Rostro-Caudal Specificity of Corticospinal Tract Projections in Mice

Abstract

Rostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex and a region in the dorsolateral cortex near the barrel field (S2). Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the dorsolateral cortex. With injections of biotinylated dextran amine (BDA) into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic sacral levels. With BDA injections into the dorsolateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae and axons from lateral sensorimotor cortex terminated primarily in laminae III-V of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.

Keywords: biotinylated dextran amine; mouse; retro-AAV; sensorimotor cortex; spinal cord.

Figure 1

Rostro-caudal distribution of FG-labeled cell bodies in the cortex following injections of FG at C5. (A–E) Area of the cortex containing retrogradely labeled neurons at different rostro-caudal levels. Coordinates with respect to bregma are indicated in the upper right of each panel. Scale bars = 400 μm in A (applies to A–D); 400 μm in E. (F, G) Higher magnification view of retrogradely labeled CST neurons in the dorsomedial frontal cortex immunostained for FG and nearby section stained for cresyl violet. (H, I) Retrogradely labeled CST neurons in the dorsolateral cortex (H) and nearby section stained for cresyl violet. (J) Map of distribution of retrogradely labeled CST neurons and injection sites. Different pairs of colored lines indicate the distribution of retrogradely labeled neurons in 3 mice. Each pair of shaded colors represents the span of labeled cells on the two sides. Colored dots represent coordinates for BDA injections on a particular date represented in Figs 5–13. Some injection sites were plotted with slight offset to enable visualization of overlapping sites. J was created using a base drawing provided by Dr T. Jones, which is a modified version of Figure 2B in Tennant et al. (2010).

Figure 2

Retrograde transfection of cortical neurons following injections of retrograde-AAV/Cre at C5. (A) Visualization of tdT fluorescence in intact uncleared spinal cord and brain by fluorescence epi-illumination. (B) Coronal section illustrating native tdT fluorescence in cell bodies and dendrites of pyramidal neurons in layer V. (C) Higher magnification view reveals intense tdT fluorescence in nuclei and labeling throughout dendrites including fine dendritic arbors in layers I–II. (D) Immunostained section (FITC) illustrating tdT-positive neurons in the main sensorimotor cortex. (E) Immunostained section illustrating tdT-positive neurons in the lateral cortex S2. (F) Projection image of 3D reconstruction with light sheet microscopy after clearing and immunostaining with iDISCO. Thousands of tdT-positive CMNs are evident in layer V throughout the primary motor, secondary motor, and primary somatosensory cortical areas and S2. IC, internal capsule; Med Pyr, medullary pyramid; Pyr Dec, pyramidal decussation. (G) Labeled neurons identified by “Spots” function in Imaris. Numbers of labeled neurons are indicated. Comparison of manual counts of retrogradely labeled CMNS (H) with counts from Imaris “Spots” function (I). Shaded boxes indicate the 3 counting sites; numbers indicate the counts per site. (J) Intact brains and spinal cords from Rosa^tdTomato mice 1 year postinjection.

Figure 3

Retrograde transfection of cortical neurons following injections of retrograde-AAV/Cre at L2. (A–C) Intact brains and spinal cords from 1 Pten^ff/Rosa^tdTomato and 2 Rosa^tdTomato mice 1 year postinjection. (D–G) Immunostained sections (FITC) illustrating tdT-positive neurons at different locations with respect to bregma. (H) Projection image of 3D reconstruction with light sheet microscopy after clearing and immunostaining with iDISCO. TdT-positive CMNs are present in posterior sensorimotor cortex. (I) Labeled neurons identified by “Spots” function in Imaris. Numbers of labeled neurons on each side are indicated. Scale bar in F = 500 μm and applies to D–G.

Figure 4

Retrogradely labeled CST neurons with retro-AAV/Cre injections at L2 and retro-AAV/shPTEN-GFP at C5. (A–E) Some commercial GFP antibodies recognize tdT. (A) Native tdT fluorescence in a section stained for GFP using Invitrogen antibody. (B) Immunofluorescence for GFP in same section shown in A. (C) Native tdT fluorescence in a section stained for GFP using Abcam antibody. (D) Immunofluorescence for GFP in same section shown in C. (E) Native tdT fluorescence in a section stained for GFP using Novus antibody. (F) Immunofluorescence for GFP in same section shown in E. (G) Visualization of GFP fluorescence in intact uncleared brain and spinal cord by fluorescence epi-illumination. (H) Visualization of tdT fluorescence in the same brain and spinal cord. (I) Coronal section through posterior sensorimotor cortex approximately 1.8 mm caudal to bregma. (J) Same section illuminated to reveal native tdT fluorescence. (K) Merged image of I and J to reveal the few double-labeled neurons that were present (arrows). Scale bar in E = 100 μm and applies to A–F. Scale bar in I = 500 μm and applies to I and J; scale bar in K = 250 μm.

Figure 5

Dorsal frontal cortex projects selectively to cervical and high thoracic levels with terminal arbors in the intermediate lamina and dorsal part of the ventral horn. (A) Map of injection sites in which BDA injections were centered at 2.6–3.0 mm anterior and 0.7–0.8 mm lateral. (B) Injection site in the case with the largest number of labeled axons (0313C, 2.6 mm anterior, 0.7 mm lateral). (C) Cytoarchitecture of the area in a nearby cresyl violet-stained section. (D–G) Rostro-caudal distribution of BDA-labeled CST axons. Location of spinal cord laminae is indicated in E. Numbers in the lower part of panels D–G indicate distance from the pyramidal decussation. (H) High magnification view of BDA-labeled axons in the dCST at cervical levels contralateral and ipsilateral to the injection (DAB stain). (I) Counts of labeled at 1-mm interval. dCST, dorsal corticospinal tract in the dorsal column; dlCST, dorsolateral corticospinal tract in the dorsal part of the lateral column.

Figure 6

RFA projects selectively to cervical and high thoracic levels with terminal arbors located throughout the intermediate lamina and medial ventral horn. (A) Map of injection sites in which BDA injections were centered at 1.8 mm anterior and 1.5–1.6 mm lateral. (B) Injection site in the case with the largest number of labeled axons (1120C). (C) Cytoarchitecture of the area in a nearby cresyl violet-stained section. (D–H) Rostro-caudal distribution of BDA-labeled axons. Location of spinal cord laminae is indicated in E. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation. (I) Counts of labeled axons at 1-mm interval. vCST, labeled axons in the ventral column ipsilateral to the cortex of origin in the expected location of the ventral CST. Other abbreviations are as in Figure 5.

Figure 7

Medial anterior forelimb cortex projects selectively to cervical and mid-thoracic levels with terminal arbors located throughout the intermediate lamina and medial ventral horn. (A) Map of injection sites at 0.6–0.8 mm anterior, 0.9–1.1mm lateral. (B) BDA labeling at injection site. (C) Cresyl violet-stained section at injection site. (D–H) Cy3-labeled axons at cervical-high lumbar levels following a single injection of BDA at 0.8 anterior and 0.9–1.1 mm lateral. Location of spinal cord laminae is indicated in E. Insets show axons in the dlCST and dCST at ×2 higher magnification. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation (I) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Abbreviations are as in Figure 5.

Figure 8

Medial posterior forelimb cortex projects to cervical–sacral levels with terminal arbors throughout the intermediate lamina and medial ventral horn. (A) Map of injection sites at 0, 0.2, and 0.4 mm anterior and 1.0, 1.1, and 1.2 mm lateral. (B) BDA labeling at injection site. (C) Cresyl violet-stained section at injection site. (D–H) Cy3-labeled axons at cervical–sacral levels following a single injection of BDA at 0.4 mm anterior and 1.2 mm lateral. Location of spinal cord laminae is indicated in E. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation. (I) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Abbreviations are as in Figure 5. Asterisk in F indicates a few labeled axons in the lateral column ipsilateral to the injected cortex.

Figure 9

Anterior-lateral forelimb cortex projects selectively to cervical levels with terminal arbors in the dorsal intermediate lamina and medial ventral horn. (A) Map of injection sites 0.7–1.0 mm anterior and 2.2–2.3 mm lateral. (B) BDA labeling at injection site in the case with the most labeled axons. (C) Cresyl violet-stained section at injection site. (D–H) Cy3-labeled axons at different levels following a single injection of BDA at 1.0 mm anterior and 2.3 mm lateral. Note large number of labeled axons in the dCST ipsilateral to the cortex of origin (insets in A and B) and that, although very few axons extend to lumbar levels, there are occasional arbors in the gray matter (H, arbor). Location of spinal cord laminae is indicated in E. Insets show axons in the dlCST and dCST at ×2 higher magnification. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation. (I) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Abbreviations are as in Figure 5.

Figure 10

Posterior-lateral sensorimotor cortex projects to cervical–lumbar levels with terminal arbors in the medial portion of the dorsal horn. (A) Map of injection sites. (B) BDA labeling at injection site in the case with the most labeled axons. (C) Cresyl violet-stained section at injection site. (D–H) Cy3-labeled axons at different levels following a single injection of BDA at 0.8 mm posterior and 2.4 mm lateral. Location of spinal cord laminae is indicated in E. Insets show axons in the dlCST and dCST at ×2 higher magnification. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation. (I) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Insets show high magnification views of labeled axons. Abbreviations are as in Figure 5.

Figure 11

Posterior-medial sensorimotor cortex (hindlimb region) projects to cervical–sacral levels with terminal arbors located primarily in the dorsal horn. (A) Map of injection sites. (B) BDA labeling at injection site in the case with the most labeled axons. (C) Cresyl violet-stained section at injection site. (D–H) Cy3-labeled axons at different levels following a single injection of BDA at 0.9 mm posterior and 1.1 mm lateral. Insets show axons in the dlCST and dCST at ×2 higher magnification. Location of spinal cord laminae is indicated in G. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation. (I) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Insets show high magnification views of labeled axons. Abbreviations are as in Figure 5.

Figure 12

Caudal-most sensorimotor cortex; 1.5 mm lateral to the midline. (A) Map of injection sites. (B) BDA labeling at injection site in the case with the most labeled axons. (C) Cresyl violet-stained section at injection site. (D–H) Cy3-labeled axons at different levels following a single injection of BDA at 0.9 mm posterior and 1.1 mm lateral. Location of spinal cord laminae is indicated in G. Numbers in the lower part of panels D–H indicate distance from the pyramidal decussation. (I) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Abbreviations are as in Figure 5.

Figure 13

CST neurons in dorsolateral cortex (S2) project to high cervical levels and arborize in the medial dorsal horn. (A) Map of injection sites. (B) BDA labeling at injection site in the case with the most labeled axons. (C) Cresyl violet-stained section at injection site. (D–G) Cy3-labeled axons at different levels following a single injection of BDA at 1.0 posterior and 3.6 lateral. (H) Counts of labeled axons in the dCST in sections spaced at 1-mm interval. Abbreviations are as in Figure 5.

Figure 14

Summary of topographic specificity of CST projections. (A) Map of injection sites that give rise to CST axons that terminate mainly at cervical levels. (B) Map of injection sites that give rise to CST axons that terminate at cervical–sacral levels. (C) Map of injection sites that give rise to CST axons that extend through cervical levels without projections into the gray matter and then terminate selectively at lumbar–sacral levels. (D) Map of injection sites that give rise to CST axons that terminate in intermediate laminae with some axons extending into the ventral horn. (E) Map of injection sites that give rise to CST axons that terminate in the dorsal horn.