Androgenic Regulation of Androgen Receptor Immunoreactivityin Motoneurons of the Spinal Nucleus of the Bulbocavernosusof Male Rats

Androgenic regulation of androgen receptor (AR) immunoreactivity was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by immunohistochemistry using the polyclonal antibody, PG21. In intact controls, intense AR immunoreactivity was confined to the cell nucleus, but not in the nucleolus of SNB motoneurons, whereas cytoplasmic AR immunoreactivity was weak. Androgen withdrawal significantly reduced both the intensity of AR immunoreactivity in the nuclei and number of AR immunoreactive nuclei of the SNB motoneurons within 1 day of castration. AR immunostaining in the nucleus and cytoplasm was completely eliminated 5 or 10 days following castration. These changes were prevented by replacement of testosterone propionate (TP). The number of AR immunoreactive nuclei recovered to about half of the control levels within 20  min or 1  hr of TP administration to males 5 days after castration, although the intensity of AR immunoreactivity was almost the same as that of males 1 day following castration. Both the intensity of nuclear and cytoplasmic AR immunoreactivity and number of AR immunoreactive nuclei recovered to the control levels 2 or 6  hr after TP injection. These results suggest that androgen causes a significant up-regulation in AR expression of SNB motoneurons.     

Androgenic regulation of expression of androgen receptor protein in the perineal motoneurons of aged male rats.

Androgen might regulate expression of androgen receptors (AR) in AR-containing motoneurons in young animals. In the present study, it was examined whether expression of AR was also regulated by androgen in aged animals. Twelve male rats were castrated at 26 months of age. Five days following castration, the animals were treated with testosterone propionate (TP; six males) or vehicle (six males) and killed 2 hours later. Six sham-castrated rats served as controls. AR immunoreactivity was examined in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in these animals by immunohistochemistry using the polyclonal antibody PG21. In control animals, slightly intense AR immunoreactivity was confined to the nuclei of the motoneurons. AR immunoreactivity was completely eliminated in the motoneurons of castrated rats. In castrated, aged animals treated with TP, the intensity of AR immunoreactivity in the nuclei of SNB motoneurons was increased. Plasma levels of testosterone in castrated, aged animals 2 hours following treatment with TP were significantly greater than those in controls. These results suggest that expression of AR in motoneurons of the SNB in aged male rats is up-regulated in response to androgen and that androgen may be, at least in part, involved in the process of aging of the SNB in male rats.     

Synaptic changes in the perineal motoneurons of aged male rats

Cholera toxin-horseradish peroxidase (CT-HRP) was injected into the bulbocavernosus muscles of young (2 months of age) and old (19–20 months of age) male rats, and animals were killed 2 days later. The spinal cords containing the spinal nucleus of the bulbocavernosus (SNB) were dissected, processed with a modified tetramethylbenzidine method for visualization of retrogradely transported CT-HRP, and examined ultrastructurally. Neuronal structures apposing the membranes of 120 CT-HRP–labeled SNB motoneurons were analyzed by measuring the percentage of somatic membranes covered by synaptic contacts, synaptoid contacts, and neuron-neuron contacts. Most of the neuronal structures in the young and old SNB motoneurons consisted of synaptic contacts. The mean percentage of somatic membranes covered by synapses in old rats was significantly smaller than that in young ones. Size and number of synaptic contacts per unit length of somatic membranes in old animals were also significantly reduced. Plasma levels of testosterone in old males were significantly smaller than those in young ones. These age-related changes in synaptic inputs to SNB motoneurons and plasma levels of androgen seem to correlate with aging of the SNB system. J. Comp. Neurol. 400:103–109, 1998. © 1998 Wiley-Liss, Inc.     

Axotomy transiently down-regulates androgen receptors in motoneurons of the spinal nucleus of the bulbocavernosus

Testosterone is an important trophic factor for motoneurons in the spinal nucleus of the bulbocavernosus (SNB), and SNB motoneurons are more responsive to testosterone than are other motoneurons. Axonal injury during early postnatal life prevents the normal development of steroid-sensitivity by adult SNB motoneurons. Axonal injury also causes changes in the expression by motoneurons of a wide range of proteins, including the up-regulation of trophic factor receptors. We have used a polyclonal antibody (PG-21; G.S. Prins) to study the expression of androgen receptors in SNB motoneurons after axonal injury. PG-21 labeled motoneuronal nuclei in the lower lumbar spinal cord of rats in a pattern that matched autoradiograpic reports of androgen accumulation in this region of the nervous system. A population of numerous, small cells located dorsal to the central canal also showed evidence of androgen receptor expression. Cutting the axons of SNB motoneurons in adulthood or in development caused a decrease in androgen receptor immunoreactivity in SNB motoneurons. This is the first report that a trophic factor receptor in motoneurons is down-regulated after axonal injury, and is interesting in light of reports that testosterone treatment can facilitate motoneuronal regeneration after nerve cut. Androgen receptor levels subsequently returned to normal, regardless of the age at axotomy, providing no evidence for a lasting effect of developmental axotomy on androgen receptor levels in SNB motoneurons. Thus, axotomy-induced down-regulation of androgen receptors does not underlie the inability of SNB motoneurons to respond to androgen treatment several months after pudendal nerve cut in development.     

The role of target muscles in the expression of calcitonin gene-related peptide mRNA in the spinal nucleus of the bulbocavernosus.

Previous studies have shown that calcitonin gene-related peptide (CGRP) mRNA steady-state levels and CGRP immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) are inversely related to the gonadal steroid environment in male rats. Since both the SNB motoneurons and their target muscles, the bulbocavernosus and the levator ani, are steroid sensitive, gonadal steroids may act at either site to regulate CGRP expression. In the present study, we tested the hypothesis that gonadal steroids influence CGRP expression in SNB motoneurons through their effects on the bulbocavernosus and levator ani muscles. We determined the levels of alpha-CGRP mRNA and immunoreactive CGRP in SNB motoneurons of adult male rats following injection of the bulbocavernosus with muscle extracts from bulbocavernosus/levator ani of castrated rats, paralysis of the bulbocavernosus or pudendal nerve cuts. Following injection of the bulbocavernosus/levator ani with extracts from castrated rats, the level of CGRP expression and the number of SNB motoneurons with alpha-CGRP message were increased. These studies suggest that the bulbocavernosus/levator ani muscles from castrated rats produce a factor that increases levels of CGRP. Injections of extract prepared from the bulbocavernosus and levator ani muscles of gonadally intact rats did not change the expression of alpha-CGRP mRNA in the SNB. Paralysis of the bulbocavernosus/levator ani with a local anesthetic increased the number of SNB motoneurons expressing alpha-CGRP mRNA and CGRP immunoreactive neurons. To determine whether nerve damage accounted for the observed effects following injection of anesthetic, the pudendal nerves were cut bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of androgen receptor and p75(NTR) mRNAs and peptides in the lumbar spinal cord of the gerbil

Development of sex differences in the spinal cord appears to be largely under the control of androgen and although neurotrophins may also have a role. Spinal cords of male and female neonatal gerbils (postnatal days 1, 5, 7, 10, 23) and adult gerbils (postnatal day 150) were examined to determine the relative temporal expression of androgen receptor (AR) and the low-affinity neurotrophin receptor (p75) mRNAs within the spinal nucleus of the bulbocavernosus (SNB) and dorsolateral nucleus (DLN). Furthermore, prepubertal male gerbils were placed into one of six gonadal hormone treatment groups at weaning: Either sham castrate, castrated with gonadal hormone replacement, or castrated without gonadal hormone replacement. Ten weeks later gerbils were aldehyde-perfused, spinal cords removed and processed for presence of AR and p75 immunoreactivity (ir) in motoneurons of the SNB and DLN. During neonatal development, there were significant increases in androgen receptor mRNA within the SNB and DLN. In the SNB, the increase in androgen receptor mRNA preceded the increase in p75 mRNA. Peripubertally, significantly more SNB than DLN motoneurons contained AR- and p75-ir. These data demonstrate that AR expression occurs along the same developmental time frame as the development of the SNB and DLN and the organizational effects of androgens on their development continues through puberty in the male gerbil.     

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7–P14) received 200 μg of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs. J. Comp. Neurol. 379:88-98, 1997. © 1997 Wiley-Liss, Inc.     

BDNF regulation of androgen receptor expression in axotomized SNB motoneurons of adult male rats

Brain-derived neurotrophic factor (BDNF) prevents the axotomy-induced loss of androgen receptor-like immunoreactivity (AR-LI) in the spinal nucleus of the bulbocavernosus (SNB) motoneurons of adult male rats. In this report, we investigated the dose-response effect of BDNF on androgen receptor expression in axotomized SNB motoneurons, and examined whether delayed application of BDNF to the cut SNB axons can completely reverse the axotomy-induced loss of androgen receptor expression. We also used autoradiography to test whether axotomy decreases the ability of SNB motoneurons to accumulate androgens. SNB motoneurons were axotomized bilaterally and BDNF or PBS was applied to the proximal ends of the axons. The percentage of SNB motoneurons expressing medium or high AR-LI was the major measure of androgen receptor expression. AR-LI was significantly higher on the BDNF-treated side than on the contralateral side treated with phosphate-buffered saline (PBS) for all three doses of BDNF (1.45, 2.9, and 5.8 mg/ml) and was higher than in rats treated bilaterally with PBS. Moreover, AR-LI at the highest dose of BDNF was not different from that in intact SNB motoneurons. Delayed application of BDNF to the axotomized SNB motoneurons restored the AR-LI to the intact level. The AR-LI decreased by axotomy started to increase significantly 4 days after BDNF application and returned to the intact level by 10 days. Furthermore, axotomy significantly decreased the percentage of SNB motoneurons to accumulate tritiated testosterone or its metabolites. In conclusion, our data demonstrate that BDNF completely prevents and reverses the axotomy-induced loss of AR-LI. Moreover, decrease of AR-LI by axotomy reflects the decrease in the ability of SNB motoneurons to accumulate androgens.     

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.     

Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: III. Differential effects of the androgen dihydrotestosterone

The spinal cord of the rat contains two sexually dimorphic nuclei: the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN). These nuclei and the perineal muscles they innervate are present in males but reduced or absent in females. The sex difference in motoneuron number in these nuclei is due to an androgen-regulated motoneuron death. Developing females treated with the androgen testosterone propionate (TP) have a fully masculine number of SNB and DLN motoneurons and retain the perineal muscles they would normally have lost. Paradoxically, females treated prenatally with the androgen dihydrotestosterone propionate (DHTP) also retain the perineal musculature but as adults lack the SNB motoneurons which would normally innervate them. The SNB target muscles retained by DHTP females are anomalously innervated by motoneurons in the DLN. Counts of motoneurons and degenerating cells in the developing SNB of DHTP-treated females showed that their feminine number is the result of a failure of DHTP to prevent the death of SNB motoneurons. Furthermore, the peak number of SNB motoneurons was below that of normal females, suggesting that DHTP treatment may also have inhibited motoneuronal migration. However, DHTP treatment fully masculinized both motoneuron number and degenerating cell counts in the DLN of these females, and it is this masculinized DLN that gives rise to the anomalous projection. Taken together, these results suggest that the effects of different androgens during development are specific and complex, involving the regulation of motoneuron death, migration, and specification of peripheral projections.     

Motoneuron morphology in the dorsolateral nucleus of the rat spinal cord: Normal development and androgenic regulation

The rat lumbar spinal cord contains two sexually dimorphic motor nuclei, the spinal nucleus of the bulbocavernosus (SNB), and the dorsolateral nucleus (DLN). These motor nuclei innervate anatomically distinct perineal muscles that are involved in functionally distinct copulatory reflexes. The motoneurons in the SNB and DLN have different dendritic morphologies. The dendrites of motoneurons in the medially positioned SNB have a radial, overlapping arrangement, whereas the dendrites of the laterally positioned DLN have a bipolar and strictly unilateral organization. During development, SNB motoneuron dendrites grow exuberantly and then retract to their mature lengths. In this experiment we determined whether the adult difference in SNB and DLN motoneuron morphology was reflected in different patterns of dendritic growth during normal development. Furthermore, the development of both these nuclei is under androgenic control. In the absence of androgens, SNB dendrites fail to grow; testosterone replacement supports normal dendritic growth. Thus, we also examined the development of DLN dendrites for similar evidence of androgenic regulation. By using cholera toxin-horseradish peroxidase (BHRP) to label motoneurons retrogradely, we measured the morphology of DLN motoneurons in normal males, and in castrates treated with testosterone or oil/blank implants at postnatal day (P) 7, P28, P49, and P70. Our results demonstrate that in contrast to the biphasic pattern of dendritic development in the SNB, dendritic growth in the DLN was monotonic; the dendritic length of motoneurons increased more than 500% between P7 and P70. However, as in the SNB, development of DLN motoneuron morphology is androgen-dependent. In castrates treated with oil/blank implants, DLN somal and dendritic growth were greatly attenuated compared to those of normal or testosterone-treated males. Thus, while androgens are clearly necessary for the growth of motoneurons in both the SNB and DLN, their different developmental patterns suggest that other factors must be involved in regulating this growth. © 1993 Wiley-Liss, Inc.     

Decreased Expression of TrkB and TrkC mRNAs in Spinal Motoneurons of Aged Rats

Several studies have indicated that a decrease in availability and/or responsiveness to neurotrophin(s) may be of importance in ageing and disease-related neurodegeneration. Using in situ hybridization we have studied the mRNA expression of the full-length neurotrophin receptors TrkB and TrkC in spinal cord motoneurons of aged rats (30 months old) with symptoms of hindlimb incapacity and in young adult rats (2–3 months old). The labelling intensity for TrkB of the individual cell profile was decreased by 25% ( P < 0.001) in both the cervical and lumbar motor nuclei of aged rats. In thoracic motoneurons of aged and young adult rats the difference in expression of TrkB mRNA was smaller (down by 15%; P < 0.05). The labelling for TrkC mRNA was much weaker than that for TrkB in both aged and young adult rats, but TrkC mRNA expression also seemed to decrease. Comparison of the different motor nuclei along the spinal cord axis revealed that the motoneurons of the L6/S1 nuclei showed the strongest hybridization signal for the two Trk receptors in both aged and young adult rats. The possibility that a decrease in TrkB mRNA may contribute to age-related motor disturbances is discussed.     

Motoneuron development after deafferentation. I. Dorsal rhizotomy does not alter growth in the spinal nucleus of the bulbocavernosus (SNB)

The spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) are sexually dimorphic motor nuclei in the rat lumbar spinal cord. During postnatal development, SNB and DLN motoneurons grow substantially in measures of soma size, dendritic length, and radial dendritic extent. SNB motoneurons exhibit a biphasic pattern of dendritic growth, where there is an initial period of exuberant growth followed by a period of retraction to mature lengths by 7 weeks. In this experiment, we examined whether primary afferent input to the SNB nucleus was necessary for the normal postnatal growth of SNB motoneurons. We partially deafferented the SNB via unilateral dorsal rhizotomy of lumbosacral dorsal roots in male rats at 1 week of age. Using cholera toxin horseradish peroxidase (BHRP) to visualize SNB motoneurons, we examined SNB motoneuron morphology at 4 and 7 weeks of age. SNB motoneurons in rhizotomized males developed normally; measures of dendritic length in rhizotomized males were typically exuberant at 4 weeks of age, and declined significantly to mature lengths by 7 weeks of age. In addition, dorsal rhizotomy did not alter the development of SNB motoneuron soma size or radial dendritic extent. These results are discussed in reference to sensorimotor connections in the SNB, the extent of the deafferentation, and dendrodendritic interactions.     

Direct androgenic regulation of calcitonin gene-related peptide expression in motoneurons of rats with mosaic androgen insensitivity.

The spinal nucleus of the bulbocavernosus (SNB) and its target muscles, bulbocavernosus and levator ani (BC/LA), form a sexually dimorphic neuromuscular circuit whose development and maintenance are androgen-dependent. The mechanisms whereby androgen regulates gene expression in the SNB of adult rats are largely unknown, although a retrograde influence from the BC/LA muscles has been suggested to underlie the suppression of calcitonin gene-related peptide (CGRP) expression observed in SNB motoneurons after systemic androgen treatment. A mosaic paradigm was used to determine the site of action of androgen in the regulation of CGRP expression in SNB motoneurons. As a consequence of random X chromosome inactivation, androgenized female rats heterozygous for the tfm androgen receptor (AR) mutation (XwtXtfm-mosaics) express a mosaic of androgen-sensitive and androgen-insensitive motoneurons in the SNB, whereas the BC/LA target musculature appears to be uniformly sensitive to androgens. In adult mosaics, testosterone administration resulted in a reduction in the proportion of androgen-sensitive cells expressing CGRP, whereas no such reduction was observed in the androgen-insensitive population, indicating that neuronal AR plays an essential role in the neuromuscular regulation of CGRP expression in these motoneurons. This provides the first in vivo demonstration of AR regulation of gene expression unambiguously localized to a neuronal population.     

N-cadherin expression in motoneurons is directly regulated by androgens: a genetic mosaic analysis in rats

We have recently reported that systemic androgens regulate adult N-cadherin (N-cad) expression in spinal motoneurons. However, the mechanism through which androgen mediates this effect remains undetermined. Androgen may act directly on motoneurons to regulate N-cad expression, or indirectly, via effects on androgen-sensitive afferent or efferent structures. Here, we describe a genetic mosaic investigation of this site-of-action indeterminacy. Following developmental random X chromosome inactivation, androgenized female rats heterozygous for the tfm androgen receptor mutation (X(WT)X(tfm)) are phenotypic mosaics of androgen-sensitive wild-type (WT) and androgen-insensitive (tfm) motoneurons. We compared steroid effects on WT and tfm cells in two sexually-dimorphic motoneuron pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), as well as a less steroid responsive motoneuron pool, the sexually monomorphic retrodorsolateral nucleus (RDLN). Independent of steroid treatment, a greater proportion of wild-type cells were N-cad immunoreactive (IR) in the DLN and RDLN. Following testosterone treatment, increased N-cad expression was observed in both cell types in the DLN, but in the SNB only the androgen-competent WT cells increased N-cad expression. Testosterone treatment did not significantly alter N-cad expression in the mosaic RDLN. The results indicate both cell autonomous and cell non-autonomous androgenic regulation of N-cad expression in spinal motoneurons.