulus to the sural nerve inducing postsynaptic inhibition). Consequently, 40 conditioned and 20 unconditioned H-reflexes, evoked at 0.33 Hz, were determined during each time epoch. A complete sequence took about 3 minutes. Conditioned and unconditioned H-reflexes were randomly alternated.

  61-70 minutes (baseline vs. 21-30 minutes, baseline vs. 31-40 minutes, baseline vs. 41-50 minutes, baseline vs. 51-60 minutes, and baseline vs. 61-70 minutes (P<0.05, P<0.001, P<0.001, P<0.01, and P<0.001 respectively, Tukey test)).

Figure 1 :

 1 Figure 1: Schematic diagrams of the techniques used to test spinal inhibitions and the

Figure 2 :

 2 Figure 2: Time courses of the modifications of presynaptic inhibition and postsynaptic

Figure 3 :

 3 Figure 3: Time courses of the effects of furosemide on presynaptic inhibition of Ia fibres

Figure 4 :Figure 5 :

 45 Figure 4: Time courses of furosemide effects on post-activation depression (A) and

2

  Faculty of Physical Therapy, Mahidol University, 73170 Nakonpathom, Thailand 3 Univ. Versailles-Saint-Quentin, EA 4497, Garches, France 4 APHP Groupe Hospitalier Pitié-Salpêtrière -Service de Médecine Physique et Réadaptation 5 APHP Hôpital Raymond-Poincaré -Service d'00; Fax: +33 1 42 16 11 02; Email: alexandra.lackmy@upmc.fr Number of figures and tables: 6 figures and 1 table Number of pages: 35 pages including references and legends of Figs

Figure 1 :

 1 Figure 1: diagram adapted from Roche et al. (2009) illustrating the experimental

Figure 2 :

 2 Figure 2: representative example of unconditioned and conditioned H reflexes in ECR

Fig. 2

 2 Fig. 2 AB illustrates an unconditioned (A) and a conditioned H reflex (B) in ECR.

Fig. 2

 2 Fig. 2 CD illustrates an unconditioned (C) and a conditioned (D) H reflex in FCR. By

Figure 3 :

 3 Figure 3: Individual data showing modulation of the two types of reciprocal inhibition

Fig. 3AB :

 3AB Fig. 3AB: modulations of reciprocal inhibition from FCR to ECR when tDCS is applied to

Fig. 3CD :

 3CD Fig. 3CD: modulations of reciprocal inhibition from ECR to FCR when tDCS is applied over

Figure 4 :

 4 Figure 4: Group data representing modulations of the two types of reciprocal inhibition

Fig. 4A :

 4A Fig. 4A: modulations of reciprocal inhibition from FCR to ECR. Group data are calculated

Fig. 4B :

 4B Fig. 4B: modulations of reciprocal inhibition from FCR to ECR (taken from our previous

Figure 5 :

 5 Figure 5: Group data representing modulations of the two types of reciprocal inhibition

Fig. 5B :

 5B Fig. 5B: modulations of reciprocal inhibition from ECR to FCR. Amounts of inhibition are

Fig 5C :

 5C Fig 5C: Impact of handedness on effects induced by ipsilateral anodal tDCS on reciprocal

Figure 6 :

 6 Figure 6: schematic diagram of reciprocal inhibition between flexor and extensor:

Fig. 6B :

 6B Fig. 6B: asymmetric reciprocal inhibition at the spinal level. The thicker the lines, the

Table 1 :

 1 Figure1

the project 2 4.1 Electrophysiological recordings 4.1.1 Stimulation test: FCR H-reflex

Table 2 : Individual values (not-normalised) of pre-and postsynaptic inhibition in SCI patients

 2

	CHAPTER 3 RESULTS : PROJECT 2
	Project 2

Table 3 Individual raw data of reciprocal inhibition in all healthy subjects IPSILATERAL CONTRALATERAL ACTIVE SHAM ACTIVE SHAM Subjects Sex (F/M) Age (year) Dominant hand baseline per1 tDCS per2 tDCS post tDCS baseline per1 tDCS per2 tDCS post tDCS baseline per1 tDCS per2 tDCS post tDCS baseline per1 tDCS per2 tDCS post tDCS

 3

	1	F	R	19,63	31,95	28,78	15,17	35,90	38,76	45,02	42,05	23,53	29,54	23,81	30,23	33,84	29,89	23,30	41,05
	2	F	R	20,58	18,65	28,96	29,12	48,50	54,94	64,31	45,28	-	-	-	-	-	-	-	-
	3	M	R	39,77	45,75	41,13	46,37	28,16	34,65	33,19	38,16	-	-	-	-	-	-	-	-
	4	F	R	14,46	17,46	17,64	9,79	18,05	4,06	1,28	22,70	-	-	-	-	-	-	-	-
	5	M	R	16,92	17,81	17,27	7,78	30,18	31,18	30,50	33,47	-	-	-	-	-	-	-	-
	6	M	R	28,71	35,43	35,50	38,29	35,41	37,68	38,40	37,51	-	-	-	-	-	-	-	-
	7	F	R	27,90	25,33	31,28	36,99	26,68	23,68	34,65	23,37	-	-	-	-	-	-	-	-
	8	M	R	14,09	2,61	5,80	12,53	42,69	40,69	37,91	33,66	-	-	-	-	-	-	-	-
	9	F	R	39,54	47,22	41,05	31,24	26,68	23,68	34,65	23,37	-	-	-	-	-	-	-	-
	10	M	R	43,81	32,10	43,84	44,40	35,94	32,49	37,65	32,17	-	-	-	-	-	-	-	-
	11	M	R	24,98	19,31	17,47	20,32	16,25	11,71	17,36	19,89	18,29	40,09	33,22	26,72	18,81	19,38	20,41	26,90
	12	F	R	30,37	18,43	21,48	26,73	36,40	40,56	36,83	19,69	54,86	42,16	51,86	58,28	52,85	61,13	56,61	56,38
	13	M	R	18,61	9,71	4,93	5,93	28,73	37,93	44,58	39,24	47,46	47,65	53,16	44,55	21,08	34,82	24,09	14,65
	14	F	R	19,28	29,22	19,46	25,00	26,49	40,03	40,23	32,99	-	-	-	-	-	-	-	-
	15	M	R	6,41	2,74	13,13	2,97	24,02	19,45	18,65	38,37	-	-	-	-	-	-	-	-
	16	F	R	8,70	7,20	1,26	14,47	24,04	15,40	28,24	20,20	13,68	7,53	3,85	8,77	37,79	32,24	40,80	32,42
	17	F	R	9,52	12,35	13,86	1,19	35,94	32,49	37,65	32,17	10,96	3,76	12,28	16,30	-	-	-	-
	18	F	L	39,88	44,84	59,23	37,75	46,54	47,36	45,14	47,11	-	-	-	-	-	-	-	-
	19	F	L	26,01	20,69	21,21	29,47	53,73	66,22	59,23	46,86	38,14	43,17	47,55	57,33
	20	F	L	35,85	32,60	18,25	29,13	52,90	55,41	58,62	59,16	-	-	-	-	-	-	-	-
	21	F	L	15,65	11,93	15,09	5,83	-	-	-	-	-	-	-	-	-	-	-	-
	22	F	L	22,30	12,13	31,39	27,64	-	-	-	-	-	-	-	-	-	-	-	-
	23	F	L	16,98	4,07	-0,01	10,72	-	-	-	-	-	-	-	-	-	-	-	-
	24	F	L	29,97	20,91	16,30	15,68	-	-	-	-	-	-	-	-	-	-	-	-
	25	F	L	41,11	40,52	44,65	47,19	-	-	-	-	-	-	-	-	-	-	-	-
	26	F	R	40,99	36,71	43,98	49,24	-	-	-	-	-	-	-	-	-	-	-	-
	27	M	R	31,77	33,01	30,78	34,23	-	-	-	-	-	-	-	-	-	-	-	-
	28	F	R	38,08	30,28	32,77	22,79	-	-	-	-	-	-	-	-	-	-	-	-
	29	F	R	33,27	29,21	38,20	42,79					7,26	10,29	7,61	15,39
	30	M	R	11,51	-1,30	7,50	-3,39	-	-	-	-	25,00	11,42	15,47	26,18	-	-	-	-
	31	M	R	20,27	17,26	22,99	22,28	-	-	-	-	9,91	11,59	5,65	10,26	-	-	-	-
	32	M	R	-	-	-	-	-	-	-	-	27,95	35,24	26,22	33,15	-	-	-	-
	33	M	R	23,87	22,86	19,08	16,02					49,77	48,37	36,80	31,82

Table 4 Individual raw data of reciprocal inhibition in all stroke patients.

 4 Side of hemiplegia Dominant hand After stroke onset (month) Baseline Per1 tDCS Per2 tDCS Post tDCS Baseline Per1 tDCS Per2 tDCS Post tDCS Baseline Per1 tDCS Per2 tDCS Post tDCS Baseline Per1 tDCS Per2 tDCS Post tDCS

	IPSILATERAL		CONTRALATERAL
	ACTIVE	SHAM	ACTIVE	SHAM

Table :

 : Individual values (not-normalised) of pre-and postsynaptic inhibition, and post-activation depression in healthy subjects.

	List of Abbreviations
	ISI	Interstimulus interval
	KCC	Na+-CI-cotransporter
	LTD	Long-term depression
	LTP	Long-term potentiation
	M1 List of Abbreviations M max	Primary motor cortex Maximum motor response
	MEP	Motor evoked potential
	Acronym MN	Description Motoneuron
	MT	Motor threshold
	ADM NKCC	Abductor minimi muscle Na+-K+-2CI-cotransporter
	CCC PAD	Cation chloride cotransporter Primary afferent depolarization
	CIMT PSTH	Constraint-induced movement therapy Post-stimulus time histogram
	CS PT	Corticospinal control Perception threshold
	CNS PTN	Central nervous system Posterior tibial nerve
	CPN rTMS	Common peroneal nerve Repetitive transcranial magnetic stimulation
	E CI RS	Chloride equilibrium potential Reticulospinal pathways
	ECR Rw	Extensor carpi radialis Renshaw cell
	ECS SMA	Extracellular space Supplementary motor cortex
	EMG SCI	Electromyographic Spinal cord injury
	EPSP TA	Excitatory postsynaptic potentials Tibialis anterior
	FCR TEN	Flexor carpi radialis Transcranial electrical stimulation
	fMRI TMS	Functional magnetic resonance imaging Transcranial magnetic brain stimulation
	GABA TVR	Gamma aminobutyric acid Tonic vibration reflex
	H max tDCS	Maximum H-reflex response Transcranial direct current stimulation
	IHI PAD	Inter-hemispheric inhibition Primary afferent depolarization
	IN	Interneuron
	IPSI	Inhibitory postsynaptic potentials

Table : Individual values (not-normalised) of pre-and postsynaptic inhibition in SCI patients Table Individual raw data of reciprocal inhibition in healthy subjects Table Individual raw data of reciprocal inhibition in stroke patients.

Remerciements

Acknowledgments

The authors wish to express their gratitude to Dr Jacques Rottembourg for the use of furosemide, Max Westby (PhD) and Nachiket Nadkarni (PhD) for having scrutinized the manuscript.

Acknowledgements

The authors wish to express their gratitude to G.W. Max Westby (PhD) and to A. Hudson Australian research fellow in Neurosciences for having scrutinised the manuscript and to Mrs G. Bard, for collating references and getting the text into presentable order. This study was supported by grants from Sorbonne Universités UPMC Paris 6 (MESR, Ministère de l'Enseignement Supérieur et de la Recherche), Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance Publique-Hôpitaux de Paris (AP-HP), Institut pour la Recherche sur la Moelle Epinière (IRME 2012A00407-36) and Agence Nationale de la Recherche (ANR-12-JSV4-0007-01).

Funding

This work was supported by grants from ANR (FIHSC 2013(FIHSC -2014)), APHP (Hemipleg P070804), MESR (Er 6 UPMC), and CICIT 805. Jean-Charles Lamy is supported by a postdoctoral fellowship from FRM (SPF20101221068).

Chapter 2 Materials and methods

Additional information

Running title: spinal excitability under furosemide

Table of Contents Category: Neuroscience -developmental/plasticity/repair

Key Point Summary

• It has already been demonstrated in humans that furosemide crosses the blood-brain barrier and blocks activity in the epileptic brain.

• In this study, we demonstrated using non-invasive electrophysiological techniques in healthy human subjects that furosemide, a cation-chloride co-transporter blocker, orally-administered at doses commonly used in the clinic (40 mg), reduces the efficacy of pre-and postsynaptic inhibition of soleus motoneurons in the spinal cord.

• Furosemide can be a useful tool to detect the intrinsic functioning of inhibitory synapses and to explore in the future if the reduced inhibitory interneuronal activity which likely contributes to spasticity, also exists in humans with spinal cord injury. Words: 248

Word count: 103

Abstract