Kevin E. McKenna

The identification of a brainstem site controlling spinal sexual reflexes in male rats.

It has long been assumed that the brainstem exerts a tonic descending inhibitory influence on spinal sexual reflexes, but the source of this inhibition is unknown. Sexual responses (penile erection and ejaculatory movements) were elicited in anesthetized male rats using urethral stimuli. Using brainstem transections and electrolytic and neurotoxic lesions, we have identified a group of neurons in the paragigantocellular reticular nucleus in the ventral medulla which mediates this descending inhibition. Correlative neuroanatomical studies indicate that the inhibition is mediated via a direct projection to pelvic efferent neurons and interneurons. These results promise new approaches to the treatment of human sexual disorders.

Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barringtons nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

Central regulation of ejaculation.

Ejaculation is a reflex mediated by a spinal control center, referred to as a spinal ejaculation generator. This spinal ejaculation generator coordinates sympathetic, parasympathetic and motor outflow to induce the two phases of ejaculation, i.e., emission and expulsion. In addition, the spinal ejaculation generator integrates this outflow with inputs that are related to the summation of sexual activity prior to ejaculation that are required to trigger ejaculation. Recently, a group of spinothalamic neurons in the lumbar spinal cord (LSt cells) were demonstrated to comprise an integral part of the spinal ejaculation generator. Specifically, lesions of LSt cells completely ablate ejaculatory function. Moreover, LSt cells are activated following ejaculation, but not following other components of sexual behavior. Furthermore, based on their relationship with autonomic nuclei, motoneurons and genital sensory inputs, LSt cells are also in the ideal anatomical position to integrate sensory inputs and autonomic and motor outflow. Additionally, the spinal ejaculation generator is under inhibitory and excitatory influence of supraspinal sites, including the nucleus paragigantocellularis (nPGi), the paraventricular nucleus of the hypothalamus (PVN) and the medial preoptic area (MPOA). Finally, sensory information related to ejaculation is processed in the spinal cord and brain, possibly contributing to the rewarding properties of ejaculation. One candidate pathway for relay of ejaculation-related cues consists of LSt cells and their projections to the parvocellular subparafascicular thalamic nucleus. Moreover, neural activation specifically related to ejaculation is observed in the brain and may reflect of processing of ejaculation-related sensory cues.

A role for 5-hydroxytryptamine in descending inhibition of spinal sexual reflexes

SummaryNeurons in the region of the rostral nucleus paragigantocellularis (nPGi) mediate the inhibition of spinal sexual reflexes. Anatomical and pharmacological evidence is presented supporting a role for 5-hydroxytryptamine (5-HT) in this inhibition. Neurons in the rostral nPGi project to the ventral horn in the vicinity of the pudendal motoneurons. A significant number (78% ipsilateral) of these neurons contain 5-HT. Anterograde tracing with Phaseolus leucoagglutinin (PHA-L) confirmed the nPGi projection to pudendal motoneuron and interneuronal areas of the lumbar cord. 5-HT immunoreactive fibers and presumptive terminals surround the pudendal motoneurons. Urethral stimulation, in the anesthetized male rat, elicited penile erections, ejaculation and rhythmic contractions of the perineal muscles, we have used the term coitus reflex to describe this response. Intrathecal injection of 5-HT (4–50 µg) abolished the coitus reflex. Methysergide (1–10 mg/kg i.v.) prevented the 5-HT induced blockade of the coitus reflex. These data support the hypothesis that 5-HT is involved in the descending inhibition of spinal sexual reflexes.

CNS cell groups involved in the control of the ischiocavernosus and bulbospongiosus muscles: a transneuronal tracing study using pseudorabies virus.

Transneuronal tracing techniques were used to identify spinal and brainstem neurons involved in the control of perineal muscles in the male rat. Two penile muscles, the bulbospongiosus and ischiocavernosus muscles, were injected with Barthas strain of pseudorabies virus. After survival periods of 2, 4, and 5 days, the rats were killed and viral labeled neurons identified by immunohistochemistry. After a 2 day survival period, only pudendal motoneurons were labeled. More spinal and brainstem neurons were labeled at longer survival times. Putative spinal interneurons were found from T13 to S1. Large numbers of neurons were found in the lateral horn of the T13‐L2 and L6‐S1 segments which contain sympathetic and parasympathetic preganglionic neurons, respectively. However, retrograde labeling experiments verified that very few of the viral neurons were preganglionic neurons. Other labeled neurons were found in the intermediate cord, especially around the central canal. Relatively few labeled neurons were seen in the dorsal or ventral horn. In the brainstem, consistent labeling was seen in the ventrolateral medulla, raphe pallidus, and magnus, the A5 and locus ceruleus noradrenergic cell groups, Barringtons nucleus in the pontine tegmentum, the periaqueductal gray, and the paraventricular nucleus of the hypothalamus. The transneuronal labeling was consistent with what is currently known of the central nervous system (CNS) control of the perineal muscles.

Oxytocinergic innervation of autonomic nuclei controlling penile erection in the rat

In the rat, spinal autonomic neurons controlling penile erection receive descending pathways that modulate their activity. The paraventricular nucleus of the hypothalamus contributes oxytocinergic fibers to the dorsal horn and preganglionic sympathetic and parasympathetic cell columns. We used retrograde tracing techniques with pseudorabies virus combined with immunohistochemistry against oxytocin and radioligand binding detection of oxytocinergic receptors to evidence the oxytocinergic innervation of thoracolumbar and lumbosacral spinal neurons controlling penile erection. Spinal neurons labelled with pseudo-rabies virus transsynaptically transported from the corpus cavernosum were present in the intermediolateral cell column and the dorsal gray commissure of the thoracolumbar and lumbosacral spinal cord. Confocal laser scanning microscopic observation of the same preparations revealed close appositions between oxytocinergic varicosities and pseudorabies virus-infected neurons, suggesting strongly the presence of synaptic contacts. Electron microscopy confirmed this hypothesis. Oxytocin binding sites were present in the superficial layers of the dorsal horn, the dorsal gray commissure and the intermediolateral cell column in both the thoracolumbar and lumbosacral segments. In rats, stimulation of the paraventricular nucleus induces penile erection, but the link between the nucleus and penile innervation remains unknown. Our findings support the hypothesis that oxytocin, released by descending paraventriculo-spinal pathways, activates proerectile spinal neurons.

Stimulation of the hypothamus initiates the urethrogenital reflex in male rats

The urethrogenital (UG) reflex is a spinal sexual reflex which is tonically inhibited in the intact male rat by neurons in the nucleus paragigantocellularis (nPGi). The medial preoptic area of the hypothalamus (MPOA) is involved in the activation of male sexual behavior. The present study examines the effect of hypothalamic stimulation on the UG reflex in the intact male rat. Areas of the hypothalamus were stimulated bilaterally with either electrical stimulation or D,L-homocysteic acid (DLH) and the presence of the UG reflex examined. Stimulation of discrete aras of the hypothalamus evoked the UG reflex. The UG reflex could be initiated in the absence of genital stimulation. Microinjections of DLH into the MPOA also initiate the UG reflex. These data suggest that stimulation of neurons in the MPOA overcome the inhibition by the nPGi and facilitate spinal genital reflexes leading to ejaculation.

Lesions of the nucleus paragigantocellularis alter ex copula penile reflexes.

The effects of electrolytic lesions of the rostral nucleus paragigantocellularis (nPGi) were examined on ex copula sexual reflexes in male rats. Bilateral lesions of the nPGi significantly reduced (by 50%) the onset of the first ex copula reflex, which usually was a glans erection. In addition, the number of dorsiflexions (flips) was significantly increased. In the anesthetized spinally intact rat the urethrogenital reflex cannot be evoked. However, after chronic bilateral lesions of the rostral nPGi, half of the rats tested displayed the urethrogenital reflex prior to section of the spinal cord. These data support a role for the rostral nPGi in the descending inhibition of male sexual reflexes.

CHAPTER 33 – Neurobiology of Male Sexual Behavior

This chapter discusses the neurobiology of male sexual behavior. Sexual behavior in animals, including the courtship that precedes it, is characterized by enormous diversity. This diversity assures that mating will occur with the optimal partner at the most appropriate time and place, in order to pass parental genotypes onto the next generation. Male animals use species-specific displays to advertise their fitness and suitability as a partner. As they approach the female, they also gain information about her desirability and willingness to mate. In rodents, both partners may emit ultrasonic vocalizations, which are mutually arousing. A receptive female typically remains immobile while the male approaches from her rear, clasps her flanks with his forepaws, and initiates a series of shallow thrusts with his pelvis. Intromission is the defining event of copulation. If the male detects the females vagina, he will perform a deeper, intravaginal thrust, followed by a rapid, springing dismount. This behavior pattern is reliably associated with penile insertion and is used as the measure of intromission in rats and many other rodents. Most male mammals ejaculate only after receiving stimulation from multiple intromissions. Ejaculation is characterized behaviorally by a deeper, longer thrust, and a slow, relaxed dismount. Rhythmic contractions of skeletal and striated perineal muscles, including the bulbospongiosus, ischiocavernosus, and anal sphincter, usually accompany ejaculation. In human males and females, such muscle contractions are associated with orgasm, the subjective correlate of the culmination of sexual excitement.

5-Hydroxytryptamine2C receptors on spinal neurons controlling penile erection in the rat

The localization of 5-hydroxytryptamine2C receptors in the lumbosacral spinal cord of the rat was investigated using selective antibodies raised against the carboxyl-terminal part of the rat receptor. The distribution of immunoperoxidase labelling at the light microscope level revealed numerous labelled neurons in the gray matter, with a higher intensity in the sacral parasympathetic nucleus, the dorsal gray commissure and particularly the motoneurons of the ventral horn. Confocal microscope analysis showed that immunostaining was mainly intracellular (motoneurons), but could also be associated with the membrane of cell bodies and dendrites. Actually, electron microscope immunogold experiments demonstrated an exclusive staining of the cis-Golgi apparatus. Following pseudo-rabies virus transsynaptic retrograde labelling from the corpus cavernosum, labelled neurons were found in the sacral parasympathetic nucleus and the dorsal gray commissure of the L6-S1 segments. All virus-labelled neurons exhibited 5-hydroxytryptamine2C receptor immunoreactivity. These results indicate that all parasympathetic preganglionic neurons and their related interneurons which contribute to the innervation of cavernosal tissue bear 5-hydroxytryptamine2C receptors. In the sacral parasympathetic nucleus, most neurons which were retrogradely-labelled from the pelvic ganglion with Fast Blue also showed 5-hydroxytryptamine2C receptor immunoreactivity. In the ventral horn, motoneurons retrogradely labelled from the ischiocavernosus muscle and the bulbospongiosus muscle, both of which are involved in erection and ejaculation, were also 5-hydroxytryptamine2C receptor-immunopositive. The supraspinal serotoninergic control of erection at the lumbosacral level therefore appears to be strongly associated with the activation of 5-hydroxytryptamine2C receptors, consistent with the proerectile properties of 5-hydroxytryptamine2C agonists.