Article Figures & Data
Figures
- Figure 1.
Typical expression pattern of interneuron markers (ChAT, CB, CR, PV) in the cervical enlargement (C6–C8) of the spinal cord. Left, Montages obtained by deconvolution confocal microscopy. Right, Single- or double-labeled epifluorescence micrographs, at higher magnification. Boxes in left panel figures show the approximate locations of micrographs in the right panels. A, ChAT. A1 is a montage from a PW8 animal. A2 is spinal section stained for ChAT (secondary antibody conjugated to Cy3; red) showing interneurons throughout laminae 5–7. A3 shows ChAT interneurons that are consistently present medially in lamina 7. The arrow points to ChAT interneurons close to the central canal. B, CB. B1 is a montage from a PW3 animal. B2 shows CB-positive interneurons (secondary antibody conjugated to FITC, green) in the dorsal horn. B3 shows CB staining in the intermediate zone and ventral horn. B3 inset shows a double-labeled interneuron, with CB staining (FITC) and PV staining (Cy3), in the ventral lamina 7 (lower small box, B1). C, CR. C1 shows a montage from a PW3 animal. C2 shows CR-positive interneurons (FITC) clustered medially in lamina 7. The arrows point to CR interneurons that also stained for ChAT (Cy3; double labeled, yellow). C3. CR interneurons (Cy 3; red) located laterally in the deep dorsal horn and intermediate zone. Note that the dense CR motor pool staining in the deconvolution image reflects dense dendritic (or axonal) processes. D, PV. D1 is a montage from a PW3 animal. D2 and D3 are of the same regions. D2 shows PV (Cy3) interneurons among the dense plexus of afferent fiber labeling. D3 shows the PV labeling (FITC) in relation to ChAT staining (Cy3). Arrows point to PV-positive interneurons that were consistently lateral to the medial ChAT cell group. Scale bars: All 500 μm, except C3, 250 μm.
- Figure 2.
Regional distribution of spinal interneurons at PW4, PW8, and ≥PW12. The locations of labeled interneurons were marked at 100× magnification in at least two animals per age group. Local density of interneurons (see Materials and Methods) is represented as a color scale (i.e., heat maps). The maps are averages, generated from the deconvolution images (as in Fig. 1; left column) from individual animals. Representative examples are shown. A, ChAT. Note the increase in ChAT interneurons between PW4 and PW8. B, CB. Note that CB interneuron numbers decreased remarkably between PW4 and PW8. C, CR. D, PV. No age-dependent changes were noted for either CR or PV. Scale bar: 500 μm; range for the color scale (in cells per millimeter): A, PW4: 0–33, PW8: 0–43, adult: 0–47; B, PW4: 0–60, PW8: 0–74, adult: 0–79; C, D, all 0–33.
- Figure 3.
Unbiased measurements of the density of spinal interneurons. The optical fractionator method was used to determine changes in the density of labeled interneurons. The insets in each part show the spinal region measured (gray). Animals were grouped as immature (PW3 and PW4) and mature (≥PW8). As discussed in the Results, cluster analysis provided justification for pooling into these two age groups. Cell density was calculated, based on estimated cell counts for each animal in each age group (5 × 40 μm sections/animal, 4 animals in each group). The mean density for each group and SEM are presented. A, ChAT, within laminae 4–6. B, ChAT, within lamina 7 dorsal. C, CB, within laminae 1–3. D, CB, within laminae 4–6. E, CR, within laminae 4–6. F, PV, within laminae 4–6. Note that, for the two age groups, there were significant increases in ChAT cell density in both laminae 4–6 (A) and lamina 7 (B) and significant decreases in CB density in laminae 1–3 (C) and 4–6 (D). In contrast, E and F show the lack of a significant change in density for CR- and PV-positive neurons in the same animals. lam, Laminae.
- Figure 4.
Interneuron clusters marked by ChAT and CBPs. The left half of the figure shows the locations of clusters (see Materials and Methods for how these were obtained) fitted for all animals (2 per age group). The right two columns are semischematic representations, combining and summarizing the immature (PW3 and PW4) and mature (PW8 and >PW12) groups. The size of all spinal cords was adjusted so that the dorsoventral length of the gray matter was the same. A, ChAT neurons were consistently present only in medial lamina 7 at PW3 and PW4 (left two columns). Note the emergence of clusters in the older animals. B, CB neurons show a clear shift from a diffuse pattern in the immature animals to a focused pattern in laminae 1–3 in the mature animals. C and D show a diffuse distribution of CR and PV cells, with no apparent change with age. Rexed's laminae are defined in one of the top summary column images.
- Figure 5.
Topographic relationship between composite CST contralateral termination patterns and interneuron cluster overlays. Each composite map was constructed from four animals, two with rostrally placed BDA tracer injections and two with caudally placed injections. A–D overlay interneuron cell clusters on the composite CST distribution maps. Each cell cluster from each animal is shown as a black outline and 50% transparent fill. The greater the overlap between animals, the darker the fill. As in the analysis for Figure 4, to facilitate spatial comparisons the dorsoventral length of the gray matter was adjusted to be the same in all animals. A, ChAT. B, Calbindin. C, Calretinin. D, Parvalbumin. Scale bars: 1 mm; range for color scale: length, 0–800 μm axon length.
- Figure 6.
Targeting of CST projections to particular contralateral spinal subregions. Each panel shows the regional distribution of axons and varicosities for a representative animal. Local density is represented as a color scale. The insets between the axon and varicosity distributions in A show the locations of tracer injection sites for A–D, and the inset in E shows that for tracer injections in E. The location of the medial lamina 7 ChAT cell group for each animal is shown (thick white outline). A, PW3. B, PW4. Note arrows pointing to two distinct CST termination clusters, in lamina 6 and lamina 7. Note that the CST does not target the medial ChAT cell group at PW3 and PW4. C, PW8. CST labeling now extends into the region of the medial ChAT group. Yellow arrows point to labeling associated with the lateral CR interneurons. D, PW12. Yellow arrows point to labeling present among the CR-positive interneuron in lateral laminae 6–7. E, Adult. Arrows in E1 and E2 point to a cluster of labeling present with the caudal injection but not the rostral injection. Diagonal white arrow in E3 and E4 points to a cluster of CST terminations absent in E1 and E2. Horizontal yellow arrows in E3 and E4 point to labeling present among the CR-positive interneuron in lateral laminae 5–7. Scale bars: 1 mm; color scale range: left panels, axon length: 0–400 mm of axon length; right panels, varicosities (number per millimeter): A–C, 0–120; D, 0–60; E2, 0–115; E4, 0–80.
- Figure 7.
Development of CB and ChAT interneurons depends on M1 activity. A, Representative regional distribution of CB interneurons in the cervical spinal cord contralateral (right) and ipsilateral (left) to M1 inactivation. Cell density is represented as a color scale. B, Similar to A, but for ChAT. C, Unbiased measurement of CB interneuron density, using the optical fractionator method. Note that there is more than a doubling of neuron density on the inactive over the active side. The bar graph plots mean. Data from individual animals are shown. D, As in C, but for ChAT interneuron density. E, Ratio of ChAT to CB, calculated for each animal. Scale bar: (in B) A, B, 1 mm. Color scale range: in (B) A, 0–75 cells/mm; B, 0–30 cells/mm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
