Tom Verhovshek

Androgen Regulates Brain-Derived Neurotrophic Factor in Spinal Motoneurons and Their Target Musculature

Trophic factors maintain motoneuron morphology and function in adulthood. Brain-derived neurotrophic factor (BDNF) interacts with testosterone to maintain dendritic morphology of spinal motoneurons. In addition, testosterone regulates BDNFs receptor (trkB) in motoneurons innervating the quadriceps muscles as well as in motoneurons of the highly androgen-sensitive spinal nucleus of the bulbocavernosus (SNB). Given these interactive effects, we examined whether androgen might also regulate BDNF in quadriceps and SNB motoneurons and their corresponding target musculature. In both motoneuron populations, castration of males reduced BDNF immunolabeling, and this effect was prevented with testosterone replacement. ELISA for BDNF in the target musculature of quadriceps (vastus lateralis, VL) and SNB (bulbocavernosus, BC) motoneurons revealed that BDNF in the VL and BC muscles was also regulated by androgen. However, although castration significantly decreased BDNF concentration in the VL muscle, BDNF concentration in the BC muscle was significantly increased in castrates. Treatment of castrated males with testosterone maintained BDNF levels at those of intact males in both sets of muscles. Together, these results demonstrate that androgens regulate BDNF in both a sexually dimorphic, highly androgen-sensitive neuromuscular system as well as a more typical somatic neuromuscular system. Furthermore, in addition to the regulation of trkB, these studies provide another possible mechanism for the interactive effects of testosterone and BDNF on motoneuron morphology. More importantly, by examining both the motoneurons and the muscles they innervate, these results demonstrate that within a neural system, BDNF levels in different components are differentially affected by androgen manipulation.

Androgen regulates trkB immunolabeling in spinal motoneurons.

Neurotrophic factors and steroid hormones have been shown to have neuroprotective/neurotherapeutic effects, and it has been shown previously that brain‐derived neurotrophic factor (BDNF) and testosterone have a combinatorial effect in the maintenance of motoneurons. Given that gonadal hormones regulate the BDNF receptor, tyrosine receptor kinase B (trkB), we hypothesized that such a regulatory effect could mediate the interactive effects of BDNF and testosterone. Using immunohistochemical methods, we examined the frequency of cells immunolabeled for trkB receptors in two populations of spinal motoneurons, the hormone‐sensitive, sexually dimorphic motoneurons of the spinal nucleus of the bulbocavernosus (SNB) and the nondimorphic motoneurons innervating the muscles of the quadriceps. In both the highly androgen‐sensitive SNB motoneurons and the more typical somatic motoneurons innervating the quadriceps, the frequency of motoneurons intensely immunolabeled for trkB receptors was regulated by the presence of testosterone. Castrated animals deprived of testosterone showed a reduced frequency of intensely labeled motoneurons compared with intact animals or castrated animals given testosterone replacement. This finding suggests that the combinatorial effect of BDNF and testosterone in the maintenance of motoneurons could occur at least in part through an androgen‐mediated expression of the BDNF receptor.

N-methyl-D-aspartate receptor blockade inhibits estrogenic support of dendritic growth in a sexually dimorphic rat spinal nucleus.

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Dendritic development of SNB motoneurons requires the action of both androgens and estrogens. Estrogenic effects are limited to the initial growth of SNB dendrites through 4 weeks of age. During this postnatal period, dendritic growth in other spinal motoneurons is regulated by N‐methyl‐D‐aspartate (NMDA) receptor activation. In this study, we tested whether NMDA receptor activation was involved in SNB dendritic growth and whether the estrogenic support of SNB dendritic growth was dependent on the activation of NMDA receptors. Motoneuron morphology was assessed in normal males, intact males treated daily with the NMDA receptor antagonist MK‐801, castrated males treated with estradiol benzoate (EB), and castrated males treated with both EB and MK‐801. SNB motoneurons were retrogradely labeled with cholera toxin‐horseradish peroxidase at 4 weeks of age (when dendritic length is normally maximal) and reconstructed in three dimensions. Somal area and dendritic length of SNB motoneurons in MK‐801‐treated, intact males were below those of normal males. Dendritic growth was partially supported in EB‐treated castrates, but this growth was blocked by MK‐801 treatment. These results suggest that, as in other motoneurons, dendritic development in the SNB involves NMDA receptors and, furthermore, that the estrogen‐sensitive component of SNB dendritic development requires their activation. J. Comp. Neurol. 451:142–152, 2002.

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.

Brain-derived neurotrophic factor and androgen interactions in spinal neuromuscular systems

Neurotrophic factors and steroid hormones interact to regulate a variety of neuronal processes such as neurite outgrowth, differentiation, and neuroprotection. The coexpression of steroid hormone and neurotrophin receptor mRNAs and proteins, as well as their reciprocal regulation provides the necessary substrates for such interactions to occur. This review will focus on androgen brain-derived neurotrophic factor (BDNF) interactions in the spinal cord, describing androgen regulation of BDNF in neuromuscular systems following castration, androgen manipulation, and injury. Androgens interact with BDNF during development to regulate normally-occurring motoneuron death, and in adulthood, androgen-BDNF interactions are involved in the maintenance of several features of neuromuscular systems. Androgens regulate BDNF and trkB expression in spinal motoneurons. Androgens also regulate BDNF levels in the target musculature, and androgenic action at the muscle regulates BDNF levels in motoneurons. These interactions have important implications for the maintenance of motoneuron morphology. Finally, androgens interact with BDNF after injury, influencing soma size, dendritic morphology, and axon regeneration. Together, these findings provide further insight into the development and maintenance of neuromuscular systems and have implications for the neurotherapeutic/neuroprotective roles of androgens and trophic factors in the treatment of motoneuron disease and recovery from injury.

Trophic Effects of Brain-Derived Neurotrophic Factor Blockade in an Androgen-Sensitive Neuromuscular System

In adult male rats, androgens are necessary for the maintenance of the motoneurons and their target muscles of the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB) neuromuscular system, regulating motoneuron and muscle morphology, function, and expression of trophic factors. Castration of males results in somal, dendritic, and muscle atrophy as well as increases in brain-derived neurotrophic factor (BDNF) in the target musculature. Because BDNF can have either facilitative or inhibitory effects in other systems, we examined SNB neuromuscular morphology after BDNF blockade using a fusion protein (tyrosine kinase receptor type B IgG). Blockade of BDNF in gonadally intact males resulted in hypertrophy of SNB motoneuron dendrites and target musculature, suggesting that normal levels of BDNF are inhibitory in SNB neuromuscular system. BDNF blockade in castrated males prevented SNB motoneuron atrophy and attenuated target muscle weight loss. This is the first demonstration that the highly androgen-sensitive SNB motoneuron dendrites and target muscles can be maintained in the absence of gonadal hormones and, furthermore, that blocking BDNF can have trophic effects on skeletal muscle. These results suggest that whereas BDNF is involved in the signaling cascade mediating the androgenic support of SNB neuromuscular morphology, its action can be inhibitory. Furthermore, the elevations in BDNF after castration may be responsible for the castration-induced atrophy in SNB motoneurons and target muscles, and the trophic effects of androgens may be mediated in part through a suppression of BDNF. These results may have relevance to therapeutic approaches to the treatment of neurodegenerative disease or myopathies.

NMDA receptor binding declines differentially in three spinal motor nuclei during postnatal development.

The NMDA subtype of glutamate receptors mediates a variety of neuronal processes involved in the development of dendritic morphology. For example, NMDA receptor antagonism during the early postnatal period attenuates dendritic growth in spinal motoneurons. NMDA receptors are present in high levels in the spinal cord early in the postnatal period and decline during development, a period of extensive dendritic plasticity in the spinal cord. Previous studies have suggested that an adult pattern of distribution of NMDA receptors is established as early as postnatal day (P)21 (day of birth = P1). However, given that dendritic growth in spinal motoneurons is not complete by this age and that NMDA receptor activation is necessary for dendritic growth, we assessed NMDA receptor binding in specific spinal motor nuclei during normal development. NMDA receptors were labeled with [3H]MK-801 at P7, P14, P28, P49, and in adult male rats. Receptor binding in the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus (DLN) and retrodorsolateral nucleus (RDLN) was measured using in vitro quantitative autoradiography. NMDA receptor binding over the SNB, DLN and RDLN in intact males was initially high, and declined to adult levels. However, the time course of the decline differed across nuclei. The local decline in NMDA receptor binding observed in the SNB and DLN is coincident with the periods of dendritic growth in these nuclei, further supporting a role for NMDA receptors in the development of motoneuron dendritic morphology.

A bilateral head injury that shows graded brain damage and behavioral deficits in adultmice

Reliable animal models of traumatic brain injury (TBI) are essential to test novel hypotheses and therapeutic interventions. In this study, based on advantages of both the closed head injury (CHI) and controlled cortical impact (CCI) models, we developed a bilateral head injury model in mice. C57BL/6 mice were used in this study. A midline craniotomy (5mm diameter) was performed extending 2mm anteriorly and 3mm posteriorly from the bregma, centered over the sagittal suture. The skull flap was left in place. A cortical impact on the surface of the skull flap was performed using an electromagnetic impactor. Here we report that the injury significantly decreased the neuroscore and increased foot drops in a severity-dependent manner. Severity-related deficits in performance on a rotarod device were also found at both slow and fast accelerations. These findings suggest that our TBI model can produce graded motor deficits. In addition, Morris water maze testing showed increased latency to locate a hidden platform in a severity-dependent manner, suggesting that our model can also produce graded memory deficits. Furthermore, an adhesive removal test revealed significant increases in time-to-contact and time-to-remove the adhesive tape from the paw in a severity-dependent manner, indicating that our TBI model produced graded somatosensory and motor deficits. Histological analysis presented a clear gradation in brain tissue damage following graded brain injuries. These findings collectively suggest that the current model may offer a sensitive, reliable and clinically-relevant model for assessments of therapeutic strategies forTBI.

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.

A semicircular controlled cortical impact produces long-term motor and cognitive dysfunction that correlates well with damage to both the sensorimotor cortex and hippocampus

Animal models of traumatic brain injury (TBI) are essential for testing novel hypotheses and therapeutic interventions. Unfortunately, due to the broad heterogeneity of TBI in humans, no single model has been able to reproduce the entire spectrum of these injuries. The controlled cortical impact (CCI) model is one of the most commonly used models of contusion TBI. However, behavioral evaluations have revealed transient impairment in motor function after CCI in rats and mice. Here we report a new semicircular CCI (S-CCI) model by increasing the impact tip area to cover both the motor cortex and hippocampal regions in adult mice. Mice were subjected to S-CCI or CCI using an electromagnetic impactor (Impactor One, MyNeuroLab; semicircular tip: 3mm radius; CCI tip diameter: 3mm). We showed that S-CCI, at two injury severities, significantly decreased the neuroscore and produced deficits in performance on a rotarod device for the entire duration of the study. In contrast, the CCI induced motor deficits only at early stages after the injury, suggesting that the S-CCI model produces long-lasting motor deficits. Morris water maze test showed that both CCI and S-CCI produced persisting memory deficits. Furthermore, adhesive removal test showed significant somatosensory and motor deficits only in the S-CCI groups. Histological analysis showed a large extent of cortical contusion lesions, including both the sensory and motor cortex, and hippocampal damage in the S-CCI. These findings collectively suggest that the current model may offer sensitive, reliable, and clinically relevant outcomes for assessments of therapeutic strategies for TBI.