Early postnatal maturation in vestibulospinal pathways involved in neck and forelimb motor control

To assess the organization and functional development of vestibulospinal inputs to cervical motoneurons (MNs), we have used electrophysiology (ventral root and electromyographic [EMG] recording), calcium imaging, trans‐synaptic rabies virus (RV) and conventional retrograde tracing and immunohistochemistry in the neonatal mouse. By stimulating the VIIIth nerve electrically while recording synaptically mediated calcium responses in MNs, we characterized the inputs from the three vestibulospinal tracts, the separate ipsilateral and contralateral medial vestibulospinal tracts (iMVST/cMVST) and the lateral vestibulospinal tract (LVST), to MNs in the medial and lateral motor columns (MMC and LMC) of cervical segments. We found that ipsilateral inputs from the iMVST and LVST were differentially distributed to the MMC and LMC in the different segments, and that all contralateral inputs to MMC and LMC MNs in each segment derive from the cMVST. Using trans‐synaptic RV retrograde tracing as well as pharmacological manipulation of VIIIth nerve‐elicited synaptic responses, we found that a substantial proportion of inputs to both neck and forelimb extensor MNs was mediated monosynaptically, but that polysynaptic inputs were also significant. By recording EMG responses evoked by natural stimulation of the vestibular apparatus, we found that vestibular‐mediated motor output to the neck and forelimb musculature became more robust during the first 10 postnatal days, concurrently with a decrease in the latency of MN discharge evoked by VIIIth nerve electrical stimulation. Together, these results provide insight into the complexity of vestibulospinal connectivity in the cervical spinal cord and a cogent demonstration of the functional maturation that vestibulospinal connections undergo postnatally. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1061–1077, 2016     

Motoneuron programmed cell death in response to proBDNF

Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75(NTR) and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75(NTR) and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.     

Noradrenergic modulation of hypoglossal motoneuron excitability: developmental and putative state-dependent mechanisms

Hypoglossal (XII) motoneurons (MNs) contribute to diverse behaviors. Their innervation of the genioglossus muscle, a tongue protruder, plays a critical role in maintaining upper airway patency during breathing. Indeed, reduced activity in these motoneurons is implicated in sleep related disorders of breathing such as obstructive sleep apnea (OSA). The excitability of these MNs is modulated by multiple neurotransmitter systems. The focus of this review is on the modulation of XII MN excitability by norepinephrine (NE), which increases MN excitability through a variety of mechanisms. The level of noradrenergic drive, however, is very dynamic, varying on developmental, sleep-wake and even millisecond timescales relevant to transitions between behaviours. Here we review and provide new data on the maturation of the noradrenergic modulatory system, focusing on those elements specifically relevant to XII MN excitability including the: i) ontogeny of the noradrenergic cell group that provides the majority of the noradrenergic innervation to the XII nucleus, the Locus subcoeruleus (LsC); ii) time course over which the XII nucleus is innervated by noradrenergic nerve fibres, and; iii) ontogeny of XII MN sensitivity to NE. In the context of state-dependent changes in noradrenergic cell activity, we review mechanisms of NE action most relevant to its role in the muscle atonia of REM sleep. We conclude with a discussion of the hypothesis that the dynamics of MN modulation by NE extend to the spatial domain and recent data suggesting that noradrenergic modulation of the dendritic tree is not uniform but compartmentalized. Implications for information processing are discussed.     

Androgens rescue avian embryonic lumbar spinal motoneurons from injury-induced but not naturally occurring cell death

The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury-induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury-induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG-21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury-induced cell death and that they act directly on these cells via an androgen receptor-mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs.     

Mechanisms of insulin-like growth factor regulation of programmed cell death of developing avian motoneurons

During development of the avian neuromuscular system, lumbar spinal motoneurons (MNs) innervate their muscle targets in the hindlimb coincident with the onset and progression of MN programmed cell death (PCD). Paralysis (activity blockade) of embryos during this period rescues large numbers of MNs from PCD. Because activity blockade also results in enhanced axonal branching and increased numbers of neuromuscular synapses, it has been postulated that following activity blockade, increased numbers of MNs can gain access to muscle-derived trophic agents that prevent PCD. An assumption of the access hypothesis of MN PCD is the presence of an activity-dependent, muscle-derived sprouting or branching agent. Several previous studies of sprouting in the rodent neuromuscular system indicate that insulin-like growth factors (IGFs) are candidates for such a sprouting factor. Accordingly, in the present study we have begun to test whether the IGFs may play a similar role in the developing avian neuromuscular system. Evidence in support of this idea includes the following: (a) IGFs promote MN survival in vivo but not in vitro; (b) neutralizing antibodies against IGFs reduce MN survival in vivo; (c) both in vitro and in vivo, IGFs increase neurite growth, branching, and synapse formation; (d) activity blockade increases the expression of IGF-1 and IGF-2 mRNA in skeletal muscles in vivo; (e) in vivo treatment of paralyzed embryos with IGF binding proteins (IGF-BPs) that interfere with the actions of endogenous IGFs reduce MN survival, axon branching, and synapse formation; (f) treatment of control embryos in vivo with IGF-BPs also reduces synapse formation; and (g) treatment with IGF-1 prior to the major period of cell death (i.e., on embryonic day 6) increases subsequent synapse formation and MN survival and potentiates the survival-promoting actions of brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) administered during the subsequent 4- to 5-day period of PCD. Collectively, these data provide new evidence consistent with the role of the IGFs as activity-dependent, muscle-derived agents that play a role in regulating MN survival in the avian embryo. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 379–394, 1998     

Context matters: in vivo and in vitro influences on muscle satellite cell activity

Skeletal muscle is formed during development by the progressive specification, proliferation, migration, and fusion of myoblasts to form terminally differentiated, contractile, highly patterned myofibers. Skeletal muscle is repaired or replaced postnatally by a similar process, involving a resident myogenic stem cell population referred to as satellite cells. In both cases, the activity of the myogenic precursor cells in question is regulated by local signals from the environment, frequently involving other, non-muscle cell types. However, while the majority of studies on muscle development were done in the context of the whole embryo, much of the current work on muscle satellite cells has been done in vitro, or on satellite cell-derived cell lines. While significant practical reasons for these approaches exist, it is almost certain that important influences from the context of the injured and regenerating muscle are lost, while potential tissue culture artifacts are introduced. This review will briefly address extracellular influences on satellite cells in vivo and in vitro that would be expected to impinge on their activity.     

Androgens rescue avian embryonic lumbar spinal motoneurons from injury‐induced but not naturally occurring cell death

The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury‐induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury‐induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG‐21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury‐induced cell death and that they act directly on these cells via an androgen receptor‐mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 585–595, 1999     

ALS motor neurons exhibit hallmark metabolic defects that are rescued by SIRT3 activation

Motor neurons (MNs) are highly energetic cells and recent studies suggest that altered energy metabolism precede MN loss in amyotrophic lateral sclerosis (ALS), an age-onset neurodegenerative disease. However, clear mechanistic insights linking altered metabolism and MN death are still missing. In this study, induced pluripotent stem cells from healthy controls, familial ALS, and sporadic ALS patients were differentiated toward spinal MNs, cortical neurons, and cardiomyocytes. Metabolic flux analyses reveal an MN-specific deficiency in mitochondrial respiration in ALS. Intriguingly, all forms of familial and sporadic ALS MNs tested in our study exhibited similar defective metabolic profiles, which were attributed to hyper-acetylation of mitochondrial proteins. In the mitochondria, Sirtuin-3 (SIRT3) functions as a mitochondrial deacetylase to maintain mitochondrial function and integrity. We found that activating SIRT3 using nicotinamide or a small molecule activator reversed the defective metabolic profiles in all our ALS MNs, as well as correct a constellation of ALS-associated phenotypes.Subject terms: Neuroscience, Pathogenesis     

Constructing and deconstructing stem cell models of neurological disease

Among the disciplines of medicine, the study of neurological disorders is particularly challenging. The fundamental inaccessibility of the human neural types affected by disease prevents their isolation for in?vitro studies of degenerative mechanisms or for drug screening efforts. However, the ability to reprogram readily accessible tissue from patients into pluripotent stem (iPS) cells may now provide a general solution to this shortage of human neurons. Gradually improving methods for directing the differentiation of patient-specific stem cells has enabled the production of several neural cell types affected by disease. Furthermore, initial studies with stem cell lines derived from individuals with pediatric, monogenic disorders have validated the stem cell approach to disease modeling, allowing relevant neural phenotypes to be observed and studied. Whether iPS cell-derived neurons will always faithfully recapitulate the same degenerative processes observed in patients and serve as platforms for drug discovery relevant to common late-onset diseases remains to be determined.     

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.     

Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into gamma-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.     

Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons

Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments.     

MiR-615在平行培养的神经干细胞与运动神经元的表达比较

目的探讨MiR-615在脊髓源性神经干细胞与运动神经元间的表达特征。方法通过免疫磁珠法分离纯化胚胎大鼠脊髓运动神经元;通过神经克隆球形成技术分离纯化胚胎大鼠脊髓源性神经干细胞。采用TaqMan miR-615Assay定量检测培养的脊髓源性神经干细胞与运动神经元中miR-615的表达差异。结果通过平行培养技术分别获得了纯化的脊髓源性神经干细胞与运动神经元。定量检测结果显示,miR-615在分离的运动神经元中较在脊髓源性神经干细胞中显著高表达。结论本文提示miR-615可能在神经干细胞定向分化为运动神经元过程中发挥重要的调节作用。     

Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into all cell types of an embryoproper. Both human and mouse ES cell lines are the subject of intensive investigation for potential applications in developmental biology and medicine. ES cells from both sources differentiate in vitro into cells of ecto-, endoand meso-dermal lineages, and robust cardiomyogenic differentiation is readily observed in spontaneously differentiating ES cells when cultured under appropriate conditions. Molecular, cellular and physiologic analyses demonstrate that ES cell-derived cardiomyocytes are functionally viable and that these cell derivatives exhibit characteristics typical of heart cells in early stages of cardiac development. Because terminal heart failure is characterized by a significant loss of cardiomyocytes, the use of human ES cell-derived progeny represents one possible source for cell transplantation therapies. With these issues in mind, this review will focus on the differentiation of pluripotent embryonic stem cells into cardiomyocytes as a developmental model, and the possible use of ES cell-derived cardiomyocytes as source of donor cells.     

Computer simulation of the motoneuron pool–muscle complex. I. Input system and motoneuron pool

 The aim of the present study was to simulate the input system and the motoneuron (MN) pool of the MN pool–muscle complex (MNPMC). Input fibers, which can originate from command centers in the central nervous system or from sensory organs, activate the MN pool. They generate sequences of action potentials, the frequency of which is proportional to a time-dependent activation factor (which is an input to the model). Different connection patterns between the input fibers and motor units (MUs) are allowed. For simplicity and since no precise experimental data are available, 70 input fibers and 4 boutons per fiber and MN are simulated (this corresponds approximately to the monosynaptic group-Ia input of the cat medial gastrocnemius muscle). Each bouton generates the same conductance change in the postsynaptic membrane. The MNs are modeled with a single compartment and a homogenous membrane. According to experimental data, the membrane leakage conductance and capacitance are MU dependent. Since the precise relation is unknown: (a) the computed relation between MU contraction force and the MN leakage conductance was taken from a steady-state MNPMC model, and (b) the capacitance was assumed to be proportional to the leakage conductance. The MN membrane includes time- and voltage-dependent ionic channels (fast and slow K⁺ and low- and high-threshold Ca²⁺ channels). The density and time constant of the slow K⁺ channels and the density of the Ca²⁺ channels were fitted to approximate afterhyperpolarization characteristics and frequency-injected current relations of type-identified cat MNs. If the membrane reaches a voltage threshold the MNs generate action potentials, which were simulated by voltage pulses. The activation of the MN pool of the human first dorsal interosseus muscle was simulated with injected and synaptic currents in order to illustrate the size principle, synaptic noise, and other features of muscle activation. It is concluded that the present model reproduces the main properties of the input–output relations of different MN types within a muscle. Together with the simulation of the muscle force and the surface EMG, which will be published in subsequent papers, it will be a powerful tool for reproducing experiments on the motor system and investigating functional mechanisms of motor control. Received: 17 April 2001 / Accepted in revised form: 6 November 2001     

VEGF modulates synaptic activity in the developing spinal cord

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma‐aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1110–1122, 2014     

The spatial-temporal gradient of naturally occurring motoneuron death reflects the time of prior exit from the cell cycle and position within the lateral motor column

Embryonic lumbar spinal motoneurons (MNs) are characterized by a period of programmed cell death (PCD) that spans several days and occurs in a rostrocaudal gradient. The generation of these MNs also takes place in a temporal-spatial gradient, such that MNs within rostral lumbar segments exit the cell cycle earlier and MNs within progressively caudal regions are born later. In vitro studies have shown that the latest born spinal MNs, presumably through the possession of endogenous "survival properties," are also the last to acquire their trophic dependence. If the birth date and therefore spinal cord location of lumbar spinal MNs influence the spatial-temporal pattern of PCD, then earlier born MNs should die sooner and be located more rostrally than those generated later. Alternatively, if the time at which MNs die during development is unrelated to their prior exit from the cell cycle, those born at various phases should die throughout the period of PCD. We report here that lumbar MNs generated during the earliest part (embryonic day 2-3) of the proliferative period in the developing chick spinal cord tend to die during the earliest stages of the PCD period and that MNs born in successive 12-h intervals die at correspondingly later periods during PCD. Furthermore, the spatial progression of PCD of these subpopulations of MNs occurs in a rostrocaudal gradient. Finally, while MNs do appear to die in a mediolateral gradient during the period of MN PCD, this pattern is only partly accounted for by MNs born in consecutive intervals. These data support the notion that the timing and rostrocaudal location of MNs undergoing PCD reflect their time of exit from the cell cycle.