Motoneuron cell death in the developing lumbar spinal cord of the mouse

doi:10.1016/0165-3806(82)90192-4

Developmental Brain Research

Volume 4, Issue 4, August 1982, Pages 473-479

https://doi.org/10.1016/0165-3806(82)90192-4 Get rights and content

Abstract

Motoneuron cell death in the lumbar lateral motor column of the mouse embryo and neonate was examined to determine the timing and position of cell death with respect to events occurring in the limb. Counts of motoneurons in histological sections of the entire lumbar lateral motor column were made in mice ranging in age from 12 $12$ days of embryonic development to 20 days of neonatal life. Between 13 and 18 days of embryonic development, 67% of the motoneurons initially present in the motor column die, or approximately 3350 out of 5000 cells. Peak motoneuron cell death occurs at 14 days. No cell death occurs during the first neonatal weeks when polyneuronal muscle fiber innervation is lost. Counts of the total number of cells present at 18 days were similar to those previously reported for adult mice, suggesting that all motoneuron cell death has occurred by the end of prenatal development. In a few 13- to 14-day mouse embryos, the hindlimbs were totally filled with horseradish peroxidase (HRP) to define the boundaries and size of the lateral motor population which projects to the limb at early stages. Counts of HRP labeled motoneurons in selected lumbar cord segments were close to the total number of lateral motoneurons in the same segments. As the HRP injections were made prior to or at the onset of cell death, these observations indicate that many cells which die have sent axons into the limb.

References (41)

E.E. Brink et al.
Localization of lumbar epaxial motoneurons in the rat
Brain Res.
(1979)
K. Kozeka et al.
The three-dimensional cyto-architecture of developing murine muscle spindles
Develop. Biol.
(1981)
A. Lamb
The projection patterns of the ventral horn to the hind limb during development
Develop. Biol.
(1976)
P.A. McGrath et al.
The development of synaptic connections between different segmental motoneurons and striated muscles in an axolotl limb
Develop. Biol.
(1979)
R.W. Oppenheim et al.
Neuronal cell death in the brachial spinal cord of the chick is unrelated to the loss of polyneuronal innervation in wing muscle
Brain Res.
(1978)
S. Pockett
Elimination of polyneuronal innervation in proximal and distal leg muscles of chick embryos
Develop. Brain Res.
(1981)
M.C. Prestige et al.
Loss of axons from ventral roots during development
Brain Res.
(1972)
J.R. Slack
Interaction between foreign and regenerating axons in axolotl muscle
Brain Res.
(1978)
M. Abercrombie
Estimation of nuclear population from microtome sections
Anat. Rec.
(1946)
M.C. Brown et al.
Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation
J. Physiol. (Lond.)
(1976)

I.-W. Chu-Wang et al.

Cell death of motoneurons in the chick embryo spinal cord. I. Light and electron microscopic study of naturally occurring and induced cell loss during development

J. comp. Neurol.

(1978)

I.-W. Chu-Wang et al.

Cell death of motoneurons in the chick embryo spinal cord. II. A quantitative and qualitative analysis of degeneration in the ventral root, including evidence for axon outgrowth and limb innervation prior to cell death

J. comp. Neurol.

(1978)

M.J. Dennis

Development of the neuromuscular junction: inductive interactions between cells

Ann. Rev. Neurosci.

(1981)

D.G. Dunlap

The development of the musculature in the hind limb in the frog, Rana pipiens

J. Morph.

(1966)

A.E.H. Flanagan

Differentiation and degeneration in the motor column of foetal mouse

J. Morph.

(1969)

V. Hamburger

Cell death in the development of the lateral motor column of the chick embryo

J. comp. Neurol.

(1975)

M. Hollyday et al.

Reduction of the naturally occurring motor neuron loss by enlargement of the periphery

J. comp. Neurol.

(1976)

W.G. Hopkins et al.

Persistence of multiple innervation in mouse muscles paralysed neonatally with botulinum toxin

Neurosci. Abstr.

(1981)

M.J. Katz et al.

Evolution of the nervous system: role of ontogenetic mechanisms in the evolution of matching populations

A. Lamb

Ventral horn cell counts in a Xenopus with naturally occurring supernumerary hind limbs

J. Embryol. exp. Morphol.

(1979)

Cited by (197)

Loss of larger hypoglossal motor neurons in aged Fischer 344 rats
2023, Respiratory Physiology and Neurobiology
The intrinsic (longitudinal, transversalis and verticalis) and extrinsic (genioglossus, styloglossus, hyoglossus and geniohyoid) tongue muscles are innervated by hypoglossal motor neurons (MNs). Tongue muscle activations occur during many behaviors: maintaining upper airway patency, chewing, swallowing, vocalization, vomiting, coughing, sneezing and grooming/sexual activities. In the tongues of the elderly, reduced oral motor function and strength contribute to increased risk of obstructive sleep apnoea. Tongue muscle atrophy and weakness is also described in rats, yet hypoglossal MN numbers are unknown. In young (6-months, n = 10) and old (24-months, n = 8) female and male Fischer 344 (F344) rats, stereological assessment of hypoglossal MN numbers and surface areas were performed on 16 µm Nissl-stained brainstem cryosections. We observed a robust loss of ∼15 % of hypoglossal MNs and a modest ∼8 % reduction in their surface areas with age. In the larger size tertile of, age-associated loss of hypoglossal MNs approached ∼30 % These findings uncover a potential neurogenic locus of pathology for age-associated tongue dysfunctions.
A KO mouse model for the lncRNA Lhx1os produces motor neuron alterations and locomotor impairment
2023, iScience
Here, we describe a conserved motor neuron-specific long non-coding RNA, Lhx1os, whose knockout in mice produces motor impairment and postnatal reduction of mature motor neurons (MNs). The ER stress-response pathway result specifically altered with the downregulation of factors involved in the unfolded protein response (UPR). Lhx1os was found to bind the ER-associated PDIA3 disulfide isomerase and to affect the expression of the same set of genes controlled by this protein, indicating that the two factors act in conjunction to modulate the UPR. Altogether, the observed phenotype and function of Lhx1os indicate its important role in the control of MN homeostasis and function.
Quantifying mitochondrial volume density in phrenic motor neurons
2021, Journal of Neuroscience Methods
Citation Excerpt :
Note that these images contain a marked heterogeneity of phrenic motor neuron somal sizes, in agreement with past studies (Brandenburg et al., 2020; Fogarty et al., 2018b; Mantilla et al., 2018; Prakash et al., 2000; Rana et al., 2019a, b; Rana et al., 2020). In EM images, putative phrenic motor neurons were identified in semi-thin survey sections by their large long and short axis diameters (>15 μm), the larger cytoplasm to nuclear ratio and the presence of a nucleolus (Fig. 1B), in an identical manner used to distinguish motor neurons from other neuronal types in various ventral horn histological preparations (Banks et al., 2005; Campa and Engel, 1970; Fogarty et al., 2013b, b; Fogarty et al., 2020b; Lance-Jones, 1982; Mitchelle and Watson, 2016; Oppenheim, 1986; Steyn et al., 2013). A region of interest was created for z-stacks (0.5 μm z-step) comprising CTB-labeled phrenic motor neurons (Fig. 2A) and the MitoTracker Red labeled mitochondria (Fig. 2B).
Previous assessments of mitochondrial volume density within motor neurons used electron microscopy (EM) to image mitochondria. However, adequate identification and sampling of motor neurons within a particular motor neuron pool is largely precluded using EM. Here, we present an alternative method for determining mitochondrial volume density in identified motor neurons within the phrenic motor neuron (PhMN) pool, with greatly increased sampling.
This novel method for assessing mitochondrial volume density in PhMNs uses a combination of intrapleural injection of Alexa 488-conjugated cholera toxin B (CTB) to retrogradely label PhMNs, followed by intrathecal application of MitoTracker Red to label mitochondria. This technique was validated by comparison to 3D EM determination of mitochondrial volume density as a “gold standard”.
A mean mitochondrial volume density of ∼11 % was observed across PhMNs using the new MitoTracker Red method. This compared favourably with mitochondrial volume density (∼11 %) measurements using EM.
The range, mean and variance of mitochondrial volume density estimates in PhMNs were not different between EM and fluorescent imaging techniques.
Fluorescent imaging may be used to estimate mitochondrial volume density in a large sample of motor neurons, with results similar to EM, although EM did distinguish finer mitochondrion morphology compared to MitoTracker fluorescence. Compared to EM methods, the assessment of a larger sample size and unambiguous identification of motor neurons belonging to a specific motor neuron pool represent major advantages over previous methods.
Presynaptic Homeostasis Opposes Disease Progression in Mouse Models of ALS-Like Degeneration: Evidence for Homeostatic Neuroprotection
2020, Neuron
Citation Excerpt :
We counted alpha motor neurons across experimental genotypes and age match controls using Nissl staining of the ventral horn of the spinal cord. Alpha motor neurons where identified according to their previously defined cellular area (nuclear diameter > 9–10 μm; cell body diameter > 20 mm) and anatomical hallmarks (Guo et al., 2013; Lance-Jones, 1982). RT-PCR was performed as previously described (Celona et al., 2017; Younger et al., 2013).
Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.
Alterations in hypoglossal motor neurons due to GAD67 and VGAT deficiency in mice
2017, Experimental Neurology
Citation Excerpt :
During development, the mammalian neuromotor system undergoes a period of programmed motor neuron (MN) cell death. Over 50% of all motor neurons generated during neurogenesis are lost in the final trimester in utero (Lance-Jones, 1982; Oppenheim, 1991). The final number of surviving MNs is regulated in an activity-dependent manner, as reduced skeletal muscle activity causes increased MN survival and neuromuscular innervation (Landmesser, 1992; Banks et al., 2003), whereas increased skeletal muscle activity causes decreased MN survival and neuromuscular innervation (Oppenheim and Nunez, 1982).
There is an emerging body of evidence that glycinergic and GABAergic synaptic inputs onto motor neurons (MNs) help regulate the final number of MNs and axonal muscle innervation patterns. Using mutant glutamate decarboxylase 67 (GAD67) and vesicular inhibitory amino acid transporter (VGAT) deficient mice, we describe the effect that deficiencies of presynaptic GABAergic and/or glycinergic release have on the post-synaptic somato-dendritic structure of motor neurons, and the development of excitatory and inhibitory synaptic inputs to MNs. We use whole-cell patch clamp recording of synaptic currents in E18.5 hypoglossal MNs from brainstem slices, combined with dye-filling of these recorded cells with Neurobiotin™, high-resolution confocal imaging and 3-dimensional reconstructions. Hypoglossal MNs from GAD67- and VGAT-deficient mice display decreased inhibitory neurotransmission and increased excitatory synaptic inputs. These changes are associated with increased dendritic arbor length, increased complexity of dendritic branching, and increased density of spiny processes. Our results show that presynaptic release of inhibitory amino acid neurotransmitters are potent regulators of hypoglossal MN morphology and key regulators of synaptic inputs during this critical developmental time point.
Rescue of a mouse model of spinal muscular atrophy with respiratory distress type 1 by AAV9-IGHMBP2 is dose dependent
2016, Molecular Therapy
Citation Excerpt :
Images were captured using Leica DM 5500 B under 10× magnification. Nissl-stained α motor neurons in the anterior horns were determined according to the previously described morphological criteria,31 where α motor neurons in the gray matter at the ventral horn were identified by their large size (cell body diameter >20 μm) and quantified by double blind counting. To confirm Nissl staining, ChAT immunostaining was performed with five sections (for each animal) selected from identical regions of L2–L5 which have been used for Nissl staining.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disease occurring during childhood. The gene responsible for disease development is a ubiquitously expressed protein, IGHMBP2. Mutations in IGHMBP2 result in the loss of α-motor neurons leading to muscle atrophy in the distal limbs accompanied by respiratory complications. Although genetically and clinically distinct, proximal SMA is also caused by the loss of a ubiquitously expressed gene (SMN). Significant preclinical success has been achieved in proximal SMA using viral-based gene replacement strategies. We leveraged the technologies employed in SMA to demonstrate gene replacement efficacy in an SMARD1 animal model. Intracerebroventricular (ICV) injection of single-stranded AAV9 expressing the full-length cDNA of IGHMBP2 in a low dose led to a significant level of rescue in treated SMARD1 animals. Consistent with drastically increased survival, weight gain, and strength, the rescued animals demonstrated a significant improvement in muscle, NMJ, motor neurons, and axonal pathology. In addition, increased levels of IGHMBP2 in lumbar motor neurons verified the efficacy of the virus to transduce the target tissues. Our results indicate that AAV9-based gene replacement is a viable strategy for SMARD1, although dosing effects and potential negative impacts of high dose and ICV injection should be thoroughly investigated.

View all citing articles on Scopus

View full text

Research reportMotoneuron cell death in the developing lumbar spinal cord of the mouse

Abstract

Brain Res.

Develop. Biol.

Develop. Biol.

Develop. Biol.

Brain Res.

Develop. Brain Res.

Brain Res.

Brain Res.

Estimation of nuclear population from microtome sections

Anat. Rec.

Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation

J. Physiol. (Lond.)

Cell death of motoneurons in the chick embryo spinal cord. I. Light and electron microscopic study of naturally occurring and induced cell loss during development

J. comp. Neurol.

Cell death of motoneurons in the chick embryo spinal cord. II. A quantitative and qualitative analysis of degeneration in the ventral root, including evidence for axon outgrowth and limb innervation prior to cell death

J. comp. Neurol.

Development of the neuromuscular junction: inductive interactions between cells

Ann. Rev. Neurosci.

The development of the musculature in the hind limb in the frog, Rana pipiens

J. Morph.

Differentiation and degeneration in the motor column of foetal mouse

J. Morph.

Cell death in the development of the lateral motor column of the chick embryo

J. comp. Neurol.

Reduction of the naturally occurring motor neuron loss by enlargement of the periphery

J. comp. Neurol.

Persistence of multiple innervation in mouse muscles paralysed neonatally with botulinum toxin

Neurosci. Abstr.

Evolution of the nervous system: role of ontogenetic mechanisms in the evolution of matching populations

Ventral horn cell counts in a Xenopus with naturally occurring supernumerary hind limbs

J. Embryol. exp. Morphol.

Research report
Motoneuron cell death in the developing lumbar spinal cord of the mouse