Neuroprotective effect of testosterone treatment on motoneuron recruitment following the death of nearby motoneurons

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have previously shown that motoneuron death induces marked dendritic atrophy in surviving nearby motoneurons. Additionally, in quadriceps motoneurons, this atrophy is accompanied by decreases in motor nerve activity. However, treatment with testosterone partially attenuates changes in both the morphology and activation of quadriceps motoneurons. Testosterone has an even larger neuroprotective effect on the morphology of motoneurons of the spinal nucleus of the bulbocavernosus (SNB), in which testosterone treatment can completely prevent dendritic atrophy. The present experiment was performed to determine whether the greater neuroprotective effect of testosterone on SNB motoneuron morphology was accompanied by a greater neuroprotective effect on motor activation. Right side SNB motoneurons were killed by intramuscular injection of cholera toxin‐conjugated saporin in adult male Sprague‐Dawley rats. Animals were either given Silastic testosterone implants or left untreated. Four weeks later, left side SNB motor activation was assessed with peripheral nerve recording. The death of right side SNB motoneurons resulted in several changes in the electrophysiological response properties of surviving left side SNB motoneurons, including decreased background activity, increased response latency, increased activity duration, and decreased motoneuron recruitment. Treatment with exogenous testosterone attenuated the increase in activity duration and completely prevented the decrease in motoneuron recruitment. These data provide a functional correlate to the known protective effects of testosterone treatment on the morphology of these motoneurons, and further support a role for testosterone as a therapeutic agent in the injured nervous system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Neuroprotective Effects on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons: A Role for Microglia?

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone protects motoneurons from induced dendritic atrophy. We explored a potential mechanism for this induced atrophy and protection by testosterone, examining the microglial response to partial depletion of motoneurons. Motoneurons innervating the vastus medialis muscles of adult male rats were killed by intramuscular injection of cholera toxin‐conjugated saporin; some saporin‐injected rats were treated with testosterone. Microglia were later visualized via immunohistochemical staining, classified as monitoring or activated, and counted stereologically. Partial motoneuron depletion increased the number of activated microglia in the quadriceps motor pool, and this increase was attenuated with testosterone treatment. The attenuation in microglial response could reflect an effect of testosterone on suppressing microglia activation, potentially sparing motoneuron dendrites. Alternatively, testosterone could be neuroprotective, sparing motoneuron dendrites, secondarily resulting in reduced microglial activation. To discriminate between these hypotheses, following partial motoneuron depletion, rats were treated with minocycline to inhibit microglial activation. Motoneurons innervating the ipsilateral vastus lateralis muscle were later labeled with cholera toxin‐conjugated horseradish peroxidase, and dendritic arbors were reconstructed. Reduction of microglial activation by minocycline did not prevent induced dendritic atrophy following partial motoneuron depletion. Further, reduction of microglial activation by minocycline treatment resulted in dendritic atrophy in intact animals. Together, these findings indicate that the neuroprotective effect of testosterone on dendrites following motoneuron death is not due to inhibiting microglial activation, and that microglial activity contributes to the normal maintenance of dendritic arbors.     

Neuroprotective effects of testosterone metabolites and dependency on receptor action on the morphology of somatic motoneurons following the death of neighboring motoneurons

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone is neuroprotective, attenuating induced dendritic atrophy. In this study we examined whether the protective effects of testosterone could be mediated via its androgenic or estrogenic metabolites. Furthermore, to assess whether these neuroprotective effects were mediated through steroid hormone receptors, we used receptor antagonists to attempt to prevent the neuroprotective effects of hormones after partial motoneuron depletion. 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 rats were treated with either dihydrotestosterone or estradiol, alone or in combination with their respective receptor antagonists, 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 animals, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons. Dendritic atrophy was attenuated with both dihydrotestosterone and estradiol treatment to a degree similar to that seen with testosterone, and attenuation of atrophy was prevented by receptor blockade. Together, these findings suggest that neuroprotective effects on motoneurons can be mediated by either androgenic or estrogenic hormones and require action via steroid hormone receptors, further supporting a role for hormones as neurotherapeutic agents in the injured nervous system. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 691–707, 2017     

Testosterone metabolites differentially maintain adult morphology in a sexually dimorphic neuromuscular system

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Androgens are necessary for the development of the SNB neuromuscular system, and in adulthood, continue to influence the morphology and function of the motoneurons and their target musculature. However, estrogens are also involved in the development of the SNB system, and are capable of maintaining function in adulthood. In this experiment, we assessed the ability of testosterone metabolites, estrogens and nonaromatizable androgens, to maintain neuromuscular morphology in adulthood. Motoneuron and muscle morphology was assessed in adult normal males, sham‐castrated males, castrated males treated with testosterone, dihydrotestosterone, estradiol, or left untreated, and gonadally intact males treated with the 5α‐reductase inhibitor finasteride or the aromatase inhibitor fadrozole. After 6 weeks of treatment, SNB motoneurons were retrogradely labeled with cholera toxin‐HRP and reconstructed in three dimensions. Castration resulted in reductions in SNB target muscle size, soma size, and dendritic morphology. Testosterone treatment after castration maintained SNB soma size, dendritic morphology, and elevated target muscle size; dihydrotestosterone treatment also maintained SNB dendritic length, but was less effective than testosterone in maintaining both SNB soma size and target muscle weight. Treatment of intact males with finasteride or fadrozole did not alter the morphology of SNB motoneurons or their target muscles. In contrast, estradiol treatment was completely ineffective in preventing castration‐induced atrophy of the SNB neuromuscular system. Together, these results suggest that the maintenance of adult motoneuron or muscle morphology is strictly mediated by androgens. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 206–221, 2010.     

Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: A cholera toxin-HRP study

The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior. Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood. Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats. Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure. Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed. Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport. However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone. Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area. These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.     

Hormonally mediated plasticity of motoneuron morphology in the adult rat spinal cord: a cholera toxin-HRP study.

The dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord are sexually dimorphic groups of motoneurons that innervate striated perineal muscles involved in male copulatory behavior. Androgens control the development of these motoneurons and their target muscles, and continue to influence the system in adulthood. Given that several features of SNB motoneuron morphology have been shown to be androgen sensitive in adult male rats, we examined the effects of androgen manipulations on the morphology of motoneurons in the DLN in adult rats. Adult male rats were castrated and implanted with testosterone-filled or blank implants, or were subjected to a sham-castration procedure. Six weeks after treatment, motoneurons in the DLN were retrogradely labeled with cholera toxin-horseradish peroxidase (HRP) after injection into the ischiocavernosus (IC) muscle and their morphology assessed. Measures of the radial extent and coverage of the dendritic arbor of DLN motoneurons projecting to the IC (DLN-IC motoneurons) were similar across the groups, indicating comparable degrees of HRP transport. However, DLN-IC motoneurons in castrates with blank implants possessed both shorter dendritic lengths and smaller somas than those of castrates treated with testosterone. Castrates with testosterone implants had DLN-IC motoneurons that were significantly larger than those of sham castrates in dendritic length and soma area. These results suggest that motoneurons in the DLN, like those in the SNB, possess a significant degree of structural plasticity in adulthood which is influenced by androgens.     

Differential effects of dihydrotestosterone and estrogen on the development of motoneuron morphology in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains a sexually dimorphic motor nucleus, the spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innnervate perineal muscles involved in copulatory reflexes. Dendritic development of SNB motoneurons is biphasic and androgen dependent. During the first 4 postnatal weeks, SNB dendrites grow exuberantly, and subsequently retract to mature lengths by 7 weeks of age. After early postnatal castration, SNB dendrites fail to grow, and testosterone replacement restores this growth. In other systems, testosterone and its metabolites, dihydrotestosterone and estrogen, are important for somatic and neural sexual differentiation. The purpose of the present study was to examine the effects of castration and dihydrotestosterone or estrogen replacement on the growth of SNB motoneuron somata and dendritic arbors. Male rat pups were castrated on postnatal (P) day 7 and treated daily with either dihydrotestosterone propionate (DHTP; 2 mg) or estradiol benzoate (EB; 100 μg) until P28 or P49. By using cholera toxin horseradish peroxidase (BHRP) histochemistry, the soma size, dendritic length, dendritic extent, and arbor area of BHRP-labeled SNB motoneurons were measured and analyzed. Both DHTP and EB treatment supported the initial exuberant growth of SNB dendrites through P28, but EB treatment was ineffective in maintaining mature, adult lengths at P49. The possible sites of hormone action and functional implications of these hormonal treatments are discussed. 1994 John Wiley & Sons, Inc.     

Androgenic, but not estrogenic, protection of motoneurons from somal and dendritic atrophy induced by the death of neighboring motoneurons

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have been investigating the effects of motoneuron loss on surviving motoneurons in a lumbar motor nucleus, the spinal nucleus of the bulbocavernosus (SNB). SNB motoneurons undergo marked dendritic and somal atrophy following the experimentally induced death of other nearby SNB motoneurons. However, treatment with testosterone at the time of lesioning attenuates this atrophy. Because testosterone can be metabolized into the estrogen estradiol (as well as other physiologically active steroid hormones), it was unknown whether the protective effect of testosterone was an androgen effect, an estrogen effect, or both. In the present experiment, we used a retrogradely transported neurotoxin to kill the majority of SNB motoneurons on one side of the spinal cord only in adult male rats. Some animals were also treated with either testosterone, the androgen dihydrotestosterone (which cannot be converted into estradiol), or the estrogen estradiol. As seen previously, partial motoneuron loss led to reductions in soma area and in dendritic length and extent in surviving motoneurons. Testosterone and dihydrotestosterone attenuated these reductions, but estradiol had no protective effect. These results indicate that the neuroprotective effect of testosterone on the morphology of SNB motoneurons following partial motoneuron depletion is an androgen effect rather than an estrogen effect.     

Castration reduces motoneuron soma size but not dendritic length in the spinal nucleus of the bulbocavernosus of wild‐type and BCL‐2 overexpressing mice

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, bulbocavernosus and levator ani (BC/LA), constitute an androgen‐sensitive neuromuscular system. Testosterone regulates SNB soma size, SNB dendritic length, and BC/LA muscle mass in adult male rats. Recent evidence indicates that the cell death‐regulatory protein, Bcl‐2, may also play a role in adult neural plasticity. The present study examined whether gonadal hormones and/or the Bcl‐2 protein influence the morphology of the SNB neuromuscular system in adult B6D2F1 mice. In Experiment 1, adult wild‐type and Bcl‐2 overexpressing males were castrated and implanted with silastic capsules containing testosterone or left blank. Six weeks after castration, cholera toxin‐horseradish peroxidase was injected into the BC muscle to label SNB dendrites. Animals were killed 48 h later, and BC/LA muscle mass, SNB soma size, and SNB dendritic arbors were examined. In Experiment 2, wild‐type and Bcl‐2 overexpressing males were castrated or sham castrated, implanted with testosterone‐filled or blank capsules, and examined 12 weeks later. In both experiments, BC/LA muscle mass and SNB soma size were significantly reduced in castrates receiving blank capsules. Surprisingly, however, there was no effect of hormone manipulation on any of several measures of dendritic length. Thus, the dendritic morphology of SNB motoneurons appears to be relatively insensitive to circulating androgen levels in B6D2F1 mice. Bcl‐2 overexpression did not influence BC/LA muscle mass, SNB soma size, or SNB dendritic length, indicating that the morphology of this neuromuscular system and the response to castration are not altered by forced expression of the Bcl‐2 protein. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 403–412, 2002     

Adult plasticity in hormone-sensitive motoneuron morphology: methodological/behavioral confounds

Changes in androgen levels can alter the structure of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), a motor nucleus that innervates perineal muscles involved in copulatory behavior. While sexual activity can alter androgen levels in normal males, it has no effect on SNB motoneuron soma size or dendritic morphology (Beversdorf, Kurz, and Sengelaub, 1990). However, Breedlove (1997) reported reductions in the size of SNB somata, nuclei, and target muscles of copulating versus noncopulating castrated rats maintained on subphysiological testosterone. To reconcile the results obtained using intact versus implant paradigms, we tested the hypothesis that the implant/behavior paradigm could produce differences in hormone levels, potentially confounding sexual behavior effects on the morphology of this androgen-sensitive neuromuscular system. Young adult male rats were castrated and immediately given 5-mm Silastic implants containing crystalline testosterone. One week later, blood samples were drawn and the males were housed with receptive females (copulators) or nonreceptive females (noncopulators) or housed alone (singles). After 27 days, blood samples were drawn again, and SNB target muscles and spinal cords removed. No differences in target muscle weight or SNB somata and nuclei size were observed between copulators, noncopulators, or singles; as expected, all measures were significantly reduced relative to intact males. Radioimmunoassay showed that testosterone declined differentially over the course of the behavioral manipulation across groups, being greatest in copulators and least pronounced in single males. These data indicate that differences in sexual or housing experience can alter testosterone titers under these implant conditions, potentially confounding hormone-sensitive measures of morphology.     

Estrogenic support of motoneuron dendritic growth via the neuromuscular periphery in a sexually dimorphic motor system

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). In males, the growth of SNB dendrites is steroid-dependent: dendrites fail to grow after castration, but grow in castrates treated with androgens or estrogens. Blocking estradiol synthesis or estrogen receptors in gonadally intact males attenuates SNB dendritic growth, suggesting that estrogens are required and must be able to act at their receptors to support normal masculine dendritic growth. However, SNB motoneurons do not accumulate estrogens, suggesting that estrogens act indirectly to support SNB dendritic growth. In this experiment, we examined whether local estrogen action in the neuromuscular periphery was involved in the postnatal development of SNB motoneurons. Motoneuron morphology was assessed in gonadally intact and castrated males. Gonadally intact males were left untreated or given either blank or tamoxifen implants sutured to the target musculature, or tamoxifen interscapular implants. Castrated males were left untreated or were given estradiol by muscle or interscapular implants or systemic injection during the period of SNB dendritic growth. At postnatal day 28, when SNB dendritic length is normally maximal, SNB motoneurons were retrogradely labeled with cholera toxin-HRP and reconstructed in three dimensions. While interscapular tamoxifen implants were ineffective, blocking estrogen receptors at the target musculature resulted in attenuation of SNB dendritic growth. In contrast, while interscapular implants of estradiol were ineffective, local treatment with estradiol at the target musculature in castrated males resulted in masculinization of dendritic growth. Thus, estrogens may act by an indirect action in the neuromuscular periphery to support SNB dendritic growth.     

Seasonal variation in mammalian striated muscle mass and motoneuron morphology

Feral white-footed mice are seasonal breeders that undergo predictable cycles of reproductive function. Photoperiod-induced fluctuations in gonadal function of white-footed mice were associated with morphological changes in perineal muscles and their motoneurons. Exposure to short daylengths resulted in testicular regression, decreased perineal muscle mass, and shrinkage of somata and nuclei of motoneurons of the spinal nucleus of the bulbocavernosus (SNB). These effects were reversed by reinstatement of long daylengths. Similar reductions in muscle mass and SNB soma size were seen following gonadectomy of white-footed mice. In addition, dendritic trees of SNB motoneurons were reduced in gonadectomized mice compared with dendritic arbors of intact mice or castrates provided with testosterone capsules. Androgen-mediated annual changes in muscle mass and motoneuron morphology appear to be a natural part of this species' physiology.     

Androgen action at the target musculature regulates brain‐derived neurotrophic factor protein in the spinal nucleus of the bulbocavernosus

We have previously demonstrated that brain‐derived neurotrophic factor (BDNF) interacts with testosterone to regulate dendritic morphology of motoneurons in the highly androgen‐sensitive spinal nucleus of the bulbocavernosus (SNB). Additionally, in adult male rats testosterone regulates BDNF in SNB motoneurons and its target muscle, the bulbocavernosus (BC). Because BDNF is retrogradely transported from skeletal muscles to spinal motoneurons, we hypothesized that testosterone could regulate BDNF in SNB motoneurons by acting locally at the BC muscle. To test this hypothesis, we restricted androgen manipulation to the SNB target musculature. After castration, BDNF immunolabeling in SNB motoneurons was maintained at levels similar to those of gonadally intact males by delivering testosterone treatment directly to the BC muscle. When the same implant was placed interscapularly in castrated males it was ineffective in supporting BDNF immunolabeling in SNB motoneurons. Furthermore, BDNF immunolabeling in gonadally intact adult males given the androgen receptor blocker hydroxyflutamide delivered directly to the BC muscle was decreased compared with that of gonadally intact animals that had the same hydroxyflutamide implant placed interscapularly, or when compared with castrated animals that had testosterone implants at the muscle. These results demonstrate that the BC musculature is a critical site of action for the androgenic regulation of BDNF in SNB motoneurons and that it is both necessary and sufficient for this action. Furthermore, the local action of androgens at the BC muscle in regulating BDNF provides a possible mechanism underlying the interactive effects of testosterone and BDNF on motoneuron morphology. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 587–598, 2013     

Castration reduces motoneuron soma size but not dendritic length in the spinal nucleus of the bulbocavernosus of wild-type and BCL-2 overexpressing mice

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, bulbocavernosus and levator ani (BC/LA), constitute an androgen-sensitive neuromuscular system. Testosterone regulates SNB soma size, SNB dendritic length, and BC/LA muscle mass in adult male rats. Recent evidence indicates that the cell death-regulatory protein, Bcl-2, may also play a role in adult neural plasticity. The present study examined whether gonadal hormones and/or the Bcl-2 protein influence the morphology of the SNB neuromuscular system in adult B6D2F1 mice. In Experiment 1, adult wild-type and Bcl-2 overexpressing males were castrated and implanted with silastic capsules containing testosterone or left blank. Six weeks after castration, cholera toxin-horseradish peroxidase was injected into the BC muscle to label SNB dendrites. Animals were killed 48 h later, and BC/LA muscle mass, SNB soma size, and SNB dendritic arbors were examined. In Experiment 2, wild-type and Bcl-2 overexpressing males were castrated or sham castrated, implanted with testosterone-filled or blank capsules, and examined 12 weeks later. In both experiments, BC/LA muscle mass and SNB soma size were significantly reduced in castrates receiving blank capsules. Surprisingly, however, there was no effect of hormone manipulation on any of several measures of dendritic length. Thus, the dendritic morphology of SNB motoneurons appears to be relatively insensitive to circulating androgen levels in B6D2F1 mice. Bcl-2 overexpression did not influence BC/LA muscle mass, SNB soma size, or SNB dendritic length, indicating that the morphology of this neuromuscular system and the response to castration are not altered by forced expression of the Bcl-2 protein.     

Aromatase inhibition reduces dendritic growth in a sexually dimorphic rat spinal nucleus

The rat lumbar spinal cord contains the steroid-sensitive spinal nucleus of the bulbocavernosus (SNB), whose motoneurons innervate perineal muscles involved in copulatory reflexes. In normal males, SNB motoneuron dendrites grow exuberantly through postnatal (P) day 28. This growth is steroid dependent: Dendrites fail to grow in males castrated at P7, but grow normally in castrates treated with testosterone or its metabolites, dihydrotestosterone combined with estrogen. Treatment with either metabolite alone supports dendritic growth, but not to the level of testosterone-treated or intact males. In this study, we tested the hypothesis that aromatization of androgens to estrogens was involved in the masculine development of SNB dendrites. Motoneuron morphology was assessed in normal males and males treated daily (P7-28) with fadrozole, a potent aromatase inhibitor (0.25 mg/kg, subcutaneously) or saline vehicle (n = 4-6/group). SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase at P28 (when dendritic length is normally maximal) and reconstructed in three dimensions. Comparable labeling was seen across groups; it was equivalent in both the rostrocaudal and radial extents. However, dendritic lengths in fadrozole-treated males were significantly below those of intact or saline-treated males. Neither SNB somata size nor target muscle weight differed across groups. These results suggest that aromatization of androgens to estrogens is necessary for development of masculine SNB dendritic morphology.     

Ovariectomy attenuates dendritic growth in hormone‐sensitive spinal motoneurons

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons in male rats is biphasic, initially showing exuberant growth through 4 weeks of age followed by a retraction to mature lengths by 7 weeks of age. The initial growth is steroid dependent, attenuated by castration or aromatase inhibition, and supported by hormone replacement. Dendritic retraction is also steroid sensitive and can be prevented by testosterone treatment, but is unaffected by aromatase inhibition. Together, these results suggest a role for estrogens during the initial growth phase of SNB development. In this study, we tested whether ovarian hormones could support SNB somal and dendritic development. Motoneuron morphology was assessed in normal males and in females perinatally masculinized with dihydrotestosterone and then either ovariectomized or left intact. SNB motoneurons were retrogradely labeled with cholera toxin‐HRP at 4 or 7 weeks of age and reconstructed in three dimensions. Initial growth of SNB dendrites was reduced after ovariectomy in masculinized females. However, no differences in dendritic length were seen at 7 weeks of age between intact and ovariectomized masculinized females, and lengths in both groups were significantly lower than those of normal males. Together with previous findings, these results suggest that estrogens are involved in the early growth of SNB dendrites, but not in their subsequent retraction. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 301–314, 2001     

Castration‐induced upregulation of muscle ERα supports estrogen sensitivity of motoneuron dendrites in a sexually dimorphic neuromuscular system

The spinal cord of rats contains the sexually dimorphic motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In males, SNB dendrites fail to grow after castration, but androgen or estrogen treatment supports dendritic growth in castrated males. Estrogenic support of SNB dendrite growth is mediated by estrogen receptors (ER) in the target muscle. ERα expression in cells lacking a basal lamina (referred to as “extra‐muscle fiber cells”) of the SNB target musculature coincides with the period of estrogen‐dependent SNB dendrite growth. In the SNB target muscle, extra‐muscle fiber ERα expression declines with age and is typically absent after postnatal (P) day 21 (P21). Given that estradiol downregulates ERα in skeletal muscle, we tested the hypothesis that depleting gonadal hormones would prevent the postnatal decline in ERα expression in the SNB target musculature. We castrated male rats at P7 and assessed ERα immunolabeling at P21; ERα expression was significantly greater in castrated males compared with normal animals. Because ERα expression in SNB target muscles mediates estrogen‐dependent SNB dendrogenesis, we further hypothesized that the castration‐induced increase in muscle ERα would heighten the estrogen sensitivity of SNB dendrites. Male rats were castrated at P7 and treated with estradiol from P21 to P28; estradiol treatment in castrates resulted in dendritic hypertrophy in SNB motoneurons compared with normal males. We conclude that early castration results in an increase in ERα expression in the SNB target muscle, and this upregulation of ERα supports estrogen sensitivity of SNB dendrites, allowing for hypermasculinization of SNB dendritic arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 921–935, 2013     

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

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.     

Androgen-sensitivity of somata and dendrites of spinal nucleus of the bulbocavernosus (SNB) motoneurons in male C57BL6J mice

In rats, androgens in adulthood regulate the morphology of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), including the size of their somata and the length of their dendrites. There are conflicting reports about whether androgens exert similar influences on SNB motoneurons in mice. We castrated or sham-operated C57BL6J mice at 90 days of age and, thirty days later, injected cholera toxin conjugated horseradish peroxidase into the bulbocavernosus muscle (to label SNB motoneurons) on one side, and into intrinsic foot muscles contralaterally (to label motoneurons of the retrodorsolateral nucleus (RDLN)). Castrated mice had significantly smaller SNB somas compared to sham-operated mice while there were no differences in soma size of RDLN motoneurons. Dendritic length in C57BL6J mice, estimated in 3-dimensions, also decreased significantly after adult castration. In rats, androgens act directly through androgen receptors (AR) in SNB motoneurons to control soma size and nearly all SNB motoneurons contain AR. Since SNB somata in C57BL6J mice shrank after adult castration, we used immunocytochemistry to characterize AR expression in SNB cells as well as motoneurons in the RDLN and dorsolateral nucleus (DLN). A pattern of labeling matched that seen previously in rats: the highest percentage of AR-immunoreactive motoneurons are in the SNB (98%), the lowest in the RDLN (25%) and an intermediate number in the DLN (78%). This pattern of AR labeling is consistent with the possibility that androgens also act directly on SNB motoneurons in mice to regulate soma size in mice.     

Ovariectomy attenuates dendritic growth in hormone-sensitive spinal motoneurons

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons in male rats is biphasic, initially showing exuberant growth through 4 weeks of age followed by a retraction to mature lengths by 7 weeks of age. The initial growth is steroid dependent, attenuated by castration or aromatase inhibition, and supported by hormone replacement. Dendritic retraction is also steroid sensitive and can be prevented by testosterone treatment, but is unaffected by aromatase inhibition. Together, these results suggest a role for estrogens during the initial growth phase of SNB development. In this study, we tested whether ovarian hormones could support SNB somal and dendritic development. Motoneuron morphology was assessed in normal males and in females perinatally masculinized with dihydrotestosterone and then either ovariectomized or left intact. SNB motoneurons were retrogradely labeled with cholera toxin-HRP at 4 or 7 weeks of age and reconstructed in three dimensions. Initial growth of SNB dendrites was reduced after ovariectomy in masculinized females. However, no differences in dendritic length were seen at 7 weeks of age between intact and ovariectomized masculinized females, and lengths in both groups were significantly lower than those of normal males. Together with previous findings, these results suggest that estrogens are involved in the early growth of SNB dendrites, but not in their subsequent retraction.