High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spinal motor neurons are among the most difficult cells to observe in vivo. Larval zebrafish have transparent bodies that allow non-invasive visualization of whole cells of single spinal motor neurons, from somas to the neuromuscular synapses. This unique feature, combined with its amenability to genome editing, pharmacology, and optogenetics, enables functional analyses of ALS-associated proteins in the spinal motor neurons in vivo with subcellular resolution. Here, we review the zebrafish skeletal neuromuscular system and the optical methods used to study it. We then introduce a recently developed optogenetic zebrafish ALS model that uses light illumination to control oligomerization, phase transition and aggregation of the ALS-associated DNA/RNA-binding protein called TDP-43. Finally, we will discuss how this disease-in-a-fish ALS model can help solve key questions about ALS pathogenesis and lead to new ALS therapeutics.

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The manipulation of spinal motor neuron axonal growth promotes spinal cord repair

10.1101/2021.11.13.468456 ◽

2021 ◽

Author(s):

Feng Wang ◽

Xinya Fu ◽

Meiemei Li ◽

Xingran Wang ◽

Jile Xie ◽

...

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Axonal Regeneration ◽

Axonal Growth ◽

Treatment Method ◽

Spinal Motor Neurons ◽

Spinal Motor ◽

Cord Injury ◽

Spinal Cord Repair

The loss of motor function in patients with spinal cord injury (SCI) is primarily due to the severing of the corticospinal tract (CST). Spinal motor neurons are located in the anterior horn of the spinal cord, and as the lower neurons of the CST, they control voluntary movement. Furthermore, its intrinsic axonal growth ability is significantly stronger than that of cerebral cortex pyramid neurons, which are the upper CST neurons. Therefore, we established an axonal regeneration model of spinal motor neurons to investigate the feasibility of repairing SCI by promoting axonal regeneration of spinal motor neurons. We demonstrated that conditionally knocking out pten in mature spinal motor neurons drastically enhanced axonal regeneration in vivo, and the regenerating axons of the spinal motor neurons re-established synapses with other cells in the damaged spinal cord. Thus, this strategy may serve as a novel and effective treatment method for SCI.

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Developmental regulation of the orphan receptor COUP-TF II gene in spinal motor neurons

Development ◽

10.1242/dev.120.1.25 ◽

1994 ◽

Vol 120 (1) ◽

pp. 25-36 ◽

Cited By ~ 11

Author(s):

B. Lutz ◽

S. Kuratani ◽

A.J. Cooney ◽

S. Wawersik ◽

S.Y. Tsai ◽

...

Keyword(s):

Spinal Cord ◽

Thyroid Hormone ◽

Motor Neuron ◽

Motor Neurons ◽

Hormone Receptor ◽

Thyroid Hormone Receptor ◽

Orphan Receptor ◽

Northern Analysis ◽

Spinal Motor Neurons ◽

Spinal Motor

Members of the steroid/thyroid hormone receptor superfamily are involved in the control of cell identity and of pattern formation during embryonic development. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) can act as regulators of various steroid/thyroid hormone receptor pathways. To begin to study the role of COUP-TFs during embryogenesis, we cloned a chicken COUP-TF (cCOUP-TF II) which is highly homologous to human COUP-TF II. Northern analysis revealed high levels of cCOUP-TF II transcripts during organogenesis. Nuclear extracts from whole embryos and from embryonic spinal cords were used in electrophoretic mobility shift assays. These assays showed that COUP-TF protein is present in these tissues and is capable of binding to a COUP element (a direct repeat of AGGTCA with one base pair spacing). Analysis of cCOUP-TF expression by in situ hybridization revealed high levels of cCOUP-TF II mRNA in the developing spinal motor neurons. Since the ventral properties of the spinal cord, including the development of motor neurons, is in part established by inductive signals from the notochord, we transplanted an additional notochord next to the dorsal region of the neural tube in order to induce ectopic motor neurons. We observed that an ectopic notochord induced cCOUP-TF II gene expression in the dorsal spinal cord in a region coextensive with ectopic domains of SC1 and Islet-1, two previously identified motor neuron markers. Collectively, our studies raise the possibility that cCOUP-TF II is involved in motor neuron development.

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The zebrafish detour gene is essential for cranial but not spinal motor neuron induction

Development ◽

10.1242/dev.126.12.2727 ◽

1999 ◽

Vol 126 (12) ◽

pp. 2727-2737 ◽

Cited By ~ 4

Author(s):

A. Chandrasekhar ◽

H.E. Schauerte ◽

P. Haffter ◽

J.Y. Kuwada

Keyword(s):

Spinal Cord ◽

Cell Death ◽

Motor Neuron ◽

Motor Neurons ◽

Spinal Motor Neuron ◽

Neuronal Phenotype ◽

Spinal Motor Neurons ◽

Spinal Motor ◽

Hh Signaling ◽

Function Cell

The zebrafish detour (dtr) mutation generates a novel neuronal phenotype. In dtr mutants, most cranial motor neurons, especially the branchiomotor, are missing. However, spinal motor neurons are generated normally. The loss of cranial motor neurons is not due to aberrant hindbrain patterning, failure of neurogenesis, increased cell death or absence of hh expression. Furthermore, activation of the Hh pathway, which normally induces branchiomotor neurons, fails to induce motor neurons in the dtr hindbrain. Despite this, not all Hh-mediated regulation of hindbrain development is abolished since the regulation of a neural gene by Hh is intact in the dtr hindbrain. Finally, dtr can function cell autonomously to induce branchiomotor neurons. These results suggest that detour encodes a component of the Hh signaling pathway that is essential for the induction of motor neurons in the hindbrain but not in the spinal cord and that dtr function is required for the induction of only a subset of Hh-mediated events in the hindbrain.

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Expression of metabotropic glutamate receptor mRNAs in the human spinal cord: implications for selective vulnerability of spinal motor neurons in amyotrophic lateral sclerosis

Journal of the Neurological Sciences ◽

10.1016/s0022-510x(01)00561-5 ◽

2001 ◽

Vol 189 (1-2) ◽

pp. 65-69 ◽

Cited By ~ 24

Author(s):

Masahiko Tomiyama ◽

Tamaki Kimura ◽

Tetsuya Maeda ◽

Hiroyasu Tanaka ◽

Kenichi Furusawa ◽

...

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Metabotropic Glutamate Receptor ◽

Selective Vulnerability ◽

Human Spinal Cord ◽

Metabotropic Glutamate ◽

Spinal Motor Neurons ◽

Spinal Motor ◽

Lateral Sclerosis ◽

Receptor Mrnas

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Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons.

The Journal of Cell Biology ◽

10.1083/jcb.100.1.74 ◽

1985 ◽

Vol 100 (1) ◽

pp. 74-85 ◽

Cited By ~ 66

Author(s):

S C Papasozomenos ◽

L I Binder ◽

P K Bender ◽

M R Payne

Keyword(s):

Spinal Cord ◽

Central Nervous System ◽

Nervous System ◽

Peripheral Nervous System ◽

Dorsal Root Ganglia ◽

Motor Neurons ◽

Dorsal Root ◽

Spinal Motor Neurons ◽

Spinal Motor ◽

The Central Nervous System

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile-treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'-Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.

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The organization of spinal motor neurons in a monotreme is consistent with a six-region schema of the mammalian spinal cord

Journal of Anatomy ◽

10.1111/joa.12492 ◽

2016 ◽

Vol 229 (3) ◽

pp. 394-405 ◽

Cited By ~ 1

Author(s):

Amer Mitchelle ◽

Charles Watson

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Spinal Motor Neurons ◽

Spinal Motor

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