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Journal of Visualized Experiments : JoVE Nov 2010Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct...
Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.
Topics: Action Potentials; Animals; Larva; Motor Endplate; Motor Neurons; Muscle, Skeletal; Patch-Clamp Techniques; Zebrafish
PubMed: 21113124
DOI: 10.3791/2351 -
Genes Dec 2019Growing evidence suggests that aberrant energy metabolism could play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Despite this, studies... (Review)
Review
Growing evidence suggests that aberrant energy metabolism could play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Despite this, studies applying advanced technologies to investigate energy metabolism in ALS remain scarce. The rapidly growing field of metabolomics offers exciting new possibilities for ALS research. Here, we review existing and emerging metabolomic tools that could be used to further investigate the role of metabolism in ALS. A better understanding of the metabolic state of motor neurons and their surrounding cells could hopefully result in novel therapeutic strategies.
Topics: Amyotrophic Lateral Sclerosis; Animals; Energy Metabolism; Humans; Metabolomics; Motor Neurons
PubMed: 31817338
DOI: 10.3390/genes10121011 -
Journal of Alzheimer's Disease : JAD 2010We review the electrophysiological studies concerning the effects of caffeine on muscle, lower and upper motor neuron excitability and cognition. Several different... (Review)
Review
We review the electrophysiological studies concerning the effects of caffeine on muscle, lower and upper motor neuron excitability and cognition. Several different methods have been used, such as electromyography, recruitment analysis, H-reflex, transcranial magnetic stimulation (TMS), electroencephalography and event-related potentials. The positive effect of caffeine on vigilance, attention, speed of reaction, information processing and arousal is supported by a number of electrophysiological studies. The evidence in favor of an increased muscle fiber resistance is not definitive, but higher or lower motor neuron excitability can occur as a consequence of a greater excitation of the descending input from the brainstem and upper motor neurons. TMS can address the influence of caffeine on the upper motor neuron. Previous studies showed that cortico-motor threshold and intracortical excitatory and inhibitory pathways are not influenced by caffeine. Nonetheless, our results indicate that cortical silent period (CSP) is reduced in resting muscles after caffeine consumption, when stimulating the motor cortex with intensities slightly above threshold. We present new data demonstrating that this effect is also observed in fatigued muscle. We conclude that CSP can be considered a surrogate marker of the effect of caffeine in the brain, in particular of its central ergogenic effect.
Topics: Adult; Analysis of Variance; Caffeine; Central Nervous System Stimulants; Electrophysiology; Evoked Potentials; Fatigue; Humans; Male; Motor Neurons; Muscles; Rest; Transcranial Magnetic Stimulation; Young Adult
PubMed: 20164574
DOI: 10.3233/JAD-2010-1377 -
Biochimica Et Biophysica Acta Apr 2015Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the degeneration of spinal motor neurons and muscle atrophy. Although... (Review)
Review
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the degeneration of spinal motor neurons and muscle atrophy. Although the genetic cause of SMA has been mapped to the Survival Motor Neuron1 (SMN1) gene, mechanisms underlying selective motor neuron degeneration in SMA remain largely unknown. Here we review the latest developments and our current understanding of the molecular mechanisms underlying SMA pathogenesis, focusing on the animal model systems that have been developed, as well as new diagnostic and treatment strategies that have been identified using these model systems. This article is part of a special issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.
Topics: Animals; Disease Models, Animal; Humans; Motor Neurons; Muscular Atrophy, Spinal; Survival of Motor Neuron 1 Protein
PubMed: 25088406
DOI: 10.1016/j.bbadis.2014.07.024 -
Respiratory Physiology & Neurobiology Nov 2013In many neurological disorders that disrupt spinal function and compromise breathing (e.g. ALS, cervical spinal injury, MS), patients often maintain ventilatory capacity... (Review)
Review
In many neurological disorders that disrupt spinal function and compromise breathing (e.g. ALS, cervical spinal injury, MS), patients often maintain ventilatory capacity well after the onset of severe CNS pathology. In progressive neurodegenerative diseases, patients ultimately reach a point where compensation is no longer possible, leading to catastrophic ventilatory failure. In this brief review, we consider evidence that common mechanisms of compensatory respiratory plasticity preserve breathing capacity in diverse clinical disorders, despite the onset of severe pathology (e.g. respiratory motor neuron denervation and/or death). We propose that a suite of mechanisms, operating at distinct sites in the respiratory control system, underlies compensatory respiratory plasticity, including: (1) increased (descending) central respiratory drive, (2) motor neuron plasticity, (3) plasticity at the neuromuscular junction or spared respiratory motor neurons, and (4) shifts in the balance from more to less severely compromised respiratory muscles. To establish this framework, we contrast three rodent models of neural dysfunction, each posing unique problems for the generation of adequate inspiratory motor output: (1) respiratory motor neuron death, (2) de- or dysmyelination of cervical spinal pathways, and (3) cervical spinal cord injury, a neuropathology with components of demyelination and motor neuron death. Through this contrast, we hope to understand the multilayered strategies used to "fight" for adequate breathing in the face of mounting pathology.
Topics: Adaptation, Physiological; Animals; Humans; Motor Neurons; Nervous System Diseases; Neuronal Plasticity; Respiratory Center; Respiratory Mechanics; Spinal Cord Injuries
PubMed: 23727226
DOI: 10.1016/j.resp.2013.05.025 -
The Journal of Comparative Neurology Dec 2022Small sensory spinal injuries induce plasticity across the neuraxis, but little is understood about their effect on segmental connections or motor neuron (MN) function....
Small sensory spinal injuries induce plasticity across the neuraxis, but little is understood about their effect on segmental connections or motor neuron (MN) function. Here, we begin to address this at two levels. First, we compared afferent input distributions from the skin and muscles of the digits with corresponding MN pools to determine their spatial relationship, in both the normal state and 4-6 months after a unilateral dorsal root/dorsal column lesion (DRL/DCL), affecting digits 1-3. Second, we looked at specific changes to MN inputs and membrane properties that likely impact functional recovery. Monkeys received a targeted unilateral DRL/DCL, and 4-6 months later, cholera toxin subunit B (CT-B) was injected bilaterally into either the distal pads of digits 1-3, or related intrinsic hand muscles, to label inputs to the cord, and corresponding MNs. In controls (unlesioned side), cutaneous and proprioceptive afferents from digits 1-3 showed different distribution patterns but similar rostrocaudal spread within the dorsal horn from C1 to T2. In contrast, MNs were distributed across just two segments (C7-8). Following the lesion, sensory inputs were significantly diminished across all 10 segments, though this did not alter MN distributions. Afferent and monoamine inputs, as well as KCC2 cotransporters, were also significantly altered on the cell membrane of CT-B labeled MNs postlesion. In contrast, inhibitory neurotransmission and perineuronal net integrity were not altered at this prechronic timepoint. Our findings indicate that even a small sensory injury can significantly impact sensory and motor spinal neurons and provide new insight into the complex process of recovery.
Topics: Animals; Cholera Toxin; Haplorhini; Motor Neurons; Spinal Cord; Spinal Cord Injuries; Symporters
PubMed: 35973735
DOI: 10.1002/cne.25395 -
The Journal of Physiology Apr 2018Although adenosine 2A (A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing...
KEY POINTS
Although adenosine 2A (A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A receptor siRNA injections. A receptor siRNA injections selectively knocked down A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A receptors relevant to A receptor-induced phrenic motor facilitation. Upregulation of A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury.
ABSTRACT
Cervical spinal adenosine 2A (A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A receptors for pMF are unknown. This is an important question since the physiological outcome of A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A receptor knock-down was verified by a ∼45% decrease in A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P < 0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A receptor agonist CGS21680 was greatly attenuated in siA (21%) versus siNT treated rats (147%; P < 0.01). There were no significant effects of siA on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A receptors relevant to A receptor-induced pMF.
Topics: Action Potentials; Adenosine A2 Receptor Agonists; Animals; Cholera Toxin; Male; Motor Neurons; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A
PubMed: 29388230
DOI: 10.1113/JP275462 -
Neurotherapeutics : the Journal of the... Oct 2010Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by the progressive loss of motor neurons. Although the molecular mechanism underlying motor neuron... (Review)
Review
Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by the progressive loss of motor neurons. Although the molecular mechanism underlying motor neuron degeneration remains unknown; non-neuronal cells (including astrocytes) shape motor neuron survival in ALS. Astrocytes closely interact with neurons to provide an optimized environment for neuronal function and respond to all forms of injury in a typical manner known as reactive astrogliosis. A strong reactive astrogliosis surrounds degenerating motor neurons in ALS patients and ALS-animal models. Although reactive astrogliosis in ALS is probably both primary and secondary to motor neuron degeneration; astrocytes are not passive observers and they can influence motor neuron fate. Due to the important functions that astrocytes perform in the central nervous system; it is of key importance to understand how these functions are altered when astrocytes become reactive in ALS. Here; we review the current evidences supporting a potential toxic role of astrocytes and their viability as therapeutic targets to alter motor neuron degeneration in ALS.
Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Cell Communication; Humans; Motor Neurons; Nitric Oxide; Receptors, Death Domain; Superoxide Dismutase; Superoxide Dismutase-1
PubMed: 20880509
DOI: 10.1016/j.nurt.2010.05.012 -
Experimental Neurology Dec 2021Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection...
Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.
Topics: Animals; Cell Survival; Cervical Cord; Male; Motor Neurons; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries
PubMed: 34363808
DOI: 10.1016/j.expneurol.2021.113832 -
Current Stem Cell Research & Therapy Sep 2009Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the selective loss of both spinal and upper motor neurons. One strategy in treating ALS is... (Review)
Review
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the selective loss of both spinal and upper motor neurons. One strategy in treating ALS is to use stem cells to replace lost spinal motor neurons. However, transplanted stem cell-derived motor neurons may not survive when exposed to the harsh microenvironment in the spinal cord of ALS. In particular, dysfunctional astrocytes and overactivated microglia in ALS may limit the survival of motor neurons generated from cell replacement therapy. On the other hand, stem cells may provide large quantities of motor neurons that can be used for studying glia-mediated toxic mechanisms and potential therapies in ALS. Here we will review methods and molecular factors for directed differentiation of stem cells into spinal motor neurons, the potential uses of these models for dissecting the mechanisms underlying glia-induced motor neuron degeneration and screening for new therapeutics aimed at protecting motor neurons in ALS, as well as discuss challenges facing the development of motor neuron replacement-based cell therapies for recovery in ALS.
Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Cell Differentiation; Cell- and Tissue-Based Therapy; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Humans; Microglia; Motor Neurons; Oxidative Stress; Spinal Cord; Stem Cell Transplantation; Stem Cells
PubMed: 19492980
DOI: 10.2174/157488809789057392