Lesley Marson

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.

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.

Identification of central nervous system neurons that innervate the bladder body, bladder base, or external urethral sphincter of female rats: A transneuronal tracing study using pseudorabies virus

Transneuronal tracing techniques were used to identify the putative spinal and brainstem neurons involved in continence and voiding in the female rat. Pseudorabies virus, Barthas K strain, was injected into either the external urethral sphincter, the bladder base, or the bladder body. After 3–5 days, the rats were perfused with fixative, and virus‐labelled cells were identified by using immunohistochemistry. External urethral sphincter (EUS) injections resulted in labelling of pudendal motoneurons in the dorsolateral nucleus of L6. Putative spinal interneurons were found in the medial cord from T13 to S1 and in the lateral gray of T13–L2 and L5–S1. After both bladder base and bladder body injections, the majority of pseudorabies virus‐labelled cells were found in the lateral gray and medial cord of L6–S1. A number of those found in the intermediolateral cell column resembled the parasympathetic preganglionic neurons; the remaining neurons in the lateral and medial gray were presumed to be interneurons. Very few pseudorabies virus‐labelled cells were found rostral to T10. In the brainstem, transneuronally labelled cells were found in the parapyramidal medullary reticular formation, Barringtons nucleus, raphe magnus, raphe pallidus, subcoeruleus pars alpha, locus coeruleus, the A5 noradrenergic cell group, and ventromedial periaqueductal gray after all injection sites. Pseudorabies virus‐labelled cells were also seen in the forebrain following the longest survival times; areas consistently labelled included the lateral hypothalamus, the parvocellular region of the paraventricular nucleus, and the medial preoptic area. These studies indicate that there is a substantial overlap of central nervous system neurons that innervate the EUS and the bladder in the female. J. Comp. Neurol. 389:584–602, 1997.

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.

Immunohistochemical localization of oxytocin receptors in human brain

The neuropeptide oxytocin (OT) regulates rodent, primate and human social behaviors and stress responses. OT binding studies employing (125)I-d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine vasotocin ((125)I-OTA), has been used to locate and quantify OT receptors (OTRs) in numerous areas of the rat brain. This ligand has also been applied to locating OTRs in the human brain. The results of the latter studies, however, have been brought into question because of subsequent evidence that (125)I-OTA is much less selective for OTR vs. vasopressin receptors in the primate brain. Previously we used a monoclonal antibody directed toward a region of the human OTR to demonstrate selective immunostaining of cell bodies and fibers in the preoptic-anterior hypothalamic area and ventral septum of a cynomolgus monkey (Boccia et al., 2001). The present study employed the same monoclonal antibody to study the location of OTRs in tissue blocks containing cortical, limbic and brainstem areas dissected from fixed adult, human female brains. OTRs were visualized in discrete cell bodies and/or fibers in the central and basolateral regions of the amygdala, medial preoptic area (MPOA), anterior and ventromedial hypothalamus, olfactory nucleus, vertical limb of the diagonal band, ventrolateral septum, anterior cingulate and hypoglossal and solitary nuclei. OTR staining was not observed in the hippocampus (including CA2 and CA3), parietal cortex, raphe nucleus, nucleus ambiguus or pons. These results suggest that there are some similarities, but also important differences, in the locations of OTRs in human and rodent brains. Immunohistochemistry (IHC) utilizing a monoclonal antibody provides specific localization of OTRs in the human brain and thereby provides opportunity to further study OTR in human development and psychiatric conditions.

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.

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.