Postnatal development of neurons, interneurons and glial cells in the substantia nigra of mice

Abstract

We investigated postnatal alterations of neurons, interneurons and glial cells in the mouse substantia nigra using immunohistochemistry. Tyrosine hydroxylase (TH), neuronal nuclei (NeuN), parvalbumin (PV), neuronal nitric oxide synthase (nNOS), glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba 1), CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) immunoreactivity were measured in 1-, 2-, 4- and 8-week-old mice. In the present study, the maturation of NeuN-immunopositive neurons preceded the production of TH in the substantia nigra during postnatal development in mice. Furthermore, the maturation of nNOS-immunopositive interneurons preceded the maturation of PV-immunopositive interneurons in the substantia nigra during postnatal development. Among astrocytes, microglia and oligodendrocytes, in contrast, the development process of oligodendrocytes is delayed in the substantia nigra. Our double-labeled immunohistochemical study suggests that the neurotrophic factors such as BDNF and GDNF secreted by GFAP-positive astrocytes may play some role in maturation of neurons, interneurons and glial cells of the substantia nigra during postnatal development in mice. Thus, our findings provide valuable information on the development processes of the substantia nigra.

Fig. 1

Representative photographs of TH immunostaining (A, B) and changes in the number of TH-immunopositive neurons (C) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). B High magnification of TH-immunostaining in 8-week-old mice. Changes in the number of TH-immunopositive cells in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 when compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 50 μm. Bar (B) = 200 μm. n = 7

Fig. 2

Representative photographs of NeuN immunostaining (A) and changes in the number of NeuN-immunopositive neurons (B) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of NeuN-immunopositive cells in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 30 μm. n = 7

Fig. 3

Representative photographs of PV immunostaining (A) and changes in the number of PV-immunopositive interneurons (B) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of PV-immunopositive interneurons in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 30 μm. n = 7

Fig. 4

Representative photographs of nNOS immunostaining (A) and changes in the number of nNOS-immunopositive interneurons (B) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of nNOS-immunopositive interneurons in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 20 μm. n = 7

Fig. 5

Representative photographs of GFAP immunostaining (A) and changes in the number of GFAP-immunopositive astrocytes (B) in the substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of nNOS-immunopositive cells in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 30 μm. n = 7

Fig. 6

Representative photographs of Iba 1 immunostaining (A) and changes in the number of Iba-1-immunopositive microglia (B) in the substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of Iba 1-immunopositive cells in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.05, **P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 30 μm. n = 7

Fig. 7

Representative photographs of CNPase immunostaining (A) and changes in areas of CNPase-immunopositive oligodendroglia (B) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in areas of CNPase-immunopositive cells in the mouse substantia nigra were expressed as the areas per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.05, **P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 30 μm. n = 7

Fig. 8

Representative photographs of BDNF immunostaining (A) and changes in the number of BDNF-immunopositive neurons (B) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of BDNF-immunopositive neurons in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 50 μm. n = 7

Fig. 9

Representative photographs of GDNF immunostaining (A) and changes in the number of GDNF-immunopositive neurons (B) in the mouse substantia nigra. A 1 week (a), 2 weeks (b), 4 weeks (c) and 8 weeks (d). Changes in the number of GDNF-immunopositive neurons in the mouse substantia nigra were expressed as the number of cells per 1 mm² using a computer-associated image analyzer (WinRoof). Values were expressed as means ± SE. *P < 0.01 compared with 8-week-old mice (Fisher’s PLSD multiple comparison test). Bar (A, a–d) = 30 μm. n = 7

Fig. 10

Representative photographs of double-labeled immunostaining in the mouse substantia nigra of 2-week-old mice. A, a: Anti-BDNF (brown) and anti-NeuN (gray) antibodies; A BDNF-positive/NeuN-positive cell (arrow). a High magnification of BDNF-positive/NeuN-positive cell. B, b, c Anti-BDNF (brown) and anti-TH (gray) antibodies. B BDNF-positive/TH-positive cell (arrow), BDNF-positive/TH-negative cell (arrow head). b High magnification of BDNF-positive/TH-positive cell (arrow), c High magnification of BDNF-positive/TH-negative cell. C, d, e Anti-BDNF (brown) and anti-PV (gray) antibodies. C BDNF-positive/PV-positive cell (arrow), BDNF-positive/PV-negative cell (arrowhead). d High magnification of BDNF-positive/PV-positive cell, e High magnification of BDNF-positive/PV-negative cell. D, f, g Anti-BDNF (brown) and anti-nNOS (gray) antibodies. D BDNF-positive/nNOS-positive cell (arrow), BDNF-positive/nNOS-negative cell (arrowhead). f High magnification of BDNF-positive/nNOS-positive cell, g High magnification of BDNF-positive/nNOS-negative cell. Bar 10 μm(A–D); 10 μm(a–g). n = 3. (Color figure online)

Fig. 11

Representative photographs of double-labeled immunostaining in the mouse substantia nigra of 2-week-old mice. A, a, b Anti-BDNF (brown) and anti-GFAP (gray) antibodies. A BDNF-positive/GFAP-positive cell (arrow), BDNF-positive/GFAP-negative cell (arrowhead). a High magnification of BDNF-positive/GFAP-positive cell, b High magnification of BDNF-positive/GFAP-negative cell. B, c, d Anti-BDNF (brown) and anti-Iba1 (gray) antibodies. B BDNF-positive/Iba1-positive cell (arrow), BDNF-positive/Iba1-positive cell (arrowhead). c High magnification of BDNF-positive/Iba1-positive cell, d high magnification of BDNF-positive/Iba1-negative cell. Bar 10 μm(A, B); 10 μm(a–d). n = 3. (Color figure online)

Fig. 12

Representative photographs of double-labeled immunostaining in the mouse substantia nigra of 2-week-old mice. A, a Anti-GDNF (red) and anti-NeuN (gray) antibodies. A GDNF-positive/NeuN-positive cell (arrow), a High magnification of GDNF-positive/NeuN-positive cell. B, b, c Anti-GDNF (red) and anti-TH (gray) antibodies. B GDNF-positive/TH-positive cell (arrow), GDNF-positive/TH-negative cell (arrowhead). b High magnification of GDNF-positive/TH-positive cell, c High magnification of GDNF-positive/TH-negative cell. C, d, e Anti-GDNF (red) and anti-PV (gray) antibodies. C GDNF-positive/PV-positive cell (arrow), GDNF-positive/PV-positive cell (arrowhead). d High magnification of GDNF-positive/PV-positive cell, e High magnification of GDNF-positive/PV-negative cell. D, f, g Anti-GDNF (red) and anti-nNOS (gray) antibodies. f High magnification of GDNF-positive/nNOS-positive cell, g High magnification of GDNF-positive/nNOS-negative cell. Bar 10 μm(A–D); 10 μm(a–g). n = 3. (Color figure online)

Fig. 13

Representative photographs of double-labeled immunostaining in the mouse substantia nigra of 2-week-old mice. A, a, b Anti-GDNF (red) and anti-GFAP (gray) antibodies. A GDNF-positive/GFAP-positive cell (arrow), GDNF-positive/GFAP-negative cell (arrowhead). a High magnification of GDNF-positive/GFAP-positive cell, b High magnification of GDNF-positive/GFAP-negative cell. B, c, d Anti-GDNF (red) and anti-Iba1 (gray) antibodies. B GDNF-positive/Iba1-positive cell (arrow), GDNF-positive/Iba1-negative cell (arrowhead). c High magnification of GDNF-positive/Iba1-positive cell, d High magnification of GDNF-positive/Iba1- negative cell Bar 10 μm(A, B); 10 μm(a–d). n = 3. (Color figure online)