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.     

Motoneuron development after deafferentation: II. Dorsal rhizotomy does doe block estrogen-supported growth in the dorsolateral nucleus (DLN)

The lumbar spinal cord of the rat contains two sexually dimorphic motor nuclei, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN). Postnatally, SNB and DLN motoneurons grow substantially and reach their adult morphology by 7 weeks of age. The masculinization of SNB and DLN motoneuron dendrites depends upon steroid hormones. After early castration, the growth of SNB and DLN dendrites is markedly attenuated, but testosterone replacement restores this growth. In the SNB, initial dendritic growth is also supported in castrates treated with estrogen. By using castration and hormone replacement techniques, we examined the development of DLN motoneuron morphology in estrogen-treated castrated rats to determine if estrogen also supports the growth of DLN motoneurons. In addition, given that dorsal root ganglia may be a site of estrogen action, we tested the hypothesis that estrogen acts at primary afferents to support DLN dendritic growth. Thus, we attempted to block the potential trophic effect of estrogen by performing unilateral dorsal rhizotomies in estrogen-treated castrates. DLN motoneuron morphology was analyzed at 4 and 7 weeks of age by using cholera toxin horseradish peroxidase (BHRP) histochemistry. As found for SNB motoneurons, estrogen treatment transiently supported development. DLN motoneurons in estrogen-treated castrates developed normally through 4 weeks of age, but by 7 weeks, DLN motoneuron morphology in estrogen-treated castrates was no longer different from that in oil-treated castrates. Moreover, deafferentation via unilateral dorsal rhizotomy did not inhibit estrogen's ability to masculinize the early development of DLN motoneurons. Thus, the trophic effect of estrogen did not appear to act via the dorsal root ganglia to support the early postnatal development of DLN motoneurons.     

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.     

Neuroprotective effects of testosterone on the morphology and function of somatic motoneurons following the death of neighboring motoneurons

Motoneuron loss is a significant medical problem, capable of causing severe movement disorders or even death. We have previously shown that partial depletion of motoneurons from sexually dimorphic, highly androgen‐sensitive spinal motor populations induces dendritic atrophy in remaining motoneurons, and this atrophy is attenuated by treatment with testosterone. To test whether testosterone has similar effects in more typical motoneurons, we examined potential neuroprotective effects in motoneurons innervating muscles of the quadriceps. Motoneurons innervating the vastus medialis muscle were selectively killed by intramuscular injection of cholera toxin‐conjugated saporin. Simultaneously, some saporin‐injected rats were given implants containing testosterone 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 males, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons, and this atrophy was attenuated by testosterone treatment. To examine the functional consequences of the induced dendritic atrophy, and its attenuation with testosterone treatment, the activation of remaining quadriceps motoneurons was assessed using peripheral nerve recording. Partial motoneuron depletion resulted in decreased amplitudes of motor nerve activity, and these changes were attenuated by treatment with testosterone, providing a functional correlate to the neuroprotective effects of testosterone treatment on quadriceps motoneuron morphology. Together these findings suggest that testosterone has neuroprotective effects on morphology and function in both highly androgen‐sensitive as well as more typical motoneuron populations, further supporting a role for testosterone as a neurotherapeutic agent in the injured nervous system. J. Comp. Neurol. 512:359–372, 2009. © 2008 Wiley‐Liss, Inc.     

Morphology of rat spinal motoneurons with normal and hormonally altered specificity

Potential determinants of motoneuronal morphology were examined by using a sexually dimorphic, steroid-sensitive neuromuscular system in the rat spinal cord. In males, the spinal nucleus of the bulbocavernosus (SNB) innervates the perineal muscles bulbocavernosus (BC) and levator ani (LA), and the dorsolateral nucleus (DLN) innervates the ischiocavernosus muscle (IC). Adult females normally lack these motoneurons and the peripheral targets. Prenatal exposure of females to the androgen dihydrotestosterone propionate (DHTP) partially masculinizes this neuromuscular system and alters moto-neuron-to-muscle specificity, resulting in retained SNB target muscles anomalously innervated by motoneurons in the DLN. Because the morphology of SNB and DLN motoneurons normally differs significantly, the influence of spinal cord location and peripheral target on motoneuron morphology can be directly compared. Injection of cholera toxin conjugated to horseradish peroxidase (CTHRP) into the LA of DHTP-treated females labeled motoneurons predominantly in the SNB. These (SNB-LA) motoneurons in DHTP females were identical in all morphological measures to those of normal males. CTHRP injection into the BC of DHTP females labeled motoneurons in both the SNB and the DLN. SNB-BC motoneurons in DHTP females resembled those of normal males in process number and orientation, but were significantly smaller in dendritic length per motoneuron and in soma size. The DLN motoneurons anomalously projecting to the BC in DHTP females differed significantly from SNB-BC motoneurons in soma size and number and orientation of primary processes. However, these motoneurons were identical in all respects to DLN-IC motoneurons in DHTP females; DLN-IC motoneurons were similar to those of normal males in the orientation of their dendritic arbor, but were significantly smaller in dendritic length, soma size, and number of primary processes. These comparisons make it clear that DHTP selectively affects motoneuronal specificity and morphology in specific motoneuron classes. Further, motoneuronal morphology in the SNB/DLN system appears to be influenced more by spinal cord location than by peripheral target.     

Steroid regulation of calcitonin gene-related peptide mRNA expression in motoneurons of the spinal nucleus of the bulbocavernosus

Motoneurons express calcitonin gene-related peptide (CGRP). Previous studies have shown that CGRP immunoreactivity is regulated by testosterone in the androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In this research the effect of plasma levels of testosterone on the expression of alpha CGRP mRNA in the SNB motoneurons of adult male rats was studied with in situ hybridization. The number of motoneurons expressing alpha CGRP mRNA and the level of alpha CGRP mRNA expression was significantly higher in the SNB of castrated male rats than in the SNB of gonadally intact rats. Using a 5x background labeling criterion in castrated rats 88.1 +/- 4.5% while in intact rats 75.3 +/- 6.4% of SNB motoneurons expressed alpha CGRP mRNA. Testosterone replacement at the time of castration prevented the effect of castration on the expression of alpha CGRP mRNA in SNB motoneurons. In castrated rats, the increase in the number of SNB cells expressing CGRP was the result of increased steady state levels of alpha CGRP mRNA in all SNB neurons.     

Androgen regulation of dendritic growth and retraction in the development of a sexually dimorphic spinal nucleus

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurons whose development and maintenance are under androgenic control. Exposure to androgens early in development permanently alters SNB motoneuron number and soma size; in adulthood, androgens regulate dendritic and synaptic architecture. The present set of experiments investigates the influence of androgens on the development of SNB dendritic morphology. In normal males, SNB dendritic growth is biphasic, reaching exuberant lengths by the fourth postnatal week and then retracting to adult lengths by 7 weeks of age. This dendritic growth is androgen dependent--males castrated on postnatal day (P) 7 and given daily injections of testosterone propionate (TP) had exuberant dendritic lengths similar to those of normal males; dendritic length in oil-treated males remained at P7 levels. The early exuberant dendritic length was retained in TP-treated males through P49. The retraction of SNB dendrites after P28 is also influenced by androgens. Males castrated at P28 and given testosterone implants retained exuberant dendritic length at P49; blank-implanted males had significantly shorter dendritic lengths by P70. These results suggest that androgens are necessary for the early exuberant growth of SNB dendrites. Furthermore, the subsequent retraction of SNB dendrites may be halted when testosterone titers reach a critical level during puberty, stabilizing their adult length.     

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.     

Increase in motoneurons in the spinal nucleus of the bulbocavernosus of prepubertally castrated male Mongolian gerbils following delayed treatment with testosterone

Sexual dimorphism in the spinal nucleus of the bulbocavernosus (SNB) of the Mongolian gerbil is achieved by two periods of postnatal increase, one in the first month after birth and one at puberty. The pubertal increase in motoneuron number is of particular interest because it occurs in a nearly adult animal. The purpose of this research was threefold. The first was to determine the response of the SNB in prepubertally castrated male gerbils receiving delayed hormone replacement as adults. Testosterone propionate (TP) treatment resulted in numbers of SNB motoneurons comparable to those seen in intact males, whereas androgen metabolites were less effective. The second purpose was to determine the latency of motoneurons to appear in response to TP. New SNB motoneurons appeared within 2 days of delayed TP replacement in prepubertally castrated males, and 16 days of treatment did not further increase SNB motoneuron numbers. The response of the motoneurons to TP appeared more rapid than the response of the bulbocavernosus (BC) muscle, scent gland, and seminal vesicles. The third purpose was to determine whether the new cells were connected to a target muscle. After 16 days of TP treatment, more motoneurons were labeled in the SNB following injection of a retrograde tract tracer into the BC muscle compared with the number seen in control animals. Thus, new motoneurons appeared in the SNB of prepubertally castrated male Mongolian gerbils within 2 days of the start of delayed TP treatment and were connected to a target within 16 days of TP treatment.     

Development of a sexually dimorphic neuromuscular system in male rats after spinal transection: morphologic changes and implications for estrogen sites of action

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). In male rats, SNB motoneurons exhibit a biphasic pattern of dendritic growth, having an initial period of exuberant growth followed by a period of retraction to mature lengths by 7 weeks of age. This growth is steroid dependent: dendrites fail to grow after castration, but growth is supported in castrates treated with estradiol. In this experiment, we examined whether supraspinal afferent input by means of descending spinal tracts to the SNB was involved in the normal postnatal development of SNB motoneurons, and whether the effect of estradiol on SNB dendritic growth could be explained by an indirect action of estradiol on supraspinal afferents. Motoneuron morphology was assessed in normal males, early- or late-postnatally transected males, castrated males left untreated or treated with estradiol, and transected castrates treated with estradiol. SNB motoneurons were retrogradely labeled with cholera toxin-horseradish peroxidase during both the growth and retraction phases of dendritic development and reconstructed in three dimensions. The removal of supraspinal afferents resulted in extremely local effects within the developing SNB arbor, as well as transient alterations in somal growth. Furthermore, spinal transection did not block the trophic effect of estradiol on supporting SNB dendritic growth, indicating that estrogens do not act by means of supraspinal input to support SNB motoneuron development.     

Androgen regulates synaptic input to motoneurons of the adult rat spinal cord

Adult male rats (Sprague-Dawley) were castrated and implanted subcutaneously with Silastic capsules containing testosterone or nothing. Sham-castrated males served as controls. Four weeks following castration, cholera toxin-horseradish peroxidase (CT-HRP) was injected bilaterally into the bulbocavernosus muscles and animals were sacrificed 2 d later. The spinal cords containing the spinal nucleus of the bulbocavernosus (SNB) were dissected, processed with a modified tetramethylbenzidine (TMB) method for visualization of retrogradely transported CT-HRP, and examined at the ultrastructural level. Neuronal structures apposing the membranes of 150 TMB-labeled SNB neurons were analyzed by measuring the percentage of somatic and proximal dendritic membranes covered by synaptic contacts, synaptoid contacts, and neuron-neuron contacts. Most of the neuronal structures in the control and experimental SNB motoneurons consisted of synaptic contacts. The mean percentage of somatic and proximal dendritic membranes covered by synapses 4 weeks after castration was reduced to approximately 30% of those in control animals. However, treatment with testosterone for 4 weeks after castration prevented this decline. Castration and testosterone treatment also influenced the size and number of synaptic contacts per unit length of somatic and proximal dendritic membranes, and the incidence of neuron-neuron contacts and double synapses onto SNB motoneurons. These results indicate that androgen is critical for maintaining the organization of synaptic inputs to these spinal motoneurons in adult male rats.     

Timing and duration of dihydrotestosterone treatment affect the development of motoneuron number and morphology in a sexually dimorphic rat spinal nucleus.

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. SNB motoneurons and their perineal target muscles are present in adult males, but reduced or absent in adult females. This dimorphism is due to the presence of androgens during development. Perinatal treatment of females with testosterone (T), or a combination of dihydrotestosterone (DHT) and estrogen (E+D females) from embryonic (E) day 16 through postnatal (P) day 5, results in a masculine number of SNB motoneurons and the retention of the target muscles. Perinatal treatment with estrogen alone does not masculinize the SNB; prenatal treatment with DHT alone from E17-E22 results in a feminine number of SNB motoneurons and a significantly altered motoneuron morphology and connectivity. To determine if masculinization of the SNB involves the interaction of estrogen and DHT or results from a longer exposure to DHT alone, the number, morphology, and connectivity of SNB motoneurons in females treated with DHT both pre- and post-natally (from E16-P5) were examined. At E22, DHTP (E16-P5) females have SNB motoneuron numbers identical to E+D and normal females, but far fewer than normal males, thus indicating that T is essential for prenatal masculinization. After E22, SNB motoneuron number declines precipitously in normal females but remains stable in DHTP (E16-P5) females and E+D females, which do not differ from normal males at P10. These results demonstrate that DHT can completely masculinize SNB motoneuron number without any synergistic actions with estrogen, and suggest that the development of SNB motoneuron number is strictly an androgen-mediated event. In adulthood, horseradish peroxidase histochemistry reveals that the connectivity, dendritic length, and soma size of SNB motoneurons in DHTP (E16-P5) females are identical to those of normal males but differ significantly from those of DHTP (E17-E22) females. These data suggest that the altered connectivity in DHTP (E17-E22) females is not simply a hormone-specific effect, but the result of a truncated hormone exposure. Thus, DHT can fully masculinize SNB morphology and connectivity if given during the appropriate period of development. It is suggested that while T may be required to masculinize the SNB prenatally, DHT may be involved in masculinizing postnatal aspects of SNB development.     

Maternal licking influences dendritic development of motoneurons in a sexually dimorphic neuromuscular system

Maternal licking of pups' perineal regions affects the development of the spinal nucleus of the bulbocavernosus (SNB), a sexually dimorphic motor nucleus in the lumbar spinal cord that controls penile reflexes involved with copulation. Maternal licking influences SNB motoneuron number, with reductions in licking resulting in fewer motoneurons. Reduced maternal licking also has functional consequences in adulthood, resulting in increased latency to ejaculation and postejaculatory intromission and longer interintromission intervals. In this experiment, we assessed the potential effect of maternal licking on the development of SNB dendritic morphology. To reduce maternal licking, dams were treated with intranasal application of zinc sulfate during the first two postnatal weeks, which interferes with their ability to detect the pup odors that drive the licking behavior. At either postnatal day (P) 28 (when SNB dendritic length is normally maximal) or P49 (when SNB dendritic morphology is normally mature), SNB motoneurons were retrogradely labeled with cholera toxin-conjugated HRP, and dendritic arbor was reconstructed in three dimensions. At P28, the dendritic arbor of reduced maternal licking pups was not different from controls; however at P49, reduced licking pups showed a 23% reduction in dendritic arbor in the SNB, an effect that was especially pronounced in the rostral end of the nucleus, where reductions reached 48%. These results suggest that reductions in perineal stimulation provided by maternal licking could affect adult male copulatory behavior via alterations in SNB motoneuron morphology, and thus support maternal licking as an important factor in normal neural and behavioral development.     

Motoneurons dorsolateral to the central canal innervate perineal muscles in the mongolian gerbil

The Mongolian gerbil provides a model in which sexually dimorphic areas in the hypothalamus are correlated with sociosexual behaviors such as scent marking and male copulatory behavior. To extend this model, investigations were conducted to determine whether sexually dimorphic areas existed in the spinal cord that could be relevant to male sexual behavior. The focus of these investigations was the perineal muscles associated with the penis. Therefore, this research identified the spinal motoneurons that innervate the bulbocavernosus, levator ani, anal sphincter, and ischiocavernosus muscles of Mongolian gerbils. The motoneuron pool that innervates the bulbocavernosus, levator ani, and anal sphincter was designated the spinal nucleus of the bulbocavernosus (SNB), as for other species of rodents. The motoneuron pool innervating the ischiocavernosus was identified as the dorsolateral nucleus, again, to be consistent with the designation for other rodents. The motoneurons of the gerbil SNB were distributed dorsolateral to the central canal in the lumbosacral transition zone of the spinal column. These motoneurons are located in the region classically defined as area X of the spinal cord. The number of SNB motoneurons was sexually dimorphic, with male gerbils having about five times as many SNB motoneurons as do female gerbils. The size of SNB motoneurons was also sexually dimorphic. The SNB motoneurons of males were 1. 5 times larger than the SNB motoneurons of females. The effects of adult castration on the male SNB were also studied. After castration, the size, but not the number, of SNB motoneurons in males was significantly decreased. This decrease was prevented by testosterone treatment. The percentage of calcitonin gene-related peptide (CGRP)-immunoreactive SNB motoneurons was also affected by adult castration. The percentage of CGRP-immunoreactive motoneurons was significantly decreased after adult castration. Again, this decrease was reversed by testosterone treatment. These findings suggest that the SNB of gerbils is sexually dimorphic and is sensitive to circulating levels of gonadal steroids. The unique placement of the SNB motoneurons suggests that an alternative laminar organizational scheme may be necessary for Mongolian gerbil. © 1995 Wiley-Liss, Inc.     

Androgen stimulates neuronal plasticity in the perineal motoneurons of aged male rats

Ten aged male rats (24 months of age) were castrated and implanted subcutaneously with Silastic capsules containing testosterone (T)(5 males) or nothing (5 males). Five sham-castrated males (25 months of age) served as controls. Four weeks after castration, cholera toxin-horseradish peroxidase (CT-HRP) was injected into the bulbocavernosus muscles 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 150 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 control and experimental SNB motoneurons consisted of synaptic contacts. The mean percentage of somatic membranes covered by synapses in castrated, aged males treated with T was significantly greater than that in control or castrated animals. Size and number of synaptic contacts per unit length of somatic membranes in castrated, aged males treated with T were also significantly greater than those in control or castrated animals. Plasma levels of T in castrated, aged males treated with T were significantly greater than that in controls. These results suggest that the SNB motoneurons of aged male rats retain a considerable synaptic plasticity in response to androgen, and that androgen may be, at least in part, involved in the process of aging of the SNB system in male rats.     

Long-term castration effects motoneuron size but not number in the spinal nucleus of the bulbocavernosus in the adult male Mongolian gerbil

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurons in Rexed's Lamina X of the lumbosacral spinal cord of the Mongolian gerbil. The SNB innervates the perineal musculature, the bulbocavernosus (BC), levator ani (LA), and external anal sphincter (EAS). Recent studies demonstrated a peripubertal component to the masculinization of the gerbil SNB with an apparent increase in both motoneuron size and number after puberty as measured with a Nissl stain. However, these studies could not determine if the apparent change in motoneuron number were due to the long-term castration involved in the methodology or due to a loss of motoneuron size beyond the point of being recognizable as motoneurons. Therefore, the current study was undertaken to examine this possibility by repeating the experimental protocol from previous peripubertal studies, on adult male gerbils with the addition of retrograde tract-tracing. Adult male gerbils were castrated at postnatal day (PND) 150 and given subcutaneous implants of testosterone proprionate (TP), dihydrotestosterone (DHT), estradiol benzoate (EB), EB and DHT, no steroid, or left intact. At PND215, the animals were injected with fluorogold (FG), a retrograde tract-tracer. At PND220, the animals were aldehyde perfused transcardially. The spinal cords were sectioned and alternate sections processed for either thionin-stain or FG visualization. Results indicated that long-term castration had no effect on SNB motoneuron number but did decrease SNB motoneuron size. TP, and to a lesser extent DHT, treatment could prevent the reduction in motoneuron size, however, EB could not. This study indicates that the maintenance of the adult male gerbil SNB-BC system is androgen, not estrogen, dependent and that long-term castration does not reduce motoneuron size to the point where they can no longer be distinguished.     

Additive effects of ciliary neurotrophic factor and testosterone on motoneuron survival; differential effects on motoneuron size and muscle morphology

Testosterone and ciliary neurotrophic factor (CNTF) each enhance motoneuron survival in the spinal nucleus of the bulbocavernosus (SNB) of newborn rats. Here we directly compared the effects of CNTF and testosterone, alone and in combination, on SNB motoneuron number, SNB cell size, and morphology of the levator ani (LA) target muscle. Female rat pups were treated daily from postnatal day 1 through 6 (P1-P6) with recombinant human CNTF (hCNTF), testosterone propionate (TP), both hCNTF and TP, or neither. Effects of treatment were assessed on P7. TP and hCNTF each increased the number of SNB motoneurons and did so to a similar degree. Females treated with both hCNTF and TP had significantly more SNB cells than those receiving either hCNTF or TP alone. TP administered from P1 to P6 also increased SNB motoneuron size on P7. In contrast, hCNTF alone did not significantly affect SNB cell size, and hCNTF in combination with TP antagonized the effect of TP on motoneuron size. TP also increased LA muscle fiber number and LA fiber size, whereas hCNTF did not significantly influence LA muscle morphology in this study. Immunohistochemistry established that virtually all SNB motoneurons of both males and females express the CNTF alpha receptor (CNTFRalpha) between embryonic day 20 and postnatal day 6. Thus, effects of TP and hCNTF on SNB motoneuron survival were additive, and increases in motoneuron survival were dissociated from changes in target muscle morphology in hCNTF-treated animals. SNB motoneurons express CNTFRalpha perinatally and are therefore potential direct sites of hCNTF action.     

Characteristics of the spinal nucleus of the bulbocavernosus are influenced by genotype in the house mouse

Sex differences and differences related to genotype have been observed in the nervous system. These observations provide the opportunity to relate differences in neural structure to functional differences. The spinal nucleus of the bulbocavernosus (SNB) was examined in castrated and gonadally intact male house mice from the following 3 genotypes: C57B1/6J, DBA/2J, and the B6D2F1 (F1) hybrid (resulting from a cross between a C57B1/6J female and a DBA/2J male). The number and size of SNB neurons were determined from thionin-stained thick sections from spinal cords of these mice. The gonadally intact DBA males had significantly fewer SNB motoneurons than either the C57 or F1 males, but no strain differences were observed for the size of SNB cells. Castration of adults significantly reduced SNB neuronal number, but not somatic area, in the C57s (the maternal strain) and reduced somatic area, but not neuronal number, in the DBAs (the paternal strain). Both characteristics of the SNB were reduced by castration in the F1 hybrid. Thus, the size and number of SNB neurons appear to be inherited independently and the influence of gonadal hormones on these traits varies with genotype.     

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)     

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.