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Musculotopic organization of the motor neurons supplying the mouse hindlimb muscles: a quantitative study using Fluoro-Gold retrograde tracing

Abstract

We have mapped the motor neurons (MNs) supplying the major hindlimb muscles of transgenic (C57/BL6J-ChAT-EGFP) and wild-type (C57/BL6J) mice. The fluorescent retrograde tracer Fluoro-Gold was injected into 19 hindlimb muscles. Consecutive transverse spinal cord sections were harvested, the MNs counted, and the MN columns reconstructed in 3D. Three longitudinal MN columns were identified. The dorsolateral column extends from L4 to L6 and consists of MNs innervating the crural muscles and the foot. The ventrolateral column extends from L1 to L6 and accommodates MNs supplying the iliopsoas, gluteal, and quadriceps femoris muscles. The middle part of the ventral horn hosts the central MN column, which extends between L2 and L6 and consists of MNs for the thigh adductor, hamstring, and quadratus femoris muscles. Within these longitudinal columns, the arrangement of the different MN groups reflects their somatotopic organization. MNs innervating muscles developing from the dorsal (e.g., quadriceps) and ventral muscle mass (e.g., hamstring) are situated in the lateral and medial part of the ventral gray, respectively. MN pools belonging to proximal muscles (e.g., quadratus femoris and iliopsoas) are situated ventral to those supplying more distal ones (e.g., plantar muscles). Finally, MNs innervating flexors (e.g., posterior crural muscles) are more medial than those belonging to extensors of the same joint (e.g., anterior crural muscles). These data extend and modify the MN maps in the recently published atlas of the mouse spinal cord and may help when assessing neuronal loss associated with MN diseases.

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Abbreviations

Ad9:

Adductor MNs of lamina 9

ALS:

Amyotrophic lateral sclerosis

Ant:

Anterior

Ax9:

Axial MNs of lamina 9

BAC:

Bacterial artificial chromosome

CC:

Central canal

ChAT:

Choline acetyltransferase

CEx9:

Crural extensor MNs of lamina 9

CFl9:

Crural flexor MNs of lamina 9

Cr9:

Cremaster MNs of lamina 9

CV:

Coefficient of variation

Dig:

Digitorum

DL:

Dorsolateral

EGFP:

Enhanced green fluorescent protein

ExA9:

External anal sphincter MNs of lamina 9

Ext:

Extensor

ExU9:

External urethral sphincter MNs of lamina 9

FG:

Fluoro-Gold

FUS/TLS:

Fused in sarcoma/translocated in liposarcoma

Gl9:

Gluteal MNs of lamina 9

Hm9:

Hamstring MNs of lamina 9

HRP:

Horseradish peroxidase

IML:

Intermediolateral nucleus

Lat:

Lateral

LDCom:

Lumbar dorsal commissural nucleus

MN:

Motor neuron

Pes9:

Foot MNs of lamina 9

Post:

Posterior

Q9:

Quadriceps MNs of lamina 9

QL9:

Quadratus lumborum MNs of lamina 9

SDCom:

Sacral dorsal commissural nucleus

SOD1:

Superoxide dismutase 1

Spf:

Superficial

TDP-43:

Transactivating response element DNA binding protein-43

VL:

Ventrolateral

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Acknowledgments

This study was supported by an NHMRC (National Health & Medical Research Council) Australia Fellowship Grant awarded to Dr George Paxinos (Grant #568605).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Correspondence to Charles Watson.

Additional information

T. Bácskai and Z. Rusznák equally contributed to this paper.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1. Extent and organization of the MN group innervating the gluteus medius. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a4) 3D reconstruction of the number and distribution of MNs innervating the gluteus medius. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and L: lateral. Scale bar: 200 μm.

Online Resource2. Extent and organization of the MN group innervating the adductor magnus. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a4) 3D reconstruction of the number and distribution of MNs innervating the adductor magnus. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and M: medial. Scale bar: 200 μm.

Online Resource 3. Extent and organization of the MN group innervating the gracilis. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a4) 3D reconstruction of the number and distribution of MNs innervating the gracilis. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and M: medial. Scale bar: 200 μm.

Online Resource 4. Extent and organization of the MN group innervating the pectineus. (a1–a2) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a3) 3D reconstruction of the number and distribution of MNs innervating the pectineus. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostral) and yellow (caudal) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and M: medial. Scale bar: 200 μm.

Online Resource 5. Extent and organization of MN groups innervating the biceps femoris. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections into the anterior head of biceps femoris muscle. (b1–b2) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections into the posterior head of biceps femoris. (c1–c2) 3D reconstruction of the number and distribution of MNs innervating the anterior and posterior heads. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and M: medial. Scale bars: 200 μm.

Online Resource 6. Extent and organization of the MN group innervating the semitendinosus. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a4) 3D reconstruction of the number and distribution of MNs innervating the semitendinosus. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and M: medial. Scale bar: 200 μm.

Online Resource 7. Extent and organization of the MN group innervating the deep anterior crural muscles. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a4) 3D reconstruction of the number and distribution of MNs innervating the deep anterior crural muscles. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and L: lateral. Scale bar: 200 μm.

Online Resource 8. Extent and organization of the MN group innervating the deep posterior crural muscles. (a1–a3) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a4) 3D reconstruction of the number and distribution of MNs innervating the deep posterior crural muscles. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostralmost), blue, and yellow (caudalmost) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and L: lateral. Scale bar: 200 μm.

Online Resource 9. Extent and organization of the MN group innervating the extensor digitorum brevis. (a1–a2) Photomicrographs of transverse sections from different levels of the spinal cord (as indicated in the bottom-right corner) after FG injections. (a3) 3D reconstruction of the number and distribution of MNs innervating the extensor digitorum brevis. Gray lines mark the border of the gray matter, green circles represent the central canal, and each red dot corresponds to the cell body of a single MN. White (rostral) and yellow (caudal) arrows indicate the positions where the photomicrographs were taken. D: dorsal, C: caudal, and M: medial. Scale bar: 200 μm.

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Bácskai, T., Rusznák, Z., Paxinos, G. et al. Musculotopic organization of the motor neurons supplying the mouse hindlimb muscles: a quantitative study using Fluoro-Gold retrograde tracing. Brain Struct Funct 219, 303–321 (2014). https://doi.org/10.1007/s00429-012-0501-7

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Keywords

  • Hindlimb
  • Spinal cord
  • Motor neuron
  • Retrograde tracing
  • Musculotopic organization
  • 3D modeling