Neuroprotective effects of testosterone on the morphology and function of somatic motoneurons following the death of neighboring motoneurons

Abstract

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have previously shown that partial depletion of motoneurons from sexually dimorphic, highly androgen-sensitive spinal motor populations induces dendritic atrophy in remaining motoneurons, and this atrophy is attenuated by treatment with testosterone. To test whether testosterone has similar effects in more typical motoneurons, we examined potential neuroprotective effects in motoneurons innervating muscles of the quadriceps. Motoneurons innervating the vastus medialis muscle were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected rats were given implants containing testosterone or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with intact normal males, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons, and this atrophy was attenuated by testosterone treatment. To examine the functional consequences of the induced dendritic atrophy, and its attenuation with testosterone treatment, the activation of remaining quadriceps motoneurons was assessed using peripheral nerve recording. Partial motoneuron depletion resulted in decreased amplitudes of motor nerve activity, and these changes were attenuated by treatment with testosterone, providing a functional correlate to the neuroprotective effects of testosterone treatment on quadriceps motoneuron morphology. Together these findings suggest that testosterone has neuroprotective effects on morphology and function in both highly androgen-sensitive as well as more typical motoneuron populations, further supporting a role for testosterone as a neurotherapeutic agent in the injured nervous system.

Figure 1

Weights of the vastus medialis (top) and vastus lateralis (bottom) muscles in normal males and saporin-injected animals that were either untreated (SAP), or treated with testosterone (SAP+T). Gray bars represent weights from the right (R) and left (L) sides in normal males, filled bars represent weights from the untreated contralateral (right) side of the quadriceps muscle, and unfilled bars represent weights from the saporin injected (left) side of the quadriceps muscle. Saporin injection reduced the weight of the vastus medialis muscle but had no effect on the adjacent vastus lateralis muscle. Testosterone treatment had no effect on muscle weight. Bar heights represent means ± SEM. * indicates significantly different from normal males.

Figure 2

Numbers of thionin-stained quadriceps motoneurons in normal males and saporin-injected animals that were either untreated (SAP), or treated with testosterone (SAP+T), expressed as a ratio of motoneuron number ipsilateral to the saporin-injected muscle relative to that on the untreated side. Saporin killed approximately 24% of the ipsilateral quadriceps motoneurons, regardless of hormone treatment. Bar heights represent means ± SEM. * indicates significantly different from normal males.

Figure 3

(Left) Darkfield digital micrographs of transverse hemisections through the lumbar spinal cords of a normal male (top), a saporin-injected male (SAP, middle), and a testosterone-treated saporin male (SAP+T, bottom), after BHRP injection into the left vastus lateralis muscle. (Right) Computer-generated composites of BHRP-labeled somata and processes drawn at 480 μm intervals through the entire rostrocaudal extent of the quadriceps motor pool; these composites were selected because they are representative of their respective group average dendritic lengths. Scale bar = 500 μm.

Figure 4

(Top) Cross-sectional soma areas of quadriceps motoneurons in normal males and saporin-injected animals that were either untreated (SAP), or treated with testosterone (SAP+T). Following saporin-induced motoneuron death, the average soma areas of surviving nearby motoneurons decreased by approximately 14%, regardless of hormone treatment, but these reductions were not statistically significant. (Bottom) Dendritic lengths of quadriceps motoneurons of normal males and saporin-injected animals that were either untreated (SAP), or treated with testosterone (SAP+T). Following saporin-induced motoneuron death, surviving nearby motoneurons lost almost 64% of their dendritic length. Treatment with testosterone attenuated this dendritic atrophy. Bar heights represent means ± SEM. * indicates significantly different from normal males. † indicates significantly different from untreated saporin-injected animals.

Figure 5

Inset: Drawing of spinal gray matter divided into radial sectors for measure of quadriceps motoneuron dendritic distribution. Length per radial bin of quadriceps dendrites of normal males (black bars) and saporin-injected animals that were either untreated (SAP, unfilled bars), or treated with testosterone (SAP+T, gray bars). For graphic purposes, dendritic length measures have been collapsed into 6 bins of 60° each. Quadriceps motoneuron dendritic arbors display a non-uniform distribution, with the majority of the arbor located between 300° and 120°. Following saporin-induced motoneuron death, surviving nearby motoneurons had reduced dendritic length in every radial bin. Treatment with testosterone attenuated this reduction. Bar heights represent means ± SEM. * indicates significantly different from normal males. † indicates significantly different from untreated saporin-injected animals.

Figure 6

Inset: Drawing of spinal gray matter divided into radial sectors for measure of quadriceps motoneuron radial dendritic extent. Radial extents of quadriceps dendrites of normal males (black bars) and saporin-injected animals that were either untreated (SAP, unfilled bars), or treated with testosterone (SAP+T, gray bars). For graphic purposes, dendritic extent measures have been collapsed into 6 bins of 60° each. Following saporin-induced motoneuron death, extent measures of surviving nearby motoneurons in SAP and SAP+T animals did not differ from those of normal animals. Bar heights represent means ± SEM.

Figure 7

Weights of the vastus medialis (top) and vastus lateralis (bottom) muscles in normal males and saporin-injected animals that were either untreated (SAP), or treated with testosterone (SAP+T) used for peripheral nerve recording. Gray bars represent weights from the right (R) and left (L) sides in normal males, filled bars represent weights from the untreated contralateral (right) side of the quadriceps muscle, and unfilled bars represent weights from the saporin injected (left) side of the quadriceps muscle. Saporin injection reduced the weight of the vastus medialis muscle but had no effect on the adjacent vastus lateralis muscle. Testosterone treatment had no effect on muscle weight. Bar heights represent means ± SEM. * indicates significantly different from normal males.

Figure 8

Inset: Representative rectified trace from a normal male of quadriceps motor nerve activity following stimulation of the L2 dorsal root. Response amplitudes as a function of stimulus intensity for normal males (filled circles), and saporin-injected animals that were either untreated (SAP, open circles), or treated with testosterone (SAP+T, triangles), measured as area under the curve. Motor nerve activation was significantly reduced in saporin-injected animals, but deficits in response amplitude were attenuated with testosterone treatment. Points represent means ± SEM. * indicates significant differences between saporin-injected animals and normal males. ‡ indicates significant differences between saporin-injected animals that were treated with testosterone and normal males. † indicates significant differences between saporin-injected animals that were untreated and those treated with testosterone.