Table 1.
Subjects used in species differences analysis.
Table 2.
Subjects used in developmental analysis.
Table 3.
Subjects used in l-CST lesion analysis using adult rhesus macaque monkeys.
Figure 1.
SPP1 mRNA expression in the sensorimotor cortex of the capuchin monkey.
A: A photograph showing SPP1 mRNA expression around the central sulcus (CS) in the capuchin monkey. B, C: Nissl-stained (B) and adjacent sections in the primary motor cortex (M1) of the capuchin monkey showing the distribution of SPP1 mRNA-positive neurons (C). D: A control section hybridized with the SPP1 mRNA sense probe. E, F: Double-labeling study with SMI32, an antibody against non-phosphorylated neurofilament H. E: Localization of SPP1 mRNA-positive neurons in layer V of the capuchin monkey M1. F: SMI 32 immunoreactivity in the same section as (E). Arrows indicate SPP1-mRNA-positive neurons showing SMI 32 immunoreactivity. Double arrowheads indicate SMI 32 immunoreactive neurons with no SPP1-mRNA expression. G-J: High-magnification photomicrographs of SPP1 mRNA-positive neurons and scattergram showing the relationship between SPP1 mRNA signal intensity and size of the neuronal cell bodies in layers V (G, H) and III (I, J). Arrows and double arrowheads in (G) and (I) indicate neurons showing intense and weak signals, respectively. In (H) and (J), the number of neurons examined was 57 and 34 in layer V, and 46 and 50 in layer III. *P<0.05, ***P<0.0001, according to linear regression analysis. Squares and triangles are data points from each capuchin monkey. The solid line and dashed line are linear approximations of the data represented by the squares and triangles, respectively. S1, primary somatosensory cortex. II–VI, layers II–VI of the cerebral cortex. Scale bars = 1 mm in A; 200 µm in B–D; 50 µm in E–G, I.
Table 4.
Summary of secreted phosphoprotein 1 (SPP1) expression in the six species examined.
Figure 2.
SPP1 expression in the motor cortex of the marmoset and squirrel monkey, and the brainstem and spinal cord of New World monkeys.
A, B: Nissl-stained (A) and adjacent sections of the marmoset primary motor cortex (M1) hybridized with the SPP1 antisense probe (B). C, D: Nissl-stained (C) and adjacent sections of the squirrel monkey M1 hybridized with the SPP1 antisense probe (D). E, F: Low- (E) and high- (F) magnification photomicrographs of SPP1 mRNA-positive neurons in the red nucleus of the capuchin monkey. G, H: Low- (G) and high- (H) magnification photomicrographs of SPP1 mRNA-positive neurons in the eighth cervical spinal segment of the capuchin monkey. I, J: Low- (I) and high- (J) magnification photomicrographs of SPP1 mRNA-positive neurons in the red nucleus of the squirrel monkey. K, L: Low- (K) and high- (L) magnification photomicrographs of SPP1 mRNA-positive neurons in the eighth cervical spinal segment of the squirrel monkey. RN, red nucleus. II–VI, layers II–VI of M1. I–X, layers I–X of the spinal cord. VM, ventral medial nucleus. Capuchin, capuchin monkey. Marmo, marmoset. Squirrel, squirrel monkey. Scale bars = 200 µm in A–D; 500 µm in E, G, I, K; 50 µm in F, H, J, L.
Figure 3.
Summary of the results from the species differences analysis.
A: The density of SPP1 mRNA-positive neurons in each layer of the primary motor cortex (M1) in five animals: rat, marmoset, squirrel monkey, capuchin monkey, and rhesus macaque (the number of sections in each species was 6, 6, 10, 4, and 6, respectively). The mean density of SPP1 mRNA-positive neurons (± SE) is shown. *P<0.05, **P<0.01, according to a Friedman's one-way analysis of variance (ANOVA) with Dunn's post-hoc tests. †P<0.05, ††P<0.01, according to a Kruskal–Wallis one-way ANOVA with Dunn's post-hoc tests. B: The density of SPP1 protein-positive neurons in each layer of M1 in human (n = 6 sections). The mean density of SPP1 protein-positive neurons (± SE) is shown. *P<0.05, **P<0.01, according to a Friedman's one-way analysis of variance (ANOVA) with Dunn's post-hoc tests. C–E: Schematic diagram of the interspecies differences in SPP1 expression in the projection neurons of motor-related structures including M1, red nucleus (RN), and lower cervical spinal segments. The motor systems are organized in a functional hierarchy from bottom to top: the spinal cord, brainstem, and motor cortex, each associated with increasing levels of complexity. SPP1 was commonly expressed in motor-related areas among all species examined; however, the level of motor hierarchy in which this gene was expressed varied among species. The regions with intense or moderate SPP1 signals are shown as red-filled circles, and those with weak or no signals are shown as gray-filled circles. See Table 4 for expression data details from the RN and spinal cord of each species.
Figure 4.
SPP1 expression in the human motor cortex.
A, B: Nissl-stained (A) and adjacent sections of the human M1 showing the distribution of SPP1protein-positive neurons (B). C, D: Control section incubated without primary antibody for the SPP1 protein (C) and incubated with primary antibody preabsorbed with SPP1 protein (D). E–H: High-magnification photomicrographs of SPP1 protein-positive neurons and scattergram showing the relationship between SPP1 signal intensity and size of the neuronal cell bodies in layers V (E, F) and III (G, H). Arrows and double arrowheads in (E) and (G) indicate neurons showing intense and weak signals, respectively. In (F) and (H), the number of neurons examined was 33, 56, and 42 in layer V, and 41, 41, and 31 in layer III; **P<0.01, ***P<0.0001, according to linear regression analysis. Squares, triangles, and small rectangles in (F) and (H) are data points from each human tissue sample. The solid, dashed, and dotted lines are linear approximations of the data represented by the squares, triangles, and small rectangles, respectively. Scale bars = 200 µm in A–D; 50 µm in E, G
Figure 5.
SPP1 expression in the developing macaque monkey.
A–D: Nissl-stained and adjacent sections showing the distribution of SPP1 mRNA-positive neurons in the primary motor cortex (M1) of macaque monkey at postnatal day 182 (P182; A, B) and P730 (C, D). E, F: High-magnification photomicrographs of SPP1 mRNA-positive neurons in layer V pyramidal neurons of the macaque monkey M1 at P182 (E) and P730 (F). Arrows and double arrowheads indicate neurons with the cell body size mainly showing intense signals at P182 and P730, respectively. G, H: Nissl-stained and adjacent sections hybridized with the SPP1 antisense probe in the macaque monkey M1 at P10. I: Scattergram showing the relationship between the average size of Nissl-stained neurons with the largest cell bodies and that of SPP1-positive neurons in the macaque monkey at each developmental stage. ***P<0.0001, according to linear regression analysis. J: Scattergram showing the logarithmic correlation between age and the density of SPP1 mRNA-positive neurons in the developing macaque monkey M1. ***P<0.0001, according to Spearman's coefficient of rank correlation. Diamonds represent data points from each monkey. II–VI, layers II–VI of M1. Scale bars = 200 µm in A–D, G, H; 50 µm in E, F.
Figure 6.
Changes of SPP1 expression after lesion of the lateral corticospinal tract.
A–H: Nissl-stained sections and adjacent sections showing the distribution of SPP1 mRNA-positive neurons in the primary motor cortex (M1; A, B) and ventral premotor cortex (PMv; C, D) of the intact monkey, and in the contralesional M1 (co-M1; E, F) and PMv (co-PMv; G, H) of the lesioned monkey (early stage). II–VI, layers II–VI of the cerebral cortex. I, J: Double-labeling study with SMI 32, an antibody against non-phosphorylated neurofilament H. I: Localization of SPP1 mRNA–positive neurons in layer V of the lesioned monkey (late stage). J: SMI 32 immunoreactivity in the same section as (I). Arrows indicate SPP1 mRNA–positive neurons showing SMI 32 immunoreactivity. Double arrowheads indicate SMI 32-immunoreactive neurons with no SPP1 expression. K: A bar chart showing the average density of SPP1 mRNA–positive neurons in M1, PMv, and dorsal premotor cortex (PMd) of an intact monkey, and in the co-M1, co-PMv, and contralesional PMd (co-PMd) of the lesioned monkey, with standard error. *P<0.05, **P<0.01, according to the Mann-Whitney U test. L: Scattergram showing the relationship between food retrieval success rate with precision grip and the density of SPP1 mRNA–positive neurons in the co-PMv. **P<0.01, according to linear regression analysis. The dashed and solid lines are linear approximations of the data represented by triangles and circles, respectively. The triangles and circles are data points from each lesioned monkey at early and late recovery stages, respectively. Scale bars = 200 µm in A–H; 50 µm in I, J.