The unusual response of serotonergic neurons after CNS Injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar

Abstract

Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or growth-inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer or cortical neurons were cultured on low amounts of laminin with or without relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of growth-associated protein-43 and/or β1 integrin than cortical neurons. Blocking β1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.

Figure 1.

Serotonergic fibers persist at the lesion edge, while callosal fibers die back. a, BDA-labeled callosal fibers (green) project to the pial surface in sham animals without a lesion. b, Two days after lesion the majority of BDA-labeled fibers retracted toward the corpus callosum, with a few fibers extending more dorsally. 5-HT-labeled serotonergic fibers (red) remain near the pial surface. The three most dorsally projecting 5-HT+ (red) and BDA+ (green) fibers are marked with arrows. c, d, At 4 d (c) and 1 week (d) after lesion, the cavity has not receded and 5-HT+ fibers remain dorsal, while BDA+ fibers continue to regress toward the corpus callosum. The white arrow marks a BDA+ macrophage. e, f, At 3 weeks (e) and 5 weeks (f) after injury, the lesion has receded, forming a cavity. g, 5-HT+ fibers are present at the edge of the cavity, growing among BDA-filled macrophages and holes in the tissue. BDA+ fibers are mostly present in the corpus callosum, with few extending dorsally. h, A schematic of the lesion (gray) in coronal section (top) and dorsal view (bottom). i, Quantification of the farthest dorsal extent of 5-HT+ and BDA+ fibers. 5-HT+ fibers extend significantly farther into the lesion than callosal fibers (two-way ANOVA, F_(1,80) = 51.58, Tukey post hoc test). All time-points are significant from each other except for 1 week from 3 weeks and 3 weeks from 5 weeks (two-way ANOVA, F_(4,80) = 60.77, Tukey post hoc test). Mean ± SEM is displayed. ***p < 0.0001, **p < 0.001, *p < 0.002. Scale bar, 50 μm.

Figure 2.

Serotonergic fibers sprout over time in the lesion filled with inhibitory CSPG and growth-promoting laminin (LN). a–d corresponds to e–h. Two days after lesion, 5-HT+ fibers (red; a, e) are spread out, and CSPG (green; e) is at low levels. One week after lesion 5-HT+ fibers (b, f) are located in the same region as increased CSPG (f). Three weeks (c, g) and 5 weeks (d, h) after lesion, 5-HT+ fibers have increased density at the lesion edge in the same area as the highest levels of CSPG. i, j, LN is mostly associated with blood vessels 2 d after lesion (i), but increases 1 week after lesion (j). l, k, Five weeks after lesion LN is at high levels in the center of the lesion (l) where 5-HT is high (k). Blood vessels (RECA-1; green) (k) do not account for all of the laminin. m, Quantification of the area of 5-HT+ fibers in the lesion area, which occupy more area over time. The area of 5-HT+ fibers at 3–5 weeks is significantly higher than that at 2–4 d (Kruskal–Wallis χ²(2) = 8.650, Mann–Whitney U post hoc test). Mean ± SEM is displayed. *p < 0.005. Scale bar, 50 μm.

Figure 3.

Callosal fibers die back away from macrophages/microglia, while serotonergic fibers grow among them. a, Many macrophages/microglia labeled by ED1 (red) are also positive for BDA (green). b, c, BDA+ ending can be bulbous and dystrophic in morphology. d, ED1+ macrophage/microglia can engulf BDA+ fibers. e, 5-HT+ fibers (red) are present among the ED1+ macrophages/microglia (green). The inset shows a magnified view of serotonergic fibers, which have beads that are much smaller than the dystrophic endings of BDA+ fibers. f, Macrophages/microglia (blue) are filled with BDA (green) but not 5-HT (red). Scale bars: a, e, 50 μm; b–e, inset, f, 10 μm.

Figure 4.

Vimentin+ cells are present dorsally in the lesion, while GFAP+ cells are present ventrally. a, Sham animals do not have hypertrophic GFAP+ astrocytes (green) or vimentin+ cells (red) in the cortex. b, Two days after lesion, GFAP+ cells begin to increase in size, and there is low vimentin staining. c, Four days after lesion, vimentin+ cells are increased dorsally, while GFAP staining increases ventrally. d, One week after lesion, vimentin+ and GFAP+ cells continue to increase, with a region of overlap (yellow). e, f, After cavity formation, vimentin+ cells are present dorsally, GFAP+ cells are present ventrally, and a strong area of overlap exists where they meet. f, At higher magnification, colabeled and single-labeled cells are clearly seen. Scale bars, 50 μm.

Figure 5.

Serotonergic fibers are present at high density in the ventricular zone and ependyma of the lateral ventricle. a, b, e, f, Both 5-HT (a, e) and SERT (b, f) label the serotonergic fibers surrounding the lateral ventricle. c, g, Vimentin+ cells are also present in the same area. d, h, Merge. Images are from 3 weeks after lesion on the contralateral lateral ventricle. Scale bars: a–d, 20 μm; e–h, 10 μm.

Figure 6.

The ventricular zone and ependyma mirror the lesion environment. a–c, Vimentin+ cells line the lateral ventricle ependyma (a, c), then GFAP+ cells extend into the tissue (b, c). d, Laminin (LN) immunostaining is present in the same area as vimentin+ cells, both punctate (inset) and around large blood vessels. e, CSPG labeled by CS-56 is in the same location as GFAP+ cells, lining the lateral ventricles and extending into the corpus callosum. f, Large blood vessels labeled by RECA-1 do not contribute all of the laminin. Images are from an uninjured lateral ventricle. Scale bars: a–c, 20 μm; d–f, 50 μm.

Figure 7.

a, Quantification of time-lapse movies (see Notes) with laminin or aggrecan plus laminin substrates. On PLL, cortical neurons (n = 6) mostly are active without forward growth but can also be inactive. Serotonergic neurons (n = 6) are active without forward growth. On 1 μg/ml laminin cortical neurons (n = 8) and serotonergic neurons (n = 8) mostly grow forward (green) but can also maintain activity without forward growth (yellow). On 100 μg/ml aggrecan plus 1 μg/ml laminin, cortical neurons (n = 9) are mostly inactive and stable (red), but few are active without forward growth (yellow). Serotonergic neurons (n = 23) are mostly active without forward growth (yellow), but also could be inactive and stable (red) or forward growing (green). Cortical neurons were visualized in lower numbers because they were more difficult to grow on the aggrecan plus laminin substrate. b–f, ChABC relieves inhibition for serotonergic neurons growing on 100 μg/ml aggrecan plus 1 μg/ml laminin. After 1 DIV, 0.5 U ChABC or saline was added to the medium and cells were fixed after 1 additional day. Cortical neurons were stained with β-tubulin III (b, c), and serotonergic neurons were stained with 5-HT (d, e). f, 5-HT+ neurons treated with ChABC (n = 56) grew significantly longer neurites than did saline-treated 5-HT+ neurons (n = 23) (Kruskal–Wallis test, χ²(3) = 48.422, post hoc Mann–Whitney U test, U = 234.00) or cortical neurons treated with either saline (U = 1616; n = 155) or ChABC (U = 2198; n = 149). Mean ± SEM. **p < 0.001. Scale bar, 20 μm.

Figure 8.

a–d, Serotonergic neurons express higher levels of GAP-43. GAP-43(red) is expressed at higher levels in 5-HT+ (green) neurons (c, d) than in β-tubulin III+ (green) cortical neurons (a, b). e–m, Serotonergic neurons have higher levels of β1 integrin, and growth is blocked with a β1 inhibitor. β1 integrin (red) is expressed in low levels in β-tubulin III+ cortical cell bodies (e, f; green) and in higher levels in 5-HT+ neurons (g, h; green). Anti-β1integrin (j, l) or isotype control (i, k) antibody was applied at the time of plating on 1 μg/ml laminin, and the longest neurite was measured after 1 DIV. m, On laminin, serotonergic neurons grew significantly longer than cortical neurons (Kruskal–Wallis test, Mann–Whitney U test post hoc, U = 5905.0). Compared with isotype control, both 5-HT+ serotonergic (l) and β-tubulin III+ cortical (j) neurite lengths were decreased significantly with the β1 function-blocking antibody [Kruskal–Wallis test, Mann–Whitney U test post hoc, U = 9277.5, 5-HT, n = 211 (control), 209 (β1); and U = 7986.0, cortical, n = 147 (control), 168 (β1)]. Mean ± SEM is displayed. Scale bar, 20 μm. **p < 0.001.