The biophysical properties and distribution of voltage gated ion channels shape the spatio-temporal pattern of synaptic inputs and determine the input-output properties of the neuron. Of the various voltage-gated ion channels, persistent Na⁺ current (INaP) is of interest because of its activation near rest, slow inactivation kinetics, and consequent effects on excitability. Overshadowed by transient Na⁺ current (INaT) of large amplitude and fast inactivation, various quantitative characterizations of INaP have yet to provide a clear understanding of their role in neuronal excitability. We addressed this question using quantitative electrophysiology to compare somatic INaP and INaT in 4–7 week old Sprague-Dawley rat hippocampal CA1 pyramidal neurons. INaP was evoked with 0.4 mV/ms ramp voltage commands and INaT with step commands in hippocampal neurons from in vitro brain slices utilizing nucleated patch-clamp recording. INaP was found to have a density of 1.4 ± 0.7 pA/pF in the soma. Compared to INaT, it has a much smaller amplitude (2.38% of INaT) and distinct voltage dependence of activation (16.7 mV lower half maximal activation voltage and 41.3% smaller slope factor than those of INaT). The quantitative measurement of INaT gave the activation time constant ([tau]m) of 22.2 ± 2.3 [mu]s at 40 mV. Hexanol, which has anesthetic effects, was shown to preferentially block INaP compared to INaT with a significant voltage threshold elevation (4.6 ± 0.7 mV) and delayed 1st spike latency (221 ± 54.6 ms) suggesting reduced neuronal excitability. The number of spikes evoked by either given step current injections or [alpha]-EPSP integration was also significantly decreased. The differential blocking of INaP by halothane, a popularly used volatile anesthetic, further supports the critical role of INaP in setting voltage threshold. Taken together, the presence of INaP in the soma demonstrates an intrinsic mechanism utilized by hippocampal CA1 pyramidal neurons to regulate axonal spike initiation through different biophysical properties of the Na⁺ channel. Furthermore, INaP becomes an interesting target of intrinsic plasticity because of its profound effect on the input-output function of the neuron. / text

Atrial Fibrillation (AF) is rare amongst young people whilst epidemic in the elderly. Whilst much is known about the pathophysiology of AF, the mechanisms underlying the vulnerability to AF amongst older people in incompletely understood. Young (< 18 months, first quintile of life) and old (> 8 years, last quintile of life) Welsh mountain sheep were used to investigate changes in atrial electrophysiology with age. Old sheep were more vulnerable to induced AF than young sheep. On the surface ECG, p-wave duration increased with age suggesting increasing atrial size. The corrected sinus node recovery time increased with age, suggesting deteriorating sinus node function. These findings confirmed the validity of sheep as a model for human ageing. In isolated atrial myocytes, action potentials (APs) were recorded using the perforated patch clamp technique. AP duration increased with age, and an increase in AP amplitude was also seen at the lowest stimulation rates. Right atrial AP durations were prolonged compared to those from left atrial myocytes, and the inter-atrial difference was similar between old and young. However, when right atrial monophasic APs were recorded from anaesthetised sheep in vivo, no difference in AP duration was seen between age groups. Alternans occurred at lower stimulation rates in old compared to young myocytes and was of greater magnitude. These age-related differences were present in isolated myocytes and in vivo. Alternans mechanisms were explored by simultaneously recording APs and intracellular calcium concentration. Atrial alternans was driven by alternans of Ca2+ cycling at low stimulation rates. However, despite disabling Ca2+ cycling using thapsigargin, alternans could still be elicited from myocytes during rapid stimulation. Right atrial conduction velocity (CV) was assessed in vivo and found to increase with age. A key determinant of CV, the Na+ current INa was investigated using the whole cell patch clamp technique. INa increased with age in left atrial myocytes and recovered faster from inactivation. Protein expression was investigated using Western blotting. Expression of the Na+ channel α-subunit did not change with age. The gap junction protein Cx43 was expressed less in older subjects, but Cx40 expression was similar. This work has cast light on several aspects of atrial electrophysiology in which the effects of age have not been thoroughly investigated. The longer cellular APs seen with age decrease the wavelength of potential re-entrant circuits which could be seen as protective against AF. However, AP prolongation is also associated with afterdepolarisations which could serve to trigger AF. The increase in alternans behaviour may set the stage for wavebreak, leading to re-entrant circuit formation. The increase in CV was surprising and might be seen as protective against AF as it increases arrhythmia wavelength, and is likely to be caused by the increased INa.

Rett Syndrome (RTT) is a developmental disorder that affects numerous neuronal systems that underlie problems with breathing, movement, cognition and sleep. RTT is caused by mutations in the methyl-CpG-binding protein 2 (Mecp2) gene. MeCP2 is a ubiquitous protein that is found in all mature neurons and binds to methylated DNA to repress transcription; thus regulating protein expression levels in neurons. The mutations in Mecp2 affect a large number of proteins that are crucial for regulating neuronal activity. Despite the abnormal expression of many of these proteins, mice with a total loss of MeCP2 can live to adulthood and some people with RTT can live to a very late age as well. It is possible that mutations in the Mecp2 gene not only cause widespread defects, but also elicit neuroadaptive processes that may limit the impact of the MeCP2 dysfunction. To test this hypothesis we performed these studies in which we focused on how synaptic and membrane currents were altered to maintain normal neuronal activity in Mecp2-null mice. We show two examples from different neurons where neuroadaptations of ion channel expression allowed the neuron to remain viable. First, the properties of the nicotinic acetylcholine receptor (nAChR) current were altered in LC neurons in Mecp2-null mice. This was caused by changes in the nicotinic receptor subunit expression. Despite the changes in the nAChR current, the cholinergic modulation of LC neuron activity in WT and Mecp2-null mice were similar. Secondly, we show that the fast Na+ voltage-gated and the hyperpolarization-activated currents were altered in mesencephalic trigeminal V (Me5) propriosensory neurons. The changes in the hyperpolarization-activated current caused a smaller sag and post-inhibitory rebound. Opposite to what we expected, these cells were hyperexcitable. The hyperexcitability was due to changes in the fast Na+ voltage-gated current causing a decreased action potential threshold. Alterations in the ionic currents in Me5 neurons seem to be due to changes in subunit expression patterns. These results indicate that despite the complications caused by defects in the Mecp2 gene, neurons respond by rearranging receptor / ion channel expression. This reorganization allows neurons to remain viable despite the MeCP2 deficiency.

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La transmission synaptique dans les corticaux est généralement décrite comme un phénomène de « tout ou rien » ou digital. Un Potentiel d'Action (PA) est émis dans la cellule présynaptique, provoquant le relargage de neurotransmetteurs au niveau du bouton présynaptique et, en conséquence, une dépolarisation transitoire de la cellule postsynaptique (Potentiel Post-Synaptique Excitateur ou PPSE). Cependant, de nombreuses études ont démontrées que la forme du PA présynaptique dépend de l'activité sous liminaire précédant son émission. En effet, si la cellule présynaptique est dépolarisée durant 5 à 10 s avant l'émission du PA, ce dernier s'élargit, ce qui provoque une augmentation du relargage de neurotransmetteurs et de l'amplitude du PPSE. Ainsi, la transmission synaptique dépend d'un phénomène digital, le PA, dont la forme est modulée analogiquement. On parle de transmission Analogique-Digitale. L'élargissement du PA et l'augmentation de la transmission synaptique suite à une longue dépolarisation de la transmission synaptique est nommée Facilitation Analogique-Digital due à la Dépolarisation (FADD). Durant cette thèse, nous nous sommes posé 3 questions principales. Quel est le mécanisme biophysique de la FADD ? Existe-il des Facilitations Analogique Digitale dépendante de modulation de l'amplitude du PA et non de sa largeur ? Les modulations de la forme du PA sont-elles toutes à court terme (de la milliseconde à la seconde) ou existe-t-il des modulations de la forme du PA à long terme (plusieurs jours) ? Pour répondre à la première question, nous avons enregistré des paires de neurones CA3 de l'hippocampe et avons dépolarisé la cellule présynaptique durant 10 s avant l'émission du PA. Nous avons observé une FADD de 30 % qui était supprimée par le blocage pharmacologique des canaux potassiques axonaux Kv1. Ces canaux sont responsables de la phase de repolarisation du PA et ont la propriété de s'inactiver durant de longues dépolarisations. Nous en avons conclu qu'entre les neurones CA3, la FADD était due à l'inactivation des canaux Kv1 pendant la dépolarisation précédant le PA, ce qui provoque un ralentissement de la phase de repolarisation du PA et ainsi un élargissment du PA. Afin de répondre à la seconde question, nous avons enregistré des paires de neurones CA3 dans l'hippocampe. Nous avons observé qu'une courte hyperpolarisation (50 ms) du neurone présynaptique avant l'émission du potentiel d'action provoquait une augmentation de l'amplitude du PA entrainant un accroissement du relargage de neurotransmetteur et de la taille du PPSE. Nous avons nommé ce phénomène FADH pour Facilitation Analogique-Digitale induite par Hyperpolarisation. La FADH est due à récupération de l'inactivation de canaux sodiques responsables de l'amplitude du PA quand le neurone présynaptique est hyperpolarisé, ce qui augmente leur disponibilité. Enfin, pour répondre à la troisième question, nous avons bloqué la transmission synaptique entre les neurones CA3 durant 3 jours. Cela a entrainé une augmentation compensatoire de la transmission synaptique entre les paires de neurones CA3. Il est important de noter que cette augmentation compensatoire est due à la régulation négative des canaux Kv1 entrainant un élargissement du PA. Ainsi, la forme du PA peut-être moduler sur le long terme et participer à la plasticité synaptique. En conclusion, nous avons démontré que le PA n'a pas une forme fixée mais que cette dernière est modulée sur des échelles de temps allant de la dizaine de ms à plusieurs, permettant aux réseaux neuronaux d'élargir leur capacité de transfert d'information. / Generally, the synaptic transmission in cortical networks is described as an « all-or-none » or digital phenomenon. An Action Potential (AP) is emitted in the presynaptic cell entailing the release of neurotransmitters at presynaptic terminal and, consequently, a transient depolarization of the postsynaptic cell (Excitatory Post-Synaptic Potential or EPSP). However, several studies showed that the presynaptic AP shape depend on the subthreshold activity before his occurrence. Indeed, if the presynaptic cell is depolarized during 5 to 10 seconds before the AP emission, the AP is getting broader which leads to an increase in neurotransmitters release and EPSP amplitude. Therefore, the synaptic transmission depends on a digital phenomenon, the AP, whose shape is modulated in an analogic way, the so-called Analog-Digital transmission. The increase in AP width and synaptic transmission following a long depolarization of the presynaptic cell is named Analog Digital Facilitation induced by depolarization (d-ADF). During this thesis, we asked 3 main questions. What is the biophysic mechanism of d-ADF? Are there ADFs depending on AP amplitude modulation? Are the modulations of the AP shape all short term modulations (ms to s) or are there some long term AP shape modulations (days)? To answer the first question, we recorded pairs of hippocampal CA3 neurons and we depolarized the presynaptic cell during 10 ms before AP emission. We observed a d-ADF of 30 % which was suppressed by the phamarcological blockade of axonal potassium channels Kv1. These channels are responsible of the AP repolarization phase and have the property to inactivate during long depolarization. We concluded that the d-ADF at the CA3-CA3 synapse is due to inactivation of Kv1 channels during the depolarization preceding the AP which entails a slowing of the AP repolarization phase and a broadening of the AP. In order to answer the second question, we recorded pairs of hippocampal CA3 neurons. We observed that a short hyperpolarization of the presynaptic neuron (50 ms) before the AP emission entailed an increase of the AP amplitude leading to an increase of neurotransmitters release and EPSP amplitude. We named this phenomenon hyperpolarization induced Analog-Digital Facilitation (h-ADF). The h-ADF is due to the recovery from inactivation of sodium channels responsible of AP amplitude when the presynaptic neuron is hyperpolarized. Finally, to answer the third question, we blocked the synaptic transmission between CA3 neurons for 3 days. This provoked a compensatory increase of synaptic transmission between pairs of CA3 neurons. Interestingly, this compensatory increase is due to the downregulation of Kv1 channels leading to a broadening of the AP. Therefore, the AP shape can be modulated within days and participate to synaptic plasticity. In conclusion, we showed that the AP is not digital but that its shape is modulated within time scales going from the ms to several days, increasing information transfer ability of neuronal networks.

La locomotion se définit par des mouvements répétés et coordonnés des membres droits et gauches et des muscles antagonistes d’une même articulation. L’activité locomotrice des rongeurs est générée par des groupes de neurones localisés dans la partie antérieure de l’élargissement lombaire; ce réseau de cellules est appelé Central Pattern Generator (CPG). Au cours de cette thèse, les études entreprises chez le rat nouveau-né ont eu pour but d’étudier les mécanismes cellulaires impliqués dans la genèse du rythme locomoteur. Le courant sodique persistant (INaP) joue un rôle important dans la genèse d’activités rythmiques de plusieurs structures supraspinales et notamment celles impliquées dans la mastication et la respiration. Curieusement, son existence et son implication dans la genèse d’activités rythmiques dans les structures du CPG locomoteur spinal n’ont jamais été abordées. A l’aide d’études électrophysiologiques, la thèse démontre l’existence de INaP et le caractérise pour la première fois au sein du CPG locomoteur. Ce courant est indispensable à la genèse du rythme locomoteur et joue un rôle fondamental dans l’émergence d’activités pacemakers au sein du CPG. Ces activités pacemakers émergent dans un contexte physiologique où des fluctuations dans la composition ionique du milieu extracellulaire interviennent au cours d’une activité locomotrice. L’ensemble de ces données suggère que le « cœur » du générateur de rythme pourrait être composé d’interneurones présentant une activité pacemaker dépendante de INaP dont la modulation pourrait être un élément fondamental à la fois dans le déclenchement et la modulation de l’activité locomotrice. / Identification of the cellular mechanisms underlying the generation of the locomotor rhythm is of longstanding interest to physiologists. Hindlimb locomotor movements are generated by lumbar neuronal networks, referred to as central pattern generators (CPG). Although rhythm generation mechanisms within the CNS can vary, the activation of a subthreshold depolarizing conductance is always needed to start the firing of individual neurons. Among various subthreshold membrane conductances, the persistent sodium current (INaP) is involved in rhythmic activity of numerous supraspinal neurons such as those involved in the generation of masticatory and respiratory rhythm. The thesis was aimed at identifying and characterizing INaP in the neonatal rodent locomotor CPG, determining its importance in shaping neuronal firing properties and its role in the operation of the locomotor circuitry. Using electrophysiological studies the thesis has characterized INaP for the first time in the locomotor CPG. This current is essential to the generation of the locomotor rhythm and plays a fundamental role in the emergence of pacemaker activity within the CPG. These pacemaker activities emerge in a physiological context in which fluctuations in the ionic composition of the extracellular environment occur during locomotion. This study provides evidence that INaP generates pacemaker activities in CPG interneurons and new insights into the operation of the locomotor network with a critical implication of INaP in stabilizing the locomotor pattern.

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