Androgenic regulation of the central glia response following nerve damage.

Current research on the effects of gonadal steroids on the brain and spinal cord indicates that these agents have profound trophic effects on many aspects of neuronal functioning, including cell survival, growth and metabolism, elaboration of processes, synaptogenesis, and neurotransmission (Jones et al., 1985; Luine, 1985; Nordeen et al., 1985; Matsumoto et al., 1988a,b; Gould et al., 1990). Since many of the aspects of normal neuronal functioning altered by gonadal steroids are affected by injury to the nervous system, we initiated a series of experiments designed to exploit the trophic capabilities of steroids as therapeutic agents in neuronal injury and repair (Kujawa et al., 1989, 1991; Kujawa and Jones, 1990). Three steroid-sensitive model systems were used for these studies: the hamster facial motoneuron, the rat sciatic motoneuron, and the hamster rubrospinal motoneuron. The results of our initial series of experiments suggest that androgens, and possibly estrogens, act either directly or indirectly on the injured motoneuron and enhance elements of the neuronal reparative response that are critical to successful recovery of function. Recently, we discovered that gonadal steroids may also modulate the central glia response to nerve damage. In this review, a summary of our data identifying a therapeutic role for androgens in enhancing the reparative response of motoneurons to injury is presented. This is followed by a discussion of the effects of androgens on the glial response to injury.     

Cellular localization of androgen and estrogen receptors in mouse-derived motoneuron hybrid cells and mouse facial motoneurons

The ability of gonadal steroid hormones to augment axonal regeneration after peripheral nerve injury has been well established in rat and hamster motoneuron systems, and provides a foundation for the use of these agents as neurotherapeutics. With the advent of mouse genetics and the availability of transgenic and knockout mice, the use of mice in studies of neuroprotection is growing. It has recently been demonstrated that both androgens and estrogens rescue motoneurons (MN) from injury in mouse-derived motoneuron hybrid cells in vitro and mouse facial motoneurons (FMN) in vivo (Tetzlaff et al. [2006] J Mol Neurosci 28:53-64). To elucidate the molecular mechanisms of these effects, the present study examined the cellular localization of androgen and estrogen receptors in mouse MN in vitro and in vivo. Immunoblotting and immunocytochemistry studies established the presence of androgen receptor (AR) and estrogen receptor alpha/beta in immortalized mouse motoneuron hybrid cells and AR and estrogen receptor alpha in mouse FMN.     

Estrogen receptor expression in the facial nucleus of adult hamsters: does axotomy recapitulate development?

Testosterone propionate (TP) augments hamster facial motoneuron regeneration following axonal injury by an androgen-mediated mechanism. Although many of the trophic properties of TP are androgenic, TP can be metabolized to estradiol (E). We have recently shown that E administered in supraphysiological doses can also enhance facial nerve regeneration. The mechanism by which E alters nerve regeneration is unknown. The recent discovery of transient estrogen receptor (ER) expression in the developing rat facial motor nucleus (FMN), coupled with the concept that regeneration may recapitulate development, has led to the hypothesis that facial nerve injury may transiently induce expression of ER in the adult hamster FMN or one of its chief afferents, the principal nucleus of the trigeminal nerve (Nu5). In the present study, this hypothesis was tested using steroid hormone autoradiographic procedures. The right facial nerve was injured in castrated or castrated plus TP adult hamsters. A gonadally intact, nonaxtomized group of hamsters was also included to examine constitutive expression of ER in the FMN or Nu5. The paraventricular nucleus of the hypothalamus (PVN; positive control), FMN, and Nu5, were qualitatively and quantitatively examined for the presence of ER. As expected, ER were present in the PVN-positive control in all groups. ER were neither present nor induced with facial nerve injury or TP administration in either the FMN or Nu5. Alternate mechanisms by which E enhancement of facial nerve regeneration without ER might be explained are discussed.     

Gonadal Steroid Regulation of Growth-Associated Protein GAP-43 mRNA Expression in Axotomized Hamster Facial Motor Neurons

Treatment with testosterone propionate (TP) after nerve injury is known to accelerate both the rate of axonal regeneration and functional recovery from facial paralysis in the adult male hamster. Peripheral nerve injury is also known to increase the expression of a 43 kilodalton growth-associated protein (GAP-43). In the intact brain, GAP-43 expression is affected by gonadal steroids. We thus postulated that steroidal modulation of GAP-43 gene expression may be a component of the neurotrophic action of TP in regenerating neurons. This issue was examined in hamster facial motor neurons (FMN) which contain androgen receptors and which have been shown to respond to exogenous steroids in a number of previous studies. Castrated adult male hamsters were subjected to right facial nerve transection and treated with either TP via subcutaneous hormone capsule implants, or left untreated (no hormone replacement). At post-injury/treatment times of 0.25, 2, 4, 7, and 14 d, the brain stem regions were harvested, cryostat sections were collected through the facial motor nucleus, and in situ hybridization was done using a ³³P-labeled GAP-43 cDNA probe. Quantitative analysis of the autoradiograms by computer assisted grain counting revealed that axotomy produced a dramatic increase in GAP-43 mRNA levels in FMN by 2 d post-axotomy and that this increase remained through 14 d post-injury in both the TP-treated and the untreated group. In the nonhormone-treated group, there was a statistically significant dip in GAP-43 mRNA levels in FMN at 7 d post-operative, relative to 4 d post-operative levels. TP-treatment prevented this transient decline in GAP-43 mRNA levels in axotomized FMN.     

Gonadal steroid preservation of central synaptic input to hamster facial motoneurons following peripheral axotomy

In recent work, we have demonstrated that testosterone propionate accelerates recovery from facial nerve injury in the adult male hamster. Central synaptic stripping following peripheral motor neuron damage is a well-established component of the injury response. Gonadal steroids regulate synaptogenesis in the normal nervous system. In this study, we tested the hypothesis that testosterone propionate administration at the time of facial nerve transection alters the synaptic connectivity of injured facial motoneurons. Adult hamsters were subjected to right facial nerve transection at the level of the stylomastoid foramen. Half the animals received subcutaneous implants of testosterone propionate; the other half were sham implanted. At 5 days postoperative, the animals were killed by intracardiac perfusion-fixation, and the control and axotomized facial nuclear groups from the brainstems of nonhormone- and testosterone propionate-treated animals processed for routine transmission electron microscopy. Quantiative analysis of the synaptic ratio (percent somal membrane covered by synaptic profiles) and the average length of axosomatic synapses was accomplished. The results indicate that axotomy alone resulted in an 81% reduction in the synaptic ratio and a 26% decrease in the average synaptic length of axosomatic synapses. Exposure to testosterone propionate from the time of facial nerve transection resulted in only a 48% reduction in the synaptic ratio and a 16% decrease in the average synaptic length of axosomatic synapses following injury. Thus, testosterone propionate significantly attenuated the amount of synaptic stripping that occurred at 5 days postoperative and the decrease in average length of the remaining synapses as well. It is concluded that gonadal steroids modulate central synaptic plasticity following peripheral nerve injury. The results are discussed in light of our recent findings of steroidal effects on the central astrocyctic response to facial nerve injury as well.     

Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron) survival after facial nerve axotomy that is dependent on CD4⁺ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system)-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide) is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4⁺ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type), a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3^−/− mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2^−/− (recombination activating gene-2-deficient) mice adoptively transferred CD4⁺ T-cells isolated from CCR3^−/− mice, but not in CCR3^−/− mice adoptively transferred CD4⁺ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4⁺ T-cell- and CCR3-mediated neuroprotection after FMN injury.     

Androgen regulation of axon growth and neurite extension in motoneurons

Androgens act on the CNS to affect motor function through interaction with a widespread distribution of intracellular androgen receptors (AR). This review highlights our work on androgens and process outgrowth in motoneurons, both in vitro and in vivo. The actions of androgens on motoneurons involve the generation of novel neuronal interactions that are mediated by the induction of androgen-dependent neurite or axonal outgrowth. Here, we summarize the experimental evidence for the androgenic regulation of the extension and regeneration of motoneuron neurites in vitro using cultured immortalized motoneurons, and axons in vivo using the hamster facial nerve crush paradigm. We place particular emphasis on the relevance of these effects to SBMA and peripheral nerve injuries.     

Neurotherapeutic action of testosterone on hamster facial nerve regeneration: temporal window of effects

Neurotherapeutic or neuroprotective effects of gonadal steroids on the injured nervous system have been demonstrated in our laboratory and others. We have previously demonstrated that testosterone propionate (TP) administered systemically at supraphysiological levels accelerates both recovery from facial paralysis and regeneration rates following facial nerve injury in the hamster. Initial temporal studies of steroidal enhancement of functional recovery from facial paralysis established that steroid exposure is necessary during the first postoperative week. Furthermore, accumulated evidence suggests that TP manifests its effects on neuronal regeneration in the immediate postoperative or preregenerative phase by altering the cellular stress response. The purpose of this study was to identify the effective temporal window of TP exposure sufficient to enhance regenerative properties of injured facial motoneurons and functional recovery from facial paralysis induced by facial nerve injury. Adult castrated male hamsters received a right facial nerve crush axotomy at the stylomastoid foramen and were divided into (1) short term, (2) delayed, (3) continuous, and (4) no TP treatment groups. Short term and continuous groups were implanted with 1 subcutaneous (sc) TP capsule each immediately after axotomy, with the capsule removed at 30 min, 2, 4, or 6 h in short-term groups and allowed to remain for the duration of the experiment in the continuous group. In the delayed TP group, 1 sc TP capsule was implanted 6 h after axotomy and allowed to remain for the duration of the experiment. For regeneration rate studies, postoperative times ranged from 4 to 7 days. For the behavioral studies, observations were made for 26 days postaxotomy. The results point to a critical 6-h interval immediately after injury when TP enhances nerve outgrowth distances and augments behavioral recovery.     

Androgen receptor mRNA regulation in adult male and female hamster facial motoneurons: effects of axotomy and exogenous androgens

Testosterone propionate (TP) administration at the time of facial nerve injury in the adult hamster augments the regenerative properties of the injured facial motoneurons (FMN), with the androgen receptor (AR) playing a key role in mediating the actions of TP on facial nerve regeneration. The purpose of the present study was to determine the effects of axotomy on AR mRNA expression in FMN. This was accomplished using in situ hybridization in conjunction with a (35)S-labeled AR riboprobe. Gonadally intact adult male and gonadectomized (gdx) adult female hamsters were subjected to a right facial nerve axotomy, with the left side serving as internal, unoperated control. Half the animals were subcutaneously implanted with a 10-mm TP Silastic capsule, and the other half were sham-implanted. An additional group of nonaxotomized, gonadally intact males was also included. Postaxotomy survival times were 1, 4, and 7 days. At 1 postoperative day 1, there were no effects of axotomy on AR mRNA levels. By postoperative days 4 and 7, axotomy caused a significant decrease in AR mRNA levels in FMN of gonadally intact males, relative to either the contralateral control FMN of the same animals or FMN from the group of gonadally intact males that were not subjected to facial nerve axotomy. There were no significant differences between AR mRNA levels in contralateral control FMN and FMN from the gonadally intact group of nonaxotomized males. TP administration at the time of axotomy had no effect on AR mRNA levels in either the axotomized or contrala(teral control FMN of gonadally intact males, relative to the nonaxotomized, gonadally intact male group. Corroborating our previous work, AR mRNA levels were reduced in the contralateral control FMN of gdx females, relative to the nonaxotomized, gonadally intact male group, with axotomy having no additional effects. The data are discussed in a mechanistic framework suggesting how TP acts to augment facial nerve regeneration.     

The agonistic adrenal: melatonin elicits female aggression via regulation of adrenal androgens

Classic findings have demonstrated an important role for sex steroids as regulators of aggression, but this relationship is lacking within some environmental contexts. In mammals and birds, the adrenal androgen dehydroepiandrosterone (DHEA), a non-gonadal precursor of biologically active steroids, has been linked to aggression. Although females, like males, use aggression when competing for limited resources, the mechanisms underlying female aggression remain understudied. Here, we propose a previously undescribed endocrine mechanism regulating female aggression via direct action of the pineal hormone melatonin on adrenal androgens. We examined this in a solitary hamster species, Phodopus sungorus, in which both sexes are highly territorial across the seasons, and display increased aggression concomitant with decreased serum levels of sex steroids in short ‘winter-like'' days. Short- but not long-day females had increased adrenal DHEA responsiveness co-occurring with morphological changes in the adrenal gland. Further, serum DHEA and total adrenal DHEA content were elevated in short days. Lastly, melatonin increased DHEA and aggression and stimulated DHEA release from cultured adrenals. Collectively, these findings demonstrate that DHEA is a key peripheral regulator of aggression and that melatonin coordinates a ‘seasonal switch’ from gonadal to adrenal regulation of aggression by direct action on the adrenal glands.     

Sex steroids and their actions on the birdsong system

It is probably not surprising to most of us that the endocrine system plays a significant role in controlling the singing behavior of birds. We are familiar with the song of birds as a conspicuous acoustic feature of our environment during the avian breeding season. We often witness song when it is produced by birds (males) that are aggressively establishing and defending territories and that are advertising to available females. Thus, it is easy to imagine that song is likely to be stimulated by gonadal hormones. However, the ways in which gonadal sex steroids influence the various parts of the brain at various stages of the bird's life to influence song are complex and far from being completely understood. In this review, I will highlight some of the significant discoveries that have contributed to our view that the songbird brain is a significant and dynamic target of sex steroids. I will also describe what we have learned about properties of the endocrine system and the brain and how they each contribute to making androgens or estrogens available to particular parts of the songbird brain. Finally, I will describe some new research directions that may help answer some unresolved issues about hormonal effects on the songbird brain. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 619–631, 1997     

Alterations in Glial Fibrillary Acidic Protein (GFAP) mRNA Levels in the Hamster Facial Motor Nucleus: Effects of Axotomy and Testosterone

Testosterone propionate (TP) administered at the time of facial nerve injury in the hamster accelerates the rate of regeneration. In this study, we tested the hypothesis that the mechanism by which TP augments peripheral nerve regeneration involves regulation of glial fibrillary acidic protein (GFAP) mRNA in the facial motor nucleus. Castrated male hamsters were subjected to right facial nerve transection, with half the animals implanted subcutaneously with Silastic capsules containing exogenous TP and the remainder sham implanted. Postoperative survival times were 0.25, 1, 2, 4, 7, and 14 d. Qualitative/quantitative analyses of both film and emulsion autoradiograms were accomplished. Axotomy, with or without TP, resulted in a dramatic increase in GFAP mRNA levels by 1 d postoperative on the axotomized side, relative to controls. GFAP mRNA levels remained elevated throughout all postoperative times in both the nonhormone- and TP-treated animals. Qualitative examination of the film autoradiograms indicated a generalized decrease in the amount of GFAP mRNA in the control and axotomized nuclei of TP-treated animals when compared to the control and axotomized nuclei, respectively, of nonhormone-treated animals. Statistical comparison of the values obtained for both the film and emulsion autoradiograms confirmed this impression. Thus, while the injury-induced increases in GFAP mRNA expression were not blocked by TP, the overall extent of the increase was significantly tempered by steroid treatment. These data suggest that hormonal modulation of the astrocytic response to peripheral nerve injury may be a contributing factor in the ability of steroids to enhance the regenerative capacities of injured motor neurons.     

Gonadal steroids and astroglial plasticity

Summary 1. Recent evidence indicates that astroglia participate in the metabolism of gonadal hormones, in the synthesis of neurosteroids, and in the plastic responses of neurons to gonadal steroids. The role of astroglia on plastic responses of neural tissue to gonadal hormones and neurosteroids is examined in this review.2. Gonadal steroids and neurosteroids promote astroglia plasticity in several areas of the central nervous system, including the hypothalamus, the striatum, and the hippocampus.3. Gonadal steroids and neurosteroids modulate astroglia proliferation and the formation of reactive astroglia after brain injury.4. Astroglia is a source of trophic factors that may mediate effects of gonadal steroids on neural tissue.5. Astroglia is involved in the promotion of synaptic plastic changes by gonadal hormones.6. The effect of gonadal hormones on astroglial plasticity is dependent on specific membrane interactions with neurons and on the expression of the embryonic highly polysialylated isoform of the neural cell adhesion molecule on neuronal membranes.7. In conclusion, coordinated responses of neurons and astroglia appear to be involved in the modulation of neural function and response to injury by gonadal hormones and neurosteroids.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Effects of facial nerve injury on mouse motoneurons lacking the p75 low-affinity neurotrophin receptor

When motoneuron axons in peripheral nerves are injured, the expression of the p75 low-affinity neurotrophin receptor (p75) increases in their cell bodies and axons, as well as in the Schwann cells undergoing Wallerian degeneration in the distal excised nerve segment. To understand the role of p75 in the events following nerve injury, we have examined the survival and regeneration of motoneurons in mice lacking the p75 receptor. In adult p75 (−/−) mice, functional recovery of whiskers movement following a facial nerve crush occurred slightly earlier than in p75 (+/+) mice, and some recovery of function over a 25-day interval following a nerve cut occurred more frequently in p75 (−/−) mice. Motoneuron profile numbers were slightly reduced in p75 (−/−) mice, and there were correspondingly fewer axons in the facial nerve. At 25 days following axotomy, profile survival in the adult p75 (−/−) mice was significantly improved compared to p75 (+/+) mice (mean 85% ± standard error of the mean 3%, n = 11 vs. 67 ± 5%, n = 11 in CD-1 mice and 68.0 ± 4%, n = 6 in balb/c mice), and significantly more regenerating axons were present in the distal facial nerve. After axotomy on postnatal day 1, there was almost total loss of motoneuron profiles in the lateral facial nucleus in p75 (+/+) mice (1.7 ± 0.3% remained, n = 5), while significantly more survived in p75 (−/−) mice (17 ± 2.5%, n = 6) . We conclude that expression of p75 in motoneurons or Schwann cells following facial nerve injury is not necessary for motoneuron survival or prompt regeneration of their axons; rather, p75 may increase their risk of dying. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 1–9, 1998     

Androgen- and estrogen-independent regulation of copulatory behavior following castration in male B6D2F1 mice

Male reproductive behavior is highly dependent upon gonadal steroids. However, between individuals and across species, the role of gonadal steroids in male reproductive behavior is highly variable. In male B6D2F1 hybrid mice, a large proportion (about 30%) of animals demonstrate the persistence of the ejaculatory reflex long after castration. This provides a model to investigate the basis of gonadal steroid-independent male sexual behavior. Here we assessed whether non-gonadal steroids promote mating behavior in castrated mice. Castrated B6D2F1 hybrids that persisted in copulating (persistent copulators) were treated with the androgen receptor blocker, flutamide, and the aromatase enzyme inhibitor, letrozole, for 8 weeks. Other animals were treated with the estrogen receptor blocker, ICI 182,780, via continual intraventricular infusion for 2 weeks. None of these treatments eliminated persistent copulation. A motivational aspect of male sexual behavior, the preference for a receptive female over another male, was also assessed. This preference persisted after long-term castration in persistent copulators, and administration of ICI 182,780 did not influence partner preference. To assess the possibility of elevated sensitivity to sex steroids in brains of persistent copulators, we measured mRNA levels for genes that code for the estrogen receptor-α, androgen receptor, and aromatase enzyme in the medial preoptic area and bed nucleus of the stria terminalis. No differences in mRNA of these genes were noted in brains of persistent versus non-persistent copulators. Taken together our results suggest that non-gonadal androgens and estrogens do not maintain copulatory behavior in B6D2F1 mice which display copulatory behavior after castration.