Enhanced neurotrophin-induced axon growth in myelinated portions of the CNS in mice lacking the p75 neurotrophin receptor

Abstract

Axonal growth in the adult mammalian CNS is limited because of inhibitory influences of the glial environment and/or a lack of growth-promoting molecules. Here, we investigate whether supplementation of nerve growth factor (NGF) to the CNS during postnatal development and into adulthood can support the growth of sympathetic axons within myelinated portions of the maturing brain. We have also asked whether p75(NTR) plays a role in this NGF-induced axon growth. To address these questions we used two lines of transgenic mice overexpressing NGF centrally, with or without functional expression of p75(NTR) (NGF/p75(+/+) and NGF/p75(-/-) mice, respectively). Sympathetic axons invade the myelinated portions of the cerebellum, beginning shortly before the second week of postnatal life, in both lines of NGF transgenic mice. Despite the presence of central myelin, these sympathetic axons continue to sprout and increase in density between postnatal days 14 and 100, resulting in a dense plexus of sympathetic fibers within this myelinated environment. Surprisingly, the growth response of sympathetic fibers into the cerebellar white matter of NGF/p75(-/-) mice is enhanced, such that both the density and extent of axon ingrowth are increased, compared with age-matched NGF/p75(+/+) mice. These dissimilar growth responses cannot be attributed to differences in cerebellar levels of NGF protein or sympathetic neuron numbers between NGF/p75(+/+) and NGF/p75(-/-) mice. Our data provide evidence demonstrating that growth factors are capable of overcoming the inhibitory influences of central myelin in the adult CNS and that neutralization of the p75(NTR) may further enhance this growth response.

Fig. 1.

Mean levels of NGF protein in the cerebellum of C57Bl/6, NGF/p75^+/+, and NGF/p75^−/− mice, as measured by a two-site ELISA. The data are represented as fold differences relative to C57Bl/7 mice, and error bars indicate SD. In contrast to the low levels of NGF protein in the cerebellum of C57Bl/6 mice, levels of NGF protein are 21- and 22-fold higher in the cerebellum of age-matched NGF/p75^+/+ and NGF/p75^−/− mice, respectively. No significant difference in cerebellar NGF levels is detected between NGF/p75^+/+ and NGF/p75^−/− mice.

Fig. 2.

Coronal sections of cerebellum from C57Bl/6, NGF/p75^+/+, and NGF/p75^−/− mice at P100, stained immunohistochemically for TH. In C57Bl/6 mice (A), a diffuse network of TH-IR axons is evident in both the cerebellar gray and white matter, representing the local innervation by locus coeruleus neurons. In contrast to these observations, the cerebella of both NGF/p75^+/+(B) and NGF/p75^−/−(C) mice display a robust ingrowth of TH-IR axons. These dense plexuses of new TH-IR axons are confined predominantly to the DWM layer and ICPs of the cerebellum and are only occasionally seen extending into the gray matter layers of the cerebellum. Qualitatively, the density of TH-IR axons appears increased in the cerebellum of NGF/p75^−/− mice compared with NGF/p75^+/+ mice. The white matter portions of the cerebellum of these mice, as outlined in the box inA, are shown at two higher magnifications in Figure3. Scale bar, 500 μm.IV, Fourth ventricle.

Fig. 3.

Higher magnifications of the cerebellar DWM of C57Bl/6, NGF/p75^+/+, and NGF/p75^−/− mice. Note the fine TH-IR locus coeruleus axons, which often course perpendicular to the DWM fibers, in control C57Bl/6 mice (A, B). In contrast, the DWM of both NGF/p75^+/+ (C, D) and NGF/p75^−/− (E, F) mice display a dramatic increase in TH-IR axons, which course approximately in parallel with the intrinsic DWM fibers. Again note that the density of TH-IR axons appears increased in the DWM of NGF/p75^−/− mice relative to NGF/p75^+/+ mice. Scale bars: A, C, E, 150 μm; B, D, F, 50 μm.

Fig. 4.

Coronal sections of cerebellum from NGF/p75^+/+ (top row) and NGF/p75^−/− (bottom row) mice at P14 (A, B), P28 (C, D), and P60 (E, F), stained immunohistochemically for TH. TH-IR sympathetic axons are already observed growing into the cerebellar DWM by P14 in both NGF/p75^+/+ and NGF/p75^−/− mice. In both NGF transgenic lines of mice, the density of TH-IR axons in the cerebellar DWM appears to increase with age. It is also apparent that the density of TH-IR axons is greater in the DWM of NGF/p75^−/− mice compared with NGF/p75^+/+ mice. Scale bar, 150 μm.

Fig. 5.

Quantitation of TH-IR axon density in the cerebellar DWM of C57Bl/6, NGF/p75^+/+, and NGF/p75^−/− mice at developmental time points. There are significant increases in the percent area occupied by TH-IR axons in the DWM of NGF/p75^−/− mice relative to NGF/p75^+/+ mice at both P14 and P100 (*p < 0.001). The density of TH-IR axons is lowest in the DWM of P100 C57Bl/6 mice.

Fig. 6.

Electron photomicrograph showing the localization of TH immunoreactivity in the cerebellar deep white matter of adult NGF/p75^+/+ mice. TH-IR unmyelinated axons are seen coursing among myelinated fibers of the cerebellum, often as part of a small cluster of unmyelinated axons. TH-IR unmyelinated axons lack any glial support and are often observed in close apposition to the surface of myelinated axons and unmyelinated axons alike. Scale bar, 0.5 μm.

Fig. 7.

Coronal sections of cerebellum from acutely ganglionectomized NGF/p75^+/+ and NGF/p75^−/− mice, stained immunohistochemically for TH. The unilateral removal of the SCG allows visualization of those sympathetic fibers from the contralateral ganglion. In NGF/p75^+/+ mice (A), few TH-IR axons from the contralateral SCG extend as far as the cerebellar midline (arrow). In contrast, numerous contralateral sympathetic axons in NGF/p75^−/− mice (B) extend past the cerebellar midline (arrow) and into the upper portions of the ipsilateral DWM. Scale bar, 500 μm.

Fig. 8.

Quantitation of sympathetic axon density in the ipsilateral and contralateral DWM of both lines of transgenic mice after acute and chronic ganglionectomy. Data for NGF/p75^+/+ mice (n = 7 acute and 5 chronic ganglionectomized animals) are represented byboxes, and data for NGF/p75^−/− mice (n = 10 acute and 6 chronic ganglionectomized animals) are represented by circles. In all acutely and chronically ganglionectomized animals, there is a significant decrease in the percent area occupied by TH-IR axons in the ipsilateral DWM relative to the contralateral DWM within the same experimental group (p < 0.001). In the contralateral DWM, the density of sympathetic axons is higher in NGF/p75^−/− mice relative to NGF/p75^+/+ mice (p < 0.001), and these densities are not affected by chronic ganglionectomy. Note that the density of sympathetic axons that persist in the ipsilateral DWM, after either acute and chronic ganglionectomy, is also higher in NGF/p75^−/− mice relative to NGF/p75^+/+ mice (p < 0.001). Importantly, sympathetic fiber density increases in the ipsilateral DWM between acutely and chronically ganglionectomized NGF/p75^−/− mice but not between acutely and chronically ganglionectomized NGF/p75^+/+ mice (p < 0.01). All data are represented as mean ± SD.

Fig. 9.

Levels of NGF protein in the cerebellum of acutely and chronically ganglionectomized NGF/p75^+/+ and NGF/p75^−/− mice (n = 3 animals per experimental group), as measured by a two-site ELISA. Levels of NGF in the ipsilateral half of the cerebellum are presented as fold differences relative to the contralateral half from the same animal, and error bars indicate SD. This analysis reveals a significant increase in NGF levels only within the ipsilateral cerebellum of NGF/p75^−/− mice after acute ganglionectomy (*p < 0.001). In all other cases, no significant differences in NGF levels are detected between ipsilateral and contralateral cerebellar halves.

Fig. 10.

Morphological and neurochemical characteristics of SCG from P60 C57Bl/6, NGF/p75^+/+, and NGF/p75^−/− mice. A, Nissl-stained sections of SCG from C57Bl/6 (left), NGF/p75^+/+ (center), and NGF/p75^−/− (right) mice reveal a greater incidence of multiple nucleoli among somata of both NGF transgenic lines of mice. B, Counts of neuronal profiles in SCG of adult animals show a significant increase in the number of SCG neurons in both NGF/p75^+/+ and NGF/p75^−/− mice compared with C57Bl/6 mice (*p < 0.05). No significant difference was detected in the number of SCG neurons between NGF/p75^+/+ and NGF/p75^−/− mice.C, RT-PCR amplification of GAPDH and trkA in SCG taken from P60 C57Bl/6, NGF/p75^+/+, and NGF/p75^−/− mice. GAPDH cDNA product (∼700 bp) is detectable in SCG from C57Bl/6 (lane 1), NGF/p75^+/+ (lane 2), and NGF/p75^−/− (lane 3) mice. Likewise, trkA cDNA product (∼200 bp) is detectable in SCG from C57Bl/6 (lane 4), NGF/p75^+/+(lane 5), and NGF/p75^−/−(lane 6) mice. No trkA cDNA product is detectable in the absence of RNA template (lane 7), and GAPDH cDNA product is not detectable in the absence of the reverse transcriptase enzyme (lane 8). L, 100 bp ladder.