Exercise is neuroprotective on the morphology of somatic motoneurons following the death of neighboring motoneurons via androgen action at the target muscle

Abstract

Motoneuron loss is a severe medical problem that can result in loss of motor control and eventually death. We have previously demonstrated that partial motoneuron loss can result in dendritic atrophy and functional deficits in nearby surviving motoneurons, and that an androgen-dependent effect of exercise following injury can be neuroprotective against this dendritic atrophy. In this study, we explored where the necessary site of androgen action is for exercise-driven neuroprotective effects on induced dendritic atrophy. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected animals were given implants of the androgen receptor antagonist hydroxyflutamide, either directly at the adjacent vastus lateralis musculature ipsilateral to the saporin-injected vastus medialis or interscapularly as a systemic control. Following saporin injections, some animals were allowed free access to a running wheel attached to their home cages. Four weeks later, motoneurons innervating the same vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Dendritic arbor lengths of saporin-injected animals allowed to exercise were significantly longer than those not allowed to exercise. Androgen receptor blockade locally at the vastus lateralis muscle prevented the protective effect of exercise. These findings indicate that exercise following neural injury exerts a protective effect on motoneuron dendrites, which acts via androgen receptor action at the target muscle.

1 |. INTRODUCTION

Neurodegenerative disease or injury often results in the loss of spinal motoneurons. For example, motor neuron diseases (e.g., amyotrophic lateral sclerosis; Cleveland & Rothstein, 2001) are characterized by progressive loss of motoneurons. Alternatively, spinal cord injury (Liu et al., 1997) or damage to spinal roots (Moschilla et al., 2001) can lead to the death of motoneurons.

Importantly, after such insults surviving motoneurons show a variety of morphological and functional changes. For example, motoneurons undergo dendritic atrophy after spinal cord injury (Byers et al., 2012). Similarly, after peripheral axotomy, motoneurons show functional and biochemical changes (Bisby & Tetzlaff, 1992; Titmus & Faber, 1990) as well as dendritic atrophy (Yang et al., 2004).

We have been examining the effects of motoneuron loss on the structure and function of surviving motoneurons using a rat model of motoneuron death. We have demonstrated that surviving motoneurons respond to the loss of their neighbors with marked dendritic atrophy (Cai et al., 2017; Little et al., 2009). This induced atrophy is responsible for at least some of the movement deficits that accompany disease- or injury-related loss of motoneurons, as it results in reduced excitability of the remaining motoneurons (Little et al., 2009). Given that we currently lack the technology to replace dead motoneurons, protecting surviving motoneurons from injury-induced atrophy is an important goal.

Treatment with gonadal steroids is neuroprotective (Foecking et al., 2015). In our model, treatment with exogenous testosterone attenuates both dendritic atrophy and reduced excitability in motoneurons (Chew et al., 2019; Little et al., 2009). This effect of androgens is mediated via classical receptor activation, and systemic blockade of androgen receptors completely prevents the neuroprotective effects (Cai et al., 2017). This suggests that receptor action is a necessary driver of the neuroprotective benefits of androgens.

We have also shown that ad libitum exercise is neuroprotective against dendritic atrophy, comparable to that seen when rats are treated with testosterone (Chew & Sengelaub, 2019). Furthermore, male rats castrated prior to beginning exercise show dendritic lengths similar to those of males who receive no therapeutic intervention, indicating that the presence of gonadal hormones is a necessary component of exercise’s attenuation of dendritic atrophy (Chew & Sengelaub, 2020).

However, it is not clear where hormones act in to produce this neuroprotection. Skeletal muscles express gonadal hormone receptors (Dubé et al., 1976), and hormonal manipulations at the muscle cause changes in protein content (Verhovshek & Sengelaub, 2013) or the morphology (Huguenard et al., 2011; Rand & Breedlove, 1995) of the innervating motoneurons. In the present study, we tested whether the androgen-dependent neuroprotective effects of exercise against induced dendritic atrophy following partial motoneuron depletion is dependent on androgen receptor action at the muscle.

2 |. METHODS

In rats, the quadriceps muscles are innervated by motoneurons located in the lateral motor column in the L2 spinal segment (Nicolopoulos-Stournaras & Iles, 1983). The distribution of somata and dendritic arbors of the motoneurons innervating the individual muscles of the ipsilateral quadriceps complex overlap extensively (Nicolopoulos-Stournaras & Iles, 1983; Sengelaub et al., 2006), making it possible to partially and selectively deplete this motor population and study the effects of that depletion on the surviving motoneurons.

2.1 |. Animals

Adult male Sprague–Dawley rats (approximately 100 days old; Envigo, Indianapolis) were maintained on a 12:12-hr light/dark cycle with unlimited access to food and water. We used the toxin saporin, conjugated to the cholera toxin B subunit, to kill motoneurons. Saporin is a ribosome-inactivating protein, killing cells by irreversibly halting protein synthesis (Stirpe et al., 1983, 1992). Cholera toxin-conjugated saporin is retrogradely transported from the injected muscle, killing the innervating motoneurons within 3–6 days (Fargo & Sengelaub, 2004).

Rats were anesthetized with isoflurane, and motoneurons innervating the left vastus medialis (VM) muscle were selectively killed by intramuscular injection of cholera toxin-conjugated saporin (2 μl, 0.1%; Advanced Targeting Systems, Inc., San Diego, CA). Some rats were not treated further (n = 6), whereas others were immediately allowed free access to exercise wheels (width = 11.2 cm; diameter = 37 cm; circumference = 116 cm) attached to their home cages (n = 10).

To assess whether exercise is neuroprotective due to androgen action at the muscle, we used a technique similar to that of Rand and Breedlove (1995) wherein a highly localized treatment with an androgen receptor antagonist is applied directly to the muscle. Such treatment can affect the dendritic morphology of the innervating motoneurons (Rand & Breedlove, 1995). Importantly, this approach delivers an effective treatment directly to the muscle, but does not provide a dose large enough to produce systemic effects. Thus, additional groups of saporin-injected, exercised rats were treated with the nonsteroidal androgen receptor antagonist hydroxyflutamide (hFLUT; 2-hydroxy-flutamide; LKT Laboratories, St. Paul, MN) immediately after saporin injection and prior to placement in their cages. Some saporin-injected rats had Silastic implants (12.5 × 3.5 × 1.5 mm) impregnated with hydroxyflutamide (0.2 mg) sutured onto the left vastus lateralis (VL) muscle (n = 12). Another group of saporin-injected, exercised rats were given identical implants placed subcutaneously in the interscapular area to control for potential systemic effects (n = 17).

Wheel revolutions were tracked daily to ensure that rats were engaging in exercise throughout the recovery period. A group of untreated and unexercised animals (n = 5) was included. Because some of the animals in the study were not included in all analyses due to histological or histochemical compromise, group sizes for each analysis are reported individually below (overall n = 50).

2.2 |. Histochemical and histological processing

Four weeks after saporin injection, animals were reanesthetized, and the left VL muscle (ipsilateral to the saporin-injected VM muscle in saporin animals) was exposed and injected with horseradish peroxidase conjugated to the cholera toxin B subunit (BHRP; 2 μl, 0.2%; Invitrogen, Carlsbad, CA). BHRP labeling permits population-level quantitative analysis of motoneuron somal and dendritic morphologies (Goldstein et al., 1990; Kurz et al., 1986, 1991). Forty-eight hours after BHRP injection, a period that ensures optimal labeling of motoneurons (Goldstein et al., 1990; Kurz et al., 1986, 1991), animals were weighed, given an overdose of urethane (approximately 0.25 g/100 g body weight), and perfused intracardially with saline followed by cold fixative (1% paraformaldehyde/1.25% glutaraldehyde). To confirm the specificity of the saporin injections, the VM and VL muscles were removed bilaterally immediately after perfusion and weighed. The lumbar portion of the spinal cord of each animal was removed, postfixed in the same fixative for 5 hr, and then, transferred to sucrose phosphate buffer (10% w/v, pH 7.4) overnight for cryoprotection. Spinal cords were then embedded in gelatin, frozen, and sectioned transversely at 40 μm; all sections were collected into four alternate series. One series was stained with thionin for use in cell counts. For visualization of BHRP, the remaining series were immediately reacted using a modified tetramethylbenzidine protocol (Mesulam, 1982), mounted on gelatin-coated slides, and counterstained with thionin.

2.3 |. Motoneuron number and morphology

2.3.1 |. Motoneuron counts

To identify the appropriate area within the lateral motor column for motoneuron counts, we used the method of Little et al. (2009). For each animal, the range of sections in which BHRP-labeled motoneurons were present was identified. Motoneurons were then counted in the appropriate matching sections in the unreacted, thionin-stained series. For each animal, estimates of the total number of motoneurons in the left and right lateral motor columns were obtained using the optical disector method using Stereo Investigator (MBF Bioscience, Williston, VT). Counts were made at x937.5 under brightfield illumination. Quadriceps motoneurons are easily recognizable as large, darkly staining, and multipolar cells.

A counting frame (110 μm × 80 μm) was moved systematically throughout an area of each ventral horn (~500 μm × 500 μm, defined by the actual distribution of BHRP-labeled somata) in each section within the identified range. Only motoneurons in which there was a clear nucleus and nucleolus were counted, provided they did not contact the forbidden lines of the counting frame; motoneuron nucleoli were counted as they appeared while focusing through the z-axis, and nucleoli in the first focal plane (i.e., “tops”) were excluded to avoid double counting. The length of the disector was approximately 16 μm, which was adequate for visualizing nucleoli in multiple focal planes. Motoneuron counts were derived from a mean of 10.84 sections spaced 480 μm apart and distributed uniformly through the rostrocaudal extent of the quadriceps motoneuron pool range. This sampling scheme produced an average estimated coefficient of error (CE) of 0.061. Cell counts for each animal were corrected for the proportion of sections sampled, and then, expressed as a ratio (motoneuron number on the saporin-injected side relative to that on the untreated side) to quantify the magnitude of motoneuron depletion examined (untreated, n = 5; SAP, n = 6; SAP + EXERCISE, n = 10; SAP + EXERCISE+hFLUT_MUS, n = 12; SAP + EXERCISE+hFLUT_SCAP, n = 17).

Using similar methods, the number of BHRP-labeled motoneurons was assessed in all sections of the reacted series through the entire rostrocaudal extent of their distribution for all animals. Counts of labeled quadriceps motoneurons were made under brightfield illumination, where somata could be visualized and cytoplasmic inclusion of BHRP reaction product confirmed (untreated, n = 5; SAP, n = 6; SAP + EXERCISE, n = 6; SAP + EXERCISE+hFLUT_MUS, n = 9; SAP + EXERCISE+hFLUT_SCAP, n = 10).

2.3.2 |. Dendritic length

For each animal, dendritic lengths in a single representative set of alternate series were measured under darkfield illumination through the rostrocaudal extent of the quadriceps motoneuron dendritic arbor (untreated, n = 5; SAP, n = 6; SAP + EXERCISE, n = 6; SAP + EXERCISE+hFLUT_MUS, n = 9; SAP + EXERCISE+hFLUT_SCAP, n = 10). BHRP-labeled fibers were traced in sections 480 μm apart in three dimensions using Neurolucida (MBF Bioscience, Williston, VT) at a final magnification of x250. Average dendritic length per labeled motoneuron was estimated by summing the measured dendritic lengths of the series of sections, multiplying to correct for sampling, then, dividing by the total number of labeled motoneurons in that series. This method does not attempt to assess the actual total dendritic length of labeled motoneurons (Goldstein & Sengelaub, 1993), but has been shown to be a sensitive and reliable indicator of changes in dendritic morphology in both normal development (Goldstein et al., 1990, 1993; Goldstein & Sengelaub, 1993), and following a variety of experimental manipulations, including after the death of neighboring motoneurons (Cai et al., 2017; Chew et al., 2019; Chew & Sengelaub, 2019; Little et al., 2009).

2.3.3 |. Dendritic distribution

To assess potential dendritic redistributions, a set of axes oriented radially around the center of the collective labeled somata was placed on the composite dendritic arbor created for each animal in the length analysis, dividing the arbor into 12 bins of 30° each. The portion of each animal’s dendritic arbor per labeled motoneuron contained within each location was then determined. This method provides a sensitive measure of dendritic redistribution after injury (Byers et al., 2012).

2.3.4 |. Dendritic extent

The comparability of BHRP labeling across groups was assessed by quantifying both the rostrocaudal and the radial extent of quadriceps motoneuron dendritic arbors. The rostrocaudal extent of the dendritic arbor was determined by recording the rostrocaudal distance spanned by labeled dendrites for each animal. The maximal extent of the arbor in the transverse plane was also measured for each animal, using the same radial axes described for the dendritic distribution analysis: for each bin, the linear distance between the center of the quadriceps motor pool and the most distal BHRP-filled process was measured. Radial dendritic extent is independent of overall dendritic length and reflects the maximal linear distance (in the transverse plane) of BHRP transport to the most distal dendritic processes.

All procedures were performed in accordance with the Indiana University Animal Care and Use Guidelines. All data were analyzed by t tests or analyses of variance followed by post hoc analyses using Fisher’s least significant difference (LSD). Digital light micrographs were obtained using an MDS 290 digital camera system (Eastman Kodak Company, Rochester, NY). Brightness and contrast of these images were adjusted in Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).

3 |. RESULTS

3.1 |. Running performance

Animals ran consistently over the 4 weeks they were allowed access to running wheels, averaging 4.26 ± 0.25 (mean ± SEM) kilometers per day. Injection of saporin or delivery of hydroxyflutamide implants had no effect on exercise, and daily distance run in animals allowed to run did not differ across groups [F(2, 32) = 0.85, ns]. Overall, animals ran an average cumulative total of 126.59 ± 7.40 km over the 4 weeks of ad libitum exercise.

3.2 |. Muscle weights

Group difference in body weight were present [F(4,47) = 12.94, p < .0001], and thus, raw muscle weights were corrected for body mass to assess potential effects of saporin and/or exercise on muscle weight (Figure 1). In untreated animals, the corrected weights of the right (0.17 ± 0.01) and left (0.17 ± 0.01) VM muscles were similar [t(4) = 2.14, ns]. There was a significant effect of group on the weights of the uninjected (right) VM muscle [F(4,47) = 6.61, p < .001]; animals allowed to exercise had larger corrected muscle weights (0.19 ± 0.002) than animals not allowed to exercise [0.16 ± 0.008; average increase of 18%; LSDs, p < .05]. Uninjected VM weights across exercised groups did not differ from each other [LSDs, ns].

FIGURE 1.

Weights of the vastus medialis and vastus lateralis muscles corrected by body weight in untreated animals and saporin-injected animals that either received no further treatment (SAP), were given ad libitum exercise (SAP + EXERCISE), or were given ad libitum exercise and received a hydroxyflutamide implant at either the ipsilateral vastus lateralis (SAP + EXERCISE+hFLUT_mus) or scapula (SAP + EXERCISE+hFLUT_scap) at 4 weeks after saporin injection. Gray bars represent weights from the right (R) and left (L) sides in untreated animals. Black bars represent weights from the untreated contralateral (right) leg, and white bars represent weights from the saporin-injected (left) leg of saporin-injected animals. Saporin injection reduced the weight of the injected vastus medialis muscle; neither exercise nor androgen receptor blockade had an effect on left vastus medialis weight in saporin-injected animals. Saporin animals who exercised showed minor hypertrophy of the (right) vastus medialis contralateral to the saporin-injected muscle. Weights of both the left and right vastus lateralis were not affected by saporin injection or exercise. Bar heights represent means ± SEM. * indicates significantly different from untreated animals. † indicates significantly different from untreated saporin-injected animals

Injection of saporin into the left VM resulted in muscle atrophy in the saporin groups [overall average of 68% reduction in weight; F(4,47) = 43.80, p < .0001]. Compared to those of untreated animals, saporin-injected animals had VM weights that were 74% lighter (LSD, p < .0001). Exercise did not prevent saporin-induced weight loss in the left VM; compared to those of untreated animals, saporin-injected rats who were allowed to exercise had VM weights that were 64% lighter (LSD, p < .0001). Muscle weights across saporin groups did not differ from each other [F(3,43) = 1.37, ns].

The effect of saporin injection on quadriceps weight was specific to the injected muscle. In untreated animals, the corrected weights of the right (0.40 ± 0.02) and left (0.41 ± 0.02) VL muscles were similar [paired t test, t(4) = 0.43, ns]. The weights of the VL muscles on the untreated side did not differ across groups [F(4,47) = 1.27, ns]. Most importantly, the weights of the VL muscles adjacent of the saporin-injected VM muscles also did not differ across groups [F(4,47) = 0.95, ns].

3.3 |. Motoneuron counts

In untreated animals, the number of motoneurons within the identified quadriceps range did not differ between the left (251.2 ± 14) and right (237.6 ± 25) motor column [paired t test, t(4) = 0.63, ns]. Injection of saporin into the left VM resulted in the death of ipsilateral quadriceps motoneurons, significantly reducing the number of motoneurons in the left motor column relative to that of the right [F(4,45) = 3.03, p < .03; Figure 2]. Unilateral injection of saporin into the left VM resulted in a 21% reduction in the relative number of motoneurons compared with that of untreated animals (LSD, p < .02). Neither exercise nor hormone treatment prevented the saporin-induced reduction in motoneuron number (overall average of 23% reduced; LSDs, p < .001 compared to untreated animals).

FIGURE 2.

Numbers of quadriceps motoneurons in untreated animals and saporin-injected animals that either received no further treatment (SAP), were only given ad libitum exercise (SAP + EXERCISE), or were given ad libitum exercise and received a hydroxyflutamide implant at either the vastus lateralis ipsilateral to saporin injection (SAP + EXERCISE+hFLUT_mus) or interscapularly (SAP + EXERCISE+hFLUT_scap) at 4 weeks after saporin injection, expressed as a ratio of motoneuron number ipsilateral to the saporin-injected muscle relative to that on the untreated side. Saporin killed approximately 22% of the ipsilateral quadriceps motoneurons, regardless of subsequent treatment. Bar heights represent means ± SEM. * indicates significantly different from untreated animals

3.4 |. Motoneuron morphometry

Injection of BHRP successfully labeled quadriceps motoneurons in all groups (Figure 3). The dendritic arbor of quadriceps motoneurons was strictly unilateral, with extensive ramification along the ventrolateral margins of the gray matter and in the lateral funiculus, as well as throughout the ventral horn. An average of 30.33 (± 2.98) motoneurons per animal was labeled with BHRP, and this did not vary across groups [F(4,31) = 1.98, ns].

FIGURE 3.

Darkfield digital micrographs of transverse hemisections through the lumbar spinal cords and computer-generated reconstructions of BHRP-labeled somata and processes of an untreated animal (A,B), and saporin-injected animals with either no further treatment (C,D), given only ad libitum exercise (E,F), or given ad libitum exercise in addition to a hydroxyflutamide implant either at the left vastus lateralis ipsilateral to saporin injection (G,H) or placed interscapularly (I,J) after BHRP injection into the left vastus lateralis muscle. Computer-generated composites of BHRP labeling were 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

3.4.1 |. Dendritic length

Surviving quadriceps motoneurons underwent marked dendritic atrophy (Figure 4). Dendritic length was decreased by 64% in saporin-injected animals who received no further treatment compared to that of untreated animals [LSD, p < .0001; overall test for the effect of group on dendritic length F(4,31) = 12.69, p < .0001]. Compared to untreated animals, dendritic lengths were significantly shorter in all saporin-injected animals, regardless of exercise status or androgen receptor blockade (LSDs, p < .001).

FIGURE 4.

Dendritic lengths of quadriceps motoneurons in untreated animals and saporin-injected animals that either received no further treatment (SAP), were only given ad libitum exercise (SAP + EXERCISE), or were given ad libitum exercise and received a hydroxyflutamide implant at the vastus lateralis ipsilateral to saporin injection (SAP + EXERCISE+hFLUT_mus) or interscapularly (SAP + EXERCISE+hFLUT_scap) at 4 weeks after saporin injection. Following saporin-induced motoneuron death, surviving neighboring motoneurons lost almost 64% of their dendritic length and exercise attenuated this dendritic atrophy. Androgen receptor blockade at the innervated vastus lateralis prevented the attenuation of atrophy by exercise, while identical implants placed interscapularly did not. Bar heights represent means ± SEM. * indicates significantly different from untreated animals. † indicates significantly different from untreated saporin-injected animals

Exercise attenuated dendritic atrophy in saporin-injected animals, with dendritic length being reduced on average by only 28%. The dendritic lengths of saporin-injected animals who exercised were 97% longer than those without exercise (LSD, p < .02).

Androgen receptor blockade at the target muscle prevented the beneficial effects of exercise on dendrites. Dendritic lengths in exercised saporin animals with local hydroxyflutamide treatment at the quadriceps were significantly shorter (34%) than those of exercised, but unimplanted, saporin animals (LSD, p < .01). Furthermore, dendritic lengths in exercised saporin animals with local hydroxyflutamide treatment at the quadriceps were not significantly different from those of saporin animals who received no exercise (LSD, ns). Importantly, the attenuation of dendritic atrophy by exercise in saporin-injected animals was not affected by the androgen receptor blockade delivered interscapularly; dendritic lengths in exercised saporin animals with hydroxyflutamide implants placed interscapularly did not differ from those of exercised, but unimplanted, saporin animals (LSD, ns). Furthermore, dendritic lengths in exercised saporin animals with androgen receptor blockade interscapularly had dendritic lengths that were significantly longer (34%) than those of exercised saporin animals with blockade at the quadriceps (LSD, p < .05).

3.4.2 |. Dendritic distribution

Dendritic length was nonuniform across radial bins, and a repeated measures ANOVA revealed a significant effect of radial location [F(11,341) = 18.15, p < .0001; Figure 5]. Consistent with the results seen in total dendritic length analysis, there was also a significant effect of group [F(4,341) = 14.59, p < .0001]. There were reductions in dendritic length throughout the radial distribution, ranging from 38% (180° to 240°) to 79% (60° to 120°) in saporin-injected animals compared with untreated animals [F(1,99) = 26.19, p < .0007]. Saporin-injected animals allowed to exercise showed attenuated reductions, with reductions in dendritic length ranging from no change (180°–300°) to only 54% (60°–120°) compared to untreated animals [F(1,99) = 9.44, p < .02]. Throughout most of the radial distribution, dendritic lengths per bin in exercised saporin-injected animals were longer than those of saporin-injected animals who received no further treatment [F(1,110) = 9.70, p < .02], with increases ranging from 61% (180°–240°) to 120% (240°–300°).

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 in untreated animals (white bars), and saporin-injected animals that either received no further treatment (SAP, black bars), were given ad libitum exercise (SAP + EXERCISE, gray bars), or were given ad libitum exercise and received a hydroxyflutamide implant at the vastus lateralis ipsilateral to saporin injection (SAP + EXERCISE+hFLUT_mus;, light gray bars) or interscapularly (SAP + EXERCISE+hFLUT_scap, dark gray bars). For graphic purposes, dendritic length measures have been collapsed into six bins of 60° each. Quadriceps motoneuron dendritic arbors display a nonuniform distribution, with the majority of the arbor located between 300° and 120°. Following saporin-induced motoneuron death, surviving neighboring motoneurons had reduced dendritic length throughout the radial distribution. Exercise attenuated this reduction, but had no effect in intact animals. Bar heights represent means ± SEM. * indicates significantly different from untreated animals. † indicates significantly different from untreated saporin-injected animals

Exercised saporin animals who received androgen receptor blockade at the quadriceps showed reductions in dendritic length ranging from 39% (180°–240°) to 68% (60°–120°) compared to untreated animals [F(1,132) = 10.00, p < .0001], and these reductions were not significantly different from those seen in saporin animals who received no further treatment [F(1,143) =0.957, ns]. Dendritic lengths per bin in exercised saporin animals who received androgen receptor blockade at the quadriceps were shorter than those of exercised saporin animals who received no androgen receptor blockade [F(1,143) = 9.11, p < .01], with reductions ranging from 23% (300°–360°) to 57% (240°–300°).

Exercised saporin animals who received androgen receptor blockade delivered interscapularly showed reductions in dendritic length ranging from 19% (180°–240°) to 52% (60°–120°) compared to untreated animals [F(1,154) = 12.93, p < .0001], and these reductions were not significantly different from those seen in saporin animals who received no further treatment [F(1,143) = 3.61, ns] or exercised saporin animals who received no androgen receptor blockade [F(1,165) = 0.053, ns].

3.4.3 |. Dendritic extent

In agreement with the nonuniform dendritic distribution of quadriceps motoneurons (Figure 5), radial extent differed across bins (Figure 6), and a repeated measures ANOVA revealed a significant effect of location [F(11,341) = 26.72, p < .0001]. There was also a significant effect of group [F(4, 341) = 3.23, p < .03] due to saporin-induced reductions in dendritic extent; extent was reduced on average 26% across the saporin-treated groups compared to untreated animals. However, unlike dendritic lengths (see above), radial extent did not differ across saporin-treated groups [F(3,279) = 0.741, ns].

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 in untreated animals (white bars), and saporin-injected animals that either received no further treatment (SAP, black bars), were given ad libitum exercise (SAP + EXERCISE, gray bars), or were given ad libitum exercise and received a hydroxyflutamide implant at the vastus lateralis ipsilateral to saporin injection (SAP + EXERCISE+hFLUT_mus, light gray bars) or interscapularly (SAP + EXERCISE+hFLUT_scap, dark gray bars). For graphic purposes, dendritic extent measures have been collapsed into six bins of 60° each. Bar heights represent means ± SEM. * indicates significantly different from untreated animals. † indicates significantly different from untreated saporin-injected animals

Rostrocaudal dendritic extent spanned 3,776.0 ± 546.3 μm in untreated animals. Saporin injection, exercise, or hydroxyflutamide implantation had no effect, and rostrocaudal extent did not differ across groups [overall average 3,695.5 ± 125.8 μm; F(4,31) = 0.06, ns].

4 |. DISCUSSION

Surviving motoneurons respond to the death of neighboring motoneurons with marked dendritic atrophy (Little et al., 2009). Treatment with testosterone, mediated by classical receptor activation, is protective against this atrophy (Cai et al., 2017). Exercise is also protective against dendritic atrophy in surviving motoneurons, and this effect has been demonstrated to be dependent on androgens (Chew & Sengelaub, 2020). In this study, we demonstrate that the neuroprotective effects of exercise are dependent on androgen receptor action specifically at the target muscle.

4.1 |. Specificity of saporin injections

Saporin injection into the VM reduced muscle weight and the number of innervating motoneurons. This induced death was specific to the motoneurons innervating the saporin-injected VM muscle; there were no changes in the number of BHRP-labeled motoneurons projecting to the adjacent VL. This is important for interpreting the effects seen on the morphology of surviving motoneurons, as induced motoneuron death results in dendritic atrophy of surviving motoneurons (Little et al., 2009). The unchanged number of motoneurons innervating the VL indicates that the changes in their dendritic morphology we observed (see below) cannot be due to accidental spread of saporin in the periphery (and subsequent death of VL motoneurons).

Neither exercise nor hydroxyflutamide treatment prevented saporin-induced decreases in the weight of the injected VM muscle, nor did they prevent saporin-induced motoneuron death. Thus, the beneficial effects of exercise on the morphology of neighboring surviving motoneurons, and the occlusion of these benefits with androgen receptor blockade at the muscle, cannot be attributed to differences resulting from the degree of peripheral damage or an attenuation of the ability of saporin to kill motoneurons.

Animals allowed to exercise, regardless of androgen receptor antagonist implantation, showed a modest increase in the weight of the uninjected (right) VM. This hypertrophy could be a response to the saporin-induced reduction of the left VM, compensating for the contralateral reduction in force production and weight bearing of the left VM (Tsumiyama et al., 2014). However, no such effect was observed in either the right VL or, more importantly, in the VL adjacent to the saporin-injected muscle. Thus, it is unlikely that the protective effects of exercise on dendrites are related to changes in muscle weight.

Neither saporin injection nor implantation with hydroxyflutamide impaired the ability of the rats to exercise. Running wheel performance in saporin-treated animals did not differ from that of intact animals, and animals in both groups ran an overall cumulative distance of roughly 124 km over the 4 weeks of treatment. Similarly, running wheel performance in implanted animals did not differ from that of intact or unimplanted saporin animals, indicating that differences in dendritic length were not due to differences in amount of exercise.

4.2 |. Specificity of androgen receptor blockade

Androgen receptor blockade at the muscle, but not interscapularly, prevented the protective effects of exercise on dendritic length. Because the amount of hydroxyflutamide in the implants was the same, but interscapular implants were ineffective in preventing the protection of dendritic length by exercise, it cannot be the case that the effects of androgen receptor blockade were the result of systemic exposure. Thus, we can confidently state that the target musculature is the necessary site of androgen action.

Identifying the muscle as the necessary site of action does not inform what cell types or biomolecular mechanisms are responsible. For example, androgens could be acting at muscle fibers, myoblasts, satellite cells, and adipose tissue in skeletal muscle, all of which are known to express androgen receptors (Dubois et al., 2012; Monks et al., 2004), and our current study did not examine which of these cells types could be involved in this neuroprotective mechanism. Monks et al. (2004) noted that androgen receptor expression in myonuclei and fibroblasts was enriched near the neuromuscular junction, and this synaptic enrichment could allow for the androgenic regulation of synapse-specific genes for proteins critical in the maintenance of the innervating motoneurons.

4.3 |. Causes and protection from dendritic atrophy

Saporin-induced motoneuron death resulted in a pronounced dendritic atrophy in surviving quadriceps motoneurons (Little et al., 2009). This dendritic atrophy is not the result of the loss of afferent fibers from the saporin-injected muscle (Cai et al., 2017), or the increase in activated microglia in the quadriceps motor pool following saporin-induced motoneuron death (Chew et al., 2019). We have speculated that the induced death of motoneurons could result in the release of toxins [e.g., inflammatory cytokines (IL-6, IL-1β, and TNF-α), purines (ATP), glutamate, and matrix metalloproteinases (MMPs)] into the extracellular space, in close proximity to the dendrites of the surviving motoneurons resulting in direct damage (Chew et al., 2019; Fargo & Sengelaub, 2004). Such local changes in the cellular microenvironment would be consistent with the general atrophy seen throughout the dendritic distribution.

The present study provides compelling evidence that the neuroprotective effects of exercise are dependent on androgen action at the musculature. These results are consistent with similar findings of the target musculature as the critical site of action for hormonal effects in the innervating motoneurons, regulating neuron number and dendrogenesis during development, or morphology and protein expression in adulthood (Foecking et al., 2015; Matsumoto, 1997; Rand & Breedlove, 1995; Verhovshek et al., 2013). For example, transgenic upregulation of androgen receptor expression in somatic musculature confers androgen sensitivity to the innervating motoneurons (Huguenard et al., 2011). Castration of male rats with enriched androgen receptor in the musculature results in dendritic atrophy in motoneurons populations and this atrophy is reversed with androgen replacement; motoneuron morphology is unaffected in wild-type animals.

Androgen action at the target musculature has also been shown to regulate the expression of brain-derived neurotrophic factor (BDNF) in motoneurons (Verhovshek et al., 2013), and BDNF is critical in maintaining normal dendritic length in motoneurons (Verhovshek & Sengelaub, 2010). Thus, it is possible that both exercise and testosterone’s neuroprotective effects following partial motoneuron depletion are due to testosterone binding at the target muscle, signaling the muscle and/or the innervating motoneurons to produce trophic factors which protect motoneurons after injury (Chew & Sengelaub, 2019; English et al., 2014).

Exercise results in elevations in serum testosterone (Kindermann et al., 1982; Sato & Iemitsu, 2015; Wood et al., 2012), although intensity, duration, prior conditioning, time point of measurement (e.g., immediately vs. hours after exercise; Vingren et al., 2010), or type of training (Sato & Iemitsu, 2015; Tremblay et al., 2004) all contribute to how testosterone concentrations change in response to exercise. Androgens also positively autoregulate expression of their cognate receptor (Mora et al., 1996), suggesting that increases in serum testosterone concentration can also lead to increased expression of androgen receptors. Thus, it is plausible that exercise-driven increases in serum testosterone can drive increased expression of androgen receptors in tissue, including the target muscle. This potential increase in androgen receptor expression at the muscle suggests that exercise may be able to modulate the efficacy of supplemental testosterone treatment compared to sedentary animals. If true, this would demonstrate an effective synergy of both behavioral and hormonal treatments following neural injury.

4.4 |. Limitations

Inclusion of both sexes when examining biological changes is important, as there may be differences between the sexes that change how the results of experiments are interpreted and generalized. One notable shortcoming in this study, and an important consideration in extrapolating the findings, was the use of only male animals. Only male animals were used for this study due to our prior establishment of the exercise-driven neuroprotection in males (Chew & Sengelaub, 2019) and its elimination after orchidectomy (Chew & Sengelaub, 2020). We have previously demonstrated that treatment with testosterone is effective in attenuating dendritic atrophy in females (Wilson et al., 2009), but have not yet investigated whether exercise is neuroprotective in this injury model.

There have been many studies that have shown differences in voluntary wheel running in males and female rodents (Rosenfeld, 2017). For example, female rats run substantially greater distances than male rats regardless of feeding regime (ad libitum versus restricted; Jones et al., 1990). It is interesting to speculate that this difference could lead to a greater neuroprotective effect of exercise after partial motoneuron depletion. Exercise has also been shown to be neurotherapeutic in both sexes following axotomy, resulting in greater axon regeneration in treadmill-trained mice compared to their sedentary counterparts (Wood et al., 2012). However, there are sex differences in the exercise regimens that facilitate axon regeneration, with males benefiting from slow, continuous walking but females benefiting from an interval training paradigm requiring short, intense bursts of near-maximum running speed (Wood et al., 2012). This neurotherapeutic effect of exercise in both sexes has also been shown to be androgen dependent, as simultaneous treatment with flutamide causes the exercise effect to disappear in both sexes (Thompson et al., 2014). Thus, it will be important for future studies to examine potential sex differences in the neuroprotective effects of exercise after partial motoneuron depletion.

4.5 |. Comparability of BHRP labeling

The possibility that confounds arising from saporin injection could affect retrograde transport is an important consideration, in that such an artifact could potentially result in apparent alterations in dendritic morphology. No difference in rostrocaudal extents of quadriceps dendrites were observed, but radial extents in saporin-treated animals were shorter than those of untreated animals. This result most likely reflects the saporin-induced atrophy of quadriceps motoneurons dendrites rather than a transport artifact, which (because rostrocaudal extent was not affected) would necessarily have had to occur selectively in the transverse plane. Further, radial extent did not differ across saporin-injected groups, indicating that the ability of quadriceps motoneuron dendrites to transport BHRP out to the most distal, highest order branches was not affected. However, differences in overall dendritic lengths were present in these groups, being longer in unimplanted saporin animals allowed to exercise, and in those with interscapular hydroxyflutamide implants. Therefore, we believe that the dendritic labeling across groups was comparable, and thus, the shorter dendritic lengths we observed generally in saporin groups reflect dendritic atrophy. Further, the differences in dendritic length across saporin groups due to exercise and/or hydroxyflutamide implantation reflect the resultant effects on dendritic morphology.

5 |. CONCLUSION

We have previously established that exercise is neuroprotective on motoneuron dendrites following the induced death of their neighbors (Chew & Sengelaub, 2019) and that this effect is dependent on the presence of androgens (Chew & Sengelaub, 2020). Here, we tested whether the neuroprotective effect of exercise is driven by androgen receptors at the muscle. Our findings indicate that androgen receptor action at the muscle is necessary for exercise-mediated neuroprotection of motoneurons following the death of their neighbors.

Both exercise and testosterone upregulate antioxidant activity (Powers et al., 1994), the presence of heat shock proteins in skeletal muscle (Kregel, 2002; Salo et al., 1991), and the cytoskeletal protein β-tubulin in neurons (Jones & Oblinger, 1994). These upregulations are theorized to be adaptive mechanisms in response to the oxidative stress and other biochemical changes experienced during exercise, as androgens have been directly implicated in the positive effects of exercise after injury (English et al., 2014; Thompson et al., 2014; Wood et al., 2012) and exercise has commonly been associated with testosterone and its role in anabolic muscle growth (Bhasin et al., 2001, 2003). Both exercise (Gómez-Pinilla et al., 2002) and androgens (Verhovshek et al., 2013) have also been implicated in the regulation of BDNF expression, and BDNF plays a critical role in maintenance of dendritic arbors (Verhovshek & Sengelaub, 2010) and the promotion of axon regeneration (Wilhelm et al., 2012). This suggests a mechanism by which exercise, androgens, and neurotrophins interact to protect motoneurons and promote recovery following neural injury. Understanding the cellular and molecular mechanisms that operate in normal and injured neurons is likely to provide important insights for developing therapeutic interventions for nervous system trauma and neurodegenerative diseases. Such uses could include the use of the muscle, rather than the motoneurons themselves, as a site of therapeutic intervention for certain neurodegenerative disease or neural injuries.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.