Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

<p>Abstract</p> <p>Background</p> <p>Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.</p> <p>Methods</p> <p>Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.</p> <p>Results</p> <p>The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both <it>in vitro </it>and <it>in vivo </it>after transplantation into regenerating muscle of immunodeficient mice.</p> <p>Conclusions</p> <p>Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess <it>in vivo </it>the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.</p

Polysynaptic Potentiation at Different Levels of Rat Olfactory Pathways Following Learning

This study was aimed at investigating the consequences of learning on late polysynaptic components of evoked field potential signals recorded in parallel at different levels of the olfactory pathways. For this, evoked field potentials induced by electrical stimulation of the olfactory bulb were recorded simultaneously in the anterior piriform cortex, the posterior piriform cortex, the lateral entorhinal cortex, and the dentate gyrus. The different parameters of late components were measured in each site before and after completion of associative learning in anesthetized rats. In the learning task, rats were trained to associate electrical stimulation of one olfactory bulb electrode with the delivery of sucrose (positive reward) and stimulation of a second olfactory bulb electrode with the delivery of quinine (negative reward). In this way, stimulation of the same olfactory bulb electrodes used for inducing field potentials served as a discriminative cue in the learning paradigm. The data confirmed previous observation that learning was associated with a lowering in late-component-1 intensity of induction in the posterior piriform cortex. The use of simultaneous recording allowed us to further specify the consequences of learning on late-component distribution in the studied network. Indeed the data showed that whereas before learning, late component 1 was rather uniformly distributed among the recorded sites; following learning, its expression was facilitated preferentially in the posterior piriform cortex and lateral entorhinal cortex. Furthermore, learning was accompanied by the emergence of a new late component (late component 2), which occurred simultaneously in the four recording sites. The possible involvement of potentiation of polysynaptic components in recognition and/or consolidation processes will be discussed

Ultrasonic Vocalizations Emission across Development in Rats: Coordination with Respiration and Impact on Brain Neural Dynamics

International audienceRats communicate using ultrasonic vocalizations (USV) throughout their life when confronted with emotionally stimulating situations, either negative or positive. The context of USV emission and the psychoacoustic characteristics of the vocalizations change greatly between infancy and adulthood. Importantly, the production of USV is tightly coordinated with respiration, and respiratory rhythm is known to influence brain activity and cognitive functions. This review goes through the acoustic characteristics and mechanisms of production of USV both in infant and adult rats and emphasizes the tight relationships that exist between USV emission and respiration throughout the rat’s development. It further describes how USV emission and respiration collectively affect brain oscillatory activities. We discuss the possible association of USV emission with emotional memory processes and point out several avenues of research on USV that are currently overlooked and could fill gaps in our knowledge

Entorhinal cortex stimulation modulates amygdala and piriform cortex responses to olfactory bulb inputs in the rat

International audienceThe rodent olfactory bulb sends direct projections to the piriform cortex and to two structures intimately implicated in memory processes, the entorhinal cortex and the amygdala. The piriform cortex has monosynaptic projections with the amygdala and the piriform cortex and is therefore in a position to modulate olfactory input either directly in the piriform cortex, or via the amygdala. In order to investigate this hypothesis, field potential signals induced in anesthetized rats by electrical stimulation of the olfactory bulb or the entorhinal cortex were recorded simultaneously in the piriform cortex (anterior part and posterior part) and the amygdala (basolateral nucleus and cortical nucleus). Single-site paired-pulse stimulation was used to assess the time courses of short-term inhibition and facilitation in each recording site in response to electrical stimulation of the olfactory bulb and entorhinal cortex. Paired-pulse stimulation of the olfactory bulb induced homosynaptic inhibition for short interpulse interpulse intervals (20-30 ms) in all the recording sites, with a significantly lower degree of inhibition in the anterior piriform cortex than in the other structures. At longer intervals (40-80 ms), paired-pulse facilitation was observed in all the structures. Paired-pulse stimulation of the entorhinal cortex mainly resulted in inhibition for the shortest interval duration (20 ms) in anterior piriform cortex, posterior piriform cortex and amygdala basolateral but not cortical nucleus. Double-site paired-pulse stimulation was then applied to determine if stimulation of the entorhinal cortex can modulate responses to olfactory bulb stimulation. For short interpulse intervals (20 ms) heterosynaptic inhibition was observed in anterior piriform cortex, posterior piriform cortex and amygdala basolateral but not cortical nucleus. The level of inhibition was greater in the basolateral nucleus than in the other structures. Taken together these data suggest that the entorhinal cortex exerts a main inhibitory effect on the olfactory input via the amygdala basolateral nucleus and to a lesser extent the piriform cortex. The potential role of these effects on the processing of olfactory information is discussed

An investigation of some temporal aspects of olfactory coding with the model of multi-site electrical stimulation of the olfactory bulb in the rat

International audienceElectrical stimulation of the olfactory bulb was used to investigate some temporal aspects of olfactory coding, with reference to respiration. Food-deprived rats implanted with permanent electrodes were trained to use bulbar multi-site stimulation patterns as discriminative stimuli for predicting the nature of an incoming reinforcement. Electrical pulse trains (100 Hz) were periodically delivered in phase with precisely defined moments of the respiratory cycle (during inspiration or expiration). Temporal aspects of olfactory coding were first considered through the measurement of the minimum duration of a stimulus necessary to identify this stimulus. The results showed that a bulbar stimulation lasting for 30 ms (3 pulses), and delivered during inspiration, was clearly identified by the rats. Stimulus identification induced a discriminative respiratory response which could manifest itself as early as the first cycle concomitant with the beginning of stimulation. It was then shown that a bulbar electrical stimulation pattern was identified with the same latency whether it occurred during expiration or during inspiration. Moreover, the perceptive events induced in those two conditions of stimulation were not different enough to be discriminated by the animals. The findings are discussed within the framework of olfactory information processing

On the ability of rats to discriminate between microstimulations of the olfactory bulb in different locations

International audienceAn investigation was made into the ability of rats to discriminate between electrical stimulations applied to the mitral cell layer of the olfactory bulb in different locations. Water-deprived rats implanted with permanent electrodes were trained to use single- or multi-site microstimulations as discriminative stimuli for selecting a palatable solution without tasting it in a two-choice test. Spontaneous reactions of the animals to stimulation with sinusoidal currents higher than 3 microA per electrode resembled sensory arousal. All rats were found to discriminate between the effects of concurrent microstimulations applied to bulbar sites separated by 500 micron. Changing the current intensity in the range 4-20 microA had no detectable effect on the discrimination. Discrimination was still possible, with a few exceptions, when electrodes were separated by 250 micron and even when they were closely adjacent. Spatial resolution of discrimination seemed not to vary in different regions along the rostrocaudal axis of the bulb. The discrimination of patterns of simultaneous stimulation at several sites was also investigated. Different multi-site patterns were easily distinguished, even when their respective components were closely adjacent or when some components occupied the same area. The findings are discussed with reference to the concept of spatial coding of odours in the olfactory bulb

A study of the effects of noradrenaline in the rat olfactory bulb using evoked field potential response

International audienceIn the rat, the main olfactory bulb receives a strong noradrenergic (NA) input from the locus coeruleus which is critical for different types of olfactory learning. However, the resulting effect of NA modulation on on the olfactory bulb electrical activity and its pharmacology are not well understood. In this study, we investigated the action of NA on the bulbar neuronal population using evoked field potentials (EFP) elicited antidromically in the olfactory bulb of anesthetized rats, by stimulation of the lateral olfactory tract (LOT). EFPs in response to single and paired-pulse stimulation of the LOT were collected before, during and until 2 h after a 10 min perfusion of pharmacological agents through a push-pull cannula. Four concentrations of NA were tested ranging from 10(-5) M to 10(-2) M. NA induced a reversible dose-dependent effect. The major effect was observed at 10(-3) M. It consisted of an increase in Component 2 amplitude (depolarization of granules cell dendrites) and a decrease in Component 3 amplitude (depolarization of granule cell bodies). In parallel, paired-pulse inhibition of mitral cells by granule cells was increased. The alpha 1 agonist phenylephrine (10(-3) M) mimicked most of the effects of NA whereas the alpha 1 antagonist prazosin (10(-3) M) blocked its main action. Isoproterenol (beta agonist, 10(-3) M) and clonidine (alpha 2 agonist, 10(-3) M) could not reproduce the effects of NA. Thus mainly through the activation of alpha 1 receptors, NA enhances synaptic activation of granule cells and increases feed-back inhibition of mitral cells. Consequences of such effects in the context of learning and memory are discussed

Ecologically relevant neurobehavioral assessment of the development of threat learning

International audienceAs altricial infants gradually transition to adults, their proximate environment changes. In three short weeks, pups transition from a small world with the caregiver and siblings to a complex milieu rich in dangers as their environment expands. Such contrasting environments require different learning abilities and lead to distinct responses throughout development. Here, we will review some of the learned fear conditioned responses to threats in rats during their ontogeny, including behavioral and physiological measures that permit the assessment of learning and its supporting neurobiology from infancy through adulthood. In adulthood, odor-shock conditioning produces robust fear learning to the odor that depends upon the amygdala and related circuitry. Paradoxically, this conditioning in young pups fails to support fear learning and supports approach learning to the odor previously paired with shock. This approach learning is mediated by the infant attachment network that does not include the amygdala. During the age range when pups transition from the infant to the adult circuit (10-15 d old), pups have access to both networks: odor-shock conditioning in maternal presence uses the attachment circuit but the adult amygdala-dependent circuit when alone. However, throughout development (as young as 5 d old) the attachment associated learning can be overridden and amygdala-dependent fear learning supported, if the mother expresses fear in the presence of the pup. This social modulation of the fear permits the expression of defense reactions in life threatening situations informed by the caregiver but prevents the learning of the caregiver itself as a threat