Spinal Nucleus Of The Bulbocavernosus Neurons Research Articles

Sexual Differentiation of the Nervous System: Where the Action Is

Sexual Differentiation of the Nervous System: Where the Action Is

Neuronal Size in the Spinal Nucleus of the Bulbocavernosus: Direct Modulation by Androgen in Rats with Mosaic Androgen Insensitivity

The motoneurons of the spinal nucleus of the bulbocavernosus (SNB) and its target muscles, the bulbocavernosus and levator ani, form a sexually dimorphic circuit that is developmentally dependent on androgen exposure and exhibits numerous structural and functional changes in response to androgen exposure in adulthood. Castration of male adult rats causes shrinkage of SNB somata, and testosterone replacement reverses this effect, but the site at which androgen is acting to cause this change is undetermined. We exploited the X-chromosome residency of the androgen receptor (AR) gene to generate androgenized female rats that were heterozygous for the testicular feminization mutant (tfm) AR mutation and that, as a consequence of ontogenetic random X-inactivation, expressed a blend of androgen-sensitive wild-type cells and tfm-affected androgen-insensitive cells in the SNB. Chronic testosterone treatment of adult mosaics increased soma sizes only in androgen-competent wild-type SNB cells. The size of tfm-affected SNB somata in the same animals did not differ from the size of either the wild-type or tfm-affected SNB neurons in control mosaics that did not receive androgen treatment in adulthood. Because the muscle targets of the SNB are known to be uniformly androgen-sensitive in tfm mosaics, this mosaic analysis provides unambiguous evidence that androgenic effects on motoneuron soma size are mediated locally in the SNB. It is possible that the neuronal AR plays a permissive role in coordinating the actions of androgen.

Critical Periods of Sensitivity of Sexually Dimorphic Spinal Nuclei to Prenatal Testosterone Exposure in Female Rats

Male rats normally have more neurons than do females in two nuclei of the lumbar spinal cord, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN). Female rats exposed to testosterone propionate (TP) on the 2 days of gestation (Days 18 and 19) when males normally experience a surge in plasma testosterone showed a maximal increase in both SNB and DLN neuronal number. TP exposure just prior to, or following, Days 18 and 19 led to smaller increments. Administration of a small (5 μg) dose of TP after birth, while having no effect by itself, synergized with prenatal TP to enhance the number of SNB neurons. DLN neurons were less responsive to postnatal TP. The somal and nuclear size of SNB, but not DLN, neurons was increased by perinatal TP. Paradoxically, the number of DLN neurons with large somas (1358 μm2or larger) was reduced by perinatal TP, a finding congruent with a previous report that females and feminized males have more of these large DLN neurons than control males. Our data suggest an exquisite sensitivity of the developing spinal nuclei to the timing of hormonal surges normally found in fetal males. Exposure to androgens during a brief prenatal period is needed to assure responsiveness to the low amounts of androgen circulating during neonatal ontogeny, when the process of sexual differentiation is completed.

Prenatal flutamide alters sexually dimorphic nuclei in the spinal cord of male rats

The effects of prenatal exposure to the antiandrogen flutamide on two sexually dimorphic nuclei of the lumbar spinal cord, the dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB), were investigated. Rat dams were given daily injections of 5 mg flutamide or vehicle alone from day 11 through 21 of pregnancy. The spinal cords and perineal morphology of their male and female offspring were examined in adulthood. Flutamide reduced the number of SNB and DLN neurons, reduced the somal and nuclear area of SNB neurons, and reduced the weight of the perineal muscles in males. Flutamide produced no effect in females. No sexual dimorphism was found in the mean somal area of DLN neurons, but a sexual dimorphism was found in the distribution of somal areas in our samples; females had proportionately more large neurons than males. Flutamide-treated males also had proportionately more large neurons than control males but fewer than females. A sexual dimorphism was found in the nuclear areas of DLN neurons but flutamide did not influence this trait.

Target-dependent hormonal control of neuron size in the rat spinal nucleus of the bulbocavernosus

The spinal nucleus of the bulbocavernosus (SNB) in the rat is a cluster of sexually dimorphic motoneurons that innervate perineal muscles. In adult male rats, the size of SNB neurons is reduced following castration, and this effect is reversed by treatment with testosterone. However, androgen receptors are present in the perineal muscles as well as in SNB neurons. Therefore, it is not clear whether the neuronal size is regulated by direct action of the hormone on SNB neurons or by a target-derived factor that may be controlled via hormonal action on the innervated muscle. To address this question, the peripheral (pudendal) nerve of SNB neurons on one side was cut and united to the grafted soleus muscle, which lacks androgen sensitivity. On the control side, the pudendal nerve was similarly cut but was allowed to reinnervate the perineal muscles. The size of SNB neurons was measured on both sides after a postoperative period of 10 weeks, during which the animal had been castrated or treated with testosterone after castration. The size of SNB neurons that had reinnervated perineal muscles was reduced following castration and enlarged by testosterone treatment. In contrast, the size of SNB neurons that had innervated the soleus muscle remained unaltered in response to testosterone manipulation. It is concluded that hormonal regulation of the size of SNB neurons in adult rats is mediated by their target muscles.

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 αCGRP mRNA in the SNB motoneurons of adult male rats was studied with in situ hybridization. The number of motoneurons expressing αCGRP mRNA and the level of αCGRP mRNA expression was significantly higher in the SNB of castrated male rats than in the SNB of gonadally intact rats. Using a 5× background labeling criterion in castrated rats 88.1 ± 4.5% while in intact rats 75.3 ± 6.4% of SNB motoneurons expressed αCGRP mRNA. Testosterone replacement at the time of castration prevented the effect of castration on the expression of αCGRP 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 αCGRP mRNA in all SNB neurons.

The effect of castration on calcitonin gene-related peptide in spinal motor neurons.

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic nucleus of about 200 androgen-accumulating motor neurons in lumbar segments 5 and 6 of the rat spinal cord. In the present work we have used immunohistochemistry to investigate the change in calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in these motor neurons induced by long-term castration. CGRP-LI was found in the cell bodies of the SNB motor neurons and in thin varicose fibers coursing through the nucleus. In the intact animals 48.4 +/- 3.9% (n = 8) of the SNB neurons were CGRP-LI positive while in the castrates this ratio increased to 80.0 +/- 1.7% (n = 9, p less than 0.001). Animals castrated for 4 weeks and then implanted with testosterone-containing Silastic capsules for an additional 4 weeks had the same percentage of CGRP-LI-containing SNB motor neurons as intact animals. These results demonstrate that a reduction in circulating testosterone levels increases the number of SNB motor neurons that express CGRP-LI implying that androgen stimulation inhibits the expression of CGRP-LI in SNB motor neurons.

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.

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 (F 1) 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 F 1 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 F 1 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.

Hormonal control of a developing neuromuscular system. I. Complete Demasculinization of the male rat spinal nucleus of the bulbocavernosus using the anti-androgen flutamide

Prenatal treatment of male rats with the anti-androgen, flutamide (FL), demasculinizes the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) by reducing the number of SNB neurons, the size of the somas and nuclei of SNB neurons, and the size of their target muscles in adulthood. However, FL does not affect mounting or the traditional, postural measure of intromission, indicating that the SNB system does not play a major role in the mediation of these particular behaviors. Postnatal testosterone propionate (TP) treatment of male rats castrated on the day of birth results in more male copulatory behaviors in adulthood and masculinizes all measures of the SNB system. The postnatal masculinization by TP is more pronounced in males treated prenatally with FL, for morphological but not behavioral measures. The combined treatment of prenatal FL and day 1 castration without TP therapy results in a male with a completely demasculinized SNB system. Specifically, such males have SNB neurons that are as scarce and as small as those of females and, like females, they lack the target muscles of the SNB. These results support the hypothesis that perinatal androgens normally direct the sexually dimorphic development of the SNB and its target muscles.

Hormonal control of a developing neuromuscular system. II. Sensitive periods for the androgen-induced masculinization of the rat spinal nucleus of the bulbocavernosus

The spinal nucleus of the bulbocavernosus (SNB) and its target muscles are reduced or absent in normal female rats (Breedlove, S. M., and A. P. Arnold (1980) Science 210: 564-566). We now report that prenatal treatment of females with testosterone propionate (TP) significantly increases the number of SNB neurons found in adulthood. Dihydrotesterone propionate (DHTP) treatment just after but not before birth also masculinizes the number of SNB neurons in females. SNB soma size is significantly masculinized, i.e., enlarged, by administration of androgen prenatally or as late as 7 to 11 days after birth, even though this late postnatal treatment has no effect on the number of SNB cells. Following TP treatment in adulthood, the androgenized females did not display the postural correlates of male copulatory behavior more often than did control females. From these results we infer the following. (1) Androgens act both before and after birth to influence the sexually dimorphic development of the SNB system. (2) There are different sensitive periods for the masculinization of SNB neuronal number and neuronal size, indicating that these two dimorphic characteristics of the SNB are masculinized by somewhat independent mechanisms. (3) TP and DHTP may act via separate mechanisms to alter the number of SNB neurons. (4) Aromatized metabolites of testosterone are not necessary for masculinization of the SNB system. (5) Virilization of the SNB system does not ensure the masculinization of the traditionally defined measures of male copulatory behavior in rodents.

Sexually dimorphic motor nucleus in the rat lumbar spinal cord: Response to adult hormone manipulation, absence in androgen-insensitive rats

There is a sexually dimorphic motor nucleus in the fifth and sixth lumbar segments of the rat spinal cord, consisting of motoneurons innervating two striated perineal muscles, the levator ani and the bulbocavernosus. This nucleus, which is diminished or absent in female rats, has been named the spinal nucleus of the bulbocavernosus (SNB)3. We now report that the number of neurons in the SNB of either male or female rats is not altered by adult gonadectomy or treatment with testosterone propionate. However, the size of individual SNB neurons is increased in the presence of androgen in either sex. Genetically male rats with the testicular feminization mutation which results in reduced receptors have a markedly feminine SNB. These results support the hypothesis that the sexually dimorphic nature of the SNB depends on neither the adult hormonal state nor the presence of a Y chromosome, but on the interaction of androgens with their receptors early in development.