Androgen mitigates axotomy-induced decreases in calbindin expression in motor neurons

Abstract

Androgens can rescue axotomized motor neurons from cell death. Here we examine a possible mechanism for this trophic action in juvenile Xenopus laevis: regulation of a calcium-binding protein, calbindin, after axotomy. Western analysis revealed that a monoclonal antibody to calbindin D specifically recognizes a single approximately 28 kDa band in X. laevis CNS and rat cerebellum. Retrograde transport of peroxidase combined with immunohistochemistry demonstrated that somata, axons, and synaptic terminals of laryngeal motor neurons in nucleus (N.) IX-X of X. laevis are calbindin-positive. The number of calbindin-positive cells was compared in the intact and axotomized sides of N.IX-X of gonadectomized males that were either hormonally untreated or DHT-treated for 1 month. Although axotomy decreased the number of calbindin-positive cells by 86% in hormonally untreated males, the decrease was only 56% in DHT-treated animals. Compared with hormonally untreated animals, the number of calbindin-labeled cells in N.IX-X of DHT-treated males was increased in both the intact (14%) and axotomized sides (75%). We conclude that axotomy decreases and that DHT enhances calbindin immunoreactivity in N.IX-X. Axotomy-induced decrease in calbindin immunoreactivity precedes cell loss in N.IX-X and may impair the capacity of motor neurons to regulate cytoplasmic calcium. Androgen-mediated maintenance of calbindin expression is thus a candidate cellular mechanism for trophic maintenance of hormone target neurons.

Fig. 1.

A, Intact motor neurons in N.IX–X of a DHT-treated animal double-labeled with HRP (brownreaction) and calbindin (green reaction). N.IX–X is heterogeneous with respect to calbindin expression: some HRP-labeled motor neurons express calbindin (arrowheads); some motor neurons do not (stars). Some cells with motor neuron-like morphology were not labeled with HRP (v). B, Detail of a motor neuron in N.IX–X; calbindin immunoreactivity is localized to both cytoplasm and dendrites. C, Axons from motor neurons of N.IX–X were immunolabeled with calbindin in the laryngeal nerve (arrowhead). D, Within the larynx, unmyelinated axons and Schwann cells associated with myelinated axons (arrowhead) were immunolabeled with calbindin antibody.E, Synaptic terminals at the laryngeal neuromuscular junction were immunoreactive for calbindin antibody. Scale bars:A, 50 μm; B, 10 μm; C, 100 μm; D, E, 25 μm.

Fig. 2.

Western blot of total proteins extracted from rat cerebellum or Xenopus brain. Proteins were resolved in 12% SDS-PAGE and transferred to nitrocellulose. Blots were probed with (+) or without (−) calbindin antibody; one band of ∼28 kDa, according to molecular weight markers (right), was labeled in both species.

Fig. 3.

Intact and axotomized sides of N.IX–X in hormonally untreated and DHT-treated animals. Sections were immunolabeled with calbindin antibody, which produced abrown cytoplasmic reaction (A–D), and adjacent sections were stained with cresyl violet (A′–D′). In N.IX–X of hormonally untreated animals, axotomy caused a striking decrease in calbindin immunoreactivity (compare A with B) and an increase in cresyl violet staining of cells (compare A′ withB′). In DHT-treated animals, axotomy caused a decrease in calbindin immunoreactivity (compare C withD) in cells of N.IX–X but had no effect on cresyl violet staining (compare C′ with D′). Scale bar, 200 μm.

Fig. 4.

Number of calbindin-immunopositive cells and number of cresyl violet-stained cells (mean ± SD;n = 4 animals per group) counted in the intact and axotomized N.IX–X of hormonally untreated (black bars) and DHT-treated (white bars) animals 1 month after axotomy. There were no significant differences in the number of cresyl violet-stained cells. There was a significant main effect of hormone treatment (p < 0.05) and axotomy (p < 0.01) on the number of calbindin-positive cells, but there was no significant interaction between treatments. For the axotomized side, the numberof calbindin-immunoreactive cells in the intact sides/number in the axotomized sides × 100 is displayed within thebars.