Le recrutement des canaux de libération du calcium (Ca2+), par la libération du Ca2+ induite par le Ca2+ (LCIC), évalué par l'introduction de 8 mM bapta dans le myoplasme de la fibre musculaire coupée de la grenouille

Dans les muscles squelettiques, les senseurs de voltage (DHPRs), situés dans la membrane des tubules transverses, subissent un changement de conformation suite à une dépolarisation. Ceci provoque l'ouverture des canaux de libération de Ca (RyRs) dans la membrane du réticulum sarcoplasmique (RS) via un couplage mécanique. Ces deux systèmes membranaires sont apposés, et les RyRs forment une rangée double où un RyR sur deux est couplé à un DHPR. À de faibles dépolarisations, peu de DHPRs sont activés et un site de libération de Ca est isolé de l'influence du Ca libéré par un site voisin. La première partie du projet de recherche consistait à évaluer si un site de libération de Ca est composé soit d'un RyR couplé à son DHPR, soit d'un RyR activé par le voltage avec des RyRs voisins recrutés par libération du Ca induite par le Ca (LCIC). La deuxième partie du projet était d'étudier ce qui se passe à de grandes dépolarisations, lorsqu'une grande densité de DHPR est activée. L'hypothèse était que comme 1 RyR sur 2 est couplé à un DHPR, les RyRs non-couplés pourraient être activés par LCIC seulement à de grandes dépolarisations, grâce à une composante additionnelle de la libération du Ca."--Résumé abrégé par UM

Les mécanismes synaptiques et intrinsèques qui sous-tendent l’activité des cellules réticulospinales (RS) en réponse à une stimulation sensorielle de type cutané chez la lamproie

Chez diverses espèces animales, les informations sensorielles peuvent déclencher la locomotion. Ceci nécessite l’intégration des informations sensorielles par le système nerveux central. Chez la lamproie, les réseaux locomoteurs spinaux sont activés et contrôlés par les cellules réticulospinales (RS), système descendant le plus important. Ces cellules reçoivent des informations variées provenant notamment de la périphérie. Une fois activées par une brève stimulation cutanée d’intensité suffisante, les cellules RS produisent des dépolarisations soutenues de durées variées impliquant des propriétés intrinsèques calcium-dépendantes et associées à l’induction de la nage de fuite. Au cours de ce doctorat, nous avons voulu savoir si les afférences synaptiques ont une influence sur la durée des dépolarisations soutenues et si l’ensemble des cellules RS partagent des propriétés d’intégration similaires, impliquant possiblement les réserves de calcium internes. Dans un premier temps, nous montrons pour la première fois qu’en plus de dépendre des propriétés intrinsèques des cellules réticulospinales, les dépolarisations soutenues dépendent des afférences excitatrices glutamatergiques, incluant les afférences spinales, pour perdurer pendant de longues périodes de temps. Les afférences cutanées ne participent pas au maintien des dépolarisations soutenues et les afférences inhibitrices glycinergique et GABAergiques ne sont pas suffisantes pour les arrêter. Dans un deuxième temps, nous montrons que suite à une stimulation cutanée, l’ensemble des cellules RS localisées dans les quatre noyaux réticulés possèdent un patron d’activation similaire et elles peuvent toutes produire des dépolarisations soutenues dont le maintien ne dépend pas des réserves de calcium internes. Enfin, les résultats obtenus durant ce doctorat ont permis de mieux comprendre les mécanismes cellulaires par lesquels l’ensemble des cellules RS intègrent une brève information sensorielle et la transforment en une réponse soutenue associée à une commande motrice.In various animal species, sensory information can initiate locomotion. This relies on the integration of sensory inputs by the central nervous system. In lampreys, the spinal locomotor networks are activated and controlled by the reticulospinal cells (RS) which constitute the main descending system. In turn, RS cells receive information coming from various synaptic inputs such as the sensory afferents. Once activated by a brief cutaneous stimulation of sufficient strength, RS cells display sustained depolarizations of various durations that rely on calcium-dependant intrinsic properties and lead to the onset of escape swimming. During the course of this Ph.D, we aimed at determining whether synaptic inputs can modulate the duration of the sustained depolarizations and if the different populations of RS cells share the same integrative properties, possibly involving the internal calcium stores. First, our results show for the first time that excitatory glutamatergic inputs, including ascending spinal feedback, contribute to prolong the sustained depolarizations for long periods of time. Cutaneous inputs do not contribute to maintain the sustained depolarizations and inhibitory glycinergic and GABAergic inputs are not sufficient to stop them. Second, we show that in response to cutaneous stimulation, the RS located in the four reticular nuclei display a similar activation pattern and can all produce sustained depolarizations which do not depend on internal calcium release to be maintained. Finally, the results obtained during this Ph.D allowed us to better understand the cellular mechanisms by which the RS cells integrate and transform a brief sensory information into a sustained response associated with a motor command

Le recrutement des canaux de libération du calcium (Ca2+), par la libération du Ca2+ induite par le Ca2+ (LCIC), évalué par l'introduction de 8 mM bapta dans le myoplasme de la fibre musculaire coupée de la grenouille

Dans les muscles squelettiques, les senseurs de voltage (DHPRs), situés dans la membrane des tubules transverses, subissent un changement de conformation suite à une dépolarisation. Ceci provoque l'ouverture des canaux de libération de Ca (RyRs) dans la membrane du réticulum sarcoplasmique (RS) via un couplage mécanique. Ces deux systèmes membranaires sont apposés, et les RyRs forment une rangée double où un RyR sur deux est couplé à un DHPR. À de faibles dépolarisations, peu de DHPRs sont activés et un site de libération de Ca est isolé de l'influence du Ca libéré par un site voisin. La première partie du projet de recherche consistait à évaluer si un site de libération de Ca est composé soit d'un RyR couplé à son DHPR, soit d'un RyR activé par le voltage avec des RyRs voisins recrutés par libération du Ca induite par le Ca (LCIC). La deuxième partie du projet était d'étudier ce qui se passe à de grandes dépolarisations, lorsqu'une grande densité de DHPR est activée. L'hypothèse était que comme 1 RyR sur 2 est couplé à un DHPR, les RyRs non-couplés pourraient être activés par LCIC seulement à de grandes dépolarisations, grâce à une composante additionnelle de la libération du Ca."--Résumé abrégé par UM

Extra activation component of calcium release in frog muscle fibres

In addition to activating more Ca2+ release sites via voltage sensors in the t-tubular membranes, it has been proposed that more depolarised voltages enhance activation of Ca2+ release channels via a voltage-dependent increase in Ca-induced Ca2+ release (CICR). To test this, release permeability signals in response to voltage-clamp pulses to two voltages, –60 and –45 mV, were compared when Δ[Ca2+] was decreased in two kinds of experiments. (1) Addition of 8 mm of the fast Ca2+ buffer BAPTA to the internal solution decreased release permeability at –45 mV by > 2-fold and did not significantly affect Ca2+ release at –60 mV. Although some of this decrease may have been due to a decrease in voltage activation at –45 mV – as assessed from measurements of intramembranous charge movement – the results do tend to support a Ca-dependent enhancement with greater depolarisations. (2) Decreasing SR (sarcoplasmic reticulum) Ca content ([CaSR]) should decrease the Ca2+ flux through an open channel and thereby Δ[Ca2+]. Decreasing [CaSR] from > 1000 μm (the physiological range) to < 200 μm decreased release permeability at –45 mV relative to that at –60 mV by > 6-fold, an effect shown to be reversible and not attributable to a decrease in voltage activation at –45 mV. These results indicate a Ca-dependent triggering of Ca2+ release at more depolarised voltages in addition to that expected by voltage control alone. The enhanced release probably involves CICR and appears to involve another positive feedback mechanism in which Ca2+ release speeds up the activation of voltage sensors

Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca2+ release during the quasi-steady level of release in twitch fibers from frog skeletal muscle

Experiments were performed to characterize the properties of the intrinsic Ca2+ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([CaT]SR and [Ca2+]SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca2+ indicator). Results indicate SR Ca2+ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca2+. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca2+]SR and [CaT]SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca2+ permeability of the SR, namely d[CaT]SR/dt ÷ [Ca2+]SR (denoted release permeability), in experiments in which only [CaT]SR or [Ca2+]SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca2+ release of 2.3 SR Ca2+ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [CaT]SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca2+]SR inhibits release when [CaT]SR declines to a low level

Initiation of locomotion in lampreys

The spinal circuitry underlying the generation of basic locomotor synergies has been described in substantial detail in lampreys and the cellular mechanisms have been identified. The initiation of locomotion, on the other hand, relies on supraspinal networks and the cellular mechanisms involved are only beginning to be understood. This review examines some of the findings relative to the neural mechanisms involved in the initiation of locomotion of lampreys. Locomotion can be elicited by sensory stimulation or by internal cues associated with fundamental needs of the animal such as food seeking, exploration, and mating. We have described mechanisms by which escape swimming is elicited in lampreys in response to mechanical skin stimulation. A rather simple neural connectivity is involved, including sensory and relay neurons, as well as the brainstem rhombencephalic reticulospinal cells, which act as command neurons. We have shown that reticulospinal cells have intrinsic membrane properties that allow them to transform a short duration sensory input into a long-lasting excitatory command that activates the spinal locomotor networks. These mechanisms constitute an important feature for the activation of escape swimming. Other sensory inputs can also elicit locomotion in lampreys. For instance, we have recently shown that olfactory signals evoke sustained depolarizations in reticulospinal neurons and chemical activation of the olfactory bulbs with local injections of glutamate induces fictive locomotion. The mechanisms by which internal cues initiate locomotion are less understood. Our research has focused on one particular locomotor center in the brainstem, the mesencephalic locomotor region (MLR). The MLR is believed to channel inputs from many brain regions to generate goal-directed locomotion. It activates reticulospinal cells to elicit locomotor output in a graded fashion contrary to escape locomotor bouts, which are all-or-none. MLR inputs to reticulospinal cells use both glutamatergic and cholinergic transmission; nicotinic receptors on reticulospinal cells are involved. MLR excitatory inputs to reticulospinal cells in the middle (MRRN) are larger than those in the posterior rhombencephalic reticular nucleus (PRRN). Moreover at low stimulation strength, reticulospinal cells in the MRRN are activated first, whereas those in the PRRN require stronger stimulation strengths. The output from the MLR on one side activates reticulospinal neurons on both sides in a highly symmetrical fashion. This could account for the symmetrical bilateral locomotor output evoked during unilateral stimulation of the MLR in all animal species tested to date. Interestingly, muscarinic receptor activation reduces sensory inputs to reticulospinal neurons and, under natural conditions, the activation of MLR cholinergic neurons will likely reduce sensory inflow. Moreover, exposing the brainstem to muscarinic agonists generates sustained recurring depolarizations in reticulospinal neurons through pre-reticular effects. Cells in the caudal half of the rhombencephalon appear to be involved and we propose that the activation of these muscarinoceptive cells could provide additional excitation to reticulospinal cells when the MLR is activated under natural conditions. One important question relates to sources of inputs to the MLR. We found that substance P excites the MLR, whereas GABA inputs tonically maintain the MLR inhibited and removal of this inhibition initiates locomotion. Other locomotor centers exist such as a region in the ventral thalamus projecting directly to reticulospinal cells. This region, referred to as the diencephalic locomotor region, receives inputs from several areas in the forebrain and is likely important for goal-directed locomotion. In summary, this review focuses on the most recent findings relative to initiation of lamprey locomotion in response to sensory and internal cues in lampreys

Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres

Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+]SR) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrin's properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One – the EGTA–phenol red method – monitored the content of total Ca in the SR ([CaT]SR) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mm), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [CaT]SR versus [Ca2+]SR as [Ca2+]SR varied from its resting level ([Ca2+]SR,R) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca–calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only ∼25% of released Ca2+ comes from calsequestrin and ∼75% comes from the free pool. The value of [Ca2+]SR,R was close to the KD for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+]SR,R is near 1 mm in cut fibres. These results along with the known rapid kinetics of the Ca–calsequestrin binding reaction indicate that calsequestrin's properties are optimized to buffer [Ca2+]SR during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation–contraction coupling process, they do indicate that passive buffering of [Ca2+]SR is a very important function of calsequestrin