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Embryonic development of rhythmogenic motor networks – RhythmDev

Anatomical and functional establishment of the central command for rhythmic activities during embryonic development

Our project «RhythmDev« aims at examining during the embryonic development the neurobiological bases underlying the development of neural networks controlling rhythmic behaviors such as locomotion and respiration.

Improve knowledge in the perinatal development of neural networks generating rhythmically organized motor activities.

By examining neural networks controlling the generation of rhythmic activities (locomotion and respiration) during the perinatal period we focus on a developmental period during which maturational processes have to occur for the neural networks to be fully functional at birth. Thus our results should help defining what are these maturational processes and therefore what could be the longterm functional consequences in pathological conditions. <br />In addition, we aim at comparing the observations made using animal models with those made on newborn babies (last part of the project), with the ultimate goal of being able to propose diagnosis for early anomalies in the development of neural networks leading to motor deficiencies in infants. <br />

In order to study the neural basis for the establishment of functional neural networks as in vitro models we are using reduced preparations (isolated hindbrains, spinal cords, transverse slices) of the central nervous system from mouse embryos and newborn mice (wildtype or transgenic) at different developmental stages. These preparations spontaneously generate rhythmically organized activities. Neuronal activities are monitored either via electrophysiological recordings, via calcium imaging, or a combination of both.

During these first 23 months, experiments examining the respiratory neural network aimed at showing that this network becomes multifunctional (controls different types of respiratory activities) already at embryonic ages. This is associated with the development of specific membrane and synaptic properties. We are now investigating when neurons exhibiting pacemaker properties appear in the network and characterizing their membrane properties and their functional role in respiratory rhythmogenesis.
Our experiments performed on the locomotor spinal networks showed changes in spontaneous activities (SA) that switch from a non-oscillatory to an oscillatory status during development. Furthermore, these oscillations, that are in phase at P0, start to alternate from P2 onwards. The maturation of SA inside one spinal cord segment precedes the one occurring between segments.
Also, we identified a new type of pacemaker neurons within the network controlling locomotion, which is robust, highly expressed and whose bursting capacities rely on the persistent sodium current (INap), a conductance required for locomotor rhythmogenesis. In order to determine which sodium channel is involved in INap we are now using a transgenic mice deleted for the Nav1.6 gene. Our preliminary data show that after the first post-natal week, the dynamic (locomotion) and static (posture) motricity deteriorates rapidly. These deficits are associated with an alteration of neuronal discharge pattern within the locomotor network (interneurons and motoneurons), which is linked to the expression of an abnormal INap.

Our goal is to improve knowledge in the mechanisms involved in the functional establishment of the rhythmic neural networks in order to also improve the therapeutical approaches used in case of premature birth and functional deficiencies at birth.

- Chapuis et al., Journal of Physiology (London): 592, 2169-2181, 2014.
- Bouhadfane et al., Journal of Neuroscience: 33, 15626-15641, 2013.
- Viemari J-C et al. Médecine et Sciences: 2013 10:875-82.

- 4 international meetings at

This proposal aims to investigate, during late phases of embryonic development, the cellular, synaptic and integrative mechanisms underlying the functional development of neural networks that generate rhythmically-organized motor behavior. Two rhythmogenic networks, so-called "Central Pattern Generators" (CPGs), will be investigated in parallel: the respiratory and locomotor CPGs located in the brainstem and spinal cord, respectively. These two networks, which control important physiological functions, are particularly amenable for in vitro neurophysiological study since they are already capable of spontaneous operation when isolated in reduced embryonic CNS preparations. Here, we propose to test the unifying and novel hypothesis that the functional maturation of these CPGs is specifically linked to the maturation of inhibitory synaptic signaling involving chloride ions, together with the establishment of neuronal subpopulations endowed with endogenous pacemaker properties, with both these processes being activity-dependent. By recording the activity of individual neurons and cell populations (via patch clamp and extracellular recordings coupled with calcium imaging) from in vitro preparations (transverse brainstem slices, isolated brainstem-spinal cord) of wild type and genetically-modified mice, we will explore the cellular and network substrates of the rhythmic motor patterns generated by the two CPGs at different embryonic ages. Specifically, by characterizing chloride co-transporter expression with immunohistochemistry and western blot analysis, and examining chloride-mediated synaptic inputs electrophysiologically, we will establish whether the functional maturation of these networks derives from changes in chloride-dependent connectivity that is known to switch from an excitatory action at early embryonic stages to inhibition later in development. Furthermore, although the respiratory and locomotor CPGs are both known to contain pacemaker neurons, their contribution to network operation remains poorly understood. We will therefore employ electrophysiology and, functional imaging to investigate this neuron phenotype, with a view to establishing a correlation between the integration of endogenous pacemaker neurons into the two CPGs and the developmental emergence of patterned motor activity. We will next explore the extent to which these maturational processes are activity-dependent and involving extrinsic modulatory influences (serotonin) and acute trophic factor signaling (BDNF). In a further step, the establishment of a functional interaction between the two CPGs during the prenatal period will be sought and its developmental role examined. Finally, in collaboration with the neonatology department at La Timone hospital in Marseille, we will seek to place our findings on the mouse model within a clinical context by exploring how the maturation of breathing and locomotor behaviors is affected in human infants that are born prematurely or were subjected prenatally to drug-related insult.
Therefore, by associating our respective expertise on two different behavioral systems, and by taking advantage of our complementary experimental methodologies and the different transgenic mouse lines available in our respective labs, we hope to improve understanding of the maturational processes and central mechanisms underlying the functional configuration of rhythmogenic neural networks during embryonic development. By comparing, whenever possible, our in vitro animal model data with observations made on newborn humans, our ultimate goal is the formulation of clinical diagnostics for the early detection of abnormalities in network development that might lead to future motor deficits.

Project coordinator

Madame Muriel THOBY-BRISSON (Institut de Neurosciences Cognitives et Intégratives d'Aquitaine) – muriel.thoby-brisson@u-bordeaux2.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INCIA Institut de Neurosciences Cognitives et Intégratives d'Aquitaine
CNRS DR12_INT Centre National de la !recherche Scientifique/Délégation Provence et Corse_Institut des Neurosciences de la Timone

Help of the ANR 511,889 euros
Beginning and duration of the scientific project: November 2012 - 36 Months

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