Effects of potassium supplementation were evaluated in four studies in endurance horses during races and treadmill exercise. In the first and second studies a potassium-free experimental formula was compared to potassium rich commercial formulas. The first study showed that supplementation increased plasma [K+], and that the extra sodium in the potassium-free experimental formulas helped to attenuate acidosis at the end of the ride. In the second study supplementation also increased plasma [K+], however speeds were lower and no increases were observed in plasma concentrations during the race. Supplementation of potassium during recovery helped to restore plasma [K+]. Higher plasma [Ca++] was found in horses supplied with experimental feeds, due to a lower dietary cation anion balance (DCAB). Three eliminated horses had heart rate arrhythmias and labile heart rates accompanied with higher plasma [K+] and lower [Ca++] than finishers. Also horses supplied with the experimental sodium-rich formula were less dehydrated than the ones receiving commercial formulas. The third study involved an 80 km endurance exercise test on the treadmill, and plasma [K+] was affected by potassium supplementation during exercise and recovery. The supply of potassium caused higher plasma [K+] helping to restore body stores. Also chloride supply in the electrolyte formulas maintained plasma [Cl-] levels during exercise and affected plasma concentrations during recovery. The fourth study showed that potassium supply affects plasma concentration, but also increases lactate production and glucose during sub-maximal exercise. A potassium-free electrolyte supply caused higher plasma [Ca++] during exercise. Higher sodium supply in the potassium-free electrolytes improved hydration during exercise. These studies show that potassium should supplemented after exercise and but not be done during exercise because of the risk of increased neuromuscular excitability. / Ph. D.

Mes travaux de thèse ont porté sur l’étude de deux modèles murins d’épilepsies génétiques de l’enfance liées à des mutations des canaux Nav1.1 (impliqués dans l’excitabilité des neurones inhibiteurs) : le Syndrome de Dravet (SD), une épilepsie pharmaco-résistante sévère, et l’Epilepsie Génétique avec Convulsions Fébriles Plus (EGCF+), présentant un phénotype plus modéré.Ils se sont décomposés en 3 axes : - La première partie mettant en évidence un phénomène d’épileptogenèse dans ces modèles murins.- La seconde permettant d’identifier des conditions expérimentales d’induction d'activités épileptiformes spécifiques du modèle murin du SD sur des tranches de cerveau.- La dernière consistant à mettre au point une stratégie pour réduire l’hyperexcitabilité cérébrale / During my thesis, I studied two murine models of childhood genetic epilepsies, caused by mutations of Nav1.1 channels (involved in the excitability of inhibitory neurons): Dravet Syndrome (DS), a severe and drug resistant epilepsy, and Genetic Epilepsy with Febrile Seizures Plus (GEFS+), characterized by a milder phenotype.My work is divided into three parts:- The first one revealed a process of epileptogenesis in these murine models.- In the second, I identified experimental conditions to induce epileptiform activities which are specific of the DS model in brain slices, which could allow pharmacological screens ex-vivo.- The third one was aimed at developing a new strategy to reduce cerebral hyperexcitability

The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts various neuromodulatory functions in mammalian brain. Enhancement of synaptic activity, mediation of chronic inflammatory and neuropathic pain, and neuroprotection in cerebral ischemia reperfusion injury constitute some of the exemplary functions of PACAP. However, it remains unclear whether PACAP signaling can directly influence the function of critical voltage-gated ion channels, which could profoundly alter the excitability of neurons. Voltage-gated K+ (Kv) channels are critical regulators of neuronal excitability. The major Kv channel in the dendrites of mammalian neurons, Kv4.2, contributes most of the fast-activating and rapidly-inactivating K+ currents (IA), and is a key regulator of dendritic excitability, as well as modulation of synaptic inputs. In addition, the major somatic Kv channel Kv2.1 that contributes the bulk of slow-activating and non-inactivating K+ currents (IK), acts as an integrator of neuronal inputs and limits high frequency firing in neurons. As such, it provides homeostatic control of excitability under hyperexcitable and ischemic conditions. Both these Kv channels are known to undergo extensive post-translational modifications mainly by phosphorylation that alters their localization and biophysical properties. PACAP can activate its specific receptor PAC1 that could result in downstream activation of various kinases including protein kinase A (PKA), protein kinase C (PKC), extracellular signal-regulated kinase (ERK1/2). Therefore, I hypothesize that PACAP activation of PAC1 receptor can cause phosphorylation-dependent modulation of somatodendritic Kv4.2 and Kv2.1 channels, resulting in altered neuronal excitability. First, I identified the various PAC1 receptor isoforms expressed in rat and mouse brain and elucidated that their activation by PACAP caused downstream PKA- and PKC-dependent signaling pathways, ultimately converging on ERK1/2 activation. Further, PACAP caused reduction in IA that was mediated by phosphorylation-dependent internalization of the channel protein from the plasma membrane. These effects were mediated by direct phosphorylation of the channel by ERK1/2 at the cytoplasmic C-terminus of the channel. Although PACAP did not significantly alter the voltage-dependence of Kv4.2 channel activation/inactivation, I observed distinct ERK1/2- and PKA-dependent changes in the extent and kinetics of channel inactivation. Next, I observed that PACAP induced dephosphorylation of the Kv2.1 channel in CHN that was mediated by protein phosphatase 2A (PP2A), and was dependent on PKC activation but was independent of the effects of PACAP on Kv4.2 currents. Rapid but reversible dephosphorylation of Kv2.1 was also observed following induction of ischemia in neurons by oxygen-glucose deprivation (OGD). PACAP prolonged the dephosphorylation of Kv2.1 following in vitro ischemia-reperfusion and also reduced neuronal death. My results therefore suggest a novel PACAP/PAC1-PKC-PP2A-Kv2.1 signaling axis that provides neuroprotection during ischemia reperfusion injury. In summary, my results suggest that PACAP can induce direct phosphorylation-dependent modulation of the Kv4.2 and Kv2.1 channel localization and function in mammalian brain neurons. The effect of PACAP on these two critical somatodendritic ion channels occurs via distinct signaling - convergent PKA-PKC-ERK-mediated phosphorylation of Kv4.2 channel, and PKC-PP2A-mediated dephosphorylation of the Kv2.1 channel. Such distinct modulations of these ion channels are presumably responsible for the multifarious roles of PACAP in the central nervous system.

En état de douleur chronique, la sensibilisation centrale s’accompagne d’une modification de l’équilibre excitation/inhibition en faveur d’une excitation accrue de la corne dorsale de la moelle épinière. Cet équilibre implique des molécules d’adhérence telles que les neuroligines postsynaptiques (NLs). Dans une première partie de notre travail de thèse, nous avons étudié la régulation éventuelle de ces protéines dans un modèle de douleur neuropathique (Spinal Nerve Ligation, SNL) chez le rat. Nos données ont montré une surexpression inattendue de la NL2, généralement associée à l’inhibition, alors que l’expression de la NL1, généralement associée à l’excitation, ne change pas. Le blocage de l’expression de NL2 in vivo par application intrathécale de siRNA, a produit des effets anti-nociceptifs réversant de façon significative l’allodynie mécanique observée chez les rats SNL. L’étude ultérieure des partenaires pré- et postsynaptiques de NL2, a démontré une co-variation spécifique avec PSD95, une protéine d’échafaudage des synapses excitatrices. De plus, une approche par co-immunoprécipitation a mis en évidence une augmentation significative des interactions protéiques NL2 /PSD95 chez les rats SNL. Enfin, cette association inhabituelle en condition neuropathique, est apparue liée à la surexpression spécifique de NL2(-), un variant d’épissage de NL2 normalement minoritaire en condition physiologique. La surexpression, l’augmentation d’association avec PSD95, et l’effet pro-nociceptif inattendu de la NL2 « inhibitrice » en condition de douleur neuropathique, indiquent une permutation fonctionnelle de la NL2 de l’inhibition vers l’excitation modifiant le rapport synaptique en faveur d’une excitation globale plus élevée dans la corne dorsale.Dans une deuxième partie du travail, nous avons exploré le rôle des molécules d’adhérence NLs dans la sensibilisation spinale associée à un autre type de douleur chronique, à savoir la douleur cancéreuse, sur un modèle de cancer de l’os chez le rat. L’étude de l’expression des NLs et de leurs partenaires, a montré une augmentation d’expression spécifique de la NL1 et de S-SCAM, une autre protéine d’échafaudage des synapses excitatrices. D’autre part, d’après la littérature, ce modèle se caractérise par une importante activation gliale dans les cornes dorsales de la moelle épinière, se traduisant notamment par une astrogliose massive. Cependant, nous avons montré que dans le modèle utilisé, il n’y avait aucune variation ni de marqueurs classiques de l’activation astrocytaire (GFAP, S100β), ni des marqueurs microgliaux (OX-42 et Iba1). Au contraire, tous ces paramètres étaient effectivement augmentés dans la corne dorsale ipsilatérale d’animaux neuropathiques. Ces résultats suggèrent que, contrairement à ce qui a été décrit précédemment, la douleur cancéreuse d’origine osseuse n’est pas nécessairement corrélée à une surexpression spinale des marqueurs de la glie réactive, tandis que la douleur neuropathique l’est.En conclusion, nos résultats obtenus dans le modèle de douleur cancéreuse montrent un phénotype concernant des molécules impliquées dans la formation, la spécification et la modulation des synapses, bien différent de celui que nous avons mis en évidence dans le modèle de douleur neuropathique. Nous montrons notamment dans les deux modèles, une implication bien distincte des molécules d’adhérence NLs et de la glie confortant les données de la littérature indiquant que ces deux grandes catégories de douleur chronique ont chacune une signature propre. De plus, nos résultats ouvrent la perspective d’identifier de nouveaux diagnostics et/ou de nouvelles possibilités thérapeutiques, en ciblant spécifiquement les NLs. / In chronic pain states, central sensitization is associated with a modification in the excitation/inhibition balance toward increased excitation in the spinal dorsal horn. This balance involves adhesion molecules such as the postsynaptic Neuroligins (NLs). In a first part of our thesis work, we investigated the putative regulation of these proteins in the Spinal Nerve Ligation (SNL) model of neuropathy in the rat. Our data showed an unexpected upregulation of NL2, usually associated to inhibition, whereas expression of NL1, usually associated to excitation, did not change. The in vivo expression blockade of NL2 by intrathecal injection of siRNAs, produced specific antinociceptive effects, significantly reversing the SNL-induced mechanical allodynia. Subsequent study of pre- and postsynaptic NL2 partners, demonstrated a specific co-variation with PSD95, a scaffolding protein of excitatory synapses. Moreover, a co-immunoprecipitation approach showed a significant increase of NL2/PSD95 protein interactions in SNL rats. Finally, this unusual association in neuropathic conditions, appeared to be linked to specific over-expression of NL2(-), a NL2 splice variant usually a minority in physiological conditions. Over-expression, increased association with PSD95, and unexpected pronociceptive effect of the “inhibitory” NL2 in neuropathic pain condition, suggest a functional shift of NL2 from inhibition to excitation changing the synaptic ratio toward higher overall excitation in the dorsal horn.In a second part of our work, we investigated the role of the NLs adhesion molecules in spinal sensitization associated with another type of chronic pain, namely cancer pain, using a rat model of bone cancer. The study of the expression of NLs and partners, showed a specific increase in the expression of NL1 and S-SCAM, another postsynaptic scaffolding protein at excitatory synapses. Moreover, according to the literature, this model is characterized by a strong glial activation in the spinal dorsal horn, identified especially by a massive astrogliosis. However, we showed that in the bone cancer model used, there was no variation, neither in the classical markers of astrocyte activation (GFAP, S100β), nor in microglial markers (OX-42 et Iba1). On the contrary, all these parameters did actually increase in the ipsilateral dorsal horn of SNL neuropathic rats. These results suggest that, at odd with what was previously described, bone cancer pain is not necessarily correlated with a spinal overexpression of markers of reactive glia, whereas neuropathic pain is.In conclusion, our results obtained with the cancer pain model, show that the molecules involved in the formation, specification and modulation of synapses, yield a phenotypes clearly different to the one evidenced in the model of neuropathic pain. More particularly, we show in the two models, a well distinct involvement of the NL adhesion molecules and of glia, reinforcing reports from the literature, which indicate that the two important categories of chronic pain, cancer and neuropathic, each have a peculiar signature. Moreover, our results raise the possibility that new diagnosis and/or new therapeutic possibilities may emerge from targeting NL expression

<p dir="ltr">The <i>SCN2A</i><i> </i>gene encodes for the neuronal sodium channel Na_V1.2, which mediates action potential initiation and propagation (Sanders et al., 2018). This protein is expressed mainly in the proximal axonal initial segment (AIS) and soma of glutamatergic excitatory cortical neurons (Kruth, Grisolano, Ahern, & Williams, 2020). <i>SCN2A</i> pathogenic variants have been associated with epilepsy. An example is the recurrent Nav1.2-L1342P variant, a heterozygous missense variant (Begemann et al., 2019) identified in five patients worldwide presenting an early-onset severe seizure phenotype that remains hard to treat with current medications (Que et al., 2021). Additionally, it is one of the few rare <i>SCN2A</i> variants that can impact brain structure (Miao et al., 2020).</p><p dir="ltr">Given that no disease-modifying treatment exists, there is an urgent need to generate novel tools to probe at variant-specific disease mechanisms, evaluate therapeutic interventions, and study interactions with other cell types. Previously, we demonstrated that hiPSC-derived 2D neuronal monolayers carrying the CRISPR/Cas9-edited Nav1.2-L1342P variant display a distinct hyperexcitability phenotype (Que et al., 2021). Despite these findings, questions persist regarding the Nav1.2-L1342P variant's influence on neurodevelopment in more physiologically relevant 3D models, such as organoids.</p><p dir="ltr">To address this, in Chapter 2 of this study, we generated human-induced pluripotent stem cell-derived cortical organoids carrying the epilepsy-related Nav1.2-L1342P variant to study its effect on neuronal hyperexcitability, neurodevelopment and other disease phenotypes. Our data suggests that Nav1.2-L1342P cortical organoid neurons display<b> </b>enhanced repetitive action potential firings, intrinsic excitability, enhanced calcium signaling, increased network neuronal firing, and excitatory postsynaptic currents (EPSCs), suggesting a marked hyperexcitability phenotype and enhanced excitatory neurotransmission. Moreover, cortical organoids with the Nav1.2-L1342P variant display significant changes in synaptic, glutamatergic, and development-related pathways. We also observed that Nav1.2-L1342P variant impacts cortical organoid synaptic and neuronal content.</p><p dir="ltr">The impact of the Nav1.2-L1342P variant was also demonstrated in the 2D-cortical neuron monolayer model, presenting a noticeable reduction in neuronal complexity, thus offering intriguing insights into their effect on neuronal morphology and developmental processes. Our findings recapitulate the hyperexcitable phenotype trends previously observed in the 2D-cortical neuron monolayer platform (Que et al., 2021) and provide evidence of non-autonomous cell development changes due to the Nav1.2-L1342P variant.</p><p dir="ltr">Chapter 3 of this dissertation established a co-culture of hiPSC-derived neurons and microglia, the brain's resident immune cells. Microglia originate from a different lineage (yolk sac) and are not naturally present in hiPSC-derived neuronal cultures. Therefore, they must be added to neuronal cultures to yield a heterogeneous environment. Microglia are also one of the few cell types able to respond to neuronal hypo and hyperexcitability changes. This unique capability prompted us to study how microglia responded to human neurons carrying a disease-causing variant and influenced neuronal excitability.</p><p dir="ltr">We found that microglia display increased branch length and enhanced process-specific calcium signal when co-cultured with the Nav1.2-L1342P neurons, recapitulating phenomena previously observed in rodent seizure models (Eyo et al., 2014; Nebeling et al., 2023). Moreover, the presence of microglia significantly lowered the repetitive action potential firing and current density of sodium channels in neurons carrying the variant, demonstrating the microglial capacity to influence and ameliorate the neuronal activity of the Nav1.2-L1342P mutant neurons. We hypothesized that this effect could be attributed to the increased release of glutamate or small molecules by the Nav1.2-L1342P mutant neurons, which could likely be triggering microglial responses. Additionally, we showed that co-culturing with microglia reduced sodium channel expression within the axon initial segment (AIS) of Nav1.2-L1342P neurons, explaining, in part, the mechanism behind the reduction of sodium current density.</p><p dir="ltr">Taken together, our observations with 2D cortical neurons and 3D cortical organoids revealed marked hyperexcitability and developmental changes associated with the Nav1.2-L1342P variant. Our work also reveals the critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing SCN2A variant.</p>

Le Syndrome de l’X Fragile (SXF) est la forme héréditaire de retard mental la plus fréquente et la cause la mieux caractérisée de troubles du spectre autistique (TSA). Elle est causée par une mutation causant l’inactivation du gène Fmr1 (codant pour la protéine FMRP). La sensibilité accrue aux stimuli sensoriels est une caractéristique importante du SXF et des TSA, mais les mécanismes sous-jacents sont encore mal compris. Nous avons constaté que la suppression du gène Fmr1 entrainait une hyperexcitabilité sensorielle dans le modèle murin du SXF. Les souris Fmr1KO nécessitaient significativement moins d'informations tactiles pour l'exploration haptique, et les représentations évoquées par les informations tactiles provenant des vibrisses dans le cortex somatosensoriel primaire (S1) se propageaient à une vitesse plus élevée chez les souris Fmr1KO par rapport aux souris témoins sauvages.Au niveau cellulaire, il a été montré que les ARNm de plusieurs sous-unités de canaux ioniques (par exemple HCN1, KCNMA1) jouant un rôle clé dans le traitement de l'information dendritique / neuronale étaient des cibles de la protéine FMRP (Liao et al, 2008; Darnell et al, 2011). Sur la base de ces observations, nous avons étudié les canalopathies comme une caractéristique importante du SXF. Nous avons testé de possibles dysfonctionnement des canaux ioniques, et leurs conséquences sur le traitement de l'information dendritique dans les neurones pyramidaux du néocortex de la couche 5 chez les souris Fmr1KO, en utilisant une combinaison d’approches électrophysiologiques et d’imagerie calcique bi-photonique. Nos résultats ont montré que les dendrites des neurones pyramidaux du S1 étaient hyperexcitables, facilitant ainsi le couplage des entrées d’information synaptique à la génération de potentiel d'action en sortie dans les neurones. Cette altération était, au moins en partie, attribuable à un dysfonctionnement des canaux Ih et BKCa et a été partiellement restaurée par l'activation pharmacologique des canaux BKCa. Ces résultats plaident en faveur d'un rôle nouveau et crucial des canalopathies dans l'expression de l'hyperexcitabilité sensorielle dans le SXF. / Fragile X Syndrome (FXS) is the most common form of inherited mental retardation syndrome and most well characterized cause of Autism Spectrum Disorders (ASD), and it is caused by a silencing mutation of the gene Fmr1 (encoding the protein FMRP). Increased sensitivity to sensory stimuli is a prominent feature of FXS and ASD, but its underlying mechanisms are poorly understood. We found that deletion of the Fmr1 gene results in somatosensory hyper-excitability in a mouse model for FXS. Fmr1 knockout (Fmr1KO) mice required significantly less tactile information for haptic exploration, and touch-evoked whisker representations in the primary somatosensory cortex (S1) spread with increased velocity in Fmr1KO mice compared to wild-type control. At the cellular level, it has been shown that the mRNAs of several ion channel subunits (e.g. HCN1, KCNMA1) playing key roles in dendritic/neuronal information processing are regulated by FMRP (Liao et al., 2008; Darnell et al., 2011). Based on these observations, we investigated channelopathies as a prominent feature of FXS. We probed ion channel dysfunction, and its consequence for dendritic information processing in neocortical pyramidal neurons of layer 5 in Fmr1KO mice, using a combination of electrophysiological and 2-photon calcium imaging approaches. Our results showed that dendrites of S1 pyramidal neurons were hyper-excitable, facilitating the coupling of synaptic input to the generation of action potential output in these neurons. This defect was, at least in part, attributable to a dysfunction of Ih channels and BKCa channels and was partially rescued by pharmacological activation of BKCa channels. These findings argue for a novel and critical role for channelopathies in the expression of sensory hyper-excitability in FXS.

Tinnitus is a common and often severely disabling symptom that is characterized by the perceived sensation of sound in the absence of an external stimulus. Traditional treatment approaches have limited efficacy. It is assumed, that tinnitus is connected with dysfunctional activation of neuronal plasticity induced by altered sensory and somatosensory input. Adaptive neuroplastic processes alter the balance between excitatory and inhibitory function of the auditory system at several levels. Functional imaging studies in tinnitus patients have revealed increased neronal activity of primary auditory cortex (PAC). Repetitive transcranial magnetic stimulation (rTMS) induces changes of neuronal activity that outlast the stimulation period. Low-frequency rTMS over the PAC region results in a decrease of cortical activity by inducing long term depression (LTD) and leads to reduced tinnitus perception. The aim of this study was to assess in prospective randomized placebo- controlled study the ability of active low-frequency rTMS guided by frameless stereotaxy to affect symptoms of chronic tinnitus compared to placebo stimulation. Treatment outcome was assessed by subjective specific questionnaires; Tinnitus Handicap Inventory (THI), Tinnitus Questionnaire (TQ) and Visual analogue scales (VAS1, VAS2)...

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Les modèles kainate et pentylènetétrazole représentent deux modèles d’épilepsie du lobe temporal dont les conséquences à long terme sont différentes. Le premier est un modèle classique d’épileptogénèse avec crises récurrentes spontanées tandis que le second se limite aux crises aigües. Nous avons d’abord caractérisé les différents changements survenant dans les circuits excitateurs et inhibiteurs de l’hippocampe adulte de rats ayant subi des crises à l’âge immature. Ensuite, ayant observé dans le modèle fébrile une différence du pronostic lié au genre, nous avons voulu savoir si cette différence était aussi présente dans des modèles utilisant des neurotoxines. L’étude électrophysiologique a démontré que les rats KA et PTZ, mâles comme femelles, présentaient une hyperactivité des récepteurs NMDA au niveau des cellules pyramidales du CA1, CA3 et DG. Les modifications anatomiques sous-tendant cette hyperexcitabilité ont été étudiées et les résultats ont montré une perte sélective des interneurones GABAergiques contenant la parvalbumine dans les couches O/A du CA1 des mâles KA et PTZ. Chez les femelles, seul le DG était légèrement affecté pour les PTZ tandis que les KA présentaient, en plus du DG, des pertes importantes au niveau de la couche O/A. Les évaluations cognitives ont démontré que seuls les rats PTZ accusaient un déficit spatial puisque les rats KA présentaient un apprentissage comparable aux rats normaux. Cependant, encore une fois, cette différence n’était présente que chez les mâles. Ainsi, nos résultats confirment qu’il y a des différences liées au genre dans les conséquences des convulsions lorsqu’elles surviennent chez l’animal immature. / Kainate and pentylenetetrazole models represent two animal models of temporal lobe epilepsy in which long-term consequences differ. The first model is a classical model of epileptogenesis with spontaneous recurrent seizures while the second one is limited to acute seizures. We wanted to characterize the difference in changes which occur in excitatory and inhibitory systems of the hippocampus of adult males and females having suffered an episode of status epilepticus during the immature stage of life. Besides having noticed a difference between genders in the febrile model, our second objective was to see if this difference was also present in models using neurotoxins. Electrophysiology recordings indicated that KA and PTZ rats (both male and female) showed a hyperactivity of NMDA receptors in CA1, CA3 and DG pyramidal cells. Anatomical modifications causing hyperactivity were studied and results show a selective loss of specific GABA interneurons PV in the O/A layer of CA1 region of the hippocampus in KA and PTZ male rats. However in female rats, only the DG layer was slightly affected in PTZ while female KA presented losses in both DG and O/A layers. Cognitive evaluation indicated that only PTZ rats showed a spatial impairment since KA rats had a similar learning pattern as controls. However, once again, that difference was observed only in males and not in females. In summary, our results confirmed that there is a difference between genders regarding brain damages after having suffered an episode of status epilepticus during the immature stage.